Investigating molecular mechanisms by which shear stress regulates endothelial cell function in skeletal muscle.

摘要

The growth of new capillaries from pre-existing ones, termed angiogenesis, occurs in adult skeletal muscle in response to many stimuli. One such stimulus is increased blood flow, which exposes capillaries to increased shear stress. This causes a type of angiogenesis called luminal splitting. Luminal splitting is characterized by the growth of internal endothelial cell projections that bridge the lumen and divide the capillary in two. Increased blood flow also initiates arteriolar remodeling. While the morphological changes in response to increased shear stress have been well described, the molecular mechanisms driving these changes have not been well characterized. The purpose of my dissertation was to investigate two proteins that are regulated by shear stress: tissue inhibitor of metalloproteinase (TIMP)-1 and vascular endothelial growth factor (VEGF). TIMP-1 is an inhibitor of matrix metalloproteinase (MMP)-2, which is repressed during luminal splitting and activated during arteriole expansion. We hypothesized that TIMP-1 protein would be increased by shear stress and be regulated at the transcriptional level. We also hypothesized that TIMP-1 delation would have a detrimental effect on the normal vascular response to increased blood flow. VEGF is the most well-known regulator of angiogenesis, and is upregulated by increased shear stress. Recent work has shown that VEGF produced in muscle is required for exercise-induced angiogenesis, while VEGF produced in endothelial cells is important for cell survival but not necessarily angiogenesis. We hypothesized that muscle-derived VEGF would not regulate the endothelial response to shear stress. We found that TIMP-1 is increased by shear stress, and that this increase is regulated by the transcription factor Sp-1 and by the transforming growth factor (TGF)-beta1 signaling pathway. We examined the effects of increased blood flow on TIMP-1 -/-mice and found that luminal splitting does not occur, and that exaggerated vessel enlargement occurs in smooth-muscle actin-positive vessels. We also found that vessel permeability and proliferation are increased when TIMP-1 is deleted, suggesting a role for TIMP-1 in maintaining vascular integrity. Myocyte-specific VEGF deleted mice also had an impaired vascular response to increased blood flow. When muscle-derived VEGF was deleted, the capillary network did not expand and the arterial response was different than that of wild type mice. These studies have identified TIMP-1 and VEGF as being important regulators of the vascular response to increased shear stress in skeletal muscle, and have indicated some signaling pathways that regulate and are regulated by these proteins.