Kelly Stevanov

Induction of Vascular Progenitor Cells From Endothelial Cells Stimulates Coronary Collateral Growth

Rationale: A well-developed coronary collateral circulation improves the morbidity and mortality of patients following an acute coronary occlusion. Although regenerative medicine has great potential in stimulating vascular growth in the heart, to date there have been mixed results, and the ideal cell type for this therapy has not been resolved. Objective: To generate induced vascular progenitor cells (iVPCs) from endothelial cells, which can differentiate into vascular smooth muscle cells (VSMCs) or endothelial cells (ECs), and test their capability to stimulate coronary collateral growth. Methods and Results: We reprogrammed rat ECs with the transcription factors Oct4, Klf4, Sox2, and c-Myc. A population of reprogrammed cells was derived that expressed pluripotent markers Oct4, SSEA-1, Rex1, and AP and hemangioblast markers CD133, Flk1, and c-kit. These cells were designated iVPCs because they remained committed to vascular lineage and could differentiate into vascular ECs and VSMCs in vitro. The iVPCs demonstrated better in vitro angiogenic potential (tube network on 2-dimensional culture, tube formation in growth factor reduced Matrigel) than native ECs. The risk of teratoma formation in iVPCs is also reduced in comparison with fully reprogrammed induced pluripotent stem cells (iPSCs). When iVPCs were implanted into myocardium, they engrafted into blood vessels and increased coronary collateral flow (microspheres) and improved cardiac function (echocardiography) better than iPSCs, mesenchymal stem cells, native ECs, and sham treatments. Conclusions: We conclude that iVPCs, generated by partially reprogramming ECs, are an ideal cell type for cell-based therapy designed to stimulate coronary collateral growth.

Mitochondrial Oxidative Stress Corrupts Coronary Collateral Growth by Activating Adenosine Monophosphate Activated Kinase-α Signaling

Objective—Our goal was to determine the mechanism by which mitochondrial oxidative stress impairs collateral growth in the heart. Approach and Results—Rats were treated with rotenone (mitochondrial complex I inhibitor that increases reactive oxygen species production) or sham-treated with vehicle and subjected to repetitive ischemia protocol for 10 days to induce coronary collateral growth. In control rats, repetitive ischemia increased flow to the collateral-dependent zone; however, rotenone treatment prevented this increase suggesting that mitochondrial oxidative stress compromises coronary collateral growth. In addition, rotenone also attenuated mitochondrial complex I activity and led to excessive mitochondrial aggregation. To further understand the mechanistic pathway(s) involved, human coronary artery endothelial cells were treated with 50 ng/mL vascular endothelial growth factor, 1 µmol/L rotenone, and rotenone/vascular endothelial growth factor for 48 hours. Vascular endothelial growth factor induced robust tube formation; however, rotenone completely inhibited this effect (P<0.05 rotenone versus vascular endothelial growth factor treatment). Inhibition of tube formation by rotenone was also associated with significant increase in mitochondrial superoxide generation. Immunoblot analyses of human coronary artery endothelial cells with rotenone treatment showed significant activation of adenosine monophosphate activated kinase (AMPK)-&agr; and inhibition of mammalian target of rapamycin and p70 ribosomal S6 kinase. Activation of AMPK-&agr; suggested impairments in energy production, which was reflected by decrease in O2 consumption and bioenergetic reserve capacity of cultured cells. Knockdown of AMPK-&agr; (siRNA) also preserved tube formation during rotenone, suggesting the negative effects were mediated by the activation of AMPK-&agr;. Conversely, expression of a constitutively active AMPK-&agr; blocked tube formation. Conclusions—We conclude that activation of AMPK-&agr; during mitochondrial oxidative stress inhibits mammalian target of rapamycin signaling, which impairs phenotypic switching necessary for the growth of blood vessels.