ABSTRACT
Thrombospondins (TSPs) are multifunctional proteins that are deposited in the extracellular matrix where they directly affect the function of vascular and other cell types. TSP-4, one of the 5 TSP family members, is expressed abundantly in tendon and muscle. We have examined the effect of TSP-4 deficiency on tendon collagen and skeletal muscle morphology and function. In Thbs4(-/-) mice, tendon collagen fibrils are significantly larger than in wild-type mice, and there is no compensatory over-expression of TSP-3 and TSP-5, the two TSPs most highly homologous to TSP-4, in the deficient mice. TSP-4 is expressed in skeletal muscle, and higher levels of TSP-4 protein are associated with the microvasculature of red skeletal muscle with high oxidative metabolism. Lack of TSP-4 in medial soleus, red skeletal muscle with predominant oxidative metabolism, is associated with decreased levels of several specific glycosaminoglycan modifications, decreased expression of a TGFß receptor beta-glycan, decreased activity of lipoprotein lipase, which associates with vascular cell surfaces by binding to glycosaminoglycans, and decreased uptake of VLDL. The soleus muscle is smaller and hind- and fore-limb grip strength is reduced in Thbs4(-/-) mice compared to wild-type mice. These observations suggest that TSP-4 regulates the composition of the ECM at major sites of its deposition, tendon and muscle, and the absence of TSP-4 alters the organization, composition and physiological functions of these tissues.
Subject(s)
Collagen/metabolism , Extracellular Matrix Proteins/physiology , Muscle, Skeletal/physiology , Tendons/physiology , Thrombospondins/genetics , Thrombospondins/physiology , Animals , Blotting, Western , DNA Primers/genetics , Endothelial Cells/metabolism , Extracellular Matrix Proteins/metabolism , Glycosaminoglycans/metabolism , Heparitin Sulfate/metabolism , Immunohistochemistry , Lipoproteins, VLDL/metabolism , Mice , Mice, Knockout , Microscopy, Confocal , Microscopy, Electron, Transmission , Muscle Strength/physiology , Muscle, Skeletal/metabolism , Tendons/metabolism , Thrombospondins/metabolismABSTRACT
OBJECTIVE: Vascular diabetic complications are associated with abnormal extracellular matrix and dysfunction of vascular cells, which later result in aberrant angiogenesis and development of atherosclerotic lesions. The tissue and cell specificity of the effects of high glucose are well recognized, but the underlying cell type-specific molecular mechanisms controlled by glucose are still unclear. We sought to identify cell type-specific mechanisms by which high glucose regulates transcription of genes in vascular cells. METHODS AND RESULTS: Thrombospondin-1 is a potent antiangiogenic protein associated with development of several diabetic complications and regulated by high glucose in multiple cell types. We report that distinct cell type-specific mechanisms regulate thrombospondin-1 gene (THBS1) transcription in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in response to high glucose: although a proximal fragment of 280 nucleotides is sufficient to drive transcription in ECs, THBS1 was regulated cooperatively by interaction between proximal (-272 to -275) and distal (-1016 to -1019) promoter elements in VSMCs. Transcription factors activated by high glucose in VSMCs were cell type-specific. The formation of a single complex interacting with both distal and proximal glucose-responsive elements of THBS1 promoter in VSMCs was confirmed using gel-shift assays, binding sequence decoy oligomers, and specific mutant promoter fragments. CONCLUSIONS: Transcriptional response of vascular cells to high glucose is cell type-specific and involves activation of distinct transcription factors, providing a basis for tissue-specific changes of vasculature in diabetics.
