Type II collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) are capable of decreasing the levels of alpha 1(II) procollagen mRNA and suppressing the synthesis of type II collagen in cultured human chondrocytes. Data reported here show that these effects of IL-1 and IFN-gamma on the expression of the human type II collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three types of experimental evidence: (1) in nuclear run-off assays, preincubation of chondrocytes with either IL-1 or IFN-gamma decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) indicated that the suppression of alpha 1(II) procollagen mRNA by IL-1 could not be ascribed to decreased mRNA stability; and (3) a plasmid (pCAT-B/4.0) containing 4.0 kb of 5'-flanking sequences of COL2A1 (-577/+3428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT-B/4.0 was observed in human chondrocytes incubated with an insulin-containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT-B/4.0 in these cells was inhibited by either IL-1 or IFN-gamma. Furthermore, expression of pCAT-B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL-1 or IFN-gamma is mediated primarily at the transcriptional level.

1Vascular ageing is accelerated in patients with diabetes. However, the underlying mechanism remains unclear. Here, we show that high glucose induces activation of apoptosis signal-regulating kinase 1 (ASK1), an apoptosisinducing signal that mediates endothelial cell senescence induced by hyperglycemia. High glucose induced a timedependent increase in the levels of ASK1 expression and its activity in human umbilical vein endothelial cells (HUVECs). Incubation of endothelial cells with high glucose increased the proportion of cells expressing senescence-associated ␤-galactosidase (SA-␤-gal) activity. However, transfection with an adenoviral construct including a dominant negative form of ASK1 gene significantly inhibited SA-␤-gal activity induced by high glucose. In addition, infection with an adenoviral construct expressing the constitutively active ASK1 gene directly induced an increase in the levels of SA-␤-gal activity. Activation of the ASK1 signal also enhanced plasminogen activator inhibitor-1 (PAI-1) expression in HUVECs. Induction of senescent endothelial cells in aortas and elevation of plasma PAI-1 levels were observed in streptozotocin (STZ) diabetic mice, whereas these changes induced by STZ were attenuated in ASK1-knockout mice. Our results suggest that hyperglycemia accelerates endothelial cell senescence and upregulation of PAI-1 expression through activation of the ASK1 signal. Thus, ASK1 may be a new therapeutic target to prevent vascular ageing and thrombosis in diabetic patients.

Interleukin-1 induced a time-dependent release of high levels of nitric oxide from rat vascular smooth muscle cells up to 96 hours. A time-dependent release of lactate dehydrogenase was also induced by Interleukin-1 from 72 to 96 hours after its stimulation. In situ nick end-labeling assay revealed that incubation for 48 hours with interleukin-1 induced a positive staining of fragmented nuclei. However, NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, inhibited both lactate dehydrogenase release and DNA fragmentation induced by interleukin-1. Furthermore, sodium nitroprusside, a nitric oxide donor, also induced lactate dehydrogenase release and DNA fragmentation. Fluorescent staining of DNA revealed patches of irregularly dispersed, brightly staining, and condensed chromatin in rat vascular smooth muscle cells treated with sodium nitroprusside. Flow cytometric analysis with monoclonal antibody against human Fas revealed that expression of Fas was upregulated by sodium nitroprusside in human vascular smooth muscle cells. Methylene blue, an inhibitor of soluble guanylate cyclase, did not affect sodium nitroprusside-induced upregulation of Fas. Furthermore, 8-bromo-guanosine 3':5'-cyclic monophosphate, an analogue of cGMP, did not upregulate Fas expression. These findings indicate that nitric oxide released from vascular smooth muscle cells may induce apoptosis in vascular smooth muscle cells themselves and also induced upregulation of Fas via a cGMP-independent mechanism. Thus, nitric oxide could trigger the remodeling of atherosclerotic plaques.

Active forms of vitamin D3, 1 alpha-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3, were administered in an open-design study to 40 patients with psoriasis vulgaris in three ways: to 17 patients 1 alpha-hydroxyvitamin D3 was given orally at a dose of 1.0 micrograms/day for 6 months, to four patients 1 alpha,25-dihydroxyvitamin D3 was given orally at a dose of 0.5 microgram/day for 6 months, and 19 patients were given 1 alpha,25-dihydroxyvitamin D3 applied topically at concentration of 0.5 microgram/g of base for 8 weeks. Improvement was observed at the end of the individual study periods in 13 (76%) patients in Group 1 with a mean period of treatment (+/- SD) of 2.7 +/- 0.6 months, in one patient in Group 2 at 3 months after the start of treatment, and in 16 (84%) patients in Group 3 when the chemical was applied for 3.3 +/- 1.2 weeks. No side-effects were observed in any of these trials. These data suggest that psoriasis may respond to active metabolites of vitamin D3 and that abnormalities in vitamin D metabolism or in responsiveness of the skin cells to active metabolites of vitamin D may be involved in the pathogenesis of this skin disease.