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1.
Methods Mol Biol ; 2375: 217-228, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34591311

RESUMO

In vitro blood-brain barrier (BBB) models have been widely used to simulate in vivo models due to their low cost, feasibility, and repeatability. To serve as a valid substitute, the in vitro BBB should have the similar barrier function as that in vivo. This chapter summarizes the detailed methods for quantifying the barrier function, e.g., the permeability of the BBB to water, ions, and solutes for an in vitro BBB generated on the Transwell filter.


Assuntos
Barreira Hematoencefálica , Transporte Biológico , Permeabilidade Capilar , Permeabilidade
2.
Methods Mol Biol ; 2409: 197-205, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34709643

RESUMO

A growing body of evidence demonstrates that endothelial cells (ECs) play a prominent role in immune-enhanced pathology seen in dengue virus (DENV) infection that might contribute to vascular permeability and hemorrhagic manifestations in severe dengue cases. However, it remains a question of whether DENV infection of ECs directly causes permeability or if extra-endothelial factors such as immune cell activation or antibody-dependent enhancement (ADE) are required. In this chapter, we detail the measurement of the transendothelial electrical resistance (TEER), a quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial monolayers and show that DENV infection of ECs does not cause endothelial permeability in vitro.


Assuntos
Vírus da Dengue , Dengue , Permeabilidade Capilar , Impedância Elétrica , Células Endoteliais , Humanos
3.
Methods Mol Biol ; 2409: 207-222, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34709644

RESUMO

This chapter will discuss reliable and relatively easy and fast strategies to evaluate the integrity of endothelial cell monolayers when infected by dengue virus (DENV). Human brain microvascular endothelial cells (HBMEC) were exploited here as general model of vessel wall core, but it may also be used as an in vitro simplified model of blood brain barrier (BBB). The integrity of endothelial cells monolayer can be inferred using a transwell culture system by: (1) measuring transendothelial electrical resistance (TEER) using a Voltohmmeter; (2) analyzing the monolayer permeability to fluorescent-conjugated proteins and fluorimetric assay; (3) investigating virus extravasation by quantitative RT-PCR and plaque conventional assay. The rational to use those strategies is that vascular alterations are often observed during dengue infection, being associated to disease severity. The vasculature core consists of a barrier of endothelial cells, which are tightly adhered by the expression of adhesion molecules and tight junctions. This structure must be preserved in order to control the flux of cells and metabolites from the circulation to the tissues and to maintain vascular homeostasis. Therefore, experimental assays that allow evaluation of endothelial integrity can be useful platforms to further understand disease pathogenesis and screen pharmaceutical interventions to control vascular disturbance.


Assuntos
Células Endoteliais , Barreira Hematoencefálica , Encéfalo , Permeabilidade Capilar , Células Cultivadas , Impedância Elétrica , Humanos , Permeabilidade
4.
FASEB J ; 36(1): e22110, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34918393

RESUMO

Dengue virus (DENV) is a cause of vascular endothelial dysfunction and vascular leakage, which are characterized as hallmarks of dengue hemorrhagic fever or dengue shock syndrome, which become a severe global health emergency with substantial morbidity and mortality. Currently, there are still no promising therapeutics to alleviate the dengue-associated vascular hemorrhage in a clinical setting. In the present study, we first observed that heme oxygenase-1 (HO-1) expression level was highly suppressed in severe DENV-infected patients. In contrast, the overexpression of HO-1 could attenuate DENV-induced pathogenesis, including plasma leakage and thrombocytopenia, in an AG129 mouse model. Our data indicate that overexpression of HO-1 or its metabolite biliverdin can maintain endothelial integrity upon DENV infection in vitro and in vivo. We further characterized the positive regulatory effect of HO-1 on the endothelial adhesion factor vascular endothelial-cadherin to decrease DENV-induced endothelial hyperpermeability. Subsequently, we confirmed that two medicinal plant-derived compounds, andrographolide, and celastrol, widely used as a nutritional or medicinal supplement are useful to attenuate DENV-induced plasma leakage through induction of the HO-1 expression in DENV-infected AG129 mice. In conclusion, our findings reveal that induction of the HO-1 signal pathway is a promising option for the treatment of DENV-induced vascular pathologies.


Assuntos
Permeabilidade Capilar , Vírus da Dengue/metabolismo , Endotélio Vascular/enzimologia , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Dengue Grave/enzimologia , Animais , Linhagem Celular , Vírus da Dengue/genética , Modelos Animais de Doenças , Heme Oxigenase-1/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Dengue Grave/genética
5.
Methods Mol Biol ; 2384: 247-255, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34550579

RESUMO

Primary monkey brain capillary endothelial cell cultures, with rat pericytes and astrocytes, provide an assay system for predicting the ability of oxytocin (OT) to cross the blood-brain barrier (BBB), using a commercially available in vitro BBB kit. The integrity of the in vitro "BBB," which has a high transendothelial electrical resistance (TEER), can be established approximately 4 days after preparations for experiments. Dominant endothelial transport of OT is from the upper (luminal blood side) to lower (abluminal brain side) chambers, dose-dependently. OT is transported by the receptor for advanced glycation end-products (RAGE) in endothelial cells, which is evidenced using the RAGE knockdown system with short hairpin RNA (shRNA) treatment. This in vitro assay system is useful for further assessment of OT transport across the BBB.


Assuntos
Barreira Hematoencefálica , Animais , Astrócitos , Permeabilidade Capilar , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais , Ocitocina , Permeabilidade , Ratos , Receptor para Produtos Finais de Glicação Avançada
6.
Biol Pharm Bull ; 44(10): 1371-1379, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34602545

RESUMO

The vascular permeability of the endothelium is finely controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions. In the majority of normal adult tissues, endothelial cells in blood vessels maintain vascular permeability at a relatively low level, while in response to inflammation, they limit vascular barrier function to induce plasma leakage and extravasation of immune cells as a defense mechanism. Thus, the dynamic but also simultaneously tight regulation of vascular permeability by endothelial cells is responsible for maintaining homeostasis and, as such, impairments of its underlying mechanisms result in hyperpermeability, leading to the development and progression of various diseases including coronavirus disease 2019 (COVID-19), a newly emerging infectious disease. Recently, increasing numbers of studies have been unveiling the important role of Rap1, a small guanosine 5'-triphosphatase (GTPase) belonging to the Ras superfamily, in the regulation of vascular permeability. Rap1 enhances VE-cadherin-mediated endothelial cell-cell junctions to potentiate vascular barrier functions via dynamic reorganization of the actin cytoskeleton. Importantly, Rap1 signaling activation reportedly improves vascular barrier function in animal models of various diseases associated with vascular hyperpermeability, suggesting that Rap1 might be an ideal target for drugs intended to prevent vascular barrier dysfunction. Here, we describe recent progress in understanding the mechanisms by which Rap1 potentiates VE-cadherin-mediated endothelial cell-cell adhesions and vascular barrier function. We also discuss how alterations in Rap1 signaling are related to vascular barrier dysfunction in diseases such as acute pulmonary injury and malignancies. In addition, we examine the possibility of Rap1 signaling as a target of drugs for treating diseases associated with vascular hyperpermeability.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Junções Intercelulares/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Humanos
7.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638621

RESUMO

Previously, we showed that mice treated with cyclophosphamide (CTX) 4 days before intravenous injection of breast cancer cells had more cancer cells in the lung at 3 h after cancer injection than control counterparts without CTX. At 4 days after its injection, CTX is already excreted from the mice, allowing this pre-treatment design to reveal how CTX may modify the lung environment to indirectly affect cancer cells. In this study, we tested the hypothesis that the increase in cancer cell abundance at 3 h by CTX is due to an increase in the adhesiveness of vascular wall for cancer cells. Our data from protein array analysis and inhibition approach combined with in vitro and in vivo assays support the following two-prong mechanism. (1) CTX increases vascular permeability, resulting in the exposure of the basement membrane (BM). (2) CTX increases the level of matrix metalloproteinase-2 (MMP-2) in mouse serum, which remodels the BM and is functionally important for CTX to increase cancer abundance at this early stage. The combined effect of these two processes is the increased accessibility of critical protein domains in the BM, resulting in higher vascular adhesiveness for cancer cells to adhere. The critical protein domains in the vascular microenvironment are RGD and YISGR domains, whose known binding partners on cancer cells are integrin dimers and laminin receptor, respectively.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Ciclofosfamida/farmacologia , Metaloproteinase 2 da Matriz/sangue , Microambiente Tumoral/efeitos dos fármacos , Animais , Membrana Basal/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Permeabilidade Capilar/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Integrina beta1/metabolismo , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Knockout , Domínios Proteicos , Microambiente Tumoral/fisiologia
8.
Elife ; 102021 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-34694226

RESUMO

Severe acute respiratory syndrome (SARS)-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5), and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein-protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by coronavirus disease (COVID-19). While validating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and ß-catenin are affected by nsp2, nsp5_c145a, and nsp7 consistent with the model prediction. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-19.


Assuntos
Permeabilidade Capilar , Endotélio Vascular/metabolismo , Interações Hospedeiro-Patógeno , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , Animais , COVID-19/virologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Mapas de Interação de Proteínas , Proteínas de Junções Íntimas/metabolismo
9.
FASEB J ; 35(11): e22008, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34679191

RESUMO

An impaired blood-retinal barrier (BRB) leads to diabetic macular edema (DME), which is a major complication of Diabetic retinopathy (DR). Mediators such as inflammation cause BRB breakdown. However, the explicit mechanism of its disruption is largely unknown. In this study, we identified tumor necrosis factor ligand-related molecule 1A (TL1A) as a crucial factor which protect retinal endothelial cells integrity in DR. By providing both human and mouse data, we show that TL1A is significantly decreased in the retinas of DME patients and diabetic rodents. We further demonstrate that the loss of TL1A accelerated diabetes-induced retinal barrier breakdown. TL1A supplementation protects the diabetic retina against BRB breakdown. Mechanistically, TL1A stabilize intracellular junctions and protect vascular integrity by blocking SHP1-Src-regulated VE-cadherin phosphorylation. Collectively, our findings reveal that loss of TL1A in the retina leads to increased vascular permeability in DR, and that TL1A treatment is of potential therapeutic interest for the treatment of DME.


Assuntos
Barreira Hematorretiniana/metabolismo , Permeabilidade Capilar , Retinopatia Diabética/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/fisiologia , Animais , Células Endoteliais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Vasos Retinianos
10.
Am J Physiol Heart Circ Physiol ; 321(5): H985-H1003, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34559580

RESUMO

Ventilation with gases containing enhanced fractions of oxygen is the cornerstone of therapy for patients with hypoxia and acute respiratory distress syndrome. Yet, hyperoxia treatment increases free reactive oxygen species (ROS)-induced lung injury, which is reported to disrupt autophagy/mitophagy. Altered extranuclear activity of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), plays a protective role in ROS injury and autophagy in the systemic and coronary endothelium. We investigated interactions between autophagy/mitophagy and TERT that contribute to mitochondrial dysfunction and pulmonary injury in cultured rat lung microvascular endothelial cells (RLMVECs) exposed in vitro, and rat lungs exposed in vivo to hyperoxia for 48 h. Hyperoxia-induced mitochondrial damage in rat lungs [TOMM20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], which was paralleled by increased markers of inflammation [myeloperoxidase (MPO), IL-1ß, TLR9], impaired autophagy signaling (Beclin-1, LC3B-II/1, and p62), and decreased the expression of TERT. Mitochondrial-specific autophagy (mitophagy) was not altered, as hyperoxia increased expression of Pink1 but not Parkin. Hyperoxia-induced mitochondrial damage (TOMM20) was more pronounced in rats that lack the catalytic subunit of TERT and resulted in a reduction in cellular proliferation rather than cell death in RLMVECs. Activation of TERT or autophagy individually offset mitochondrial damage (MTT). Combined activation/inhibition failed to alleviate hyperoxic-induced mitochondrial damage in vitro, whereas activation of autophagy in vivo decreased mitochondrial damage (MTT) in both wild type (WT) and rats lacking TERT. Functionally, activation of either TERT or autophagy preserved transendothelial membrane resistance. Altogether, these observations show that activation of autophagy/mitophagy and/or TERT mitigate loss of mitochondrial function and barrier integrity in hyperoxia.NEW & NOTEWORTHY In cultured pulmonary artery endothelial cells and in lungs exposed in vivo to hyperoxia, autophagy is activated, but clearance of autophagosomes is impaired in a manner that suggests cross talk between TERT and autophagy. Stimulation of autophagy prevents hyperoxia-induced decreases in mitochondrial metabolism and sustains monolayer resistance. Hyperoxia increases mitochondrial outer membrane (TOMM20) protein, decreases mitochondrial function, and reduces cellular proliferation without increasing cell death.


Assuntos
Células Endoteliais/enzimologia , Hiperóxia/complicações , Lesão Pulmonar/enzimologia , Pulmão/irrigação sanguínea , Microvasos/enzimologia , Mitocôndrias/enzimologia , Mitofagia , Telomerase/metabolismo , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Permeabilidade Capilar , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Técnicas de Inativação de Genes , Mediadores da Inflamação/metabolismo , Lesão Pulmonar/etiologia , Lesão Pulmonar/genética , Lesão Pulmonar/patologia , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Microvasos/patologia , Mitocôndrias/genética , Mitocôndrias/patologia , Ratos Sprague-Dawley , Ratos Transgênicos , Receptores de Superfície Celular/metabolismo , Telomerase/deficiência , Telomerase/genética , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo
11.
FASEB J ; 35(10): e21941, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34559928

RESUMO

Thromboxane receptor (TP) mediates nasal obstruction, a typical symptom of allergic rhinitis. Since it has been reported that several types of eicosanoids, such as non-enzymatic oxidation product of arachidonic acid isoprostane, act as a TP ligand, there is a possibility that some other eicosanoids contribute to the TP-mediated nasal obstruction. The aim of this study is to investigate the mechanisms of TP-mediated nasal obstruction. Intranasal challenges of ovalbumin (OVA) induced nasal obstruction in mice. Pharmacological blockade of TP receptor but not thromboxane A2 synthase inhibited OVA-induced nasal obstruction. Simultaneous analysis of eicosanoids in nasal lavage fluid and the responses in trans-endothelial resistance suggested that 8-iso-prostaglandin E2 (PGE2 ) can be a candidate for TP ligand. Intranasal challenge of 8-iso-PGE2 induced vascular hyperpermeability and nasal obstruction in TP receptor-dependent manner. Wholemount immunostaining of nasal septum mucosa revealed that 8-iso-PGE2 increased plasma leakage accompanied by distention of venous sinusoids. This study shows that 8-iso-PGE2 is a contributor in TP-mediated nasal obstruction in mice.


Assuntos
Dinoprostona/análogos & derivados , Modelos Animais de Doenças , Isoprostanos/farmacologia , Obstrução Nasal/induzido quimicamente , Obstrução Nasal/complicações , Receptores de Tromboxanos/metabolismo , Rinite Alérgica/complicações , Rinite Alérgica/metabolismo , Administração Intranasal , Animais , Permeabilidade Capilar/efeitos dos fármacos , Dinoprostona/administração & dosagem , Dinoprostona/farmacologia , Feminino , Isoprostanos/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais/efeitos dos fármacos
12.
FASEB J ; 35(10): e21952, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34555210

RESUMO

Cyclophosphamide (CP) has been widely used in the treatment of various malignancies and autoimmune diseases, but acrolein, a byproduct of CP, causes severe hemorrhagic cystitis as the major side effect of CP. On the other hand, a large amount of prostacyclin (PGI2 ) is produced in bladder tissues, and PGI2 has been shown to play a critical role in bladder homeostasis. PGI2 is biosynthesized from prostaglandin (PG) H2 , the common precursor of PGs, by PGI2 synthase (PTGIS) and is known to also be involved in inflammatory responses. However, little is known about the roles of PTGIS-derived PGI2 in bladder inflammation including CP-induced hemorrhagic cystitis. Using both genetic and pharmacological approaches, we here revealed that PTGIS-derived PGI2 -IP (PGI2 receptor) signaling exacerbated CP-induced bladder inflammatory reactions. Ptgis deficiency attenuated CP-induced vascular permeability and chemokine-mediated neutrophil migration into bladder tissues and then suppressed hemorrhagic cystitis. Treatment with RO1138452, an IP selective antagonist, also suppressed CP-induced cystitis. We further found that cystitis-related nociceptive behavior was also relieved in both Ptgis-/- mice and RO1138452-treated mice. Our findings may provide new drug targets for bladder inflammation and inflammatory pain in CP-induced hemorrhagic cystitis.


Assuntos
Ciclofosfamida/efeitos adversos , Cistite/induzido quimicamente , Cistite/prevenção & controle , Epoprostenol/deficiência , Dor/prevenção & controle , Bexiga Urinária , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito , Cistite/complicações , Sistema Enzimático do Citocromo P-450/deficiência , Progressão da Doença , Epoprostenol/metabolismo , Feminino , Hemorragia/complicações , Hemorragia/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Tamanho do Órgão/efeitos dos fármacos , Dor/induzido quimicamente , Dor/complicações , Prostaglandina-E Sintases , Bexiga Urinária/efeitos dos fármacos
13.
Cells ; 10(9)2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34571834

RESUMO

We previously reported that claudin-5, a tight junctional protein, mediates lung vascular permeability in a murine model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Recently, it has been reported that haloperidol, an antipsychotic medication, dose-dependently increases expression of claudin-5 in vitro and in vivo, in brain endothelium. Notably, claudin-5 is highly expressed in both brain and lung tissues. However, the effects of haloperidol on EC barrier function are unknown. We hypothesized that haloperidol increases lung EC claudin-5 expression and attenuates agonist-induced lung EC barrier disruption. Human pulmonary artery ECs were pretreated with haloperidol at variable concentrations (0.1-10 µM) for 24 h. Cell lysates were subjected to Western blotting for claudin-5, in addition to occludin and zona occludens-1 (ZO-1), two other tight junctional proteins. To assess effects on barrier function, EC monolayers were pretreated for 24 h with haloperidol (10 µM) or vehicle prior to treatment with thrombin (1 U/mL), with measurements of transendothelial electrical resistance (TER) recorded as a real-time assessment of barrier integrity. In separate experiments, EC monolayers grown in Transwell inserts were pretreated with haloperidol (10 µM) prior to stimulation with thrombin (1 U/mL, 1 h) and measurement of FITC-dextran flux. Haloperidol significantly increased claudin-5, occludin, and ZO-1 expression levels. Measurements of TER and FITC-dextran Transwell flux confirmed a significant attenuation of thrombin-induced barrier disruption associated with haloperidol treatment. Finally, mice pretreated with haloperidol (4 mg/kg, IP) prior to the intratracheal administration of LPS (1.25 mg/kg, 16 h) had increased lung claudin-5 expression with decreased lung injury as assessed by bronchoalveolar lavage (BAL) fluid protein content, total cell counts, and inflammatory cytokines, in addition to lung histology. Our data confirm that haloperidol results in increased claudin-5 expression levels and demonstrates lung vascular-protective effects both in vitro and in vivo in a murine ALI model. These findings suggest that haloperidol may represent a novel therapy for the prevention or treatment of ALI and warrants further investigation in this context.


Assuntos
Células Endoteliais/efeitos dos fármacos , Haloperidol/farmacologia , Pulmão/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Impedância Elétrica , Células Endoteliais/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo
14.
Phytother Res ; 35(11): 6389-6400, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34541711

RESUMO

Anti-angiogenic medicines have been evaluated as anticancer therapies, however, their use remains limited in clinical practice due to associated adverse effects. Asiatic acid (AA) is known to have broad-spectrum anticancer properties, however, its effects on angiogenesis in breast cancer remain to be fully established. In this study, we analyzed the inhibitory effects of AA on angiogenesis using human umbilical vein endothelial cells (HUVECs) cultured in vitro and on the growth and metastasis of a subcutaneous breast cancer 4T1 tumor model and a lung metastasis model in vivo. AA significantly inhibited HUVECs proliferation, migration, and tube formation in vitro. In vivo, AA significantly reduced the microvascular density and blood vascular permeability in breast cancer tumors and inhibited growth and lung metastasis. AA inhibited the expression of vascular endothelial growth factor (VEGF) in HUVECs and subsequently downregulated the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream target proteins including ERK1/2, Src, and FAK. These results indicate that AA significantly inhibits angiogenesis and blood vessel permeability through the VEGF/VEGFR2 signal axis to inhibit the growth and metastasis of breast cancer. Our data strongly demonstrate the potential applications of AA in the treatment of breast cancer.


Assuntos
Neoplasias da Mama , Fator A de Crescimento do Endotélio Vascular , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Neoplasias da Mama/tratamento farmacológico , Permeabilidade Capilar , Movimento Celular , Proliferação de Células , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Neovascularização Patológica/tratamento farmacológico , Triterpenos Pentacíclicos , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
15.
Am J Physiol Renal Physiol ; 321(5): F600-F616, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34541901

RESUMO

Following our previous reports on mesangial sclerosis and vascular proliferation in diabetic nephropathy (DN) (Kriz W, Löwen J, Federico G, van den Born J, Gröne E, Gröne HJ. Am J Physiol Renal Physiol 312: F1101-F1111, 2017; Löwen J, Gröne E, Gröne HJ, Kriz W. Am J Physiol Renal Physiol 317: F399-F410, 2019), we now describe the advanced stages of DN terminating in glomerular obsolescence and tubulointerstitial fibrosis based on a total of 918 biopsies. The structural aberrations emerged from two defects: 1) increased synthesis of glomerular basement membrane (GBM) components by podocytes and endothelial cells leading to an accumulation of GBM material in the mesangium and 2) a defect of glomerular vessels consisting of increased leakiness and an increased propensity to proliferate. Both defects may lead to glomerular degeneration. The progressing compaction of accumulated worn-out GBM material together with the retraction of podocytes out of the tuft and the collapse and hyalinosis of capillaries results in a shrunken tuft that fuses with Bowman's capsule (BC) to glomerular sclerosis. The most frequent pathway to glomerular decay starts with local tuft expansions that result in contacts of structurally intact podocytes to the parietal epithelium initiating the formation of tuft adhesions, which include the penetration of glomerular capillaries into BC. Exudation of plasma from such capillaries into the space between the parietal epithelium and its basement membrane causes the formation of insudative fluid accumulations within BC spreading around the glomerular circumference and, via the glomerulotubular junction, onto the tubule. Degeneration of the corresponding tubule develops secondarily to the glomerular damage, either due to cessation of filtration in cases of global sclerosis or due to encroachment of the insudative spaces. The degenerating tubules induce the proliferation of myofibroblasts resulting in interstitial fibrosis.NEW & NOTEWORTHY Based on analysis of 918 human biopsies, essential derangement in diabetic nephropathy consists of accumulation of worn-out glomerular basement membrane in the mesangium that may advance to global sclerosis. The most frequent pathway to nephron dropout starts with the penetration of glomerular capillaries into Bowman's capsule (BC), delivering an exudate into BC that spreads around the entire glomerular circumference and via the glomerulotubular junction onto the tubule, resulting in glomerular sclerosis and chronic tubulointerstitial damage.


Assuntos
Nefropatias Diabéticas/patologia , Glomerulonefrite/patologia , Néfrons/patologia , Biópsia , Cápsula Glomerular/patologia , Capilares/patologia , Permeabilidade Capilar , Nefropatias Diabéticas/metabolismo , Progressão da Doença , Células Endoteliais/patologia , Fibrose , Membrana Basal Glomerular/patologia , Glomerulonefrite/metabolismo , Humanos , Microscopia Eletrônica de Transmissão , Neovascularização Patológica , Néfrons/metabolismo , Néfrons/ultraestrutura , Podócitos/patologia
16.
Dis Model Mech ; 14(11)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34542605

RESUMO

Vascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, including cancer, stroke, cardiovascular disease, retinal conditions and COVID-19-associated pulmonary edema, sepsis and acute lung injury. Understanding temporal molecular regulation of VEGF-induced vascular permeability will facilitate developing therapeutics to inhibit vascular permeability, while preserving tissue-restorative angiogenesis. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. We show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9-generated Stat3 knockout zebrafish. Intercellular adhesion molecule 1 (ICAM-1) expression is transcriptionally regulated by STAT3, and VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved antimicrobial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse and human endothelium. Collectively, our findings suggest that VEGF/VEGFR-2/JAK2/STAT3 signaling regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability. This article has an associated First Person interview with the first author of the paper.


Assuntos
Permeabilidade Capilar , Endotélio Vascular/metabolismo , Fator de Transcrição STAT3/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Sistemas CRISPR-Cas , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Janus Quinase 2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Peixe-Zebra
17.
Am J Physiol Cell Physiol ; 321(4): C671-C680, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34469202

RESUMO

Hypoxia-induced pulmonary microvascular endothelial cell (PMVEC) monolayers hyperpermeability is vital for vascular leakage, which participates in vascular diseases, such as acute lung injury (ALI) and high-altitude pulmonary edema (HAPE). We previously observed that PMVEC permeability was markedly elevated in hypoxia when cocultured with primary type II alveolar epithelial cells (AECII) in which isthmin1 (ISM1) was highly upregulated. However, whether the upregulation of ISM1 plays a role in hypoxia-induced PMVEC hyperpermeability is unclear. In this study, we assessed the role of AECII-derived ISM1 in hypoxia-induced PMVEC hyperpermeability with an AECII/PMVEC coculture system and uncovered the underlying mechanism whereby hypoxia stimulates ISM1 gene expression. We found that ISM1 gene expression was upregulated in cultured AECII cells exposed to hypoxia (3% O2) and that AECII-derived ISM1 participated in hypoxia-induced hyperpermeability of PMVEC monolayers, as small interference RNA (siRNA)-mediated knockdown of ISM1 in AECII markedly attenuated the increase in PMVEC permeability in coculture system under hypoxia. In addition, we confirmed that ISM1 was regulated by hypoxia-inducible factor-1α (HIF1α) according to the evidence that silencing of HIF1α inhibited the hypoxia-mediated upregulation of ISM1. Mechanismly, overexpression of HIF1α transcriptionally activated ISM1 gene expression by directly binding to the conserved regulatory elements upstream of the ism1 locus. We identified a novel HIF-1-target gene ISM1, which involves in hyperpermeability of pulmonary microvascular endothelial cell monolayers under hypoxia. Our in vitro cell experiments implied that the upregulated ISM1 derived from alveolar epithelium might be a vital modulator in hypoxia-induced endothelial hyperpermeability and thereby implicates with hypoxic pulmonary-related diseases.


Assuntos
Células Epiteliais Alveolares/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pulmão/irrigação sanguínea , Microvasos/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , Técnicas de Cocultura , Impedância Elétrica , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos Endogâmicos C57BL , Comunicação Parácrina , Soroalbumina Bovina/metabolismo , Transdução de Sinais , Ativação Transcricional , Regulação para Cima
19.
Am J Physiol Regul Integr Comp Physiol ; 321(5): R732-R741, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34549626

RESUMO

Although hypertension disrupts the blood-brain barrier (BBB) integrity within the paraventricular nucleus of hypothalamus (PVN) and increases the leakage into the brain parenchyma, exercise training (T) was shown to correct it. Since there is scarce and contradictory information on the mechanism(s) determining hypertension-induced BBB deficit and nothing is known about T-induced improvement, we sought to evaluate the paracellular and transcellular transport across the BBB within the PVN in both conditions. Spontaneously hypertensive rats (SHR) and WKY submitted to 4-wk aerobic T or sedentary (S) protocol were chronically catheterized for hemodynamic recordings at rest and intra-arterial administration of dyes (Rhodamine-dextran 70 kDa + FITC-dextran 10 kDa). Brains were harvesting for FITC leakage examination, qPCR evaluation of different BBB constituents and protein expression of caveolin-1 and claudin-5, the main markers of transcytosis and paracellular transport, respectively. Hypertension was characterized by increased arterial pressure and heart rate, augmented sympathetic modulation of heart and vessels, and reduced cardiac parasympathetic control, marked FITC extravasation into the PVN which was accompanied by increased caveolin-1 gene and protein expression, without changes in claudin-5 and others tight junctions' components. SHR-T vs. SHR-S showed a partial pressure reduction, resting bradycardia, improvement of autonomic control of the circulation simultaneously with correction of both FITC leakage and caveolin-1 expression; there was a significant increase in claudin-5 expression. Caveolin-1 content was strongly correlated with improved autonomic control after exercise. Data indicated that within the PVN the transcytosis is the main mechanism governing both hypertension-induced BBB leakage, as well as the exercise-induced correction.


Assuntos
Barreira Hematoencefálica/metabolismo , Capilares/metabolismo , Permeabilidade Capilar , Caveolina 1/metabolismo , Claudina-5/metabolismo , Terapia por Exercício , Hipertensão/terapia , Núcleo Hipotalâmico Paraventricular/irrigação sanguínea , Condicionamento Físico Animal , Junções Íntimas/metabolismo , Transcitose , Animais , Barreira Hematoencefálica/fisiopatologia , Capilares/fisiopatologia , Sistema Cardiovascular/inervação , Caveolina 1/genética , Claudina-5/genética , Modelos Animais de Doenças , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Esforço Físico , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Sistema Nervoso Simpático/fisiopatologia
20.
J Cell Sci ; 134(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34345888

RESUMO

Dysfunction of vascular barriers is a critical step in inflammatory diseases. Endothelial tight junctions (TJs) control barrier function, and the cytoplasmic adaptor protein cingulin connects TJs to signalling pathways. However, local events at TJs during inflammation are largely unknown. In this study, we investigate the local response of TJ adaptor protein cingulin and its interaction with Rho guanine nucleotide exchange factor H1 (GEF-H1, also known as ARHGEF2) upon vascular barrier disruption to find a new approach to counteract vascular leak. Based on transendothelial-electrical-resistance (TEER) measurements, cingulin strengthened barrier integrity upon stimulation with histamine, thrombin and VEGF. Cingulin also attenuated myosin light chain 2 (MLC2; also known as MYL2) phosphorylation by localising GEF-H1 to cell junctions. By using cingulin phosphomutants, we verified that the phosphorylation of the cingulin head domain is required for its protective effect. Increased colocalisation of GEF-H1 and cingulin was observed in the vessels of vasculitis patients compared to those in healthy skin. Our findings demonstrate that cingulin can counteract vascular leak at TJs, suggesting the existence of a novel mechanism in blood endothelial cells that protects barrier function during disease.


Assuntos
Células Endoteliais , Junções Íntimas , Permeabilidade Capilar , Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Junções Íntimas/metabolismo
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