Transcriptomic Analysis of Extracellular Vesicles in the Search for Novel Plasma and Thrombus Biomarkers of Ischemic Stroke Etiologies.

Accurate etiologic diagnosis provides an appropriate secondary prevention and better prognosis in ischemic stroke (IS) patients; still, 45% of IS are cryptogenic, urging us to enhance diagnostic precision. We have studied the transcriptomic content of plasma extracellular vesicles (EVs) (n = 21) to identify potential biomarkers of IS etiologies. The proteins encoded by the selected genes were measured in the sera of IS patients (n = 114) and in hypertensive patients with (n = 78) and without atrial fibrillation (AF) (n = 20). IGFBP-2, the most promising candidate, was studied using immunohistochemistry in the IS thrombi (n = 23) and atrium of AF patients (n = 13). In vitro, the IGFBP-2 blockade was analyzed using thromboelastometry and endothelial cell cultures. We identified 745 differentially expressed genes among EVs of cardioembolic, atherothrombotic, and ESUS groups. From these, IGFBP-2 (cutoff > 247.6 ng/mL) emerged as a potential circulating biomarker of embolic IS [OR = 8.70 (1.84-41.13) p = 0.003], which was increased in patients with AF vs. controls (p < 0.001) and was augmented in cardioembolic vs. atherothrombotic thrombi (p < 0.01). Ex vivo, the blockage of IGFBP-2 reduced clot firmness (p < 0.01) and lysis time (p < 0.001) and in vitro, diminished endothelial permeability (p < 0.05) and transmigration (p = 0.06). IGFBP-2 could be a biomarker of embolic IS and a new therapeutic target involved in clot formation and endothelial dysfunction.

Role of factor XIII in ischemic stroke: a key molecule promoting thrombus stabilization and resistance to lysis.

BACKGROUND: Active coagulation factor XIII (FXIII) catalyzing crosslinking of fibrin and other hemostatic factors plays a key role in clot stability and lysis. OBJECTIVES: To evaluate the effect of FXIII inhibition in a mouse model of ischemic stroke (IS) and the role of activated FXIII (FXIIIa) in clot formation and lysis in patients with IS. METHODS: A ferric chloride IS murine model was performed before and after administration of a FXIIIa inhibitor (FXIIIinh). Thromboelastometry in human and mice blood was used to evaluate thrombus stiffness and lysis with FXIIIinh. FXIIIa-dependent fibrin crosslinking and lysis with fibrinolytic drugs (tissue plasminogen activator and tenecteplase) were studied on fibrin plates and on thrombi and clotted plasma of patients with IS. Finally, circulating and thrombus FXIIIa were measured in 85 patients with IS. RESULTS: FXIIIinh administration before stroke induction reduced infarct size, α2-antiplasmin (α2AP) crosslinking, and local microthrombosis, improving motor coordination and fibrinolysis without intracranial bleeds (24 hours). Interestingly, FXIII blockade after stroke also reduced brain damage and neurologic deficit. Thromboelastometry in human/mice blood with FXIIIinh showed delayed clot formation, reduced clot firmness, and shortened tissue plasminogen activator lysis time. FXIIIa fibrin crosslinking increased fibrin density and lysis resistance, which increased further after α2AP addition. FXIIIinh enhanced ex vivo lysis in stroke thrombi and fibrin plates. In patients with IS, thrombus FXIII and α2AP were associated with inflammatory and hemostatic components, and plasma FXIIIa correlated with thrombus α2AP and fibrin. CONCLUSION: Our results suggest a key role of FXIIIa in thrombus stabilization, α2AP crosslinking, and lysis resistance, with a protective effect of FXIIIinh in an IS experimental model.

Trimethylamine N-Oxide as a Mediator Linking Peripheral to Central Inflammation: An In Vitro Study.

In this study, the plausible role of trimethylamine N-oxide (TMAO), a microbiota metabolite, was investigated as a link between peripheral inflammation and the inflammation of the central nervous system using different cell lines. TMAO treatment favored the differentiation of adipocytes from preadipocytes (3T3-L1 cell line). In macrophages (RAW 264.7 cell line), which infiltrate adipose tissue in obesity, TMAO increased the expression of pro-inflammatory cytokines. The treatment with 200 µM of TMAO seemed to disrupt the blood-brain barrier as it induced a significant decrease in the expression of occludin in hCMECs. TMAO also increased the expression of pro-inflammatory cytokines in primary neuronal cultures, induced a pro-inflammatory state in primary microglial cultures, and promoted phagocytosis. Data obtained from this project suggest that microbial dysbiosis and increased TMAO secretion could be a key link between peripheral and central inflammation. Thus, TMAO-decreasing compounds may be a promising therapeutic strategy for neurodegenerative diseases.

Role of LCN2 in a murine model of hindlimb ischemia and in peripheral artery disease patients, and its potential regulation by miR-138-5P.

BACKGROUND AND AIMS: Peripheral arterial disease (PAD) is a leading cause of morbimortality worldwide. Lipocalin-2 (LCN2) has been associated with higher risk of amputation or mortality in PAD and might be involved in muscle regeneration. Our aim is to unravel the role of LCN2 in skeletal muscle repair and PAD. METHODS AND RESULTS: WT and Lcn2-/- mice underwent hindlimb ischemia. Blood and crural muscles were analyzed at the inflammatory and regenerative phases. At day 2, Lcn2-/- male mice, but not females, showed increased blood and soleus muscle neutrophils, and elevated circulating pro-inflammatory monocytes (p < 0.05), while locally, total infiltrating macrophages were reduced (p < 0.05). Moreover, Lcn2-/- soleus displayed an elevation of Cxcl1 (p < 0.001), and Cxcr2 (p < 0.01 in males), and a decrease in Ccl5 (p < 0.05). At day 15, Lcn2 deficiency delayed muscle recovery, with higher density of regenerating myocytes (p < 0.04) and arterioles (αSMA+, p < 0.025). Reverse target prediction analysis identified miR-138-5p as a potential regulator of LCN2, showing an inverse correlation with Lcn2 mRNA in skeletal muscles (rho = -0.58, p < 0.01). In vitro, miR-138-5p mimic reduced Lcn2 expression and luciferase activity in murine macrophages (p < 0.05). Finally, in human serum miR-138-5p was inversely correlated with LCN2 (p ≤ 0.001 adjusted, n = 318), and associated with PAD (Odds ratio 0.634, p = 0.02, adjusted, PAD n = 264, control n = 54). CONCLUSIONS: This study suggests a possible dual role of LCN2 in acute and chronic conditions, with a probable role in restraining inflammation early after skeletal muscle ischemia, while being associated with vascular damage in PAD, and identifies miR-138-5p as one potential post-transcriptional regulator of LCN2.

Loss of the matrix metalloproteinase-10 causes premature features of aging in satellite cells.

Aged muscles accumulate satellite cells with a striking decline response to damage. Although intrinsic defects in satellite cells themselves are the major contributors to aging-associated stem cell dysfunction, increasing evidence suggests that changes in the muscle-stem cell local microenvironment also contribute to aging. Here, we demonstrate that loss of the matrix metalloproteinase-10 (MMP-10) in young mice alters the composition of the muscle extracellular matrix (ECM), and specifically disrupts the extracellular matrix of the satellite cell niche. This situation causes premature features of aging in the satellite cells, contributing to their functional decline and a predisposition to enter senescence under proliferative pressure. Similarly, reduction of MMP-10 levels in young satellite cells from wild type animals induces a senescence response, while addition of the protease delays this program. Significantly, the effect of MMP-10 on satellite cell aging can be extended to another context of muscle wasting, muscular dystrophy. Systemic treatment of mdx dystrophic mice with MMP-10 prevents the muscle deterioration phenotype and reduces cellular damage in the satellite cells, which are normally under replicative pressure. Most importantly, MMP-10 conserves its protective effect in the satellite cell-derived myoblasts isolated from a Duchenne muscular dystrophy patient by decreasing the accumulation of damaged DNA. Hence, MMP-10 provides a previously unrecognized therapeutic opportunity to delay satellite cell aging and overcome satellite cell dysfunction in dystrophic muscles.

The thrombus proteome in stroke reveals a key role of the innate immune system and new insights associated with its etiology, severity, and prognosis.

BACKGROUND: Nowadays little is known about the molecular profile of the occluding thrombus of patients with ischemic stroke. OBJECTIVES: To analyze the proteomic profile of thrombi in patients who experienced an ischemic stroke in order to gain insights into disease pathogenesis. METHODS: Thrombi from an exploratory cohort of patients who experienced a stroke were obtained by thrombectomy and analyzed by sequential window acquisition of all theoretical spectra-mass spectrometry. Unsupervised k-means clustering analysis was performed to stratify patients who experienced a stroke. The proteomic profile was associated with both the neurological function (National Institute of Health Stroke Scale [NIHSS]) and the cerebral involvement (Alberta Stroke Program Early CT Score [ASPECTS]) prior to thrombectomy and the clinical status of patients at 3 months using the modified Rankin Scale. In an independent cohort of 210 patients who experienced a stroke, the potential role of neutrophils in stroke severity was interrogated. RESULTS: Proteomic analysis identified 580 proteins in thrombi, which were stratified into 4 groups: hemostasis, proteasome and neurological diseases, structural proteins, and innate immune system and neutrophils. The thrombus proteome identified 3 clusters of patients with distinctive severity, prognosis, and etiology of the stroke. A protein signature clearly distinguished atherothrombotic and cardioembolic strokes. Several proteins were significantly correlated with the severity of the stroke (NIHSS and ASPECTS). Functional proteomic analysis highlighted the prominent role of neutrophils in stroke severity. This was in line with the association of neutrophil activation markers and count with NIHSS, ASPECTS, and the modified Rankin Scale score 90 days after the event. CONCLUSION: The use of sequential window acquisition of all theoretical spectra-mass spectrometry in thrombi from patients who experienced an ischemic stroke has provided new insights into pathways and players involved in its etiology, severity, and prognosis. The prominent role of the innate immune system identified might pave the way for the development of new biomarkers and therapeutic approaches in this disease.

NADPH Oxidase 5 (NOX5) Overexpression Promotes Endothelial Dysfunction via Cell Apoptosis, Migration, and Metabolic Alterations in Human Brain Microvascular Endothelial Cells (hCMEC/D3).

NADPH oxidases (NOX) constitute the main reactive oxygen species (ROS) source in blood vessels. An oxidative stress situation due to ROS overproduction can lead into endothelial dysfunction, a molecular mechanism that precedes cardiovascular diseases (CVDs) such as atherosclerosis, myocardial infarction, and stroke. NOX5 is the last discovered member of the NOX family, studied in a lesser extent due to its absence in the rodent genome. Our objective was to describe the phenotypic alterations produced by an oxidative stress situation derived from NOX5 overexpression in an endothelial in vitro model. The in vitro model consists of the hCMEC/D3 cell line, derived from brain microvascular endothelium, infected with a recombinant NOX5-ß adenovirus. After an initial proteomic analysis, three phenotypic alterations detected in silico were studied: cell proliferation and apoptosis, general and mitochondrial metabolism, and migration capacity. NOX5 infection of hCMEC/D3 generates a functional protein and an increase in ROS production. This model produced changes in the whole cell proteome. The in silico analysis together with in vitro validations demonstrated that NOX5 overexpression inhibits proliferation and promotes apoptosis, metabolic alterations and cell migration in hCMEC/D3 cells. NOX5 overexpression in endothelial cells leads to phenotypic changes that can lead to endothelial dysfunction, the onset of atherosclerosis, myocardial infarction, and stroke.

Association between Circulating Levels of 25-Hydroxyvitamin D3 and Matrix Metalloproteinase-10 (MMP-10) in Patients with Type 2 Diabetes.

Background: Matrix metalloproteinase-10 (MMP-10) levels increase progressively starting from early diabetic kidney disease (DKD) stages. Vitamin D3 (vitD3) deficit is associated with a higher risk of diabetic microangiopathy. Reduced MMP-10 expression has been observed after exposure to vitD3. Aim: to assess how vitD3 status is related to MMP-10 levels in patients with Type 2 diabetes (T2D). Methods: 256 patients with T2D were included in this cross-sectional study. Demographic, clinical and serum MMP-10 and 25-hydroxyvitamin D3 (25(OH)D3) levels were collected from each patient. The association between MMP-10 and (25(OH)D3) levels was assessed using a correlation analysis and fitting a multivariate linear regression model. Results: Serum MMP-10 levels were inversely correlated with circulating 25(OH)D3 (rho = −0.25; p < 0.001). In the subgroup analysis this correlation was significant in patients with DKD (rho = −0.28; p = 0.001) and in subjects with vitD3 deficit (rho = −0.24; p = 0.005). In the regression model adjusted for kidney function, body adiposity, smoking and vitD supplementation MMP-10 levels were 68.7 pg/mL lower in patients with 25(OH)D3 > 20 ng/mL, with respect to ≤20 ng/mL (p = 0.006). Conclusions: vitD3 repletion status is an independent predictor of MMP-10 levels in T2D patients. Perhaps, high 25(OH)D3 values should be targeted in these patients in order to prevent vascular complications.

Matrix Metalloproteinase 10 Contributes to Choroidal Neovascularisation.

Age-related macular degeneration (AMD) is currently the main cause of severe visual loss among older adults in developed countries. The pathophysiology has not been clarified, but oxidative stress is believed to play a major role. Matrix metalloproteinases (MMP) may play a prominent role in several steps of the pathophysiology of AMD, especially in its neovascular form; therefore, there is of great interest in understanding their role in choroidal neovascularisation. This study aimed to elucidate the role of MMP10 in the development of choroidal neovascularisation (CNV). We have demonstrated that MMP10 was expressed by retinal pigment epithelium cells and endothelial cells of the neovascular membrane, in cell culture, mouse and human retina. MMP10 expression and activity increased under oxidative stress conditions in ARPE-19 cells. MMP10-/- mice developed smaller laser-induced areas of CNV. Furthermore, to exclude a systemic MMP10 imbalance in these patients, plasma MMP10 concentrations were assessed in an age- and sex-matched sample of 52 control patients and 52 patients with neovascular AMD and no significant differences were found between the groups, demonstrating that MMP10 induction is a local phenomenon. Our findings suggest that MMP10 participates in the development of choroidal neovascularisation and promotes MMP10 as a possible new therapeutic target.

Hemostatic Biomarkers and Volumetry Help to Identify High-Risk Abdominal Aortic Aneurysms.

Predicting the progression of small aneurysms is a main challenge in abdominal aortic aneurysm (AAA) management. The combination of circulating biomarkers and image techniques might provide an alternative for risk stratification. We evaluated the association of plasma TAT complexes (TAT) and D-dimer with AAA severity in 3 groups of patients: group 1, without AAA (n = 52), group 2, AAA 40−50 mm (n = 51) and group 3, AAA > 50 mm (n = 50). TAT (p < 0.001) and D-dimer (p < 0.001) were increased in patients with AAA (groups 2 and 3) vs. group 1. To assess the association between baseline TAT and D-dimer concentrations, and AAA growth, aortic diameter and volume (volumetry) were measured by computed tomography angiography (CTA) in group 2 at recruitment (baseline) and 1-year after inclusion. Baseline D-dimer and TAT levels were associated with AAA diameter and volume variations at 1-year independently of confounding factors (p ≤ 0.044). Additionally, surgery incidence, recorded during a 4-year follow-up in group 2, was associated with larger aneurysms, assessed by aortic diameter and volumetry (p ≤ 0.036), and with elevated TAT levels (sub-hazard ratio 1.3, p ≤ 0.029), while no association was found for D-dimer. The combination of hemostatic parameters and image techniques might provide valuable tools to evaluate AAA growth and worse evolution.

Lipocalin-2 and Calprotectin Potential Prognosis Biomarkers in Peripheral Arterial Disease.

OBJECTIVE: Peripheral arterial disease (PAD) is the most prevalent cardiovascular (CV) condition globally. Despite the high CV risk of PAD patients, no reliable predictors of adverse clinical evolution are yet available. In this regard, previous transcriptomic analyses revealed increased expression of calprotectin (S100A8/A9) and lipocalin-2 (LCN2) in circulating extracellular vesicles (EVs) of patients with PAD. The aim of this study was to determine the prognostic value of LCN2 and calprotectin for CV risk assessment in PAD. METHODS: LCN2 and the S100A9 subunit of calprotectin were examined in human femoral plaques by immunohistochemistry and qPCR. LCN2 and calprotectin were determined by ELISA in PAD (CHN cohort, n = 331, Fontaine II-IV, serum), and PAD diagnosed by population based screening (VIVA trial, n = 413, the majority Fontaine 0-I, plasma). Patients were followed up for a mean of four years, recording the primary outcomes; CV death or amputation in the CHN cohort and CV death or major lower limb events (MALE) in the VIVA population. Secondary outcomes were all cause death or amputation, and all cause death or MALE, respectively. RESULTS: LCN2 and S100A9 were detected in human plaques in regions rich in inflammatory cells. LCN2 and calprotectin levels were 70% and 64% lower in plasma than in serum. In the CHN cohort, high serum levels of LCN2 and calprotectin increased the risk of primary and secondary outcomes 5.6 fold (p < .001) and 1.8 fold (p = .034), respectively, after covariable adjustment. Similarly, elevated plasma levels of LCN2 and calprotectin increased by three fold the risk of primary and secondary outcomes (p < .001) in the VIVA cohort. Moreover, addition of the combined variable to basal models, considering clinically relevant risk factors, improved reclassification for the primary outcome in both cohorts (p ≤ .024). CONCLUSION: Combined assessment of the inflammatory biomarkers LCN2 and calprotectin might be useful for risk stratification in advanced and early PAD.

Role of Extracellular Vesicles as Potential Diagnostic and/or Therapeutic Biomarkers in Chronic Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the first cause of death worldwide. In recent years, there has been great interest in the analysis of extracellular vesicles (EVs), including exosomes and microparticles, as potential mediators of biological communication between circulating cells/plasma and cells of the vasculature. Besides their activity as biological effectors, EVs have been also investigated as circulating/systemic biomarkers in different acute and chronic CVDs. In this review, the role of EVs as potential diagnostic and prognostic biomarkers in chronic cardiovascular diseases, including atherosclerosis (mainly, peripheral arterial disease, PAD), aortic stenosis (AS) and aortic aneurysms (AAs), will be described. Mechanistically, we will analyze the implication of EVs in pathological processes associated to cardiovascular remodeling, with special emphasis in their role in vascular and valvular calcification. Specifically, we will focus on the participation of EVs in calcium accumulation in the pathological vascular wall and aortic valves, involving the phenotypic change of vascular smooth muscle cells (SMCs) or valvular interstitial cells (IC) to osteoblast-like cells. The knowledge of the implication of EVs in the pathogenic mechanisms of cardiovascular remodeling is still to be completely deciphered but there are promising results supporting their potential translational application to the diagnosis and therapy of different CVDs.

CM-352 Efficacy in a Mouse Model of Anticoagulant-Associated Intracranial Hemorrhage.

BACKGROUND: Intracranial hemorrhage (ICH) is one of the major devastating complications of anticoagulation. Matrix metalloproteinase (MMP) inhibition has been proposed as a novel pharmacological approach for ICH treatment. OBJECTIVES: We evaluated the effects of CM-352 (MMP-fibrinolysis inhibitor) in an experimental ICH model associated with oral anticoagulants as compared with clinically used prothrombin complex concentrate (PCC). METHODS: ICH was induced by collagenase injection into the striatum of wild type (C57BL/6J) anticoagulated mice (warfarin or rivaroxaban) and Mmp10 -/- mice. Hematoma volume and neurological deficits were measured 24 hours later by diaminobenzidine staining and different behavioral tests. Circulating plasminogen activator inhibitor-1 (PAI-1) activity and interleukin-6 (IL-6) were measured in plasma samples and local inflammation was assessed by neutrophil infiltration. Finally, fibrinolytic effects of MMP-10 and rivaroxaban were evaluated by thromboelastometry and thrombin-activatable fibrinolysis inhibitor (TAFI) activation assays. RESULTS: Only PCC reduced hemorrhage volume and improved functional outcome in warfarin-ICH, but both PCC and CM-352 treatments diminished hemorrhage volume (46%, p < 0.01 and 64%, p < 0.001, respectively) and ameliorated functional outcome in rivaroxaban-ICH. We further demonstrated that CM-352, but not PCC, decreased neutrophil infiltration in the hemorrhage area at 24 hours. The effect of CM-352 could be related to MMP-10 inhibition since Mmp10 -/- mice showed lower hemorrhage volume, better neurological score, reduced IL-6 levels and neutrophil infiltration, and increased PAI-1 after experimental ICH. Finally, we found that CM-352 reduced MMP-10 and rivaroxaban-related fibrinolytic effects in thromboelastometry and TAFI activation. CONCLUSION: CM-352 treatment, by diminishing MMPs and rivaroxaban-associated fibrinolytic effects, might be a novel antihemorrhagic strategy for rivaroxaban-associated ICH.

Deficiency of MMP-10 Aggravates the Diseased Phenotype of Aged Dystrophic Mice.

Matrix metalloproteinases (MMPs) have been implicated in the progression of muscular dystrophy, and recent studies have reported the role of MMP-10 in skeletal muscle pathology of young dystrophic mice. Nevertheless, its involvement in dystrophin-deficient hearts remains unexplored. Here, we aimed to investigate the involvement of MMP-10 in the progression of severe muscular dystrophy and to characterize MMP-10 loss in skeletal and cardiac muscles of aged dystrophic mice. We examined the histopathological effect of MMP-10 ablation in aged mdx mice, both in the hind limb muscles and heart tissues. We found that MMP-10 loss compromises survival rates of aged mdx mice, with skeletal and cardiac muscles developing a chronic inflammatory response. Our findings indicate that MMP-10 is implicated in severe muscular dystrophy progression, thus identifying a new area of research that could lead to future therapies for dystrophic muscles.

Association of SDF1 and MMP12 with Atherosclerosis and Inflammation: Clinical and Experimental Study.

BACKGROUND: Atherosclerosis is the main etiology of cardiovascular diseases (CVD), associated to systemic inflammation. Matrix metalloproteinases (MMPs) are related to atherosclerosis progression through the SDF1/CXCR4 axis promoting macrophages recruitment within the vascular wall. The goal was to assess new circulatory inflammatory markers in relation to atherosclerosis. METHODS: Measurement of SDF1, MMP12 and CRP in blood samples of 298 prospective patients with cardiovascular risk. To explore atherosclerosis progression, CXCR4/SDF1 axis and MMP12 expression were determined by RT-qPCR and by immunohistochemistry in the aorta of accelerated and delayed atherosclerosis mice models (Apoe-/- and Apoe-/-Mmp10-/-). RESULTS: SDF1, MMP12 and CRP were elevated in patients with clinical atherosclerosis, but after controlling by confounding factors, only SDF1 and CRP remained increased. Having high levels of both biomarkers showed 2.8-fold increased risk of presenting clinical atherosclerosis (p = 0.022). Patients with elevated SDF1, MMP12 and CRP showed increased risk of death in follow-up (HR = 3.2, 95%CI: 1.5-7.0, p = 0.004). Gene and protein expression of CXCR4 and MMP12 were increased in aortas from Apoe-/- mice. CONCLUSIONS: The combination of high circulating SDF1, MMP12 and CRP identified patients with particular inflammatory cardiovascular risk and increased mortality. SDF1/CXCR4 axis and MMP12 involvement in atherosclerosis development suggests that they could be possible atherosclerotic targets.

Urinary Extracellular Vesicles for Diabetic Kidney Disease Diagnosis.

Diabetic kidney disease (DKD) is the leading cause of end stage renal disease (ESRD) in developed countries, affecting more than 40% of diabetes mellitus (DM) patients. DKD pathogenesis is multifactorial leading to a clinical presentation characterized by proteinuria, hypertension, and a gradual reduction in kidney function, accompanied by a high incidence of cardiovascular (CV) events and mortality. Unlike other diabetes-related complications, DKD prevalence has failed to decline over the past 30 years, becoming a growing socioeconomic burden. Treatments controlling glucose levels, albuminuria and blood pressure may slow down DKD evolution and reduce CV events, but are not able to completely halt its progression. Moreover, one in five patients with diabetes develop DKD in the absence of albuminuria, and in others nephropathy goes unrecognized at the time of diagnosis, urging to find novel noninvasive and more precise early diagnosis and prognosis biomarkers and therapeutic targets for these patient subgroups. Extracellular vesicles (EVs), especially urinary (u)EVs, have emerged as an alternative for this purpose, as changes in their numbers and composition have been reported in clinical conditions involving DM and renal diseases. In this review, we will summarize the current knowledge on the role of (u)EVs in DKD.

Molecular and Cellular Mechanisms of Delayed Fracture Healing in Mmp10 (Stromelysin 2) Knockout Mice.

The remodeling of the extracellular matrix is a central function in endochondral ossification and bone homeostasis. During secondary fracture healing, vascular invasion and bone growth requires the removal of the cartilage intermediate and the coordinate action of the collagenase matrix metalloproteinase (MMP)-13, produced by hypertrophic chondrocytes, and the gelatinase MMP-9, produced by cells of hematopoietic lineage. Interfering with these MMP activities results in impaired fracture healing characterized by cartilage accumulation and delayed vascularization. MMP-10, Stromelysin 2, a matrix metalloproteinase with high homology to MMP-3 (Stromelysin 1), presents a wide range of putative substrates identified in vitro, but its targets and functions in vivo and especially during fracture healing and bone homeostasis are not well defined. Here, we investigated the role of MMP-10 through bone regeneration in C57BL/6 mice. During secondary fracture healing, MMP-10 is expressed by hematopoietic cells and its maximum expression peak is associated with cartilage resorption at 14 days post fracture (dpf). In accordance with this expression pattern, when Mmp10 is globally silenced, we observed an impaired fracture-healing phenotype at 14 dpf, characterized by delayed cartilage resorption and TRAP-positive cell accumulation. This phenotype can be rescued by a non-competitive transplant of wild-type bone marrow, indicating that MMP-10 functions are required only in cells of hematopoietic linage. In addition, we found that this phenotype is a consequence of reduced gelatinase activity and the lack of proMMP-9 processing in macrophages. Our data provide evidence of the in vivo function of MMP-10 during endochondral ossification and defines the macrophages as the lead cell population in cartilage removal and vascular invasion. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The Role of Circulating Biomarkers in Peripheral Arterial Disease.

Peripheral arterial disease (PAD) of the lower extremities is a chronic illness predominantly of atherosclerotic aetiology, associated to traditional cardiovascular (CV) risk factors. It is one of the most prevalent CV conditions worldwide in subjects >65 years, estimated to increase greatly with the aging of the population, becoming a severe socioeconomic problem in the future. The narrowing and thrombotic occlusion of the lower limb arteries impairs the walking function as the disease progresses, increasing the risk of CV events (myocardial infarction and stroke), amputation and death. Despite its poor prognosis, PAD patients are scarcely identified until the disease is advanced, highlighting the need for reliable biomarkers for PAD patient stratification, that might also contribute to define more personalized medical treatments. In this review, we will discuss the usefulness of inflammatory molecules, matrix metalloproteinases (MMPs), and cardiac damage markers, as well as novel components of the liquid biopsy, extracellular vesicles (EVs), and non-coding RNAs for lower limb PAD identification, stratification, and outcome assessment. We will also explore the potential of machine learning methods to build prediction models to refine PAD assessment. In this line, the usefulness of multimarker approaches to evaluate this complex multifactorial disease will be also discussed.

Inside the Thrombus: Association of Hemostatic Parameters With Outcomes in Large Vessel Stroke Patients.

Background: Actual clinical management of ischemic stroke (IS) is based on restoring cerebral blood flow using tissue plasminogen activator (tPA) and/or endovascular treatment (EVT). Mechanical thrombectomy has permitted the analysis of thrombus structural and cellular classic components. Nevertheless, histological assessment of hemostatic parameters such as thrombin-activatable fibrinolysis inhibitor (TAFI) and matrix metalloproteinase 10 (MMP-10) remains unknown, although their presence could determine thrombus stability and its response to thrombolytic treatment, improving patient's outcome. Methods: We collected thrombi (n = 45) from large vessel occlusion (LVO) stroke patients (n = 53) and performed a histological analysis of different hemostatic parameters [TAFI, MMP-10, von Willebrand factor (VWF), and fibrin] and cellular components (erythrocytes, leukocytes, macrophages, lymphocytes, and platelets). Additionally, we evaluated the association of these parameters with plasma levels of MMP-10, TAFI and VWF activity and recorded clinical variables. Results: In this study, we report for the first time the presence of MMP-10 and TAFI in all thrombi collected from LVO patients. Both proteins were localized in regions of inflammatory cells, surrounded by erythrocyte and platelet-rich areas, and their content was significantly associated (r = 0.41, p < 0.01). Thrombus TAFI was lower in patients who died during the first 3 months after stroke onset [odds ratio (OR) (95%CI); 0.59 (0.36-0.98), p = 0.043]. Likewise, we observed that thrombus MMP-10 was inversely correlated with the amount of VWF (r = -0.30, p < 0.05). Besides, VWF was associated with the presence of leukocytes (r = 0.37, p < 0.05), platelets (r = 0.32, p < 0.05), and 3 months mortality [OR (95%CI); 4.5 (1.2-17.1), p = 0.029]. Finally, plasma levels of TAFI correlated with circulating and thrombus platelets, while plasma MMP-10 was associated with cardiovascular risk factors and functional dependence at 3 months. Conclusions: The present study suggests that the composition and distribution of thrombus hemostatic components might have clinical impact by influencing the response to pharmacological and mechanical therapies as well as guiding the development of new therapeutic strategies.

Association of calprotectin with other inflammatory parameters in the prediction of mortality for ischemic stroke.

BACKGROUND: Inflammatory response plays an important role in many processes related to acute ischemic stroke (AIS). Calprotectin (S100A8/S100A9), released by monocytes and neutrophils, is a key protein in the regulation of inflammation and thrombosis. The purpose of this study is to evaluate the association of circulating calprotectin with other inflammatory biomarkers and AIS prognosis, as well as the calprotectin content in stroke thrombi. METHODS: Among the 748 patients treated at a comprehensive stroke center between 2015 and 2017, 413 patients with confirmed acute ischemic injury were prospectively evaluated. Patients with systemic inflammation or infection at onset were excluded. Plasma calprotectin was measured by ELISA in blood samples of AIS patients within the first 24 h. Univariate and multivariate logistic regression models were performed to evaluate its association with mortality and functional independence (FI) at 3 months (defined as modified Rankin Scale < 3) and hemorrhagic transformation (HT) after ischemic stroke. Further, S100A9 was localized by immunostaining in stroke thrombi (n = 44). RESULTS: Higher calprotectin levels were associated with 3-month mortality, HT, and lower 3-month FI. After adjusting for potential confounders, plasma calprotectin remained associated with 3-month mortality [OR (95% CI) 2.31 (1.13-4.73)]. Patients with calprotectin ≥ 2.26 µg/mL were 4 times more likely to die [OR 4.34 (1.95-9.67)]. Addition of calprotectin to clinical variables led to significant improvement in the discrimination capacity of the model [0.91 (0.87-0.95) vs 0.89 (0.85-0.93); p < 0.05]. A multimarker approach demonstrated that patients with increased calprotectin, CRP, and NLR had the poorest outcome with a mortality rate of 42.3% during follow-up. S100A9 protein, as part of the heterodimer calprotectin, was present in all thrombi retrieved from AIS patients. Mean S100A9 content was 3.5% and tended to be higher in patients who died (p = 0.09). Moreover, it positively correlated with platelets (Pearson r 0.46, p < 0.002), leukocytes (0.45, p < 0.01), and neutrophil elastase (0.70, p < 0.001) thrombus content. CONCLUSIONS: Plasma calprotectin is an independent predictor of 3-month mortality and provides complementary prognostic information to identify patients with poor outcome after AIS. The presence of S100A9 in stroke thrombi suggests a possible inflammatory mechanism in clot formation, and further studies are needed to determine its influence in resistance to reperfusion.