Cellular Senescence, Mitochondrial Dysfunction, and Their Link to Cardiovascular Disease.

Cardiovascular diseases (CVDs), a group of disorders affecting the heart or blood vessels, are the primary cause of death worldwide, with an immense impact on patient quality of life and disability. According to the World Health Organization, CVD takes an estimated 17.9 million lives each year, where more than four out of five CVD deaths are due to heart attacks and strokes. In the decades to come, an increased prevalence of age-related CVD, such as atherosclerosis, coronary artery stenosis, myocardial infarction (MI), valvular heart disease, and heart failure (HF) will contribute to an even greater health and economic burden as the global average life expectancy increases and consequently the world's population continues to age. Considering this, it is important to focus our research efforts on understanding the fundamental mechanisms underlying CVD. In this review, we focus on cellular senescence and mitochondrial dysfunction, which have long been established to contribute to CVD. We also assess the recent advances in targeting mitochondrial dysfunction including energy starvation and oxidative stress, mitochondria dynamics imbalance, cell apoptosis, mitophagy, and senescence with a focus on therapies that influence both and therefore perhaps represent strategies with the most clinical potential, range, and utility.

Senescent cardiomyocytes contribute to cardiac dysfunction following myocardial infarction.

Myocardial infarction is a leading cause of morbidity and mortality. While reperfusion is now standard therapy, pathological remodelling leading to heart failure remains a clinical problem. Cellular senescence has been shown to contribute to disease pathophysiology and treatment with the senolytic navitoclax attenuates inflammation, reduces adverse myocardial remodelling and results in improved functional recovery. However, it remains unclear which senescent cell populations contribute to these processes. To identify whether senescent cardiomyocytes contribute to disease pathophysiology post-myocardial infarction, we established a transgenic model in which p16 (CDKN2A) expression was specifically knocked-out in the cardiomyocyte population. Following myocardial infarction, mice lacking cardiomyocyte p16 expression demonstrated no difference in cardiomyocyte hypertrophy but exhibited improved cardiac function and significantly reduced scar size in comparison to control animals. This data demonstrates that senescent cardiomyocytes participate in pathological myocardial remodelling. Importantly, inhibition of cardiomyocyte senescence led to reduced senescence-associated inflammation and decreased senescence-associated markers within other myocardial lineages, consistent with the hypothesis that cardiomyocytes promote pathological remodelling by spreading senescence to other cell-types. Collectively this study presents the demonstration that senescent cardiomyocytes are major contributors to myocardial remodelling and dysfunction following a myocardial infarction. Therefore, to maximise the potential for clinical translation, it is important to further understand the mechanisms underlying cardiomyocyte senescence and how to optimise senolytic strategies to target this cell lineage.

Nucleic Acid Programmable Protein Arrays (NAPPA) for the Discovery of Autoantibodies in Osteoarthritis.

Since a decade, the nucleic acid protein programmable array (NAPPA) technology has provided researchers with a high-throughput proteomic technique for deciphering immune signatures and screening for biomarkers, among other applications. In osteoarthritis (OA), the activation of proinflammatory pathways of innate immunity due to cellular stress response and cartilage degradation is now considered to be one of the pathophysiological drivers of the disease, perpetuating the catabolic process and the inflammation of the joint. Therefore, the identification of immunosignatures in OA would allow to detect autoantibodies (AAbs) as a new source of disease-specific biomarkers. Herein, we describe a proteomic strategy combining NAPPA and in situ protein expression coupled with enzyme-linked immunoassays (ELISA) to search for novel serum AAbs in OA.

Autophagy Activation by Resveratrol Reduces Severity of Experimental Rheumatoid Arthritis.

SCOPE: Previous work reported that dietary supplementation with resveratrol lowers synovial hyperplasia, inflammatory and oxidative damage in an antigen-induced arthritis (AIA) model. Here, it is investigated whether resveratrol can regulate the abnormal synovial proliferation by inducing autophagy and controlling the associated inflammatory response. METHODS AND RESULTS: Animals treated with resveratrol 8 weeks before AIA induction show the highest significant signal for microtubule-associated protein 1 light chain 3 by confocal microscopy. Besides, resveratrol significantly reduces p62 expression, but it does not increase the signal of beclin-1. Also, active caspase-3 expression, as well as poly(ADP-ribose) polymerase, is upregulated in the AIA group, and is significantly reduced in resveratrol-treated AIA group. Resveratrol also mitigates angiopoietin-1 and vascular endothelial growth factor signals. Finally, resveratrol significantly reduces the serum levels of IL-1ß, C reactive protein, and prostaglandin E2, as well as nuclear factor κB synovial tissue expression, which shows a significant correlation with p62 expression. CONCLUSION: Dietary supplementation with resveratrol induces the noncanonical autophagy pathway and limits the cross-talk with inflammation, which in consequence modulates the synovial hyperplasia. Preventive strategies that incorporate dietary intervention with resveratrol may offer a potential therapeutic alternative to drugs to influence the risk of rheumatoid arthritis and influence its course.

Predictive modeling of therapeutic response to chondroitin sulfate/glucosamine hydrochloride in knee osteoarthritis.

BACKGROUND: In the present study, we explored potential protein biomarkers useful to predict the therapeutic response of knee osteoarthritis (KOA) patients treated with pharmaceutical grade Chondroitin sulfate/Glucosamine hydrochloride (CS+GH; Droglican, Bioiberica), in order to optimize therapeutic outcomes. METHODS: A shotgun proteomic analysis by iTRAQ labelling and liquid chromatography-mass spectrometry (LC-MS/MS) was performed using sera from 40 patients enrolled in the Multicentre Osteoarthritis interVEntion trial with Sysadoa (MOVES). The panel of proteins potentially useful to predict KOA patient's response was clinically validated in the whole MOVES cohort at baseline (n = 506) using commercially available enzyme-linked immunosorbent assays kits. Logistic regression models and receiver-operating-characteristics (ROC) curves were used to analyze the contribution of these proteins to our prediction models of symptomatic drug response in KOA. RESULTS: In the discovery phase of the study, a panel of six putative predictive biomarkers of response to CS+GH (APOA2, APOA4, APOH, ITIH1, C4BPa and ORM2) were identified by shotgun proteomics. Data are available via ProteomeXchange with identifier PXD012444. In the verification phase, the panel was verified in a larger set of KOA patients (n = 262). Finally, ITIH1 and ORM2 were qualified by a blind test in the whole MOVES cohort at baseline. The combination of these biomarkers with clinical variables predict the patients' response to CS+GH with a specificity of 79.5% and a sensitivity of 77.1%. CONCLUSIONS: Combining clinical and analytical parameters, we identified one biomarker that could accurately predict KOA patients' response to CS+GH treatment. Its use would allow an increase in response rates and safety for the patients suffering KOA.

Discovery of an autoantibody signature for the early diagnosis of knee osteoarthritis: data from the Osteoarthritis Initiative.

OBJECTIVE: To find autoantibodies (AAbs) in serum that could be useful to predict incidence of radiographic knee osteoarthritis (KOA). DESIGN: A Nucleic-acid Programmable Protein Arrays (NAPPA) platform was used to screen AAbs against 2125 human proteins in sera at baseline from participants free of radiographic KOA belonging to the incidence and non-exposed subcohorts of the Osteoarthritis Initiative (OAI) who developed or not, radiographic KOA during a follow-up period of 96 months. NAPPA-ELISA were performed to analyse reactivity against methionine adenosyltransferase two beta (MAT2ß) and verify the results in 327 participants from the same subcohorts. The association of MAT2ß-AAb levels with KOA incidence was assessed by combining several robust biostatistics analysis (logistic regression, Receiver Operating Characteristic and Kaplan-Meier curves). The proposed prognostic model was replicated in samples from the progression subcohort of the OAI. RESULTS: In the screening phase, six AAbs were found significantly different at baseline in samples from incident compared with non-incident participants. In the verification phase, high levels of MAT2ß-AAb were significantly associated with the future incidence of KOA and with an earlier development of the disease. The incorporation of this AAb in a clinical model for the prognosis of incident radiographic KOA significantly improved the identification/classification of patients who will develop the disorder. The usefulness of the model to predict radiographic KOA was confirmed on a different OAI subcohort. CONCLUSIONS: The measurement of AAbs against MAT2ß in serum might be highly useful to improve the prediction of OA development, and also to estimate the time to incidence.

Analysis of Endogenous Peptides Released from Osteoarthritic Cartilage Unravels Novel Pathogenic Markers.

Osteoarthritis (OA) is a pathology characterized by the loss of articular cartilage. In this study, we performed a peptidomic strategy to identify endogenous peptides (neopeptides) that are released from human osteoarthritic tissue, which may serve as disease markers. With this aim, secretomes of osteoarthritic and healthy articular cartilages obtained from knee and hip were analyzed by shotgun peptidomics. This discovery step led to the identification of 1175 different peptides, corresponding to 101 proteins, as products of the physiological or pathological turnover of cartilage extracellular matrix. Then, a targeted multiple reaction monitoring-mass spectrometry method was developed to quantify the panel of best marker candidates on a larger set of samples (n = 62). Statistical analyses were performed to evaluate the significance of the observed differences and the ability of the neopeptides to classify the tissue. Eight of them were differentially abundant in the media from wounded zones of OA cartilage compared with the healthy tissue (p < 0.05). Three neopeptides belonging to Clusterin and one from Cartilage Oligomeric Matrix Protein showed a disease-dependent decrease specifically in hip OA, whereas two from Prolargin (PRELP) and one from Cartilage Intermediate Layer Protein 1 were significantly increased in samples from knee OA. The release of one peptide from PRELP showed the best metrics for tissue classification (AUC = 0.834). The present study reveals specific neopeptides that are differentially released from knee or hip human osteoarthritic cartilage compared with healthy tissue. This evidences the intervention of characteristic pathogenic pathways in OA and provides a novel panel of peptidic candidates for biomarker development.

Multiplexed mass spectrometry monitoring of biomarker candidates for osteoarthritis.

The methods currently available for the diagnosis and monitoring of osteoarthritis (OA) are very limited and lack sensitivity. Being the most prevalent rheumatic disease, one of the most disabling pathologies worldwide and currently untreatable, there is a considerable interest pointed in the verification of specific biological markers for improving its diagnosis and disease progression studies. Considering the remarkable development of targeted proteomics methodologies in the frame of the Human Proteome Project, the aim of this work was to develop and apply a MRM-based method for the multiplexed analysis of a panel of 6 biomarker candidates for OA encoded by the Chromosome 16, and another 8 proteins identified in previous shotgun studies as related with this pathology, in specimens derived from the human joint and serum. The method, targeting 35 different peptides, was applied to samples from human articular chondrocytes, healthy and osteoarthritic cartilage, synovial fluid and serum. Subsequently, a verification analysis of the biomarker value of these proteins was performed by single point measurements on a set of 116 serum samples, leading to the identification of increased amounts of Haptoglobin and von Willebrand Factor in OA patients. Altogether, the present work provides a tool for the multiplexed monitoring of 14 biomarker candidates for OA, and verifies for the first time the increased amount of two of these circulating markers in patients diagnosed with this disease. SIGNIFICANCE: We have developed an MRM method for the identification and relative quantification of a panel of 14 protein biomarker candidates for osteoarthritis. This method has been applied to analyze human articular chondrocytes, articular cartilage, synovial fluid, and finally a collection of 116 serum samples from healthy controls and patients suffering different degrees of osteoarthritis, in order to verify the biomarker usefulness of the candidates. HPT and VWF were validated as increased in OA patients.