Peripheral arterial disease and diabetes: effects on endothelial progenitor cell differentiation and nitric oxide metabolism
Creators: name: Basile, Ilaria <1979>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2008
Subjects: BIO/10 Biochimica

description: In the recent years it is emerged that peripheral arterial disease (PAD) has become a growing health
problem in Western countries. This is a progressive manifestation of atherothrombotic vascular
disease, which results into the narrowing of the blood vessels of the lower limbs and, as final
consequence, in critical leg ischemia. PAD often occurs along with other cardiovascular risk factors,
including diabetes mellitus (DM), low-grade inflammation, hypertension, and lipid disorders.
Patients with DM have an increased risk of developing PAD, and that risk increases with the
duration of DM. Moreover, there is a growing population of patients identified with insulin
resistance (IR), impaired glucose tolerance, and obesity, a pathological condition known as
“metabolic syndrome”, which presents increased cardiovascular risk.
Atherosclerosis is the earliest symptom of PAD and is a dynamic and progressive disease arising
from the combination of endothelial dysfunction and inflammation. Endothelial dysfunction is a
broad term that implies diminished production or availability of nitric oxide (NO) and/or an
imbalance in the relative contribution of endothelium-derived relaxing factors. The secretion of
these agents is considerably reduced in association with the major risks of atherosclerosis,
especially hyperglycaemia and diabetes, and a reduced vascular repair has been observed in
response to wound healing and to ischemia. Neovascularization does not only rely on the
proliferation of local endothelial cells, but also involves bone marrow-derived stem cells, referred to
as endothelial progenitor cells (EPCs), since they exhibit endothelial surface markers and
properties. They can promote postnatal vasculogenesis by homing to, differentiating into an
endothelial phenotype, proliferating and incorporating into new vessels. Consequently, EPCs are
critical to endothelium maintenance and repair and their dysfunction contributes to vascular disease.
The aim of this study has been the characterization of EPCs from healthy peripheral blood, in terms
of proliferation, differentiation and function. Given the importance of NO in neovascularization and
homing process, it has been investigated the expression of NO synthase (NOS) isoforms, eNOS,
nNOS and iNOS, and the effects of their inhibition on EPC function. Moreover, it has been
examined the expression of NADPH oxidase (Nox) isoforms which are the principal source of
ROS in the cell. In fact, a number of evidences showed the correlation between ROS and NO
metabolism, since oxidative stress causes NOS inactivation via enzyme uncoupling. In particular, it
has been studied the expression of Nox2 and Nox4, constitutively expressed in endothelium, and
Nox1.
The second part of this research was focused on the study of EPCs under pathological conditions.
Firstly, EPCs isolated from healthy subject were cultured in a hyperglycaemic medium, in order to
evaluate the effects of high glucose concentration on EPCs. Secondly, EPCs were isolated from the
peripheral blood of patients affected with PAD, both diabetic or not, and it was assessed their
capacity to proliferate, differentiate, and to participate to neovasculogenesis. Furthermore, it was
investigated the expression of NOS and Nox in these cells.
Mononuclear cells isolated from peripheral blood of healthy patients, if cultured under
differentiating conditions, differentiate into EPCs. These cells are not able to form capillary-like
structures ex novo, but participate to vasculogenesis by incorporation into the new vessels formed
by mature endothelial cells, such as HUVECs. With respect to NOS expression, these cells have
high levels of iNOS, the inducible isoform of NOS, 3-4 fold higher than in HUVECs. While the
endothelial isoform, eNOS, is poorly expressed in EPCs. The higher iNOS expression could be a
form of compensation of lower eNOS levels. Under hyperglycaemic conditions, both iNOS and
eNOS expression are enhanced compared to control EPCs, as resulted from experimental studies in
animal models.
In patients affected with PAD, the EPCs may act in different ways. Non-diabetic patients and
diabetic patients with a higher vascular damage, evidenced by a higher number of circulating
endothelial cells (CECs), show a reduced proliferation and ability to participate to vasculogenesis.
On the other hand, diabetic patients with lower CEC number have proliferative and vasculogenic
capacity more similar to healthy EPCs. eNOS levels in both patient types are equivalent to those of
control, while iNOS expression is enhanced. Interestingly, nNOS is not detected in diabetic patients,
analogously to other cell types in diabetics, which show a reduced or no nNOS expression.
Concerning Nox expression, EPCs present higher levels of both Nox1 and Nox2, in comparison
with HUVECs, while Nox4 is poorly expressed, probably because of uncompleted differentiation
into an endothelial phenotype. Nox1 is more expressed in PAD patients, diabetic or not, than in
controls, suggesting an increased ROS production. Nox2, instead, is lower in patients than in
controls. Being Nox2 involved in cellular response to VEGF, its reduced expression can be
referable to impaired vasculogenic potential of PAD patients.