Eric Ispanovic

HIF-1α and HIF-2α play a central role in stretch-induced but not shear-stress-induced angiogenesis in rat skeletal muscle

Angiogenesis, which is essential for the physiological adaptation of skeletal muscle to exercise, occurs in response to the mechanical forces of elevated capillary shear stress and cell stretch. Increased production of VEGF is a characteristic of endothelial cells undergoing either stretch‐ or shear‐stress‐induced angiogenesis. Because VEGF production is regulated by hypoxia inducible factors (HIFs), we examined whether HIFs play a significant role in the angiogenic process initiated by these mechanical forces. Rat extensor digitorum longus (EDL) muscles were overloaded to induce stretch, or exposed to the dilator prazosin to elevate capillary shear stress, and capillaries from these muscles were isolated by laser capture microdissection for RNA analysis. HIF‐1α and HIF‐2α transcript levels increased after 4 and 7 days of stretch, whereas a transient early induction of HIF‐1α and HIF‐2α transcripts was detected in capillaries from prazosin‐treated muscles. Skeletal muscle microvascular endothelial cells exposed to 10% stretch in vitro showed an elevation in HIF‐1α and HIF‐2α mRNA, which was preceded by increases in HIF‐binding activity. Conversely, HIF‐1α and HIF‐2α mRNA were reduced significantly, and HIF‐α proteins were undetectable, after 24 h exposure to elevated shear stress (16 dyn cm−2 (16 ×10−5 N cm−2). Given the disparate regulation of HIFs in response to these mechanical stimuli, we tested the requirement of HIF‐α proteins in stretch‐ and shear‐stress‐induced angiogenesis by impeding HIF accumulation through use of the geldanamycin derivative 17‐DMAG. Treatment with 17‐DMAG significantly impaired stretch‐induced, but not shear‐stress‐induced, angiogenesis. Together, these results illustrate that activation of HIF‐1α and HIF‐2α contributes significantly to stretch‐ but not to shear‐stress‐induced capillary growth.

Cdc42 and RhoA have opposing roles in regulating membrane type 1-matrix metalloproteinase localization and matrix metalloproteinase-2 activation.

Proteolysis of the basement membrane and interstitial matrix occurs early in the angiogenic process and requires matrix metalloproteinase (MMP) activity. Skeletal muscle microvascular endothelial cells exhibit robust actin stress fibers, low levels of membrane type 1 (MT1)-MMP expression, and minimal MMP-2 activation. Depolymerization of the actin cytoskeleton increases MT1-MMP expression and MMP-2 activation. Rho family GTPases are regulators of actin cytoskeleton dynamics, and their activity can be modulated in response to angiogenic stimuli such as vascular endothelial growth factor (VEGF). Therefore, we investigated their roles in MMP-2 and MT1-MMP production. Endothelial cells treated with H1152 [an inhibitor of Rho kinase (ROCK)] induced stress fiber depolymerization and an increase in cortical actin. Both MMP-2 and MT1-MMP mRNA increased, which translated into greater MMP-2 protein production and activation. ROCK inhibition rapidly increased cell surface localization of MT1-MMP and increased PI3K activity, which was required for MMP-2 activation. Constitutively active Cdc42 increased cortical actin polymerization, phosphatidylinositol 3-kinase activity, MT1-MMP cell surface localization, and MMP-2 activation similarly to inhibition of ROCK. Activation of Cdc42 was sufficient to decrease RhoA activity. Capillary sprout formation in a three-dimensional collagen matrix was increased in cultures treated with RhoAN19 or Cdc42QL and, conversely, decreased in cultures treated with dominant negative Cdc42N17. VEGF stimulation also induced activation of Cdc42 while inhibiting RhoA activity. Furthermore, VEGF-dependent activation of MMP-2 was reduced by inhibition of Cdc42. These results suggest that Cdc42 and RhoA have opposing roles in regulating cell surface localization of MT1-MMP and MMP-2 activation.

Static Strain Stimulates Expression of Matrix Metalloproteinase-2 and VEGF in Microvascular Endothelium Via JNK- and ERK-Dependent Pathways

VEGF and MMP protein production are both required for exercise‐induced capillary growth in skeletal muscle. The underlying process by which muscle activity initiates an angiogenic response is not established, but it is known that mechanical forces such as muscle stretch are involved. We hypothesized that stretch of skeletal muscle microvascular endothelial cells induces production of MMP‐2 and VEGF through a common signal pathway. Endothelial cells were grown on Bioflex plates and exposed to 10% static stretch for up to 24 h. MMP‐2 protein level was measured by gelatin zymography and VEGF, MMP‐2, and MT1‐MMP mRNA levels were quantified by real‐time quantitative PCR. ERK1/2 and JNK phosphorylation and VEGF protein levels were assessed by Western blotting. Effects of mitogen‐activated protein kinases (MAPKs) (ERK1/2, JNK) and reactive oxygen species (ROS) on stretch‐induced expression of MMP‐2 and VEGF were tested using pharmacological inhibitors. Stretching of endothelial cells for 24 h caused significant increases in MMP‐2 protein and mRNA level, but no change in MT1‐MMP mRNA. While MMP‐2 protein production was enhanced by H2O2 in unstretched cells, ROS inhibition during stretch did not diminish MMP‐2 mRNA or protein production. Inhibition of JNK suppressed stretch‐induced MMP‐2 protein and mRNA, but inhibition of ERK had no effect. In contrast, inhibition of ERK but not JNK attenuated the stretch‐induced increase in VEGF mRNA. Our results demonstrate that differential regulation of MMP‐2 and VEGF by MAPK signal pathways contribute to stretch‐induced activation of microvascular endothelial cells. J. Cell. Biochem. 100: 750–761, 2007.