Summary. Adult endothelial as well as smooth muscle progenitor cells are engaged in the complex pathophysiology of atherosclerosis including primary remodeling with development and progression of atherosclerotic plaques as well as secondary complications associated with ischemia, endothelial damage, neointimal growth and transplant arteriosclerosis. These adult vascular precursor cells correspond to similar embryonic stem cell-derived progeny and are primarily located in bone marrow and peripheral blood. Recently, specific investigation on their recruitment emerged as a novel fundamental in the pathogenesis of arterial remodeling, plaque stability and angiogenesis. This multifaceted process of mobilization and homing is regulated by numerous chemokines, adhesion molecules and growth factors that guide and control the trafficking of vascular progenitor cells to the arterial wall after injury or during ischemia.
Although endothelial progenitor cells (EPCs) in bone marrow and peripheral blood have been intensively studied within the past decade, their exact definition remains rather controversial. This paradox is based in part on the plasticity of distinct adult cell sub-populations (CD34+/−, CD14+/−) in acquiring endothelial phenotype [1–4]. Thus, one central question still concerns the correct marker combination for defining of circulating putative EPCs that can give rise to functional endothelial outgrowth in vitro. Most of the studies have focused on surface expression of hematopoietic, progenitor and endothelial marker such as CD133, CD34 and VEGFR2 [1–4]. However, by using cell sorting it has been recently revealed that functional endothelial outgrowth was entirely derived from circulating CD34+CD45− mononuclear cells that were positive for VEGFR2, but negative for CD133 . Parallel to VEGFR2, these CD34+CD45− cells also expressed endothelial markers such as VE-cadherin and CD146. Hence, exclusion of CD45+ leukocytes and assessment of CD34+VEGFR2+ mononuclear cells appears accurate and up-to date definition for the circulating EPC. Furthermore, CD14+ myeloid sub-sets in peripheral blood (CD14+CD3410, CD14+VEGFR2+CXCR2+/− and Tie2+CD1410CD16+) were recently recognized to be functional in angiogenesis contributing to neovascularization and endothelial regeneration through several different mechanisms, e.g. incorporation into neo-endothelium or paracrine secretion of angiogenic cytokines [1,2,6].
The most commonly used procedures to elicit endothelial outgrowth cells from peripheral blood require plating of separated mononuclear cells on fibronectin-coated dishes in specific endothelial growth medium. This process results in adherent colony-forming-units and spindle-shaped cells after 5–7 days and these ‘early’ endothelial outgrowth cells share common properties of monocytic and endothelial origin and secret angiogenic growth factors [7,8]. During prolonged culture period for 3–4 weeks, some of these cells gradually form monolayer islands and convert into a ‘late’ proliferative endothelial outgrowth cells with morphological and functional characteristics of mature endothelial cells . Such endothelial outgrowth from peripheral blood has the potential to be used not only for functional assessment in vitro but also for achieving higher cell number for autologous cell-based therapy. Accordingly, combined transplantation of early and late endothelial outgrowth cells markedly improves the neovascularization of ischemic tissue .
Smooth muscle progenitor cells (SPCs) are recognized as circulating cells that express markers of mesenchymal/smooth muscle lineage, such as endoglin (CD105), calponin, α-SMA, SM-MHC and SM22 [1,11]. These cells can also be clonally expanded from bone marrow or peripheral blood mononuclear cells. Similar to the EPCs, circulating human myeloid CD14+ SPCs have been shown to be a subset of CD14+CD105+ cells .
Mobilization and homing of vascular progenitor cells
The bone marrow is a major store of adult progenitor cells, including, for example, c-kit+ hematopoietic and vascular precursors. Under quiescent conditions, these cells are maintained inactive by contacting the bone marrow stroma. During the process of mobilization (after shedding of soluble kit-ligand), the major pool of c-kit+ progenitor cells translocate from the steady-state stromal niche into the vascular bone marrow compartment. This process is matrix metalloproteinase-9 dependent and undergoes activation by signals from the periphery. During mobilization, stromal cell derived factor-1 (SDF-1 also known as CXCL12) binds to the CXC chemokine receptor 4 (CXCR4), and regulates mobilization of stem/progenitor cells with relevance in vasculogenesis and angiogenesis . The gene expression of CXCL12 is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1). The expression of HIF-1 is up-regulated in injured arteries and recruitment of regenerative CXCR4+ progenitor cells is mediated by hypoxic gradients via HIF-1-induced expression of CXCL12 [12–14].
Similar to hypoxia in the context of tissue damage, vascular trauma also mediates transient increase in circulating EPCs . Nitric oxide (NO) is another locally produced mobilizing agent for EPCs . In a positive feedback loop, CXCL12 induces the release of NO in endothelial cells . Furthermore, VEGF, erythropoietin and G-CSF exert mobilizing action on EPCs . Across their pleiotropic activities statins, as well thiazolidindiones have also been reported to induce transient increase of circulating EPCs, and promote their incorporation into neo-endothelium, an effect that is therapeutically implicated in diminishing neointimal formation [16,17].
Once mobilized in the circulation, EPCs can home to areas of endothelial injury and ischemia. This homing is a complex process primarily regulated by interaction of soluble or surface arrested angiogenic CC- and CXC-chemokines (CCL2, CXCL1, CXCL7, CXCL12) with their respective receptors (CCR2, CXCR2, CXCR4) on the EPC surface [18,19]. Furthermore, the homing of EPCs to denuded matrix proteins, or platelet aggregates after endothelial denudation, is mediated via beta-inetgrins and P-selectin . Finally, EPCs are able to degrade extracellular matrix by secretion of proteolytic enzymes which is supportive in the homing during neovascularization of ischemic tissue [10,20].
Progenitor cell trafficking and remodeling of the arterial wall
It has been reported that infused or endogenously mobilized EPCs support endothelial regeneration, thus attenuating neointimal hyperplasia after carotid injury in mice [1,16,18,19]. These cells were able to engraft at the site of arterial injury and to promote differentiation into endothelial cells. Furthermore, EPCs are crucially involved in post-natal vasculogenesis and neovascularization of ischemic tissue by creating new vessels and secreting angiogenic growth factors [1–4]. Hence, not only engraftment and differentiation towards functional endothelial cells but also paracrine mechanisms appear supportive in the EPC-related vascular maintenance.
Studies of the therapeutic effects of EPCs have examined administration of autologous angiogenic bone marrow cells or cultured EPCs and show promise in the treatment of acute as well chronic myocardial ischemia (reviewed ). Other published data, however, demonstrate that infusion of EPCs increases plaque size and decreases plaque stability in Apoe−/− mice . This observation could be explained in part by the above angiogenic, as well proteolytic and pro-inflammatory, properties of EPC subtypes (e.g. CD14+). Thus, therapeutic use of EPCs requires context-dependent attention and evaluation. For instance, wide-spread mobilization of EPCs could potentially promote plaque destabilization during advanced atherosclerosis, while the local stimulation of the EPC homing after arterial injury or during ischemia could support endothelial repair and tissue regeneration.
Recent studies employing transplantation of bone marrow in mice revealed that bone marrow-derived cells contribute to neointimal growth after arterial injury . Similarly, donor-derived SMCs were identified within the atherosclerotic vessel wall in patients after sex-mismatched bone marrow transplantation . Recent data, however, found that SMCs in advanced atherosclerotic lesions originate from the vessel wall, and healing after plaque rupture involves local but not circulating SMCs [25,26]. In contrast to EPCs, therapeutic infusion of SPCs demonstrated a stabilizing effect on atherosclerotic plaques in mouse model of advanced atherosclerosis, and further suggested that reduced SPC numbers contribute to plaque destabilization and rupture . In human transplant arteriopathy, neointimal SMCs are mainly derived from the recipients, but not from the donor bone marrow cells . The premise that SPCs are derived from local tissue sources is further supported by the finding that sca-1+linneg progenitor cells within the adventitia in an allograft model can migrate to the neointima where they express smooth muscle specific proteins and transform into functional SMCs . Thus, it appears that healing of atherosclerotic plaques and remodeling in transplant arteriopathy mainly occur by SMCs of local vascular wall origin, while bone marrow-derived or circulating SPCs are primarily involved in the pathology of neointimal growth following arterial injury. As a final clinical point, two studies revealed increased number of circulating SPCs in patients with stable coronary artery disease and reduced number in those with acute coronary syndromes [11,27]. These findings suggest a protective role for circulating SPCs in stabilizing atherosclerotic plaques.
Within the spectrum of vascular disease, SPCs likely have discordant effects: it is likely that generalized influx of SPCs into existing plaques may prevent fragility and rupture of the fibrous cap while local recruitment of mesenchymal-like stem cells and particularly SPCs may promote pathological arterial remodeling, intimal thickening, an neointimal growth after arterial injury or coronary intervention [30,31]. Hence, reducing local adhesion and accumulation of SPCs concomitant with improving of EPC recruitment may help achieve adequate regeneration after arterial injury. This could be supported by selective coating of stents with EPC-attracting peptides or specific antibodies and further extended by the use of ex vivo expanded endogenous EPCs for local therapeutic application or endothelialization of vascular grafts [1,18,32].
Vascular progenitor cells have been recently recognized as putative players in the pathology of primary atherosclerosis and secondary arterial remodeling after injury. Generalized mobilization of EPCs may associate with plaque destabilization, while directed local EPC homing at compartments of ischemia or arterial injury may even support tissue regeneration and endothelial repair. Conversely, a widespread recruitment of SPCs during advanced atherosclerosis may serve to maintain stable plaque phenotype, whereas local inhibition of SPC adhesion after arterial injury critically assist in attenuating of neointimal progression (Fig. 1). On the other hand, migratory hematopoietic progenitor cells have recently been found to proliferate within extramedullary tissues, giving rise to tissue-resident myeloid cells, preferentially to dendritic cells upon exposure to Toll-like receptor agonists. Thus, these cells may not only survey peripheral organs to foster the local production of innate immune cells but may also contribute to the homeostasis of healthy vessels and under conditions of vascular inflammation . Hence, a selective control of progenitor cell trafficking in the arterial wall will help in maintaining vascular homeostasis. This appears crucial across the clinical application of vascular progenitor cells not only in terms of prognostic/diagnostic cardiovascular biomarker but also in terms of devise therapeutics in clinical medicine.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.