Kensuke Ohta

Angiotensin II induces cardiac phenotypic modulation and remodeling in vivo in rats

Cardiac phenotypic modulation and remodeling appear to be involved in the pathophysiology of cardiac hypertrophy and heart failure. We undertook this study to examine whether angiotensin II (Ang II) in vivo, independent of blood pressure, contributes to cardiac phenotypic modulation and remodeling. A low dose (200 ng/kg per minute) of Ang II was continuously infused into rats by osmotic minipump for 24 hours or 3 or 7 days to examine the effects on the expression of cardiac phenotype-related or fibrosis-related genes. This Ang II dose caused a small and gradual increase in blood pressure over 7 days. Left ventricular mRNAs for skeletal alpha-actin, beta-myosin heavy chain, atrial natriuretic polypeptide, and fibronectin were already increased by 6.9-, 1.8-, 4.8-, and 1.5-fold, respectively, after 24 hours of Ang II infusion and by 6.9-, 3.3-, 7.5-, and 2.5-fold, respectively, after 3 days, whereas ventricular alpha-myosin heavy chain and smooth muscle alpha-actin mRNAs were not significantly altered by Ang II infusion. Ventricular transforming growth factor-beta 1 and types I and III collagen mRNA levels did not increase at 24 hours and began to increase by 1.4-, 2.8-, and 2.1-fold, respectively, at 3 days. An increase in left ventricular weight occurred 3 days after Ang II infusion. Treatment with TCV-116 (3 mg/kg per day), a nonpeptide selective angiotensin type 1 receptor antagonist, completely inhibited the above-mentioned Ang II-induced increases in ventricular gene expressions and weight. Hydralazine (10 mg/kg per day), which completely normalized blood pressure, did not block cardiac hypertrophy or increased cardiac gene expressions by Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular Endothelial Growth Factor–Expressing Mesenchymal Stem Cell Transplantation for the Treatment of Acute Myocardial Infarction

Objective—Vascular endothelial growth factor (VEGF) plays an important role in inducing angiogenesis. Mesenchymal stem cells (MSCs) may have potential for differentiation to several types of cells, including myocytes. We hypothesized that transplantation of VEGF-expressing MSCs could effectively treat acute myocardial infarction (MI) by providing enhanced cardioprotection, followed by angiogenic effects in salvaging ischemic myocardium. Methods and Results—The human VEGF165 gene was transfected to cultured MSCs of Lewis rats using an adenoviral vector. Six million VEGF-transfected and LacZ-transfected MSCs (VEGF group), LacZ-transfected MSCs (control group), or serum-free medium only (medium group) were injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. At 1 week after MI, MSCs were detected by X-gal staining in infarcted region. High expression of VEGF was immunostained in the VEGF group. At 28 days after MI, infarct size, left ventricular dimensions, ejection fraction, E wave/A wave ratio and capillary density of the infarcted region were most improved in the VEGF group, compared with the medium group. Immunohistochemically, &agr;-smooth muscle actin–positive cells were most increased in the VEGF group. Conclusions—This combined strategy of cell transplantation with gene therapy could be a useful therapy for the treatment of acute MI.

Transmission of symptomatic parvovirus B19 infection by fibrin sealant used during surgery

Human parvovirus B19 infection has been shown to be transmissible by blood and blood products and to result in transient aplastic crisis in patients with rapid red cell turnover. We report three cases of iatrogenic parvovirus B19 infection resulting from the use of the same batch of fibrin sealant under operation. Fibrin sealant, which is a typical haemostatic agent produced from blood, has been used during surgery. Human parvovirus is resistant to existing virus‐inactivating techniques, suggesting that infection may occur from blood products contaminated with it. Use of recombinant products for these proteins may thus be necessary.

Role of angiotensin II in renal injury of deoxycorticosterone acetate-salt hypertensive rats.

To investigate the role of angiotensin II (Ang II) in hypertension-induced tissue injury, we gave TCV-116 (1 mg/kg per day PO), a nonpeptide Ang II type I receptor antagonist, or enalapril (10 mg/kg per day PO) to deoxycorticosterone acetate (DOCA)-salt hypertensive rats for 3 weeks and examined the effects on tissue mRNA levels for transforming growth factor-beta 1 (TGF-beta 1) and extracellular matrix components. Tissue mRNA levels were measured by Northern blot analysis. Renal mRNA levels for TGF-beta 1; types I, III, and IV collagen; and fibronectin in DOCA-salt hypertensive rats were increased by severalfold (P < .01) compared with sham-operated rats. In the aorta of DOCA-salt hypertensive rats, TGF-beta 1 and fibronectin mRNA levels were increased, but types I, III, and IV collagen mRNAs did not increase. In the heart, increased mRNA was found only for fibronectin. Thus, these gene expressions are regulated in a tissue-specific manner. TCV-116 or enalapril did not lower blood pressure in DOCA-salt hypertensive rats. However, the increase in renal mRNAs for TGF-beta 1 and extracellular matrix components in DOCA-salt hypertensive rats was significantly inhibited by treatment with TCV-116 or enalapril, which was associated with a significant decrease in urinary protein and albumin excretions and histological improvement of renal lesions. In contrast, in the aorta and heart these gene expressions were not affected by TCV-116 or enalapril. Thus, local Ang II may contribute to renal injury of DOCA-salt hypertension by stimulating the gene expression of TGF-beta 1 and extracellular matrix components.

Angiotensin II Type 1 Receptor Blockade Inhibits the Expression of Immediate-Early Genes and Fibronectin in Rat Injured Artery

BACKGROUND Vascular injury activates various kinds of genes, including proto-oncogenes, growth factors, and extracellular matrix proteins. However, the significance of activation of these genes in neointimal formation is poorly understood. Angiotensin II type 1 (AT1) receptor antagonist is shown to prevent neointimal formation after vascular injury, although the mechanism is unclear. To understand the molecular mechanism of vascular thickening, we examined the effects of AT1 receptor blockade on the gene expression of proto-oncogenes, transforming growth factor-beta 1 (TGF-beta 1), and extracellular matrix proteins after vascular injury. METHODS AND RESULTS Endothelial denudation of the left common carotid artery in Sprague-Dawley rats was performed with a Fogarty 2F balloon catheter. TCV-116 (10 mg.kg-1.d-1), a selective nonpeptide AT1 receptor antagonist, or vehicle was administered orally to rats from 1 day before to 14 days after balloon injury. Injured left and uninjured right common carotid arteries were removed from rats at 1, 6, and 24 hours and 3, 7, and 14 days after balloon injury. Tissue mRNA levels were measured with Northern blot analysis using specific cDNA probes and corrected for 18S ribosomal RNA value. Arterial mRNAs for c-fos, c-jun, jun B, jun D, and Egr-1 increased significantly at 1 hour after balloon injury and decreased rapidly. At 6 hours, ornithine decarboxylase (ODC) mRNA expression reached the maximal levels. TGF-beta 1 and fibronectin mRNA levels started to increase at 6 hours after injury and remained enhanced until 7 days after injury. On the other hand, collagen types I, III, and IV and laminin mRNA levels were not significantly increased over 7 days. Treatment with TCV-116 significantly inhibited the induction of mRNAs for c-fos, c-jun, Egr-1, ODC, and fibronectin in injured artery, whereas the increase in TGF-beta 1 gene expression after injury was not prevented by TCV-116. Immunohistological studies indicated that TCV-116 decreased not only the intimal thickening but also the amount of these extracellular matrix proteins in the intima. CONCLUSIONS The results indicate that AT1 receptor blockade inhibits the induction of immediate-early genes, ODC, and fibronectin in rat injured artery. Thus, inhibition of intimal thickening by AT1 receptor blockade may be mediated at least in part by suppression of multiple genes related to cell growth and migration in the very early phase after vascular injury.

Exogenous administration of mesenchymal stem cells ameliorates dextran sulfate sodium-induced colitis via anti-inflammatory action in damaged tissue in rats.

AIMS Mesenchymal stem cells (MSCs) may modulate inflammatory responses resulting in improvement in inflammatory diseases, as well as tissue regeneration via cellular differentiation. We examined the therapeutic effects of exogenously administered MSCs in dextran sulfate sodium (DSS)-induced colitis in rats. MAIN METHODS Experimental colitis was produced in inbred male Lewis rats by administration of 4% DSS in drinking water for 7 days. MSCs (5x10(6) cells) which were isolated from whole marrow cells and cultured in an optimal medium for MSC outgrowth were administered to the treated rats via the tail vein on days 0, 2, and 4. On day 7, we evaluated colon length, histological changes, and colonic various mRNA expressions by RT-PCR. Localization of MSCs was evaluated using a green-fluorescent cell linker dye. To evaluate the anti-inflammatory action of MSCs, we assayed LPS-induced TNF-alpha secretion in a co-culture of MSCs and monocytes (THP-1 cells) using ELISA. KEY FINDINGS MSCs reduced in bloody stools, weight loss, colon shortening, and microscopic injuries. In the rectum of MSCs-treated rats, mRNA expression of TNF-alpha, IL-1beta, and COX-2 decreased to 40, 15, and 15% of their respective control levels. MSCs significantly suppressed mRNA expression of VEGF, HGF, and b-FGF to 40, 25, and 25% of their respective control levels. Green-fluorescent-labeled MSCs were found only within the lamina propria in inflamed regions. LPS-induced TNF-alpha secretion by THP-1 cells was significantly suppressed by co-culture with MSCs dose-dependently. SIGNIFICANCE We conclude that exogenous MSCs accumulated in inflamed tissues and ameliorated DSS-induced colitis via a local anti-inflammatory action.

Platelet recovery after eradication of Helicobacter pylori in patients with idiopathic thrombocytopenic purpura

Abstract. The association between Helicobacter pylori (H. pylori) infection and idiopathic thrombocytopenic purpura (ITP) has been reported by several groups. We investigated the prevalence of H. pylori infection and the effectiveness of its eradication in Japanese patients with ITP. H. pylori infection was found in 21 of 30 patients (70.0%) by 13C urea breath test and presence of serum antibodies to H. pylori. H. pylori was eradicated in 18 of the 21 infected patients (85.7%) by administration of a proton pump inhibitor and two kinds of antibiotics. In only one patient was medication discontinued due to skin rash on the 4th day of treatment. Platelet recovery was obtained in ten patients (55.6%). In two patients with treatment failure, platelet recovery was obtained after successful re-eradication. In three patients without H. pylori infection, platelet counts did not significantly increase with the same treatment. On the other hand, eradication therapy did not affect platelet counts in patients with gastric ulcer. In conclusion, H. pylori eradication can be used for initial treatment with tolerable adverse effects in some ITP patients.

Effects of an AT1 receptor antagonist, an ACE inhibitor and a calcium channel antagonist on cardiac gene expressions in hypertensive rats

1 This study was undertaken to determine whether the AT1 receptor directly contributes to hypertension‐induced cardiac hypertrophy and gene expressions. 2 Stroke‐prone spontaneously hypertensive rats (SHRSP) were given orally an AT1 receptor antagonist (losartan, 30 mg kg−1 day−1), an angiotensin converting enzyme inhibitor (enalapril 10 mg kg−1 day−1), a dihydropyridine calcium channel antagonist (amlodipine, 5 mg kg−1 day−1), or vehicle (control), for 8 weeks (from 16 to 24 weeks of age). The effects of each drug were compared on ventricular weight and mRNA levels for myocardial phenotype‐ and fibrosis‐related genes. 3 Left ventricular hypertrophy of SHRSP was accompanied by the increase in mRNA levels for two foetal phenotypes of contractile proteins (skeletal α‐actin and β‐myosin heavy chain (β‐MHC)), atrial natriuretic polypeptide (ANP), transforming growth factor‐β‐1 (TGF‐β1) and collagen, and a decrease in mRNA levels for an adult phenotype of contractile protein (α‐MHC). Thus, the left ventricle of SHRSP was characterized by myocardial transition from an adult to a foetal phenotype and interstitial fibrosis at the molecular level. 4 Although losartan, enalapril and amlodipine lowered blood pressure of SHRSP to a comparable degree throughout the treatment, losartan caused regression of left ventricular hypertrophy of SHRSP to a greater extent than amlodipine (P < 0.01). 5 Losartan significantly decreased mRNA levels for skeletal α‐actin, ANP, TGF‐β1 and collagen types I, III and IV and increased α‐MHC mRNA in the left ventricle of SHRSP. Amlodipine did not alter left ventricular ANP, α‐MHC and collagen types I and IV mRNA levels of SHRSP. 6 The effects of enalapril on left ventricular hypertrophy and gene expressions of SHRSP were similar to those of losartan, except for the lack of inhibition of collagen type I expression by enalapril. 7 Unlike the hypertrophied left ventricle, there was no significant difference between losartan and amlodipine in the effects on non‐hypertrophied right ventricular gene expressions of SHRSP. 8 Our results show that hypertension causes not only left ventricular hypertrophy but also molecular transition of myocardium to a foetal phenotype and interstitial fibrosis‐related molecular changes. These hypertension‐induced left ventricular molecular changes may be at least in part mediated by the direct action of local angiotensin II via the AT1 receptor.

Contribution of local renin-angiotensin system to cardiac hypertrophy, phenotypic modulation, and remodeling in TGR (mRen2)27 transgenic rats.

BACKGROUND The transgenic rat TGR(mRen2)27, carrying the mouse Ren-2 gene, is a new model to elucidate the role of the local renin-angiotensin system in vivo. However, the role of the local renin-angiotensin system in the heart remains to be determined in TGR(mRen2)27. METHODS AND RESULTS TGR(mRen2)27 were treated with various antihypertensive drugs for 6 weeks to examine the effects on cardiac hypertrophy and gene expression. Cardiac mRNAs were examined by Northern blot analysis. In TGR(mRen2)27, left ventricular hypertrophy was associated with a decrease in alpha-myosin heavy chain expression of 31% and an increase in skeletal alpha-actin and atrial natriuretic polypeptide expression by 2.6- and 21-fold, respectively (P < .05), thereby showing the shift of myocardium to a fetal phenotype. Furthermore, cardiac collagen and laminin expressions were increased in TGR(mRen2)27 (P < .05), suggesting the occurrence of cardiac remodeling. Although treatment of TGR(mRen2)27 with a high dose of TCV-116 (angiotensin AT1 receptor antagonist) or manidipine (calcium antagonist) combined with atenolol (beta 1-adrenergic receptor blocker) completely normalized blood pressure, TCV-116 regressed cardiac hypertrophy and suppressed the changes in cardiac mRNA levels of TGR(mRen2)27 much more potently than manidipine with atenolol. Furthermore, the inhibitory effects of a low dose of TCV-116 on cardiac hypertrophy and altered gene expressions of TGR(mRen2)27 were greater than those of doxazosin (alpha 1-adrenergic receptor blocker) combined with atenolol, despite their similar hypotensive effects. CONCLUSIONS Our present observations provide evidence that the cardiac renin-angiotensin system in TGR(mRen2)27 is responsible for cardiac hypertrophy, phenotypic modulation, and remodeling.

Transforming Growth Factor-β1 Expression and Phenotypic Modulation in the Kidney of Hypertensive Rats

We have previously reported that renal mRNA levels for transforming growth factor-beta 1, fibronectin, and collagens were increased in 32-week-old stroke-prone spontaneously hypertensive rats (SHRSP) with severe nephrosclerosis. To elucidate the mechanism of hypertension-induced nephrosclerosis, we examined gene expression and localization of transforming growth factor-beta 1 and cellular phenotype in the kidney of 25-week-old SHRSP with moderate renal damage. Renal mRNA was measured by Northern blot analysis. The localization of transforming growth factor-beta 1 and cellular phenotype was determined by immunohistochemistry. In the kidney of 25-week-old SHRSP, renal transforming growth factor-beta 1 mRNA was elevated compared with Wistar-Kyoto rats (WKY), whereas renal collagen mRNAs of SHRSP were not increased. Immunoreactive transforming growth factor-beta 1 in SHRSP was mainly localized in glomerular cells. Furthermore, alpha-smooth muscle actin and desmin were significantly expressed in SHRSP glomerular cells, in contrast to negligible expression of these proteins in WKY. alpha-Smooth muscle actin staining was also observed in interstitial cells, and vimentin, another phenotypic marker, was expressed in atrophic tubular cells of SHRSP, despite no staining of these proteins in WKY. Furthermore, all these phenotypic changes in SHRSP were associated with increased cell proliferation, as shown by the increased number of proliferating cell nuclear antigen-positive cells. Treatment of SHRSP with cilazapril and nifedipine (from the age of 13 to 25 weeks) prevented the increase in transforming growth factor-beta 1 expression and the cellular phenotypic modulation and was accompanied by a reduction of urinary albumin excretion and inhibition of cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)