Cartilage specimens from osteoarthritis (OA)-affected patients spontaneously released PGE 2 at 48 h in ex vivo culture at levels at least 50-fold higher than in normal cartilage and 18-fold higher than in normal cartilage ϩ cytokines ϩ endotoxin. The superinduction of PGE 2 production coincides with the upregulation of cyclooxygenase-2 (COX-2) in OA-affected cartilage. Production of both nitric oxide (NO) and PGE 2 by OA cartilage explants is regulated at the level of transcription and translation. Dexamethasone inhibited only the spontaneously released PGE 2 production, and not NO, in OA-affected cartilage. The NO synthase inhibitor H N G -monomethyl-L -arginine monoacetate inhibited OA cartilage NO production by Ͼ 90%, but augmented significantly (twofold) the spontaneous production of PGE 2 in the same explants. Similarly, addition of exogenous NO donors to OA cartilage significantly inhibited PGE 2 production. Cytokine ϩ endotoxin stimulation of OA explants increased PGE 2 production above the spontaneous release. Addition of L -NMMA further augmented cytokine-induced PGE 2 production by at least fourfold. Inhibition of PGE 2 by COX-2 inhibitors (dexamethasone or indomethacin) or addition of exogenous PGE 2 did not significantly affect the spontaneous NO production. These data indicate that human OA-affected cartilage in ex vivo conditions shows ( a ) superinduction of PGE 2 due to upregulation of COX-2, and ( b ) spontaneous release of NO that acts as an autacoid to attenuate the production of the COX-2 products such as PGE 2 . These studies, together with others, also suggest that PGE 2 may be differentially regulated in normal and OA-affected chondrocytes. ( J. Clin. Invest. 1997. 99:1231-1237.)

Tetracyclines have recently been shown to have ''chondroprotective'' effects in inf lammatory arthritides in animal models. Since nitric oxide (NO) is spontaneously released from human cartilage affected by osteoarthritis (OA) or rheumatoid arthritis in quantities sufficient to cause cartilage damage, we evaluated the effect of tetracyclines on the expression and function of human OA-affected nitric oxide synthase (OA-NOS) and rodent inducible NOS (iNOS). Among the tetracycline group of compounds, doxycycline > minocycline blocked and reversed both spontaneous and interleukin 1␤-induced OA-NOS activity in ex vivo conditions. Similarly, minocycline > doxycycline inhibited both lipopolysaccharide-and interferon-␥-stimulated iNOS in RAW 264.7 cells in vitro, as assessed by nitrite accumulation. Although both these enzyme isoforms could be inhibited by doxycycline and minocycline, their susceptibility to each of these drugs was distinct. Unlike acetylating agents or competitive inhibitors of L-arginine that directly inhibit the specific activity of NOS, doxycycline or minocycline has no significant effect on the specific activity of iNOS in cell-free extracts. The mechanism of action of these drugs on murine iNOS expression was found to be, at least in part, at the level of RNA expression and translation of the enzyme, which would account for the decreased iNOS protein and activity of the enzyme. Tetracyclines had no significant effect on the levels of mRNA for ␤-actin and glyceraldehyde-3-phosphate dehydrogenase nor on levels of protein of ␤-actin and cyclooxygenase 2 expression. These studies indicate that a novel mechanism of action of tetracyclines is to inhibit the expression of NOS. Since the overproduction of NO has been implicated in the pathogenesis of arthritis, as well as other inf lammatory diseases, these observations suggest that tetracyclines should be evaluated as potential therapeutic modulators of NO for various pathological conditions.

Vascular endothelial growth factor (VEGF) receptorFlk-1/KDR in endothelial cells is activated during vasculogenesis and angiogenesis upon ligand-receptor interaction. Activated Flk-1/KDR has been shown to recruit Src homology 2 domain-containing signaling molecules that are known to serve as links to the activation of the mitogen-activated protein (MAP) kinase signaling pathway. To define the functional significance of phosphatidylinositol (PI) 3-kinase in VEGF signaling, we have examined its role in human umbilical vein endothelial cell (HUVEC) cycle progression. We show herein that p85, the regulatory subunit of PI 3-kinase, is constitutively associated with Flk-1/KDR. The treatment of HUVECs with VEGF promoted tyrosine autophosphorylation of Flk-1/KDR and also induced phosphorylation of p85. This was followed by an increase in the PI 3-kinase activity, which was sensitive to wortmannin, a potent PI 3-kinase inhibitor. VEGF also induced a striking activation of MAP kinase in a time-dependent manner. Inhibition studies with both a dominant-negative p85 mutant and the PI 3-kinase inhibitor, wortmannin, were employed to show for the first time that VEGF-stimulated PI 3-kinase modulates MAP kinase activation and nuclear events such as transcription from c-fos promoter and entry into the synthesis (S)-phase. Our data demonstrate the importance of PI 3-kinase as a necessary signaling component of VEGF-mediated cell cycle progression.

Platelet-derived growth factor signaling critically regulates postinfarction repair. Both PDGFR-beta- and PDGFR-alpha-mediated pathways promote collagen deposition in the infarct. Activation of PDGF-B/PDGFR-beta is also involved in recruitment of mural cells by neovessels, regulating maturation of the infarct vasculature. Acquisition of a mural coat and maturation of the vasculature promotes resolution of inflammation and stabilization of the scar.