RESUMO
Left ventricular (LV) dysfunction is an early, clinically detectable sign of cardiomyopathy in type 2 diabetes mellitus (T2DM) that precedes the development of symptomatic heart failure. Preclinical models of diabetic cardiomyopathy are essential to develop therapies that may prevent or delay the progression of heart failure. This study examined the molecular, structural, and functional cardiac phenotype of two rat models of T2DM induced by a high-fat diet (HFD) with a moderate- or high-sucrose content (containing 88.9 or 346 g/kg sucrose, respectively), plus administration of low-dose streptozotocin (STZ). At 8 wk of age, male Sprague-Dawley rats commenced a moderate- or high-sucrose HFD. Two weeks later, rats received low-dose STZ (35 mg/kg ip for 2 days) and remained on their respective diets. LV function was assessed by echocardiography 1 wk before end point. At 22 wk of age, blood and tissues were collected postmortem. Relative to chow-fed sham rats, diabetic rats on a moderate- or high-sucrose HFD displayed cardiac reactive oxygen species dysregulation, perivascular fibrosis, and impaired LV diastolic function. The diabetes-induced impact on LV adverse remodeling and diastolic dysfunction was more apparent when a high-sucrose HFD was superimposed on STZ. In conclusion, a high-sucrose HFD in combination with low-dose STZ produced a cardiac phenotype that more closely resembled T2DM-induced cardiomyopathy than STZ diabetic rats subjected to a moderate-sucrose HFD.NEW & NOTEWORTHY Left ventricular dysfunction and adverse remodeling were more pronounced in diabetic rats that received low-dose streptozotocin (STZ) and a high-sucrose high-fat diet (HFD) compared with those on a moderate-sucrose HFD in combination with STZ. Our findings highlight the importance of sucrose content in diet composition, particularly in preclinical studies of diabetic cardiomyopathy, and demonstrate that low-dose STZ combined with a high-sucrose HFD is an appropriate rodent model of cardiomyopathy in type 2 diabetes.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Cardiomiopatias Diabéticas , Insuficiência Cardíaca , Disfunção Ventricular Esquerda , Ratos , Masculino , Animais , Estreptozocina/efeitos adversos , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Experimental/induzido quimicamente , Ratos Sprague-Dawley , Dieta Hiperlipídica/efeitos adversos , FenótipoRESUMO
Brain inflammation and apoptosis contribute to neuronal damage and loss following ischaemic stroke, leading to cognitive and functional disability. It is well-documented that the human gene-2 (H2)-relaxin hormone exhibits pleiotropic properties via its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1), including anti-inflammatory and anti-apoptotic effects, thus making it a potential therapeutic for stroke. Hence, the current study investigated whether post-stroke H2-relaxin administration could improve functional and histological outcomes. 8-12-week-old male C57BL/6 mice were subjected to sham operation or photothrombotic stroke and intravenously-administered with either saline (vehicle) or 0.02, 0.2 or 2 mg/kg doses of recombinant H2-relaxin at 6, 24 and 48 h post-stroke. Motor function was assessed using the hanging wire and cylinder test pre-surgery, and at 24 and 72 h post-stroke. Brains were removed after 72 h and infarct volume was assessed via thionin staining, and RXFP1 expression, leukocyte infiltration and apoptosis were determined by immunofluorescence. RXFP1 was identified on neurons, astrocytes and macrophages, and increased post-stroke. Whilst H2-relaxin did not alter infarct volume, it did cause a dose-dependent improvement in motor function at 24 and 72 h post-stroke. Moreover, 2 mg/kg H2-relaxin significantly decreased the number of apoptotic cells as well as macrophages and neutrophils within the ischaemic hemisphere, but did not alter T or B cells numbers. The anti-inflammatory and anti-apoptotic effects of H2-relaxin when administered at 6 h post-cerebral ischaemia may provide a novel therapeutic option for patients following ischaemic stroke.
Assuntos
Isquemia Encefálica , AVC Isquêmico , Relaxina , Acidente Vascular Cerebral , Camundongos , Animais , Humanos , Masculino , Relaxina/farmacologia , Relaxina/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/química , Receptores de Peptídeos/metabolismo , Camundongos Endogâmicos C57BL , Acidente Vascular Cerebral/tratamento farmacológico , Encéfalo/metabolismo , Apoptose , Infarto , Anti-InflamatóriosRESUMO
BACKGROUND AND AIMS: Atherosclerosis is associated with a reduction in the bioavailability and/or bioactivity of endogenous nitric oxide (NO). Dietary nitrate has been proposed as an alternate source when endogenous NO production is reduced. Our previous study demonstrated a protective effect of dietary nitrate on the development of atherosclerosis in the apoE-/- mouse model. However most patients do not present clinically until well after the disease is established. The aims of this study were to determine whether chronic dietary nitrate supplementation can prevent or reverse the progression of atherosclerosis after disease is already established, as well as to explore the underlying mechanism of these cardiovascular protective effects. METHODS: 60 apoE-/- mice were given a high fat diet (HFD) for 12 weeks to allow for the development of atherosclerosis. The mice were then randomized to (i) control group (HFD + 1 mmol/kg/day NaCl), (ii) moderate-dose group (HFD +1 mmol/kg/day NaNO3), or (iii) high-dose group (HFD + 10 mmol/kg/day NaNO3) (20/group) for a further 12 weeks. A group of apoE-/- mice (n = 20) consumed a normal laboratory chow diet for 24 weeks and were included as a reference group. RESULTS: Long-term supplementation with high dose nitrate resulted in ~ 50% reduction in plaque lesion area. Collagen expression and smooth muscle accumulation were increased, and lipid deposition and macrophage accumulation were reduced within atherosclerotic plaques of mice supplemented with high dose nitrate. These changes were associated with an increase in nitrite reductase as well as activation of the endogenous eNOS-NO pathway. CONCLUSION: Long-term high dose nitrate significantly attenuated the progression of established atherosclerosis in the apoE-/- mice fed a HFD. This appears to be mediated in part through a XOR-dependent reduction of nitrate to NO, as well as enhanced eNOS activation via increased Akt and eNOS phosphorylation.
Assuntos
Aterosclerose , Placa Aterosclerótica , Animais , Camundongos , Apolipoproteínas E/genética , Aterosclerose/prevenção & controle , Aterosclerose/metabolismo , Dieta Hiperlipídica/efeitos adversos , Suplementos Nutricionais , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitratos , Óxido Nítrico , Placa Aterosclerótica/prevenção & controleRESUMO
Fibrosis is a hallmark of several cardiovascular diseases. The relaxin family peptide receptor 1 (RXFP1) agonist, relaxin, has rapidly occurring anti-fibrotic actions which are mediated through RXFP1 and angiotensin II receptor crosstalk on renal and cardiac myofibroblasts. Here, we investigated whether this would allow relaxin to indirectly activate angiotensin II type 2 receptor (AT2 R)-specific signal transduction in primary human cardiac myofibroblasts (HCMFs). The anti-fibrotic effects of recombinant human relaxin (RLX; 16.8 nM) or the AT2 R-agonist, Compound 21 (C21; 1 µM), were evaluated in TGF-ß1-stimulated HCMFs, in the absence or presence of an RXFP1 antagonist (1 µM) or AT2 R antagonist (0.1 µM) to confirm RXFP1-AT2 R crosstalk. Competition binding for RXFP1 was determined. Western blotting was performed to determine which AT2 R-specific protein phosphatases were expressed by HCMFs; then, the anti-fibrotic effects of RLX and/or C21 were evaluated in the absence or presence of pharmacological inhibition (NSC95397 (1 µM) for MKP-1; okadaic acid (10 nM) for PP2A) or siRNA-knockdown of these phosphatases after 72 hours. The RLX- or C21-induced increase in ERK1/2 and nNOS phosphorylation, and decrease in α-SMA (myofibroblast differentiation) and collagen-I expression by HCMFs was abrogated by pharmacological blockade of RXFP1 or the AT2 R, confirming RXFP1-AT2 R crosstalk in these cells. HCMFs were found to express AT2 R-dependent MKP-1 and PP2A phosphatases, while pharmacological blockade or siRNA-knockdown of either phosphatase also abolished RLX and/or C21 signal transduction in HCMFs (all P < .05 vs RLX or C21 alone). These findings demonstrated that RLX can indirectly activate AT2 R-dependent phosphatase activity in HCMFs by signaling through RXFP1-AT2 R crosstalk, which have important therapeutic implications for its anti-fibrotic actions.
Assuntos
Fibrose/tratamento farmacológico , Fibrose/metabolismo , Coração/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Relaxina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Óxido Nítrico Sintase Tipo I/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
ABSTRACT: Nitroxyl (HNO), the 1 electron-reduced and protonated form of nitric oxide (NOâ¢), has emerged as a nitrogen oxide with a suite of vasoprotective properties and therapeutic advantages over its redox sibling. Although HNO has garnered much attention due to its cardioprotective actions in heart failure, its ability to modulate vascular function, without the limitations of tolerance development and NO⢠resistance, is desirable in the treatment of vascular disease. HNO serves as a potent vasodilator and antiaggregatory agent and has an ability to limit vascular inflammation and reactive oxygen species generation. In addition, its resistance to scavenging by reactive oxygen species and ability to target distinct vascular signaling pathways (Kv, KATP, and calcitonin gene-related peptide) contribute to its preserved efficacy in hypertension, diabetes, and hypercholesterolemia. In this review, the vasoprotective actions of HNO will be compared with those of NOâ¢, and the therapeutic utility of HNO donors in the treatment of angina, acute cardiovascular emergencies, and chronic vascular disease are discussed.
Assuntos
Fármacos Cardiovasculares/uso terapêutico , Doenças Cardiovasculares/tratamento farmacológico , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Óxidos de Nitrogênio/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Humanos , Inibidores da Agregação Plaquetária/uso terapêutico , Transdução de Sinais , Vasodilatadores/uso terapêuticoRESUMO
Reactive oxygen species (ROS) dysregulation is a hallmark of cardiovascular disease, characterised by an imbalance in the synthesis and removal of ROS. ROS such as superoxide (â¢O2-), hydrogen peroxide (H2O2), hydroxyl (OHâ¢) and peroxynitrite (ONOO-) have a marked impact on cardiovascular function, contributing to the vascular impairment and cardiac dysfunction associated with diseases such as angina, hypertension, diabetes and heart failure. Central to the vascular dysfunction is a reduction in bioavailability and/or physiological effects of vasoprotective nitric oxide (NOâ¢), leading to vasoconstriction, inflammation and vascular remodelling. In a cardiac context, increased ROS generation can also lead to modification of key proteins involved in cardiac contractility. Whilst playing a key role in the pathogenesis of cardiovascular disease, ROS dysregulation also limits the clinical efficacy of current therapies, such as nitrosovasodilators. As such, alternate therapies are sought. This review will discuss the impact of ROS dysregulation on the therapeutic utility of NO⢠and its redox sibling, nitroxyl (HNO). Both nitric oxide (NO) and nitroxyl (HNO) donors signal through soluble guanylyl cyclase (sGC). NO binds to the Fe(II) form of sGC and nitroxyl possibly to both sGC heme and thiol groups. In the vasculature, nitroxyl can also signal through voltage-dependent (Kv) and ATP-sensitive (KATP) K+ channels as well as calcitonin gene-related peptide (CGRP). In the heart, HNO directly targets critical thiols to increase myocardial contractility, an effect not seen with NO. The qualitative effects via elevation of cGMP are similar, i.e. lusitropic in the heart and inhibitory on vasoconstriction, inflammation, aggregation and vascular remodelling. Of pathophysiological significance is the fact the efficacy of NO donors is impaired by ROS, e.g. through chemical scavenging of NO, to generate reactive nitrogen oxide species (RNOS), whilst nitroxyl is apparently not.
Assuntos
Óxido Nítrico , Irmãos , Humanos , Peróxido de Hidrogênio , Óxidos de Nitrogênio , Oxirredução , Espécies Reativas de OxigênioRESUMO
Macrophages, which accumulate in tissues during inflammation, may be polarized toward pro-inflammatory (M1) or tissue reparative (M2) phenotypes. The balance between these phenotypes can have a substantial influence on the outcome of inflammatory diseases such as atherosclerosis. Improved biomarkers of M1 and M2 macrophages would be beneficial for research, diagnosis, and monitoring the effects of trial therapeutics in such diseases. To identify novel biomarkers, we have characterized the global proteomes of THP-1 macrophages polarized to M1 and M2 states in comparison with unpolarized (M0) macrophages. M1 polarization resulted in increased expression of numerous pro-inflammatory proteins including the products of 31 genes under the transcriptional control of interferon regulatory factor 1 (IRF-1). In contrast, M2 polarization identified proteins regulated by components of the transcription factor AP-1. Among the most highly upregulated proteins under M1 conditions were the three interferon-induced proteins with tetratricopeptide repeats (IFITs: IFIT1, IFIT2, and IFIT3), which function in antiviral defense. Moreover, IFIT1, IFIT2, and IFIT3 mRNA were strongly upregulated in M1 polarized human primary macrophages and IFIT1 was also expressed in a subset of macrophages in aortic sinus and brachiocephalic artery sections from atherosclerotic ApoE-/- mice. On the basis of these results, we propose that IFITs may serve as useful markers of atherosclerosis and potentially other inflammatory diseases.
Assuntos
Fator Regulador 1 de Interferon/genética , Macrófagos/imunologia , Proteínas/análise , Proteômica/métodos , Repetições de Tetratricopeptídeos , Animais , Aterosclerose/diagnóstico , Aterosclerose/patologia , Biomarcadores/análise , Humanos , Inflamação/diagnóstico , Inflamação/patologia , Macrófagos/química , Camundongos , Camundongos Knockout , Proteínas/genética , Células THP-1 , Regulação para Cima/genéticaRESUMO
Nitric oxide (NO) plays a pivotal role in the maintenance of cardiovascular homeostasis. A reduction in the bioavailability of endogenous NO, manifest as a decrease in the production and/or impaired signaling, is associated with many cardiovascular diseases including hypertension, atherosclerosis, stroke and heart failure. There is substantial evidence that reactive oxygen species (ROS), generated predominantly from NADPH oxidases (Nox), are responsible for the reduced NO bioavailability in vascular and cardiac pathologies. ROS can compromise NO function via a direct inactivation of NO, together with a reduction in NO synthesis and oxidation of its receptor, soluble guanylyl cyclase. Whilst nitrovasodilators are administered to compensate for the ROS-mediated loss in NO bioactivity, their clinical utility is limited due to the development of tolerance and resistance and systemic hypotension. Moreover, efforts to directly scavenge ROS with antioxidants has had limited clinical efficacy. This review outlines the therapeutic utility of NO-based therapeutics in cardiovascular diseases and describes the source and impact of ROS in these pathologies, with particular focus on the interaction with NO. Future therapeutic approaches in the treatment of cardiovascular diseases are highlighted with a focus on nitroxyl (HNO) donors as an alternative to traditional NO donors and the development of novel Nox inhibitors.
Assuntos
Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/metabolismo , Homeostase/fisiologia , Humanos , Doadores de Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
OBJECTIVE: To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension. METHODS: Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4mg/d,s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10days post-surgery, mice commenced treatment with anakinra (75mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys. RESULTS: By 10days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3±2.4mmHg) compared to control mice (121.7±2.7mmHg; n=18, P<0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by â¼20mmHg (n=16, P<0.05), but had no effect in controls. In 1K/DOCA/salt-treated mice, anakinra modestly reduced (â¼30%) renal expression of some (CCL5, CCL2; n=7-8; P<0.05) but not all (ICAM-1, IL-6) inflammatory markers, and had no effect on immune cell infiltration (n=7-8, P>0.05). Anakinra reduced renal collagen content (n=6, P<0.01) but paradoxically appeared to exacerbate the renal and glomerular hypertrophy (n=8-9, P<0.001) that accompanied 1K/DOCA/salt-induced hypertension. CONCLUSION: Despite its anti-hypertensive and renal anti-fibrotic actions, anakinra had minimal effects on inflammation and leukocyte infiltration in mice with 1K/DOCA/salt-induced hypertension. Future studies will assess whether the anti-hypertensive actions of anakinra are mediated by protective actions in other BP-regulating or salt-handling organs such as the arteries, skin and brain.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Fibrose/tratamento farmacológico , Hipertensão Renal/tratamento farmacológico , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Nefropatias/tratamento farmacológico , Rim/efeitos dos fármacos , Animais , Anti-Hipertensivos/farmacologia , Biomarcadores/metabolismo , Acetato de Desoxicorticosterona/farmacologia , Fibrose/metabolismo , Hipertensão Renal/induzido quimicamente , Hipertensão Renal/metabolismo , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Rim/metabolismo , Nefropatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cloreto de Sódio na Dieta/farmacologiaRESUMO
Nitroxyl anion (HNO) donors are currently being assessed for their therapeutic utility in several cardiovascular disorders including heart failure. Here, we examine their effect on factors that precede atherosclerosis including endothelial cell and monocyte activation, leucocyte adhesion to the endothelium and macrophage polarization. Similar to the NO donor glyceryl trinitrate (GTN), the HNO donors Angeli's salt (AS) and isopropylamine NONOate (IPA/NO) decreased leucocyte adhesion to activated human umbilical vein endothelial cells (HUVECs) and mouse isolated aorta. This reduction in adhesion was accompanied by a reduction in intercellular adhesion molecule-1 (ICAM-1) and the cytokines monocyte chemoattractant protein 1 (MCP-1) and interleukin 6 (IL-6) which was inhibitor of nuclear factor κB (NFκB) α (IκBα)- and subsequently NFκB-dependent. Intriguingly, the effects of AS on leucocyte adhesion, like those on vasodilation, were found to not be susceptible to pharmacological tolerance, unlike those observed with GTN. As well, HNO reduces monocyte activation and promotes polarization of M2 macrophages. Taken together, our data demonstrate that HNO donors can reduce factors that are associated with and which precede atherosclerosis and may thus be useful therapeutically. Furthermore, since the effects of the HNO donors were not subject to tolerance, this confers an additional advantage over NO donors.
Assuntos
Aterosclerose/tratamento farmacológico , Polaridade Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Óxidos de Nitrogênio/administração & dosagem , Animais , Aorta/efeitos dos fármacos , Aorta/imunologia , Aorta/fisiopatologia , Aterosclerose/imunologia , Aterosclerose/fisiopatologia , Quimiocina CCL2/imunologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Molécula 1 de Adesão Intercelular/imunologia , Interleucina-6/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologiaRESUMO
Increased vascular stiffness and reduced endothelial nitric oxide (NO) bioavailability are characteristic of diabetes. Whether these are evident at a more moderate levels of hyperglycaemia has not been investigated. The objectives of this study were to examine the association between the level of glycaemia and resistance vasculature phenotype, incorporating both arterial stiffness and endothelial function. Diabetes was induced in male Sprague Dawley rats with streptozotocin (STZ; 55mg/kg i.v.) and followed for 8 weeks. One week post STZ, diabetic rats were allocated to either moderate (â¼20mM blood glucose, 6-7U/insulins.c. daily) or severe hyperglycaemia (â¼30mM blood glucose, 1-2U/insulins.c. daily as required). At study end, rats were anesthetized, and the mesenteric arcade was collected. Passive mechanical wall properties were assessed by pressure myography. Responses to the endothelium-dependent vasodilator acetylcholine (ACh) were assessed using wire myography. Our results demonstrated for the first time that mesenteric arteries from both moderate and severely hyperglycaemic diabetic rats exhibited outward hypertrophic remodelling and increased axial stiffness compared to arteries from non-diabetic rats. Secondly, mesenteric arteries from severely (â¼30mM blood glucose), but not moderately hyperglycaemic (â¼20mM blood glucose) rats exhibit a significant reduction to ACh sensitivity compared to their non-diabetic counterparts. This endothelial dysfunction was associated with significant reduction in endothelium-derived hyperpolarisation and endothelium-dependent NO-mediated relaxation. Interestingly, endothelium-derived nitroxyl (HNO)-mediated relaxation was intact. Therefore, moderate hyperglycaemia is sufficient to induce adverse structural changes in the mesenteric vasculature, but more severe hyperglycaemia is essential to cause endothelial dysfunction.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Angiopatias Diabéticas/etiologia , Endotélio Vascular/fisiopatologia , Artérias Mesentéricas/fisiopatologia , Remodelação Vascular , Rigidez Vascular , Animais , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/fisiopatologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Hemoglobinas Glicadas/metabolismo , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/metabolismo , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Resistência Vascular , Vasodilatação , Vasodilatadores/farmacologiaRESUMO
Macrophages accumulate in blood vessels during hypertension. However, their contribution to vessel remodeling is unknown. In the present study, we examined the polarization state of macrophages (M1/M2) in aortas of mice during hypertension and investigated whether antagonism of chemokine receptors involved in macrophage accumulation reduces vessel remodeling and blood pressure (BP). Mice treated with ANG II (0.7 mg·kg(-1)·day(-1), 14 days) had elevated systolic BP (158 ± 3 mmHg) compared with saline-treated animals (122 ± 3 mmHg). Flow cytometry revealed that ANG II infusion increased numbers of CD45(+)CD11b(+)Ly6C(hi) monocytes and CD45(+)CD11b(+)F4/80(+) macrophages by 10- and 2-fold, respectively. The majority of macrophages were positive for the M2 marker CD206 but negative for the M1 marker inducible nitric oxide synthase. Expression of other M2 genes (arginase-1, Fc receptor-like S scavenger receptor, and receptor-1) was elevated in aortas from ANG II-treated mice, whereas M1 genes [TNF and chemokine (C-X-C motif) ligand 2] were unaltered. A PCR array to identify chemokine receptor targets for intervention revealed chemokine (C-C motif) receptor 2 (CCR2) to be upregulated in aortas from ANG II-treated mice, while flow cytometry identified Ly6C(hi) monocytes as the main CCR2-expressing cell type. Intervention with a CCR2 antagonist (INCB3344; 30 mg·kg(-1)·day(-1)), 7 days after the commencement of ANG II infusion, reduced aortic macrophage numbers. INCB334 also reduced aortic collagen deposition, elastin loss, and BP in ANG II-treated mice. Thus, ANG II-dependent hypertension in mice is associated with Ly6C(hi) monocyte and M2 macrophage accumulation in the aorta. Inhibition of macrophage accumulation with a CCR2 antagonist prevents ANG II-induced vessel fibrosis and elevated BP, highlighting this as a promising approach for the future treatment of vessel remodeling/stiffening in hypertension.
Assuntos
Aorta/patologia , Pressão Sanguínea , Elastina/metabolismo , Hipertensão/patologia , Macrófagos/metabolismo , Angiotensina II/toxicidade , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos Ly/genética , Antígenos Ly/metabolismo , Aorta/efeitos dos fármacos , Aorta/metabolismo , Arginase/genética , Arginase/metabolismo , Colágeno/genética , Colágeno/metabolismo , Elastina/genética , Fibrose/metabolismo , Fibrose/patologia , Hipertensão/etiologia , Hipertensão/metabolismo , Macrófagos/classificação , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismoRESUMO
Pre-clinical studies have identified nitroxyl (HNO), the reduced congener of nitric oxide (NOâ¢), as a potent vasodilator which is resistant to tolerance development. The present study explores the efficacy of HNO in human blood vessels and describes, for the first time, a vasodilator for humans that is not susceptible to tolerance. Human radial arteries and saphenous veins were obtained from patients undergoing coronary artery graft surgery and mounted in organ baths. Repeated vasodilator responses to the HNO donor Angeli's salt (AS) and NO⢠donor glyceryl trinitrate (GTN) were determined. AS- and GTN-induced concentration-dependent vasorelaxation of both human radial arteries (AS pEC50: 6.5 ± 0.2; -log M) and saphenous veins (pEC50: 6.7 ± 0.1) with similar potency. In human radial arteries, GTN-induced relaxation was reduced by the NO⢠scavenger hydroxocobalamin (HXC; P<0.05) but was unaffected by the HNO scavenger L-cysteine. Alternately, AS was unaffected by HXC but was reduced by L-cysteine (5-fold shift, P<0.05). The sGC (soluble guanylate cyclase) inhibitor ODQ abolished responses to both AS and GTN in arteries and veins (P<0.05). Inhibition of voltage-dependent potassium channels (Kv channels) with 4-AP also significantly reduced responses to AS (pEC50: 5.5) and GTN, suggesting that the relaxation to both redox congeners is cGMP- and Kv channel-dependent. Critically, a concentration-dependent development of tolerance to GTN (1 and 10 µM; P<0.05), but not to AS, was observed in both saphenous veins and radial arteries. Like GTN, the HNO donor AS causes vasorelaxation of human blood vessels via activation of a cGMP-dependent pathway. Unlike GTN, however, it does not develop tolerance in human blood vessels.
Assuntos
Doadores de Óxido Nítrico/farmacologia , Nitritos/farmacologia , Óxidos de Nitrogênio/farmacologia , Nitroglicerina/farmacologia , Artéria Radial/efeitos dos fármacos , Veia Safena/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Tolerância a Medicamentos , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Humanos , Técnicas In Vitro , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Artéria Radial/fisiologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Veia Safena/fisiologia , Guanilil Ciclase SolúvelRESUMO
Nitroxyl (HNO), the redox congener of nitric oxide, has numerous vasoprotective actions including an ability to induce vasodilation and inhibit platelet aggregation. Given HNO is resistant to scavenging by superoxide and does not develop tolerance, we hypothesised that HNO would retain its in vivo vasodilatory action in the setting of hypertension. The in vitro and in vivo vasodilator properties of the HNO donors Angeli's salt (AS) and isopropylamine/NONOate (IPA/NO) were compared with the NO donor diethylamine/NONOate (DEA/NO) in spontaneously hypertensive rats (SHR) and normotensive [Wistar-Kyoto (WKY) rats]. AS (10, 50, and 200 µg/kg), IPA/NO (10, 50, and 200 µg/kg), and DEA/NO (1, 5, and 20 µg/kg) caused dose-dependent depressor responses in conscious WKY rats of similar magnitude. Depressor responses to AS and IPA/NO were significantly attenuated (P < 0.01) after infusion of the HNO scavenger N-acetyl-l-cysteine (NAC), confirming that AS and IPA/NO function as HNO donors in vivo. In contrast, responses to DEA/NO were unchanged following NAC infusion. Depressor responses to AS and IPA/NO in conscious SHR retained their sensitivity to the inhibitory effects of NAC (P < 0.01), yet those to DEA/NO in SHR were significantly (P < 0.05) enhanced following NAC infusion. Importantly, depressor responses to AS, IPA/NO, and DEA/NO were preserved in hypertension and vasorelaxation to AS and DEA/NO, in isolated aorta, unchanged in SHR as compared with WKY rats. This study has shown for the first time that HNO donors exert antihypertensive effects in vivo and may, therefore, offer a therapeutic alternative to traditional nitrovasodilators in the treatment of cardiovascular disorders such as hypertension.
Assuntos
Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Doadores de Óxido Nítrico/administração & dosagem , Óxidos de Nitrogênio/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Animais , Pressão Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Wistar , Resultado do TratamentoRESUMO
Nitroxyl (HNO) is a redox congener of NO. We now directly compare the antihypertrophic efficacy of HNO and NO donors in neonatal rat cardiomyocytes and compare their contributing mechanisms of actions in this setting. Isopropylamine-NONOate (IPA-NO) elicited concentration-dependent inhibition of endothelin-1 (ET1)-induced increases in cardiomyocyte size, with similar suppression of hypertrophic genes. Antihypertrophic IPA-NO actions were significantly attenuated by l-cysteine (HNO scavenger), Rp-8-pCTP-cGMPS (cGMP-dependent protein kinase inhibitor), and 1-H-(1,2,4)-oxodiazolo-quinxaline-1-one [ODQ; to target soluble guanylyl cyclase (sGC)] but were unaffected by carboxy-PTIO (NO scavenger) or CGRP8-37 (calcitonin gene-related peptide antagonist). Furthermore, IPA-NO significantly increased cardiomyocyte cGMP 3.5-fold (an l-cysteine-sensitive effect) and stimulated sGC activity threefold, without detectable NO release. IPA-NO also suppressed ET1-induced cardiomyocyte superoxide generation. The pure NO donor diethylamine-NONOate (DEA-NO) reproduced these IPA-NO actions but was sensitive to carboxy-PTIO rather than l-cysteine. Although IPA-NO stimulation of purified sGC was preserved under pyrogallol oxidant stress (in direct contrast to DEA-NO), cardiomyocyte sGC activity after either donor was attenuated by this stress. Excitingly IPA-NO also exhibited acute antihypertrophic actions in response to pressure overload in the intact heart. Together these data strongly suggest that IPA-NO protection against cardiomyocyte hypertrophy is independent of both NO and CGRP but rather utilizes novel HNO activation of cGMP signaling. Thus HNO acutely limits hypertrophy independently of NO, even under conditions of elevated superoxide. Development of longer-acting HNO donors may thus represent an attractive new strategy for the treatment of cardiac hypertrophy, as stand-alone and/or add-on therapy to standard care.
Assuntos
Cardiomegalia/tratamento farmacológico , Fármacos Cardiovasculares/uso terapêutico , GMP Cíclico/metabolismo , Hidrazinas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Óxidos de Nitrogênio/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Antioxidantes/farmacologia , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Proteína Quinase Dependente de GMP Cíclico Tipo I/antagonistas & inibidores , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Relação Dose-Resposta a Droga , Endotelina-1/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Doadores de Óxido Nítrico/farmacologia , Pirogalol/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Guanilil Ciclase Solúvel , Fatores de TempoRESUMO
Stroke is a leading cause of mortality and morbidity with a paucity of effective pharmacological treatments. We have previously identified insulin-regulated aminopeptidase (IRAP) as a potential target for the development of a new class of drugs for the treatment of stroke, as global deletion of this gene in mice significantly protected against ischemic damage. In the current study, we demonstrate that small molecular weight IRAP inhibitors reduce infarct volume and improve neurological outcome in a hypertensive animal model of ischemic stroke. The effects of two structurally distinct IRAP inhibitors (HFI419 or SJM164) were investigated in a model of stroke where the middle cerebral artery was transiently occluded with endothelin-1 in the conscious spontaneously hypertensive rat. IRAP inhibitor was administered into the lateral ventricle at 2 or 6 h after stroke, with subsequent doses delivered at 24, 48 and 70 h post-stroke. Functional outcomes were assessed prior to drug treatment, and on day 1 and 3 post-stroke. Histological analyses and neuroinflammatory cytokine profiling were conducted at 72 and 24 h post-stroke respectively. IRAP inhibitor treatment following stroke significantly reduced infarct volume and improved neurological and motor deficits. These protective effects were maintained even when the therapeutic window was extended to 6 h. Examination of the cellular architecture at 72 h post-stroke demonstrated that IRAP expression was upregulated in CD11b positive cells and activated astrocytes. Furthermore, IRAP inhibitor treatment significantly increased gene expression for interleukin 6 and C-C motif chemokine ligand 2 in the ischemic core. This study provides proof-of-principle that selective inhibition of IRAP activity with two structurally distinct IRAP inhibitors reduces infarct volume and improves functional outcome even when the first dose is administered 6 h post-stroke. This is the first direct evidence that IRAP inhibitors are a class of drug with potential use in the treatment of ischemic stroke.
Assuntos
Cistinil Aminopeptidase , AVC Isquêmico , Animais , Camundongos , Ratos , Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/metabolismo , Infarto , AVC Isquêmico/tratamento farmacológico , Neuroproteção , Ratos Endogâmicos SHRRESUMO
In cardiovascular disease, pathological and protective roles are reported for the Th2 cytokines IL-4 and IL-13, respectively. We hypothesised that differential effects on macrophage function are responsible. Type I and II receptor subunit (IL-2Rγ, IL-4Rα and IL-13Rα1) and M2 marker (MRC-1, CCL18, CCL22) expression was assessed via RT-qPCR in IL-4- and IL-13-treated human primary macrophages. Downstream signalling was evaluated via STAT1, STAT3 and STAT6 inhibitors, and IL-4- and IL-13-induced reactive oxygen species (ROS) generation assessed. IL-4 and IL-13 exhibited equivalent potency and efficacy for M2 marker induction, which was attenuated by STAT3 inhibition. Both cytokines enhanced PDBu-stimulated superoxide generation however this effect was 17% greater with IL-4 treatment. Type I IL-4 receptor expression was increased on M1-like macrophages but did not lead to a differing ability of these cytokines to modulate M1-like macrophage superoxide production. Overall, this study did not identify major differences in the ability of IL-4 and IL-13 to modulate macrophage function, suggesting that the opposing roles of these cytokines in cardiovascular disease are likely to be via actions on other cell types. Future studies should directly compare IL-4 and IL-13 in vivo to more thoroughly investigate the therapeutic validity of selective targeting of these cytokines.
Assuntos
Doenças Cardiovasculares , Interleucina-13 , Humanos , Doenças Cardiovasculares/metabolismo , Citocinas/metabolismo , Interleucina-13/farmacologia , Interleucina-13/metabolismo , Interleucina-4/farmacologia , Interleucina-4/metabolismo , Macrófagos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismoRESUMO
The hormone, relaxin (RLX), exerts various organ-protective effects independently of etiology. However, its complex two-chain and three disulphide bonded structure is a limitation to its preparation and affordability. Hence, a single chain-derivative of RLX, B7-33, was developed and shown to retain the anti-fibrotic effects of RLX in vitro and in vivo. Here, we determined whether B7-33 could retain the other cardioprotective effects of RLX, and also compared its therapeutic efficacy to the ACE inhibitor, perindopril. Adult male 129sv mice were subjected to isoprenaline (ISO; 25 mg/kg/day, s.c)-induced cardiomyopathy, then s.c-treated with either RLX (0.5 mg/kg/day), B7-33 (0.25 mg/kg/day; equivalent dose corrected for MW) or perindopril (1 mg/kg/day) from days 7-14 post-injury. Control mice received saline instead of ISO. Changes in animal body weight (BW) and systolic blood pressure (SBP) were measured weekly, whilst cardiomyocyte hypertrophy and measures of vascular dysfunction and rarefaction, left ventricular (LV) inflammation and fibrosis were assessed at day 14 post-injury. ISO-injured mice had significantly increased LV inflammation, cardiomyocyte hypertrophy, fibrosis, vascular rarefaction and aortic contractility in the absence of any changes in BW or SBP at day 14 post-injury. Both B7-33 and RLX equivalently reduced LV fibrosis and normalised the ISO-induced LV inflammation and cardiomyocyte hypertrophy, whilst restoring blood vessel density and aortic contractility. Comparatively, perindopril lowered SBP and the ISO-induced LV inflammation and vascular rarefaction, but not fibrosis or hypertrophy. As B7-33 retained the cardioprotective effects of RLX and provided rapid-occurring anti-fibrotic effects compared to perindopril, it could be considered as a cost-effective cardioprotective therapy.
Assuntos
Cardiomiopatias , Rarefação Microvascular , Relaxina , Camundongos , Animais , Masculino , Perindopril/farmacologia , Perindopril/uso terapêutico , Relaxina/farmacologia , Rarefação Microvascular/tratamento farmacológico , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/prevenção & controle , Modelos Teóricos , Inflamação/tratamento farmacológico , Hipertrofia/tratamento farmacológicoRESUMO
Despite recent therapeutic advances, pulmonary hypertension (PH) remains a fatal disease due to the development of right ventricular (RV) failure. At present, no treatments targeted at the right ventricle are available, and RV function is not widely considered in the preclinical assessment of new therapeutics. Several small animal models are used in the study of PH, including the classic models of exposure to either hypoxia or monocrotaline, newer combinational and genetic models, and pulmonary artery banding, a surgical model of pure RV pressure overload. These models reproduce selected features of the structural remodelling and functional decline seen in patients and have provided valuable insight into the pathophysiology of RV failure. However, significant reversal of remodelling and improvement in RV function remains a therapeutic obstacle. Emerging animal models will provide a deeper understanding of the mechanisms governing the transition from adaptive remodelling to a failing right ventricle, aiding the hunt for druggable molecular targets. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.
Assuntos
Insuficiência Cardíaca , Hipertensão Pulmonar , Disfunção Ventricular Direita , Animais , Modelos Animais de Doenças , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Monocrotalina , Artéria Pulmonar , Disfunção Ventricular Direita/tratamento farmacológico , Função Ventricular DireitaRESUMO
BACKGROUND AND PURPOSE: The risk of fatal cardiovascular events is increased in patients with type 2 diabetes mellitus (T2DM). A major contributor to poor prognosis is impaired nitric oxide (NOâ¢) signalling at the level of tissue responsiveness, termed NO⢠resistance. This study aimed to determine if T2DM promotes NO⢠resistance in the heart and vasculature and whether tissue responsiveness to nitroxyl (HNO) is affected. EXPERIMENTAL APPROACH: At 8 weeks of age, male Sprague-Dawley rats commenced a high-fat diet. After 2 weeks, the rats received low-dose streptozotocin (two intraperitoneal injections, 35 mg·kg-1 , over two consecutive days) and continued on the same diet. Twelve weeks later, isolated hearts were Langendorff-perfused to assess responses to the NO⢠donor diethylamine NONOate (DEA/NO) and the HNO donor Angeli's salt. Isolated mesenteric arteries were utilised to measure vascular responsiveness to the NO⢠donors sodium nitroprusside (SNP) and DEA/NO, and the HNO donor Angeli's salt. KEY RESULTS: Inotropic, lusitropic and coronary vasodilator responses to DEA/NO were impaired in T2DM hearts, whereas responses to Angeli's salt were preserved or enhanced. Vasorelaxation to Angeli's salt was augmented in T2DM mesenteric arteries, which were hyporesponsive to the relaxant effects of SNP and DEA/NO. CONCLUSION AND IMPLICATIONS: This is the first evidence that inotropic and lusitropic responses are preserved, and NO⢠resistance in the coronary and mesenteric vasculature is circumvented, by the HNO donor Angeli's salt in T2DM. These findings highlight the cardiovascular therapeutic potential of HNO donors, especially in emergencies such as acute ischaemia or heart failure.