We investigated the role of c-Kit and the membrane-bound ligand (mbKitL) in endothelial progenitor cell (EPC) recruitment by microvascular endothelial cells (ECs). We demonstrated that inflammatory activation induced the expression of the mbKitL on ECs both in vitro and in vivo, and that recruitment of EPCs depended on c-Kit/mbKitL interaction. Depletion of endogenous c-Kit or inhibition of c-Kit enzymatic activity by imatinib mesylate prevented adhesion of EPCs to activated ECs both in vitro and in vivo, indicating that a functional c-Kit on EPCs is essential. We also demonstrate that Akt was the downstream molecule regulating cell adhesion. A potential role of the c-Kit/mbKitL interaction in pathological settings is sustained by the expression of the mbKitL on ECs lining intraplaque neovessels. Thus, our results provide new insights into the mechanisms underlying EPC recruitment and the bases for novel strategies to hinder pathological angiogenesis.

IntroductionIn the past, it has been assumed that new blood vessels originate from sprouting cells and cooption of neighboring preexisting vessels. 1 It has recently been proposed, however, that both vascular healing and tumor angiogenesis are also supported by mobilization and recruitment of other cells, including the bone marrow (BM)-derived cells denoted as endothelial progenitor cells (EPCs). 2,3 The existence of a BM reservoir of EPCs has attracted interest, especially as target for therapeutic intervention, both in chronic inflammatory diseases and cancer. However, a consensus on the identity and biologic activity of EPCs is still controversial. In general, 2 types of EPCs have been described to date: early and late EPCs. 4 Although they share common features, they have distinct features with respect to morphology, proliferative potential, and in vitro functional characteristics. [5][6][7][8][9][10] Because early EPCs do not adopt a typical endothelial phenotype in vitro and enhance neovascularization in an indirect paracrine fashion in vivo, they have been redefined as circulating angiogenic cells (CAC). 4 More recently, it has been reported that CD45 ϩ CAC retain some myeloid progenitor activity and are unable to originate perfused vessels in vivo. 10 Thus, the best way to distinguish between EPCs and hemopoietic-derived CAC seems by their function rather than by their phenotype.The positive contribution of hemopoietic-derived cells in inflammatory and tumor neoangiogenesis has been extensively demonstrated. [11][12][13] The release of angiogenic mediators, including IL-3, by activated T cells has been reported to contribute to this process. [14][15][16][17][18] Early in vivo and in vitro studies have implicated interleukin (IL) in the survival, proliferation, and differentiation of hemopoietic progenitor/stem cells (HSC) as well as of mature cells. 19,20 However, a clinical study revealed that administration of IL-3 also increased BM vascularization, 21 and it has recently been demonstrated that IL-3 is involved in inflammatory and tumor angiogenesis. 22,23 IL-3 exerts its biologic effects by activating the signal transducer and activator of transcription 5 (STAT5) signaling pathway, both in hemopoietic 24 and vascular tissues 22 as revealed by the intrinsic defective IL-3 responsiveness of STAT5-deficient HSC 25 and endothelial cells (EC). 26 The 2 highly homologous proteins STAT5A and STAT5B, 27 activated by Janus tyrosine kinase 2 (JAK2) 28 or c-Src kinases, 29 act as signaling components between the plasma membrane and the nucleus, and as transcriptional factors, by regulating the expression of genes involved in different cell functions including proliferation, survival, and differentiation. 27 During development, specification of EC into arterial or venous cells represents the main determinant of vascular diversity. Physiologic parameters were considered the main factors in establishing arterial and venous identity. 30 However, more recently, it has been shown that the membrane ligand EphrinB2 and ...

Objective-Circulating angiogenic cells (CACs) expansion is a multistage process requiring sequential activation of transcriptional factors, including STAT5. STAT5, in concert with peroxisome proliferator-activated receptors (PPARs), seems to induce discrete biological responses in different tissues. In the present study we investigated the role of STAT5 and PPAR␥ in regulating CAC expansion in normal and diabetic settings. Methods and Results-Normal

Lipid abnormalities and oxidative stress, by stimulating mesangial cell (MC) proliferation, can contribute to the development of diabetes-associated renal disease. In this study we investigated the molecular events elicited by oxidized low density lipoproteins (ox-LDL) in MC. We demonstrate that in MC cultured in the presence of ox-LDL, survival and mitogenic signals on Akt and Erk1/2 MAPK pathways are induced, respectively. Moreover, as shown by the expression of the dominant negative Rac-1 construct, we first report that ox-LDL-mediated cell survival and cell cycle progression depend on Rac-1 GTPase-mediated reactive oxygen species production and on epidermal growth factor receptor transactivation. By silencing Akt and blocking Erk1/2 MAPK pathways, we also demonstrate that these signals are downstream to Rac-1/reactive oxygen species production and epidermal growth factor receptor activation. Finally, by endogenous depletion of ␤4 integrin, expressed in MC, we provide evidence that the expression of this adhesion molecule is essential for ox-LDL-mediated MC dysfunction. Our data identify a novel signaling pathway involved in oxidative stress-induced diabetes-associated renal disease and provide the rationale for therapeutically targeting ␤4 integrin.Abnormalities in lipoprotein metabolism are commonly observed in patients with renal disease. Specifically, hyperlipidemia and glomerular lipid deposition of atherogenic lipoproteins (low density lipoproteins (LDL), 2 and their oxidized variants, ox-LDL) are implicated in key pathobiological processes involved in the development of glomerular diseases, including mesangial cell (MC) hypercellularity (1). The relevance of mitogen intracellular signaling in mesangial proliferative disease has only recently been recognized (2). Indeed, accumulation of atherogenic lipoproteins within the glomerulus, by activating membrane receptor protein-tyrosine kinases (RPTK), such as the epidermal growth factor receptor (EGFR), triggers MAPK cascades leading to cyclin/cyclin-dependent kinase activation of DNA synthesis and MC proliferation (2). Moreover, in smooth muscle cells, ox-LDL (3) has been shown to trigger the phosphatidylinositol 3-kinase/Akt signaling pathway (4). These data thus suggest that atherogenic lipoproteins may act as one of the major endogenous modulators for mitogenic signaling response within the glomerulus.Although superoxide anions and hydrogen peroxide are generally considered to be toxic, recent evidence suggests that the production of the reactive oxygen species (ROS) might be an integral component of membrane receptor signaling (5). In mammalian cells, a vast array of intracellular stimuli have been shown to induce a transient increase in the intracellular ROS concentrations, and specific inhibition of ROS generation results in a complete blockage of stimulant-dependent signaling (5). In particular, growth factor-mediated ROS generation seems to be necessary for propagation of downstream mitogenic and antiapoptotic signals (5).Rho family GTPases are imp...

Risk factors associated with cardiovascular diseases reduce the availability of endothelial progenitor cells (EPC) by affecting their mobilization and integration into injured vascular sites. The existence of a bone marrow reservoir of EPC has attracted interest, especially as target for therapeutic intervention in different pathological settings. Among the cardiovascular risk factors, hypertension has been shown to be a strongest predictor of EPC migratory impairment. However, at present, data concerning EPC biology are still limited. In this article we provide an overview of data relevant to their potential clinical implications in cardiovascular disorders. In addition, the recent advances in understanding the role of EPC in the pathophysiology of hypertension are discussed.