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Recent studies reported that circulating microRNAs (miRNAs) can target different metalloproteases (MMPs) involved in matrix remodeling and plaque vulnerability. Consequently, they might have a role in the diagnosis and prognosis of coronary artery disease. To quantify circulating miRNAs (miRNA126, miRNA146, and miRNA21) suggested to have possible cardiovascular implications, as well as levels of MMP-1 and MMP-9, and to determine their association with left ventricular (LV) function and with arterial function, in patients with either ST-segment elevation acute myocardial infarction (STEMI) or stable ischemic heart disease (SIHD). A total of 90 patients with coronary artery disease (61% men, 58 ± 12 years), including 60 patients with STEMI and 30 patients with SIHD, were assessed within 24 h of admission, by measuring serum microRNAs, and serum MMP-1 and MMP-9. LV function was assessed by measuring ejection fraction (EF) by 2D and 3D echocardiography, and global longitudinal strain (GLS) by speckle tracking. Arterial function was assessed by echo tracking, CAVI, and peripheral Doppler. Circulating levels of miRNA146, miRNA21, and MMP1 were significantly increased in patients with STEMI vs. SIHD (p = 0.0001, p = 0.0001, p = 0.04, respectively). MiRNA126 negatively correlated with LVEF (r = -0.33, p = 0.01) and LV deformation parameters (r = -0.31, p = 0.03) in patients with STEMI and negatively correlated with ABI parameters (r = -0.39, p = 0.03, r = -0.40, p = 0.03, respectively) in patients with SIHD. MiRNA146 did not have any significant correlations, while higher values of miRNA21 were associated with lower values of GLS in STEMI patients and with higher values of GLS in SIHD patients. Both MMP1 and MMP9 correlated negatively with LVEF (r = -0.27, p = 0.04, r = -0.40, p = 0.001, respectively) and GLS in patients with STEMI, and positively with arterial stiffness in patients with SIHD (r = 0.40 and r = 0.32, respectively; both p < 0.05). MiRNA126, miRNA21, and both MMP1 and MMP9 are associated with LV and arterial function parameters in patients with acute coronary syndrome. Meanwhile, they inversely correlate with arterial function in patients with chronic atherosclerotic disease. However, further studies are needed to establish whether these novel biomarkers have diagnosis and prognosis significance.
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Parathyroid hormone (PTH) is a key regulator of calcium, phosphate and vitamin D metabolism. Although it has been reported that aortic valve calcification was positively associated with PTH, the pathophysiological mechanisms and the direct effects of PTH on human valvular cells remain unclear. Here we investigated if PTH induces human valvular endothelial cells (VEC) dysfunction that in turn could impact the switch of valvular interstitial cells (VIC) to an osteoblastic phenotype. Human VEC exposed to PTH were analyzed by qPCR, western blot, Seahorse, ELISA and immunofluorescence. Our results showed that exposure of VEC to PTH affects VEC metabolism and functions, modifications that were accompanied by the activation of p38MAPK and ERK1/2 signaling pathways and by an increased expression of osteogenic molecules (BMP-2, BSP, osteocalcin and Runx2). The impact of dysfunctional VEC on VIC was investigated by exposure of VIC to VEC secretome, and the results showed that VIC upregulate molecules associated with osteogenesis (BMP-2/4, osteocalcin and TGF-ß1) and downregulate collagen I and III. In summary, our data show that PTH induces VEC dysfunction, which further stimulates VIC to differentiate into a pro-osteogenic pathological phenotype related to the calcification process. These findings shed light on the mechanisms by which PTH participates in valve calcification pathology and suggests that PTH and the treatment of hyperparathyroidism represent a therapeutic strategy to reduce valvular calcification.
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Estenose da Valva Aórtica , Calcinose , Valva Aórtica/patologia , Estenose da Valva Aórtica/patologia , Calcinose/patologia , Diferenciação Celular/genética , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese , Hormônio Paratireóideo/metabolismo , FenótipoRESUMO
Background: Valvular endothelial cells (VEC) have key roles in maintaining valvular integrity and homeostasis, and dysfunctional VEC are the initiators and major contributors to aortic valve disease in diabetes. Previous studies have shown that HG stimulated an inflammatory phenotype in VEC. Inflammation was shown to induce endothelial to mesenchymal transition (EndMT), a process extensively involved in many pathologies, including calcification of the aortic valve. However, the effect of HG on EndMT in VEC is not known. In addition, there is evidence that endothelin (ET) is a proinflammatory agent in early diabetes and was detected in aortic stenosis, but it is not known whether HG induces ET and endothelin receptors and whether endothelin modulates HG-dependent inflammation in VEC. This study aims to evaluate HG effects on EndMT, on endothelin and endothelin receptors induction in VEC and their role in HG induced VEC inflammation. Methods and Results: We developed a new 3D model of the aortic valve consisting of a hydrogel derived from a decellularized extracellular cell matrix obtained from porcine aortic root and human valvular cells. VEC were cultured on the hydrogel surface and VIC within the hydrogel, and the resulted 3D construct was exposed to high glucose (HG) conditions. VEC from the 3D construct exposed to HG exhibited: attenuated intercellular junctions and an abundance of intermediate filaments (ultrastructural analysis), decreased expression of endothelial markers CD31 and VE-cadherin and increased expression of the mesenchymal markers α-SMA and vimentin (qPCR and immunocytochemistry), increased expression of inflammatory molecules ET-1 and its receptors ET-A and ET-B, ICAM-1, VCAM-1 (qPCR and Immunocytochemistry) and augmented adhesiveness. Blockade of ET-1 receptors, ET-A and ET-B reduced secretion of inflammatory biomarkers IL-1ß and MCP-1 (ELISA assay). Conclusions: This study demonstrates that HG induces EndMT in VEC and indicates endothelin as a possible target to reduce HG-induced inflammation in VEC.
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Neutrophils have been classically viewed as a homogenous population. Recently, neutrophils were phenotypically classified into pro-inflammatory N1 and anti-inflammatory N2 sub-populations, but the functional differences between the two subtypes are not completely understood. We aimed to investigate the phenotypic and functional differences between N1 and N2 neutrophils, and to identify the potential contribution of the S100A9 alarmin in neutrophil polarization. We describe distinct transcriptomic profiles and functional differences between N1 and N2 neutrophils. Compared to N2, the N1 neutrophils exhibited: i) higher levels of ROS and oxidative burst, ii) increased activity of MPO and MMP-9, and iii) enhanced chemotactic response. N1 neutrophils were also characterized by elevated expression of NADPH oxidase subunits, as well as activation of the signaling molecules ERK and the p65 subunit of NF-kB. Moreover, we found that the S100A9 alarmin promotes the chemotactic and enzymatic activity of N1 neutrophils. S100A9 inhibition with a specific small-molecule blocker, reduced CCL2, CCL3 and CCL5 chemokine expression and decreased MPO and MMP-9 activity, by interfering with the NF-kB signaling pathway. Together, these findings reveal that N1 neutrophils are pro-inflammatory effectors of the innate immune response. Pharmacological blockade of S100A9 dampens the function of the pro-inflammatory N1 phenotype, promoting the alarmin as a novel target for therapeutic intervention in inflammatory diseases.
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Calgranulina B/fisiologia , Perfilação da Expressão Gênica , Agentes de Imunomodulação/farmacologia , Neutrófilos/fisiologia , Sulfonamidas/farmacologia , Animais , Polaridade Celular , Quimiocinas/análise , Feminino , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/classificação , Neutrófilos/efeitos dos fármacos , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Calcific aortic valve disease (CAVD), a degenerative disease characterized by inflammation, fibrosis and calcification, is accelerated in diabetes. Hyperglycemia contributes to this process by mechanisms that still need to be uncovered. We have recently developed a 3D model of the human aortic valve based on gelatin methacrylate and revealed that high glucose (HG) induced osteogenic molecules and increased calcium deposits in a pro-osteogenic environment. To further understand the events leading to calcification in diabetic conditions in CAVD, we analyzed here the inflammatory and remodeling mechanisms induced by HG in our 3D model. We exposed valvular endothelial cells (VEC) and interstitial cells (VIC) to normal glucose (NG) or HG for 7 and 14 days, then we isolated and separated the cells by anti-CD31 immunomagnetic beads. The changes induced by HG in the 3D model were investigated by real-time polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. Our results showed that HG induced expression of different cytokines, cell adhesion molecules and matrix metalloproteinases in VEC and VIC. In addition, protein kinase C was increased in VEC and VIC, indicating molecular mechanisms associated with HG induced inflammation and remodeling in both valvular cells. These findings may indicate new biomarkers and targets for therapy in diabetes associated with CAVD.
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Calcific aortic valve disease (CAVD)-the most common valvular heart disease-is accelerated in diabetes and has no pharmacotherapy. Although it is known that early CAVD is associated with inflammation and osteogenesis, the molecular mechanisms involved in diabetes-associated CAVD still need to be uncovered. In this context, we have developed a 3D construct based on gelatin populated with human valvular endothelial cells (VEC) and valvular interstitial cells (VIC) and evaluated the effect of high glucose (HG) concentration on osteogenic molecules expression and on calcification mechanisms. First, we characterized the 3D model and assessed VIC remodelling properties at different time-points. Then, we exposed it to normal glucose (NG) or high glucose (HG) for 7, 14 and 21 days after which the cells were isolated, separated and investigated individually. Our results showed that encapsulated VIC actively remodel the hydrogel, as demonstrated by an increased expression of extracellular matrix (ECM) proteins and matrix metalloproteinases (MMPs). Moreover, exposure of the construct to HG triggered bone morphogenetic protein (BMP) and TGF-ß signalling pathways, up-regulating expression of osteogenic molecules-BMP-2/-4, osteocalcin, osteopontin, SMADs and Runt-related transcription factor (Runx-2)-and increased calcium deposits in an osteogenic environment. These findings underline the potential of the developed 3D model as a suitable system to investigate the mechanisms of human CAVD and may help to better understand the calcification mechanisms in CAVD associated to diabetes.
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Estenose da Valva Aórtica/genética , Valva Aórtica/patologia , Calcinose/genética , Glucose/toxicidade , Modelos Biológicos , Valva Aórtica/efeitos dos fármacos , Proteína Morfogenética Óssea 2/metabolismo , Cálcio/metabolismo , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alicerces Teciduais/química , Fator de Crescimento Transformador beta/metabolismo , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/genéticaRESUMO
Diabetes contributes directly to the development of cardiovascular aortic valve disease. There is currently no drug therapy available for a dysfunctional valve and this urges the need for additional research to identify distinctive mechanisms of cardiovascular aortic valve disease evolution. The aim of this study was to evaluate changes of valvular aortic lesions induced in a hyperlipemic ApoE-/- mouse model by early type 1 diabetes onset (at 4 and 7 days after streptozotocin induction). The haemodynamic valve parameters were evaluated by echography and blood samples and aortic valves were collected. Plasma parameters were measured, and inflammatory, remodelling and osteogenic markers were evaluated in the aortic valves. Next, correlations between all parameters were determined. The results showed early aortic valve dysfunction detected by echography after 1 week of diabetes; lesions were found in the aortic root. Moreover, increased expression of cell adhesion molecules, extracellular matrix remodelling and osteogenic markers were detected in hyperlipemic ApoE-/- diabetic mice. Significant correlations were found between tissue valve biomarkers and plasmatic and haemodynamic parameters. Our study may help to understand the mechanisms of aortic valve disease in the diabetic milieu in order to discover and validate new biomarkers of cardiovascular aortic valve disease in diabetes and reveal new possible targets for nanobiotherapies.
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Valva Aórtica , Aterosclerose/complicações , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Doenças das Valvas Cardíacas/etiologia , Animais , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Glicemia/metabolismo , Moléculas de Adesão Celular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Hemoglobinas Glicadas/metabolismo , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/patologia , Doenças das Valvas Cardíacas/fisiopatologia , Hemodinâmica , Mediadores da Inflamação/metabolismo , Lipídeos/sangue , Masculino , Camundongos Knockout para ApoE , Osteogênese , Fatores de TempoRESUMO
Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease-related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC-MAC cross-talk was achieved using trans-well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross-talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP-1 and signalling molecules. We found that upon cross-talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP-1 and MMP-9; (ii) significant increase in the enzymatic activity of MMP-9; (iii) higher levels of soluble MCP-1 chemokine which is functionally active and involved in MMPs up-regulation; (iv) activated PKCα signalling pathway which, together with NF-kB are responsible for MMP-1 and MMP-9 up-regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP-1 released by cell cross-talk in diabetic conditions binds to CCR2 and triggers MMP-1 and MMP-9 over-expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients.