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1.
Microvasc Res ; 133: 104103, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33181170

RESUMO

Diabetic retinopathy (DR) is a disease that causes blindness due to vascular leakage or abnormal angiogenesis. Hepatocyte growth factor (HGF) is increased in the serum or vitreous fluid in proliferative diabetic retinopathy (PDR) patients, although the effect of HGF on the blood vessels remains unclear. This study focused on the effect of HGF on pericyte (PC) survival and endothelial cell (EC) permeability. It was demonstrated that HGF was increased in the diabetic mouse retina. However, HGF prevented PC apoptosis caused by TNF-α, which increased in the diabetic retinas both in vitro and in vivo. In addition, HGF was involved in PC survival by increasing the Akt signaling pathway. Moreover, HGF strengthened the EC tight junction in co-cultures of PCs and ECs by promoting PC survival, thereby reducing EC permeability. These results suggest that HGF may play a role in the prevention of increased vascular leakage by inhibiting the PC loss that occurs in DR to some extent. However, careful HGF reduction in DR might avoid an increase in PC loss.


Assuntos
Apoptose/efeitos dos fármacos , Retinopatia Diabética/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Pericitos/efeitos dos fármacos , Vasos Retinianos/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pericitos/metabolismo , Pericitos/patologia , Permeabilidade , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/patologia
2.
J Vis Exp ; (161)2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32804163

RESUMO

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.


Assuntos
Arteríolas/diagnóstico por imagem , Capilares/diagnóstico por imagem , Imageamento Tridimensional , Pericitos/fisiologia , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Capilares/efeitos dos fármacos , Capilares/fisiologia , Cateterismo , Corantes Fluorescentes/metabolismo , Hemodinâmica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Perfusão , Pericitos/efeitos dos fármacos , Pinacidil/farmacologia , Pressão , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Aglutininas do Germe de Trigo/metabolismo
3.
Clin Hemorheol Microcirc ; 75(3): 313-323, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32224529

RESUMO

It has been reported that the beta-adrenergic receptor blocker (propranolol) and the a-adrenergic receptor (AR) blocker (phentolamine) both can inhibit human endothelial cell (EC) angiogenesis in vitro. However, it is unknown whether this inhibition also acts on pericytes. The present study aimed to determine how pericytes react to treatment with an a-/ß- AR blocker. In the study, cell proliferation assays and scratch assay were performed to assess the effect of phentolamine or propranolol on cell proliferation and migration. Western blot and ELISA were employed to determine changes in VEGF-A and Ang-1 expression levels. The results indicated that the nonselective a-/ß- AR blocker inhibited the proliferation, migration, and secretion of pericytes. The use of the nonselective a-/ß- AR blocker might have an impact on vascularization and vascular maturation. Our research suggests the rational use of nonselective a-/ß- AR blockers to treat angiogenesis-dependent diseases.


Assuntos
Antagonistas Adrenérgicos alfa/uso terapêutico , Antagonistas Adrenérgicos beta/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Pericitos/efeitos dos fármacos , Antagonistas Adrenérgicos alfa/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Movimento Celular , Proliferação de Células , Humanos
4.
Sci Rep ; 10(1): 2939, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076044

RESUMO

Anti-vascular endothelial growth factor (VEGF) therapy shows antitumor activity against various types of solid cancers. Several resistance mechanisms against anti-VEGF therapy have been elucidated; however, little is known about the mechanisms by which the acquired resistance arises. Here, we developed new anti-VEGF therapy-resistant models driven by chronic expression of the mouse VEGFR2 extracellular domain fused with the human IgG4 fragment crystallizable (Fc) region (VEGFR2-Fc). In the VEGFR2-Fc-expressing resistant tumors, we demonstrated that the FGFR2 signaling pathway was activated, and pericytes expressing high levels of FGF2 were co-localized with endothelial cells. Lenvatinib, a multiple tyrosine kinase inhibitor including VEGFR and FGFR inhibition, showed marked antitumor activity against VEGFR2-Fc-expressing resistant tumors accompanied with a decrease in the area of tumor vessels and suppression of phospho-FGFR2 in tumors. Our findings reveal the key role that intercellular FGF2 signaling between pericytes and endothelial cells plays in maintaining the tumor vasculature in anti-VEGF therapy-resistant tumors.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Transdução de Sinais , Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Modelos Biológicos , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo
5.
Sci Rep ; 10(1): 2477, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051471

RESUMO

Oxidative stress has been associated with the etipathogenesis of Diabetic retinopathy (DR). Studies have shown that DJ-1 plays an important role in regulating the reactive oxygen species (ROS) production and resistance to oxidative stress-induced apoptosis. This study aimed to investigate whether DJ-1 upregulates oxidative stress and prevents damage to retinal capillary pericytes by increasing antioxidant capacity through the Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Nrf2 is a redox-sensitive transcription factor that encode antioxidant enzymes and phase II metabolic enzymes, activation of Nrf2 functions is one of the critical defensive mechanisms against oxidative stress in many tissues. Our results showed after DJ-1 overexpression, apoptosis of rat retinal pericytes (RRPs) decreased, the ratio of B-cell lymphoma-2 (Bcl-2) to BCL2-Associated X Protein (BAX) increased, the production of ROS decreased, and the protein expression and activity of manganese superoxide dismutase (MnSOD, also called SOD2) and catalase (CAT) increased. DJ-1 overexpression activated Nrf2 expression, however, after Nrf2 silencing, apoptosis of RRPs increased, the ratio of Bcl-2 to BAX decreased, the production of ROS increased, the protein expression of MnSOD and CAT decreased, and the expression of heme oxygenase-1 (HO-1), NADP(H) quinone oxidoreductase (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) decreased. These data suggest that enhancement of the Nrf2 pathway is a potential protective strategy for the treatment of DR. Therefore, DJ-1 may prevent high glucose-induced oxidative stress and RRPs apoptosis through the Nrf2 signaling pathway, thereby preventing the early onset and progression of DR.


Assuntos
Retinopatia Diabética/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Pericitos/metabolismo , Proteína Desglicase DJ-1/metabolismo , Animais , Apoptose , Catalase/genética , Catalase/metabolismo , Células Cultivadas , Glucose/metabolismo , Glucose/toxicidade , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , NADP/genética , NADP/metabolismo , Fator 2 Relacionado a NF-E2/genética , Pericitos/efeitos dos fármacos , Proteína Desglicase DJ-1/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Regulação para Cima , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
6.
Sci Rep ; 10(1): 977, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969665

RESUMO

As a clinical manifestations of diabetic retinopathy (DR), pericytes (PCs) loss from the capillary walls is thought to be an initial pathological change responsible for the breakdown of the blood-retinal barrier (BRB). This study was performed to investigate the effects of ursodeoxycholic acid (UDCA) in PC depletion mice by injection of an antibody against platelet-derived growth factor reception-ß (PDGFR-ß clone APB5). To assess the integrity of the retinal vessels, their density, diameters, vessel branching points, and number of acellular capillaries were evaluated. While all types of retinal vessels became enlarged in APB5-induced mice, treatment with UDCA rescued the vasculature; the vessel density, diameter of the veins and capillaries, and vessel branching points were significantly lower in mice treated with UDCA. Although APB5-induced mice displayed progressive exacerbation of retinal edema, whole retinal thickness upon treatment with UDCA was significantly decreased. Additionally, UDCA reduced the expression of F4/80+ macrophages in the APB5-induced retina according to immunofluorescent labeling. UDCA also reduced the increased expression of angiogenic factors and inflammatory mediators (vascular endothelial growth factor, intercellular adhesion molecule-1, and monocyte chemotactic protein-1). These findings suggest that UDCA can be used to prevent the progression of and treat DR.


Assuntos
Retinopatia Diabética/tratamento farmacológico , Pericitos/efeitos dos fármacos , Vasos Retinianos/efeitos dos fármacos , Ácido Ursodesoxicólico/uso terapêutico , Animais , Quimiocina CCL2/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Pericitos/metabolismo , Pericitos/patologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Ácido Ursodesoxicólico/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
J Endocrinol ; 245(1): 79-92, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31999623

RESUMO

Reproductive tract inflammatory disease (RTID) commonly occurs after the traumatic events of parturition and adversely affects follicular function. This study is the first to describe the cellular and steroidogenic characteristics of corpora lutea from cattle with RTID and the effects of pathogen-associated molecular patterns (PAMPs) on luteal angiogenesis and function in vitro. Luteal weight (P < 0.05) and progesterone content (P < 0.05) were reduced (1.2-fold) in cows with RTID, accompanied by reduced CYP11A (P < 0.05), HSD3B (P < 0.01) and STAR (P < 0.01) protein expression. Immunohistochemistry revealed that luteal vascularity (VWF) and pericyte (ACTA2) coverage were >3-fold lower in RTID cows (P < 0.05). To link these observations to bacterial infection and determine specificity of action, a physiologically relevant luteal angiogenesis culture system examined the effects of PAMPs on endothelial cell (EC) network formation and progesterone production, in the presence of pro-angiogenic factors. Luteal EC networks were reduced ≤95% (P < 0.05) by lipopolysaccharide (LPS, toll-like receptor (TLR) 4 agonist) but not by TLR2 agonists lipoteichoic acid or peptidoglycan. Conversely, progesterone production and steroidogenic protein expression were unaffected by PAMPs (P > 0.05). Moreover, the adverse effect of LPS on luteal EC networks was dose-dependent and effective from 1 ng/mL (P < 0.05), while few EC networks were present above 10 ng/mL LPS (P < 0.001). LPS reduced proliferation (P < 0.05) and increased apoptosis of EC (P < 0.001). The specific TLR4 inhibitor TAK242 reversed the effects of LPS on EC networks. In conclusion, luteal vasculature is adversely sensitive to LPS acting via TLR4, therefore ovarian exposure to LPS from any Gram-negative bacterial infection will profoundly influence subsequent reproductive potential.


Assuntos
Corpo Lúteo/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Células Lúteas/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Doenças Uterinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Bovinos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Corpo Lúteo/irrigação sanguínea , Corpo Lúteo/metabolismo , Família 11 do Citocromo P450/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Células Lúteas/metabolismo , Padrões Moleculares Associados a Patógenos/metabolismo , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Receptor 4 Toll-Like/metabolismo , Doenças Uterinas/fisiopatologia
8.
Neurochem Res ; 45(2): 310-321, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31776970

RESUMO

Docosahexaenoic acid (DHA) can alleviate cerebral ischemia/reperfusion injury by reducing blood-brain barrier permeability and maintaining its integrity, accompanied by an increased Ang-1/Ang-2 ratio; however, the underlying mechanisms of these effects remain unclear. Src-suppressed C kinase substrates (SSeCKS), a substrate of protein kinase C, plays an important role in maintaining cell junctions and cell morphology and regulating cell permeability. However, whether DHA can increase SSeCKS expression and then mediate the Ang-1/Ang-2 ratio still needs to be studied. Human cerebrovascular pericytes (HBVPs) cultured in vitro were divided into groups, treated with or without DHA along with SSeCKS siRNA to knockdown SSeCKS expression, and then subjected to 24 h of hypoxia followed by 6 h of reoxygenation. Cell viability; lactate dehydrogenase (LDH) release; and Ang-1, Ang-2 and VEGF activity were detected by using ELISA kits. The apoptosis rate was assessed by TUNEL flow cytometry. Expression of the SSeCKS, Ang-1, Ang-2 and VEGF proteins was evaluated by western blotting. Pretreatment with 10 µM or 40 µM DHA efficiently attenuated hypoxia/reoxygenation (H/R) injury by activating SSeCKS to increase the Ang-1/Ang-2 ratio and downregulate VEGF expression in HBVPs, as evidenced by decreased LDH release and apoptotic rates and increased HBVPs viability. Meanwhile, after we used SSeCKS siRNA to knock down SSeCKS protein expression, the protective effect of DHA on HBVPs following H/R injury was reversed. In conclusion, DHA can activate SSeCKS to increase the Ang-1/Ang-2 ratio and downregulate VEGF expression in HBVPs, thus reducing H/R injury.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Hipóxia Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Pericitos/efeitos dos fármacos , Proteínas de Ancoragem à Quinase A/genética , Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Apoptose/efeitos dos fármacos , Encéfalo/citologia , Proteínas de Ciclo Celular/genética , Hipóxia Celular/fisiologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Diab Vasc Dis Res ; 17(1): 1479164119878427, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31726874

RESUMO

Thiamine prevents high glucose-induced damage in microvasculature, and progression of retinopathy and nephropathy in diabetic animals. Impaired thiamine availability causes renal damage in diabetic patients. Two single-nucleotide polymorphisms in SLC19A3 locus encoding for thiamine transporter 2 are associated with absent/minimal diabetic retinopathy and nephropathy despite long-term type 1 diabetes. We investigated the involvement of thiamine transporter 1 and thiamine transporter 2, and their transcription factor specificity protein 1, in high glucose-induced damage and altered thiamine availability in cells of the inner blood-retinal barrier. Human endothelial cells, pericytes and Müller cells were exposed to hyperglycaemic-like conditions and/or thiamine deficiency/over-supplementation in single/co-cultures. Expression and localization of thiamine transporter 1, thiamine transporter 2 and transcription factor specificity protein 1 were evaluated together with intracellular thiamine concentration, transketolase activity and permeability to thiamine. The effects of thiamine depletion on cell function (viability, apoptosis and migration) were also addressed. Thiamine transporter 2 and transcription factor specificity protein 1 expression were modulated by hyperglycaemic-like conditions. Transketolase activity, intracellular thiamine and permeability to thiamine were decreased in cells cultured in thiamine deficiency, and in pericytes in hyperglycaemic-like conditions. Thiamine depletion reduced cell viability and proliferation, while thiamine over-supplementation compensated for thiamine transporter 2 reduction by restoring thiamine uptake and transketolase activity. High glucose and reduced thiamine determine impairment in thiamine transport inside retinal cells and through the inner blood-retinal barrier. Thiamine transporter 2 modulation in our cell models suggests its major role in thiamine transport in retinal cells and its involvement in high glucose-induced damage and impaired thiamine availability.


Assuntos
Retinopatia Diabética/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Ependimogliais/efeitos dos fármacos , Glucose/toxicidade , Proteínas de Membrana Transportadoras/metabolismo , Pericitos/efeitos dos fármacos , Vasos Retinianos/efeitos dos fármacos , Tiamina/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Microambiente Celular , Técnicas de Cocultura , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Humanos , Proteínas de Membrana Transportadoras/genética , Pericitos/metabolismo , Pericitos/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Transcetolase/metabolismo
10.
Cell Mol Neurobiol ; 40(1): 113-121, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31414300

RESUMO

Metastasis of lung cancer to the brain is associated with poor outcomes and limited therapeutic options. The blood-brain barrier (BBB) plays a major role in brain metastasis. However, little is known about the role of pericytes in brain metastasis formation. This study aimed to reveal the interaction between pericytes and cancer cells. We established in vitro BBB models with rat primary cultured BBB-related cells (endothelial cells, astrocytes, and pericytes) and investigated the relationship between BBB-related cells and metastatic cancer cell lines. We observed a significant decrease in transendothelial electrical resistance with metastatic cancer cells in monolayer and coculture models with astrocytes. In contrast, the coculture model with pericytes showed inhibition of the decrease in transendothelial electrical resistance with metastatic cancer cells. In addition, the expression of tight junction protein was preserved only in the coculture model with pericytes. The conditioned medium of pericytes with metastatic cancer cells suppressed the proliferation of the cancer cells significantly. This study revealed that brain pericytes are the major regulators of the resistance of the BBB to lung cancer metastasis to the brain. Pericytes exert an anti-metastatic effect and thus have potential for the preventive treatment of brain metastasis.


Assuntos
Neoplasias Encefálicas/secundário , Neoplasias Pulmonares/patologia , Pericitos/patologia , Células A549 , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Impedância Elétrica , Humanos , Pericitos/efeitos dos fármacos , Ratos , Proteínas de Junções Íntimas/metabolismo
11.
Cardiovasc Res ; 116(3): 686-697, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173066

RESUMO

AIMS: The progressive accumulation of cells in pulmonary vascular walls is a key pathological feature of pulmonary arterial hypertension (PAH) that results in narrowing of the vessel lumen, but treatments targeting this mechanism are lacking. The C-X-C motif chemokine 12 (CXCL12) appears to be crucial in these processes. We investigated the activity of two CXCL12 neutraligands on experimental pulmonary hypertension (PH), using two complementary animal models. METHODS AND RESULTS: Male Wistar rats were injected with monocrotaline (MCT) or were subjected to SU5416 followed by 3-week hypoxia to induce severe PH. After PH establishment, assessed by pulsed-wave Doppler echocardiography, MCT-injected or SU5416 plus chronic hypoxia (SuHx) rats were randomized to receive CXCL12 neutraligands chalcone 4 or LIT-927 (100 mg/kg/day), the C-X-C motif chemokine receptor 4 (CXCR4) antagonist AMD3100 (5 mg/kg/day), or vehicle, for 2 or 3 weeks, respectively. At the end of these treatment periods, echocardiographic and haemodynamic measurements were performed and tissue samples were collected for protein expression and histological analysis. Daily treatment of MCT-injected or SuHx rats with established PH with chalcone 4 or LIT-927 partially reversed established PH, reducing total pulmonary vascular resistance, and remodelling of pulmonary arterioles. Consistent with these observations, we found that neutralization of CXCL12 attenuates right ventricular hypertrophy, pulmonary vascular remodelling, and decreases pulmonary artery smooth muscle cell (PA-SMC) proliferation in lungs of MCT-injected rats and SuHx rats. Importantly, CXCL12 neutralization with either chalcone 4 or LIT-927 inhibited the migration of PA-SMCs and pericytes in vitro with a better efficacy than AMD3100. Finally, we found that CXCL12 neutralization decreases vascular pericyte coverage and macrophage infiltration in lungs of both MCT-injected and SuHx rats. CONCLUSION: We report here a greater beneficial effect of CXCL12 neutralization vs. the conventional CXCR4 blockade with AMD3100 in the MCT and SuHx rat models of severe PH, supporting a role for CXCL12 in the progression of vascular complications in PH and opening to new therapeutic options.


Assuntos
Chalconas/farmacologia , Quimiocina CXCL2/antagonistas & inibidores , Hipertensão Pulmonar/tratamento farmacológico , Artéria Pulmonar/efeitos dos fármacos , Pirimidinonas/farmacologia , Remodelação Vascular/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL2/metabolismo , Modelos Animais de Doenças , Compostos Heterocíclicos/farmacologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Pericitos/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Ratos Wistar , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Transdução de Sinais
12.
Aging (Albany NY) ; 11(23): 11391-11415, 2019 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-31811815

RESUMO

The pathophysiological mechanism of white matter hyperintensities of cerebral small vessel disease (CSVD) includes an impaired blood-brain barrier (BBB) with increased permeability. Neuroinflammation likely contributes to the disruption of the BBB in CSVD. Therefore, understanding the molecular mechanism of how neuroinflammation causes BBB damage is essential to preventing BBB disruption in CSVD. Matrix metalloproteinase 9 (MMP-9) contributes to BBB damage in neuroinflammatory diseases. In this study, we observed that interleukin-1ß (IL-1ß)-induced MMP-9 secretion in pericytes increased BBB permeability to sodium fluorescein (Na-F) by damaging the disruption of VE-cadherin, occludin, claudin-5, and zonula occludin-1 (ZO-1). Melatonin reduced BBB permeability to Na-F and inhibited the disruption of the adherens and tight junction proteins. Melatonin also downregulated MMP-9 and upregulated tissue inhibitor of metalloproteinases 1 (TIMP-1) gene expression, which decreased the MMP-9/TIMP-1 ratio. In addition, nuclear translocation of NF-κB/p65 induced by IL-1ß in pericytes upregulated MMP-9 expression, which was inhibited by the NF-κB inhibitor PDTC. However, the NOTCH3 inhibitor DAPT significantly inhibited NF-κB/p65 translocation to the nucleus, while melatonin in combination with DAPT significantly prevented NF-κB/p65 translocation than DAPT alone. Our results suggest that melatonin reduced MMP-9-induced permeability of the BBB. Melatonin reduced MMP-9 expression and activity, which was induced by IL-1ß through the regulation of the NOTCH3/NF-κB signaling pathway in pericytes, suggesting that pericytes regulate BBB integrity and function.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Melatonina/farmacologia , NF-kappa B/metabolismo , Receptor Notch3/metabolismo , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/fisiologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 9 da Matriz/genética , NF-kappa B/genética , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Permeabilidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch3/genética , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo
13.
Neuroscience ; 422: 12-20, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31705893

RESUMO

Oncostatin M (OSM) is a cytokine of the interleukin (IL)-6 family members. It induces blood-brain barrier (BBB) dysfunction by activating Janus-activated kinase (JAK) and signal transducer and activator of transcription (STAT) 3 pathways in brain endothelial cells. Brain pericytes located around microvessels are one of the BBB constituents. Pericytes work as a boundary surface between the blood circulation and brain parenchyma, and their functions are altered under pathophysiological conditions, leading to BBB dysregulation. However, it remains unknown whether pericytes are associated with OSM-induced BBB dysfunction. We demonstrated that pericyte exposure to OSM (100 ng/mL) elevated phosphorylation of STAT3, a main OSM signaling pathway, and that pericytes expressed OSM receptors (OSMRs) including OSMRß and glycoprotein 130. These results suggest that pericytes are able to respond to OSM. To determine the effects of OSM-reactive pericytes on BBB functions, rat brain endothelial cell (RBEC) monolayers were cultured with OSM-treated pericytes. The presence of pericytes exposed to 100 ng/mL of OSM for 48 h aggravated both the elevated permeability to sodium fluorescein and the lowered transendothelial electrical resistance which were induced by OSM in RBECs. This OSM-reactive pericyte-induced aggravation of lowered RBEC barrier function was reversed by ruxolitinib, a JAK inhibitor. These findings suggest that activated JAK/STAT3 signaling in pericytes contributes to OSM-produced BBB breakdown. Thus, OSM-reactive pericytes may have to be considered a characteristic machinery in the formation and progression of BBB breakdown under pathological conditions associated with increased OSM levels.


Assuntos
Barreira Hematoencefálica/fisiopatologia , Janus Quinases/metabolismo , Oncostatina M/farmacologia , Oncostatina M/fisiologia , Fator de Transcrição STAT3/metabolismo , Animais , Receptor gp130 de Citocina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Oncostatina M/antagonistas & inibidores , Subunidade beta de Receptor de Oncostatina M/metabolismo , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Pirazóis/farmacologia , Ratos , Transdução de Sinais
14.
Proc Natl Acad Sci U S A ; 116(47): 23551-23561, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31685607

RESUMO

Angiogenesis frequently occurs in the context of acute or persistent inflammation. The complex interplay of proinflammatory and proangiogenic cues is only partially understood. Using an experimental model, permitting exposure of developing blood vessel sprouts to multiple combinations of diverse biochemical stimuli and juxtacrine cell interactions, we present evidence that a proinflammatory cytokine, tumor necrosis factor (TNF), can have both proangiogenic and antiangiogenic effects, depending on the dose and the presence of pericytes. In particular, we find that pericytes can rescue and enhance angiogenesis in the presence of otherwise-inhibitory high TNF doses. This sharp switch from proangiogenic to antiangiogenic effect of TNF observed with an escalating dose of this cytokine, as well as the effect of pericytes, are explained by a mathematical model trained on the biochemical data. Furthermore, this model was predictive of the effects of diverse combinations of proinflammatory and antiinflammatory cues, and variable pericyte coverage. The mechanism supports the effect of TNF and pericytes as modulating signaling networks impinging on Notch signaling and specification of the Tip and Stalk phenotypes. This integrative analysis elucidates the plasticity of the angiogenic morphogenesis in the presence of diverse and potentially conflicting cues, with immediate implications for many physiological and pathological settings.


Assuntos
Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Pericitos/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Comunicação Celular , Técnicas de Cultura de Células , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação , Lisofosfolipídeos/farmacologia , Modelos Biológicos , Neovascularização Patológica/patologia , Pericitos/efeitos dos fármacos , Receptores Notch/fisiologia , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Engenharia Tecidual , Fator de Necrose Tumoral alfa/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/fisiologia
15.
Metabolism ; 101: 153998, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31666193

RESUMO

BACKGROUND: The incidence of growth hormone deficiency (GHD) in adamantinomatous craniopharyngioma (aCP) is significantly higher than in other sellar region tumors, but the possible mechanism is still elusive. A high level of inflammatory responses is another feature of aCP. We investigated the internal connection between interleukin-1α (IL-1α) and GHD, while focusing on its biological activities in pituitary fibrosis. MATERIALS AND METHODS: To diagnosis of GHD, the Body Mass Index (BMI), Insulin Like Growth Factor-1(IGF-1) and peak growth hormone (GH) values after insulin stimulation test of 15 aCP patients were recorded. Histological staining was performed on the aCP samples. Levels of 9 proinflammatory cytokines in tumor tissue and cell supernatant were detected using Millipore bead arrays. The effect of IL-1α on GH secretion was evaluated in vivo and in vitro. Western blot, qRT-PCR and cell functional assays were used to explore the potential mechanism through which IL-1α acts on GH secretion. The stereotactic ALZET osmotic pump technique was used to simulate aCP secretion of proinflammatory cytokines in rats. Recombinant IL-1α (rrIL-1α) and conditioned media (CM) prepared from the supernatant of aCP cells was infused directly into the intra-sellar at a rate of 1 µl/h over 28 days, and then the effects of IL-1α treatment on pathological changes of pituitary gland and GH secretion were measured. To further confirm whether IL-1α affects GH secretion through IL-1R1, an IL-1R1 blocker (IL-1R1a, 10 mg/kg body weight, once daily) was administered subcutaneously from the first day until day 28. RESULTS: There was a significant positive correlation between pituitary fibrosis and GHD (rS = 0.756, P = 0.001). A number of cytokines, in particular IL-1α, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1), were elevated in tumor tissue and cell supernatant. Only IL-1α showed a significant difference between the GHD group and the No-GHD group (P < 0.001, F = 6.251 in tumor tissue; P = 0.003, F = 1.529 in cell supernatant). IL-1α significantly reduced GH secretion in coculture of GH3 and pericytes. The activation of pericytes induced by IL-1α was mediated by the IL-1R1 signaling pathway. In vivo, IL-1α induces pituitary fibrosis, further leading to a decreased level of GH. This pathological change was antagonized by IL-1R1a. CONCLUSION: This study found that the cross talk between aCP cells and stroma cells in the pituitary, i.e. pericytes, is an essential factor in the formation of GHD, and we propose that neutralization of IL-1α signaling might be a potential therapy for GHD in aCP.


Assuntos
Comunicação Celular , Craniofaringioma/patologia , Hormônio do Crescimento Humano/deficiência , Interleucina-1alfa/farmacologia , Pericitos/efeitos dos fármacos , Adulto , Animais , Craniofaringioma/etiologia , Citocinas/metabolismo , Feminino , Fibrose , Hormônio do Crescimento Humano/efeitos dos fármacos , Hormônio do Crescimento Humano/metabolismo , Humanos , Inflamação , Masculino , Pericitos/citologia , Hipófise/metabolismo , Hipófise/patologia , Ratos
16.
Aging (Albany NY) ; 11(22): 10167-10182, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31740626

RESUMO

Pericytes, important elements of the blood-brain barrier (BBB), play critical roles in maintaining BBB integrity and modulating hemostasis, angiogenesis, inflammation and phagocytic function. We investigated whether pericytes are involved in the recombinant tissue plasminogen activator (rt-PA)-induced inflammatory response, which disrupts the BBB, and investigated the potential mechanisms. Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) were employed to mimic hypoxic-ischemic conditions. Rt-PA was intravenously injected into mice 1 h after 1 h MCAO, and Rt-PA was added to the culture medium after 4 h OGD. Rt-PA treatment aggravated the disruption of the BBB compared with hypoxia treatment, and etanercept (TNF-α inhibitor) combined with rt-PA alleviated the rt-PA-induced BBB disruption in vivo and in vitro. Rt-PA treatment increased the TNF-α and MCP-1 levels and decreased the TGF-ß, p-Smad2/3 and PDGFR-ß levels compared with hypoxia treatment in vivo and vitro. TGF-ß combined with rt-PA decreased TNF-α and MCP-1 secretion and alleviated BBB disruption compared with rt-PA; these changes were abrogated by TPO427736 HCL (a TGF-ß/p-Smad2/3 pathway inhibitor) cotreatment in vitro. Rt-PA did not decrease TGF-ß and p-Smad2/3 expression in PDGFR-ß-overexpressing pericytes after OGD. These findings identify PDGFR-ß/TGF-ß/p-Smad2/3 signaling in pericytes as a new therapeutic target for the treatment of rt-PA-induced BBB damage.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Fibrinolíticos/farmacologia , Inflamação/metabolismo , Pericitos/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/farmacologia , Inibidores do Fator de Necrose Tumoral/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Quimiocina CCL2/metabolismo , Etanercepte/farmacologia , Camundongos , Pericitos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
17.
Eur Rev Med Pharmacol Sci ; 23(18): 7749-7756, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31599400

RESUMO

OBJECTIVE: To elucidate the function of miRNA-138-5p in the early diabetic retinopathy (DR) and the potential mechanism. MATERIALS AND METHODS: DR model in rats was first established by streptozotocin (STZ) injection. MiRNA-138-5p expression in rat retinal tissues was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Besides, its expression in retinal capillary endothelial cells (EC) and pericytes (RP) was also detected. Cell counting kit-8 (CCK-8) assay was performed to evaluate proliferative potentials of EC and RP cells. The target gene of miRNA-138-5p was predicted by bioinformatics and further confirmed by dual-luciferase reporter gene assay. Rescue experiments were carried out to verify whether the target gene could reverse the regulatory effect of miRNA-138-5p on the proliferation of EC and RP cells. RESULTS: MiRNA-138-5p was lowly expressed in retinal tissues of DR rats, as well as in EC and RP cells. Overexpression of miRNA-138-5p suppressed the proliferative rate of EC and RP cells, and miRNA-138-5p knockdown obtained the opposite trends. NOVA1 was verified to be the target gene of miRNA-138-5p by dual-luciferase reporter gene assay and RIP assay, which was highly expressed in retinal tissues of DR rats, EC, and RP cells. MiRNA-138-5p knockdown markedly upregulated the mRNA and protein levels of NOVA1 in EC and RP cells. Of note, the inhibitory effect of miRNA-138-5p overexpression on proliferative potentials of EC and RP cells was reversed by NOVA1 overexpression. On the contrary, miRNA-138-5p knockdown accelerated their proliferative potentials and was further reversed by NOVA1 knockdown. CONCLUSIONS: MiRNA-138-5p was lowly expressed in retinal tissues of DR rats, as well as in EC and RP cells. MiRNA-138-5p regulates the early DR by promoting cell proliferation via targeting NOVA1.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Retinopatia Diabética/genética , MicroRNAs/genética , Estreptozocina/farmacologia , Animais , Antibióticos Antineoplásicos/administração & dosagem , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , China/epidemiologia , Biologia Computacional/métodos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Injeções Intraperitoneais , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Substâncias Protetoras , RNA Mensageiro/genética , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Ratos , Retina/citologia , Retina/metabolismo , Sincalida/metabolismo , Estreptozocina/administração & dosagem , Regulação para Cima
18.
Arch Toxicol ; 93(11): 3249-3260, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31552474

RESUMO

Sunitinib malate is a multi-targeted tyrosine kinase inhibitor used extensively for treatment of human tumors. However, cardiovascular adverse effects of sunitinib limit its clinical use. It is pivotal to elucidate molecular targets that mediate sunitinib-induced cardiotoxicity. Sirtuin 3 (Sirt3) is an effective mitochondrial deacetylase that has been reported to regulate sensitivity of different types of cells to chemotherapies, but roles of Sirt3 in sunitinib-induced cardiotoxicity have not been investigated. In the present study, we established wild type, Sirt3-knockout, and Sirt3-overexpressing mouse models of sunitinib (40 mg kg-1 day-1 for 28 days)-induced cardiotoxicity and examined cardiovascular functions and pathological changes. We further cultured wild type, Sirt3-knockout, and Sirt3-overexpressing primary mouse cardiac pericytes and analyzed sunitinib (10 µMol for 48 h)-induced alterations in cellular viability, cell death processes, and molecular pathways. Our results show that sunitinib predominantly induced hypertension, left ventricular systolic dysfunction, and cardiac pericyte death accompanied with upregulation of Sirt3 in cardiac pericytes, and these cardiotoxicities were significantly attenuated in Sirt3-knockout mice, but aggravated in Sirt3-overexpressing mice. Mechanistically, sunitinib induced cardiac pericyte death through inhibition of GSTP1/JNK/autophagy pathway and Sirt3 interacted with and inhibited GSTP1, further inhibiting the pathway and aggravating sunitinib-induced pericyte death. Conclusively, we demonstrate that Sirt3 promotes sensitivity to sunitinib-induced cardiotoxicity via GSTP1/JNK/autophagy pathway. Our results suggest Sirt3 might be a potential target for developing cardioprotective therapies for sunitinib-receiving patients.


Assuntos
Antineoplásicos/toxicidade , Glutationa S-Transferase pi/metabolismo , Hipertensão/induzido quimicamente , MAP Quinase Quinase 4/metabolismo , Pericitos/efeitos dos fármacos , Sirtuína 3/genética , Sunitinibe/toxicidade , Animais , Autofagia/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Cardiotoxicidade , Sobrevivência Celular/efeitos dos fármacos , Coração/efeitos dos fármacos , Hipertensão/metabolismo , Hipertensão/patologia , Camundongos , Camundongos Knockout , Pericitos/metabolismo , Pericitos/patologia , Transdução de Sinais
19.
Sci Rep ; 9(1): 13800, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31551436

RESUMO

The endothelium represents the inner cell layer of blood vessels and is supported by smooth muscle cells and pericytes, which form the vessel structure. The endothelium is involved in the pathogenesis of many diseases, including the development of atherosclerosis. Due to direct blood contact, the blood vessel endothelium is inevitably exposed to genotoxic substances that are systemically taken up by the body, including benzo[a]pyrene, which is a major genotoxic component in cigarette smoke and a common environmental mutagen and human carcinogen. Here, we evaluated the impact of benzo[a]pyrene diol epoxide (BPDE), which is the reactive metabolite of benzo[a]pyrene, on the three innermost vessel cell types. Primary human endothelial cells (HUVEC), primary human smooth muscle cells (HUASMC) and primary human pericytes (HPC) were treated with BPDE, and analyses of cytotoxicity, cellular senescence and genotoxic effects were then performed. The results showed that HUVEC were more sensitive to the cytotoxic activity of BPDE than HUASMC and HPC. We further show that HUVEC display a detraction in the repair of BPDE-induced adducts, as determined through the comet assay and the quantification of BPDE adducts in post-labelling experiments. A screening for DNA repair factors revealed that the nucleotide excision repair (NER) proteins ERCC1, XPF and ligase I were expressed at lower levels in HUVEC compared with HUASMC and HPC, which corresponds with the impaired NER-mediated removal of BPDE adducts from DNA. Taken together, the data revealed that HUVEC exhibit an unexpected DNA repair-impaired phenotype, which has implications on the response of the endothelium to genotoxicants that induce bulky DNA lesions, including the development of vascular diseases resulting from smoking and environmental pollution.


Assuntos
Benzo(a)pireno/efeitos adversos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Pericitos/efeitos dos fármacos , Linhagem Celular , Ensaio Cometa/métodos , DNA/genética , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/genética , Dano ao DNA/efeitos dos fármacos , Compostos de Epóxi/efeitos adversos , Humanos , Mutagênicos/efeitos adversos
20.
Neurosci Lett ; 712: 134475, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31491466

RESUMO

The inability to achieve adequate intracellular antiretroviral concentrations may contribute to HIV persistence within the brain and to neurocognitive deficits in opioid abusers. To investigate, intracellular antiretroviral concentrations were measured in primary human astrocytes, microglia, pericytes, and brain microvascular endothelial cells (BMECs), and in an immortalized brain endothelial cell line (hCMEC/D3). HIV-1 Tat and morphine effects on intracellular antiretroviral concentrations also were evaluated. After pretreatment for 24 h with vehicle, HIV-1 Tat, morphine, or combined Tat and morphine, cells were incubated for 1 h with equal concentrations of a mixture of tenofovir, emtricitabine, and dolutegravir at one of two concentrations (5 µM or 10 µM). Intracellular drug accumulation was measured using LC-MS/MS. Drug penetration differed depending on the drug, the extracellular concentration used for dosing, and cell type. Significant findings included: 1) Dolutegravir (at 5 µM or 10 µM) accumulated more in HBMECs than other cell types. 2) At 5 µM, intracellular emtricitabine levels were higher in microglia than other cell types; while at 10 µM, emtricitabine accumulation was greatest in HBMECs. 3) Tenofovir (5 or 10 µM extracellular dosing) displayed greater accumulation inside HBMECs than in other cell types. 4) After Tat and/or morphine pretreatment, the relative accumulation of antiretroviral drugs was greater in morphine-exposed HBMECs compared to other treatments. The opposite effect was observed in astrocytes in which morphine exposure decreased drug accumulation. In summary, the intracellular accumulation of antiretroviral drugs differed depending on the particular drug involved, the concentration of the applied antiretroviral drug, and the cell type targeted. Moreover, morphine, and to a lesser extent Tat, exposure also had differential effects on antiretroviral accumulation. These data highlight the complexity of optimizing brain-targeted HIV therapeutics, especially in the setting of chronic opioid use or misuse.


Assuntos
Analgésicos Opioides/farmacologia , Antirretrovirais/farmacologia , Encéfalo/efeitos dos fármacos , Morfina/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Cromatografia Líquida , Emtricitabina/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Oxazinas , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Piperazinas , Piridonas , Espectrometria de Massas em Tandem , Tenofovir/farmacologia
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