There are growing data to suggest that tissue hypoxia represents a critical force that drives adult vasculogenesis. Vascular endothelial growth factor (VEGF) expression is dramatically up-regulated by hypoxia and results in enhanced neovascularization. Although the role of VEGF in angiogenesis has been well characterized, its role in adult vasculogenesis remains poorly understood. We used two distinct murine bone marrow transplantation (BMT) models to demonstrate that increased VEGF levels at the site of tumor growth promoted vasculogenesis in vivo. This effect of VEGF was downstream of its effect to enhance either mobilization or survival of circulating endothelial progenitor cells (EPCs). Both VEGFR1 (flt1) and VEGFR2 (flk1) are expressed on culture expanded human EPCs. Previous studies suggest that the effect of VEGF on endothelial cell migration is primarily mediated via VEGFR2; however, VEGF-induced EPC migration in vitro was mediated by both receptors, suggesting that VEGF-VEGFR1 interactions in EPCs are distinct from differentiated endothelial cells. We used specific blocking antibodies to these receptors to demonstrate that VEGFR1 plays an important role in human EPC recruitment to tumors. These findings were further supported by our finding that tumor-associated placental growth factor (PlGF), a VEGFR1-specific agonist, increased tumor vasculogenesis in a murine BMT model. We further showed that both VEGF receptors were necessary for the formation of functional vessels derived from exogenously administered human ex vivo expanded EPCs. Our data suggest local VEGF and/or PlGF expression promote vasculogenesis; VEGF plays a role in EPC recruitment and subsequent formation of functional vessels.

Atherosclerotic plaques express high levels of small proline-rich repeat protein (SPRR3), a previously characterized component of the cornified cell envelope of stratified epithelia, where it is believed to play a role in cellular adaptation to biomechanical stress. We investigated the physiological signals and underlying mechanism(s) that regulate atheroma-enriched SPRR3 expression in vascular smooth muscle cells (VSMCs). We showed that SPRR3 is expressed by VSMCs in both human and mouse atheromas. In cultured arterial VSMCs, mechanical cyclic strain, but neither shear stress nor lipid loading induced SPRR3 expression. Furthermore, this upregulation of SPRR3 expression was dependent on VSMC adherence to type I collagen. To link the mechanoregulation of SPRR3 to specific collagen/integrin interactions, we used blocking antibodies against either integrin ␣1 or ␣2 subunits and VSMCs from mice that lack specific collagen receptors. Our results showed a dependence on the ␣1␤1 integrin for SPRR3 expression induced by cyclic strain. Furthermore, we showed that integrin ␣1 but not ␣2 subunits were expressed on VSMCs within mouse lesions but not in normal arteries. Therefore, we identified the enrichment of the mechanical strain-regulated protein SPRR3 in VSMCs of both human and mouse atherosclerotic lesions whose expression is dependent on the collagen-binding integrin ␣1␤1 on VSMCs. These data suggest that SPRR3 may play a role in VSMC adaptation to local biomechanical stress within the plaque microenvironment.

No abstract

Objective Atherosclerosis is the primary driver of cardiovascular disease, the leading cause of death worldwide. Identification of naturally occurring atheroprotective genes has become a major goal for development of interventions that will limit atheroma progression and associated adverse events. To this end, we have identified SPRR3 (small proline-rich repeat protein 3) as selectively upregulated in vascular smooth muscle cells (VSMCs) of atheroma-bearing arterial tissue versus healthy arterial tissue. In this study, we sought to determine the role of SPRR3 in atheroma pathophysiology. Approach and Results We found that atheroprone ApoE-null mice lacking SPRR3 developed significantly greater atheroma burden. To determine the cellular driver(s) of this increase, we evaluated SPRR3-dependent changes in bone-marrow-derived cells, endothelial cells (ECs), and VSMCs. Bone marrow transplant of SPRR3-expressing cells into SPRR3−/− ApoE−/− recipients failed to rescue atheroma burden. Similarly, ECs did not exhibit a response to SPRR3 loss. However, atheromas from SPRR3-deficient mice exhibited increased TUNEL-positive VSMCs compared with control. Cell death in SPRR3-deficient VSMCs was significantly increased in vitro. Conversely, SPRR3-overexpressing VSMCs exhibited reduced apoptosis compared with control. We also observed a PI3K/Akt-dependent positive association between SPRR3 expression and levels of active Akt in VSMCs. The survival advantage seen in SPRR3-overexpressing VSMCs was abrogated following the addition of a PI3K/Akt pathway inhibitor. Conclusions These results indicate that SPRR3 protects the lesion from VSMC loss by promoting survival signaling in plaque VSMCs, thereby significantly decreasing atherosclerosis progression. As the first identified atheroma-specific VSMC pro-survival factor, SPRR3 represents a potential target for lesion-specific modulation of VSMC survival.