Sortilin enhances fibrosis and calcification in aortic valve disease by inducing interstitial cell heterogeneity.

AIMS: Calcific aortic valve disease (CAVD) is the most common valve disease, which consists of a chronic interplay of inflammation, fibrosis, and calcification. In this study, sortilin (SORT1) was identified as a novel key player in the pathophysiology of CAVD, and its role in the transformation of valvular interstitial cells (VICs) into pathological phenotypes is explored. METHODS AND RESULTS: An aortic valve (AV) wire injury (AVWI) mouse model with sortilin deficiency was used to determine the effects of sortilin on AV stenosis, fibrosis, and calcification. In vitro experiments employed human primary VICs cultured in osteogenic conditions for 7, 14, and 21 days; and processed for imaging, proteomics, and transcriptomics including single-cell RNA-sequencing (scRNA-seq). The AVWI mouse model showed reduced AV fibrosis, calcification, and stenosis in sortilin-deficient mice vs. littermate controls. Protein studies identified the transition of human VICs into a myofibroblast-like phenotype mediated by sortilin. Sortilin loss-of-function decreased in vitro VIC calcification. ScRNA-seq identified 12 differentially expressed cell clusters in human VIC samples, where a novel combined inflammatory myofibroblastic-osteogenic VIC (IMO-VIC) phenotype was detected with increased expression of SORT1, COL1A1, WNT5A, IL-6, and serum amyloid A1. VICs sequenced with sortilin deficiency showed decreased IMO-VIC phenotype. CONCLUSION: Sortilin promotes CAVD by mediating valvular fibrosis and calcification, and a newly identified phenotype (IMO-VIC). This is the first study to examine the role of sortilin in valvular calcification and it may render it a therapeutic target to inhibit IMO-VIC emergence by simultaneously reducing inflammation, fibrosis, and calcification, the three key pathological processes underlying CAVD.

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Promotes Macrophage Activation via LDL Receptor-Independent Mechanisms.

BACKGROUND: Activated macrophages contribute to the pathogenesis of vascular disease. Vein graft failure is a major clinical problem with limited therapeutic options. PCSK9 (proprotein convertase subtilisin/kexin 9) increases low-density lipoprotein (LDL)-cholesterol levels via LDL receptor (LDLR) degradation. The role of PCSK9 in macrophage activation and vein graft failure is largely unknown, especially through LDLR-independent mechanisms. This study aimed to explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion. METHODS: We used Ldlr-/- mice to examine the LDLR-independent roles of circulating PCSK9 in experimental vein grafts. Adeno-associated virus (AAV) vector encoding a gain-of-function mutant of PCSK9 (rAAV8/D377Y-mPCSK9) induced hepatic PCSK9 overproduction. To explore novel inflammatory targets of PCSK9, we used systems biology in Ldlr-/- mouse macrophages. RESULTS: In Ldlr-/- mice, AAV-PCSK9 increased circulating PCSK9, but did not change serum cholesterol and triglyceride levels. AAV-PCSK9 promoted vein graft lesion development when compared with control AAV. In vivo molecular imaging revealed that AAV-PCSK9 increased macrophage accumulation and matrix metalloproteinase activity associated with decreased fibrillar collagen, a molecular determinant of atherosclerotic plaque stability. AAV-PCSK9 induced mRNA expression of the pro-inflammatory mediators IL-1ß (interleukin-1 beta), TNFα (tumor necrosis factor alpha), and MCP-1 (monocyte chemoattractant protein-1) in peritoneal macrophages underpinned by an in vitro analysis of Ldlr-/- mouse macrophages stimulated with endotoxin-free recombinant PCSK9. A combination of unbiased global transcriptomics and new network-based hyperedge entanglement prediction analysis identified the NF-κB (nuclear factor-kappa B) signaling molecules, lectin-like oxidized LOX-1 (LDL receptor-1), and SDC4 (syndecan-4) as potential PCSK9 targets mediating pro-inflammatory responses in macrophages. CONCLUSIONS: Circulating PCSK9 induces macrophage activation and vein graft lesion development via LDLR-independent mechanisms. PCSK9 may be a potential target for pharmacologic treatment for this unmet medical need.

Progression of Mitral Regurgitation in Rheumatic Valve Disease: Role of Left Atrial Remodeling.

Introduction: Mitral regurgitation (MR) is the most common valve abnormality in rheumatic heart disease (RHD) often associated with stenosis. Although the mechanism by which MR develops in RHD is primary, longstanding volume overload with left atrial (LA) remodeling may trigger the development of secondary MR, which can impact on the overall progression of MR. This study is aimed to assess the incidence and predictors of MR progression in patients with RHD. Methods: Consecutive RHD patients with non-severe MR associated with any degree of mitral stenosis were selected. The primary endpoint was a progression of MR, which was defined as an increase of one grade in MR severity from baseline to the last follow-up echocardiogram. The risk of MR progression was estimated accounting for competing risks. Results: The study included 539 patients, age of 46.2 ± 12 years and 83% were women. At a mean follow-up time of 4.2 years (interquartile range [IQR]: 1.2-6.9 years), 54 patients (10%) displayed MR progression with an overall incidence of 2.4 per 100 patient-years. Predictors of MR progression by the Cox model were age (adjusted hazard ratio [HR] 1.541, 95% CI 1.222-1.944), and LA volume (HR 1.137, 95% CI 1.054-1.226). By considering competing risk analysis, the direction of the association was similar for the rate (Cox model) and incidence (Fine-Gray model) of MR progression. In the model with LA volume, atrial fibrillation (AF) was no longer a predictor of MR progression. In the subgroup of patients in sinus rhythm, 59 had an onset of AF during follow-up, which was associated with progression of MR (HR 2.682; 95% CI 1.133-6.350). Conclusions: In RHD patients with a full spectrum of MR severity, progression of MR occurs over time is predicted by age and LA volume. LA enlargement may play a role in the link between primary MR and secondary MR in patients with RHD.

Wnt Site Signaling Inhibitor Secreted Frizzled-Related Protein 3 Protects Mitral Valve Endothelium From Myocardial Infarction-Induced Endothelial-to-Mesenchymal Transition.

Background The onset and mechanisms of endothelial-to-mesenchymal transition (EndMT) in mitral valve (MV) leaflets following myocardial infarction (MI) are unknown, yet these events are closely linked to stiffening of leaflets and development of ischemic mitral regurgitation. We investigated whether circulating molecules present in plasma within days after MI incite EndMT in MV leaflets. Methods and Results We examined the onset of EndMT in MV leaflets from 9 sheep with inferior MI, 8 with sham surgery, and 6 naïve controls. Ovine MVs 8 to 10 days after inferior MI displayed EndMT, shown by increased vascular endothelial cadherin/α-smooth muscle actin-positive cells. The effect of plasma on EndMT in MV endothelial cells (VECs) was assessed by quantitative polymerase chain reaction, migration assays, and immunofluorescence. In vitro, post-MI plasma induced EndMT marker expression and enhanced migration of mitral VECs; sham plasma did not. Analysis of sham versus post-MI plasma revealed a significant drop in the Wnt signaling antagonist sFRP3 (secreted frizzled-related protein 3) in post-MI plasma. Addition of recombinant sFRP3 to post-MI plasma reversed its EndMT-inducing effect on mitral VECs. RNA-sequencing analysis of mitral VECs exposed to post-MI plasma showed upregulated FOXM1 (forkhead box M1). Blocking FOXM1 reduced EndMT transcripts in mitral VECs treated with post-MI plasma. Finally, FOXM1 induced by post-MI plasma was downregulated by sFRP3. Conclusions Reduced sFRP3 in post-MI plasma facilitates EndMT in mitral VECs by increasing the transcription factor FOXM1. Restoring sFRP3 levels or inhibiting FOXM1 soon after MI may provide a novel strategy to modulate EndMT in the MV to prevent ischemic mitral regurgitation and heart failure.

Prothymosin Alpha: A Novel Contributor to Estradiol Receptor Alpha-Mediated CD8+ T-Cell Pathogenic Responses and Recognition of Type 1 Collagen in Rheumatic Heart Valve Disease.

BACKGROUND: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. METHODS: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcuspyogenes proteomes were used to identify mimic epitopes. RESULTS: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8+ T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8+ T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8+ T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8+ T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and Spyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8+ T-cell activation in vitro. CONCLUSIONS: ProTα-dependent CD8+ T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8+ T cells, suggesting mechanisms provoking autoimmunity.

T-Cell Subpopulations Exhibit Distinct Recruitment Potential, Immunoregulatory Profile and Functional Characteristics in Chagas versus Idiopathic Dilated Cardiomyopathies.

Chronic Chagas cardiomyopathy (CCC) is one of the deadliest cardiomyopathies known and the most severe manifestation of Chagas disease, which is caused by infection with the parasite Trypanosoma cruzi. Idiopathic dilated cardiomyopathies (IDC) are a diverse group of inflammatory heart diseases that affect the myocardium and are clinically similar to CCC, often causing heart failure and death. While T-cells are critical for mediating cardiac pathology in CCC and IDC, the mechanisms underlying T-cell function in these cardiomyopathies are not well-defined. In this study, we sought to investigate the phenotypic and functional characteristics of T-cell subpopulations in CCC and IDC, aiming to clarify whether the inflammatory response is similar or distinct in these cardiomyopathies. We evaluated the expression of systemic cytokines, determined the sources of the different cytokines, the expression of their receptors, of cytotoxic molecules, and of molecules associated with recruitment to the heart by circulating CD4+, CD8+, and CD4-CD8- T-cells from CCC and IDC patients, using multiparameter flow cytometry combined with conventional and unsupervised machine-learning strategies. We also used an in silico approach to identify the expression of genes that code for key molecules related to T-cell function in hearts of patient with CCC and IDC. Our data demonstrated that CCC patients displayed a more robust systemic inflammatory cytokine production as compared to IDC. While CD8+ T-cells were highly activated in CCC as compared to IDC, CD4+ T-cells were more activated in IDC. In addition to differential expression of functional molecules, these cells also displayed distinct expression of molecules associated with recruitment to the heart. In silico analysis of gene transcripts in the cardiac tissue demonstrated a significant correlation between CD8 and inflammatory, cytotoxic and cardiotropic molecules in CCC transcripts, while no correlation with CD4 was observed. A positive correlation was observed between CD4 and perforin transcripts in hearts from IDC but not CCC, as compared to normal tissue. These data show a clearly distinct systemic and local cellular response in CCC and IDC, despite their similar cardiac impairment, which may contribute to identifying specific immunotherapeutic targets in these diseases.

Distinct CD4-CD8- (Double-Negative) Memory T-Cell Subpopulations Are Associated With Indeterminate and Cardiac Clinical Forms of Chagas Disease.

CD4-CD8- (double-negative, DN) T cells are critical orchestrators of the cytokine network associated with the pathogenic inflammatory response in one of the deadliest cardiomyopathies known, Chagas heart disease, which is caused by Trypanosoma cruzi infection. Here, studying the distribution, activation status, and cytokine expression of memory DN T-cell subpopulations in Chagas disease patients without cardiac involvement (indeterminate form-IND) or with Chagas cardiomyopathy (CARD), we report that while IND patients displayed a higher frequency of central memory, CARD had a high frequency of effector memory DN T cells. In addition, central memory DN T cells from IND displayed a balanced cytokine profile, characterized by the concomitant expression of IFN-γ and IL-10, which was not observed in effector memory DN T cells from CARD. Supporting potential clinical relevance, we found that the frequency of central memory DN T cells was associated with indicators of better ventricular function, while the frequency of effector memory DN T cells was not. Importantly, decreasing CD1d-mediated activation of DN T cells led to an increase in IL-10 expression by effector memory DN T cells from CARD, restoring a balanced profile similar to that observed in the protective central memory DN T cells. Targeting the activation of effector memory DN T cells may emerge as a strategy to control inflammation in Chagas cardiomyopathy and potentially in other inflammatory diseases where these cells play a key role.

Systemic cytokines, chemokines and growth factors reveal specific and shared immunological characteristics in infectious cardiomyopathies.

Heart disease is a major cause of death worldwide. Chronic Chagas cardiomyopathy (CCC) caused by infection with Trypanosoma cruzi leading to high mortality in adults, and rheumatic heart disease (RHD), resulting from infection by Streptococcus pyogenes affecting mainly children and young adults, are amongst the deadliest heart diseases in low-middle income countries. Despite distinct etiology, the pathology associated with both diseases is a consequence of inflammation. Here we compare systemic immune profile in patients with these cardiopathies, to identify particular and common characteristics in these infectious heart diseases. We evaluated the expression of 27 soluble factors, employing single and multivariate analysis combined with machine-learning approaches. We observed that, while RHD and CCC display higher levels of circulating mediators than healthy individuals, CCC is associated with stronger immune activation as compared to RHD. Despite distinct etiologies, univariate analysis showed that expression of TNF, IL-17, IFN-gamma, IL-4, CCL4, CCL3, CXCL8, CCL11, CCL2, PDGF-BB were similar between CCC and RHD, consistent with their inflammatory nature. Network analysis revealed common inflammatory pathways between CCC and RHD, while highlighting the broader reach of the inflammatory response in CCC. The final multivariate model showed a 100% discrimination power for the combination of the cytokines IL-12p70, IL-1Ra, IL-4, and IL-7 between CCC and RHD groups. Thus, while clear immunological distinctions were identified between CCC and RHD, similarities indicate shared inflammatory pathways in these infectious heart diseases. These results contribute to understanding the pathogenesis of CCC and RHD and may impact the design of immune-based therapies for these and other inflammatory cardiopathies that may also share immunological characteristics.

Integration of Functional Imaging, Cytometry, and Unbiased Proteomics Reveals New Features of Endothelial-to-Mesenchymal Transition in Ischemic Mitral Valve Regurgitation in Human Patients.

Background: Following myocardial infarction, mitral regurgitation (MR) is a common complication. Previous animal studies demonstrated the association of endothelial-to-mesenchymal transition (EndMT) with mitral valve (MV) remodeling. Nevertheless, little is known about how MV tissue responds to ischemic heart changes in humans. Methods: MVs were obtained by the Cardiothoracic Surgical Trials Network from 17 patients with ischemic mitral regurgitation (IMR). Echo-doppler imaging assessed MV function at time of resection. Cryosections of MVs were analyzed using a multi-faceted histology and immunofluorescence examination of cell populations. MVs were further analyzed using unbiased label-free proteomics. Echo-Doppler imaging, histo-cytometry measures and proteomic analysis were then integrated. Results: MVs from patients with greater MR exhibited proteomic changes associated with proteolysis-, inflammatory- and oxidative stress-related processes compared to MVs with less MR. Cryosections of MVs from patients with IMR displayed activated valvular interstitial cells (aVICs) and double positive CD31+ αSMA+ cells, a hallmark of EndMT. Univariable and multivariable association with echocardiography measures revealed a positive correlation of MR severity with both cellular and geometric changes (e.g., aVICs, EndMT, leaflet thickness, leaflet tenting). Finally, proteomic changes associated with EndMT showed gene-ontology enrichment in vesicle-, inflammatory- and oxidative stress-related processes. This discovery approach indicated new candidate proteins associated with EndMT regulation in IMR. Conclusion: We describe an atypical cellular composition and distinctive proteome of human MVs from patients with IMR, which highlighted new candidate proteins implicated in EndMT-related processes, associated with maladaptive MV fibrotic remodeling.

Histopathological Characterization of Mitral Valvular Lesions from Patients with Rheumatic Heart Disease. / Caracterização Histológica das Lesões da Valva Mitral de Pacientes com Cardiopatia Reumática.

BACKGROUND: The underlying mechanisms by which rheumatic heart disease (RHD) lead to severe valve dysfunction are not completely understood. OBJECTIVE: The present study evaluated the histopathological changes in mitral valves (MV) seeking an association between the pattern of predominant valvular dysfunction and histopathological findings. METHODS: In 40 patients who underwent MV replacement due to RHD, and in 20 controls that underwent heart transplant, histological aspects of the excised MV were analyzed. Clinical and echocardiographic data were also collected. Histological analyses were performed using hematoxylin-eosin staining. Inflammation, fibrosis, neoangiogenesis, calcification and adipose metaplasia were determined. A p value<0.05 was considered to be statistically significant. RESULTS: The mean age of RHD patients was 53±13 years, 36 (90%) were female, whereas the mean age of controls was 50±12 years, similar to the cases, with the majority of males (70%). The rheumatic valve endocardium presented greater thickness than the controls (1.3±0.5 mm versus 0.90±0.4 mm, p=0.003, respectively), and a more intense inflammatory infiltrate in the endocardium (78% versus 36%; p=0.004), with predominance of mononuclear cells. Moderate to marked fibrosis occurred more frequently in rheumatic valves than in control valves (100% vs. 29%; p<0.001). Calcification occurred in 35% of rheumatic valves, especially among stenotic valves, which was associated with the mitral valve area (p=0.003). CONCLUSIONS: Despite intense degree of fibrosis, the inflammatory process remains active in the rheumatic mitral valve, even at late disease with valve dysfunction. Calcification predominated in stenotic valves and in patients with right ventricular dysfunction.

FUNDAMENTOS: Os mecanismos subjacentes pelos quais a doença cardíaca reumática (DCR) levam à disfunção valvar grave não são totalmente compreendidos. OBJETIVO: O presente estudo avaliou as alterações histopatológicas nas valvas mitrais (VM) buscando uma associação entre o padrão de disfunção valvar predominante e os achados histopatológicos. MÉTODOS: Em 40 pacientes submetidos à troca da VM devido a DCR e em 20 controles submetidos a transplante cardíaco, foram analisados os aspectos histológicos da VM excisada. Dados clínicos e ecocardiográficos também foram coletados. As análises histológicas foram realizadas usando coloração com hematoxilina-eosina. Determinou-se inflamação, fibrose, neoangiogênese, calcificação e metaplasia adiposa. Valores de p<0,05 foram considerados estatisticamente significativos. RESULTADOS: A idade média dos pacientes com DCR foi de 53±13 anos, sendo 36 (90%) do sexo feminino, enquanto a idade média dos controles foi de 50±12 anos, semelhante aos casos, sendo a maioria do sexo masculino (70%). O endocárdio valvar reumático apresentou espessura maior que os controles (1,3±0,5 mm versus 0,90±0,4 mm, p=0,003, respectivamente), e infiltrado inflamatório mais intenso no endocárdio (78% versus 36%; p=0,004), com predominância de células mononucleares. Ocorreu fibrose moderada a acentuada mais frequentemente em válvulas reumáticas do que em válvulas controle (100% vs. 29%; p<0,001). Ocorreu calcificação em 35% das valvas reumáticas, principalmente entre as valvas estenóticas, associada à área valvar mitral (p=0,003). CONCLUSÕES: Apesar do intenso grau de fibrose, o processo inflamatório permanece ativo na valva mitral reumática, mesmo em doença tardia com disfunção valvar. A calcificação predominou em valvas estenóticas e em pacientes com disfunção ventricular direita.

Caracterização Histológica das Lesões da Valva Mitral de Pacientes com Cardiopatia Reumática / Histopathological Characterization of Mitral Valvular Lesions from Patients with Rheumatic Heart Disease

Resumo Fundamentos: Os mecanismos subjacentes pelos quais a doença cardíaca reumática (DCR) levam à disfunção valvar grave não são totalmente compreendidos. Objetivo: O presente estudo avaliou as alterações histopatológicas nas valvas mitrais (VM) buscando uma associação entre o padrão de disfunção valvar predominante e os achados histopatológicos. Métodos: Em 40 pacientes submetidos à troca da VM devido a DCR e em 20 controles submetidos a transplante cardíaco, foram analisados os aspectos histológicos da VM excisada. Dados clínicos e ecocardiográficos também foram coletados. As análises histológicas foram realizadas usando coloração com hematoxilina-eosina. Determinou-se inflamação, fibrose, neoangiogênese, calcificação e metaplasia adiposa. Valores de p<0,05 foram considerados estatisticamente significativos. Resultados: A idade média dos pacientes com DCR foi de 53±13 anos, sendo 36 (90%) do sexo feminino, enquanto a idade média dos controles foi de 50±12 anos, semelhante aos casos, sendo a maioria do sexo masculino (70%). O endocárdio valvar reumático apresentou espessura maior que os controles (1,3±0,5 mm versus 0,90±0,4 mm, p=0,003, respectivamente), e infiltrado inflamatório mais intenso no endocárdio (78% versus 36%; p=0,004), com predominância de células mononucleares. Ocorreu fibrose moderada a acentuada mais frequentemente em válvulas reumáticas do que em válvulas controle (100% vs. 29%; p<0,001). Ocorreu calcificação em 35% das valvas reumáticas, principalmente entre as valvas estenóticas, associada à área valvar mitral (p=0,003). Conclusões: Apesar do intenso grau de fibrose, o processo inflamatório permanece ativo na valva mitral reumática, mesmo em doença tardia com disfunção valvar. A calcificação predominou em valvas estenóticas e em pacientes com disfunção ventricular direita.

Abstract Background: The underlying mechanisms by which rheumatic heart disease (RHD) lead to severe valve dysfunction are not completely understood. Objective: The present study evaluated the histopathological changes in mitral valves (MV) seeking an association between the pattern of predominant valvular dysfunction and histopathological findings. Methods: In 40 patients who underwent MV replacement due to RHD, and in 20 controls that underwent heart transplant, histological aspects of the excised MV were analyzed. Clinical and echocardiographic data were also collected. Histological analyses were performed using hematoxylin-eosin staining. Inflammation, fibrosis, neoangiogenesis, calcification and adipose metaplasia were determined. A p value<0.05 was considered to be statistically significant. Results: The mean age of RHD patients was 53±13 years, 36 (90%) were female, whereas the mean age of controls was 50±12 years, similar to the cases, with the majority of males (70%). The rheumatic valve endocardium presented greater thickness than the controls (1.3±0.5 mm versus 0.90±0.4 mm, p=0.003, respectively), and a more intense inflammatory infiltrate in the endocardium (78% versus 36%; p=0.004), with predominance of mononuclear cells. Moderate to marked fibrosis occurred more frequently in rheumatic valves than in control valves (100% vs. 29%; p<0.001). Calcification occurred in 35% of rheumatic valves, especially among stenotic valves, which was associated with the mitral valve area (p=0.003). Conclusions: Despite intense degree of fibrosis, the inflammatory process remains active in the rheumatic mitral valve, even at late disease with valve dysfunction. Calcification predominated in stenotic valves and in patients with right ventricular dysfunction.

Proinflammatory Matrix Metalloproteinase-1 Associates With Mitral Valve Leaflet Disruption Following Percutaneous Mitral Valvuloplasty.

Mitral regurgitation (MR) is a major complication of the percutaneous mitral valvuloplasty (PMV). Despite high technical expertise and cumulative experience with the procedure, the incidence rate of severe MR has not decreased. Although some of MR can be anticipated by echocardiographic analysis; leaflet tearing, which leads to the most dreaded type of MR, remains unpredictable. Irregular valvular collagen remodeling is likely to compromise tissue architecture and increase the tearing risk during PMV balloon inflation. In this study, we evaluated histological and molecular characteristics of excised mitral valves from patients with rheumatic mitral stenosis (MS) who underwent emergency surgery after PMV due to severe MR caused by leaflet tear. Those findings were compared with patients who underwent elective mitral valve replacement surgery owing to severe MS, in whom PMV was not indicated. In vitro assay using peripheral blood mononuclear cells was performed to better understand the impact of the cellular and molecular alterations identified in leaflet tear mitral valve specimens. Our analysis showed that focal infiltration of inflammatory cells contributes to accumulation of MMP-1 and IFN-γ in valve leaflets. Moreover, we showed that IFN-γ increase the expression of MMP-1 in CD14+ cells (monocytes) in vitro. Thus, inflammatory cells contribute to unevenly remodel collagen resulting in variable thickening causing abnormalities in leaflet architecture making them more susceptible to laceration.

Immunopathology and modulation induced by hookworms: From understanding to intervention.

Hookworm infection is considered the most prevalent human soil-transmitted helminth infection affecting approximately 500 million people and accounting for 3.2 million disability-adjusted life years lost annually. As with many other neglected tropical diseases, no international surveillance mechanisms that show accurate data on the prevalence of hookworm infection are in place, thus hindering strategies to control parasite transmission. In this review, we unravel the current knowledge in immunopathology and immunoregulation of hookworm infection and present discoveries in drug therapies based on the capability of hookworms to regulate inflammation to treat allergic, inflammatory and metabolic diseases. Additionally, we highlight potential vaccine development and treatments and propose avenues for further inquiry.

Human CD8+ T Cells Release Extracellular Traps Co-Localized With Cytotoxic Vesicles That Are Associated With Lesion Progression and Severity in Human Leishmaniasis.

Cell death plays a fundamental role in mounting protective and pathogenic immunity. Etosis is a cell death mechanism defined by the release of extracellular traps (ETs), which can foster inflammation and exert microbicidal activity. While etosis is often associated with innate cells, recent studies showed that B cells and CD4+ T cells can release ETs. Here we investigate whether CD8+ T cells can also release ETs, which might be related to cytotoxicity and tissue pathology. To these ends, we first employed an in vitro system stimulating human CD8+ T cells isolated from healthy volunteers with anti-CD3/anti-CD28. Using time-frame video, confocal and electron microscopy, we demonstrate that human CD8+ T cells release ETs upon stimulation (herein LETs - lymphocyte extracellular traps), which display unique morphology and functional characteristics. CD8+ T cell-derived LETs form long strands that co-localize with CD107a, a marker of vesicles containing cytotoxic granules. In addition, these structures connect the LET-releasing cell to other neighboring cells, often resulting in cell death. After demonstrating the release of LETs by human CD8+ T cells in vitro, we went on to study the occurrence of CD8-derived LETs in a human disease setting. Thus, we evaluated the occurrence of CD8-derived LETs in lesions from patients with human tegumentary leishmaniasis, where CD8+ T cells play a key role in mediating pathology. In addition, we evaluated the association of these structures with the intensity of the inflammatory infiltrate in early and late cutaneous, as well as in mucosal leishmaniasis lesions. We demonstrated that progression and severity of debilitating and mutilating forms of human tegumentary leishmaniasis are associated with the frequency of CD8+ T cells in etosis, as well as the occurrence of CD8-derived LETs carrying CD107a+ vesicles in the lesions. We propose that CD8+ T cell derived LETs may serve as a tool for delivering cytotoxic vesicles to distant target cells, providing insights into mechanisms of CD8+ T cell mediated pathology.

Mitral Regurgitation After Percutaneous Mitral Valvuloplasty: Insights Into Mechanisms and Impact on Clinical Outcomes.

OBJECTIVES: The aim of this study was to assess the incidence, mechanisms, and outcomes of mitral regurgitation (MR) after percutaneous mitral valvuloplasty (PMV). BACKGROUND: Significant MR continues to be a major complication of PMV, with a wide range in clinical presentation and prognosis. METHODS: Consecutive patients with mitral stenosis undergoing PMV were prospectively enrolled. MR severity was evaluated by using quantitative echocardiographic criteria, and its mechanism was characterized by 3-dimensional transesophageal echocardiography, divided broadly into 4 categories based on the features contributing to the valve damage. B-type natriuretic peptide levels were obtained before and 24 h after the procedure. Endpoints estimated cardiovascular death or mitral valve (MV) replacement due to predominant MR. RESULTS: A total of 344 patients, ages 45.1 ± 12.1 years, of whom 293 (85%) were women, were enrolled. Significant MR after PMV was found in 64 patients (18.6%). The most frequent mechanism of MR was commissural, which occurred in 22 (34.4%) patients, followed by commissural with posterior leaflet in 16 (25.0%), leaflets at central scallop or subvalvular damage in 15 (23.4%), and central MR in 11 (17.2%). During the mean follow-up period of 3 years (range 1 day to 10.6 years), 60 patients reached the endpoint. The event-free survival rates were similar among patients with mild or commissural MR, whereas patients with damaged central leaflet scallop or subvalvular apparatus had the worst outcome, with an event-free survival rate at 1 year of only 7%. Long-term outcome was predicted by net atrioventricular compliance (Cn) at baseline and post-procedural variables, including valve area, mean gradient, and magnitude of decrease in B-type natriuretic peptide levels, adjusted for the mechanism of MR. CONCLUSIONS: Significant MR following PMV is a frequent event, mainly related to commissural splitting, with favorable clinical outcome. Parameters that express the relief of valve obstruction and the mechanism by which MR develops were predictors of long-term outcomes.

S100A9-RAGE Axis Accelerates Formation of Macrophage-Mediated Extracellular Vesicle Microcalcification in Diabetes Mellitus.

OBJECTIVE: Vascular calcification is a cardiovascular risk factor and accelerated in diabetes mellitus. Previous work has established a role for calcification-prone extracellular vesicles in promoting vascular calcification. However, the mechanisms by which diabetes mellitus provokes cardiovascular events remain incompletely understood. Our goal was to identify that increased S100A9 promotes the release of calcification-prone extracellular vesicles from human macrophages in diabetes mellitus. Approach and Results: Human primary macrophages exposed to high glucose (25 mmol/L) increased S100A9 secretion and the expression of receptor for advanced glycation end products (RAGE) protein. Recombinant S100A9 induced the expression of proinflammatory and osteogenic factors, as well as the number of extracellular vesicles with high calcific potential (alkaline phosphatase activity, P<0.001) in macrophages. Treatment with a RAGE antagonist or silencing with S100A9 siRNA in macrophages abolished these responses, suggesting that stimulation of the S100A9-RAGE axis by hyperglycemia favors a procalcific environment. We further showed that an imbalance between Nrf-2 (nuclear factor 2 erythroid related factor 2) and NF-κB (nuclear factor-κB) pathways contributes to macrophage activation and promotes a procalcific environment. In addition, streptozotocin-induced diabetic Apoe-/-S100a9-/- mice and mice treated with S100a9 siRNA encapsulated in macrophage-targeted lipid nanoparticles showed decreased inflammation and microcalcification in atherosclerotic plaques, as gauged by molecular imaging and comprehensive histological analysis. In human carotid plaques, comparative proteomics in patients with diabetes mellitus and histological analysis showed that the S100A9-RAGE axis associates with osteogenic activity and the formation of microcalcification. CONCLUSIONS: Under hyperglycemic conditions, macrophages release calcific extracellular vesicles through mechanisms involving the S100A9-RAGE axis, thus contributing to the formation of microcalcification within atherosclerotic plaques.

Innate and adaptive immunity in cardiovascular calcification.

Despite the focus placed on cardiovascular research, the prevalence of vascular and valvular calcification is increasing and remains a leading contributor of cardiovascular morbidity and mortality. Accumulating studies provide evidence that cardiovascular calcification is an inflammatory disease in which innate immune signaling becomes sustained and/or excessive, shaping a deleterious adaptive response. The triggering immune factors and subsequent inflammatory events surrounding cardiovascular calcification remain poorly understood, despite sustained significant research interest and support in the field. Most studies on cardiovascular calcification focus on innate cells, particularly macrophages' ability to release pro-osteogenic cytokines and calcification-prone extracellular vesicles and apoptotic bodies. Even though substantial evidence demonstrates that macrophages are key components in triggering cardiovascular calcification, the crosstalk between innate and adaptive immune cell components has not been adequately addressed. The only therapeutic options currently used are invasive procedures by surgery or transcatheter intervention. However, no approved drug has shown prophylactic or therapeutic effectiveness. Conventional diagnostic imaging is currently the best method for detecting, measuring, and assisting in the treatment of calcification. However, these common imaging modalities are unable to detect early subclinical stages of disease at the level of microcalcifications; therefore, the vast majority of patients are diagnosed when macrocalcifications are already established. In this review, we unravel the current knowledge of how innate and adaptive immunity regulate cardiovascular calcification; and put forward differences and similarities between vascular and valvular disease. Additionally, we highlight potential immunomodulatory drugs with the potential to target calcification and propose avenues in need of further translational inquiry.