261Decisive role of microRNA-494 in smooth muscle cell proliferation and vascular remodeling

Background Proliferation of vascular smooth muscle cells triggered by cytokines and growth factors is a main driver in the development of vascular proliferative diseases such as atherosclerosis and in-stent-restenosis after angioplasty. MicroRNAs (miR) are small noncoding RNAs that can inhibit the expression of multiple genes simultaneously. However, the contribution of microRNAs to the differential gene regulation that triggers vascular remodeling processes is not well understood. Methods and results Neointima formation was induced by a wire-mediated injury of the femoral artery in C57BL/6 mice. Microarray analysis of the developing neointimal lesion showed a strong reduction of miR-494 (0.411±0.04; p<0.05) at 7 days after injury. In order to investigate the expression levels of miRs in vascular cells, human coronary artery smooth muscle cells (SMC), human coronary artery endothelial cells and human monocytes were analyzed via microarray analysis. Intriguingly, miR-494 was found to be predominantly expressed in SMC via microarray and qPCR analysis. The regulation of miR-494 expression was further analyzed after stimulation of SMC with 10%FCS. Following this mitogenic stimulation, mir-494 expression dropped robustly and significantly in a time-dependent manner at 6, 9, and 24 hours. To investigate the functional impact of miR-494 on SMC proliferation, miR-494 was overexpressed using miR-494-mimics (20μM). Overexpression of miR-494 significantly reduced the FCS-induced proliferation of SMC as assessed by BrdU-incorporation. In silico analyses of potential target genes for miR-494 identified ROCK1 and Survivin, both important molecules in the mitogenic response of SMC to cytokines and growth factors, as potential targets of miR-494. Indeed, ROCK1 and Survivin were found down-regulated on the mRNA and protein level after transfection of SMC with miR-494 mimics and both mulecules could be identified as direct targets using luciferase reporter assays. Following the specific inhibition of miR-494 by local application (in a perivascular thermos-responsive, self-degrading pluronic gel) of an in vivo stabilized Pre-miR-494 after wire-induced injury of the mouse femoral artery, SMC proliferation was significantly reduced, as assessed by Ki67 immunofluorescence (26.3% vs 11.2%; p<0.05). Consistently, local application of Pre-miR-494 significantly reduced neointima formation (neointima/media ratio 2.31 in control vs 1.01 in treated animals; p<0.01). Conclusion Our results show that mir-494 is strongly down-regulated in proliferating SMC in vitro as well as during neointimal lesion formation in vivo. Moreover, overexpression/ reconstitution of miR-494 levels effectively prevented SMC proliferation and neointima formation, indicating an important functional role of miR-494 in these processes. Hence, miR-494 may represent an attractive SMC-specific target for future therapeutic interventions for the treatment of vascular proliferative diseases.

Abstract 19241: Downregulation of microRNA-494 Facilitates Smooth Muscle Cell Proliferation and Vascular Remodeling

Introduction: Proliferation of vascular smooth muscle cells (VSMC) plays a crucial role in the development of vascular proliferative diseases such as atherosclerosis and in-stent-restenosis. MicroRNAs (miR) are small noncoding RNAs that modulate gene expression in a posttranscriptional manner. Hypothesis: in this project we aimed to determine the functional impact of a micro-RNA which is strongly regulated during vascular remodeling. Methods and Results: Neointima formation was induced by a wire-mediated injury of the femoral artery in C57BL/6 mice. Microarray analysis of the developing neointimal lesion showed a strong reduction of miR-494 (0.411±0.04) at 7 days after injury as compared to uninjured controls, which could be confirmed by qPCR. Interestingly, miR-494 was found to be predominantly expressed in SMC as determined by in situ hybridization. The regulation of miR-494 expression was further analyzed in vitro after stimulation of SMC with 10% FCS. Following this mitogenic stimulation, mir-494 expression dropped robustly and significantly in a time-dependent manner at 6, 9, and 24 hours. To investigate the functional impact of miR-494 on SMC proliferation, miR-494 was overexpressed using miR-494-mimics (20μM). Overexpression of miR-494 significantly reduced the FCS-induced proliferation of SMC as assessed by BrdU-incorporation and total cell counts. In silico analyses of potential target genes for miR-494 identified survivin, an important molecule for cell cycle regulation, as potential target of miR-494. Indeed, survivin was found down-regulated on the mRNA and protein level after transfection of SMC with miR-494 mimics and could be confirmed as direct target using luciferase reporter assays. Conclusions: Taken together, our results show that mir-494 is strongly down-regulated in proliferating SMC in vitro as well as during neointimal lesion formation in vivo. Moreover, the anti-proliferative effect of miR-494-reconstitution reveals a crucial functional role of miR-494 in the mitogenic response of SMC. Thus, miR-494 might represent a novel and promising SMC-specific target for future therapeutic strategies in the treatment of vascular proliferative diseases.

Abstract 468: The Role of Ca2+/PKA Signaling Enhancer Protein Phosphatase Inhibitor 1 in the Control of Ca2+ Cycling and Signaling in VSMCs

Vascular remodeling is associated with trans-differentiation of contractile vascular smooth muscle cells (VSMC) towards a proliferating/synthetic phenotype. We have recently demonstrated (Bobe et al., 2011) that the Ca2+ cycling in “contractile” VSMC requires the expression of the fast isoform of sarco/endo plasmic ATPase (SERCA2a), whereas the Ca2+ cycling in “proliferating” ones is associated with the expression of the ubiquitous isoform SERCA2b only. Phospholamban (PLB), a negative regulator of SERCA2 activity, is inhibited by PKA phosphorylation and activated by protein phosphatase 1 (PP1) dependent dephosphorylation. Inhibitor-1 (I-1), a highly specific inhibitor of PP1, enhances both PKA-dependent PLB phosphorylation and SERCA activity. The goal of this study was to elucidate the role of PKA signaling enhancer I-1 in the control of VSMC Ca2+ cycling. In humans and rodents, the expression of I-1 was found to be specific for contractile VSMC, whereas its target PP1 was highly expressed in synthetic VSMC. Consequently, PLB phosphorylation was decreased in synthetic VSMC whereas the expression of total PLB remained unchanged. Genetic deletion of I-1 in mice model (I-1 KO) resulted in lack of PLB phosphorylation in VSMC of adult animals. Despite the fact that SERCA2a was expressed, VSMCs from I-1 deficient mice were locked in the synthetic state. Consistent with this, the adult I-1 deficient mice developed a vascular proliferative disorder and excessive neointimal proliferation after vascular injury. Adenovirus-directed gene transfer of constitutively active I-1 (I-1c) significantly increased PLB phosphorylation and Ca2+ uptake in both synthetic and contractile VSMCs, however, there was no effect on the type of Ca2+ transient, which appears to be a SERCA2 isoform-dependent characteristic. Therefore, I-1c prevented proliferation and remodeling of contractile VSMC, but had no effect on synthetic VSMC, which predominantly express SERCA2b. In conclusion, synergistic action of I-1 and SERCA2a is necessary for acquisition of the VSMC contractile phenotype. Gene transfer of I-1c may be considered as a promising therapeutic strategy for preventing vascular proliferative diseases.

Can microRNAs control vascular smooth muscle phenotypic modulation and the response to injury?

Vascular smooth muscle cell (VSMC) migration and proliferation are critical events in vascular proliferative diseases. Recent studies have established microRNAs (miRNAs) as important mediators for the modulation of VSMC phenotype by targeting transcription factors and the cytoskeleton, which act as molecular switches for VSMC differentiation. The importance of miRNAs for VSMC development, differentiation, and function is evident by the fact that loss of the miRNA processing enzyme Dicer in VSMCs results in embryonic lethality due to severe vascular abnormalities. Similar abnormalities are observed in adult miR-143/145 knockout mice, indicating that these miRNAs are important for VSMC differentiation and function. However, since miR-143/145 knockout is not embryonically lethal, additional miRNA must be required during embryonic development of VSMCs. In addition, specific miRNAs such as miR-145, miR-21, and miR-221 have been found to regulate neointimal hyperplasia following vascular injury, which provides interesting possibilities for future therapeutical targets against vascular disease. Herein, we summarize recent advances regarding the role of miRNAs in VSMC phenotype modulation and response to injury.

Expression and function of periostin-like factor in vascular smooth muscle cells

In injured blood vessels activated vascular smooth muscle cells (VSMCs) migrate from the media to the intima, proliferate and synthesize matrix proteins. This results in occlusion of the lumen and detrimental clinical manifestations. We have identified a novel isoform of the periostin family of proteins referred to as periostin-like factor (PLF). PLF expression in VSMCs was increased following treatment with mitogenic compounds, suggesting that PLF plays a role in VSMC activation. Correspondingly, proliferation of the cells was significantly reduced with anti-PLF antibody treatment. PLF expression increased VSMC migration, an essential cellular process leading to vascular restenosis after injury. PLF protein was localized to neointimal VSMC of rat and swine balloon angioplasty injured arteries, as well as in human arteries with transplant restenosis, supporting the hypothesis that PLF is involved in VSMC activation and vascular proliferative diseases. Taken together, these data suggest a role for PLF in the regulation of vascular proliferative disease.