Autocrine TGF-beta1 mediates angiotensin II-induced proliferative response of cerebral vessels in vivo.

BACKGROUND: Angiotensin II (Ang) has been shown to induce expression of transforming growth factor-beta1 (TGF-beta1) in cardiovascular cells in vitro, but the regulation of TGF-beta1 by Ang has not been shown in cerebral vessels in vivo. Here, we tested the hypothesis that Ang promotes proliferative and fibrogenic responses in cerebral vessels through autocrine production and signaling of TGF-beta1 by vascular smooth muscle cells (VSMC). METHODS: Rats were implanted with miniosmotic pumps that delivered a low dose of Ang (9 microg/kg/h subcutaneously for 4 to 28 days). To test for autocrine production and signaling of TGF-beta1 by VSMC, we suppressed TGF-beta1 gene expression in VSMC by infusing antisense oligodeoxynucleotide (AS-ODN) versus sense oligodeoxynucleotide (S-ODN) into the cisterna magna. RESULTS: Systemic infusion of Ang for 28 days caused upregulation of proliferative cell nuclear antigen (PCNA) and of collagen type I in endothelium and VSMC of basilar arteries, with these changes observed as early as day 4, before the onset of hypertension. Also by day 4, significant increases in expression of TGF-beta1, TGF-beta receptors I and II, and phospho-Smad3 were observed in endothelial and VSMC layers, but plasma levels of TGF-beta1 were unchanged. With AS-ODN, but not S-ODN, TGF-beta1 was significantly reduced in VSMC but not in endothelial layers of the basilar artery, and PCNA and collagen upregulation in VSMC were essentially eliminated. CONCLUSIONS: Autocrine TGF-beta1 signaling in VSMC is required for Ang-induced proliferative and fibrogenic responses in cerebral vessels in vivo.

Interaction of ACTH synthetic fragments with rat adrenal cortex membranes.

Synthetic peptide, corresponding to the amino acid sequence 11-24 of human adrenocorticotropic hormone (ACTH), was labeled with tritium (specific activity of 22 Ci/mmol). [(3)H]ACTH (11-24) was found to bind to rat adrenal cortex membranes with high affinity and specificity (K(d) = 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) have been synthesized and their ability to inhibit the specific binding of [(3)H]ACTH (11-24) to adrenocortical membranes has been investigated. Unlabeled fragment ACTH 15-18 (KKRR) was found to replace in a concentration-dependent manner [(3)H]ACTH (11-24) in the receptor-ligand complex (K(i) = 2.3 +/- 0.2 nM). ACTH (15-18) was labeled with tritium (specific activity of 20 Ci/mmol). [(3)H]ACTH (15-18) was found to bind to rat adrenal cortex membranes with high affinity (K(d) = 2.1 +/- 0.1 nM). The specific binding of [(3)H]ACTH (15-18) was inhibited by unlabeled ACTH (11-24) (K(i) = 2.2 +/- 0.1 nM). ACTH (15-18) at the concentration range of 1-1000 nM did not affect the adenylate cyclase activity in adrenocortical membranes.

Tumor necrosis factor-related apoptosis-inducing ligand enhances collagen production by human lung fibroblasts.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/APO-2L) is a member of the tumor necrosis factor family that induces apoptosis in a variety of transformed cell lines and in normal human hepatocytes and brain cells. Soluble TRAIL at high concentrations was found to induce apoptotic death in normal human lung fibroblasts, whereas at low concentrations it was found to stimulate collagen production by these cells. Collagen alpha2(I) mRNA expression was assessed by semiquantitative reverse transcriptase/polymerase chain reaction; total soluble collagen was measured in culture supernatants by the Sircol assay. Both alpha2(I) collagen mRNA level and total soluble collagen secretion were increased upon TRAIL stimulation, with peak response (> 4-fold increase in mRNA level) at 1 ng/ml TRAIL. Analysis of the transcriptional response in TRAIL-stimulated fibroblasts, using DNA microarray hybridization, revealed an augmented expression of a number of genes involved in tissue remodeling, including those related to the transforming growth factor-beta (TGF-beta) pathway. DNA microarray results for the increase in TGF-beta1 mRNA level were confirmed by Northern blot analysis and by measurements of total active TGF-beta1 in culture supernatants. In addition, pan-specific TGF-beta antibody was shown to inhibit TRAIL-stimulated collagen mRNA and protein expression. These data suggest that TRAIL can enhance extracellular matrix synthesis in fibroblasts by triggering TGF-beta production that acts in an autocrine manner.

Beta-endorphin-like peptide SLTCLVKGFY reduces the production of 11-oxycorticosteroids by rat adrenal cortex through nonopioid beta-endorphin receptors.

Beta-endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid beta-endorphin receptors on rat adrenal cortex membranes (Kd = 31.6 +/- 0.2 nM, Bmax = 37.4 +/- 2.2 pmol/mg protein). Immunorphin at concentrations of 10(-9) to 10(-6) M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10-100 microg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.