Role of peroxiredoxin 6 in the chondroprotective effects of microvesicles from human adipose tissue-derived mesenchymal stem cells.

BACKGROUND: Osteoarthritis (OA) is a joint disease characterized by cartilage degradation, low-grade synovitis and subchondral bone alterations. In the damaged joint, there is a progressive increase of oxidative stress leading to disruption of chondrocyte homeostasis. The modulation of oxidative stress could control the expression of inflammatory and catabolic mediators involved in OA. We have previously demonstrated that extracellular vesicles (EVs) present in the secretome of human mesenchymal stem cells from adipose tissue (AD-MSCs) exert anti-inflammatory and anti-catabolic effects in OA chondrocytes. In the current work, we have investigated whether AD-MSC EVs could regulate oxidative stress in OA chondrocytes as well as the possible contribution of peroxiredoxin 6 (Prdx6). METHODS: Microvesicles (MV) and exosomes (EX) were isolated from AD-MSC conditioned medium by differential centrifugation with size filtration. The size and concentration of EVs were determined by resistive pulse sensing. OA chondrocytes were isolated from knee articular cartilage of advanced OA patients. 4-Hydroxynonenal adducts, IL-6 and MMP-13 were determined by enzyme-linked immunosorbent assay. Expression of Prdx6 and autophagic markers was assessed by immunofluorescence and Western blotting. Prdx6 was downregulated in AD-MSCs by transfection with a specific siRNA. RESULTS: MV and to a lesser extent EX significantly reduced the production of oxidative stress in OA chondrocytes stimulated with IL-1ß. Treatment with MV resulted in a dramatic upregulation of Prdx6. MV also enhanced the expression of autophagy marker LC3B. We downregulated Prdx6 in AD-MSCs by using a specific siRNA and then MV were isolated. These Prdx6-silenced MV failed to modify oxidative stress and the expression of autophagy markers. We also assessed the possible contribution of Prdx6 to the effects of MV on IL-6 and MMP-13 production. The reduction in the levels of both mediators induced by MV was partly reverted after Prdx6 silencing. CONCLUSION: Our results indicate that EVs from AD-MSCs regulate the production of oxidative stress in OA chondrocytes during inflammation. Prdx6 may mediate the antioxidant and protective effects of MV.The translational potential of this article: This study gives insight into the protective properties of EVs from AD-MSCs in OA chondrocytes. Our findings support the development of novel therapies based on EVs to prevent or treat cartilage degradation.

Current and emerging fixed combination therapies in glaucoma: a safety and tolerability review.

INTRODUCTION: Fixed dose combinations (FCs) represent a potentially valuable treatment strategy in glaucoma management. Fixed combinations not only improve adherence by reducing the medication burden, but also decrease the total amount of potentially deleterious preservatives an eye is exposed to. AREAS COVERED: We provide a critical review of selected evidence on both the safety and tolerability of presently available and emerging glaucoma FCs. There is convincing short-term safety and tolerability evidence on intraocular pressure (IOP)-lowering FCs compared to that of monotherapies and, to a lesser degree, to that of concomitant, equivalent combination therapies. In contrast, there is a scarcity of trials evaluating the long-term efficacy and safety of glaucoma FCs and no conclusive data on the reduction of adverse events with FCs. EXPERT OPINION: It is vital for clinicians to carefully weigh the efficacy, safety, tolerability, and adherence of IOP-lowering FCs. Given the number of currently available and emerging FC therapy options in glaucoma, as well as the complexities of incorporating them in the various combination therapy regimens, successful stepwise therapy remains often elusive.

Optical coherence tomography analysis of filtering blebs after long-term, functioning trabeculectomy and XEN® stent implant.

PURPOSE: The purpose of this study was to use Triton® SweptSource OCT to evaluate the morphology of blebs formed when eyes are treated with XEN® implants and to compare these with the blebs in successfully functioning eyes after trabeculectomy (TB) and with eyes of healthy controls. METHODS: A cross-sectional, observational study. We analyzed 25 eyes, 15 after TB and 10 with XEN® implants, comparing them with 23 healthy eyes (controls). We evaluated the conjunctival morphology of the eyes using AS-OCT. The main parameters evaluated were bleb height, sub-epithelial fibrosis, epithelial thickness, and changes in intraocular pressure (IOP). RESULTS: We found that the filtering blebs formed in eyes in which a XEN® stent was implanted were significantly flatter (bleb height 417 ± 183 µm) than the blebs formed in TB eyes (bleb height 618 ± 256 µm, p < 0.05). Moreover, sub-epithelial fibrosis did not develop in any of the blebs produced by the XEN stent, whereas some fibrosis was evident in 40% of the blebs that formed after TB (p < 0.05). The epithelium was thicker when the XEN implant was used (65 ± 18.5 µm) than when eyes underwent TB (60 ± 17.7 µm), and it was thicker than in control eyes (51 ± 9.7 µm, p < 0.05). Moreover, the decrease in the IOP induced by the XEN® stent (- 8.5 ± 5.3 mmHg) was similar to that produced by TB (- 8.8 ± 5.2 mmHg, p > 0.05). CONCLUSIONS: Filtering blebs obtained after the introduction of a XEN® stent were morphologically distinct to those produced by TB, and they are more similar to the healthy conjunctiva.

Outcomes of Trabeculectomy and Phacotrabeculectomy With Collagen Matrix Implant (Ologen) and Low-dose Mitomycin C: 2-Year Follow-up.

PURPOSE: The purpose of this article is to analyze the results achieved in lowering intraocular pressure (IOP) after trabeculectomy or combined surgery (phacotrabeculectomy) with low-dose mitomycin C (MMC) using the Ologen Collagen Matrix (Ologen CM) implant. MATERIALS AND METHODS: This retrospective study included 58 eyes from 47 consecutive patients with glaucoma who underwent filtering surgery alone or combined with cataract surgery. The study group included 29 eyes that underwent trabeculectomy (14 eyes) or phacotrabeculectomy (15 eyes) with low-dose MMC (0.1 mg/mL×1 min) and subconjunctival Ologen CM implant at the end of surgery. The control group included 29 eyes, 12 that underwent trabeculectomy and 17 that underwent phacotrabeculectomy, with the same MMC dose but without the collagen matrix implant. All surgical procedures were performed by the same surgeon. The follow-up period for the patients was 2 years. RESULTS: We found statistically significant differences between the 2 groups in the age of the patients (P=0.02). We found no statistically significant differences in the baseline IOP (P=0.37) or preoperative IOP (P=0.5), nor in the visual field damage measured with mean deviation (P=0.2). The number of hypotensive medications used preoperatively was higher in the study group (P=0.0001). At 1 and 2 years after surgery, we only found statistically significant differences in favor of the study group in patients who underwent phacotrabeculectomy (P=0.0008 and 0.02, respectivily). CONCLUSION: The Ologen CM implant can be considered as an adjunct to MMC in patients undergoing filtering surgery combined with phacoemulsification to improve postoperative IOP results over the long term.

Extracellular Vesicles from Adipose-Derived Mesenchymal Stem Cells Downregulate Senescence Features in Osteoarthritic Osteoblasts.

Osteoarthritis (OA) affects all articular tissues leading to pain and disability. The dysregulation of bone metabolism may contribute to the progression of this condition. Adipose-derived mesenchymal stem cells (ASC) are attractive candidates in the search of novel strategies for OA treatment and exert anti-inflammatory and cytoprotective effects on cartilage. Chronic inflammation in OA is a relevant factor in the development of cellular senescence and joint degradation. In this study, we extend our previous observations of ASC paracrine effects to study the influence of conditioned medium and extracellular vesicles from ASC on senescence induced by inflammatory stress in OA osteoblasts. Our results in cells stimulated with interleukin- (IL-) 1ß indicate that conditioned medium, microvesicles, and exosomes from ASC downregulate senescence-associated ß-galactosidase activity and the accumulation of γH2AX foci. In addition, they reduced the production of inflammatory mediators, with the highest effect on IL-6 and prostaglandin E2. The control of mitochondrial membrane alterations and oxidative stress may provide a mechanism for the protective effects of ASC in OA osteoblasts. We have also shown that microvesicles and exosomes mediate the paracrine effects of ASC. Our study suggests that correction of abnormal osteoblast metabolism by ASC products may contribute to their protective effects.

Anti-senescence and Anti-inflammatory Effects of the C-terminal Moiety of PTHrP Peptides in OA Osteoblasts.

Osteoarthritis (OA) is characterized by degenerative changes in the whole joint leading to physical disability in the elderly population. This condition is associated with altered bone metabolism in subchondral areas suggesting that therapeutic strategies aimed at modifying bone cell metabolism may be of interest. We have investigated the effects of several parathyroid hormone-related protein (PTHrP)-derived peptides (1-37): (N-terminal), (107-111) and (107-139) (C-terminal) on senescence features induced by inflammatory stress in human OA osteoblasts. Incubation of these primary cells with interleukin(IL)-1ß led to an increased expression of senescence markers senescence-associated-ß-galactosidase activity, γH2AX foci, p16, p21, p53, and caveolin-1. PTHrP (107-111) and PTHrP (107-139) significantly reduced all these parameters. Both peptides decreased the production of IL-6 and prostaglandin E2 which was the consequence of cyclo-oxygenase-2 downregulation. PTHrP (107-139) also reduced tumor necrosis factor-α release. These anti-inflammatory effects would be related to the reduction of nuclear factor-κB activation by both peptides and activator protein-1 by PTHrP (107-139). The three PTHrP peptides favored osteoblastic function although the C-terminal domain of PTHrP was more efficient than its N-terminal domain. Our data support an anti-senescence and anti-inflammatory role for the C-terminal moiety of PTHrP with potential applications in chronic inflammatory conditions such as OA.

Paracrine effects of human adipose-derived mesenchymal stem cells in inflammatory stress-induced senescence features of osteoarthritic chondrocytes.

Aging and exposure to stress would determine the chondrocyte phenotype in osteoarthritis (OA). In particular, chronic inflammation may contribute to stress-induced senescence of chondrocytes and cartilage degeneration during OA progression. Recent studies have shown that adipose-derived mesenchymal stem cells exert paracrine effects protecting against degenerative changes in chondrocytes. We have investigated whether the conditioned medium (CM) from adipose-derived mesenchymal stem cells may regulate senescence features induced by inflammatory stress in OA chondrocytes. Our results indicate that CM down-regulated senescence markers induced by interleukin-1ß including senescence-associated ß-galactosidase activity, accumulation of γH2AX foci and morphological changes with enhanced formation of actin stress fibers. Treatment of chondrocytes with CM also decreased the production of oxidative stress, the activation of mitogen-activated protein kinases, and the expression of caveolin-1 and p21. The effects of CM were related to the reduction in p53 acetylation which would be dependent on the enhancement of Sirtuin 1 expression. Therefore, CM may exert protective effects in degenerative joint conditions by countering the premature senescence of OA chondrocytes induced by inflammatory stress.

Perimetric progression using the Visual Field Index and the Advanced Glaucoma Intervention Study score and its clinical correlations / Progresión perimétrica utilizando el índice del campo visual y la puntuación del estudio de intervención del glaucoma avanzado, y sus correlaciones clínicas

Purpose: To evaluate the association between clinical parameters and the diagnosis of progression using VFI (Visual Field Index) and AGIS (Advanced Glaucoma Intervention Study) score in primary open angle glaucoma. Methods: Retrospective study of 517 visual fields of 78 eyes with primary open angle glaucoma analyzed with VFI and AGIS score. Clinical data registered included: age, sphere, pachimetry, basal intraocular pressure (IOP), and IOP during the follow up. Results: Only the AGIS score diagnosis of progression was associated with the clinical parameters registered. Among the analyzed data, the mean IOP during follow up (p=0.0005) and IOP at the third month of follow up (p=0.004) were statistically associated with progression using the AGIS criteria. Conclusion: The diagnosis of perimetric progression using the AGIS score in the current study was closer to the real functional progression than the diagnosis using the VFI, as the former was associated with known risk factors for progression in glaucoma (AU)

Objetivo: Evaluar la asociación entre los parámetros clínicos y el diagnóstico de progresión utilizando el Índice del Campo Visual (VFI) y la Puntuación del Estudio de Intervención del Glaucoma en el glaucoma primario de ángulo abierto. Métodos: Estudio retrospectivo de 517 campos visuales de 78 ojos con glaucoma primario de ángulo abierto analizados con VFI y la puntuación AGIS. Los datos clínicos registrados incluyeron: edad, esfera, paquimetría, presión intraocular (PIO) basal y PIO durante el seguimiento. Resultados: Únicamente el diagnóstico de la progresión de la puntuación AGIS se asoció a los parámetros clínicos evaluados. Entre los datos analizados, la PIO media durante el seguimiento (p=0,0005) y la PIO al tercer mes de seguimiento (p=0,004) se asociaron estadísticamente a la progresión, utilizando los criterios AGIS. Conclusión: El diagnóstico de la progresión perimétrica utilizando la puntuación AGIS en el estudio actual se acercó más a la progresión funcional real que el diagnóstico utilizando el VFI, ya que la primera se asoció a los factores de riesgo conocidos para la progresión del glaucoma (AU)

Effect of topical prostaglandin analogues on corneal hysteresis.

PURPOSE: To evaluate possible changes in corneal hysteresis (CH) after topical treatment with a prostaglandin analogue in medication-naïve eyes. METHODS: This was a prospective, observational cohort study. Sixty-eight eyes of 68 patients were prospectively included who were newly diagnosed with primary open-angle glaucoma or ocular hypertension in our institution. All patients were treatment-naïve. Patients were evaluated at baseline and after 6 months of treatment with latanoprost in the eye with the lower intraocular pressure (IOP) measured by Goldmann applanation tonometry (GAT). The ocular response analyzer was used to measure CH. RESULTS: CH increased significantly (p = 0.0001) from 8.96 ± 2.3 mmHg to 9.79 ± 1.97 mmHg, and this increase was correlated significantly (p = 0.0001, r = 0.64, r(2) = 0.41) with the basal CH. We identified a weak but significant (r(2) = 0.06, p = 0.01) relationship between the basal CH and the drug-induced reduction of the GAT IOP. Nevertheless, the increase in the drug-induced CH was not correlated with the decrease in the GAT IOP. CONCLUSION: Treatment with latanoprost increases CH. The CH increase was not correlated with the drug-induced decrease in the GAT IOP, which suggested a direct effect of latanoprost on the viscoelastic corneal properties.

Perimetric progression using the Visual Field Index and the Advanced Glaucoma Intervention Study score and its clinical correlations.

PURPOSE: To evaluate the association between clinical parameters and the diagnosis of progression using VFI (Visual Field Index) and AGIS (Advanced Glaucoma Intervention Study) score in primary open angle glaucoma. METHODS: Retrospective study of 517 visual fields of 78 eyes with primary open angle glaucoma analyzed with VFI and AGIS score. Clinical data registered included: age, sphere, pachimetry, basal intraocular pressure (IOP), and IOP during the follow up. RESULTS: Only the AGIS score diagnosis of progression was associated with the clinical parameters registered. Among the analyzed data, the mean IOP during follow up (p = 0.0005) and IOP at the third month of follow up (p = 0.004) were statistically associated with progression using the AGIS criteria. CONCLUSION: The diagnosis of perimetric progression using the AGIS score in the current study was closer to the real functional progression than the diagnosis using the VFI, as the former was associated with known risk factors for progression in glaucoma.

Heme oxygenase-1 mediates protective effects on inflammatory, catabolic and senescence responses induced by interleukin-1ß in osteoarthritic osteoblasts.

Osteoarthritis (OA) is a chronic degenerative joint disease showing altered bone metabolism. Osteoblasts contribute to the regulation of cartilage metabolism and bone remodeling. We have shown previously that induction of heme oxygenase-1 (HO-1) protects OA cartilage against inflammatory and degradative responses. In this study, we investigated the effects of HO-1 induction on OA osteoblast metabolism. HO-1 was induced with cobalt protoporphyrin IX (CoPP) and by transduction with LV-HO-1. In osteoblasts stimulated with interleukin (IL)-1ß, CoPP enhanced mineralization, the expression of a number of markers of osteoblast differentiation such as Runx2, bone morphogenetic protein-2, osteocalcin, and collagen 1A1 and 1A2, as well as the ratio osteoprotegerin/receptor activator of nuclear factor-κB ligand. HO-1 induction significantly reduced the expression of matrix metalloproteinase (MMP)-1, MMP-2 and MMP-3, and the production of pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6 whereas IL-10 levels increased. HO-1 also exerted inhibitory effects on prostaglandin (PG)E(2) production which could be dependent on cyclooxygenase-2 and microsomal PGE synthase-1 down-regulation. The activity of senescence-associated ß-galactosidase and the expression of the senescence marker caveolin-1 were significantly decreased after HO-1 induction. The inhibition of nuclear factor-κB activation induced by IL-1ß in OA osteoblasts may contribute to some HO-1 effects. Our results have shown that HO-1 decreases the production of relevant inflammatory and catabolic mediators that participate in OA pathophysiology thus eliciting protective effects in OA osteoblasts.

Control of cell migration and inflammatory mediators production by CORM-2 in osteoarthritic synoviocytes.

BACKGROUND: Osteoarthritis (OA) is the most widespread degenerative joint disease. Inflamed synovial cells contribute to the release of inflammatory and catabolic mediators during OA leading to destruction of articular tissues. We have shown previously that CO-releasing molecules exert anti-inflammatory effects in animal models and OA chondrocytes. We have studied the ability of CORM-2 to modify the migration of human OA synoviocytes and the production of chemokines and other mediators sustaining inflammatory and catabolic processes in the OA joint. METHODOLOGY/PRINCIPAL FINDINGS: OA synoviocytes were stimulated with interleukin(IL)-1ß in the absence or presence of CORM-2. Migration assay was performed using transwell chambers. Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. CORM-2 reduced the proliferation and migration of OA synoviocytes, the expression of IL-8, CCL2, CCL20, matrix metalloproteinase(MMP)-1 and MMP-3, and the production of oxidative stress. We found that CORM-2 reduced the phosphorylation of extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2 and to a lesser extent p38. Our results also showed that CORM-2 significantly decreased the activation of nuclear factor-κB and activator protein-1 regulating the transcription of chemokines and MMPs in OA synoviocytes. CONCLUSION/SIGNIFICANCE: A number of synoviocyte functions relevant in OA synovitis and articular degradation can be down-regulated by CORM-2. These results support the interest of this class of agents for the development of novel therapeutic strategies in inflammatory and degenerative conditions.

Haem oxygenase-1 down-regulates high mobility group box 1 and matrix metalloproteinases in osteoarthritic synoviocytes.

OBJECTIVES: Activation of osteoarthritic synoviocytes by pro-inflammatory cytokines results in the release of biochemical mediators such as MMPs and high mobility group box 1 (HMGB1). Extracellular HMGB1 can play an important role in joint diseases as a mediator of synovitis. We have shown previously that haem oxygenase-1 (HO-1) exerts protective effects during inflammatory responses. In this study, we have examined whether HO-1 induction would be an effective strategy to control MMP and HMGB1 production in osteoarthritic synoviocytes. METHODS: Osteoarthritic synoviocytes were obtained by digestion with collagenase and cultured until third passage. HO-1 was induced by cobalt protoporphyrin IX (CoPP). Lentiviral HO-1 vector (LV-HO-1) was also used for HO-1 overexpression. HO-1 gene silencing was achieved by using a specific small interfering RNA. Gene expression was analysed by quantitative PCR and protein expression by western blot, ELISA and IF. MMP activity was studied by fluorometric procedures. RESULTS: Induction of HO-1 by CoPP in the presence of IL-1beta decreased the expression of MMP-1 and -3, and MMP activity. IL-1beta stimulation of synoviocytes increased HMGB1 expression, its translocation into the cytoplasm and secretion. HO-1 induction exerted inhibitory effects on these processes. The consequences of HO-1 induction were counteracted by HO-1 gene silencing, whereas transfection with LV-HO-1 confirmed the effects of pharmacological HO-1 induction. CONCLUSIONS: We have provided direct evidence that HO-1 down-regulates MMP-1, -3 and HMGB1 in osteoarthritic synoviocytes. HO-1 may be a potential strategy to control inflammatory and degradative processes in the progression of OA.

Heme oxygenase-1 induction modulates microsomal prostaglandin E synthase-1 expression and prostaglandin E(2) production in osteoarthritic chondrocytes.

Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) may participate in the pathogenesis of cartilage damage in osteoarthritis (OA) through the production of catabolic enzymes and inflammatory mediators. Induction of heme oxygenase-1 (HO-1) has previously been shown to exert anti-inflammatory effects in different cell types. We have investigated whether HO-1 induction may modify chondrocyte viability and the production of relevant mediators such as oxidative stress and prostaglandin E(2) (PGE(2)) elicited by IL-1beta in OA chondrocytes. Chondrocytes were isolated from OA cartilage and used in primary culture. Cells were stimulated with IL-1beta in the absence or presence of the HO-1 inducer cobalt protoporphyrin IX (CoPP). Gene expression was assessed by quantitative real-time PCR, protein levels by ELISA and Western blot, apoptosis by laser scanning cytometry using annexin V-FITC and TUNEL assays, and oxidative stress by LSC with dihydrorhodamine 123. HO-1 induction by CoPP enhanced chondrocyte viability and aggrecan content while inhibiting apoptosis and oxidative stress generation. PGE(2) is produced in OA chondrocytes stimulated by IL-1beta by the coordinated induction of cyclooxygenase-2 and microsomal PGE synthase 1 (mPGES-1). The production of PGE(2) was decreased by HO-1 induction as a result of diminished mPGES-1 protein and mRNA expression. Transfection with HO-1 small interfering RNA counteracted CoPP effects. In addition, the activation of nuclear factor-kappaB and early growth response-1 was significantly reduced by CoPP providing a basis for its anti-inflammatory effects. These results confirm the protective role of HO-1 induction in OA chondrocytes and suggest the potential interest of this strategy in degenerative joint diseases.

Reconstruction of the medial patellofemoral ligament for patellar instability using a semitendinosus autograft.

Between 1999 and 2001 thirty knees underwent a semitendinosus tendon plasty to recreate the medial patellofemoral ligament for recurrent patellar dislocation. The mean follow-up was 38 months. The mean improvement of the patellofemoral congruence angle after surgery was 14 +/- 7 degrees. All patients ended up with a full range of motion, except one patient, whose flexion was limited to 120 degrees due to superficial wound infections. Dislocation did not recur. According to the Larsen and Lauridsen outcome score the clinical results were excellent in 27 patients, good in 2 and fair in one. In conclusion this procedure is indicated for the chronic dislocation and cases of severe femoral dysplasia with marked laxity. The procedure assures the stabilisation of the patella, although it doesn't restore the patellofemoral congruence angle to normal values.