In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mg of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mg of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential. Gene Therapy (2006) 13, 225-234.
Hypoxia causes increased expression of several proteins that have the potential to promote neovascularization. Vascular endothelial growth factor (VEGF) is up-regulated by hypoxia in the retina and plays a central role in the development of several types of ocular neovascularization, but the effects of other hypoxia-regulated proteins are less clear. Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, have hypoxia response elements in the promoter regions of their genes and are increased in hypoxic liver and heart. In this study, we found that SDF-1 and CXCR4 are increased in hypoxic retina, with SDF-1 localized in glial cells primarily near the surface of the retina and CXCR4 localized in bone marrow-derived cells. Glial cells also expressed CXCR4, which suggested the possibility of autocrine stimulation, but influx of bone marrow-derived cells is the major source of increased levels of CXCR4. High levels of VEGF in the retina in the absence of hypoxia also increased levels of Cxcr4 and Sdf1 mRNA. CXCR4 antagonists reduced influx of bone marrow-derived cells into ischemic retina and strongly suppressed retinal neovascularization, VEGF-induced subretinal neovascularization, and choroidal neovascularization. These data suggest that SDF-1 and CXCR4 contribute to the involvement of bone marrow-derived cells and collaborate with VEGF in the development of several types of ocular neovascularization. They provide new targets for therapeutic intervention that may help to bolster and supplement effects obtained with VEGF antagonists.
Objective-Myocardin is a coactivator of serum response factor (SRF) required for vascular smooth muscle cell (VSMC) differentiation. HERP1 is a transcriptional repressor, which is abundantly expressed in vascular system and is known to function as a target gene of Notch. However, the role of HERP1 in the pathogenesis of vascular lesions remains unknown. The present study characterizes the expression of HERP1 in normal and diseased vessels, and tests the hypothesis that HERP1 inhibits SRF/myocardin-dependent SMC gene expression. Methods and Results-Immunohistochemistry revealed that HERP1 and myocardin expression was localized to SMC in the neointima of balloon-injured rat aorta and in human coronary atherosclerotic lesions. Expression of both HERP1 and myocardin was elevated in cultured VSMCs compared with medial SMC. Overexpressed HERP1 inhibited the myocardin-induced SMC marker gene expression in 10T1/2 cells. HERP1 protein interfered with the SRF/CArG-box interaction in vivo and in vitro. Immunoprecipitation assays showed that HERP1 physically interacts with SRF. Conclusions-HERP1 expression was associated with the SMC proliferation and dedifferentiation in vitro and in vivo.HERP1 may play a role in promoting the phenotypic modulation of VSMCs during vascular injury and atherosclerotic process by interfering with SRF binding to CArG-box through physical association between HERP1 and SRF. Key Words: HERP1 Ⅲ myocardin Ⅲ serum response factor Ⅲ smooth muscle cells P henotypic modulation of vascular smooth muscle cells (VSMCs) from contractile to synthetic forms plays a pivotal role in the pathogenesis of vascular diseases including atherosclerosis and restenosis after angioplasty. 1 It is wellestablished that VSMC phenotype is regulated by a complex array of local environmental cues including humoral factors, cell-cell and cell-matrix interactions, inflammatory stimuli, and mechanical stresses. Such complex stimuli downregulate a number of genes required for the contractile phenotype in synthetic VSMCs. These include smooth muscle myosin heavy chain (SM-MHC), SM22␣, caldesmon, and calponin. Because the genes encoding these proteins are differentially expressed depending on the proliferative state of VSMCs, transcription factors regulated by numerous stimuli are responsible at least in part for the distinct pattern of gene expression seen in synthetic VSMCs.There is mounting evidence that most SMC marker proteins such as SM-MHC and SM22␣ are controlled by serum response factor (SRF), which binds to a sequence known as a CArG box and recruits a potent coactivator, myocardin, for SMC differentiation. 1 When myocardin is ectopically expressed in nonmuscle cells, it can induce SMC differentiation. 2,3 Most importantly, mouse embryos deficient for myocardin show no evidence of vascular SMC, indicating myocardin as a necessary and sufficient factor for SMC differentiation in vivo. 4 These observations, in conjunction with downregulation of SMC marker genes in synthetic VSMC, led us to speculate that myocardin express...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.