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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.
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
3.
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
4.
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
5.
J Dermatol Sci ; 103(1): 25-32, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34148739

RESUMO

BACKGROUND: Mucopolysaccharide polysulfate (MPS) is a heparinoid and MPS-containing formulations are widely used as moisturizers for dry skin and to treat peripheral vascular insufficiency. Although MPS has therapeutic effects in skin diseases with microvascular abnormalities, the effects of MPS on microvascular function remain incompletely understood. OBJECTIVE: The aim of this study was to evaluate the functional activities of MPS on human pericytes (HPC) and human dermal microvascular endothelial cells (HDMEC) in vitro, and on microvascular permeability of the skin. METHODS: The protein expression of angiopoietin (Ang)-1 in HPC, and platelet-derived growth factor-BB (PDGF-BB) and phosphorylated tyrosine-protein kinase receptor 2 (Tie2) in HDMEC were measured in the presence or absence of MPS. The vascular barrier was evaluated by the expressions of claudin-5 and vascular endothelial (VE)-cadherin, and transendothelial electrical resistance (TEER). RESULTS: In HPC, MPS dose-dependently enhanced Ang-1 secretion, which activated Tie2 in HDMEC. In HDMEC, MPS significantly increased the production of PDGF-BB, which is important for the recruitment of HPC to the vascular endothelium, and significantly increased the phosphorylation of Tie2, which results in the activation of the Ang-1/Tie2 signaling . MPS significantly increased the expression of tight junction protein claudin-5 and TEER in the HDMEC. Moreover, the intradermal injection of MPS prevented vascular endothelial growth factor-induced increase in vascular permeability in mouse skin. CONCLUSION: We found that MPS promoted microvascular stabilization and barrier integrity in HDMEC via Ang-1/Tie2 activation. These results suggest that MPS might improve microvascular abnormalities in various diseases accompanied by disturbances in Ang-1/Tie2 signaling.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Emolientes/farmacologia , Endotélio Vascular/efeitos dos fármacos , Glicosaminoglicanos/farmacologia , Microvasos/efeitos dos fármacos , Angiopoietina-1/metabolismo , Animais , Becaplermina/metabolismo , Células Endoteliais , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Humanos , Injeções Intradérmicas , Camundongos , Microvasos/citologia , Microvasos/metabolismo , Modelos Animais , Pericitos , Fosforilação/efeitos dos fármacos , Receptor TIE-2/metabolismo , Pele/irrigação sanguínea , Pele/efeitos dos fármacos , Pele/metabolismo , Dermatopatias Vasculares/tratamento farmacológico
6.
Int J Biol Macromol ; 183: 695-706, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-33932419

RESUMO

Implantation of biomaterials and hybrid constructs in tissue engineering approaches presents major limitations such as inflammatory reaction and the lack of vasculature integration. Therefore, new strategies are needed to enhance implant function, immune protection, and revascularization. In this work, we developed fibrous meshes composed of fucoidan (Fu), a sulfated polysaccharide extracted from brown algae, and polycaprolactone (PCL), a synthetic biodegradable polymer, using the airbrush technique. The chemical characterization by FTIR, EDS, and XPS confirmed the presence of the two polymers in the structure of airbrushed nanofibrous meshes (ANFM). Moreover, these nanofibrous exhibited good wettability and mechanical properties envisaging their application as templates for biomaterials and cell culture. The developed ANFM were directly cultured with human pulmonary microvascular endothelial (HPMEC-ST1.6R) cells for up to 7 days. Biological results demonstrated that ANFM comprising Fu promoted cellular attachment, spreading, and proliferation of human endothelial cells. The angiogenic potential of ANFM was further evaluated by onplantation of PCL and PCL/Fu ANFM in chick chorioallantoic membrane (CAM). In ovo and ex ovo results showed that the incorporation of Fu increased the pro-angiogenic potential of ANFM. Altogether, the results suggest that airbrush biocomposite meshes could be used as a biomaterial substrate to promote vascularization.


Assuntos
Indutores da Angiogênese/farmacologia , Membrana Corioalantoide/irrigação sanguínea , Pulmão/irrigação sanguínea , Poliésteres/química , Polissacarídeos/farmacologia , Indutores da Angiogênese/química , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Microvasos/citologia , Microvasos/efeitos dos fármacos , Nanofibras , Polissacarídeos/química , Telas Cirúrgicas , Engenharia Tecidual
7.
Methods Mol Biol ; 2311: 185-193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033087

RESUMO

Studies of blood-brain barrier (BBB) require developing of a novel and convenient in vitro endothelial cell model. We isolated primary human and rodent brain microvascular endothelial cells and developed methods for culturing, characterization, and high-efficiency transfection of endothelial cells. Here, we describe the improved methods to obtain in vitro human and rodent BBB models to study expression of endogenous and exogenous genes of interest.


Assuntos
Barreira Hematoencefálica/fisiologia , Encéfalo/irrigação sanguínea , Separação Celular , Células Endoteliais/fisiologia , Microvasos/citologia , Transfecção , Animais , Barreira Hematoencefálica/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Endoteliais/metabolismo , Feto , Idade Gestacional , Humanos , Camundongos , Ratos
8.
PLoS One ; 16(4): e0249814, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33852594

RESUMO

The blood-brain barrier (BBB) keeps pathogens and toxins out of the brain but also impedes the entry of pharmaceuticals. Human cerebral microvascular endothelial cells (hCMECs) and astrocytes are the main functional cell components of the BBB. Although available commercially as cryopreserved cells in suspension, improvements in their cryopreservation and distribution as cryopreserved monolayers could enhance BBB in vitro studies. Here, we examined the response to slow cooling and storage in liquid nitrogen of immortalized hCMEC/D3 cells and human primary astrocytes in suspension and in monolayers. HCMEC/D3 cells in suspension cryopreserved in 5% dimethyl sulfoxide (DMSO) and 95% fetal bovine serum or in 5% DMSO and 6% hydroxyethyl starch (HES) showed post-thaw membrane integrities above 90%, similar to unfrozen control. Cryopreservation did not affect the time-dependent ability of hCMEC/D3 cells to form tubes on Matrigel. Primary astrocytes in suspension cryopreserved in the presence of 5% DMSO and 6% HES had improved viability over those cryopreserved in 10% DMSO. Monolayers of single cultures or co-cultures of hCMEC/D3 cells and astrocytes on fibronectin-coated Rinzl coverslips retained membrane integrities and metabolic function, after freezing in 5% DMSO, 6% HES, and 2% chondroitin sulfate, that were comparable to those of unfrozen controls even after overnight incubation. Rinzl is better than glass or Thermanox as an underlying solid substrate for cryopreserving hCMEC/D3 monolayers. Cryopreserved hCMEC/D3 monolayers expressed the junction proteins ZO-1 and claudin-5 similar to their unfrozen counterparts. Hence, we describe improved cryopreservation protocols for hCMEC/D3 cells and astrocytes in suspension, and a novel protocol for the cryopreservation of monolayers of hCMEC/D3 cells and astrocytes as single cultures or co-cultures that could expand their distribution for research on disease modeling, drug screening, and targeted therapy pertaining to the BBB.


Assuntos
Astrócitos/citologia , Barreira Hematoencefálica/citologia , Criopreservação/métodos , Astrócitos/metabolismo , Células Cultivadas , Claudina-1/genética , Claudina-1/metabolismo , Dimetil Sulfóxido/química , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Microvasos/citologia , Nitrogênio/química , Soroalbumina Bovina/química , Amido/análogos & derivados , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
9.
Food Funct ; 12(6): 2715-2725, 2021 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-33667286

RESUMO

The microvasculature endothelium accurately regulates the passage of molecules across the gut-vascular barrier (GVB), which plays an essential role in intestinal immunity. Naringenin is reported to have therapeutic potential against several intestinal disorders. However, the effect of naringenin on GVB disruption has been rarely studied. This study aims to investigate the effect of naringenin on GVB function and the potential mechanism. In the present study, the in vitro GVB disruption of rat intestinal microvascular endothelial cells (RIMVEC) was induced by 50 ng mL-1 of tumor necrosis factor-α (TNF-α). The integrity of the in vitro GVB was determined by Evans blue (EB)-albumin efflux assay and trans-endothelial electrical resistance (TER). Meanwhile, the expression of tight junction proteins and the related NF-κB, MLCK/p-MLC and NLRP3 pathways were determined using enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), western blot analysis and immunofluorescence. The results show that naringenin (100 µM) inhibits TNF-α-induced interleukin (IL)-6 hypersecretion, alleviates GVB disruption and mitigates the change in the tight junction protein expression pattern. Naringenin inhibits the GVB-disruption-associated activation of the MLCK/p-MLC system and TLR4/NF-κB/NLRP3 pathways. Furthermore, naringenin shows a similar effect to that of NF-κB inhibitor Bay 11-7082 in reducing the TNF-α-induced activation of NLRP3, p-MLC and secondary GVB disruption. The results suggest that naringenin evidently alleviates TNF-α-induced in vitro GVB disruption via the maintenance of a tight junction protein pattern, partly with the inhibition of the NF-κB-mediated MLCK/p-MLC and NLRP3 pathway activation.


Assuntos
Flavanonas/farmacologia , Mucosa Intestinal , Microvasos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Microvasos/citologia , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas de Junções Íntimas/metabolismo
10.
Pharm Res ; 38(1): 97-111, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33532991

RESUMO

PURPOSE: P-glycoprotein (P-gp) at the blood-brain barrier (BBB) precludes the brain penetration of many xenobiotics and mediates brain-to-blood clearance of ß-amyloid, which accumulates in the Alzheimer's disease (AD) brain. Zinc and copper are reported to modulate BBB expression and function of P-gp; however, the impact of exogenous iron, which accumulates in AD, on P-gp dynamics remains unknown. METHODS: P-gp protein and MDR1 transcript levels were assessed in immortalised human cerebral microvascular endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC; 250 µM, 72 h), by Western blotting and RT-qPCR, respectively. P-gp function was assessed using rhodamine-123 and [3H]-digoxin accumulation. Intracellular reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate and intracellular iron levels quantified using a ferrozine assay. RESULTS: FAC treatment significantly reduced P-gp protein (36%) and MDR1 mRNA (16%) levels, with no significant change in rhodamine-123 or [3H]-digoxin accumulation. While P-gp/MDR1 downregulation was associated with elevated ROS and intracellular iron, MDR1 downregulation was not attenuated with the antioxidant N-acetylcysteine nor the iron chelators desferrioxamine and deferiprone, suggesting the involvement of a ROS-independent mechanism or incomplete iron chelation. CONCLUSIONS: These studies demonstrate that iron negatively regulates P-gp expression at the BBB, potentially impacting CNS drug delivery and brain ß-amyloid clearance.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Barreira Hematoencefálica/patologia , Compostos Férricos/metabolismo , Ferro/metabolismo , Fármacos Neuroprotetores/farmacocinética , Compostos de Amônio Quaternário/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Células Endoteliais/patologia , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Compostos Férricos/análise , Humanos , Ferro/análise , Microvasos/citologia , Microvasos/patologia , Fármacos Neuroprotetores/administração & dosagem , Compostos de Amônio Quaternário/análise , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo
11.
Methods Mol Biol ; 2235: 27-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576968

RESUMO

Pericytes are mural cells closely associated with endothelial cells in capillaries and microvessels. They are precursors of mesenchymal stem/stromal cells that have historically been retrospectively characterized in culture. We established a protocol, described in this chapter, to characterize and isolate pericytes from multiple human organs by flow cytometry and fluorescence-activated cell sorting. This prospective purification of pericytes brings us a step forward in the development of strategies for their use in the clinic.


Assuntos
Citometria de Fluxo/métodos , Pericitos/citologia , Pericitos/transplante , Capilares/citologia , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Cultivadas , Células Endoteliais/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Microvasos/citologia , Pericitos/metabolismo , Fenótipo
12.
Methods Mol Biol ; 2235: 37-45, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576969

RESUMO

Pericytes are found in all vascularized organs and are defined anatomically as perivascular cells that closely surround endothelial cells in capillaries and microvessels and are embedded within the same basement membrane. They have been shown to have diverse physiological and pathological functions including regulation of blood pressure, and tissue regeneration and scarring. Fundamental to understanding the role these cells play in these diverse processes is the ability to accurately identify and localize them in vivo. To do this, we have developed multicolor immunohistochemistry protocols described in this chapter.


Assuntos
Imuno-Histoquímica/métodos , Pericitos/citologia , Pericitos/transplante , Capilares/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Humanos , Microvasos/citologia , Pericitos/metabolismo , Fenótipo
13.
Methods Mol Biol ; 2235: 47-59, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33576970

RESUMO

We report the use of self-assembled peptide (F2/S) hydrogels and cellular metabolomics to identify a number of innate molecules that are integral to the metabolic processes which drive cellular differentiation of multipotent pericyte stem cells. The culture system relies solely on substrate mechanics to induce differentiation in the absence of traditional differentiation media and therefore is a non-invasive approach to assessing cellular behavior at the molecular level and identifying key metabolites in this process. This novel approach demonstrates that simple metabolites can provide an alternative means to direct stem cell differentiation and that biomaterials can be used to identify them simply and quickly.


Assuntos
Metabolômica/métodos , Pericitos/citologia , Pericitos/transplante , Animais , Materiais Biocompatíveis/metabolismo , Capilares/citologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Humanos , Hidrogéis/química , Microvasos/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Peptídeos/química , Pericitos/metabolismo , Fenótipo
14.
J Surg Res ; 261: 226-235, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33460967

RESUMO

BACKGROUND: RNA-binding motif protein 3 (RBM3) is an important cold shock protein, which also responds to hypothermia or hypoxia. RBM3 is involved into multiple physiologic processes, such as promoting cell survival. However, its expression and function in acute lung injury (ALI) have not been reported. METHODS: A mouse ALI model was established by lipopolysaccharides (LPS) treatment. The RBM3 and cold inducible RNA-binding protein mRNA levels were examined by RT-qPCR, and MMP9 mRNA stability was determined by actinomycin D assay. RBM3 and MMP9 mRNA was tested by RNA immunoprecipitation (RIP assay). RBM3 overexpression or silent stable cell lines were established using recombinant lentivirus and subsequently used for cell survival and tight junction measurements. RESULTS: In this study, we found that RBM3, rather than cold inducible RNA-binding protein, was upregulated in lung tissue of ALI mice. RBM3 was increased in human pulmonary microvascular endothelial cells (HPMVECs) in response to LPS treatment, which is modulated by the NF-κB signaling pathway. Furthermore, RBM3 could reduce cell apoptosis induced by LPS, probably through suppressing p53 expression. Because increased permeability of HPMVECs leads to pulmonary edema in ALI, we subsequently examined the effect of RBM3 on cell tight junctions. Unexpectedly, RBM3 decreased the expression of tight junction protein zonula occludens-1 and increased cell permeability, and RBM3 overexpression increased MMP9 mRNA stability. Furthermore, RIP assay confirmed the interaction between RBM3 and MMP9 mRNA, possibly explaining the contribution of RBM3 to increase cell permeability. CONCLUSIONS: RBM3 seems to act as a "double-edged sword" in ALI, that RBM3 alleviates cell apoptosis but increases HPMVEC permeability in ALI.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Células Endoteliais/fisiologia , Proteínas de Ligação a RNA/metabolismo , Junções Íntimas/fisiologia , Animais , Apoptose , Linhagem Celular , Humanos , Lipopolissacarídeos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Microvasos/citologia , Microvasos/fisiologia , NF-kappa B/metabolismo , Estabilidade de RNA
16.
Biomed Pharmacother ; 133: 110999, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33227710

RESUMO

Abnormal angiogenesis is associated with intraocular diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, and current therapies for these eye diseases are not satisfactory. The purpose of this study was to determine whether capilliposide B (CPS-B), a novel oleanane triterpenoid saponin derived from Lysimachia capillipes Hemsl, can inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis signaling events and cellular responses in primary human retinal microvascular endothelial cells (HRECs). Our study revealed that the capilliposide B IC50 for HRECs was 8.5 µM at 72 h and that 1 µM capilliposide B specifically inhibited VEGF-induced activation of VEGFR2 and its downstream signaling enzymes Akt and Erk. In addition, we discovered that this chemical effectively blocked VEGF-stimulated proliferation, migration and tube formation of the HRECs, suggesting that capilliposide B is a promising prophylactic for angiogenesis-associated diseases such as proliferative diabetic retinopathy.


Assuntos
Indutores da Angiogênese/farmacologia , Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Saponinas/farmacologia , Triterpenos/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Microvasos/citologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasos Retinianos/citologia , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
17.
Biochim Biophys Acta Gen Subj ; 1865(2): 129796, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33212230

RESUMO

BACKGROUND: Iron excess is a risk factor for cardiovascular diseases and it is important to understand the effect of iron on vascular permeability, particularly for the transport of large metabolic hormones such as adiponectin. METHODS: We used 2-dimensional monolayers of cultured human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) as well as 3-dimensional microvascular networks to measure transendothelial flux. RESULTS: Iron supplementation reduced transendothelial electric resistance (TEER). Flux analysis indicated that under control conditions permeability of 70 kDa dextran and oligomeric forms of adiponectin were restricted in comparison with a 3 kDa dextran, however upon iron treatment permeability of the larger molecules was increased. The increased permeability and size-dependent trans-endothelial movement in response to iron was also observed in 3-dimensional microvascular networks. Mechanistically, the alteration in barrier functionality was associated with increased oxidative stress in response to iron since alterations in TEER and permeability were rescued when reactive oxygen species production was attenuated by pre-treatment with the antioxidant N-acetyl cysteine.]. CONCLUSIONS: Iron supplementation induced ROS production resulting in increased transendothelial permeability. GENERAL SIGNIFICANCE: Altogether, this suggests that the oxidative stress associated with iron excess could play an important role in the regulation of endothelial functionality, controlling hormone action in peripheral tissues by regulating the first rate-limiting step controlling hormone access to target tissues.


Assuntos
Adiponectina/metabolismo , Células Endoteliais/metabolismo , Ferro/metabolismo , Microvasos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Permeabilidade Capilar , Linhagem Celular , Impedância Elétrica , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Dispositivos Lab-On-A-Chip , Microvasos/citologia , Permeabilidade
18.
Neuromolecular Med ; 23(1): 184-198, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33067719

RESUMO

Ergothioneine (ET) is a naturally occurring antioxidant that is synthesized by non-yeast fungi and certain bacteria. ET is not synthesized by animals, including humans, but is avidly taken up from the diet, especially from mushrooms. In the current study, we elucidated the effect of ET on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induces a dose-dependent loss of cell viability and an increase in apoptosis and necrosis in the endothelial cells. A relocalization of the tight junction proteins, zonula occludens-1 (ZO-1) and claudin-5, towards the nucleus of the cells was also observed. These effects were significantly attenuated by ET. In addition, 7KC induces marked increases in the mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX2), as well as COX2 enzymatic activity, and these were significantly reduced by ET. Moreover, the cytoprotective and anti-inflammatory effects of ET were significantly reduced by co-incubation with an inhibitor of the ET transporter, OCTN1 (VHCL). This shows that ET needs to enter the endothelial cells to have a protective effect and is unlikely to act via extracellular neutralizing of 7KC. The protective effect on inflammation in brain endothelial cells suggests that ET might be useful as a nutraceutical for the prevention or management of neurovascular diseases, such as stroke and vascular dementia. Moreover, the ability of ET to cross the blood-brain barrier could point to its usefulness in combatting 7KC that is produced in the CNS during neuroinflammation, e.g. after excitotoxicity, in chronic neurodegenerative diseases, and possibly COVID-19-related neurologic complications.


Assuntos
Antioxidantes/farmacologia , COVID-19/complicações , Células Endoteliais/efeitos dos fármacos , Ergotioneína/farmacologia , Cetocolesteróis/toxicidade , Doenças do Sistema Nervoso/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Antioxidantes/farmacocinética , Apoptose/efeitos dos fármacos , Transporte Biológico , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Linhagem Celular , Colesterol/metabolismo , Claudina-5 , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Citocinas/biossíntese , Citocinas/genética , Avaliação Pré-Clínica de Medicamentos , Ergotioneína/farmacocinética , Humanos , Microvasos/citologia , Doenças do Sistema Nervoso/etiologia , Fármacos Neuroprotetores/farmacocinética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas de Transporte de Cátions Orgânicos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Simportadores , Proteína da Zônula de Oclusão-1
19.
Methods Mol Biol ; 2206: 193-203, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754819

RESUMO

The capability of forming functional blood vessel networks is critical for the characterization of endothelial cells. In this chapter, we will review a modified in vivo vascular network forming assay by replacing traditional mouse tumor-derived Matrigel with a well-defined collagen-fibrin hydrogel. The assay is reliable and does not require special equipment, surgical procedure, or a skilled person to perform. Moreover, investigators can modify this method on-demand for testing different cell sources, perturbation of gene functions, growth factors, and pharmaceutical molecules, and for the development and investigation of strategies to enhance neovascularization of engineered human tissues and organs.


Assuntos
Bioensaio/métodos , Vasos Sanguíneos/citologia , Microvasos/citologia , Neovascularização Fisiológica/fisiologia , Animais , Colágeno/metabolismo , Combinação de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibrina/metabolismo , Humanos , Hidrogéis/metabolismo , Laminina/metabolismo , Camundongos , Camundongos Nus , Proteoglicanas/metabolismo , Engenharia Tecidual/métodos
20.
J Cell Biochem ; 122(1): 116-129, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32748513

RESUMO

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro- or microvasculature) play a role in the formation of microvessel-like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor-ß1, and interleukin-8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel-like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins' aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.


Assuntos
Fosfatase Alcalina/metabolismo , Movimento Celular , Colágeno/metabolismo , Endotélio Vascular/fisiologia , Fibroblastos/fisiologia , Microvasos/fisiologia , Neovascularização Fisiológica , Fosfatase Alcalina/química , Sítios de Ligação , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Endotélio Vascular/citologia , Fibroblastos/citologia , Humanos , Técnicas In Vitro , Microvasos/citologia , Conformação Proteica
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