Calorie restriction increases telomerase activity, enhances autophagy, and improves diastolic dysfunction in diabetic rat hearts.

The aims of this study were to investigate the impact of caloric restriction (CR) on cardiac telomere biology in an animal model of diabetes and to examine the signal transduction involved in cell senescence as well as cardiac function. Male 8-week-old Otsuka Long-Evans Tokushima fatty (OLETF) diabetic rats were divided into two groups: a group fed ad libitum (OLETF-AL) and a group fed with CR (OLETF-CR: 30% energy reduction). Long-Evans Tokushima Otsuka (LETO) non-diabetic rats were used as controls. LETO rats were also divided into two groups: a CR (LETO-CR) group and a group fed AL (LETO-AL). At 40 weeks of age, the body weight was decreased by 9.7% and the insulin resistance was less in OLETF-CR rats. Telomerase activity in OLETF-CR rats was significantly increased, and telomerase reverse transcriptase was more highly expressed in those rats. However, the telomere length (TL) was not different between AL- and CR-treated rats of each strain. The protein expressions for FoxO1 and FoxO3 were increased in OLETF-AL rats, but the levels of phosphorylated (p)-Akt were decreased compared to those in OLETF-CR rats. Autophagic LC3II signals revealed significant increases in OLETF-CR rats. Echocardiography showed that OLETF-CR improved the left ventricular diastolic dysfunction without changes in the left ventricular dimension. This study revealed that CR increases cardiac telomerase activity without TL attrition, and significantly ameliorates diastolic dysfunction. These findings suggest that cardiac telomerase activity may play an important role in the maintenance of normal cardiac function.

TNF-alpha employs a protein-tyrosine phosphatase to inhibit activation of hepatocyte growth factor receptor and hepatocyte growth factor-induced endothelial cell proliferation.

TNF-alpha impairs endothelial cell growth and angiogenesis. The anti-angiogenic effects of TNF-alpha have mainly been explained by its modulating vascular endothelial growth factor (VEGF)-specific angiogenic pathway. Hepatocyte growth factor (HGF) also promotes the growth of vascular endothelial cells and the development of new blood vessels through interaction with its specific receptor, c-met. However, it is little known whether TNF-alpha interacts with the HGF system or not. In this study, we examined the effect of TNF-alpha on HGF receptor function. In human umbilical venous endothelial cells (HUVEC), TNF-alpha acutely inhibited the phosphorylation and activation of c-met induced by HGF. The ability of TNF-alpha to inhibit HGF-induced c-met activity was impaired by sodium orthovanadate, suggesting that the inhibitory effect of TNF-alpha was mediated by a protein-tyrosine phosphatase. Treatment of HUVEC with TNF-alpha impairs the ability of HGF to activate MAPK and Akt, and this effect was blocked by SOV. HGF-induced c-met responses specifically associated with endothelial cell proliferation and mitogen-activated protein kinase activation were also inhibited by TNF-alpha, and these were reversed by sodium orthovanadate. HGF-induced SHP-1 (a cytoplasmic protein-tyrosine phosphatase) and pretreatment of HUVEC with TNF-alpha prior to HGF treatment resulted in substantial increase in the amount of SHP-1. These data suggest that TNF-alpha employs a protein-tyrosine phosphatase and may exert its anti-angiogenic function in part by modulating the HGF-specific angiogenic pathway in pathological settings.

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.

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.

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.

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.

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.

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.

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.

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.

Nifedipine prevents apoptosis of endothelial cells induced by oxidized low-density lipoproteins.

Calcium channel blockade has been shown to inhibit experimental atherosclerosis, and early clinical trials suggest that it also reduces atherosclerosis in humans. However, the mechanisms underlying the direct protective effect of calcium channel blockade on endothelial cell injury are not fully understood. The apoptosis of endothelial cells induced by oxidized low-density lipoproteins (oxLDL) may provide a mechanistic clue to the "response-to-injury" hypothesis of atherogenesis. Here we report that the calcium channel blocker, nifedipine, prevents the apoptosis of human umbilical venous endothelial cells (HUVECs) induced by oxLDL via downregulation of the endothelial receptor for oxidized LDL (LOX-1) and inhibition of CPP32-like protease activity. The incubation of HUVEC with oxLDL increased LOX-1 mRNA levels and CPP32-like protease activity, and induced apoptosis. Preincubation of HUVEC with nifedipine before incubation with oxLDL significantly suppressed the increase in LOX-1 mRNA levels and CPP32-like protease activity, preventing apoptosis in a dose-dependent manner. These results suggest that nifedipine blocks the suicide pathway leading to the apoptosis of endothelial cells by decreasing LOX-1 mRNA levels and CPP32-like protease activity. Thus, nifedipine seems to play a protective role against the "response-to-injury" hypothesis of atherogenesis.

Increased proliferation of endothelial cells with overexpression of soluble TNF-alpha receptor I gene.

Vascular endothelial growth factor (VEGF) can overcome a potential anti-angiogenic effect of TNF-alpha by inhibiting endothelial apoptosis induced by this cytokine. Soluble TNF-alpha receptor I (sTNFRI) is an extracellular domain of TNFRI and antagonizes the activity of TNF-alpha. Here we report that sTNFRI is able to stimulate the growth of endothelial cells not by antagonizing TNF-alpha. Exogenously added recombinant human sTNFRI stimulated significantly more cell growth of human umbilical venous endothelial cells (HUVEC) with a low dose (50-200 pg/ml) compared with smooth muscle cells. In contrast, monoclonal antibody against TNF-alpha did not stimulate growth of human HUVEC. The sTNFRI expression plasmid (pcDNA3.1 plasmid) was introduced into the cell culture using OPTI-MEM, lipofectin and transferrin. Growth of HUVEC transfected with sTNFRI vector also increased significantly compared with those transfected with control vector. HUVEC transfected with sTNFRI vector increased the extracellular domain of TNFRI mRNA levels, but did not affect the intracellular domain of TNFRI mRNA levels. Accumulation of sTNFRI significantly increased in conditioned medium from HUVEC transfected with sTNFRI vector compared with those transfected with control vector. HUVEC transfected with sTNFRI vector not only increased sTNFRI but also prevented shedding of sTNFRI from TNFRI. The TNF-alpha -induced internucleosomic fragmentation was also significantly prevented in HUVEC transfected with sTNFRI vector compared with those transfected with control vector. These results suggest that instead of growth factors such as VEGF, local transfection of the sTNFRI gene may have potential therapeutic value in vascular diseases in which TNF-alpha is also usually highly expressed.