Increased capillary shear stress induces angiogenesis in skeletal muscle, but the signaling mechanisms underlying this response are not known. We hypothesize that shear stress-dependent activation of vascular endothelial growth factor receptor 2 (VEGFR2) causes p38 and ERK1/2 phosphorylation, which contribute to shear stress-induced angiogenesis. Skeletal muscle microvascular endothelial cells were sheared (12 dynes/cm 2 , 0.5-24 hours). VEGFR2-Y1214 phosphorylation increased in response to elevated shear stress and VEGF stimulation. p38 and ERK1/2 phosphorylation increased at 2 hours of shear stress, but only p38 remained phosphorylated at 6 and 24 hours of shear stress. VEGFR2 inhibition abrogated p38, but not ERK1/2 phosphorylation. VEGF production was increased in response to shear stress at 6 hours, and this increased production was abolished by p38 inhibition. Male Sprague-Dawley rats were administered prazosin (50 mg/L drinking water, 1, 2, 4 or 7 days) to induce chronically elevated capillary shear stress in skeletal muscle. In some experiments, mini-osmotic pumps were used to dispense p38 inhibitor SB203580 or its inactive analog SB202474, to the extensor digitorum longus (EDL) of control and prazosin treated rats. Immunostaining and Western blotting showed increases in p38 phosphorylation in capillaries from rats treated with prazosin for 2 days but returned to basal levels at 4 and 7 days. p38 inhibition abolished the increase in capillary to muscle fibre ratio seen after 7 days of prazosin treatment. Our data suggest that p38 activation is necessary for shear stress-dependent angiogenesis.

Microvascular endothelial cells embedded within three-dimensional (3D) type I collagen matrixes assemble into cellular networks, a process that requires the upregulation of membrane type 1 (MT1) matrix metalloproteinase (MMP) and MMP-2. The purpose of this study was to identify the signaling pathways responsible for the transcriptional activation of MT1-MMP and MMP-2 in endothelial cells in 3D collagen lattices. We hypothesized that the 3D type I collagen induction of MT1-MMP and MMP-2 is mediated by the mitogen-activated protein kinase family of enzymes. Here, we show that 3D type I collagen elicits a persistent increase in ERK1/2 and JNK activation and a decrease in p38 activation. Inhibition of ERK1/2 or JNK disrupted endothelial network formation in 3D type I collagen lattices, whereas inhibition of p38 promoted network formation. mRNA levels of both MT1-MMP and MMP-2 were attenuated by ERK1/2 inhibition but unaffected by either JNK or p38 inhibition. By contrast, expression of constitutively active MEK was sufficient to stimulate MMP-2 production in a monolayer of endothelial cells cultured on type I collagen. These results provide evidence that signaling through both ERK1/2 and JNK regulates endothelial assembly into cellular networks but that the ERK1/2 signaling cascade specifically regulates network formation and the production of both MT1-MMP and MMP-2 genes in response to 3D type I collagen.

Angiogenesis (the growth of new capillaries) occurs in adults in response to physiological stimuli such as wound healing and exercise. The mitogen-activated protein kinase c-jun N-terminal kinase (JNK) has a controversial role in the process of angiogenesis, with previous evidence supporting JNK as both a positive and negative regulator of blood vessel growth. The purpose of this study was to clarify the role of JNK in the angiogenesis process. Phosphorylated JNK was observed in cultured endothelial cells, and levels were constant regardless of extracellular matrix composition. Using SP600125, inhibition of JNK attenuated sprout growth in 3D capillary sprout cultures. Inhibition of JNK reduced endothelial cell proliferation and migration in vitro. JNK inhibition and siRNA knockdown of c-jun (a downstream target of JNK) decreased protein levels of the transcription factor Egr-1, a regulator of genes involved in proliferation and migration. Matrix metalloproteinase-2 (MMP-2) production, and activity also, was reduced in sprout cultures treated with SP600125. c-Jun silencing decreased both MMP-2 and membrane type-1 (MT1)-MMP mRNA in endothelial cells, implicating both JNK and c-jun as activators of proteolysis. Taken together, these results provide evidence that JNK and its downstream target c-jun positively regulate angiogenesis via activation of endothelial cell proliferation, migration and proteolysis.

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 H(2)O(2) 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.

Elevated shear stress within the skeletal muscle microvasculature is implicated in the induction of a longitudinal splitting form of angiogenesis, which is characterized by the lack of basement membrane breakage. We investigated whether the transcriptional regulator, Ets-1, is responsive to changes in hemodynamic forces and if so, whether Ets-1 controls microvascular endothelial cell integrity by inducing the expression of inhibitors of matrix degrading proteases. Rats were treated with prazosin for 2, 4 and 7 days to increase in microvascular shear stress in hindlimb skeletal muscles. In complimentary in vitro experiments, rat microvascular skeletal muscle endothelial cells were exposed to laminar shear stress (15 dyne/cm 2 ) for 0.5, 2, and 24 hours. TaqMan PCR analysis of laser microdissected capillaries isolated from EDL muscles demonstrated transient (after 2 days) induction of Ets-1 gene expression. In cultured cells, a transient up-regulation of Ets-1 mRNA was observed after 2hr shear stimulation, accompanied by increased phosphorylation of Ets-1 and enhanced Ets-1 DNA binding activity. This response was modulated by ERK1/2 and p38 MAP kinases, but was not dependent on NOS or COX-2 activity. PAI-1, TIMP-1 and TIMP-3 mRNA were elevated significantly in prazosin treated EDL, and in response to shear stimulation in vitro. In cultured endothelial cells, Ets-1 RNA interference abolished the shear-induced increases in Ets-1, PAI-1, TIMP-1 and TIMP-3 mRNA expression. These results suggest that enhanced laminar shear stress may act to preserve the integrity of microvascular walls in part through Ets-1-dependent induction of protease inhibitors.