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Diabetes and Endothelial Dysfunction: A Clinical Perspective

Departments of Internal Medicine (J.C.-E.) and Obstetrics and Gynecology (M.C.), College of Medicine, University of Vermont, Burlington, Vermont 05401

	Abstract

Top Abstract I. Introduction II. Endothelial Cell (EC)... III. Endothelial Dysfunction and... IV. Reversal of Endothelial... V. Summary and Conclusions References

 
The main etiology for mortality and a great percent of morbidity in patients with diabetes mellitus is atherosclerosis. A hypothesis for the initial lesion of atherosclerosis is endothelial dysfunction, defined pragmatically as changes in the concentration of the chemical messengers produced by the endothelial cell and/or by blunting of the nitric oxide-dependent vasodilatory response to acetylcholine or hyperemia. Endothelial dysfunction has been documented in patients with diabetes and in individuals with insulin resistance or at high risk for developing type 2 diabetes. Factors associated with endothelial dysfunction in diabetes include activation of protein kinase C, overexpression of growth factors and/or cytokines, and oxidative stress. Several therapeutic interventions have been tested in clinical trials aimed at improving endothelial function in patients with diabetes. Insulin sensitizers may have a beneficial effect in the short term, but the virtual absence of trials with cardiovascular end-points preclude any definitive conclusion. Two trials offer optimism that treatment with ACE inhibitors may have a positive impact on the progression of atherosclerosis. Although widely used, the effect of hypolipidemic agents on endothelial function in diabetes is not clear. The role of antioxidant therapy is controversial. No data have been published regarding the effects of hormonal replacement therapy on endothelial dysfunction in postmenopausal women with type 2 diabetes.

I. Introduction

II. Endothelial Cell Dysfunction

A. Normal endothelial cell function

B. Endothelial dysfunction

III. Endothelial Dysfunction and Diabetes

A. Insulin effects on the vasculature

B. Endothelial dysfunction in type 1 diabetes

C. Endothelial dysfunction in type 2 diabetes

IV. Reversal of Endothelial Dysfunction: Lessons from Human Clinical Trials

A. Insulin sensitizers

B. ACE inhibitors

C. Hypolipidemic therapy

D. Arginine supplementation and antioxidants

V. Summary and Conclusions

	I. Introduction

Top Abstract I. Introduction II. Endothelial Cell (EC)... III. Endothelial Dysfunction and... IV. Reversal of Endothelial... V. Summary and Conclusions References

 
THE MAIN etiology for death and for a great percent of morbidity in patients with diabetes (type 1 or type 2) is vascular disease (1, 2). Type 2 diabetes affects small (microangiopathy) or large vessels (macroangiopathy). Microvascular disease is the hallmark of retinopathy, neuropathy, and nephropathy, whereas macroangiopathy in diabetes is manifested by accelerated atherosclerosis, which affects vital organs (heart and brain). Atherosclerosis in patients with type 2 diabetes is multifactorial and includes a very complex interaction including hyperglycemia, hyperlipidemia, oxidative stress, accelerated aging, hyperinsulinemia and/or hyperproinsulinemia, and alterations in coagulation and fibrinolysis (3). A current hypothesis for the initial lesion of atherosclerosis involves changes in endothelial cell (EC) function (4). Endothelial dysfunction has been documented in patients with type 2 diabetes (5, 6, 7, 8, 9, 10) and also in individuals with type 1 diabetes especially when there is clinically manifest microalbuminuria (11, 12, 13, 14, 15, 16, 17). Recent data demonstrate that endothelial dysfunction may also be present in individuals who have insulin resistance (e.g., obese patients) (18) or who are at high risk for developing type 2 diabetes [i.e., impaired glucose tolerance (IGT), metabolic syndrome] (19) and in patients with former gestational diabetes (20).

This review will present a synopsis of our current understanding of endothelial dysfunction in patients with diabetes; special emphasis will be directed to patients with type 2 diabetes.

	II. Endothelial Cell (EC) Dysfunction

Top Abstract I. Introduction II. Endothelial Cell (EC)... III. Endothelial Dysfunction and... IV. Reversal of Endothelial... V. Summary and Conclusions References

 
A. Normal EC function
The EC lines the internal lumen of all the vasculature and serves as an interface between circulating blood and vascular smooth muscle cells (VSMC). In addition to serving as a physical barrier between the blood and tissues, the EC facilitates a complex array of functions in intimate interaction with the VSMC, as well as cells within the blood compartment as depicted in Fig. 1.

View larger version (35K): [in this window] [in a new window]   Figure 1. Microanatomy of a small vessel. The endothelium (EC) confers a lining to all the vasculature. It interacts directly with the VSMC of the vessels and with the blood cells as well as with the plasma components. Via several chemical mediators, the EC is in fact a regulator of the VSMC and plays a key role in maintaining hemostasis and blood fluidity. IEL, Internal elastic lamina.

Endothelial-NOS (eNOS) type III is essential for the control of vascular tone in response to several stimuli, including mechanical (e.g., shear stress) and receptor dependent (e.g., acetylcholine) and receptor independent (e.g., calcium ionophore) (29). NO produced by NOS type III in the endothelium diffuses to the vascular smooth muscle (VSM) where it activates the enzyme guanylate cyclase. The concomitant increase in cyclic GMP then induces relaxation of the VSM. Thus, the net effect of an increase in NO is vasodilation (Fig. 2). NO production by the NOS type III is also basally produced and in some circulations (e.g., cerebral), basal NO production is substantial. The continual vasodilation produced by basal NO production has a role in regulation of blood pressure as well. Many studies have demonstrated that systemic infusion of NOS inhibitors elevate blood pressure.

View larger version (15K): [in this window] [in a new window]   Figure 2. Endothelial cell as a regulator of the smooth muscle cells. The EC produces NO, gas that diffuses into the VSMC and activates the enzyme guanylate cyclase which produces cyclic GMP. The latter induces muscle relaxation, which is physiologically translated into vasodilation. The immediate precursor of NO is the amino acid arginine and the key enzyme in its production is NOS.

2. Measurement of NO-mediated vasodilation. Typically, NO-dependent vasodilatation is probed by the vasodilatory response to infusion of a compound (e.g., acetylcholine or methacholine), which increases the synthesis and release of NO via a receptor-mediated response that is calcium dependent (9, 29, 37, 38, 39) or in response to reactive hyperemia which stimulates shear stress-induced NO production. This response is compared with the vasodilation evoked by specific chemical compounds that directly act on VSMC (e.g., sodium nitroprusside). The difference in vasodilation observed between the two conditions can be considered endothelium-dependent vasodilation. In addition, specific inhibitors of NOS [e.g., nitro-L-arginine (L-NNA)] have been used to further probe EC function in vivo (13, 33, 40, 41, 42).

3. Angiotensin II (ANG-II). The EC also produces mediators that induce vasoconstriction, including endothelin (43, 44, 45), prostaglandins (46, 47), and ANG-II (48, 49, 50, 51) and regulates vascular tone by maintaining a balance between vasodilation (NO production) and vasoconstriction (e.g., A-II generation). ANG-II is produced in local tissues by the EC (52, 53). The key enzyme that regulates the local generation of ANG-II is angiotensin converting enzyme (ACE). This proteolytic enzyme is synthesized by the EC, expressed in the surface of the EC, and exerts activity upon the blood-borne angiotensin I. Angiotensin I is produced by cleavage of a precursor macromolecule (angiotensinogen) effected by plasma renin, another proteolytic enzyme produced in the kidney. ANG-II binds to and regulates VSMC tone via specific angiotensin (ANG) receptors. Depending upon the specific receptor activated, ANG-II can exert regulatory effects upon several VSMC functional activities including contraction (i.e., vasoconstriction) and growth, proliferation, and differentiation. Overall, the actions of ANG-II oppose those of NO.

As reviewed above, NO is a product of the enzyme NOS, which responds to specific activators and inhibitors. NOS also is regulated by local concentrations of bradykinin (54). This peptide acts with b2 receptors on the EC cell surface membrane, increasing the generation of NO via NOS activation. Interestingly, the local concentrations of bradykinin are regulated by the activity of ACE. ACE breaks down bradykinin into inactive peptides (52, 55). Hence, high ACE concentrations will antagonize NO activity not only by increasing ANG-II generation but also, and possibly most importantly, by decreasing concentrations of bradykinin.

A model of regulation of vascular tone (and lumen regulation) in which ACE plays a key role has emerged in recent years (Fig. 3). This model predicts that high ACE activity will result in vasoconstriction because of a decrease in NO generation and increased generation of ANG-II. This results in contraction of VSMCs and decreased lumen diameter. Moreover, sustained activity of this enzyme will presumably be associated with an increase in the growth, proliferation, and differentiation of the VSMC as well as a decrease in the antiproliferative action of NO coupled with a decrease in local fibrinolysis and an increase in platelet aggregation.

View larger version (26K): [in this window] [in a new window]   Figure 3. The role of ACE in endothelial cell function. The EC membrane holds the ACE which, when overactive or overexpressed, produces a large amount of ANG-II. The latter acts directly on the VSMC by attaching to specific receptors located on the membrane of the VSMC. Many of the actions of ANG-II are antagonistic to those of NO as depicted on the figure. ACE activation also leads into faster catabolism of bradykinin.

View larger version (10K): [in this window] [in a new window]   Figure 4. Local coagulation and fibrinolysis are regulated by the endothelial cell. The main inhibitor of the fibrinolytic system is PAI-1, which has been documented to be elevated in disease states with insulin resistance (e.g., obesity, diabetes).

B. Endothelial dysfunction
Since the actions of the EC are multiple and involve several systems, alterations in EC function may affect one or more of these systems, either simultaneously or at distinct time periods. Thus, no single definition of EC dysfunction covers the whole array of possible disruption in normal function. In consequence, endothelial dysfunction has been defined pragmatically. It basically involves either an increase (or a decrease) in any of the EC-related chemical messenger and/or by alteration in any of the functional changes listed earlier (Table 1). Some examples of EC dysfunction include an increased permeation of macromolecules (22, 63, 64), increased or decreased production of vasoactive factors producing abnormal vasoconstriction/vasodilation (22, 30, 43, 65), and increased prothrombotic and/or procoagulant activity (66). However, the most commonly accepted EC dysfunction alteration pertains to abnormalities in the regulation of the lumen of vessels. In this context, EC dysfunction has been defined by blunting of the vasodilatory response to acetylcholine or hyperemia, both of which are known to produce NO-dependent vasodilation. In some specific circumstances, endothelial dysfunction has been defined by a paradoxical vasoconstrictive response to acetylcholine or similar pharmacological agents (i.e., metacholine). At the heart of the definition of EC dysfunction is the measurement of EC function. This review will focus on the methods that are available for measurement of EC function in vivo in humans. The methodology available for in vitro measurement is beyond the scope of this manuscript.

1. How do we measure in vivo EC function in humans?
As explained before, the range of EC function(s) may differ according to the type of vessel(s) affected as well as the tissue or organ perfused. EC action may affect one or several functions, either simultaneously or in a temporal sequence and thus cannot be considered a single, discrete, and uniquely defined entity. In consequence, there is no gold standard for measurement of EC dysfunction. In general, EC function is measured experimentally by 1) methods that assess the functional consequences of EC activity, alone or complemented by 2) measurement of the concentration of those chemical mediators that mediate EC function.

The approach to measuring EC function in vivo stems from the fact that the most widely recognized function of the EC pertains to its effects on vascular tone. The EC produces chemical mediators that may induce contraction or relaxation of the adjacent VSM (i.e., vasoconstriction or vasodilation). The vasodilatory molecules include several peptides, hormones as well as NO (30, 31, 32, 33, 52, 67) and an unidentified endothelium-dependent hyperpolarizing factor (EDHF) (68). From the viewpoint of lumen regulation, NO occupies a prominent role and is considered one of the most important molecules that the EC produces to regulate vascular tone.

From the clinical perspective, EC function has been estimated by measuring invasively (55, 69, 70, 71) (i.e., coronary catheterization) or noninvasively (72, 73) (i.e., ultrasound) changes in blood flow. Thus, physiologically, in vivo, EC function is defined in humans as an increase in blood flow or in the diameter of a vessel in response to agents that increase the concentration of NO. This can be correlated with a decrease in blood flow evoked by a decrease in the local concentrations of NO after production of the latter is blocked by L-NNA. The response must be attributable to the EC and not to dysfunction of the SMC. The latter is probed by measuring the blood flow response (or the diameter of the vessel) in response to chemical agents that act directly on the SMC (e.g., nitroprusside). Blood flow and/or vessel diameter can be measured in human beings using a wide array of techniques. A discussion of pros and cons of these techniques is beyond the scope of this review, but a summary of those currently available is presented in Table 2.

	III. Endothelial Dysfunction and Diabetes

Top Abstract I. Introduction II. Endothelial Cell (EC)... III. Endothelial Dysfunction and... IV. Reversal of Endothelial... V. Summary and Conclusions References

 
Diminished capacity of NOS to generate NO has been demonstrated experimentally when ECs are exposed either in vitro or in vivo to a diabetic environment (75, 87, 88, 89, 90, 91, 92, 93, 94). The EC is then a target of the diabetic milieu and endothelial dysfunction is thought to play an important role in the vasculopathy of this disease state. A large body of evidence in humans indicates that endothelial dysfunction is closely associated to microangiopathy and atherosclerosis in both types 1 and 2 diabetes mellitus (11). This association is particularly true in those patients with type 1 diabetes who have either early (microalbuminuria) or late (macroalbuminuria) nephropathy. In these patients, a great variety of markers indicate endothelial dysfunction: poor EC-dependent vasodilation, increased blood levels of von Willebrand factor (vWF), thrombomodulin, selectin, PAI-1, type IV collagen, and t-PA (11, 12, 13, 95, 96, 97, 98, 99, 100). Once established, EC dysfunction can, in turn, induce alterations in vessels that worsen vasculopathy and progress disease. Of note is that arteries and arterioles are not considered commonly as target tissues/organs of insulin action. However, in recent years a body of evidence has accumulated that supports the hypothesis that vessels are insulin responsive.

A. Insulin effects on the vasculature
Several years ago Jialal and colleagues (101) described the presence of receptors for insulin, IGF-I, and IGF-II on cells from micro- and macrovessels with binding characteristics that are similar to those in other cells. Interestingly, these investigators suggested that the finding of large numbers of IGF-I and IGF-II receptors on ECs supported a physiological role for these growth factors and proposed the hypothesis that they may be involved in vascular complications associated with diabetes. In a previous publication, this same group (102) had demonstrated that the concentrations of receptors for aortic smooth muscle cells were 10-fold fewer than other cell types. Moreover, insulin stimulated glucose incorporation into glycogen and stimulated DNA replication in retinal ECs and pericytes and aortic smooth muscle cells, but had no effect on aortic endothelium. These data suggested that a differential response to insulin may exist between endothelium of micro- and macrovasculature and that retinal capillary endothelium and retinal pericytes are both very insulin-sensitive tissues. Insulin deficiency and chronic hyperglycemia can increase the concentration of the membrane-bound protein kinase C (PKC) and total diacylglycerol (DAG) levels. Insulin administration and consequently euglycemia can prevent the increase in PKC activities and DAG levels (103). Significant information regarding insulin signaling in the vascular tissues has emerged recently.

Insulin signaling on the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways were compared in vascular tissues of lean and obese Zucker rats (104). As anticipated, insulin-stimulated tyrosine phosphorylation of insulin receptor ß-subunits in microvessels of obese rats was significantly decreased compared with lean rats as well as insulin-induced tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2). Moreover, the association of the p85 subunit to the IRS proteins and the IRS-associated PI 3-kinase activities stimulated by insulin in the aorta of obese rats were significantly decreased compared with the lean rats as was the serine phosphorylation of Akt. These in vitro results were comparable to in vivo studies using the euglycemic clamp technique. In marked contrast, these investigators found that insulin-stimulated tyrosine phosphorylation of MAP kinase (ERK-1/2) was equal in isolated microvessels of lean and obese rats, although basal tyrosine phosphorylation of ERK-1/2 was higher in the obese rats. Thus, insulin has a direct effect on vascular tissues using signaling similar to other tissues. A selective resistance to PI 3-kinase (but not to MAP kinase pathway) in the vascular tissues of obese Zucker rats was found which the investigators hypothesize may be of importance in the vascular disease of insulin resistance states. More recently, using physiological concentrations of insulin, King’s group (105) found that the hormone increased the levels of eNOS mRNA, protein, and its activity by 2-fold after 2–8 h of incubation of ECs. Interestingly, this effect of insulin was seen in microvessels isolated from Zucker lean insulin-sensitive rats but not from insulin-resistant Zucker fatty rats. PKC activators inhibited both the activation by insulin of PI-3 kinase and eNOS mRNA levels. These investigators concluded that insulin may have not only an acute vasodilatory effect but also chronically modulate vascular tone. Moreover, they postulated that activation of PKC in the vascular tissues may be a primary event that leads to endothelial dysfunction in insulin-resistant states.

B. Endothelial dysfunction in type 1 diabetes
It is not clear from the literature whether EC dysfunction is the consequence of the diabetic milieu in type 1 diabetes or a marker of vascular damage. In a experimental model of type 1 diabetes, endothelial function was evaluated directly in rats (106) with chronically implanted flow probes. The responses to acetylcholine and sodium nitroprusside were not altered significantly; moreover, neither endotheliummediated vasodilation nor responsiveness to NO was impaired, and hyperglycemia did not directly or significantly impair endothelium-mediated relaxation in this model of insulin-dependent diabetes mellitus. In a recent and well designed study (13), endothelium-dependent and endothelium-independent vasodilatation, endothelium-dependent hemostatic factors, and vasoconstrictor responses were determined in type 1 diabetic patients during euglycemia with and without microvascular complications. Forearm endothelium-dependent and endothelium-independent vasodilatation and adrenergic responsiveness were unaltered in type 1 diabetic patients with and without microvascular complications. Relative to healthy control subjects, endothelium-dependent vasodilatation was depressed during a repeated ACh challenge (with L-arginine coinfusion) in the diabetic patients without complications or with microalbuminuria. In contrast, this vasodilatation was enhanced in the patients with retinopathy. Elevation of the endothelium-derived tissue factor plasminogen inhibitor was the most consistent marker of endothelial damage of all the endothelial markers measured in these group of patients but showed no correlation with the presence or absence of microvascular complications. Clarkson et al. (107) compared 80 young adults with insulin-dependent diabetes with 80 matched nondiabetic control subjects. Flow-mediated dilation was significantly impaired in diabetic subjects (5.0 ± 3.7% vs. 9.3 ± 3.8% in control subjects, P < 0.001). Using multivariate analysis, flow-mediated dilation was inversely related to duration of diabetes (r = –0.26, P < 0.05) and low density lipoprotein cholesterol (LDL-C) levels (r = –0.38, P < 0.005). Thus, in this study, EC dysfunction was found as an early manifestation of vascular disease but late in the course of type 1 diabetes.

One cross-sectional study assessed endothelial adhesion molecules in patients with type 1 diabetes (n = 70), with varying degrees of metabolic control and status of diabetic late complications, and compared them to those in age-matched healthy subjects (n = 70) (108). Concentrations of cICAM-1 and cVCAM-1 were elevated in insulin-dependent diabetes mellitus (IDDM) whereas cELAM-1 did not differ between the groups. The levels of cVCAM-1 were more markedly elevated in IDDM patients with diabetic retinopathy than in those patients with micro- or macroalbuminuria, whereas no difference in cICAM-1 and cELAM-1 was apparent regarding the clinical status of diabetic microangiopathy. No correlations were found between hemoglobin A1c and cAMs. This relation to the duration of diabetes was not found by Johnstone et al. (14), who measured vascular reactivity in the forearm resistance vessels of 15 patients with IDDM and 16 age-matched normal subjects. These investigators stated that no patient had hypertension or dyslipidemia. The vasodilative response to methacholine was less in diabetic than in normal subjects, but forearm blood flow (FBF) responses to nitroprusside and verapamil and the forearm vasoconstrictor responses to phenylephrine were similar in diabetic and healthy subjects. In diabetic subjects, endothelium-dependent vasodilation correlated inversely with serum insulin concentration but not with glucose concentration, glycosylated hemoglobin, or duration of diabetes.

In another study (108A ), the plasma fibronectin 30-kDa domain was measured in 44 type 1 diabetic patients and in 20 healthy subjects. A significantly raised mean concentration of a free N-terminal fibronectin 30-kDa domain was found in plasma of diabetic patients with proliferative retinopathy as compared with healthy persons, and a positive correlation was observed between free N-terminal fibronectin and vWF in plasma of all examined subjects (r = 0.62). A similar correlation was present between fibronectin and the degree of albuminuria (r = 0.56). However, no relationship was found between fibronectin and the degree of control of diabetes. Thus, in these two cross-sectional studies, type 1 diabetes has been associated more with the presence of microvascular disease than with the diabetic milieu.

It is the general consensus that the occurrence of EC dysfunction in type 1 diabetes signifies a very high risk of micro- and macroangiopathy and, although the diabetic state predisposes to EC dysfunction in this disease, is not sufficient to cause it. More likely, other agents (genes, environment) are likely to play a role in determining those patients that will develop aggressive angiopathy and hence EC dysfunction. Irrespective of whether EC dysfunction is a cause or a consequence of vascular injury in type 1 diabetes, therapeutic efforts aimed at restoring EC to normal will more likely have an affect on the natural history of vasculopathy in type 1 diabetes.

C. Endothelial dysfunction in type 2 diabetes
The role of endothelial dysfunction in type 2 diabetes is more complicated than that for type 1. The effects of aging, hyperlipidemia, hypertension, and other factors add to the complexity of the problem. In contrast to patients with type 1 diabetes, endothelial dysfunction can also occur in patients with type 2 diabetes even when the patients have normal urinary albumin excretion. In fact, markers of endothelial dysfunction are often elevated years before any evidence of microangiopathy becomes evident (11, 109, 110, 111, 112, 113, 114, 115). A major pathophysiological alteration of type 2 diabetes is insulin resistance. As a result, a great research effort has been focused on defining the possible contribution of insulin resistance to endothelial dysfunction.

1. Endothelial dysfunction and insulin resistance. There is a growing body of evidence accumulating to demonstrate the coexistence of insulin resistance and endothelial dysfunction. Insulin-induced vasodilation, which is partially mediated by NO (116) release, is impaired in obese individuals who do not have type 2 diabetes but who display insulin resistance (18). Moreover, the obese state, a model of human insulin resistance, is associated with high levels of endothelin in plasma (117). PAI-1 concentrations in blood also are high in patients with otherwise uncomplicated obesity; a drastic decrease in PAI-1 levels has been noted by our laboratory in response to moderate weight loss (118). Recently, it has been demonstrated that women with previous gestational diabetes have evidence of endothelial dysfunction (20, 119, 120). In women with polycystic ovary syndrome, high levels of PAI-1 have been found which improve with any therapeutic intervention that improves insulin sensitivity (121, 122, 123, 124). Further, evidence suggests that endothelial dysfunction occurs in a concomitant manner with insulin resistance and antedates overt hyperglycemia in patients with type 2 diabetes. Steinberg et al. (125) recently demonstrated that elevated free fatty acid levels in blood (produced in normal individuals by simultaneous infusion of triglycerides and heparin) induced endothelial dysfunction. FFA are classically elevated in obese patients, patients with type 2 diabetes, and, in general, in those individuals who display features of the syndrome of insulin resistance. Thus, data support the hypothesis that the metabolic abnormalities of insulin resistance may lead to endothelial dysfunction.

More recently, Caballero et al. (126) demonstrated early abnormalities in vascular reactivity and biochemical markers of EC activation in individuals at risk of developing type 2 diabetes. These investigators measured the increase in blood flow in the microcirculation (laser Doppler flowmetry) and in the macrocirculation (ultrasound) in four groups of individuals: 1) healthy normoglycemic subjects with no history of type 2 diabetes in a first-degree relative (controls); 2) healthy normoglycemic subjects with a history of type 2 diabetes in one or both parents (relatives); 3) subjects with IGT; and 4) patients with type 2 diabetes without vascular complications. Moreover, these investigators measured plasma concentrations of endothelin-1 (ET-1), vWF, soluble intercellular adhesion molecule (sICAM), and soluble vascular cell adhesion molecule (sVCAM) were also measured as indicators of EC activation. The vasodilatory responses to acetylcholine were reduced in groups 2, 3, and 4 compared with controls. The plasma levels of ET-1 were significantly higher in these three groups. On stepwise multivariate analysis, age, sex, fasting plasma glucose, and body mass index (BMI) were the most important contributing factors to the variation of vascular reactivity. However, all clinical and biochemical measures explained only 32–37% of the variation in vascular reactivity. These results suggest that abnormalities in vascular reactivity and biochemical markers of EC activation are present early in individuals at risk of developing type 2 diabetes, even at a stage when normal glucose tolerance exists and that factors in addition to insulin resistance may be instrumental in the EC dysfunction of individuals at high risk of developing type 2 diabetes.

The insulin resistance syndrome encompasses more than a subnormal response to insulin-mediated glucose disposal. Patients with this syndrome also frequently display elevated blood pressure, hyperlipidemia, and dysfibinolysis, even without any clinically demonstrable alteration in plasma glucose concentrations. Of note, endothelial dysfunction also has been demonstrated in patients with hypertension (85, 127, 128, 129, 130, 131, 132, 133), which is one of the features of the syndrome of insulin resistance. It is tempting to speculate that loss of endothelial-dependent vasodilation and increased vasoconstrictors might be etiological factors of hypertension. Moreover, loss of activity and/or quantity of endothelium-bound protein lipase activity may contribute to hyperlipidemia, which is typical of the insulin resistance syndrome. A synergistic interaction and vicious cycle may exist in which endothelial dysfunction contributes to insulin resistance and vice versa.

2. Endothelial dysfunction, dysfibinolysis, and insulin resistance. Under normal conditions, the blood is constantly in a balance between a "basal on-going" activation of coagulation and compensatory fibrinolysis. A current hypothesis states that, in patients with endothelial dysfunction, PAI-1 levels are elevated, which in turn inhibits dissolution of fibrin deposits on the luminal side of the vessel wall. Several investigators have suggested that PAI-1 plays a major role in the generation and/or progression of atherosclerosis. Our laboratory and many others have found high levels of PAI-1 in disease states in which insulin resistance is a prominent pathophysiological feature (122, 134, 135, 136, 137, 138, 139). Examples of this are type 2 diabetes, upper body obesity, and polycystic ovary syndrome. These disease states also are associated with accelerated atherosclerosis, which supports the hypothesis that high levels of PAI-1 may play a role in initiation and/or progression of macrovascular disease. It has been proposed that the hyper (pro)-insulinemia of insulin resistance might be implicated as an etiological alteration for the high blood levels of PAI-1 (140). Several in vitro, in vivo, and animal models are strongly supportive of this hypothesis (141, 142, 143, 144, 145, 146, 147, 148, 149). However, other studies have found that infusion of insulin directly in humans for up to 6 h is not associated with an increase in the blood levels of PAI-1 (150, 151). Recently, we found that simulation of the diabetic environment in normal individuals by exogenous insulin infusions, resulting in hyperinsulinemia, hyperglycemia, and hyperlipidemia, was associated with an increase in the blood concentrations of PAI-1 (152). We concluded from that study that the dysfibrinolysis of type 2 diabetes is most likely a multifactorial process, which includes changes in hormonal (insulin) and substrate (lipids, glucose) concentrations.

3. Cellular and molecular basis for EC dysfunction in diabetes and insulin resistance. The biochemical and cellular factors that are associated with endothelial dysfunction in diabetes are listed in Table 3 and summarized as follows:

2. Overexpression of growth factors (154, 155) has also been implicated as a link between diabetes and proliferation of both ECs and VSM, possibly promoting neovascularization. Levels of these growth factors are increased in animal diabetic models, but the temporal sequence is not well defined and, therefore, these issues require further investigation.

3. Chronic hyperglycemia (156) leads to nonenzymatic glycation of proteins and macromolecules and, hence, this biochemical reaction has been implicated in many of the chronic complications of diabetes. Changes in the properties of protein and DNA as well as antigenic changes have been demonstrated to occur as a consequence of nonenzymatic glycation. Independent of chronic effects of hyperglycemia, acute glucose exposure dilates arteries with intrinsic tone and impairs cerebrovascular reactivity to changes in intravascular pressure via an endothelium-mediated mechanism that involves NO and prostaglandins (157).

4. Hyperglycemia increases the flux of glucose through the glycolytic pathway, increasing de novo synthesis of DAG (158, 159, 160). Increased DAG has been shown to occur in both EC and VSM, leading to increased PKC activity. Both DAG and PKC are important intracellular signaling molecules involved in wide variety of cellular responses, including modulating vasoconstriction (94). Increased PKC-induced contraction has been demonstrated in rat mesenteric arteries exposed to elevated glucose (94).

5. The EC is very susceptible to damage by oxidative stress. The diabetic state is typified by an increased tendency for oxidative stress (109, 115, 161, 162, 163, 164, 165) and high levels of oxidized lipoproteins, especially the so-called small, dense LDL-C. High levels of fatty acids and hyperglycemia have both been shown to induce an increased level of oxidation of phospholipids as well as proteins. A proposed hypothesis suggests that this might be one of the etiological factors in inducing endothelial dysfunction in type 2 diabetes.

6. The diabetic state in humans is associated with a prothrombotic tendency as well as increased platelet aggregation as already discussed above. This may be related to several factors, including diminished NO production (10) and decreased fibrinolytic activity related to high levels of PAI-1 levels found in the blood of patients with this disease (166). Remarkably, this defect may be an acquired one. As described before, we have recently demonstrated that mimicking the diabetic environment in normal individuals by simultaneous infusion of glucose and intravenous fat emulsion induces an increase in the blood concentrations of PAI-1. Assuming that the latter represents a market for endothelial dysfunction, we may conclude that the metabolic abnormalities characteristic of type 2 diabetes induce endothelial dysfunction. In addition to decreased fibrinolysis, the diabetic state is also associated with an increase in the activation of the coagulation cascade by various mechanisms such as nonenzymatic glycation, formation of advanced glycosylation end-products (AGE) (156, 167, 168), and decreased heparan sulfate synthesis. Although there is no direct link between activation of the coagulation cascade and endothelial dysfunction in humans, it is possible to speculate that repeated activation of the coagulation cascade may cause overstimulation of ECs and induce endothelial dysfunction.

7. Tumor necrosis factor (TNF) has been implicated as a link between insulin resistance, diabetes, and endothelial dysfunction (169). Increased expression of this factor in human obesity supports the hypothesis that elevated TNF induces insulin resistance. TNF also can induce the synthesis of other cytokines, which alone or in concert with others, may alter endothelial function.

8. In type 2 diabetes, insulin levels tend to be either normal or elevated. The effects of hyperinsulinemia per se on endothelial function, however, have not been extensively studied. The hypothesis has been advanced in recent years that insulin and/or insulin precursors may be atherogenic; however, the data from the recently published United Kingdom Prospective Diabetes Study (UKPDS) suggest that this is not the case (170).

	IV. Reversal of Endothelial Dysfunction: Lessons from Human Clinical Trials

Top Abstract I. Introduction II. Endothelial Cell (EC)... III. Endothelial Dysfunction and... IV. Reversal of Endothelial... V. Summary and Conclusions References

 
Several therapeutic interventions have been tested in clinical trials aimed at improving endothelial function. We will review those that are most relevant for the patients with diabetes and/or insulin resistance, such as those testing the effects of insulin sensitizers, antioxidants, and ACE inhibitors. It is beyond the scope of this paper to review the large literature regarding the possible effects of hypolipidemic agents since none of those trials have focused directly on the effects of these agents in affecting endothelial function in patients with diabetes. Moreover, estrogen replacement therapy has been shown to improve endothelial function; however, as of the day of writing this review, no specific trial on women with diabetes was found in the literature.

A. Insulin sensitizers
As reviewed earlier, insulin-resistant states have been found to be associated with endothelial dysfunction. Thus, investigators have tested the possibility that therapeutic agents that increase insulin sensitivity may also improve endothelial function. Several studies using cell preparations provide support for this hypothesis. Pasceri et al. (171) found that troglitazone (activator of the peroxisome proliferator receptor- and also insulin sensitizer) inhibits in vitro the expression of VCAM-1 and ICAM-1 in activated ECs. This drug also significantly reduced monocyte/macrophage homing to atherosclerotic plaques. In separate studies (172, 173) it was found that this agent also reduced in a dose-dependent manner the expression of VCAM-1, ICAM-1, and E-selectin induced by different amounts of oxidized LDL and tumor necrosis factor. These studies and others (174) provided a rationale for testing the hypothesis that insulin sensitizers may have a beneficial effect on endothelial function in patients with diabetes or insulin resistance.

In consequence, some clinical trials have been conducted to investigate the nonhypoglycemic effects of this class of new antidiabetic drugs on endothelial function. In a short trial, Murakami et al. (175) reported that administration of troglitazone was associated with a substantial reduction in the frequency of episodes of angina in patients with coronary artery disease and type 2 diabetes. Moreover, these investigators found that the decrease in episodes of pain was correlated with angiographic (coronary) improvement in endothelial function.

Avena et al. (176) studied patients with peripheral vascular disease and IGT (but not overt type 2 diabetes; "occult diabetes") and normal controls matched for age and gender. Brachial artery (BA) flow was measured before and after 5 min of BA occlusion during fasting and at 30 min, 1 h, and 2 h after the administration of 75 g of glucose [oral glucose tolerance test (OGTT)]. The evaluation was repeated 2 and 4 months after administration of troglitazone (400 mg/day). The so-called "occult diabetic" group had an abnormal response to hyperemia before the treatment with troglitazone with almost no change in flow in response to BA occlusion. After 4 months of therapy with insulin sensitizer, BA flow normalized both while fasting and after oral glucose intake during the OGTT. Cominacini et al. (172) tested the effect of troglitazone (200 mg once daily) in a randomized, placebocontrolled, parallel group study in 29 patients with type 2 diabetes. The results of this study strongly supported a beneficial role for troglitazone since it was associated with an increase in the resistance of LDL to be oxidized compared with the group receiving placebo, and the serum of the patients treated with this medication had lower toxic effects on ECs in vitro. Moreover, plasma E-selectin levels decreased from 56.5 ± 2.33 to 43.7 ± 1.77 µg/liter (no change in the placebo group, P < 0.01). These investigators concluded that in type 2 diabetes, troglitazone may slow down the development of atherosclerosis by modifying LDL-related atherogenic events.

However, not all trials have reached the same conclusions. Tack et al. (177) used a randomized, double-blind, cross-over design to study the effects of troglitazone (400 mg/day). Fifteen obese subjects participated in the trial. A very comprehensive vascular evaluation was performed at the end of each period, which included forearm vasodilator responses to intraarterially administered acetylcholine and sodium nitroprusside; insulin sensitivity and insulin-induced vascular and neurohumoral responses (clamp); and vasoconstrictor responses to NC-monomethyl-L-arginine (L-NMMA) during hyperinsulinemia. These patients also had ambulatory 24-h blood pressure monitoring. The participants were insulin resistant compared with lean subjects, and troglitazone improved whole-body and forearm glucose uptake (from 1.09 ± 0.54 to 2.31 ± 0.69 µmol dl^–1 x min^–1), P = 0.006). Insulin-induced vasodilatation was blunted in obese subjects [percent increase in FBF in lean 66.5 ± 23.0%, vs. 10.1 ± 11.3% in obese, P = 0.04], but did not improve during troglitazone. Vascular responses to acetylcholine, sodium nitroprusside, and L-NMMA did not differ between the obese and lean group, nor between both treatment periods in the obese individuals. These investigators concluded that in insulin-resistant obese subjects, endothelial vascular function is normal despite impaired vasodilator responses to insulin. Moreover, these authors found that troglitazone improved insulin sensitivity but it had no effects on endothelium-dependent and -independent vascular responses.

In summary, the data regarding effects of insulin sensitizers on endothelial function in patients with diabetes or insulin-resistant states is suggestive of a beneficial effect at least in the short term, accepting that the opinion is not uniform since a well designed trial did not show any link between improvement in insulin sensitivity and vascular function. Unfortunately, we do not have long-term trials with well defined cardiovascular endpoints to draw any definitive conclusion. Therefore, at this stage, we feel that the hypothesis that insulin sensitizers may prevent or delay atherosclerosis in patients with type 2 diabetes or in those with the syndrome of insulin resistance deserves testing.

B. ACE inhibitors
Therapeutically, it has been established that acute and short-term inhibition of ACE in patients with established vascular disease improves EC function, as defined by an improved vascular tone, especially in response to the infusion of agents that increase NO synthesis. The TREND (Trial on Reversing Endothelial Dysfunction) study (178) investigated the theory that inhibition of ACE with quinapril might improve endothelial dysfunction in normotensive patients with coronary artery disease and no heart failure, cardiomyopathy, or major lipid abnormalities. After 6 months of therapy, the quinapril-treated group showed significant net improvement, compared with the placebo arm, in vasodilatory response to incremental concentrations of acetylcholine. The investigators speculated that the benefits of ACE inhibition were due to attenuation of the contractile and superoxide-generating effects of ANG-II, as well as enhancement of EC release of NO in response to diminished breakdown of bradykinin.

Whether the improvement of EC function documented by TREND is correlated with improvements in atherosclerosis is not clear. In this respect, the QUIET (Quinapril Ischemic Event Trial) (179) studied 1,750 patients with normal left ventricular function who were undergoing coronary angiography and angioplasty and who were randomized to 20 mg/day of quinapril vs. placebo and followed for 3 yr for cardiac end points. For those who received a second angiography in follow up, 119 of 243 placebo-treated patients (49%) had progression of atherosclerosis. Among the 234 quinapril-treated patients that could be evaluated, there were 111 progressors (47%) (P > 0.1). In the placebo group, 44 patients (19%) experienced new stenosis development and 50 (22%) in the quinapril group (P = NS). Thus, no definitive answer for a role for ACE inhibition as an antiatherosclerosis treatment is yet available. However, it must be pointed out that the participants in the QUIET trial already had advanced atherosclerosis, which in itself may affect EC function. Moreover, advanced atherosclerosis may not be amenable to regression even when EC function is corrected.

More recently, the HOPE trial assessed the role of an ACE inhibitor, ramipril, in patients who were at high risk for cardiovascular events but who did not have left ventricular dysfunction or heart failure. A total of 9,297 high-risk patients (55 yr of age or older) who had evidence of vascular disease or diabetes plus one other cardiovascular risk factor and who were not known to have a low ejection fraction or heart failure were randomly assigned to receive ramipril (10 mg once per day orally) or matching placebo for a mean of 5 yr. The primary outcome was a composite of myocardial infarction, stroke, or death from cardiovascular causes. A total of 651 patients who were assigned to receive ramipril (14.0%) reached the primary end point, as compared with 826 patients who were assigned to receive placebo (17.8%) (relative risk, 0.78; 95% confidence interval, 0.70–0.86; P < 0.001). Treatment with ramipril reduced the rates of death from cardiovascular causes (6.1%, as compared with 8.1% in the placebo group; relative risk, 0.74; P < 0.001), myocardial infarction (9.9% vs. 12.3%; relative risk, 0.80; P < 0.001), stroke (3.4% vs. 4.9%; relative risk, 0.68; P < 0.001), death from any cause (10.4% vs. 12.2%; relative risk, 0.84; P = 0.005), revascularization procedures (16.3% vs. 18.8%; relative risk, 0.85; P < 0.001), cardiac arrest (0.8% vs. 1.3%; relative risk, 0.63; P = 0.03), heart failure (9.1% vs. 11.6%; relative risk, 0.77; P < 0.001), and complications related to diabetes (6.4% vs. 7.6%; relative risk, 0.84; P = 0.03). The conclusion of the study was as follows: Ramipril significantly reduces the rates of death, myocardial infarction, and stroke in a broad range of high-risk patients who are not known to have a low ejection fraction or heart failure, suggesting that use of ACE inhibition may prevent the progression of initially clinically silent atherosclerosis.

O’Driscoll et al. (180) reported on the acute and short-term response of the EC to inhibition of ACE in patients with type 1 diabetes. Pretreatment acetylcholine responses were depressed in diabetic patients relative to the normal subjects; however, no difference between the groups was evident in response to nitroprusside. Acute ACE inhibition (with intrabrachial enalapril) enhanced acetylcholine response in diabetic patients (P < 0.005), with further improvement evident after 1 month of therapy (P < 0.001). ACE inhibition did not affect sodium nitroprusside response. Thus, short-term ACE inhibition improved EC function in patients with type 1 diabetes.

Mullen et al. (181) investigated the effect of 6 months of ACE inhibition on endothelial function in type 1 diabetes. BA flow-mediated dilation (FMD), an endothelium-dependent stimulus, and response to glyceryl trinitrate (GTN) were assessed by high-resolution external vascular ultrasound at baseline and after 12 and 24 weeks of treatment. Treatment with enalapril had no significant effect on FMD (P = 0.67) or response to the endothelial-independent dilator GTN (P = 0.45). Thus, the impairment of endothelium-dependent dilation in young subjects with type 1 diabetes was not improved by treatment with the ACE inhibitor. Moreover, McFarlane et al. (181A ) evaluated the effect of ACE inhibition before and after 12 weeks of treatment in 20 patients with type 1 diabetes and known endothelial dysfunction. ACE inhibition had no significant effect on endothelial function in this group. In summary, in patients with type 1 diabetes, inhibition of ACE may improve EC function acutely; however, chronic inhibition does not seem to be associated with a noticeable improvement of EC function.

The effects of inhibition of ACE on EC function in patients with type 2 diabetes and/or insulin resistance are not clear. O’Driscoll et al. (182) investigated the effects on endothelium-dependent and -independent vasodilator function after treatment with enalapril (10 mg twice daily for 4 weeks) in 10 subjects with type 2 diabetes. Enalapril increased the response to the endothelium-dependent vasodilator, acetylcholine (P < 0.02) and the vasoconstrictor response to the NOS inhibitor, L-NMMA (P < 0.002). No difference was evident in the response to sodium nitroprusside. ACE inhibition improved stimulated and basal NO-dependent endothelial function in type 2 diabetes in this short-term trial. In contrast, Bijlstra et al. (183) could not document a beneficial effect on endothelium-dependent blood flow after 6 months treatment with the ACE inhibitor, perindopril, in 10 patients with type 2 diabetes and hypertension. Other therapeutic interventions aimed at restoring EC function by enhancing enhancers of insulin sensitivity or antioxidant treatments (39, 110, 184) have met with disparate results or no effects.

C. Hypolipidemic therapy
As previously discussed, among other factors, hyperlipidemia and increased levels of oxidized LDL are important pathogenic mechanisms of endothelial dysfunction in patients with diabetes. Since it has been shown that treatment with hypolipidemic agents improves endothelial function in nondiabetic, hyperlipidemic individuals (185), it becomes very tempting to extrapolate the conclusions from these findings to the diabetic population. The latter is supported by a cross-sectional study which found that lipid concentrations in blood were a major determinant of the endothelial function in patients with type 2 diabetes independent of other metabolic alterations and microalbuminuria (186).

Very few controlled trials to examine the effect of hypolipidemic therapy on endothelial function in diabetic patients have been published. Mullen et al. (187) investigated the effects of oral L-arginine (7 g/day) and a reductase inhibitor [Lipitor (Pfizer, Inc.) 40 mg/day] on endothelial function in patients with type I diabetes mellitus, using a 6-month, double-blind, 2 x 2 factorial study. Of note is that the 84 participants in this study were all normocholesterolemic at baseline. Endothelial function was estimated by BA dilation in response to changes in blood flow and vessel smooth muscle function by the response to trinitrate. The use of Lipitor resulted in a 48 ± 10% decrease in serum LDL-C levels and was associated with a significant increase in BA dilation induced by flow. In contrast, L-arginine therapy had no significant effect on endothelial function. These authors concluded that treatment with Lipitor may have an impact on the progression of atherosclerosis in these patients, even when the serum cholesterol levels were normal.

The so-called "statins" are widely used in the treatment of hypercholesterolemia in individuals with type 2 diabetes. However, very scant information exists as to the effect of these medications on endothelial function in this disease. A small trial of a statin in type 2 diabetic patients did not result in a significant improvement in endothelial function (188). In this study, 21 individuals with type 2 diabetes and hypercholesterolemia were treated with simvastatin (10 mg/day) for 24 weeks and compared with a group of controls. Endothelial function was evaluated ultrasonographically as the response of the diameter of the BA in response to flow. These investigators confirmed that the patients with type 2 diabetes displayed endothelial dysfunction. However, the reduction in cholesterol levels achieved with simvastatin was not associated with an improvement in the latter. More detailed information on the effects of reducing triglyceride concentration has been provided by a well controlled, although short-term trial. Evans et al. (189) examined the effects of fibrate therapy on endothelial function and oxidative stress in patients with type 2 diabetes mellitus. Twenty patients were enrolled in a 3-month, double-blind, placebo-controlled study. Endothelial function (FMD) and oxidative stress (paramagnetic resonance spectroscopy) were measured after fasting and 4 h postprandially. Fasting and postprandial endothelial function were significantly higher (from 3.8 ± 1.8% and 1.8 ± 1.3% to 4.8 ± 1.1% and 3.4 ± 1.1%; P < 0.05). This improvement occurred in a setting in which there was a fall in fasting and postprandial triglycerides (3. 1 ± 2.1 and 6.6 ± 4.1 mmol/liter to 1.5 ± 0.8 and 2.8 ± 1.3 mmol/liter, P < 0. 05). There were no changes in total or LDL-C. There were postprandial increases in oxidative stress at baseline, which were significantly attenuated by ciprofibrate Thus, short-term (3 month) fibrate therapy improved fasting and postprandial endothelial function in type 2 diabetes in association with an improvement in serum triglyceride concentration.

D. Arginine supplementation and antioxidants
Given that L-arginine is a substrate for NOS, it is easy to assume that L-arginine supplementation would activate NOS and produce more NO with greater vasodilation. This hypothesis has been tested in many diverse systems including reversal of endothelial dysfunction associated with chronic heart failure, cyclosporin-induced endothelial damage, and diabetes (190, 191, 192, 193). However, not all studies have demonstrated that L-arginine supplementation has an effect on NO production and vasodilation (194). In fact, it is not clear how elevated L-arginine plasma levels could produce more NOS activation. The Michaelis-Menten constant (K_m) for NOS is very low compared with circulating L-arginine levels that are on the order of 80–120 µmol/liter, suggesting that in the normal state, L-arginine is present at concentrations that would support maximum enzymatic turnover of arginine to citrulline and NO (190). Along these lines, one study demonstrated that the concentration of arginine in both plasma and aortic tissue was decreased in streptozocin (STZ) rats (191). It is possible then that in the cases in which L-arginine supplementation restored normal endothelial function, arginine levels were low to begin with. However, it is difficult to know what intracellular L-arginine levels actually are, and there is some evidence that arginine transporters can concentrate arginine against a concentration gradient (190). Therefore, even measuring arginine levels would not necessarily permit accurate conclusions about intracellular arginine levels.

The lay press is inundated with a vast amount of information that suggests that chronic administration of antioxidants and/or vitamins may be beneficial in improving cardiovascular risk. Preliminary evidence for vitamin E or C and/or other antioxidant therapy to improve vascular function in patients with diabetes has recently emerged.

In one study (195) the effects of short-term dietary supplementation with tomato juice (source of lycopene), vitamin E, and vitamin C on susceptibility of LDL to oxidation and circulating levels of C-reactive protein (C-RP) and cell adhesion molecules were measured in patients with type 2 diabetes. Fifty-seven patients with well controlled type 2 diabetes were randomized to receive tomato juice (500 ml/day), vitamin E (800 U/day), vitamin C (500 mg/day), or placebo treatment for 4 weeks. Lycopene administration (tomato juice) and vitamin E were both associated with resistance of LDL to oxidation, but only the latter group showed a decrease in C-Reactive Protein. Levels of cell adhesion molecules and plasma glucose did not change significantly during the study. These authors suggested that their findings may be relevant to strategies aimed at reducing risk of myocardial infarction in patients with diabetes.

Reaven et al. (196) concluded that vitamin E (1, 600 IU/day, 10 weeks) decreased the susceptibility of LDL to oxidation in comparison with placebo (lag time, 243 ± 46 vs. 151 ± 22 min, P < 0.01; 3 h TBARS, 24 ± 12 vs. 66 ±18 nmol malondialdehyde/mg LDL, P < 0.05). Vitamin E had this effect in both buoyant and dense LDL subfractions. This protection occurred in a setting in which glycemic indexes did not change and protein glycation was not affected.

The hypothesis that endothelial function and LDL oxidation may be linked was tested by Pinkney et al. (197). These investigators studied 46 patients with type 1 diabetes without nephropathy and compared results to 39 controls using a 3-month duration, randomized, placebo-controlled double-blind trial of vitamin E, 500 U/day. Endothelial function was estimated by FMD in the forearm by high-resolution ultrasound. LDL oxidation by Cu²⁺ was measured in vitro. FMD did not differ in type 1 diabetic and nondiabetic subjects, nor did indices of lipid peroxidation and in vitro LDL oxidation. Vitamin E supplementation increased the plasma vitamin E levels and mildly enhanced FMD in these patients with type 1 diabetes but this occurred in the absence of changes in LDL oxidation, suggesting that FMD changes may not be mediated by reduced oxidation of LDL.

Our laboratory found that intracellular levels of vitamin C are reduced in the patient with type 1 diabetes, especially in those with poor control (198). Histologically, the microvascular lesions of scurvy have a striking similarity to those seen in longstanding diabetes. Hence, a possible beneficial effect of supplementation with this vitamin is an appealing therapeutic alternative. Indeed, several short-term studies have demonstrated an acute beneficial effect of ascorbic acid (vitamin C) on vascular function, especially in smokers (199, 200, 201, 202) and after ingestion of meals with a high fat content (203). However, the effect of vitamin C is not chronically sustained, at least in smokers (204). Based on these observations, the hypothesis that the antioxidant, vitamin C, could improve endothelium-dependent vasodilation in forearm resistance vessels of patients with non-insulin-dependent diabetes mellitus was tested recently by Ting et al. (39). These investigators studied 10 subjects with diabetes and 10 age-matched, nondiabetic control subjects. FBF was determined by venous occlusion plethysmography, and endothelium-dependent vasodilation was assessed by intraarterial infusion of methacholine before and during concomitant intraarterial administration of vitamin C (24 mg/min). In diabetic subjects, endothelium-dependent vasodilation to methacholine was augmented by simultaneous infusion of vitamin C (P = 0.002); in contrast, in nondiabetic subjects, vitamin C administration did not alter endothelium-dependent vasodilation. The data from this study indeed support the hypothesis that acute administration of vitamin C improves the endothelial function associated with the diabetic state; however, no insight into chronic effects can be deducted from this study. Moreover, no conclusions can be drawn about the specificity of the effect of vitamin C since no other vitamin or drug was tested. Despite an intensive search, we could not find any long-term trial of vitamin C supplementation in patients with diabetes (type 1 or 2). Therefore, no conclusion can be drawn at the present time regarding the possible use of this vitamin to prevent atherosclerosis and/or microvascular disease in patients with diabetes.

	V. Summary and Conclusions

Top Abstract I. Introduction II. Endothelial Cell (EC)... III. Endothelial Dysfunction and... IV. Reversal of Endothelial... V. Summary and Conclusions References

 
Endothelial dysfunction has been demonstrated in type 1 and type 2 diabetes. In the former, this alteration appears temporally linked to vascular disease and is more likely a consequence of the metabolic alterations that also explain the microangiopathy of this disease. In type 2 diabetes, EC dysfunction is detectable very early in the course of the disease, even before overt hyperglycemia ensues, and may play a key role in the etiopathology of the vasculopathy associated with this disease. The hypothesis that endothelial dysfunction may be causative of some of the features of the syndrome of insulin resistance deserves further research. Two published trials (TREND, HOPE) offer some optimism that treatment of endothelial dysfunction with ACE inhibitors may have an impact on the progression of atherosclerosis. The role of insulin sensitizers and/or antioxidant therapy remains to be further explored as well as the effect of hypolipidemic therapy. Of special note is that there are no data regarding the effects of estrogen replacement on endothelial function in postmenopausal women with diabetes.

	Footnotes

 
Address reprint requests to: Jorge Calles-Escandon, MD, Associate Professor of Medicine, University of Vermont, College of Medicine, Given Building C-344A, Burlington, Vermont, 05401. E-mail: jcallese{at}zoo.uvm.edu

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