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1.
Adv Exp Med Biol ; 1155: 391-406, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468417

RESUMO

Heat stress is an environmental factor that causes severe economic loss to the current intensive breeding industry and induces huge impact on the long-term growth in livestock and poultry industry. Many animal experiments confirmed that heat stress is a major cause of heat stroke death, which is due to severe damage to endothelial cells. In order to provide a theoretical basis for the treatment or mitigation of heat stress related diseases in broilers, the effect of taurine on injury and apoptosis of aortic endothelial cells in broilers under heat stress was investigated in the present study. Ten days healthy broilers were sacrificed, then aortic tissue was used to isolate and cultivate primary broiler aortic endothelial cells. The third to the fifth generations of cells were used in the experiment. The cells were randomly divided into five groups, including control group (C), heat stress group (HS), low taurine (HS+LTau) group, mild taurine (HS+MTau) group and high taurine (HS+HTau) group. Cells in all groups were cultivated for 24 h in cell incubator (37 °C, 5% CO2). Then the heat stress group cells were cultivated in a 43 °C thermostatic water bath for 6 h under heat stress, and then re-incubated under 37 °C for 1 h. The results showed that compared with the control group, expression levels of Bax, Caspase-9, Caspase-3, Cyt-c, P53 and other pro-apoptosis factors in HS groups were significantly increased (P < 0.05), while expression levels of anti-apoptosis factor Bcl-2 showed a significant decrease (P < 0.05). Compared with HS group, expression levels of Bcl-2 in endothelial cells were significantly increased by taurine administration (P < 0.05), while expression of Bax, Caspase-9, Caspase-3, Cyt-c and P53 were significantly increased by taurine (P < 0.05). In summary, the present data indicated that taurine could protect against injury and apoptosis of aortic endothelial cells under heat stress by inhibiting the activation of mitochondria-mediated apoptotic pathways.


Assuntos
Apoptose/efeitos dos fármacos , Galinhas , Células Endoteliais/efeitos dos fármacos , Resposta ao Choque Térmico , Taurina/farmacologia , Animais , Aorta/citologia , Células Cultivadas , Células Endoteliais/citologia , Distribuição Aleatória
2.
Carbohydr Polym ; 217: 152-159, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31079672

RESUMO

Composite biomaterials offer a new approach for engineering novel, minimally-invasive scaffolds with properties that can be modified for a range of soft tissue applications. In this study, a new way of controlling the gelation of alginate hydrogels using Ga-based glass particles is presented. Through a comprehensive analysis, it was shown that the setting time, mechanical strength, stiffness and degradation properties of this composite can all be tailored for various applications. Specifically, the hydrogel generated through using a glass particle, wherein toxic aluminium is replaced with biocompatible gallium, exhibited enhanced properties. The material's stiffness matches that of soft tissues, while it displays a slow and tuneable gelation rate, making it a suitable candidate for minimally-invasive intra-vascular injection. In addition, it was also found that this composite can be tailored to deliver ions into the local cellular environment without affecting platelet adhesion or compromising viability of vascular cells in vitro.


Assuntos
Alginatos/química , Materiais Biocompatíveis/química , Gálio/química , Vidro/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Alginatos/isolamento & purificação , Alginatos/toxicidade , Animais , Aorta/citologia , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/toxicidade , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Força Compressiva , Módulo de Elasticidade , Células Endoteliais/efeitos dos fármacos , Hidrogel de Polietilenoglicol-Dimetacrilato/síntese química , Hidrogel de Polietilenoglicol-Dimetacrilato/toxicidade , Miócitos de Músculo Liso/efeitos dos fármacos , Engenharia Tecidual/métodos , Tecidos Suporte/química
3.
J Toxicol Sci ; 44(5): 327-333, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31068538

RESUMO

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, and metal-binding protein that protects cells from the cytotoxic effects of heavy metals and reactive oxygen species. Previously, we found that transcriptional induction of endothelial MT-1A was mediated by not only the metal-regulatory transcription factor 1 (MTF-1)-metal responsive element (MRE) pathway but also the nuclear factor-erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile responsive element (ARE) pathway, whereas that of MT-2A was mediated only by the MTF-1-MRE pathway, using the organopnictogen compounds tris(pentafluorophenyl)stibane, tris(pentafluorophenyl)arsane, and tris(pentafluorophenyl)phosphane as molecular probes in vascular endothelial cells. In the present study, we investigated the binding sites of MTF-1 and Nrf2 in the promoter regions of MTs in cultured bovine aortic endothelial cells treated with these organopnictogen compounds. We propose potential mechanisms underlying transcriptional induction of endothelial MT isoforms. Specifically, both MRE activation by MTF-1 and that of ARE in the promoter region of the MT-2A gene by Nrf2 are involved in transcriptional induction of MT-1A, whereas only MRE activation by MTF-1 or other transcriptional factor(s) is required for transcriptional induction of MT-2A in vascular endothelial cells.


Assuntos
Células Endoteliais/efeitos dos fármacos , Metalotioneína/genética , Fosfinas/toxicidade , Animais , Aorta/citologia , Bovinos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Células Endoteliais/metabolismo , Fator 2 Relacionado a NF-E2/genética , Isoformas de Proteínas/genética , Fatores de Transcrição/genética , Transcrição Genética
4.
Cell Physiol Biochem ; 52(6): 1339-1360, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31050282

RESUMO

BACKGROUND/AIMS: Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration. METHODS: We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²âº mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis. RESULTS: We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²âº signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes. CONCLUSION: Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries.


Assuntos
Receptor Tipo 1 de Melanocortina/metabolismo , Aorta/citologia , Sinalização do Cálcio/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , AMP Cíclico/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Antígeno Ki-67/metabolismo , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Melanocortina/genética , alfa-MSH/farmacologia
5.
Int Heart J ; 60(3): 688-694, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31105154

RESUMO

The prevalence and extent of immunoglobulin G4 (IgG4)-positive cell infiltration were investigated in 282 surgical samples of aortic wall and aortic valve. Tissue infiltration of IgG4-positive cells was observed in 24 (17.3%) of 139 aortic valve samples and 46 (32%) of 143 aortic wall samples, and the condition of IgG4-positive cell infiltration > 30/hpf together with IgG4/CD138 ratio > 40% was observed in 2 (1.4%) of aortic valve samples and 14 (9.8%) of aortic wall samples. Among 275 patients, preoperative serum IgG4 level was available in 48 patients (50 samples), and it was > 135 mg/dL in only one patient. Of these 48 patients with serum IgG4 measurement, 29 patients had aortic valve stenosis and 12 had aortic aneurysm. Compared with 23 aortic stenosis patients without tissue infiltration of IgG4-positive cells in the aortic valve, six patients with IgG4-positive cell infiltration had a more prevalent smoking history (26% versus 83%) and borderline significantly higher serum IgG4 (median, 24.5 mg/dL versus 55.5 mg/dL), although either preoperative peak pressure gradient between left ventriculum and aorta or aortic valve area did not differ significantly between groups. Compared with six aortic aneurysm patients without tissue infiltration of IgG4-positive cells in the aortic wall, six patients with IgG4-positive cell infiltration had borderline significantly higher serum IgG4 (median, 28.9 mg/dL versus 68.2 mg/dL). The current study showed that tissue IgG4-positive infiltration is not a rare occurrence in the aortic stenosis and aortic aneurysm. Clinical significance of tissue IgG4-postive cell infiltration in these patients requires further investigation.


Assuntos
Aneurisma Aórtico/imunologia , Estenose da Valva Aórtica/imunologia , Doença Relacionada a Imunoglobulina G4/sangue , Imunoglobulina G/sangue , Plasmócitos/patologia , Idoso , Idoso de 80 Anos ou mais , Aorta/anatomia & histologia , Aorta/citologia , Aorta/diagnóstico por imagem , Aorta/cirurgia , Aneurisma Aórtico/sangue , Aneurisma Aórtico/patologia , Valva Aórtica/citologia , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/cirurgia , Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/patologia , Ecocardiografia/métodos , Feminino , Humanos , Doença Relacionada a Imunoglobulina G4/imunologia , Doença Relacionada a Imunoglobulina G4/patologia , Masculino , Pessoa de Meia-Idade , Plasmócitos/imunologia , Período Pré-Operatório , Estudos Retrospectivos
6.
J Ethnopharmacol ; 237: 149-158, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-30880260

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Alpinia zerumbet (FAZ), a dry and ripe fruit of Alpinia zerumbet (Pers.) Burtt. et Smith, is widely used as a spice to treat cardiovascular diseases in clinic as a miao folk medicine in Guizhou Province of China. Essential oil extracted from FAZ (EOFAZ) is the key bioactive ingredients. AIM OF THE STUDY: This study aimed to examine the effects and mechanisms of EOFAZ on lipopolysaccharide (LPS)-induced endothelial cell injury, inflammation and apoptosis in vitro and in vivo. MATERIALS AND METHODS: For the in vitro study, LPS-treated human aortic endothelial cells were used to perform PCR, western blot analysis and immunofluorescence. For the in vivo study, male mouse were divided into four groups, vehicle control group and LPS group received 0.5% Tween-80 in saline; and two EOFAZ groups receive different dose of EOFAZ (90 mg kg -1·day-1, 180 mg kg -1·day-1) respectively. Each group was fed for 7 days by intragastrical administration at daily base. Then, except vehicle control group received saline, mice in other three groups were administered with LPS (1 mg kg -1, dissolved in saline) by intraperitoneal injection. 24 h later, Aorta tissue was collected and frozen immediately in liquid N2, stored at -80 °C for western blot analysis. RESULTS: We found that EOFAZ completely prevented LPS-induced HAEC activation and inflammation in vitro and in vivo, as assessed by expression of endothelial adhesion molecules, ICAM-1 and VCAM-1. Similarly, EOFAZ significantly blunted LPS-induced endothelial injury, as tested by MTT assay, LDH release and caspase-3 activation. We further demonstrated that TLR4-dependent NF-κB signaling may be involved in the process. CONCLUSION: EOFAZ protected against LPS-induced endothelial cell injury and inflammation likely via inhibition of TLR4-dependent NF-κB signaling.


Assuntos
Alpinia , Anti-Inflamatórios/farmacologia , Células Endoteliais/efeitos dos fármacos , Óleos Voláteis/farmacologia , Animais , Aorta/citologia , Células Cultivadas , Células Endoteliais/metabolismo , Frutas , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos , Masculino , Camundongos , NF-kappa B/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
7.
Biochem Soc Trans ; 47(2): 591-601, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30902922

RESUMO

The first definitive blood cells during embryogenesis are derived from endothelial cells in a highly conserved process known as endothelial-to-haematopoietic transition (EHT). This conversion involves activation of a haematopoietic transcriptional programme in a subset of endothelial cells in the major vasculature of the embryo, followed by major morphological changes that result in transitioning cells rounding up, breaking the tight junctions to neighbouring endothelial cells and adopting a haematopoietic fate. The whole process is co-ordinated by a complex interplay of key transcription factors and signalling pathways, with additional input from surrounding tissues. Diverse model systems, including mouse, chick and zebrafish embryos as well as differentiation of pluripotent cells in vitro, have contributed to the elucidation of the details of the EHT, which was greatly accelerated in recent years by sophisticated live imaging techniques and advances in transcriptional profiling, such as single-cell RNA-Seq. A detailed knowledge of these developmental events is required in order to be able to apply it to the generation of haematopoietic stem cells from pluripotent stem cells in vitro - an achievement which is of obvious clinical importance. The aim of this review is to summarise the latest findings and describe how these may have contributed towards achieving this goal.


Assuntos
Endotélio/citologia , Animais , Aorta/citologia , Aorta/metabolismo , Endotélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Gônadas/citologia , Gônadas/metabolismo , Hematopoese/genética , Hematopoese/fisiologia , Humanos , Mesonefro/citologia , Mesonefro/metabolismo
8.
Emerg Microbes Infect ; 8(1): 232-241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866776

RESUMO

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused the public health emergency. Recently, we have proved a novel small animal tree shrew was susceptive to ZIKV infection and presented the most common rash symptoms as ZIKV patients. Here we further cultured the primary cells from different tissues of this animal to determine the tissue tropism of ZIKV infection in vitro. The results showed that the primary cells from tree shrew kidney, lung, liver, skin and aorta were permissive to ZIKV infection and could support viral replication by the detection of viral specific RNA intra- and extra-cells. In comparing, the skin fibroblast and vascular endothelial cells were highly permissive to ZIKV infection with high releasing of active virus particles in supernatants proved by its infectivity in established neonatal mouse model. The expressions of ZIKV envelop and nonstructural protein-1, and the effects and strong immune response of primary tree shrew cells were also detected followed by ZIKV infection. These findings provide powerful in vitro cell-level evidence to support tree shrew as animal model of ZIKV infection and may help to explain the rash manifestations in vivo.


Assuntos
Modelos Animais de Doenças , Tupaiidae/virologia , Infecção por Zika virus/virologia , Zika virus/patogenicidade , Animais , Aorta/citologia , Aorta/virologia , Células Cultivadas , Cercopithecus aethiops , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Rim/citologia , Rim/virologia , Fígado/citologia , Fígado/virologia , Pulmão/citologia , Pulmão/virologia , Pele/citologia , Pele/virologia , Células Vero , Replicação Viral
9.
Int J Mol Sci ; 20(6)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884801

RESUMO

The small GTPase Rho and its downstream effector, Rho-kinase (ROCK), regulate various cellular functions, including organization of the actin cytoskeleton, cell adhesion and migration. A pro-inflammatory lipid mediator, lysophosphatidic acid (LPA), is a potent activator of the Rho/ROCK signalling pathway and has been shown to induce the expression of chemokines and cell adhesion molecules (CAMs). In the present study, we aimed to elucidate the precise mechanism by which ROCK regulates LPA-induced expressions and functions of chemokines and CAMs. We observed that ROCK blockade reduced LPA-induced phosphorylation of IκBα and inhibited NF-κB RelA/p65 phosphorylation, leading to attenuation of RelA/p65 nuclear translocation. Furthermore, small interfering RNA-mediated ROCK isoform knockdown experiments revealed that LPA induces the expression of monocyte chemoattractant protein-1 (MCP-1) and E-selectin via ROCK2 in human aortic endothelial cells (HAECs). Importantly, we found that ROCK2 but not ROCK1 controls LPA-induced monocytic migration and monocyte adhesion toward endothelial cells. These findings demonstrate that ROCK2 is a key regulator of endothelial inflammation. We conclude that targeting endothelial ROCK2 is potentially effective in attenuation of atherosclerosis.


Assuntos
Aterosclerose/genética , Células Endoteliais/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Quinases Associadas a rho/genética , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Aorta/citologia , Aorta/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Adesão Celular/genética , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Selectina E/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/genética , Monócitos/efeitos dos fármacos , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/genética , Quinases Associadas a rho/metabolismo
10.
Eur J Pharmacol ; 853: 169-183, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30768980

RESUMO

The aim of this study was to investigate the relaxation effect of farrerol on rat aortic vascular smooth muscle cells (VSMCs) and its underlying mechanism. VSMCs were cultured primarily and were used to examine the relaxation effect of farrerol. Cells surface and length were measured by dynamic observation, or by rhodamine-phalloidin labeling and hematoxylin-eosin staining. Cells contractive activity were tested using collagen gel contraction assay. The [Ca2+]in was measured with molecular probe fluo-4-AM. The mRNA and protein expression of regulatory proteins for contraction were measured. In addition, rat aortic VSMCs were transfected with lentivirus-mediated α1D-adrenoceptor gene-shRNA, then the effect of farrerol were detected by the above experimental methods. The results revealed that 10 µΜ AngⅡ promoted cell contraction, increased [Ca2+]in and enhanced collagen contraction in rat aortic VSMCs. 10 µΜ AngⅡ not only increased expression of myosin light chain kinase (MLCK) and smooth muscle protein 22α (SM22α), but also increased phosphorylation level of myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1). The above effects induced by AngⅡ could be significantly inhibited by farrerol in a concentration dependent manner. When the cells were transfected with lentivirus mediated α1D-adrenoceptor gene-shRNA, the effects of farrerol on changes induced by AngⅡ in rat aortic VSMCs were markedly reversed. In conclusion, farrerol could produce relaxtion effect in rat aortic VSMCs precontracted by 10 µΜ AngⅡ, which was involved in downregulation expression of MLCK and SM22α, and inhibition phosphorylation level of MYPT1 and MLC via activating α1D-adrenoceptor gene.


Assuntos
Anti-Hipertensivos/farmacologia , Aorta/efeitos dos fármacos , Aorta/fisiologia , Cromonas/farmacologia , Músculo Liso Vascular/citologia , Receptores Adrenérgicos alfa 1/metabolismo , Vasodilatação/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Aorta/citologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos
11.
J Pharm Pharmacol ; 71(6): 988-995, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30809816

RESUMO

OBJECTIVES: TGF-ß through hyperelongation of glycosaminoglycan (GAG) chains leads to binding of low-density lipoproteins to the proteoglycans. The vasoactive peptide, endothelin-1 (ET-1), plays a key role in the development of atherosclerosis. This study addressed the question whether ET-1 by activating the Rho kinase and cytoskeletal rearrangement can transactivate the TGF-ß receptor leading to phosphorylation of the transcription factor Smad2 and increased expression of the GAG chain synthesizing enzyme such as chondroitin synthase-1 (CHSY-1) in bovine aortic endothelial cells (BAECs). METHODS: In this study, intermediates in ET-1-induced Smad2C phosphorylation and the protein level of CHSY-1 were identified and quantified by Western blotting. KEY FINDINGS: Endothelin-1 caused time-dependent phosphorylation of Smad2C which was inhibited in the presence of the endothelin B receptor antagonist, BQ788. The response to ET-1 was inhibited by the Rho/ROCK kinase antagonist, Y27632 and by cytochalasin D, an inhibitor of actin polymerization but the ET-1-mediated pSmad2C was not inhibited by the matrix metalloproteinase (MMP) inhibitor, GM6001. ET-1 increased CHSY-1 protein level, which was inhibited in the presence of BQ788, cytochalasin D and Y27632. CONCLUSIONS: Endothelin-1 signalling via the ETB receptor utilizes cytoskeletal rearrangement and Rho kinase but not MMPs leading to TßRI transactivation signalling and phosphorylation of Smad2C and through this pathway increased the level of CHSY-1.


Assuntos
Células Endoteliais/metabolismo , Endotelina-1/metabolismo , N-Acetilgalactosaminiltransferases/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Amidas/farmacologia , Animais , Aorta/citologia , Western Blotting , Bovinos , Células Cultivadas , Citocalasina D/farmacologia , Oligopeptídeos/farmacologia , Fosforilação/fisiologia , Piperidinas/farmacologia , Piridinas/farmacologia , Receptor de Endotelina B/efeitos dos fármacos , Receptor de Endotelina B/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Tempo , Ativação Transcricional/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Quinases Associadas a rho/metabolismo
12.
J Immunol ; 202(3): 694-703, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30598511

RESUMO

Many nonlymphoid cell types express at least two, if not all three, subunits of the IL-2R; although, compared with lymphocytes, relatively little is known about how IL-2 affects the function of nonlymphoid cells. The limited information available suggests that IL-2 has a substantial impact on cells such as gastrointestinal epithelial cells, endothelial cells, and fibroblasts. In a previous report from our laboratory, we noted that IL-2 and IL-2Rß-deficient mice lose smooth muscle cells over time, eventually resulting in aneurysmal aortas and ectatic esophagi. This finding, combined with our work showing that IL-2 surrounds vascular smooth muscle cells by association with perlecan, led us to ask whether vascular smooth muscle cells express an IL-2R. Toward this end, we reported the expression of IL-2Rß on human and murine vascular smooth muscle cells. We now report that vascular smooth muscle cells express all three subunits of the IL-2R, and that expression of IL-2Rα varies with vascular smooth muscle cell phenotype. Furthermore, we show that, through a functional IL-2R, IL-2 initiates signaling pathways and impacts vascular smooth muscle cell function. Finally, we demonstrate that IL-2 expression increases upon initiation of conditions that promote intimal hyperplasia, suggesting a mechanism by which the IL-2/IL-2R system may impact this widespread vascular pathology.


Assuntos
Subunidade alfa de Receptor de Interleucina-2/genética , Interleucina-2/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Aorta/citologia , Artérias Carótidas/metabolismo , Artérias Carótidas/transplante , Movimento Celular , Proliferação de Células , Células Cultivadas , Humanos , Hiperplasia/metabolismo , Interleucina-2/farmacologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Células Jurkat , Camundongos , Músculo Liso Vascular/citologia , Coelhos , Transdução de Sinais
13.
Toxicol In Vitro ; 56: 156-162, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30677511

RESUMO

Aortic aneurysm (AA) is a common disease that is associated with the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). VSMC cells can be directly exposed to environmental endocrine disruptors (EEDs) such as bisphenol A (BPA). However, the effects of BPA on the biological functions of VSMC are not well studied. Our present study found that nanomolar bisphenol A (BPA) can increase the proliferation of VSMC, which was further evidenced by the results that BPA can increase the expression of proliferating cell nuclear antigen (PCNA). The expression of Angiotensin II (Ang II), which is associated with the proliferation and inflammation of VSMCs, was upregulated after BPA treatment. While losartan, an Ang II receptor antagonist, can attenuate BPA induced cell proliferation, suggesting the essential role of Ang II in BPA induced cell proliferation. BPA treatment can also increase the expression of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) via an Ang II dependent manner. Both estrogen receptor α (ERα) and G protein-coupled estrogen receptor (GPER) can be detected in VSMCs. Blocking the functions of ERα and GPER by their specific inhibitors can attenuate the BPA induced proliferation of VSMCs and expression of Ang II. Consistently, BPA induced expression of TNFα and IL-6 was also attenuated by inhibitors of ERα and GPER. BPA can increase the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) though GPER. The inhibitor of ERK1/2 can abolish BPA induced upregulation of Ang II. Collectively, our present study suggested that BPA can trigger the proliferation of VSMCs via both ERα and GPER dependent manners.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Fenóis/toxicidade , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Angiotensina II/genética , Angiotensina II/metabolismo , Animais , Aorta/citologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citocinas/genética , Humanos , Miócitos de Músculo Liso/metabolismo , Ratos
14.
Biochem Biophys Res Commun ; 509(1): 89-95, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30579596

RESUMO

Glycosaminoglycans (GAGs) play an integral role in low-density lipoprotein (LDL) retention in the vascular intimal layer and have emerged as attractive therapeutic targets for atherosclerosis. GAG biosynthesis involves the cooperation of numerous enzymes. Chondroitin sulfate N-acetylgalactosaminyltransferase-2 (ChGn-2) is a vital Golgi transferase that participates in enzymatic elongation of GAGs. Here, we investigated the effects of ChGn-2 gene deletion on the development of atherosclerosis. Partial carotid artery ligation was performed on ChGn-2-/-/LDLr-/- and ChGn-2+/+/LDLr-/- mice to induce diffuse intimal thickening (DIT). Aortic smooth muscle cells (ASMCs) were isolated to investigate cellular LDL binding and migration. Histological analysis of human coronary artery sections revealed that ChGn-2 was expressed in early and advanced atherosclerotic lesions. Deletion of the ChGn-2 gene significantly reduced LDL retention in the DIT mouse model. Furthermore, LDL binding, visualized using rhodamine-labeled LDLs, was dramatically reduced. Interestingly, a functional assay of ASMCs prepared from ChGn-2-/- mice displayed abrogation of platelet-derived growth factor (PDGF)-mediated migration via reduced PDGF receptor phosphorylation. Taken together, these findings indicate that ChGn-2 is functionally involved in the progression of atherosclerosis both in its early and advanced stages. Therefore, ChGn-2 may serve as a plausible target to treat atherosclerotic-related diseases in the future.


Assuntos
Aorta/patologia , Aterosclerose/patologia , Lipoproteínas/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Adulto , Idoso , Animais , Aorta/citologia , Aorta/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Movimento Celular , Células Cultivadas , Deleção de Genes , Humanos , Lipoproteínas/análise , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , N-Acetilgalactosaminiltransferases/genética , Fosforilação , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Adulto Jovem
15.
Tissue Eng Part A ; 25(5-6): 399-415, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30582419

RESUMO

IMPACT STATEMENT: The generation of a small-caliber arterial graft, utilizing a large vessel of a small animal, such as the aorta of the rat or rabbit, for clinical use in the peripheral arterial tree, can widen the options for arterial prostheses. This in vivo study demonstrated the ability of the decellularization protocol that was used to produce a noncytotoxic acellular small-caliber arterial graft, with sufficient biomechanical and biological integrity to withstand the demanding flow and pressure environment of the rat aorta. This work also demonstrated the superiority of the decellularized homograft over its intact counterpart, in terms of lower immunogenicity.


Assuntos
Aorta/citologia , Aorta/imunologia , Materiais Biocompatíveis/farmacologia , Animais , Antígenos CD/metabolismo , Aorta/efeitos dos fármacos , Fenômenos Biomecânicos , Imuno-Histoquímica , Masculino , Modelos Animais , Ratos , Transplante Homólogo
16.
Chem Phys Lipids ; 218: 149-157, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30582896

RESUMO

Liposomal delivery systems (LDSs) have been at the forefront of medicinal nanotechnology for over three decades. Increasing LDS association to target cells and cargo delivery is crucial to bolstering overall nanodrug efficacy. Our laboratory aims to develop LDSs for molecular therapeutics aimed at vascular pathology. We have previously established a liposome platform that is an effective delivery system for RNA interference in vascular cell types by using polyethylene glycol (PEG) decorated liposomes bearing an octa-arginine (R8) cell penetrating peptide (CPP). Further tailoring liposome membranes to mimic vascular cell membrane lipid constituents may be a promising strategy for increasing cargo delivery. Here we aimed to develop liposomal formulations that could make use of diacylglycerol (DAG) and phosphatidylserine (PS), naturally occurring lipid species that are known to influence vascular cell function, as a facile and efficient means to increase nanodrug efficacy without compromising clinical viability. We investigated the ability of DAG and PS to amplify the cellular uptake of our previously established LDS platform loaded with small interfering ribonucleic acid (siRNA) cargo. Cellular fluorescence microscopy experiments were performed in conjunction with quantitative cell association assays and cytotoxicity assays to analyze the effect of DAG/PS on the differential delivery of fluorescently-tagged liposomes to vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) and on liposomal-mediated toxicity. In these studies, significant, dose-dependent increases in association to target cells were observed, as well as cell-type specific effects on cell viability. The stability and encapsulation-efficiency of the DAG/PS-modified LDSs were analyzed by standard nanoparticle characterization methods, and siRNA transfection efficacy was quantified to gauge delivery potential as a function of DAG/PS modification. Our results suggest that the signaling lipids tested here imbue our LDS architectures with increased therapeutic potential, without compromising stability, encapsulation efficiency, or biocompatibility, thus presenting a natural strategy to increase nanodrug efficacy and specificity.


Assuntos
Diglicerídeos/química , Sistemas de Liberação de Medicamentos , Células Endoteliais/química , Músculo Liso Vascular/química , Nanopartículas/química , Fosfatidilserinas/química , RNA Interferente Pequeno/farmacologia , Aorta/citologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Portadores de Fármacos/química , Humanos , Lipossomos/química , Estrutura Molecular , RNA Interferente Pequeno/química , Relação Estrutura-Atividade
17.
J Thorac Cardiovasc Surg ; 157(1): 109-119.e2, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30528439

RESUMO

OBJECTIVES: Fluoroquinolone (FQ) antibiotics are associated with adverse aortic clinical events. We assessed human aortic myofibroblast-mediated extracellular matrix (ECM) dysregulation as a possible cellular mechanism underlying FQ-associated aortopathy. METHODS: Human aortic myofibroblasts were isolated from patients with aortopathy undergoing elective ascending aortic resection (N = 9). The capacity for extracellular matrix degradation in cells exposed to FQ was assessed by multiplex analysis of secreted matrix metalloproteinases relative to tissue inhibitors of matrix metalloproteinases (TIMPs). Direct evaluation of extracellular matrix degradation was investigated in human aortic cells using a 3-dimensional gelatin-fluorescein isothiocyanate fluorescence microgel assay. Aortic cellular collagen-1 expression following FQ exposure was determined by immunoblotting and immunofluorescent staining. Cell apoptosis, necrosis, and metabolic viability was determined by annexin-V, propidium iodide staining, and water-soluble tetrazolium salt (WST1) assay. RESULTS: FQ exposure significantly decreased aortic cell TIMP-1 (P = .004) and TIMP-2 (P = .0004) protein expression compared with vehicle control. The ratio of matrix metalloproteinase-9/TIMP-2 was increased suggesting an increased capacity for extracellular matrix degradation (P = .01). In collagen gels, we show a trend toward increased aortic myofibroblast-mediated collagen fiber degradation with FQ exposure (P = .09). Similarly, FQ exposure attenuated collagen-1 expression as assessed by immunoblotting (P = .002) and immunofluorescence (P = .02). Cell apoptosis, necrosis, and metabolic viability was not significantly influenced by FQ exposure. CONCLUSIONS: For the first time, we document a putative mechanism underlying FQ-associated aortopathy whereby decreased TIMP expression with impaired compensatory collagen-1 expression results in human aortic myofibroblast-mediated extracellular matrix dysregulation. These novel data may provide a cellular and molecular mechanism to explain the established clinical association between FQ exposure and acute aortic events.


Assuntos
Aorta/citologia , Doenças da Aorta/induzido quimicamente , Matriz Extracelular/efeitos dos fármacos , Fluoroquinolonas/efeitos adversos , Miofibroblastos/efeitos dos fármacos , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Doenças da Aorta/fisiopatologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular/fisiologia , Feminino , Imunofluorescência , Humanos , Immunoblotting , Masculino , Pessoa de Meia-Idade , Miofibroblastos/fisiologia , Inibidores Teciduais de Metaloproteinases/antagonistas & inibidores
18.
Am J Physiol Cell Physiol ; 316(2): C210-C222, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30566394

RESUMO

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Specifically, endothelial apoptosis plays pivotal roles in atherosclerosis whereas prevention of endothelial apoptosis is a prerequisite for neovascularization in tumors and metastasis. Endothelial biology is intertwined with the composition of subendothelial basement membrane proteins. Apoptosis was induced by addition of tumor necrosis factor-α to cycloheximide-sensitized endothelial cells. Cells were either grown on polystyrene culture plates or on plates precoated with healthy basement membrane proteins (collagen IV, fibronectin, or laminin) or collagen I. Our results reveal that proteins of healthy basement membrane alleviate cytokine-induced apoptosis whereas precoating with collagen type I had no significant effect on apoptosis by addition of tumor necrosis factor-α to cycloheximide-sensitized endothelial cells compared with cells cultured on uncoated plates. Yet, treatment with transforming growth factor-ß1 significantly reduced the rate of apoptosis endothelial cells grown on collagen I. Detailed analysis reveals differences in intracellular signaling pathways for each of the basement membrane proteins studied. We provide additional insights into the importance of basement membrane proteins and the respective cytokine milieu on endothelial biology. Exploring outside-in signaling by basement membrane proteins may constitute an interesting target to restore vascular function and prevent complications in the atherosclerotic cascade.


Assuntos
Aorta/metabolismo , Apoptose/fisiologia , Membrana Basal/metabolismo , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Aorta/citologia , Membrana Basal/citologia , Células Cultivadas , Vasos Coronários/citologia , Matriz Extracelular/metabolismo , Humanos
19.
Int J Mol Sci ; 19(12)2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30563079

RESUMO

Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound 991 and AICAR had no substantial inhibitory effect on insulin-stimulated Akt phosphorylation in ECs. Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Aorta/enzimologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pironas/farmacologia , Tiofenos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Aorta/citologia , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo
20.
Int J Nanomedicine ; 13: 8037-8049, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30568444

RESUMO

Purpose: To evaluate the adverse vascular effects of nanoparticles (NPs) in vitro, extensive studies have investigated the toxicity of NPs on endothelial cells, but the knowledge of potential toxicity on human smooth-muscle cells (SMCs) is currently limited. Methods: This study compared the toxicity of TiO2, ZnO, and Ag NPs to human aortic SMCs. Results: Only ZnO NPs significantly induced cytotoxicity, accompanied by increased intracellular reactive oxygen species, Zn ions, and endoplasmic reticulum stress biomarkers (DDIT3 expression and p-Chop proteins). All the NPs significantly promoted the release of soluble VCAM1 and soluble sICAM1, but not IL6, which suggested that metal-based NPs might promote inflammatory responses. Furthermore, KLF4 expression (a transcription factor for SMC-phenotype switch) was significantly induced by TiO2 NPs and modestly by ZnO NPs, but the expression of CD68 remained unaltered. Conclusion: Our data indicated that ZnO NPs were more cytotoxic to human aortic SMCs than TiO2 and Ag NPs at the same mass concentrations, which might have been associated with intracellular reactive oxygen species, Zn ions, and endoplasmic reticulum stress.


Assuntos
Aorta/citologia , Nanopartículas Metálicas/toxicidade , Miócitos de Músculo Liso/citologia , Prata/toxicidade , Titânio/toxicidade , Óxido de Zinco/toxicidade , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Endocitose , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Íons , Fatores de Transcrição Kruppel-Like/metabolismo , Nanopartículas Metálicas/ultraestrutura , Modelos Biológicos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo
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