Sildenafil (Viagra) attenuates ischemic cardiomyopathy and improves left ventricular function in mice

We tested the hypothesis that chronic treatment with sildenafil attenuates myocardial infarction (MI)-induced heart failure. Sildenafil has potent protective effects against necrosis and apoptosis following ischemia-reperfusion in the intact heart and cardiomyocytes. ICR mice underwent MI by left anterior descending coronary artery ligation and were treated with sildenafil (0.71 mg/kg bid) or saline for 4 wk. Infarct size (IS) was measured 24 h postinfarction, and apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Left ventricular end-diastolic diameter (LVEDD) and fractional shortening (FS) were measured by echocardiography. Sildenafil reduced IS (40.0 +/- 4.6%) compared with that in saline (69.6 +/- 4.1%, P < 0.05). NG-nitro-l-arginine methyl ester, a nitric oxide synthase (NOS) inhibitor (15 mg/kg bid), blocked the protective effect of sildenafil (IS, 60.2 +/- 1.6%, P < 0.05 vs. sildenafil). Western blot analysis revealed a significant increase in endothelial NOS/inducible NOS proteins 24 h post-MI after treatment with sildenafil versus saline. Apoptosis decreased from 2.4 +/- 0.3% with saline to 1.2 +/- 0.1% with sildenafil (P < 0.05) on day 7 and from 2.0 +/- 0.2% with saline to 1.2 +/- 0.1% with sildenafil on day 28 (P < 0.05), which was associated with an early increase in the Bcl-2-to-Bax ratio. LVEDD increased from baseline value of 3.6 +/- 0.1 to 5.2 +/- 0.2 and to 5.5 +/- 0.1 mm on days 7 and 28, respectively, with saline (P < 0.05) but was attenuated to 4.4 +/- 0.2 and 4.4 +/- 0.1 mm following sildenafil treatment on days 7 and 28, respectively (P > 0.05 vs. baseline). FS significantly improved post-MI with sildenafil. A marked decline in cardiac hypertrophy was observed with sildenafil, which paralleled a reduction in pulmonary edema. Survival rate was lower with saline (36%) compared with sildenafil (93%, P < 0.05). Sildenafil attenuates ischemic cardiomyopathy in mice by limiting necrosis and apoptosis and by preserving left ventricular function possibly through a nitric oxide-dependent pathway.     

Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction

Matrix metalloproteinase-2 (MMP-2) is prominently overexpressed both after myocardial infarction (MI) and in heart failure. However, its pathophysiological significance in these conditions is still unclear. We thus examined the effects of targeted deletion of MMP-2 on post-MI left ventricular (LV) remodeling and failure. Anterior MI was produced in 10- to 12-wk-old male MMP-2 knockout (KO) and sibling wild-type (WT) mice by ligating the left coronary artery. By day 28, MI resulted in a significant increase in mortality in association with LV cavity dilatation and dysfunction. The MMP-2 KO mice had a significantly better survival rate than WT mice (56% vs. 85%, P < 0.05), despite a comparable infarct size (50 +/- 3% vs. 51 +/- 3%, P = not significant), heart rate, and arterial blood pressure. The KO mice had a significantly lower incidence of LV rupture (10% vs. 39%, P < 0.05), which occurred within 7 days of MI. The KO mice exerted less LV cavity dilatation and improved fractional shortening after MI by echocardiography. The LV zymographic MMP-2 level significantly increased in WT mice after coronary artery ligation; however, this was completely prevented in KO mice. In contrast, the increase in the LV zymographic MMP-9 level after MI was similar between KO and WT mice. MMP-2 activation is therefore considered to contribute to an early cardiac rupture as well as late LV remodeling after MI. The inhibition of MMP-2 activation may therefore be a potentially useful therapeutic strategy to manage post-MI hearts.     

Inhibition of matrix metalloproteinases improves left ventricular function in mice lacking osteopontin after myocardial infarction

Osteopontin (OPN) plays an important role in left ventricular (LV) remodeling after myocardial infarction (MI) by promoting collagen synthesis and accumulation. This study tested the hypothesis that MMP inhibition modulates post-MI LV remodeling in mice lacking OPN. Wild-type (WT) and OPN knockout (KO) mice were treated daily with MMP inhibitor (PD166793, 30 mg/kg/day) starting 3 days post-MI. LV functional and structural remodeling was measured 14 days post-MI. Infarct size was similar in WT and KO groups with or without MMP inhibition. M-mode echocardiography showed greater increase in LV end-diastolic (LVEDD) and end-systolic diameters (LVESD) and decrease in percent fractional shortening (%FS) and ejection fraction in KO-MI versus WT-MI. MMP inhibition decreased LVEDD and LVESD, and increased %FS in both groups. Interestingly, the effect was more pronounced in KO-MI group versus WT-MI (P < 0.01). MMP inhibition significantly decreased post-MI LV dilation in KO-MI group as measured by Langendorff-perfusion analysis. MMP inhibition improved LV developed pressures in both MI groups. However, the improvement was significantly higher in KO-MI group versus WT-MI (P < 0.05). MMP inhibition increased heart weight-to-body weight ratio, myocyte cross-sectional area, fibrosis and septal wall thickness only in KO-MI. Percent apoptotic myocytes in the non-infarct area was not different between the treatment groups. Expression and activity of MMP-2 and MMP-9 in the non-infarct area was higher in KO-MI group 3 days post-MI. MMP inhibition reduced MMP-2 activity in KO-MI with no effect on the expression of TIMP-2 and TIMP-4 14 days post-MI. Thus, activation of MMPs contributes to reduced fibrosis and LV dysfunction in mice lacking OPN.     

Sustained therapeutic hypercapnia attenuates pulmonary arterial Rho-kinase activity and ameliorates chronic hypoxic pulmonary hypertension in juvenile rats

Sustained therapeutic hypercapnia prevents pulmonary hypertension in experimental animals, but its rescue effects on established disease have not been studied. Therapies that inhibit Rho-kinase (ROCK) and/or augment nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling can reverse or prevent progression of chronic pulmonary hypertension. Our objective in the present study was to determine whether sustained rescue treatment with inhaled CO(2) (therapeutic hypercapnia) would improve structural and functional changes of chronic hypoxic pulmonary hypertension. Spontaneously breathing pups were exposed to normoxia (21% O(2)) or hypoxia (13% O(2)) from postnatal days 1-21 with or without 7% CO(2) (Pa(CO(2)) elevated by ～25 mmHg) or 10% CO(2) (Pa(CO(2)) elevated by ～40 mmHg) from days 14 to 21. Compared with hypoxia alone, animals exposed to hypoxia and 10% CO(2) had significantly (P < 0.05) decreased pulmonary vascular resistance, right-ventricular systolic pressure, right-ventricular hypertrophy, and medial wall thickness of pulmonary resistance arteries as well as decreased lung phosphodiesterase (PDE) V, RhoA, and ROCK activity. Rescue treatment with 10% CO(2), or treatment with a ROCK inhibitor (15 mg/kg ip Y-27632 twice daily from days 14 to 21), also increased pulmonary arterial endothelial nitric oxide synthase and lung NO content. In contrast, cGMP content and cGMP-dependent protein kinase (PKG) activity were increased by exposure to 10% CO(2), but not by ROCK inhibition with Y-27632. In vitro exposure of pulmonary artery smooth muscle cells to hypercapnia suppressed serum-induced ROCK activity, which was prevented by inhibition of PKG with Rp-8-Br-PET-cGMPS. We conclude that sustained hypercapnia dose-dependently inhibited ROCK activity, augmented NO-cGMP-PKG signaling, and led to partial improvements in the hemodynamic and structural abnormalities of chronic hypoxic PHT in juvenile rats. Increased PKG content and activity appears to play a major upstream role in CO(2)-induced suppression of ROCK activity in pulmonary arterial smooth muscle.     

PDE5A inhibition attenuates bleomycin-induced pulmonary fibrosis and pulmonary hypertension through inhibition of ROS generation and RhoA/Rho kinase activation

Pulmonary hypertension frequently complicates interstitial lung disease, where it is associated with a high mortality. Patients with this dual diagnosis often fare worse than those with pulmonary arterial hypertension (PAH) alone and respond poorly to standard PAH therapy, often dying of right ventricular (RV) failure. We hypothesize that nitric oxide synthase (NOS) uncoupling is important in the pathogenesis of interstitial lung disease-associated pulmonary hypertension, and this process can be abrogated by phosphodiesterase type 5 (PDE5) inhibition to improve pulmonary vascular remodeling and right ventricular function. Intratracheal bleomycin (4 U/kg) or saline control was administered to C57/BL6 mice after anesthesia. After recovery, animals were fed a diet of sildenafil (100 mg.kg(-1).day(-1)) or vehicle for 2 wk when they underwent hemodynamic measurements, and tissues were harvested. Survival was reduced in animals treated with bleomycin compared with controls and was improved with sildenafil (100.0 vs. 73.7 vs. 84.2%, P < 0.05). RV/LV+S ratio was higher in bleomycin-alone mice with improvement in ratio when sildenafil was administered (33.00 +/- 0.01% vs. 20.98 +/- 0.01% P < 0.05). Histology showed less pulmonary vascular and RV fibrosis in the group cotreated with sildenafil. Bleomycin was associated with a marked increase in superoxide generation by DHE histological staining and luminol activity in both heart and lung. Treatment with sildenafil resulted in a concomitant reduction in superoxide levels in both heart and lung. These data demonstrate that PDE5 inhibition ameliorates RV hypertrophy and pulmonary fibrosis associated with intratracheal bleomycin in a manner that is associated with improved NOS coupling and a reduction in reactive oxygen species signaling.     

Accelerated LV remodeling after myocardial infarction in TIMP-1-deficient mice: effects of exogenous MMP inhibition

Alterations in matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have been implicated in adverse left ventricular (LV) remodeling after myocardial infarction (MI). However, the direct mechanistic role of TIMPs in the post-MI remodeling process has not been completely established. The goal of this project was to define the effects of altering endogenous MMP inhibitory control through combined genetic and pharmacological approaches on post-MI remodeling in mice. This study examined the effects of MMP inhibition (MMPi) with PD-166793 (30 mg.kg(-1).day(-1)) on LV geometry and function (conductance volumetry) after MI in wild-type (WT) mice and mice deficient in the TIMP-1 gene [TIMP-1 knockout (TIMP1-KO)]. At 3 days after MI (coronary ligation), mice were randomized into four groups: WT-MI/MMPi (n = 10), TIMP1-KO-MI/MMPi (n = 10), WT-MI (n = 22), and TIMP1-KO-MI (n = 23). LV end-diastolic volume (EDV) and ejection fraction were determined 14 days after MI. Age-matched WT (n = 20) and TIMP1-KO (n = 28) mice served as reference controls. LVEDV was similar under control conditions in WT and TIMP1-KO mice (36 +/- 2 and 40 +/- 2 microl, respectively) but was greater in TIMP1-KO-MI than in WT-MI mice (48 +/- 2 vs. 61 +/- 5 microl, P < 0.05). LVEDV was reduced from MI-only values in WT-MI/MMPi and TIMP1-KO-MI/MMPi mice (42 +/- 2 and 36 +/- 2 microl, respectively, P < 0.05) but was reduced to the greatest degree in TIMP1-KO mice (P < 0.05). LV ejection fraction was reduced in both groups after MI and increased in TIMP1-KO-MI/MMPi, but not in WT-MI/MMPi, mice. These unique results demonstrated that myocardial TIMP-1 plays a regulatory role in post-MI remodeling and that the accelerated myocardial remodeling induced by TIMP-1 gene deletion can be pharmacologically "rescued" by MMP inhibition. These results define the importance of local endogenous control of MMP activity with respect to regulating LV structure and function after MI.     

Diminished glucagon suppression after β-cell reduction is due to impaired α-cell function rather than an expansion of α-cell mass

Impaired suppression of glucagon levels after oral glucose or meal ingestion is a hallmark of type 2 diabetes. Whether hyperglucagonemia after a β-cell loss results from a functional upregulation of glucagon secretion or an increase in α-cell mass is yet unclear. CD-1 mice were treated with streptozotocin (STZ) or saline. Pancreatic tissue was collected after 14, 21, and 28 days and examined for α- and β-cell mass and turnover. Intraperitoneal (ip) glucose tolerance tests were performed at day 28 as well as after 12 days of subcutaneous insulin treatment, and glucose, insulin, and glucagon levels were determined. STZ treatment led to fasting and post-challenge hyperglycemia (P < 0.001 vs. controls). Insulin levels increased after glucose injection in controls (P < 0.001) but were unchanged in STZ mice (P = 0.36). Intraperitoneal glucose elicited a 63.1 ± 4.1% glucagon suppression in control mice (P < 0.001), whereas the glucagon suppression was absent in STZ mice (P = 0.47). Insulin treatment failed to normalize glucagon levels. There was a significant inverse association between insulin and glucagon levels after ip glucose ingestion (r(2) = 0.99). β-Cell mass was reduced by ～75% in STZ mice compared with controls (P < 0.001), whereas α-cell mass remained unchanged (P > 0.05). α-Cell apoptosis (TUNEL) and replication (Ki67) were rather infrequently noticed, with no significant differences between the groups. These studies underline the importance of endogenous insulin for the glucose-induced suppression of glucagon secretion and suggest that the insufficient decline in glucagon levels after glucose administration in diabetes is primarily due to a functional loss of intraislet inhibition of α-cell function rather than an expansion of α-cell mass.     

Urocortin 1 administration from onset of rapid left ventricular pacing represses progression to overt heart failure

Urocortin 1 (Ucn1) may be involved in the pathophysiology of heart failure (HF), but the impact of Ucn1 administration on progression of the disease is unknown. The aim of this study was to investigate the effects of Ucn1 in sheep from the onset of cardiac overload and during the subsequent development of HF. Eight sheep underwent two 4-day periods of HF induction by rapid left ventricular pacing (225 beats/min) in conjunction with continuous infusions of Ucn1 (0.1 microg.kg(-1).h(-1) iv) and a vehicle control (0.9% saline). Compared with control, Ucn1 attenuated the pacing-induced decline in cardiac output (2.43 +/- 0.46 vs. 3.70 +/- 0.89 l/min on day 4, P < 0.01) and increases in left atrial pressure (24.9 +/- 1.0 vs. 11.9 +/- 1.1 mmHg, P < 0.001) and peripheral resistance (38.7 +/- 9.4 vs. 25.2 +/- 6.1 mmHg.l(-1).min, P < 0.001). Ucn1 wholly prevented increases in plasma renin activity (4.02 +/- 1.17 vs. 0.87 +/- 0.1 nmol.l(-1).h(-1), P < 0.001), aldosterone (1,313 +/- 324 vs. 413 +/- 174 pmol/l, P < 0.001), endothelin-1 (3.8 +/- 0.5 vs. 2.0 +/- 0.1 pmol/l, P < 0.001), and vasopressin (10.8 +/- 4.1 vs. 1.8 +/- 0.2 pmol/l, P < 0.05) during pacing alone and blunted the progressive increases in plasma epinephrine (2,132 +/- 697 vs. 1,250 +/- 264 pmol/l, P < 0.05), norepinephrine (3.61 +/- 0.73 vs. 2.07 +/- 0.52 nmol/l, P < 0.05), and atrial (P < 0.05) and brain (P < 0.01) natriuretic peptide levels. Ucn1 administration also maintained urine sodium excretion (0.75 +/- 0.34 vs. 1.59 +/- 0.50 mmol/h on day 4, P < 0.05) and suppressed pacing-induced declines in creatinine clearance (P < 0.05). These findings indicate that Ucn1 treatment from the onset of cardiac overload has the ability to repress the ensuing hemodynamic and renal deterioration and concomitant adverse neurohumoral activation, thereby delaying the development of overt HF. These data strongly support a use for Ucn1 as a therapeutic option early in the course of the disease.     

Effect of glutamine analogue-acivicin on tumor induced angiogenesis in Ehrlich ascites carcinoma

The inhibition of tumor growth and tumor induced angiogenesis by the glutamine antimetabolite acivicin was evaluated in 6-7 weeks old male Swiss albino mice bearing Ehrlich ascites carcinoma (EAC) transplanted by intraperitoneal (ip) injections of EAC cells. Treatment involving ip injections with two different doses of acivicin (0.05 and 0.41microg/g body weight/day) in saline revealed decrease in tumor volumes and reduced number of blood vessels on peritoneal wall after 10 and 15 days of treatment when compared to control (i.e. injected with saline only). Vascular hyperpermeability was found to be lesser in the treated groups of mice than the control as indicated by the FITC- D and colloidal carbon assay. Serum VEGF level was found to decrease in the drug treated groups both after 10 and 15 days of treatment. The results thus suggest that acivicin may suppress tumoral angiogenesis through regulation of VEGF level.     

Regulation of cGMP-dependent protein kinase expression by Rho and Kruppel-like transcription factor-4

Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury.     

Effects of pre-, peri-, and postmyocardial infarction treatment with losartan in rats: effect of dose on survival, ventricular arrhythmias, function, and remodeling

Angiotensin receptor blockers (ARBs) reduce adverse left ventricular (LV) remodeling and improve LV function and survival when started postmyocardial infarction (MI). ARBs also reduce ventricular arrhythmias during ischemia-reperfusion injury when started pre-MI. No information exists regarding their efficacy and safety when started pre-MI and continued peri- and post-MI. We evaluated whether the ARB losartan improves the outcome when started pre-MI and continued peri- and post-MI. Male Wistar rats (n = 502) were treated for 7 days pre-MI with losartan at a high dose (30 mg.kg(-1).day(-1)), progressively increasing dose (3 mg.kg(-1).day(-1) increased to 10 mg.kg(-1).day(-1) 10 days and 30 mg.kg(-1).day(-1) 20 days post-MI), or no treatment. Ambulatory systolic blood pressure and Holter monitoring were performed for 24 h post-MI. Echocardiography was done 30 days post-MI, and LV remodeling, cardiac hemodynamics, and fetal gene expression were assessed 38 days post-MI. High-dose losartan reduced 24-h post-MI survival compared with the progressive dose and control (21.9% vs. 36.6% and 38.1%, P = 0.033 and P = 0.009, respectively). This was associated with greater hypotension in the high dose and no change in ventricular arrhythmias in all groups. In 24-h post-MI survivors, the progressive dose group had reduced mortality from 24 h to 38 days (8.5% vs. 28.6% for control vs. 38.9% for high dose, P = 0.032 and P = 0.01, respectively). Survivors of both losartan groups demonstrated improved LV remodeling, cardiac hemodynamics, preserved GLUT-4, and reduced cardiac fetal gene expression. Pretreatment with ARBs does not reduce 24-h post-MI ventricular arrhythmias or survival, and high doses increase mortality by causing excessive hypotension. In 24-h post-MI survivors, progressively increasing doses of losartan have multiple beneficial effects, including improved survival.     

SPARC mediates early extracellular matrix remodeling following myocardial infarction

Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that functions in the extracellular processing of newly synthesized collagen. Collagen deposition to form a scar is a key event following a myocardial infarction (MI). Because the roles of SPARC in the early post-MI setting have not been defined, we examined age-matched wild-type (WT; n=22) and SPARC-deficient (null; n=25) mice at day 3 post-MI. Day 0 WT (n=28) and null (n=20) mice served as controls. Infarct size was 52 ± 2% for WT and 47 ± 2% for SPARC null (P=NS), indicating that the MI injury was comparable in the two groups. By echocardiography, WT mice increased end-diastolic volumes from 45 ± 2 to 83 ± 5 μl (P < 0.05). SPARC null mice also increased end-diastolic volumes but to a lesser extent than WT (39 ± 3 to 63 ± 5 μl; P < 0.05 vs. day 0 controls and vs. WT day 3 MI). Ejection fraction fell post-MI in WT mice from 57 ± 2 to 19 ± 1%. The decrease in ejection fraction was attenuated in the absence of SPARC (65 ± 2 to 28 ± 2%). Fibroblasts isolated from SPARC null left ventricle (LV) showed differences in the expression of 22 genes encoding extracellular matrix and adhesion molecule genes, including fibronectin, connective tissue growth factor (CTGF; CCN2), matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-2 (TIMP-2). The change in fibroblast gene expression levels was mirrored in tissue protein extracts for fibronectin, CTGF, and MMP-3 but not TIMP-2. Combined, the results of this study indicate that SPARC deletion preserves LV function at day 3 post-MI but may be detrimental for the long-term response due to impaired fibroblast activation.     

Improved erectile function after Rho-kinase inhibition in a rat castrate model of erectile dysfunction

Androgens are reported to act as strong modulators of erectile function influencing both nitric oxide and vasoconstrictor signaling. Castration results in a depressed erectile response that is associated with a loss of nitric oxide production and increased responsiveness to constrictive agents. The increased vasoconstrictor response may be a result of an active RhoA/Rho-kinase signaling pathway. We report here results of studies designed to test the hypothesis that inhibition of the Rho-kinase pathway restores erectile function in a castrate model by relaxing the smooth muscle. Mean arterial (MAP) and corpus cavernosal (CCP) pressures were monitored during intracavernosal injection of the Rho-kinase inhibitor Y-27632. Castration reduced the maximal erectile response (CCP/MAP) by 33%, and testosterone replacement restored the response (intact, 0.736 +/- 0.040; castrate, 0.492 +/- 0.022; testosterone, 0.681 +/- 0.073). Injection of Y-27632 increased CCP in all experimental groups; it also left shifted the voltage response curve and increased the maximal CCP/MAP response (intact, 0.753 +/- 0.091; castrate, 0.782 +/- 0.081; testosterone treated, 0.894 +/- 0.033). Y-27632 dose dependently relaxed phenylephrine-stimulated cavernosal tissues. Cavernosal tissues showed increased RhoA and Rho-kinase protein levels after castration. Our data support the hypothesis that an active Rho/Rho-kinase pathway contributes to the reduced erectile response after castration due to an upregulation of RhoA/Rho-kinase protein levels and that inhibition of this pathway may serve as an effective treatment for erectile dysfunction.     

Effects of growth hormone (GH) on mRNA levels of uncoupling proteins 1, 2, and 3 in brown and white adipose tissues and skeletal muscle in obese mice.

We have investigated whether GH treatment influences the expression of UCP1, 2 and 3 mRNA in a KK-Ay obese mouse model. KK-Ay mice (n = 10) and C57Bl/6J control mice (n = 10) were injected subcutaneously with human GH (1.0 mg/kg/day and 3.5 mg/kg/day) for 10 days, and compared with mice injected with physical saline. The KK-Ay obese mice weighed significantly less (p < 0.01 : 1.0 mg/kg/day, p < 0.05 : 3.5 mg/kg/day) and had smaller inguinal subcutaneous and perimetric white adipose tissue (WAT) pads (p < 0.05 : 3.5 mg/kg/day), but increased skeletal muscle weight (p < 0.05). The brown adipose tissue (BAT) weight did not change significantly. Not only plasma free fatty acid and glucose levels but also plasma insulin levels decreased. The reduced HOMA-IR (homeostasis model assessment-insulin resistance) values suggested that insulin resistance was improved by GH treatment. UCP1 mRNA levels increased after the 3.5 mg GH treatment by 2.8-fold (p < 0.01 vs. saline controls) and 2.0-fold (p < 0.05 vs. 1 mg GH treatment) in BAT, and by 6.0-fold in subcutaneous WAT (p < 0.05 vs. controls). UCP2 mRNA levels increased 2.2-fold (p < 0.05 vs. control) and 2.1-fold (p < 0.05 vs. 1 mg GH treatment) in BAT, and 2.0-fold (p < 0.05 vs. controls) in skeletal muscle. One mg GH administration also stimulated UCP1 mRNA expression by 2.5-fold (p < 0.05 vs. controls) and UCP3 mRNA expression by 2.8-fold (p < 0.05 vs. controls) in the muscle. On the other hand, lean mice showed no significant difference in body composition or plasma parameters. UCP1, 2 and 3 mRNA expression in lean mice did not show any significant change after treatment with GH. We conclude that GH treatment increased mRNA levels for not only UCP1, but also UCP 2 and 3 in BAT, WAT and muscle in a KK-Ay obese mouse model. These findings suggest that GH-induced thermogenesis may contribute to the reduction in WAT and energy expenditure.     

The Time Window for Therapy with Peptide Nanofibers Combined with Autologous Bone Marrow Cells in Pigs after Acute Myocardial Infarction

Background

We previously showed that injection of peptide nanofibers (NF) combined with autologous bone marrow mononuclear cells (MNC) immediately after coronary artery ligation improves cardiac performance in pigs. To evaluate the clinical feasibility, this study was performed to determine the therapeutic time window for NF/MNC therapy in acute myocardial infarction (MI).

Methods and Results

A total of 45 adult minipigs were randomly grouped into 7 groups: sham or MI plus treatment with NS (normal saline), or NF or MNC alone at 1 day (1D) post-MI, or NF/MNC at 1, 4, or 7 days post-MI (N≥6). Cardiac function was assessed by echocardiography and ventricular catheterization. Compared with the NS control, pigs treated with NF/MNC at 1 day post-MI (NF/MC-1D) had the greatest improvement in left ventricle ejection fraction (LVEF; 55.1±1.6%; P<0.01 vs. NS) 2 months after MI. In contrast, pigs treated with either NF/MNC-4D or NF/MNC-7D showed 48.9±0.8% (P<0.05 vs. NS) and 43.5±2.3% (n.s. vs. NS) improvements, respectively. The +dP/dt and -dP/dt, infarct size and interstitial collagen content were also improved in the NF/MNC-1D and -4D groups but not in the -7D group. Mechanistically, MNC quality and the states of systemic inflammation and damaged heart tissue influence the therapeutic efficiency of NF/MNC therapy, as revealed by another independent study using 16 pigs.

Conclusions

Injection of NF/MNC at 1 or 4 days, but not at 7 days post-MI, improves cardiac performance and prevents ventricular remodeling, confirming the importance of early intervention when using this therapy for acute MI.     

Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.     

Combination Therapy with Atorvastatin and Amlodipine Suppresses Angiotensin II-Induced Aortic Aneurysm Formation

Background

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease. It is controversial whether statin and calcium channel blockers (CCBs) has an inhibitory effect on the expansion of AAA. Some studies reported that CCBs have an inhibitory effect on Rho-kinase activity. Rho-kinase plays an important role in the pathogenesis of various cardiovascular diseases. However, there is no study reporting of the association between Rho-kinase and human AAAs.

Methods and Results

Experimental AAA was induced in Apolipoprotein E-deficient (ApoE^-/-) mice infused with angiotensin II (AngII) for 28 days. They were randomly divided into the following 5 groups; saline infusion alone (sham), AngII infusion alone, AngII infusion plus atorvastatin (10 mg/kg/day), AngII infusion plus amlodipine (1 mg/kg/day), and AngII infusion plus combination therapy with atorvastatin (10 mg/kg/day) and amlodipine (1 mg/kg/day). The combination therapy significantly suppressed AngII-induced increase in maximal aortic diameter as compared with sham, whereas each monotherapy had no inhibitory effects. The combination therapy significantly reduced AngII-induced apoptosis and elastin degradation at the AAA lesion, whereas each monotherapy did not. Moreover, Rho-kinase activity, as evaluated by the extent of phosphorylation of myosin-binding subunit (a substrate of Rho-kinase) and matrix metalloproteinase activity were significantly increased in the AngII-induced AAA lesion as compared with sham, both of which were again significantly suppressed by the combination therapy. In human aortic samples, immunohistochemistory revealed that the activity and expression of Rho-kinase was up-regulated in AAA lesion as compared with abdominal aorta from control subjects.

Conclusions

Rho-kinase is up-regulated in the aortic wall of human AAA. The combination therapy with amlodipine and Atorvastatin, but not each monotherapy, suppresses AngII-induced AAA formation in mice in vivo, for which Rho-kinase inhibition may be involved.     

Rho-Kinase Inhibition Ameliorates Metabolic Disorders through Activation of AMPK Pathway in Mice

Background

Metabolic disorders, caused by excessive calorie intake and low physical activity, are important cardiovascular risk factors. Rho-kinase, an effector protein of the small GTP-binding protein RhoA, is an important cardiovascular therapeutic target and its activity is increased in patients with metabolic syndrome. We aimed to examine whether Rho-kinase inhibition improves high-fat diet (HFD)-induced metabolic disorders, and if so, to elucidate the involvement of AMP-activated kinase (AMPK), a key molecule of metabolic conditions.

Methods and Results

Mice were fed a high-fat diet, which induced metabolic phenotypes, such as obesity, hypercholesterolemia and glucose intolerance. These phenotypes are suppressed by treatment with selective Rho-kinase inhibitor, associated with increased whole body O₂ consumption and AMPK activation in the skeletal muscle and liver. Moreover, Rho-kinase inhibition increased mRNA expression of the molecules linked to fatty acid oxidation, mitochondrial energy production and glucose metabolism, all of which are known as targets of AMPK in those tissues. In systemic overexpression of dominant-negative Rho-kinase mice, body weight, serum lipid levels and glucose metabolism were improved compared with littermate control mice. Furthermore, in AMPKα2-deficient mice, the beneficial effects of fasudil, a Rho-kinase inhibitor, on body weight, hypercholesterolemia, mRNA expression of the AMPK targets and increase of whole body O₂ consumption were absent, whereas glucose metabolism was restored by fasudil to the level in wild-type mice. In cultured mouse myocytes, pharmacological and genetic inhibition of Rho-kinase increased AMPK activity through liver kinase b1 (LKB1), with up-regulation of its targets, which effects were abolished by an AMPK inhibitor, compound C.

Conclusions

These results indicate that Rho-kinase inhibition ameliorates metabolic disorders through activation of the LKB1/AMPK pathway, suggesting that Rho-kinase is also a novel therapeutic target of metabolic disorders.     

Electrotonic remodeling following myocardial infarction in dogs susceptible and resistant to sudden cardiac death.

Passive electrical remodeling following myocardial infarction (MI) is well established. These changes can alter electrotonic loading and trigger the remodeling of repolarization currents, a potential mechanism for ventricular fibrillation (VF). However, little is known about the role of passive electrical markers as tools to identify VF susceptibility post-MI. This study investigated electrotonic remodeling in the post-MI ventricle, as measured by myocardial electrical impedance (MEI), in animals prone to and resistant to VF. MI was induced in dogs by a two-stage left anterior descending (LAD) coronary artery ligation. Before infarction, MEI electrodes were placed in remote (left circumflex, LCX) and infarcted (LAD) myocardium. MEI was measured in awake animals 1, 2, 7, and 21 days post-MI. Subsequently, VF susceptibility was tested by a 2-min LCX occlusion during exercise; 12 animals developed VF (susceptible, S) and 12 did not (resistant, R). The healing infarct had lower MEI than the normal myocardium. This difference was stable by day 2 post-MI (287 +/- 32 Omega vs. 425 +/- 62 Omega, P < 0.05). Significant differences were observed between resistant and susceptible animals 7 days post-MI; susceptible dogs had a wider electrotonic gradient between remote and infarcted myocardium (R: 89 +/- 60 Omega vs. S: 180 +/- 37 Omega). This difference increased over time in susceptible animals (252 +/- 53 Omega at 21 days) due to post-MI impedance changes on the remote myocardium. These data suggest that early electrotonic changes post-MI could be used to assess later arrhythmia susceptibility. In addition, passive-electrical changes could be a mechanism driving active-electrical remodeling post-MI, thereby facilitating the induction of arrhythmias.