Simvastatin attenuates the additive effects of TNF-α and IL-18 on the connexin 43 up-regulation and over-proliferation of cultured aortic smooth muscle cells.

Statin therapy is known to down-regulate inflammatory activities in atheromatous tissues of animals. The aims of this study were to examine the regulatory role of interleukin-18 (IL-18) in the connexin 43 (Cx43) and the proliferation of cultured aortic smooth muscle cells (SMCs) as well as to elucidate the underlying therapeutic mechanism of simvastatin. Vytorin therapy significantly alleviated high-cholesterol diet-induced hypercholesterolemia, suppressed neointimal hyperplasia, macrophage infiltration, and Cx43 and IL-18 expression in rabbit aortic walls. In vitro study using an aortic SMC line showed that IL-18 up-regulated constitutive Cx43 expression and potentiated tumor necrosis factor-α (TNF-α)-triggered Akt and MAPK signaling pathways. Simvastatin treatment alone reduced constitutive Cx43 levels and prevented the TNF-α-induced IL-18 up-regulation. Mechanistic investigation using kinase-specific inhibitors showed that simvastatin pretreatment attenuated TNF-α-elicited Akt and ERK1/2 phosphorylation, whereas PI3K and all MAPK activities were also implied in the additive effect of TNF-α and IL-18 on Cx43 up-regulation. Proliferation assay indicated that IL-18 stimulated SMC proliferation and synergized the TNF-α-stimulated cell proliferation. Likewise, simvastatin treatment suppressed the SMC over-proliferation induced not only by TNF-α alone, but also by simultaneous treatment with TNF-α and IL-18. The suppression of simvastatin in SMC proliferation was not mediated through mitochondrial related pro-apoptogenesis under both scenarios. In conclusion, simvastatin attenuates the additive effects of TNF-α and IL-18 on Cx43 up-regulation and over-proliferation of aortic SMCs, mainly through the blockade of Akt signaling pathway. These findings may fortify the rationale underlying the atheroprotective mechanism of statin therapy.

Extracorporeal shock wave therapy reverses ischemia-related left ventricular dysfunction and remodeling: molecular-cellular and functional assessment.

An optimal treatment for patients with diffuse obstructive arterial disease unsuitable for catheter-based or surgical intervention is still pending. This study tested the hypothesis that extracorporeal shock wave (ECSW) therapy may be a therapeutic alternative under such clinical situation. Myocardial ischemia was induced in male mini-pigs through applying an ameroid constrictor over mid-left anterior descending artery (LAD). Twelve mini-pigs were equally randomized into group 1 (Constrictor over LAD only) and group 2 (Constrictor over LAD plus ECSW [800 impulses at 0.09 mJ/mm(2)] once 3 months after the procedure). Results showed that the parameters measured by echocardiography did not differ between two groups on days 0 and 90. However, echocardiography and left ventricular (LV) angiography showed higher LV ejection fraction and lower LV end-systolic dimension and volume in group 2 on day 180 (p<0.035). Besides, mRNA and protein expressions of CXCR4 and SDF-1α were increased in group 2 (p<0.04). Immunofluorescence staining also showed higher number of vWF-, CD31-, SDF-1α-, and CXCR4-positive cells in group 2 (all p<0.04). Moreover, immunohistochemical staining showed notably higher vessel density but lower mean fibrosis area, number of CD40-positive cells and apoptotic nuclei in group 2 (all p<0.045). Mitochondrial protein expression of oxidative stress was lower, whereas cytochrome-C was higher in group 2 (all p<0.03). Furthermore, mRNA expressions of MMP-9, Bax and caspase-3 were lower, whereas Bcl-2, eNOS, VEGF and PGC-1α were higher in group 2 (all p<0.01). In conclusion, ECSW therapy effectively reversed ischemia-elicited LV dysfunction and remodeling through enhancing angiogenesis and attenuating inflammation and oxidative stress.

Autologous bone marrow cell implantation attenuates left ventricular remodeling and improves heart function in porcine myocardial infarction: an echocardiographic, six-month angiographic, and molecular-cellular study.

BACKGROUND: We investigated the potential benefits and the underlying mechanisms of autologous bone marrow-derived mononuclear cell (BMDMNC) implantation in a porcine model of acute anterior wall myocardial infarction (AAWMI) by studying 6-month left ventricular (LV) function and LV remodeling. METHODS: After being aspirated from the iliac crest and cultured for 1 week, BMDMNCs were implanted immediately after AAWMI induction through the left anterior descending artery ligation. Thirty male mini-pigs (16-18 kg) were equally divided into group 1 [AAWMI plus saline injection into infarct-ischemia area (IA)], group 2 (AAWMI plus 3.0 × 107 BMDMNC transplantation into non-IA), group 3 (AAWMI plus 3.0 × 107 BMDMNC transplantation into IA), group 4 (sham control plus 3.0 × 107 BMDMNC transplantation into LV myocardium), and group 5 (normal control). RESULTS: By day 90, echocardiography demonstrated an increased LV end-diastolic and end-systolic dimensions but reduced LV ejection fraction (LVEF) in groups 1 and 2 than in other groups (all p < 0.01). Six-month angiographic study showed a lower LVEF and wall motion score but a higher mitral regurgitation in groups 1 and 2 than in other groups (all p < 0.01). In IA and peri-infarct area, the number of small vessels and mRNA expressions of endothelial nitric oxide synthase, Bcl-2, interleukin (IL)-10, and peroxisome proliferator-activated receptor-γ coactivator-1α were lower, whereas the number of apoptotic nuclei, caspase-3, Bax, endothelin-1, IL-8, and matrix metalloproteinase was higher in groups 1 and 2 than in other groups (all p < 0.01). CONCLUSIONS: Autologous BMDMNC transplantation into IA rather non-IA improves LV function and reduces LV remodeling via eliciting a broad-spectrum of molecular-cellular defensive mechanisms.

Six-month angiographic study of immediate autologous bone marrow mononuclear cell implantation on acute anterior wall myocardial infarction using a mini-pig model.

This study investigated six-month angiographic results of autologous bone marrow mononuclear cell (BMMNC) transplantation immediately following acute myocardial infarction (AMI) in a mini-pig model.AMI was induced by left anterior descending artery ligation. Twenty-four mini-pigs were equally divided into group 1 [AMI plus saline injection in infarcted area (IA)], group 2 (AMI plus BMMNC transplantation into non-IA), group 3 (AMI plus BMMNC implantation into IA), and group 4 (sham control). One-week cultured BMMNCs (3.0 x 10(7)) were immediately transplanted following AMI induction. Angiographic studies over 6 months demonstrated that mitral regurgitation (MR) was lower in groups 3 and 4 than in groups 1 and 2 (all P < 0.01). Wall motion scores and left ventricular ejection fraction (LVEF) were higher in groups 3 and 4 than in groups 1 and 2 (all P < 0.05). Collateral circulation was higher in group 3 than in groups 1 and 2 ( P < 0.01). The wall thickness of the IA was higher, whereas the heart weight was lower in group 3 than in groups 1 and 2 (all P < 0.01).Immediate autologous BMMNC transplantation into IA is superior to saline-treated only or BMMNC transplantation into non-IA following AMI for reducing MR and improving LVEF.

Bone marrow-derived mononuclear cell therapy alleviates left ventricular remodeling and improves heart function in rat-dilated cardiomyopathy.

OBJECTIVES: This study hypothesized that bone marrow-derived mononuclear cell (BMDMNC) therapy may improve cardiac function through preventing cell death, alleviating left ventricular (LV) remodeling, and enhancing angio-/vasculo-genesis, as well as preserving LV contractility in a rat model of dilated cardiomyopathy (DCM). DESIGN: A model of DCM in Sprague-Dawley rats was used to investigate the effects of BMDMNC therapy on inflammatory and oxidative response, energy depression, cellular apoptosis, expressions of protein kinase C-(PKC)-epsilon, and connexin43 protein (Cx43) in LV myocardium and heart function. SETTING: An animal model research laboratory at Kaohsiung Chang Gung Memorial Hospital. MEASUREMENTS: The rats were divided into group 1 (normal control, n = 8), group 2 (saline-treated DCM, n = 10), and group 3 (1.2 x 10 BMDMNC implanted into LV anterior wall on day 35 after DCM induction, n = 10). The DCM and normal control rats were killed on day 90 following DCM induction. RESULTS: The results demonstrated that Cx43 protein expression and messenger RNA expressions of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, endothelial nitric oxide synthase, and interleukin-10 were higher, whereas messenger RNA expressions of endothelin-1 and matrix metalloproteinase-9 were lower in groups 1 and 3 than in group 2 (all p < 0.05). Additionally, expressions of PKC-epsilon in plasma membrane and mitochondria and LV function were conserved in group 1 and improved in group 3, whereas cardiomyocyte apoptosis, mitochondrial oxidative stress, and fibrosis of LV myocardium were reduced in groups 1 and 3 than in group 2 (all p < 0.005). CONCLUSION: BMDMNC therapy in DCM significantly improves LV function by limiting cellular apoptosis, inflammatory and oxidative responses, and by up-regulating expressions of Cx43, PKC-epsilon, and energy transcription factors.

Diabetes mellitus downregulates expression of connexin43 in rat aortic medial smooth muscle cells and can be reversed by simvastatin and losartan therapy.

BACKGROUND: Diabetes mellitus (DM) plays a crucial role in the pathogenesis of initiation and propagation of atherosclerosis. Although previous studies have suggested that interactions between cells form the framework for understanding the pathogenesis of atherosclerosis, little is known about how DM impacts intercellular communication within arteries, which occurs via connexin43 (Cx43) gap junctions (GJs). This study tested the hypothesis that DM suppresses expression of Cx43 GJs, and that this suppression can be abrogated via simvastatin or losartan treatment. METHODS: An experimental model of DM (induced by streptozocin 60 mg/kg body weight) in adult male rats (n = 24) was utilized to investigate Cx43 expression in the aorta. These rats were divided into group I (insulin therapy only), group II (insulin plus simvastatin 20 mg/kg/day) and group III (insulin plus losartan 20 mg/kg/day). Twenty-four diabetic rats and 8 healthy rats (group IV) were sacrificed 3 weeks after DM induction for Western blot and immunofluorescence analysis. RESULTS: By day 21, the blood sugar level was significantly higher than the respective baseline values in groups I, II and III (all values of p < 0.0001). Additionally, the final blood sugar levels of groups I-III were significantly higher than that of group IV (p < 0.0001). The final body weight in group IV was significantly higher than that in groups I-III (all values of p < 0.0001). Experimental results demonstrated that Cx43 expression in the aortic wall did not differ among groups II-IV (p > 0.1). However, compared with groups II-IV, Cx43 expression in the aortic wall was significantly mitigated in group I (all values of p < 0.05). Western blot results showed that relative density of Cx43 to beta-actin was significantly higher in groups II-IV than in group I (p < 0.01). CONCLUSIONS: DM markedly suppressed expression of Cx43 in rat aortic walls. Both simvastatin and losartan treatment significantly reversed the effects of DM on integrity of Cx43 expression.

Cilostazol therapy attenuates monocrotaline-induced pulmonary arterial hypertension in rat model.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance caused by a proliferation of vascular endothelial and smooth muscle cells, resulting in occlusion of the lumen of small pulmonary arteries. Cilostazol, with its antiproliferative effects on vascular endothelial and smooth muscle cells, may ameliorate monocrotaline (MCT)-induced PAH in rats. METHODS AND RESULTS: Male Sprague - Dawley rats (n=10/each group) were randomized to receive MCT (75 mg/kg) only (group 1), MCT plus cilostazol (20 mg .kg(-1 ). day(-1)) (group 2) and saline injection only (group 3). Hemodynamic measurement on day 28 following MCT treatment indicated the development of significant PAH on MCT-treated groups (p<0.0001). Cilostazol was given to group 2 orally on days 28-90. By day 90 following MCT treatment, the right ventricular (RV) systolic blood pressure and RV hypertrophy were significantly higher in group 1 than in groups 2 and 3 (all values of p<0.01). Additionally, connexin43 and endothelial nitric oxide synthase gene expressions of lung and RV, and Bcl-2 protein expression of RV, were significantly lower in group 1 than in groups 2 and 3 (all values of p<0.01). Furthermore, the number of alveolar sac and small arterioles of the lung were significantly lower in group 1 than in groups 2 and 3 (all values of p<0.01). CONCLUSION: Cilostazol therapy effectively attenuates of MCT-induced PAH.

Autologous transplantation of bone marrow-derived endothelial progenitor cells attenuates monocrotaline-induced pulmonary arterial hypertension in rats.

OBJECTIVES: Bone marrow-derived endothelial progenitor cells have been shown to circulate to damaged vascular endothelium and differentiate into mature endothelial cells. This study investigated whether bone marrow-derived endothelial progenitor cell therapy ameliorates monocrotaline (MCT)-induced pulmonary arterial hypertension in a rat model. DESIGN: Male Sprague-Dawley rats were randomized to receive MCT (75 mg/kg) only (group 1), MCT plus autologous bone marrow-derived endothelial progenitor cell (1.2 x 10(6) cells) transplantation (group 2), and saline injection only (group 3). Mononuclear cells were obtained from femoral bone marrow of group 2 rats and isolated by Ficoll gradient centrifugation. The cells were cultured for 21 days in endothelial culture medium. SETTING: An animal research laboratory at Kaohsiung Chang Gung Memorial Hospital. MEASUREMENTS: Hemodynamics, ventricular weight, expressions of connexin43, endothelial nitric oxide synthase messenger RNA gene, Bcl-2, and number of alveolar sacs and small lung arterioles were measured. RESULTS: Hemodynamic measurements on day 28 after MCT treatment revealed the development of significantly increased pulmonary arterial hypertension in MCT-treated groups (p < .0001). The bone marrow-derived endothelial progenitor cells were intravenously transplanted in group 2 on day 28 after MCT-induced pulmonary arterial hypertension. By day 90 after MCT treatment, the right ventricular systolic blood pressure and right ventricular hypertrophy were significantly increased in group 1 compared with groups 2 and 3 (all p values <.01). In addition, connexin43 and endothelial nitric oxide synthase messenger RNA gene expressions of lung and right ventricle and Bcl-2 protein expression of right ventricle were significantly lower in group 1 than in groups 2 and 3 (all p values <.01). Furthermore, the number of alveolar sacs and small lung arterioles were significantly lower in group 1 than in groups 2 and 3 (all p values <.01). CONCLUSIONS: Autologous bone marrow-derived endothelial progenitor cell transplantation effectively ameliorates MCT-induced pulmonary arterial hypertension.

Prognostic value of activated toll-like receptor-4 in monocytes following acute myocardial infarction.

This study tested the hypothesis that activated toll-like receptor-4 (TLR-4) is closely related to combined major adverse clinical outcomes (MACO) [defined as advanced Killip score (> or = 3), overt congestive heart failure (CHF) (New York Heart Association functional class > or = 2) or 30-day death] in patients with ST-segment elevation (ST-se) acute myocardial infarction (AMI) undergoing primary percutaneous coronary intervention (PCI). We conducted a prospective cohort study in 43 consecutive patients with ST-se AMI of onset < 12 hours who were undergoing primary PCI. Blood samples for TLR-4 and serum level of tumor necrosis factor-alpha (TNF-alpha) were collected from 43 patients at 24 hours after AMI and from 20 normal outpatients. The experimental results revealed significantly higher baseline levels of TLR-4, TNF-alpha and white blood cell (WBC) count in the study patients than in normal control subjects (all P < 0.0001). Additionally, baseline levels of TLR-4, TNF-alpha , creatinine, peak level of CK-MB, and multiple vessel disease were significantly higher, whereas left ventricular performance was notably lower in patients (n = 18) with occurrence of MACO than in patients (n = 25) without occurrence of MACO (all P < 0.05). Furthermore, the level of lipopolysaccharide (LPS)-stimulated LTR-4 was significantly increased in MACO patients than in those without MACO (P < 0.0001). Moreover, LPS-stimulated TLR-4 was the most independent predictor of 30-day MACO (P < 0.01). In patients with ST-se AMI, activated TLR-4 is independently predictive of 30-day MACO.

Impact of diabetes on cardiomyocyte apoptosis and connexin43 gap junction integrity: role of pharmacological modulation.

The integrity of myocardial structures plays a crucial role in signal transductions and cardiac function. The aim of this study was to test the hypothesis that diabetes mellitus (DM) exerts adverse effects on the integrity of gap junctions (GJs) and induces cellular apoptosis in rat cardiomyocytes that can be abolished by simvastatin or losartan therapy. An experimental model of DM (induced by streptozocin 60 mg/kg body weight) in adult male rats (n = 24) was utilized to investigate the integrity of GJs containing connexin43 (Cx43) and the incidence of cellular apoptosis in the left ventricular myocardium. These rats were divided into 3 groups; group I (insulin therapy only), group II (insulin plus simvastatin 20 mg/kg/day), and group III (insulin plus losartan 20 mg/kg/day). Diabetic rats and 8 healthy rats (group IV) were sacrificed at 3 weeks following DM induction for immunofluorescence analysis. The experimental results demonstrated that the number of intact Cx43 GJs and the integrated area (mum(2)) constituted by clusters of Cx43 spots were significantly higher in groups II and IV than in group III, and in groups II-IV than in group I (all P values < 0.05). Additionally, the number of apoptotic bodies was remarkably higher in group I than in groups II-IV, and notably higher in groups II-III than in group IV (all P values < 0.05). Simvastatin is more effective than losartan at inhibiting the effects of DM on the integrity of myocardial ultrastructures. Both drugs effectively prevent cellular apoptosis in diabetic rat heart.

Impact of simvastatin and losartan on antiinflammatory effect: in vitro study.

Antiinflammatory properties of losartan are currently unclear. This study tested the hypothesis that losartan itself has an antiinflammatory effect comparable to that of simvastatin. Human umbilical vein endothelial cells (HUVECs) were (1) incubated with culture medium alone, (2) incubated with added C-reactive protein (CRP) (25, 50, 75, and 100 microg/mL) for stimulation, and (3) pretreated with losartan (stepwise increased dose: 100, 300, 500, and 750 micromol/L) and simvastatin (stepwise increased dose: 25, 50, 75, and 100 micromol/L) for 4 hours before adding CRP for stimulation. Surface expression of vascular cell adhesion molecule-1 (VCAM-1) was determined by flow cytometry. Supernatant levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) were measured by ELISA. Experimental results showed that the effect of CRP on VCAM-1 expression and supernatant levels of MCP-1 and IL-6 increases stepwise as CRP concentrations increase from 25 to 50 to 75 to 100 microg/mL (all P < 0.001). The effect of CRP on VCAM-1 expression in HUVECs and supernatant levels of MCP-1 and IL-6 were significantly suppressed by 25 micromol/L simvastatin with stepwise increased suppression as simvastatin dose increased to 50, 75, and 100 micromol/L (all P < 0.0001). However, losartan did not significantly suppress CRP's effect on VCAM-1 expression in HUVECs (P > 0.5). Moreover, losartan did not suppress CRP's effect on MCP-1 and IL-6 secretion unless a high dose (> or =500 micromol/L) of losartan was used. Compared with simvastatin, losartan had less effect on suppression of CRP-mediated inflammation.