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1.
Arterioscler Thromb Vasc Biol ; 41(9): e427-e439, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34261328

RESUMO

Objective: Atheromatous fibrous caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We tested whether the recruitment mechanisms are the same as in embryonic artery development, which relies prominently on Notch signaling to form the subendothelial medial SMC layers. Approach and Results: Notch elements were expressed in regions of fibrous cap in human and mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice where the Notch pathway was inactivated in SMCs by conditional knockout of the essential effector transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). The recruitment of cap SMCs was significantly reduced without major effects on plaque size. Lineage tracing revealed the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. Conversely, to analyze whether the loss of Notch signaling is required for SMC-derived cells to accumulate in atherogenesis, we studied atherosclerosis in mice with constitutive activation of Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development. Conclusions: Sequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the cap forms as a neo-media that restores the connection between endothelium and subendothelial SMCs, transiently disrupted in early atherogenesis.


Assuntos
Aterosclerose/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica , Receptores Notch/metabolismo , Túnica Média/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Artérias/metabolismo , Artérias/patologia , Aterosclerose/genética , Aterosclerose/patologia , Linhagem da Célula , Células Cultivadas , Progressão da Doença , Fibrose , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , Ratos , Receptores Notch/genética , Transdução de Sinais , Túnica Média/patologia
2.
Biomolecules ; 11(6)2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073580

RESUMO

Background: Vitamin D (vitD) deficiency is highly prevalent in patients with pulmonary arterial hypertension (PAH). Moreover, PAH-patients with lower levels of vitD have worse prognosis. We hypothesize that recovering optimal levels of vitD in an animal model of PAH previously depleted of vitD improves the hemodynamics, the endothelial dysfunction and the ionic remodeling. Methods: Male Wistar rats were fed a vitD-free diet for five weeks and then received a single dose of Su5416 (20 mg/Kg) and were exposed to vitD-free diet and chronic hypoxia (10% O2) for three weeks to induce PAH. Following this, vitD deficient rats with PAH were housed in room air and randomly divided into two groups: (a) continued on vitD-free diet or (b) received an oral dose of 100,000 IU/Kg of vitD plus standard diet for three weeks. Hemodynamics, pulmonary vascular remodeling, pulmonary arterial contractility, and K+ currents were analyzed. Results: Recovering optimal levels of vitD improved endothelial function, measured by an increase in the endothelium-dependent vasodilator response to acetylcholine. It also increased the activity of TASK-1 potassium channels. However, vitD supplementation did not reduce pulmonary pressure and did not ameliorate pulmonary vascular remodeling and right ventricle hypertrophy. Conclusions: Altogether, these data suggest that in animals with PAH and severe deficit of vitD, restoring vitD levels to an optimal range partially improves some pathophysiological features of PAH.


Assuntos
Endotélio Vascular/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Hipertensão Arterial Pulmonar , Deficiência de Vitamina D , Vitamina D , Animais , Endotélio Vascular/patologia , Masculino , Hipertensão Arterial Pulmonar/tratamento farmacológico , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Ratos , Ratos Wistar , Vitamina D/farmacocinética , Vitamina D/farmacologia , Deficiência de Vitamina D/tratamento farmacológico , Deficiência de Vitamina D/metabolismo , Deficiência de Vitamina D/patologia
3.
Hypertension ; 76(4): 1134-1146, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32829658

RESUMO

K+ channels play a fundamental role regulating membrane potential of pulmonary artery (PA) smooth muscle cells and their impairment is a common feature in pulmonary arterial hypertension (PAH). K+ voltage-gated channel subfamily Q (KCNQ1-5) or Kv7 channels and their regulatory subunits subfamily E (KCNE) regulatory subunits are known to regulate vascular tone, but whether Kv7 channel function is impaired in PAH and how this can affect the rationale for targeting Kv7 channels in PAH remains unknown. Here, we have studied the role of Kv7/KCNE subunits in rat PA and their possible alteration in PAH. Using the patch-clamp technique, we found that the total K+ current is reduced in PA smooth muscle cells from pulmonary hypertension animals (SU5416 plus hypoxia) and Kv7 currents made a higher contribution to the net K+ current. Likewise, enhanced vascular responses to Kv7 channel modulators were found in pulmonary hypertension rats. Accordingly, KCNE4 subunit was highly upregulated in lungs from pulmonary hypertension animals and patients. Additionally, Kv7 channel activity was enhanced in the presence of Kv1.5 and TASK-1 channel inhibitors and this was associated with an increased KCNE4 membrane abundance. Compared with systemic arteries, PA showed a poor response to Kv7 channel modulators which was associated with reduced expression and membrane abundance of Kv7.4 and KCNE4. Our data indicate that Kv7 channel function is preserved and KCNE4 is upregulated in PAH. Therefore, compared with other downregulated channels, the contribution of Kv7 channels is increased in PAH resulting in an enhanced sensitivity to Kv7 channel modulators. This study provides insight into the potential usefulness of targeting Kv7 channels in PAH.


Assuntos
Canal de Potássio KCNQ1/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Animais , Proliferação de Células/fisiologia , Humanos , Hipóxia/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Ratos
4.
Am J Physiol Lung Cell Mol Physiol ; 319(4): L627-L640, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32726132

RESUMO

Vitamin D (VitD) receptor regulates the expression of several genes involved in signaling pathways affected in pulmonary hypertension (PH). VitD deficiency is highly prevalent in PH, and low levels are associated with poor prognosis. We investigated if VitD deficiency may predispose to or exacerbate PH. Male Wistar rats were fed with a standard or a VitD-free diet for 5 wk. Next, rats were further divided into controls or PH, which was induced by a single dose of Su-5416 (20 mg/kg) and exposure to hypoxia (10% O2) for 2 wk. VitD deficiency had no effect on pulmonary pressure in normoxic rats, indicating that, by itself, it does not trigger PH. However, it induced several moderate but significant changes characteristic of PH in the pulmonary arteries, such as increased muscularization, endothelial dysfunction, increased survivin, and reduced bone morphogenetic protein (Bmp) 4, Bmp6, DNA damage-inducible transcript 4, and K+ two-pore domain channel subfamily K member 3 (Kcnk3) expression. Myocytes isolated from pulmonary arteries from VitD-deficient rats had a reduced whole voltage-dependent potassium current density and acid-sensitive (TASK-like) potassium currents. In rats with PH induced by Su-5416 plus hypoxia, VitD-free diet induced a modest increase in pulmonary pressure, worsened endothelial function, increased the hyperreactivity to serotonin, arterial muscularization, decreased total and TASK-1 potassium currents, and further depolarized the pulmonary artery smooth muscle cell membrane. In human pulmonary artery smooth muscle cells from controls and patients with PH, the active form of VitD calcitriol significantly increased KCNK3 mRNA expression. Altogether, these data strongly suggest that the deficit in VitD induces pulmonary vascular dysfunction.


Assuntos
Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Deficiência de Vitamina D/metabolismo , Animais , Humanos , Pulmão/metabolismo , Pulmão/fisiopatologia , Masculino , Potenciais da Membrana/fisiologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Ratos Wistar , Vitamina D/metabolismo
5.
Arthritis Rheumatol ; 72(3): 477-487, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31509349

RESUMO

OBJECTIVE: Pulmonary arterial hypertension (PAH), one of the major complications of systemic sclerosis (SSc), is a rare disease with unknown etiopathogenesis and noncurative treatments. As mice deficient in P-selectin glycoprotein ligand 1 (PSGL-1) develop a spontaneous SSc-like syndrome, we undertook this study to analyze whether they develop PAH and to examine the molecular mechanisms involved. METHODS: Doppler echocardiography was used to estimate pulmonary pressure, immunohistochemistry was used to assess vascular remodeling, and myography of dissected pulmonary artery rings was used to analyze vascular reactivity. Angiotensin II (Ang II) levels were quantified by enzyme-linked immunosorbent assay, and Western blotting was used to measure Ang II type 1 receptor (AT1 R), AT2 R, endothelial cell nitric oxide synthase (eNOS), and phosphorylated eNOS expression in lung lysates. Flow cytometry allowed us to determine cytokine production by immune cells and NO production by endothelial cells. In all cases, there were 4-8 mice per experimental group. RESULTS: PSGL-1-/- mice showed lung vessel wall remodeling and a reduced mean ± SD expression of pulmonary AT2 R (expression ratio [relative to ß-actin] in female mice age >18 months: wild-type mice 0.799 ± 0.508 versus knockout mice 0.346 ± 0.229). With aging, female PSGL-1-/- mice had impaired up-regulation of estrogen receptor α (ERα) and developed lung vascular endothelial dysfunction coinciding with an increase in mean ± SEM pulmonary Ang II levels (wild-type 48.70 ± 5.13 pg/gm lung tissue versus knockout 78.02 ± 28.09 pg/gm lung tissue) and a decrease in eNOS phosphorylation, leading to reduced endothelial NO production. These events led to a reduction in the pulmonary artery acceleration time:ejection time ratio in 33% of aged female PSGL-1-/- mice, indicating pulmonary hypertension. Importantly, we found expanded populations of interferon-γ-producing PSGL-1-/- T cells and B cells and a reduced presence of regulatory T cells. CONCLUSION: The absence of PSGL-1 induces a reduction in Treg cells, NO production, and ERα expression and causes an increase in Ang II in the lungs of female mice, favoring the development of PAH.


Assuntos
Hipertensão Pulmonar/genética , Glicoproteínas de Membrana/deficiência , Escleroderma Sistêmico/genética , Angiotensina II/metabolismo , Animais , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/biossíntese , Remodelação Vascular/genética
6.
J Physiol Biochem ; 75(4): 519-529, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31432395

RESUMO

Endothelial dysfunction plays a central role in the pathophysiology of pulmonary arterial hypertension (PAH). MicroRNAs (miRNAs) are small single-strand and non-coding RNAs that negatively regulate gene function by binding to the 3'-untranslated region (3'-UTR) of specific mRNAs. microRNA-1 (miR-1) is upregulated in plasma from idiopathic PAH patients and in lungs from an experimental model of PAH. However, the role of miRNA-1 on endothelial dysfunction is unknown. The aim of this study was to analyze the effects of miR-1 on endothelial function in rat pulmonary arteries (PA). Endothelial function was studied in PA from PAH or healthy animals and mounted in a wire myograph. Some PA from control animals were transfected with miR-1 or scramble miR. Superoxide anion production by miR-1 was quantified by dihydroethidium (DHE) fluorescence in rat PA smooth muscle cells (PASMC). Bioinformatic analysis identified superoxide dismutase-1 (SOD1), connexin-43 (Cx43), caveolin 2 (CAV2) and Krüppel-like factor 4 (KLF4) as potential targets of miR-1. The expression of SOD1, Cx43, CAV2, and KLF4 was determined by qRT-PCR and western blot in PASMC. PA incubated with miR-1 presented decreased endothelium-dependent relaxation to acetylcholine. We also found an increase in the production of O2- and decreased expression of SOD1, Cx43, CAV2, and KLF4 in PASMC induced by miR-1, which may contribute to endothelial dysfunction. In conclusion, these data indicate that miR-1 induces endothelial dysfunction, suggesting a pathophysiological role in PAH.


Assuntos
MicroRNAs/fisiologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Animais , Células Cultivadas , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/patologia , Ratos , Ratos Wistar
7.
J Am Heart Assoc ; 8(15): e013111, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31339057

RESUMO

Background Inflammation underlies many forms of pulmonary hypertension (PH), including that resulting from Schistosoma infection, a major cause of PH worldwide. Schistosomiasis-associated PH is proximately triggered by embolization of parasite eggs into the lungs, resulting in localized type 2 inflammation. However, the role of CD4+ T cells in this disease is not well defined. Methods and Results We used a mouse model of schistosomiasis-associated PH, induced by intraperitoneal egg sensitization followed by intravenous egg challenge, with outcomes including right ventricle systolic pressure measured by cardiac catheterization, and cell density and phenotype assessed by flow cytometry. We identified that embolization of Schistosoma eggs into lungs of egg-sensitized mice increased the perivascular density of T-helper 2 (Th2) CD4+ T cells by recruitment of cells from the circulation and triggered type 2 inflammation. Parabiosis confirmed that egg embolization is required for localized type 2 immunity. We found Th2 CD4+ T cells were necessary for Schistosoma-induced PH, given that deletion of CD4+ T cells or inhibiting their Th2 function protected against type 2 inflammation and PH following Schistosoma exposure. We also observed that adoptive transfer of Schistosoma-sensitized CD4+ Th2 cells was sufficient to drive type 2 inflammation and PH. Conclusions Th2 CD4+ T cells are a necessary and sufficient component for the type 2 inflammation-induced PH following Schistosoma exposure.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Hipertensão Pulmonar/imunologia , Hipertensão Pulmonar/parasitologia , Pneumonia/imunologia , Pneumonia/parasitologia , Esquistossomose/complicações , Esquistossomose/imunologia , Células Th2/imunologia , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL
8.
Mol Nutr Food Res ; 63(18): e1900044, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31144780

RESUMO

SCOPE: The aim of the present study is to investigate the potential protective effect of a cocoa-rich diet on functional and structural vascular alterations in diabetes and the mechanism involved. METHODS AND RESULTS: Male Zucker diabetic fatty (ZDF) rats are fed on a standard (ZDF-C) or cocoa-rich diet (ZDF-Co) from week 10 to 20 of life. Diabetic ZDF-C rats showed increased blood pressure and enhanced aortic stiffness, as demonstrated by the increased pulse pressure and the augmented aortic medial thickness with loss and disruption of elastic fibres. Interestingly, cocoa intake strongly avoided all these adverse effects and reduced aortic oxidative stress. Mechanistically, cocoa diet prevented sirtuin-1 (SIRT-1) depletion and increased NADPH oxidases (NOXs) and reactive oxygen species production as well as reduced active nuclear factor E2 related factor 2 (Nrf2) and their antioxidant products. CONCLUSION: The results demonstrate for the first time that a cocoa-rich diet strongly prevents aortic stiffening and remodeling in diabetic animals and avoids aortic oxidative stress. It is suggested that this effect could be mediated via its effects on SIRT-1, NOXs, and Nrf2.


Assuntos
Chocolate , Diabetes Mellitus Experimental/dietoterapia , Estresse Oxidativo/efeitos dos fármacos , Remodelação Vascular/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cacau , Diabetes Mellitus Experimental/fisiopatologia , Masculino , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ratos Zucker , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/metabolismo , Remodelação Vascular/fisiologia
9.
Br J Pharmacol ; 176(13): 2131-2145, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30883701

RESUMO

BACKGROUND AND PURPOSE: The NO/cGMP pathway represents a major physiological signalling controlling tone in pulmonary arteries (PA), and drugs activating this pathway are used to treat pulmonary arterial hypertension. Kv channels expressed in PA smooth muscle cells (PASMCs) are key determinants of vascular tone. We aimed to analyse the contribution of Kv 1.5 and Kv 7 channels in the electrophysiological and vasodilating effects evoked by NO donors and the GC stimulator riociguat in PA. EXPERIMENTAL APPROACH: Kv currents were recorded in isolated rat PASMCs using the patch-clamp technique. Vascular reactivity was assessed in a wire myograph. KEY RESULTS: The NO donors diethylamine NONOate diethylammonium (DEA-NO) and sodium nitroprusside hyperpolarized the membrane potential and induced a bimodal effect on Kv currents (augmenting the current between -40 and -10 mV and decreasing it at more depolarized potentials). The hyperpolarization and the enhancement of the current were suppressed by Kv 7 channel inhibitors and by the GC inhibitor ODQ but preserved when Kv 1.5 channels were inhibited. Additionally, DEA-NO enhanced Kv 7.5 currents in COS7 cells expressing the KCNQ5 gene. Riociguat increased Kv currents at all potentials ≥-40 mV and induced membrane hyperpolarization. Both effects were prevented by Kv 7 inhibition. Likewise, PA relaxation induced by NO donors and riociguat was attenuated by Kv 7 inhibitors. CONCLUSIONS AND IMPLICATIONS: NO donors and riociguat enhance Kv 7 currents, leading to PASMC hyperpolarization. This mechanism contributes to NO/cGMP-induced PA vasodilation. Our study identifies Kv 7 channels as a novel mechanism of action of vasodilator drugs used in the treatment of pulmonary arterial hypertension.


Assuntos
GMP Cíclico/fisiologia , Canais de Potássio KCNQ/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Óxido Nítrico/fisiologia , Artéria Pulmonar/fisiologia , Animais , Células COS , Chlorocebus aethiops , Hidrazinas/farmacologia , Canal de Potássio Kv1.5/fisiologia , Masculino , Miócitos de Músculo Liso/fisiologia , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Artéria Pulmonar/citologia , Ratos Wistar , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
10.
PLoS One ; 14(1): e0211281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30689673

RESUMO

Diabetes is a very strong predictor of chronic systemic vascular diseases and acute cardiovascular events. Recently, associations between metabolic disorders and pulmonary hypertension have also been reported in both humans and animal models. In order to get some further insight into the relationship of pulmonary hypertension with obesity, insulin resistance and hyperglycemia, herein we have used the Zucker diabetic fatty rats (ZDF/clr-lepr fa) at 20 weeks fed a standard diet and compared to their lean Zucker littermates (ZL). ZDF rats were obese, had elevated plasma glucose levels and insulin resistance, i.e. a clinically relevant model of type 2 diabetes. They presented elevated systolic, diastolic and mean pulmonary arterial pressures and a parallel increase in the Fulton index. Systemic arterial pressures were also increased but the left ventricle plus septum weight was similar in both groups and the heart rate was reduced. Wall media thickening was observed in the small pulmonary arteries from the ZDF rats. Isolated pulmonary arteries mounted in a wire myograph showed similar vasoconstrictor responses to phenylephrine and 5-HT and similar responses to the endothelium-dependent vasodilator acetylcholine. However, the iNOS inhibitor 1400W enhanced the vasoconstrictor responses in ZDF but not in ZL rats. The protein expression of eNOS and iNOS was not significantly different in the lungs of the two groups. The lung expression of Bmpr2 mRNA was downregulated. However, the mRNA expression of Kcna5, Kcnk3, Kcnq1, Kcnq4 or Kcnq5, which encode for the potassium channels Kv1.5, TASK-1, Kv7.1, Kv7.4 and Kv7.5, respectively, was similar in ZL and ZDF rats. In conclusion, ZDF rats show increased pulmonary arterial pressure, right ventricular hypertrophy, pulmonary arterial medial thickening and downregulated lung Bmpr2 despite leptin resistance. These changes were mild but are consistent with the view that diabetes is a risk factor for pulmonary hypertension.


Assuntos
Pressão Arterial/fisiologia , Artéria Pulmonar/fisiologia , Amidinas/farmacologia , Animais , Benzilaminas/farmacologia , Glicemia/análise , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Citocinas/sangue , Regulação da Expressão Gênica , Hemodinâmica , Pulmão/metabolismo , Pulmão/patologia , Obesidade/patologia , Obesidade/veterinária , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ratos , Ratos Zucker , Vasoconstrição/efeitos dos fármacos
11.
J Physiol ; 597(4): 1185-1197, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29717493

RESUMO

KEY POINTS: The expression of miR-1 is increased in lungs from the Hyp/Su5416 PAH rat model. Pulmonary artery smooth muscle cells from this animal model are more depolarized and show decreased expression and activity of voltage-dependent potassium channel (Kv)1.5. miR-1 directly targets Kv1.5 channels, reduces Kv1.5 activity and induces membrane depolarization. Antagomir-1 prevents Kv1.5 channel downregulation and the depolarization induced by hypoxia/Su5416 exposition. ABSTRACT: Impairment of the voltage-dependent potassium channel (Kv) plays a central role in the development of cardiovascular diseases, including pulmonary arterial hypertension (PAH). MicroRNAs are non-coding RNAs that regulate gene expression by binding to the 3'-untranslated region region of specific mRNAs. The present study aimed to analyse the effects of miR-1 on Kv channel function in pulmonary arteries (PA). Kv channel activity was studied in PA from healthy animals transfected with miR-1 or scrambled-miR. Kv currents were studied using the whole-cell configuration of the patch clamp technique. The characterization of the Kv1.5 currents was performed with the selective inhibitor DPO-1. miR-1 expression was increased and Kv1.5 channels were decreased in lungs from a rat model of PAH induced by hypoxia and Su5416. miR-1 transfection increased cell capacitance, reduced Kv1.5 currents and induced membrane depolarization in isolated pulmonary artery smooth muscle cells. A luciferase reporter assay indicated that KCNA5, which encodes Kv1.5 channels, is a direct target gene of miR-1. Incubation of PA with Su5416 and hypoxia (3% O2 ) increased miR-1 and induced a decline in Kv1.5 currents, which was prevented by antagomiR-1. In conclusion, these data indicate that miR-1 induces pulmonary artery smooth muscle cell hypertrophy and reduces the activity and expression of Kv channels, suggesting a pathophysiological role in PAH.


Assuntos
Hipertensão Pulmonar/metabolismo , Canal de Potássio Kv1.5/metabolismo , MicroRNAs/metabolismo , Artéria Pulmonar/metabolismo , Potenciais de Ação , Animais , Células COS , Hipóxia Celular , Chlorocebus aethiops , Regulação para Baixo , Hipertensão Pulmonar/etiologia , Indóis/toxicidade , Canal de Potássio Kv1.5/genética , Masculino , MicroRNAs/genética , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiopatologia , Pirróis/toxicidade , Ratos , Ratos Wistar
12.
Am J Physiol Lung Cell Mol Physiol ; 315(5): L711-L723, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30136611

RESUMO

Human immunodeficiency virus (HIV) infection is an established risk factor for pulmonary arterial hypertension (PAH); however, the pathogenesis of HIV-related PAH remains unclear. Since K+ channel dysfunction is a common marker in most forms of PAH, our aim was to analyze whether the expression of HIV proteins is associated with impairment of K+ channel function in the pulmonary vascular bed. HIV transgenic mice (Tg26) expressing seven of the nine HIV viral proteins and wild-type (WT) mice were used. Hemodynamic assessment was performed by echocardiography and catheterization. Vascular reactivity was studied in endothelium-intact pulmonary arteries. K+ currents were recorded in freshly isolated pulmonary artery smooth muscle cells (PASMC) using the patch-clamp technique. Gene expression was assessed using quantitative RT-PCR. PASMC from Tg26 mice had reduced K+ currents and were more depolarized than those from WT. Whereas voltage-gated K+ channel 1.5 (Kv1.5) currents were preserved, pH-sensitive noninactivating background currents ( IKN) were nearly abolished in PASMC from Tg26 mice. Tg26 mice had reduced lung expression of Kv7.1 and Kv7.4 channels and decreased responses to the Kv7.1 channel activator L-364,373 assessed by vascular reactivity and patch-clamp experimental approaches. Although we found pulmonary vascular remodeling and endothelial dysfunction in Tg26 mice, this was not accompanied by changes in hemodynamic parameters. In conclusion, the expression of HIV proteins in vivo impairs pH-sensitive IKN and Kv7 currents. This negative impact of HIV proteins in K+ channels was not sufficient to induce PAH, at least in mice, but may play a permissive or accessory role in the pathophysiology of HIV-associated PAH.


Assuntos
HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Hipertrofia Ventricular Direita/patologia , Músculo Liso Vascular/patologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Artéria Pulmonar/patologia , Transgenes/fisiologia , Animais , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Hipertrofia Ventricular Direita/metabolismo , Masculino , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Artéria Pulmonar/metabolismo , Vasoconstrição
13.
Sci Rep ; 8(1): 9681, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29946072

RESUMO

We have analysed whether pulmonary arterial hypertension (PAH) alters the rat faecal microbiota. Wistar rats were injected with the VEGF receptor antagonist SU5416 (20 mg/kg s.c.) and followed for 2 weeks kept in hypoxia (10% O2, PAH) or injected with vehicle and kept in normoxia (controls). Faecal samples were obtained and microbiome composition was determined by 16S rRNA gene sequencing and bioinformatic analysis. No effect of PAH on the global microbiome was found (α- or ß-diversity). However, PAH-exposed rats showed gut dysbiosis as indicated by a taxonomy-based analysis. Specifically, PAH rats had a three-fold increase in Firmicutes-to-Bacteroidetes ratio. Within the Firmicutes phylum, there were no large changes in the relative abundance of the bacterial families in PAH. Among Bacteroidetes, all families were less abundant in PAH. A clear separation was observed between the control and PAH clusters based on short chain fatty acid producing bacterial genera. Moreover, acetate was reduced in the serum of PAH rats. In conclusion, faecal microbiota composition is altered as a result of PAH. This misbalanced bacterial ecosystem might in turn play a pathophysiological role in PAH by altering the immunologic, hormonal and metabolic homeostasis.


Assuntos
Microbioma Gastrointestinal/fisiologia , Hipertensão Pulmonar/microbiologia , Hipertensão Pulmonar/fisiopatologia , Animais , Biologia Computacional , Hemodinâmica/fisiologia , Masculino , RNA Ribossômico 16S/genética , Ratos , Ratos Wistar
14.
PLoS One ; 13(1): e0191239, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29364918

RESUMO

INTRODUCTION: Current treatment with vasodilators for pulmonary hypertension associated with respiratory diseases is limited by their inhibitory effect on hypoxic pulmonary vasoconstriction (HPV) and uncoupling effects on ventilation-perfusion (V'/Q'). Hypoxia is also a well-known modulator of the nitric oxide (NO) pathway, and may therefore differentially affect the responses to phosphodiesterase 5 (PDE5) inhibitors and soluble guanylyl cyclase (sGC) stimulators. So far, the effects of the sGC stimulator riociguat on HPV have been poorly characterized. MATERIALS AND METHODS: Contraction was recorded in pulmonary arteries (PA) in a wire myograph. Anesthetized rats were catheterized to record PA pressure. Ventilation and perfusion were analyzed by micro-CT-SPECT images in rats with pulmonary fibrosis induced by bleomycin. RESULTS: The PDE5 inhibitor sildenafil and the sGC stimulator riociguat similarly inhibited HPV in vitro and in vivo. Riociguat was more effective as vasodilator in isolated rat and human PA than sildenafil. Riociguat was ≈3-fold more potent under hypoxic conditions and it markedly inhibited HPV in vivo at a dose that barely affected the thromboxane A2 (TXA2) mimetic U46619-induced pressor responses. Pulmonary fibrosis was associated with V'/Q' uncoupling and riociguat did not affect the V'/Q' ratio. CONCLUSION: PDE5 inhibitors and sGC stimulators show a different vasodilator profile. Riociguat was highly effective and potentiated by hypoxia in rat and human PA. In vivo, riociguat preferentially inhibited hypoxic than non-hypoxic vasoconstriction. However, it did not worsen V'/Q' coupling in a rat model of pulmonary fibrosis.


Assuntos
Hipóxia/tratamento farmacológico , Hipóxia/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiopatologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Citrato de Sildenafila/farmacologia , Vasodilatadores/farmacologia , Relação Ventilação-Perfusão/efeitos dos fármacos , Idoso , Animais , Modelos Animais de Doenças , Ativadores de Enzimas/farmacologia , Feminino , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Inibidores da Fosfodiesterase 5/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/fisiopatologia , Ratos , Ratos Wistar , Guanilil Ciclase Solúvel/metabolismo , Vasoconstrição/efeitos dos fármacos
15.
Pulm Circ ; 7(3): 643-653, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28447910

RESUMO

Pulmonary arterial hypertension (PAH) is a progressive and debilitating condition. Despite promoting vasodilation, current drugs have a therapeutic window within which they are limited by systemic side effects. Nanomedicine uses nanoparticles to improve drug delivery and/or reduce side effects. We hypothesize that this approach could be used to deliver PAH drugs avoiding the systemic circulation. Here we report the use of iron metal organic framework (MOF) MIL-89 and PEGylated MIL-89 (MIL-89 PEG) as suitable carriers for PAH drugs. We assessed their effects on viability and inflammatory responses in a wide range of lung cells including endothelial cells grown from blood of donors with/without PAH. Both MOFs conformed to the predicted structures with MIL-89 PEG being more stable at room temperature. At concentrations up to 10 or 30 µg/mL, toxicity was only seen in pulmonary artery smooth muscle cells where both MOFs reduced cell viability and CXCL8 release. In endothelial cells from both control donors and PAH patients, both preparations inhibited the release of CXCL8 and endothelin-1 and in macrophages inhibited inducible nitric oxide synthase activity. Finally, MIL-89 was well-tolerated and accumulated in the rat lungs when given in vivo. Thus, the prototypes MIL-89 and MIL-89 PEG with core capacity suitable to accommodate PAH drugs are relatively non-toxic and may have the added advantage of being anti-inflammatory and reducing the release of endothelin-1. These data are consistent with the idea that these materials may not only be useful as drug carriers in PAH but also offer some therapeutic benefit in their own right.

16.
Thorax ; 72(5): 460-471, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27701117

RESUMO

BACKGROUND: Pulmonary hypertension (PH) is frequently observed in patients with acute respiratory distress syndrome (ARDS) and it is associated with an increased risk of mortality. Both acid sphingomyelinase (aSMase) activity and interleukin 6 (IL-6) levels are increased in patients with sepsis and correlate with worst outcomes, but their role in pulmonary vascular dysfunction pathogenesis has not yet been elucidated. Therefore, the aim of this study was to determine the potential contribution of aSMase and IL-6 in the pulmonary vascular dysfunction induced by lipopolysaccharide (LPS). METHODS: Rat or human pulmonary arteries (PAs) or their cultured smooth muscle cells (SMCs) were exposed to LPS, SMase or IL-6 in the absence or presence of a range of pharmacological inhibitors. The effects of aSMase inhibition in vivo with D609 on pulmonary arterial pressure and inflammation were assessed following intratracheal administration of LPS. RESULTS: LPS increased ceramide and IL-6 production in rat pulmonary artery smooth muscle cells (PASMCs) and inhibited pulmonary vasoconstriction induced by phenylephrine or hypoxia (HPV), induced endothelial dysfunction and potentiated the contractile responses to serotonin. Exogenous SMase and IL-6 mimicked the effects of LPS on endothelial dysfunction, HPV failure and hyperresponsiveness to serotonin in PA; whereas blockade of aSMase or IL-6 prevented LPS-induced effects. Finally, administration of the aSMase inhibitor D609 limited the development of endotoxin-induced PH and ventilation-perfusion mismatch. The protective effects of D609 were validated in isolated human PAs. CONCLUSIONS: Our data indicate that aSMase and IL-6 are not simply biomarkers of poor outcomes but pathogenic mediators of pulmonary vascular dysfunction in ARDS secondary to Gram-negative infections.


Assuntos
Hipertensão Pulmonar/metabolismo , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Artéria Pulmonar/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielina Fosfodiesterase/farmacologia , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Células Cultivadas , Ceramidases/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Humanos , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Masculino , Músculo Liso Vascular/citologia , Norbornanos , Ratos , Ratos Wistar , Tiocarbamatos , Tionas/farmacologia , Vasoconstrição/efeitos dos fármacos
17.
Molecules ; 21(12)2016 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-27999410

RESUMO

BACKGROUND: We hypothesized that treatment with quercetin could result in improved hemodynamics, lung inflammatory parameters and mortality in a rat model of hemorrhagic shock. METHODS: Rats were anesthetized (80 mg/kg ketamine plus 8 mg/kg xylazine i.p.). The protocol included laparotomy for 15 min (trauma), hemorrhagic shock (blood withdrawal to reduce the mean arterial pressure to 35 mmHg) for 75 min and resuscitation by re-infusion of all the shed blood plus lactate Ringer for 90 min. Intravenous quercetin (50 mg/kg) or vehicle were administered during resuscitation. RESULTS: There was a trend for increased survival 84.6% (11/13) in the treated group vs. the shock group 68.4% (13/19, p > 0.05 Kaplan-Meier). Quercetin fully prevented the development of lung edema. The activity of aSMase was increased in the shock group compared to the sham group and the quercetin prevented this effect. However, other inflammatory markers such as myeloperoxidase activity, interleukin-6 in plasma or bronchoalveolar fluid were similar in the sham and shock groups. We found no bacterial DNA in plasma in these animals. CONCLUSIONS: Quercetin partially prevented the changes in blood pressure and lung injury in shock associated to hemorrhage and reperfusion.


Assuntos
Quercetina/uso terapêutico , Choque Hemorrágico/tratamento farmacológico , Choque Traumático/tratamento farmacológico , Animais , Pressão Arterial/efeitos dos fármacos , Biomarcadores/sangue , Edema/prevenção & controle , Hemodinâmica , Inflamação/complicações , Inflamação/tratamento farmacológico , Interleucina-6/química , Soluções Isotônicas/uso terapêutico , Masculino , Peroxidase/química , Edema Pulmonar/complicações , Edema Pulmonar/tratamento farmacológico , Ratos , Ratos Wistar , Reperfusão , Ressuscitação , Lactato de Ringer , Choque Hemorrágico/complicações , Choque Traumático/complicações
18.
Clin Sci (Lond) ; 130(20): 1823-36, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27413020

RESUMO

PPARß/δ activation protects against endothelial dysfunction in diabetic models. Elevated glucose is known to impair cAMP-induced relaxation and Kv channel function in coronary arteries (CA). Herein, we aimed to analyse the possible protective effects of the PPARß/δ agonist GW0742 on the hyperglycaemic-induced impairment of cAMP-induced relaxation and Kv channel function in rat CA. As compared with low glucose (LG), incubation under high glucose (HG) conditions attenuated the relaxation induced by the adenylate cyclase activator forskolin in CA and this was prevented by GW0742. The protective effect of GW0742 was supressed by a PPARß/δ antagonist. In myocytes isolated from CA under LG, forskolin enhanced Kv currents and induced hyperpolarization. In contrast, when CA were incubated with HG, Kv currents were diminished and the electrophysiological effects of forskolin were abolished. These deleterious effects were prevented by GW0742. The protective effects of GW0742 on forskolin-induced relaxation and Kv channel function were confirmed in CA from type-1 diabetic rats. In addition, the differences in the relaxation induced by forskolin in CA incubated under LG, HG or HG + GW0742 were abolished by the Kv7 channel inhibitor XE991. Accordingly, GW0742 prevented the down-regulation of Kv7 channels induced by HG. Finally, the preventive effect of GW0742 on oxidative stress and cAMP-induced relaxation were overcome by the pyruvate dehydrogenase kinase 4 (PDK4) inhibitor dichloroacetate (DCA). Our results reveal that the PPARß/δ agonist GW0742 prevents the impairment of the cAMP-mediated relaxation in CA under HG. This protective effect was associated with induction of PDK4, attenuation of oxidative stress and preservation of Kv7 channel function.


Assuntos
Vasos Coronários/metabolismo , AMP Cíclico/metabolismo , Hiperglicemia/metabolismo , Canal de Potássio KCNQ1/metabolismo , PPAR delta/metabolismo , PPAR beta/metabolismo , Animais , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiopatologia , Diabetes Mellitus Experimental , Humanos , Hiperglicemia/genética , Canal de Potássio KCNQ1/genética , Masculino , PPAR delta/genética , PPAR beta/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Tiazóis/administração & dosagem , Vasodilatação/efeitos dos fármacos
19.
Cardiovasc Res ; 106(1): 98-108, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25616413

RESUMO

AIMS: Voltage-gated potassium channels encoded by KCNQ genes (Kv7 channels) are emerging as important regulators of vascular tone. In this study, we analysed the contribution of Kv7 channels to the vasodilation induced by hypoxia and the cyclic AMP pathway in the coronary circulation. We also assessed their regional distribution and possible impairment by diabetes. METHODS AND RESULTS: We examined the effects of Kv7 channel modulators on K+ currents and vascular reactivity in rat left and right coronary arteries (LCAs and RCAs, respectively). Currents from LCA were more sensitive to Kv7 channel inhibitors (XE991, linopirdine) and activators (flupirtine, retigabine) than those from RCA. Accordingly, LCAs were more sensitive than RCAs to the relaxation induced by Kv7 channel enhancers. Likewise, relaxation induced by the adenylyl cyclase activator forskolin and hypoxia, which were mediated through Kv7 channel activation, were greater in LCA than in RCA. KCNQ1 and KCNQ5 expression was markedly higher in LCA than in RCA. After incubation with high glucose (HG, 30 mmol/L), myocytes from LCA, but not from RCA, were more depolarized and showed reduced Kv7 currents. In HG-incubated LCA, the effects of Kv7 channel modulators and forskolin were diminished, and the expression of KCNQ1 and KCNQ5 was reduced. Finally, vascular responses induced by Kv7 channel modulators were impaired in LCA, but not in RCA, from type 1 diabetic rats. CONCLUSION: Our results reveal that the high expression and function of Kv7 channels in the LCA and their down-regulation by diabetes critically determine the sensitivity to key regulators of coronary tone.


Assuntos
Circulação Coronária/fisiologia , Vasos Coronários/fisiologia , Diabetes Mellitus Experimental/fisiopatologia , Regulação para Baixo/fisiologia , Hiperglicemia/fisiopatologia , Canais de Potássio KCNQ/fisiologia , Canal de Potássio KCNQ1/fisiologia , Animais , Vasos Coronários/efeitos dos fármacos , AMP Cíclico/fisiologia , Diabetes Mellitus Experimental/induzido quimicamente , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Glucose/farmacologia , Hipóxia/fisiopatologia , Canais de Potássio KCNQ/efeitos dos fármacos , Canal de Potássio KCNQ1/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estreptozocina/efeitos adversos , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia
20.
PLoS One ; 9(12): e114492, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25460361

RESUMO

Quercetin is a dietary flavonoid which exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in humans and animal models of systemic hypertension. We hypothesized that oral quercetin treatment might be protective in a rat model of pulmonary arterial hypertension. Three weeks after injection of monocrotaline, quercetin (10 mg/kg/d per os) or vehicle was administered for 10 days to adult Wistar rats. Quercetin significantly reduced mortality. In surviving animals, quercetin decreased pulmonary arterial pressure, right ventricular hypertrophy and muscularization of small pulmonary arteries. Classic biomarkers of pulmonary arterial hypertension such as the downregulated expression of lung BMPR2, Kv1.5, Kv2.1, upregulated survivin, endothelial dysfunction and hyperresponsiveness to 5-HT were unaffected by quercetin. Quercetin significantly restored the decrease in Kv currents, the upregulation of 5-HT2A receptors and reduced the Akt and S6 phosphorylation. In vitro, quercetin induced pulmonary artery vasodilator effects, inhibited pulmonary artery smooth muscle cell proliferation and induced apoptosis. In conclusion, quercetin is partially protective in this rat model of PAH. It delayed mortality by lowering PAP, RVH and vascular remodeling. Quercetin exerted effective vasodilator effects in isolated PA, inhibited cell proliferation and induced apoptosis in PASMCs. These effects were associated with decreased 5-HT2A receptor expression and Akt and S6 phosphorylation and partially restored Kv currents. Therefore, quercetin could be useful in the treatment of PAH.


Assuntos
Hipertensão Pulmonar/tratamento farmacológico , Quercetina/uso terapêutico , Animais , Perfilação da Expressão Gênica , Hipertensão Pulmonar/genética , Hipertrofia Ventricular Direita/fisiopatologia , Masculino , Potenciais da Membrana , Ratos , Ratos Wistar , Análise de Sobrevida
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