Discovery of microvascular miRNAs using public gene expression data: miR-145 is expressed in pericytes and is a regulator of Fli1

International audienceBACKGROUND: A function for the microRNA (miRNA) pathway in vascular development and angiogenesis has been firmly established. miRNAs with selective expression in the vasculature are attractive as possible targets in miRNA-based therapies. However, little is known about the expression of miRNAs in microvessels in vivo. Here, we identified candidate microvascular-selective miRNAs by screening public miRNA expression datasets. METHODS: Bioinformatics predictions of microvascular-selective expression were validated with real-time quantitative reverse transcription PCR on purified microvascular fragments from mouse. Pericyte expression was shown with in situ hybridization on tissue sections. Target sites were identified with 3' UTR luciferase assays, and migration was tested in a microfluid chemotaxis chamber. RESULTS: miR-145, miR-126, miR-24, and miR-23a were selectively expressed in microvascular fragments isolated from a range of tissues. In situ hybridization and analysis of Pdgfb retention motif mutant mice demonstrated predominant expression of miR-145 in pericytes. We identified the Ets transcription factor Friend leukemia virus integration 1 (Fli1) as a miR-145 target, and showed that elevated levels of miR-145 reduced migration of microvascular cells in response to growth factor gradients in vitro. CONCLUSIONS: miR-126, miR-24 and miR-23a are selectively expressed in microvascular endothelial cells in vivo, whereas miR-145 is expressed in pericytes. miR-145 targets the hematopoietic transcription factor Fli1 and blocks migration in response to growth factor gradients. Our findings have implications for vascular disease and provide necessary information for future drug design against miRNAs with selective expression in the microvasculature

Myofibroblasts and the Vascular Endothelium : Impact of Fibrin Degradation Products and miRNA on Vascular Motility and Function

Angiogenesis is the formation of new blood vessels from pre-existing vasculature and is important during development as well as wound healing and tissue remodeling. Angiogenesis also occurs during pathological conditions such as diabetic retinopathy and cancer. This thesis is centered on the biology of endothelial cells, lining the blood vessels, and myofibroblasts, important for wound healing. We investigated an endothelial cell specific gene, ExoC3l2, and its role in VEGFR2 signaling and migration. EXOC3L2 co-localize with members of the exocyst complex, involved in vesicular transport, as well as VEGFR2. Reducing the level of EXOC3L2 in microvascular endothelial cells results in reduced VEGFR2 signaling and subsequently reduced chemotactic response to VEGF-A. MicroRNA (miRNA) have been shown to be regulators of gene transcription and cell type specific miRNAs have been identified. We investigated two miRNAs, miR-145 and miR-24. miR-145 is expressed in pericytes and fibroblasts but was shown to regulate fli1, an endothelial transcription factor. miR-145 overexpression reduced chemotaxis in both fibroblasts and endothelial cells, as did suppression of the endogenous miR-145 level in fibroblasts. miR-24 in contrast is expressed by endothelial cells and are able to target Ndst1, important for heparan sulfate (HS) sulfation. Sulfation of HS is important for many processes, amongst them growth factor signaling. Overexpression of miR-24 resulted in lower sulfation of HS chains, decreasing the ability of HS to interact with VEGF-A. Overexpressing miR-24 resulted in disturbed chemotaxis, similar to suppressing Ndst1 using siRNA. Myofibroblast recruitment is an important step in wound healing. The myofibroblasts contract the wound, synthesize new extracellular matrix and contribute to revascularization by looping angiogenesis. Maturation from resting fibroblast to myofibroblast is dependent on TGF-β. We found that fibrin fragment E (FnE), a degradation product of fibrin, potentiated the response of fibroblasts to TGF-β thus enhancing TGF-β-induced myofibroblast differentiation. FnE was also found to influence the migration of fibroblasts. These responses are dependent on integrins and toll-like receptors. These findings may serve to further increase the understanding of angiogenesis and wound healing to develop new therapies against pathological conditions.

Myofibroblasts and the Vascular Endothelium : Impact of Fibrin Degradation Products and miRNA on Vascular Motility and Function

Angiogenesis is the formation of new blood vessels from pre-existing vasculature and is important during development as well as wound healing and tissue remodeling. Angiogenesis also occurs during pathological conditions such as diabetic retinopathy and cancer. This thesis is centered on the biology of endothelial cells, lining the blood vessels, and myofibroblasts, important for wound healing. We investigated an endothelial cell specific gene, ExoC3l2, and its role in VEGFR2 signaling and migration. EXOC3L2 co-localize with members of the exocyst complex, involved in vesicular transport, as well as VEGFR2. Reducing the level of EXOC3L2 in microvascular endothelial cells results in reduced VEGFR2 signaling and subsequently reduced chemotactic response to VEGF-A. MicroRNA (miRNA) have been shown to be regulators of gene transcription and cell type specific miRNAs have been identified. We investigated two miRNAs, miR-145 and miR-24. miR-145 is expressed in pericytes and fibroblasts but was shown to regulate fli1, an endothelial transcription factor. miR-145 overexpression reduced chemotaxis in both fibroblasts and endothelial cells, as did suppression of the endogenous miR-145 level in fibroblasts. miR-24 in contrast is expressed by endothelial cells and are able to target Ndst1, important for heparan sulfate (HS) sulfation. Sulfation of HS is important for many processes, amongst them growth factor signaling. Overexpression of miR-24 resulted in lower sulfation of HS chains, decreasing the ability of HS to interact with VEGF-A. Overexpressing miR-24 resulted in disturbed chemotaxis, similar to suppressing Ndst1 using siRNA. Myofibroblast recruitment is an important step in wound healing. The myofibroblasts contract the wound, synthesize new extracellular matrix and contribute to revascularization by looping angiogenesis. Maturation from resting fibroblast to myofibroblast is dependent on TGF-β. We found that fibrin fragment E (FnE), a degradation product of fibrin, potentiated the response of fibroblasts to TGF-β thus enhancing TGF-β-induced myofibroblast differentiation. FnE was also found to influence the migration of fibroblasts. These responses are dependent on integrins and toll-like receptors. These findings may serve to further increase the understanding of angiogenesis and wound healing to develop new therapies against pathological conditions.

Myofibroblasts and the Vascular Endothelium : Impact of Fibrin Degradation Products and miRNA on Vascular Motility and Function

Angiogenesis is the formation of new blood vessels from pre-existing vasculature and is important during development as well as wound healing and tissue remodeling. Angiogenesis also occurs during pathological conditions such as diabetic retinopathy and cancer. This thesis is centered on the biology of endothelial cells, lining the blood vessels, and myofibroblasts, important for wound healing. We investigated an endothelial cell specific gene, ExoC3l2, and its role in VEGFR2 signaling and migration. EXOC3L2 co-localize with members of the exocyst complex, involved in vesicular transport, as well as VEGFR2. Reducing the level of EXOC3L2 in microvascular endothelial cells results in reduced VEGFR2 signaling and subsequently reduced chemotactic response to VEGF-A. MicroRNA (miRNA) have been shown to be regulators of gene transcription and cell type specific miRNAs have been identified. We investigated two miRNAs, miR-145 and miR-24. miR-145 is expressed in pericytes and fibroblasts but was shown to regulate fli1, an endothelial transcription factor. miR-145 overexpression reduced chemotaxis in both fibroblasts and endothelial cells, as did suppression of the endogenous miR-145 level in fibroblasts. miR-24 in contrast is expressed by endothelial cells and are able to target Ndst1, important for heparan sulfate (HS) sulfation. Sulfation of HS is important for many processes, amongst them growth factor signaling. Overexpression of miR-24 resulted in lower sulfation of HS chains, decreasing the ability of HS to interact with VEGF-A. Overexpressing miR-24 resulted in disturbed chemotaxis, similar to suppressing Ndst1 using siRNA. Myofibroblast recruitment is an important step in wound healing. The myofibroblasts contract the wound, synthesize new extracellular matrix and contribute to revascularization by looping angiogenesis. Maturation from resting fibroblast to myofibroblast is dependent on TGF-β. We found that fibrin fragment E (FnE), a degradation product of fibrin, potentiated the response of fibroblasts to TGF-β thus enhancing TGF-β-induced myofibroblast differentiation. FnE was also found to influence the migration of fibroblasts. These responses are dependent on integrins and toll-like receptors. These findings may serve to further increase the understanding of angiogenesis and wound healing to develop new therapies against pathological conditions.