Shih-Hurng Loh

Suppression of the development of hypertension by the inhibitor of inducible nitric oxide synthase

Our previous study demonstrated that the aortic inducible nitric oxide synthase (iNOS) expression and the plasma nitrite level in spontaneously hypertensive rats (SHR) were greater than that in age‐matched Wistar‐Kyoto rats (WKY). We subsequently hypothesized that the over‐expression of iNOS might play an important role in the pathogenesis of hypertension in SHR. In the present study, pyrrolidinedithiocarbamate (PDTC, 10 mg kg−1 day−1, p.o., antioxidant and nuclear factor‐κ B inhibitor) and aminoguanidine (15 mg kg−1 day−1, p.o., selective inhibitor of iNOS) was used to treat SHR and WKY from age of 5 weeks through 16 weeks. We found that PDTC and aminoguanidine significantly suppressed the development of hypertension and improved the diminished vascular responses to acetylcholine in SHR but not in WKY. Likewise, the increase of iNOS expression, nitrotyrosine immunostaining, nitric oxide production and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with PDTC and aminoguanidine. In conclusion, this study demonstrated that both PDTC and aminoguanidine significantly attenuated the development of hypertension in SHR. The results suggest that PDTC suppresses iNOS expression due to its anti‐oxidant and/or nuclear factor‐κ B inhibitory properties. However, the effect of aminoguanidine was predominantly mediated by inhibition of iNOS activity, thereby reducing peroxynitrite formation. We propose that the development of a more specific and potent inhibitor of iNOS might be beneficial in preventing pathological conditions such as the essential hypertension.

Increasing expression of extracellular matrix metalloprotease inducer in ovary tumors : Tissue microarray analysis of immunostaining score with clinicopathological parameters

Summary: Ovary cancer invasion is responsible for both local tissue destruction and distant metastasis. Invasion is largely mediated by matrix metalloproteases that are thought to be induced by tumor cell-derived extracellular matrix metalloprotease inducer (EMMPRIN) in surrounding fibroblasts. We hypothesized that EMMPRIN isoverexpressed in ovary tumors. Immunohistochemical analysis of EMMPRIN was performed in tissue microarrays of ovary neoplasms including 84 cases of serous adenocarcinoma, 23 cases of mucinous adenocarcinoma, 10 cases of endometrioid adenocarcinoma, 12 cases of yolk sac tumor, 12 cases of clear cell carcinoma, 8 cases of dysgerminoma, 8 cases of granulosa cell tumor, 6 cases of transitional cell carcinoma, and 6 cases of Brenner tumor. All malignant ovary tumors showed significant immunohistochemical expression of EMMPRIN. The EMMPRIN scores in malignant ovary tumors were significantly higher than their nontumor counterparts (313 ± 28 for serous adenocarcinoma; 308 ± 25 for mucinous adenocarcinoma; 187 ± 19 for endometrioid adenocarcinoma; 265 ± 23 for yolk sac tumors; 87 ± 13 for clear cellcarcinoma; 126 ± 15 for dysgerminoma; 243 ± 26 for granulosa cell tumor; 87 ± 16 for transitional cell carcinoma). The EMMPRIN score was significantly higher in serous adenocarcinomas than in serous adenomas and serous borderline tumors and was correlated with nodal stage. Our findings show for the first time that EMMPRIN is overexpressed in all malignant ovary tumors.

Effect of Hoe 694, a novel Na+ -H+ exchange inhibitor, on intracellular pH regulation in the guinea-pig ventricular myocyte

1 Hoe 694 (3‐methylsulphonyl‐4‐piperidinobenzoyl, guanidine hydrochloride) is a Na+/H+ exchange (NHE) inhibitor exhibiting cardioprotective properties during ischaemia and reperfusion in animal hearts. We have (i) tested the selectivity of Hoe 694 for NHE over other pHi‐regulating mechanisms in the myocardium, and (ii) tested if the functionally important NHE isoform contributing to intracellular pH regulation in heart is NHE‐1, as suggested from molecular biology studies of this protein. 2 pHi was recorded by fluorescence microscopy with carboxy SNARF‐1, AM‐loaded into single ventricular myocytes of guinea‐pig. 3 In nominally HCO3−‐free media, recovery of pHi from an intracellular acid load is mediated by NHE, and was inhibited by Hoe 694, amiloride (an NHE inhibitor) or dimethyl amiloride (DMA, a high affinity NHE inhibitor) with potency values of 2.05, 87.3 and 1.96 μm respectively, giving the potency series: Hoe 694 ⋍ DMA > > amiloride. This potency series, and the potency values (corrected for drug competition with extracellular Na+) match those determined previously for cloned NHE‐1 expressed in mutant fibroblasts. In the absence of extracellular Na+ (to inhibit NHE), Hoe 694 had no effect on pHi. 4 In 5% CO2/HCO3−‐buffered solution containing DMA, pHi recovery from acidosis is mediated by Na+‐HCO3− symport and was unaffected by Hoe 694. The drug also had no effect on pHi recovery from an alkali‐load, a process largely mediated by Cl−‐HCO3− exchange. Finally, the fall of pHi upon adding extracellular Na‐lactate is assisted by H+‐lactate symport, and this too was unaffected by Hoe 694. 5 We conclude (i) Hoe 694 has no detectable inhibitory potency for pH‐regulating carriers in heart other than NHE. (ii) native NHE functioning during pHi‐regulation in the cardiomyocyte is the NHE‐1 isoform. These data strengthen the case for NHE‐1 being the receptor for mediating the cardioprotective effects of Hoe 694.

Increasing expression of serine protease matriptase in ovarian tumors: tissue microarray analysis of immunostaining score with clinicopathological parameters

Matriptase is a type II transmembrane serine protease expressed by cells of surface epithelial origin, including epithelial ovarian tumor cells. Matriptase cleaves and activates proteins implicated in the progression of cancer and represents a potential prognostic and therapeutic target. The aim of this study was to examine the expression of matriptase in ovarian tumors and to assign clinicopathological correlations. Immunohistochemical analysis of matriptase was performed in tissue microarrays of 164 ovarian neoplasms including 84 serous adenocarcinomas, 23 mucinous adenocarcinomas, 10 endometrioid adenocarcinomas, six yolk sac tumors, 12 clear cell carcinomas, six dysgerminomas, eight granulosa cell tumors, four transitional cell carcinomas, five fibromas, and six Brenner tumors. All ovarian tumors except the fibromas and Brenner tumors showed significant expression of matriptase. The matriptase scores were significantly higher in the tumors than in their nontumor counterparts (304±26 for serous adenocarcinoma; 361±28 for mucinous adenocarcinoma; 254±17 for endometrioid adenocarcinoma; 205±19 for yolk sac tumor; 162±16 for clear cell carcinoma; 109±11 for dysgerminoma; 105±9 for granulosa cell tumor; and 226±18 for transitional cell carcinoma). Matriptase scores in serous adenocarcinoma were correlated with TNM stage and FIGO stage. Our findings demonstrate for the first time that matriptase is overexpressed in many malignant ovarian tumors. It may be a novel biomarker for diagnosis and treatment of malignant ovarian tumors.

Ionic mechanisms responsible for the antiarrhythmic action of dehydroevodiamine in guinea-pig isolated cardiomyocytes.

1 Dehydroevodiamine alkaloid (DeHE), an active ingredient of a Chinese herbal medicine Wu‐Chu‐Yu (Evodiae frutus), has been shown to decrease aterial blood pressure in experimental animals and prolong action potential duration in cardiac cells. The aim of the present study was to explore the ionic basis of its possible antiarrhythmic effects. 2 Guinea‐pig atrial and ventricular myocytes were isolated enzymatically and the ionic currents were recorded under whole‐cell patch‐clamp with single suction pipettes. 3 DeHE at a concentration of 0.1 μm inhibited reversibly the time‐dependent outward K current (delayed rectifier, Ik) and the Na‐dependent inward current (INa). 4 In low‐K (1 mm) and high‐Ca (9 mm) solution, DeHE also depressed the delayed afterdepolarizations (DAD) and the transient inward current (Iti) induced by 2 μm strophanthidin. On the other hand, DeHE occasionally induced early afterdepolarizations and slow response action potentials at a depolarized level. 5 At higher concentrations (1 μm and above), the L‐type Ca current (ICa,L) was moderately inhibited. 6 The present findings indicate that DeHE may depress triggered arrhythmias in Ca‐overloaded guinea‐pig cardiac myocytes through its inhibitory actions on INa, Iti and, to a smaller extent, ICa. DeHE may also exert class III antiarrhythmic effect through a reduction of outward K currents (Ik) across the sarcolemma.

Urotensin II prevents cardiomyocyte apoptosis induced by doxorubicin via Akt and ERK.

Urotensin II, a potent vasoactive peptide, is upregulated in the heart under pathological conditions. Previous in vitro studies show that urotensin II promotes cardiomyocyte hypertrophy, particularly via through cell survival/antiapoptotic pathways Akt and extracellular signal-regulated kinases (ERK). Cardiomyopathies secondary to cardiomyocyte apoptosis are limiting factors for treating cancer with doxorubicin. In this study, we hypothesized that urotensin II might exert a protective effect on doxorubicin-induced cardiomyocyte apoptosis. To test this hypothesis, we evaluated the effect of urotensin II on doxorubicin-induced cardiomyocyte apoptosis, and explored its intracellular mechanism(s). Rat neonatal cardiomyocytes were treated with the vehicle, doxorubicin (1μM), urotensin II, or urotensin II plus doxorubicin. Myocyte apoptosis was evaluated based on caspase-3 activity and the DNA strand break level using terminal deoxynucleotidyl transferase deoxyuridine triphosphate-mediated nick-end labeling (TUNEL) staining. Western blot analysis was employed to determine the related protein expression and flow cytometry assay was used to determine the TUNEL-positive cells. Akt and ERK phosphorylation was enhanced by urotensin II treatment in cardiomyocytes. Urotensin II-mediated Akt and ERK phosphorylation could be prevented by treatment with the urotensin II antagonist, urantide. Furthermore, urotensin II administration increased the cell viability and lowered apoptotic cell death induced by doxorubicin, as indicated by a decrease in trypan blue-positive cells, and reduced TUNEL staining and caspase-3 activity. These protective effects of urotensin II were abolished by prior inhibition of phosphatidylinositol 3-kinase/Akt by LY294002 (2-[4-morpholinyl]-8-phenyl-4H-1-benzopyran-4-one), and ERK by U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene). These findings suggest that urotensin II protects cardiomyocytes from doxorubicin-induced apoptosis partly via Akt and ERK, which may promote cell survival and cardioprotection.

Off-pump technique in coronary artery bypass grafting in elderly patients.

Background:  The use of cardiopulmonary bypass (CPB) during coronary artery bypass grafting (CABG) is associated with substantial morbidity and mortality, especially in the elderly. The purpose of this study was to evaluate the feasibility of beating heart coronary artery revascularization in patients aged at least 80 years.

Hydrogen peroxide-induced intracellular acidosis and electromechanical inhibition in the diseased human ventricular myocardium

Accumulation of oxygen free radicals is an important mediator of post-ischemia/reperfusion cardiac dysfunction. However, oxidative injury has not been well characterized in human cardiac tissues. In the present study, we superfused hydrogen peroxide (H(2)O(2)) into the diseased human ventricle in order to assess the effects of oxygen free radicals on the electromechanical parameters and the intracellular pH (pH(i)), and to test the ability of certain potential cardioprotective agents, including scavengers of hydrogen peroxide (dibenzamidostilbene disulfonic acid; DBDS), the.OH free radical (N-(mercaptopropionyl)-glycine; N-MPG), and the HOCl free radical (L-methionine), to protect against oxidative injury. Disease human ventricular tissues were obtained from patients undergoing heart transplantation. Electrophysiological experiments were performed using a traditional micropipette, while the pH(i) was measured by microspectrofluorimetry. We found that (a) H(2)O(2) (30 microM-3 mM) induced a significant dose-dependent intracellular acidosis, (b) H(2)O(2) (30 microM-3 mM) had a notable dose-dependent biphasic effect on the contractile force (an increase, followed by a decrease), while moderate concentrations of H(2)O(2) also inhibited the generation of action potential and increased the diastolic resting force significantly, and (c) N-MPG caused significant block of both the intracellular acidosis and the electromechanical inhibition induced by 3 mM H(2)O(2), whereas L-methionine and DBDS did not. Our data suggest that the toxic effects of H(2)O(2) are caused mainly through the generation of.OH, which is attributed to the intracellular acidosis seen in the diseased human ventricle.

Triptolide Transcriptionally Represses HER2 in Ovarian Cancer Cells by Targeting NF-κB

Triptolide (TPL) inhibits the proliferation of a variety of cancer cells and has been proposed as an effective anticancer agent. In this study, we demonstrate that TPL downregulates HER2 protein expression in oral, ovarian, and breast cancer cells. It suppresses HER2 protein expression in a dose- and time-dependent manner. Transrepression of HER2 promoter activity by TPL is also observed. The interacting site of TPL on the HER2 promoter region is located between −207 and −103 bps, which includes a putative binding site for the transcription factor NF-κB. Previous reports demonstrated that TPL suppresses NF-κB expression. We demonstrate that overexpression of NF-κB rescues TPL-mediated suppression of HER2 promoter activity and protein expression in NIH3T3 cells and ovarian cancer cells, respectively. In addition, TPL downregulates the activated (phosphorylated) forms of HER2, phosphoinositide-3 kinase (PI3K), and serine/threonine-specific protein kinase (Akt). TPL also inhibits tumor growth in a mouse model. Furthermore, TPL suppresses HER2 and Ki-67 expression in xenografted tumors based on an immunohistochemistry (IHC) assay. These findings suggest that TPL transrepresses HER2 and suppresses the downstream PI3K/Akt-signaling pathway. Our study reveals that TPL can inhibit tumor growth and thereby may serve as a potential chemotherapeutic agent.

Effect of S20787, a novel Cl--HCO3- exchange inhibitor, on intracellular pH regulation in guinea pig ventricular myocytes.

S20787 has recently been proposed to be a selective Cl--HCO3- anion exchange (AE) inhibitor in rat cardiomyocytes. The AE transporter mediates sarcolemmal acid influx but is only one part of the cardiac cells dual acid loading mechanism, the other part being a sarcolemmal Cl--OH- exchanger (CHE). We have therefore (1) investigated the differential effects of S20787 on the AE and CHE transporters in isolated guinea pig ventricular myocytes and (2) re-examined the influence of the drug on other sarcolemmal acid transporters by monitoring its effect on intracellular pH (pH(i)) recovery from alkali or acid loads. The pH(i) was measured using microspectrofluorimetry (carboxy-SNARF-1). The results indicate that CHE activity was unaffected by the drug (1-20 microM), whereas up to 78% of AE activity was blocked (K(i) = 3.9 microM). Thus, S20787 targets only the AE component of the dual acid influx system. Activities of other acid-transporting carriers, such as Na+-H+ exchange, Na+-HCO3- co-transport and the monocarboxylic acid transporter, were unaffected by the drug. The inhibitory efficacy of S20787 for AE in guinea pig cardiomyocytes appears to be considerably higher (approximately 78%) than proposed previously for rat cardiomyocytes (50%). This is most likely because, in both cells, a significant fraction (20-30%) of acid influx is mediated through the S20787-insensitive CHE transporter. Previous studies made no allowance for the CHE component, which would result in an underestimation. S20787 is thus a highly selective AE inhibitor which may be useful as an experimental tool and a potential cardiac protective agent in the heart.