A HEV-restricted sulfotransferase is expressed in rheumatoid arthritis synovium and is induced by lymphotoxin-alpha/beta and TNF-alpha in cultured endothelial cells.

BackgroundThe recruitment of lymphocytes to secondary lymphoid organs relies on interactions of circulating cells with high endothelial venules (HEV). HEV are exclusive to these organs under physiological conditions, but they can develop in chronically-inflamed tissues. The interaction of L-selectin on lymphocytes with sulfated glycoprotein ligands on HEV results in lymphocyte rolling, which represents the initial step in lymphocyte homing. HEV expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, GST-3, LSST or CHST4), an HEV-restricted sulfotransferase, is essential for the elaboration of L-selectin functional ligands as well as a critical epitope recognized by MECA-79 mAb.ResultsWe examined the expression of GlcNAc6ST-2 in relationship to the MECA-79 epitope in rheumatoid arthritis (RA) synovial vessels. Expression of GlcNAc6ST-2 was specific to RA synovial tissues as compared to osteoarthritis synovial tissues and localized to endothelial cells of HEV-like vessels and small flat-walled vessels. Double MECA-79 and GlcNAc6ST-2 staining showed colocalization of the MECA-79 epitope and GlcNAc6ST-2. We further found that both TNF-alpha and lymphotoxin-alphabeta induced GlcNAc6ST-2 mRNA and protein in cultured human umbilical vein endothelial cells.ConclusionThese observations demonstrate that GlcNAc6ST-2 is induced in RA vessels and provide potential cytokine pathways for its induction. GlcNAc6ST-2 is a novel marker of activated vessels within RA ectopic lymphoid aggregates. This enzyme represents a potential therapeutic target for RA

A HEV-restricted sulfotransferase is expressed in rheumatoid arthritis synovium and is induced by lymphotoxin-α/β and TNF-α in cultured endothelial cells

BACKGROUND: The recruitment of lymphocytes to secondary lymphoid organs relies on interactions of circulating cells with high endothelial venules (HEV). HEV are exclusive to these organs under physiological conditions, but they can develop in chronically-inflamed tissues. The interaction of L-selectin on lymphocytes with sulfated glycoprotein ligands on HEV results in lymphocyte rolling, which represents the initial step in lymphocyte homing. HEV expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, GST-3, LSST or CHST4), an HEV-restricted sulfotransferase, is essential for the elaboration of L-selectin functional ligands as well as a critical epitope recognized by MECA-79 mAb. RESULTS: We examined the expression of GlcNAc6ST-2 in relationship to the MECA-79 epitope in rheumatoid arthritis (RA) synovial vessels. Expression of GlcNAc6ST-2 was specific to RA synovial tissues as compared to osteoarthritis synovial tissues and localized to endothelial cells of HEV-like vessels and small flat-walled vessels. Double MECA-79 and GlcNAc6ST-2 staining showed colocalization of the MECA-79 epitope and GlcNAc6ST-2. We further found that both TNF-α and lymphotoxin-αβ induced GlcNAc6ST-2 mRNA and protein in cultured human umbilical vein endothelial cells. CONCLUSION: These observations demonstrate that GlcNAc6ST-2 is induced in RA vessels and provide potential cytokine pathways for its induction. GlcNAc6ST-2 is a novel marker of activated vessels within RA ectopic lymphoid aggregates. This enzyme represents a potential therapeutic target for RA

TGF-β antagonist attenuates fibrosis but not luminal narrowing in experimental tracheal stenosis.

Introduction/Objective: Acquired tracheal stenosis (ATS) is an unusual disease often secondary to prolonged mechanical trauma. ATS pathogenesis involves inflammation and subsequent fibrosis with narrowing of the tracheal lumen. TGF-β represents a pivotal factor in most fibrotic processes and therefore, a potential target in this context. The aim of this study is to analyze the role of TGF-β as a target for anti-fibrotic interventions in tracheal stenosis. Methods: Human stenotic tracheobronchial tissues from patients with benign airway stenosis and normal controls from pneumonectomy specimens were analyzed. Tracheal stenosis was induced in adult NZ rabbits by a circumferential thermal injury to the mucosa during open surgery and re-anastomosis. Rabbits were treated postoperatively with a peritracheal collagen sponge containing a TGF-β peptide antagonist (p17) or vehicle. Fibrosis was determined by Masson’s trichrome staining, and α-SMA+ Results: Human and rabbit stenotic tissues showed extensive submucosal fibrosis, characterized by significantly increased α-SMA myofibroblasts, CTGF and p-Smad2/3 expression by immunohistochemistry. + myofibroblasts and CTGF expression. In human stenotic lesions, increased p-Smad2/3+ nuclei were also observed. p17 treatment significantly reduced the fibrotic thickness as well as the density of α-SMA+ myofibroblasts and CTGF+ Conclusion: ATS is characterized by a TGF-β dependent fibrotic process but reduction of the fibrotic component by TGF-β1 antagonist therapy was not sufficient to improve tracheal narrowing, suggesting that fibrosis may not be the main contributor to luminal stenosis. cells in rabbit stenotic lesions but failed to improve the luminal area.pre-print1304 K

CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans

The chemokine CXCL12 (also known as stromal cell-derived factor, SDF-1) is constitutively expressed by stromal resident cells and is involved in the homeostatic and inflammatory traffic of leukocytes. Binding of CXCL12 to glycosaminoglycans on endothelial cells (ECs) is supposed to be relevant to the regulation of leukocyte diapedesis and neoangiogenesis during inflammatory responses. To improve our understanding of the relevance of this process to rheumatoid arthritis (RA), we have studied the mechanisms of presentation of exogenous CXCL12 by cultured RA ECs. RA synovial tissues had higher levels of CXCL12 on the endothelium than osteoarthritis (OA) tissues; in both, CXCL12 colocalized to heparan sulfate proteoglycans (HSPGs) and high endothelial venules. In cultured RA ECs, exogenous CXCL12α was able to bind in a CXCR4-independent manner to surface HSPGs. Desulfation of RA EC HSPGs by pretreatment with sodium chlorate, or by replacing in a synthetic CXCL12α the residues Lys24 and Lys27 by Ser (CXCL12α-K2427S), decreased or abrogated the ability of the chemokine to bind to RA ECs. Ex vivo, synovial ECs from patients with either OA or RA displayed a higher CXCL12-binding capacity than human umbilical vein ECs (HUVECs), and in HUVECs the binding of CXCL12 was increased on exposure to tumor necrosis factor-α or lymphotoxin-α(1)β(2). Our findings indicate that CXCL12 binds to HSPGs on ECs of RA synovium. The phenomenon relates to the interaction of HSPGs with a CXCL12 domain with net positive surface charge located in the first β strand, which encompasses a canonical BXBB HSPG-binding motif. Furthermore, we show that the attachment of CXCL12 to HSPGs is upregulated by inflammatory cytokines. Both the upregulation of a constitutive chemokine during chronic inflammation and the HSPG-dependent immobilization of CXCL12 in EC surfaces are potential sites for therapeutic intervention

One-year, efficacy and safety open label study, with a single injection of a new hyaluronan for knee OA: the SOYA trial

[Abstract] Purpose. To assess the efficacy and safety of a single injection of a new formulation of hyaluronic acid (MPS-HA2%) in patients with symptomatic knee osteoarthritis after 12 months’ follow-up. Patients and Methods. Prospective, single-arm, multicentre, open-label, 12-month follow-up study. Patients with Kellgren–Lawrence (KL) 2–3 and visual analogue scale (VAS) pain scores of ≥40–< 80 mm received a single injection of MPS-HA2%. The primary outcome was the reduction in VAS pain scores from baseline, and the secondary outcomes were the Western Ontario and McMaster (WOMAC) Universities Osteoarthritis Index, the minimum clinically important improvement (MCII), and patient and investigator global assessments (PGA, IGA) measured on 5-point Likert scale. Adverse events were recorded throughout the study for safety purposes. Results. A total of 101 patients (mean age: 68 years; 74% female; and 78% overweight) were included. The mean reduction in pain at 12 months was 37.7%; the total WOMAC score improved by 36.5% and the pain, stiffness and physical function subscores returned improvements of 32.1%, 34.1% and 32.7%, respectively (p=0.0001 with respect to baseline). At 12 months, a statistically significant 62.2% of patients obtained an improvement equal to or greater than the MCII. The mean PGA score at baseline was 2.44 and 1.46 at 12 months (p<0.05), and the mean IGA scores at equivalent timepoints were 2.29 and 1.48 (p<0.05). Fourteen patients received a second injection at the 6-month follow-up visit. Eight patients reported a total of 12 treatment-related adverse events that were local, non-serious and of mild-to-moderate intensity. Conclusion. With just a single intra-articular injection, this not controlled trial suggests that MPS-HA2% is effective 12 months after the procedure in most cases. Patient tolerability and safety were both optimal (NCT03852914)

Immature Blood Vessels in Rheumatoid Synovium Are Selectively Depleted in Response to Anti-TNF Therapy

BACKGROUND:Angiogenesis is considered an important factor in the pathogenesis of Rheumatoid Arthritis (RA) where it has been proposed as a therapeutic target. In other settings, active angiogenesis is characterized by pathologic, immature vessels that lack periendothelial cells. We searched for the presence of immature vessels in RA synovium and analyzed the dynamics of synovial vasculature along the course of the disease, particularly after therapeutic response to TNF antagonists. METHODOLOGY/PRINCIPAL FINDINGS:Synovial arthroscopic biopsies from RA, osteoarthritis (OA) and normal controls were analyzed by double labeling of endothelium and pericytes/smooth muscle mural cells to identify and quantify mature/immature blood vessels. To analyze clinicopathological correlations, a cross-sectional study on 82 synovial biopsies from RA patients with variable disease duration and severity was performed. A longitudinal analysis was performed in 25 patients with active disease rebiopsied after anti-TNF-alpha therapy. We found that most RA synovial tissues contained a significant fraction of immature blood vessels lacking periendothelial coverage, whereas they were rare in OA, and inexistent in normal synovial tissues. Immature vessels were observed from the earliest phases of the disease but their presence or density was significantly increased in patients with longer disease duration, higher activity and severity, and stronger inflammatory cell infiltration. In patients that responded to anti-TNF-alpha therapy, immature vessels were selectively depleted. The mature vasculature was similarly expanded in early or late disease and unchanged by therapy. CONCLUSION/SIGNIFICANCE:RA synovium contains a significant fraction of neoangiogenic, immature blood vessels. Progression of the disease increases the presence and density of immature but not mature vessels and only immature vessels are depleted in response to anti-TNFalpha therapy. The different dynamics of the mature and immature vascular fractions has important implications for the development of anti-angiogenic interventions in RA

Topical application of a peptide inhibitor of transforming growth factor-beta1 ameliorates bleomycin-induced skin fibrosis

Transforming growth factor-beta (TGF-beta) plays a crucial role in the pathogenesis of skin fibrotic diseases. Systemic TGF-beta inhibitors effectively inhibit fibrosis in different animal models; however, systemic inhibition of TGF-beta raises important safety issues because of the pleiotropic physiological effects of this factor. In this study, we have investigated whether topical application of P144 (a peptide inhibitor of TGF-beta1) ameliorates skin fibrosis in a well-characterized model of human scleroderma. C3H mice received daily subcutaneous injections of bleomycin for 4 wk, and were treated daily with either a lipogel containing P144 or control vehicle. Topical application of P144 significantly reduced skin fibrosis and soluble collagen content. Most importantly, in mice with established fibrosis, topical treatment with P144 lipogel for 2 wk significantly decreased skin fibrosis and soluble collagen content. Immunohistochemical studies in P144-treated mice revealed a remarkable suppression of connective tissue growth factor expression, fibroblast SMAD2/3 phosphorylation, and alpha-smooth muscle actin positive myofibroblast development, whereas mast cell and mononuclear cell infiltration was not modified. These data suggest that topical application of P144, a peptide inhibitor of TGF-beta1, is a feasible strategy to treat pathological skin scarring and skin fibrotic diseases for which there is no specific therapy

Senescent synovial fibroblasts accumulate prematurely in rheumatoid arthritis tissues and display an enhanced inflammatory phenotype

[Abstract] Background Accumulation of senescent cells has been associated with pro-inflammatory effects with deleterious consequences in different human diseases. The purpose of this study was to analyze cell senescence in human synovial tissues (ST), and its impact on the pro-inflammatory function of synovial fibroblasts (SF). Results The expression of the senescence marker p16INK4a (p16) was analyzed by immunohistochemistry in rheumatoid arthritis (RA), osteoarthritis (OA), and normal ST from variably aged donors. The proportion of p16(+) senescent cells in normal ST from older donors was higher than from younger ones. Although older RA and OA ST showed proportions of senescent cells similar to older normal ST, senescence was increased in younger RA ST compared to age-matched normal ST. The percentage of senescent SA-β-gal(+) SF after 14 days in culture positively correlated with donor’s age. Initial exposure to H2O2 or TNFα enhanced SF senescence and increased mRNA expression of IL6, CXCL8, CCL2 and MMP3 and proteins secretion. Senescent SF show a heightened IL6, CXCL8 and MMP3 mRNA and IL-6 and IL-8 protein expression response upon further challenge with TNFα. Treatment of senescent SF with the senolytic drug fenofibrate normalized IL6, CXCL8 and CCL2 mRNA expression. Conclusions Accumulation of senescent cells in ST increases in normal aging and prematurely in RA patients. Senescence of cultured SF is accelerated upon exposure to TNFα or oxidative stress and may contribute to the pathogenesis of synovitis by increasing the production of pro-inflammatory mediators.Instituto de Salud Carlos III; FIS 16/00032Insituto de Salud Carlos III; RETICS RD16/0012 RIE

Hif-1α knockdown reduces glycolytic metabolism and induces cell death of human synovial fibroblasts under normoxic conditions

[Abstract] Increased glycolysis and HIF-1α activity are characteristics of cells under hypoxic or inflammatory conditions. Besides, in normal O2 environments, elevated rates of glycolysis support critical cellular mechanisms such as cell survival. The purpose of this study was to analyze the contribution of HIF-1α to the energy metabolism and survival of human synovial fibroblasts (SF) under normoxic conditions. HIF-1α was silenced using lentiviral vectors or small-interfering RNA (siRNA) duplexes. Expression analysis by qRT-PCR and western blot of known HIF-1α target genes in hypoxia demonstrated the presence of functional HIF-1α in normoxic SF and confirmed the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a HIF-1α target even in normoxia. HIF-1α silencing induced apoptotic cell death in cultured SF and, similarly, treatment with glycolytic, but not with OXPHOS inhibitors, induced SF death. Finally, in vivo HIF-1α targeting by siRNA showed a significant reduction in the viability of human SF engrafted into a murine air pouch. Our results demonstrate that SF are highly dependent on glycolytic metabolism and that HIF-1α plays a regulatory role in glycolysis even under aerobic conditions. Local targeting of HIF-1α provides a feasible strategy to reduce SF hyperplasia in chronic arthritic diseases.Instituto de Salud Carlos III; FIS 12/439Instituto de Salud Carlos III; RETICS RD12/009Instituto de Salud Carlos III; CP13/00014Comunidad de Madrid; RAPHYME-CM S2010/BMD235