Anti-inflammatory interventions: a promising pathophysiological approach in the treatment of acute myocardial infarction?*

Wim K. Lagrand1,4, Remco Nijmeijer1,2,4, Paul A.J. Krijnen2,4,
Hans W.M. Niessen2,4, Cees A. Visser1,4, C. Erik Hack3,5

Departments of 1Cardiology, 2Pathology, and 3Clinical Chemistry,
VU University Medical Center, Amsterdam, The Netherlands
4ICaR-VU, Amsterdam, The Netherlands
5CLB, Sanquin Blood Supply Foundation and Department of Pathophysiology of Plasma Proteins, Amsterdam, The Netherlands
Correspondence: Dr Wim K. Lagrand, VU University Medical Center, Department of Cardiology, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Tel: +31 20 444 2244, fax +31 20 444 2446, e-mail wk.lagrand@vumc.nl

Introduction
Acute myocardial infarction (AMI) is one of the major causes of mortality and morbidity in the Western world. Mortality after AMI is due to arrhythmia, acute heart failure, and cardiac rupture, whereas morbidity often results from chronic heart failure. AMI is considered to be caused by myocardial cell death from oxygen depletion resulting from acute coronary occlusion by thrombus formation on preexisting atherosclerotic lesions. An important prognostic determinant is the total amount of myocardial necrosis, i.e. infarct size. Studies in animals have shown that irreversible myocardial cell injury starts about 30 min after occlusion of the coronary vessel and proceeds for hours. It is, however, remarkable that the primary oxygen deficit is only in part responsible for the total extent of myocardial necrosis (infarct size). The local processes in infarcted myocardium point to elicitation of immunologic reactions of both the a-specific and specific immunologic system. The local inflammatory response ensuing in the infarcted myocardium is characterized by the local production of chemotactic factors, the infiltration and activation of neutrophils, the local production of cytokines (such as tumor necrosis factor-alpha [TNFa], interleukin [IL]-6, and IL-8), elicitation of the acute-phase response, expression of adhesion molecules, and local activation of the complement system.[1] The later phase of myocardial cell injury, in part, results from these acute inflammatory reactions ensuing in the ischemic myocardium, as infarct size can be effectively reduced by anti-inflammatory agents. For example, corticosteroids given as late as 6 h after coronary occlusion can reduce infarct size by up to 35% in comparison with untreated control animals.[2]

Reperfusion injury
Early reperfusion of ischemic myocardium is a major goal in the treatment of AMI, since reperfusion results in an overall reduction in infarct size and a better prognosis in time. However, reperfusion of ischemic myocardium itself may induce inflammatory reactions, which, amongst others, involve further activation of complement and neutrophils, and the generation of oxygen radicals.[3,4] These inflammatory reactions may damage the cardiac tissue and limit the beneficial effects of a restored circulation (‘reperfusion injury’). Reperfusion therapy in AMI can therefore be regarded as a ‘double-edged sword’.
All observations, described above, have resulted in an increase in interest in the subject, since intervention in the inflammatory processes may provide new possibilities for (additional) treatment in patients with AMI.
In this manuscript we discuss potential therapeutic approaches for anti-inflammatory interventions in AMI. In Table I we have grouped and summarized studies in which anti-inflammatory interventions during AMI were investigated.

Corticosteroids
Corticosteroids such as prednisone and dexamethasone are potent inhibitors of the inflammatory response. As early as 1953, Johnson et al[5] reported the cardioprotective effects of cortisone by its ability to limit myocardial damage during myocardial infarction in dogs. In subsequent animal studies the infarct size-reducing effects of corticosteroids were also observed.[2,6,7] Several clinical trials with both positive and inconclusive results followed thereafter.[29,30] Corticosteroids, however, presumably because of their effects on cytokine and growth factor production, impair wound healing processes. Probably due to these effects, a higher incidence of left ventricular rupture was reported in some clinical trials in patients receiving high-dose corticosteroid therapy.[31,32] Because of these negative effects on wound healing and scar tissue formation, corticosteroids were considered inappropriate for the treatment of AMI in humans.

Neutrophils
During AMI, polymorphonuclear cells (PMN) infiltrate, accumulate, and degranulate in the infarcted parts of the myocardium. Results from animal studies point to an important role for PMN in the inflammatory reactions during AMI. Activated PMN are able to generate oxygen radicals and proteolytic enzymes, thereby exacerbating myocardial tissue injury.[8,9] Reduction, depletion, or inactivation of PMN during AMI indeed resulted in a significant reduction in myocardial necrosis in several animal models of AMI (Table I).[8–11] Also, inhibition of the infiltration of PMN into the infarcted myocardium, by inhibition of intercellular adhesion molecule-1 (ICAM-1) upregulation on endothelium, significantly reduced neutrophil activity locally in the ischemic myocardium in ischemia-reperfusion experiments, resulting in cardioprotection.[25]

Table I. Infarct reduction by inhibition of inflammation during AMI.



The complement system
The complement system consists of more than 30 serum and cellular proteins linked in three biochemical cascades: the classical, the alternative, and the mannan-binding lectin pathway (Figure 1).


Figure 1. The complement system.

All three pathways end in one final common pathway resulting in the formation of the membrane attack complex (MAC). Complement activation occurs during myocardial ischemia and infarction, which was first demonstrated by Hill and Ward[33] who showed that complement activation products generated in the infarcted myocardium were responsible for the infiltration of neutrophils. Later studies suggest that ischemic myocardium indeed activates complement: plasma levels of activated complement components increase in patients following AMI,[34] and several complement components become localized in the infarcted area during the course of AMI, as has been shown in animals as well as in humans.[3,13,35,36] Complement and its activation products, in particular C5a, have the ability to provoke stimulation, aggregation, and degranulation of PMN. Thus complement activation may be responsible for the progressive leukocyte capillary plugging during myocardial ischemia, which may impair full restoration of the capillary flow upon reperfusion, the so-called ‘no-reflow’ phenomenon.[37]
How the complement system during myocardial ischemia is activated is still unclear. Ammonia,[38] reperfusion,[39] and mitochondrial constituents[40] are possible activators, but thrombolytic agents are also able to activate complement.[41] In our own studies we have obtained evidence that C-reactive protein (CRP) is involved since CRP is able to activate complement in vitro as well as in vivo,[42] and CRP colocalizes with activated complement in infarcted sites of the myocardium during AMI.[43] Recently, Griselli et al[44] demonstrated that such a role for CRP in cardiovascular disease is very likely.
A detrimental role for complement is suggested by the presence of MAC on damaged muscle fibers in ischemic myocardial areas.[45] More conclusive evidence for such a role was obtained in rabbits deficient in complement factor C6, which cannot assemble a fully active MAC. These animals have a reduced infarct size in cardiac ischemia-reperfusion models compared with C6-sufficient rabbits.[46] MAC may mediate effects via various mechanisms. The pore-forming ability of the MAC on the cell membrane can cause direct cell lysis.[47] MAC, inserted in the cell membrane, facilitates the movement of the electrolytes across the cell membrane, resulting in a rapid increase in intracellular Ca2+ concentration, which enhances the rate of cell death.[48] A sudden influx of calcium may also be harmful to the cell in other ways, for example, by activation of calcium-dependent phospholipases, increase in ATPase activity, and the uncoupling of oxidative phosphorylation in mitochondria.[49] In addition to these direct cytotoxic effects, MAC at sublytic concentrations may also have a number of other effects on target cells, such as induction of cytokines and changes in prostaglandin production.[50] Thus, activation of complement by ischemic myocardium has pathogenic significance: complement activation products like the anaphylatoxins and MAC may have deleterious effects on the myocardium by mechanisms dependent on, and independent of, neutrophils, which may result in vasoconstriction, an impaired microcirculation, an increase in coronary perfusion pressure, ischemia, contractile failure of the myocardium, tachycardia, and impairment of atrioventricular conduction.[1,51,52]
Complement activation can also facilitate activation of the coagulation cascade. For example, MAC insertion in cell membranes is accompanied by the formation of membrane vesicles on the cell surface. These vesicles express binding sites for factor Va and support prothrombinase activity.[53] Complement activation with subsequent MAC formation can also result in the upregulation of tissue factor activity.[54] All these effects promote coagulation and are therefore potentially harmful in AMI.
The deleterious effects of complement activation products on the myocardium have been substantiated by observations that in animal models, complement inhibition before or shortly after permanent occlusion of a coronary vessel significantly reduces the amount of myocardial necrosis.[12,13] Administration of cobra venom factor (CoVF) in vivo rapidly produces profound and sustained depletion of C3 and C5.[55,56] As for a long time it was the only agent available to manipulate complement in animals, in most experimental studies of AMI, CoVF was used to inhibit complement activation. Cardioprotective effects of CoVF, resulting in reduced myocardial tissue damage, were also demonstrated in animal ischemia-reperfusion experiments.[12,13] These cardioprotective effects were accompanied by reduced deposition of complement factors, except for C4, in the ischemic myocardium.[13] Positive hemodynamic effects of CoVF, such as an increase in blood flow in the jeopardized area, with subsequent increase in oxygen utilization, have also been demonstrated.[57]
Administration of soluble complement receptor type 1 (sCR1) in rats exposed to ischemia-reperfusion of the heart, reduced both infarct size and the deposition of MAC in the infarcted myocardium.[14,15] Furthermore, infiltration of leukocytes in the ischemic myocardial areas was significantly attenuated in comparison with control animals,[14,15] as was also confirmed in a later study.[16] In the latter study, administration of sCR1 resulted in improved hemodynamic variables, eg better postischemic contractile function, after reperfusion of the ischemic myocardium.[16]
Inhibition of C5 activation, by a monoclonal antibody directed against C5, was found to prevent the formation of C5a and MAC in an ischemia-reperfusion model in rats.[22] This monoclonal antibody was shown to reduce infarct size significantly and reduce PMN infiltration locally in the infarcted myocardium. Interestingly, these phenomena were accompanied by reduced apoptosis in the ischemic area.[22] Administration of an antibody that specifically inhibits the activity of C5a resulted in improved hemodynamic parameters and less tissue injury (necrosis) after ischemia-reperfusion.[21] As expected, the deposition of MAC was not significantly changed by anti-C5a, in line with the specificity of the monoclonal antibody. The anti-C5a also inhibited (in vitro) neutrophil cytotoxic activity but not neutrophil accumulation in the ischemic myocardium, indicating that fragments of the complement system other than C5a contribute to this phenomenon.[21]
C1 esterase inhibitor (C1-INH) is a primary inhibitor of the classical pathway of the complement system. C1-INH is also an important regulator of the intrinsic pathway of coagulation. Hence, among complement inhibitors, C1-INH is unique in that it also inhibits other inflammatory systems. Moreover, this inhibitor does not impair the alternative pathway and does not prevent all defense functions of complement. Buerke et al[17,19] showed that C1-INH significantly reduced infarct size in an ischemia-reperfusion model in cats. Contractility of the heart was improved in comparison with control animals. Furthermore, PMN accumulation was shown to be reduced in the ischemic area. Intracoronary C1-INH treatment during ischemia-reperfusion reduced circulating C3 and slightly attenuated C5a plasma concentrations. This was accompanied by a significant reduction in plasma markers of myocardial cellular injury such as creatine kinase and troponin-T. No differences were observed with respect to global hemodynamic parameters, but local myocardial contractility was markedly improved in the ischemic zone in C1-INH-treated animals.[18,20] In our own studies, we have observed beneficial effects of C1-INH (reduction in infarct size by up to 40%) in a dog model for AMI, not only in ischemia-reperfusion, but particularly under conditions of permanent occlusion (Kleine et al, manuscript submitted for publication).
All studies discussed above show that inhibition of the complement system may markedly limit myocardial infarct size and improve myocardial function after AMI. Unfortunately, only limited data are available regarding a potentially detrimental role of complement inhibition in the formation of scar tissue in the infarcted myocardium after AMI, since insufficient scar formation would eliminate the clinical use of complement inhibitors in AMI such as was observed with corticosteroids (as discussed above). In one study, CoVF was shown to reduce slightly the ventricle wall thickness of the infarcted area in rats 3 weeks after induction of AMI.[12] Regarding C1-INH, we found no effect on scar formation in dogs treated with C1-INH 3 months after experimental AMI (Kleine et al, manuscript submitted for publication). In a limited clinical trial with C1-INH in patients with AMI, we observed promising effects with respect to infarct size (Hermens et al, manuscript in preparation). As described above, inhibition of complement activation reduces myocardial infarct size considerably in animals both after permanent coronary occlusion and during induction by reperfusion of ischemic myocardium.[14,21] Whether the molecular mechanisms underlying this ischemia-reperfusion-induced complement activation are similar to those occurring during permanent occlusion, remains to be established.

Adhesion molecules
Adhesion molecules are expressed on endothelial and inflammatory cells during AMI. This expression is probably initiated by the inflammatory reactions ensuing in the infarcted myocardium. P-, L- and E-selectins, CD11/ -CD18 and other (vascular and intercellular) adhesion molecules are expressed, both on endothelial and inflammatory cells (PMN). ICAM-1 effectively promotes the adherence of activated leukocytes, including PMN. Complement, in particular anaphylatoxin C5a, is able to upregulate ICAM-1 by endothelial cells.[58] ICAM-1 expression in ischemic myocardium is upregulated upon reperfusion.[59] In postmortem studies in humans who died following AMI we found increased ICAM-1 expression by nonviable cardiomyocytes in areas containing deposits of complement factors.[60] Moreover, the presence of CD66b on cardiomyocytes strongly suggested degranulation of PMN in these ICAM-1-positive areas, which was not observed in ICAM-1-negative areas (Figure 2).

Figure 2A. Localization of CD66b in extravascular nonadherent PMN. In this area of myocardial infarction cardiomyocytes stained positively for C3d but negatively for ICAM-1 (magnification ´250). Figure 2B. Localization of CD66b in PMN adhering to infarcted cardiomyocytes that stained positively for both C3d and ICAM-1. Positive staining for CD66b also was found in cardiomyocytes, unrelated to (adherent) PMN (magnification ´250).

 

Hence, ICAM-1 upregulation by cardiomyocytes may be an important event in the processes ultimately leading to the death of these cells. Although the precise trigger for ICAM-1 expression by ischemic cardiomyocytes is not known, the time sequence between complement deposition and the expression of ICAM-1 as well as the observation that ICAM-1 expression is strictly restricted to complement-positive areas, suggest that complement plays a role.
Blockade of ICAM-1 during reperfusion was shown to be cardioprotective through limitation of infarct size by inhibiting neutrophil adhesion to coronary endothelium.[26] Inhibition of neutrophil accumulation in the infarcted myocardium by use of an anti-CD18 monoclonal antibody resulted in a significantly smaller infarct size.[23] Reduction in endothelial P-selectin expression by N,N,N-trimethylsphingosine also significantly attenuated myocardial necrotic injury.[24]

Cytokines
During myocardial ischemia and infarction several cytokines are released by macrophages, endothelial cells, and fibroblasts of the jeopardized tissue. The main cytokines involved are IL-6, IL-8, and TNFa).61 IL-6 is the main cytokine initiating the acute-phase response. The acute-phase response is a well-known clinical phenomenon consisting of leukocytosis, fever, alterations in metabolism of many organs, and changes in plasma concentrations of various so-called acute-phase proteins.[62] The function of the acute-phase response is not well understood: possible hypotheses are prevention of ongoing tissue damage, neutralization of the inflammatory agent, or activation of tissue repair processes. IL-8 is a very potent chemoattractant and is thought to play an important role in the activation and transmigration into interstitium of neutrophils. Boyle et al[27] demonstrated that a specific monoclonal antibody that neutralizes IL-8 activity was able to reduce the degree of myocardial necrosis in rabbits subjected to ischemia-reperfusion. In agreement with the results described for anti-IL-8, Li et al[28] observed significant cardioprotective effects of anti-TNFa in a similar rabbit model for AMI.

Mechanisms of cell death
Cell death during AMI with reperfusion not only occurs via necrosis (‘accidental cell death’) but may also result from apoptosis (‘programmed cell death’).[63] In contrast to ‘accidental cell death’, apoptosis is energy-requiring and highly orchestrated by several regulatory proteins. The expression of two such proteins, bcl-2 and Bax, has been studied in human hearts from patients who died following AMI. Bcl-2, an apoptotic inhibitor, was found in the border zone of myocardial infarction. Bax, when overexpressed, a protein with proapoptotic abilities, was also found in these areas but especially in older infarctions. The Bax/bcl-2 ratio in these areas may therefore be an indicator of the extent of apoptotic activity.[64]
The link between inflammation and the different mechanisms of cell death is not fully understood. Recent experimental studies identified, for example, the MAC of the complement system as proapoptotic.[65] To what extent the MAC, and other inflammatory phenomena, contribute to apoptosis in ischemic myocardium remains to be determined. In line with this, the exact clinical and therapeutic implications of apoptotic phenomena for humans with AMI are still unclear. Inhibition of the caspase system, however, was found to result in a marked reduction in myocardial tissue damage.[63]

Conclusions
During myocardial ischemia, inflammatory reactions are elicited locally in the infarcted myocardium. These reactions comprise complicated interactions between ischemic cardiomyocytes, inflammatory cells (such as PMN), cytokines, complement factors, acute-phase proteins, and adhesion molecules. Whether the inflammatory reactions in humans with AMI with reperfusion may differ from those without reperfusion is still unclear, but in both situations the inflammatory reactions may considerably contribute to the final myocardial tissue injury. Anti-inflammatory interventions have been demonstrated to be effective in reducing overall infarct size. It is, however, notable that most results originate from animal studies, the results of which cannot be translated directly to the human situation.
Future studies should reveal whether anti-inflammatory interventions are efficacious and safe in humans with AMI. Analysis of these studies should be accurately performed since the corticosteroid studies in the 1970s have demonstrated that anti-inflammatory therapies may have very severe detrimental consequences.

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Section of Cardiovascular Sciences, Methodist Hospital, Houston, Texas.

Experimental models of acute ischemic myocardial injury indicate that the inflammatory response after the ischemic event contributes to tissue damage. This is especially apparent with reperfusion of the ischemic tissue. In such models some therapeutic strategies designed to reduce neutrophil accumulation or function have resulted in apparently beneficial effects. Although such findings are encouraging, interventions into these pathological processes using specific molecular targets will require greater understanding of specific mechanisms. Current evidence indicates that potential sites of therapeutic intervention will be found in pathways leading to complement activation, generation of lipid-derived mediators, adhesion of neutrophils to endothelial cells and cardiac myocytes, and activation of neutrophil secretory processes releasing, for example, proteolytic enzymes and reactive oxygen. Understanding the dynamic interplay between the mediators, adhesion pathways, and secretory processes that results in myocardial damage will allow a rational approach to controlling the detrimental inflammatory consequences of ischemia and reperfusion.

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Myocardial concentrations of C1q, a subunit of the first component of complement, were measured 5-120 minutes after ligation of a coronary artery in dogs injected with 125I-labeled human C1q and 131I-labeled human albumin. The 131I-labeled human serum albumin was used as a plasma protein marker. Ischemic regions of myocardium were defined by measuring regional myocardial blood flow by the reference sample method at intervals after coronary artery occlusion. Significant accumulations of 125I-C1q were demonstrated in the ischemic myocardium after coronary artery occlusions lasting 45 minutes. Some localization of C1q in ischemic myocardium was observed after a 15-minute occlusion, but the accumulations of C1q achieved in this case were not statistically significant. After coronary artery occlusions lasting greater than or equal to 45 minutes, left ventricular concentrations of C1q correlated reciprocally with regional myocardial blood flow. Moreover, high concentrations of C1q persisted in formerly ischemic segments after reperfusion. Radiolabeled neutrophils also accumulated selectively in ischemic segments relatively rich in C1q. It is suggested that complement activation may initiate the neutrophil-dependent portion of ischemic injury, delineated in recent years, that is associated with free radical release by phagocytic cells.

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4: Circ Res 1992 Dec;71(6):1518-24 Related Articles, Books, LinkOut

Kinetics of C5a release in cardiac lymph of dogs experiencing coronary artery ischemia-reperfusion injury.

Dreyer WJ, Michael LH, Nguyen T, Smith CW, Anderson DC, Entman ML, Rossen RD.

Department of Pediatrics, Methodist Hospital, Houston, Tex.

Previous studies of myocardial ischemia suggest that complement activation may play a central role in the inflammatory response during reperfusion. Our previous work has demonstrated neutrophil chemotactic activity to be present in reperfusion canine cardiac lymph after myocardial ischemia and infarction. To evaluate the contribution of the complement-dependent anaphylatoxin C5a to this neutrophil chemotactic activity, rabbit antiserum to canine C5a was prepared. At dilutions > 1:500 but < 1:2,000, the antiserum abolished the ability of zymosan-activated dog serum to induce a ruffled, bipolar morphology in isolated neutrophils used as a bioassay of chemotactic stimulation. This antiserum did not affect similar morphological changes in neutrophils exposed to platelet activating factor (10(-7)-10(-6) M) or recombinant human interleukin-8 (10(-9)-10(-8) M); thus, we deemed it functionally specific for canine C5a. In a pattern similar to what we previously reported, cardiac lymph collected before a 1-hour ligation of the left circumflex coronary artery had little ability to alter the morphology of canine neutrophils (shape change index, 11.3 +/- 4.6, mean +/- SEM; n = 7), but by 1 hour of reperfusion, lymph activated neutrophils significantly in five of seven dogs (mean shape change index, 72.6 +/- 17.7; p < 0.01). At 2 hours of reperfusion, neutrophil activation by lymph occurred in six of seven dogs (mean shape change index, 103.1 +/- 22.2). At 3 hours of reperfusion, cardiac lymph of only three of six dogs caused neutrophil activation, and at 4 hours of reperfusion, this activity was evident in lymph from only two of five dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

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Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog.

Romson JL, Hook BG, Kunkel SL, Abrams GD, Schork MA, Lucchesi BR.

Accumulation of polymorphonuclear neutrophils during the acute inflammatory response may exacerbate tissue injury through the release of activated oxygen products or proteolytic enzymes or both. To assess the role of neutrophils in acute myocardial infarction, circulating neutrophil levels in dogs were reduced by 77 +/- 2% (mean +/- SEM) by administering rabbit antiserum to dog neutrophils. Acute myocardial infarction was induced in open-chest anesthetized dogs by 90 minutes of left circumflex coronary artery occlusion followed by 6 hours of reperfusion. Dogs treated with neutrophil antiserum (n = 8) developed myocardial infarcts that were an average of 43% smaller than infarcts in dogs treated with nonimmune rabbit serum (n = 7) (27.0 +/- 4.5% vs 47.1% +/- 7.5% of the area at risk, p less than 0.05). In a saline-treated control group (n = 8), infarct size was 48.0 +/- 4.7% of the area at risk, a value not significantly different from that of the nonimmune serum group but significantly greater than that in the neutrophil antiserum dogs (p less than 0.05). There were no major hemodynamic differences between groups. Histopathologic examination revealed that infarcted myocardium from dogs given saline or treated with nonimmune serum had a substantial neutrophilic infiltrate, which was virtually absent in infarcted tissue from dogs treated with neutrophil antiserum. These observations suggest that neutrophil accumulation in response to myocardial ischemia may be responsible for a substantial portion of the irreversible myocardial injury resulting from temporary coronary artery occlusion.

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9: Am Heart J 1986 Oct;112(4):682-90 Related Articles, Books, LinkOut

Reduction of myocardial infarct size by neutrophil depletion: effect of duration of occlusion.

Jolly SR, Kane WJ, Hook BG, Abrams GD, Kunkel SL, Lucchesi BR.

Experiments were performed in the dog to examine the effects of neutropenia on ultimate infarct size resulting from short (90 minutes) or prolonged (4 hours) circumflex coronary artery occlusion. Sheep antiserum to canine neutrophils was used to produce neutropenia. Control animals received nonimmune serum. Neutrophil infiltration into myocardial infarcts was examined using histopathologic techniques and a semiquantitative scoring system. In 90-minute occlusions with 24-hour reperfusion, neutropenia was associated with the development of significantly smaller infarcts: normopenic group, 43.2% +/- 3.3% (n = 7) vs. neutropenic group, 26.6% +/- 3.7% (n = 10) of the area at risk, means +/- SEM. However, in 4-hour occlusion with 6-hour reperfusion experiments, the tendency of neutrophil depletion to reduce infarct size did not reach statistical significance (46.4% +/- 7.2% vs. 31.5% +/- 6.0% of the area at risk, normopenic vs. neutropenic) despite differences in neutrophil infiltration into the reperfused region. The observed differences in ultimate infarct size could not be attributed to differences in myocardial oxygen consumption. The results suggest that a significant amount of myocardial infarction induced by a limited duration of coronary artery occlusion followed by reperfusion is neutrophil dependent and appears to be less important in determining the fate of myocardium subjected to more prolonged periods of ischemia followed by reperfusion.

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10: J Clin Invest 1988 Feb;81(2):624-9 Related Articles, Books, LinkOut

Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (anti-Mo1, anti-CD11b) that inhibits leukocyte adhesion.

Simpson PJ, Todd RF 3rd, Fantone JC, Mickelson JK, Griffin JD, Lucchesi BR.

Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0010.

A monoclonal antibody (904) that binds to a leukocyte cell adhesion-promoting glycoprotein, (Mo1; CD11b/CD18) was administered (1 mg/kg, iv.) to open chest anesthetized dogs 45 min after the induction of regional myocardial ischemia. Ischemia was produced by occluding the left circumflex coronary artery (LCX) for 90 min and then reperfusing for 6 h. There was no difference between control and antibody treated groups with respect to arterial blood pressure, heart rate, or LCX blood flow. Administration of antibody produced no observable effect on circulating neutrophil counts, suggesting that antibody-bound neutrophils were not cleared from the circulation. The mean size of myocardial infarct expressed as percentage of the area at risk of infarction that resulted was reduced by 46% with anti-Mo1 treatment (25.8 +/- 4.7%, n = 8) compared to control (47.6 +/- 5.7%, n = 8; P less than 0.01). The area at risk of infarction was similar between groups. Circulating (serum) antibody excess was confirmed in all 8 anti-Mo1 treated dogs by immunofluorescence analysis. Analysis of ST segment elevation on the electrocardiogram as an indicator of the severity of ischemia suggests that the anti-Mo1 reduces infarct size independent of the severity of ischemia. An additional group of dogs (n = 5) was tested with a control monoclonal antibody of the same subtype (murine IgG1) and was found to produce no significant reduction in myocardial infarct size. Accumulation of neutrophils within the myocardium was significantly attenuated with 904 treatment when analyzed by histological methods. These data demonstrate that administration of anti-Mo1 monoclonal antibody after the induction of regional myocardial ischemia results in reduced myocardial reperfusion injury as measured by ultimate infarct size.

PMID: 3339135 [PubMed - indexed for MEDLINE]
 
11: Circulation 1989 Dec;80(6):1816-27 Related Articles, Books, LinkOut

Neutrophil depletion limited to reperfusion reduces myocardial infarct size after 90 minutes of ischemia. Evidence for neutrophil-mediated reperfusion injury.

Litt MR, Jeremy RW, Weisman HF, Winkelstein JA, Becker LC.

Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, MD 21205.

Reperfusion of ischemic myocardium may accelerate necrosis of injured myocytes. To determine the role of neutrophil leukocytes in this process, we examined whether neutrophil depletion during reperfusion could modify infarct size in anesthetized dogs. The proximal circumflex coronary artery was occluded for 90 minutes and then reperfused for 2 hours via an extracorporeal circuit with either whole blood (n = 11) or with blood depleted of neutrophils by leukocyte filters (n = 11). The leukocyte filters caused near-total neutropenia in blood reperfusing the ischemic myocardium (7 +/- 7 neutrophils/microliters compared with 2,551 +/- 317/microliters in controls, mean +/- SEM; p less than 0.001. Infarct size was measured by planimetry of myocardial slices stained with triphenyltetrazolium chloride (TTC), and the accuracy of TTC for identifying necrotic myocardium was verified by electron microscopy. The size of the ischemic risk region was the same in the control (41.6 +/- 1.0%) and neutropenic (41.8 +/- 2.1%) groups. Collateral blood flow to the risk region was the same in control (0.15 +/- 0.03 ml/min/g) and neutropenic (0.13 +/- 0.03 ml/min/g) groups. Among dogs with collateral flow less than 0.2 ml/min/g, infarct size was reduced in the neutropenic group (27.7 +/- 6.7% of risk region, n = 8), compared with control dogs (52.5 +/- 5.7%; n = 7; p = 0.02). Multiple linear regression described the relation between infarct size, risk region size, and collateral flow in the control group, and the same regression relation was used to predict infarct size for the neutropenic group. Mean predicted infarct size in the neutropenic group (n = 11) was 16.8 +/- 3.4% of left ventricle, whereas mean observed infarct size was 9.6 +/- 3.1% (p less than 0.01). The extent of the no-reflow zone (absence of thioflavin-S-fluorescence) was also less in the neutropenic than the control group (2.2 +/- 0.8% vs. 8.1 +/- 2.7% of the risk region, p less than 0.05). Neutropenia limited to the reperfusion period is associated with significant reductions in the extent of the infarct and no-reflow zones after 90 minutes of ischemia. These findings support the hypothesis that reperfusion necrosis occurs after prolonged myocardial ischemia and indicate that neutrophil leukocytes are important mediators of such reperfusion injury.

PMID: 2598440 [PubMed - indexed for MEDLINE]
 
12: J Clin Invest 1978 Mar;61(3):661-70 Related Articles, Books, LinkOut

Reduction by cobra venom factor of myocardial necrosis after coronary artery occlusion.

Maroko PR, Carpenter CB, Chiariello M, Fishbein MC, Radvany P, Knostman JD, Hale SL.

PMID: 641147 [PubMed - indexed for MEDLINE]
 
13: Circulation 1988 Dec;78(6):1449-58 Related Articles, Books, LinkOut

Complement and neutrophil activation in the pathogenesis of ischemic myocardial injury.

Crawford MH, Grover FL, Kolb WP, McMahan CA, O'Rourke RA, McManus LM, Pinckard RN.

Department of Medicine, University of Texas Health Science Center, San Antonio 78284-7872.

Complement depletion with cobra venom factor (CVF) before coronary artery ligation has been previously shown to reduce subsequent ischemic myocardial tissue injury in the baboon; however, whether complement depletion after the initiation of acute myocardial ischemia affords similar myocardial preservation is not known. Both complement depletion with CVF or the administration of certain nonsteroidal anti-inflammatory drugs, including ibuprofen, are thought to decrease myocardial infarct size by reducing polymorphonuclear leukocytic (PMN) infiltration; nevertheless, complement activation also could alter tissue injury by PMN-independent actions. Thus, the relative effects of CVF administered after coronary artery ligation on the subsequent development of myocardial tissue injury were assessed in a baboon myocardial infarction model. The animals were randomized into three treatment groups (n = 6): either CVF (125 units/kg) or saline was given 30 minutes after coronary artery ligation, and ibuprofen (12.5 mg/kg) was administered 30 minutes and 4 hours after ligation. The extent of ischemic myocardial injury was assessed 24 hours later. Relative to saline-treated baboons, both CVF and ibuprofen reduced PMN infiltration (36 +/- 4 vs. 24 +/- 4 and 24 +/- 4 PMN/mm2, respectively; mean +/- SEM) and histological evidence of transmural myocardial infarction (100% vs. 47% and 53%, respectively) in electrocardiographically designated, expected infarct sites. In both saline- and ibuprofen-treated animals, there was extensive localization of C4, C3, and C5 in all infarct sites; in contrast, there was only C4 localization in the CVF-treated baboons. When expected infarct sites were assessed for creatine kinase content as an indicator of tissue injury, there was significantly less epicardial and endocardial creatine kinase depletion in the CVF-treated animals (31.7 +/- 5.6% and 39.3 +/- 4.8%) than in the saline-treated animals (54.1 +/- 5.4% and 59.0 +/- 4.7%; p = 0.012 and 0.011, respectively). The percent creatine kinase depletion in the ibuprofen-treated animals was intermediate between the two other groups. These results suggest that depletion of complement after coronary ligation has beneficial effects in reducing tissue injury that cannot be explained solely on the basis of reducing PMN infiltration into the ischemic myocardium.

PMID: 3191598 [PubMed - indexed for MEDLINE]
 
14: Trans Assoc Am Physicians 1990;103:64-72 Related Articles, Books, LinkOut

Recombinant soluble CR1 suppressed complement activation, inflammation, and necrosis associated with reperfusion of ischemic myocardium.

Weisman HF, Bartow T, Leppo MK, Boyle MP, Marsh HC Jr, Carson GR, Roux KH, Weisfeldt ML, Fearon DT.

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

In summary, conversion of wild-type CR1 to a soluble form (sCR1) creates a potent inhibitor of complement activation by both the classical and alternative pathways by inhibiting the C3/C5 convertases. In the rat reperfusion infarct model, sCR1 significantly suppresses complement activation at the endothelial surface of capillaries and venules. This suppression of complement activation is accompanied by reduced accumulation of leukocytes within the infarct zone, perhaps because of reduction of the generation of C5a, which promotes expression of leukocyte adhesion receptors and leukocyte chemotaxis. In addition, formation of the C5b-9 attack complex, which may contribute to direct endothelial injury, was suppressed by sCR1. The inhibition of complement activation and leukocyte infiltration by sCR1 explains the observed significant reduction in myocardial necrosis after ischemia and reperfusion. These studies have identified sCR1 as a potential agent for therapeutic intervention in diseases associated with complement-dependent tissue injury.

PMID: 2132543 [PubMed - indexed for MEDLINE]
 
15: Science 1990 Jul 13;249(4965):146-51 Related Articles, Books, LinkOut

Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis.

Weisman HF, Bartow T, Leppo MK, Marsh HC Jr, Carson GR, Concino MF, Boyle MP, Roux KH, Weisfeldt ML, Fearon DT.

Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.

The complement system is an important mediator of the acute inflammatory response, and an effective inhibitor would suppress tissue damage in many autoimmune and inflammatory diseases. Such an inhibitor might be found among the endogenous regulatory proteins of complement that block the enzymes that activate C3 and C5. Of these proteins, complement receptor type 1 (CR1; CD35) has the most inhibitory potential, but its restriction to a few cell types limits its function in vivo. This limitation was overcome by the recombinant, soluble human CR1, sCR1, which lacks the transmembrane and cytoplasmic domains. The sCR1 bivalently bound dimeric forms of its ligands, C3b and methylamine-treated C4 (C4-ma), and promoted their inactivation by factor I. In nanomolar concentrations, sCR1 blocked complement activation in human serum by the two pathways. The sCR1 had complement inhibitory and anti-inflammatory activities in a rat model of reperfusion injury of ischemic myocardium, reducing myocardial infarction size by 44 percent. These findings identify sCR1 as a potential agent for the suppression of complement-dependent tissue injury in autoimmune and inflammatory diseases.

PMID: 2371562 [PubMed - indexed for MEDLINE]
 
16: Circulation 1993 Dec;88(6):2812-26 Related Articles, Books, LinkOut

Soluble complement receptor type 1 inhibits the complement pathway and prevents contractile failure in the postischemic heart. Evidence that complement activation is required for neutrophil-mediated reperfusion injury.

Shandelya SM, Kuppusamy P, Herskowitz A, Weisfeldt ML, Zweier JL.

Department of Medicine, Johns Hopkins Medical-Institutions, Francis Scott Key Medical Center, Baltimore, MD 21224.

BACKGROUND. Complement-mediated neutrophil activation has been hypothesized to be an important mechanism of reperfusion injury. It has been proposed that soluble complement receptor 1 (sCR1), a potent inhibitor of both classical and alternative complement pathways, may prevent the complement-dependent activation of polymorphonuclear leukocytes (PMNs) that occurs within postischemic myocardium and thereby inhibit PMN-derived free radical generation and prevent postischemic contractile failure. Therefore, we performed studies to determine the effects of sCR1 on contractile function, PMN adhesion, complement deposition, and PMN-derived free radical generation in the postischemic heart. METHODS AND RESULTS. Studies were performed in an isolated rat heart model in which the isolated effects of given cellular or humoral factors could be determined. Plasma and PMNs were present to study the effects of sCR1 on contractile function, coronary flow, leukocyte adhesion, complement deposition, and PMN-derived free radical generation. Isolated rat hearts were perfused by the method of Langendorff (n = 10 in each group) and subjected to 20 minutes of global ischemia and reperfusion with PMNs and plasma in the presence or absence of sCR1. Left ventricular developed pressure (LVDP), coronary flow (CF), left ventricular end-diastolic pressure (LVEDP), and rate-pressure product (RPP) were measured during the preischemic period, during 1-minute control infusion of PMNs and plasma, and on reflow following 20 minutes of global ischemia. During the preischemic control infusion, no significant alterations in the physiologic parameters were observed, and there was no measurable free radical generation. Reperfusion with sCR1 markedly improved the recovery of postischemic contractile function. LVDP after 45 minutes of reperfusion was 76 +/- 9.8% compared with 32 +/- 6.2% (P < .001). In addition, significant improvements in LVEDP, RPP, and CF were observed in hearts treated with sCR1. Additional experiments were also performed to determine the effect of sCR1 on complement-mediated PMN activation. Measurements of PMN-derived free radical generation were performed in both isolated PMNs and the coronary effluent of hearts using electron paramagnetic resonance spectroscopy (EPR) with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). EPR measurements in both isolated PMNs and coronary effluent demonstrated that sCR1 blocked complement-mediated free radical generation from the PMNs. Increased accumulation of PMNs was observed both in hearts treated with sCR1 and in those not treated with sCR1. Immunohistochemical staining of the postischemic myocardial tissue demonstrated marked complement deposition on the endothelial surface of small arterioles and capillaries, which was prevented by sCR1 treatment. Thus, sCR1 did not prevent PMN adhesion but did prevent complement deposition with activation of the PMN oxidative burst. CONCLUSIONS. The potent complement inhibitor sCR1 was found to be effective at preventing postischemic myocardial contractile dysfunction and enhancing the recovery of coronary flow. This study demonstrated that complement activation occurs in postischemic myocardium and is necessary for activation of the neutrophil oxidative burst with the generation of reactive oxygen free radicals. The process of neutrophil adhesion, however, was not affected by sCR1 and was independent of complement factors. These findings demonstrate the sCR1 is a highly potent agent at preventing complement-mediated PMN activation and secondary free radical generation in the postischemic heart. This genetically engineered protein appears to be a promising therapeutic agent in the prevention of myocardial reperfusion injury.

PMID: 8252695 [PubMed - indexed for MEDLINE]
 
17: Circulation 1995 Jan 15;91(2):393-402 Related Articles, Books, LinkOut

Comment in:


Cardioprotective effects of a C1 esterase inhibitor in myocardial ischemia and reperfusion.

Buerke M, Murohara T, Lefer AM.

Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pa. 19107.

BACKGROUND: Myocardial injury after ischemia and reperfusion can be attributed largely to the effects of polymorphonuclear leukocytes (PMN). The complement system plays an important role as a chemotactic agent, affecting adhesion molecule expression and neutrophil accumulation. METHODS AND RESULTS: In the present study, the cardioprotective effects of C1 esterase inhibitor (C1 INH) were examined in a feline model of myocardial ischemia and reperfusion (90 minutes of ischemia followed by 270 minutes of reperfusion). C1 INH (15 mg/kg) administered 10 minutes before reperfusion significantly attenuated myocardial necrosis compared with vehicle (10 +/- 2% and 29 +/- 2% necrosis as a proportion of area at risk, respectively; P < .01). Myocardial preservation was also related to reduced plasma accumulation of creatine kinase activity. C1 INH treatment resulted in improved recovery of cardiac contractility and preservation of coronary vascular endothelial function, as assessed by relaxation in response to acetylcholine, compared with contractility and preservation of endothelial function in vehicle-treated animals (69 +/- 6% and 20 +/- 4% relaxation, respectively; P < .01). In addition, cardiac myeloperoxidase activity (an index of PMN accumulation) in the ischemic area was significantly reduced after C1 INH treatment. Furthermore, immunohistochemical analysis of ischemic-reperfused myocardial tissue demonstrated deposition of the first component of the classic complement pathway, C1q, on cardiac myocytes and coronary vessels. CONCLUSIONS: Blocking of the classic complement pathway by C1 INH appears to be an effective means of preserving ischemic myocardium from reperfusion injury. The mechanism of this cardioprotective effect appears to be inhibition of PMN-endothelium interaction; this inhibition leads to preservation of normal endothelial function, which results in reduced cardiac necrosis.

PMID: 7805243 [PubMed - indexed for MEDLINE]

 
18: Circulation 1997 Feb 4;95(3):701-8 Related Articles, Books, LinkOut

Intracoronary application of C1 esterase inhibitor improves cardiac function and reduces myocardial necrosis in an experimental model of ischemia and reperfusion.

Horstick G, Heimann A, Gotze O, Hafner G, Berg O, Boehmer P, Becker P, Darius H, Rupprecht HJ, Loos M, Bhakdi S, Meyer J, Kempski O.

Johannes Gutenberg-University Mainz, Second Medical Clinic, Germany.

BACKGROUND: Myocardial injury from ischemia can be aggravated by reperfusion of the jeopardized area. The precise underlying mechanisms have not been clearly defined, but proinflammatory events, including complement activation, leukocyte adhesion, and infiltration and release of diverse mediators, probably play important roles. The present study addresses the possibility of reducing reperfusion damage by the application of C1 esterase inhibitor (C1-INH). METHODS AND RESULTS: Cardioprotection by C1-INH 20 IU/kg IC was examined in a pig model with 60 minutes of coronary occlusion, followed by 120 minutes of reperfusion. C1-INH was administered during the first 5 minutes of coronary reperfusion Compared with the NaCl controls, C1-INH reduced myocardial injury (48.8 +/- 7.8% versus 73.4 +/- 4.0% necrosis of area at risk, P < or = .018). C1-INH treatment significantly reduced circulating C3a and slightly attenuated C5a plasma concentrations. Myocardial protection was accompanied by reduced plasma concentration of creatine kinase and troponin-T. C1-INH had no effect on global hemodynamic parameters, but local myocardial contractility was markedly improved in the ischemic zone. In the short-axis view, 137 degrees of the anteroseptal region showed significantly improved wall motion at early and 29 degrees at late reperfusion with C1-INH treatment. CONCLUSIONS: C1-INH significantly protects ischemic tissue from reperfusion damage, reduces myocardial necrosis, and improves local cardiac function.

PMID: 9024160 [PubMed - indexed for MEDLINE]
 
19: J Pharmacol Exp Ther 1998 Jul;286(1):429-38 Related Articles, Books, LinkOut

Blocking of classical complement pathway inhibits endothelial adhesion molecule expression and preserves ischemic myocardium from reperfusion injury.

Buerke M, Prufer D, Dahm M, Oelert H, Meyer J, Darius H.

II. Department of Medicine, Johannes Gutenberg University, Mainz, Germany.

Myocardial injury after ischemia (I) and reperfusion (R) is related to leukocyte activation with subsequent release of cytokines and oxygen-derived free radicals as well as complement activation. In our study, the cardioprotective effects of exogenous C1 esterase inhibitor (C1 INH) were examined in a rat model of myocardial I + R (i.e., 20 min + 24 hr or 48 hr). The C1 INH (10, 50 and 100 U/kg) administered 2 min before reperfusion significantly attenuated myocardial injury after 24 hr of R compared to vehicle treated rats (P < .001). Further, cardiac myeloperoxidase activity (i.e., a marker of PMN [polymorphonuclear leukocyte] accumulation) in the ischemic area was significantly reduced after C1 INH treatment compared to vehicle treated animals (0.81 +/- 0.1, 0.34 +/- 0.13, 0.13 +/- 0.1 vs. 1.44 +/- 0.3 U/100 mg tissue, P < .001). In addition, C1 INH (100 U/kg) significantly attenuated myocardial injury and neutrophil infiltration even after 48 hr of reperfusion compared to vehicle treatment. Immunohistochemical analysis of ischemic-reperfused myocardial tissue demonstrated activation of classical complement pathway by deposition of C1q on cardiac myocytes and cardiac vessels. In addition, expression of the endothelial adhesion molecules P-selectin and intercellular adhesion molecule 1 (ICAM-1) was observed after reperfusion of the ischemic myocardium. In this regard, C1 INH administration abolished expression of P-selectin and ICAM-1 on the cardiac vasculature after myocardial ischemia and reperfusion. Blocking the classical complement pathway by exogenous C1 INH appears to be an effective means to preserve ischemic myocardium from injury after 24 and 48 hr of reperfusion. The mechanisms of this cardioprotective effect appears to be due to blocking of complement activation and reduced endothelial adhesion molecule expression with subsequent reduced PMN-endothelium interaction, resulting in diminished cardiac necrosis.

PMID: 9655888 [PubMed - indexed for MEDLINE]
 
20: Methods Find Exp Clin Pharmacol 1995 Oct;17(8):499-507 Related Articles, Books, LinkOut

Cardioprotective effects of selective inhibition of the two complement activation pathways in myocardial ischemia and reperfusion injury.

Murohara T, Guo JP, Delyani JA, Lefer AM.

Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA.

The complement (C) system-mediated neutrophil activation, adhesion to the coronary endothelium and accumulation into cardiac tissue are key steps in the pathogenesis of myocardial ischemia-reperfusion (MI/R) injury. We examined the differential role of the classical and the alternative complement pathway in MI/R injury in vivo. Rats were subjected to 20 min of myocardial ischemia followed by 24 h of reperfusion. Either a classical pathway inhibitor [C1 esterase inhibitor (C1-INH) (15 mg/kg)] or an alternative pathway inhibitor soluble complement receptor 1 (sCR1)[des-LHR-A](15 mg/kg) or their vehicle were administered intravenously 1 min prior to reperfusion, and myocardial necrosis (creatine kinase loss) and neutrophil accumulation, cardiac myeloperoxidase activity, were examined. C1-INH significantly attenuated cardiac creatine kinase loss compared to MI/R rats given only vehicle (p < 0.05) 24 h after reperfusion. An alternative pathway inhibitor, sCR1 [des-LHR-A] attenuated myocardial injury to a lesser extent, although it was not significantly different from the value for C1-INH or vehicle. Besides cardiac myeloperoxidase activity, the ischemic cardiac tissue was significantly attenuated by both C1-INH and sCR1[desLHR-A] (p < 0.05 vs. vehicle). Both the classical and alternative pathways may contribute to MI/R injury via a neutrophil-dependent mechanism in vivo. Selective inhibition of the classical pathway of complement activation seems to be slightly more effective in limiting necrotic MI/R injury than the selective alternative pathway inhibition in this 24 h model of reperfusion injury, but equal doses of each inhibitor attenuated neutrophil accumulation.

PMID: 8749222 [PubMed - indexed for MEDLINE]
 
21: Am J Physiol 1995 Jan;268(1 Pt 2):H448-57 Related Articles, Books, LinkOut

Limitation of reperfusion injury by a monoclonal antibody to C5a during myocardial infarction in pigs.

Amsterdam EA, Stahl GL, Pan HL, Rendig SV, Fletcher MP, Longhurst JC.

Division of Cardiovascular Medicine, University of California, Davis 95616.

The complement system has been implicated in reperfusion injury during acute myocardial infarction. We therefore attempted to reduce reperfusion injury with a monoclonal antibody (MAb) to the complement component, C5a. In 13 control pigs and 9 pigs pretreated with this MAb, ischemia was induced by a 50-min occlusion of the left anterior descending coronary artery, followed by 3 h of reperfusion. Infarct area (as percent of risk area) was reduced from 58 +/- 5% in controls to 38 +/- 7% (P < 0.05) in MAb-treated animals. Heart rate-systolic blood pressure product, left ventricular (LV) first derivative of pressure, LV end-diastolic pressure, and coronary blood flow were similar (P > 0.05) in the two groups. At 15 min of reperfusion, immunoreactive factor Bb began to increase significantly (P < 0.05) in regional coronary venous plasma, consistent with activation of the alternative complement pathway. The anti-C5a MAb did not attenuate formation of the membrane attack complex (C5b-9) as assessed by a hemolytic complement assay. Myocardial myeloperoxidase activity, a marker of tissue neutrophil concentration, was similar in the risk regions of the two groups, suggesting that neutrophil infiltration was unaltered by the MAb. However, in vitro the MAb (15 and 30 micrograms/ml) reduced C5a-stimulated neutrophil aggregation (67.4 and 70.9%), chemotaxis (52.5 and 81.4%), degranulation (66.7 and 75.8%), and superoxide generation (26.7 and 100%). In conclusion, myocardial infarction-reperfusion is associated with activation of the alternative complement pathway. Furthermore, a MAb to C5a that inhibits neutrophil cytotoxic activity, but neither the membrane attack complex nor myocardial neutrophil accumulation, decreases infarct size in pigs. These data suggest an important role of the alternative complement pathway and C5a in the propagation of ischemia cardiac damage during reperfusion.

PMID: 7840295 [PubMed - indexed for MEDLINE]
 
22: Circulation 1998 Jun 9;97(22):2259-67 Related Articles, Books, LinkOut

Myocardial infarction and apoptosis after myocardial ischemia and reperfusion: role of the terminal complement components and inhibition by anti-C5 therapy.

Vakeva AP, Agah A, Rollins SA, Matis LA, Li L, Stahl GL.

Haartman Institute, Department of Bacteriology and Immunology, University of Helsinki, Finland.

BACKGROUND: Myocardial ischemia and reperfusion (MI/R)-induced tissue injury involves necrosis and apoptosis. However, the precise contribution of apoptosis to cell death, as well as the mechanism of apoptosis induction, has not been delineated. In this study, we sought to define the contribution of the activated terminal complement components to apoptosis and necrosis in a rat model of MI/R injury. METHODS AND RESULTS: Monoclonal antibodies (mAbs; 18A and 16C) raised against the rat C5 complement component bound to purified rat C5 (ELISA). 18A effectively blocked C5b-9-mediated cell lysis and C5a-induced chemotaxis of rat polymorphonuclear leukocytes (PMNs), whereas 16C had no complement inhibitor activity. A single dose (20 mg/kg i.v.) of 18A blocked >80% of serum hemolytic activity for >4 hours. Administration of 18A before myocardial ischemia (30 minutes) and reperfusion (4 hours) significantly reduced (91%) left ventricular free wall PMN infiltration compared with 16C treatment. Treatment with 18A 1 hour before ischemia or 5 minutes before reperfusion significantly reduced infarct size compared with 16C treatment. A significant reduction in infarct size (42%) was also observed in 18A-treated rats after 30 minutes of ischemia and 7 days of reperfusion. DNA ladders and DNA labeling (eg, TUNEL assay) demonstrated a dramatic reduction in MI/R-induced apoptosis in 18A-treated compared with 16C-treated rats. CONCLUSIONS: Anti-C5 therapy in the setting of MI/R significantly inhibits cell apoptosis, necrosis, and PMN infiltration in the rat despite C3 deposition. We conclude that the terminal complement components C5a and C5b-9 are key mediators of tissue injury in MI/R.

PMID: 9631876 [PubMed - indexed for MEDLINE]
 
23: Br J Pharmacol 1994 Apr;111(4):1123-8 Related Articles, Books, LinkOut

Effect of duration of ischaemia on reduction of myocardial infarct size by inhibition of neutrophil accumulation using an anti-CD18 monoclonal antibody.

Williams FM, Kus M, Tanda K, Williams TJ.

Department of Applied Pharmacology, National Heart & Lung Institute, London.

1. Neutrophil accumulation is a characteristic feature of the inflammatory response in myocardial tissue which has undergone a period of ischaemia. The aim of this study was to examine whether inhibition of myocardial neutrophil infiltration, using an antibody to the CD18 leukocyte adhesion molecule, was effective in reducing infarct size in anaesthetized rabbits. 2. Anaesthetized rabbits underwent coronary artery occlusion (CAO) for periods of 30 or 45 min followed by reperfusion for 3 h. Animals were treated intravenously 10 min prior to reperfusion with IB4, a monoclonal antibody to CD18 (1 mg kg-1) or saline (1 ml kg-1). In one group undergoing 45 min CAO, a control antibody, OKMI (1 mg kg-1) was given. 3. Following either 30 or 45 min of CAO, administration of IB4 resulted in a < 75% inhibition in neutrophil accumulation in the area at risk myocardium (AR) compared with control animals. 4. With the 30 min occlusion period, IB4 significantly reduced myocardial infarct size, 27.2 +/- 3.2% vs 67.4 +/- 5.6% in the saline control group (n = 5 P < 0.01). In contrast, IB4 did not reduce infarct size following a 45 min period of ischaemia. 5. In the same animals administration of IB4 significantly inhibited oedema formation in skin elicited by intradermal administration of the neutrophil chemoattractant f-Met-Leu-Phe, but had no effect on coronary microvascular plasma protein leakage in the AR. 6. Our results indicate that infiltrating neutrophils exacerbate tissue injury following a relatively short, 30 min period of myocardial ischaemia in the rabbit.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 7913371 [PubMed - indexed for MEDLINE]
 
24: J Leukoc Biol 1996 Mar;59(3):317-24 Related Articles, Books, LinkOut

Myocardial protection by N,N,N-trimethylsphingosine in ischemia reperfusion injury is mediated by inhibition of P-selectin.

Scalia R, Murohara T, Delyani JA, Nossuli TO, Lefer AM.

Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA.

Polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia/reperfusion (MI/R) injury. We examined the cardioprotective effects of N,N,N-trimethylsphingosine (TMS) in a murine model of MI (20 min) and R (24 h) injury in vivo, focusing on leukocyte-endothelial interactions. TMS is a synthetic N-methylated sphingosine derivative that has protein kinase C inhibitory activity and has been shown to prevent leukocyte activation. TMS (18 microgram/kg), administered intravenously 1 min prior to reperfusion, significantly attenuated myocardial necrotic injury assessed by myocardial creatine kinase loss compared with MI/R rats receiving only vehicle (P<0.001). Cardiac myeloperoxidase activity, an index of PMN accumulation in the ischemic myocardium, was also significantly attenuated by TMS compared with rats receiving vehicle (P<0.001). We further examined whether TMS can attenuate leukocyte-endothelial interaction by intravital microscopy. TMS significantly attenuated NG-nitro-L-arginine-methyl ester (L-NAME)-stimulated PMN rolling and adherence to the rat microvascular endothelium. This action of TMS appears to be mediated by reduction of P-selectin expression because immunohistochemical analysis demonstrated that TMS significantly attenuated endothelial P-selectin expression in the L-NAME-superfused rat mesenteric microvasculature. Similarly, TMS markedly attenuated rapid P-selectin expression in rat platelets stimulated with either thrombin or L-NAME assessed by flow cytometry. In conclusion, TMS seems to be an effective cardioprotective agent by inhibiting early leukocyte-endothelial interaction, thus preventing leukocyte accumulation in the ischemic reperfused myocardium.

PMID: 8604008 [PubMed - indexed for MEDLINE]
 
25: Cardiovasc Res 1995 Jul;30(1):47-54 Related Articles, Books, LinkOut

Protection of ischemic/reperfused canine myocardium by CL18/6, a monoclonal antibody to adhesion molecule ICAM-1.

Hartman JC, Anderson DC, Wiltse AL, Lane CL, Rosenbloom CL, Manning AM, Humphrey WR, Wall TM, Shebuski RJ.

Upjohn Company, Kalamazoo, MI 49001, USA.

OBJECTIVE: A blocking monoclonal antibody to intercellular adhesion molecule-1 (ICAM-1), CL18/6, previously has been demonstrated to inhibit neutrophil attachment to isolated vascular endothelium and cardiomyocytes. Due to the well known participation of ICAM-1 in the inflammatory responses associated with myocardial ischemia/reperfusion injury, we investigated if CL18/6 could attenuate myocardial ischemia/reperfusion injury in vivo. METHODS: Saline (3-5 ml, i.v., n = 6), non-blocking control MAb CL18/1D8 or CL18/6 (both 0.5 mg kg-1, i.v., n = 4) were administered prior to coronary occlusion (1 h) and subsequent reperfusion (5 h) produced by inflation of a coronary balloon angioplasty catheter in isoflurane-anesthetized, closed-chest dogs. Heart rate and arterial pressure were measured, and regional myocardial blood flow (rMBF), and myeloperoxidase activity (MPO) to index local neutrophil sequestration, were determined. Myocardial infarct size (IS) was evaluated using the tetrazolium staining technique and expressed as a percent of area at risk (AR). RESULTS: Changes in heart rate and arterial pressure were insignificant throughout the experiment. rMBF (mean +/- s.e.m.) in the ischemic subendocardium for each treatment group was: Saline (0.07 +/- 0.02 ml min-1 g-1); CL18/1D8 (0.04 +/- 0.02); CL18/6 (0.06 +/- 0.02). IS/AR% was: saline (37 +/- 3%); CL18/1D8 (39 +/- 9%); CL18/6 (15 +/- 4%*); * = significantly different from CL18/1D8 and saline, P < 0.05. MPO assayed from AR immediately adjacent to the infarct was significantly reduced below infarct MPO only in the CL18/6 treated group-36%). CONCLUSIONS: The results indicate that CL18/6 antagonism of ICAM-1 provided cardioprotection associated with reduced neutrophil activity in vulnerable myocardium, and suggest that ICAM-1 mediated neutrophil sequestration in endangered cardiac tissue is an important mechanism of myocardial ischemia/reperfusion injury.

PMID: 7553723 [PubMed - indexed for MEDLINE]
 
26: J Leukoc Biol 1997 Sep;62(3):292-300 Related Articles, Books, LinkOut

Monoclonal antibody to ICAM-1 preserves postischemic blood flow and reduces infarct size after ischemia-reperfusion in rabbit.

Zhao ZQ, Lefer DJ, Sato H, Hart KK, Jefforda PR, Vinten-Johansen J.

Department of Cardiothoracic Surgery, Bowman Gray School of Medicine of Wake Forest University, Medical Center Boulevard, Winston-Salem, North Carolina, USA.

Neutrophils are pivotal in the pathogenesis of reperfusion injury leading to myocardial infarction. Firm adhesion of PMN to endothelium may be initiated by the interaction between constitutively expressed intercellular adhesion molecule-1 (ICAM-1) on endothelium and beta2 integrin (CD11b/CD18) on neutrophils. We tested the hypothesis that a monoclonal antibody (mAb RR1/1) against ICAM-1 would preserve postischemic myocardial blood flow and attenuate myocardial injury in an anesthetized rabbit model of coronary occlusion and reperfusion. Either mAb RR1/1 or isotypematched control mAb (R3.1) was injected 10 min before reperfusion. Postischemic myocardial blood flow in the area at risk (Ar) and necrotic area was significantly improved with mAb RR1/1 treatment compared with vehicle and mAb R3.1 during the reperfusion period. RR1/1 had no effect on nonischemic zone blood flow. The Ar as a percent of left ventricle was comparable between groups. Infarct size (TTC) as a percent of Ar was significantly reduced by mAb RR1/1 compared with saline vehicle and mAb R3.1. Plasma creatine kinase activity confirmed the reduction of infarct size in mAb RR1/1 group. In in vitro studies, 40 microg/mL mAb RR1/l, which approximates the plasma concentration of 2 mg/kg mAb RR1/1, markedly inhibited platelet-activating factor-stimulated neutrophil adherence to rabbit aortic endothelium. We conclude that blockade of ICAM-1 during reperfusion reduces postischemic perfusion defects and attenuates the progression of myocardial injury leading to necrosis. This cardioprotection by mAb RR1/1 may be due to inhibition of neutrophil adhesion to the coronary endothelium.

PMID: 9307067 [PubMed - indexed for MEDLINE]
 
27: J Thorac Cardiovasc Surg 1998 Jul;116(1):114-21 Related Articles, Books, LinkOut

Comment in:


Inhibition of interleukin-8 blocks myocardial ischemia-reperfusion injury.

Boyle EM Jr, Kovacich JC, Hebert CA, Canty TG Jr, Chi E, Morgan EN, Pohlman TH, Verrier ED.

Department of Surgery, University of Washington, Seattle 98195, USA.

INTRODUCTION: Interleukin-8 is thought to play a role in neutrophil activation and transcapillary migration into the interstitium. Because neutrophils are principal effector cells in acute myocardial ischemia-reperfusion injury, we postulated that the inhibition of interleukin-8 activity with a neutralizing monoclonal antibody directed against rabbit interleukin-8 (ARIL8.2) would attenuate the degree of myocardial injury encountered during reperfusion. METHODS: In New Zealand White rabbits, the large branch of the marginal coronary artery supplying most of the left ventricle was occluded for 45 minutes, followed by 2 hours of reperfusion. Fifteen minutes before reperfusion, animals were given an intravenous bolus of either 2 mg/kg of ARIL8.2 or 2 mg/kg anti-glycoprotein-120, an isotype control antibody that does not recognize interleukin-8. At the completion of the 120-minute reperfusion period, infarct size was determined. RESULTS: In the area at risk for infarction, 44.3% +/- 4% of the myocardium was infarcted in the anti-glycoprotein-120 group compared with 24.8% +/- 9% in the ARIL8.2 group (p < 0.005). In control animals, edema and diffuse infiltration of neutrophils were observed predominantly in the infarct zone and the surrounding area at risk. Tissue myeloperoxidase determinations did not differ significantly between groups, indicating that the cardioprotective effect of ARIL8.2 was independent of an effect on neutrophil infiltration. CONCLUSIONS: A specific monoclonal antibody that neutralizes interleukin-8 significantly reduces the degree of necrosis in a rabbit model of myocardial ischemia-reperfusion injury.

PMID: 9671905 [PubMed - indexed for MEDLINE]

 
28: Am Heart J 1999 Jun;137(6):1145-52 Related Articles, Books, LinkOut

Kinetics of tumor necrosis factor alpha in plasma and the cardioprotective effect of a monoclonal antibody to tumor necrosis factor alpha in acute myocardial infarction.

Li D, Zhao L, Liu M, Du X, Ding W, Zhang J, Mehta JL.

Department of Medicine, University of Florida and VA Medical Center, Gainesville, FL, USA.

BACKGROUND: Inflammation plays a critical role in acute myocardial infarction (AMI) and tumor necrosis factor alpha (TNF-alpha) is a potent inflammatory trigger. This study was designed to examine the kinetics of TNF-alpha in plasma in patients with AMI and the potential benefit of inhibition of TNF-alpha monoclonal antibody in AMI. METHODS AND RESULTS: TNF-alpha levels in plasma were measured in 42 patients with AMI. TNF-alpha levels were elevated at 4 hours after onset of chest pain and declined to control values at 48 hours. TNF-alpha levels were higher in patients with Killip III and IV than in those with Killip I and II (P <.01). To examine the pathogenic role of TNF-alpha, New Zealand White rabbits were treated with buffer or a TNF-alpha monoclonal antibody before left anterior descending artery (LAD) ligation. Treatment with the TNF-alpha monoclonal antibody decreased area of necrosis, number of circulating endothelial cells, and lipid peroxidation product malonaldehyde bis(dimethyl acetal). There was a significant correlation of TNF-alpha levels with peak CK-MB in AMI patients, and area of necrosis, MDA, and circulating endothelial cells in rabbits (all P <.05). CONCLUSIONS: TNF-alpha release early in the course of AMI contributes to myocardial injury and dysfunction. Treatment with the monoclonal antibody against TNF-alpha can be cardioprotective, particularly in the setting of heart failure in patients with AMI.

Publication Types:
  • Clinical Trial


PMID: 10347344 [PubMed - indexed for MEDLINE]

29. Dall JL, Peng AA. A trial of hydrocortisone in acute myocardial infarction. Lancet. 1963;ii:1097–1098.

 
30: Chest 1972 May;61(5):488-91 Related Articles, Books, LinkOut

Use of hydrocortisone in the treatment of acute myocardial infarction. Summary of a clinical trial in 446 patients.

Barzilai D, Plavnick J, Hazani A, Einath R, Kleinhaus N, Kanter Y.

PMID: 5046847 [PubMed - indexed for MEDLINE]
 
31: Br Heart J 1985 Jul;54(1):11-6 Related Articles, Books, LinkOut

Rupture of the myocardium. Occurrence and risk factors.

Dellborg M, Held P, Swedberg K, Vedin A.

The occurrence of myocardial rupture was studied in a well defined unselected population of patients with acute myocardial infarction, and the group of patients who died of rupture of the heart were compared with two control groups. Of a total of 3960 patients, 1746 (44%) fulfilled the diagnostic criteria for acute myocardial infarction. Rupture was defined solely on the basis of the presence of a pathological passage through part of the myocardium, either the free wall of the left ventricle or the septum, found at necropsy or during operation. Two controls were selected for each patient and matched for age and sex, one (control group A) with acute myocardial infarction having died in hospital but not of rupture (non-rupture cardiac death) and one (control group B) with acute myocardial infarction having survived the hospital stay. Necropsy was performed in 75% of all fatal cases with acute myocardial infarction. The total hospital mortality was 19%, the highest mortality being among women over 70 years (29%). Ruptures (n = 56) were found in 17% of the hospital deaths, or 3.2% of all cases of acute myocardial infarction. Women aged less than 70 had the highest incidence of rupture, 42% of deaths being due to rupture. The mean age for patients with rupture and controls was 70.5 years. The median time after admission to death was approximately 50 hours for patients and control group A. Thirty per cent of the patients with rupture occurred within 24 hours of the initial symptoms occurring.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 4015910 [PubMed - indexed for MEDLINE]
 
32: Circulation 1976 Mar;53(3 Suppl):I204-6 Related Articles, Books, LinkOut

Deleterious effects of methylprednisolone in patients with myocardial infarction.

Roberts R, DeMello V, Sobel BE.

PMID: 1253361 [PubMed - indexed for MEDLINE]
 
33: J Exp Med 1971 Apr 1;133(4):885-900 Related Articles, Books, LinkOut

The phlogistic role of C3 leukotactic fragments in myocardial infarcts of rats.

Hill JH, Ward PA.

PMID: 4993831 [PubMed - indexed for MEDLINE]
 
34: Circulation 1990 Jan;81(1):156-63 Related Articles, Books, LinkOut

The complement system in ischemic heart disease.

Yasuda M, Takeuchi K, Hiruma M, Iida H, Tahara A, Itagane H, Toda I, Akioka K, Teragaki M, Oku H, et al.

First Department of Internal Medicine, Osaka City University Medical School, Japan.

The mechanisms by which tissue injury after acute myocardial infarction (AMI) occurs has not been fully elucidated. Recent evidence in experimental models has suggested involvement of the complement system in microvascular and macrovascular injury subsequent to AMI. With respect to angina pectoris, whether or not the complement system is activated is not clear. The present study assessed the role of complement as a mediator of myocardial inflammation by quantifying products of complement activation, including C3d, C4d, Bb, and SC5b-9 complexes, in 31 patients with AMI, 17 patients with unstable angina pectoris, 19 patients with stable angina pectoris, and 20 normal volunteers. The plasma C3d levels increased in patients with AMI and in those with unstable angina pectoris (p less than 0.01). The plasma levels of C4d, Bb, and SC5b-9 increased only in patients with AMI (p less than 0.01). The plasma SC5b-9 level was related to peak creatine phosphokinase (r = 0.71) and inversely related to the ejection fraction (r = -0.71). The plasma SC5b-9 level of patients with congestive heart failure was higher than that of patients without congestive heart failure in AMI. These results show that activation of complement system occurs after AMI and show an association of myocardial damage with complement activation. With respect to angina pectoris, the complement system is mildly activated in patients with unstable angina pectoris; however, the cardiac function of patients with unstable angina pectoris is not damaged. The complement system of patients with stable angina pectoris is not activated.

PMID: 2297823 [PubMed - indexed for MEDLINE]
 
35: Am J Pathol 1994 Jun;144(6):1357-68 Related Articles, Books, LinkOut

Time course of complement activation and inhibitor expression after ischemic injury of rat myocardium.

Vakeva A, Morgan BP, Tikkanen I, Helin K, Laurila P, Meri S.

Department of Bacteriology and Immunology, University of Helsinki, Finland.

Activation of the complement (C) system has been documented in both experimental and clinical studies of myocardial infarction, but the exact time course and mechanisms leading to C activation have remained unclear. Our earlier postmortem study on human beings showed that formation of the membrane attack complex (MAC) of C was associated with loss of CD59 (protectin), an important sarcolemmal regulator of MAC, from the infarcted area. The recent discovery of a rat analogue of CD59 has now allowed the first experimental evaluation of the temporal and spatial relationship between C component deposition and loss of CD59 in acute myocardial infarction (AMI). After ligating the left coronary artery in rats the earliest sign of C activation, focal deposition of C3, was observed at 2 hours. Deposition of the early (C1, C3) and late pathway (C8, C9) components in the AMI lesions occurred at 3 hours. Glycophosphoinositol-anchored rat CD59 was expressed in the sarcolemmal membranes of normal cardiomyocytes. In Western blot analysis extracts of normal rat heart CD59 appeared as a band of 21 kd of molecular weight under nonreducing conditions. Loss of CD59 in the AMI lesions was observed in association with deposits of MAC from day one onward. Our results show that C activation universally accompanies AMI in vivo. It is initiated within 2 hours after coronary artery obstruction via deposition of C3, which may be due to generation of the alternative pathway C3 convertase in the ischemic area. Deposition of C1 and late C components also starts during the early hours (2 to 4 hours) after ischemia. Subsequent loss of the protective CD59 antigen may initiate postinjury clearance of the irreversibly damaged tissue.

PMID: 7515561 [PubMed - indexed for MEDLINE]
 
36: Clin Exp Immunol 1990 Jul;81(1):132-6 Related Articles, Books, LinkOut

Quantitative measurement of SC5b-9 and C5b-9(m) in infarcted areas of human myocardium.

Hugo F, Hamdoch T, Mathey D, Schafer H, Bhakdi S.

Institute of Medical Microbiology, University of Giessen, West Germany.

Previous immunohistochemical work has indicated that terminal C5b-9 complement complexes are selectively deposited in infarcted areas of human myocardium. In the present study, we sought to quantify C5b-9 levels in myocardial tissue, and to differentiate between the membrane-bound C5b-9 (m) and the cytolytically inactive SC5b-9 complex. Paired tissue specimens from infarcted and non-infarcted myocardium were obtained from 36 autopsies. The homogenized and washed tissues were extracted with n-octyl-beta-D-glucopyranoside (octylglucoside) detergent, and the concentrations of C5b-9 in the extracts were determined by ELISA. Membrane-derived C5b-9 (m) and SC5b-9 were differentiated from each other on the basis of their characteristic sedimentation behaviour in sucrose density gradients. It was found that infarcted myocardial tissue contained on average an approximately three-fold higher concentration of C5b-9, compared with non-infarcted tissue. This increase was due in part to an increase in levels of C5b-9 (m). The results corroborate previous immunohistochemical data and show that complement activation occurs to completion with the generation of potentially cytotoxic C5b-9 complexes in infarcted myocardial tissues.

PMID: 2379317 [PubMed - indexed for MEDLINE]
 
37: Am J Pathol 1983 Apr;111(1):98-111 Related Articles, Books, LinkOut

Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog.

Engler RL, Schmid-Schonbein GW, Pavelec RS.

After several hours of ischemia an incomplete return of blood flow has been reported in brain, kidney, skeletal muscle, and heart. The mechanisms responsible for the no-reflow phenomenon have been unclear, and perivascular edema, platelet or red cell plugs, and interstitial hemorrhage have been implicated. In the present study evidence is provided that leukocyte entrapment in capillaries might contribute to no reflow. Leukocytes are large and stiff cells, which adhere to vascular endothelium naturally and are known to alter in their adherence properties under a variety of conditions. Accordingly, 11 open-chest dogs were studied, 1-5 hours after left anterior descending coronary artery occlusion. Reperfusion (9 dogs) at 75 mm Hg arterial pressure was accomplished with Ringer's lactate and carbon suspension as a marker for capillary patency. In non-ischemic tissue, 98% of the capillaries contained carbon, rare leukocytes, and few erythrocytes, whereas tissue from the distribution of the occluded artery was heterogeneous: 60% of the capillaries had no carbon, high hematocrits, and approximately one leukocyte per unbranched capillary; 40% demonstrated reflow and no leukocytes. A significant correlation between capillaries without carbon (no reflow) and the frequency of leukocytes remaining in these capillaries indicated that leukocytes were present in obstructed capillaries. Furthermore, the frequency of leukocytes remaining after the washout with lactate was ten times greater than in normal arrested heart muscle without washout. Our results suggest that progressive leukocyte capillary plugging during myocardial ischemia contributes to preventing full restoration of capillary flow upon reperfusion.

PMID: 6837725 [PubMed - indexed for MEDLINE]
 
38: J Clin Invest 1989 Aug;84(2):665-71 Related Articles, Books, LinkOut

The erythrocyte as instigator of inflammation. Generation of amidated C3 by erythrocyte adenosine deaminase.

Hostetter MK, Johnson GM.

Department of Pediatrics, University of Minnesota Medical School, Minneapolis 55455.

Myocardial ischemia is characterized by the liberation of adenosine and by complement-mediated inflammation. We have reported that amidated C3, formed when ammonia (NH3) disrupts the thiolester bond of C3, serves as an alternative pathway convertase, generates C5b-9, and stimulates phagocytic oxidative metabolism. We investigated whether the deamination of adenosine by adenosine deaminase in hematopoietic cells might liberate sufficient ammonia to form amidated C3 and thereby trigger complement-mediated inflammation at ischemic sites. In the presence of 4 mM adenosine, NH3 production per erythrocyte (RBC) was equal to that per neutrophil (PMN) (3.3 X 10(-15) mol/cell per h). Because RBC outnumber PMN in normal blood by a thousandfold, RBC are the major source of NH3 production in the presence of adenosine. NH3 production derived only from the deamination of adenosine by the enzyme adenosine deaminase and was abolished by 0.4 microM 2'-deoxycoformycin, a specific inhibitor of adenosine deaminase. When purified human C3 was incubated with 5 X 10(8) human RBC in the presence of adenosine, disruption of the C3 thiolester increased more than twofold over that measured in C3 incubated with buffer, or in C3 incubated with RBC (P less than 0.05). The formation of amidated C3 was abolished by the preincubation of RBC with 2'-deoxycoformycin (P less than 0.001). Amidated C3 elicited statistically significant release of superoxide, myeloperoxidase, and lactoferrin from PMN. Thus, the formation of amidated C3 by RBC deamination of adenosine triggers a cascade of complement-mediated inflammatory reactions.

PMID: 2788175 [PubMed - indexed for MEDLINE]
 
39: Cardiovasc Res 1994 Apr;28(4):437-44 Related Articles, Books, LinkOut

The complement system in myocardial ischaemia/reperfusion injury.

Kilgore KS, Friedrichs GS, Homeister JW, Lucchesi BR.

University of Michigan Medical School, Department of Pharmacology, Ann Arbor 48109-0626.

Publication Types:
  • Review
  • Review, Academic


PMID: 8181029 [PubMed - indexed for MEDLINE]

 
40: Circ Res 1989 Mar;64(3):607-15 Related Articles, Books, LinkOut

Molecular basis of complement activation in ischemic myocardium: identification of specific molecules of mitochondrial origin that bind human C1q and fix complement.

Kagiyama A, Savage HE, Michael LH, Hanson G, Entman ML, Rossen RD.

Laboratory for Immunology Research, Veterans Administration Medical Center, Houston, TX 77211.

Mitochondria may be a source of molecules that activate complement during ischemic injury to myocardium, providing therewith a stimulus for infiltration of polymorphonuclear leukocytes. To identify specific molecules that activate the classical complement pathway, detergent lysates of canine cardiac mitochondria were fractionated by polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose paper (NCP). The NCP replicas of the gels were incubated with isolated C1q and fresh sera as a source of complement, washed briefly, and overlaid with sensitized sheep erythrocytes (RBC) in agarose. A cluster of four to six molecules between 45 and 53 kDa as well as four others, 34, 30, 26, and 23 kDa, consumed complement thereby preventing complement-mediated lysis of sensitized sheep RBC in the agarose overlay. Additional molecules reactive with C1 were identified by their ability to bind isolated human C1q and to serve as assembly sites for later acting complement components. Sites of localization of complement were demonstrated by incubating NCP replicas of fractionated mitochondria with antisera specific for C1q, C3, C5, and C9, followed by peroxidase-conjugated anti-immunoglobulin and substrate. A total of 12 C1q binding molecules ranging in size from 67 kDa to 23 kDa, which can fix later acting complement components, were identified. At least two of these reacted with antisera prepared against canine cardiac lymph collected in the first 3-4 hours after a 45-minute coronary artery occlusion. These studies present direct evidence that specific molecules, released from subcellular fractions of myocardial cells rich in mitochondria, can activate the complement cascade.

PMID: 2783892 [PubMed - indexed for MEDLINE]
 
41: Circulation 1994 Dec;90(6):2666-70 Related Articles, Books, LinkOut

Activation of complement and kinin systems after thrombolytic therapy in patients with acute myocardial infarction. A comparison between streptokinase and recombinant tissue-type plasminogen activator.

Agostoni A, Gardinali M, Frangi D, Cafaro C, Conciato L, Sponzilli C, Salvioni A, Cugno M, Cicardi M.

Institute of Internal Medicine, University of Milan, Italy.

BACKGROUND: We have previously shown that treatment with streptokinase induces abrupt complement activation and transient neutropenia in patients with acute myocardial infarction (AMI). The purpose of this study was to compare the effects of two different thrombolytic agents--streptokinase (SK) and recombinant tissue-type plasminogen activator (rTPA)--on activation of the complement and kinin systems in plasma of patients with AMI. METHODS AND RESULTS: Forty-one patients with AMI who were eligible for thrombolytic therapy were studied. Twenty-three patients were treated with streptokinase (1.5 million IU IV over 60 minutes) and 18 were treated with rTPA (8 with bolus of 10 mg IV, followed by 50 mg infused over 60 minutes and then 40 mg infused over 120 minutes; 10 patients were administered rTPA and heparin according to the accelerated infusion protocol indicated by the GUSTO study). C4a and C3a were measured by radioimmunoassay, soluble terminal complement components (SC5b-9) and anti-SK IgG antibodies were measured by ELISA. Cleaved high molecular weight kininogen (HK) was quantitated in plasma by SDS-PAGE and immunoblotting analysis. C4a levels were significantly and similarly increased in both groups, whereas the levels of C3a and SC5b-9 after rTPA infusion were only slightly elevated and were significantly lower than after SK. No differences were observed between patients treated with slow or accelerated rTPA regimens. The titer of antibodies to SK was highly correlated with the levels of C3a and SC5b-9, whereas a lesser correlation was observed with C4a. Treatment with rTPA did not induce the transient neutropenia observed after SK infusion. The cleavage products of HK were significantly greater after SK than after rTPA infusion. CONCLUSIONS: Our results show that both thrombolytic agents activate the classic complement pathway and that plasmin could be the common trigger for this phenomenon. A significant activation of the complement common pathway (from C3 to terminal components) was observed only with SK infusion and is attributable to the rapid formation of immunocomplexes between SK and anti-SK antibodies present in plasma as a consequence of previous streptococcal infections. The minimal activation of C5 component of the common pathway explains the absence of leukopenia in patients treated with rTPA. Cleavage of HK, larger after SK than after rTPA infusion, represents a condition enhancing the generation of bradykinin by kallikrein. The recent experimental data that indicate a damaging effect of complement activation on the infarcted zone and the contrasting favorable effect consequent to bradykinin formation raise some questions about the clinical importance of the different biological consequences of SK versus rTPA.

PMID: 7994806 [PubMed - indexed for MEDLINE]
 
42: Circulation 1999 Jul 6;100(1):96-102 Related Articles, Books, LinkOut

Comment in:


C-reactive protein as a cardiovascular risk factor: more than an epiphenomenon?

Lagrand WK, Visser CA, Hermens WT, Niessen HW, Verheugt FW, Wolbink GJ, Hack CE.

Departments of Cardiology, Pathology and Internal Medicine, Free University Hospital, Amsterdam, The Netherlands.

BACKGROUND: Circulating levels of C-reactive protein (CRP) may constitute an independent risk factor for cardiovascular disease. How CRP as a risk factor is involved in cardiovascular disease is still unclear. METHODS AND RESULTS: By reviewing available studies, we discuss explanations for the associations between CRP and cardiovascular disease. CRP levels within the upper quartile/quintile of the normal range constitute an increased risk for cardiovascular events, both in apparently healthy persons and in persons with preexisting angina pectoris. High CRP responses after acute myocardial infarction indicate an unfavorable outcome, even after correction for other risk factors. This link between CRP and cardiovascular disease has been considered to reflect the response of the body to the inflammatory reactions in the atherosclerotic (coronary) vessels and adjacent myocardium. However, because CRP localizes in infarcted myocardium (with colocalization of activated complement), we hypothesize that CRP may directly interact with atherosclerotic vessels or ischemic myocardium by activation of the complement system, thereby promoting inflammation and thrombosis. CONCLUSIONS: CRP constitutes an independent cardiovascular risk factor. Unraveling the molecular background of this association may provide new directions for prevention of cardiovascular events.

Publication Types:

  • Review
  • Review, Tutorial


PMID: 10393687 [PubMed - indexed for MEDLINE]

 
43: Circulation 1997 Jan 7;95(1):97-103 Related Articles, Books, LinkOut

C-reactive protein colocalizes with complement in human hearts during acute myocardial infarction.

Lagrand WK, Niessen HW, Wolbink GJ, Jaspars LH, Visser CA, Verheugt FW, Meijer CJ, Hack CE.

Department of Cardiology, Free University Hospital, Amsterdam, Netherlands. cardiol@azvu.nl

BACKGROUND: Rises in circulating C-reactive protein (CRP), the prototypical acute-phase protein in humans, correlate with clinical outcome in patients with myocardial ischemia and infarction. We hypothesized that these correlations might reflect active participation of CRP in the local inflammatory response ensuing in the jeopardized myocardium because on binding to a ligand, CRP is able to activate the classic pathway of complement, and in addition, complement activation has been shown to occur locally in infarcted myocardium. METHODS AND RESULTS: To verify our hypothesis, we investigated localization of CRP in relation to deposition of complement in tissue specimens of infarcted and healthy heart tissue obtained from 17 patients who had died after acute myocardial infarction. CRP was found to be deposited only in infarcted regions and not in normal-appearing areas of the myocardium, being colocalized with depositions of C4 and C3 activation fragments of the complement system. Deposition of CRP and complement in infarcted myocardium appeared to be time dependent, because it was found in all infarctions except for one of young age (< 12 hours old) and two of greater age (> 1 year old), whereas another tissue specimen of an infarct < 12 hours old showed only moderate but positive staining for both CRP and complement in comparison with older infarctions. CONCLUSIONS: We conclude that in humans, CRP may localize in infarcted heart tissue and suggest that this acute-phase protein promotes local complement activation, and hence tissue damage, in acute myocardial infarction.

PMID: 8994423 [PubMed - indexed for MEDLINE]
 
44: J Exp Med 1999 Dec 20;190(12):1733-40 Related Articles, Books, LinkOut

C-reactive protein and complement are important mediators of tissue damage in acute myocardial infarction.

Griselli M, Herbert J, Hutchinson WL, Taylor KM, Sohail M, Krausz T, Pepys MB.

Immunological Medicine Unit, Division of Medicine, Department of Histopathology, Hammersmith Hospital, London W12 ONN, United Kingdom.

Myocardial infarction in humans provokes an acute phase response, and C-reactive protein (CRP), the classical acute phase plasma protein, is deposited together with complement within the infarct. The peak plasma CRP value is strongly associated with postinfarct morbidity and mortality. Human CRP binds to damaged cells and activates complement, but rat CRP does not activate complement. Here we show that injection of human CRP into rats after ligation of the coronary artery reproducibly enhanced infarct size by approximately 40%. In vivo complement depletion, produced by cobra venom factor, completely abrogated this effect. Complement depletion also markedly reduced infarct size, even when initiated up to 2 h after coronary ligation. These observations demonstrate that human CRP and complement activation are major mediators of ischemic myocardial injury and identify them as therapeutic targets in coronary heart disease.

PMID: 10601349 [PubMed - indexed for MEDLINE]
 
45: Virchows Arch 1997 Apr;430(4):327-32 Related Articles, Books, LinkOut

Membrane attack complex of complement and 20 kDa homologous restriction factor (CD59) in myocardial infarction.

Tada T, Okada H, Okada N, Tateyama H, Suzuki H, Takahashi Y, Eimoto T.

Department of Pathology, Nagoya City University Medical School, Japan.

In order to investigate the mechanism of deposition of the complement membrane attack complex (MAC) in cardiomyocytes in areas of human myocardial infarction, the 20 kDA homologous restriction factor of complement (HRF20; CD59) and complement components (Clq. C3d and MAC) were analysed immunohistochemically using specific antibodies. Myocardial tissues obtained at autopsy from nine patients who died of acute myocardial infarction were fixed in acetone and embedded in paraffin. The ages of the infarcts ranged from about 3.5 h to 12 days. In cases of myocardial infarction of 20 h or less, MAC deposition was shown in the infarcted cardiomyocytes without loss of HRF20. Where the duration was 4 days or more, the cardiomyocytes with MAC deposition in the infarcted areas also showed complete loss of HRF20. Outside the infarcts, HRF20 in the cardiomyocytes was well preserved without MAC deposition. The present study suggests that the initial MAC deposition in dead cardiomyocytes can occur as a result of degradation of plasma-membrane by a mechanism independent of complement-mediated injury to the membrane. Loss of HRF20 from dead cardiomyocytes may not be the initial cause of MAC deposition, but may accelerate the deposition process of MAC in later stages of infarction.

PMID: 9134044 [PubMed - indexed for MEDLINE]
 
46: Cardiovasc Res 1996 Aug;32(2):294-305 Related Articles, Books, LinkOut

Comment in:


Influence of the terminal complement-complex on reperfusion injury, no-reflow and arrhythmias: a comparison between C6-competent and C6-deficient rabbits.

Ito W, Schafer HJ, Bhakdi S, Klask R, Hansen S, Schaarschmidt S, Schofer J, Hugo F, Hamdoch T, Mathey D.

Department of Cardiology, University Hospital Eppendorf, Hamburg, Germany.

OBJECTIVE: The complement system has been suggested to play a role in reperfusion injury which may result from an enhanced destruction of myocardial tissue or from an impairment of reflow. We investigated the influence of the C5b-9 complement complex on infarct size, reflow and arrhythmogenesis. METHODS: Twenty-eight C6-competent rabbits and 18 rabbits with congenital C6 deficiency were subjected to either 30 min or 2 h of coronary artery occlusion followed by reperfusion. C6 deficiency was confirmed by the complement titration test and immunohistology. The triphenyl tetrazolium chloride method was used to delineate infarct size. Reflow into infarcted areas was evaluated histologically after an in vivo injection of propidium iodide which served as an early fluorescence microscopic marker of damaged myocardium subjected to reflow. Continuous ECG monitoring allowed the recording of arrhythmias. RESULTS: After 30 min of coronary artery occlusion infarct size was significantly smaller in C6-deficient rabbits (5.0 +/- 2% of the risk region) as compared to C6-competent rabbits (28.4 +/- 8.5%, P = 0.0371). The extent of reflow into damaged myocardium was nearly the same in both animal groups at this time (38 +/- 9 vs. 39 +/- 7% of the risk region). After 2 h of coronary artery occlusion, infarct size was not different between both animal groups, but the extent of reflow into damaged myocardium was significantly smaller in C6-competent rabbits than in C6-deficient rabbits (25 +/- 4 vs. 40 +/- 4%; P = 0.0185). Two of the 18 C6-deficient rabbits had ventricular arrhythmias (Lown II-IV), none of which was fatal. Eleven of the 28 C6-competent animals had major ventricular arrhythmias which were fatal in 6 rabbits. CONCLUSIONS: These results suggest that the lytic C5b-9 complement complex leads to reperfusion injury in the early phase (30 min) of ischaemia, resulting in a larger infarct. After 2 h of ischaemia, complement activation enhances the no-reflow phenomenon but does not affect infarct size. Finally, the C6 status seems to influence the susceptibility to ventricular arrhythmias after coronary artery occlusion, independent of reperfusion.

PMID: 8796116 [PubMed - indexed for MEDLINE]

 
47: Nature 1982 Aug 5;298(5874):534-8 Related Articles, Books, LinkOut

Formation of transmembrane tubules by spontaneous polymerization of the hydrophilic complement protein C9.

Tschopp J, Muller-Eberhard HJ, Podack ER.

The ninth component of complement C9 can undergo circular polymerization in the fluid phase and on lipid membranes. The concomitant hydrophilic-amphiphilic transition is the result of a conformational reorganization of C9 and allows insertion of poly C9 into membranes in the form of a transmembrane protein channel. The ultrastructure of poly C9 resembles that of membrane lesions caused by complement.

PMID: 7099251 [PubMed - indexed for MEDLINE]
 
48: J Immunol 1987 Mar 1;138(5):1530-6 Related Articles, Books, LinkOut

Nucleated cell killing by complement: effects of C5b-9 channel size and extracellular Ca2+ on the lytic process.

Kim SH, Carney DF, Hammer CH, Shin ML.

For C5b-9 channels to mediate cytolysis of a nucleated cell, a sufficient number of channels must be formed in the plasma membrane to override the compensatory mechanisms that nucleated cells might employ to survive. It is well known that nucleated cells are relatively resistant to lysis by complement in comparison to erythrocytes, and it is now evident that this resistance is due, in part, to the ability of nucleated cells to rapidly eliminate C5b-9 from the cell surface. The ability of nucleated cells to eliminate complement complexes is related to physiochemical properties of the complex, such as channel diameter, which in turn affect Ca2+ fluxes that stimulate metabolic processes involved in the elimination process. Paradoxically, these same channel properties that stimulate the defense response may also be responsible for the lethal effects of complement. To further study the role of channel size on cytolysis of nucleated cells by C5b-9, we examined the lytic efficiency of larger C5b-9 channels containing several C9 molecules in comparison with smaller C5b-9 channels containing fewer C9. We have obtained data to indicate that although the larger channels were more cytolytically potent, the channel size had little influence on the rate of cell death. In contrast, the rate of lysis of erythrocytes was substantially slower when smaller C5b-9 channels were present. In evaluating the effect of the extracellular Ca2+ concentration, [Ca2+]o, on nucleated cell lysis in the presence of a lytic number of C5b-9 complexes, it was observed that when the [Ca2+]o was increased the rate of cell death also increased. These findings suggest that lysis of nucleated cells by C5b-9, unlike erythrocytes, may not be entirely due to colloid osmotic deregulation.

PMID: 2433349 [PubMed - indexed for MEDLINE]
 
49: Circ Res 1986 Apr;58(4):539-51 Related Articles, Books, LinkOut

A phosphorus-31 nuclear magnetic resonance study of the metabolic, contractile, and ionic consequences of induced calcium alterations in the isovolumic rat heart.

Hoerter JA, Miceli MV, Renlund DG, Jacobus WE, Gerstenblith G, Lakatta EG.

Isolated adult rat hearts perfused in an isovolumic mode were used to study the effects of sodium-potassium pump inhibition and sodium-calcium exchange alterations on the tissue content of adenosine triphosphate, phosphocreatine, inorganic phosphate, and intracellular pH, all measured by phosphorus-31 nuclear magnetic resonance spectroscopy. Rates of oxygen consumption, contractile function, and the cell contents of calcium, sodium, and potassium also were determined. The inhibition of sodium-potassium adenosine triphosphatase, either by the reduction in perfusate potassium from 5.9 to 1 millimolar or less, or by the addition of 10(-4) molar ouabain, transiently increased systolic pressure. This was followed by a decrease in systolic pressure, an increase in diastolic pressure, and eventual inexcitability. This contractile profile was accompanied by a persistent increase in oxygen consumption, a monotonic decline in cellular adenosine triphosphate and phosphocreatine content, the development of marked intracellular acidosis, a gain in cell sodium and calcium content, and a reduction in cell potassium. Quite similar metabolic changes were also observed when cell calcium was increased after a reduction in perfusate sodium. These metabolic and contractile effects could be prevented or reversed by decreasing perfusate calcium. The results emphasize the profound role of calcium in modulating cell oxygen consumption, energy balance, pH, excitability, and force production. These data are discussed in light of changes in the myocardial energy supply/demand balance, as well as from the viewpoint of the known competition between mechanisms for mitochondrial calcium transport vs. high-energy phosphate production.

PMID: 2421940 [PubMed - indexed for MEDLINE]
 
50: Am J Pathol 2000 Jun;156(6):2091-101 Related Articles, Books, LinkOut

Complement C5b-9-mediated arachidonic acid metabolism in glomerular epithelial cells : role of cyclooxygenase-1 and -2.

Takano T, Cybulsky AV.

Department of Medicine, McGill University Health Centre, Montreal, Quebec, Canada. ttomok@po-box.mcgill.ca

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. This study addresses the role of cyclooxygenase (COX)-1 and -2 in C5b-9-mediated eicosanoid production in GEC. Unstimulated rat GEC in culture primarily express COX-1. When stimulated with sublytic C5b-9, COX-2 was significantly up-regulated, whereas COX-1 was not affected. Compared with control, complement-treated GEC produced 32% more prostaglandin (PG) E(2) in the presence of exogenous substrate, and the increase was abolished with the COX-2-selective inhibitor, NS-398. Release of arachidonic acid from GEC phospholipids via C5b-9-induced activation of cytosolic phospholipase A(2) was associated with a marked stimulation of PGE(2) production, which was inhibited by 60% with NS-398. The results in cultured GEC were extended to GEC injury in vivo by examining COX-1 and -2 expression in PHN. Glomeruli from rats with PHN expressed significantly more COX-1 and COX-2, as compared with normal rats. PGE(2) production in glomeruli of rats with PHN was about twofold greater than in control glomeruli, and the increase was partially inhibited with NS-398. Thus, in GEC in culture and in vivo, C5b-9-induced eicosanoid production is regulated by both isoforms of COX. The inducible COX-2 may be an important novel mediator of C5b-9-induced glomerular injury.

PMID: 10854230 [PubMed - indexed for MEDLINE]
 
51: Circ Res 1990 Mar;66(3):596-607 Related Articles, Books, LinkOut

Thromboxane A2 and peptidoleukotrienes contribute to the myocardial ischemia and contractile dysfunction in response to intracoronary infusion of complement C5a in pigs.

Ito BR, Roth DM, Engler RL.

Department of Medicine, Veterans Administration Medical Center, San Diego, CA 92161.

Intracoronary infusions of activated complement C5a result in myocardial ischemia, contractile dysfunction, and leukocyte accumulation. The hypothesis was tested that the generation of the coronary vasoconstrictors, thromboxane A2 and the 5-lipoxygenase leukotrienes (LTC4 and LTD4), contributes to the C5a-induced decrease in coronary blood flow and contractile function. The left anterior descending coronary artery in anesthetized swine was cannulated and servo pump-perfused with arterial blood at constant pressure and measured flow. Regional subendocardial contractile function was assessed with sonomicrometry. The interventricular vein was cannulated for sampling of coronary venous blood for leukocyte count. The responses in left anterior descending coronary artery blood flow and percent segment shortening to intracoronary infusions of LTC4 (1 microgram), LTD4 (1 microgram), thromboxane agonist U46619 (7.5 micrograms), and C5a (500 ng) were assessed before and after 1) LTD4/LTE4 receptor blockade with leukotriene receptor blocker LY171883 (10 mg/kg i.v.) (n = 5), 2) thromboxane A2/prostaglandin H2 receptor blockade with thromboxane receptor blocker BM13505 (2 mg/kg i.v.) (n = 5), and 3) combined thromboxane and leukotriene receptor blockade (n = 5). In the absence of receptor blockade, intracoronary C5a decreased coronary flow (50-60%) and regional segment function (60-70%) compared with the preinfusion levels. This was accompanied by a fall in coronary venous blood leukocyte levels by 5-6 x 10(6) cells/ml in the absence of alterations in arterial blood leukocyte count. Intracoronary injections of LTD4, LTC4, or U46619 also resulted in prompt decreases in coronary blood flow (50-60%) and segment function (70-80%) from preinfusion levels. Leukotriene receptor blockade with LY171883 abolished these responses to LTD4 and LTC4. Administration of LY171883 also attenuated (p less than 0.05) the myocardial response to C5a; coronary flow and segment function decreased by approximately 28% from preinfusion levels. Thromboxane receptor blockade with BM13505 eliminated the response in coronary flow and segment function to intracoronary U46619. Similar to LY171883, administration of BM13505 blunted (p less than 0.05) the C5a-induced decreases in coronary flow and contractile function, which fell by approximately 20-25% from the preinfusion level. After the combined LTD4/LTE4 receptor and thromboxane A2/prostaglandin H2 receptor blockade, intracoronary C5a resulted in little change in both coronary blood flow and segment shortening. In contrast to the flow and function effects, the C5a-induced myocardial leukocyte extraction was not decreased by leukotriene and/or thromboxane receptor blockade.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 2137727 [PubMed - indexed for MEDLINE]
 
52: Proc Natl Acad Sci U S A 1985 Feb;82(3):886-90 Related Articles, Books, LinkOut

Cardiac dysfunction caused by purified human C3a anaphylatoxin.

del Balzo UH, Levi R, Polley MJ.

The purpose of this investigation was to define the cardiac effects of complement-derived C3a anaphylatoxin, in view of the possibility that cardiac dysfunction may occur as a result of complement activation. Purified human C3a was administered by intracoronary bolus injections into isolated guinea pig hearts. As a function of dose, C3a caused tachycardia, impairment of atrioventricular conduction, left ventricular contractile failure, coronary vasoconstriction, and histamine release. These effects were abolished by cleavage of the COOH-terminal arginine by carboxypeptidase B. The magnitude of C3a-induced tachycardia correlated with the amount of endogenous cardiac histamine released into the coronary effluent. Whereas the tachycardia was markedly reduced by the histamine H2 antagonist cimetidine, the contractile failure and the coronary vasoconstriction caused by C3a were antagonized by the leukotriene antagonist FPL 55712 and by the cyclooxygenase inhibitor indomethacin, respectively. This suggests that histamine, leukotrienes, and vasoactive prostanoates may mediate the various cardiac effects of C3a. Our findings indicate that C3a anaphylatoxin has marked cardiac effects at concentrations that are likely to be attained with a degree of C3 activation commonly seen in various disease states. Thus, our data are compatible with the hypothesis that generation of anaphylatoxins may induce cardiac dysfunction in clinical conditions.

PMID: 2579381 [PubMed - indexed for MEDLINE]
53: J Biol Chem 1990 Mar 5;265(7):3809-14 Related Articles, Books, LinkOut

Complement proteins C5b-9 induce vesiculation of the endothelial plasma membrane and expose catalytic surface for assembly of the prothrombinase enzyme complex.

Hamilton KK, Hattori R, Esmon CT, Sims PJ.

Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City.

Assembly of the terminal complement proteins C5b-9 on human endothelial cells results in increased cytosolic calcium and nonlytic secretion of high molecular weight multimers of von Willebrand factor from intracellular storage granules. We now demonstrate that this C5b-9-induced secretory response is accompanied by vesiculation of membrane particles from the endothelial surface which express binding sites for factor Va and support prothrombinase activity. Exposure of factor Va binding sites after C5b-9 assembly was accompanied by greater than 2-fold increase in prothrombinase activity, which was not observed for cells exposed to C5b-8 (in the absence of C9). By contrast, only a 3-16% increase in prothrombinase activity was observed when these cells were maximally stimulated to secrete by either histamine, thrombin, or the Ca2+ ionophore A23187. Increased prothrombinase activity after C5b-9 was not accompanied by a change in thrombomodulin activity, and was unrelated to cell lysis, the complement-treated cells remaining greater than 99% viable. Endothelial prothrombinase activity was predominately associated with small membrane vesicles (less than 1 microns diameter) released from the cell monolayer. Analysis by fluorescence-gated flow cytometry revealed that these vesicles incorporate the C5b-9 proteins and express binding sites for factor Va. The capacity of the C5b-9 proteins to induce vesiculation of the endothelial plasma membrane and thereby expose catalytic surface for the prothrombinase enzyme complex may contribute to fibrin deposition associated with immune endothelial injury.

PMID: 2105954 [PubMed - indexed for MEDLINE]
54: Blood 1990 Jul 15;76(2):361-7 Related Articles, Books, LinkOut

Consecutive enzyme cascades: complement activation at the cell surface triggers increased tissue factor activity.

Carson SD, Johnson DR.

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-6495.

Complement activation at the cell surface initiates cell damage through a series of reactions occurring at the cell membrane and, after assembly of the terminal membrane attack complex, produces leakage of cytoplasmic contents from the cell. It has been documented that chemical or physical damage to cell membranes can cause a rapid increase in the expression of tissue factor procoagulant activity. In this study, antibody-mediated complement activation at the cell surface resulted in increased tissue factor activity, which correlated with cytolysis, as measured by 51-chromium release. Therefore, complement fixation on the cell surface can have a direct and immediate stimulatory effect on the coagulation cascade at the point of its initiation, with formation of a fibrin clot requiring only three consecutive proteolytic reactions after immunologically mediated cell damage.

PMID: 2369639 [PubMed - indexed for MEDLINE]
 
55: J Immunol Methods 1979;30(2):105-17 Related Articles, Books, LinkOut

An improved method for the isolation from Naja naja venom of cobra factor (CoF) free of phospholipase A.

Pepys MB, Tompkins C, Smith AD.

An improved method is reported for the isolation from cobra (Naja naja) venom of cobra factor (CoF), the anticomplementary protein which is derived from cobra C3. Sequential chromatography on DEAE-Sepharose, Sephacryl-S200, and finally hydroxylapatite yielded 6.25 mg CoF per gram of crude venom. The purified CoF had 1 unit of functional anticomplementary activity per 1--2 micrograms of protein, and was homogeneous on gradient and non-reduced sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). In SDS-PAGE after reduction with mercaptoethanol there were two major bands (M.W. 75,000 and 51,000 daltons), three minor bands (M.W. 29--31,500 daltons) and two trace bands (36,500 and 41,500 daltons). By analogy with mammalian C3 it is suggested that the CoF consists of two polypeptide chains linked by disulphide bridges, one of which undergoes cleavage of the peptide chain at several points either in vivo or in vitro.

PMID: 574149 [PubMed - indexed for MEDLINE]
 
56: Immunology 1975 Feb;28(2):369-77 Related Articles, Books, LinkOut

Studies in vivo of cobra factor and murine C3.

Pepys MB.

The effect of the isolated C3-cleaving factor (CoF) of cobra venom on murine C3 in vivo and in vitro was studied. Optimal quantities of 100-200 units (0.5 minus 1.0 mg) of CoF per kg administered to mice by intraperitoneal injection in divided doses caused plasma C3 levels to fall to less than 5 per cent of normal from 1 to at least 4 days afterwards. Passive anti-CoF serum promptly abrogated the in vivo plasma C3 depletion, and under optimal conditions C3 levels reached 50 per cent of normal after approximately 15 hours. Injection of as little as 20 mug per mouse of CoF in saline induced a precipitating anti-CoF antibody response which prevented subsequent depletion of plasma C3 by CoF. The in vivo half-life of 125I-labelled CoF in normal mice estimated by whole body elimination and clearance from the blood was 24 hours. The presence in vivo of antibodies to CoF caused rapid clearance from the blood and elimination of 125I-labelled CoF, and also localization of some CoF in the spleen, liver and kidneys.

PMID: 804439 [PubMed - indexed for MEDLINE]
 
57: Basic Res Cardiol 1987 Jan-Feb;82(1):57-65 Related Articles, Books, LinkOut

Effect of complement depletion on O2 supply and consumption in ischemic dog myocardium.

Grover GJ, Weiss HR.

The purpose of this study was to determine whether depletion of serum complement can decrease the severity of an ischemic episode by improving regional O2 supply and consumption parameters in the ischemic region of the heart. Fourteen anesthetized dogs with serum complement intact or depleted (100 U/kg cobra venom factor given 8 hrs before) were subjected to left anterior descending coronary artery (LAD) occlusion for 6 hrs. Myocardial blood flows were determined before and 6 hrs after LAD occlusion using radioactive microspheres. Regional arterial and venous O2 saturations were determined using microspectrophotometry. In control animals, flow decreased from 122 +/- 42 to 13 +/- 14 ml/min/100 g (mean +/- SD) in the occluded LAD region. With complement depletion, LAD occlusion resulted in a flow reduction in the ischemic region (38 +/- 29 ml/min/100 g), but to a lesser degree than seen in the same region in control animals, especially in the subendocardium. O2 consumption was decreased in the ischemic region of both treatment groups, though O2 consumption was higher in this region in complement depleted animals compared to the values in control animals. The O2 supply/consumption ratio was decreased similarly in the ischemic region of control and complement depleted groups. Thus, with complement depletion, flow to the ischemic zone was improved but this region was still flow restricted. The flow increase during complement depletion was sufficient to allow an increased O2 utilization in the ischemic region.

PMID: 3593182 [PubMed - indexed for MEDLINE]
 
58: J Clin Invest 1996 Jul 15;98(2):503-12 Related Articles, Books, LinkOut

Requirement and role of C5a in acute lung inflammatory injury in rats.

Mulligan MS, Schmid E, Beck-Schimmer B, Till GO, Friedl HP, Brauer RB, Hugli TE, Miyasaka M, Warner RL, Johnson KJ, Ward PA.

Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

The complement activation product, C5a, may play a key role in the acute inflammatory response. Polyclonal antibody to rat C5a was used to define the requirements for C5a in neutrophil-dependent inflammatory lung injury after systemic activation of complement by cobra venom factor (CVF) or after intrapulmonary deposition of IgG immune complexes. In the CVF model, intravenous infusion (but not intratracheal instillation) of anti-C5a produced a dose-dependent reduction in lung permeability and in lung content of myeloperoxidase. In C6-deficient rats, CVF infusion caused the same level of lung injury (measured by leak of 125I-albumin) as found in C6-sufficient rats. In the IgG immune complex model of lung injury, anti-C5a administered intratracheally (but not intravenously) reduced in a dose-dependent manner both the increase in lung vascular permeability as well as the buildup of lung myeloperoxidase. Treatment with anti-C5a greatly suppressed upregulation of lung vascular intercellular adhesion molecule-1 (ICAM-1). This was correlated with a substantial drop in levels of TNFalpha in bronchoalveolar fluids. These data demonstrate the requirement for C5a in the two models of injury. In the IgG immune complex model, C5a is required for the full production of TNFalpha and the corresponding upregulation of lung vascular ICAM-1.

PMID: 8755663 [PubMed - indexed for MEDLINE]
 
59: J Clin Invest 1993 Sep;92(3):1504-16 Related Articles, Books, LinkOut

Regulation of intercellular adhesion molecule-1 (ICAM-1) in ischemic and reperfused canine myocardium.

Kukielka GL, Hawkins HK, Michael L, Manning AM, Youker K, Lane C, Entman ML, Smith CW, Anderson DC.

Speros P. Martel Laboratory, Department of Pediatrics, Methodist Hospital, Houston, Texas.

Previous studies in vitro have shown an important role for intercellular adhesion molecule-1 (ICAM-1) in adherence interactions of canine neutrophils with canine jugular vein endothelial cells and in cytotoxicity of canine neutrophils for adult cardiac myocytes. To evaluate the regulation of ICAM-1 in myocardial inflammation and its role in the pathogenesis of myocardial ischemia and reperfusion, a series of in vivo and ex vivo studies were performed in canine animals. Systemic administration of LPS elicited ICAM-1 mRNA in several tissues, including myocardium, which demonstrated increasing ICAM-1 staining on intercalated discs of cardiac myocytes. In ischemia and reperfusion protocols: (a) ICAM-1 mRNA was found in ischemic segments within 1 h of reperfusion and in both ischemic and normally perfused segments by 24 h of reperfusion; (b) expression of ICAM-1 was detected in cardiac myocytes in the ischemic region by 6 h of reperfusion; increased expression was seen thereafter as a function of time; (c) post-ischemic (but not preischemic) cardiac lymph collected at intervals from 1 to 24 h after reperfusion elicited ICAM-1 mRNA, ICAM-1 expression, and ICAM-1-dependent neutrophil adhesion in canine jugular vein endothelial cells and in cardiac myocytes with peak cytokine activity seen by 1 h; (d) extravascular localization of neutrophils was detected in ischemic areas only, and was associated with endothelium bearing high levels of ICAM-1 within 1 h of reperfusion; infiltration increased thereafter in association with increasing levels of ICAM-1 mRNA in myocardial segments and increasing levels of ICAM-1 expression on cardiac myocytes. These findings provide the first direct evidence for inflammatory regulation of ICAM-1 in ischemic and reperfused canine myocardium. They support the hypothesis that ICAM-1 participates in neutrophil-mediated myocardial damage.

PMID: 8104198 [PubMed - indexed for MEDLINE]
 
60: Cardiovasc Res 1999 Mar;41(3):603-10 Related Articles, Books, LinkOut

Upregulation of ICAM-1 on cardiomyocytes in jeopardized human myocardium during infarction.

Niessen HW, Lagrand WK, Visser CA, Meijer CJ, Hack CE.

Department of Pathology, Free University Hospital, Amsterdam, The Netherlands. pathol@azvu.nl

OBJECTIVE: Impaired perfusion of the myocardium induces a local inflammatory response. In animal models, there is ample evidence that polymorphonuclear leucocytes (PMNs) infiltrating infarcted myocardium contribute significantly to infarct size. METHODS: To explore a possible role for PMNs in the tissue damage of human myocardial infarction, we investigated localization of intercellular adhesion molecule-1 (ICAM-1) and CD66b (previously clustered as CD67), a marker of degranulation of human PMNs, in relation to deposition of complement in tissue specimens of infarcted and healthy parts of the heart obtained from 20 patients, who had died following acute myocardial infarction. RESULTS: ICAM-1 was transiently expressed by endothelium and for a longer period (few days) on myofibers of infarcted myocardium. This expression only occurred in parts that stained positive for complement. PMN infiltration exclusively occurred in areas with ICAM-1 expression, but not every ICAM-1-positive area contained PMN infiltrates. CD66b was found in PMNs but was also fixed to the plasma membrane of myofibers that stained positive for complement and ICAM-1. CONCLUSION: These findings indicate that, in infarcted human myocardium, PMNs are degranulated, possibly upon interaction with ICAM-1 and activated complement.

PMID: 10435032 [PubMed - indexed for MEDLINE]
 
61: Eur J Immunol 1988 May;18(5):717-21 Related Articles, Books, LinkOut

Induction of rat acute-phase proteins by interleukin 6 in vivo.

Geiger T, Andus T, Klapproth J, Hirano T, Kishimoto T, Heinrich PC.

Biochemisches Institut, Universitat Freiburg, FRG.

Recombinant human interleukin 6 (rhIL 6) was injected i.p. into male Wistar rats to investigate its role as a mediator of the acute-phase response. Hepatic mRNA levels of beta-fibrinogen, alpha 2-macroglobulin, cysteine proteinase inhibitor, alpha 1-acid glycoprotein and albumin were measured at different times after the administration of rhIL 6. Maximal increases of mRNA concentrations were observed already 4 h after the injection of rhIL 6 leading to 4.8-, 19.7-, 10- and 16-fold stimulations in mRNA levels of beta-fibrinogen, alpha 2-macroglobulin, cysteine proteinase inhibitor or alpha 1-acid glycoprotein, respectively. The rhIL 6-induced stimulation of acute-phase protein mRNA was much more rapid than the acute-phase induction after turpentine, where maximal mRNA levels were found between 16 and 24 h. For all acute-phase proteins studied, the stimulation of mRNA synthesis was found to be dependent on the dose of rhIL 6 injected. In the case of alpha 2-macroglobulin mRNA a sex-specific induction by rhIL 6 was found. Only male rats showed an acute-phase response, whereas in female rats an acute-phase reaction of alpha 2-macroglobulin mRNA was not inducible by IL 6. The increases in mRNA levels of the acute-phase proteins studied were followed by corresponding changes of the proteins in the serum determined by rocket immunoelectrophoresis. It is concluded that IL 6 represents a potent mediator of the acute-phase response in the rat.

PMID: 2454191 [PubMed - indexed for MEDLINE]
 
62: Immunol Today 1994 Feb;15(2):74-80 Related Articles, Books, LinkOut

Comment in:


The acute phase response.

Baumann H, Gauldie J.

Dept of Molecular and Cell Biology, Roswell Park Cancer Institute, Buffalo, NY 14263.

Adult mammals respond to tissue damage by implementing the acute phase response, which comprises a series of specific physiological reactions. This review outlines the principal cellular and molecular mechanisms that control initiation of the tissue response at the site of injury, the recruitment of the systemic defense mechanisms, the acute phase response of the liver and the resolution of the acute phase response.

Publication Types:

  • Review
  • Review, Tutorial


PMID: 7512342 [PubMed - indexed for MEDLINE]

 
63: Br J Pharmacol 2000 May;130(2):197-200 Related Articles, Books, LinkOut

Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury.

Mocanu MM, Baxter GF, Yellon DM.

The Hatter Institute, Division of Cardiology, University College Hospitals & Medical School, Grafton Way, London, WC1E 6DE.

Ischaemia-reperfusion injury causes cell death by both necrosis and apoptosis. Caspase activation is a major event in apoptosis. We therefore examined the effect of caspase inhibitors during reperfusion upon myocardial infarction. Rat isolated hearts were subjected to 35 min coronary occlusion and 120 min reperfusion. Treatment groups were perfused with caspase inhibitors during early reperfusion. We assessed a non-selective caspase inhibitor (Z-VAD. fmk, 0.1 microM), a caspase-8 inhibitor (Z-IETD.fmk, 0.07 microM), a caspase-9 inhibitor (Z-LEHD.fmk, 0.07 microM) and a caspase-3 inhibitor (Ac-DEVD.cmk, 0.07 microM). All caspase inhibitors limited infarct size (infarct-risk ratio per cent: control 38.5+/-2.6; Z-VAD. fmk 24.6+/-3.4; Z-LEHD.fmk 19.3+/-2.4; Z-IETD.fmk 23.0+/-5.4; Ac-DEVD.cmk 27.8+/-3.3; P<0.05 when compared with control value, 1-way ANOVA). We conclude that caspase inhibition during early reperfusion protects myocardium against lethal reperfusion injury.

PMID: 10807653 [PubMed - indexed for MEDLINE]
 
64: Circulation 1996 Oct 1;94(7):1506-12 Related Articles, Books, LinkOut

Expression of bcl-2 protein, an inhibitor of apoptosis, and Bax, an accelerator of apoptosis, in ventricular myocytes of human hearts with myocardial infarction.

Misao J, Hayakawa Y, Ohno M, Kato S, Fujiwara T, Fujiwara H.

Second Department of Medicine, Gifu University School of Medicine, Japan.

BACKGROUND: In general, myocyte death in myocardial infarctions (MIs) is attributed to necrosis, but recently the involvement of apoptosis has been suggested. The ratio of bcl-2 protein, an inhibitor of apoptosis, to Bax protein, an inducer of apoptosis, determines survival or death after an apoptotic stimulus. We speculated that bcl-2 or Bax expression is induced by ischemia and that it may be related to myocyte death in human hearts. METHODS AND RESULTS: We studied immunohistochemically 37 autopsied human hearts (acute MI, n = 15; old MI, n = 12; normal hearts as a control, n = 10) with the use of bcl-2 and Bax antibodies. There were no myocytes with positive bcl-2 immunoreactivity in the controls or hearts with old MI. However, myocytes with positive bcl-2 immunoreactivity were seen in 9 of 15 hearts (60%) with acute MI, in that it was localized only in salvaged areas surrounding the infarcted tissues. Myocytes with slightly positive Bax immunoreactivity were observed in the control hearts. In the salvaged myocytes surrounding the infarcted tissues, Bax was overexpressed in 2 of 15 hearts (13%) with acute MI but in 10 of 12 hearts (83%) with old MI. CONCLUSIONS: bcl-2 protein is induced in salvaged myocytes at the acute stage of infarction, but Bax protein is overexpressed at the old stage. The expression of bcl-2 and the overexpression of Bax may play an important pathophysiological role in the protection or acceleration of the apoptosis of human myocytes after ischemia and/or reperfusion.

PMID: 8840837 [PubMed - indexed for MEDLINE]
 
65: Am J Physiol Renal Physiol 2000 May;278(5):F747-57 Related Articles, Books, LinkOut

C5b-9 membrane attack complex mediates endothelial cell apoptosis in experimental glomerulonephritis.

Hughes J, Nangaku M, Alpers CE, Shankland SJ, Couser WG, Johnson RJ.

Division of Nephrology, Department of Medicine, University of Washington, Seattle, Washington, 98195, USA. jeremyh@u.washington.edu

We studied the role of the C5b-9 membrane attack complex in two models of inflammatory glomerulonephritis (GN) initiated by acute glomerular endothelial injury in Piebold-viral-Glaxo (PVG) complement-sufficient rats (C+), C6-deficient rats (C6-), and rats systematically depleted of complement with cobra venom factor (CVF). GN was induced by performing a left nephrectomy and selectively perfusing the right kidney with either 1) the lectin concanavalin A (Con A) followed by complement-fixing anti-Con A (Con A GN) or 2) purified complement-fixing goat anti-rat glomerular endothelial cell (GEN) antibody [immune-mediated thrombotic microangiopathy (ITM)]. Comparable levels of GEN apoptosis were detected in C+ animals in both models. CVF administration reduced GEN apoptosis by 10- to 12-fold. GEN apoptosis was C5b-9 dependent because PVG C6- rats were protected from GEN loss. Furthermore, functional inhibition of the cell surface complement regulatory protein CD59 by renal perfusion with anti-CD59 antibody in ITM resulted in a 3.5-fold increase in GEN apoptosis. Last, in Con A GN, abrogation of GEN apoptosis preserved endothelial integrity and renal function. This study demonstrates the specific role of C5b-9 in the induction of GEN apoptosis in experimental inflammatory GN, a finding with implications for diseases associated with the presence of antiendothelial cell antibodies.

PMID: 10807586 [PubMed - indexed for MEDLINE]

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