Keiko Tsuchida

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 μg 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.

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 μg 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 (Mol Cell Biochem 266: 127–132, 2004)

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.

Intramuscular Gene Transfer of Soluble Tumor Necrosis Factor-α 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)-&agr; inhibits the proliferative response of endothelial cells through inactivation of receptors for vascular endothelial growth factor (VEGF). Soluble TNF-&agr; receptor 1 (sTNFR1) is an extracellular domain of TNFR1 and an antagonist to TNF-&agr;. 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 &mgr;g of sTNFR1 or LacZ plasmid was injected into 3 different sites of the adductor muscle immediately after the induction of ischemia. TNF-&agr; bioactivity in ischemic adductors increased in rats receiving LacZ plasmid compared with sham-operated rats. However, sTNFR1 plasmid significantly suppressed the increase in TNF-&agr; 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-&agr;, which might impair angiogenesis. Suppression of TNF-&agr; 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.

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‐α release early in the course of AMI contributes to myocardial injury, and TNF‐α induces apoptosis in cardiomyocytes. Soluble TNF‐alpha receptor 1 (sTNFR1) is an antagonist to TNF‐α. 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‐α bioactivity in the myocardium and the apoptosis of cardiomyocytes. These findings suggest that the anti‐TNF‐α 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.

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.

Secretion of preβ HDL increases with the suppression of cholesteryl ester transfer protein in Hep G2 cells

Abstract Preβ HDL are small, protein rich lipoproteins that are predominantly composed of apo A-I, without apo A-II. Preβ HDL are secreted from the liver as nascent HDL and/or are produced in the incubated plasma by cholesteryl ester transfer protein (CETP). However, the role of CETP in the secretion of HDL from the liver has yet to be determined. In the present study, we examined the effect of the suppression of hepatic CETP by antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the secretion of apo A-I using a Hep G2 cell culture. The ODNs against CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for the regulation of liver gene expression. Hep G2 cells were cultured in DMEM supplemented with 10% FBS. After 2 days, the medium was changed to DMEM with EGF and the cells were divided into three groups. The control group received saline, while the sense group was mixed with the sense ODNs complex and the antisense group was mixed with the antisense ODNs complex, respectively, for 2 days. Both the hepatic CETP mRNA and the CETP mass in the medium in the antisense group decreased significantly more than in the sense and the control groups (CETP mass: 1.697±0.410 ng/mg cell protein vs. 2.367±0.22 and 2.360±0.139, n =3 in each determination). In contrast, both the hepatic apo A-I mRNA and the apo A-I mass in the medium in the antisense group were significantly higher than those in the sense and the control groups (apo A-I mass; 1.877±0.215 μg/mg cell protein vs. 1.213±0.282 and 1.097±0.144, n =3 in each determination). The increase in apo A-I was mainly due to the increase in preβ apo A-I. These findings may partly explain why HDL and apo A-I increase in patients with CETP deficiency, while also indicating the possibility that the original level of preβ HDL is sufficient in such patients.

Increased proliferation of endothelial cells with overexpression of soluble TNF-α 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.

Reduction of plasma angiotensin II to normal levels by antisense oligodeoxynucleotides against liver angiotensinogen cannot completely attenuate vascular remodeling in spontaneously hypertensive rats

Objective The exact role of angiotensinogen (AGT) in vascular remodeling has yet to be determined. In the present study, we examined the effects of reducing plasma AGT by intravenous injections with antisense oligodeoxynucleotides (ODNs) against AGT targeted to the liver on vascular remodeling in spontaneously hypertensive rats (SHRs). Design and methods The ODNs against rat AGT were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for regulating liver gene expression. Male SHRs (n = 18) and age-matched male Wistar-Kyoto (WKY) rats (n = 4) were used for this study. All animals were fed a standard rat diet throughout the experiment. At 10 weeks of age, the SHRs were divided into three groups (n = 6); systolic blood pressure (SBP) was similar in each group. The control group received saline, the sense group was injected with the sense ODN complex and the antisense group was injected with the antisense ODN complex. WKY rats were fed for the same period of time. The ASOR-poly(L)lysine-ODN complex was injected into the tail veins twice a week. Results At the end of the treatment, a reduction in AGT mRNA levels in the liver and plasma AGT was observed only in the animals injected with antisense ODNs. Antisense ODNs significantly reduced the plasma angiotensin II (Ang II) concentrations to levels similar to those observed in WKY rats. Antisense ODNs significantly reduced the SBP (180.7 ± 4.4 mmHg) and media cross-sectional areas of the aorta (1.11 ± 0.02 mm2), which were still larger than those seen in WKY rats (140.3 ± 2.1 mmHg, 0.84 ± 0.02 mm2), compared with the SHRs injected with sense ODNs (225.2 ± 4.4 mmHg, 1.24 ± 0.02 mm2) and control SHRs (223.7 ± 4.8 mmHg, 1.25 ± 0.02 mm2). The aortic angiotensin-converting enzyme (ACE) activity and collagen concentrations, which were significantly higher than those seen in WKY rats, did not significantly change among the SHR groups. The aortic AGT, ACE, angiotensin II type 1 (AT1) receptor and angiotensin II type 2 (AT2) receptor mRNA also did not significantly change among the SHR groups. Conclusion On the basis of these findings, plasma AGT is thus considered to play a role in the development of hypertrophy of smooth muscle in the aorta of SHRs, it is thought to have only a slight effect, however, on the remodeling of the matrix tissue when the suppression of hypertension is insufficient.