Abstract Objectives (1) To evaluate clinical and molecular cardiovascular disease (CVD) signs and their relationship with psoriatic arthritis (PsA) features and (2) to identify a clinical patient profile susceptible to benefit from methotrexate (MTX) and/or apremilast regarding CVD risk. Methods This cross‐sectional study included 100 patients with PsA and 100 age‐matched healthy donors. In addition, an exploratory cohort of 45 biologically naïve patients treated for 6 months with apremilast, MTX or combined therapy according to routine clinical practice was recruited. Extensive clinical and metabolic profiles were obtained. Ninety‐nine surrogate CVD‐related molecules were analysed in plasma and peripheral blood mononuclear cells (PBMCs). Hard cluster analysis was performed to identify the clinical and molecular phenotypes. Mechanistic studies were performed on adipocytes. Results Cardiometabolic comorbidities were associated with disease activity and long‐term inflammatory status. Thirty‐five CVD‐related proteins were altered in the plasma and PBMCs of PsA patients and were associated with the key clinical features of the disease. Plasma levels of some of the CVD‐related molecules might distinguish insulin‐resistant patients (MMP‐3, CD163, FABP‐4), high disease activity (GAL‐3 and FABP‐4) and poor therapy outcomes (CD‐163, LTBR and CNTN‐1). Hard cluster analysis identified two phenotypes of patients according to the rates of cardiometabolic comorbidities with distinctive clinical and molecular responses to each treatment. Conclusions (1) Novel CVD‐related proteins associated with clinical features could be emerging therapeutic targets in the context of PsA and (2) the pleiotropic action of apremilast could make it an excellent choice for the management of PsA patients with high CVD risk, targeting metabolic alterations and CVD‐related molecules.
Endothelial dysfunction (ED) is well known as a process that can lead to atherosclerosis and is frequently presented in radiographic axial spondyloarthritis (r-axSpA) patients. Here, we investigated cellular and molecular mechanisms underlying r-axSpA-related ED, and analyzed the potential effect of peripheral blood mononuclear cells (PBMCs) in promoting endothelial injury in r-axSpA. A total of 30 r-axSpA patients and 32 healthy donors (HDs) were evaluated. The endothelial function, inflammatory and atherogenic profile, and oxidative stress were quantified. In vitro studies were designed to evaluate the effect of PBMCs from r-axSpA patients on aberrant endothelial activation. Compared to HDs, our study found that, associated with ED and the plasma proatherogenic profile present in r-axSpA, PBMCs from these patients displayed a pro-oxidative, proinflammatory, and proatherogenic phenotype, with most molecular changes noticed in lymphocytes. Correlation studies revealed the relationship between this phenotype and the microvascular function. Additional in vitro studies confirmed that PBMCs from r-axSpA patients promoted endothelial injury. Altogether, this study suggests the relevance of r-axSpA itself as a strong and independent cardiovascular risk factor, contributing to a dysfunctional endothelium and atherogenic status by aberrant activation of PBMCs. Lymphocytes could be the main contributors in the development of ED and subsequent atherosclerosis in this pathology.
Introduction RA patients are at higher risk of cardiovascular disease, influenced by therapies. Studying their cardiovascular and cardiometabolic proteome can unveil biomarkers and insights into related biological pathways. Methods This study included two cohorts of RA patients: newly diagnosed individuals (n=25) and those with established RA (disease duration >25 years, n=25). Both cohorts were age and sex-matched with a control group (n=25). Additionally, a longitudinal investigation was conducted on a cohort of 25 RA patients treated with methotrexate and another cohort of 25 RA patients treated with tofacitinib for 6 months. Clinical and analytical variables were recorded, and serum profiling of 184 proteins was performed using the Olink technology platform. Results RA patients exhibited elevated levels of 75 proteins that might be associated with cardiovascular disease. In addition, 24 proteins were increased in RA patients with established disease. Twenty proteins were commonly altered in both cohorts of RA patients. Among these, elevated levels of CTSL1, SORT1, SAA4, TNFRSF10A, ST6GAL1 and CCL18 discriminated RA patients and HDs with high specificity and sensitivity. Methotrexate treatment significantly reduced the levels of 13 proteins, while tofacitinib therapy modulated the expression of 10 proteins. These reductions were associated with a decrease in DAS28. Baseline levels of SAA4 and high levels of BNP were associated to the non-response to methotrexate. Changes in IL6 levels were specifically linked to the response to methotrexate. Regarding tofacitinib, differences in baseline levels of LOX1 and CNDP1 were noted between non-responder and responder RA patients. In addition, response to tofacitinib correlated with changes in SAA4 and TIMD4 levels. Conclusion In summary, this study pinpoints molecular changes linked to cardiovascular disease in RA and proposes candidate protein biomarkers for distinguishing RA patients from healthy individuals. It also highlights how methotrexate and tofacitinib impact these proteins, with distinct alterations corresponding to each drug’s response, identifying potential candidates, as SAA4, for the response to these therapies.
Disease severity, progression and response to therapy might be worse in obese rheumatoid arthritis (RA) patients, but paradoxically, obesity also might protect from radiographic joint damage. Thus, the intricate relationship between obesity and RA needs urgent clarification. The aim of this study was to assess the influence of obesity on the onset and development of RA and to determine whether arthritis could modify the adipose tissue biology and whether conventional Disease Modifying Anti-Rheumatic Drugs (cDMARDs) can modulate these alterations. Two strategies were followed: (1) clinical profiling of two cohorts of RA: non-obese and obese patients; and (2) mechanistic studies carried out in both a collagen-induced arthritis (CIA) in an obese mouse model and 3T3-L1 adipocytes treated with cDMARDs (leflunomide, methotrexate, and hydroxychloroquine). In our cohort of RA patients with low-moderate disease activity, the presence of obesity was not related to a higher activity of the disease; actually, disease activity score 28-erythrocyte sedimentation rate (DAS28-ESR) was reduced in the obese RA patients. However, the induction of arthritis promoted transcriptomic changes in the adipose tissue under obesity condition in the obese CIA model. Treatment with hydroxychloroquine reduced weight and insulin resistance, accompanied by beneficial metabolic effects in the adipose tissue. These molecular changes in adipose tissue were also observed after methotrexate administration. In sum, arthritis might affect directly the inflammatory burden and metabolic alterations associated with obesity in adipose tissue. Clinicians should be cautious measuring the activity of the disease in obesity and managing the best therapeutic options for the metabolic comorbidities of these patients, where the combination of hydroxychloroquine and methotrexate should be considered to improve adipose tissue dysfunction in obese RA.
Radiographic assessment plays a crucial role in diagnosing and monitoring axial Spondyloarthritis (axSpA). Structural changes, such as erosions, sclerosis, and syndesmophytes, provide insights into the severity and impact of the disease on the axial skeleton. Understanding the molecular mechanisms driving radiographic progression is essential for developing targeted therapies and improving patient outcomes. Identifying biomarkers associated with radiographic progression can aid in early detection, risk stratification, and the development of personalized treatment strategies for patients with axSpA.
Objectives:
1) to assess the radiographic progression of axSpA over a span of five years and 2) to examine the clinical and molecular aspects associated with radiographic progression in axSpA.
Methods:
a cohort of 70 axSpA patients was recruited at the Reina Sofia Hospital (Córdoba, Spain). Clinical and analytical variables were recorded. Blood extraction was carried out for the subsequent analysis of the serum levels of 184 proteins using Olink technology. The enriched biological functions of the identified altered proteins were assessed using the STRING platform. Structural damage, assessed using the "modified Stoke Ankylosing Spinal Score" (mSASSS), was examined at the time of the visit and in the preceding five years. Radiographic progression was quantified as Δ mSASSS (mSASSS at visit – mSASSS on the preceding 5 years). AxSpA progressors were determined using the following formula: Δ mSASSS/ years (with a cut-off value of > 1 mSASSS). Statistically significant analyses were defined by a p-value < 0.01.
Results:
out of 70 axSpA patients, 26 exhibited an annual increment of more than 1 point in mSASSS, categorizing them as progressors, while the remaining 44 were classified as non-progressors. There were no differences in age, sex, disease duration, disease activity, acute phase reactants, and HLA-B27 positivity among groups of progressors or non-progressors. However, levels of alkaline phosphatase (ALP) were significantly elevated in axSpA progressors compared to non-progressors. Remarkably, molecular analyses identified 12 proteins that were significantly differentially elevated in the progressor group compared to non-progressors (GRN, IL-2RA, KLK-6, IL-18BP, LTBR, IGFBP-7, TNFSF-13B, GDF-15, IL-10RA, IL-2, IL2Rβ, and IL-10). Furthermore, these proteins exhibited significant correlations with Δ mSASSS over the preceding 5 years. Additionally, levels of IL2RA, IL2Rβ, GDF-15, and IL-18BP significantly correlated with ALP. These findings may imply the potential engagement of these proteins in the development of structural damage and, consequently, radiographic progression in axSpA. The biological enrichment analysis of the altered proteins in progressors compared to non-progressors unveiled a significant protein-protein interaction enrichment (p-value=1.14e-09). Among the prominently enriched biological processes were the positive regulation of plasma cell differentiation, the regulation of T cell homeostatic proliferation, and the regulation of B cell apoptotic processes. These findings imply an exaggerated immune system, potentially linked to structural damage. Notably, IL-2 stood out significantly across all enrichment analyses, encompassing molecular functions, cellular components, KEGG pathways, and reactome pathways.
Conclusion:
1) The progression of structural damage in axSpA is intricately linked with the levels of ALP, a routine clinical follow-up variable; 2) a distinctive molecular signature associated with radiographic progression has unveiled novel candidate biomarkers indicative of the evolving structural damage landscape in axSpA; 3) the identified proteins exhibit enrichments in biological functions suggestive of a significant immune component potentially influencing structural damage. Notably, the IL-2 signalling pathways may play a pivotal role, underscoring their potential importance in the molecular mechanisms orchestrating radiographic progression in axSpA.
REFERENCES:
NIL.
Acknowledgements:
Supported the "Instituto de Salud Carlos III" (PMP21/00119 and RICOR-RD21/0002/0033) co-financed by the European Union.
patients with Spondyloarthritis (SpA) have an increased risk of cardiovascular disease. Taking into account the strong relationship between inflammation and CVD, there is an urgent need to identify different molecular drivers of CVD signs and their association with inflammation.
Objectives
to investigate the alteration of CVD-related proteins in the plasma of SpA patients, their association with clinical features, and to evaluate their potential role as biomarkers for the identification of persistent inflammation.
Methods
a cross-sectional study including 120 patients with SpA and 30 age-sex-matched healthy donors (HDs) was carried out. Clinical and laboratory parameters and CVD risk factors were recorded. To measure the presence of persistent inflammation, levels of c-reactive protein (CRP) were collected retrospectively for 5 years previous to the study, a patient was considered to have persistent inflammation with increased CRP in at least 50% of the measures during the previous 5 years. Levels of 92 proteins with a recognized role in CVD were analyzed in the plasma using proximity extension assay (PEA) technology (Olink Target 96 CVD III panel, Cobiomic Biosciences).
Results
SpA patients showed higher rates of CVD comorbidities compared to HDs. Plasma levels of TNF-R1, RARRES-2, CHI3L1, PGLYRP-1, CTSD, UPAR, IL2RA, TIMP-4, CTSB, GDF-15, MMP-9, and PDGF-A were significantly increased in SpA compared to HDs. Specifically, these proteins are also related to biological processes such as neutrophil degranulation, immune response, cell activation, atherosclerosis, apoptosis, and inflammatory response. Besides, a significant alteration of these CVD-related proteins in SpA was also associated with the presence of arterial hypertension, insulin resistance, obesity, hyperuricemia, and high levels of acute phase reactants. 36% of SpA patients displayed persistence of inflammation. Interestingly, SpA patients with persistent inflammation showed higher levels of ankylosing spondylitis disease activity (ASDAS) score, CRP, glucose, complement component 3, and lower levels of HDL-cholesterol and apolipoprotein A compared to SpA patients with non-persistent inflammation, suggesting the relationship of inflammation with metabolic alterations. In addition, 8 out of 12 CVD-related proteins altered in SpA patients were specifically changed in patients with persistent inflammation: MMP-9, RARRES-2, PGLYRP-1, UPAR, TNF-R1, PDGF-A, IL-2RA and GDF-15, highlighting levels of MMP-9 protein as a potential biomarker for persistent inflammation in SpA (AUC=0.796 p>0.0001).
Conclusion
1) SpA patients show an altered CVD proteome profile which is strongly associated with CVD risk factors and clinical inflammatory markers, 2) SpA patients with persistent inflammation display a deeper alteration in their plasma CVD protein pattern suggesting the link between subclinical CVD risk and the chronic inflammation, and 3) this study identifies novel potential biomarkers to distinguish SpA patients with persistent inflammation. Funded by ISCIII (PI20/00079, PMP21/00119, and RICOR-RD21/0002/0033) co-financed by ERDF.
The anti-cyclic citrullinated protein antibodies (anti-CCPs) are the most specific auto-antibodies associated with Rheumatoid Arthritis (RA). To date, no study has evaluated the direct effect of the anti-CCPs in the leukocytes and their relationship with the atherogenesis and cardiovascular disease (CVD) observed in these patients
Objectives
To evaluate the effect of the in vitro treatment with anti-CCPs antibodies in the induction of the pro-oxidative and inflammatory state observed in RA leukocytes, and its relationship with the chronic inflammation and early atherogenesis that commonly characterize this disease
Methods
53 RA patients and 31 healthy donors were included in this study. Carotid intima-media thickness (CIMT) was evaluated as atherosclerosis marker; other markers of CVD were also studied. Several pro-coagulant factors, leukocyte activation markers, peroxides and peroxynitrite levels, intracellular glutathione (GSH) and mitochondrial membrane potential (MMP) were analysed in monocytes, lymphocytes and neutrophils by flow cytometry. Oxidative stress (total antioxidant capacity, nitric oxide and protein tyrosine nitration) and inflammation plasma markers (several interleukins, IFNg, VEGF, MCP-1, MIP-a, MMP-13, sP-selectina and tPA) were analyzed. To elucidate the cellular origin of the inflammatory molecules altered in the plasma from RA patients, RT-PCR of the three cell populations was performed. Finally, anti-CCPs antibodies were isolated from plasma of RA patients and in vitro treatment of healthy monocytes, lymphocytes and neutrophils was conducted
Results
Inflammatory factors such as IL-2, IL-6, IL-8, IL-10, IL-17a, MCP-1, MIP-A, sP-selectin and nitric oxide levels were found greatly elevated in RA plasma versus controls. The antioxidant capacity and protein tyrosine nitration were lower in RA plasma compared to controls. Additionally, high titles of anti-CCPs were associated to an increased expression of the pro-thrombotic and inflammatory markers, high oxidative stress and pathological CIMT. RA monocytes and neutrophils had elevated levels of peroxynitrites and peroxides, considerable depolarization of the MM and lower GSH activity compared with controls. They also showed an increased membrane presence of the cellular activation markers. RT-PCR of the proinflammatory molecules showed that each cell subtype presented a specific expression pattern. The in vitro treatment with anti-CCPs antibodies from RA patients supported the results observed in vivo in RA patients and confirmed the different effects caused by the anti-CCPs in the leukocytes
Conclusions
1)The anti-CCPs antibodies directly induce inflammation and oxidative stress in the leukocytes from RA patients. 2)The anti-CCPs effects are different depending on the cell type, being the monocytes the cell type most clearly involved in the thrombotic, oxidative and inflammatory status, while lymphocytes were mainly associated to the inflammation and neutrophils to the oxidative stress observed in RA 3)Understanding the specific effects induced by anti-CCPs antibodies would help to develop a cellular targeted therapy preventing the atherosclerosis and CVD in RA patients
Acknowledgements
Supported by: P08-CVI-04234, PI12/01511, Spanish Rheumatology Society
Abstract Background Rheumatoid arthritis ( RA ) patients are at increased risk of insulin resistance ( IR ); however, the specific mechanisms mediating this association are currently unknown. Objective To investigate whether the inflammatory activity associated with RA accounts for the observed defective glucose metabolism and lipid metabolism in these patients. Methods We followed two main strategies: (i) extensive metabolic profiling of a RA cohort of 100 patients and 50 healthy control subjects and (ii) mechanistic studies carried out in both a collagen‐induced arthritis mouse model and 3T3‐L1 adipocytes treated with conditioned serum from RA patients. Results Following the exclusion of obese and diabetic subjects, data from RA patients demonstrated a strong link between the degree of systemic inflammation and the development of IR . These results were strengthened by the observation that induction of arthritis in mice resulted in a global inflammatory state characterized by defective carbohydrate and lipid metabolism in different tissues. Adipose tissue was most susceptible to the RA ‐induced metabolic alterations. These metabolic effects were confirmed in adipocytes treated with serum from RA patients. Conclusions Our results show that the metabolic disturbances associated with RA depend on the degree of inflammation and identify inflammation of adipose tissue as the initial target leading to IR and the associated molecular disorders of carbohydrate and lipid homeostasis. Thus, we anticipate that therapeutic strategies based on tighter control of inflammation and flares could provide promising approaches to normalize and/or prevent metabolic alterations associated with RA .
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