α6-Integrin Subunit Plays a Major Role in the Proangiogenic Properties of Endothelial Progenitor Cells

Abstract

Objective-Alpha6 integrin subunit (␣6) expression is increased by proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor. This increase correlates with enhanced in vitro tube formation by endothelial cells and their progenitors called Endothelial Colony-Forming Cells (ECFCs). We thus studied the role of ␣6 in vasculogenesis induced by human ECFCs, in a mouse model of hindlimb ischemia. Methods and Results-We used small interfering RNA (siRNA) to inhibit ␣6 expression on the surface of ECFCs. For in vivo studies, human ECFCs were injected intravenously into a nude mouse model of unilateral hind limb ischemia. Transfection with siRNA ␣6 abrogated neovessel formation and reperfusion of the ischemic hind limb induced by ECFCs (PϽ0.01 and PϽ0.001, respectively). It also inhibited ECFC incorporation into the vasculature of the ischemic muscle (PϽ0.001). In vitro, siRNA ␣6 inhibited ECFC adhesion (PϽ0.01), pseudotube formation on Matrigel, migration, and AKT phosphorylation (PϽ0.0001), with no effect on cell proliferation or apoptosis. Key Words: adhesion molecules Ⅲ angiogenesis Ⅲ ischemia Ⅲ peripheral arterial disease Ⅲ vascular biology T he ␣6-integrin subunit (␣6) is a 140-kDa protein that can associate with ␤1-or ␤4-integrin subunits. Integrin ␣6␤1 is a receptor for laminin, the main component of the basement membrane, but can also bind other extracellular matrix (ECM) proteins, such as the angiogenic inducer CYR61. 1 It is expressed on platelets, monocytes/macrophages, neutrophils, and endothelial cells. Integrin ␣6␤4 primarily binds laminin; its expression is restricted to epithelial tissues, endothelia, and peripheral nerves; and it is responsible for adhesion junctions called hemidesmosomes. Conclusion-␣6In mice, deletion of the gene coding for ␣6 leads to an absence of hemidesmosomes and, consequently, to severe skin blistering and neonatal death. 2 In humans, defects in ␣6 result in hemidesmosome deficiency, causing epidermolysis bullosa or pyloric atresia and, in most cases, early postnatal death. 3,4 No obvious vascular abnormalities have been reported in such patients or in ␣6 knockout mice, suggesting that ␣6 does not have a major role in vasculogenesis during embryogenesis. In contrast, ␣6 is involved in the angiogenic properties of mature cells. For example, ␣6 is required for the formation of vascular networks in vitro by human brain microvascular endothelial cells 5 and human umbilical vein endothelial cells. 6 Regarding immature cells, activation of CD117-positive cells localized in the subepicardium of adult human heart is associated with ␣6 expression. 7 Cell transplantation experiments on irradiated mice have shown that ␣6 plays a role in hematopoietic stem cell homing to bone marrow. 8 Because hematopoietic cells and endothelial cells might arise from a common precursor (the hemangioblast), we investigated the possible role of ␣6 in the homing of endothelial progenitor cells (EPCs).EPCs are marrow-derived circulating cells involved in postnata...

Vascular Incorporation of Endothelial Colony-Forming Cells Is Essential for Functional Recovery of Murine Ischemic Tissue Following Cell Therapy

“…21 Cord blood-derived ECFCs have previously been reported to enhance neovascularization using in vivo hindlimb ischemia models. 7,[22][23][24] However, the underlying mechanisms have remained elusive to date. Therefore, we set out to selectively eliminate ECFCs 2 weeks after infusion into a hindlimb ischemia model to assess whether selective killing of KDR ϩ ECFCs would lead to disruption of a newly created vascular network and subsequent decline in limb perfusion.…”

Acidic preconditioning improves the proangiogenic responses of endothelial colony forming cells

“…EPC can support neovessel formation in vivo through three different mechanisms [45]: (1) they form new vessels by themselves, (2) they insert into the murine endothelial lining of the murine vasculature, or (3) they exert a paracrine effect. Since we have previously demonstrated in the same model of ischemia that ECFC are integrated into the new murine vessels [46], it is conceivable that preconditioned ECFC exhibit a similar behavior and contribute structurally to this process. Nevertheless, although it has been reported that late outgrowth EPC such as ECFC exert no significant paracrine angiogenic effect [26,45] the possibility that acidosis increases the paracrine action of these cells is being currently studied in our laboratory.…”

The Role of Endothelial Progenitor Cells in Postnatal Vasculogenesis: Implications for Therapeutic Neovascularization and Wound Healing

“…a 6 b 1 is another b 1 -integrin that promotes EPC homing to the sites of ischemia and vascular remodeling and has been shown to be essential for skin homeostasis and regulates endothelial tube formation. 27 It is a laminin-binding integrin and is regulated by factors such as VEGF and bFGF, which are the major growth factors that regulate wound healing. 28,29 EPC adhesion to denuded vessels is also mediated by integrins a v b 3 and a v b 5 .…”