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Biomedical subjects

Naoki Makino

Publications and source records attributed to Naoki Makino.

At least 19 recordsLinked to original sources
PubMed2006

Transient receptor potential (TRP) protein 7 acts as a G protein-activated Ca2+ channel mediating angiotensin II-induced myocardial apoptosis.

Transient receptor potential (TRP) proteins have been identified as cation channels that are activated by agonist-receptor coupling and mediate various cellular functions. TRPC7, a homologue of TRP channels, has been shown to act as a Ca2+ channel activated by G protein-coupled stimulation and to be abundantly expressed in the heart with an as-yet-unknown function. We studied the role of TRPC7 in G protein-activated signaling in HEK293 cells and cultured cardiomyocytes in vitro transfected with FLAG-tagged TRPC7 cDNA and in Dahl salt-sensitive rats with heart failure in vivo. TRPC7-transfected HEK293 cells showed an augmentation of carbachol-induced intracellular Ca2+ transient, which was attenuated under a Ca2+-free condition or in the presence of SK&F96365 (a Ca2+-permeable channel blocker). Upon stimulation with angiotensin II (Ang II), cultured neonatal rat cardiomyocytes transfected with TRPC7 exhibited a significant increase in apoptosis detected by TUNEL staining, accompanied with a decrease in the expression of atrial natriuretic factor and destruction of actin fibers, as compared with non-transfected cardiomyocytes. Ang II-induced apoptosis was inhibited by CV-11974 (Candesartan; Ang II type 1 [AT1] receptor blocker), SK&F96365, and FK506 (calcineurin inhibitor). In Dahl salt-sensitive rats, apoptosis and TRPC7 expression were increased in the failing myocardium, and a long-term treatment with temocapril, an angiotensin-converting enzyme inhibitor, suppressed both. Our findings suggest that TRPC7 could act as a Ca2+ channel activated by AT1 receptors, leading to myocardial apoptosis possibly via a calcineurin-dependent pathway. TRPC7 might be a key initiator linking AT1-activation to myocardial apoptosis, and thereby contributing to the process of heart failure.

Angiotensin II↗
PubMed2006

SiRNA targeting SHP-1 accelerates angiogenesis in a rat model of hindlimb ischemia.

Vascular endothelial growth factor (VEGF) receptor-2 (KDR/flk-1) has a tyrosine kinase domain, and once activated, induces the autophosphorylation of the tyrosine residues, which is essential for angiogenesis. SHP-1, a cytoplasmic protein tyrosine phosphatase, plays a negative regulatory role in signal transduction pathways by dephosphorylation of the receptors to which it binds. Thus, therapeutic angiogenesis designed to inhibit expression of SHP-1 would be beneficial in hindlimb ischemia. In in vitro, the inhibition of SHP-1 by SHP-1 siRNA impaired the ability of TNF to block the tyrosine phosphorylation of KDR/flk-1 induced by VEGF and showed an increase in endothelial cell growth. In in vivo, SHP-1 mRNA, SHP-1 protein levels and VEGF were increased in a rat model of hindlimb ischemia. Upon injection to the ischemic adductor muscle, vector-based siRNA reduced SHP-1, increased phosphorylation of KDR/flk-1, and markedly increased capillary density. Our data demonstrated in vivo the potential use of siRNA targeting SHP-1 as therapy for peripheral ischemic diseases.

Animals↗
PubMed2006

Peroxisome proliferator-activated receptor-gamma ligands attenuate brain natriuretic peptide production and affect remodeling in cardiac fibroblasts in reoxygenation after hypoxia.

Cardiac fibroblasts (CFs) participate in cardiac remodeling after hypoxic cardiac damage, and remodeling is thought to be mediated by CF synthesis of brain natriuretic peptide (BNP). It is unknown whether the peroxisome proliferator-activated receptors (PPARs), which mediate cellular signaling for growth and migration, affect BNP synthesis and whether PPARs participate in regulation of extracellular matrix protein (ECM) expression for remodeling. We examined the production of BNP in cultured neonatal ventricular CFs and its signaling system on collagen synthesis and on activation of matrix metalloproteinases (MMPs) in reoxygenation after hypoxia. BNP mRNA was detected in CFs, and a specific BNP protein, BNP1-32, was secreted into the media. Abundance of collagen I and III was increased in the media at reoxygenation. mRNA and protein levels for MMP-2 and the tissue inhibitor of metalloproteinase (TIMP)-1 were enhanced in CFs at reoxygenation. These observations also were noted in CFs after incubation with angiotensin II (10 microM) for 24 h. Pretreatment with pioglitaozone (0.1-10 microM) attenuated BNP mRNA and protein abundance of collagen III, MMP-2, and TIMP-1 in CFs at reoxygenation. The secreted BNP was also decreased by pioglitaozone in the media. Furthermore, PPAR activators inhibited reoxygenation-induced activation of nuclear factor (NF)-kappaB. These results demonstrate that PPAR activators inhibit BNP synthesis in CFs and imply that PPAR activators may regulate ECM remodeling partially through the NF-kappaB-mediated pathway.

Angiotensin II↗
PubMed2005

Increased productivity of tumor necrosis factor-alpha in helper T cells in patients with systolic heart failure.

BACKGROUND AND AIMS: Whereas increased circulating proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), play an important role in heart failure, where and how TNF-alpha production is upregulated remains largely unknown. We studied the productivity of TNF-alpha in peripheral lymphocytes and underlying mechanisms in patients with heart failure. METHODS: Symptomatic NYHA II-IV patients with chronic heart failure with systolic dysfunction (n=39, aged 74+/-11, ejection fraction [EF]<==50%) were compared with asymptomatic NYHA I patients (n=18, aged 72+/-10, EF>50%) and normal subjects (n=15, aged 67+/-11). Lymphocyte subsets (CD3, CD4, and CD8) and intracellular production of TNF-alpha in peripheral leukocytes were quantified by immunofluorescent flow cytometry, and relationships between these parameters and circulating proinflammatory cytokines were analyzed. RESULTS: Subpopulation of TNF-alpha-producing CD4 was larger in NYHA II-IV patients (23.7% [18.0-28.6]) than in normal subjects (17.1% [6.5-19.5], p<0.05) and was correlated with plasma TNF-alpha levels (r=0.26, p<0.05), EF (r=-0.26, p<0.05), CD4/CD8 ratios (r=0.42, p<0.001), and subpopulation of TNF-alpha-producing monocytes (r=0.47, p<0.0001). Plasma levels of soluble CD14 and interleukin-12 (IL-12) were significantly higher in NYHA II-IV patients than in normal subjects (1971 ng/mL [1740-2375] vs. 1607 ng/mL [1530-1930], p<0.01; and 121 pg/mL [62-230] vs. 62 pg/mL [54-99], p<0.05, respectively), and plasma IL-12 levels were correlated with plasma TNF-alpha levels (r=0.41, p<0.001). CONCLUSIONS: Increased productivity of TNF-alpha in helper T cells, associated with their dominance over cytotoxic T cells, may partially contribute to an increase in circulating TNF-alpha levels in heart failure.

Aged↗
PubMed2005

RNA interference targeting SHP-1 attenuates myocardial infarction in rats.

The Src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) plays a key role in apoptosis and decreases phosphorylation of Akt. Apoptosis of cardiomyocytes is thought to contribute to the increased area of acute myocardial infarction (AMI), and Akt activation exerts a powerful cardioprotective effect after ischemia. Thus, a therapeutic strategy designed to inhibit expression of SHP-1 would be beneficial in AMI. Here we report that siRNA targeting SHP-1 reduced infarct size in a rat model of AMI. Upon injection into the ischemic left ventricular wall, the vector-based siRNA significantly suppressed the increase in the SHP-1 mRNA and the SHP-1 protein levels. The siRNA vector also significantly reduced the SHP-1 that bound to Fas-R. The SHP-1 siRNA vector increased phospho-Akt and reduced DNA fragmentation and caspase activity compared with the scramble siRNA vector. Finally, the area of myocardial infarction was significantly smaller with the SHP-1 siRNA vector than with the scramble siRNA vector at 2 days after LCA ligation. In conclusion, SHP-1 in the heart increased from the early stage of AMI, and this increase was thought to contribute to the increased area of myocardial infarction. Suppression of SHP-1 with the SHP-1 siRNA vector markedly reduced the infarct size in AMI.

Animals↗
PubMed2004

Successful treatment of pure red cell aplasia with cyclosporin A and erythropoietin after thymectomy in a 88-year old woman.

An 88-year old Japanese female with pure red cell aplasia was treated safely and effectively by a combination of thymectomy, cyclosporin A, and erythropoietin. The thymoma was histologically classified as lymphocytic type or cortical type, which are uncommon in cases of a thymoma accompanied by pure red cell aplasia. Immunohistochemical analysis of the thymoma and bone marrow revealed a predominance of CD8(+) cells. Thymectomy alone was ineffective, but cyclosporin A treatment subsequent to thymectomy was safe and effective and resulted in the disappearance of a Vbeta12 bearing T-cell clone in the bone marrow. Additional treatment with erythropoietin enhanced the effects of cyclosporin A and restored the patient's hemoglobin to normal levels. The beneficial effect of cyclosporin A may be attributed not to a broad immunomodulatory effect, but to a local effect on a limited T-cell subset.

Aged, 80 and over↗
PubMed2004

In vivo transfer of soluble TNF-alpha receptor 1 gene improves cardiac function and reduces infarct size after myocardial infarction in rats.

Increased circulating and cardiac TNF-alpha levels during myocardial ischemia have been found in both experimental animals and patients with ischemic heart disease and advanced heart failure. Soluble TNF-alpha receptor 1 (sTNFR1) is an antagonist to TNF-alpha. In the present study, we examined whether sTNFR1 improves cardiac function in rats after myocardial infarction. Male Wistar rats were subjected to left coronary artery (LCA) ligation. Immediately after the ligation, a total of 200 microg of either the sTNFR1 or LacZ plasmid was injected into three different sites in the left ventricular wall. From 1 to 21 days after LCA ligation, TNF-alpha bioactivity in the heart was higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase. The LV diastolic dimension was significantly lower, and the fractional shortening was significantly higher in rats treated with the sTNFR1 plasmid than in those treated with the LacZ plasmid. At 21 days after LCA ligation, the LV end-diastolic pressure was also significantly lower in the rats treated with the sTNFR1 plasmid. In addition, the sTNFR1 expression plasmid had significantly reduced the infarct size. In conclusion, TNF-alpha bioactivity in the heart increased during the early stage of infarction and remained elevated. This elevation seemed partially responsible for the impairment of LV function and the increased infarct size. Suppression of TNF-alpha bioactivity from the early stage of infarction with the sTNFR1 plasmid improved cardiac function and reduced infarct size.

Animals↗
PubMed2004

Intramuscular gene transfer of soluble tumor necrosis factor-alpha receptor 1 activates vascular endothelial growth factor receptor and accelerates angiogenesis in a rat model of hindlimb ischemia.

BACKGROUND: In a pathological setting, tumor necrosis factor (TNF)-alpha inhibits the proliferative response of endothelial cells through inactivation of receptors for vascular endothelial growth factor (VEGF). Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellular domain of TNFR1 and an antagonist to TNF-alpha. In the present study, we examined the effect of sTNFR1 expression plasmid on receptor for VEGF (KDR/flk-1) and angiogenesis in a rat model of hindlimb ischemia. METHODS AND RESULTS: The left femoral artery was exposed and excised to induce limb ischemia. A total of 400 microg of sTNFR1 or LacZ plasmid was injected into 3 different sites of the adductor muscle immediately after the induction of ischemia. TNF-alpha bioactivity in ischemic adductors increased in rats receiving LacZ plasmid compared with sham-operated rats. However, sTNFR1 plasmid significantly suppressed the increase in TNF-alpha bioactivity. KDR/flk-1 mRNA and tyrosine phosphorylation of KDR/flk-1 were significantly increased in the muscles injected with sTNFR1 plasmid compared with those injected with LacZ plasmid. VEGF increased both in muscles injected with sTNFR1 plasmid and in muscles injected with LacZ plasmid but did not differ significantly between them. At 21 days after the induction of ischemia, the sTNFR1 plasmid-transfected muscles showed significantly increased capillary density compared with LacZ plasmid-transfected muscles. CONCLUSIONS: In a rat model of hindlimb ischemia, VEGF increased but activation of KDR/flk-1 was suppressed, possibly by TNF-alpha, which might impair angiogenesis. Suppression of TNF-alpha with sTNFR1 plasmid upregulated KDR/flk-1 and accelerated angiogenesis. Local transfection of the sTNFR1 gene can be a new strategy for therapeutic angiogenesis in peripheral ischemic diseases.

Animals↗
PubMed2004

Effects of soluble TNF-alpha receptor 1 on apoptosis induced by oxidized LDL in endothelial cells.

Apoptosis-inducing agents have been reported to cause rapid shedding of tumor necrosis factor receptor 1 (TNFR1) in endothelial cells (EC). Oxidized LDL (oxLDL) has also been known to induce apoptosis of EC and to inhibit proliferation of EC. In the present study, we show that oxLDL also causes shedding of TNFR1 in EC and that EC transfected with soluble TNFR1 (sTNFR1 ), which is an extracellular domain of TNFR1, can antagonize the toxicity induced by oxLDL. These results suggest that transfection with the sTNFR1 gene plays a protective role against the injury of EC induced by oxLDL. We speculate therefore that sTNFR1 can be a new strategy for treatment of atherogenesis possibly by preventing shedding of TNFR1.

Apoptosis↗
PubMed2004

Local delivery of soluble TNF-alpha receptor 1 gene reduces infarct size following ischemia/reperfusion injury in rats.

Apoptosis in the myocardium is linked to ischemia/reperfusion injury, and TNF-alpha induces apoptosis in cardiomyocytes. A significant amount of TNF-alpha is detected after ischemia and reperfusion. Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellular domain of TNF-alpha receptor 1 and is an antagonist to TNF-alpha. In the present study, we examined the effects of sTNFR1 on infarct size in acute myocardial infarction (AMI) following ischemia/reperfusion. Male Wistar rats were subjected to left coronary artery (LCA) ligation. After 30 min of LCA occlusion, the temporary ligature on the LCA was released and blood flow was restored. Immediately after reperfusion, a total of 200 microg of sTNFR1 or LacZ plasmid was injected into three different sites of the left ventricular wall. At 6 h, 1 and 2 days after reperfusion, the TNF-alpha bioactivity in the myocardium was significantly higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase in the TNF-alpha bioactivity. The sTNFR1 plasmid significantly reduced DNA fragmentation and caspase activity compared to the LacZ plasmid. Finally, the sTNFR1 expression-plasmid treatment significantly reduced the area of myocardial infarction at 2 days after ischemia/reperfusion compared to LacZ plasmid. In conclusion, the TNF-alpha bioactivity in the heart increased from the early stage of ischemia/reperfusion, and this increase was thought to contribute in part to the increased area of myocardial infarction. Suppression of TNF-alpha bioactivity with the sTNFR1 plasmid reduced the infarct size in AMI following ischemia and reperfusion.

Animals↗
PubMed2004

A protein tyrosine phosphatase inhibitor accelerates angiogenesis in a rat model of hindlimb ischemia.

Vascular endothelial growth factor (VEGF) receptor-2 (KDR/flk-1) has a tyrosine kinase domain and, once activated, induces the autophosphorylation of the tyrosine residues. The phosphorylated KDR/flk-1 can be a substrate for intracellular protein tyrosine phosphatases (PTPs). In the present study, we have examined whether the PTP inhibitor sodium orthovanadate (SOV) activates KDR/flk-1 and accelerates angiogenesis in a rat model of hindlimb ischemia. The left femoral artery was exposed and excised to induce limb ischemia. The PTP activity in ischemic adductors increased, whereas SOV significantly suppressed the increase in the activity. Tyrosine phosphorylation of KDR/flk-1 and Akt phosphorylation significantly increased in the muscles injected with SOV compared with those injected with saline. The amount of VEGF increased in both the muscles injected with SOV and those injected with the saline but did not differ significantly. At 21 days after the induction of ischemia, immunohistochemical studies demonstrated that muscles injected with SOV showed significantly increased capillary density compared with those injected with saline. In a rat model of hindlimb ischemia, not only VEGF but also PTP, which might impair angiogenesis, increased. SOV activated KDR/flk-1 and accelerated angiogenesis. Thus, a PTP inhibitor can be a new drug for therapeutic angiogenesis in peripheral ischemic diseases.

Angiogenesis Inducing Agents↗
PubMed2003

Chronic inhibition of Rho kinase blunts the process of left ventricular hypertrophy leading to cardiac contractile dysfunction in hypertension-induced heart failure.

The Gq-RhoA-Rho kinase pathway, activated by neurohormonal factors such as angiotensin II (Ang II), has been proposed to be one of the important signaling pathways involved in the progression of left ventricular (LV) hypertrophy to heart failure. We tested the hypothesis that chronic inhibition of Rho kinase prevents this process. Heart failure was induced in Dahl salt-sensitive (DS) rats fed an 8% NaCl diet from 8 until 17 weeks of age. Y-27632 (5 mg/kg per day), a selective Rho kinase inhibitor, was applied orally to DS rats starting at 10 weeks of age for 7 weeks (DS/Y+). DS rats without Y-27632 (DS/Y-) and Dahl salt-resistant (DR) rats fed the 8% NaCl diet were regarded as non-therapeutic and normotensive controls, respectively. At 17 weeks of age, there was no significant difference in the blood pressure of DS/Y- and DS/Y+ rats. DS/Y- rats exhibited: (1) increases in LV mass, cross-sectional area (CSA) of cardiomyocytes, and interstitial fibrosis; (2) contractile dysfunction, i.e. decreases in LV ejection fraction and % fractional shortening, and prolongation of time to peak tension as well as to 50% relaxation in the twitch contraction of isolated papillary muscle; and (3) increases in the protein expression of Galphaq and Rho kinase in the myocardial membrane fraction. In DS/Y+ rats, the degree of myocardial hypertrophy was significantly inhibited in association with improved contractile function, without a decrease in the degree of interstitial fibrosis. Our results suggest the possibility that the Gq-Rho kinase pathway plays an important role in the process of hypertension-induced LV hypertrophy leading to contractile dysfunction.

Amides↗
PubMed2003

Beneficial effects of angiotensin-converting enzyme inhibition on sarcoplasmic reticulum function in the failing heart of the Dahl rat.

Inhibition of angiotensin-converting enzyme (ACE) retards the process of myocardial remodeling and contractile dysfunction that leads to heart failure. However, the intracellular mechanisms by which ACE inhibition preserves myocardial contractility are largely unclear. Using a model of heart failure induced by hypertension in Dahl salt-sensitive (DS) rats, the mechanisms by which ACE inhibitors (ACEI) exert a beneficial effect on myocardial contractility were studied. Dahl salt-resistant (DR) rats, DS rats not given temocapril (DS/T-), and DS rats treated with temocapril (10 mg/kg per day from 10 to 17 weeks of age, DS/T+) were fed an 8% NaCl diet from 8 to 17 weeks of age (n=8, each group). Echocardiography, hemodynamic measurement, histology, contraction of isolated skinned papillary muscle, and Western blot analysis were carried out. At an elevated final blood pressure similar to that of the DS/T- rats, DS/T+ rats exhibited (1) a decrease in left ventricular (LV) mass associated with decreases in both cardiomyocyte size and interstitial fibrosis; (2) improvement of both systolic and diastolic LV function; and (3) an increase in caffeine contraction after constant Ca(2+)-loading with 8-bromo-cAMP into the sarcoplasmic reticulum (SR) associated with an increase in Ser16-phosphorylated phospholamban, as compared with the DS/T- rats. In addition to inhibition of myocardial remodeling, a restoration of the Ca(2+)-handling ability of the SR by normalized phosphorylated phospholamban may contribute to the improved LV contractile function achieved by chronic treatment with an ACEI.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2002

In vivo gene transfer of soluble TNF-alpha receptor 1 alleviates myocardial infarction.

Apoptosis is the major independent form of cardiomyocyte cell death in acute myocardial infarction (AMI). TNF-alpha release early in the course of AMI contributes to myocardial injury, and TNF-alpha induces apoptosis in cardiomyocytes. Soluble TNF-alpha receptor 1 (sTNFR1) is an antagonist to TNF-alpha. However, the effect of sTNFR1 on AMI remains unclear. Here we report that direct injection of an sTNFR1 expression plasmid DNA to the myocardium reduces infarct size in experimental rat AMI. Treatment with sTNFR1 expression plasmid DNA reduced the TNF-alpha bioactivity in the myocardium and the apoptosis of cardiomyocytes. These findings suggest that the anti-TNF-alpha therapy by sTNFR1 can be a new strategy for treatment of AMI.

Animals↗
PubMed2002

Effects of calmodulin and okadaic acid on myofibrillar Ca2+ sensitivity in cardiac myocytes.

Whereas it has been established that the phosphorylation of 20 kD regulatory myosin light chain (MLC20) is a key regulator of contraction in smooth muscle, troponin complex has been thought to be that of myofibrillar Ca2+ sensitivity in cardiac muscle. To elucidate the role of the phosphorylation of cardiac regulatory myosin light chain (MLC2) in the regulation of cardiac muscle contraction, we observed effects of calmodulin and okadaic acid, a protein phosphatase inhibitor, on myofibrillar Ca2+ sensitivity as estimated by pCa50 values obtained from pCa-tension relationships using beta-escin-skinned cardiomyocytes from Wistar rat hearts, in relation to changes in the phosphorylation of myofibrillar regulatory proteins. Whereas myofibrillar Ca2+ sensitivity tended to be progressively decreased by repeated Ca2+-activation in the absence of calmodulin (pCa50; from 5.91 to 5.86, n = 5), calmodulin (2.5 microM) significantly increased myofibrillar Ca2+ sensitivity (pCa50; from 5.92 to 6.03, n = 5, p < 0.05). Okadaic acid over 3 microM enhanced Ca2+-activated force, which was inhibited by 50 microM trifluoperazine, a calmodulin antagonist. Okadaic acid (3 microM) significantly increased myofibrillar Ca2+ sensitivity (pCa50; from 5.96 to 6.11, n = 6, p < 0.05). Whereas the phosphorylation level of troponin I was not changed by 3 microM okadaic acid, that of MLC2 was significantly increased by the same dose of okadaic acid (from 12 to 31%, n = 4, p < 0.05). These results suggest that MLC2 phosphorylation plays a partial role in the regulation of myofibrillar Ca2+ sensitivity in cardiac muscle.

Animals↗