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Departments of Internal Medicine (J.C.-E.) and Obstetrics and Gynecology (M.C.), College of Medicine, University of Vermont, Burlington, Vermont 05401
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Abstract |
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 Top Abstract I. IntroductionII. Endothelial Cell (EC)...III. Endothelial Dysfunction and...IV. Reversal of Endothelial...V. Summary and ConclusionsReferences |
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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
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I. Introduction |
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TopAbstractI. IntroductionII. Endothelial Cell (EC)...III. Endothelial Dysfunction and...IV. Reversal of Endothelial...V. Summary and ConclusionsReferences |
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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.
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II. Endothelial Cell (EC) Dysfunction |
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TopAbstractI. IntroductionII. Endothelial Cell (EC)...III. Endothelial Dysfunction and...IV. Reversal of Endothelial...V. Summary and ConclusionsReferences |
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(21, 22, 23). The system exerts effects on
both the surrounding VSMC and the cells in the blood that lead to one
or more of the following alterations: 1) vasodilation or
vasoconstriction to regulate organ blood, 2) growth and/or changes in
the phenotypic characteristics of VSMC, 3) proinflammatory or
antiinflammatory changes, and 4) maintenance of fluidity of blood and
avoidance of bleeding (22, 24, 25, 26, 27, 28). Thus, as summarized in Table 1 the cellular effects of the EC maintain a balance of opposing
physiological and molecular effects. It is conceptualized currently as
maintaining a balance of opposing forces with the end result of
maintaining a proper blood supply to tissues and regulating
inflammation and coagulation.
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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.
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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.
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). The coagulation cascade will not be
detailed in this review. In essence, the coagulation explosion has the
ultimate function of generating active thrombin (56). Thrombin is a
proteolytic enzyme, and fibrinogen is its natural and most abundant
substrate. Upon activation of thrombin, fibrinogen is transformed into
fibrin with the release of fibrinopeptides A and B. Fibrin then
undergoes polymerization and cross-linking, creating a stable clot.
Thereafter, the clot is dissolved upon the action of another
proteolytic enzyme, plasmin, which is the main effector of the
fibrinolytic system. The transformation of the plasmin precursor,
plasminogen, to plasmin results from specific activators.
Physiologically (as well as pharmacologically) the most important
activator of the conversion of plasminogen to plasmin is tissue
plasminogen activator (t-PA). This peptide has a critical role in the
dissolution of clots and maintenance of vessel lumen and has been used
therapeutically in the treatment of events in which acute occlusion by
thrombi is a precipitating event of life-threatening disease states
(i.e., myocardial infarction, stroke, massive pulmonary
embolism). Several positive and negative activators regulate t-PA
activity. Physiologically, the most important regulator of t-PA is the
peptide, plasminogen activator inhibitor (PAI) (57). There are four
types of the PAI, of which type 1 (PAI-1) seems to play the most
preeminent role.
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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.
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III. Endothelial Dysfunction and Diabetes |
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TopAbstractI. IntroductionII. Endothelial Cell (EC)...III. Endothelial Dysfunction and...IV. Reversal of Endothelial...V. Summary and ConclusionsReferences |
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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:
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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).
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IV. Reversal of Endothelial Dysfunction: Lessons from Human Clinical Trials |
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TopAbstractI. IntroductionII. Endothelial Cell (EC)...III. Endothelial Dysfunction and...IV. Reversal of Endothelial...V. Summary and ConclusionsReferences |
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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
(Km) 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.
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