Abstract Background The genetic background of pulmonary arterial hypertension (PAH) in the Spanish pediatric population could be different from the genetic background described in other countries. Besides, clinical implications of a positive genetic result in the PAH pediatric population are not fully understood yet, and could result in the “reclassification” of patients from one PAH group to another type among the current pediatric classification. Material and methods Patients under 19 years at diagnosis included in the REHIPED registry from January 2011 to December 2021 were included. Clinical variables and genetical results were recorded. Succesive NGS panels involving up to 35 genes were used. After the results of the genetic testing, we analyzed differences in survival, and if patients “moved” to another category in the current Nice classification. Results In the selected cohort of 98 patients (56.1% female), median age at diagnosis was 7.1 years (IQr 1.5–14.7), and ethnicity as follows: Caucasian (81.6%), Romani (8.2%), others (10.2%) (Table 1). Before the genetic testing, patients had been classified as Idiopathic (53.1%), Congenital Heart Disease-PAH (30.6%), Heritable (5.1%), Pulmonary veno-oclusive disease (PVOD) 6.1%, and Multisystemic disorder associated with PAH (5.1%). Pathogenic or likely pathogenic variants were found in 44 of the screened patients (44.9%): BMPR2 (12 cases), EIF2AK4 (9), TBX4 (n=4), MECP2 (n=3), KCNK3 (n=2), FOXF1 (n=2), NFU1 (N=4), ACVRL1 (n=1), BMPR1B (n=1), CLBCI (n=1), GBE1 (n=1), GDF2 (n=1), SOX17 (n=1), VHL (n=1), and digenic pathogenic variant in ABCC8/SMAD1 (n=1). After genetic analysis, 28 patients (28.6%) were “reclassified” (Fig. 1, panel A), with HPAH, PVOD and multisystemic disorders increasing up to 18.4%, 8.2%, and 12.2%, respectively. Worse Survival from death or lung transplantation was observed in heritable PVOD and multisystemic disorders (Fig. 1, panel B). Conclusions The Spanish pediatric PAH population showed higher prevalence of EIF2AK4 than other pediatric registries. Genetic testing resulted in the “reclassification” of a significant number of patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Rio Hortega grant from the Spanish Ministry of Science and Innovation (Instituto de Salud Carlos III).

Abstract Background Pulmonary arterial hypertension (PAH) is a rare and severe disease, genetically predisposed in a high proportion of patients. PAH is subclassified in different subtypes depending on the underlying condition. Gene variants are more frequent among heritable or idiopathic forms. Nevertheless, pathogenic variants have been described across the entire spectrum of this disease. Evidence regarding genetics in pediatric PAH is scarce [1]. Purpose Our aim is to describe the prevalence of significant gene mutations among a pediatric PAH cohort and to define specific data in the different subtypes. Methods Samples for genetic studies were obtained from blood tests of patients included in the Spanish National Registry of Pediatric Pulmonary Hypertension (REHIPED). Guardians signed informed consent before the inclusion in the study. Qualitative variables were compared by Chi-square test. Quantitative variables were assessed by Kruskal-Wallis, considering the asymmetric distribution of variables. STATA 14.0 was used for analyses. Results Sixty four patients were included between 2011 and 2021. Median age of the entire sample was 7.1 years (2.0–12.6) and 42.2% of them were male. There were significant differences in the age at diagnosis and race between the different included groups (table). Pathogenic or likely pathogenic variants were more frequent in familial pulmonary venooclusive disease (PVOD) and familial PAH cases. A similar percentage of mutations were found in idiopathic cases and in PAH associated with congenital heart disease (Figure). Gene variants in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) were the most frequent mutations in the PAH familial cohort and there was also the most frequent finding in congenital heart disease and sporadic PAH, in conjunction with the TBX4 gene. Homozygous or compound heterozygous EIF2AK4 (eukaryotic translation initiation factor 2 a kinase 4) mutations were found in all the patients diagnosed with PVOD. Heritable PAH and PVOD cases were diagnosed more frequently after family screening. Conclusions This study shows a comparable proportion of pathogenic-likely pathogenic gene mutations in patients diagnosed of pulmonary arterial hypertension associated with congenital heart disease and idiopathic cases, with similar distribution of specific genes. BMPR2 and TBX 4 were the most frequent gene variants in this pediatric PAH population. BMPR2 and EIF2AK4 are the most common mutations in familial PAH and PVOD subtypes, respectively. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ACU holds a Rio Hortega Grant from the Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation.JAT and NG hold grants from FEDER (Federaciόn Española de Enfermedades Raras) and from the FCHP. Table 1. Characteristics of PAH subtypesFigure 1. PAH and ACMG classification.

Background Oxidative stress results in the disruption of normal physiologic signaling leading to inflammatory changes, cartilage degradation and osteoarthritis (OA) progression (1). Recent advances have revealed the role of cell-derived microvesicles (MV) as a new mechanism of cell-to-cell communication with potential therapeutic applications. We have shown previously the antiinflammatory effects of human adipose tissue-derived mesenchymal stem cells (AD-MSC) conditioned medium in OA chondrocytes (2). Objectives We have isolated the MV fraction from the secretome of AD-MSC to investigate its activity on oxidative stress and inflammation in OA chondrocytes stimulated with interleukin (IL)-1β. Furthermore, we have characterized the MV protein content by proteomic analysis. Methods AD-MSC were isolated from fat of patients who undergone abdominoplasty (without obesity). MV were isolated from AD-MSC conditioned medium by differential centrifugation with size filtration. MV size and concentration were determined by resistive pulse sensing. Proteomic analysis was performed by LC-MS/MS, with ProteinPilot and PeakView software and the bioinformatic tools UNIPROT and PANTHER. OA chondrocytes were isolated from knee specimens of advanced OA patients, stimulated with IL-1β (10 ng/mL) and treated with MV (3.6x107 particles/mL) for 24h. Accumulation of 4-hydroxy-2-nonenal (HNE)-modified proteins and cytokines were measured by ELISA, NO production and MMP activity by fluorometry. Expression of specific proteins was evaluated by confocal microscopy or immunostaining. The data were analysed by ANOVA followed by Dunnett9s test. Results MV reduced the accumulation of HNE-modified proteins, a biomarker of oxidative stress–induced lipid peroxidation, in OA chondrocytes stimulated with IL-1 β. The production of NO, IL-6 and TNFα, as well as MMP activity were also significantly reduced by MV treatment, whereas IL-10 and collagen II were enhanced. Proteomic analysis of MV showed high levels (5.89-fold upregulation) of peroxiredoxin 6 (Prdx6), a member of the peroxiredoxin family of antioxidant proteins which is downregulated in OA cartilage (3). MV treatment increased the expression of Prdx6 in OA chondrocytes suggesting a protective role against oxidative stress in these cells. Conclusions MV from AD-MSC downregulate the production of oxidative stress and inflammatory mediators in OA chondrocytes. Prdx6, an antioxidant enzyme, may contribute to the protective effects of MV. Our data support the interest of these MV to develop new therapeutic approaches. References Loeser RF et al. (2002) Detection of nitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin-1beta and with chondrocyte resistance to insulin-like growth factor 1. Arthritis Rheum 46: 2349–2357. Platas J et al. (2016) Paracrine effects of human adipose-derived mesenchymal stem cells in inflammatory stress-induced senescence features of osteoarthritic chondrocytes. Aging 8: 1703–17. Ikeda D et al. (2013) iTRAQ-based proteomics reveals novel biomarkers of osteoarthritis. Biomarkers 18: 565–572. Acknowledgements Funded by SAF2013–48724-R (MINECO, FEDER) and PROMETEOII/2014/071 (Generalitat Valenciana). Disclosure of Interest None declared

Purpose: Adipose-derived mesenchymal stem cells (ASC) exhibit a high potential for cell therapy besides exerting anti-inflammatory paracrine effects. Premature cellular senescence is known to occur in chronic inflammatory diseases, including osteoarthritis (OA). In addition to natural senescence of ageing, different stresses such as exposure to cytokines or oxidative stress induce senescence. We have investigated the ability of conditioned media from adipose-derived mesenchymal stem cells (ASC-CM) to regulate stress-induced senescence occurring in OA chondrocytes stimulated with interleukin-1β (IL-1β). Methods: Adipose tissue from patients subjected to abdominal lipectomy surgery, was used for ASC isolation by collagenase treatment. Cells were incubated in DMEM/F12 containing 15% human serum. Cell phenotype was analyzed by flow cytometry with specific antibodies anti-CD105-PE, anti-CD90-PerCP-eFluo 710, anti-CD34-APC, and anti-CD45-PE (International Society of Cellular Therapy), and cellular viability with propidium iodide. The conditioned medium (ASC-CM) was collected after 48h of culture, centrifuged and stored at -80ºC in sterile conditions. Cartilage specimens were obtained from patients with diagnosis of advanced OA. Protocols were approved by the Institutional Ethical Committee. Chondrocytes were used in primary culture. Isolated chondrocytes were stimulated with 10 ng/ml IL-1β for different times. Protein expression and oxidative stress were investigated by ELISA and Western Blot. Senescence-associated β-galactosidase activity was measured by immunocytochemistry. Detection of γ-H2A.X foci by immunofluorescence. Results: Our results indicate that ASC population was ≥ 95% positive for CD90 and CD105 and <1% positive for CD45 and CD34 along the entire culture period, indicating a phenotype of mesenchymal stem cell. IL-1β enhanced senescence-associated to heterochromatic foci as well as senescence-associated β-galactosidase activity in OA chondrocytes but both effects were significantly decreased by ASC-CM treatment. In addition, oxidative stress, and increased p53 acetylation and p21 and caveolin-1 expression were observed in OA chondrocytes in the presence of IL-1β whereas ASC-CM counteracted these changes and augmented Sirt-1 expression. The effect of ASC-CM on senescence was abolished by a Sirt-1 siRNA demonstrating the dependence of this protective effect on Sirt-1 activation. Conclusions: These findings show that ASC-CM contribute to the down-regulation of important senescence markers in OA chondrocytes. Our data suggest that ASC-CM may have therapeutic potential in the treatment of joint disease by countering premature senescence of chondrocytes induced by pro-inflammatory cytokines.

Purpose: Extracellular high mobility group box 1 (HMGB1) has been demonstrated to participate in inflammatory processes in joint diseases.Activation of osteoarthritic synoviocytes by proinflammatory cytokines results in HMGB1 translocation and release.We have shown recently a potential modulation of HMGB1 by heme oxygenase-1 (HO-1).In this study, we have examined the interactions between both pathways in osteoarthritic synoviocytes.Methods: Synovial tissue samples were obtained from 15 osteoarthritic patients undergoing total knee joint replacement.Synoviocytes were obtained by digestion with collagenase and cultured until third passage.Cells were treated with human recombinant HMGB1 (15 ng/ml) in the presence or absence of IL-1β (100 U/ml).HO-1 was induced by cobalt protoporphyrin IX (CoPP).Lentiviral HO-1.flag vector was also used for HO-1 overexpression.HO-1 gene silencing was achieved by using a specific siRNA.Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA.HMGB1 translocation into the cytoplasm was studied by immunofluorescence.Results: Treatment of synoviocytes with HMGB1 down-regulated HO-1.This effect was potentiated by IL-1β and accompanied by a significant increase in matrix metalloproteinase (MMP)-1 and MMP-3 gene expression and activity.Conversely, induction of HO-1 by CoPP in the presence of IL-1 β led to reduced expression of both protein and mRNA of HMGB1.These effects were accompanied by a significant reduction in gene expression of MMP-1 and MMP-3, and MMP activity.The consequences of HO-1 induction were counteracted by HO-1 gene silencing.Interestingly, we observed a marked reduction in the translocation of HMGB1 from the nucleus into the cytoplasm.Transfection with lentiviral HO-1.flag vector confirmed the inhibitory effect of HO-1 on HMGB1 translocation.Conclusions: We have provided direct evidence that HO-1 inhibits the translocation of HMGB1 into the cytoplasm.Our data indicate a reciprocal negative regulation between HO-1 and HMGB1 pathways in osteoarthritic synoviocytes.

437 –Table 1. Components of Factors retained by PCA using data from the medial tibial plateaus Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Parameters Parameters Parameters Parameters Parameters Articular Cartilage (AC) Area (0.94) CCD Area (0.86) Subchondral Bone (SCB) Area (0.71) Area of Weight-Bearing Meniscus (0.87) Axial Osteophyte (OP) Size

S86Osteoarthritis and Cartilage Vol.16 Supplement 4 subsequently treated with tumour necrosis factor-a (TNF-a) in combination with Actinomycin D to induce apoptosis.Apoptotic chondrocytes in cartilage sections were identified using an indirect immunohistochemical staining technique to detect expression of active caspase-3.Haematoxylin and eosin/safranin-o stained sections were used to score cellularity and structural differences between samples.Results: Prior to culture, (mean±standard deviation) chondrocyte viability was 80.7% (3.5).The extent of chondrocyte apoptosis induced by TNF-a/Actinomycin D varied markedly according to the joint type that the cartilage was sampled from.For MCP joints, the extent of overall chondrocyte apoptosis was significantly higher (P < 0.001) in TNFa/Actinomycin D-stimulated explants (26.7%, 10.3) than that observed in unstimulated control samples (9.6%, 7.5).Chondrocytes from PIP and DIP joint cartilage did not respond significantly to apoptotic stimulation (P > 0.05); apoptosis in both control and stimulated explants was virtually identical.Significant variations in cellularity and thickness were evident between cartilages of different joint types.Cartilage from DIP and PIP joints was significantly thicker than that of the MCP joint (P < 0.001 and P < 0.05 respectively).Moreover, MCP joint AC was significantly more cellular than both PIP and DIP joints (P < 0.001).Conclusions: Data in this study demonstrate that chondrocytes from three equine joint types with varying prevalence's of OA differ significantly in terms of susceptibility to apoptosis induction.This may provide a possible explanation for the joint-specific nature of the disease.Jointtype dependent differences in cartilage thickness and cellularity may also have contributory roles.

HCCK the B max values being 121550±45550 and 84880±5380 sites/cell, respectively.From these data the calculated K D values were 1.1±0.06(HFLS) and 3.1±1.7 (HCCK) nM.Inhibition curves were performed with antagonists (1 pM-1 mM) or unlabelled BK at the [ 3 H]BK binding (1.5 nM).K i values for BK were 0.93±0.12(HFLS) and 0.83±0.12(HCCK) nM.Both antagonists displayed high affinity values: MEN16132 K i values of 1.35±0.05and 1.75±0.35nM in HFLS and HCCK, respectively, and icatibant was 3-to 5-fold less potent, K i values being 4.6±1.7 and 8.8±2.9 nM.BK induced IP accumulation both in HFLS (EC 50 0.73±0.08nM) and HCCK (EC 50 0.64±0.05nM), and the induced maximal effect was 11±3 (HFLS) and 15±1.5 (HCCK) fold over the basal response.The antagonists potency was evaluated by preincubating cells with MEN16132 or icatibant at different concentrations (0.1 nM-10 mM) for 15 min before the agonist concentration-response curve (0.03 nM-100 mM).Both antagonists displayed a concentration-dependent antagonism, by shifting to the right the BK curve, but MEN16132 displayed a greater potency in respect to icatibant.The calculated pK B values were 9.93±0.09(HFLS) and 10.44±0.16(HCCK) for MEN16132, and 8.1±0.03(HFLS) and 8.2±0.16(HCCK) for icatibant.Conclusions: This is the first study quantifying B 2 receptors, the affinity and potency of BK and its antagonists in HCCK.Current data highlight the pharmacological profile of MEN16132 as a very potent bradykinin antagonist, in respect with icatibant, both in HFLS and HCCK.

Osteoarthritis and Cartilage Vol. 15, Supplement C C199 RANTES, IL-6, MIP-1, MDC, and MMP-9, whereas Pd or Dp alone at these concentrations had little or no effect.Dp is known to increase cAMP in mononuclear cells by multiple mechanisms including inhibition of phosphodiesterases (PDEs).The cAMP activator forskolin synergized with Pd suggesting elevated cAMP plays a key role in the synergistic response.The PKA inhibitor H89 did not block the synergistic suppression of RANTES and MDC suggesting that synergy is PKA independent.To evaluate dissociation of anti-inflammatory responses from a GC-mediated toxicity such as adrenal insufficiency, we evaluated expression of proopiomelanocortin (POMC) in the murine corticotroph cell line AtT-20/D16v-F2 by RT-PCR.As expected, Pd treatment resulted in dose-dependent reduction of POMC expression to a maximum of 40%, and Dp treatment did not alter this dose-dependent reduction.Similarly, Dp did not increase the Pd-induced transcription of a reporter gene controlled by four glucocorticoid response elements expressed in the T-cell line CCRF-CEM HSB-2.Amplification of Pd anti-inflammatory activity by Dp was therefore demonstrated not to extend to models of trans-activating and cis-repressing side effects of the steroid.These data suggest the unique set of molecular targets modulated by Dp drives the dissociated activity profile of CRx-102.Conclusions: Treatment with CRx-102 suppresses a unique set of cytokines, chemokines and MMPs that have been implicated in the pathogenesis of OA and RA.The components of CRx-102 impact cAMP-and GCR-regulated signaling pathways to synergistically suppress key inflammatory mediators including TNFα, IL-6, RANTES, and MMP-9.The amplification of Pd antiinflammatory activity by Dp does not extend to traditional cisrepression, or trans-activation GRE-regulated side effects.These data show that combining Pd and Dp results in a synergistic multi-target mechanism that inhibits key effectors underlying OA and RA and suggests that CRx-102 may be a valuable new therapeutic agent to treat arthritis.

We have studied the potential anti-inflammatory effects of 20 2-tosylamino and 2-tosyliminopyrimidine new derivatives in human neutrophils. We have evaluated their interference with some leukocyte functions and 5-lipoxygenase activity. All the compounds reduced neutrophil degranulation process at concentrations in the microM range. Besides, compounds with a phenolic substitution inhibited leukotriene B(4) biosynthesis in neutrophils and decreased the cell-free 5-lipoxygenase activity. This study demonstrates that 2-tosylamino and 2-tosyliminopyrimidine derivatives can reduce the activation of neutrophil cells which may have relevance for the modulation of the inflammatory response.

Many in vitro studies have used cell cultures to focus on the relationships between cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) isoforms. We have investigated the time-course of regulation and the role of COX-2 and iNOS in a model of experimental inflammation in mice, the air pouch injected with zymosan. This study demonstrates that there is an early acute phase (4 h) mediated mainly by eicosanoids, with high levels of prostaglandin E2 (PGE2) produced by cyclo-oxygenase-1. In addition, in the later phase (from 12 h) there is a participation of nitric oxide (NO) and PGE2 accompanied by co-induction of both iNOS and COX-2. These enzymes were detected in migrating leukocytes as well as in macrophages lining the air pouch. Administration of NS398 or indomethacin inhibited PGE2 levels and COX activity, but also nitrite levels and iNOS activity, which was accompanied by a reduction in iNOS expression. Aminoguanidine inhibited nitrite levels and iNOS activity in addition to exerting inhibitory effects on the COX pathway. Treatment of animals with dexamethasone reduced nitrite and PGE2 concentrations in air pouch exudates, as well as iNOS and COX-2 expression in migrating cells. Our results indicate that PGE2 and NO may play in vivo mutual modulatory roles in the inflammatory response caused by zymosan injection into the mouse air pouch, a suitable model to study drugs acting on those pathways.

A ditriazine derivative (4,10-dichloropyrido[5,6:4,5]thieno[3,2-d':3, 2-d]-1,2,3-ditriazine (DTD)) inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B(4) production, without any effect on 5-lipoxygenase activity. This compound reduced nitric oxide (NO) and prostaglandin E(2) production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of inducible NO synthase, cyclo-oxygenase-2 or cyclo-oxygenase-1 was observed. DTD significantly reduced mouse paw oedema induced by carrageenan and also markedly reduced NO and prostaglandin E(2) levels in exudates from 24-h zymosan-stimulated mouse air pouch. Western blot analysis showed that DTD reduced the expression of inducible NO synthase and cyclo-oxygenase-2. Our results indicate that DTD exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and prostaglandin E(2) production, which could be due to a decreased expression of inducible NO synthase and cyclo-oxygenase-2.

The inhibitory effect of some isoxazolpyrimidine derivatives on iNOS and COX-2 endotoxin induction in mouse peritoneal macrophages has been studied. Three of these compounds inhibited nitrite and PGE2 accumulation in a concentration dependent-manner at microM range. None of these active compounds affected iNOS, COX-2, COX-1 or PLA2 activities, although some reduced iNOS or COX-2 expression. Besides, no effect was observed on human neutrophil inflammatory responses (LTB4 biosynthesis and superoxide or elastase release). Active compounds were assayed by oral administration in the mouse air pouch model, where they inhibited nitrite accumulation without affecting PGE2 levels or leukocyte migration.

In a previous work, we tested a series of chalcone derivatives as possible anti‐inflammatory compounds. We now investigate the effects of three of those compounds, CH1, CH8 and CH12, on nitric oxide and prostanoid generation in mouse peritoneal macrophages stimulated with lipopolysaccharide and in the mouse air pouch injected with zymosan, where they showed a dose‐dependent inhibition with inhibitory concentration 50% values in the μM range. This effect was not the consequence of a direct inhibitory action on enzyme activities. Our results demonstrated that chalcone derivatives inhibited de novo inducible nitric oxide synthase and cyclooxygenase‐2 synthesis, being a novel therapeutic approach for inflammatory diseases.

Ultraviolet-A radiation has weak effects on the release of inflammatory mediators by skin cells due to the poor overlap between UVA wavelengths and the absorption spectra of the relevant chromophores of key biomolecules. However, this situation could be very different in the presence of a photosensitizing drug. To investigate this issue, we have irradiated human skin cells (keratinocytes and fibroblasts) in the presence of fenofibric acid (the active phototoxic metabolite of fenofibrate). The results of this research show a dual effect on the production/release of inflammatory mediators: the synthesis of the proinflammatory cytokine interleukin-6 becomes strongly inhibited at photosensitizer concentrations that clearly stimulate the production of prostaglandins (PGE2) by skin cells. We have found evidences showing that the de novo synthesis of cytokines is inhibited in photosensitized cells due to the fact that cellular mRNA is degraded. Interestingly, when the medium taken from irradiated cultures is added to nonexposed cells, a significant stimulation of cytokine synthesis is observed that can be inhibited by anti-PGE2 antibodies. These observations may be relevant in vivo, where prostaglandins released by photosensitized skin cells could stimulate cytokine synthesis by underlying, nonirradiated cells.