Nutrition, Metabolism & Cardiovascular Diseases (2006) 16, S39eS45

www.elsevier.com/locate/nmcd

Endothelial dysfunction in type 2

diabetes mellitus
Angelo Avogaro*, Gian Paolo Fadini, Alessandra Gallo,
Elisa Pagnin, Saula de Kreutzenberg

Department of Clinical and Experimental Medicine, Division of Metabolic Diseases,

University of Padova, School of Medicine, Via Giustiniani 2, 35128 Padova, Italy

Received 18 October 2005; received in revised form 20 October 2005; accepted 29 October 2005

KEYWORDS Abstract Aims: Vascular endothelial cells play a major role in maintaining cardio-
Endothelium; vascular homeostasis in health. Diabetes mellitus (DM) substantially impairs the
Diabetes; vasodilating properties of the endothelium and leads to endothelial dysfunction,
Protein kinase C; which can thus be considered the first step in the progression of cardiovascular dis-
Diabetic ease. The aim of the present study is to illustrate possible mechanisms responsible
complications; for endothelial dysfunction in DM.
Cardiovascular disease Data synthesis: We have shown that NADPH oxidase gene expression is increased
in circulating lymphomonocytes from patients with DM, and that this increased
gene expression is dependent upon metabolic control. Hyperglycemia can mediate
its adverse effects through the activation of protein kinase C. We have shown an
increase in membrane-associated PKC beta 2 activity in monocytes from patients
with DM. This activity was reduced by 40% in the euglycemic condition. Finally,
we show a reduction of the circulating endothelial progenitor cells, a subset of
bone marrow-derived endothelial-oriented stem cells, which can give rise to
mature endothelial cells.
Conclusion: Endothelial dysfunction, the initial step of the atherosclerotic process,
is reversible. Thus, major efforts should be made to control not only hyperglycemia
but also the other risk factors for cardiovascular disease, in order to prevent the
onset of all these processes that eventually leads the diabetic patient to premature
death.
ª 2005 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: þ39 049 8212178; fax: þ39 049 8754179.
E-mail address: angelo.avogaro@unipd.it (A. Avogaro).

0939-4753/$ - see front matter ª 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.numecd.2005.10.015
S40
Introduction
Vascular endothelial cells play a major role in
maintaining cardiovascular homeostasis in health.
In addition to providing a physical barrier between
the vessel wall and lumen, the endothelium
secretes a number of mediators that regulate
platelet aggregation, coagulation, fibrinolysis and
vascular tone. The term endothelial dysfunction
refers to a condition in which the endothelium
loses its physiological properties: the tendency to
promote vasodilation, fibrinolysis and antiaggrega-
tion. Endothelial cells secrete several mediators
that can alternatively mediate either vasoconstric-
tion, such as endothelin-1 and thromboxane A2, or
vasodilation such nitric oxide (NO), prostacyclin
and endothelium-derived hyperpolarizing factor
(EDHF) [1]. NO is the major contributor to endo-
thelium-dependent relaxation in conduit arteries
whereas the contribution of EDHF predominates
in smaller resistance vessels. Diabetes mellitus
(DM) substantially impairs the vasodilating proper-
ties of the endothelium and leads to endothelial
dysfunction, which can thus be considered the first
step in the progression of cardiovascular disease
(CVD) [2].
Nitric oxide synthesis and
biological effects
NO, which is the most powerful vasodilating
compound, derives from L-arginine: the reaction
is catalyzed by a family of enzymes, called the
NO synthases (NOS). Three NOS isoforms have
been identified: endothelial NOS (eNOS), neuronal
NOS (nNOS) and inducible or inflammatory NOS
(iNOS). In endothelial cells, eNOS generates NO
only if one essential cofactor such as tetrahydro-
biopterin (BH4) is optimally present within the
cells. Enzyme activity can be regulated by post-
translational modifications: these modifications
occur through the phosphorylation of Ser1179,
which increases the activity of the enzyme [1].
Several kinases can phosphorylate this site, includ-
ing protein kinase A, protein kinase C, and serine/
threonine kinase Akt/PKB. On the contrary eNOS
activity can be downregulated by other proteins,
including caveolin-1, the major coating protein of
caveolae, vesicles found throughout endothelial
cells, which are implicated not only in signal trans-
duction but also in endothelial barrier function and
mechanotransduction [3]. In order to induce
vasodilation in the vascular smooth muscle cells,
NO must activate NO-sensitive guanylyl cyclase
A. Avogaro et al.
(NO-sensitive GC), which is the most important re-
ceptor for the signalling molecule NO. This leads to
the conversion of GTP to cyclic guanosine mono-
phosphate (cGMP) [4]. The NO/cGMP signalling
cascade is crucial in the cardiovascular system,
where it controls smooth muscle relaxation, and
inhibition of platelet aggregation; cyclic nucleo-
tide phosphodiesterases hydrolyze cGMP and thus
terminate their action. NO has several important
effects on the vasculature: first, it maintains basal
tone by relaxing vascular smooth muscle cells; it
also inhibits platelet adhesion, activation, secre-
tion, and aggregation and promotes platelet disag-
gregation, in part through a cGMP-dependent
mechanism. In addition to these effects, endothe-
lial-derived NO inhibits leukocyte adhesion to the
endothelium and smooth muscle cell migration
and proliferation [2]: therefore NO is a powerful
inhibitor of the earlier phases of atherosclerosis
and all the mechanisms that lead to neointimal
proliferation after vascular injury.
Molecular mechanisms underlying
endothelial dysfunction in
diabetes mellitus
Oxidative reactions are crucial in all the events
that lead to atherogenesis, including endothelial
dysfunction. In endothelium exposed to agents
that damage the vasculature there is stimulation
of several enzymes that can produce reactive
oxygen species (ROS): the enzymes of the mito-
chondrial electron transport chain, xanthine
oxidase, cyclooxygenases, lipoxygenases, myelo-
peroxidases, cytochrome P450 monooxygenase,
uncoupled NOS, heme oxygenases, peroxidases,
and NAD(P)H oxidases. In turn, ROS can be pro-
duced intracellularly, extracellularly, or in specific
intracellular compartments. Among these en-
zymes, nicotinamide adenine dinucleotide/NADPH
oxidase is relevant because it is a major vascular
source of ROS [5]. There is evidence that strong
correlations exist among NADPH oxidase activity,
atherosclerotic risk factors, and endothelial
dysfunction. We have recently shown that NADPH
oxidase gene expression is increased in circulating
lymphomonocytes from patients with diabetes
mellitus (DM), and that this increased gene expres-
sion is dependent upon metabolic control [6]. In-
terestingly, NADPH activity is increased not only
by factors that damage vascular endothelium,
but possibly by insulin itself [7,8]. ROS generation
is enhanced in the blood vessels of hypertensive
animal models and in atherosclerotic lesions in
Endothelium and diabetes
humans and animals: this process appears to pro-
mote vascular proliferation, hypertrophy and
remodelling, and is involved in the progression of
atherosclerosis. Insulin resistance, which most
often precedes the onset of type 2 diabetes, and
increased FFA levels cause oxidative stress due
to increased mitochondrial uncoupling. Moreover,
oxidative stress plays a key role in the pathogene-
sis of late diabetic complications. Hyperglycemia
leads to mitochondrial dysfunction and activation
of stress pathways. Both in vitro and in vivo studies
reveal that oxidative stress due to hyperglycemia
occurs before late complications become clinically
evident. This indicates that oxidative stress plays
a crucial role in the pathogenesis of late diabetic
complications [9].
Hyperglycemia also leads to generation of ad-
vanced glycation endproducts (AGE), the products
of non-enzymatic glycation of proteins and lipids
that accumulate in the vessel wall. They are signal
transduction ligands for the receptor for AGE
(RAGE) [10]. AGEs accumulate in the vessel wall
exposed to hyperglycemia and alter the structural
integrity of the vessel wall and underlying base-
ment membrane and are able to quench nitric
oxide. This contributes substantially to negatively
affect endothelial function [11].
Hyperglycemia is one of the major causal
factors in the development of diabetic vascular
complications and can mediate their adverse
effects through multiple pathways. One of those
mechanisms is the activation of PKC by hypergly-
cemia-induced increases in diacylglycerol (DAG)
level, partly due to de novo synthesis [12]. There is
increasing evidence that PKC activation is impor-
tant in diabetes-related endothelial dysfunction;
impaired NO-vasodilation and increased ET-1 re-
lease involved PKC-mediated inhibition of eNOS
[11]. We have shown an increase in membrane-
associated PKC beta 2 activity in monocytes from
patients with DM; this activity was reduced by
40% in the euglycemic condition [13]. PKC activa-
tion also mediates the overexpression of adhesion
molecules such as ICAM, VCAM and E-selectin. PKC
also play a crucial role in mediating vascular
smooth muscle cell contractility thus mediating
vascular vasoconstriction in response to risk fac-
tors such as hyperglycemia.
Atherosclerosis is also regarded as a progressive
disease arising from the combination of endothe-
lial dysfunction and inflammation. Indeed, inflam-
mation has been recognized as a keypoint in the
cluster of the insulin resistance syndrome, includ-
ing diabetes, obesity, hypertension and hyperlipe-
mia [14,15]. Much of the inflammatory reactions
are mediated by the endothelium, located at the
S41
interface of blood and tissue, which maintains
a balance between these two compartments.
With disruption of this balance, mediated by in-
flammation, the vascular wall becomes susceptible
to atheroma formation [16]. In response to DM,
blood leukocytes attach to the endothelium where
there is an exaggerated production of ROS [17]. In
their turn, ROS induce several inflammatory cyto-
kines such that systemic oxidative stress also
means systemic inflammation. Accumulating evi-
dence suggests a major link between C reactive
protein (CRP) and atherosclerosis. CRP, which is
produced in the liver in response to pro-inflamma-
tory states, potently downregulates eNOS tran-
scription and destabilizes eNOS mRNA, with
resultant decreases in both basal and stimulated
NO release [18]. CRP also stimulates endothelin-1
and interleukin-6 release, upregulates adhesion
molecules, and stimulates MCP-1. Thus, CRP is
not only a marker of endothelial dysfunction/
cardiovascular risk [19,20] but also takes part in
its pathogenesis [21]. The inflammatory response
involved in the initiation and progression of ath-
erosclerotic lesions involves many mediators that
activate NF-kB, which has specific target sequen-
ces on many immune and inflammatory gene
promoters [22].
There is general agreement that hyperglycemia
impairs endothelium-dependent vasodilation in
vivo, in humans [23e27]. Interestingly, even in
normoglycemic subjects who are prone to develop
DM and insulin resistance syndrome, impaired en-
dothelial function has been observed during an
oral glucose tolerance test [28]. This has led to
the hypothesis that endothelial dysfunction may
precede the development of overt DM, and that
a prolonged and repeated exposure to postprandial
hyperglycemia may play an important role in the
development of atherosclerosis, even in those
who have normal fasting plasma glucose levels.
With endothelial function being defined as the
blood flow response to acetylcholine, insulin resis-
tance itself is characterized by endothelial dys-
function [29]. In DM, endothelial dysfunction
appears a consistent although not widespread
finding; our previous finding of a normal vascular
response to acetylcholine in DM without other
known risk factors for CHD suggests that the endo-
thelial dysfunction in DM cannot be attributed to
a single, but possibly, to a variety of factors such
as the diameter of LDL particles, total cholesterol,
leukocyte count, von Willebrand factor and serum
AGEs [30]. However, there is general agreement
that hyperglycemia and DM lead to an impairment
of NO production and activity. We have recently
shown that the fraction of L-arginine converted
S42
to NO is lower in DM patients than in normal sub-
jects using a stable isotope approach [31]. It has
also been shown that an inappropriate production
of asymmetric dimethylarginine (ADMA) can alter
endothelial function in patients with DM: this
endogenous competitor for NOS increases after
a fatty meal in DM and its circulating levels corre-
late with a reduction of vascular function [32].
The role of circulating endothelial
progenitor cells in DM
Circulating endothelial progenitor cells (EPCs) are
considered a subset of bone marrow-derived en-
dothelial-oriented stem cells, expressing both
hematopoietic immature surface markers, such
as CD34 and CD133, and endothelial lineage
markers [33]. EPCs give rise to mature endothelial
cells, as demonstrated in pioneer in vitro studies
and recently confirmed in vivo. EPCs originally
reside in the bone marrow and are mobilized to
peripheral blood by a number of stimuli, including
tissue ischemia, physical exercise, cytokines and
certain drugs provided with pleiotropic cardiovas-
cular effects [34]. Currently, it is suggested that
increase in EPCs may be one of the mechanisms
by which these stimuli would ameliorate vascular
function: once in the bloodstream, EPCs could
help to maintain endothelial integrity and stimu-
late angiogenesis in ischemic organs.
On the contrary, reduction in circulating EPCs
has been demonstrated in the presence of risk
factors for atherosclerosis, endothelial dysfunction,
hypercholesterolemia, aging, smoking, chronic re-
nal failure, coronary artery disease, cerebral
vascular disease and DM [35]. Low levels of circu-
lating EPCs may be associated with both endothe-
lial dysfunction and poor collateral development.
Indeed, the concept that EPCs are involved both
in early and advanced events of the development
of cardiovascular diseases is of crucial importance
for diabetology since DM is widely associated with
endothelial dysfunction as well as with poor new
vessel generation [36].
In diabetes, atherosclerotic vascular disease is
characterized by high prevalence, early develop-
ment and rapid progression. The severity of macro-
vascular complications in DM seems to be due to
profound impaired collateralization of vascular
ischemic beds, but mechanisms that hinder
ischemia-induced neovascularization remain elu-
sive. In DM collateralization results are insufficient
to overcome the loss of blood flow through occluded
arteries, leading to ischemia, and often to limb
A. Avogaro et al.
amputation. Thus, emerging evidence indicating
that bone marrow-derived EPCs participate in
postnatal neovascularization led to the hypothesis
that alterations in EPC number or function could
be involved in the pathogenesis of vascular com-
plications in DM. Lambiase et al. have shown that
poor coronary collateral development, which is
typical of DM, is related to low levels of circulating
EPCs [37].
A possible role for EPCs in diabetic vascular
disease was first investigated in diabetic mice with
experimental hindlimb ischemia, in which infusion
of human CD34-positive leukocytes was able to
accelerate blood flow restoration [38]. Tepper
et al. showed that PBMC-derived EPCs isolated
from patients with DM displayed a proliferation
rate in culture decreased by 48% compared to con-
trol subjects, a weaker adherence to activated
human umbilical vein endothelial cells (HUVEC)
and a 2.5-fold reduced incorporation into vascular
structures in vitro [39]. The rate of EPC prolifera-
tion from plated PBMCs in patients with DM was
inversely correlated with the levels of glycated
hemoglobin, suggesting a possible relation be-
tween glucose control and EPC function. Almost
identical results have been reported by Loomans
in type 1 diabetic patients [40]. EPC reduction in
diabetes has been hailed as a novel concept in
the pathogenesis of macrovascular complications.
We have recently demonstrated that patients
with DM and peripheral arterial disease have a pro-
found reduction of circulating EPCs in comparison
with controls, with patients with vascular disease
but without DM and with patients with DM but
without vascular disease, thus strengthening the
hypothesis that EPC reduction may have a role in
the pathogenesis of vascular disease in DM. We
also confirmed the negative relation between
EPC levels and the number of risk factors and
showed a strong positive correlation between
EPCs from patients with limb ischemia and the
ankle-brachial index [41].
Antidiabetic therapy improves
endothelial function
Glycemic control remains the major intervention
for prevention of both micro- and macrovascular
disease. However, in the UKPDS study, the 12%
reduction in all diabetes-related endpoints
achieved by intensive insulin treatment was mainly
due to a 25% decrease in microvascular complica-
tions, while myocardial infarction showed a non-
statistical 16% reduction [42]. Stronger predictors
Endothelium and diabetes
of macrovascular complications were factors such
as LDL cholesterol and blood pressure, that may
be related more to insulin resistance than to glyce-
mic control.
Nonetheless, the correction of both hypergly-
cemia and insulin resistance improves endothelial
function. Although weight loss ameliorates insulin
sensitivity, a parallel sharp improvement in endo-
thelial function has not been observed. However,
weight loss decreases serum levels of inflammatory
markers and enhances vasodilatation in response
to L-arginine [43].
Oral agents that improve insulin sensitivity can
ameliorate vascular function: metformin has been
reported to improve endothelial function in pa-
tients with type DM in one placebo-controlled trial
[44], while, in another study, 8 weeks of treatment
with glibenclamide, metformin and glimepiride all
had similar effects on endothelial function [45].
We have recently showed that this positive effect
of metformin on endothelial function may reside
in its capability to inhibit the PKC b2 activation
in endothelial cells exposed to high glucose con-
centration [46].
The thiazolidinediones seem to be promising
drugs to correct endothelial dysfunction: they are
provided with pleiotropic actions on the cardio-
vascular system, such as anti-hypertensive, anti-
thrombotic and anti-inflammatory effects. The
anti-inflammatory capacity derives from de-
creased ROS generation by mononuclear cells and
to reduce lipid peroxidation [47]. These effects are
observed in obese individuals both with and with-
out DM. Another mechanism that may mediate
the improvement of vascular reactivity by TZDs is
suppression of FFAs through inhibition of lipolysis
in adipose tissue.
Finally, insulin therapy has been demonstrated
to improve or reverse vascular dysfunction as
extensively shown by the group of Yki-Jarvinen
[48,49].
Both insulin deficiency and insulin resistance are
associated with endothelial dysfunction. Indeed,
insulin is a weak vasodilator and emerging data
support the notion that metabolic-vascular cou-
pling is fundamental for physiological insulin
action. Anti-atherogenic effects of insulin are
believed to originate not only from correction of
hyperglycemia and its negative vascular conse-
quences, but also from direct anti-inflammatory
effects. For example, insulin therapy suppresses
NF-kB and reduces inflammatory markers in acute
myocardial infarction [50,51]. These favorable in-
sulin pathways, which are mediated by the activa-
tion of PI3K/Akt, act on glucose, lipid and protein
metabolism as well as on vascular biology.
S43
On the contrary, insulin has also been shown to
exert pro-atherogenic effects. In fact, chronically
elevated insulin levels, such those reached in the
setting of insulin resistance and when the hormone
is chronically administered, force insulin to act as
a growth factor through the MAP kinase pathway,
thus promoting the atherogenetic process [52].
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