Peroxisome proliferator-activated receptor γ1 expression is diminished in human osteoarthritic cartilage and is downregulated by interleukin-1β in articular chondrocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in the regulation of many cellular processes. We and others have previously shown that PPARγ activators display anti-inflammatory and chondroprotective properties in vitro and improve the clinical course and histopathological features in an experimental animal model of osteoarthritis (OA). However, the expression and regulation of PPARγ expression in cartilage are poorly defined. This study was undertaken to investigate the quantitative expression and distribution of PPARγ in normal and OA cartilage and to evaluate the effect of IL-1β, a prominent cytokine in OA, on PPARγ expression in cultured chondrocytes. Immunohistochemical analysis revealed that the levels of PPARγ protein expression were significantly lower in OA cartilage than in normal cartilage. Using real-time RT-PCR, we demonstrated that PPARγ1 mRNA levels were about 10-fold higher than PPARγ2 mRNA levels, and that only PPARγ1 was differentially expressed: its levels in OA cartilage was 2.4-fold lower than in normal cartilage (p < 0.001). IL-1 treatment of OA chondrocytes downregulated PPARγ1 expression in a dose- and time-dependent manner. This effect probably occurred at the transcriptional level, because IL-1 decreases both PPARγ1 mRNA expression and PPARγ1 promoter activity. TNF-α, IL-17, and prostaglandin E2 (PGE2), which are involved in the pathogenesis of OA, also downregulated PPARγ1 expression. Specific inhibitors of the mitogen-activated protein kinases (MAPKs) p38 (SB203580) and c-Jun N-terminal kinase (SP600125), but not of extracellular signal-regulated kinase (PD98059), prevented IL-1-induced downregulation of PPARγ1 expression. Similarly, inhibitors of NF-κB signaling (pyrrolidine dithiocarbamate, MG-132, and SN-50) abolished the suppressive effect of IL-1. Thus, our study demonstrated that PPARγ1 is downregulated in OA cartilage. The pro-inflammatory cytokine IL-1 may be responsible for this downregulation via a mechanism involving activation of the MAPKs (p38 and JNK) and NF-κB signaling pathways. The IL-1-induced downregulation of PPARγ expression might be a new and additional important process by which IL-1 promotes articular inflammation and cartilage degradation

Hydrodynamic Delivery of Chitosan-Folate-DNA Nanoparticles in Rats with Adjuvant-Induced Arthritis

50 kDa chitosan was conjugated with folate, a specific tissue-targeting ligand. Nanoparticles such as chitosan-DNA and folate-chitosan-DNA were prepared by coacervation process. The hydrodynamic intravenous injection of nanoparticles was performed in the right posterior paw in normal and arthritic rats. Our results demonstrated that the fluorescence intensity of DsRed detected was 5 to 12 times more in the right soleus muscle and in the right gastro muscle than other tissue sections. β-galactosidase gene expression with X-gal substrate and folate-chitosan-plasmid nanoparticles showed best coloration in the soleus muscle. Treated arthritic animals also showed a significant decrease in paw swelling and IL-1β and PGE2 concentration in serum compared to untreated rats. This study demonstrated that a nonviral gene therapeutic approach using hydrodynamic delivery could help transfect more efficiently folate-chitosan-DNA nanoparticles in vitro/in vivo and could decrease inflammation in arthritic rats

Alterations of metabolic activity in human osteoarthritic osteoblasts by lipid peroxidation end product 4-hydroxynonenal

4-Hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues, but its role in bone metabolism is ill-defined. In this study, we tested the hypothesis that alterations in OA osteoblast metabolism are attributed, in part, to increased levels of HNE. Our data showed that HNE/protein adduct levels were higher in OA osteoblasts compared to normal and when OA osteoblasts were treated with H(2)O(2). Investigating osteoblast markers, we found that HNE increased osteocalcin and type I collagen synthesis but inhibited alkaline phosphatase activity. We next examined the effects of HNE on the signaling pathways controlling cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) expression in view of their putative role in OA pathophysiology. HNE dose-dependently decreased basal and tumour necrosis factor-α (TNF-α)-induced IL-6 expression while inducing COX-2 expression and prostaglandin E(2 )(PGE(2)) release. In a similar pattern, HNE induces changes in osteoblast markers as well as PGE(2 )and IL-6 release in normal osteoblasts. Upon examination of signaling pathways involved in PGE(2 )and IL-6 production, we found that HNE-induced PGE(2 )release was abrogated by SB202190, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Overexpression of p38 MAPK enhanced HNE-induced PGE(2 )release. In this connection, HNE markedly increased the phosphorylation of p38 MAPK, JNK2, and transcription factors (CREB-1, ATF-2) with a concomitant increase in the DNA-binding activity of CRE/ATF. Transfection experiments with a human COX-2 promoter construct revealed that the CRE element (-58/-53 bp) was essential for HNE-induced COX-2 promoter activity. However, HNE inhibited the phosphorylation of IκBα and subsequently the DNA-binding activity of nuclear factor-κB. Overexpression of IKKα increased TNF-α-induced IL-6 production. This induction was inhibited when TNF-α was combined with HNE. These findings suggest that HNE may exert multiple effects on human OA osteoblasts by selective activation of signal transduction pathways and alteration of osteoblastic phenotype expression and pro-inflammatory mediator production

Efficient Nonviral Gene Therapy Using Folate-Targeted Chitosan-DNA Nanoparticles In Vitro

Nonviral cationic polymers like chitosan can be combined with DNA to protect it from degradation. The chitosan is a biocompatible, biodegradable, nontoxic, and cheap polycationic polymer with low immunogenicity. The objective of this study was to synthesize and then assess different chitosan-DNA nanoparticles and to select the best ones for selective in vitro transfection in human epidermoid carcinoma (KB) cell lines. It revealed that different combinations of molecular weight, the presence or absence of folic acid ligand, and different plasmid DNA sizes can lead to nanoparticles with various diameters and diverse transfection efficiencies. The intracellular trafficking, nuclear uptake, and localization are also studied by confocal microscopy, which confirmed that DNA was delivered to cell nuclei to be expressed