Renal ischemia induces tropomyosin dissociation-destabilizing microvilli microfilaments.

Ischemic-induced cell injury results in rapid duration-dependent actin-depolymerizing factor (ADF)/cofilin-mediated disruption of the apical microvilli microfilament cores. Because intestinal microvillar microfilaments are bound and stabilized in the terminal web by the actin-binding protein tropomyosin, we questioned whether a protective effect of tropomyosin localization to the terminal web of the proximal tubule microfilament cores is disrupted during ischemic injury. With tropomyosin-specific antibodies, we examined rat cortical sections under physiological conditions and following ischemic injury by confocal microscopy. In addition, Western blot analysis of cortical extracts and urine was undertaken. Our studies demonstrated the presence of tropomyosin isoforms in the proximal tubule microvillar terminal web under physiological conditions and their dissociation in response to 25 min of ischemic injury. This correlated with the excretion of tropomyosin-containing plasma membrane vesicles in urine from ischemic rats. In addition, we noted increased tropomyosin Triton X-100 solubility following ischemia in cortical extracts. These studies suggest tropomyosin binds to and stabilizes the microvillar microfilament core in the terminal web under physiological conditions. With the onset of ischemic injury, we propose that tropomyosin dissociates from the microfilament core providing access to microfilaments in the terminal web for F-actin binding, severing and depolymerizing actions of ADF/cofilin proteins.

Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells.

BACKGROUND: The delivery of autologous cells to increase angiogenesis is emerging as a treatment option for patients with cardiovascular disease but may be limited by the accessibility of sufficient cell numbers. The beneficial effects of delivered cells appear to be related to their pluripotency and ability to secrete growth factors. We examined nonadipocyte stromal cells from human subcutaneous fat as a novel source of therapeutic cells. METHODS AND RESULTS: Adipose stromal cells (ASCs) were isolated from human subcutaneous adipose tissue and characterized by flow cytometry. ASCs secreted 1203+/-254 pg of vascular endothelial growth factor (VEGF) per 10(6) cells, 12 280+/-2944 pg of hepatocyte growth factor per 10(6) cells, and 1247+/-346 pg of transforming growth factor-beta per 10(6) cells. When ASCs were cultured in hypoxic conditions, VEGF secretion increased 5-fold to 5980+/-1066 pg/10(6) cells (P=0.0016). The secretion of VEGF could also be augmented 200-fold by transfection of ASCs with a plasmid encoding VEGF (P<0.05). Conditioned media obtained from hypoxic ASCs significantly increased endothelial cell growth (P<0.001) and reduced endothelial cell apoptosis (P<0.05). Nude mice with ischemic hindlimbs demonstrated marked perfusion improvement when treated with human ASCs (P<0.05). CONCLUSIONS: Our experiments delineate the angiogenic and antiapoptotic potential of easily accessible subcutaneous adipose stromal cells by demonstrating the secretion of multiple potentially synergistic proangiogenic growth factors. These findings suggest that autologous delivery of either native or transduced subcutaneous ASCs, which are regulated by hypoxia, may be a novel therapeutic option to enhance angiogenesis or achieve cardiovascular protection.