Axonal guidance and vascular patterning share several guidance cues, including proteins in the netrin family. We demonstrate that netrins stimulate proliferation, migration, and tube formation of human endothelial cells in vitro and that this stimulation is independent of known netrin receptors. Suppression of netrin1a messenger RNA in zebrafish inhibits vascular sprouting, implying a proangiogenic role for netrins during vertebrate development. We also show that netrins accelerate neovascularization in an in vivo model of ischemia and that they reverse neuropathy and vasculopathy in a diabetic murine model. We propose that the attractive vascular and neural guidance functions of netrins offer a unique therapeutic potential.

Background— Sonic hedgehog (Shh) is a prototypical morphogen known to regulate epithelial-mesenchymal interaction during embryonic development. Recent observations indicate that exogenous administration of Shh can induce angiogenesis and may accelerate repair of ischemic myocardium and skeletal muscle. Because angiogenesis plays a pivotal role in wound repair, we hypothesized that activation of the hedgehog pathway may promote a favorable effect on microvascular remodeling during cutaneous wound healing and thereby accelerate wound closure. Because diabetes is associated with impaired wound healing, we tested this hypothesis in a diabetic model of cutaneous wound repair. Methods and Results— In Ptc1-LacZ mice, cutaneous injury resulted in LacZ expression, indicating that expression of the Shh receptor Patched was induced and therefore that the Shh signaling pathway was intact postnatally and upregulated in the process of wound repair. In diabetic mice, topical gene therapy with the use of naked DNA encoding for Shh resulted in significant local gene expression and acceleration of wound recovery. The acceleration in wound healing was notable for increased wound vascularity. In bone marrow transplantation models, the enhanced vascularity of the wound was shown to be mediated, at least in part, by enhanced recruitment of bone marrow–derived endothelial progenitor cells. In vitro, Shh promoted production of angiogenic cytokines from fibroblasts as well as proliferation of dermal fibroblasts. Furthermore, Shh directly promoted endothelial progenitor cell proliferation, migration, adhesion, and tube formation. Conclusions— These findings suggest that a simple strategy of topically applied Shh gene therapy may have significant therapeutic potential for enhanced wound healing in patients with impaired microcirculation such as occurs in diabetes. (Circulation. 2006;113:2413-2424.)

Delayed reendothelialization contributes to restenosis after angioplasty and stenting in diabetes. Prior data have shown that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to endothelial recovery after arterial injury. We investigated the hypothesis that the EPC contribution to reendothelialization may be impaired in diabetes, resulting in delayed reendothelialization. Reendothelialization was significantly reduced in diabetic mice compared with nondiabetic mice in a wire-induced carotid denudation model. The EPC contribution to neoendothelium was significantly reduced in Tie2/LacZ BM-transplanted diabetic versus nondiabetic mice. BM from diabetic and nondiabetic mice was transplanted into nondiabetic mice, revealing that reendothelialization was impaired in the recipients of diabetic BM. To examine the relative roles of denuded artery versus EPCs in diabetes, we injected diabetic and nondiabetic EPCs intravenously after arterial injury in diabetic and nondiabetic mice. Diabetic EPCs recruitment to the neoendothelium was significantly reduced, regardless of the diabetic status of the recipient mice. In vitro, diabetic EPCs exhibited decreased migration and adhesion activities. Vascular endothelial growth factor and endothelial NO synthase expressions were also significantly reduced in diabetic EPCs. Notably, thrombospondin-1 mRNA expression was significantly upregulated in diabetic EPCs, associating with the decreased EPC adhesion activity in vitro and in vivo. Reendothelialization is impaired by malfunctioning EPCs in diabetes. Diabetic EPCs have phenotypic differences involving thrombospondin-1 expression compared with nondiabetic EPCs, revealing potential novel mechanistic insights and therapeutic targets to improve reendothelialization and reduce restenosis in diabetes.

Background— Estradiol (E ₂ ) modulates the kinetics of circulating endothelial progenitor cells (EPCs) and favorably affects neovascularization after ischemic injury. However, the roles of estrogen receptors α (ERα) and β (ERβ) in EPC biology are largely unknown. Methods and Results— In response to E ₂ , migration, tube formation, adhesion, and estrogen-responsive element–dependent gene transcription activities were severely impaired in EPCs obtained from ERα-knockout mice (ERαKO) and moderately impaired in ERβKO EPCs. The number of ERαΚΟ EPCs (42.4±1.5; P <0.001) and ERβKO EPCs (55.4±1.8; P =0.03) incorporated into the ischemic border zone was reduced as compared with wild-type (WT) EPCs (72.5±1.3). In bone marrow transplantation (BMT) models, the number of mobilized endogenous EPCs in E ₂ -treated mice was significantly reduced in ERαKO BMT (WT mice transplanted with ERαKO bone marrow) (2.03±0.18%; P =0.004 versus WT BMT) and ERβKO BMT (2.62±0.07%; P =0.02 versus WT) compared with WT BMT (2.87±0.13%) (WT to WT BMT as control) mice. Capillary density at the border zone of ischemic myocardium also was significantly reduced in ERαKO BMT and ERβKO BMT compared with WT mice (WT BMT, 1718±75/mm ² ; ERαKO BMT, 1107±48/mm ² ; ERβKO BMT, 1567±50/mm ² ). ERα mRNA was expressed more abundantly on EPCs compared with ERβ. Moreover, vascular endothelial growth factor was significantly downregulated on ERαKO EPCs compared with WT EPCs both in vitro and in vivo. Conclusions— Both ERα and ERβ contribute to E ₂ -mediated EPC activation and tissue incorporation and to preservation of cardiac function after myocardial infarction. ERα plays a more prominent role in this process. Moreover, ERα contributes to upregulation of vascular endothelial growth factor, revealing possible mechanisms of an effect of E ₂ on EPC biology. Finally, these data provide additional evidence of the importance of bone marrow–derived EPC phenotype in ischemic tissue repair.