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1.
J Biomed Sci ; 28(1): 74, 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34749728

RESUMO

BACKGROUND: Toll-like receptor-2 (TLR2) promotes vascular smooth muscle cell (VSMC) transdifferentiation to chondrocytes and calcification in a p38 MAPK-dependent manner. Vascular 5-methoxytryptophan (5-MTP) is a newly identified factor with anti-inflammatory actions. As 5-MTP targets p38 MAPK for its actions, we postulated that 5-MTP protects against vascular chondrogenesis and calcification. METHODS: High-fat diet-induced advanced atherosclerosis in mice were performed to investigate the effect of 5-MTP on atherosclerotic lesions and calcification. VSMCs were used to determine the role of 5-MTP in VSMC chondrogenic differentiation and calcification. Alizarin red S and Alcian blue staining were used to measure VSMC calcification and chondrogenic differentiation, respectively. RESULTS: 5-MTP was detected in aortic tissues of ApoE-/- mice fed control chow. It was reduced in ApoE-/- mice fed high-fat diet (HFD), but was restored in ApoE-/-Tlr2-/- mice, suggesting that HFD reduces vascular 5-MTP production via TLR2. Intraperitoneal injection of 5-MTP or its analog into ApoE-/- mice fed HFD reduced aortic atherosclerotic lesions and calcification which was accompanied by reduction of chondrogenesis and calcium deposition. Pam3CSK4 (Pam3), ligand of TLR2, induced SMC phenotypic switch to chondrocytes. Pretreatment with 5-MTP preserved SMC contractile proteins and blocked Pam3-induced chondrocyte differentiation and calcification. 5-MTP inhibited HFD-induced p38 MAPK activation in vivo and Pam3-induced p38 MAPK activation in SMCs. 5-MTP suppressed HFD-induced CREB activation in aortic tissues and Pam3-induced CREB and NF-κB activation in SMCs. CONCLUSIONS: These findings suggest that 5-MTP is a vascular arsenal against atherosclerosis and calcification by inhibiting TLR2-mediated SMC phenotypic switch to chondrocytes and the consequent calcification. 5-MTP exerts these effects by blocking p38 MAPK activation and inhibiting CREB and NF-κB transactivation activity.


Assuntos
Aterosclerose/prevenção & controle , Calcinose/prevenção & controle , Condrogênese , Dieta Hiperlipídica/efeitos adversos , Triptofano/análogos & derivados , Animais , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , Calcinose/metabolismo , Calcinose/fisiopatologia , Camundongos , Triptofano/metabolismo
2.
Am J Cardiol ; 156: 108-113, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34344508

RESUMO

Aortic valve calcium (AVC) is a strong predictor of aortic stenosis (AS) severity and is typically calculated by multidetector computed tomography (MDCT). We propose a novel method using pixel density quantification software to objectively quantify AVC by two-dimensional (2D) transthoracic echocardiography (TTE) and distinguish severe from non-severe AS. A total of 90 patients (mean age 76 ± 10 years, 75% male, mean AV gradient 32 ± 11 mmHg, peak AV velocity 3.6 ± 0.6 m/s, AV area (AVA) 1.0 ± 0.3 cm2, dimensionless index (DI) 0.27 ± 0.08) with suspected severe aortic stenosis undergoing 2D echocardiography were retrospectively evaluated. Parasternal short axis aortic valve views were used to calculate a gain-independent ratio between the average pixel density of the entire aortic valve in short axis at end diastole and the average pixel density of the aortic annulus in short axis (2D-AVC ratio). The 2D-AVC ratio was compared to echocardiographic hemodynamic parameters associated with AS, MDCT AVC quantification, and expert reader interpretation of AS severity based on echocardiographic AVC interpretation. The 2D-AVC ratio exhibited strong correlations with mean AV gradient (r = 0.72, p < 0.001), peak AV velocity (r = 0.74, p < 0.001), AVC quantified by MDCT (r = 0.71, p <0.001) and excellent accuracy in distinguishing severe from non-severe AS (area under the curve = 0.93). Conversely, expert reader interpretation of AS severity based on echocardiographic AVC was not significantly related to AV mean gradient (t = 0.23, p = 0.64), AVA (t = 2.94, p = 0.11), peak velocity (t = 0.59, p = 0.46), or DI (t = 0.02, p = 0.89). In conclusion, these data suggest that the 2D-AVC ratio may be a complementary method for AS severity adjudication that is readily quantifiable at time of TTE.


Assuntos
Estenose da Valva Aórtica/diagnóstico , Valva Aórtica/diagnóstico por imagem , Calcinose/diagnóstico , Cálcio/metabolismo , Ecocardiografia/métodos , Idoso , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/fisiopatologia , Calcinose/metabolismo , Calcinose/fisiopatologia , Feminino , Seguimentos , Hemodinâmica/fisiologia , Humanos , Masculino , Estudos Retrospectivos , Índice de Gravidade de Doença
3.
Biomed Pharmacother ; 139: 111674, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243610

RESUMO

OBJECTIVES: In calcific aortic valve disease (CAVD), the valve interstitial cells (VIC) osteogenic phenotype changes can lead to thickening and calcification of the valve leaflets,eventually lead to restricted valve movement and life-threatening. This study aims to investigate the effect and mechanism of dihydrotanshinone I (DHI) on osteogenic medium (OM) induced osteogenic phenotypic transition of porcine valve interstitial cells (PVICs), which can provide theoretical and scientific basis for clinical intervention in CAVD. METHODS AND RESULTS: Immunohistochemical methods were used to detect the expression of osteogenic indicators Runx2, OPN and inflammation indicators IL-1ß and p-NF-κB in valve specimens of CAVD patients(N = 3) and normal controls(N = 1). PVICs stimulated by osteoblastic medium (OM) were treated with or without DHI. CCK8, ALP and Alizarin Red S staining were used to detect cell growth and calcification, respectively. The results showed that under the treated with DHI, compared with OM, the formation of calcium nodules was reduced, and the expression of calcification-related markers Runx2 and OPN were down-regulated, which quantified by qRT-PCR and western blot. In addition, on the basis of OM induction, DHI also inhibited the phosphorylation of the NF-κB/ERK1/2 and SMAD1/5/8 signaling pathway. CONCLUSION: DHI (10 µM) treatment can reverse the osteogenic phenotypic transition of PVICs induced by osteogenic medium, and the mechanism may be related to NF-κB、ERK 1/2 and Smad1/5/8 pathways.


Assuntos
Estenose da Valva Aórtica/tratamento farmacológico , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/patologia , Calcinose/tratamento farmacológico , Furanos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Fenantrenos/farmacologia , Quinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Animais , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/metabolismo , Calcinose/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Humanos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Suínos
4.
Eur Heart J ; 42(30): 2935-2951, 2021 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-34179958

RESUMO

AIMS: The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited. Here, we evaluated the role and therapeutic value of dual-specificity phosphatase 26 (DUSP26) in CAVD. METHODS AND RESULTS: Microarray profiling of human calcific aortic valves and normal controls demonstrated that DUSP26 was significantly up-regulated in calcific aortic valves. ApoE-/- mice fed a normal diet or a high cholesterol diet (HCD) were infected with adeno-associated virus serotype 2 carrying DUSP26 short-hairpin RNA to examine the effects of DUSP26 silencing on aortic valve calcification. DUSP26 silencing ameliorated aortic valve calcification in HCD-treated ApoE-/- mice, as evidenced by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and decreased levels of osteogenic markers (Runx2, osterix, and osteocalcin) in the aortic valves. These results were confirmed in osteogenic medium-induced human valvular interstitial cells. Immunoprecipitation, liquid chromatography-tandem mass spectrometry, and functional assays revealed that dipeptidyl peptidase-4 (DPP4) interacted with DUSP26 to mediate the procalcific effects of DUSP26. High N6-methyladenosine levels up-regulated DUSP26 in CAVD; in turn, DUSP26 activated DPP4 by antagonizing mouse double minute 2-mediated ubiquitination and degradation of DPP4, thereby promoting CAVD progression. CONCLUSION: DUSP26 promotes aortic valve calcification by inhibiting DPP4 degradation. Our findings identify a previously unrecognized mechanism of DPP4 up-regulation in CAVD, suggesting that DUSP26 silencing or inhibition is a viable therapeutic strategy to impede CAVD progression.


Assuntos
Estenose da Valva Aórtica , Valva Aórtica/patologia , Calcinose , Fosfatases de Especificidade Dupla , Fosfatases da Proteína Quinase Ativada por Mitógeno , Animais , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/metabolismo , Calcinose/genética , Calcinose/metabolismo , Células Cultivadas , Dipeptidil Peptidase 4 , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Humanos , Camundongos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Ubiquitinação
5.
Lab Invest ; 101(9): 1267-1280, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34117364

RESUMO

Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and ß-glycerophosphate (ßG), and direct monitoring of the mineralization process was conducted using Raman micro-spectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to ßG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with ßG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples.


Assuntos
Neoplasias da Mama , Calcinose , Análise Espectral Raman/métodos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Calcinose/diagnóstico por imagem , Calcinose/metabolismo , Calcinose/patologia , Fosfatos de Cálcio/química , Fosfatos de Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos
6.
Cell Prolif ; 54(6): e13018, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33945189

RESUMO

BACKGROUND: Endothelial-to-mesenchymal transition (EndMT) is a common pathophysiology in valvular calcification (VC) among non-chronic kidney disease (CKD) patients. However, few studies were investigated in CKD-induced VC. Parathyroid hormone (PTH) was considered to be an important component of EndMT in CKD-induced cardiovascular diseases. Therefore, determining whether PTH could induce valvular EndMT and elucidating corresponding mechanism involved further study. METHODS: Performing a 5/6 nephrectomy with a high phosphorus diet was done to construct VC models in rats with CKD. miRNA sequencing was used to ascertain changes in microRNA in human umbilical vein endothelial cells (HUVECs) intervened by PTH. VC was observed by Von Kossa staining and scanning electron microscope. RESULTS: PTH induced valvular EndMT in VC. Global microRNA expression profiling of HUVECs was examined in PTH versus the control in vitro, in which miR-29a-5p was most notably decreased and was resumed by PTHrP(7-34) (PTH-receptor1 inhibitor). Overexpression of miR-29a-5p could inhibit PTH-induced EndMT in vitro and valvular EndMT in vivo. The dual-luciferase assay verified that γ-secretase-activating protein (GASP) served as the target of miR-29a-5p. miR-29a-5p-mimics, si-GSAP and DAPT (γ-secretase inhibitor) inhibited PTH-induced γ-secretase activation, thus blocking Notch1 pathway activation to inhibit EndMT in vitro. Moreover, Notch1 pathway activation was observed in VC. Blocking Notch1 pathway activation via AAV-miR-29a and DAPT inhibited valvular EndMT. In addition, blocking Notch1 pathway activation was also shown to alleviate VC. CONCLUSION: PTH activates valvular EndMT via miR-29a-5p/GSAP/Notch1 pathway, which can contribute to VC in CKD rats.


Assuntos
Estenose da Valva Aórtica/etiologia , Valva Aórtica/patologia , Calcinose/etiologia , MicroRNAs/genética , Hormônio Paratireóideo/metabolismo , Receptor Notch1/metabolismo , Insuficiência Renal Crônica/complicações , Animais , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Calcinose/genética , Calcinose/metabolismo , Calcinose/patologia , Endotélio/metabolismo , Endotélio/patologia , Transição Epitelial-Mesenquimal , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , MicroRNAs/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Notch1/genética , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Transdução de Sinais , Transcriptoma , Regulação para Cima
7.
J Physiol Biochem ; 77(3): 461-468, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34043161

RESUMO

Little is known about the biological functions and underlying mechanisms of long non-coding RNA AFAP1-AS1 in degenerative calcified aortic valve disease (DCAVD). This study aims to explore whether AFAP1-AS1 regulates macrophage polarization in aortic valve calcification. Macrophage polarization and AFAP1-AS1 expression were detected in normal and calcified aortic valves of DCAVD patients. To explore the effect of AFAP1-AS1 on macrophage polarization, gain and loss of function were performed in THP-1 cells, and the percentage of M1 and M2 and the expressions of M1 and M2 markers were analyzed. Meanwhile, osteogenic differentiation was examined in valve interstitial cells (VICs). Compared with normal valves, there were more M1, less M2, and high AFAP1-AS1 expressions in calcified aortic valves, which may indicate a relationship between AFAP1-AS1 and macrophage polarization. AFAP1-AS1 overexpression promoted M1 polarization in lipopolysaccharide (LPS) and interferon gamma (IFN-γ)-treated THP-1 cells but inhibited M2 polarization, as well as augmented VIC osteogenic differentiation. On the contrary, the silence of AFAP1-AS1 could induce macrophage to M2-type and inhibit VIC osteogenic differentiation. These results elucidate that AFAP1-AS1 can promote M1 macrophages polarization to aggravate VIC osteogenic differentiation, playing a role in aortic valve calcification.


Assuntos
Estenose da Valva Aórtica/metabolismo , Valva Aórtica/patologia , Calcinose/metabolismo , Macrófagos/citologia , Osteogênese , RNA Longo não Codificante/fisiologia , Idoso , Valva Aórtica/metabolismo , Diferenciação Celular , Polaridade Celular , Células Cultivadas , Feminino , Humanos , Ativação de Macrófagos , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células
8.
FEBS J ; 288(22): 6528-6542, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34009721

RESUMO

Calcific aortic valve disease (CAVD) is the most prevalent valvulopathy worldwide. Growing evidence supports a role for viral and cell-derived double-stranded (ds)-RNA in cardiovascular pathophysiology. Poly(I:C), a dsRNA surrogate, has been shown to induce inflammation, type I interferon (IFN) responses, and osteogenesis through Toll-like receptor 3 in aortic valve interstitial cells (VIC). Here, we aimed to determine whether IFN signaling via Janus kinase (JAK)/Signal transducers and activators of transcription (STAT) mediates dsRNA-induced responses in primary human VIC. Western blot, ELISA, qPCR, calcification, flow cytometry, and enzymatic assays were performed to evaluate the mechanisms of dsRNA-induced inflammation and calcification. Poly(I:C) triggered a type I IFN response characterized by IFN-regulatory factors gene upregulation, IFN-ß secretion, and STAT1 activation. Additionally, Poly(I:C) promoted VIC inflammation via NF-κB and subsequent adhesion molecule expression, and cytokine secretion. Pretreatment with ruxolitinib, a clinically used JAK inhibitor, abrogated these responses. Moreover, Poly(I:C) promoted a pro-osteogenic phenotype and increased VIC calcification to a higher extent in cells from males. Inhibition of JAK with ruxolitinib or a type I IFN receptor blocking antibody blunted Poly(I:C)-induced calcification. Mechanistically, Poly(I:C) promoted VIC apoptosis in calcification medium, which was inhibited by ruxolitinib. Moreover, Poly(I:C) co-operated with IFN-γ to increase VIC calcification by synergistically activating extracellular signal-regulated kinases and hypoxia-inducible factor-1α pathways. In conclusion, JAK/STAT signaling mediates dsRNA-triggered inflammation, apoptosis, and calcification and may contribute to a positive autocrine loop in human VIC in the presence of IFN-γ. Blockade of dsRNA responses with JAK inhibitors may be a promising therapeutic avenue for CAVD.


Assuntos
Estenose da Valva Aórtica/tratamento farmacológico , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/patologia , Calcinose/tratamento farmacológico , Inflamação/tratamento farmacológico , Inibidores de Janus Quinases/farmacologia , Nitrilas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA de Cadeia Dupla/antagonistas & inibidores , Adolescente , Adulto , Idoso , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Calcinose/metabolismo , Calcinose/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Inibidores de Janus Quinases/química , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Nitrilas/química , Pirazóis/química , Pirimidinas/química , RNA de Cadeia Dupla/metabolismo , Adulto Jovem
9.
Sci Rep ; 11(1): 11269, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050203

RESUMO

Coronary calcifications are an obstacle for successful percutaneous treatment of coronary artery disease patients. The optimal method for delineating calcifications extent is coronary optical coherence tomography (OCT). To identify calcification on OCT and subsequently tailor the appropriate treatment, requires expertise in both image acquisition and interpretation. Image acquisition consists from system calibration, blood clearance by a contrast agent along with synchronization of the pullback process. Accurate interpretation demands careful review by the operator of a segment of 50-75 mm of the coronary vessel at steps of 5-10 frames per mm accounting for 375-540 images in each OCT run, which is time consuming and necessitates some expertise in OCT analysis. In this paper we developed a new deep learning algorithm to assist the physician to identify and quantify coronary calcifications promptly, efficiently and accurately. Our algorithm achieves an accuracy of 0.9903 ± 0.009 over the test set at size of 1500 frames and even managed to find calcifications that were not recognized manually by the physician. For the best knowledge of the authors our algorithm achieves high accuracy which was never achieved in the past.


Assuntos
Doença das Coronárias/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Calcificação Vascular/diagnóstico por imagem , Algoritmos , Calcinose/diagnóstico por imagem , Calcinose/metabolismo , Meios de Contraste , Angiografia Coronária/métodos , Doença da Artéria Coronariana/diagnóstico por imagem , Vasos Coronários/diagnóstico por imagem , Bases de Dados Factuais , Aprendizado Profundo , Coração/diagnóstico por imagem , Humanos , Aprendizado de Máquina , Tomografia de Coerência Óptica/métodos
10.
Nat Commun ; 12(1): 2344, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879794

RESUMO

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (-) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.


Assuntos
Doenças Transmissíveis/genética , Doenças Transmissíveis/metabolismo , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Fotoquímica/métodos , RNA/química , RNA/metabolismo , Calcinose/genética , Calcinose/metabolismo , Cistos do Sistema Nervoso Central/genética , Cistos do Sistema Nervoso Central/metabolismo , Reagentes para Ligações Cruzadas , Enterovirus Humano D/genética , Furocumarinas , Genoma Viral , Humanos , Leucoencefalopatias/genética , Leucoencefalopatias/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Processos Fotoquímicos , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA Viral/química , RNA Viral/genética
11.
Sci Rep ; 11(1): 7486, 2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33820929

RESUMO

Fetuin-A is an inhibitor of ectopic calcification that is expressed mainly in hepatocytes and is secreted into the circulation after posttranslational processing, including glycosylation and phosphorylation. The molecular weight (MW) of fully modified fetuin-A (FM-fetuin-A) is approximately 60 kDa in an immunoblot, which is much higher than the estimated MW by amino acid sequence. Under conditions of calcification stress such as advanced stage chronic kidney disease, fetuin-A prevents calcification by forming colloidal complexes, which are referred to as calciprotein particles (CPP). Since the significance of CPP in this process is unclear, we investigated the effect of synthetic secondary CPP on the level of FM-fetuin-A in HepG2 cells. Secondary CPP increased the level of FM-fetuin-A in dose- and time-dependent manners, but did not affect expression of mRNA for fetuin-A. Treatment with O- and/or N-glycosidase caused a shift of the 60 kDa band of FM-fetuin-A to a lower MW. Preincubation with brefeldin A, an inhibitor of transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus, completely blocked the secondary CPP-induced increase in FM-fetuin-A. Treatment with BAPTA-AM, an intracellular calcium chelating agent, also inhibited the CPP-induced increase in the FM-fetuin-A level. Secondary CPP accelerate posttranslational processing of fetuin-A in HepG2 cells.


Assuntos
Calcinose/metabolismo , Processamento de Proteína Pós-Traducional , alfa-2-Glicoproteína-HS/metabolismo , Brefeldina A/farmacologia , Cálcio/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Células Hep G2 , Humanos , Fosforilação , Fosfosserina/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , alfa-2-Glicoproteína-HS/genética
12.
Sci Rep ; 11(1): 8271, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859255

RESUMO

Spontaneous mineralization of the nucleus pulposus (NP) has been observed in cases of intervertebral disc degeneration (IDD). Inflammatory cytokines have been implicated in mineralization of multiple tissues through their modulation of expression of factors that enable or inhibit mineralization, including TNAP, ANKH or ENPP1. This study examines the underlying factors leading to NP mineralization, focusing on the contribution of the inflammatory cytokine, TNF, to this pathologic event. We show that human and bovine primary NP cells express high levels of ANKH and ENPP1, and low or undetectable levels of TNAP. Bovine NPs transduced to express TNAP were capable of matrix mineralization, which was further enhanced by ANKH knockdown. TNF treatment or overexpression promoted a greater increase in mineralization of TNAP-expressing cells by downregulating the expression of ANKH and ENPP1 via NF-κB activation. The increased mineralization was accompanied by phenotypic changes that resemble chondrocyte hypertrophy, including increased RUNX2 and COL10A1 mRNA; mirroring the cellular alterations typical of samples from IDD patients. Disc organ explants injected with TNAP/TNF- or TNAP/shANKH-overexpressing cells showed increased mineral content inside the NP. Together, our results confirm interactions between TNF and downstream regulators of matrix mineralization in NP cells, providing evidence to suggest their participation in NP calcification during IDD.


Assuntos
Calcinose/genética , Calcinose/metabolismo , NF-kappa B/metabolismo , Núcleo Pulposo/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Bovinos , Células Cultivadas , Expressão Gênica/genética , Humanos , Mediadores da Inflamação/efeitos adversos , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , NF-kappa B/genética , Proteínas de Transporte de Fosfato/genética , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética
13.
Can J Cardiol ; 37(7): 1016-1026, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33677100

RESUMO

Aortic stenosis is one of the most common cardiovascular diseases in the world. Extensive work on the underlying pathophysiology responsible for calcific aortic valve disease and its progression to aortic stenosis has described a complex process involving inflammation, lipid deposition, mineralisation, and genetic factors such as elevated lipoprotein(a). With the advancement of gene silencing technology and development of novel therapeutic agents, we may now be closer than ever to having medical therapies that prevent, or at least slow the progression of aortic stenosis. In this review, we highlight the pathophysiology and risk factors of calcific aortic valve disease, along with current, potential, and emerging novel medical therapies. We also provide potential explanations for the failure of statin trials and suggest new avenues for research and new randomised trials in this area.


Assuntos
Estenose da Valva Aórtica , Valva Aórtica/patologia , Calcinose , Conduta do Tratamento Medicamentoso/tendências , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/prevenção & controle , Estenose da Valva Aórtica/terapia , Calcinose/etiologia , Calcinose/metabolismo , Calcinose/prevenção & controle , Calcinose/terapia , Progressão da Doença , Drogas em Investigação/farmacologia , Terapia Genética/métodos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia
14.
Ann Thorac Surg ; 112(6): 1962-1972, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-33545156

RESUMO

BACKGROUND: Calcium accumulation and fibrotic activities are principal mechanisms for calcific aortic valve disease (CAVD). Active complement products are observed in human stenotic aortic valves. Runt-related transcription factor 2 (Runx-2) is involved in tissue calcification. We hypothesized that complement upregulates Runx-2 to induce profibrogenic change in human aortic valve interstitial cells (AVICs). METHODS: AVICs were isolated from 6 normal and 6 CAVD donor valves. Cells were treated with complement cocktails. Profibrogenic activities and associated signaling molecules were analyzed by Western blot assay and collagen staining. RESULTS: Complement time and dose dependently enhanced profibrogenic activities in AVICs, and complement exposure also induced total collagen deposition in AVICs. Complement-induced profibrogenic responses were associated with increased Runx-2 expression and phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Genetic silencing of Runx-2 decreased both matrix metalloproteinase 9 (MMP-9) and collagen I levels. Pharmacological inhibition of ERK1/2 decreased complement-mediated MMP-9, collagen I, and Runx-2 expression as well as total collagen deposition in human AVICs. Further, treating AVICs with heat-deactivated complement resulted in reduced MMP-9, collagen I, and Runx-2 levels compared with active complement treatment. CONCLUSIONS: Complement induced profibrogenic activities in AVICs by activation of ERK1/2-mediated Runx-2 signaling pathways. This study demonstrates a potential role for complement-mediated CAVD pathogenesis, establishing a possible therapeutic target to limit CAVD progression.


Assuntos
Estenose da Valva Aórtica/genética , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Calcinose/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Idoso , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Western Blotting , Calcinose/metabolismo , Calcinose/patologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA/genética , Transdução de Sinais
15.
Theranostics ; 11(3): 1129-1146, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33391525

RESUMO

Rationale: Vascular microcalcification increases the risk of rupture of vulnerable atherosclerotic lesions. Inhibition of ERK1/2 reduces atherosclerosis in animal models while its role in vascular calcification and the underlying mechanisms remains incompletely understood. Methods: Levels of activated ERK1/2, DKK1, LRP6 and BMP2 in human calcific aortic valves were determined. ApoE deficient mice received ERK1/2 inhibitor (U0126) treatment, followed by determination of atherosclerosis, calcification and miR-126-3p production. C57BL/6J mice were used to determine the effect of U0126 on Vitamin D3 (VD3)-induced medial arterial calcification. HUVECs, HAECs and HASMCs were used to determine the effects of ERK1/2 inhibitor or siRNA on SMC calcification and the involved mechanisms. Results: We observed the calcification in human aortic valves was positively correlated to ERK1/2 activity. At cellular and animal levels, U0126 reduced intimal calcification in atherosclerotic lesions of high-fat diet-fed apoE deficient mice, medial arterial calcification in VD3-treated C57BL/6J mice, and calcification in cultured SMCs and arterial rings. The reduction of calcification was attributed to ERK1/2 inhibition-reduced expression of ALP, BMP2 and RUNX2 by activating DKK1 and LRP6 expression, and consequently inactivating both canonical and non-canonical Wnt signaling pathways in SMCs. Furthermore, we determined ERK1/2 inhibition activated miR-126-3p production by facilitating its maturation through activation of AMPKα-mediated p53 phosphorylation, and the activated miR-126-3p from ECs and SMCs played a key role in anti-vascular calcification actions of ERK1/2 inhibition. Conclusions: Our study demonstrates that activation of miR-126-3p production in ECs/SMCs and interactions between ECs and SMCs play an important role in reduction of vascular calcification by ERK1/2 inhibition.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , MicroRNAs/metabolismo , Transdução de Sinais/fisiologia , Calcificação Vascular/metabolismo , Animais , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Calcinose/metabolismo , Calcinose/patologia , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Calcificação Vascular/patologia
16.
J Pharmacol Sci ; 145(2): 213-221, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33451756

RESUMO

Since aortic valve stenosis (AVS) is the most frequent and serious valvular heart disease in the elderly, and is accompanied by irreversible valve calcification, medicinal prevention of AVS is important. Although we recently demonstrated that human aortic valve interstitial cells (HAVICs) obtained from patients with AVS were highly sensitive to ectopic calcification stimulation, the cell types contributing to calcification are unknown. We aimed to immunocytochemically characterize HAVICs and identify their contribution to valve calcification. HAVICs were isolated from patients with AVS and cultured on non-coated dishes. Immunocytochemical features and HAVIC differentiation were analyzed in passage 1 (P1). The immunohistochemical features of the calcified aortic valve were analyzed. Most cultured P1 HAVICs were CD73-, CD90-, and CD105-positive, and CD45-and CD34-negative. HAVICs were vascular endothelial growth factor receptor 2 (VEGFR2)-positive; however, approximately half were α-smooth muscle actin (SMA)-positive, colonized, and easily differentiated into osteoblastic cells. Calcified aortic valve immunohistochemistry showed that all cells were positive for VEGFR2 and partly α-SMA. Further, VEGFR2-positive cells were more sensitive to tumor necrosis factor-α-induced ectopic calcification with or without α-SMA positivity. We conclude that HAVICs obtained from patients with AVS are VEGFR2-positive undifferentiated mesenchymal cells and may contribute to aortic valve ectopic calcification.


Assuntos
Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Valva Aórtica/citologia , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Calcinose/metabolismo , Calcinose/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Actinas/metabolismo , Idoso , Estenose da Valva Aórtica/etiologia , Calcinose/etiologia , Células Cultivadas , Feminino , Humanos , Masculino , Fator de Necrose Tumoral alfa/metabolismo
17.
FASEB J ; 35(2): e21288, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33484482

RESUMO

Transient-receptor-potential cation channel, subfamily M, member 3 (TRPM3) serves as a polymodal calcium sensor in diverse mammalian cell-types. Mutation of the human TRPM3 gene (TRPM3) has been linked with inherited forms of early-onset cataract with or without other eye abnormalities. Here, we have characterized the ocular phenotypes of germline "knock-in" mice that harbor a human cataract-associated isoleucine-to-methionine mutation (p.I65M) in TRPM3 (Trpm3-mutant) compared with germline "knock-out" mice that functionally lack TRPM3 (Trpm3-null). Despite strong expression of Trpm3 in lens epithelial cells, neither heterozygous (Trpm3+/- ) nor homozygous (Trpm3-/- ) Trpm3-null mice developed cataract; however, the latter exhibited a mild impairment of lens growth. In contrast, homozygous Trpm3-M/M mutants developed severe, progressive, anterior pyramid-like cataract with microphthalmia, whereas heterozygous Trpm3-I/M and hemizygous Trpm3-M/- mutants developed anterior pyramidal cataract with delayed onset and progression-consistent with a semi-dominant lens phenotype. Histochemical staining revealed abnormal accumulation of calcium phosphate-like deposits and collagen fibrils in Trpm3-mutant lenses and immunoblotting detected increased αII-spectrin cleavage products consistent with calpain hyper-activation. Immunofluorescent confocal microscopy of Trpm3-M/M mutant lenses revealed fiber cell membrane degeneration that was accompanied by accumulation of alpha-smooth muscle actin positive (α-SMA+ve) myofibroblast-like cells and macrosialin positive (CD68+ve) macrophage-like cells. Collectively, our mouse model data support an ocular disease association for TRPM3 in humans and suggest that (1) Trpm3 deficiency impaired lens growth but not lens transparency and (2) Trpm3 dysfunction resulted in progressive lens degeneration and calcification coupled with pro-fibrotic (α-SMA+ve) and immune (CD68+ve) cell responses.


Assuntos
Calcinose/metabolismo , Catarata/metabolismo , Canais de Cátion TRPM/metabolismo , Actinas/metabolismo , Animais , Calcinose/genética , Calcinose/patologia , Cálcio/metabolismo , Calpaína/metabolismo , Catarata/genética , Catarata/patologia , Colágeno/metabolismo , Fibrose , Heterozigoto , Cristalino/metabolismo , Cristalino/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Canais de Cátion TRPM/genética
18.
Nat Microbiol ; 6(4): 455-466, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33510473

RESUMO

The most frequent fetal birth defect associated with prenatal Zika virus (ZIKV) infection is brain calcification, which in turn may potentially affect neurological development in infants. Understanding the mechanism could inform the development of potential therapies against prenatal ZIKV brain calcification. In perivascular cells, bone morphogenetic protein (BMP) is an osteogenic factor that undergoes maturation to activate osteogenesis and calcification. Here, we show that ZIKV infection of cultivated primary human brain pericytes triggers BMP2 maturation, leading to osteogenic gene expression and calcification. We observed extensive calcification near ZIKV+ pericytes of fetal human brain specimens and in vertically transmitted ZIKV+ human signal transducer and activator of transcription 2-knockin mouse pup brains. ZIKV infection of primary pericytes stimulated BMP2 maturation, inducing osteogenic gene expression and calcification that were completely blocked by anti-BMP2/4 neutralizing antibody. Not only did ZIKV NS3 expression alone induce BMP2 maturation, osteogenic gene expression and calcification, but purified NS3 protease also effectively cleaved pro-BMP2 in vitro to generate biologically active mature BMP2. These findings highlight ZIKV-induced calcification where the NS3 protease subverts the BMP2-mediated osteogenic signalling pathway to trigger brain calcification.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Encéfalo/patologia , Calcinose/patologia , Feto/patologia , Serina Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Infecção por Zika virus/patologia , Zika virus/patogenicidade , Animais , Proteína Morfogenética Óssea 2/metabolismo , Encéfalo/metabolismo , Encéfalo/virologia , Calcinose/metabolismo , Calcinose/virologia , Cálcio/metabolismo , Células Cultivadas , Feto/virologia , Humanos , Transmissão Vertical de Doenças Infecciosas , Camundongos , Camundongos Transgênicos , Osteogênese/genética , Pericitos , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Zika virus/enzimologia , Infecção por Zika virus/metabolismo , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
19.
Nat Commun ; 12(1): 549, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483479

RESUMO

Intrauterine growth restriction (IUGR) is associated with reduced kidney size at birth, accelerated renal function decline, and increased risk for chronic kidney and cardiovascular diseases in adults. Precise mechanisms underlying fetal programming of adult diseases remain largely elusive and warrant extensive investigation. Setting up a mouse model of hypoxia-induced IUGR, fetal adaptations at mRNA, protein and cellular levels, and their long-term functional consequences are characterized, using the kidney as a readout. Here, we identify fetuin-A as an evolutionary conserved HIF target gene, and further investigate its role using fetuin-A KO animals and an adult model of ischemia-reperfusion injury. Beyond its role as systemic calcification inhibitor, fetuin-A emerges as a multifaceted protective factor that locally counteracts calcification, modulates macrophage polarization, and attenuates inflammation and fibrosis, thus preserving kidney function. Our study paves the way to therapeutic approaches mitigating mineral stress-induced inflammation and damage, principally applicable to all soft tissues.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Retardo do Crescimento Fetal/metabolismo , Rim/metabolismo , Traumatismo por Reperfusão/fisiopatologia , alfa-2-Glicoproteína-HS/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Calcinose/genética , Calcinose/metabolismo , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/genética , Fibrose , Hipóxia , Inflamação/genética , Inflamação/metabolismo , Rim/patologia , Ativação de Macrófagos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , alfa-2-Glicoproteína-HS/genética
20.
Am J Cardiol ; 140: 103-109, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33144156

RESUMO

In patients who underwent transcatheter aortic valve implantation (TAVI), vascular disease is associated with increased risk of mortality. Thoracic aortic calcification (TAC), an objective surrogate of vascular disease, could be a predictor of mortality after TAVI. We aimed to analyze the association between TAC burden and 1-year all-cause mortality in patients who underwent TAVI in a US population. From July 2015 through July 2017, a retrospective review of TAVI procedures was performed at Baylor Scott & White-The Heart Hospital, Plano, Texas. Patients were analyzed for comorbidities, cardiac risk factors, and 30-day and 1-year all-cause mortality. Restricted cubic splines analysis was used to define low, moderate, and high TAC categories. The association between TAC and survival was evaluated using unadjusted and adjusted Cox models. A total of 431 TAVI procedures were performed, of which TAC was measured in 374 (81%) patients. Median (interquartile range) age was 82 (77, 87) years, and 51% were male. Median (interquartile range) STS PROM was 5.6 (4.1, 8.2) %. Overall 30-day and 1-year all-cause mortality was 1% and 10%, respectively. TAC was categorized as low (<1.6 cm3), moderate (1.6 to 2.9 cm3), and high (>2.9 cm3). At 1 year, all-cause mortality was 16% in patients with high TAC compared with 6% in the low and moderate TAC categories (p = 0.008). Unadjusted and adjusted Cox regression analysis showed a significant increase in mortality for patients with high TAC compared with low TAC (hazard ratio 2.98, 95% confidence interval [1.34-6.63]), but not significant compared with moderate TAC group. TAC is a predictor of late mortality after TAVI. In conclusion, adding TAC to preoperative evaluation may provide an objective, reproducible, and potentially widely available tool that can help in shared decision-making.


Assuntos
Aorta Torácica/diagnóstico por imagem , Doenças da Aorta/diagnóstico , Estenose da Valva Aórtica/cirurgia , Calcinose/diagnóstico , Cálcio/metabolismo , Tomografia Computadorizada Multidetectores/métodos , Substituição da Valva Aórtica Transcateter/métodos , Idoso , Idoso de 80 Anos ou mais , Aorta Torácica/metabolismo , Doenças da Aorta/metabolismo , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/cirurgia , Estenose da Valva Aórtica/diagnóstico , Calcinose/epidemiologia , Calcinose/metabolismo , Feminino , Seguimentos , França/epidemiologia , Humanos , Masculino , Sistema de Registros , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida/tendências
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