**2. Flow cytometric characteristics of EPC**

The arterial vessel wall is mostly composed of endothelial cells (ECs), vascular smooth muscle cells (VSMCs), adventitial connective tissue and macrophages. Endothelial impairment is believed to be a major contributor to atherosclerosis or restenosis after percutaneous coronary intervention (PCI). Reendothelialization with ECs can effectively inhibit VSMC migration and proliferation and decrease neointimal thickening. It is for this reason that we studied a mechanism to achieve a rapid reendothelialization, through, for example, autologous translators of endothelial progenitor cells (EPC), mature or immature, as a fundamental hypothesis in the prevention of these two pathologies: atherosclerosis and restenosis, which derive in the same

196 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

EPCs are divided into different evolutionary stages from mother cells to mature ECs. Both early and late EPCs can repair blood vessels, but late EPCs that have a strong proliferation capacity are more involved in the formation of new vessels or angiogenesis. By measuring EPC in patients by flow cytometry in patients by flow cytometry, we found that in patients with atherosclerosis are decreased compared to control subjects without atherosclerosis [4–6]. Several studies show that EPCs can be recruited to sites of endothelial injury then mature in site, changing cluster of differentiation (CD), and playing a major role in reendothelialization [7–9].

Atherosclerosis is an inflammatory disease with leukocyte infiltration, accumulation of smooth muscle cells, and formation of neointima. Damage of the endothelial monolayer triggers the development of thrombosis with consequent occlusion versus arterial subocclusion. Recent studies demonstrated the recruitment and incorporation of EPC into atherosclerotic lesions and therefore provided evidence supporting the role of vascular cells in the pathophysiology of atherosclerosis. Moreover, there is evidence that EPC are capable of regenerat-

The EPCs can mediate vascular repair and attenuate atherosclerosis progression even in the continued presence of vascular injury. Although the mechanisms involved are still not clear, EPCs seem to contribute to the restoration of the endothelial monolayer [12]. In addition to bone marrow, spleen-derived EPCs also have the capacity to repair damaged endothelium [13]. EPCs derived from spleen homogenates also enhanced reendothelialization and reduce neointima formation after induction of endothelial cell damage using the carotid artery model [14].

Other models have also been used, such as the balloon injury model, mobilization of circulating EPCs, and accelerated repair of the nude endothelium [15]. In addition, autologous EPCs that overexpress endothelial nitric oxide synthase (eNOS) ameliorates endothelial integrity when transplanted into mice after carotid artery balloon injury. Increased NO bioavailability significantly strengthens the vasoprotective properties of the reconstituted endothelium,

Transfer of progenitor cells is not always beneficial. ApoE KO mice receiving mononuclear bone marrow cells, following induced hind limb ischemia, showed increased neovascularization, accelerated atherosclerotic plaque formation, and lesion size compared to control groups [17]. In an alternate study, because of proinflammatory properties of these cells, as reduction in IL-10 levels in the atherosclerotic aortas was observed accelerated atherosclerosis along with reduced plaque stability [18]. Similarly, even though implantation of an arteriovenous anti-CD34-ePTFE

clinical entity: acute coronary syndrome.

ing cells, vascular grafts, and native vessels [10, 11].

leading to inhibition of neointimal hyperplasia [16].

EPCs are identified by expression of CD34, CD133, or VEGFR2. Their accurate characterization is very difficult, because as these cells may originate from multiple precursors: the hemangioblast, nonhematopoietic mesenchymal precursors, such as the bone marrow, monocytic cells, and also tissue resident stem cells. Two methods for isolation of EPCs from the peripheral blood have been described [22]:


How was it exposed beforehand, there are two distinct phenotypes: early EPCs and late outgrowth EPCs [26, 27] which differ fundamentally from each other their proliferation potential. The first, that are derived from monocytic cells, have low proliferative capacity but express of eNOS and they fail to form perfused vessels in vivo. The late outgrowth EPCs have a high proliferation rate and can be maintained in culture extensively. These cells play a key role in angiogenesis [22]. Some studies further identified these cells as CD34<sup>+</sup> CD45<sup>−</sup> precursors [28] and clarified their origin from the peripheral blood monocytes. CD14<sup>+</sup> cells seem to give rise to early EPCs, whereas late EPCs develop exclusively from the CD14<sup>−</sup> subpopulation [29].

In experimental studies, where EPCs are infused into ischemic lower limbs, only a small number of these can be seen in capillaries of the patient, although the perfusion improves considerably [30–33]. This suggests the potential release paracrine of angiogenic factors. This supportive function of EPCs may be crucial in ensuring the survival of tissue-residing cells and enhancing blood vessel formation and tissue repair. Early outgrowth EPCs produce higher levels of growth factors [34, 35]. To summary, it can say that EPC phenotype vary depending on their origin and their clutters of differentiation, with different functions:


## **3. Angiogenesis in the vessel wall**

An interesting question could be: How do the vessel wall progenitor cells migrate to the endothelial and intima layer of the vessel? The answer is the vasa vasorum. These play a significant role in transporting cells to the intimal region and have positively correlated with the development of atherosclerosis [35, 36]. In atherosclerotic lesions abundant microvessels can be observed. The vasa vasorum are considered to significantly contribute to:


The real thing is that decreased blood supply through the adventitial vasa vasorum can trigger atherogenic intima thickening [37, 38]. Using the Lac-Z mice, Xu et al. [10] provided unique insights into the formation of these microvessels. It was clearly demonstrated that endothelial cells of microvessels within allografted vessels were derived from bone marrow progenitor cells (**Figure 1**). These results suggest a potentially dual role of EPCs in transplant atherosclerosis, protective through the repair of the denuded endothelium and promoting plaque

**Figure 1.** EPC origins. EPCs could be released from bone marrow, fat tissues, vessel wall, especially adventitia and spleen, liver, and intestine, where they form a circulating EPC pool. They can then contribute to the repair of damaged vessels in pathological conditions.

angiogenesis. Some studies have shown the potential detrimental EPC transplantation as lung cancer or multiple myeloma [38]. Additional experiments are required to fully delineate the functional significance of stem cell incorporation into the microvasculature and define the role of progenitors in tipping the balance between atheroprotection and atherogenesis.
