Further insights about the beneficial effects of n-3 fatty acids in the early molecular events of renal fibrosis in vitro.

BACKGROUND: Accumulating experimental and clinical evidence reveals beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) in kidney disease by modulating inflammation and fibrosis mechanisms that lead to renal failure. METHODS: EPA, DHA (n-3 PUFAs) and AA (n-6 PUFA) effects, compared to those of AngII, on renal fibrotic processes at the extracellular matrix (ECM) level were verified in human mesangial cells in vitro, by means of RT-PCR, mitogenic assay and Western-blot analysis. RESULTS: Unlike AngII, EPA and DHA enhanced the expression of MMP2 and DN, a TGFbeta inhibitor, while decreasing mitogenic factors such as PDGF and bFGF, and cell proliferation. Moreover, n-3 PUFAs elicited Bax expression in AngII-treated cells and downregulated COX-2--an enzyme involved in the inflammatory cascade. The mechanism of action could implicate PPARgamma activation, as this transcription factor was shown to translocate to the nucleus upon n-3 PUFA treatment. CONCLUSIONS: These results complement our previous reports demonstrating that EPA and DHA prevent ECM accumulation and inflammation that typify the fibrotic process, providing new insights into the cellular and molecular mechanisms underlying their beneficial effects. We confirm that n-3 PUFAs could effectively counteract kidney fibrosis development providing a rationale for their use in clinical settings.

The tumor necrosis factor-{alpha}-blocking agent infliximab inhibits interleukin 1beta (IL-1beta) and IL-6 gene expression in human osteoblastic cells.

OBJECTIVE: Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine involved in the pathogenesis of several rheumatic diseases, including rheumatoid arthritis (RA), associated with systemic bone loss and subchondral bone erosions. TNF-alpha-blocking agents such as infliximab have been successful in treatment of disease-modifying antirheumatic drug-resistant rheumatic diseases. Infliximab therapy in RA also had beneficial effects on local bone destruction and bone mineral density. We assessed effects of infliximab treatment on the bone tissue compartment and cytokine profile expression in vitro. METHODS: Osteoblast-like cells were exposed for 24 h to sera of RA patients collected at baseline and after 1 month (T1) and 3 years (T2) of infliximab treatment. Total RNA was extracted, and expression of interleukin 1beta (IL-1beta), IL-6, and osteoprotegerin (OPG) was measured by RT-PCR. RESULTS: IL-1beta gene expression was significantly reduced by the T1 serum, and the same decrease was elicited by the T2 serum. IL-6 downregulation was evident with the T2 serum. OPG was unaffected. CONCLUSION: The finding of downregulation of inflammatory cytokines was interesting, particularly IL-6, which plays a crucial role in arthritis-related bone loss due to its involvement in osteoclast recruitment and activation. These results may represent a biological explanation and a link for the clinical observation of the beneficial effects of anti-TNF-alpha agents on the progression of rheumatic diseases at the bone level.

EPA and DHA suppress AngII- and arachidonic acid-induced expression of profibrotic genes in human mesangial cells.

BACKGROUND: There is some evidence suggesting a close relationship between polyunsaturated fatty acids (PUFAs) and renal inflammation and fibrosis, which are crucial stages in chronic kidney disease. METHODS: To verify the role of PUFAs in renal fibrosis processes, we investigated the effects of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) on the gene expression of TGFbeta, fibronectin (FN), connective tissue growth factor (CTGF) and type IV collagen (COLIV) in human mesangial cells, in the absence or presence of angiotensin II (AngII), using reverse transcriptase PCR. RESULTS: The addition of AA to mesangial cell cultures induced a significant up-regulation of TGFbeta, FN, CTGF and COLIV expression, similar to that induced by AngII, while EPA and DHA had no stimulatory effects. The coincubation of cells with AngII and AA potentiated AngII-induced gene expression; on the contrary, the coexposure of cells to EPA or DHA suppressed the AngII- and AA-induced up-regulation of TGFbeta, FN, CTGF and COLIV. CONCLUSION: We conclude that the PUFAs have different effects, dependent on their chemical structure, on the AngII-TGFbeta system, a major regulator of the renal fibrotic process. Our in vitro results may provide new therapeutic options toward interrupting the irreversible process of renal fibrosis and ameliorating chronic renal injury.

Relationship between angiogenesis and inflammation in experimental arthritis.

Background. Angiogenesis is involved in rheumatoid arthritis (RA) leading to leucocyte recruitment and inflammation in the synovium. Furthermore, synovial inflammation itself further potentiates endothelial proliferation and angiogenesis. In this study, we aimed at evaluating the reciprocical relationship between synovial inflammation and angiogenesis in a RA model, namely collagen-induced arthritis (CIA). Methods. CIA was induced by immunization of DBA/1 mice with collagen type II in adjuvant. Endothelial cells were detected using a GSL-1 lectin-specific immunohistochemical staining on knee joint sections. Angiogenesis, clinical scores and histological signs of arthritis were evaluated from the induction of CIA until the end of the experiment. Angiogenesis was quantified by counting both the isolated endothelial cells and vessels stained on each section. To evaluate the effect of increased angiogenesis on CIA, VEGF gene transfer was performed using an adeno-associated virus encoding VEGF (AAV-VEGF), by intra-muscular or intra-articular injection in mice with CIA. Results. We showed an increase in synovial angiogenesis from day 6 to day 55 after CIA induction, and, moreover, joint vascularization and clinical scores of arthritis were correlated (p < 0.0001, r = 0.61). Vascularization and histological scores were also correlated (p = 0.0006, r = 0.51). Systemic VEGF overexpression in mice with CIA was followed by an aggravation of arthritis as compared to AAV-lacZ control group (p < 0.0001). In contrast, there was no difference in clinical scores between control mice and mice injected within the knee with AAV-VEGF, even if joint vascularization was higher in this group than in all other groups (p = 0,05 versus non-injected group). Intra-articular AAV-VEGF injections induced more severe signs of histological inflammation and bone destruction than AAV-Lac Z or no injection. Conclusion. Angiogenesis and joint inflammation evolve in parallel during collagen-induced arthritis. Furthermore, this work shows that exogenous VEGF can aggravate CIA. It is direct evidence that the increase in joint vascularization leads to an exacerbation of arthritis. Taken together, these results emphasize the role of angiogenesis in inflammatory arthritis. It also suggests an early involvement of angiogenesis in joint inflammation.

Immune response against gene therapy vectors: influence of synovial fluid on adeno-associated virus mediated gene transfer to chondrocytes.

Intraarticular gene transfer with adeno-associated virus (AAV) vectors may allow efficient therapeutic transgene expression within the joint. In an effort to understand potential obstacles (particularly immunity against AAV vectors) to intraarticular gene therapy better, our objective was to determine whether synovial fluid (SF) influenced AAV-mediated gene transfer to chondrocytes. SF and sera from 21 patients with joint diseases were collected. Neutralizing activity against AAV/interleukin-4 (IL-4) was determined by assessing the ability of SF or serum to inhibit AAV/IL-4 transduction to the C20A4 chondrocytes. IgGs were purified from SF by salt-dependent chromatography. Anti-AAV IgG levels were determined by ELISA in the SF. SF and sera from all the patients inhibited AAV-mediated gene transfer to chondrocytes. Six SF out of 21 exerted a stronger inhibition. Serum from healthy patients were also inhibitory. Purified IgGs from SF exhibited inhibition patterns similar to those seen with whole SF. Anti-AAV IgG were found in SF from 13 patients out of 18. Moreover, in the SF, anti-AAV IgG level was correlated with the neutralizing activity (p < 0.001, r = 0.716). A correlation was observed between levels of inhibition by the SF and serum (P < 0.0001, r = 0.813). Inhibition of AAV/IL-4 infection of C20A4 cells by SF and sera was abolished by increasing the number of AAV/IL-4 particles. SF from patients with joint disease consistently inhibited AAV infection of chondrocytes in vitro. This effect was ascribable to IgG, most probably directed against AAV. In the future, these data may be useful for tailoring intraarticular AAV-mediated gene therapy to individual patients.

Laboratory investigations in osteoarthritis.

Progress in the knowledge of pathogenic mechanisms and a better definition of the disease, together with the availability of new technologies, have recently improved the value of laboratory investigations in osteoarthritis (OA). The main objectives of these findings are early diagnosis, assessment of disease activity and severity, and evaluation of therapeutic effects. In this context, biochemical markers are potentially useful, as they are non-invasive and non-expansive. However, among the numerous substances increasingly proposed for these purposes, very few may be considered as true disease markers in OA; COMP, antigenic keratan sulphate, hyaluronic acid, YKL-40, type III collagen N-propeptide and urinary glucosyl-galactosyl pyridinoline seem to be the most promising. However, serum or urinary determinations of these molecules are difficult to interpret adequately, due to their complex metabolism. Careful analysis of synovial fluid, mainly directed to leukocyte count and crystal detection, is still essential for diagnosis, but also for the evaluation of the levels of important markers of local inflammation, such as metalloproteinases and cytokines, which seem to be crucial in the pathogenesis of OA.