Early detection of diabetic and non-diabetic subjects with increased cardiovascular risk: new risk indicators

Dr A. Jager9(1), Dr C.D.A. Stehouwer(1,2,3)
(1)Institute for Research in Extramural Medicine, (2)Institute for Cardiovascular Research, and (3)Department of Internal Medicine, University Hospital,
Vrije Universiteit, Amsterdam, The Netherlands

Cardiovascular disease continues to be the principal cause of death in the USA, Europe and a large part of Asia.[1,2] However, over the last decade, mortality from cardiovascular disease has shown an annual decline which may be largely due to improvements in the treatment and secondary prevention of myocardial infarction.[3] Nevertheless, congestive heart failure, a progressive disease, has emerged as one of the leading cardiovascular disorders in developed countries and is expected to become a major disease burden by the year 2020.[4] This situation emphasizes the necessity of risk reduction among asymptomatic subjects in order to prevent clinical symptoms of cardiovascular disease, i.e. primary prevention.

Diabetes mellitus probably affects 5–7% of Western populations. It has been estimated that its prevalence will double to reach 200 million worldwide over the next 15 years, due to lifestyle changes, ageing and better treatment facilities giving a longer lifespan.[5,6] Diabetes mellitus is associated with a three- to fourfold higher risk of cardiovascular mortality[7–9] and an approximately 5- to 10-year reduced life-expectancy compared with non-diabetic subjects.[10] The annual decline in cardiovascular mortality observed in the general population during the past 30 years has been almost absent in the diabetic population.[11] Consequently, the incidence and prevalence of cardiovascular disease among diabetic subjects will increase dramatically. Conventional cardiovascular risk factors are known to have a similar impact on diabetic as well as non-diabetic subjects,[12] and at all risk factor levels diabetic subjects have a two to three times higher absolute risk of a serious cardiovascular event than do non-diabetics.[12] Moreover, diabetic subjects have less favorable outcomes after myocardial infarction than do non-diabetic subjects.[13] In view of these considerations, focus on primary prevention among diabetic subjects might be even more important than among non-diabetic subjects.
The overall objective of cardiovascular disease prevention both in asymptomatic subjects and in subjects with clinically established cardiovascular disease is the same: to reduce the risk of subsequent cardiovascular events. There are, however, differences in treatment strategies used for primary and secondary prevention. In general, the use of intensive treatment strategies is more justified for secondary prevention than for primary prevention since benefits are easier to establish for secondary prevention. To decide whether drug therapy is also indicated for primary prevention, it is important to identify those asymptomatic subjects who have a relatively high absolute risk of cardiovascular disease (Figure 1).

Atherothrombosis
Atherothrombosis is a slowly progressive degenerative disease of large and middle-sized elastic and muscular arteries. The disease begins with formation of fatty streaks in adolescence and when progressed unabated develops into fibrous plaques and complicated lesions, culminating in thrombotic occlusions and cardiovascular events in later life. The most commonly held view of the pathogenesis of atherothrombosis is described in the so-called response-to-injury hypothesis first postulated by Ross and Harker in 1976.[18] In the latest update of this hypothesis, endothelial dysfunction is proposed to be the initiating factor leading to a series of specific cellular and molecular responses that can best be described as an inflammatory disease.[19]
Intact endothelium has important bioactive properties, i.e. it actively regulates vascular tone, permeability to macromolecules and leukocytes, the balance between coagulation and fibrinolysis, the composition of the subendothelial matrix, and the proliferation of vascular smooth muscle cells.[20–22] To carry out these functions, endothelium produces a variety of regulatory mediators (e.g. nitric oxide, prostanoids, endothelin, angiotensin II, tissue-type plasminogen activator, plasminogen activator inhibitor-1, vWF and several cytokines), components of the extra-cellular matrix (e.g. heparan sulfate proteoglycans, collagen and laminin) and adhesion molecules (e.g. vascular cell adhesion molecule-1 [VCAM-1] and E-selectin). When the properties of intact endothelium change, either in the basal state or after stimulation, in such a way that normal organ function is no longer preserved, endothelial dysfunction is considered to be present.
Early manifestations of endothelial dysfunction include the appearance of specific adhesive molecules on the surface of the endothelial cells. Monocytes and T-lymphocytes attach to the these molecules and transmigrate to the subendothelial space. Once in the arterial wall, monocytes become macrophages where they act as antigen-presenting cells to T-lymphocytes, as scavenger cells to remove noxious materials, and as a source of growth-regulatory molecules and cytokines. In all stages of atherothrombosis, macrophages and T-lymphocytes are present in the arterial wall, suggesting that atherothrombosis is an ongoing low-grade inflammatory disease.[18,19]
Taken together, the initiating endothelial dysfunction and, subsequently, the ongoing low-grade inflammation of the arterial vessel wall are important (early) features of the atherothrombotic process. Therefore, markers reflecting endothelial dysfunction or low-grade inflammation might be useful for stratification of risk of cardiovascular disease.

Microalbuminuria
Albumin is a relatively large negatively charged protein (molecular weight 69 kDa, size 36 Å), which is usually excreted in small amounts in the urine (<20 µg/min). Slightly raised urinary albumin excretion, known as microalbuminuria,[23] is defined as urinary albumin excretion 20–200 µg/min or 30–300 mg/24 h.[24]
The filter through which albumin must pass before entering the urine, the glomerular capillary wall, is size- and charge-selective. Microalbuminuria is thought to be a consequence of increased albumin leakage through the glomerular capillary wall as a result of increased permeability of the wall, increased intraglomerular pressure, or both.[25,26] Hyperglycemia and high blood pressure are generally accepted to be the main risk factors for developing microalbuminuria.[27,28] Both can increase intraglomerular pressure.[29] Moreover, hyperglycemia can alter the charge selectivity of the glomerular capillary wall, thereby increasing its permeability.[30] In a healthy kidney, over 99% of filtered albumin is reabsorbed by mechanisms that are probably close to saturation. A small increase in albumin filtered by the glomerulus will lead to an excessive supply of albumin to the renal tubulus. Although a compensatory increase of tubular reabsorption has been suggested, this might only be present in an early stage of increased albumin filtration.[31] As a consequence, increased filtered albumin will lead to increased albumin excretion in the urine.[32]
The prevalence of microalbuminuria is low in the general population and rises with age, resulting in a prevalence of about 8–10% in the general elderly population.[28,33] The prevalence of microalbuminuria among diabetic and hypertensive subjects is much higher than that in the general population, i.e. approximately 30%[28,34] and 20%,[28,34] respectively.

Microalbuminuria as a cardiovascular risk indicator
Microalbuminuria is a well-recognized, strong and independent risk marker of cardiovascular disease among diabetic subjects.[35] In a systematic review, Dinneen and Gerstein[36] showed that microalbuminuria among Type 2 diabetic subjects was associated with a 2.4-fold (95% CI 1.8–3.1) increased risk of cardiovascular death compared with normoalbuminuria. In addition, elevated urinary albumin excretion has been found to be an independent risk marker for cardiovascular disease among non-diabetic subjects.[37,38] Whether microalbuminuria is also a risk marker among hypertensive subjects is still the subject of debate.[39,40] In a 10-year follow-up study, Samuelsson et al.[41] showed that macroalbuminuria (i.e. albumin excretion above the microalbuminuria threshold) was associated with an approximately threefold increased cardiovascular risk among hypertensive males. Recently, we were the first to show that the presence of microalbuminuria was also associated with a threefold increased risk of cardiovascular mortality among hypertensive subjects.[42]
In view of the strong and independent association between microalbuminuria and deterioration of renal function, the American Diabetes Association recommends yearly routine urinalysis for the detection of microalbuminuria among diabetic subjects.[24] However, although microalbuminuria is strongly associated with a decline in renal function among Type 1 diabetic subjects,[23,43] among Type 2 diabetic subjects microalbuminuria is much more strongly related to risk of cardiovascular disease.[36] Among 503 Type 2 diabetic subjects followed for 10 years, 2% died from uremia whereas 56% died from cardiovascular disease.[35] In other words, many Type 2 diabetic subjects will die of cardiovascular disease before renal failure develops. Thus, screening for microalbuminuria among Type 2 diabetic subjects should be used primarily as a means to stratify risk of cardiovascular disease.

Pathophysiological mechanisms
The underlying pathophysiological mechanism through which microalbuminuria is related to cardiovascular disease is unclear, although several mechanisms have been proposed which can be categorized into three main hypotheses (Figure 2). First, microalbuminuria could simply be a marker of an underlying pathophysiological process causing atherothrombosis (Figure 2). Several processes have been suggested. Microalbuminuria could reflect a systemic transvascular leakage of albumin[44,45] caused by alterations in the extracellular matrix, which might predispose to greater penetration of atherogenic lipoprotein particles into the arterial wall.[46] Alternatively, microalbuminuria could reflect generalized endothelial dysfunction (without necessarily involving other layers of the vessel wall)[47]. Accordingly, microalbuminuria is associated with increased plasma levels of proteins secreted by or shed from injured endothelium, such as vWF (see below)[48], thrombomodulin[49] and fibronectin.[50] Microalbuminuria could also reflect chronic low-grade inflammatory activity since it has been found to be associated with levels of proinflammatory cytokines.[51] Furthermore, among Type 2 diabetic subjects, microalbuminuria has been found to be associated with impaired fibrinolytic activity[52] and with a procoagulant state.[53] Finally, microalbuminuria has been proposed to be part of the insulin resistance syndrome,[54,55] a cluster of cardiovascular risk factors (i.e. glucose intolerance, insulin resistance, hypertension, obesity and dyslipidemia), and might as such increase the risk of cardiovascular disease. Thus, microalbuminuria could reflect several pathophysiological pathways causing atherothrombosis.
The second hypothesis suggests that microalbuminuria might reflect a certain risk factor which has not yet been discovered (Figure 2). Hyperhomocysteinemia may be a candidate since an increased homocysteine level has been found to be associated with risk of atherothrombotic disease[56,57] on the one hand and with the presence of microalbuminuria on the other.[58] A decreased level of cardioprotective proteins such as apolipoprotein A-I is another candidate.[59] These proteins could be lost in the urine along with albumin.
The third hypothesis proposes that microalbuminuria could reflect a certain susceptibility for developing atherothrombotic diseases (Figure 2). This hypothesis is based on the idea that some subjects may have a more pronounced inherent response to risk factors, thus predisposing them to increased cardiovascular risk.

Figure. 2. Proposed pathophysiological pathways through which microalbuminuria might be related to cardiovascular events.

In summary, microalbuminuria is a strong independent indicator of increased cardiovascular risk among non-diabetic and Type 2 diabetic subjects. Therefore, microalbuminuria can be used for stratification of risk of cardiovascular disease. Once microalbuminuria is present, cardiovascular risk factor reduction should be more ‘aggressive’. Finally, it is of great importance to unravel the pathophysiological mechanism behind the association between microalbuminuria and cardiovascular risk in order to develop more specific treatments.

von Willebrand factor (vWF)
vWF, a multimeric glycoprotein, is secreted mainly by endothelial cells and megakaryocytes. vWF is continuously released into the bloodstream in small amounts. This results in plasma vWF concentrations of 50–150% (i.e. 0.5–1.5 U/ml)48, as assessed by highly-specific electroimmunopheresis or ELISA. Once in the bloodstream, vWF mediates platelet adhesion to the subendothelium and serves as a carrier protein for Factor VIII.
Levels of vWF can increase rapidly in response to acute endothelial injury, as a result of acute release of stored vWF from endothelial storage bodies, the so-called Weibel-Palade bodies. Plasma vWF concentration can also increase slowly due to an increase in vWF secretion in response to abnormal environments such as diabetes mellitus, hypertension, renal failure or malignancies.[48,60,61]
To date, there is no consensus about which cut-off level should be used to define a high vWF concentration. The cut-off level which is used most often is a vWF level above 150%.

von Willebrand factor (vWF) as a cardiovascular risk indicator or risk factor
Increased levels of vWF have been found to be associated with the presence of peripheral,[62] cerebral,[63] and coronary artery atherothrombotic disease.[64] Furthermore, vWF levels are higher among subjects with (compared to those without) cardiovascular risk factors, e.g. hypertension,[65] hypercholesterolemia,[66] smoking[67]and diabetes.[68–71] Moreover, prospective studies have shown high levels of vWF to be associated with cardiovascular mortality among patients recently presenting with clinical manifestations of cardiovascular disease[63,64,72] and among Type 2 diabetic subjects.[69]
So far, similar prospective data among healthy subjects or subjects at high risk of developing atherothrombosis are lacking (except for one study among Type 2 diabetic subjects[69]).

Pathophysiological mechanisms
The pathophysiological explanation for the association between increased cardiovascular risk and high vWF levels is not entirely clear (Table 1). The most commonly held view is that increased levels of vWF reflect generalized endothelial dysfunction.[48,60,73] Accordingly, injury to endothelial cells has been shown to increase the secretion of vWF both in vitro and in vivo.[48,60] Alternatively, it has been hypothesized that vWF, as an acute-phase reactant,[74] reflects endothelial activation and stimulation (without necessarily implying endothelial dysfunction) and as such reflects a low-grade inflammatory state. On the other hand, the bioactive properties of vWF might by themselves increase cardiovascular risk. (Note that if that were in fact the case, high vWF levels would be a risk factor rather than a risk indicator.) Plasma vWF has a key role in platelet adhesion, thrombus formation and coagulation. As a consequence, increased levels of vWF could induce a procoagulant and prothrombotic state,[75] thereby explaining the high risk of reinfarction among survivors of myocardial infarction with high vWF levels.[72] Alternatively, increased vWF levels could increase plasma viscosity. vWF multimers are the largest known soluble human plasma protein molecules and by virtue of their size may increase plasma viscosity.[76] Moreover, high viscosity has been found to be related to increased risk of cardiovascular disease.[77–79] Thus, increased vWF levels are strongly associated with risk of cardiovascular mortality among subjects with advanced atherothrombosis. Although similar associations are likely to be present among other subgroups with high cardiovascular risk, prospective studies are necessary to confirm this.

C-reactive protein (CRP)
CRP, an acute-phase reactant, is a marker of inflammation which is often assessed in clinical practice to monitor inflammatory responses.[80] The synthesis of CRP in the liver is largely regulated by cytokines, in particular interleukin-6, which are secreted by activated leukocytes, fibroblasts and endothelial cells. The physiological role of CRP is not fully understood, although it has been suggested that CRP contributes to the inflammatory response.[81,82] In healthy subjects, plasma CRP concentrations assessed by highly sensitive assays[83,84] are generally low, i.e. 95% of the general population have a CRP level <3 mg/l and 99% a level <10 mg/l. In response to inflammatory stimuli, CRP levels can rise five- to more than 100-fold within 6 h. The plasma half-life and the fractional catabolic rate of CRP are constant in almost all conditions. Consequently, plasma CRP levels are thought to be determined only by the rate of synthesis, which basically reflects the presence, extent and activity of disease. Therefore, slightly increased, but conventionally normal, CRP levels may reflect a chronic, low-grade inflammatory state.

C-reactive protein (CRP) as a cardiovascular risk indicator or risk factor
Increased levels of CRP have been found to be associated with lipid levels, obesity, diabetes and pack-years of smoking.[51,85,86] In addition, levels of CRP have been found to be higher among subjects with (compared with those without) (sub)clinical cardiovascular disease.[85] Moreover, several prospective studies have demonstrated a clear association between slightly increased CRP levels and risk of cardiovascular events among subjects at high risk of atherothrombotic events,[87] those with stable and unstable angina,[85] those with prior myocardial infarction,[88] and among apparently healthy men89 and women.[86] For example, among subjects free of clinical cardiovascular disease, risk of cardiovascular events was about three times higher among those with CRP levels in the upper quartile compared with those with CRP levels in the lower three quartiles.[90] Levels of CRP are higher among Type 2 diabetic subjects than among non-diabetic subjects.[91] To date, no prospective data on CRP and cardiovascular disease in Type 2 diabetes have been published.

Pathophysiological mechanisms
It has been hypothesized that a chronic low-grade inflammation is the mechanism through which CRP is associated with increased risk of cardiovascular events (Table 1).[89] 

Table 1. Proposed pathophysiological pathways through which high plasma levels von Willebrand factor (vWF), C-reactive protein (CRP) and soluble vascular cell adhesion molecule-1 (sVCAM-1) may be related to risk of cardiovascular events.

Alternatively, CRP might have bioactive properties which by themselves could counterregulate the inflammatory response. Both pro- and anti-inflammatory properties of CRP have been proposed.[81,82] In vitro studies have shown that high levels of CRP could diminish the first step of neutrophil extravasation by downregulating the expression of L-selectin on the neutrophil surface[82] and thus attenuate the normal inflammatory response. On the other hand, raised CRP levels can activate complement via the classic pathway[81] and thus have proinflammatory properties. These data, however, should be interpreted with caution since the concentrations of CRP used in these experiments were often in a supraphysiological range.
Taken together, measurement of CRP levels may provide a novel method for assessing cardiovascular risk among healthy subjects as well as among subjects with clinical manifestations of atherothrombosis.

Soluble vascular cell adhesion molecule-1 (sVCAM-1)
Membrane-bound VCAM-1 is a member of the immunoglobulin superfamily, one of the main four classes of adhesion molecule receptors. Membrane-bound VCAM-1 is a ligand for leukocyte integrins and is thought to allow tethering and rolling of monocytes and lymphocytes as well as firm attachment and transendothelial migration of leukocytes,[92,93] both of which are important early in the atherothrombotic process. In the normal situation, there is a constitutively low expression of membrane-bound VCAM-1 on endothelial cells, smooth muscle cells, tissue macrophages,[94,95] lymphoid dendritic cells and renal tubular cells.[96]
Soluble forms (sVCAM-1) have been detected in plasma.[97] The release of sVCAM-1 in the bloodstream is reported to be in parallel with the expression of membrane-bound VCAM-1 on endothelial cells.[98] Plasma levels of sVCAM-1 can be assessed by highly-sensitive ELISA techniques. The concentration of sVCAM-1 normally present in plasma of healthy subjects has been found to be 400–600 ng/ml.[99] This concentration, however, is highly dependent on the ELISA technique used.
Membrane-bound VCAM-1 synthesis can be upregulated several-fold in response to stimuli such as cytokines,[100] modified lipoproteins101 and advanced glycation endproducts.[102] Furthermore, plasma levels of sVCAM-1 can rise in response to increasing blood pressure induced by the cold pressor test, suggesting that increased pressure on endothelial cells can also upregulate VCAM-1 synthesis or increase its shedding from the cell membrane.[103] Finally, the presence of oxidants has been suggested to play a crucial role in the upregulation of VCAM-1 synthesis.[104] Interestingly, De Mattia et al.[105] showed that treatment with antioxidants decreased plasma sVCAM-1 levels among Type 2 diabetic subjects.

Soluble vascular cell adhesion molecule-1 (sVCAM-1) as a cardiovascular risk indicator or risk factor
In vitro experiments showed endothelial expression of VCAM-1 to be an early manifestation of experimental cholesterol-induced atherothrombosis.[106] Increased VCAM-1 expression has also been found to be present on human atherothrombotic plaques.[107] In addition, sVCAM-1 levels are associated with cardiovascular risk factors, i.e. hypertension,104 impaired glucose tolerance[108] and hypertriglyceridemia.[109] Recent cross-sectional studies have shown significant positive associations between sVCAM-1 levels and carotid artery intimal-medial thickness[110,111] and the severity of peripheral arterial disease obtained by angiography.[110,112] Two prospective studies, however, could not demonstrate high levels of sVCAM-1 to be associated with risk of cardiovascular events among apparently healthy subjects[113] or among subjects with peripheral arterial disease.[114]
Levels of sVCAM-1 have been found to be higher among Type 2 diabetic subjects than among non-diabetic subjects.[71,99,111] Recently, Otsuki et al.[115] found sVCAM-1 levels to be positively associated with intimal plus medial complex thickness of the carotid arteries among diabetic but not among non-diabetic subjects. They reasoned that increased sVCAM-1 levels might be a specific indicator of atherothrombosis among diabetic subjects only. If so, levels of advanced glycation endproducts, which are increased among Type 2 diabetic subjects compared with non-diabetic subjects, might play an important role, since they can strongly stimulate VCAM-1 synthesis. It would therefore be of particular interest to investigate the predictive value of sVCAM-1 for future cardiovascular disease among diabetic subjects.

Pathophysiological mechanisms
The most commonly held view is that increased plasma sVCAM-1 levels reflect increased membrane-bound VCAM-1 levels and thus reflect progressive formation of atherosclerotic lesions (Table 1).[116] Alternatively, increased levels of sVCAM-1 could reflect generalized endothelial dysfunction, since plasma levels most likely originate mainly from endothelial cells and are closely correlated with vWF levels.[110,113] Third, increased sVCAM-1 levels might simply be a marker of an acute-phase response, reflecting the progressive low-grade inflammation of the vessel wall. Accordingly, several cytokines which can induce an acute-phase reaction in response to proinflammatory antigens strongly increase the expression of VCAM-1 on cultured endothelial cells.[117] Fourth, increased levels of sVCAM-1 might be explained not only by an increased synthesis/shedding but also by impaired clearance of sVCAM-1 molecules. Although little is known about the route of elimination of these molecules, an important role of the kidney has been suggested.[118] Finally, sVCAM-1 itself may have bioactive properties related to cardiovascular risk. For example, Koch et al.[119]  have recently shown that sVCAM-1 has proangiogenic properties.
Thus, there is growing evidence that increased levels of sVCAM-1 are associated with the degree of atherothrombosis, although prospective data are not conclusive. Future prospective studies should focus on the importance of increased plasma sVCAM-1 level for future risk of cardiovascular events, especially among diabetic subjects.

Conclusion
Current clinical practice follows the strategy of matching the intensity of treatment of individual patients to their risk of cardiovascular disease. Guidelines for primary prevention of coronary heart disease in clinical practice, however, are mostly based on the use of risk tables which include only conventional risk factors.[120,121] Although a rough risk estimate can be obtained from these tables, more precise risk stratification is needed. Therefore, we have described four promising new risk indicators which may provide increased precision in risk estimation, i.e. microalbuminuria and increased plasma levels of vWF, CRP and sVCAM-1

REFERENCES
1. Breslow JL. Cardiovascular disease burden increases, NIH funding decreases. Nat Med 1997; 3: 600–601.
2. Braunwald E. Shattuck lecture — cardiovascular medicine at the turn of the millennium: triumphs, concerns, and opportunities. N Engl J Med 1997; 337: 1360–1369.
3. Rosamond WD, Chambless LE, Folsom AR et al. Trends in the incidence of myocardial infarction and in mortality due to coronary heart disease, 1987 to 1994. N Engl J Med 1998; 339: 861–867.
4. Feuerstein GZ, Ruffolo RR Jr. Congestive heart failure and genomic medicine: a look into the 21st century. Cardiovasc Drug Ther 1997; 11: 713–717.
5. Orchard T. Diabetes: a time for excitement — and concern. Hopeful signs exist that the ravages of diabetes can be tamed. Br Med J 1998; 317: 691–692.
6. Clark CM Jr. How should we respond to the worldwide diabetes epidemic? Diabetes Care 1998; 21: 475–476.
7. Kannel WB, McGee DL. Diabetes and cardiovascular disease: the Framingham study. J Am Med Assoc 1979; 241: 2035–2038.
8. Jarrett RJ, Shipley MJ. Type 2 (non-insulin-dependent) diabetes mellitus and cardiovascular disease — putative association via common antecedents: further evidence from the Whitehall study. Diabetologia 1988; 31: 737–740.
9. Uusitupa MIJ, Niskanen LK, Siitonen O et al. Five-year incidence of atherosclerotic vascular disease in relation to general risk factors, insulin level, and abnormalities in lipoprotein composition in non-insulin-dependent and nondiabetic subjects. Circulation 1990; 82: 27–36.
10. Yudkin JS. How can we best prolong life? Benefits of coronary risk factor reduction in non-diabetic and diabetic subjects. Br Med J 1993; 306: 1313–1318.
11. Gu K, Cowie CC, Harris MI. Diabetes and decline in heart disease mortality in US adults. J Am Med Assoc 1999; 281: 1291–1297.
12. Stamler J, Vaccaro O, Neaton JD, Wentworth D. Diabetes, other risk factors, and 12-year cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial. Diabetes Care 1993; 16: 434–444.
13. Woodfield SL, Lundergan CF, Reiner JS et al. Angiographic findings and outcome in diabetic patients treated with thrombolytic therapy for acute myocardial infarction: the GUSTO-I experience. J Am Coll Cardiol 1996; 28: 1661–1669.
14. Rosengren A, Welin L, Tsipogianni A, Wilhelmsen L. Impact of cardiovascular risk factors on coronary heart disease and mortality among middle aged diabetic men: a general population study. Br Med J 1989; 299: 1127–1131.
15. Jarrett RJ, Shipley MJ. Mortality and associated risk factors in diabetes. Acta Endocrinol 1985; 110(S272): 21–26.
16. Hopkins PN, Williams RR. Identification and relative weight of cardiovascular risk factors. Cardiol Clin 1986; 4: 3–31.
17. Tunstall-Pedoe H, Woodward M, Tavendale R et al. Comparison of the prediction by 27 different factors of coronary heart disease and death in men and women of the Scottish Heart Health Study: cohort study. Br Med J 1997; 315: 722–729.
18. Ross R, Harker L. Hyperlipidemia and atherosclerosis. Science 1976; 193: 1094–1100.
19. Ross R. Atherosclerosis — an inflammatory disease. N Engl J Med 1999; 340: 115–126.
20. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288: 373–376.
21. Ryan US. Endothelium as a transducing surface. J Mol Cell Cardiol 1989; 21(S1): 85–90.
22. Vane JR, Anggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med 1990; 323: 27–36.
23. Viberti GC, Hill RD, Jarrett RJ et al. Microalbuminuria as a predictor of clinical nephropathy in insulin-dependent diabetes mellitus. Lancet 1982; i: 1430–1432.
24. The American Diabetes Association. Consensus development conference on the diagnosis and management of nephropathy in patients with diabetes mellitus. American Diabetes Association and the National Kidney Foundation. Diabetes Care 1994; 17(11): 1357–1361.
25. Brenner BM, Hostetter TH, Humes HD. Molecular basis of proteinuria of glomerular origin. N Engl J Med 1978; 298: 826–833.
26. Jensen JS, Borch-Johnsen K, Deckert T et al. Reduced glomerular size- and charge-selectivity in clinically healthy individuals with microalbuminuria. Eur J Clin Invest 1995; 25: 608–614.
27. Mogensen CE. Microalbuminuria, blood pressure and diabetic renal disease: origin and development of ideas. Diabetologia 1999; 42: 263–285.
28. Jager A, Kostense P, Nijpels G et al. Microalbuminuria is strongly associated with NIDDM and hypertension, but not with the insulin resistance syndrome: the Hoorn Study. Diabetologia 1998; 41: 694–700.
29. Ljungman S. Microalbuminuria in essential hypertension. Am J Hypertens 1990; 3: 956–960.
30. Shimomura H, Spiro RG. Studies on macromolecular components of human glomerular basement membrane and alterations in diabetes. Decreased levels of heparan sulfate proteoglycan and laminin. Diabetes 1987; 36: 374–381.
31. Tucker BJ, Rasch R, Blantz RC. Glomerular filtration and tubular reabsorption of albumin in preproteinuric and proteinuric diabetic rats. J Clin Invest 1993; 92: 686–694.
32. Viberti GC, Wiseman MJ. The kidney in diabetes: significance of the early abnormalities. Clin Endocrinol 1986; 15: 753–782.
33. Jensen JS, Feldt-Rasmussen B, Borch-Johnsen K, Jensen G. Urinary albumin excretion in a population based sample of 1011 middle aged non-diabetic subjects. The Copenhagen City Heart Study Group. Scand J Clin Lab Invest 1993; 53: 867–872.
34. Alzaid AA. Microalbuminuria in patients with NIDDM: an overview. Diabetes Care 1996; 19: 79–89.
35. Schmitz A, Vaeth M. Microalbuminuria: a major risk factor in non-insulin-dependent diabetes. A 10-year follow-up study of 503 patients. Diabetic Med 1988; 5: 126–134.
36. Dinneen SF, Gerstein HC. The association of microalbuminuria and mortality in non-insulin-dependent diabetes mellitus. A systematic overview of the literature. Arch Intern Med 1997; 157: 1413–1418.
37. Damsgaard EM, Froland A, Jorgensen OD, Mogensen CE. Microalbuminuria as predictor of increased mortality in elderly people. Br Med J 1990; 300: 297–300.
38. Yudkin JS, Forrest RD, Jackson CA. Microalbuminuria as predictor of vascular disease in non-diabetic subjects. Islington Diabetes Survey. Lancet 1988; ii: 530–533.
39 Parving HH. Microalbuminuria in essential hypertension and diabetes mellitus. J Hypertens Suppl 1996; 14: S89–94.
40. Stehouwer CD, Jager A, Donker AJ. Microalbuminuria in essential hypertension: of limited value as an indicator of patients with a high risk for complications. Ned Tijdschr Geneeskd 1997; 141: 1649–1653.
41. Samuelsson O, Wilhelmsen L, Elmfeldt D et al. Predictors of cardiovascular morbidity in treated hypertension: results from the Primary Preventive Trial in Göteborg, Sweden. J Hypertens 1985; 3: 167–176.
42. Jager A, Kostense PJ, Ruhe HG et al. Microalbuminuria and peripheral arterial disease are independent predictors of cardiovascular and all-cause mortality, especially among hypertensive subjects. Five-year follow-up of the Hoorn Study. Arterioscler Thromb Vasc Biol 1999; 19: 617–624.
43. Mogensen CE, Christensen CK. Predicting diabetic nephropathy in insulin-dependent leakiness in clinically healthy subjects. Clin Sci 1995; 88: 629–633.
46 Stender S, Zilversmit DB. Transfer of plasma lipoprotein components and of plasma proteins into aortas of cholesterol-fed rabbits. Molecular size as a determinant of plasma lipoprotein influx. Arteriosclerosis 1981; 1: 38–49.
47. Stehouwer CD, Nauta JJ, Zeldenrust GC et al. Urinary albumin excretion, cardiovascular disease, and endothelial dysfunction in non-insulin-dependent diabetes mellitus. Lancet 1992; 340: 319–323.
48. Stehouwer CDA. Von Willebrand factor, dysfunction of the vascular endothelium, and the development of renal and vascular complication in diabetes. In: Mogensen CE, ed. The kidney and hypertension in diabetes mellitus. Boston/Dordrecht/ London: Kluwer, 1997; 155–163.
49. Aso Y, Inukai T, Takemura Y. Mechanisms of elevation of serum and urinary concentrations of soluble thrombomodulin in diabetic patients: possible application as a marker for vascular endothelial injury. Metabolism 1998; 47: 362–365.
50. De Giorgio LA, Bartolomei G, Gironi A et al. Increased plasma fibronectin concentration in diabetic patients with microalbuminuria. Diabetes Care 1988; 11: 527–530.
51 Yudkin JS, Stehouwer CDA, Emeis JJ, Coppack SW. C-reactive protein in healthy subjects: associations with obesity, insulin resistance and endothelial dysfunction. A potential role for cytokines originating from adipose tissue? Arterioscler Thromb Vasc Biol 1999; 19: 972–978.
52. Mormile A, Veglio M, Gruden G et al. Physiological inhibitors of blood coagulation and prothrombin fragment F 1 + 2 in type 2 diabetic patients with normoalbuminuria and incipient nephropathy. Acta Diabetol 1996; 33: 241–245.
53. Kario K, Matsuo T, Kobayashi H et al. Activation of tissue factor-induced coagulation and endothelial cell dysfunction in non-insulin-dependent diabetic patients with microalbuminuria. Arterioscler Thromb Vasc Biol 1995; 15: 1114–1120.
54. Groop L, Ekstrand A, Forsblom C et al. Insulin resistance, hypertension and microalbuminuria in patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 1993; 36: 642–647.
55. Haffner SM, Gonzales C, Valdez RA et al. Is microalbuminuria part of the prediabetic state? The Mexico City Diabetes Study. Diabetologia 1993; 36: 1002–1006.
56. Nygard O, Nordrehaug JE, Refsum H et al. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 1997; 337: 230–236.
57. Stehouwer CDA, Gall M-A, Hougaard P et al. Plasma homocysteine concentration predicts mortality in non-insulindependent diabetic patients with and without microalbuminuria. Kidney Int 1999; 55: 308–314.
58. Hoogeveen EK, Kostense PJ, Jager A et al. Serum homocysteine level and protein intake are related to risk of microalbuminuria: the Hoorn Study. Kidney Int 1998; 54: 203–209.
59. Niskanen L, Uusitupa M, Sarlund H et al. Microalbuminuria predicts the development of serum lipoprotein abnormalities favouring atherogenesis in newly diagnosed type 2 (non-insulin-dependent) diabetic patients. Diabetologia 1990; 33: 237–243.
60. Mannucci PM. von Willebrand factor: a marker of endothelial damage? Arterioscler Thromb Vasc Biol 1998; 18: 1359–1362.
61. Stehouwer CDA, Lambert J, Donker AJM, van Hinsbergh VWM. Endothelial dysfunction and pathogenesis of diabetic angiopathy. Cardiovasc Res 1997; 34: 55–68.
62. Blann AD, Seigneur M, Steiner M et al. Circulating endothelial cell markers in peripheral vascular disease: relationship to the location and extent of atherosclerotic disease. Eur J Clin Invest 1997; 27: 916–921.
63. Catto AJ, Carter AM, Barrett JH et al. Von Willebrand factor and factor VIII:C in acute cerebrovascular disease. Relationship to stroke, subtype and mortality. Thromb Haemostasis 1997; 77: 1104–1108.
64. Cortellaro M, Boschetti C, Cofrancesco E et al. The PLAT study: hemostatic function in relation to atherothrombotic ischemic events in vascular disease patients. Principal results. Arterioscler Thromb 1992; 12: 1063–1070.
65. Blann AD, Naqvi T, Waite M, McCollum CN. von Willebrand factor and endothelial damage in essential hypertension. J Hum Hypertens 1993; 7: 107–111.
66. Blann AD, Davis A, Miller JP, McCollum CN. von Willebrand factor and soluble E-selectin in hyperlipidemia: relationship to lipids and vascular disease. Am J Hematol 1997; 55: 15–23.
67. Blann AD, McCollum CN. Adverse influence of cigarette smoking on the endothelium. Thromb Haemostasis 1993; 70: 707–711.
68. Chen JW, Gall M-A, Deckert M et al. Increased serum concentration of von Willebrand factor in non-insulin-dependent diabetic patients with and without diabetic nephropathy. Br Med J 1995; 311: 1405–1406.
69. Standl E, Balletshofer B, Dahl B et al. Predictors of 10-year macrovascular and overall mortality in patients with NIDDM: the Munich General Practitioner Project. Diabetologia 1996; 39: 1540–1545.
70. Galajda P, Martinka E, Mokan M, Kubisz P. Endothelial markers in diabetes mellitus. Thromb Res 1997; 85: 63–65.
71. Steiner M, Reinhardt KM, Krammer B et al. Increased levels of soluble adhesion molecules in type 2 (non-insulin dependent) diabetes mellitus are independent of glycaemic control. Thromb Haemostasis 1994; 72: 979–984.
72. Jansson JH, Nilsson TK, Johnson O. von Willebrand factor in plasma: a novel risk factor for recurrent myocardial infarction and death. Br Heart J 1991; 66: 351–355.
73. Lip GY, Blann A. von Willebrand factor: a marker of endothelial dysfunction in vascular disorders? Cardiovasc Res 1997; 34: 255–265.
74. Pottinger BE, Read RC, Paleolog EM et al. von Willebrand factor is an acute phase reactant in man. Thromb Res 1989; 53: 387–394.
75. Juhan-Vague I, Pyke SDM, Alessi MC et al. Fibrinolytic factors and the risk of myocardial infarction or sudden death in patients with angina pectoris. Circulation 1996; 94: 2057–2063.
76. Blann A, Bignell A, McCollum C. von Willebrand factor, fibrinogen and other plasma proteins as determinants of plasma viscosity. Atherosclerosis 1998; 139: 317–322.
77. Junker R, Heinrich J, Ulbrich H et al. Relationship between plasma viscosity and the severity of coronary heart disease. Arterioscler Thromb Vasc Biol 1998; 18: 870–875.
78. Bonithon-Kopp C, Levenson J, Scarabin PY et al. Longitudinal associations between plasma viscosity and cardiovascular risk factors in a middle-aged French population. Atherosclerosis 1993; 104: 173–182.
79. Lowe GD, Lee AJ, Rumley A et al. Blood viscosity and risk of cardiovascular events: the Edinburgh Artery Study. Br J Haematol 1997; 96: 168–173.
80. Ballou SP, Kushner I. C-reactive protein and the acute phase response. Adv Intern Med 1992; 37: 313–336.
81. Bruins P, te Velthuis H, Yazdanbakhsh AP et al. Activation of the complement system during and after cardiopulmonary bypass surgery: postsurgery activation involves C-reactive protein and is associated with postoperative arrhythmia. Circulation 1997; 96: 3542–3548.
82. Zouki C, Beauchamp M, Baron C, Filep JG. Prevention of in vitro neutrophil adhesion to endothelial cells through shedding of L-selectin by C-reactive protein and peptides derived from C-reactive protein. J Clin Invest 1997; 100: 522–529.
83. Macy EM, Hayes TE, Tracy RP. Variability in the measurement of C-reactive protein in healthy subjects: implications for reference intervals and epidemiological applications. Clin Chem 1997; 43: 52–58.
84. Wilkins J, Gallimore JR, Moore EG, Pepys MB. Rapid automated high sensitivity enzyme immunoassay of C-reactive protein. Clin Chem 1998; 44: 1358–1361.
85. Haverkate F, Thompson SG, Pyke SDM et al. Production of C-reactive protein and risk of coronary events in stable and unstable angina. Lancet 1997; 349: 462–466.
86 Hak AE, Stehouwer CDA, Bots ML et al. Associations of C-reactive protein with measures of obesity, insulin resistance and subclinical atherosclerosis in healthy middle-aged women. Arterioscler Thromb Vasc Biol 1999; 19. In press.
87. Kuller LH, Tracy RP, Shaten J, Meilahn EN. Relation of C-reactive protein and coronary heart disease in the MRFIT nested case-control study. Am J Epidemiol 1996; 144: 537–547.
88. Ridker PM, Rifai N, Pfeffer MA et al. Inflammation, pravastatin, and the risk of coronary events after myocardial infarction in patients with average cholesterol levels. Circulation 1998; 98: 839–844.
89. Ridker PM, Cushman M, Stampfer MJ et al. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med 1997; 336: 973–979.
90. Ridker PM, Haughie P. Prospective studies of C-reactive protein as a risk factor for cardiovascular disease. J Invest Med 1998; 46: 391–395.
91. Pickup JC, Mattock MB, Chusney GD, Burt D. NIDDM as a disease of the innate immune system: association of acute-phase reactants and interleukin-6 with metabolic syndrome X. Diabetologia 1997; 40: 1286–1292.
92. Alon R, Kassner PD, Carr MW et al. The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J Cell Biol 1995; 128: 1243–1253.
93. Gerszten RE, Luscinskas FW, Ding HT et al. Adhesion of memory lymphocytes to vascular cell adhesion molecule-1-transduced human vascular endothelial cells under simulated physiological flow conditions in vitro. Circ Res 1996; 79: 1205–1215.
94. O’Brien KD, McDonald TO, Chait A et al. Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation 1996; 93: 672–682.
95. Libby P, Li H. Vascular cell adhesion molecule-1 and smooth muscle cell activation during atherogenesis. J Clin Invest 1993; 92: 538–539.
96. Stad RK, Buurman WA. Current views on structure and function of endothelial adhesion molecules. Cell Adhes Commun 1994; 2: 261–268.
97. Gearing AJ, Hemingway I, Pigott R et al. Soluble forms of vascular adhesion molecules, E-selectin, ICAM-1, and VCAM-1: pathological significance. Ann N Y Acad Sci 1992; 667: 324–331.
98. Pigott R, Dillon LP, Hemingway IH, Gearing AJ. Soluble forms of E-selectin, ICAM-1 and VCAM-1 are present in the supernatants of cytokine activated cultured endothelial cells. Biochem Biophys Res Commun 1992; 187: 584–589.
99. Albertini JP, Valensi P, Lormeau B et al. Elevated concentrations of soluble E-selectin and vascular cell adhesion molecule-1 in NIDDM. Effect of intensive insulin treatment. Diabetes Care 1998; 21: 1008–1013.
100. Osborn L, Hession C, Tizard R et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 1989; 59: 1203–1211.
101. De Caterina R, Cybulsky MI, Clinton SK et al. The omega-3 fatty acid docosahexaenoate reduces cytokine-induced expression of proatherogenic and proinflammatory proteins in human endothelial cells. Arterioscler Thromb 1994; 14: 1829–1836.
102. Vlassara H, Fuh H, Donnelly T, Cybulsky M. Advanced glycation endproducts promote adhesion molecule (VCAM-1, ICAM-1) expression and atheroma formation in normal rabbits. Mol Med 1995; 1: 447–456.
103. Buemi M, Allegra A, Aloisi C et al. Cold pressor test raises serum concentrations of ICAM-1, VCAM-1 and E-selectin in normotensive and hypertensive patients. Hypertension 1997; 30: 84–847.
104. Marui N, Offermann MK, Swerlick R et al. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest 1993; 92: 1866–1874.
105. De Mattia G, Bravi MC, Laurenti O et al. Reduction of oxidative stress by oral N-acetyl-L-cysteine treatment decreases plasma soluble vascular cell adhesion molecule-1 concentrations in non-obese, non-dyslipidaemic, normotensive, patients with non-insulin-dependent diabetes. Diabetologia 1998; 41: 1392–1396.
106. Cybulsky MI, Gimbrone MA Jr. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 1991; 251: 788–791.
107. Davies MJ, Gordon JL, Gearing AJ et al. The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J Pathol 1993; 171: 223–229.
108. Ferri C, Desideri G, Baldoncini R et al. Early activation of vascular endothelium in nonobese, nondiabetic essential hypertensive patients with multiple metabolic abnormalities. Diabetes 1998; 47: 660–667.
109. Hackman A, Abe Y, Insull W et al. Levels of soluble cell adhesion molecules in patients with dyslipidemia. Circulation 1996; 93: 1334–1338.
110. De Caterina R, Basta G, Lazzerini G et al. Soluble vascular cell adhesion molecule-1 as a biohumoral correlate of atherosclerosis. Arterioscler Thromb Vasc Biol 1997; 17: 2646–2654.
111. Kawamura T, Umemura T, Kanai A et al. The incidence and characteristics of silent cerebral infarction in elderly diabetic patients: association with serum-soluble adhesion molecules. Diabetologia 1998; 41: 911–917.
112. Peter K, Nawroth P, Conradt C et al. Circulating vascular cell adhesion molecule-1 correlates with the extent of human atherosclerosis in contrast to circulating intercellular adhesion molecule-1, E-selectin, P-selectin, and thrombomodulin. Arterioscler Thromb Vasc Biol 1997; 17: 505–512.
113. Hwang SJ, Ballantyne CM, Sharrett AR et al. Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: the Atherosclerosis Risk In Communities (ARIC) study. Circulation 1997; 96: 4219–4225.
114. Blann AD, Seigneur M, Steiner M et al. Circulating ICAM-1 and VCAM-1 in peripheral artery disease and hypercholesterolaemia: relationship to the location of atherosclerotic disease, smoking, and in the prediction of adverse events. Thromb Haemostasis 1998; 79: 1080–1085.
115. Otsuki M, Hashimoto K, Morimoto Y et al. Circulating vascular cell adhesion molecule-1 (VCAM-1) in atherosclerotic NIDDM patients. Diabetes 1997; 46: 2096–2101.
116. Jang Y, Lincoff AM, Plow EF, Topol EJ. Cell adhesion molecules in coronary artery disease. J Am Coll Cardiol 1994; 24: 1591–1601.
117. Ahmad M, Theofanidis P, Medford RM. Role of activating protein-1 in the regulation of the vascular cell adhesion molecule-1 gene expression by tumor necrosis factor-alpha. J Biol Chem 1998; 273: 4616–4621.
118. Bonomini M, Reale M, Santarelli P et al. Serum levels of soluble adhesion molecules in chronic renal failure and dialysis patients. Nephron 1998; 79: 399–407.
119. Koch AE, Halloran MM, Haskell CJ et al. Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1. Nature 1995; 376: 517–519.
120. Yudkin JS, Chaturvedi N. Developing risk stratification charts for diabetic and nondiabetic subjects. Diabetic Med 1999; 16: 219–227.
121. Wood D. European and American recommendations for coronary heart disease prevention. Eur Heart J 1998; 19 (suppl A): A12–A19.