Mian-Shin Tan

Native and oxidized low-density lipoproteins enhance superoxide production from diabetic rat glomeruli.

Oxygen free radicals have been implicated in mediating diabetic complications, and patients with diabetic nephropathy frequently show increased levels of circulating and oxidized low–density lipoproteins (LDL). In the present study, we measured the superoxide production of glomeruli isolated from poorly controlled diabetic (streptozotocin) rats sacrificed 1 week and 1, and 3 months after the induction of diabetes. The animals were stimulated with native and oxidized LDL isolated from normal humans with normolipidemia. The superoxide ion was measured by using a spectrophotometer. The results demonstrated that the poorly controlled diabetic rat glomeruli showed a significantly higher production of superoxide than normal glomeruli under basal conditions, and this production increased further with the progression of diabetes. Stimulation with either LDL or oxidized LDL enhanced superoxide production by diabetic glomeruli, with oxidized LDL being more potent than LDL. Our results suggest that oxidized LDL may play important roles in the pathogenesis of diabetic nephropathy through enhanced generation of oxygen free radicals.

Effects of increased pulmonary flow on the expression of endothelial nitric oxide synthase and endothelin-1 in the rat

The purpose of the study was to assess whether increased pulmonary flow and subsequent development of pulmonary vascular remodelling could alter the expression of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) in the rat lung. Nine 42-day-old Wistar rats underwent abdominal aortocaval shunt to increase pulmonary blood flow for 12 weeks. The shunt resulted in significant medial hypertrophy of pulmonary artery without significant alterations in pulmonary or systemic blood pressure. Using competitive reverse transcription-PCR, significant increases in the preproET-1 mRNA expression and eNOS mRNA expression in the lungs of rats with abdominal aortocaval shunt were detected. Increased eNOS protein in the lung of shunt rats was also found by Western blot analysis. However, the plasma ET-1 concentration in the pulmonary artery (sham: 5+/-0.7 pg/ml; shunt: 6+/-0.8 pg/ml) or the lung ET-1 content (sham: 218+/-41 ng/g protein; shunt: 224+/-40 ng/g protein) was unchanged. There was an elevated immunohistochemical expression of eNOS, but not ET-1, in the pulmonary vascular endothelium in rats with the shunt. These results suggest that eNOS and ET-1 may be involved in remodelling prior to the development of pulmonary hypertension.

Decrease of Renal Endothelin 1 Content and Gene Expression in Diabetic Rats with Moderate Hyperglycemia

To investigate the intrarenal endothelin 1 (ET-1) synthesis in streptozocin (STZ) diabetic rats with moderate hyperglycemia, we measured plasma ET-1, renal ET-1 mRNA, and renal tissue ET-1 levels. The renal ET-1 mRNA expression progressively decreased from the 2nd to the 6th week after induction of diabetes by STZ. The renal ET-1 mRNA expression and the renal tissue ET-1 content were significantly reduced in 8 diabetic rats with a mean blood glucose level of 21.0 +/- 0.4 mM as compared with 7 normal rats sacrificed at the 6th week after STZ or citric buffer injection. The reduction of renal ET-1 and mRNA levels was ameliorated in 9 diabetic rats with a mean blood glucose level of 6.9 +/- 0.7 mM after strict glycemic control by insulin treatment. Kidney weight and glomerular filtration rate in moderately hyperglycemic rats were significantly increased as compared with normal rats at the 6th week after STZ injection. The mean plasma ET-1 levels in moderately hyperglycemic diabetic rats were not different from those of the other two groups. This study demonstrates that moderate hyperglycemia in diabetic rats is associated with a reduction in renal ET-1 synthesis. Whether decreased renal ET-1 synthesis is an adaptive phenomenon of a renal hemodynamic change during the early stage of diabetes is worthy of further investigation.

Oxidized low-density lipoprotein stimulates endothelin-1 release and mRNA expression from rat mesangial cells

Evidence indicates that the glomerular injuries and renal hemodynamic abnormalities in hyperlipidemia are caused by the interaction of low-density lipoprotein (LDL) and oxidized LDL (Ox-LDL) with mesangial cells. Experiments were designed to investigate whether the synthesis of mesangial cell endothelin-1 (ET-1), a potent renal vasoconstrictor and mitogen for mesangial cells, is modulated by LDL and Ox-LDL. Using competitive semi-quantitative reverse transcription polymerase chain reaction (PCR), we report that the expression of cultured rat mesangial cell ET-1 mRNA was increased after treatment with Ox-LDL but not native LDL. Ox-LDL stimulated the release of ET-1 peptide into the culture medium in a time- and concentration-dependent manner. The maximal effect was observed at a concentration of 100 microg/ml, and a higher dose of Ox-LDL was found to be cytotoxic to the mesangial cells. Our results suggest that ET-1 released by Ox-LDL stimulation may be an important pathogenetic factor contributing to the renal hemodynamic alterations and progressive chronic renal diseases induced by hypercholesterolemia.

Attenuation of pulmonary hypertension secondary to left ventricular dysfunction in the rat by Rho-kinase inhibitor fasudil.

Pulmonary hypertension (PH) in left ventricular dysfunction is attributable not only to backward failure of the left ventricle, but also to increased pulmonary vascular resistance (PVR) in some patients. Recently, Rho‐kinase has been known as a potent growth stimulator and mediator of vasoconstriction, and Rho‐kinase inhibitors could ameliorate PVR, little is known about the role of Rho‐kinase in left ventricular dysfunction‐induced PH. We utilized the ascending aortic‐banded rat and assessed the effect of Rho‐kinase inhibitor fasudil on the development of PH secondary to left ventricular dysfunction. Subsequently, in rats subjected to aortic banding for 6 weeks, there were increases in mean pulmonary arterial pressure, pulmonary arteriolar medial thickness, active RhoA, Rho‐kinase II, Rho‐kinase activity, endothelial nitric oxide synthase (eNOS) and endothelin‐1(ET‐1) concomitant with decreased levels in NO and cGMP in the lung. Treatment with fasudil at a dose of 30 mg/kg/day from days 1 to 28 or from days 29 to 42 decreased the mean pulmonary arterial pressure by 57% and 56%, right ventricular hypertrophy by 31% and 30%, pulmonary arteriolar medial thickness by 50% and 50%, and pulmonary expression of Rho‐kinase II by 41% and 28%, respectively, as well as augmented pulmonary expression of eNOS by 16% and 31% and NO by 50% and 76%, respectively, when compared with the vehicle controls. In conclusion, these results suggest that inhibition of Rho‐kinase may provide therapeutic potential for preventing and attenuating the development of PH in left ventricular dysfunction. Further translational study in human is needed to substantiate the findings. Pediatr Pulmonol. 2011; 46:45–59.

Effects of sildenafil on pulmonary hypertension and levels of ET-1, eNOS, and cGMP in aorta-banded rats.

Sildenafil, an oral phosphodiesterase Type 5 inhibitor, has vasodilatory effects through a cGMP-dependent mechanism. We previously showed that aortic banding could result in left ventricular overloading and pulmonary hypertension (PH). In this study, we investigated whether early administration of sildenafil, either immediately after or 2 weeks after aortic banding, could ameliorate the development of PH and alter gene expression of endothelin (ET)-1 and endothelial nitric oxide synthase (eNOS), and alter the levels of cGMP in rats undergoing an ascending aortic banding. Rats (n = 32) were divided into sham-operated and banding groups with or without treatment. The banded rats were further divided into three groups: (i) receiving saline on Days 1–28 (AOB28; n = 8), (ii) receiving saline on Days 1–14 followed by treatment with 50 mg/kg/day sildenafil on Days 15–28 (AOB28/Sil15–28; n = 8), and (iii) receiving 50 mg/kg/day sildenafil on days 1–28 (AOB28/Sil1–28; n = 8). The sham-operated rats were administrated saline on Days 1–28 (n = 8). Four weeks after banding, there was a significant development of PH with pulmonary vascular remodeling. Although both sildenafil-treatment groups had significant increases in cGMP and had reductions in the thickening in the medial layer of pulmonary arteriole, notable attenuation of PH occurred only in the AOB28/Sil1–28 group. PreproET-1 and eNOS messenger RNA (mRNA) expressions were measured by competitive reverse transcription polymerase chain reaction, and eNOS protein was determined by Western blotting. Sildenafil did not alter the elevated ET-1 or preproET-1 mRNA in banded rats. Interestingly, pulmonary eNOS increased in the AOB28/Sil1–28 group. In conclusion, early treatment with sildenafil inhibited the rise in pulmonary arterial pressure and pulmonary vascular remodeling in PH secondary to heart failure, and cGMP, but not ET-1, might be involved. Clinically, early repeated administration of sildenafil may offer an alternative in protecting against PH in heart failure.

Statins ameliorate pulmonary hypertension secondary to left ventricular dysfunction through the Rho‐kinase pathway and NADPH oxidase

Pulmonary hypertension (PH) is a devastating disorder, for which no therapy is curative. It has been reported that pulmonary vascular remodeling, associated with increasing mean pulmonary arterial pressure and upregulated expression of endothelial nitric oxide synthase (eNOS), endothelin‐1 (ET‐1), RhoA/RhoH‐kinase results in the development of PH. Oxidative stress and the RhoA/Rho‐kinase pathway are also thought to be involved in the pathophysiology of PH. Statins are 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors (HMG‐CoA reductase inhibitors) with pleiotropic effects and are potential agents for the treatment of PH. In this study, we investigated the beneficial effects of simvastatin on the development of PH secondary to left ventricular dysfunction.

The effects of debanding on the lung expression of ET-1, eNOS, and cGMP in rats with left ventricular pressure overload.

Pulmonary hypertension (PH) usually develops secondary to left ventricular (LV) dysfunction; therefore, it is also called retrograde PH. To investigate our hypothesis that PH is at least partially reversible, as in some congenital heart diseases, in a rat model we investigated whether release of constriction could attenuate pulmonary vascular remodeling and change the expression of endothelin (ET)-1 and endothelial nitric oxide synthase (eNOS). We used rats with LV dysfunction produced by an ascending aortic banding. In this study, there were four groups enrolled: 4-weeks banded (AOB1–28; n = 7), 7-weeks banded (AOB1–49; n = 7), debanded groups (AOB1–28/DeB29–49; n = 7), and rats receiving a sham operation (n = 7). Subsequently, there was significant attenuation of medial hypertrophy in pulmonary arterioles and reversal of PH in the AOB1–28/DeB29–49 group (sham, 19 ± 1.3 mm Hg; AOB1–28, 31 ± 2.7 mm Hg; AOB1–49, 32 ± 2.7 mm Hg; and AOB1–28/DeB29–49, 20 ± 1.3 mm Hg). PreproET-1 mRNA and eNOS mRNA were measured by competitive reverse transcriptase (RT) polymerase chain reaction (PCR), and eNOS was measured by Western blotting. Compared with the banded groups, debanding significantly decreased pulmonary preproET-1 mRNA, pulmonary ET-1 (sham, 210 ± 12 pg/g protein; AOB1–28, 242 ± 12 pg/g protein; AOB1–49, 370 ± 49 pg/g protein; and AOB1–28/DeB29–49, 206 ± 1.9 pg/g protein), and plasma ET-1 levels (sham, 10.1 ± 1.5 pg/ml; AOB1–28, 13.4 ± 2.0 pg/ml; AOB1–49, 15.4 ± 2.0 pg/ml; and AOB1–28/DeB29–49, 10.3 ± 0.9 pg/ml protein). Debanding could not, however, alter pulmonary eNOS, eNOS mRNA, or cGMP. These findings suggest that pulmonary vascular remodeling, increased pulmonary arterial pressure, and upregulation of ET-1 gene expression are all reversible. We infer that it is the upregulated gene expression of ET-1, not eNOS, that is closely related to the development of the PH secondary to 4 weeks of aortic banding.

Protective effects of a dual endothelin converting enzyme/neutral endopeptidase inhibitor on the development of pulmonary hypertension secondary to cardiac dysfunction in the rat

Endothelium‐derived nitric oxide (NO) and endothelin (ET)‐1 interact to regulate the vascular tone in pulmonary hypertension (PH). We investigated the protective effects of an orally active, dual endothelin converting enzyme (ECE)/neutral endopeptidase (NEP) inhibitor/CGS 26393 on pulmonary vascular remodeling and pulmonary expressions of ET‐1 and endothelial nitric oxide synthase (eNOS) during the development of PH secondary to cardiac dysfunction. Significant increases in the mean pulmonary arterial pressure, pulmonary arteriolar medial thickness, and pulmonary expression of ET‐1 were seen in rats subjected to aortic banding for 4 weeks, compared with sham‐operated rats. Treatment with CGS 26393 (30 mg/kg, twice daily, p.o.) began on 1 day after aortic banding. CGS 26393 treated rats had lower mean pulmonary arterial pressure (15 ± 1 mmHg, mean ± SEM, P < 0.05) compared to vehicle‐treated rats (37 ± 1 mmHg). It also normalized pulmonary arteriolar medial thickness and reduced the levels of pulmonary ET‐1 and big ET‐1 by 55% (P < 0.05) and 28% (P < 0.01), respectively, when compared with vehicle‐treated animals. Meanwhile, the expressions of eNOS mRNA and eNOS protein and cGMP levels in the lung of CGS 26393‐treated rats were increased by 62% (P < 0.05), 100% (P < 0.05), and 32% (P < 0.01), respectively, compared to the vehicle‐treated rats. These results suggest that CGS 26393 could offer preventive effects on the development of PH by ameliorating pulmonary remodeling, decreasing ET‐1 production, and up‐regulating eNOS and cGMP in aorta‐banded rats. However, the molecular mechanisms by which treatment with CGS 26393 results in altered expressions of eNOS and cGMP awaits further investigation. Pediatr Pulmonol. 2010;45:1076–1085.