Objective: To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.Methods: IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.Results: Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.Conclusion: This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.

We investigated the effects of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated matrix metalloproteinase (MMP)‐13 production in human chondrocytes from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). Secreted levels of MMP‐13 in conditioned media were detected by immunoblotting, while intracellular MMP‐13 synthesis in articular cartilage was evaluated by immunofluorescence microscopic analysis. Mitogen‐activated protein kinases (MAPKs), p38, extracellular signal‐regulated kinases (ERK), and c‐jun NH2‐terminal kinase (JNK) were assessed by Western blotting. IL‐1β (2 ng/ml) stimulates the secretion of MMP‐13 in both OA and RA chondrocytes. Inhibition studies using specific MAPK inhibitors revealed that IL‐1β induced MMP‐13 via p38 in both OA and RA chondrocytes. HA down‐regulates IL‐1β‐stimulated MMP‐13 and phosphorylated p38 (p‐p38) in a dose‐dependent manner (0.1, 1, 2, and 4 mg/ml). When used at 4 mg/ml, HA inhibits p‐p38 phosphorylation by more than 60%. In response to IL‐1β, RA chondrocytes express a higher level of p‐p38 than that of OA chondrocytes. Inhibition of CD44, using a blocking antibody, significantly reversed the inhibitory effect of HA on both MMP‐13 and p‐p38. Our study clearly shows that HA inhibits IL‐1β‐induced MMP‐13 via its principal receptor, CD44, and subsequent intracellular p38 MAPK signaling in OA and RA chondrocytes. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:258–264, 2011

Objective. In rheumatoid arthritis (RA), it is well known that rheumatoid synovial fibroblasts (RSF) produce matrix metalloproteinases (MMPs) when stimulated with proinflammatory cytokines such as interleukin-1β (IL-1β), which causes joint destruction. We have previously shown that hyaluronan (HA) inhibits IL-1β actions in RSF via CD44, the principal HA receptor. However, CD44 mediates HA effects only partially, and intracellular events after the HA binding to its receptors remain unclear. We investigated the role of intercellular adhesion molecule-1 (ICAM-1), another cell surface receptor for HA, and the intracellular signalling pathways in the actions of HA. Methods. RSF were isolated from rheumatoid synovial tissues by enzymatic digestion and cultured in monolayers. The confluent cells were incubated for 48 h with IL-1β, IL-1β in the presence of HA, or IL-1β in the presence of HA with pretreatment with anti-ICAM-1 antibody. Secretion of MMP-1 and MMP-3 was analysed by immunoblotting and immunofluorescence cytochemistry. Immunofluorescence cytochemistry was also performed to evaluate binding of HA to ICAM-1. The phosphorylation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) was analysed by immunoblotting. Results. Production of MMP-1 and MMP-3 by RSF was stimulated by IL-1β. HA at ≥2 mg/ml significantly inhibited MMP production induced by IL-1β in a dose-dependent manner. Moreover, pretreatment with anti-ICAM-1 antibody at 50 μg/ml significantly blocked the effects of HA on the actions of IL-1β on RSF, as shown by immunoblotting and immunofluorescence cytochemistry. Another immunofluorescence cytochemistry study demonstrated that HA bound RSF via ICAM-1. Inhibition studies revealed the requirement of NF-κB, p38 and c-jun NH₂-terminal kinase (JNK) for IL-1β-induced MMP production. IL-1β activated all three pathways, whereas HA down-regulated their phosphorylation. Pretreatment with anti-ICAM-1 antibody reversed the inhibitory effects of HA on the activation of NF-κB and p38 without affecting JNK. Conclusion. HA suppresses IL-1β-enhanced MMP-1 and MMP-3 synthesis in RSF via ICAM-1 through down-regulation of NF-κB and p38. Intra-articular injection of HA of high molecular weight may work through such a mechanism in RA joints.

The aged population is currently at its highest level in human history and is expected to increase further in the coming years. In humans, aging is accompanied by impaired angiogenesis, diminished blood flow and altered metabolism, among others. A cellular mechanism that impinges upon these manifestations of aging can be a suitable target for therapeutic intervention. Here we identify cell surface receptor CD47 as a novel age-sensitive driver of vascular and metabolic dysfunction. With the natural aging process, CD47 and its ligand thrombospondin-1 were increased, concurrent with a reduction of self-renewal transcription factors OCT4, SOX2, KLF4 and cMYC (OSKM) in arteries from aged wild-type mice and older human subjects compared to younger controls. These perturbations were prevented in arteries from aged CD47-null mice. Arterial endothelial cells isolated from aged wild-type mice displayed cellular exhaustion with decreased proliferation, migration and tube formation compared to cells from aged CD47-null mice. CD47 suppressed ex vivo sprouting, in vivo angiogenesis and skeletal muscle blood flow in aged wild-type mice. Treatment of arteries from older humans with a CD47 blocking antibody mitigated the age-related deterioration in angiogenesis. Finally, aged CD47-null mice were resistant to age- and diet-associated weight gain, glucose intolerance and insulin desensitization. These results indicate that the CD47-mediated signaling maladapts during aging to broadly impair endothelial self-renewal, angiogenesis, perfusion and glucose homeostasis. Our findings provide a strong rationale for therapeutically targeting CD47 to minimize these dysfunctions during aging.

Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability.