Jin-Sook Kwon

Trichostatin A prevents neointimal hyperplasia via activation of Krüppel like factor 4.

The proliferation of vascular smooth muscle cells (VSMCs) is an integral part of the mechanism of vascular diseases such as restenosis. Post-translational modifications by histone deacetylase (HDAC) inhibitors play an important role in the regulation of gene expression by inducing cell cycle arrest. However, the role and mechanism of the HDAC inhibitor trichostatin A (TSA) on neointimal proliferation remain unknown. In this study, we investigated the effect and mechanism whereby TSA prevents the proliferation of VSMCs and neointimal hyperplasia induced by balloon injury in rat carotid artery. Local administration of TSA significantly prevented neointimal hyperplasia. TSA dramatically inhibited the proliferation and DNA synthesis of VSMCs in response to FBS or PDGF-BB. Overexpression of Krüppel like factor 4 (KLF4) blocked the cell proliferation and DNA synthesis, as determined by the MTT and [³H]thymidine incorporation assays, whereas knockdown of KLF4 resulted in an increase in VSMC proliferation. In VSMCs, TSA increased the mRNA level and protein expression of KLF4. Treatment with TSA or transfection of KLF4 increased the expression of both p21 and p27 and promoter activity. In addition, the anti-proliferative activity of TSA was recovered in KLF4-knockdown cells. These data demonstrate that TSA inhibits neointimal thickening and VSMC proliferation via activation of the KLF4/p21/p27 signaling pathway.

Inhibitory effect of fenofibrate on neointima hyperplasia via G0/G1 arrest of cell proliferation

We have previously reported that fenofibrate displayed a potent antithrombotic effect by the inhibition of platelet aggregation. The present study was designed to investigate the effects of fenofibrate on the neointimal hyperplasia and its possible molecular mechanism. Neointimal hyperplasia was measured in balloon-inflated-induced vascular injury model of male Sprague-Dawley rats and cell proliferation was measured in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Fenofibrate-treated group showed a significant reduction in neointimal formation (0.07±0.04mm(2)) from the control (0.13±0.04mm(2)). Fenofibrate significantly inhibited platelet-derived growth factor (PDGF)-BB-induced cell counting and [(3)H]-thymidine incorporation into DNA. Fenofibrate suppressed the PDGF-BB-inducible progression through G(0)/G(1) to S phase of cell cycle. Moreover, fenofibrate inhibited not only phosphorylation of retinoblastoma (Rb) protein and expression of cyclin D/E, CDK 2/4 and proliferating cell nuclear antigen (PCNA) proteins but also mitogen-activated protein kinase (MAPK) signaling pathways such as ERK 1/2, p38 and JNK phosphorylation. In conclusion, the present study demonstrates that fenofibrate significantly inhibits neointimal formation via G(0)/G(1) arrest of PDGF-BB-induced cell proliferation in association with the inhibition of MAPK, which resulted in the downregulation of expressions of cyclin D/E, CDK 2/4 and PCNA proteins, suggesting that fenofibrate may be useful for individuals with a high risk of thrombotic or cardiovascular diseases.

Engineering and Visualization of Bacteria for Targeting Infarcted Myocardium

Optimization of the specific affinity of cardiac delivery vector could significantly improve the efficiency of gene/protein delivery, yet no cardiac vectors to date have sufficient target specificity for myocardial infarction (MI). In this study, we explored bacterial tropism for infarcted myocardium based on our previous observations that certain bacteria are capable of targeting the hypoxic regions in solid tumors. Out of several Escherichia coli or Salmonella typhimurium strains, the S. typhimurium defective in the synthesis of ppGpp (ΔppGpp S. typhimurium) revealed accumulation and selective proliferation in the infarcted myocardium without spillover to noncardiac tissue. The Salmonellae that were engineered to express a variant of Renilla luciferase gene (RLuc8), under the control of the E. coli arabinose operon promoter (P(BAD)), selectively targeted and delivered RLuc8 in the infarcted myocardium only upon injection of L-arabinose. An examination of the infarct size before and after infection, and estimations of C-reactive protein (CRP) and procalcitonin indicated that intravenous injection of ΔppGpp S. typhimurium did not induce serious local or systemic immune reactions. This current proof-of-principle study demonstrates for the first time the capacity of Salmonellae to target infarcted myocardium and to serve as a vehicle for the selective delivery of therapeutic agents in MI.

B cell translocation gene, a direct target of miR-142-5p, inhibits vascular smooth muscle cell proliferation by down-regulating cell cycle progression

Vascular smooth muscle cell (VSMC) proliferation plays a key role in neointimal hyperplasia and restenosis. Here we report the role of the microRNA miR‐142‐5p and its downstream target genes on the proliferation of cultured VSMCs. miR‐142‐5p promoted VSMC proliferation by down‐regulating B cell translocation gene 3 (BTG3). We found that BTG3 inhibited the expression of cell cycle regulatory genes and cell growth. As shown by luciferase reporter assay, miR‐142‐5p bound directly to the 3′‐untranslated region of BTG3. Overexpression of miR‐142‐5p induced expression of cell cycle regulatory genes. Thus, BTG3, a novel, direct target of miR‐142‐5p, negatively regulates VSMC proliferation.

Enhancer of polycomb1 lessens neointima formation by potentiation of myocardin-induced smooth muscle differentiation.

OBJECTIVE Previously, we reported that enhancer of polycomb1 (Epc1) induces skeletal muscle differentiation through the serum response factor (SRF). Considering that SRF plays a critical role in vascular smooth muscle cell (VSMC) differentiation, we expected that Epc1 also works in VSMCs. Here we examined the effect of Epc1 on neointima formation after arterial balloon injury and the underlying mechanism. METHODS Epc1 expression was examined in carotid artery injury or VSMC models. Interaction with myocardin (Myocd), a master regulator of smooth muscle differentiation, was examined by immunoprecipitation or promoter analysis with smooth muscle (SM) 22α promoter. Finally, we investigated whether local delivery of Epc1 regulated neointimal formation after injury. RESULTS Epc1 expression was down-regulated during proliferation induced by platelet-derived growth factor BB, whereas it was upregulated during differentiation in VSMCs. Forced expression of Epc1 induced VSMC differentiation. Epc1 physically interacted with Myocd to synergistically activate SM22α promoter activity. Transient transfection of Epc1 enhanced the physical interaction between Myocd and SRF, whereas that interaction was reduced when A10 cells were treated with siRNA for Epc1. Local delivery of Epc1 significantly reduced neointima formation induced by balloon injury. CONCLUSIONS Our results indicate that Epc1 induces VSMC differentiation by interacting with Myocd to induce SRF-dependent smooth muscle genes. We propose that Epc1 acts as a novel negative regulator of neointima formation after carotid injury.

Regulation of MMP/TIMP by HUVEC transplantation attenuates ventricular remodeling in response to myocardial infarction

AIMS We elucidated the therapeutic potential of human umbilical vein endothelial cells (HUVECs) for ameliorating progressive heart failure in a myocardial infarction (MI) rat model. MAIN METHODS MI was induced by ligation of left anterior descending artery, and HUVEC was transplanted 1week after MI. Cardiac function was evaluated by echocardiography, and histological analyses were performed. KEY FINDINGS Phosphate-buffered saline (MI-V, n=5) or HUVEC (MI-HV, n=5) were injected into the border zone and infarcted area 7days after ligation of the left coronary artery in rats. The MI-HV group showed attenuation of left ventricular (LV) remodeling compared with the MI-V group. In the infarcted myocardium, a few of injected HUVEC was retained up to 28days. The ratios of matrix metalloproteinase (MMP)-2 or MMP-9 to tissue inhibitor of metalloproteinase (TIMP)-1 or TIMP-3 were decreased in the MI-HV group compared with the MI-V group. In vivo zymography analysis showed that HUVEC transplantation decreased the activities of MMP-2 and MMP-9. In immunohistochemistry, decreased MMP-2 and increased TIMP-1 and TIMP-3 expression were observed at 48h after HUVEC transplantation. These effects on MMP/TIMP balance were inhibited by L-NAME administration (an eNOS inhibitor, 10mg/kg). NOS inhibition decreased the protein expressions of TIMP-1 and TIMP-3 but did not change the protein expressions of MMP-2 and MMP-9. SIGNIFICANCE Our data suggest that altered balance between MMP and TIMP by HUVEC transplantation contributed to attenuation of ventricular remodeling after MI via eNOS.