Endothelial Dysfunction, Inflammation, and Apoptosis in Diabetes Mellitus

Abstract

Endothelial dysfunction is regarded as an important factor in the pathogenesis of vascular disease in obesity-related type 2 diabetes. The imbalance in repair and injury (hyperglycemia, hypertension, dyslipidemia) results in microvascular changes, including apoptosis of microvascular cells, ultimately leading to diabetes related complications. This review summarizes the mechanisms by which the interplay between endothelial dysfunction, inflammation, and apoptosis may cause (micro)vascular damage in patients with diabetes mellitus.

Full text

Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2010, Article ID 792393, 15 pages
doi:10.1155/2010/792393
Review Article
Endothelial Dysfunction, Inflammation, and Apoptosis in
Diabetes Mellitus
Inge A. M. van den Oever,1Hennie G. Raterman,2Mike T. Nurmohamed,1, 3
and Suat Simsek3, 4
1Department of Rheumatology, Jan van Breemen Institute, Amsterdam, The Netherlands
2Department of Rheumatology, VU University Medical Center, Amsterdam, The Netherlands
3Department of Internal Medicine, VU University Medical Center, Amsterdam, The Netherlands
4Department of Internal Medicine, Medical Center Alkmaar, Wilhelminalaan 12, 1815 JD Alkmaar, The Netherlands
Correspondence should be addressed to Suat Simsek, s.simsek@mca.nl
Received 1 December 2009; Accepted 22 March 2010
Academic Editor: Oreste Gualillo
Copyright © 2010 Inge A. M. van den Oever et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Endothelial dysfunction is regarded as an important factor in the pathogenesis of vascular disease in obesity-related type 2 diabetes.
The imbalance in repair and injury (hyperglycemia, hypertension, dyslipidemia) results in microvascular changes, including
apoptosis of microvascular cells, ultimately leading to diabetes related complications. This review summarizes the mechanisms by
which the interplay between endothelial dysfunction, inflammation, and apoptosis may cause (micro)vascular damage in patients
with diabetes mellitus.
1. Introduction
The rapidly increasing prevalence of diabetes mellitus world-
wide is one of the most serious and challenging health
problems in the 21st century.
The number of people with diabetes grows faster than
expected. In 2007, 246 million people (roughly 6%) were
affected worldwide and it is estimated that this will increase
to 380 million, or 7.3% by 2025. Furthermore, it is estimated
that there are even more people (308 million or 8.1%)
with impaired glucose tolerance (IGT). These people have
a significant risk of developing type 2 diabetes mellitus
(T2DM).
Diabetes is a metabolic disorder which is characterized
by hyperglycemia and glucose intolerance due to insulin
deficiency, impaired effectiveness of insulin action or, both.
Type 1 diabetes mellitus (T1DM) is caused by cellular-
mediated autoimmune destruction of pancreatic islet beta-
cells leading to loss of insulin production. It usually starts
during childhood, but can occur at all ages. T2DM accounts
for 90%–95% of all diabetes and is more common in people
older than 45 who are overweight. There is strong evidence
that genetics plays an important role as well. However, the
prevalence of T2DM is becoming higher in children and
young adults because of the higher rate of obesity in this
population.
Central obesity and insulin resistance next to diabetes,
high cholesterol and high blood pressure form the most
important risk factors for cardiovascular disease (CVD).
CVD is the major cause of death in people with T2DM.
Diabetes is also the leading cause of blindness, renal failure,
and lower limb amputations [1,2].
Dysfunction of the endothelium is regarded as an
important factor in the pathogenesis of vascular disease
in diabetes mellitus [3–5]. The endothelium is the active
inner monolayer of the blood vessels, forming an interface
or barrier between circulating blood in the lumen and
the rest of the vessel wall, and plays a critical role in
vascular homeostasis. It actively regulates vascular tone
and permeability, the balance between coagulation and
fibrinolysis, the inflammatory activity and cell proliferation.
The endothelium even affects the functions of other cell
types, such as vascular smooth muscle cells (VSMC’s),
platelets, leukocytes, retinal pericytes, renal mesangial cells,

2Mediators of Inflammation
Relaxing
eNOS
L-citrulline
L-arginine
NO
O2
Leucocyte adhesion
VCAM-1
MCP-1
Guanylate
cyclase cGMP
Pulsatile bloodflow
VSMC
Vasodilation
Antiinflammatory
Antioxidant
Antiproliferative
Antiapoptotic
NF-κB
Antiplatelet
Permeability decreasing
Figure 1: Properties and production process of NO (nitric oxide) as important factor in endothelial function.
and macrophages, amongst others through the production of
several chemical mediators [3–8]. In health, endothelial cell
injury is mitigated by endogenous reparative processes.
An imbalance in repair and injury resulting in early
microvascular changes, including apoptosis of microvascular
cells, can be seen in both experimental diabetic animal
models and humans with diabetes. Several studies indicate
that microvascular cell apoptosis plays an important role in
the development of early lesions [6,8,9].
We will review the role of endothelial dysfunction
and especially inflammation-induced apoptosis of endothe-
lial cells in obesity-related diabetes mellitus and its co-
morbidities.
2. Endothelial Function and Dysfunction
To maintain vascular homeostasis, the endothelium pro-
duces components of the extracellular matrix such as colla-
gen and a variety of regulatory chemical mediators, including
nitric oxide (NO), prostanoids (prostacycline), endothelin-
1 (ET-1), angiotensin II (ANG-II), tissue-type plasminogen
activator (t-PA), plasminogen activator inhibitor-1 (PAI-1),
von Willebrand factor (vWF), adhesion molecules (VCAM,
LAM, ICAM), and cytokines, among them Tumor Necrosis
Factor α(TNFα)[10](Figure 1).
The endothelium has a prominent role in maintaining
blood fluidity and restoration of vessel wall integrity to
avoid bleeding. It regulates fibrinolysis by producing t-
PA and its inhibitor PAI-1 and limits activation of the
coagulation cascade by thrombomodulin/protein C, hep-
arin sulphate/antithrombin and tissue factor/tissue factor
inhibitor interactions. Through release of promoters and
inhibitors of growth and differentiation of the VSMC,
such as platelet-derived growth factor (PDGF) and ANG-II,
endothelium also has an impact on vascular remodeling [11].
ANG-II exerts regulatory effects on several VSMC
activities including contraction, growth, proliferation, and
differentiation. By the production of adhesion molecules like
leukocyte adhesion molecule (LAM), intracellular adhesion
molecule (ICAM), and vascular cell adhesion molecule
(VCAM), inflammatory cells are attracted and anchored,
thereby playing a regulatory inflammatory role [12,13].
Endothelial dysfunction is the change of these properties,
either in the basal state or after stimulation, that is inap-
propriate with regard to the preservation of organ function.
The kind of changes that occur, can depend on the type of
injury and may depend on the intrinsic properties of the
endothelium (venous versus arterial endothelium).
Under physiological circumstances, there is a balanced
release of endothelial-derived relaxing factors such as nitric
oxide (NO) and prostacyclin (PGI2), and contracting
factors such as endothelin-1 (ET-1), prostaglandins, and
angiotensin II (ANG-II). In endothelial dysfunction, this
balance is altered, predisposing the onset and progres-
sion of atherosclerosis [14]. Risk factors such as hyperc-
holesterolemia, dyslipidemia, smoking, and diabetes initiate
atherosclerosis through endothelial activation and therefore
through endothelial dysfunction. Endothelial dysfunction is
expressed in increased interactions with leukocytes, smooth
muscle growth, vasoconstriction, impaired coagulation, vas-
cular inflammation, thrombosis, and atherosclerosis [15].
A very important mediator synthesized by endothelial
cells is nitric oxide (NO), because of its vasodilatory,
antiplatelet, antiproliferative, permeability-decreasing, anti-
inflammatory, and antioxidant properties [16]. NO inhibits
rolling and adhesion of leucocytes as well as cytokine-
induced expression of vascular cell adhesion molecule-1

Mediators of Inflammation 3
(VCAM-1) and monocyte chemotactic protein-1 (MCP-1)
[17], probably through the inhibition of the transcription
factor nuclear factor κB(NF-κB) [14,18,19].
NO is produced through the conversion of the amino
acid l-arginine to l-citrulline by the enzyme NO-synthase
(NOS). There are several isoforms: NOS1 isolated from
the brain, NOS2, or iNOS, produced by macrophages and
NOS3 or eNOS from endothelial cells. eNOS is activated by
the pulsatile flow of blood through vessels. eNOS produces
NO which diffuses to the vascular smooth muscle (VSM)
where it activates the enzyme guanylate cyclase which in turn
increases cyclic GMP and thereby induces relaxation of the
VSM. In this way it maintains the diameter of the blood vessel
ensuring optimal perfusion of tissues. NOS is regulated by
bradykinin, which acts with b2 receptors on the endothelial
cell surface membrane, increasing the production of NO via
NOS activation. The local concentrations of bradykinin are
regulated by the activity of angiotensin converting enzyme
(ACE), by breaking down bradykinin into inactive peptides
[20,21].
Endothelial dysfunction is associated with decreased
NO availability, either through loss of NO production or
through loss of NO biological activity [22]. NO production
is diminished in cells which are subject to oxidative stress.
Oxidative stress is caused by three factors: (1) an increase in
oxidant generation, (2) a decrease in antioxidant protection,
(3) a failure to repair oxidative damage. Cell damage is
induced by reactive oxygen species (ROS), which are either
free radicals, reactive anions containing oxygen atoms, or
molecules containing oxygen atoms that can either produce
free radicals or are chemically activated by them. Examples
are hydroxyl radical, super oxide, hydrogen peroxide, and
peroxynitrite. Normally these ROS are scavenged by different
intra- and extra cellular mechanisms, but in a situation of
oxidative stress these mechanisms are insufficient to cope
with the exaggerated generation of ROS. NO may react with
some ROS species to form peroxynitrite, in turn increasing
the oxidative stress in the cell. Several cardiovascular risk
factors like hyperglycemia, insulin resistance, dyslipidemia,
inflammation, and also cigarette smoking may induce oxida-
tive stress [5,19].
Oxidative stress is an important factor which can induce
cell apoptosis. In the next part, we will explain the process of
apoptosis.
3. Apoptosis
Apoptosis is the process in which a cell plays an active role
in its own death. This is why it is also called cell suicide.
Apoptosis differs from necrosis in the level of control of the
process. Apoptosis is a controlled and regulated process and
involves individual cells. Necrosis is an uncontrolled process
of cell lysis leading to inflammation and destruction of tissue
areas or even whole organs, which can cause serious health
problems. Apoptosis, or programmed cell death, is a normal
component of the development and health of multicellular
organisms and continues throughout adult life. Apoptosis
and proliferation are responsible for shaping tissues and
organs in developing embryos. During adult life, apoptosis
is a protection mechanism which eliminates old, useless,
and damaged cells. In healthy organisms apoptosis and
cell proliferation are in balance. In diseases such as cancer
there is an imbalance whereby cells have undergone certain
mutations that prevent them from undergoing apoptosis. In
neurodegenerative diseases such as Parkinson’s disease apop-
tosis is thought to account for the excessive loss of neurons.
There are several mechanisms through which apoptosis
can be induced in cells. There are extrinsic signals such
as the binding of death inducing ligands to cell surface
receptors also called death receptors. Some of these ligands
are expressed on the surface of cytotoxic T lymphocytes, for
example, when a cell is infected by a virus. Apoptosis can also
be induced by intrinsic signals, that are produced following
cellular stress. Cellular stress can be caused by oxidative
stress through free radicals, deprivation of growth factor, or
exposure to radiation or chemicals. The sensitivity of cells
to these stimuli can vary depending on a number of factors,
such as the expression of pro- and antiapoptotic proteins, the
severity of the stimulus and the stage of the cell cycle.
Very important death inducing ligands are the Fas ligand,
TNFαand TRAIL (TNF related apoptosis inducing ligand).
When they bind their specific death receptor, apoptotic
signals are transmitted in the cell and a caspase cascade is
activated within seconds of ligand binding, inducing apop-
tosis in a very rapid way. The general signaling pathway that
is activated through death receptor binding begins with the
generation of ceramide, produced by acid sphingomyelinase.
Ceramide release promotes lipid raft fusion which results
in clustering of death receptors. This is important because
it helps amplify the apoptotic signaling. A conformational
change in the intracellular domains of the death receptors
reveals the presence of a death domain which allows the
recruitment of various apoptotic proteins to the receptor.
This is called the death inducing signaling complex (DISC).
As a final step, the DISC recruits and activates procaspase 8.
Caspase 8 initiates the apoptosis of the cell.
The sensitivity of cells to apoptotic stimuli can depend
on the balance of pro- and antiapoptotic bcl-2 proteins. Bcl-
2 and bcl-XL are antiapoptotic, while Bad, Bax and Bid
are proapoptotic proteins [23,24]. The proapoptotic bcl-2
proteins are often found in the cytosol acting as sensors of
cellular damage or stress. In case of cell stress they relocate to
the surface of mitochondria where the antiapoptotic proteins
are located. This interaction between pro- and antiapoptotic
proteins leads to the formation of Permeability Transition
pores (PTP) in the mitochondrial membranes [25]. Recent
evidence implies that there may also be a mitochondrial
apoptotic pathway distinct from that activated by proaptotic
bcl-2 family proteins, dependent on cyclophilin D [26]. The
mitochondria contains proapoptotic proteins such as Apop-
tosis Inducing Factor (AIF), Smac/DIABLO, and cytochrome
C, which are released through these pores, which in turn
leads to the formation of the apoptosome and the activation
of the caspase cascade [27,28].
Once cytochrome C is released into the cytosol, it
interacts with apoptotic peptidase activating factor-1 (APAF-
1) and this leads to the recruitment of procaspase 9 into
a multiprotein complex called the apoptosome. Activation

4Mediators of Inflammation
Insulin resistance
Adipose
tissue
PI3K
Oxidative
stress
Endothelial
dysfunction
HDL
Trig ly ce ri des
FFA
Endothelial cell
apoptosis
Impaired capillary recruitment
TNFα
Akt eNOS
JNK
IRS-1
NO
L-arginine
Endothelial cell
Figure 2: Mechanisms of insulin resistance and adipose tissue in relation to endothelial dysfunction and apoptosis.
of caspase 9 through formation of the apoptosome causes
apoptosis.
Nitric oxide has been demonstrated to inhibit apoptosis
in a number of cell types including endothelial cells. The
antiapoptotic effects can be mediated through mechanisms
such as nitrosylation and inactivation of caspase 1, 3 and
8. Other mechanisms include activating p53, upregulating
heat shock protein 70, and upregulating antiapoptotic
proteins Bcl-2 and Bcl-XL. Through activation of cGMP
signaling, caspase activity is suppressed, cGMP-dependent
protein kinases are activated and possibly the expression of
antiapoptotic proteins increases. Apoptosis and especially
apoptosis of endothelial cells may be highly significant in the
development of diabetes and atherosclerosis [29].
4. Endothelial Cell Dysfunction and
Apoptosis in Diabetes
Dysfunction of endothelium in diabetes mellitus is character-
ized by changes in proliferation, barrier function, adhesion
of other circulating cells, and sensitivity to apoptosis.
Furthermore, it is suggested that diabetes mellitus modifies
angiogenic and synthetic properties of endothelial cells [30–
36].
There is a lot of evidence that endothelial dysfunction is
closely connected to the development of diabetic retinopathy,
nephropathy, and atherosclerosis in both T1DM and T2DM
[4,37]. But, what are the specific mechanisms that cause
this close association between diabetes and endothelial
dysfunction? Large clinical trials in both T1DM and T2DM
have shown that hyperglycemia plays a big part in the
pathogenesis of microvascular complications and is a major
causal factor in the development of endothelial dysfunction
and endothelial cell apoptosis [5,38,39]. However, the
exact mechanism of hyperglycemia-related tissue damage
and clinical complications remains unclear. There is also
a significant role for insulin and especially insulin resis-
tance, as increasing evidence implies that the obesity-related
progression of insulin resistance to T2DM parallels the
progression of endothelial dysfunction to atherosclerosis.
Still this relationship has been difficult to prove because
insulin resistance is often accompanied by a cluster of other
risk factors as mentioned above.
5. Endothelial Dysfunction and
Apoptosis in T2DM
The role of endothelial dysfunction in T2DM is very
complicated, due to the many independent factors involved,
including ageing, obesity, hyperlipidemia, hypertension, low
grade inflammation, insulin resistance, and hyperglycemia
[40]. All of these factors are associated with the metabolic
syndrome, which usually precedes T2DM. The relationship
of endothelial dysfunction and all of these factors is not
completely understood despite extensive research. Even the
question whether endothelial dysfunction is a consequence
or the cause of all the changes occurring in the metabolic
syndrome and diabetes cannot be answered easily. In the next
few paragraphs we will discuss the relation between endothe-
lial dysfunction and the individual factors mentioned above,
starting with insulin resistance.
5.1. Insulin Resistance. Insulin resistance is defined as the
decreased ability of insulin to promote glucose uptake in
skeletal muscle and adipose tissue and the decreased hepatic
output of glucose. This may be present years before the
development of abnormal plasma glucose levels becomes
evident [41,42](Figure 2).
Insulin resistance is associated with an increased free
fatty acids (FFA) release from adipose tissue, which results
in dyslipidemia, including VLDL-hypertriglyceridemia, high
plasma FFA, and low HDL-cholesterol concentrations. High

Mediators of Inflammation 5
FFA levels and hypertriglyceridemia are associated with
endothelial dysfunction. FFA-mediated endothelial dysfunc-
tion is probably caused by reduced availability of L-arginine
and/or NO and oxidative stress [43]. It has been proven
that increased saturated and polyunsaturated FFA concen-
trations, except for oleic acid, directly induce cell cycle arrest
and apoptosis in vascular endothelial cells [44].
Insulin is a vasoactive hormone and enhances muscle
blood flow and vasodilation via stimulation of NO produc-
tion. The increased blood flow caused by insulin, differs
among different types of vessels. Insulin can also redirect
blood flow in skeletal muscles so that more glucose can
be uptaken by muscle cells. This process is called capillary
recruitment. In T2DM, hypertension and obesity, insulin’s
vasodilator actions are impaired, probably for a large part
because of low NO action. Normally, stimulation of NO pro-
duction by insulin is mediated by signaling pathways involv-
ing activation of Phosphoinositide-3 (PI-3) kinase leading
to phosphorylation of eNOS. It is suggested that endothelial
dysfunction and impaired capillary recruitment can cause
insulin resistance because the microvascular endothelium
can not react properly to insulin and glucose disposal is
decreased. This is called endothelial insulin resistance. How
metabolic and endothelial insulin resistance originate and
their exact relationship are not fully understood. Both TNFα
and nonesterified acids (NEFAs) can cause metabolic and
endothelial insulin resistance. Inflammatory cytokines like
TNFα, can act as mediators of insulin resistance by impairing
the tyrosine kinase activity of both the insulin receptor
(IR) and insulin receptor substrate (IRS-1), thus inhibit-
ing insulin signaling. It is suggested that a bidirectional
relationship exists between hyperinsulinemia and low-grade
chronic inflammation, by which hyperinsulinemia can lead
to vascular inflammation and vascular inflammation causes
insulin resistance and finally compensatory hyperinsuline-
mia. At normal physiological concentrations insulin exerts
prevailing antiinflammatory effects, while hyperinsulinemia
increases levels of oxidative stress and inflammation. A
recent study with Human Umbilical Vein Endothelial Cells
(HUVECs) shows that insulin, at pathophysiological concen-
trations alone or in combination with low concentrations
of TNFα, has the ability to promote VCAM-1 expression,
through increasing the steady state levels of mRNA via the
activation of transcription factors, such as NF-κB, which
has been linked to VCAM-1 transactivation before. This
way, hyperinsulinemia leads to increased monocytoid cell
adhesion to HUVECs [5,19,45].
A very important effect of insulin resistance is the fact
that the normal route for insulin to activate the PI-3 kinase
and Akt-dependent signaling pathways is impaired, whereas
hyperinsulinemia overactivates Mitogen activated protein
kinases (MAPK)-pathways, thereby creating an imbalance
between PI-3 kinase and MAP-kinase-dependent functions
of insulin. This probably leads to decreased NO production
and increased ET-1 secretion, characteristic of endothelial
dysfunction. Through activation of the MAP-kinase signal-
ing pathways, hyperinsulinemia promotes secretion of ET-1,
activates cation pumps, and increases expression of VCAM-
1 and E-selectin [46]. ET-1, a vasoconstrictor, can increase
serine phosphorylation of IRS-1, causing a decreased activity
of PI-3 kinase in vascular smooth muscle cells. Moreover, ET-
1 may also impair insulin-stimulated translocation of GLUT-
4 in adipocytes [47,48].
5.2. Hypertension. Hypertension induces endothelial activa-
tion and probably also endothelial dysfunction and is a major
determinant of microangiopathy and atherothrombosis in
diabetes. Hypertension is associated with insulin resistance
and this relation can partly be explained by decreased
capillary density and impaired capillary recruitment seen
in insulin resistant states. Another explanation is the fact
that NO availability is diminished and ET-1 availability is
increased in both insulin resistance and hypertension. The
exact link between diabetes and hypertension is not fully
known [49].
5.3. Obesity. The adipose tissue has become known to
be a highly active endocrine organ, releasing hormones,
cytokines, and enzymes with the tendency to impair insulin
sensitivity. It is an important modulator of endothelial
function via secretion of a variety of hormones, including
adiponectin, resistin, leptin, PAI-1, angiotensin, estradiol,
and the cytokines TNFαand interleukin-6 (IL-6). Plasma
adiponectin levels are reduced in people with obesity
and also in people with diseases associated with obesity,
like T2DM and coronary artery disease. Adiponectin has
antiinflammatory features and is inversely related to BMI,
oxidized LDL, insulin resistance, and atherosclerosis [19]. It
plays an important role in fatty acid metabolism and glucose
homeostasis. Low adiponectin levels are associated with an
increased oxidative state in the arterial wall and systemic
oxidative stress. In endothelial cells, adiponectin increases
the production of nitric oxide and suppresses oxidative stress
and the inflammatory signaling cascades via AMP-activated
protein kinases (AMPK) and the cyclic AMP-protein kinase
A-linked pathway [50]. Moreover, it reduces the attachment
of monocytes to endothelial cells and inhibits the expression
of adhesion molecules [5,51].
The role of resistin in insulin resistance and diabetes
is controversial since a number of studies have shown that
resistin levels increase with increased central adiposity and
other studies have demonstrated a significant decrease in
resistin levels in increased adiposity. PAI-1 is present in
increased levels in obesity and the metabolic syndrome. It
has been linked to the increased occurrence of thrombosis
in patients with these conditions.
Angiotensin II is also present in adipose tissue and has an
important effect on endothelial function. When angiotensin
II binds the angiotensin II type 1 receptor on endothelial
cells, it stimulates the production of ROS via NADPH
oxidase, increases expression of ICAM-1 and increases ET-
1 release from the endothelium [52–54]. Angiotensin also
activates JNK and MAPK pathways in endothelial cells, which
leads to increased serine phosphorylation of IRS-1, impaired
PI-3 kinase activity and finally endothelial dysfunction
and probably apoptosis. This is one of the explanations
why an ACE inhibitor and angiotensin II type 1 receptor

6Mediators of Inflammation
blockers (ARBs) protect against cardiovascular comorbidity
in patients with diabetes and vice versa [55].
Insulin receptor substrate 1 (IRS-1) is a protein down-
stream of the insulin receptor, which is important for
signaling to metabolic effects like glucose uptake in fat cells
and NO-production in endothelial cells. IRS-1 in endothelial
cells and fat cells can be downregulated by stressors like
hyperglycemia and dyslipidemia, causing insulin resistance
and endothelial dysfunction. A low adipocyte IRS-1 expres-
sion may thereby be a marker for insulin resistance [19,56,
57].
5.4. Inflammation. Nowadays atherosclerosis is considered
to be an inflammatory disease and the fact that atheroscle-
rosis and resulting cardiovascular disease is more prevalent
in patients with chronic inflammatory diseases like rheuma-
toid arthritis, systemic lupus erythematosus and ankylosing
spondylitis than in the healthy population supports this
statement. Inflammation is regarded as an important inde-
pendent cardiovascular risk factor and is associated with
endothelial dysfunction.
Interestingly, a study performed by bij van Eijk et al.
shows that patients with active ankylosing spondylitis, an
inflammatory disease, also have impaired microvascular
endothelium-dependent vasodilatation and capillary recruit-
ment in skin, which improves after TNFα-blocking therapy
with etanercept [58].
The existence of chronic inflammation in diabetes is
mainly based on the increased plasma concentrations of
C-reactive protein (CRP), fibrinogen, interleukin-6 (IL-
6), interleukin-1 (IL-1), and TNFα[59–61]. Inflammatory
cytokines increase vascular permeability, change vasoregu-
latory responses, increase leukocyte adhesion to endothe-
lium, and facilitate thrombus formation by inducing pro-
coagulant activity, inhibiting anticoagulant pathways and
impairing fibrinolysis via stimulation of PAI-1. NF-κB
consists of a family of transcription factors, which regulate
the inflammatory response of vascular cells, by transcription
of various cytokines which causes an increased adhesion of
monocytes, neutrophils, and macrophages, resulting in cell
damage. On the other hand, NF-κB is also a regulator of
genes that control cell proliferation and cell survival and
protects against apoptosis, amongst others by activating the
antioxidant enzyme superoxide dismutase (SOD) [62]. NF-
κBisactivatedbyTNFαand IL-1 next to hyperglycemia,
AGEs, ANG-II, oxidized lipids, and insulin. Once activated,
NF-κB translocates from the cytoplasm to the nucleus
to activate gene transcription. NF-κB-regulated genes are
VCAM-1, E-selectin, ICAM-1, IL-1, IL-6, IL-8, tissue factor,
PAI-1, and NOS.
The TNF-family of cytokines plays an important role
in regulating the immune response, inflammation, and
apoptosis. The first cytokine discovered is TNFα,which
is produced by neutrophils, macrophages, and adipocytes
and can induce other powerful cytokines such as IL-6,
which in turn regulates the expression of C-reactive protein
(CRP). CRP increases the expression of endothelial ICAM-
1, VCAM-1, E-selectin, MCP-1 and increases the secretion of
ET1. Moreover, CRP decreases eNOS expression and elevates
the expression of angiotensin receptor type 1 in the vessel
wall [63,64].
TNFαcan induce insulin resistance and this is probably
a part of the explanation why insulin resistance, endothelial
dysfunction, and atherothrombosis are so closely related.
Recent studies indicate that TNFαis likely involved in the
pathogenesis of diabetic nephropathy and retinopathy. A
very recent study with T1DM and T2DM rats shows that
TNFαplays an important role in microvascular apoptosis in
diabetes. When the diabetic rats were treated with pegsuner-
cept,aTNFαinhibitor, a significant reduction of the number
of endothelial cells that expressed activated caspase-3 by 76%
to 80% occurred. TNFαinhibition decreases intercellular
adhesion molecule 1 (ICAM-1) levels and NF-κBactivityin
diabetic retina. Another study in diabetic rats demonstrated
that increased levels of TNFαconsequently enhanced FOXO-
1 mRNA levels, nuclear translocation, and DNA binding
in retinas of T1DM and T2DM rats. It also showed that
the transcription factor FOXO-1, which regulates cell death;
prevents cell cycle progression, modulates differentiation in
various cell types, plays a critical role in diabetes-induced
apoptosis and retinal microvascular cell loss [65]. It is
possible that TNFαupregulation may contribute to increased
apoptosis detected in other diabetes associated complications
and TNFαinhibition may be a potential therapeutic option
in preventing this comorbidity [66].
Tumor necrosis factor alpha-Related Apoptosis-Inducing
Ligand (TRAIL), also known as APO2L, is another member
of the TNF family of cytokines and is a type II membrane
protein. The effects induced by TRAIL are mediated by
interactionswithcellsurfaceTRAILreceptors.FiveTRAIL
receptors have been found so far in humans. When TRAIL
binds TRAIL-R1 (DR4) and TRAIL-R2 (DR5) apoptotic sig-
nals are transduced. TRAIL-R3 (DcR1), TRAIL-R4 (DcR2),
and osteoprogeterin (OPG) lack an intracellular death
domain and can not induce apoptosis. Uniquely, TRAIL
can exert anticancer activity, while causing no or minimal
organ toxicity and inflammation. TRAIL acts among others
onnuclearfactorkappaB(NF-κB). TRAIL induces the
release of NO by vascular endothelial cells [67]. Studies have
shown that OPG is remarkably increased in diabetic patients
and even more so in patients with cardiovascular disease,
like coronary artery disease or abdominal aortic aneurysm
[68,69]. In a study with SZT-induced rats and a control
group of healthy rats the OPG/TRAIL ratio was markedly
increased in the diabetic animals with respect to the control
animals. The next remarkable observation in this study was
the ability of insulin to downregulate TRAIL expression in
rat aortas in vivo.
Further investigation of the role of insulin in the TRAIL
expression in diabetes was done with VSMCs in vitro. This
showed the same result: a decrease of surface TRAIL expres-
sion. High glucose levels did not show any significant effect
on TRAIL surface expression in both studies. These findings
suggest that the downregulation of TRAIL expression may
play a role in diabetic vasculopathy. A possible explanation
for these results is the upregulation of the transcription
factor early growth response protein 1 (Egr-1), which in
turn downregulates TRAIL expression in endothelial cells, by

Mediators of Inflammation 7
Hyperglycemia
Mitochondria
ROS
Oxidative stress
Polyol pathway DAG/PKC pathway AGE formation
Endothelial dysfunction
Diabetic complications
Hexosamine pathway
Figure 3: Mechanisms of hyperglycemia which are supposed to cause endothelial dysfunction and in the end diabetic complications.
both hyperglycemia and insulin. A supportive finding for this
hypothesis is the fact that VEGF receptor 1 (FLT1) and PAI-1,
both known Egr-1 responsive genes, are also increased in the
presence of glucose and insulin. Thus, Egr-1 upregulation,
which is frequently observed in atherosclerosis, is likely to be
involved in insulin-mediated TRAIL downregulation [70].
Plasma levels of C-reactive protein (CRP) are increased
in both T1DM and T2DM. CRP plays a significant role in
atherogenesis in endothelial cells, next to vascular smooth
muscle cells and macrophages, and several studies have
revealed that CRP levels predict cardiovascular disease
[71]. CRP causes numerous proinflammatory and pro-
atherogenic effects in endothelial cells, such as decreased NO
and prostacyclin, increased ET-1, cell adhesion molecules,
MCP-1, IL-8, and PAI-1 [5].
Another important contribution to chronic inflamma-
tion in diabetes is caused by primed peripheral poly-
morphonuclear leukocytes (PMNs). In a small study with
T2DM patients and a control group, it was shown that
T2DM patients are exposed to oxidative stress and chronic
inflammation partially because of the primed state of their
PMNs, amongst others because these primed PMNs release
superoxide significantly faster than normal control PMNs.
Apoptosis in primed PMNs was also higher in the diabetic
patients, probably partly because of intracellular factors such
as high cytosolic calcium concentrations [72]. At the same
time apoptosis of normal PMNs of the control group was
significantly higher in diabetic serum, suggesting leucoclastic
activity of diabetic serum. This was confirmed by the findings
of Abu El-Asrar et al. [73]. This study also observed a
decrease in plasma gluthathione (GSH), an intra- and extra
cellular antioxidant, which neutralizes oxidants, including
hydrogen peroxide and superoxide, by converting them to
other oxidized forms [61].
5.5. Dyslipidemia. Dyslipidemia is characterized by low
HDL-cholesterol levels and an excess of small, dense LDL
and is associated with obesity, insulin resistance and diabetes
in general. An increase in postprandial triacylglycerol-rich
lipoproteins, like chilomicrons and -LDL particles, enhances
oxidative stress and consequently causes endothelial dysfunc-
tion and increased apoptosis [74].
6. Hyperglycemia and Endothelial Dysfunction
There have been various mechanisms discovered that can
explain how hyperglycemia causes vascular complications.
There are several pathways which get activated through
hyperglycemia and can potentiate each other. The basis for
the activation of these pathways is most likely the overpro-
duction of ROS in mitochondria induced by hyperglycemia
(Figure 3).
6.1. The Polyol/Sorbitol/Aldose Reductase Pathway. In a lot
of cells excess glucose is reduced to sorbitol by aldose
reductase. Sorbitol is later metabolized to fructose by sorbitol
dehydrogenase, the polyol pathway. At the same time it
increases the oxidation of NADPH to NADP+ and the
reduction of NAD+ to NADH, the co-factors, which in
turn decreases NO bioavailability [75]. This causes a redox
imbalance that resembles tissue hypoxia and is therefore
called hyperglycemic pseudohypoxia. It also increases the
formation of methylglyoxal and AGEs. All these pro-
cesses enhance oxidative stress [76]. The increased sorbitol
accumulation increases osmotic stress and decreases other
osmolytes such as myo-inositol and taurine. A study in
rat and human retinas produced evidence that the polyol
pathway may have an important role in diabetic retinopathy.
It also proved that the aldose reductase inhibitor (sorbinol)
prevents vascular processes, culminating in the development
of acellular capillaries [5,75,77]. This may imply that
the polyol pathway can cause endothelial cell apoptosis.
However, the full impact of this pathway in the endothelial
dysfunction is not completely understood yet.
6.2. The DAG/PKC Pathway. The hyperglycemia induced
activation of the diacylglycerol (DAG)-protein kinase C

8Mediators of Inflammation
(PKC) pathway has multiple adverse effects on the vascular
function. Hyperglycemia increases the levels of DAG, which
in turn activates PKC. In hyperglycemic circumstances
DAG is synthesized from the glycolytic intermediates dihy-
droxyacetone phosphate (DHAP) and glycerylaldehyde-3-
phosphate, by a de novo pathway [78]. Oxidants like H2O2
can also activate the DAG/PKC pathway. There are at least
eleven PKC isoforms. In vascular cells the isoform PKC-
beta-II is most frequently activated [79]. The pathogenic
consequences of PKC activation include dysregulation of
the vascular permeability through the induction of vascular
endothelial growth factor (VEGF) in smooth muscle cells
[80], dysregulation of blood flow by decreasing endothelial
NOS activity and/or increasing ET-1 synthesis [81], base-
ment membrane thickening through Transforming Growth
Factor-beta (TGF-β)-mediated increased production of type
IV collagen and fibronectin, increased expression of PAI-
1 which causes impaired fibrinolysis and activation of
superoxide producing enzymes like NADPH as well as
an increased expression of a dysfunctional, superoxide-
producing, uncoupled endothelial NOS, thus increasing
oxidative stress [5].
Recently, Geraldes et al. have identified a new signaling
pathway by which hyperglycemia causes increased vascular
cell pathology and apoptosis resulting in diabetic retinopathy
in mouse retinas. They proved that hyperglycemia, especially
in pericytes, activates PKC-δ, probably through an increase
in transcription of the gene encoding PKC-δ. This as well
as activation of p38αMAPK leads to increased expression
of Scr homology-2 domain-containing phosphatase-1 (SHP-
1), which subsequently induces apoptosis via deactivation of
platelet-derived growth factor β(PDGF-β)[82].
6.3. Non-Enzymatic Glycation End Products (AGE). Non-
enzymatic glycation products are a complex and heteroge-
neous group of compounds which accumulate in plasma and
tissues in diabetes and renal failure. There is emerging evi-
dence that these compounds play a role in the pathogenesis
of chronic complications associated with diabetes and renal
failure. Earlier research in both diabetic animals and humans
revealed an association between the accumulation of AGE-
modified proteins and the severity of microvascular compli-
cations. The second evidence stems from the fact that typical
microvascular complications develop following injections of
AGE-modified proteins in non-diabetic animals [83].
The advanced glycation end-products (AGE) concept
proposes that chemical modification and cross linking of
tissue proteins, lipids, and DNA affect their structure,
function and turnover, contributing to a gradual decline
in tissue function and to the pathogenesis of diabetic
complications. Nonenzymatic glycation of proteins is a
condensation reaction between the carbonyl group of free
glucose and the N-terminus of reactive-protein amino
groups, like lysine or arginine, yielding Schiff-base interme-
diates that undergo Amadori rearrangement to form stable
proteinglucose adducts, for example glycated hemoglobin
A1c (HbA1c) and fructosamine (fructoselysine). Amadori-
modified matrix proteins are increased in diabetes. Because
Amadori-adducts are relatively stable, only a small fraction
undergoes rearrangements to irreversible AGEs. At first it
was believed that AGEs are only formed on long-lived extra
cellular molecules, because of the slow rate of reaction of
glucose with proteins. However, other sugars like glucose-
6-phosphate and glyceraldehyde-3-phophate can also create
AGEs with intracellular and short-lived molecules and at
a much faster rate than glucose. AGEs can arise from the
decomposition of Amadori products, from fragmentation
products of the polyol pathway, and as glycoxidative products
which all react with protein amino groups. When oxidation
is involved, the so-called glycoxidation products such as
pentosidine and carboxymethyllysine are formed. It has
recently been found that glucose can probably autoxidize to
form reactive carbonyl compounds (glyoxal, methylglyoxal
and 3-deoxyglucosone) which may react with protein to form
glycoxidation products. In endothelial cells methylglyoxal
is probably the main AGE formed. AGEs can interfere
with the endothelial function in several ways. They can
act as oxidants and cause generation of reactive oxygen
species (ROS). AGEs can decrease arterial elasticy and AGE
modified type I and IV collagen can prevent normal matrix
formation and cross-linking. Interactions of mononuclear
cells and macromolecules like LDL with the endothelial wall
are stimulated by AGE-modified matrix, through increased
expression of endothelial adhesion molecules. AGEs can also
impair the binding of heparan sulfate to the extra cellular
matrix, which results in a loss of anionic sites and thus in
an increase in endothelial permeability. Early diabetic micro
angiopathy is characterized by vasodilation, increased blood
flow, and increased capillary permeability. AGE-modified
proteins may lead to all these changes.
When AGEs get into the blood circulation they are
highly reactive but are often detoxified by various enzymes.
When they are not eliminated by the kidneys, recirculating
AGE peptides can generate new AGEs reacting with plasma
or tissue components. At this stage glycation accelerates
the progress of deterioration. Age-modified plasma pro-
teins can bind to AGE receptors (RAGE =AGE-receptor,
macrophage scavenger receptor A) on different cell types like
endothelial cells, where it can adversely affect the expression
of thrombomodulin, tissue factor, and VCAM-1 genes.
RAGE-binding mediates signal transduction via a receptor-
mediated induction of ROS and activation of transcription
factors NF-κB and p21-ras, leading to apoptosis [84].
The nonenzymatic glycation of LDL (gLDL) and its role
in the pathogenesis of atherosclerosis is a popular subject
in studies of late. Due to hyperglycemia, LDL glycation is
increased in diabetic patients, however nonenzymatic glyca-
tion of LDL happens naturally in all individuals. The modifi-
cation of LDL by glycation leads to a decreased recognition of
LDL by the LDL receptor (LDL-R) and in turn increases the
relative circulation time of the lipoprotein, which may result
in increased particle oxidation, the formation of AGEs, and
the activation of alternative uptake mechanisms by non—
LDL-R—mediated pathways. Additionally, gLDL prevents
shear stress-mediated L-arginine uptake and nitric oxide
formation and causes increased production of plasminogen-
activator inhibitor 1 and prostaglandins, while inhibiting

Mediators of Inflammation 9
the expression of tissue plasminogen activator in endothelial
cells [85–87]. Finally gLDL reduces proliferation and triggers
apoptosis in HUVECs [44].
It has been proposed that these processes could con-
tribute to the increased susceptibility of diabetic patients to
atherosclerosis and coronary heart disease.
So measurement of the products of nonenzymatic glyca-
tion has a two-fold meaning: on one hand, measurement of
early glycation products can estimate the extent of exposure
to glucose and the subjects of previous metabolic control;
on the other hand, measurement of intermediate and late
products of the glycation reaction is a precious instrument
in verifying the relationship between glycation products and
tissue modifications.
6.4. Hyperglycemia and Oxidative Stress. A single unify-
ing mechanism of the above mentioned pathways has
recently been found. The increased production of superoxide
anion radicals by mitochondrial electron transport chain
plays a key role in the activation of the above path-
ways. Hyperglycemia-induced superoxide overproduction
inhibits GADPH activity by 66%, which is a consequence
of poly ADP-ribosylation of GADPH by poly ADP-ribose
polymerase (PARP), which in turn is activated by DNA
strand-breaks synthesized by mitochondrial superoxide over-
production. This overproduction particularly happens in
mitochondria that have been uncoupled by the flux of NADH
from the hyperglycemia-enhanced glycolysis. GADPH inhi-
bition causes accumulation of glycolysis intermediates. In
aortic endothelial cells, the hyperglycemia induced increased
mitochondrial superoxide production and prevented eNOS
activity and expression [88]. In addition to mitochondrial
uncoupling there are other mechanisms that can contribute
to superoxide production in diabetes, namely, uncoupling of
eNOS, increased peroxidation and glycoxidation, activation
of NADPH oxidases, decreased clearance of superoxide, and
impaired antioxidant status [61]. Increased production of
ROS causes oxidative stress. Oxidative stress is probably a key
event in endothelial dysfunction since inhibition of hyper-
glycemia, induced, ROS production prevents activation of
the aldose reductase, hexosamine pathways, PKC activation,
and AGE formation [77,89]. ROS at low concentrations can
function as signaling molecules and participate as signaling
intermediates in the regulation of fundamental cell activities,
such as cell growth and cell adaptation responses. At higher
concentrations they can cause oxidative stress, cellular injury,
and apoptosis [7,90]. ROS can effect many signaling path-
ways, including G-proteins, protein kinases, ion channels
and transcription factors. Finally ROS can modify endothe-
lial function by a variety of mechanisms, like peroxidation
of membrane lipids, activation of NF-κB, and decreasing the
availability of NO [91]. A recently published study showed
that transient exposure of cultured human aortic endothelial
to hyperglycemia induces persistent epigenetic changes in
the promoter of the NF-κB p65 subunit. In the proximal
promoter region of p65, increased monomethylation of
histone 3 lysine 4 by the histone methyltransferase Set 7
caused a continuing increase in p65 gene expression, leading
to a sustained increase in the expression of the NF-κB-
responsive proatherogenic genes MCP-1 and VCAM-1. The
cause of these changes was found in the increased generation
of methylglyoxal and hyperglycemic-induced ROS formation
by the mitochondrial electron transport chain. This means
that transient hyperglycemia can cause persistent atherogenic
effects during normoglycemia by inducing long lasting chro-
matin remodeling and vascular epigenetic changes. These
results provide a molecular basis for better understanding of
the variation in risk for diabetic complications, which can
not be explained by HbA1c [92].
Oxidative stress is known to induce senescence prema-
turely in fibroblasts. Cellular senescence or cellular ageing is
the phenomenon where normal diploid differentiated cells
lose the ability to divide. This phenomenon is also known
as “replicative senescence” or the “Hayflick phenomenon”. In
response to DNA damage (including shortened telomeres)
cells either age or go into apoptosis if the damage cannot
be repaired. There is strong evidence as mentioned above,
that oxidative stress is increased in diabetic patients. Other
studies have revealed that endothelial cells in atherosclerotic
lesions show features of cellular senescence, like senescence
associated β-galactosidase (SA-β-gal) staining and telomere
shortening. Expression of inflammatory cytokines and adhe-
sion molecules is upregulated in senescent endothelial cells.
Furthermore, nitric oxide production is significantly reduced
in these cells. More importantly, senescence enhances vascu-
lar inflammation and thrombosis in vessels, promoting the
development of cardiovascular events. There is also evidence
that senescence is more accelerated in patients with diabetes
compared to healthy individuals. One study demonstrated
that high glucose induced premature cellular senescence in
HUVECs through the activation of the Apoptosis Signal-
Regulating Kinase 1 (ASK1). Activation of ASK-1 also
upregulated PAI-1 expression in the HUVECs and this plus
senescence was also observed in aortas of STZ-diabetic wild
type mice, whereas this was not seen in STZ-diabetic ASK-1
knock-out mice. PAI-1 is known to play an important role in
the pathogenesis of atherosclerosis and thrombosis [93].
7. Hyperglycemia and Apoptosis
The number of (in vitro) studies delivering evidence that
hyperglycemia can induce endothelial cell apoptosis [30,
90,94] has increased extensively over the last few years.
These studies have focused mainly on human or animal
endothelial cells of kidney, retina, myocardium, and human
umbilical vein endothelial cells (HUVECs). Thanks to these
studies, the mechanisms by which hyperglycemia initiates
apoptosis are better understood. These mechanisms include
oxidative stress, increased intracellular Ca2+, mitochondrial
dysfunction otherwise known as the mitochondria apoptosis
pathway, changes in intracellular fatty acid metabolism,
activation of Mitogen activated protein kinases (MAPK) sig-
naling pathways, and impaired phosphorylation activation of
the protein kinase Akt [24,31](Figure 4).
One specific study with HUVECs demonstrated that ele-
vated glucose induces apoptosis and downregulates VEGF in
HUVECs by inhibiting p42/44 MAP kinase activation. High
glucose also significantly increased Bax protein but did not

10 Mediators of Inflammation
ROS
Oxidative
stress
Bax
Bax/Bcl-2
ratio
Procaspase 3 Caspase 3
Endothelial cell
apoptosis
MEKK1 P38 MAPK
JNK/SAPK pathway
ERK pathway
Mitochodrial apoptotic pathway
PTpore
Mitochondrium
Akt activation
AMPK Fatty acid oxidation
Malonyl-coA
Hyperglycemia
Ca2+
Ca2+
Figure 4: Mechanisms by which hyperglycemia is supposed to induce endothelial cell apoptosis.
affect Bcl-2, thereby elevating the Bax/Bcl-2 ratio which acti-
vates cleavage of procaspase 3 into active caspase-3, in turn
triggering apoptosis in HUVECs. When VEGF was added
to the HUVECs exposed to high glucose, apoptosis was
prevented through inhibition of elevated ROS generation,
calcium overload and activation of the mitochondria apop-
tosis pathway. VEGF significantly decreased Bax expression
without affecting the Bcl-2 level and attenuated the increase
in caspase 3 activity. VEGF in HUVECs could also decrease
H2O2production at 48 hours high glucose stimulation,
suggesting that it inhibits the ROS/NF-κB/JNK/Caspase-3
pathway [24].
One earlier study with human aortic endothelial cells and
bovine aortic endothelial cells exposed to high D-glucose
also showed a significant increase in the Bax/Bcl-2 ratio
followed by an increase in caspase-3 activity and cell death.
They proved that Bax inserts the mitochondrial membranes,
triggering a transformation of mitochondrial function after
high D-glucose treatment of the human aortic endothelial
cells. This study also demonstrated that high D-glucose
leads to phosphorylation of p38 Mitogen-Activated Protein
Kinase (p38 MAPK) mediated by MEK-kinase1 (MEKK1)
downstream of bax-caspase proteases and thereby causes
apoptosis of aortic endothelial cells [27].
Another study with HUVECs also investigated the role of
the three MAPK pathways: the extra cellular signal-regulated
kinases (ERK), the c-Jun NH 2-Terminal Kinase /stress-
activated protein kinases (JNK/SAPK), and p38 MAPK. They
found that high glucose triggers apoptosis via ROS through
activating JNK/SAPK. This study showed no significant
role for the other two MAPK pathways [95]. Later in
2005 this research group found that hyperglycemia induces
ROS generation through a PI3K-dependent pathway. They
observed that hyperglycemia causes a PI3K/Akt-dependent
upregulation of Cyclooxygenase 2 (COX-2) expression and
thereby an increase of prostaglandin E2 (PGE2) production
and subsequently a caspase-3 activation and facilitation of
apoptosis in HUVECs. These findings were supported by the
fact that LY294002 or wortmann (both PI3K/Akt inhibitors)
prevented the COX-2 mediated PGE2 production and sub-
sequently the caspase-3 activity, and apoptosis. Inhibition
of COX-2 with a selective COX-2 inhibitor NS398 also
inhibited PGE2 production, caspase-3 activity and apoptosis
in HUVECs treated with high glucose levels. Moreover they
found that hyperglycemia could trigger NF-κBactivation
and that dominant-negative IkBαcould prevent COX-2
expression and apoptosis, implying that NF-κBactivation
can lead to COX-2 mediated PGE2 production and apoptosis
in HUVECs exposed to hyperglycemia [96].
There are several studies with HUVECs that prove that
high glucose-induced apoptosis is associated with an increase
in Ca2+ current, resulting from Ca2+ entrymediatedbystore-
operated channels. An increased amount of cellular Ca2+
causes more mitochondrial Ca2+ uptake. Ca2+ accumulation
in mitochondria is one of the primary causes for mitochon-
drial permeability transition, through the opening of the PT-
pore and this is an important key factor in the apoptotic
pathway [24].
The involvement of the intracellular fatty acid meta-
bolism is suggested by a study in which HUVECs were
treated with high glucose concentrations for 24 hours and
showed inhibition of fatty acid oxidation, increases in fatty
acid esterification and the concentration of malonyl-CoA
before apoptosis was induced. This finding suggests a causal
relation of alterations in intracellular fatty acid and apoptosis
in hyperglycemia. Decreases in mitochondrial membrane

Mediators of Inflammation 11
potential and cellular ATP content also preceded apoptosis.
All these metabolic alterations are associated with an increase
in caspase-3 activity and an impaired ability of insulin at
a physiological concentration to activate Akt. Finally an
antiapoptotic role for AMPK is suggested in this study
because incubation of the HUVECs with 5-aminoimidazole-
4-carboxamide-riboside (AICAR), an AMPK activator,
prevented all of the above changes. Likewise, a similar
decrease in caspase-3 activity was observed when
AMPK activity was increased by infecting HUVEC with
constitutively active AMPK using an adenoviral vector [33].
Recently, a study with human pancreatic islet microvas-
cular endothelial cells (MECs) proved that sustained hyper-
glycemia progressively affects cellular survival and prolif-
eration and increases apoptosis of cultured MECs. After
24 to 48 hours, apoptosis was detected in high glucose
both by DNA fragmentation and activation of the caspase
family. In this study they found that the islet MECs,
under conditions of sustained hyperglycemia, showed a
progressively reduced phosphorylation of Akt, suggesting an
interference with the pathways involved in Akt activation.
Hyperglycemia also downregulated the tyrosine phospho-
rylated form of the transmembrane protein nephrin. It is
known that phosphorylated nephrin associates with PI3K
and activates the multifunctional Akt-dependent pathways.
This suggests that hyperglycemia-induced apoptosis of islet
endothelium likely involves the nephrin-mediated signaling
cascade, wherein phosphorylation of the tyrosine sites within
the intracytoplasmic C terminal domain of nephrin activates
mitogen-activated protein kinase p38 and JNK and thereby
the transcription factor activating protein-1 (AP-1)/c-Jun,
which modulates apoptosis. The study with islet MECs also
detected an increased production of the proinflammatory
cytokine IL-1β, which can induce Fas expression enabling
Fas-mediated apoptosis [31].
8. Conclusion
The relation between diabetic micro- and macroangiopathy
and endothelial dysfunction is complex and is still a subject
of extensive research. Especially in type 2 diabetes a lot
of factors are involved including hyperglycemia, hyperin-
sulinemia, insulin resistance, dyslipidemia, hypertension,
and obesity, which all influence each other and probably
intensify each others actions. More insights into the exact
mechanisms underlying endothelial dysfunction may lead
to important treatment strategies which can significantly
reduce the morbidity and mortality rate caused by endothe-
lial dysfunction especially in diabetes patients. Although
apoptosis is a natural phenomenon in all multicellular
organisms, an increased and accelerated rate of apoptosis of
endothelial cells is probably a crucial factor in diabetic co-
morbidity. There are many pathways involved in activating
endothelial cell apoptosis and all of these pathways can be
activated in multiple ways. A common mechanism causing
endothelial dysfunction and endothelial cell apoptosis is
oxidative stress. Several studies show contradictory results
regarding a possible role for antioxidants in the treatment to
prevent micro- and macroangiopathy. However a treatment
aimed at reducing oxidative stress in endothelial cells may
be an answer to this major problem, especially since diabetes
will soon become an even bigger health problem involving
more than 5% of the world population.
Abbreviations
ACE: Angiotensin Converting Enzyme
AGE: Advanced Glycation End-products
AIF: Apoptosis Inducing Factor
ANG-II: Angiotensin II
AMP: Adenosine Monophosphate
AMPK: AMP-activated Protein Kinases
APAF-1: Apoptotic Peptidase Activating Factor 1
ARB: Angiotensin II type 1 Receptor Blocker
ASK-1: Apoptosis Signal Regulating Kinase 1
BMI: Body Mass Index
cGMP: Cyclic Guanosine MonoPhosphate
COX: Cyclooxygenase
CRP: C-Reactive Protein
CVD: Cardio Vascular Disease
DISC: Death Inducing Signaling Complex
DNA: Deoxyribonucleic Acid
Egr-1: EarlyGrowthResponseProtein1
EMP: Endothelial Micro Particle
ERK: Extra cellular signal-Regulated Kinase
ET-1: Endothelin-1
FADD: Fas Associated Death Domain
FasL: Fas-ligand or CD95-ligand
FFA: Free Fatty Acids
FOXO-1: Forkhead box O1
GLUT-4: Glucose Transporter-4
GADP: Glyceraldehyde 3-Phosphate
GSH: Gluthathione
HbA1c: Hemoglobin A1c
HDL: High Density Lipoproteins
HUVEC: Human Umbilical Vein Endothelial Cell
ICAD: Inactive Caspase Activated DNase
ICAM: Intracellular Adhesion Molecule
IL-6: Interleukine-6
IR: Insulin Receptor
IRS-1: Insulin Receptor-Substrate-1
IGT: Impaired Glucose Tolerance
JNK: c-Jun NH 2-Terminal Kinase
LAM: Leucocyte Adhesion Molecule
MAPK: Mitogen-Activated Protein Kinase
MCP-1: Monocyte Chemotactic Protein-1
MEC: Microvascular Endothelial Cell
MEKK1: Methyl Ethyl Ketone Kinase-1
MP: Micro Particle
mRNA: messenger Ribonucleic Acid
NADP(H): Nicotinamide adenine dinucleotide
phosphate
NEFA: Non-Esterified Fatty Acid
NO: Nitric Oxide
NOS: Nitric Oxide Synthase
NF-κB: Nuclear Factor κB
OPG: Osteoprogesterin
PARP: Poly ADP-Ribose Polymerase

12 Mediators of Inflammation
PAI-1: Plasminogen Activator Inhibitor-1
PMN: Polymorphonuclear Leukocyte
PTP: Permeability Transition Pore
PDGF: Platelet-Derived Growth Factor
PGE2: Prostaglandin E2
PGI2: Prostacyclin
PI-3K: Phosphoinositide-3 Kinase
ROS: Reactive Oxygen Species
SAβ-gal: β-Galactosidase
SAPK: Stress-Activated Protein Kinases
SOD: Superoxide Dismutase
STZ: Streptozotocin
T1DM: Type 1 Diabetes Mellitus
T2DM: Type 2 Diabetes Mellitus
TNFα: Tumor Necrosis Factor-α
t-PA: Tissue-type Plasminogen Activator
TRAIL: TNF-Related apoptosis Inducing Ligand
VCAM: Vascular Cell Adhesion Molecule
VEGF: Vascular Endothelial Growth Factor
VLDL: Very Low Density Lipoproteins
VSMC/VSM: Vascular Smooth Muscle (Cell)
vWF: Von Willebrand Factor.
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