Sisi Chen

Inhibition of neointimal hyperplasia in the rat carotid artery injury model by a HMGB1 inhibitor

OBJECTIVE High mobility group box 1 protein (HMGB1) is expressed in atherosclerotic lesions. However, its role in vascular system is unknown. In this study, we explore whether the inhibition of HMGB1 attenuates neointimal formation in animal models. METHODS AND RESULTS Experiments were performed with VSMCs from thoracic aorta of SD rats in vitro, and a rat carotid artery balloon injury model in vivo. HMGB1 levels were increased after stimulation of angiotensin II (Ang II) and 10% serum in cultured VSMCs. HMGB1 inhibitor (glycyrrhizin) significantly inhibited the proliferation and migration of Ang II-treated VSMCs, which was accompanied with decreased oxidative stress and inflammation. The underlying mechanisms were related with the promotion of antioxidant systems activity and deactivation of p38 MAPK/NF-κB signaling pathway, respectively. Furthermore, inhibition of HMGB1 blunted Notch signaling pathway during VSMCs phenotypic transition, and correspondingly restored VSMCs differentiated phenotype under 10% serum stimulation. In vivo study, HMGB1 expression was elevated after artery injury. Meanwhile, glycyrrhizin treatment suppressed HMGB1 expression, which was accompanied with blunted inflammation and oxidative stress after 7 days of balloon injury. Moreover, the area of neointimal to media area ratio was significantly decreased in glycyrrhizin group compared with injury group at 14 days after balloon injury. CONCLUSIONS Inhibition of HMGB1 activity attenuated VSMCs activation and neointimal formation after carotid injury. Therefore, blockage of HMGB1 might represent a novel therapeutic strategy for vascular injury.

Lentivirus-mediated RNAi targeting CREB binding protein attenuates neointimal formation and promotes re-endothelialization in balloon injured rat carotid artery.

Background/Aims: Lentiviral vectors provide a promising strategy for the treatment of cardiovascular diseases, owing to their ability to govern efficient and durable gene transfer. However, relatively few studies have been addressed on restenosis after balloon or stent associated arterial injury. We previously found that CREB binding protein (CBP), a powerful transcriptional coactivator, regulated cell proliferation and apoptosis in vascular endothelial and smooth muscle cells. Therefore, we investigated whether inhibition of CBP by lentivirus-mediated small interfering RNA can reduce neointimal formation after arterial injury. Methods: The carotid arteries from Sprague-Dawley rats were injured by balloon catheter, followed by incubating with 100 µl lentivirus expressing CBP or negative control (NC)-specific short hairpin RNAs (shRNAs) or PBS solution for 30 minutes. The rats were euthanized for real-time PCR, Western blot, immunohistochemical staining, and morphometric analysis at 4 weeks after balloon injury and in vivo gene transfer. Results: Lentiviral shRNA targeting CBP markedly reduced CBP expression. Moreover, CBP siRNA showed potent inhibition on balloon injury-induced Nuclear factor kappaB (NF-ĸB) acetylation. Compared with controls, the significant decrease of neointimal formation by CBP siRNA was accompanied by reduced cell proliferation in the neointima of injured arteries. However, no changes in medial area were observed among these different groups. Interestingly, endothelial cell marker CD31 immunostaining and morphometric analysis both showed that CBP knockdown significantly accelerated re-endothelialization. Conclusions: These findings suggest that CBP is involved in the control of neointimal formation and re-endothelialization via regulating NF-ĸB acetylation. Lentivirus-mediated CBP silencing may represent a novel therapeutic approach for the prevention of restenosis after vascular interventions.

CREB-binding protein silencing inhibits thrombin-induced endothelial progenitor cells angiogenesis

Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence from our group suggested that CREB-binding protein (CBP) plays an important role in thrombin-induced EPCs migration. However, whether CBP could regulate EPCs angiogenic properties is unknown. In the present study, we investigated whether CBP silencing could inhibit thrombin-induced EPCs angiogenesis. EPCs isolated from the bone marrow of Sprague–Dawley rats were cultured and identified, and then were treated by thrombin alone or combined with CBP-shRNA lentivirus. The effect of CBP silencing on EPCs proliferation was assessed using BrdU incorporation assay. Cell adhesion and tube formation were detected to evaluate the angiogenic functions. Finally, mRNA and protein expression of relevant angiogenic genes were examined by real-time PCR, western-blot, and enzyme-linked immunoassay respectively. Luciferase reporter gene assay was performed to evaluate NF-κB activity. Administration of thrombin significantly promoted EPCs proliferation and adhesion. Thrombin also increased the tube formation in Matrigel assay. However, these effects of thrombin were abolished by CBP gene silencing. CBP silencing also abrogated thrombin-induced increases of integrin β2 expression. In thrombin-induced EPCs, CBP silencing significantly decreased the secretion of VEGF, IL-6 and suppressed NF-κB activity. In conclusion, thrombin-induced EPCs proliferation, adhesion, and tube formation were inhibited by CBP silencing, indicating that CBP plays an important role in thrombin-induced EPCs neovascularization.

Down-regulation of CREB-binding protein expression blocks thrombin-mediated endothelial activation by inhibiting acetylation of NF-κB

OBJECTIVES CREB-binding protein (CBP) belongs to a unique class of transcription co-activators possessing histone acetyltransferase (HAT) activity. The aim of the present study was to evaluate the role of CBP in thrombin-induced endothelial activation, and also explore the underlying mechanism. METHODS Leukocyte-endothelial adhesion was calculated as the proportion of the labeled-neutrophils that adhered to ECs relative to all neutrophils applied. Levels of adhesion molecules were analyzed by real-time RT-PCR and western blot. Electrophoretic mobility shift assay and NF-κB reporter assay were performed to evaluate NF-κB activation. Acetylation of NF-κB was measured with immunoprecipitation and western blot assay. To detect the CBP-HAT activity, acetyl residues on an acetylated histone H4 was analyzed. RESULTS Leukocyte-endothelial adhesion induced by thrombin was markedly attenuated in endothelial cells with CBP knockdown. The decreased adhesion was paralleled by the reduction of vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin. Furthermore, CBP silencing suppressed thrombin-mediated NF-κB activation, and this inhibitory effect was associated with decreased acetylation of NF-κB and CBP-HAT activity. CONCLUSIONS Our results indicate that CBP is involved in the regulation of endothelial activation via NF-κB-dependent pathway. Down-regulation of CBP may play a role in returning ECs from a pre-inflammatory status to a quiescent state in the pathogenesis of atherosclerosis.

Transcriptional regulation of platelet-derived growth factor-B chain by thrombin in endothelial cells: involvement of Egr-1 and CREB-binding protein

Thrombin and platelet-derived growth factor-B chain (PDGF-B) are key factors in the stimulation of atherosclerosis. The effect of thrombin on PDGF-B production has been characterized. However, the underlying mechanism is still far clear. Here, we investigate the transcription factors and regulators that are involved in PDGF-B production caused by thrombin in endothelial cells (ECs). Levels of PDGF were analyzed by real-time RT-PCR and ELISA, while levels of early growth response-1 (Egr-1) were analyzed by real-time RT-PCR and western blot. To evaluate the function of CBP and Egr-1 involved in regulation of PDGF-B, small interfering RNA (siRNA) were used to down-regulate their expression in mRNA and protein level. Interaction of Egr-1 and CBP was measured with immunoprecipitation and western blot. Thrombin induced an early and transient up-regulation of transcription factor early Egr-1, which was followed by a delayed increase of PDGF-B. siRNA against Egr-1-inhibited thrombin-induced PDGF-B production. Furthermore, thrombin could enhance the interaction of Egr-1 with its co-activator CREB-binding protein (CBP). CBP knockdown attenuated this interaction, and led to a reduction of PDGF-B expression induced by thrombin. Our results suggest that CBP might be one of the main interaction targets for Egr-1, and the transient activation of Egr-1 and recruitment of CBP are required for thrombin-induced PDGF-B in ECs.

GW24-e2386 Down-regulation of histone demethylase KDM3A attenuates balloon injury-induced neointimal hyperplasia in diabetic rats through modulation of epigenetic histone lysine 9 di-methylation

Objectives Diabetic patients continued to develop vascular complications despite achieving normal glucose level, suggesting metabolic memory which was associated with histone H3 lysine (H3K9) methylation through gene repression. Here we hypothesised that H3K9 di-methylation (H3K9me2) might be involved in vascular remodelling under diabetic condition; and histone demethylase KDM3A, an important regulator for H3K9me2, might play a key role in these pathologies. Methods To generate diabetic model, male SD rats were fed with 60% fat diet for 4 weeks followed by intraperitoneal injection with 40mg/kg streptozotocin for another 2 weeks. The carotid arteries from diabetic rats (glucose 15-33 mM) were injured by balloon catheter to induced neointimal hyperplasia, and transfected with adenovirus encoding KDM3A gene or control adenovirus (Ad-GFP) for KDM3A overexpression. A KDM3A siRNA was delivered by lentivirus to down-regulate KDM3A level; the control lentivirus is Lenti-NC. Vascular smooth muscle cells (VSMCs) were isolated from thoracic aorta of SD rats. Microarray analysis was performed to identify altered gene profile in carotid arteries after balloon injury. Chip assay was used to evaluate the H3K9me2 level at the promoter region of certain genes. Results H3K9me2 level was decreased by 51.3% in carotid arteries from diabetic rats compared to normal rats, which was associated with 1.32-fold increase of KDM3A mRNA during neointimal formation. After 28-day balloon injury, the area of neointimal to media (I/M) ratio was further enhanced by KDM3A overexpression compared with Ad-GFP control group (1.58 ± 0.30 vs. 0.86 ± 0.13, p < 0.05). However, KDM3A knockdown significantly decreased I/M ratio as compared to Lenti-NC (0.64 ± 0.18 vs. 1.45 ± 0.19, p < 0.05). Microarray analysis revealed ROCK2- and AGTR1-dependent vascular remodelling pathways was mediated by KDM3A after balloon injury at 7 days. KDM3A, ROCK2 and AGTR1 were up-regulated 4.17-fold, 1.57-fold, 4.14-fold, respectively, in KDM3A overexpression group as compared to Ad-GFP; whereas KDM3A knockdown reduced the expression of these 3 genes by 42.5%, 21.3%, 28.5%, respectively (p < 0.05 vs. Lenti-NC). The KDM3A effects were further verified in VSMCs. HG (30mM)- and Ang II (1μM)-stimulated proliferation and migration of VSMCs was further enhanced in KDM3A overexpression group. However, the PCNA level, a proliferation index, was markedly blunted by KDM3A knockdown compared with Lenti-NC group (0.20 ± 0.02 vs. 0.15 ± 0.03, p < 0.05). Similar inhibitory effect of migration was also observed after KDM3A knockdown. Western blot indicated HG-induced expression of ROCK2 and downstream target p-MYPT-1 was further enhanced by KDM3A overexpression, but oppositely abolished by 41.4% and 31.9%, respectively, after knocking-down KDM3A (p < 0.05 vs. Lenti-NC). AGTR1 levels was also pronouncedly elevated after KDM3A overexpression, but blocked by 52.8% after KDM3A knockdown under 1μM Ang II stimulation (p < 0.05 vs. Lenti-NC); the downstream target ERK1/2 activity changed in the same tendency. More importantly, KDM3A knockdown increases H3K9m2 in the proximal promoter region of ROCK2 and AGTR1 genes in cultured VSMCs by Chip assay (p < 0.05 vs. Lenti-NC). Conclusions H3K9me2 may be a major underlying mechanism for vascular remolding in diabetes, and histone demethylase KDM3A may represent a promising therapeutic approach for the prevention of coronary artery disease combined with diabetes.