Aliskiren enhances protective effects of valsartan against type 2 diabetic nephropathy in mice.

OBJECTIVES: Addition of aliskiren, a direct renin inhibitor, to losartan provides additive reduction of urinary albumin excretion in type 2 diabetic patients. However, the detailed effect of aliskiren on type 2 diabetic nephropathy is still unknown. This study was undertaken to examine the efficacy of aliskiren and the combination of aliskiren with valsartan on type 2 diabetic nephropathy. METHODS: db/db mice were treated with aliskiren (3 mg/kg per day), valsartan (5 or 10 mg/kg per day), combined aliskiren (3 mg/kg per day) and valsartan (5 mg/kg per day), and hydralazine (80 mg/kg per day), for 6 weeks, and the protective effects against diabetic nephropathy were compared among each group. RESULTS: Aliskiren significantly attenuated albuminuria and glomerular mesangial matrix expansion in db/db mice, which was associated with the improvement of the increased glomerular transforming growth factor-beta and type IV collagen expressions, the increased macrophage infiltration, and the decreased glomerular nephrin expression of db/db mice. These protective effects of aliskiren in db/db mice were attributed to the attenuation of p22(phox)-related nicotinamide adenine dinucleotide phosphate oxidase-induced superoxide. Addition of aliskiren to valsartan treatment provided more beneficial effects on all the above-mentioned parameters than valsartan monotherapy. CONCLUSION: Aliskiren protected against type 2 diabetic nephropathy, through pleiotropic effects, and significantly enhanced the protective effects of valsartan against diabetic nephropathy in db/db mice.

TNNI3K could be a novel molecular target for the treatment of cardiac diseases.

Recently, regenerative medicine using the transplantation of embryonic stem cells and bone marrow stem cells has been a great success but still has many unconfirmed problems including its clinical evaluation. The aim of this article is to review current literature and some patents regarding molecular therapeutic agents including using MAP kinase TNNI3K for the treatment and diagnosis of acute myocardial ischemia or infarction. TNNI3K is a novel cardiac troponin I-interacting kinase gene and its overexpression may promote cardiac myogenesis, improve cardiac performance, and attenuate ischemia-induced ventricular remodeling. The modulation of embryonal stem cells with high TNNI3K activity using a TNNI3K active peptide could be a useful therapeutic approach for ischemic cardiac diseases. For overexpressing TNNI3K or enhancing TNNI3K activity in cardiac precursor cells, the engraftment of bone marrow cells or embryonic stem cells can effectively promote cardiac myogenesis, beating frequency, and contractile functions, and decrease "silent" (no contraction) cardiac cells after cell transplantion, indicating that the overexpression of TNNI3K can increase the success rate of transplanting embryonic stem cells or bone marrow cells into ischemic hearts for the treatment of ischemic cardiac diseases. Although previous investigations showing that TNNI3K may be involved in the development of cardiac hypertrophy, it is still unclear whether TNNI3K has a role in cardiac hypertrophy or what mechanism is involved in the effects of TNNI3K. To confirm this, further investigations need to be undertaken.

Overexpression of TNNI3K, a cardiac-specific MAP kinase, promotes P19CL6-derived cardiac myogenesis and prevents myocardial infarction-induced injury.

TNNI3K is a new cardiac-specific MAP kinase whose gene is localized to 1p31.1 and that belongs to a tyrosine kinase-like branch in the kinase tree of the human genome. In the present study we investigated the role of TNNI3K in the cardiac myogenesis process and in the repair of ischemic injury. Pluripotent P19CL6 cells with or without transfection by pcDNA6-TNNI3K plasmid were used to induce differentiation into beating cardiomyocytes. TNNI3K promoted the differentiation process, judging from the increasing beating mass and increased number of alpha-actinin-positive cells. TNNI3K improved cardiac function by enhancing beating frequency and increasing the contractile force and epinephrine response of spontaneous action potentials without an increase of the single-cell size. TNNI3K suppressed phosphorylation of cardiac troponin I, annexin-V(+) cells, Bax protein, and p38/JNK-mediated apoptosis. Intramyocardial administration of TNNI3K-overexpressing P19CL6 cells in mice with myocardial infarction improved cardiac performance and attenuated ventricular remodeling compared with injection of wild-type P19CL6 cells. In conclusion, our study clearly indicates that TNNI3K promotes cardiomyogenesis, enhances cardiac performance, and protects the myocardium from ischemic injury by suppressing p38/JNK-mediated apoptosis. Therefore, modulation of TNNI3K activity would be a useful therapeutic approach for ischemic cardiac disease.

Pravastatin enhances beneficial effects of olmesartan on vascular injury of salt-sensitive hypertensive rats, via pleiotropic effects.

OBJECTIVE: This work was undertaken to investigate comparative effect of AT1 receptor blocker (ARB), 3-hydroxy-3-methylglutaryl (HMG) coenzymeA (CoA) reductase inhibitor (statin), and their combination on vascular injury of salt-sensitive hypertension. METHODS AND RESULTS: Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats) were treated with (1) vehicle, (2) hydralazine (5 mg/kg/d), (3) olmesartan (0.5 mg/kg/d), (4) pravastatin (100 mg/kg/d), and (5) combined olmesartan and pravastatin for 4 weeks. Olmesartan or pravastatin significantly and comparably improved vascular endothelium-dependent relaxation to acetylcholine, coronary arterial remodeling, and eNOS activity of DS rats. Olmesartan prevented vascular eNOS dimer disruption or the downregulation of dihydrofolate reductase (DHFR) more than pravastatin, whereas Akt phosphorylation was enhanced by pravastatin but not olmesartan, indicating differential pleiotropic effects between olmesartan and pravastatin. Add-on pravastatin significantly enhanced the improvement of vascular endothelial dysfunction and remodeling by olmesartan in DS rats. Moreover, pravastatin enhanced the increase in eNOS activity by olmesartan, being associated with additive effects of pravastatin on phosphorylation of Akt and eNOS. CONCLUSIONS: Olmesartan and pravastatin exerted beneficial vascular effects in salt-sensitive hypertension, via differential pleiotropic effects. Pravastatin enhanced vascular protective effects of olmesartan. Thus, the combination of ARB with statin may be the potential therapeutic strategy for vascular diseases of salt-sensitive hypertension.

Enhancement of cardiac oxidative stress by tachycardia and its critical role in cardiac hypertrophy and fibrosis.

OBJECTIVE: To examine the mechanism and significance of tachycardia-induced cardiac damage, using azelnidipine, a relatively new dihydropyridine calcium channel blocker which does not increase heart rate. METHODS: Comparing azelnidipine and amlodipine, we examined the cardiac effects and the direct effects on a sinus node/atrial preparation in stroke-prone spontaneously hypertensive rats (spSHRs). By pacing the right atrium, we examined the effect of tachycardia per se on cardiac oxidative stress. Using apocynin, a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, we investigated the role of oxidative stress in cardiac remodelling. RESULTS: Azelnidipine suppressed cardiac hypertrophy, fibrosis, NADPH oxidase and superoxide in spSHRs more potently than amlodipine, and was associated with lower heart rates than amlodipine. Azelnidipine caused a greater reduction than amlodipine in the beat rate of the sinus node/atrial preparation of spSHRs. A 10 or 20% increase in heart rate, independent of blood pressure or sympathetic nerve activity, significantly enhanced cardiac NADPH oxidase activity, superoxide and activated mitogen-activated protein kinases. Reduction of cardiac oxidative stress by apocynin led to the suppression of cardiac hypertrophy, inflammation and fibrosis in spSHRs, beyond its hypotensive effect. CONCLUSIONS: Our work provided evidence that the increase in heart rate per se, independent of sympathetic nerve activity, enhances cardiac oxidative stress and activates mitogen-activated protein kinases, which seem to be responsible for cardiac remodelling. Azelnidipine, without causing an increase in heart rate, has the potential to be useful for the treatment of cardiac remodelling.

Effects of intramyocardial administration of slow-release basic fibroblast growth factor on angiogenesis and ventricular remodeling in a rat infarct model.

BACKGROUND: Basic fibroblast growth factor (bFGF) stimulates neoangiogenesis. Incorporation into biodegradable gelatin hydrogels provides the sustained release of bFGF. The effects of intramyocardial injections of slow-release bFGF on neoangiogenesis in a rat model of infarction were investigated. METHODS AND RESULTS: Myocardial infarction was induced in rats using coronary artery ligation. A total of 124 rats received an intramyocardial injection of 20 microg of bFGF, the same amount of bFGF incorporated into gelatin hydrogel (bFGF + gel), gelatin hydrogel (gel) or saline. Ventricular function was evaluated by echocardiography 2 or 4 weeks later. Morphometric and histological analyses were used to evaluate infarct size, vascular density and myocardial apoptosis. Capillary density in the infarct border zone was higher in the bFGF and bFGF + gel groups than in the saline and gel groups at 4 weeks (p<0.001). Arteriolar density was higher in the bFGF + gel group than in the other 3 groups (p<0.05). The bFGF and bFGF + gel groups contained fewer apoptotic cardiomyocytes in the border zone than the saline and gel groups (p<0.01). The bFGF+gel group had thicker (p<0.05) and less expanded infarcts (p<0.01) compared with the saline group at 4 weeks. CONCLUSIONS: Incorporation of bFGF in gelatin hydrogels enhanced the effects of bFGF on arteriogenesis, ventricular remodeling and cardiac function.

Urokinase-immobilization suppresses inflammatory responses to polyurethane tubes implanted in rabbit muscles.

Urokinase and plasmin appear to have antiinflammatory activity in some injury models, and urokinase immobilization has been clinically used to prevent thrombus formation in various implants, including intravenous indwelling catheters and subcutaneous drainage tubes. In the present study, polyurethane tubes were embedded in rabbit muscle for 3 months and the effect of urokinase immobilization on inflammatory responses to the implanted tubes was studied at 1 week, 1 month, and 3 months. Mononuclear leukocyte accumulation occurred around implanted polyurethane tubes and peaked after 1 month, but was reduced significantly by urokinase immobilization. The treatment also lessened as well as delayed eosinophil accumulation, but did not affect fibrosis caused by implanted tubes. These results indicate suppressive effects of urokinase immobilization on polyurethane-elicited inflammatory responses and suggest that an approach to develop persistently active urokinase immobilization is rational for successful long-term device implantation.

Coculture of Th cells with interleukin (IL)-7 in the absence of antigenic stimuli induced T-cell anergy reversed by IL-15.

Interleukin-7 (IL-7) is an important survival factor for T cells. We report here for the first time that it has another important role, facilitating T-cell clonal unresponsiveness, or anergy. The anergy was induced by a 20-day coculture of activated-human CD4(+) T-cell clones with IL-7 and irradiated peripheral blood mononuclear cells without antigenic stimuli. T-cell survival, but not T-cell anergy induction, was dependent on direct cell contacts between T cells and irradiated peripheral blood mononuclear cells. The anergic T cells exhibited no or very low expression of IL-7 receptor alpha chain (IL-7Ralpha), IL-2 receptor alpha chain (IL-2Ralpha), and common gamma chain (gammac), and did not express cytotoxic T-lymphocyte-associated protein 4, but expressed IL-15Ralpha. Coculture for 3 to 9 days of anergic T cells with a T-cell-activating cytokine IL-15, but not IL-2, restored the responsiveness of IL-7-induced anergic T cells together with reexpressions of IL-7Ralpha, IL-2Ralpha, and gammac. The anergy induction by IL-7 and restoration of responsiveness by IL-15 suggest novel mechanisms for regulation of helper T-cell responses, induction of peripheral tolerance, and breakdown of T-cell self-tolerance.

Effects of sasanquasaponin on ischemia and reperfusion injury in mouse hearts.

We investigated effects of sasanquasaponin (SQS), a traditional Chinese herb's effective component, on ischemia and reperfusion injury in mouse hearts and the possible role of intracellular Cl- homeostasis on SQS's protective effects during ischemia and reperfusion. An in vivo experimental ischemia model was made in mice (weight 27-45 g) using ligation of left anterior descending coronary artery, and in vitro models were made in perfused hearts by stopping flow or in isolated ventricular myocytes by hypoxia. The in vivo results showed that SQS inhibited cardiac arrhythmias during ischemia and reperfusion. Incidence of arrhythmias during ischemia and reperfusion, including ventricular premature beats and ventricular fibrillation, was significantly decreased in the SQS-pretreated group (P<0.05). Results in perfused hearts showed that SQS suppressed the arrhythmias, prevented against ischemia-induced decrease in contract force and promoted the force recovery from reperfusion. Furthermore, intracellular Cl- concentrations ([Cl-]i) were measured using a MQAE fluorescence method in isolated ventricular myocytes in vitro. SQS slightly decreased [Cl-]i in non-hypoxic myocytes and delayed the hypoxia/reoxygenation-induced increase in [Cl-]i during ischemia and reperfusion (P<0.05). Our results showed that SQS protected against ischemia/reperfusion-induced cardiac injury in mouse hearts and that modulation of intracellular Cl- homeostasis by SQS would play a role in its anti-arrhythmia effects during ischemia and reperfusion.

Modulation of intracellular Cl- homeostasis by lectin-stimulation in Jurkat T lymphocytes.

We investigated changes in intracellular Cl(-) concentration ([Cl(-)](i)) during lectin-induced activation and proliferation in human Jurkat T lymphocytes. [Cl(-)](i) was measured using Cl(-) fluorescence dye (N-(6-methoyquinolyl) acetoxy-acetyl-ester, MQAE) methods. Lectins, phytohemagglutinin and concanavalin A, dose-dependently increased [Cl(-)](i) and triggered intracellular Cl(-) oscillation in human Jurkat T lymphocytes. However, some mitochondria metabolism inhibitors, such as m-chlorocarbonylcyanide phenylhydrazone (CCP) and 2,4-dinitrophenol, increased [Cl(-)](i) without triggering any Cl(-) oscillation. Furthermore, both lectins and metabolism inhibitors-induced elevation in [Cl(-)](i) were blocked by removal of extracellular Cl(-) from perfusion solution or by application of anthracene-9-carboxylate, a blocker of Cl(-) channels. Since an extracellular Cl(-)-free condition and application of 9-AC also inhibited PHA-induced proliferation, we suggested that elevation of [Cl(-)](i) via activation of Cl(-) channels and increase in incidence of Cl(-) oscillation would play an important role in modulation of Jurkat T cell activation and proliferation.

[The relationship between intracellular chloride concentration and ischemia reperfusion-induced arrhythmias in myocardial cells].

We investigated the effects of simulated ischemia on intracellular Cl- concentration ([Cl-]i) in guinea pig ventricular myocardial cells and possible role of the [Cl-]i on the ischemia/reperfusion-induced arrhythmias in perfused rat hearts. Our results provided direct evidence that the [Cl-]i in ventricular muscle was increased under ischemic conditions, which suggested that activation of the Cl-(-)HCO3- exchanger by ischemia would partially contribute to the elevation of [Cl-]i. Application of stilbene derivatives or lowering Cl- concentration in perfusion solution delayed the onset of ischemia-induced deterioration in action potentials, pHi, [Cl-]i, and suppressed the incidence of ischemia/reperfusion-induced arrhythmias. The conclusion was made to emphasize the important role of intracellular Cl- homeostasis in cardiac physiology and pathogenesis of myocardial ischemia/reperfusion injury.