Hitoshi Horimoto

Implantation of Mesenchymal Stem Cells Overexpressing Endothelial Nitric Oxide Synthase Improves Right Ventricular Impairments Caused by Pulmonary Hypertension

Background— Pulmonary hypertension (PH) is a life-threatening disease. Bone marrow cell transplantation is reported to reduce the development of PH by increasing vascular beds in pulmonary circulation. However, adenoviral overexpression of endothelial nitric oxide synthase (eNOS) in the lung is also known to reduce PH. Because mesenchymal stem cells (MSCs) are potential cell sources for neovascularization, the implantation of MSCs overexpressing eNOS (MSCs/eNOS) may further improve the surgical results. We evaluated the efficacy of MSCs/eNOS implantation in monocrotaline (MCT)-induced PH rats. Methods and Results— MSCs were isolated from rat bone marrow. PH was induced in rats by subcutaneous injection of MCT. One week after MCT administration, the rats received 3 different treatments: MSCs (MSC group), MSCs/eNOS (MSC/eNOS group), or nontreatment (PH group). As the negative control, rats received saline instead of MCT (control group). Right ventricular (RV) hypertrophy and the elevation of RV systolic pressure (RVSP) were evaluated 3 weeks after MCT administration. Moreover, the effects of MSCs/eNOS on survival were investigated in PH induced by MCT 3 weeks earlier. RVSP in both the MSC and MSC/eNOS groups was significantly lower than the PH group. RVSP in the MSC/eNOS group was significantly lower than the MSC group. The RV weight to body weight ratio was significantly lower in the MSC and MSC/eNOS groups than the PH group. The survival time of rats receiving MSCs/eNOS was significantly longer than the nontreatment rats. Conclusion— Intravenous implantation of MSCs/eNOS may offer ameliorating effects on PH-related RV impairment and survival time.

Preischemic infusion of alpha-human atrial natriuretic peptide elicits myoprotective effects against ischemia reperfusion in isolated rat hearts.

Carperitide, a synthetic alpha-human atrial natriuretic peptide (ANP) is a newly developed drug for the treatment of heart failure. However, effects of carperitide on susceptibility to ischemia reperfusion injury are left to be determined. Isolated rat hearts were subjected to Langendorff perfusion. Six hearts received 0.1 μM of carperitide for 10 min, 6 hearts received 1 mM of a NO synthetase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) for 5 min before the infusion of carperitide, 6 hearts received 0.02 μM of a PKC synthetase inhibitor chelerythrine chloride for 5 min before the infusion of carperitide, 6 hearts received 100 μM of a selective mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-dehydroxydecanoate (5HD) before the infusion of carperitide, 6 hearts received 10 μM of a soluble guanylate cyclase inhibitor methylene blue for 5 min before the infusion of carperitide, and 6 hearts served as a control with no drug infusion. All hearts were then subjected to 20 min of global ischemia followed by 120 min of reperfusion. Left ventricular pressures and coronary flow were measured throughout the experiment and infarct size was detected at the end of experiment. Both plasma and tissue cGMP levels were also determined. The results showed: (1) Carperitide significantly reduced infarct size compared to control (26.1 ± 2.8 vs. 42.7 ± 2.3%, carperitide vs. control, p < 0.05). This effect was reversed by L-NAME, chelerythrine and 5HD, but not methylene blue. (2) Plasma cGMP levels were increased in carperitide-treated group. This effect was reversed by L-NAME (0.16 ± 0.03 vs. 1.04 ± 0.09* vs. 0.28 ± 0.02 nmol/L, control vs. carperitide vs. L-NAME, *p < 0.01 vs. control). We conclude that preischemic infusion of carperitide exerts cardioprotective effects possibly through NO-PKC dependent pathway followed by mitochondrial KATP channel activation.

Mitochondrial ATP-Sensitive Potassium Channel Plays a Dominant Role in Ischemic Preconditioning of Rabbit Heart

Background: The ATP-sensitive potassium (KATP) channel has been shown to be important in the ischemic preconditioning (IPC) response. Recently, the mitochondrial rather than the sarcolemmal KATP channel has been focused on due to its energy-modulating property. Hence, this study was undertaken to elucidate the role of the mitochondrial KATP channel in IPC by modulating the mitochondrial KATP channel in isolated perfused rabbit hearts. Methods: Seven hearts served as a control with no interventions. Seven hearts underwent IPC consisting of two 5-min cycles of global ischemia followed by 5 min of reperfusion. Seven hearts received the selective mitochondrial KATP channel blocker 5-dehydroxydecanoate (5-HD, 100 µM) for 5 min before IPC, and 7 hearts received the selective mitochondrial KATP channel opener diazoxide (50 µM) for 5 min. Then, all hearts were subjected to 1 h of left anterior descending coronary artery ischemia and 1 h of reperfusion. Left ventricular pressures, monophasic action potentials and coronary flow were measured throughout the experiment and infarct size was detected at the end of experiment. Results: (1) The mitochondria-selective KATP channel opener diazoxide reduced infarct size as compared to control (p < 0.05); (2) IPC reduced infarct size and preserved postischemic diastolic function as compared to control (p < 0.05), and (3) the mitochondria-selective KATP channel blocker 5-HD reversed these effects. Conclusion: The mitochondrial ATP-sensitive potassium channel may be a potential site of cardioprotection.

Intra-Arterial Bone Marrow Cell Transplantation Induces Angiogenesis in Rat Hindlimb Ischemia

Background: Bone marrow (BM) cells have been shown to augment local angiogenesis by differentiating vessels themselves and/or secreting paracrinally angiogenic growth factors. Herein, the angiogenic effects of intra-arterial BM mononuclear cell (BM-MNC) transplantation were evaluated in a rat ischemic hindlimb model. Methods: Unilateral hindlimb ischemia was created by excising the femoral artery and its branch in Lewis rats. BM-MNCs were isolated by centrifugation through a Histopaque density gradient. One week after excision of the unilateral femoral artery, BM-MNCs (5 × 106 cells, Group A, n = 6) or PBS (Group B, n = 7) were injected into the ischemic thigh skeletal muscles at the six points with a gauge needle. Another injection of BM-MNCs (3 × 107 cells, Group C, n = 6) or PBS (Group D, n = 7) was administered via the indwelling catheter in the right common iliac artery. Results: Four weeks after the BM-MNC transplantation, angiographic examination revealed the development of collateral vessels in both BM-MNC-transplanted groups. The difference in skin temperature between right and left hindlimbs was significantly reduced in both BM-MNC-transplanted groups (0.93 ± 0.15 vs. 2.84 ± 0.35 vs. 1.20 ± 0.26 vs. 2.61 ± 0.37°C, Group A vs. Group B vs. Group C vs. Group D, p < 0.05). Moreover, immunohistochemical analysis demonstrated that capillary endothelial cells were increased in both BM-MNC-transplanted groups. Conclusion: BM-MNC implantation was able to induce functional neovascularization in rat ischemic hindlimb. The intra-arterial administration offered similar levels of angiogenic activity as intramuscular injection.

Nifedipine enhances the cardioprotective effect of an angiotensin-II receptor blocker in an experimental animal model of heart failure

This study was designed to examine the hypothesis that a calcium channel blocker nifedipine (CCB) could enhance the cardioprotective effect of an angiotensin-II receptor blocker candesartan (ARB) in the treatment for heart failure. Isoproterenol (ISP) was injected into male rats at 300 mg/kg to produce progressive heart failure. Three months later, the rats were divided into 4 groups and treated for 4 weeks with 1) vehicle (n=20), 2) ARB at 0.2 mg/kg/day (n=6), 3) CCB at 10 mg/kg/day (n=6), or 4) both drugs (n=8). Rats injected with saline served as controls (n=13). ISP caused severe myocardial degeneration and decreased the capillary density (Dcap) of the left ventricular (LV) myocardium (mean±SD: 2,197±627 vs. 2,847 ±298 N/mm2 for normal controls), while increasing plasma thiobarbituric acid-reactive substances (TBARS; 3.6±1.1 vs. 1.9±0.5 nmol/ml). Although ARB therapy preserved cardiac morphology, it had little effect on Dcap or oxidative stress. On the other hand, CCB decreased plasma TBARS and 4-hydroxy-2-nonenal protein expression in LV myocardium. Furthermore, the combination of CCB and ARB increased Dcap and preserved the ultrastructure of LV myocardium, so this combination may be a useful option for the treatment of heart failure.

Repetitive preischemic infusion of phosphodiesterase III inhibitor olprinone elicits cardioprotective effects in the failing heart after myocardial infarction

Beta-adrenergic (BA) signaling including cAMP-protein kinase A (PKA) pathway has been implicated in the mechanism of ischemic preconditioning (IPC). However, effect of IPC on the failing heart, in which BA signaling is supposed to be altered, is left to be determined. To assess a role of BA signaling in IPC, levels of beta2-adrenergic receptor (B2AR) mRNA were quantified by real time RT-PCR, and in vivo intracardiac function was evaluated in post-MI heart. The effect of IPC on post-MI heart was then determined with an isolated heart perfusion system. Finally, cardioprotective effect of repetitive preischemic infusion of phosphodiesterase III inhibitor olprinone (30 μM), which is known to increase myocardial cAMP levels, was evaluated with/without PKA inhibitor H-89 (2 μM). B2AR mRNA levels in post-MI heart were significantly reduced compared to non-MI heart. IPC was not effective in post-MI heart. Repetitive preischemic infusion of olprinone increased peak developed pressure (94.6 ± 6.3 vs. 62.8 ± 4.9%, OLP vs. control, p < 0.05) and decreased infect size (15.2 ± 0.4 vs. 33.5 ± 2.5%, OLP vs. control, p < 0.01). These effects were abolished by H-89. These results may indicate that repetitive preischemic infusion of olprinone mimics IPC through cAMP-PKA pathway in post-MI heart, and that BA signaling plays a crucial role in IPC response.

Synthetic vascular prosthesis impregnated with mesenchymal stem cells overexpressing endothelial nitric oxide synthase.

Background— Endothelial dysfunction is known to exaggerate coronary artery disease, sometimes leading to irreversible myocardial damage. In such cases, repetitive coronary revascularization including coronary artery bypass grafting is needed, which may cause a shortage of graft conduits. On the other hand, endothelial nitric oxide synthase (eNOS) is an attractive target of cardiovascular gene therapy. The vascular prostheses, of which the inner surfaces are covered with mesenchymal stem cells (MSCs) overexpressing eNOS, are expected to offer feasible effects of NO and angiogenic effects of MSCs on the native coronary arterial beds, as well as improvement of self-patency. Herein, we attempted to develop small caliber vascular prostheses generating the bioactive proteins. Also, we attempted to transduce eNOS cDNA into MSCs. Methods and Results— The MSCs were isolated from rat bone marrow and transduced with each adenovirus harboring rat eNOS cDNA and &bgr;-galactosidase (&bgr;-gal) (eNOS/MSCs and &bgr;-gal/MSCs). The &bgr;-gal/MSCs were impregnated into vascular prostheses, then the expressions of &bgr;-gal on the inner surfaces of them were evaluated by 5-bromo-4-chloro-3-indolyl &bgr;-d-galactoside staining. The NOS activity of eNOS/MSCs was assayed by monitoring the conversion of 3H-arginine to 3H-citrulline. The inner surfaces of the vascular prostheses were covered with MSCs expressing &bgr;-gal. The amount of the 3H-citrulline increased, and eNOS/MSCs were determined to generate enzymatic activity of eNOS. This activity was completely inhibited by NG-nitro-l-arginine methyl ester. Conclusions— The inner surface of expanded polytetrafluoroethylene vascular prostheses seeded with lacZ gene-transduced MSCs exhibited recombinant proteins. Development of eNOS/MSC-seeded vascular prostheses would promise much longer graft patency and vasculoprotective effects.

HMG-CoA reductase inhibitor cerivastatin prolonged rat cardiac allograft survival by blocking intercellular signals

BACKGROUND The development of atherosclerotic cardiovascular complications caused by hyperlipidemia is a common and serious problem for long-term survivors of organ transplantation. However, adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and lymphocyte function-associated antigen (LFA)-1 are involved in allograft rejection, possibly by providing costimulatory signals. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor cerivastatin has been shown to suppress ICAM-1 expression in acute inflammatory responses. METHODS In this study, we evaluated the immunosuppressive effects of cerivastatin in rat cardiac allografts. The hearts of Fischer rats were transplanted heterotopically into Lewis rats. Cerivastatin (2 mg/kg) was administrated intraperitoneally to recipients for 7 consecutive days from the day before transplantation. RESULTS Graft survival in the cerivastatin-treated group (n = 8) was significantly longer than in controls (n = 10) (24.6 +/- 2.2 days vs 10.2 +/- 1.3 days, p < 0.05). Mixed lymphocyte reaction (MLR) showed that on Day 8 after grafting, the proliferative response of alloreactive T cells against F344 alloantigen in cerivastatin-treated rats was significantly more suppressed than in Lewis rats. The Interleukin-2 concentration of supernatant in MLR cultures in the cerivastatin-treated group was lower than in the control group. Immunohistochemical analysis showed that the percentage of CD4-positive cells to infiltrating mononuclear cells was less prominent in the cerivastatin-treated group (9.8% +/- 2.2%) than in the control group (20.9% +/- 3.2%). CONCLUSIONS The HMG-CoA reductase inhibitor cerivastatin effectively suppressed acute graft rejection, possibly by blocking intercellular signals via ICAM/LFA-1, and cerivastatin may be a candidate for treating patients with hyperlipidemia who undergo organ transplantation.

Activation of β2-adrenergic receptor plays a pivotal role in generating the protective effect of ischemic preconditioning in rat hearts

Background. Ischemic preconditioning (IPC) protects hearts against ischemia by reducing infarct size. However, IPC does not preserve cardiac function, such as left ventricular peak developed pressure (LVPDP). Moreover, IPC fails to protect the post-myocardial infarct (MI) heart. Design. Rat hearts were transfected with β2-adrenergic receptor (B2AR) cDNA by the hemagglutinating virus of Japan–liposome method. After the gene transfer, the hearts were perfused in a Langendorff mode and preconditioned with two cycles of 5 min of ischemia and reperfusion. After 20 min of global ischemia, the hearts were reperfused under aerobic conditions for 90 min. LVPDP was measured as an indicator of the cardiac function. Results. LVPDP of ischemic hearts was well preserved by the combination treatment of IPC and gene transfer of B2AR, but not IPC or gene transfer of B2AR alone. Moreover, the treatment was beneficial to even the post-MI heart. On the contrary, gene transfer of β-adrenergic receptor kinase 1 (BARK1) reduced the protective effect of IPC. We also found that the mRNA ratio of B2AR and BARK1 was well correlated with the preservation of the LVPDP. Conclusions. The combination treatment of IPC and gene transfer of B2AR protects cardiac function against ischemia and it shows the beneficial effect also in post-MI hearts.

Potent adenylate cyclase agonist forskolin restores myoprotective effects of ischemic preconditioning in rat hearts after myocardial infarction.

BACKGROUND The purpose of this study was to determine whether ischemic preconditioning (IPC) provides myoprotective effects in post-myocardial infarction (MI) hearts, and whether beta adrenergic signaling is involved in IPC. METHODS Rats were subjected to either ligation of the left anterior descending coronary artery (LAD) resulting in MI, or a sham operation. Two weeks later, hearts were isolated and perfused. Six groups (n = 7 each) were studied: group 1, control (sham operation); group 2, sham operation + IPC; group 3, post-MI; group 4, post-MI + IPC; group 5, post-MI + forskolin; group 6, post-MI + forskolin + IPC. IPC consisted of two cycles of 5 minutes of global ischemia. The adenylate cyclase agonist forskolin (1.0 x 10(-5) M) was administered in post-MI hearts either alone (group 5) or for 5 minutes before IPC (group 6). All hearts were then subjected to 20 minutes of global ischemia followed by 120 minutes of reperfusion, after which infarct size was measured. Concentrations of endogenous catecholamines and myocardial mRNA expression of beta 2 adrenergic receptor were measured in the post-MI model. RESULTS (1) IPC reduced infarct size in shams, from 34.7 +/- 5.2% in group 1 to 21.4 +/- 3.8% in group 2, but did not affect infarct size in post-MI hearts (group 3 versus group 4). (2) Forskolin combined with IPC reduced infarct size in post-MI hearts to 29.3 +/- 3.4% (group 6), but not in group 5 where the value was 39.3 +/- 4.8%. (3) Beta 2 adrenergic receptor mRNA expression in post-MI hearts was significantly decreased as compared with sham-operated animals. CONCLUSIONS The results indicate that downregulation of beta adrenergic receptors in post-MI hearts may be associated with ineffectiveness of IPC, and that beta adrenergic signaling, especially in relation to adenylate cyclase activation, may be required to generate the IPC response in post-MI hearts.