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1.
Nat Biomed Eng ; 5(8): 847-863, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34385693

RESUMO

The therapeutic efficacy of stem cells transplanted into an ischaemic brain depends primarily on the responses of the neurovascular unit. Here, we report the development and applicability of a functional neurovascular unit on a microfluidic chip as a microphysiological model of ischaemic stroke that recapitulates the function of the blood-brain barrier as well as interactions between therapeutic stem cells and host cells (human brain microvascular endothelial cells, pericytes, astrocytes, microglia and neurons). We used the model to track the infiltration of a number of candidate stem cells and to characterize the expression levels of genes associated with post-stroke pathologies. We observed that each type of stem cell showed unique neurorestorative effects, primarily by supporting endogenous recovery rather than through direct cell replacement, and that the recovery of synaptic activities is correlated with the recovery of the structural and functional integrity of the neurovascular unit rather than with the regeneration of neurons.


Assuntos
AVC Isquêmico/terapia , Dispositivos Lab-On-A-Chip , Transplante de Células-Tronco , Astrócitos/citologia , Astrócitos/metabolismo , Barreira Hematoencefálica/química , Barreira Hematoencefálica/metabolismo , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Microglia/citologia , Microglia/metabolismo , Microvasos/citologia , Modelos Biológicos , Neurônios/citologia , Neurônios/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-34348917

RESUMO

INTRODUCTION: This study aims to determine whether high glucose condition and dynamic O-linked N-acetylglucosamine (O-GlcNAc) modification can promote the proliferation and migration of human retinal microvascular endothelial cells (HRMECs) and whether Runt-related transcription factor 1 (RUNX1) could mediate the glucose and O-GlcNAc-driven proliferation and migration of HRMECs. RESEARCH DESIGN AND METHODS: Western blot analysis was used to detect the O-GlcNAc modification level and RUNX1 level in cells and retina tissues, cell growth was studied by cell counting kit-8 assay, cell proliferation was detected by immunofluorescence staining. Then, cell migration and tube formation were investigated by scratch-wound assay, Transwell assay, and tube-forming assay. The changes of retinal structure were detected by H&E staining. The O-GlcNAc modification of RUNX1 was detected by immunoprecipitation. RESULTS: High glucose increases pan-cellular O-GlcNAc modification and the proliferation and migration of HRMECs. Hence, O-GlcNAc modification is critical for the proliferation and migration of HRMECs. RUNX1 mediates the glucose and O-GlcNAc-driven proliferation and migration in HRMECs. RUNX1 can be modified by O-GlcNAc, and that the modification is enhanced in a high glucose environment. CONCLUSIONS: The present study reveals that high glucose condition directly affects retinal endothelial cells (EC) function, and O-GlcNAc modification is critical for the proliferation and migration of HRMECs, RUNX1 may take part in this mechanism, and maybe the function of RUNX1 is related to its O-GlcNAc modification level, which provides a new perspective for studying the mechanism of RUNX1 in diabetic retinopathy.


Assuntos
Acetilglucosamina , Subunidade alfa 2 de Fator de Ligação ao Core , Células Endoteliais/citologia , Movimento Celular , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Glucose , Humanos , Retina/citologia
3.
FASEB J ; 35(9): e21840, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34423881

RESUMO

With an aging world population, there is an increased risk of fracture and impaired healing. One contributing factor may be aging-associated decreases in vascular function; thus, enhancing angiogenesis could improve fracture healing. Both bone morphogenetic protein 2 (BMP-2) and thrombopoietin (TPO) have pro-angiogenic effects. The aim of this study was to investigate the effects of treatment with BMP-2 or TPO on the in vitro angiogenic and proliferative potential of endothelial cells (ECs) isolated from lungs (LECs) or bone marrow (BMECs) of young (3-4 months) and old (22-24 months), male and female, C57BL/6J mice. Cell proliferation, vessel-like structure formation, migration, and gene expression were used to evaluate angiogenic properties. In vitro characterization of ECs generally showed impaired vessel-like structure formation and proliferation in old ECs compared to young ECs, but improved migration characteristics in old BMECs. Differential sex-based angiogenic responses were observed, especially with respect to drug treatments and gene expression. Importantly, these studies suggest that NTN1, ROBO2, and SLIT3, along with angiogenic markers (CD31, FLT-1, ANGPT1, and ANGP2) differentially regulate EC proliferation and functional outcomes based on treatment, sex, and age. Furthermore, treatment of old ECs with TPO typically improved vessel-like structure parameters, but impaired migration. Thus, TPO may serve as an alternative treatment to BMP-2 for fracture healing in aging owing to improved angiogenesis and fracture healing, and the lack of side effects associated with BMP-2.


Assuntos
Envelhecimento , Células da Medula Óssea/citologia , Proteína Morfogenética Óssea 2/farmacologia , Células Endoteliais/efeitos dos fármacos , Pulmão/citologia , Neovascularização Fisiológica/efeitos dos fármacos , Caracteres Sexuais , Trombopoetina/farmacologia , Indutores da Angiogênese/metabolismo , Animais , Biomarcadores/metabolismo , Movimento Celular , Proliferação de Células , Células Endoteliais/citologia , Feminino , Consolidação da Fratura/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
4.
Molecules ; 26(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34443317

RESUMO

Endothelial cell injury is an early event in systemic sclerosis (SSc) pathogenesis and several studies indicate oxidative stress as the trigger of SSc-associated vasculopathy. Here, we show that circulating factors present in sera of SSc patients increased reactive oxygen species (ROS) production and collagen synthesis in human pulmonary microvascular endothelial cells (HPMECs). In addition, the possibility that iloprost, a drug commonly used in SSc therapy, might modulate the above-mentioned biological phenomena has been also investigated. In this regard, as compared to sera of SSc patients, sera of iloprost-treated SSc patients failed to increased ROS levels and collagen synthesis in HPMEC, suggesting a potential antioxidant mechanism of this drug.


Assuntos
Colágeno/biossíntese , Células Endoteliais/efeitos dos fármacos , Iloprosta/farmacologia , Microvasos/citologia , Estresse Oxidativo/efeitos dos fármacos , Escleroderma Sistêmico/sangue , Soro/metabolismo , Adulto , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Espécies Reativas de Oxigênio/metabolismo
5.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360818

RESUMO

Liver sinusoids are lined by liver sinusoidal endothelial cells (LSEC), which represent approximately 15 to 20% of the liver cells, but only 3% of the total liver volume. LSEC have unique functions, such as fluid filtration, blood vessel tone modulation, blood clotting, inflammatory cell recruitment, and metabolite and hormone trafficking. Different subtypes of liver endothelial cells are also known to control liver zonation and hepatocyte function. Here, we have reviewed the origin of LSEC, the different subtypes identified in the liver, as well as their renewal during homeostasis. The liver has the exceptional ability to regenerate from small remnants. The past decades have seen increasing awareness in the role of non-parenchymal cells in liver regeneration despite not being the most represented population. While a lot of knowledge has emerged, clarification is needed regarding the role of LSEC in sensing shear stress and on their participation in the inductive phase of regeneration by priming the hepatocytes and delivering mitogenic factors. It is also unclear if bone marrow-derived LSEC participate in the proliferative phase of liver regeneration. Similarly, data are scarce as to LSEC having a role in the termination phase of the regeneration process. Here, we review what is known about the interaction between LSEC and other liver cells during the different phases of liver regeneration. We next explain extended hepatectomy and small liver transplantation, which lead to "small for size syndrome" (SFSS), a lethal liver failure. SFSS is linked to endothelial denudation, necrosis, and lobular disturbance. Using the knowledge learned from partial hepatectomy studies on LSEC, we expose several techniques that are, or could be, used to avoid the "small for size syndrome" after extended hepatectomy or small liver transplantation.


Assuntos
Células Endoteliais , Hepatectomia , Hepatócitos , Falência Hepática/patologia , Regeneração Hepática , Fígado , Animais , Células Endoteliais/citologia , Células Endoteliais/patologia , Hepatócitos/citologia , Hepatócitos/patologia , Humanos , Fígado/citologia , Fígado/patologia
6.
Nat Biomed Eng ; 5(8): 880-896, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34426676

RESUMO

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.


Assuntos
Células Endoteliais/transplante , Coração/fisiologia , Infarto do Miocárdio/terapia , Miócitos de Músculo Liso/transplante , Regeneração , Animais , Ácido Ascórbico/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Reprogramação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Junções Comunicantes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Neovascularização Fisiológica , Transcriptoma
7.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445519

RESUMO

Cardiovascular disease is the leading cause of morbidity and mortality in diabetes. Recent clinical studies indicate that sodium-glucose co-transporter 2 (SGLT2) inhibitors improve cardiovascular outcomes in patients with diabetes. The mechanism underlying the beneficial effect of SGLT2 inhibitors is not completely clear but may involve direct actions on vascular cells. SGLT2 inhibitors increase the bioavailability of endothelium-derived nitric oxide and thereby restore endothelium-dependent vasodilation in diabetes. In addition, SGLT2 inhibitors favorably regulate the proliferation, migration, differentiation, survival, and senescence of endothelial cells (ECs). Moreover, they exert potent antioxidant and anti-inflammatory effects in ECs. SGLT2 inhibitors also inhibit the contraction of vascular smooth muscle cells and block the proliferation and migration of these cells. Furthermore, studies demonstrate that SGLT2 inhibitors prevent postangioplasty restenosis, maladaptive remodeling of the vasculature in pulmonary arterial hypertension, the formation of abdominal aortic aneurysms, and the acceleration of arterial stiffness in diabetes. However, the role of SGLT2 in mediating the vascular actions of these drugs remains to be established as important off-target effects of SGLT2 inhibitors have been identified. Future studies distinguishing drug- versus class-specific effects may optimize the selection of specific SGLT2 inhibitors in patients with distinct cardiovascular pathologies.


Assuntos
Complicações do Diabetes/prevenção & controle , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Remodelação Vascular/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Complicações do Diabetes/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Óxido Nítrico/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico
8.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361779

RESUMO

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.


Assuntos
Cavéolas/efeitos dos fármacos , Colesterol/química , Células Endoteliais/efeitos dos fármacos , Lipossomos/química , Microdomínios da Membrana/efeitos dos fármacos , Transfecção/métodos , Animais , Cavéolas/química , Cavéolas/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Clatrina/metabolismo , DNA/química , DNA/metabolismo , Endocitose/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Filipina/química , Filipina/farmacologia , Expressão Gênica , Lipossomos/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Nistatina/química , Nistatina/farmacologia , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacologia , Pinocitose/efeitos dos fármacos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos
9.
ACS Appl Mater Interfaces ; 13(33): 39018-39029, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34397215

RESUMO

Targeting nanoparticles as drug delivery platforms is crucial to facilitate their cellular entry. Docking of nanoparticles by targeting ligands on cell membranes is the first step for the initiation of cellular uptake. As a model system, we studied brain microvascular endothelial cells, which form the anatomical basis of the blood-brain barrier, and the tripeptide glutathione, one of the most effective targeting ligands of nanoparticles to cross the blood-brain barrier. To investigate this initial docking step between glutathione and the membrane of living brain endothelial cells, we applied our recently developed innovative optical method. We present a microtool, with a task-specific geometry used as a probe, actuated by multifocus optical tweezers to characterize the adhesion probability and strength of glutathione-coated surfaces to the cell membrane of endothelial cells. The binding probability of the glutathione-coated surface and the adhesion force between the microtool and cell membrane was measured in a novel arrangement: cells were cultured on a vertical polymer wall and the mechanical forces were generated laterally and at the same time, perpendicularly to the plasma membrane. The adhesion force values were also determined with more conventional atomic force microscopy (AFM) measurements using functionalized colloidal probes. The optical trapping-based method was found to be suitable to measure very low adhesion forces (≤ 20 pN) without a high level of noise, which is characteristic for AFM measurements in this range. The holographic optical tweezers-directed functionalized microtools may help characterize the adhesion step of nanoparticles initiating transcytosis and select ligands to target nanoparticles.


Assuntos
Membrana Celular/metabolismo , Células Endoteliais/metabolismo , Glutationa/metabolismo , Nanopartículas/metabolismo , Pinças Ópticas , Fenômenos Biofísicos , Barreira Hematoencefálica/metabolismo , Encéfalo , Adesão Celular , Membrana Celular/ultraestrutura , Células Endoteliais/citologia , Galactosamina/química , Humanos , Ligantes , Microscopia de Força Atômica , Nanopartículas/química , Polietilenoglicóis/química , Polímeros/metabolismo , Propriedades de Superfície , Transcitose
10.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360602

RESUMO

The primary function of the endothelial cells (EC) lining the inner surface of all vessels is to regulate permeability of vascular walls and to control exchange between circulating blood and tissue fluids of organs. The EC actin cytoskeleton plays a crucial role in maintaining endothelial barrier function. Actin cytoskeleton reorganization result in EC contraction and provides a structural basis for the increase in vascular permeability, which is typical for many diseases. Actin cytoskeleton in non-muscle cells presented two actin isoforms: non-muscle ß-cytoplasmic and γ-cytoplasmic actins (ß-actins and γ-actins), which are encoded by ACTB and ACTG1 genes, respectively. They are ubiquitously expressed in the different cells in vivo and in vitro and the ß/γ-actin ratio depends on the cell type. Both cytoplasmic actins are essential for cell survival, but they perform various functions in the interphase and cell division and play different roles in neoplastic transformation. In this review, we briefly summarize the research results of recent years and consider the features of the cytoplasmic actins: The spatial organization in close connection with their functional activity in different cell types by focusing on endothelial cells.


Assuntos
Actinas/metabolismo , Citoplasma/metabolismo , Células Endoteliais/fisiologia , Animais , Células Endoteliais/citologia , Humanos
11.
Am J Physiol Cell Physiol ; 321(3): C535-C548, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34288724

RESUMO

Extracellular vesicles (EVs) contain biological molecules and are secreted by cells into the extracellular milieu. The endothelial sodium channel (EnNaC) plays an important role in modulating endothelial cell stiffness. We hypothesized EVs secreted from human aortic endothelial cells (hAoECs) positively regulate EnNaC in an autocrine-dependent manner. A comprehensive lipidomic analysis using targeted mass spectrometry was performed on multiple preparations of EVs isolated from the conditioned media of hAoECs or complete growth media of these cells. Cultured hAoECs challenged with EVs isolated from the conditioned media of these cells resulted in an increase in EnNaC activity when compared with the same concentration of media-derived EVs or vehicle alone. EVs isolated from the conditioned media of hAoECs but not human fibroblast cells were enriched in MARCKS-like protein 1 (MLP1). The pharmacological inhibition of the negative regulator of MLP1, protein kinase C, in cultured hAoECs resulted in an increase in EV size and release compared with vehicle or pharmacological inhibition of protein kinase D. The MLP1-enriched EVs increased the density of actin filaments in cultured hAoECs compared with EVs isolated from human fibroblast cells lacking MLP1. We quantified 141 lipids from glycerolipids, glycerophospholipids, and sphingolipids in conditioned media EVs that represented twice the number found in control media EVs. The concentrations of sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine were higher in conditioned media EVs. These results provide the first evidence for EnNaC regulation in hAoECs by EVs and provide insight into a possible mechanism involving MLP1, unsaturated lipids, and bioactive lipids.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Lisofosfatidilcolinas/metabolismo , Proteínas dos Microfilamentos/genética , Fosfatidiletanolaminas/metabolismo , Esfingomielinas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Aorta/citologia , Aorta/metabolismo , Comunicação Autócrina , Proteínas de Ligação a Calmodulina/metabolismo , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Vesículas Extracelulares/química , Expressão Gênica , Glicerofosfolipídeos/metabolismo , Humanos , Lipidômica/métodos , Lisofosfatidilcolinas/farmacologia , Proteínas dos Microfilamentos/metabolismo , Fosfatidiletanolaminas/farmacologia , Cultura Primária de Células , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Esfingomielinas/farmacologia
12.
Mol Med Rep ; 24(4)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34328190

RESUMO

Pulmonary microvascular endothelial cell (PMVEC) apoptosis is the initial stage of adult pulmonary hypertension (PH), which involves high pulmonary arterial pressure and pulmonary vascular remodeling. However, the mechanism regulating PMVEC apoptosis and its involvement in the early stages of neonatal hypoxic PH (HPH) pathogenesis are currently unclear. The present study aimed to investigate the effects of heat shock protein 70 (HSP70) on hypoxia­induced apoptosis in PMVECs. PMVECs isolated from neonatal Sprague­Dawley rats were transfected with lentivirus with or without HSP70, or treated with the synthetic HSP70 inhibitor N­formyl­3,4­methylenedioxy­benzylidene-g-butyrolactam under hypoxic conditions (5% O2) for 24, 48 or 72 h. PMVEC apoptosis was evaluated by performing flow cytometry and mitochondrial membrane potential (MMP) assays. The expression levels of HSP70, hypoxia­inducible factor­1α (HIF­1α) and apoptosis­associated proteins were determined by conducting reverse transcription­quantitative PCR and western blotting. Following 24, 48 or 72 h of hypoxia, the apoptotic rates of PMVECs were significantly elevated compared with cells under normoxic conditions. The MMP was significantly reduced, whereas the mRNA and protein expression levels of HIF­1α, cytochrome c (cyt C), caspase­3 and HSP70 were enhanced by hypoxia compared with those under normoxic conditions. Additionally, the mRNA and protein expression levels of B­cell lymphoma 2 (Bcl­2) were significantly downregulated in the hypoxia group compared with those in the normoxia group. In hypoxic PMVECs, HSP70 overexpression decreased the apoptotic rate and the expression levels of cyt C, downregulated the expression levels of caspase­3 and HIF­1α, and increased the MMP and the expression levels of Bcl­2. HSP70 inhibition resulted in the opposite outcomes compared with those of HSP70 overexpression. Therefore, the results of the present study suggested that HSP70 may inhibit mitochondrial pathway­mediated apoptosis in isolated neonatal rat PMVECs in early­stage hypoxia, which may be associated with HSP70­mediated HIF­1α downregulation. Overall, HSP70 may be protective against neonatal HPH through the HSP70/HIF­1α pathway.


Assuntos
Apoptose/genética , Células Endoteliais/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Hipertensão Pulmonar/metabolismo , Microvasos/metabolismo , Animais , Animais Recém-Nascidos , Caspase 3/genética , Caspase 3/metabolismo , Hipóxia Celular , Regulação para Baixo , Células Endoteliais/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metaloproteinases da Matriz/metabolismo , Microvasos/citologia , Mitocôndrias/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/genética , Regulação para Cima
13.
ACS Appl Mater Interfaces ; 13(30): 35431-35443, 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34304556

RESUMO

Stent implantation is the primary method used to treat coronary heart disease. However, it is associated with complications such as restenosis and late thrombosis. Despite surface modification being an effective way to improve the biocompatibility of stents, the current research studies are not focused on changes in the vascular microenvironment at the implantation site. In the present study, an adaptive drug-loaded coating was constructed on the surface of vascular stent materials that can respond to oxidative stress at the site of vascular lesions. Two functional molecules, epigallocatechin gallate (EGCG) and cysteine hydrochloride, were employed to fabricate a coating on the surface of 316L stainless steel. In addition, the coating was used as a drug carrier to load pitavastatin calcium. EGCG has antioxidant activity, and pitavastatin calcium can inhibit smooth muscle cell proliferation. Therefore, EGCG and pitavastatin calcium provided a synergistic anti-inflammatory effect. Moreover, the coating was cross-linked using disulfide bonds, which accelerated the release of the drug in response to reactive oxygen species. A positive correlation was observed between the rate of drug release and the degree of oxidative stress. Collectively, this drug-loaded oxidative stress-responsive coating has been demonstrated to significantly inhibit inflammation, accelerate endothelialization, and reduce the risk of restenosis of vascular stents in vivo.


Assuntos
Stents Farmacológicos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Animais , Catequina/administração & dosagem , Catequina/análogos & derivados , Catequina/química , Catequina/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Reestenose Coronária/prevenção & controle , Cistamina/administração & dosagem , Cistamina/química , Liberação Controlada de Fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Quinolinas/administração & dosagem , Quinolinas/química , Quinolinas/farmacologia , Coelhos , Ratos Sprague-Dawley , Aço Inoxidável/química
14.
Methods Mol Biol ; 2319: 61-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331243

RESUMO

The blood vascular system is a tree-like hierarchical branching structure and needs to function even before fully established. Abnormal formation of blood vessels results in embryonic lethality and also contributes to the pathogenesis of a number of human diseases, including cancer metastasis. To understand the molecular events associated with blood vessel formation, we established a fluorescence staining-based protocol on mouse embryonic skin. We harvested mouse embryonic skin and performed whole-mount staining. The reconstructed three-dimensional vascular structure provided detailed information on angiogenesis.


Assuntos
Células Endoteliais/citologia , Imuno-Histoquímica/métodos , Neovascularização Fisiológica , Pele/irrigação sanguínea , Pele/citologia , Coloração e Rotulagem/métodos , Animais , Células Endoteliais/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Pele/crescimento & desenvolvimento , Pele/metabolismo
15.
Methods Mol Biol ; 2319: 69-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331244

RESUMO

There is increasing interest in the study of the mammalian lymphatic system, including the lymphatic endothelial cells (LECs) that make up lymphatic vessels. The ability to isolate primary LECs from tissue of normal and genetically modified mice permits detailed analysis of this unique cell type. Here, we describe a robust protocol for the isolation and in vitro expansion of LECs from mouse lung by antibody-based magnetic separation.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Endoteliais/citologia , Separação Imunomagnética/métodos , Pulmão/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Meios de Cultura/química , Células Endoteliais/metabolismo , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos
16.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299317

RESUMO

Decidualization is a crucial step for human reproduction, which is a prerequisite for embryo implantation, placentation and pregnancy maintenance. Despite rapid advances over recent years, the molecular mechanism underlying decidualization remains poorly understood. Here, we used the mouse as an animal model and generated a single-cell transcriptomic atlas of a mouse uterus during decidualization. By analyzing the undecidualized inter-implantation site of the uterus as a control, we were able to identify global gene expression changes associated with decidualization in each cell type. Additionally, we identified intercellular crosstalk between decidual cells and niche cells, including immune cells, endothelial cells and trophoblast cells. Our data provide a valuable resource for deciphering the molecular mechanism underlying decidualization.


Assuntos
Decídua/citologia , Decídua/metabolismo , Útero/citologia , Útero/metabolismo , Animais , Comunicação Celular/genética , Comunicação Celular/imunologia , Decídua/imunologia , Implantação do Embrião/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Humanos , Camundongos , Modelos Animais , Placentação/genética , Gravidez , Manutenção da Gravidez/genética , RNA-Seq , Análise de Célula Única , Células Estromais/citologia , Células Estromais/metabolismo , Transcriptoma , Trofoblastos/citologia , Trofoblastos/metabolismo , Útero/imunologia
17.
Nat Commun ; 12(1): 4155, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230480

RESUMO

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Pericárdio/citologia , Pericárdio/metabolismo , Animais , Quimiocinas , Vasos Coronários/metabolismo , Embrião de Mamíferos , Transição Epitelial-Mesenquimal , Expressão Gênica , Coração , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso , Proteínas Nucleares , Pericárdio/embriologia , Fator de Resposta Sérica , Transdução de Sinais , Transativadores , Fatores de Transcrição/metabolismo , Transcriptoma
18.
Methods Mol Biol ; 2320: 81-88, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302650

RESUMO

The present protocol describes a method to generate cylindrical engineered cardiac tissues (ECTs) composed of cardiovascular cell lineages induced from human induced pluripotent stem cells (hiPSCs). Cardiomyocytes, endothelial cells, and vascular mural cells induced from hiPSCs are mixed with gel matrix and poured into a tissue mold with posts. By culture day 14, the mixed culture matures into a cylindrical ECT which beats spontaneously and synchronously. Cardiomyocytes align to the long axis of the ECT. The ECTs generated by the present method may be regarded as a surrogate of human myocardium and be served as researches in cardiac regenerative medicine, disease modeling, drug discovery, and cardiac toxicity tests.


Assuntos
Linhagem da Célula/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Humanos , Engenharia Tecidual/métodos
19.
Nat Biomed Eng ; 5(8): 830-846, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34127820

RESUMO

The neurovascular unit, which consists of vascular cells surrounded by astrocytic end-feet and neurons, controls cerebral blood flow and the permeability of the blood-brain barrier (BBB) to maintain homeostasis in the neuronal milieu. Studying how some pathogens and drugs can penetrate the human BBB and disrupt neuronal homeostasis requires in vitro microphysiological models of the neurovascular unit. Here we show that the neurotropism of Cryptococcus neoformans-the most common pathogen causing fungal meningitis-and its ability to penetrate the BBB can be modelled by the co-culture of human neural stem cells, brain microvascular endothelial cells and brain vascular pericytes in a human-neurovascular-unit-on-a-chip maintained by a stepwise gravity-driven unidirectional flow and recapitulating the structural and functional features of the BBB. We found that the pathogen forms clusters of cells that penetrate the BBB without altering tight junctions, suggesting a transcytosis-mediated mechanism. The neurovascular-unit-on-a-chip may facilitate the study of the mechanisms of brain infection by pathogens, and the development of drugs for a range of brain diseases.


Assuntos
Barreira Hematoencefálica/metabolismo , Cryptococcus neoformans/fisiologia , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Barreira Hematoencefálica/química , Barreira Hematoencefálica/microbiologia , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Matriz Extracelular/química , Humanos , Hidrogéis/química , Meningite/microbiologia , Meningite/patologia , Microvasos/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Transcitose
20.
Lab Invest ; 101(9): 1142-1152, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34103662

RESUMO

Numerous studies have revealed that hyperglycemia is a pivotal driver of diabetic vascular complications. However, the mechanisms of hyperglycemia-induced endothelial dysfunction in diabetes remain incompletely understood. This study aims to expound on the underlying mechanism of the endothelial dysfunction induced by hyperglycemia from the perspective of long non-coding RNAs (lncRNA). In this study, a downregulation of SNHG15 was observed in the ischemic hind limb of diabetic mice and high glucose (HG)-treated HUVECs. Functionally, the overexpression of SNHG15 promoted cell proliferation, migration, and tube formation, and suppressed cell apoptosis in HG-treated HUVECs. Mechanistically, SNHG15 reduced thioredoxin-interacting protein (TXNIP) expression by enhancing ITCH-mediated ubiquitination of TXNIP. TXNIP overexpression abrogated the protective effect of lncRNA SNHG15 overexpression on HG-induced endothelial dysfunction. The following experiment further confirmed that SNHG15 overexpression promoted angiogenesis of the ischemic hind limb in diabetic mice. In conclusion, SNHG15 is a novel protector for hyperglycemia-induced endothelial dysfunction via decreasing TXNIP expression.


Assuntos
Proteínas de Transporte , Hiperglicemia/metabolismo , RNA Longo não Codificante , Tiorredoxinas , Ubiquitinação/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
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