Tang J

Mitofusin 2 Triggers Vascular Smooth Muscle Cell Apoptosis via Mitochondrial Death Pathway

Previous studies have shown that mitofusin 2 (Mfn-2) (or hyperplasia suppressor gene [HSG]) inhibits vascular smooth muscle cell (VSMC) proliferation. Here, we demonstrate that Mfn-2 is a primary determinant of VSMC apoptosis. First, oxidative stress with H2O2, inhibition of protein kinase C with staurosporine, activation of protein kinase A with forskolin, and serum deprivation concurrently elevate Mfn-2 expression and induce VSMC apoptosis. Second, overexpression of Mfn-2 also triggers apoptosis of VSMCs in culture and in balloon-injured rat carotid arteries, thus contributing to Mfn-2–mediated prevention of neointima formation after angioplasty. Third, Mfn-2 silencing protects VSMCs against H2O2 or Mfn-2 overexpression–induced apoptosis, indicating that upregulation of Mfn-2 is necessary and sufficient for oxidative stress–mediated VSMC apoptosis. The Mfn-2 proapoptotic effect is independent of its role in mitochondrial fusion but mainly mediated by inhibition of Akt signaling and the resultant activation of the mitochondrial apoptotic pathway, as manifested by decreased Akt phosphorylation, increased mitochondrial Bax/Bcl-2 ratio, cytochrome c release, and activation of caspases-9 and caspase-3. Furthermore, Mfn-2–induced apoptosis was blocked by overexpression of an active phosphoinositide 3-kinase mutant or Bcl-xL or inhibition of caspase-9 but not caspases-8. Thus, in addition to its antiproliferative effects, Mfn-2 constitutes a primary determinant of VSMC apoptosis.

Mitofusin-2 Is a Major Determinant of Oxidative Stress-mediated Heart Muscle Cell Apoptosis

An inexorable loss of terminally differentiated heart muscle cells is a crucial causal factor for heart failure. Here, we have provided several lines of evidence to demonstrate that mitofusin-2 (Mfn-2; also called hyperplasia suppressor gene), a member of the mitofusin family, is a major determinant of oxidative stress-mediated cardiomyocyte apoptosis. First, oxidative stress with H2O2 led to concurrent increases in Mfn-2 expression and apoptosis in cultured neonatal rat cardiomyocytes. Second, overexpression of Mfn-2 to a level similar to that induced by H2O2 was sufficient to trigger myocyte apoptosis, which is associated with profound inhibition of Akt activation without altering ERK1/2 signaling. Third, Mfn-2 silencing inhibited oxidative stress-induced apoptosis in H9C2 cells, a cardiac muscle cell line. Furthermore, Mfn-2-induced myocyte apoptosis was abrogated by inhibition of caspase-9 (but not caspase-8) and by overexpression of Bcl-xL or enhanced activation of phosphatidylinositol 3-kinase-Akt, suggesting that inhibition of Akt signaling and activation of the mitochondrial death pathway are essentially involved in Mfn-2-induced heart muscle cell apoptosis. These results indicate that increased cardiac Mfn-2 expression is both necessary and sufficient for oxidative stress-induced heart muscle cell apoptosis, suggesting that Mfn-2 deregulation may be a crucial pathogenic element and a potential therapeutic target for heart failure.

Adenovirus-expressed human hyperplasia suppressor gene induces apoptosis in cancer cells

Hyperplasia suppressor gene (HSG), also called human mitofusin 2, is a novel gene that markedly suppresses the cell proliferation of hyperproliferative vascular smooth muscle cells from spontaneously hypertensive rat arteries. This gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network. In this report, we showed that an adenovirus vector encoding human HSG (Ad5-hHSG) had an antitumor activity in a wide range of cancer cell lines. We further focused on the lung cancer cell line A549 and the colon cancer cell line HT-29 and then observed that Ad5-hHSG induced apoptosis both in vitro and in vivo. Confocal laser scanning microscopy and electron microscopy revealed that cells infected with Ad5-hHSG formed dose-dependent perinuclear clusters of fused mitochondria. Adenovirus-mediated hHSG overexpression induced apoptosis, cell cycle arrest, mitochondrial membrane potential (ΔΨm) reduction and release of cytochrome c, caspase-3 activation, and cleavage of PARP in vitro. Overexpression of hHSG also significantly suppressed the growth of subcutaneous tumors in nude mice both ex vivo and in vivo. In addition, Ad5-hHSG increased the sensitivity of these cell lines to two chemotherapeutic agents, VP16 and CHX, and radiation. These results suggest that Ad5-hHSG may serve as an effective therapeutic drug against tumors. [Mol Cancer Ther 2008;7(1):222–32]

A dynorphin peptide induces hypotension by stimulating the release of atrial natriuretic peptide from rat atrium

Intravenous injection of dynorphin A-(1-10) amide (Dyn, 81-324 nmol/kg) induced a dose-dependent hypotensive effect in the rat. This effect was antagonized by pretreatment with immunoglobulin G, purified from a specific antiserum raised against alpha-human atrial natriuretic peptide (anti-hANP-IgG), as well as by high doses of naloxone (2 or 10 mg/kg). In addition, a 12-fold increase in plasma level of atrial natriuretic peptide-like immunoreactivity (ANP-IR) was found following Dyn administration, which was accompanied by a significant decrease of atrial ANP-IR. These results suggest that the stimulated release of ANP-IR from the atrium may constitute one of the mechanisms for the depressor effect of dynorphin peptides.

Atriopeptin and spontaneous hypertension in rats

The involvement of atriopeptin in hypertension was investigated in spontaneously hypertensive rats (SHR). It was found that intravenous injection of atriopeptin III (20-80 nmol/kg) markedly decreased the mean arterial pressure in anesthetized SHR in a dose dependent manner. The heart rate was not significantly affected. The contents of atriopeptin immunoreactive material in the rat atrium and plasma were measured with radioimmunoassay. Both the atrium and plasma contents of atriopeptin immunoreactive material were found to be significantly higher in SHR than in the normotensive control Wistar Kyoto (WKY), indicating an increase in the biosynthesis and release of atriopeptin in SHR. Whether this change was a compensatory response induced by hypertension remains to be investigated.

Characterization and localization of atriopeptin in rat atrium

An antiserum specific for atriopeptin was used to characterize and localize atriopeptin-like immunoreactive material in rat atrium by radioimmunoassay and immunohistochemical techniques. The antiserum recognizes atriopeptin I, atriopeptin III, and alpha-human atrial natriuretic polypeptide, but does not recognize met-enkephalin, cholecystokinin, dynorphin A, bradykinin, substance P, or beta-endorphin. A high content of atriopeptin was found in crude extracts of rat atria, as compared to ventricles, and the atriopeptin-like immunoreactive material was found to be located exclusively in granules within atrial cardiocytes. Fractionation of the immunoreactive material by gel filtration and reverse-phase HPLC revealed the presence of multiple atriopeptins.

Augmentation of revascularization and prevention of plasma leakage by angiopoietin-1 and vascular endothelial growth factor co-transfection in rats with experimental limb ischaemia.

Background — Angiopoietin–1 (Ang1) and vascular endothelial growth factor (VEGF) are endothelial cell-specific growth factors. In the current study, we tested the hypothesis that co-expression of Ang1 and VEGF could have an effect on development of leakage-resistant vessels. Methods and results — Expression plasmids, pcD2 (control plasmid), pcD2/Ang1, pcD2/VEGF121, or pcD2/Ang1 +pcD2/VEGF121, were injected intramuscularly into an ischaemic hindlimb rat model, followed by electroporation. Collateral vessel development and skeletal muscle atrophy were assessed before and 7, 14, 28 days after treatment. Capillary density was significantly increased in the rats transfected with Ang1 or VEGF compared with that in the rats transfected with pcD2 alone (P < 0.05). Rats transfected with Ang1 + VEGF had the highest capillary density (P < 0.05).The mean perimeter ratio of ligated hindlimb to non-ligated hindlimb was lower with pcD2 treatment rats compared with that in the rats transfected with Ang1, VEGF or Ang1 +VEGF (P < 0.01). Limb necrosis was observed in two of 33 control rats, but none in the Ang1 and/or VEGF gene-transferred rats.The amount of extravasated Evans blue in rat ligated hindlimb muscle (7 days after treatment) treated with both Angl and VEGF was significantly less compared with that in the rats transfected with VEGF alone (P < 0.01). Conclusions —The current study demonstrated that co-expression of Ang1 and VEGF genes in the ischaemic muscle effectively develops leakage-resistant vessels in the rat model. Therefore, this approach may provide a more appropriate therapeutic strategy in ischaemic vascular diseases.

Therapeutic actions of α-human atrial natriuretic polypeptide in 16 clinical cases

Abstract α-Human atrial natriuretic polypeptide (α-hANP) was applied to 16 clinical patients,6 patients with essential hypertension, 7 patients with congestive heart failure and 3 patients with cirrhosis. Following intravenous bolus injection of 400μg of synthetic α-hANP, a hypotensive effect of very rapid onset was found, which was more potent in the hypertensive patients than in the normotensive cases. Cardiac functions were improved significantly with a similar time course as the depressor response in the cases of heart failure or hypertension. Hemodynamic observations showed a marked increase in cardiac output, cardiac index, stroke volume, ejection fraction and ejection rate, and a concomitant decrease of the pressure in the right side of the heart and pulmonary circulation in these subjects. In addition, the renal response to α-hANP induced obvious increases in urine volume, electrolytes and creatinine excretions in all the subjects. Finally, plasma levels of aldosterone, Argvasopressin and noradrenaline were also altered by α-hANP. No significant side effects were registered. The above result confirms the therapeutic actions of α-hANP in human subjects and opens the possibility to research α-hANP as a powerful pharmacological tool as well as potential new medicine for human disorders.

Gene suture — A novel method for intramuscular gene transfer and its application in hypertension therapy

In this report, reporter gene beta-galactosidase (LacZ) was chosen to compare two different intramuscular gene transfer methods, direct injection and gene suture. Evidence showed that gene suture can produce a higher foreign gene express efficiency in skeletal muscle compared with the direct injection method. The highly efficient eukaryotic expressing vectors of human atrial natriuretic factor (ANF) were constructed (pcD2/pAdVAntage/hANF and pcDNA3/hANF), and in vivo ANF gene delivery was performed by intramuscular gene suture. The effects of ANF gene transfer on blood pressure and renal sodium and water excretion were studied in three models of hypertensive animals. Results showed that a marked decrease of mean arterial pressure (MAP) and a significant increase of urine volume and urinary sodium excretion was produced in rats receiving the hANF construct due to the local expression of ANF and its secretion into plasma. Taken together, these results indicate that gene suture may represent a novel gene delivery modality in gene therapy.

Hemodynamic Effects of Centrally Administered Adrenomedullin (13–52) in Anesthetized Rats

This study was conducted to investigate the effect of intracerebroventricular administration of adrenomedullin (13-52) [ADM(13-52)], a novel hypotensive peptide, on the hemodynamic parameters of anesthetized rats. ADM(13-52) was administered centrally in a dose of 0.4-3.2 nmol/kg. It provoked marked, prolonged and dose-dependent increases in mean arterial blood pressure, heart rate, stroke volume, cardiac index, left ventricular pressure, left ventricular dp/dtmax and dp/dtmin, but reduction in total peripheral resistance index. In addition, intracerebroventricular administration of ADM(13-52; 1.6 nmol/kg) provoked a marked increase in renal sympathetic nerve activity. Intracerebroventricular administration of artificial cerebrospinal fluid had no effect on the hemodynamic parameters and renal sympathetic nerve activity. The results indicate that ADM(13-52) exerts a central action on the cardiovascular system. The mechanisms of hemodynamic changes induced by central ADM(13-52) were preliminarily analyzed in this study. ADM might play a role in the central control of the cardiovascular system, although the confirmed mechanisms and the physiological implications are undetermined.