Pretreatment of Endothelial Progenitor Cells with Osteopontin Enhances Cell Therapy for Peripheral Vascular Disease

Abstract

Tissue necrosis resulting from critical limb ischemia (CLI) leads to amputation in a significant number of patients. Autologous cell therapy using angiogenic cells such as endothelial progenitor cells (EPCs) holds promise as a treatment for CLI but a limitation of this treatment is that the underlying disease etiology that resulted in CLI may also contribute to dysfunction of the therapeutic EPCs. This study aimed to elucidate the mechanism of EPC dysfunction using diabetes mellitus as a model and to determine whether correction of this defect in dysfunctional EPCs ex vivo would improve the outcome after cell transplantation in the murine hind limb ischemia model. EPC dysfunction was confirmed in a homogenous population of patients with type 1 diabetes mellitus and a microarray study was preformed to identify dysregulated genes. Notably, the secreted proangiogenic protein osteopontin (OPN) was significantly downregulated in diabetic EPCs. Furthermore, OPN-deficient mice showed impaired recovery following hind limb ischemia, suggesting a critical role for OPN in postnatal neovascularization. EPCs isolated from OPN KO mice showed decreased ability to adhere to endothelial cells as well as impaired angiogenic potential. However, this dysfunction was reversed upon exposure to recombinant OPN, suggesting that OPN may act in an autocrine manner on EPCs. Indeed, exposure of OPN knockout (KO) EPCs to OPN was sufficient to induce the secretion of angiogenic proteins (IL-6, TGF-α, and FGF-α). We also demonstrated that vascular regeneration following hind limb ischemia in OPN KO mice was significantly improved upon injection of EPCs preexposed to OPN. We concluded that OPN acts in an autocrine manner on EPCs to induce the secretion of angiogenic proteins, thereby playing a critical role in EPC-mediated neovascularization. Modification of cells by exposure to OPN may improve the efficacy of autologous EPC transplantation via the enhanced secretion of angiogenic proteins.

Modulation of inflammation and angiogenesis and changes in ECM GAG-activity via dual delivery of nucleic acids

“…At 14 days a similar increase in angiogenic factors was observed, with the exception of FGF and NRP-2 that are unchanged compared with the control and an increase in osteopontin (OPN) and matrix metalloproteinase-8 (MMP-8) expression. Both OPN [59,60] and MMP-8 [61] have been proven to play important roles in angiogenesis in vitro and in vivo. The reduction in IL-10 expression, coupled with no change in IL-4 and IL-13 expression, indicates macrophages may be in a more pro-inflammatory phenotype compared with the control.…”

Autologous circulating angiogenic cells treated with osteopontin and delivered via a collagen scaffold enhance wound healing in the alloxan-induced diabetic rabbit ear ulcer model

“…Early EPCs or CACs are decreased in number and are dysfunctional in people with diabetes mellitus [30]. We have shown that the exposure of CACs to OPN rescues the angiogenic potential of OPN knockout CACs and restores blood flow in limb ischemia [18]. Using this strategy to restore the disease associated dysfunction associated with diabetic CACs, the wound healing benefit of CACs exposed to OPN was tested.…”

“…CACs isolated from people with diabetes demonstrate reduced angiogenic potential as demonstrated by reduced tubule formation in the matrigel assay. Diabetic CACs demonstrate reduced adhesion to matrix proteins and reduced migration [17,18]. Our group has recently observed decreased expression of the matricellular protein osteopontin (OPN) in CACs from patients with type 1 diabetes mellitus in the absence of microvascular or macrovascular complications.…”

“…Our group has recently observed decreased expression of the matricellular protein osteopontin (OPN) in CACs from patients with type 1 diabetes mellitus in the absence of microvascular or macrovascular complications. OPN is a secreted glycoprotein that is involved in cell migration, cell survival, regulation of immune cell function, inhibition of calcification, control of tumor cell phenotype and has been implicated in the process of neovascularization in various cancer models and peripheral ischemia [18]. …”

“…The exposure of CACs from OPN knockout mice to recombinant OPN augments angiogenesis in a mouse hind limb ischemia model [18]. Autologous CACs can be manipulated ex vivo to augment cellular function.…”