Extracellular Matrix Turnover Research Articles

Extracellular matrix turnover proteins as risk markers in people with type 2 diabetes and microalbuminuria

BackgroundThis post-hoc study investigated whether biomarkers reflecting extracellular matrix (ECM) turnover predicted cardiovascular disease (CVD), mortality, and progression of diabetic kidney disease (DKD) in individuals with type 2 diabetes (T2D) and microalbuminuria. MethodsSerum levels of specific ECM turnover biomarkers were assessed in 192 participants with T2D and microalbuminuria from an observational study conducted at Steno Diabetes Center Copenhagen from 2007 to 2008. Endpoints included CVD events, mortality, and DKD progression, defined as decline in estimated glomerular filtration rate (eGFR) of >30 %. ResultsParticipants had a mean age of 59 years, with 75 % males. Over a median follow-up of 4.9 to 6.3 years, the study recorded 38 CVD events, 24 deaths, and 40 DKD events. Elevated levels of a degradation fragment of collagen type I (C1M) were associated with an increased risk of >30 % eGFR decline, although this association was not independent of other risk factors. No significant associations were found between other ECM turnover biomarkers and DKD progression, mortality, or CVD risk. ConclusionElevated C1M levels were linked to DKD progression in individuals with T2D and microalbuminuria, but not independently of other risk factors. None of the ECM turnover biomarkers were associated with CVD or mortality.

Aged Tendons Exhibit Altered Mechanisms of Strain-Dependent Extracellular Matrix Remodeling.

Aging is a primary risk factor for degenerative tendon injuries, yet the etiology and progression of this degeneration are poorly understood. While aged tendons have innate cellular differences that support a reduced ability to maintain mechanical tissue homeostasis, the response of aged tendons to altered levels of mechanical loading has not yet been studied. To address this question, we subjected young and aged murine flexor tendon explants to various levels of in vitro tensile strain. We first compared the effect of static and cyclic strain on matrix remodeling in young tendons, finding that cyclic strain is optimal for studying remodeling in vitro. We then investigated the remodeling response of young and aged tendon explants after 7 days of varied mechanical stimulus (stress deprivation, 1%, 3%, 5%, or 7% cyclic strain) via assessment of tissue composition, biosynthetic capacity, and degradation profiles. We hypothesized that aged tendons would show muted adaptive responses to changes in tensile strain and exhibit a shifted mechanical setpoint, at which the remodeling balance is optimal. Interestingly, we found that 1% cyclic strain best maintains native physiology while promoting extracellular matrix (ECM) turnover for both age groups. However, aged tendons display fewer strain-dependent changes, suggesting a reduced ability to adapt to altered levels of mechanical loading. This work has a significant impact on understanding the regulation of tissue homeostasis in aged tendons, which can inform clinical rehabilitation strategies for treating elderly patients.

Neo-epitope detection identifies extracellular matrix turnover in systemic inflammation and sepsis: an exploratory study

BackgroundSepsis is associated with high morbidity and mortality, primarily due to systemic inflammation-induced tissue damage, resulting organ failure, and impaired recovery. Regulated extracellular matrix (ECM) turnover is crucial for maintaining tissue homeostasis in health and in response to disease-related changes in the tissue microenvironment. Conversely, uncontrolled turnover can contribute to tissue damage. Systemic Inflammation is implicated to play a role in the regulation of ECM turnover, but the relationship between the two is largely unclear.MethodsWe performed an exploratory study in 10 healthy male volunteers who were intravenously challenged with 2 ng/kg lipopolysaccharide (LPS, derived from Escherichia coli) to induce systemic inflammation. Plasma samples were collected before (T0) and after (T 1 h, 3 h, 6 h and 24 h) the LPS challenge. Furthermore, plasma was collected from 43 patients with septic shock on day 1 of ICU admission. Circulating neo-epitopes of extracellular matrix turnover, including ECM degradation neo-epitopes of collagen type I (C1M), type III (C3M), type IV (C4Ma3), and type VI (C6M), elastin (ELP-3) and fibrin (X-FIB), as well as the ECM synthesis neo-epitopes of collagen type III (PRO-C3), collagen type IV (PRO-C4) and collagen type VI (PRO-C6) were measured by ELISA. Patient outcome data were obtained from electronic patient records.ResultsTwenty-four hours after LPS administration, all measured ECM turnover neo-epitopes, except ELP-3, were increased compared to baseline levels. In septic shock patients, concentrations of all measured ECM neo-epitopes were higher compared to healthy controls. In addition, concentrations of C6M, ELP-3 and X-FIB were higher in patients with septic shock who ultimately did not survive (N = 7) compared to those who recovered (N = 36).ConclusionECM turnover is induced in a model of systemic inflammation in healthy volunteers and was observed in patients with septic shock. Understanding interactions between systemic inflammation and ECM turnover may provide further insight into mechanisms underlying acute and persistent organ failure in sepsis.

Circulating biomarkers and progression of idiopathic pulmonary fibrosis: data from the INMARK trial

BackgroundWe used data from the INMARK trial to investigate associations between circulating biomarkers of extracellular matrix (ECM) turnover, inflammation, and epithelial dysfunction and disease progression in subjects with idiopathic pulmonary fibrosis (IPF).MethodsSubjects with IPF and FVC ≥80% predicted were randomised 1:2 to receive nintedanib 150 mg bid or placebo for 12 weeks followed by open-label nintedanib for 40 weeks. Associations between baseline biomarker levels and the proportion of subjects with disease progression (decline in FVC ≥10% predicted or death) over 52 weeks were assessed in subjects randomised to placebo using logistic regression. Associations between baseline demographic/clinical characteristics and biomarker levels and disease progression over 52 weeks were analysed using multivariate models.ResultsOf 230 subjects who received placebo for 12 weeks then open-label nintedanib for 40 weeks, 70 (30.4%) had disease progression over 52 weeks. Baseline levels of CRPM, C3M, CRP, KL-6 and SP-D were not significantly associated with disease progression over 52 weeks in analyses corrected for multiple comparisons. In models including only baseline demographic/clinical characteristics, 61.2% to 64.2% of subjects were correctly classified as having or not having disease progression over 52 weeks. When both demographic/clinical characteristics and biomarker levels were included in the models, 50.0% to 64.5% of the test set were correctly classified.ConclusionsAmong subjects with IPF and preserved FVC, multivariate models based on demographic/clinical characteristics and biomarker levels at baseline did not provide an accurate prediction of which patients would progress.

Extracellular Matrix Interactome in Modulating Vascular Homeostasis and Remodeling.

The ECM (extracellular matrix) is a major component of the vascular microenvironment that modulates vascular homeostasis. ECM proteins include collagens, elastin, noncollagen glycoproteins, and proteoglycans/glycosaminoglycans. ECM proteins form complex matrix structures, such as the basal lamina and collagen and elastin fibers, through direct interactions or lysyl oxidase-mediated cross-linking. Moreover, ECM proteins directly interact with cell surface receptors or extracellular secreted molecules, exerting matricellular and matricrine modulation, respectively. In addition, extracellular proteases degrade or cleave matrix proteins, thereby contributing to ECM turnover. These interactions constitute the ECM interactome network, which is essential for maintaining vascular homeostasis and preventing pathological vascular remodeling. The current review mainly focuses on endogenous matrix proteins in blood vessels and discusses the interaction of these matrix proteins with other ECM proteins, cell surface receptors, cytokines, complement and coagulation factors, and their potential roles in maintaining vascular homeostasis and preventing pathological remodeling.

Bile duct matrix metalloproteinase-7 expression: a new modality for diagnosis of biliary atresia

BackgroundBiliary atresia (BA) is an obliterative cholangiopathy of infancy that results in cholestasis and liver fibrosis. This fibrosis is due to an imbalance in extracellular matrix (ECM) breakdown and deposition. The mechanism by which the progressive injury occurs is not fully elucidated. Matrix metalloproteinases (MMPs) are involved in ECM turnover but also have non-ECM-related functions. Matrix metalloproteinase 7 (MMP7) has been suggested as a promising biomarker in diagnosing BA.ObjectiveThe aim of this study was to assess the hepatic expression of MMP-7 in infants with BA.Patients and methodsThe study was a retrospective-prospective case–control study that included 50 patients who were categorized into two groups, BA group (25 patients) and non-BA cholestatic patients as a control group (25 patients). Liver biochemistry, liver biopsy, histopathology, and immunohistochemical staining for primary antibody MMP-7 were performed for all studied patients.ResultsBile duct MMP7 expression was significantly higher in infants with BA than in non-BA cholestasis (P = 0.003), While the hepatic MMP-7 intensity did not differ significantly between both groups (P > 0.05). Bile duct expression of MMP-7 had a significant positive correlation with the BA Score (P = 0.017), while hepatic MMP-7 intensity had a significant positive correlation with alanine transaminase levels (P = 0.007) and a significant negative correlation with γ glutamyl transferase in the BA group (P = 0. 038). There was no statistically significant difference among different stages of fibrosis as regards the median of the hepatic MMP-7 intensity score and MMP-7 bile duct expression in infants with BA. There was no statistically significant difference between infants with successful and failed Kasai as regard the hepatic MMP-7 intensity and its bile duct expression.ConclusionBile duct expression of MMP-7 measured by immunohistochemistry is useful for the diagnosis of BA, but it is limited in predicting the stage of liver fibrosis and the outcome of Kasai portoenterostomy (KPE).

Longitudinal plasma proteomics reveals biomarkers of alveolar-capillary barrier disruption in critically ill COVID-19 patients

The pathobiology of respiratory failure in COVID-19 consists of a complex interplay between viral cytopathic effects and a dysregulated host immune response. In critically ill patients, imatinib treatment demonstrated potential for reducing invasive ventilation duration and mortality. Here, we perform longitudinal profiling of 6385 plasma proteins in 318 hospitalised patients to investigate the biological processes involved in critical COVID-19, and assess the effects of imatinib treatment. Nine proteins measured at hospital admission accurately predict critical illness development. Next to dysregulation of inflammation, critical illness is characterised by pathways involving cellular adhesion, extracellular matrix turnover and tissue remodelling. Imatinib treatment attenuates protein perturbations associated with inflammation and extracellular matrix turnover. These proteomic alterations are contextualised using external pulmonary RNA-sequencing data of deceased COVID-19 patients and imatinib-treated Syrian hamsters. Together, we show that alveolar capillary barrier disruption in critical COVID-19 is reflected in the plasma proteome, and is attenuated with imatinib treatment. This study comprises a secondary analysis of both clinical data and plasma samples derived from a clinical trial that was registered with the EU Clinical Trials Register (EudraCT 2020–001236–10, https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001236-10/NL) and Netherlands Trial Register (NL8491, https://www.trialregister.nl/trial/8491).

Cellular behavior and extracellular matrix turnover in bovine annulus fibrosus cells under hydrostatic pressure and deviatoric strain.

Intervertebral disc herniation is a common spinal disorder that is often treated with discectomy when conservative measures fail. To devise therapeutic strategies for tears in the annulus fibrosus (AF), the regenerative capability of AF cells under spinal loading needs to be addressed. We hypothesized that the compressive loading associated with deformation in AF cells reduces synthetic and degradative activities in extracellular matrix and cell proliferation. We evaluated expression of key matrix molecules and cell proliferation by RT-PCR and immunohistochemistry by inner and outer bovine AF cells incubated under hydrostatic pressure (HP), arc-bending strain (Strain), and combined HP and Strain (HP/Strain) mimicking spinal loading. Inner AF cells showed significantly increased levels of aggrecan core protein, chondroitin sulfate N-acetylgalactosaminyltransferase-1, and tissue inhibitor of metalloproteinases-2 by 6 days under HP (p < 0.05), with a tendency toward increased matrix metalloproteinase-13. Outer AF cells demonstrated a significant decline in collagen type-2 under Strain and HP/Strain (p < 0.05) and a tendency toward suppression of collagen type-1 and elastin expression compared to HP and unloaded control. On the other hand, proliferating cell nucleus antigen increased significantly under Strain and HP/Strain in inner AF and declined under unloaded and HP in outer AF (p < 0.05). Immunohistology findings supported reductions in gene expressions of matrix molecules. Thus, changes in HP/Strain in AF appear to diminish synthetic and degradative activities while increasing cell proliferation. To promote regeneration, continuous overloading should be avoided, as it converts the synthetic activity to a state in which tissue repair is limited.

Dysregulation of extracellular matrix and Lysyl Oxidase in Ehlers-Danlos syndrome type IV skin fibroblasts

BackgroundEhlers-Danlos syndrome Type IV (aka Vascular Ehlers Danlos, or vEDS) is a dominantly inherited mutation in the Collagen 3A1 gene (COL3A1). The disease is characterized by tissue friability and age-related susceptibility to arterial aneurysm, dissection and rupture as well as uterine and bowl tears. These clinical manifestations result in major surgical intervention and decreased life expectancy. Understanding how mutations in COL3A1 impact the structure and function of the extracellular matrix (ECM) is important to managing the disease and finding treatments.ResultsSkin fibroblasts from vEDS subjects heterozygous for the p.G588S pathogenic variant in the COL3A1 gene and a normal individual were cultured and studied. Proteomics analysis identified dozens of upregulated proteins related to extracellular matrix dysregulation that is characteristic of fibrosis. Gene expression libraries from cultured primary fibroblasts were screened for messenger RNA (mRNA) markers of ECM degradation. The proteomics and targeted gene expression array results were largely consistent with dysregulation of the extracellular matrix in vEDS. The data show upregulation of multiple Collagen proteins and genes, other ECM components, and enzymes related to ECM processing and turn-over. vEDS fibroblasts expressed significantly more cross linked C-Telopeptide of Collagen III (CTXIII) than normal fibroblasts, indicative of Collagen III degradation and turn-over. Further, the expression and activity of Lysyl Oxidase (LOX), an enzyme that initiates covalent cross-linking of soluble collagen and elastin into protease resistant fibers, is elevated in vEDS fibroblasts compared to normal fibroblasts.ConclusionTogether, these findings suggest dysregulated ECM deposition and processing, reminiscent of a state of fibrosis. Therapeutics that target the dysregulated ECM proteins or help replace damaged tissue may improve clinical outcomes.

Clearance of extracellular human amyloid-β aggregates in C. elegans by nutraceutical and pharmaceutical interventions.

Numerous anti-amyloid therapies have seen recent clinical development and approval, such as the monoclonal antibodies aducanumab and lecanemab. However, in Alzheimer's disease patients, amyloid-β (Aβ) plaques are found embedded in the extracellular matrix and surrounded by collagens, which might hinder these antibodies from targeting the plaques. We reasoned that various different nutraceutical and pharmaceutical agents might induce collagen and extracellular matrix turnover and removal of these collagen-embedded amyloid-β (Aβ) plaques. To address this idea, here, we used a transgenic C. elegans strain, LSD2104 , expressing fluorescent human Aβ 1-42 as an in-vivo model for secreted amyloid aggregation in the extracellular matrix. We performed a screen of various nutraceuticals and pharmaceuticals along with different combinations, and we found that quercetin 350 µM and rifampicin 75 µM successfully cleared the extracellular amyloid plaque burden compared to the 0.2% DMSO control group, with a combination of the two agents producing the maximum effect compared to either drug alone. These results may implicate the exploration of combination therapeutics of nutraceuticals and pharmaceuticals in the clearance of amyloid-β (Aβ) plaques in Alzheimer's disease.

Sex as a biological variable for cardiovascular physiology.

There have been ongoing efforts by federal agencies and scientific communities since the early 1990s to incorporate sex and/or gender in all aspects of cardiovascular research. Scientific journals provide a critical function as change agents to influence transformation by encouraging submissions for topic areas, and by setting standards and expectations for articles submitted to the journal. As part of ongoing efforts to advance sex and gender in cardiovascular physiology research, the American Journal of Physiology-Heart and Circulatory Physiology recently launched a call for papers on Considering Sex as a Biological Variable. This call was an overwhelming success, resulting in 78 articles published in this collection. This review summarizes the major themes of the collection, including Sex as a Biological Variable Within: Endothelial Cell and Vascular Physiology, Cardiovascular Immunity and Inflammation, Metabolism and Mitochondrial Energy, Extracellular Matrix Turnover and Fibrosis, Neurohormonal Signaling, and Cardiovascular Clinical and Epidemiology Assessments. Several articles also focused on establishing rigor and reproducibility of key physiological measurements involved in cardiovascular health and disease, as well as recommendations and considerations for study design. Combined, these articles summarize our current understanding of sex and gender influences on cardiovascular physiology and pathophysiology and provide insight into future directions needed to further expand our knowledge.

Effect of Sparc knockout on the extracellular matrix of mouse periodontal ligament cells

The periodontal ligament (PDL) is a critical component in maintaining tooth stability. It is composed of cells and an extracellular matrix (ECM), each with unique roles in tissue function and homeostasis. Secreted protein acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, plays a crucial role in regulating ECM assembly and turnover, alongside facilitating cellular-ECM interactions. In the present study, mass spectrometry-based proteomics was used to assess the impacts of Sparc-knockout (KO) on PDL-derived cells. Results demonstrated that Sparc-KO significantly reduces ECM production and alters its composition with increased levels of type I collagen. Despite this increase in Sparc-KO, type I collagen was not likely to be effectively integrated into the fibrils due to collagen cross-linking impairment. Furthermore, the pathway and process enrichment analyses suggested that SPARC plays a protective role against ECM degradation by antagonistically interacting with cell-surface collagen receptors. These findings provide detailed insights into the multifaceted role of SPARC in ECM organization, including its impact on ECM production, collagen regulation, and interactions with various cellular compartments. A better understanding of these complex mechanisms is crucial for comprehending the causes of periodontal disease and tissue regeneration, where precise control of ECM organization is necessary.

Mechanism of pro-MMP9 activation in co-culture of pro-inflammatory macrophages and cardiomyocytes

ObjectiveA wide range of cardiac diseases is associated with inflammation. “Inflamed” heart tissue is infiltrated with pro-inflammatory macrophages which extensively secrete matrix metalloproteinase 9 (MMP9), a regulator of extracellular matrix turnover. As MMP9 is released from macrophages in a latent form, it requires activation. The present study addresses the role of cardiomyocytes in the course of this activation process. Methods and resultsIn mono- and co-cultures of pro-inflammatory rat macrophages (bone marrow-derived and peritoneal) and cardiomyocytes (H9C2 cell line) gelatin zymography demonstrated that activated macrophages robustly secreted latent pro-MMP9, whereas cardiomyocytes could not produce the enzyme. Co-culturing of the two cell species was critical for pro-MMP9 activation and was also accompanied by processing of cardiomyocyte-secreted pro-MMP2. A cascade of pro-MMP9 activation was initiated on macrophage membrane with pro-MMP2 cleavage. Namely, pro-inflammatory macrophages expressed an active membrane type 1 MMP (MT1MMP), which activated pro-MMP2, which in turn converted pro-MMP9. Downregulation of MT1MMP in macrophages by siRNA abolished activation of both pro-MMP2 and pro-MMP9 in co-culture. In addition, both cell species secreted MMP13 as a further pro-MMP9 activator. In co-culture, activation of pro-MMP13 occurred on membranes of macrophages and was enhanced in presence of active MMP2. Using incubations with recombinant MMPs and isolated macrophage membranes, we demonstrated that while both MMP2 and MMP13 individually had the ability to activate pro-MMP9, their combined action provided a synergistic effect. ConclusionActivation of pro-MMP9 in a co-culture of pro-inflammatory macrophages and cardiomyocytes was the result of a complex interaction of several MMPs on the cell membrane and in the extracellular space. Both cell types contributed critically to pro-MMP9 processing.

Extracellular Matrix Deposition Defines the Duration of Cell Sheet Assembly from Human Adipose-Derived MSC

Cell sheet (CS) engineering using mesenchymal stromal cells (MSC) draws significant interest for regenerative medicine and this approach translates to clinical use for numerous indications. However, little is known of factors that define the timing of CS assembly from primary cultures. This aspect is important for planning CS delivery in autologous and allogeneic modes of use. We used a comparative in vitro approach with primary donors’ (n = 14) adipose-derived MSCs and evaluated the impact of healthy subject’s sex, MSC culture features (population doubling time and lag-phase), and extracellular matrix (ECM) composition along with factors related to connective tissue formations (α-SMA and FAP-α) on CS assembly duration. Using qualitative and quantitative analysis methods, we found that, in seeded MSCs, high contents of collagen I and collagen IV had a direct correlation with longer CS assembly duration. We found that short lag-phase cultures faster turned to a ready-to-use CS, while age, sex, fibronectin, laminin, α-SMA, and FAP-α failed to provide a significant correlation with the timing of assembly. In detachable CSs, FAP-α was negatively correlated with the duration of assembly, suggesting that its concentration rose over time and contributed to MSC activation, transitioning to α-SMA-positive myofibroblasts and ECM turnover. Preliminary data on cell density and collagen I deposition suggested that the TGF-β1 signaling axis is of pivotal importance for ECM composition and construct maturation.

Single cell RNA sequencing to dissect transcriptional signature of cardiac fibroblasts activation in experimental autoimmune myocarditis

Abstract Introduction Dilated cardiomyopathy (DCM) is a condition that evolves from inflammation of the myocardium, and it is often associated with a poor prognosis and fatal outcome. Experimental autoimmune myocarditis (EAM) represents a CD4+ T cell-dependent animal model of acute myocarditis followed by the development of post-inflammatory DCM. Purpose The aim of the study was to investigate the transcriptional changes in cardiac fibroblasts during disease progression in a mouse model of EAM at the single cell level. Methods 6–8-week-old BALB/c mice (or reporter mice expressing EGFP under the collagen type I promoter) were immunized with αMyHC peptide together with Complete Freunds’ Adjuvant. Live cardiac cells were sorted from healthy hearts (d0), at acute myocarditis (d19), during resolution of inflammation (d25), and at the DCM stage (d35) using the BD FACSAria II Cell Sorter. Single-cell cDNA libraries were prepared using 10x Genomics technology, and the sequencing was performed on an Illumina NovaSeq 6000 platform, targeting 50000 reads per cell. Single cell RNA sequencing data was analyzed using RStudio software and Seurat packages. At d0 and d19 of EAM, bulk RNAseq was performed on live cardiac EGFP+CD45-CD34- fibroblasts. Results Following a quality control check, we included in the analysis 25058 cells. We identified 13 distinct main cell populations, including fibroblasts, lymphoid and myeloid immune cells, endothelial cells, smooth muscle cells, and cardiomyocytes (Fig. 1A). Differential expression analysis revealed that fibroblasts in acute myocarditis (d19) showed activation of genes related to immune processes such as cytokine-cytokine receptor interaction, IL-17, and TNF-alpha signaling pathways. Subset analysis on 4901 cardiac fibroblasts resulted in 5 distinct fibroblast subsets, FB1–FB5 (Fig. 1B). A major fibroblast subset found in the heart (FB1) expanded during the course of EAM (Fig. 1C). Of note, cells expressing the myofibroblastic signature (FB4) were identified at d19 only (Fig. 1C). Fibroblast subset FB1 activated expression of proinflammatory genes, such as Ly6a, Ly6c1, Cxcl14, Ccl2, and Ccl7, particularly during the inflammatory phase of EAM (d19, Fig. 1D). Surprisingly, the expression of profibrotic genes, including Col1a1, Col3a1, and Postn in FB1, was downregulated during the progression of EAM (Fig. 1E). Bulk RNA sequencing analysis confirmed that cardiac fibroblasts in acute myocarditis (d19) activated immune processes (chemokine production), but also showed dysregulated expression of genes involved in cytoskeletal re-organization and extracellular matrix turnover. Conclusions Our data indicated that acute inflammation in the heart activated a pro-inflammatory transcriptional response in cardiac fibroblasts and induced a fibroblast-to-myofibroblast transition. Understanding changes in transcriptomic signatures on a single cell level can help to develop new therapies in inflammatory heart diseases.

Biological profiling in the spectrum of acute coronary syndrome: characterizing patients from MINOCA to NSTEMI with different features of the culprit plaque

Abstract Background In the last two decades, the development of procedures and tools such as coronary angiography and optical coherence tomography (OCT) have made it possible to highlight several aspects of acute coronary syndromes (ACS), identifying multiple subgroups such as patients with myocardial infarction with non-obstructive coronary arteries (MINOCA) and patients with Non-ST-Elevation Myocardial Infarction (NSTEMI) and ruptured fibrous cap (RFC) or intact fibrous cap (IFC) culprit lesions. The aim of the study is to better characterize the ACS spectrum by evaluating the biological profile of non-characterized NTSEMI, RFC-NSTEMI, IFC-NSTEMI, and MINOCA patients. Methods We enrolled 56 consecutive patients presenting with ACS, specifically 16 patients with non-characterized NSTEMI, 12 patients with RFC-NSTEMI, 8 patients with IFC-NSTEMI, and 20 patients with MINOCA. Biological samples were collected and stored. We performed gene expression analysis through qRT-PCR technique for 21 genes. Results Our data displayed several differences in gene expression levels involved in inflammation, cellular adhesion, extracellular matrix turnover, and oxidative stress between the groups under examination. We observed no significant difference between non-characterized NSTEMI and MINOCA for all the studied genes. Alongside, MINOCA displayed significantly lower levels of the following genes than RFC and IFC patients: CD44, GPX1, ICAM1, PLA2G7, SOD1, and VEGFA. Furthermore, when compared with IFC patients, MINOCA showed lower levels of EDN1, LGALS8, MMP1, and TNF, and non-characterized NSTEMI showed a lower gene expression of CD31, ICAM1, and PI16. Finally, CD44, EDN1, GPX1, LGALS8, NOS3, SOD1, TFRC, and VEGFA were more expressed in RFC and IFC compared to non-characterized NSTEMI patients. Conclusions Our preliminary data, besides exploring new pathways in MINOCA patients, reflects the need for characterization and patients’ stratifications in the clinical management of ACS, since no differences were observed between non-characterized NSTEMI and MINOCA. This result may indicate that a one size fits all approach is not the most suitable since a plaque could be an innocent bystander and should be treated accordingly. A precision medicine approach that recognizes the presence of obstructive plaque and defines the type of culprit plaque (i.e., RFC or IFC) could identify better treatment, for a tailored therapy, allowing better prognosis and quality of life.Figure

LIGHT/TNFSF14 levels in carotid atherosclerotic plaques are associated with symptomatic cerebrovascular disease

Abstract Background Plaque rupture is the underlying cause of most cardiovascular events, such as stroke and myocardial infarction. Co-stimulatory molecules, the immune checkpoints, are emerging as strong candidates to battle atherosclerosis progression. The co-stimulatory molecule LIGHT (tumour necrosis factor 14; TNFSF14) exists in foam cell-rich regions of atherosclerotic plaques, but whether it has a driving role in promoting a high-risk plaque phenotype is not known. Purpose This study investigates the association between intra-plaque LIGHT and plaque vulnerability. Methods Plaque levels of LIGHT/TNFSF14 were measured in homogenised carotid endarterectomy plaques by Proximity Extension Assay (PEA) in subjects from the Carotid Plaque Imaging Cohort (CPIP; N=201). Homogenates were further examined by multi-plex cytokine analysis and ELISA, and tissue plaque sections through histology/immunohistochemistry. Results Plaque levels of LIGHT were associated with the occurrence of pre-operative cerebrovascular symptoms (p=0.00014). Furthermore, LIGHT correlated strongly with the histological plaque vulnerability index (p=5x10-15): a ratio between the sum of destabilising versus stabilising plaque components (i.e. macrophages, intra-plaque haemorrhage and lipids versus smooth muscle cells and collagen) as well as with necrotic core size and the inflammatory cytokines interleukin(IL)-1β, IL-6, chemokine (C-C motif) ligand (CCL)-2, CCL4 and CCL5. Moreover, plaque LIGHT levels correlated with several components of the extracellular matrix turnover machinery, including cleaved collagen, the collagen cross-linking proteoglycans fibromodulin and lumican, the matricellular protein COMP (cartilage oligomeric matrix protein), and the remodelling regulator ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs-7). Levels also correlated with matrix metalloproteinases (MMPs) 1, 2, 9 and 10, and tissue inhibitor of metalloproteinase (TIMP)-1. Conclusion Expression of LIGHT in atherosclerotic plaques does not only correlate with markers of plaque destabilisation¸ but is also significantly elevated in plaques from symptomatic, compared to asymptomatic, patients. These results connect LIGHT content to a rupture-prone plaque phenotype and suggest the LIGHT signalling pathway as a potential therapeutic target to promote atherosclerotic plaque stability.

Relaxin as a biological marker and therapeutic target in heart failure

An important task of modern cardiology is the search and study of new cardiovascular biological markers that can help in the early diagnosis of heart diseases, serve as a tool to assess the effectiveness of treatment, and act as a prognostic marker and risk stratification criterion. This literature review aimed to consider relaxin (RLN) as a new diagnostic and prognostic cardiovascular biological marker. RLN is a natural peptide hormone with a molecular weight of approximately 6000 daltons. RLN2 is the main circulating form of RLN in the blood. Although human RLN2 was originally discovered as a hormone mainly secreted by the corpus luteum of the ovary, it is also synthesized in various tissues in non-pregnant women and men and is considered one of the most pleiotropic hormones of the human body, which perform various activities beyond reproduction.&#x0D; The availability of serelaxin (a recombinant molecule identical to human RLN2) has made it possible to study the effects of RLN2 on the cardiovascular system, kidneys, liver, and brain and to evaluate it in several randomized placebo-controlled clinical trials. RLN2 exerts many cardioprotective effects on the heart and vessels and has been proposed as a therapeutic target for cardiovascular diseases such as heart failure, atrial fibrillation, coronary heart disease, myocardial infarction, and arterial hypertension. Through the activation of its cognate receptor and subsequent flow of several molecular signaling pathways in the cardiovascular system, RLN2 can induce vasodilation and angiogenesis, increase arterial compliance and cardiac output, reduce vascular resistance, exert antifibrotic effects through regulation and remodeling of extracellular matrix turnover, reduce inflammation and apoptosis, inhibit oxidative stress, induce chronotropic and inotropic effects, and inhibit ventricular and atrial ectopic activity.&#x0D; Further studies are needed to demonstrate the potential use of RLN as an additional laboratory tool for diagnosis, risk stratification, and prediction of cardiovascular events in patients with heart failure. The effects of relaxin on morbidity and mortality in patients with heart failure have yet to be evaluated in more detail.

Abstract 14391: Biomarkers, Proteomics, and Echocardiography Lend Insights into Cardiovascular Death in HFrEF: A VICTORIA Substudy

Background: Using data from the VICTORIA ECHO substudy in high-risk patients with heart failure with reduced ejection fraction (HFrEF), we evaluated the potential complementary role of cardiac function, biomarkers, and proteomics in association with cardiovascular death (CVD), a key component of the primary composite (HF hospitalization or CVD). Hypothesis: Measurement of cardiac biomarkers, proteins, and ECHO parameters may provide insights into the mechanism of CVD in HFrEF. Methods: At baseline, left ventricular ejection fraction (LVEF) and LV end-systolic volume index (LVESVI) on ECHO, 6 quantitative biomarkers, and 92 proteins (Olink CVD III panel) were assessed by blinded core labs. Adjusted (MAGGIC) associations and HRs (95% CIs) are presented. Proteomics associations were adjusted for false discovery rate. Results: Of 583 patients, 96 (16.5%) had CVD (21.6/100 pt-years) a median 8 months (IQR 6-11) from randomization. See Table for MAGGIC-adjusted HRs for LVEF, LVESVI, and biomarkers. NT-proBNP was most strongly associated with CVD (HR per doubling 1.53 [1.32-1.77]). Proteins significantly associated with CVD were cathepsin D (2.32 [1.62-3.32]), matrix metalloproteinase-2 (2.31 [1.45-3.68]), IL-2 receptor (1.74 [1.40-2.14]), transferrin receptor (1.64 [1.31-2.04]) and tumor necrosis superfamily (1.88 [1.41-2.50]) (Fig). Conclusions: After MAGGIC adjustment, a rise in LVESVI, but not LVEF, was associated with CVD. Upregulation of selected biomarkers/proteins related to myocardial wall stress, extracellular matrix turnover, inflammation, and oxidative stress were also associated with CVD and may serve as novel future therapeutic targets in HFrEF.

Whole Genome Expression Profiling of Semitendinosus Tendons from Children with Diplegic and Tetraplegic Cerebral Palsy.

Cerebral palsy (CP) is the most common movement disorder in children, with a prevalence ranging from 1.5 to 4 per 1000 live births. CP is caused by a non-progressive lesion of the developing brain, leading to progressive alterations of the musculoskeletal system, including spasticity, often leading to the development of fixed contractures, necessitating tendon lengthening surgery. Total RNA-sequencing analysis was performed on semitendinosus tendons from diplegic and tetraplegic CP patients subjected to tendon lengthening surgery compared to control patients undergoing anterior cruciate ligament reconstructive surgery. Tetraplegic CP patients showed increased expression of genes implicated in collagen synthesis and extracellular matrix (ECM) turnover, while only minor changes were observed in diplegic CP patients. In addition, tendons from tetraplegic CP patients showed an enrichment for upregulated genes involved in vesicle-mediated transport and downregulated genes involved in cytokine and apoptotic signaling. Overall, our results indicate increased ECM turnover with increased net synthesis of collagen in tetraplegic CP patients without activation of inflammatory and apoptotic pathways, similar to observations in athletes where ECM remodeling results in increased tendon stiffness and tensile strength. Nevertheless, the resulting increased tendon stiffness is an important issue in clinical practice, where surgery is often required to restore joint mobility.