Katsuo Kanmatsuse

Antiischemic effects of nicorandil during coronary angioplasty in humans

SummaryThe present study was undertaken on 10 patients with angina undergoing percutaneous transluminal coronary angioplasty. The angioplasty procedure consisted of two successive 30-second balloon inflations at 5 minute intervals. After the first inflation, nicorandil (0.1 mg/kg) was given intravenously over a 2-minute period. The second inflation was then performed 3 minutes after the completion of drug administration. Myocardial ischemia was measured as the magnitude of ST-segment elevation on the intracoronary electrocardiogram (intracoronary ECG) recorded from the guidewire. Nicorandil significantly reduced the magnitude of ST-segment elevation. Nicorandil did not change the heart rate-blood pressure product, nor the oxygen saturation of the blood sampled from the great cardiac vein, nor the velocity of coronary blood flow in those patients with no evidence of collaterals. These results favor the conclusion that nicorandil prolongs the intrinsic ability of cardiac myocyte to withstand oxygen deprivation. This salutary effect is possibly due to a direct cellular mechanism because nicorandil did not modify the peripheral and coronary hemodynamic parameters that govern myocardial oxygen consumption.

Role of endogenous angiotensin II in the increased expression of growth factors in vascular smooth muscle cells from spontaneously hypertensive rats

In culture, vascular smooth muscle cells (VSMC) derived from spontaneously hypertensive rats (SHR) show exaggerated growth compared with cells from normotensive Wistar-Kyoto (WKY) rats. SHR-derived VSMC express higher levels of transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF) A-chain, and basic fibroblast growth factor (bFGF) mRNAs than cells from WKY rats. We have recently observed production of angiotensin II (Ang II) in homogeneous cultures of VSMC from SHR. In the current study we investigated the contribution of endogenous Ang II to increased expression of the above-mentioned growth factors in VSMC from SHR. The levels of mRNAs encoding TGF-β1, PDGF A-chain, and bFGF were determined by reverse transcription-polymerase chain reaction and were much higher in VSMC from SHR than in cells from WKY rats. The basal level of Ang II-like immunoreactivity (LI) in conditioned medium as determined by radioimmunoassay was significantly higher in VSMC from SHR than in cells from WKY rats. Isoproterenol is known to induce angiotensinogen gene significantly increased Ang II-LI in VSMC from both WKY rats and SHR. Isoproterenol also increased angiotensinogen, TGF-β1, PDGF A-chain, and bFGF mRNAs in VSMC from SHR. An angiotensin-converting enzyme inhibitor delapril significantly decreased Ang II-LI in VSMC from WKY rats and SHR. Delapril considerably decreased the levels of TGF-β1, PDGF A-chain, and bFGF mRNAs in VSMC from SHR. An Ang II type 1 receptor antagonist CV11974 decreased the levels of TGF-β1, PDGF A-chain, and bFGF mRNAs, and the levels of TGF-β1, PDGF-AA, and bFGF proteins in VSMC from SHR. These findings suggest that increased generation of Ang II is associated with enhanced expression of TGF-β1, PDGF A-chain, and bFGF, and the increases in the levels of these growth factors by endogenous Ang II may contribute to the exaggerated growth of VSMC from SHR.

Phenotypic Modulation by Fibronectin Enhances the Angiotensin II–Generating System in Cultured Vascular Smooth Muscle Cells

We previously demonstrated that homogeneous cultures of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats produce angiotensin II (Ang II) in response to increases in the levels of angiotensinogen, cathepsin D, and angiotensin-converting enzyme (ACE). The change of VSMCs from the contractile to the synthetic phenotype increased the amount of synthetic organelles, resulting in the production of proteases and growth factors. To evaluate the contribution of the synthetic phenotype to the generation of Ang II, we examined the effect of fibronectin (FN), which reportedly induces the synthetic phenotype, on the Ang II-generating system in VSMCs. Cultured VSMCs from Wistar-Kyoto rats were incubated with an active fragment of FN, Arg-Gly-Asp-Ser, for 24, 48, or 72 hours after synchronization of the cell cycle with 0. 2% calf serum for 48 hours. Immunofluorescence and protein levels of alpha-smooth muscle (SM) actin and expression of SM22alpha mRNA, apparent in the contractile phenotype, were suppressed by FN, whereas expression of matrix Gla mRNA and osteopontin mRNA and protein, apparent in the synthetic phenotype, was increased. FN (1 to 1000 microg/mL) dose-dependently increased DNA synthesis in the VSMCs, which was inhibited by the Ang II type 1 receptor antagonist CV-11974. Ang II-like immunoreactivity as determined by radioimmunoassay was significantly increased in conditioned medium from the VSMCs. In addition, mRNA for the Ang II-generating proteases cathepsin D and ACE was increased by FN. Expression of transforming growth factor-beta1, platelet-derived growth factor A-chain, and basic fibroblast growth factor mRNAs was also increased by FN. These results indicate that the changes accompanying the alteration to the synthetic phenotype in homogeneous cultures of VSMCs increase expression of proteases such as cathepsin D and ACE, which then produce Ang II, and that these changes increase expression of growth factors that then induce growth of VSMCs.

A nitric oxide synthesis inhibitor decreased prostaglandin production in rat mesenteric vasculature

Endothelial cells synthesize and release nitric oxide (NO) and prostacyclin (PGI2) which are involved in the regulation of vascular tone and blood pressure. Our objective was to evaluate the effects of inhibiting NO synthesis on vascular prostaglandin (PG) and cyclic nucleotide production, as well as the pressor response to norepinephrine (NE). Isolated mesenteric arterial beds were perfused with Krebs-Henseleit solution containing 100 microM NG-monomethyl-L-arginine (L-NMMA), 100 microM L-arginine (LA), or vehicle. After a 30 min equilibration 0.1, 0.5, 1, or 5 microM NE was infused into the superior mesenteric artery and the perfusion pressure was monitored. The basal perfusion pressure did not differ significantly between groups. The pressure-response curve was shifted to the right in the L-NMMA group vs. the LA and control groups. Perfusion was similarly performed with a Krebs-Henseleit solution containing 100 microM L-NMMA, LA, D-arginine, or vehicle. Perfusates were collected before and after NE infusion for the measurement of PGE2, 6-keto-PGF1 alpha, TxB2, cAMP, and cGMP. In the L-NMMA group the release of PGE2 and 6-keto-PGF1 alpha was decreased, and the release of cGMP was prevented. Production of cAMP did not differ between the four groups before NE infusion, and NE increased cAMP release in the L-NMMA group and controls. The results indicate that inhibition of NO synthesis by L-NMMA enhanced the pressor response to NE, possibly mediated by the decreased cGMP and PGI2 production in resistance vessels.

DNA–RNA chimeric hammerhead ribozyme to transforming growth factor-β1 mRNA inhibits the exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats

OBJECTIVEnThe purpose of this study was to develop DNA-RNA chimeric hammerhead ribozyme against transforming growth factor-beta(1) (TGF-beta(1)) mRNA as a gene therapy agent for arterial proliferative diseases.nnnMETHODSnA 38-base hammerhead ribozyme against rat TGF-beta(1) mRNA, to produce cleavage at the GUC sequence at nucleotide 825 according to the secondary structure of rat TGF-beta(1) mRNA was designed. To enhance its stability, we synthesized a DNA-RNA chimeric ribozyme with two phosphorothioate linkages at the 3-terminal. We also synthesized a mismatch ribozyme with single base change in the catalytic loop region as a control. These ribozymes were delivered into rat vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats by lipofectin-mediated transfection, and their biological effects were investigated.nnnRESULTSnAccording to in vitro cleavage studies, the synthetic ribozyme can cleave the synthetic substrate RNA into two RNA fragments. Chimeric ribozyme significantly inhibited DNA synthesis in VSMC from SHR but not in cells from WKY rats. Mismatch ribozyme showed only a little effect on growth of VSMC from SHR. Chimeric ribozyme significantly inhibited proliferation of VSMC from SHR; in contrast, the proliferation of VSMC from WKY rats was significantly increased by this chimeric ribozyme. Mismatch ribozyme did not affect proliferation of VSMC from either rat strain. Chimeric hammerhead ribozyme to rat TGF-beta(1) dose-dependently inhibited TGF-beta(1) mRNA expression detected by reverse transcription and polymerase chain reaction analysis in VSMC from both rat strains. Chimeric hammerhead ribozyme to rat TGF-beta(1) also dose-dependently inhibited TGF-beta(1) protein production detected by Western blot analysis.nnnCONCLUSIONSnThe present results demonstrated that our designed DNA-RNA chimeric hammerhead ribozyme to TGF-beta(1) mRNA might be a useful gene therapy agent for hypertensive vascular diseases.

Organization of the Human Prostacyclin Synthase Gene and Association Analysis of a Novel CA Repeat in Essential Hypertension

In 1976, Vane et al discovered prostacyclin (PGI2) that is a potent inhibitor of platelet secretion and aggregation, smooth muscle cell proliferation and vasoconstriction1–3. It is considered to play a key role in vasoprotection and its deficiency may lead to thrombosis and other vascular lesions4, 5. Biosynthesis of PGI2 is catalyzed serially by phospholipase A2 which liberates arachidonic acid from the sn-2 position of membrane phospholipids, prostaglandin H2 synthase, a bifunctional enzyme which catalyzes the conversion of arachidonic acid to prostaglandin G2 and subsequently to prostaglandin H2, and prostacyclin synthase (PGIS, EC 5.3.99.4) which catalyzes the formation of PGI2 from prostaglandin H2. PGIS has been shown to be widely distributed, predominantly in vascular endothelial and smooth muscle cells6, 7. The enzyme is a membrane-bound protein located in the endoplasmic reticulum. It exhibits spectral characteristics of cytochrome P450s but has no mono-oxygenase activity8, 9. Instead, it catalyzes an isomerization reaction and thus does not require a reductase to initiate the catalytic activity. A description has been given of the cDNA of human PGIS10 and the deduced amino acid sequence. In the present study, the genomic organization of the human PGIS gene was investigated11, and the association study of essential hypertension was carried out using a novel CA/TG dinucleotide repeat polymorphism in this gene12.

Effects of the L- and N-type calcium channel blocker cilnidipine on growth of vascular smooth muscle cells from spontaneously hypertensive rats.

&NA; Cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) show exaggerated growth compared with cells from Wistar‐Kyoto (WKY) rats. Calcium antagonists have recently been reported to have an in vivo antiproliferative effect on hypertensive cardiovascular organs. We investigated the effects of the calcium antagonist cilnidipine that blocks both L‐ and N‐type calcium channels on the growth of VSMC from SHR. Cilnidipine (1 and 10 &mgr;M) significantly inhibited basal DNA synthesis in VSMC from both rat strains; the inhibition was significantly larger in VSMC from SHR than in cells from WKY rats, and was significantly greater than effects of nifedipine. Cilnidipine (1&mgr;M) significantly inhibited serum‐stimulated DNA synthesis in VSMC from both rat strains. The inhibition was more marked in VSMC from SHR than in cells from WKY rats. Angiotensin II, platelet‐derived growth factor (PDGF)‐AA, and phorbol‐12‐myristate‐13‐acetate dose‐dependently increased DNA synthesis in VSMC from SHR but not in cells from WKY rats. Cilnidipine (1 &mgr;M) significantly suppressed this increase in DNA synthesis in VSMC from SHR. Expression of basic fibroblast growth factor (bFGF), transforming growth factor‐&bgr;1, and PDGF A‐chain mRNAs was markedly greater in VSMC from SHR than in cells from WKY rats. Cilnidipine (1 &mgr;M) significantly inhibited the expression of TGF‐&bgr;1 mRNA in VSMC from SHR but not in cells from WKY rats. These findings suggest that cilnidipine exerts its antiproliferative effects through the inhibition of DNA synthesis induced by growth‐promoting factors and by inhibiting the expression of TGF‐&bgr;1 mRNA in VSMC from SHR.

Magnetic resonance imaging of cardiac hemangiopericytoma

SummaryWe report a patient with hemangiopericytoma, a rare soft tissue sarcoma involving the left ventricle. T1- and T2-weighted magnetic resonance imaging (MRI) revealed a high signal mass invading the left ventricular wall. A biopsied specimen obtained from the metastatic subcutaneous tumor in the right popliteal fossa showed pathologic findings consistent with hemangiopericytoma.