RESUMO
Recent findings from our group demonstrated that females exhibit higher endothelial mineralocorticoid receptor (MR) expression than males, which predisposes them to aldosterone-mediated endothelial dysfunction in the context of metabolic disorders. However, whether the endothelium of female mice presents a higher propensity to MR-mediated dysfunction than that of males in the absence of comorbidities remains unknown. We therefore sought to investigate whether increasing aldosterone production endogenously with sodium restriction impairs endothelial function in otherwise healthy female mice. We fed male and female Balb/C mice a normal (0.4% NaCl; NSD) or sodium-restricted diet (0.05% NaCl; SRD) for 4 wk. Females exhibited higher baseline endothelial function (relaxation to acetylcholine) and lower vascular contractility (constriction to phenylephrine, serotonin, and KCl). However, SRD impaired endothelial-dependent relaxation and increased vascular contractility in female mice, effectively ablating the baseline sex difference. Female sex also increased baseline adrenal CYP11B2 expression; however, SRD significantly enhanced CYP11B2 expression in male and female mice and ablated the sex difference. Nitric oxide synthase (NOS) inhibition with Nω-nitro-l-arginine methyl ester hydrochloride eliminated both sex as well as diet-induced differences in endothelial dysfunction. In accordance, females demonstrated higher vascular endothelial NOS expression at baseline, which SRD significantly decreased. In addition, SRD diminished vascular NOX4 expression in female mice only. MR blockade with spironolactone-protected female mice from decreases in endothelial-dependent relaxation but not increases in vascular contractility. Utilizing sodium restriction as a method to increase plasma aldosterone levels in healthy female mice, we demonstrated that female mice are more susceptible to vascular damage via MR activation in the vascular endothelium only.NEW & NOTEWORTHY Female sex confers improved endothelial relaxation and vascular constriction responses in female Balb/C mice compared with males under baseline conditions. Sodium restriction impairs endothelial function, which is nitric oxide dependent, and increases vascular contractility in association with reduced vascular endothelial nitric oxide synthase and NOX4 expression in female mice ablating the baseline sex difference. Mineralocorticoid receptor antagonism ablates sodium restriction-induced endothelial dysfunction, but not increased vascular contractility, in female mice.
Assuntos
Aldosterona/sangue , Dieta Hipossódica , Endotélio Vascular/metabolismo , Óxido Nítrico/metabolismo , Receptores de Mineralocorticoides/metabolismo , Vasoconstrição , Vasodilatação , Glândulas Suprarrenais/enzimologia , Animais , Citocromo P-450 CYP11B2/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Masculino , Camundongos Endogâmicos BALB C , NADPH Oxidase 4/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Fatores Sexuais , Transdução de Sinais , Regulação para CimaRESUMO
People living with human immunodeficiency virus (HIV) (PLWH) have increased risk for atherosclerosis-related cardiovascular disease (CVD), the main cause of death in this population. Notwithstanding, the mechanisms of HIV-associated vascular pathogenesis are not fully elucidated. Therefore, we sought to determine whether HIV-regulatory protein Tat mediates HIV-induced endothelial dysfunction via NADPH oxidase 1 (Nox1)-dependent mechanisms. Body weight, fat mass, leptin levels, expression of reactive oxygen species (ROS)-producing enzymes and vascular function were assessed in C57BL/6 male mice treated with Tat for 3 days and 4 weeks. Aortic rings and human endothelial cells were also treated with Tat for 2-24 h in ex vivo and in vitro settings. Chronic (4 weeks) but not acute (3 days and 2-24 h) treatment with Tat decreased body weight, fat mass, and leptin levels and increased the expression of Nox1 and its coactivator NADPH oxidase Activator 1 (NoxA1). This was associated with impaired endothelium-dependent vasorelaxation. Importantly, specific inhibition of Nox1 with GKT771 and chronic leptin infusion restored endothelial function in Tat-treated mice. These data rule out direct effects of HIV-Tat on endothelial function and imply the contribution of reductions in adipose mass and leptin production which likely explain upregulated expression of Nox1 and NoxA1. The Nox1 and leptin system may provide potential targets to improve vascular function in HIV infection-associated CVD.
Assuntos
Tecido Adiposo/fisiologia , Endotélio Vascular/efeitos dos fármacos , NADPH Oxidase 1/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Leptina/sangue , Leptina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 1/antagonistas & inibidores , NADPH Oxidase 1/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The adipokine leptin, which is best-known for its role in the control of metabolic function, is also a master regulator of cardiovascular function. While leptin has been approved for the treatment of metabolic disorders in patients with congenital generalized lipodystrophy (CGL), the effects of chronic leptin deficiency and the treatment on vascular contractility remain unknown. Herein, we investigated the effects of leptin deficiency and treatment (0.3 mg/day/7 days) on aortic contractility in male Berardinelli-Seip 2 gene deficient mice (gBscl2-/-, model of CGL) and their wild-type control (gBscl2+/+), as well as in mice with selective deficiency in endothelial leptin receptor (LepREC-/-). Lipodystrophy selectively increased vascular adrenergic contractility via NO-independent mechanisms and induced hypertrophic vascular remodeling. Leptin treatment and Nox1 inhibition blunted adrenergic hypercontractility in gBscl2-/- mice, however, leptin failed to rescue vascular media thickness. Selective deficiency in endothelial leptin receptor did not alter baseline adrenergic contractility but abolished leptin-mediated reduction in adrenergic contractility, supporting the contribution of endothelium-dependent mechanisms. These data reveal a new direct role for endothelial leptin receptors in the control of vascular contractility and homeostasis, and present leptin as a safe therapy for the treatment of vascular disease in CGL.
Assuntos
Adrenérgicos/metabolismo , Aorta Torácica/metabolismo , Endotélio Vascular/metabolismo , Leptina/metabolismo , Lipodistrofia Generalizada Congênita/metabolismo , Contração Muscular/genética , Músculo Liso Vascular/metabolismo , Transdução de Sinais/genética , Adrenérgicos/administração & dosagem , Adrenérgicos/efeitos adversos , Animais , Modelos Animais de Doenças , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Leptina/administração & dosagem , Leptina/efeitos adversos , Lipodistrofia Generalizada Congênita/tratamento farmacológico , Masculino , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Resultado do TratamentoRESUMO
Protein tyrosine phosphatase 1b (Ptp1b) is a negative regulator of leptin and insulin-signalling pathways. Its targeted deletion in proopiomelanocortin (POMC) neurons protects mice from obesity and diabetes by increasing energy expenditure. Inflammation accompanies increased energy expenditure. Therefore, the present study aimed to determine whether POMC-Ptp1b deletion increases energy expenditure via an inflammatory process, which would impair endothelial function. We characterized the metabolic and cardiovascular phenotypes of Ptp1b+/+ and POMC-Ptp1b-/- mice. Clamp studies revealed that POMC-Ptp1b deletion reduced body fat and increased energy expenditure as evidenced by a decrease in feed efficiency and an increase in oxygen consumption and respiratory exchange ratio. POMC-Ptp1b deletion induced a 2.5-fold increase in plasma tumour necrosis factor α (TNF-α) levels and elevated body temperature. Vascular studies revealed an endothelial dysfunction in POMC-Ptp1b-/- mice. Nitric oxide synthase inhibition [N-nitro-L-arginine methyl ester (L-NAME)] reduced relaxation to a similar extent in Ptp1b+/+ and POMC-Ptp1b-/- mice. POMC-Ptp1b deletion decreased ROS-scavenging enzymes [superoxide dismutases (SODs)] whereas it increased ROS-generating enzymes [NADPH oxidases (NOXs)] and cyclooxygenase-2 (COX-1) expression, in aorta. ROS scavenging or NADPH oxidase inhibition only partially improved relaxation whereas COX-2 inhibition and thromboxane-A2 (TXA2) antagonism fully restored relaxation in POMC-Ptp1b-/- mice Chronic treatment with the soluble TNF-α receptor etanercept decreased body temperature, restored endothelial function and reestablished aortic COX-2, NOXs and SOD expression to their baseline levels in POMC-Ptp1b-/- mice. However, etanercept promoted body weight gain and decreased energy expenditure in POMC-Ptp1b-/- mice. POMC-Ptp1b deletion increases plasma TNF-α levels, which contribute to body weight regulation via increased energy expenditure and impair endothelial function via COX-2 and ROS-dependent mechanisms.
Assuntos
Células Endoteliais/metabolismo , Metabolismo Energético/genética , Neurônios/metabolismo , Pró-Opiomelanocortina/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Fator de Necrose Tumoral alfa/metabolismo , Tecido Adiposo/metabolismo , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Ciclo-Oxigenase 2/metabolismo , Metabolismo Energético/fisiologia , Camundongos Transgênicos , Obesidade/genética , Obesidade/metabolismo , Fator de Necrose Tumoral alfa/genética , Aumento de Peso/genéticaRESUMO
Combination antiretroviral therapy (cART) has markedly increased life expectancy in people with HIV (PWH) but has also resulted in an increased prevalence of cardiometabolic disorders, whose etiopathology remains ill-defined. Notably, the respective contribution of cART and HIV-derived proteins to obesity and vascular alterations remain poorly understood. Therefore, we investigated the individual and combined effects of HIV-proteins and of the integrase strand transfer inhibitor Dolutegravir (DTG) on body composition and vascular reactivity. Male wildtype (WT) and HIV transgenic (Tg26) mice, received DTG or vehicle for 12 weeks. Viral proteins expression in Tg26 mice lowered fat mass, increased heat production, and induced a 2-fold increase in brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression. DTG increased the expression of markers of adipogenesis in adipocytes in culture, but also reduced heat production and BAT UCP1 and UCP3 expression in Tg26 mice. DTG increased food intake, fat percentage and protected from lean mass reduction in Tg26 mice only. However, DTG did not increase body weight in either WT or Tg26 mice. Viral protein expression reduced acetylcholine (endothelium)-mediated relaxation by 14% in mesenteric arteries preconstricted with phenylephrine. However, DTG did not impair nor improve endothelium-dependent relaxation. Together, these data indicate that DTG's effects on food intake, adipogenesis and energy expenditure are insufficient to increase body weight, even in the presence of HIV-proteins, suggesting that body weight gain in PWH involves additional factors likely including other cART components and pre-existing comorbidities. Moreover, these data rule out DTG as a source of vascular disorders in PWH.
Assuntos
Modelos Animais de Doenças , Metabolismo Energético , Infecções por HIV , Inibidores de Integrase de HIV , Compostos Heterocíclicos com 3 Anéis , Camundongos Transgênicos , Oxazinas , Piperazinas , Piridonas , Animais , Metabolismo Energético/efeitos dos fármacos , Masculino , Piperazinas/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Oxazinas/farmacologia , Piridonas/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inibidores de Integrase de HIV/farmacologia , Camundongos , Proteína Desacopladora 1/metabolismo , Proteína Desacopladora 1/genética , Vasodilatação/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/virologia , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Type 1 diabetes (T1D) is a major cause of endothelial dysfunction. Although cellular bioenergetics has been identified as a new regulator of vascular function, whether glycolysis, the primary bioenergetic pathway in endothelial cells (EC), regulates vascular tone and contributes to impaired endothelium-dependent relaxation (EDR) in T1D remains unknown. METHODS: Experiments were conducted in Akita mice with intact or selective deficiency in EC PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3), the main regulator of glycolysis. Seahorse analyzer and myography were employed to measure glycolysis and mitochondrial respiration, and EDR, respectively, in aortic explants. EC PFKFB3 (Ad-PFKFB3) and glycolysis (Ad-GlycoHi) were increased in situ via adenoviral transduction. RESULTS: T1D increased EC glycolysis and elevated EC expression of PFKFB3 and NADPH oxidase Nox1 (NADPH oxidase homolog 1). Functionally, pharmacological and genetic inhibition of PFKFB3 restored EDR in T1D, while in situ aorta EC transduction with Ad-PFKFB3 or Ad-GlycoHi reproduced the impaired EDR associated with T1D. Nox1 inhibition restored EDR in aortic rings from Akita mice, as well as in Ad-PFKFB3-transduced aorta EC and lactate-treated wild-type aortas. T1D increased the expression of the advanced glycation end product precursor methylglyoxal in the aortas. Exposure of the aortas to methylglyoxal impaired EDR, which was prevented by PFKFB3 inhibition. T1D and exposure to methylglyoxal increased EC expression of HIF1α (hypoxia-inducible factor 1α), whose inhibition blunted methylglyoxal-mediated EC PFKFB3 upregulation. CONCLUSIONS: EC bioenergetics, namely glycolysis, is a new regulator of vasomotion and excess glycolysis, a novel mechanism of endothelial dysfunction in T1D. We introduce excess methylglyoxal, HIF1α, and PFKFB3 as major effectors in T1D-mediated increased EC glycolysis.
Assuntos
Diabetes Mellitus Tipo 1 , Células Endoteliais , Animais , Camundongos , Aldeído Pirúvico , Glicólise , EndotélioRESUMO
RATIONALE: The heterotypic interactions of endothelial cells and mural cells (smooth muscle cells or pericytes) are crucial for assembly, maturation, and subsequent function of blood vessels. Yet, the molecular mechanisms underlying their association have not been fully defined. OBJECTIVE: Our previous in vitro studies indicated that Notch3, which is expressed in mural cells, mediates these cell-cell interactions. To assess the significance of Notch3 on blood vessel formation in vivo, we investigated its role in retinal angiogenesis. METHODS AND RESULTS: We show that Notch3-deficient mice exhibit reduced retinal vascularization, with diminished sprouting and vascular branching. Moreover, Notch3 deletion impairs mural cell investment, resulting in progressive loss of vessel coverage. In an oxygen-induced retinopathy model, we demonstrate that Notch3 is induced in hypoxia and interestingly, pathological neovascularization is decreased in retinas of Notch3-null mice. Analysis of oxygen-induced retinopathy mediators revealed that angiopoietin-2 expression is significantly reduced in the absence of Notch3. Furthermore, in vitro experiments showed that Notch3 is sufficient for angiopoietin-2 induction, and this expression is additionally enhanced in the presence of hypoxia-inducible factor 1α. CONCLUSIONS: These results provide compelling evidence that Notch3 is important for the investment of mural cells and is a critical regulator of developmental and pathological blood vessel formation.
Assuntos
Músculo Liso Vascular/fisiologia , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Pericitos/fisiologia , Receptores Notch/genética , Angiopoietina-2/genética , Animais , Aorta/citologia , Aorta/fisiologia , Comunicação Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Mutantes , Músculo Liso Vascular/citologia , Oxigênio/toxicidade , Pericitos/citologia , Receptor Notch3 , Receptores Notch/metabolismo , Vasos Retinianos/citologia , Vasos Retinianos/fisiologiaRESUMO
BACKGROUND: Patients with preeclampsia demonstrate increases in placental leptin production in midgestation, and an associated increase in late gestation plasma leptin levels. The consequences of mid-late gestation increases in leptin production in pregnancy is unknown. Our previous work indicates that leptin infusion induces endothelial dysfunction in nonpregnant female mice via leptin-mediated aldosterone production and endothelial mineralocorticoid receptor (ECMR) activation, which is ablated by ECMR deletion. Therefore, we hypothesized that leptin infusion in mid-gestation of pregnancy induces endothelial dysfunction and hypertension, hallmarks of clinical preeclampsia, which are prevented by ECMR deletion. METHODS: Leptin was infused via miniosmotic pump (0.9 mg/kg per day) into timed-pregnant ECMR-intact (WT) and littermate-mice with ECMR deletion (KO) on gestation day (GD)11-18. RESULTS: Leptin infusion decreased fetal weight and placental efficiency in WT mice compared with WT+vehicle. Radiotelemetry recording demonstrated that blood pressure increased in leptin-infused WT mice during infusion. Leptin infusion reduced endothelial-dependent relaxation responses to acetylcholine (ACh) in both resistance (second-order mesenteric) and conduit (aorta) vessels in WT pregnant mice. Leptin infusion increased placental ET-1 (endothelin-1) production evidenced by increased PPET-1 (preproendothelin-1) and ECE-1 (endothelin-converting enzyme-1) expressions in WT mice. Adrenal aldosterone synthase (CYP11B2) and angiotensin II type 1 receptor b (AT1Rb) expression increased with leptin infusion in pregnant WT mice. KO pregnant mice demonstrated protection from leptin-induced reductions in pup weight, placental efficiency, increased BP, and endothelial dysfunction. CONCLUSIONS: Collectively, these data indicate that leptin infusion in midgestation induces endothelial dysfunction, hypertension, and fetal growth restriction in pregnant mice, which is ablated by ECMR deletion.
Assuntos
Hipertensão , Pré-Eclâmpsia , Animais , Pressão Sanguínea , Endotelina-1/metabolismo , Feminino , Humanos , Hipertensão/tratamento farmacológico , Leptina/farmacologia , Camundongos , Camundongos Knockout , Placenta/metabolismo , Pré-Eclâmpsia/tratamento farmacológico , Gravidez , Receptores de Mineralocorticoides/fisiologiaRESUMO
Leptin plays a crucial role in blood pressure (BP) regulation, notably in the context of obesity through central sympatho-mediated pressor effects. Leptin also relaxes arteries via endothelial (EC) leptin receptor (LepREC)-mediated increases in nitric oxide (NO) bioavailability. Herein, we investigated whether leptin-mediated increases in NO bioavailability represent a buffering mechanism against leptin-induced sympatho-activation. We tested the direct contribution of LepREC to BP regulation in physiological conditions and in response to chronic leptin infusion using mice deficient in LepREC. LepREC deficiency did not alter baseline metabolic profile nor leptin-induced reduction in adiposity and increases in energy expenditure. LepREC-/- mice demonstrated no increase in baseline BP and heart rate (HR) (MAP: LepREC+/+:94.7 ± 1.6, LepREC-/-:95.1 ± 1.8 mmHg; HR:LepREC+/+:492.4 ± 11.7, LepREC-/-:509.5 ± 13.4 bpm) nor in response to leptin (MAP, LepREC+/+:101.1 ± 1.7, LepREC-/-:101.7 ± 1.8 mmHg; HR, LepREC+/+:535.6 ± 11.1, LepREC-/-:539.3 ± 14.2 bpm). Moreover, baseline neurogenic control of BP and HR was preserved in LepREC-/- mice as well as leptin-mediated increases in sympathetic control of BP and HR and decreases in vagal tone. Remarkably, LepREC deficiency did not alter endothelium-dependent relaxation in resistance vessels, nor NO contribution to vasodilatation. Lastly, leptin induced similar increases in adrenergic contractility in mesenteric arteries from both LepREC+/+ and LepREC-/- mice. Collectively, these results demonstrate that the NO buffering effects of leptin are absent in resistance arteries and do not contribute to BP regulation. We provide further evidence that leptin-mediated hypertension involves increased vascular sympatho-activation and extend these findings by demonstrating for the first time that increased cardiac sympatho-activation and reduced vagal tone also contribute to leptin-mediated hypertension.
Assuntos
Hipertensão , Receptores para Leptina , Adrenérgicos , Animais , Endotélio/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Leptina/farmacologia , Masculino , Camundongos , Óxido Nítrico , Obesidade/genética , Obesidade/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismoRESUMO
Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.
Assuntos
Apolipoproteínas D/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Comunicação Parácrina , Receptores Notch/metabolismo , Transdução de Sinais , Análise de Variância , Apolipoproteínas D/genética , Proteínas de Ligação ao Cálcio/metabolismo , Adesão Celular , Células Cultivadas , Técnicas de Cocultura , Regulação para Baixo , Adesões Focais/metabolismo , Genes Reporter , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Músculo Liso Vascular/citologia , Regiões Promotoras Genéticas , Interferência de RNA , Receptor Notch3 , Receptores Notch/genética , Proteínas Serrate-Jagged , TransfecçãoRESUMO
Endothelial cells and mural cells (smooth muscle cells, pericytes, or fibroblasts) are known to communicate with one another. Their interactions not only serve to support fully functional blood vessels but also can regulate vessel assembly and differentiation or maturation. In an effort to better understand the molecular components of this heterotypic interaction, we used a 3D model of angiogenesis and screened for genes, which were modulated by coculturing of these 2 different cell types. In doing so, we discovered that NOTCH3 is one gene whose expression is robustly induced in mural cells by coculturing with endothelial cells. Knockdown by small interfering RNA revealed that NOTCH3 is necessary for endothelial-dependent mural cell differentiation, whereas overexpression of NOTCH3 is sufficient to promote smooth muscle gene expression. Moreover, NOTCH3 contributes to the proangiogenic abilities of mural cells cocultured with endothelial cells. Interestingly, we found that the expression of NOTCH3 is dependent on Notch signaling, because the gamma-secretase inhibitor DAPT blocked its upregulation. Furthermore, in mural cells, a dominant-negative Mastermind-like1 construct inhibited NOTCH3 expression, and endothelial-expressed JAGGED1 was required for its induction. Additionally, we demonstrated that NOTCH3 could promote its own expression and that of JAGGED1 in mural cells. Taken together, these data provide a mechanism by which endothelial cells induce the differentiation of mural cells through activation and induction of NOTCH3. These findings also suggest that NOTCH3 has the capacity to maintain a differentiated phenotype through a positive-feedback loop that includes both autoregulation and JAGGED1 expression.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Comunicação Parácrina , Pericitos/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Bovinos , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Proteínas de Ligação a DNA/metabolismo , Dipeptídeos/farmacologia , Perfilação da Expressão Gênica , Homeostase , Humanos , Proteína Jagged-1 , Neovascularização Fisiológica , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/genética , Inibidores de Proteases/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Receptor Notch3 , Receptores Notch/genética , Proteínas Serrate-Jagged , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transativadores/metabolismo , Fatores de Transcrição , Ativação Transcricional , Transfecção , Regulação para CimaRESUMO
Neurofibromatosis type 1 (NF1) is a heritable cancer predisposition syndrome resulting from mutations in the NF1 tumor suppressor gene. Genotype-phenotype correlations for NF1 are rare due to the large number of NF1 mutations and role of modifier genes in manifestations of NF1; however, emerging reports suggest that persons with NF1 display a distinct anthropometric and metabolic phenotype featuring short stature, low body mass index, increased insulin sensitivity, and protection from diabetes. Nf1 heterozygous (Nf1+/-) mice accurately reflect the dominant inheritance of NF1 and are regularly employed as a model of NF1. Here, we sought to identify whether Nf1+/- mice recapitulate the anthropometric and metabolic features identified in persons with NF1. Littermate 16-20 week-old male wildtype (WT) and Nf1+/- C57B/6J mice underwent nuclear magnetic resonance (NMR), indirect calorimetry, and glucose/insulin/pyruvate tolerance testing. In some experiments, tissues were harvested for NMR and histologic characterization. Nf1+/- mice are leaner with significantly reduced visceral and subcutaneous fat mass, which corresponds with an increased density of small adipocytes and reduced leptin levels. Additionally, Nf1+/- mice are highly reliant on carbohydrates as an energy substrate and display increased glucose clearance and insulin sensitivity, but normal response to pyruvate suggesting enhanced glucose utilization and preserved gluconeogenesis. Finally, WT and Nf1+/- mice subjected to high glucose diet were protected from diet-induced obesity and hyperglycemia. Our data suggest that Nf1+/- mice closely recapitulate the anthropometric and metabolic phenotype identified in persons with NF1, which will impact the interpretation of previous and future translational studies of NF1.
Assuntos
Antropometria , Genes da Neurofibromatose 1 , Heterozigoto , Neurofibromatose 1/metabolismo , Animais , Humanos , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurofibromatose 1/genética , Neurofibromatose 1/patologiaRESUMO
Background Cardiovascular disease is currently the leading cause of death in patients with human immunodeficiency virus on combination antiretroviral therapy. Although the use of the protease inhibitor ritonavir has been associated with increased prevalence of cardiovascular disease, the underlying mechanisms remain ill-defined. Herein, we tested the hypothesis that ritonavir-mediated lipoatrophy causes endothelial dysfunction via reducing endothelial leptin signaling. Methods and Results Long-term (4 weeks) but not short-term (3 days) treatment with ritonavir reduced body weight, fat mass, and leptin levels and induced endothelial dysfunction in mice. Moreover, ritonavir increased vascular NADPH oxidase 1, aortic H2O2 levels as well as interleukin-1ß, GATA3 (GATA binding protein 3), the macrophage marker (F4/80), and C-C chemokine receptor type 5 (CCR5) expression. Reactive oxygen species scavenging with tempol restored endothelial function, and both NADPH oxidase 1 and CCR5 deletion in mice protected from ritonavir-mediated endothelial dysfunction and vascular inflammation. Remarkably, leptin infusion markedly improved endothelial function and significantly reduced vascular NADPH oxidase 1, interleukin-1ß, GATA3, F4/80, and CCR5 levels in ritonavir-treated animals. Selective deficiency in endothelial leptin receptor abolished the protective effects of leptin infusion on endothelial function. Conversely, selective increases in endothelial leptin signaling with protein tyrosine phosphatase deletion blunted ritonavir-induced endothelial dysfunction. Conclusions All together, these data indicate that ritonavir-associated endothelial dysfunction is a direct consequence of a reduction in adiposity and leptin secretion, which decreases endothelial leptin signaling and leads to a NADPH oxidase 1-induced, CCR5-mediated reduction in NO bioavailability. These latter data also introduce leptin deficiency as an additional contributor to cardiovascular disease and leptin as a negative regulator of CCR5 expression, which may provide beneficial avenues for limiting human immunodeficiency virus infection.
Assuntos
Tecido Adiposo , Doenças Cardiovasculares , Células Endoteliais , NADPH Oxidase 1/metabolismo , Receptores CCR5/metabolismo , Receptores para Leptina/metabolismo , Ritonavir/farmacocinética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/prevenção & controle , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inibidores da Protease de HIV/farmacocinética , Inflamação/metabolismo , Leptina/metabolismo , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Transdução de SinaisRESUMO
BACKGROUND: Recent evidence by our laboratory demonstrates that women and female mice endogenously express higher endothelial mineralocorticoid receptor (ECMR) than males. Mounting clinical evidence also indicates that aldosterone production is higher in pathological conditions in females compared to males. However, the role for increased activation of ECMR by aldosterone in the absence of a comorbid condition is yet to be explored. The current study hypothesized that increased ECMR activation induced by elevated aldosterone production predisposes healthy female mice to endothelial dysfunction. METHOD: Vascular reactivity was assessed in aortic rings from wild-type (WT) and ECMR KO (KO) mice fed either a normal salt (NSD, 0.4% NaCl) or sodium-restricted diet (SRD, 0.05% NaCl) for 28 days. RESULTS: SRD elevated plasma aldosterone levels as well as adrenal CYP11B2 and angiotensin II type 1 receptor (AT1R) expressions in female, but not male, WT mice. In baseline conditions (NSD), endothelial function, assessed by vascular relaxation to acetylcholine, was higher while vascular contractility to phenylephrine, serotonin, and KCl lower in female than male WT mice. SRD impaired endothelial function and increased vascular contractility in female, but not male, WT mice effectively ablating the baseline sex differences. NOS inhibition with LNAME ablated endothelial relaxation to a higher extent in male than female mice on NSD and ablated differences in acetylcholine relaxation responses between NSD- and SRD-fed females, indicating a role for NO in SRD-mediated endothelial function. In association, SRD significantly reduced vascular NOX4 expression in female, but not male, mice. Lastly, selective deletion of ECMR protected female mice from SRD-mediated endothelial dysfunction and increased vascular contractility. CONCLUSION: Collectively, these data indicate that female mice develop aldosterone-induced endothelial dysfunction via endothelial MR-mediated reductions in NO bioavailability. In addition, these data support a role for ECMR to promote vascular contractility in female mice in response to sodium restriction.
Assuntos
Aldosterona/sangue , Endotélio Vascular/metabolismo , Receptores de Mineralocorticoides/metabolismo , Caracteres Sexuais , Doenças Vasculares/etiologia , Glândulas Suprarrenais/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Citocromo P-450 CYP11B2/metabolismo , Dieta Hipossódica , Modelos Animais de Doenças , Feminino , Masculino , Camundongos Knockout , NADPH Oxidase 4/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Doenças Vasculares/metabolismoRESUMO
Diabetes notably increases the risk for endothelial dysfunction, a main precursor for microvascular complications. While endoplasmic reticulum stress (ERS) and protein tyrosine phosphatase 1B (PTP1B) have been associated with endothelial dysfunction in resistance vessels, whether these mechanisms also contribute to diabetes-mediated endothelial dysfunction in conduit arteries remains unknown. Herein, we tested the hypothesis that diabetes induces macrovascular endothelial dysfunction via endothelial ERS-induced, PTP1B-mediated apoptosis. We showed that diabetes concomitantly increased the expression of PTP1B and of markers of ERS, including GRP78, XBP1, splXBP1 and CHOP in human vessels. Exposure of aortic rings from wild-type mice to the ERS inducers tunicamycin and thapsigargin markedly reduced endothelium-dependent relaxation. Global and endothelial-specific deletion of PTP1B as well as pharmacological inhibition protected aortic rings from ERS-mediated endothelial dysfunction. Nitric oxide synthase inhibition with l-NAME abolished relaxation in the presence and absence of ERS, but neither reactive oxygen species scavenging with tempol or peg-catalase, nor cyclooxygenase inhibition with indomethacin prevented ERS-mediated endothelial dysfunction. However, both p38-MAPK and JNK inhibition protected aortic rings from ERS-mediated endothelial dysfunction. In HUVECs, PTP1B deletion prevented ERS-induced PARP cleavage and apoptosis. Lastly, acute ERS inhibition in aortic rings and selective deficiency of endothelial PTP1B in mice protected mice from diabetes-induced endothelial dysfunction. Altogether, these data support the contribution of the p38/JNK-apoptosis pathway in ERS-mediated endothelial dysfunction and present endothelial PTP1B as a major regulator of endothelial cell viability in conduit vessels and a potential target for the management of macrovascular diseases in diabetes.
Assuntos
Apoptose/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Estresse do Retículo Endoplasmático/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Animais , Sobrevivência Celular , Diabetes Mellitus Experimental/complicações , Chaperona BiP do Retículo Endoplasmático , Células Endoteliais/patologia , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Communication between endothelial and mural cells (smooth muscle cells, pericytes, and fibroblasts) can dictate blood vessel size and shape during angiogenesis, and control the functional aspects of mature blood vessels, by determining things such as contractile properties. The ability of these different cell types to regulate each other's activities led us to ask how their interactions directly modulate gene expression. To address this, we utilized a three-dimensional model of angiogenesis and screened for genes whose expression was altered under coculture conditions. Using a BeadChip array, we identified 323 genes that were uniquely regulated when endothelial cells and mural cells (fibroblasts) were cultured together. Data mining tools revealed that differential expression of genes from the integrin, blood coagulation, and angiogenesis pathways were overrepresented in coculture conditions. Scans of the promoters of these differentially modulated genes identified a multitude of conserved C promoter binding factor (CBF)1/CSL elements, implicating Notch signaling in their regulation. Accordingly, inhibition of the Notch pathway with gamma-secretase inhibitor DAPT or NOTCH3-specific small interfering RNA blocked the coculture-induced regulation of several of these genes in fibroblasts. These data show that coculturing of endothelial cells and fibroblasts causes profound changes in gene expression and suggest that Notch signaling is a critical mediator of the resultant transcription.
Assuntos
Expressão Gênica , Neovascularização Fisiológica/genética , Receptores Notch/metabolismo , Transdução de Sinais/genética , Técnicas de Cocultura , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Regiões Promotoras Genéticas , Receptores Notch/genética , Transcrição Gênica , TransfecçãoRESUMO
Leptin is the current treatment for metabolic disorders associated with acquired and congenital generalized lipodystrophy (CGL). Although excess leptin levels have been associated with vascular inflammation and cardiovascular disease in the context of obesity, the effects of chronic leptin treatment on vascular function remain unknown in CGL. Here, we hypothesized that leptin treatment will improve endothelial function via direct vascular mechanisms. We investigated the cardiovascular consequences of leptin deficiency and supplementation in male gBscl2-/- (Berardinelli-Seip 2 gene-deficient) mice-a mouse model of CGL. CGL mice exhibited reduced adipose mass and leptin levels, as well as impaired endothelium-dependent relaxation. Blood vessels from CGL mice had increased NADPH Oxidase 1 (Nox1) expression and reactive oxygen species production, and selective Nox1 inhibition restored endothelial function. Remarkably, chronic and acute leptin supplementation restored endothelial function via a PPARγ-dependent mechanism that decreased Nox1 expression and reactive oxygen species production. Selective ablation of leptin receptors in endothelial cells promoted endothelial dysfunction, which was restored by Nox1 inhibition. Lastly, we confirmed in aortic tissue from older patients undergoing cardiac bypass surgery that acute leptin can promote signaling in human blood vessels. In conclusion, in gBscl2-/- mice, leptin restores endothelial function via peroxisome proliferator activated receptor gamma-dependent decreases in Nox1. Furthermore, we provide the first evidence that vessels from aged patients remain leptin sensitive. These data reveal a new direct role of leptin receptors in the control of vascular homeostasis and present leptin as a potential therapy for the treatment of vascular disease associated with low leptin levels.
Assuntos
Leptina/farmacologia , Lipodistrofia Generalizada Congênita/tratamento farmacológico , NADPH Oxidase 1/metabolismo , PPAR gama/metabolismo , Análise de Variância , Animais , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Lipodistrofia Generalizada Congênita/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismo , Valores de Referência , Resultado do TratamentoRESUMO
Compelling clinical evidence indicates that obesity and its associated metabolic abnormalities supersede the protective effects of female sex-hormones and predisposes premenopausal women to cardiovascular disease. The underlying mechanisms remain poorly defined; however, recent studies have implicated overactivation of the aldosterone-MR (mineralocorticoid receptor) axis as a cause of sex-specific cardiovascular risk in obese females. Experimental evidence indicates that the MR on endothelial cells contributes to obesity-associated, leptin-induced endothelial dysfunction in female experimental models, however, the vascular-specific mechanisms via which females are predisposed to heightened endothelial MR activation remain unknown. Therefore, we hypothesized that endogenous expression of endothelial MR is higher in females than males, which predisposes them to obesity-associated, leptin-mediated endothelial dysfunction. We found that endothelial MR expression is higher in blood vessels from female mice and humans compared with those of males, and further, that PrR (progesterone receptor) activation in endothelial cells is the driving mechanism for sex-dependent increases in endothelial MR expression in females. In addition, we show that genetic deletion of either the endothelial MR or PrR in female mice prevents leptin-induced endothelial dysfunction, providing direct evidence that interaction between the PrR and MR mediates obesity-associated endothelial impairment in females. Collectively, these novel findings suggest that progesterone drives sex-differences in endothelial MR expression and predisposes female mice to leptin-induced endothelial dysfunction, which indicates that MR antagonists may be a promising sex-specific therapy to reduce the risk of cardiovascular diseases in obese premenopausal women.
Assuntos
Endotélio Vascular/patologia , Regulação da Expressão Gênica , Obesidade/fisiopatologia , Progesterona/metabolismo , Receptores de Mineralocorticoides/genética , Animais , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Feminino , Leptina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Obesidade/genética , Distribuição Aleatória , Medição de Risco , Sensibilidade e Especificidade , Fatores Sexuais , Regulação para CimaRESUMO
Clinical studies indicate that salt-sensitive hypertension is more prevalent in women than in men. However, animal models of salt sensitivity have primarily focused on the mechanisms of salt sensitivity in male animals; therefore, elucidation of these mechanisms in female animal models is needed. We have previously shown that female Balb/C mice have higher aldosterone synthase expression and aldosterone production than males. We hypothesized that female Balb/C mice develop salt-sensitive increases in blood pressure. Seven-day feeding of a 4% NaCl high-salt (HS) diet increased blood pressure in female mice without altering blood pressure in males. Females on an HS diet displayed no apparent increases in sodium retention as assessed by 24-hour urine collection, sodium balance measure, and saline loading excretion analysis. Females on an HS diet exhibited lower renin-angiotensin system activity (plasma Ang II [angiotensin II], plasma renin activity, and ACE [angiotensin-converting enzyme] activity) compared with males but developed a salt-induced elevation in adrenal aldosterone synthase expression and retained higher aldosterone levels than males on HS. This resulted in a higher aldosterone/plasma renin activity ratio in females compared with males on HS feeding. Adrenal mRNA expression of angiotensinogen and leptin receptor was increased in female mice on an HS diet. HS impaired endothelium-dependent relaxation in female mice only. MR (mineralocorticoid receptor) inhibition (eplerenone) restored blood pressure and endothelial function in females on an HS diet. Collectively, these data indicate that Balb/C mice develop sex-discrepant salt-sensitive hypertension likely via aldosterone-MR-mediated mechanisms involving impaired endothelium-dependent relaxation in females only. This study presents the first model of spontaneous sex-specific salt sensitivity, which mimics the human pathology.
Assuntos
Aldosterona/metabolismo , Pressão Sanguínea/fisiologia , Hipertensão/metabolismo , Sistema Renina-Angiotensina/fisiologia , Cloreto de Sódio na Dieta , Glândulas Suprarrenais/metabolismo , Angiotensinogênio/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores para Leptina/metabolismo , Fatores SexuaisRESUMO
The human gastric pathogen Helicobacter pylori exhibits a remarkably small repertoire of transcriptional regulators including three complete two-component systems as well as the orphan response regulators HP1021 and HP1043. Both HP1021 and HP1043 show atypical receiver sequences and are required for the normal cell growth of H. pylori. Recently, we demonstrated that phosphorylation of HP1021 and HP1043 according to the two-component paradigm is not a prerequisite for the cell growth-associated functions of these response regulators, raising the question of how the activity of this regulatory proteins is modulated. Here, we report that strict transcriptional control of its expression is not involved in the cell-growth associated function of HP1021. We show that expression of hp1043 is controlled both on the post-transcriptional or post-translational level and by transcriptional regulation. Furthermore, we provide evidence that hp1043 can be replaced by the orthologous gene cj0355 from Campylobacter jejuni.