Arteriogenesis: nature’s way to maintain structure and metabolism in the presence of occluded arteries

Wolfgang Schaper, Matthias Heil, Frederic Pipp Max Planck Institute for Clinical and Physiological Research, Dept of Experimental Cardiology, Bad Nauheim, Germany
Correspondence: Professor Wolfgang Schaper, Max Planck Institute for Clinical and Physiological Research, Dept of Experimental Cardiology, Benekestr 2, 61231 Bad Nauheim, Germany. Tel: +49 6032 705402, fax: +49 6032 705 419, e-mail: w.schaper@kerckhoff.mpg.de

Introduction
The suffix “genesis” refers to the development of a mature cell line or tissue from a less differentiated precursor, such as in vasculogenesis where blood vessels develop from angioblasts [1]. In the case of arteriogenesis, mature arteries develop from pre-existing arterioles that, before transformation, had a different function (regulation of blood flow) and where the precursors (endothelial and smooth muscle cells) had to undergo dedifferentiation and proliferation before they could assume their new structure and function, ie, the mass transport of blood. The term “arteriogenesis” was coined because angiogenesis
was indiscriminately used to describe all kinds of vascular adaptations in the adult organism that occurred together with arterial occlusion and ischemia [2, 3]. Angiogenesis describes the sprouting of capillaries from pre-existing capillaries, but typical angiogenic growth factors such as vascular endothelial growth factor (VEGF) often do not lead to sprouting but rather to capillary enlargement [1].
In this article we will describe how arteries develop from the pre-existing arteriolar network that connects side-branches proximal to an occluded artery with those distal, or the distal ones connecting with side-branches of a juxtapositioned artery. We will show that arteriogenesis and angiogenesis differ in their mechanisms of growth but that they share the initial step of endothelial activation. In contrast to angiogenesis, ischemia is not a required environment for arteriogenesis, but physical forces such as fluid shear stress are prerequisite [3].

Clinical relevance
Numerous reports have described the life- and organ-saving potency of the collateral circulation that develops by the process of arteriogenesis in response to arterial occlusions in the heart, brain, peripheral circulation, kidney, and intestines [4–7]. Predisposing factors are the extent of the network and the intensity of the basal metabolism. If the network is well developed and the basal metabolism low, the chances of restitution of structure and function after arterial occlusion by arteriogenesis are very good, and vice versa. In the heart, an organ with high basal metabolism, acute coronary occlusion is badly tolerated, especially since the network is not well developed except in the hearts of dogs and guinea pigs [7]. Most other rodent hearts do not have an interconnecting network and experimental coronary occlusion leads to infarctions that encompass almost the entire perfusion area of the occluded arteries. However, slowly progressing stenoses leading to occlusions within several weeks enable the collateral network to expand and infarction is prevented [4]. Resting skeletal muscles exhibit a low basal metabolic rate and have a well-developed arteriolar network and hence tolerate acute arterial occlusion well: the collateral blood flow is high enough to maintain the structure. Enlargement by arteriogenic growth restores partial function relatively soon within a few days.
The advent and availability of angiogenic growth factors raised hopes for accelerating the processes of adaptive vascular growth and increasing its extent, because in most clinical cases the collateral circulation restores function only partially. It is at present not clear whether the known growth factors and their genes will fulfill these hopes, because the success rate will be compared with the standard procedures of revascularization by surgery and stent implantation. On the other hand, to reserve for gene therapy only cases that cannot be operated upon may be asking too much of any therapy.

The pre-existing arteriolar network
The pre-existing arteriolar network is a remnant of the primary capillary network of embryonic development from which arteries and their sub-branches differentiate by recruiting smooth muscle cells and by pruning away via apoptosis most of the capillary network connections [1]. Complete differentiation results in structural endarteries without arteriolar connections, a situation reached, by inference, in organs with a poor ischemia tolerance. Organs with a well-developed arteriolar network have incompletely regressed their primary network, which protects them against ischemia after arterial occlusion but limits maximal blood flow under conditions of challenged metabolism because the network absorbs physical energy. The persistence of the network is genetically determined. The white laboratory mouse strain BALB/c shows complete regression of the network in the skeletal muscles of the hindlimb whereas the black mouse strain C57/Bl6 shows incomplete regression. Consequently the black mice tolerate ischemia well and the white poorly. But the white mice show superior performance in a treadmill running test. Despite the relatively high collateral blood flow in the black mice after femoral artery occlusion, arteriogenesis occurs faster than in white mice, which strongly argues against a role of ischemia.

The role of ischemia/hypoxia
Hypoxia and, to a more limited extent, ischemia are powerful stimulators of gene expression. The heterodimeric transcription factor hypoxia-inducible factor (HIF) consists of the normoxic unstable HIF-1a monomer that becomes stable under hypoxic conditions [8–10] and dimerizes with HIF-1b, which initiates the transcription of numerous genes, the most important of which are VEGF, erythropoietin, and many enzymes of the glycolytic pathway [11, 12]. VEGF, a powerful angiogenic growth factor, has two further advantages: its mRNA becomes more stable under hypoxic conditions [13] and it has a protected side entry into the ribosome, which will guarantee its translation even under hypoxic conditions when most translational activity is shut down. VEGF upregulation by hypoxia leads to angiogenesis, ie, capillary sprouting. Shortage of oxygen is the factor common to both ischemia and hypoxia. However, the accumulation of metabolic waste, typical for ischemia, may counteract part of the increased gene expression seen with hypoxia. Reversible ischemia that does not lead to acute tissue loss or chronic atrophy does not cause a significant increase in capillary density in skeletal muscle. Deep ischemia with tissue loss and atrophy leads only to increased capillary density in white muscles and in regions of inflammation employing different pathways of angiogenesis. It is therefore not unusual to find studies in the literature where skeletal muscle ischemia first leads to a decrease in capillary density which returns partially to normal with time and which can be accelerated by growth factors. It is probably not justified to call this increased or “therapeutic” angiogenesis.
Whereas capillary sprouting and capillary dilatation occur in ischemia by one mechanism or another, ischemia is not a prerequisite for arteriogenesis. The pre-existing arteriolar network is perfused with arterial blood and will therefore never become ischemic. Since the arterioles of the network feed the surrounding tissues with arterial blood, no ischemic tissue will surround transforming arterioles. Collateral artery growth (arteriogenesis) occurs at large distances from ischemic tissue. An extreme case is femoral artery occlusion in humans where collaterals develop around the occlusion in the tissues of the thigh, whereas the ischemic center is in the foot, almost 100 cm away from the location of arteriogenesis. Collateral arteries also continue to grow and remodel a long time after cessation of ischemia. Interesting models to study this problem are again the white and black mouse strains. The black mouse does not experience deep ischemia in the lower leg (and no ischemia in the upper leg) due to a better arteriolar network that provides more collateral blood flow following acute femoral artery occlusion but nevertheless exhibits a strong and fast arteriogenic response [14]. This means that mechanisms other than ischemia are responsible for arteriogenesis.

The role of physical forces
Blood vessels, arteries, and arterioles, in particular, are under the influence of strong mechanical forces: they have to withstand high and varying pressures and flows. Diameter and wall thickness are adapted to withstand the pressure, and adaptations usually tend to maintain a physiological level of Laplace’s quotient, ie, diameter multiplied by pressure divided by wall thickness. In comparison, the forces acting on the endothelial cells by the flowing blood, ie, the viscous drag, fluid shear stress, are much weaker in terms of physical units but are nevertheless powerful determinants of vascular diameter. When an artery is occluded, the pressure in the distal stump and in the interconnecting network falls and a pressure gradient between the vascular regions proximal and distal from the occlusion develops, which accelerates blood flow in the interconnecting vessels and subsequently fluid shear stress, which is linearly related to the velocity of flow but inversely related to the cube of the radius. Although this triggers vascular growth and hence radius, shear stress falls and the remodeling stops. This is probably the reason why collaterals are normally never as good as the artery they have replaced.
There are numerous reports demonstrating the important morphogenic power of fluid shear stress on arteries in experimental animals, but clinical application is difficult. Arteriovenous shunts applied to renal patients for hemodialysis usually lead to growth of the artery, and repeated operations have to be carried out. An “experiment of nature” is the Bland-White-Garland syndrome where one of the coronary arteries originates from the pulmonary artery. This leads to ischemia after birth because one coronary artery is now perfused under a low pressure and with venous blood. Furthermore, collaterals between the right and left coronary artery are formed, which first alleviates ischemia but later creates a left-to-right shunt from the high pressure aorta via coronary artery and collaterals to the low pressure pulmonary artery. This results in ongoing growth of the collaterals and to more shunt flow, a vicious circle which leads again to ischemia and pulmonary hypertension. But above all it shows the power of fluid shear stress as a morphogen. In the clinical setting one should try to increase flow, which can be done with exercise (an accepted treatment in peripheral artery disease) or with drugs. However, vasodilatory drugs are out of fashion, probably because they are not very specific and may cause hypotension, thereby reducing the fluid shear stress. Shear stress leads to upregulation of adhesion molecules on the endothelial surface, release of chemokines (monocyte chemoattractant protein [MCP]-1), and changes in the open probability of the chloride channels, thereby leading to a loss of volume control, resulting in edema [15–18].
The other physical force, tangential or circumferential wall stress, plays a role in smooth muscle proliferation and differentiation. During the maturation phase the thin-walled collateral vessel increases its muscular coat to withstand the Laplace forces [3].

The role of monocytes
Upregulation of adhesion molecules and chemokines by endothelial cells under the influence of increased shear stress [19, 20] leads to adhesion of monocytes [3, 21], binding via their Mac-1 receptor to adhesion molecules of the endothelium, mainly intercellular adhesion molecule-1 (ICAM-1) [22]. Monocytes that are activated in this process now start to produce growth factors [23–25], penetrate the intima, and enter the perivascular space (Figure 1), where they also secrete proteases that digest the matrix and the internal elastic lamina [26, 27].

Figure 1. Monocytes/macrophages (arrowheads) and around the growing collateral arteries 3 days after occlusion. Tissue samples were harvested from rabbit thighs with femoral artery occlusion, placed during a monocyte rebound phase, and stained with the RAM11 antibody (monocytes/macrophages, green), TRITC-phalloidin (F-actin, red) and DAPI/TOTO3 (nuclear staining, blue).

It is somewhat surprising that cells which play a negative role in the genesis of atherosclerosis are important in the adaptation of arteries against the consequences of atherosclerosis, ie, arterial occlusion. This is the reason why we conducted numerous experiments to challenge the monocyte hypothesis of arteriogenesis. Animal models with monocyte deficiencies (osteopetrotic rats and mice) show a significant retardation of arteriogenesis whereas continuous infusion of MCP-1 significantly accelerates collateral artery growth (Figure 2) [28].

Antibodies against ICAM-1, when injected intravenously, prevent the adhesion of monocytes and retard arteriogenesis [29]. When the monocyte concentration in peripheral blood is reduced by treatment with the antimetabolite 5-fluorouracil, arteriogenesis is retarded. When the femoral occlusion is placed during the monocyte rebound after cessation of 5-fluorouracil and when the monocyte concentration is increased, arteriogenesis is accelerated [30]. Mice with targeted disruption of the MCP-1 receptor, chemokine receptor-2 (CCR-2), do not tolerate femoral occlusion well and blood flow does not recover for many weeks, if at all. Other factors that attract monocytes such as transforming growth factor-b and VEGF-A also stimulate arteriogenesis [31–33]. Factors that increase the lifespan of monocytes/ macrophages, such as granulocyte-macro-phage colony-stimulating factor, were recently shown to alleviate angina pectoris by stimulating the collateral circulation in a double-blind clinical trial [34].

Vascular growth factors
Endothelial and smooth muscle cells will not divide unless stimulated by a mitogen that is bound to a cell surface receptor with active transcellular signaling. Some of the growth factors now tested for therapeutic uses are not specific for vascular cells, eg, the fibroblast growth factors (FGFs), but endothelial and smooth muscle cells, when activated, externalize receptors for FGFs. In fact, this is the preferred regulatory mechanism: receptor availability is the molecular switch that enables cell division, not the availability of the ligand. Under normal physiological conditions, growth factor receptors are downregulated. However, some receptors are upregulated when the basal concentration of their ligand is increased, as with VEGF [35]. The factors FGF-1 and -2 are stored in the extracellular matrix and are available at all times. When the gene for VEGF is inactivated by targeted disruption, the embryo dies from disturbed angiogenesis [36]. This shows the importance of this gene for the development of the vascular system. Its role in the adult organism is less clear. Compared with FGF-1 and -2, VEGF is a specific endothelial mitogen, but is much less potent than the FGFs [37, 38]. However, when the FGF-1 and -2 genes are knocked out, no pathological phenotype occurs and the animals develop normally and are fertile. The situation becomes even more complex when we observe that under conditions of arteriogenesis, VEGF and its receptors are not upregulated in the growing arteries nor in the surrounding tissues [39]. Only the FGF receptor-1 is briefly upregulated hours after femoral artery occlusion in the rabbit. Whereas FGF knockout mice do not differ in their arteriogenic potency from their wild-type siblings, overexpression of FGF-2 accelerates arteriogenesis, especially when combined with physical exercise. VEGF is mildly arteriogenic, but we have shown that this is not due to its mitogenic action on endothelial cells but rather to its chemokinetic action on monocytes by binding to the VEGF receptor (VEGFR)-1 that is exclusively expressed on monocytes. Whereas VEGF-A binds to both VEGFR-1 (on monocytes) and VEGFR-2 (on endothelial cells), VEGF-E binds only to VEGFR-2 [40], and placenta growth factor only to VEGFR-1 [24]. Infusion of placenta growth factor into the proximal stump of the occluded femoral artery stimulated arteriogenesis whereas infusion of VEGF-E had almost no effect. This suggests that the arteriogenic properties of VEGF-A are largely due to its chemokine effects on monocytes.

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Max-Planck-Institute for Physiological and Clinical Research, Department of Experimental Cardiology, Bad Nauheim, Germany.

Publication Types:

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• Review, Tutorial

PMID: 8888683 [PubMed - indexed for MEDLINE]

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Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.

Wang GL, Jiang BH, Rue EA, Semenza GL.

Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21287-3914, USA.

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia.

PMID: 7539918 [PubMed - indexed for MEDLINE]

 
Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.

Jiang BH, Rue E, Wang GL, Roe R, Semenza GL.

Center for Medical Genetics, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914, USA.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes including the human erythropoietin (EPO) gene. In this study, we report structural features of the HIF-1alpha subunit that are required for heterodimerization, DNA binding, and transactivation. The HIF-1alpha and HIF-1beta (ARNT; aryl hydrocarbon receptor nuclear translocator) subunits were coimmunoprecipitated from nuclear extracts, indicating that these proteins heterodimerize in the absence of DNA. In vitro-translated HIF-1alpha and HIF-1beta generated a HIF-1/DNA complex with similar electrophoretic mobility and sequence specificity as HIF-1 present in nuclear extracts from hypoxic cells. Compared to 826-amino acid, full-length HIF-1alpha, amino acids 1-166 mediated heterodimerization with HIF-1beta (ARNT), but amino acids 1-390 were required for optimal DNA binding. A deletion involving the basic domain of HIF-1alpha eliminated DNA binding without affecting heterodimerization. In cotransfection assays, forced expression of recombinant HIF-1alpha and HIF-1beta (ARNT) activated transcription of reporter genes containing EPO enhancer sequences with intact, but not mutant, HIF-1 binding sites. Deletion of the carboxy terminus of HIF-1alpha (amino acids 391-826) markedly decreased the ability of recombinant HIF-1 to activate transcription. Overexpression of a HIF-1alpha construct with deletions of the basic domain and carboxy terminus blocked reporter gene activation by endogenous HIF-1 in hypoxic cells.

PMID: 8663540 [PubMed - indexed for MEDLINE]

 
Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit.

Huang LE, Arany Z, Livingston DM, Bunn HF.

Hematology-Oncology Division, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. Bunn@calvin.bwh.harvard.edu

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is critical for hypoxic induction of a number of physiologically important genes. We present evidence that regulation of HIF-1 activity is primarily determined by the stability of the HIF-1alpha protein. Both HIF-1alpha and HIF-1beta mRNAs were constitutively expressed in HeLa and Hep3B cells with no significant induction by hypoxia. However, the HIF-1alpha protein was barely detectable in normoxic cells, even when HIF-1alpha was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1beta protein levels remained constant, regardless of pO2. Hypoxia-induced HIF-1 binding as well as the HIF-1alpha protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension. Moreover, short pre-exposure of cells to hydrogen peroxide selectively prevented hypoxia-induced HIF-1 binding via blocking accumulation of HIF-1alpha protein, whereas treatment of hypoxic cell extracts with H2O2 had no effect on HIF-1 binding. These observations suggest that an intact redox-dependent signaling pathway is required for destablization of the HIF-1alpha protein. In hypoxic cell extracts, HIF-1 DNA binding was reversibly abolished by sulfhydryl oxidation. Furthermore, the addition of reduced thioredoxin to cell extracts enhanced HIF-1 DNA binding. Consistent with these results, overexpression of thioredoxin and Ref-1 significantly potentiated hypoxia-induced expression of a reporter construct containing the wild-type HIF-1 binding site. These experiments indicate that activation of HIF-1 involves redox-dependent stabilization of HIF-1alpha protein.

PMID: 8943284 [PubMed - indexed for MEDLINE]

 
Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5' enhancer.

Liu Y, Cox SR, Morita T, Kourembanas S.

Joint Program in Neonatology, Harvard Medical School, Boston, Mass, USA.

Vascular endothelial growth factor (VEGF) is a potent mitogen specific for endothelial cells. Its expression is dramatically induced by low oxygen tension in a variety of cell types, and it has been suggested to be a key mediator of hypoxia-induced angiogenesis. Although VEGF action is targeted to endothelial cells, it is generally believed that these cells do not express VEGF. In addition, the mechanisms by which hypoxia regulates VEGF production remain unclear. We report in the present study that pulmonary artery endothelial cells do not express VEGF under basal conditions; however, significant VEGF mRNA levels accumulate when these cells are exposed to hypoxia. Using a DNA fragment containing human VEGF promoter sequence, we identified a 28-bp element that is necessary and sufficient to upregulate transcription in response to hypoxia. This element can act as a hypoxia-specific enhancer when placed upstream or downstream from a heterologous promoter. The enhancer includes, in addition to an octamer homologous to the hypoxia-inducible factor-1 (HIF-1) consensus, a sequence that resides 3' to the consensus. Although this sequence may not be involved in the binding of HIF-1, it is absolutely required for the enhancer activity and may be the binding site for certain constitutive binding proteins. The expression of VEGF by endothelial cells in response to hypoxia may provide an important mechanism by which endothelial cell permeability and proliferation is regulated in an autocrine manner.

PMID: 7641334 [PubMed - indexed for MEDLINE]

 
Stabilization of vascular endothelial growth factor mRNA by hypoxia and hypoglycemia and coregulation with other ischemia-induced genes.

Stein I, Neeman M, Shweiki D, Itin A, Keshet E.

Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is upregulated in response to a hypoxic or hypoglycemic stress. Here we show that the increase in steady-state levels of VEGF mRNA is partly due to transcriptional activation but mostly due to increase in mRNA stability. Both oxygen and glucose deficiencies result in extension of the VEGF mRNA half-life in a protein synthesis-dependent manner. Viewing VEGF as a stress-induced gene, we compared its mode of regulation with that of other stress-induced genes. Results showed that under nonstressed conditions, VEGF shares with the glucose transporter GLUT-1 a relatively short half-life (0.64 and 0.52 h, respectively), which is extended fourfold and more than eightfold, respectively, when cells are deprived of either oxygen or glucose. In contrast, the mRNAs of another hypoxia-inducible and hypoglycemia-inducible gene, grp78, as well as that of HSP70, were not stabilized by these metabolic insults. To show that VEGF and GLUT-1 are coinduced in differentially stressed microenvironments, multicell spheroids representing a clonal population of glioma cells in which each cell layer is differentially stressed were analyzed by in situ hybridization. Cellular microenvironments conducive to induction of VEGF and GLUT-1 were completely coincidental. These findings show that two different consequences of tissue ischemia, namely, hypoxia and glucose deprivation, induce VEGF and GLUT-1 expression by similar mechanisms. These proteins function, in turn, to satisfy the tissue needs through expanding its vasculature and improving its glucose utilization, respectively.

PMID: 7565686 [PubMed - indexed for MEDLINE]

 
Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice.

Scholz D, Ziegelhoeffer T, Helisch A, Wagner S, Friedrich C, Podzuweit T, Schaper W.

Department of Exp. Cardiology, Max-Planck-Institute, Bad Nauheim, Germany. d.scholz@kerckhoff.mpg.de

The goal of this study was to examine the mechanisms of vascular growth that lead to the restoration of perfusion in a peripheral vascular disease model in mice. We monitored blood flow recovery and measured vascular growth in inbred strains of mice following femoral artery occlusion. Acute collateral blood flow to the hindlimb was lowest in Balb/C mice, causing intense ischemia, and showed a slower recovery (more than 21 days to 50% normal) than C57Bl/6 which had a 7-fold higher acute collateral flow and a fast recovery (3 days). Collateral vessels were enlarged by proliferation of ECs and SMCs. Capillary density increased in the lower limbs of Balb/Cs (1.7-fold) and of sv129s. Tissue oxygen saturation recovered faster than flow in all strains. Morphometry of mature collaterals showed a diameter increase of 2.1-2.4 fold. The increase in total vessel wall area exceeded that of the femoral artery by 1.4-fold and the common lumenal area by 1.6-fold. Infusion of the growth factor peptide FGF-2 by osmotic minipump accelerated arteriogenesis but inhibited the angiogenic response probably because it prevented ischemia. Conclusion: the speed of arteriogenesis is inversely related to the intensity of ischemia, and arteriogenesis is by far the most efficient mechanism to increase blood flow after femoral artery occlusion. De novo arteriogenesis was not observed. Copyright 2002 Elsevier Science Ltd. All rights reserved.

PMID: 12099717 [PubMed - indexed for MEDLINE]

 
Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium.

Shyy YJ, Hsieh HJ, Usami S, Chien S.

Institute for Biomedical Engineering, University of California at San Diego, La Jolla 92093-0412.

The focal distribution of atherosclerotic lesions in the arterial tree is related to the local shear stress generated by blood flow, but the molecular basis of the atherogenic response of endothelial cells in these lesion-prone areas is still unclear. We report that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells (EC). Northern blot analysis indicated that the level of MCP-1 mRNA in human umbilical vein EC (HUVEC) subjected to a shear stress of 16 dynes/cm2 (1 dyne = 10 microN) for 1.5 hr increased by 2- to 3-fold when compared with static cells. The MCP-1 gene expression decreased to the basal level at 4 hr and then declined further to become completely quiescent at 5 hr after the onset of shear. Once the gene expression was fully suppressed, it remained quiescent even after static incubation for 1.5 hr and would not respond to reshearing after this static incubation. However, if the postshearing incubation extended from 1.5 to 24 hr, the MCP-1 mRNA returned to the basal level and was then able to increase after the reapplication of shear stress. Nuclear run-on experiments showed that the shear-induced increased MCP-1 mRNA in HUVEC was regulated at the transcriptional level. By using cycloheximide, it was shown that de novo protein synthesis was not necessary for the induction of MCP-1 by shear stress. The biphasic response of MCP-1 gene expression was found in experiments in which the applied shear stress was 6, 16, or 32 dynes/cm2, and it was observed not only in HUVEC but also in HeLa cells, glioma cell lines, and skin fibroblasts. This in vitro study demonstrates that the response of MCP-1 gene to shear stress represents an immediate early gene activation and suggests that this gene is probably suppressed in EC that have been exposed to a constant shear stress.

PMID: 8197118 [PubMed - indexed for MEDLINE]

 
Fluid shear stress increases the production of granulocyte-macrophage colony-stimulating factor by endothelial cells via mRNA stabilization.

Kosaki K, Ando J, Korenaga R, Kurokawa T, Kamiya A.

Department of Orthopedic Surgery, Graduate School of Medicine, University of Tokyo, Japan.

To investigate whether the production of colony-stimulating factors (CSFs) by vascular endothelial cells is regulated by hemodynamic force, we exposed cultured human umbilical vein endothelial cells (HUVECs) to controlled levels of shear stress in a flow-loading apparatus and examined changes in the production of CSFs at both the protein and mRNA level. Exposure of HUVECs to a shear stress of 15 and 25 dyne/cm2 markedly increased the release of granulocyte-macrophage CSF (GM-CSF) detected by ELISA to 5.0 and 9.5 times, respectively, the amount released by the static controls at 24 hours, but it had no significant influence on the release of granulocyte CSF or macrophage CSF. The results of reverse transcriptase-polymerase chain reaction demonstrated that GM-CSF mRNA began to increase as early as 2 hours after initiation of 15 dyne/cm2 shear stress and continued to increase with time, reaching a peak of about four times the control levels at 24 hours. This increase in GM-CSF mRNA levels in response to shear stress depended on protein synthesis, because it was blocked by cycloheximide. Neither nuclear run-on assay or luciferase assay using a reporter gene containing GM-CSF gene promoter showed any significant change in transcription of the GM-CSF gene even after 24-hour exposure to a shear stress of 15 dyne/cm2. Actinomycin D chase experiments using a competitive polymerase chain reaction showed that shear stress extended the half-life of GM-CSF mRNA from approximately 23 to 42 minutes in HUVECs. These findings suggest that fluid shear stress increases the production of GM-CSF in HUVECs via mRNA stabilization.

PMID: 9562439 [PubMed - indexed for MEDLINE]

 
Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium.

Chappell DC, Varner SE, Nerem RM, Medford RM, Alexander RW.

Department of Medicine, Emory University, Atlanta, GA, USA.

Low and oscillatory shear stresses are major features of the hemodynamic environment of sites opposite arterial flow dividers that are predisposed to atherosclerosis. Atherosclerosis is a focal inflammatory disease characterized initially by the recruitment of mononuclear cells into the arterial wall. The specific characteristics of the hemodynamic environment that facilitate the generation of arterial inflammatory responses in the presence of, for example, hyperlipidemia are unknown. We show here that prolonged oscillatory shear stress induces expression of endothelial cell leukocyte adhesion molecules, which are centrally important in mediating leukocyte localization into the arterial wall. Vascular cell adhesion molecule-1 was upregulated an average 9-fold relative to endothelial monolayers in static culture. Intercellular adhesion molecule-1 and E-selectin exhibited 11-fold and 7.5-fold increases, respectively. Upregulation of these adhesion molecules was associated with enhanced monocyte adherence. Cytokine stimulation of surface vascular cell adhesion molecule-1 was maximally induced after 6 and 8 hours of cytokine incubation. Oscillatory shear stress for these time periods elicited respective vascular cell adhesion molecule-1 levels of 16% and 30% relative to those observed for cytokine stimulation. Surface intercellular adhesion molecule-1 induction by cytokine stimulation for 24 hours was found to be approximately five times the level detected after 24 hours of oscillatory shear stress. Experiments performed in the presence of the antioxidant N-acetylcysteine demonstrated that the expression of vascular cell adhesion molecule-1 could be almost totally abolished, whereas that of intercellular adhesion molecule-1 was typically reduced by approximately 70%. These results imply that oscillatory shear stress per se is sufficient to stimulate mononuclear leukocyte adhesion and, presumptively, migration into the arterial wall. These results further indicate that atherosclerotic lesion initiation is likely related, at least in part, to unique signals generated by oscillatory shear stress and that the mechanism of upregulation is, to some extent, redox sensitive.

PMID: 9529157 [PubMed - indexed for MEDLINE]

 
Temporal gradient in shear but not steady shear stress induces PDGF-A and MCP-1 expression in endothelial cells: role of NO, NF kappa B, and egr-1.

Bao X, Lu C, Frangos JA.

Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093-0412, USA.

Three well-defined laminar flow profiles were created to distinguish the influence of a gradient in shear and steady shear on platelet-derived growth factor A (PDGF-A) and monocyte chemoattractant protein-1 (MCP-1) expression in human endothelial cells. The flow profiles (16 dyne/cm2 maximum shear stress) were ramp flow (shear stress smoothly transited at flow onset), step flow (shear stress abruptly applied at flow onset), and impulse flow (shear stress abruptly applied for 3 s only). Ramp flow induced only minor expression of PDGF-A and did not increase MCP-1 expression. Step flow increased PDGF-A and MCP-1 mRNA levels 3- and 2-fold at 1.5 hours, respectively, relative to ramp flow. In contrast, impulse flow increased PDGF-A and MCP-1 expression 6- and 7-fold at 1.5 hours, and these high levels were sustained for at least 4 hours. These results indicate that a temporal gradient in shear (impulse flow and the onset of step flow) and steady shear (ramp flow and the steady component of step flow) stimulates and diminishes the expression of PDGF-A and MCP-1, respectively. NO synthase inhibitor NG-amino-L-arginine (L-NAA) was found to markedly enhance MCP-1 and PDGF-A expression induced by step flow, but decrease their expression induced by impulse flow, in a dose-dependent manner. NO donor spermine-NONOate (SPR/NO) dose-dependently reduced the MCP-1 and PDGF-A expression induced by impulse flow. Moreover, impulse flow was found to stimulate sustained (4 hours) I kappa B-alpha degradation and egr-1 mRNA induction. L-NAA prevented I kappa B-alpha degradation, whereas SPR/NO increased I kappa B-alpha resynthesis 2 hours after impulse flow. Both L-NAA and SPR/NO inhibited the impulse flow inducibility of egr-1 4 hours after the flow stimulation. The results show that both NO induced by steady shear and NO donor inhibit temporal gradient in shear-induced MCP-1 and PDGF-A expression by downregulation of their respective transcription factors NF kappa B and egr-1, whereas NO induced by impulse flow stimulates MCP-1 and PDGF-A expression by upregulation of the transcription factors. The above findings suggest distinct roles of temporal gradient in shear and steady shear in atherogenesis in vivo.

PMID: 10195928 [PubMed - indexed for MEDLINE]

 
Ultrastructure and molecular histology of rabbit hind-limb collateral artery growth (arteriogenesis).

Scholz D, Ito W, Fleming I, Deindl E, Sauer A, Wiesnet M, Busse R, Schaper J, Schaper W.

Department of Experimental Cardiology, Max-Planck-Institute, Bad Nauheim, Germany. d.scholz@kerckhoff.mpg.de

Previous studies in the canine heart had shown that the growth of collateral arteries occurs via proliferative enlargement of pre-existing arteriolar connections (arteriogenesis). In the present study, we investigated the ultrastructure and molecular histology of growing and remodeling collateral arteries that develop after femoral artery occlusion in rabbits as a function of time from 2 h to 240 days after occlusion. Pre-existent arteriolar collaterals had a diameter of about 50 microm. They consisted of one to two layers of smooth muscle cells (SMCs) and were morphologically indistinguishable from normal arterioles. The stages of arteriogenesis consisted of arteriolar thinning, followed by transformation of SMCs from the contractile- into the proliferative- and synthetic phenotype. Endothelial cells (ECs) and SMCs proliferated, and SMCs migrated and formed a neo-intima. Intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) showed early upregulation in ECs, which was accompanied by accumulation of blood-derived macrophages. Mitosis of ECs and SMCs started about 24 h after occlusion, whereas adhesion molecule expression and monocyte adhesion occurred as early as 12 h after occlusion, suggesting a role of monocytes in vascular cell proliferation. Treatment of rabbits with the pro-inflammatory cytokine MCP-1 increased monocyte adhesion and accelerated vascular remodeling. In vitro shear-stress experiments in cultured ECs revealed an increased phosphorylation of the focal contacts after 30 min and induction of ICAM-1 and VCAM-1 expression between 2 h and 6 h after shear onset, suggesting that shear stress may be the initiating event. We conclude that the process of arteriogenesis, which leads to the positive remodeling of an arteriole into an artery up to 12 times its original size, can be modified by modulators of inflammation.

PMID: 10782885 [PubMed - indexed for MEDLINE]

 
The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis.

Clauss M, Weich H, Breier G, Knies U, Rockl W, Waltenberger J, Risau W.

Abteilung fur Molekulare Zellbiologie, Max-Planck-Institut fur Physiologische und Klinische Forschung, D-61231 Bad Nauheim, Germany.

Two distinct receptors for vascular endothelial growth factor (VEGF), the tyrosine kinase receptors Flt-1 and Flk-1/KDR, have been described. In this study we show that monocytes, in contrast to endothelium, express only the VEGF receptor Flt-1, and that this receptor specifically binds also the VEGF homolog placenta growth factor (PlGF). Both VEGF and PlGF stimulate tissue factor production and chemotaxis in monocytes at equivalent doses. In contrast, endothelial cells expressing both the Flt-1 and the Flk-1/KDR receptors produce more tissue factor upon stimulation with VEGF than after stimulation with PlGF. Neutralizing antibodies to the KDR receptor reduce the VEGF-stimulated tissue factor induction in endothelial cells to levels obtained by stimulation with PlGF alone, but do not affect PlGF-induced tissue factor induction in endothelial cells nor the VEGF-dependent tissue factor production in monocytes. These findings strongly suggest Flt-1 as a functional receptor for VEGF and PlGF in monocytes and endothelial cells and identify this receptor as a mediator of monocyte recruitment and procoagulant activity.

PMID: 8663424 [PubMed - indexed for MEDLINE]

 
Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb.

Arras M, Ito WD, Scholz D, Winkler B, Schaper J, Schaper W.

Department of Experimental Cardiology, Max-Planck Institute for Physiological and Clinical Research, D-61231 Bad Nauheim, Germany.

We have previously shown that monocytes adhere to the vascular wall during collateral vessel growth (arteriogenesis) and capillary sprouting (angiogenesis). In this study we investigated the association of monocyte accumulation with both the production of the cytokines-basic fibroblast growth factor (bFGF) and TNF-alpha-and vessel proliferation in the rabbit after femoral artery occlusion. In particular, we studied the effects of an increase in monocyte recruitment by LPS on capillary density as well as collateral and peripheral conductance after 7 d of occlusion. Monocytes accumulated around day 3 in collateral arteries when maximal proliferation was observed, and stained strongly for bFGF and TNF-alpha. In the lower limb where angiogenesis was shown to be predominant, macrophage accumulation was also closely associated with maximal proliferation (around day 7). LPS treatment significantly increased capillary density (424+/-26.1 n/mm2 vs. 312+/-20.7 n/mm2; P < 0.05) and peripheral conductance (109+/-33.8 ml/min/100 mmHg vs. 45+/-6.8 ml/min/100 mmHg; P < 0.05) as compared with untreated animals after 7 d of occlusion. These results indicate that monocyte activation plays a major role in angiogenesis and collateral artery growth.

PMID: 9421464 [PubMed - indexed for MEDLINE]

26. Deindl E, Fernández B, Höfer IE, van Royen N, Scholz D, Schaper W. Arteriogenesis, collateral blood vessels, and their development. In: Rubanyi GM, ed. Angiogenesis in Health and Disease. New York, NY: Marcel Dekker; 1999:31–46.
 

 
Matrix metalloproteinases in vascular remodeling and atherogenesis: the good, the bad, and the ugly.

Galis ZS, Khatri JJ.

Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga, USA. zgalis@emory.edu

Vascular remodeling, defined as any enduring change in the size and/or composition of an adult blood vessel, allows adaptation and repair. On the other hand, inappropriate remodeling, including its absence, underlies the pathogenesis of major cardiovascular diseases, such as atherosclerosis and restenosis. Since degradation of the extracellular matrix scaffold enables reshaping of tissue, participation of specialized enzymes called matrix metalloproteinases (MMPs) has become the object of intense recent interest in relation to physiological ("good") and pathological ("bad") vascular remodeling. Experimental evidence acquired in vitro and in vivo suggests that the major drivers of vascular remodeling, hemodynamics, injury, inflammation, and oxidative stress, regulate MMP expression and activity. Alternatively, nonspecific MMP inhibition seems to oppose remodeling, as suggested by the inhibition of intimal thickening and outward arterial remodeling. An emerging concept is that MMP-related genetic variations may contribute to heterogeneity in the presentation and natural history of atherosclerosis. The hypothesis that MMPs contribute to weakening of atherosclerotic plaques is especially attractive for the potential development of therapeutic interventions aimed at preventing plaque disruption ("the ugly"), a major cause of acute cardiovascular events. However, the current lack of appropriate experimental tools, including availability of specific MMP inhibitors and pertinent animal models, still limits our understanding of the many actions and relative contributions of specific MMPs. Our future potential ability to control vascular remodeling via regulation of MMPs will also depend on reaching a consensus of what is indeed "good" or "bad" vascular remodeling, concepts that have continued to evolve and change.

Publication Types:

• Review
• Review, Tutorial

PMID: 11861412 [PubMed - indexed for MEDLINE]

 
Monocyte chemotactic protein-1 increases collateral and peripheral conductance after femoral artery occlusion.

Ito WD, Arras M, Winkler B, Scholz D, Schaper J, Schaper W.

Department of Experimental Cardiology, Max-Planck-Institute for Physiological and Clinical Research, Bad Nauheim, Germany. wito@alpha.kerckhoff.mpg.de

Monocytes are activated during collateral artery growth in vivo, and monocyte chemotactic protein-1 (MCP-1) has been shown to be upregulated by shear stress in vitro. In order to investigate whether MCP-1 enhances collateral growth after femoral artery occlusion, 12 rabbits were randomly assigned to receive either MCP-1, PBS, or no local infusion via osmotic minipump. Seven days after occlusion, isolated hindlimbs were perfused with autologous blood at different pressures, measuring flows at maximal vasodilation via flow probe and radioactive microspheres, as well as peripheral pressures. This allowed the calculation of collateral (thigh) and peripheral (lower limb) conductances from pressure-flow tracings (slope of the curve). Collateral growth on postmortem angiograms was restricted to the thigh and was markedly enhanced with MCP-1 treatment. Both collateral and peripheral conductances were significantly elevated in animals with MCP-1 treatment compared with the control group, reaching values of nonoccluded hindlimbs after only 1 week of occlusion (collateral conductance, 70.6 +/- 19.23 versus 25.1 +/- 2.59 mL/min per 100 mm Hg; P < .01; peripheral conductance, 119.3 +/- 22.37 versus 45.4 +/- 6.80 mL/min per 100 mm Hg; P < .05). These results suggest that activation of monocytes plays an important role in collateral growth as well as in capillary sprouting.

PMID: 9168785 [PubMed - indexed for MEDLINE]

29. Buschmann I, Hoefer I, Heil M, Schaper W. Anti-adhesion monoclonal antibodies against ICAM inhibit arteriogenesis [abstract]. JACC. 1999;33(Suppl.A):911–911.

 
Blood monocyte concentration is critical for enhancement of collateral artery growth.

Heil M, Ziegelhoeffer T, Pipp F, Kostin S, Martin S, Clauss M, Schaper W.

Department for Experimental Cardiology, Max Planck Institute for Physiological and Clinical Research, 61231 Bad Nauheim, Germany. m.heil@kerckhoff.mpg.de

Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. We tested the hypothesis that arteriogenesis is functionally linked to the concentration of circulating blood monocytes. Monocyte concentrations in peripheral blood were manipulated by single injections of the antimetabolite 5-fluorouracil (5-FU), resulting in a marked rebound effect in New Zealand White rabbits. Collateral artery growth was assessed by the use of a model of acute femoral artery ligation. Seven days after ligation, collateral conductance and the number of visible collateral arteries were increased in the rebound group. This increase was accompanied by an increased monocyte accumulation as demonstrated by immunohistology in the thigh 3 days after surgery. In a second animal model (129S2/SvHsd mice), 5-FU treatment caused a remarkable decrease in blood monocyte numbers at day 4, followed by a rebound effect at day 12. Foot blood flow, assessed by laser-Doppler imaging before and at various time points after surgery, increased from day 7 through day 21 in mice from the rebound group. In contrast, ligation during the phase of monocyte depletion resulted in a reduction of blood flow reconstitution. This inhibition could be reversed by an injection of isolated monocytes. In conclusion, we have demonstrated a functional link between the monocyte concentration in the peripheral blood and the enhancement of arteriogenesis.

PMID: 12388258 [PubMed - indexed for MEDLINE]

 
Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease.

van Royen N, Piek JJ, Buschmann I, Hoefer I, Voskuil M, Schaper W.

Max Planck Institute for Physiological and Clinical Research, Department of Experimental Cardiology, Bad Nauheim, Germany. vanroyen@med1.ukl.uni-freiburg.de

After birth two forms of vessel growth can be observed; angiogenesis and arteriogenesis. Angiogenesis refers to the formation of capillary networks. Arteriogenesis refers to the growth of preexistent collateral arterioles leading to formation of large conductance arteries that are well capable to compensate for the loss of function of occluded arteries. The process of arteriogenesis is initiated when shear stresses increase in the preexistent collateral pathways upon narrowing of a main artery. The increased shear stress leads to an upregulation of cell adhesion molecules for circulating monocytes, which accumulate subsequently around the proliferating arteries and provide the several required cytokines and growth factors. Several strategies are currently tested for their potential to stimulate the process of arteriogenesis. These strategies focus either at shear stress, at direct stimulation of endothelial and smooth muscle cell growth or at the monocytic pathway and promising results were obtained from experimental studies. However, some important questions remain to be answered before arteriogenesis can be brought from bench to bedside.

Publication Types:

• Review
• Review, Academic

PMID: 11166267 [PubMed - indexed for MEDLINE]

33. Pipp F, Heil M, Clauss M, Herold J, Tribulova S, Schaper W. Vergleich von VEGF und MCP-1 im ischämischen Kaninchenhinterlauf. Z Kardiol. 2002;91:I/5.3

 
VEGF121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of chorioallantoic membrane.

Wilting J, Birkenhager R, Eichmann A, Kurz H, Martiny-Baron G, Marme D, McCarthy JE, Christ B, Weich HA.

Anatomisches Institut II, Albert-Ludwigs-Universitat Freiburg, Germany.

We have studied the effect of VEGF(121) homodimer and VEGF(121/165) heterodimer on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. The factors were applied in doses of 2-4 micrograms and the effects were evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was studied according to the BrdU-anti-BrdU method on whole mounts and sections. The labeling density was quantified in whole mounts. The fractal dimension, D, of the vascular tree was assessed as a value for vascular bifurcation density. Both forms of VEGF induce brush-like vessel formation in the precapillary region. New capillaries are found in the stroma of the CAM, which normally does not contain capillaries. Our results show that VEGF(121) is a specific endothelial cell mitogen. A fourfold increase of BrdU-labeled endothelial cells is found after VEGF(121) application. The fractal dimension of the vascular tree increases from 1.26 in the controls to 1.44 (VEGF(121)) and 1.41 (VEGF(121/165)). The endothelial cells of the newly formed capillaries possess many mitochondria and micropinocytotic vesicles, but no fenestrations. These capillaries are obviously formed by intussusceptive microvascular growth. Signs of sprouting are almost absent. An effect on the lymphatic vessels of the CAM is not detectable. Compared to VEGF(165) and VEGF(121/165), VEGF(121) diffuses over a slightly greater distance. Using in situ hybridization, VEGF receptor-2 (flk-1/Quek1) and the homologous flt-4 (Quek2) receptor were studied in the CAM of normal quail embryos and after VEGF(121) application on the CAM of 11-day-old quail embryos. During normal development, flk-1 expression becomes restricted to vascular endothelial cells of large vessels in the stroma of the CAM. VEGF(121) application induces expression of flk-1 in capillaries that normally do not express the receptor. In the normal development of the CAM, flt-4 becomes restricted to endothelial cells of vessels that appear to be lymphatic vessels. Application of VEGF(121) does not alter flt-4 expression.

PMID: 8654896 [PubMed - indexed for MEDLINE]

 
The biology of vascular endothelial growth factor.

Ferrara N, Davis-Smyth T.

Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080, USA.

Publication Types:

• Review
• Review, Academic

PMID: 9034784 [PubMed - indexed for MEDLINE]

 
A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases.

Meyer M, Clauss M, Lepple-Wienhues A, Waltenberger J, Augustin HG, Ziche M, Lanz C, Buttner M, Rziha HJ, Dehio C.

Department of Infection Biology, Max Planck Institute for Biology, Spemannstrasse 34, D-72076 Tubingen, Germany.

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.

PMID: 9889193 [PubMed - indexed for MEDLINE]