The product of the von Hippel-Lindau gene, pVHL, targets the ␣ subunits of the heterodimeric transcription factor hypoxiainducible factor (HIF) for polyubiquitination in the presence of oxygen. The binding of pVHL to HIF is governed by the enzymatic hydroxylation of conserved prolyl residues within peptidic motifs present in the HIF␣ family members. By using a biochemical purification strategy, we have identified a human homolog of Caenorhabditis elegans Egl9 as a HIF prolyl hydroxylase. In addition, we studied the activity of a structurally diverse collection of low molecular weight inhibitors of procollagen prolyl 4-hydroxylase as potential inhibitors of the HIF hydroxylase. A model compound of this series stabilized HIF in a variety of cells, leading to the increased production of its downstream target, vascular endothelial growth factor.
Patients with scleroderma receiving Iloprost as a treatment for severe Raynaud's phenomenon report a reduction in skin tightness, suggesting that this drug inhibits skin fibrosis. Connective tissue growth factor (CTGF), a recently described profibrotic cytokine, acts downstream and in concert with TGF-β to stimulate the fibrotic process and is involved in the fibrosis seen in scleroderma. Here we show that Iloprost, acting by elevation of cAMP, blocks the induction of CTGF and the increase in collagen synthesis in fibroblasts exposed to TGF-β. The potency of Iloprost with respect to suppression of CTGF far exceeds that of other prostanoid receptor agonists, suggesting that its effect is mediated by the prostacyclin receptor IP. By sampling dermal interstitial fluid using a suction blister device, we show that CTGF levels are greatly elevated in the dermis of scleroderma patients compared with healthy controls and that Iloprost infusion causes a marked decrease in dermal CTGF levels. These studies suggest that Iloprost could be reducing the level of a key profibrotic cytokine in scleroderma patients and that endogenous production of eicosanoids may limit the fibrotic response to TGF-β.
Connective tissue growth factor (CTGF) is felt to be one of the key profibrotic factors and is a downstream effector molecule mediating the action of transforming growth factor (TGF)-beta1, a cytokine known to induce severe and progressive fibrosis. However, the in vivo fibrogenic effect of isolated CTGF expression is not well described. We used adenoviral gene transfer to transiently overexpress CTGF in rat lungs after intratracheal administration and compared it with transient overexpression of active TGF-beta1 delivered by a similar adenovirus vector. This high expression of CTGF over 6-10 days induced a moderate but reversible fibrosis. We observed an increase of fibronectin, procollagen 1a2, and endogenous CTGF gene expression at 14 days, which suggested an indirect activation by CTGF. Tissue inhibitor of metalloproteinase-1 was weakly and transiently upregulated after CTGF exposure. These same genes were robustly and persistently stimulated by TGF-beta1 from Day 3 to Day 21. This data suggested that CTGF may act as a TGF-beta1 cofactor rather than a direct fibrogenic factor. We demonstrate that CTGF overexpression can initiate fibrogenic activity but likely requires the presence of additional factors, such as tissue inhibitor of metalloproteinase-1, to maintain a nonfibrolytic environment and to cause progression of fibrosis.
Patients with scleroderma receiving Iloprost as a treatment for severe Raynaud's phenomenon report a reduction in skin tightness, suggesting that this drug inhibits skin fibrosis. Connective tissue growth factor (CTGF), a recently described profibrotic cytokine, acts downstream and in concert with TGF-β to stimulate the fibrotic process and is involved in the fibrosis seen in scleroderma. Here we show that Iloprost, acting by elevation of cAMP, blocks the induction of CTGF and the increase in collagen synthesis in fibroblasts exposed to TGF-β. The potency of Iloprost with respect to suppression of CTGF far exceeds that of other prostanoid receptor agonists, suggesting that its effect is mediated by the prostacyclin receptor IP. By sampling dermal interstitial fluid using a suction blister device, we show that CTGF levels are greatly elevated in the dermis of scleroderma patients compared with healthy controls and that Iloprost infusion causes a marked decrease in dermal CTGF levels. These studies suggest that Iloprost could be reducing the level of a key profibrotic cytokine in scleroderma patients and that endogenous production of eicosanoids may limit the fibrotic response to TGF-β.
We examined the potential of radiolabeled somatostatin analogs, 125I‐Tyr‐3‐octreotide (125I‐octreotide), 111In‐DTPA(diethylenetriaminepentaacetatic acid)‐d‐Phe‐1‐octreotide (111In‐octreotide), and 188Re‐octreotide for targeting small‐cell lung cancer (SCLC) in a mouse model. Tyr‐3‐octreotide was labeled with 125I by the chloramine T method, and 111In‐octreotide was obtained as a kit, while 188Re was eluted from a 188W/188Re generator, and octreotide was directly labeled with 188Re by reducing disulfide bonds. The 125I‐, 111In‐, and 188Re‐octreotides were injected i.v. into athymic mice bearing NCI‐H69 tumors, and the biodistributions were determined at 15 min, and 2, 4, 8, and 24 h. Tumor uptakes were 0.5±0.2, 0.3±0.1, 0.3±0.1 %ID/g, and tumor‐to‐blood ratios were 1.8, 11.9, 1.2 at 8 h for 125I‐, 111In‐, and 188Re‐octreotides, respectively. Accumulations of 111In‐octreotide in normal tissues were lower than those of 125I‐ and 188Re‐octreotides. 188Re‐octreotide can be used to localize SCLC lesions as efficiently as radioiodinated octreotide. However, 111In‐octreotide was the most suitable agent to obtain high tumor‐to‐normal tissue contrast for localizing SCLC.
In mouse hepatocytes, the gap junctional proteins connexin32 (Cx32) and connexin26 (Cx26) are expressed in the same gap junctional plaque. Expression of the major Cx32 protein is downregulated during liver regeneration and cholestasis. Here we have analyzed the acute-phase response (after experimental inflammation) and circadian connexin expression in Cx32-deficient and wild-type mouse liver. Acute-phase response was triggered by intraperitoneal injection of lipopolysaccharide (LPS). Injection of recombinant mouse interleukin-1beta (mIL-1beta), mIL-6 or tumor necrosis factor alpha (mTNF-alpha) had no inflammatory effect. Northern blot analysis of positive and negative acute-phase transcripts following stimulation with cytokine or LPS revealed no difference between Cx32-deficient livers and wild-type controls, suggesting that loss of the Cx32 gene had no effect on experimental liver inflammation. Actin, beta-fibrinogen and Cx26 transcripts were increased after endotoxin stimulation. Under conditions of hepatic acute-phase response, Cx32 transcripts were not detected in LPS-treated livers of wild-type mice. Immunoblot analysis of proteins from inflamed wild-type livers indicated a strongly diminished amount of Cx32 protein, whereas the level of Cx26 protein was increased. Although intraperitoneal injection of mIL-1, mIL-6 as well as mTNF-alpha did not induce an acute-phase response, Cx32 protein expression was diminished, suggesting that post-transcriptional downregulation of Cx32 preceded the acute-phase response. Northern blot hybridization of RNA from wild-type and Cx32-deficient mouse liver revealed a similar circadian regulation of Cx26 and GAPDH transcripts with maximal expression around 2 p.m. and a minimum after midnight.
In order to evaluate the feasibility of 188Re-labeled antibodies for radioimmunotargeting, monoclonal antibody B72.3, recognizing TAG-72, expressed on the surface membranes of colorectal cancer cells, was directly labeled with 188Re, obtained from a 188W/188Re generator, using stannous tartrate and compared with 125I-labeled B72.3. As a control, a human IgG was also radiolabeled with 188Re and 125I. Prepared antibodies for 188Re labeling could be stored as kits. Biodistribution was determined in nude mice inoculated with human colorectal carcinoma LoVo. Labeling efficiency and immunoreactivity of 188Re-B72.3 were 80.3% and 64.7%, respectively. 188Re-B72.3 localized specifically in the LoVo tumors. Although the absolute tumor accumulation level of 188Re-B72.3 was lower than 125I-B72.3, 188Re-B72.3 demonstrated higher tumor-to-blood contrast than the 125I-labeled counterpart, 2.04 +/- 0.44 vs. 1.05 +/- 0.28 at 96 hours, because of fast clearance from the blood. 188Re-B72.3 seemed efficient for the imaging and therapy of colorectal carcinoma.
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