Balvin H.L. Chua

Regulation of endothelin-1 mRNA by angiotensin II in rat heart endothelial cells

In a series of experiments carried out in cultured endothelial cells derived from rat hearts (RHE), angiotensin II (AII) is shown to stimulate preproendothelin-1 mRNA in a dose- and time-dependent manner. The induction of preproendothelin-1 mRNA is rapid, reaching a maximal level 1 h after the addition of AII (1 x 10(-8) M). The mRNA levels decline rapidly to basal levels in 4 h. The addition of Losartan (Dup 753; 1 x 10(-6) M), an AII receptor (type I) antagonist, blocks the AII effect. Calphostin C, a potent protein kinase C inhibitor, is able to abolish this effect of AII suggesting that the induction of preproendothelin-1 mRNA is mediated by a protein kinase C-dependent pathway. Since endothelial cells line the inner surface of the myocardium and blood vessels and sense the rise of AII associated with renovascular hypertension at the endothelial surface, these data suggest that endothelin which is produced by RHE cells in response to AII could be an important mediator which may play a role in modulating gene expression in AII-mediated cardiac hypertrophy.

Angiotensin II induces TIMP-1 production in rat heart endothelial cells.

Angiotensin II (AII) was found to upregulate tissue inhibitor of metalloproteineses-1 (TIMP-1) gene expression in rat heart endothelial cells in a dose and time-dependent manner. The maximal stimulation of TIMP-1 mRNA was achieved by 2 h after the addition of AII. This effect was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of cycloheximide superinduced and actinomycin D abolished the induction. These results suggest that AII stimulates TIMP-1 production by a protein kinase C dependent pathway which is dependent upon de novo RNA synthesis. Immunoprecipitation experiment showed an enhanced band of 28 kDa from the conditioned medium of AII-treated cultures. Immunoblot analysis revealed that TIMP-1 was detectable in the conditioned medium 4 h after AII stimulation. Since endothelial cells line the blood vessels and sense the rise in AII associated with hypertension, the TIMP-1 released by these cells may provide an initial trigger leading to cardiac fibrosis in angiotensin-renin dependent hypertension.

Effect of Growth Factors on Collagen Metabolism in Cultured Human Heart Fibroblasts

Using human heart fibroblasts (HHF), we studied the effect of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) on the gene expression of type I collagen, collagenase and tissue inhibitor of metalloproteinases (TIMP). Initially, treatment of HHF with bFGF alone (10 ng/ml) resulted in elevated secretion of collagenase into the culture medium. Subsequent treatment of HHF with TGF-beta in combination with bFGF suppressed collagenase secretion. Northern blot analysis reinforced this observation by revealing an enhancement of the steady-state mRNA level of collagenase in response to bFGF. In order to examine if the collagenase gene was affected by bFGF at the transcriptional level, transfection experiments were carried out with a plasmid containing collagenase promoter linked to chloramphenicol acetyltransferase gene (CAT). Basic FGF stimulated CAT activity by four-fold, indicating increased promoter activity whereas the combination of TGF-beta and bFGF resulted in decreased CAT activity. TGF-beta was shown to increase type I collagen and TIMP mRNA levels by 2.5- and 2.1-fold, respectively. These results suggest that TGF-beta and bFGF may play a pivotal role in regulating collagen metabolism in HHF.

Overexpression of CuZnSOD in coronary vascular cells attenuates myocardial ischemia/reperfusion injury

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 +/- 3% in the transgenic hearts as compared to 30 +/- 5% in the nontransgenic hearts (p <.05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.

Tumor Necrosis Factor-α Induces mRNA for Collagenase and Timp in Human Skin Fibroblasts

We demonstrated previously that growth promoting factors in general could induce the secretion of interstitial collagenase into the medium of human fibroblast cells (HF). In this study, the effect of tumor necrosis factor-ct (TNF-α) on the induction of collagenase and tissue inhibitor of metalloproteinases (TIMP) was examined. Stimulation of quiescent HF cells with 10 ng/ml TNF-α induced the secretion of Mr 57,000, 52,000 procollagenases into the medium. The collagenase activity was elevated 2.8-fold after TNF-α treatment. Northern blot analysis of the steady-state mRNA indicated a tenfold elevation of collagenase transcript after 24 h treatment with 10 ng/ml TNF-α. The increase in collagenase mRNA was due to transcriptional activation of collagenase gene activity. TIMP mRNA level increased three-fold after TNF-α treatment. The activity of TNF-α on collagenase and TIMP induction may play an important role in tissue inflammatory, repair and remodeling processes after wound and injury.

Estrone Modulates the EGF Receptor in the Liver of db/db Mouse

The genetically diabetic db/db mouse is an excellent model to study the effect of diabetes on hormone receptors. The decrease of EGF binding sites could be detected in the hepatic microsomes of diabetic mice as early as 3 weeks of age. In addition, there was an age-related decrease in the autophosphorylating activity of EGF receptor isolated from the liver of diabetic mice. Estrone feeding (0.005%) partially restored this autophosphorylating activity. Northern blot analysis showed that the hepatic EGF receptor transcripts were dramatically decreased during the progression of diabetes and could be reversed by estrone feeding. Transfection experiments carried out on HepG2 cells using EGF receptor promoter (pERCAT-6) demonstrated that addition of 2 x 10(-8) M estrone stimulated chloramphenicol acetyltransferase activity. Our results suggest that estrone modulates EGF receptor by enhancing EGF receptor transcripts and the promoter activity of this gene.

Anti-Idiotypic Antibody as a Probe for Anf Receptor

Generation of anti-idiotypic antibodies (anti-Id) is a rapid and new approach to produce anti-receptor antibodies without isolation of the receptor. This report describes the production of polyclonal anti-ANF anti-Id antibodies. These antibodies could inhibit the binding of [125I]-ANF to its receptor on aortic smooth muscle cells. Immunoblot analysis of detergent Chaps-solubilized adrenal gland membranes indicated that these anti-Id antibodies could recognize an Mr 130,000 band under nonreducing condition and an Mr 70,000 band under reducing condition. In addition, these antibodies could slightly increase the production of cyclic GMP in aortic smooth muscle cells.