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2.
Artigo em Inglês | MEDLINE | ID: mdl-31408105

RESUMO

AIMS: Cardiovascular thrombosis is responsible a quarter of deaths annually worldwide. Current imaging methods for cardiovascular thrombosis focus on anatomical identification of thrombus but cannot determine thrombus age or activity. Molecular imaging techniques hold promise for identification and quantification of thrombosis in vivo. Our objective was to assess a novel optical and positron-emitting probe targeting Factor XIIIa (ENC2015) as biomarker of active thrombus formation. METHODS AND RESULTS: Optical and positron-emitting ENC2015 probes were assessed ex vivo using blood drawn from human volunteers and passed through perfusion chambers containing denuded porcine aorta as a model of arterial injury. Specificity of ENC2015 was established with co-infusion of a factor XIIIa inhibitor. In vivo18F-ENC2015 biodistribution, kinetics, radiometabolism, and thrombus binding were characterized in rats. Both Cy5 and fluorine-18 labelled ENC2015 rapidly and specifically bound to thrombi. Thrombus uptake was inhibited by a factor XIIIa inhibitor. 18F-ENC2015 remained unmetabolized over 8 h when incubated in ex vivo human blood. In vivo, 42% of parent radiotracer remained in blood 60 min post-administration. Biodistribution studies demonstrated rapid clearance from tissues with elimination via the urinary system. In vivo,18F-ENC2015 uptake was markedly increased in the thrombosed carotid artery compared to the contralateral patent artery (mean standard uptake value ratio of 2.40 vs. 0.74, P < 0.0001). CONCLUSION : ENC2015 rapidly and selectively binds to acute thrombus in both an ex vivo human translational model and an in vivo rodent model of arterial thrombosis. This probe holds promise for the non-invasive identification of thrombus formation in cardiovascular disease.

3.
J Physiol ; 597(3): 767-780, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30537108

RESUMO

KEY POINTS: Type 1 diabetes mellitus increases cardiovascular risk; hypertension amplifies this risk, while pressure natriuresis regulates long-term blood pressure. We induced type 1 diabetes in rats by streptozotocin injection and demonstrated a substantial impairment of pressure natriuresis: acute increases in blood pressure did not increase renal medullary blood flow, tubular sodium reabsorption was not downregulated, and proximal tubule sodium reabsorption, measured by lithium clearance, was unaffected. Insulin reduced blood glucose in diabetic rats, and rescued the pressure natriuresis response without influencing lithium clearance, but did not restore medullary blood flow. Radiotelemetry showed that diastolic blood pressure was increased in diabetic rats, and its diurnal variation was reduced. Increases in medullary blood flow and decreases in distal tubule sodium reabsorption that offset acute rises in BP are impaired in early type 1 diabetes, and this impairment could be a target for preventing hypertension in type 1 diabetes. ABSTRACT: Type 1 diabetes mellitus (T1DM) substantially increases cardiovascular risk, and hypertension amplifies this risk. Blood pressure (BP) and body sodium homeostasis are linked. T1DM patients have increased total exchangeable sodium, correlating directly with BP. Pressure natriuresis is an important physiological regulator of BP. We hypothesised that pressure natriuresis would be impaired, and BP increased, in the early phase of T1DM. Male Sprague-Dawley rats were injected with streptozotocin (30-45 mg/kg) or citrate vehicle. After 3 weeks, pressure natriuresis was induced by serial arterial ligation. In non-diabetic controls, this increased fractional excretion of sodium from ∼1% to ∼25% of the filtered load (P < 0.01); in T1DM rats, the response was significantly blunted, peaking at only ∼3% (P < 0.01). Mechanistically, normal lithium clearance suggested that distal tubule sodium reabsorption was not downregulated with increased BP in T1DM rats. The pressure dependence of renal medullary perfusion, considered a key factor in the integrated response, was abolished. Insulin therapy rescued the natriuretic response in diabetic rats, restoring normal downregulation of tubular sodium reabsorption when BP was increased. However, the pressure dependence of medullary perfusion was not restored, suggesting persistent vascular dysfunction despite glycaemic control. Radiotelemetry showed that T1DM did not affect systolic BP, but mean diastolic BP was ∼5 mmHg higher than in non-diabetic controls (P < 0.01), and normal diurnal variation was reduced. In conclusion, functional impairment of renal sodium and BP homeostasis is an early manifestation of T1DM, preceding hypertension and nephropathy. Early intervention to restore pressure natriuresis in T1DM may complement reductions in cardiovascular risk achieved with glycaemic control.

4.
Endocrinology ; 159(11): 3791-3800, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30289445

RESUMO

Equine Cushing disease [pituitary pars intermedia dysfunction (PPID)] is a common condition of older horses, but its pathophysiology is complex and poorly understood. In contrast to pituitary-dependent hyperadrenocorticism in other species, PPID is characterized by elevated plasma ACTH but not elevated plasma cortisol. In this study, we address this paradox and the hypothesis that PPID is a syndrome of ACTH excess in which there is dysregulation of peripheral glucocorticoid metabolism and binding. In 14 horses with PPID compared with 15 healthy controls, we show that in plasma, cortisol levels and cortisol binding to corticosteroid binding globulin were not different; in urine, glucocorticoid and androgen metabolites were increased up to fourfold; in liver, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) expression was reduced; in perirenal adipose tissue, 11ß-HSD1 and carbonyl reductase 1 expression was increased; and tissue cortisol levels were not measurably different. The combination of normal plasma cortisol with markedly enhanced urinary cortisol metabolite excretion and dysregulated tissue-specific steroid-metabolizing enzymes suggests that cortisol clearance is increased in horses with PPID. We infer that the ACTH excess may be compensatory and pituitary pathology and autonomous secretion may be a secondary rather than primary pathology. It is possible that successful therapy in PPID may be targeted either at lowering ACTH or, paradoxically, at reducing cortisol clearance.


Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Doenças dos Cavalos/metabolismo , Hidrocortisona/metabolismo , Hipersecreção Hipofisária de ACTH/veterinária , Adeno-Hipófise Parte Intermédia/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Tecido Adiposo/metabolismo , Androgênios/metabolismo , Androgênios/urina , Animais , Carbonil Redutase (NADPH)/metabolismo , Estudos de Casos e Controles , Glucocorticoides/metabolismo , Glucocorticoides/urina , Cavalos , Hidrocortisona/urina , Fígado/metabolismo , Hipersecreção Hipofisária de ACTH/metabolismo , Transcortina/metabolismo
5.
Br J Cancer ; 119(12): 1508-1517, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30374123

RESUMO

BACKGROUND: The Wilms' tumour protein (WT1), which influences tumour development and angiogenesis, is a promising therapeutic target in breast cancer. We hypothesised that WT1 expression would vary in endothelial cells in distinct sub-classifications of breast cancer. METHODS: WT1 expression and vascular density were quantified by immunohistochemical analysis of human (n = 57) and murine breast cancers. Human tumours were sub-classified by histopathological grade, ER status and HER2 enrichment. RESULTS: WT1 was identified in endothelial (and epithelial and smooth muscle) cells in tumours and tumour-free tissues (controls) from patients and mice with breast cancer. WT1 expression was higher in tumours than in controls, but this was not due to increased endothelial WT1. Vascular WT1 in cancers decreased as histopathological grade increased. WT1 was higher in ER-positive versus ER-negative cancers. Strikingly, reduced WT1 expression in controls correlated with an increased Nottingham Prognostic Index score. CONCLUSIONS: Expression of WT1 is increased in breast cancers but this is not limited to the vascular compartment. The association between reduced WT1 in tumour-free tissue and poor prognosis suggests a protective role for WT1 in the healthy breast.


Assuntos
Neoplasias da Mama/patologia , Proteínas WT1/análise , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/química , Neoplasias da Mama/mortalidade , Feminino , Humanos , Camundongos , Gradação de Tumores , Receptor ErbB-2/análise , Receptores Estrogênicos/análise , Proteínas WT1/fisiologia
6.
Sci Rep ; 8(1): 11532, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30068994

RESUMO

Leukemia inhibitory factor (LIF), a pleiotropic cytokine belonging to the interleukin-6 family, is most often noted for its role in maintaining the balance between stem cell proliferation and differentiation. In rodents, LIF is expressed in both the fetal and adult testis; with the peritubular myoid (PTM) cells thought to be the main site of production. Given their anatomical location, LIF produced by PTM cells may act both on intratubular and interstitial cells to influence spermatogenesis and steroidogenesis respectively. Indeed, the leukemia inhibitory factor receptor (LIFR) is expressed in germ cells, Sertoli cells, Leydig cells, PTM cells and testicular macrophages, suggesting that LIF signalling via LIFR may be a key paracrine regulator of testicular function. However, a precise role(s) for testicular LIFR-signalling in vivo has not been established. To this end, we generated and characterised the testicular phenotype of mice lacking LIFR either in germ cells, Sertoli cells or both, to identify a role for LIFR-signalling in testicular development/function. Our analyses reveal that LIFR is dispensable in germ cells for normal spermatogenesis. However, Sertoli cell LIFR ablation results in a degenerative phenotype, characterised by abnormal germ cell loss, sperm stasis, seminiferous tubule distention and subsequent atrophy of the seminiferous tubules.


Assuntos
Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Células de Sertoli/metabolismo , Espermatogênese , Testículo/fisiologia , Animais , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/deficiência , Masculino , Camundongos , Camundongos Knockout
7.
Mol Ther ; 26(7): 1669-1684, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29703701

RESUMO

Pluripotent stem cell-derived differentiated endothelial cells offer high potential in regenerative medicine in the cardiovascular system. With the aim of translating the use of a human stem cell-derived endothelial cell product (hESC-ECP) for treatment of critical limb ischemia (CLI) in man, we report a good manufacturing practice (GMP)-compatible protocol and detailed cell tracking and efficacy data in multiple preclinical models. The clinical-grade cell line RC11 was used to generate hESC-ECP, which was identified as mostly endothelial (60% CD31+/CD144+), with the remainder of the subset expressing various pericyte/mesenchymal stem cell markers. Cell tracking using MRI, PET, and qPCR in a murine model of limb ischemia demonstrated that hESC-ECP was detectable up to day 7 following injection. Efficacy in several murine models of limb ischemia (immunocompromised/immunocompetent mice and mice with either type I/II diabetes mellitus) demonstrated significantly increased blood perfusion and capillary density. Overall, we demonstrate a GMP-compatible hESC-ECP that improved ischemic limb perfusion and increased local angiogenesis without engraftment, paving the way for translation of this therapy.


Assuntos
Células Endoteliais/citologia , Membro Posterior/citologia , Isquemia/terapia , Neovascularização Fisiológica/fisiologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/metabolismo , Membro Posterior/metabolismo , Humanos , Isquemia/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Pericitos/citologia , Pericitos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco/métodos
8.
Mol Cell Endocrinol ; 465: 82-91, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29024781

RESUMO

Male disadvantage in cardiovascular health is well recognised. However, the influence of androgens on atherosclerosis, one of the major causes of many life-threatening cardiovascular events, is not well understood. With the dramatic increase in clinical prescription of testosterone in the past decade, concerns about the cardiovascular side-effects of androgen supplementation or androgen deprivation therapy are increasing. Potential atheroprotective effects of testosterone could be secondary to (aromatase-mediated) conversion into oestradiol or, alternatively, to direct activation of androgen receptors (AR). Recent development of animal models with cell-specific AR knockout has indicated a complex role for androgen action in atherosclerosis. Most studies suggest androgens are atheroprotective but the precise role of AR remains unclear. Increased use of AR knockout models should clarify the role of AR in atherogenesis and, thus, lead to exploitation of this pathway as a therapeutic target.


Assuntos
Aterosclerose/metabolismo , Receptores Androgênicos/metabolismo , Animais , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Modelos Animais de Doenças , Humanos , Neointima/metabolismo , Neointima/patologia , Neovascularização Fisiológica , Remodelação Vascular
10.
Sci Rep ; 7(1): 10633, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28878267

RESUMO

Carbonyl Reductase 1 (CBR1) is a ubiquitously expressed cytosolic enzyme important in exogenous drug metabolism but the physiological function of which is unknown. Here, we describe a role for CBR1 in metabolism of glucocorticoids. CBR1 catalyzes the NADPH- dependent production of 20ß-dihydrocortisol (20ß-DHF) from cortisol. CBR1 provides the major route of cortisol metabolism in horses and is up-regulated in adipose tissue in obesity in horses, humans and mice. We demonstrate that 20ß-DHF is a weak endogenous agonist of the human glucocorticoid receptor (GR). Pharmacological inhibition of CBR1 in diet-induced obesity in mice results in more marked glucose intolerance with evidence for enhanced hepatic GR signaling. These findings suggest that CBR1 generating 20ß-dihydrocortisol is a novel pathway modulating GR activation and providing enzymatic protection against excessive GR activation in obesity.


Assuntos
Carbonil Redutase (NADPH)/metabolismo , Metabolismo Energético , Glucocorticoides/metabolismo , Obesidade/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Carbonil Redutase (NADPH)/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Estudos de Associação Genética , Variação Genética , Glucocorticoides/química , Glucocorticoides/urina , Cavalos , Humanos , Hidrocortisona/metabolismo , Hidroxicorticosteroides/metabolismo , Hidroxicorticosteroides/urina , Fígado/metabolismo , Masculino , Camundongos , Modelos Moleculares , Conformação Molecular , Obesidade/genética , Fenótipo , Ligação Proteica , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/química , Relação Estrutura-Atividade
11.
J Endocrinol ; 234(3): 291-299, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28676523

RESUMO

11ß-Hydroxysteroid dehydrogenase-1 (11ß-HSD1) predominantly converts inert glucocorticoids into active forms, thereby contributing to intracellular glucocorticoid levels. 11ß-HSD1 is dynamically regulated during inflammation, including in macrophages where it regulates phagocytic capacity. The resolution of inflammation in some disease models including inflammatory arthritis is impaired by 11ß-HSD1 deficiency or inhibition. However, 11ß-HSD1 deficiency/inhibition also promotes angiogenesis, which is beneficial in mouse models of surgical wound healing, myocardial infarction or obesity. The cell types responsible for the anti-inflammatory and anti-angiogenic roles of 11ß-HSD1 have not been characterised. Here, we generated Hsd11b1MKO mice with LysM-Cre mediated deletion of Hsd11b1 to investigate whether 11ß-HSD1 deficiency in myeloid phagocytes is pro-angiogenic and/or affects the resolution of inflammation. Resolution of inflammatory K/BxN-induced arthritis was impaired in Hsd11b1MKO mice to a similar extent as in mice globally deficient in 11ß-HSD1. This was associated with >2-fold elevation in levels of the endothelial marker Cdh5 mRNA, suggesting increased angiogenesis in joints of Hsd11b1MKO mice following arthritis. A pro-angiogenic phenotype was confirmed by measuring angiogenesis in subcutaneously implanted polyurethane sponges, in which Hsd11b1MKO mice showed 20% greater vessel density than their littermate controls, associated with higher expression of Cdh5 Thus, 11ß-HSD1 deficiency in myeloid phagocytes promotes angiogenesis. Targeting 11ß-HSD1 in macrophages may be beneficial in tissue repair.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/deficiência , Inflamação/enzimologia , Macrófagos/enzimologia , Neovascularização Patológica/enzimologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/imunologia , Animais , Humanos , Inflamação/genética , Inflamação/imunologia , Macrófagos/imunologia , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia
12.
ACS Nano ; 11(5): 4542-4552, 2017 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-28443337

RESUMO

The development of engineered nanomaterials is growing exponentially, despite concerns over their potential similarities to environmental nanoparticles that are associated with significant cardiorespiratory morbidity and mortality. The mechanisms through which inhalation of nanoparticles could trigger acute cardiovascular events are emerging, but a fundamental unanswered question remains: Do inhaled nanoparticles translocate from the lung in man and directly contribute to the pathogenesis of cardiovascular disease? In complementary clinical and experimental studies, we used gold nanoparticles to evaluate particle translocation, permitting detection by high-resolution inductively coupled mass spectrometry and Raman microscopy. Healthy volunteers were exposed to nanoparticles by acute inhalation, followed by repeated sampling of blood and urine. Gold was detected in the blood and urine within 15 min to 24 h after exposure, and was still present 3 months after exposure. Levels were greater following inhalation of 5 nm (primary diameter) particles compared to 30 nm particles. Studies in mice demonstrated the accumulation in the blood and liver following pulmonary exposure to a broader size range of gold nanoparticles (2-200 nm primary diameter), with translocation markedly greater for particles <10 nm diameter. Gold nanoparticles preferentially accumulated in inflammation-rich vascular lesions of fat-fed apolipoproteinE-deficient mice. Furthermore, following inhalation, gold particles could be detected in surgical specimens of carotid artery disease from patients at risk of stroke. Translocation of inhaled nanoparticles into the systemic circulation and accumulation at sites of vascular inflammation provides a direct mechanism that can explain the link between environmental nanoparticles and cardiovascular disease and has major implications for risk management in the use of engineered nanomaterials.


Assuntos
Nanopartículas Metálicas/administração & dosagem , Doenças Vasculares/metabolismo , Administração por Inalação , Adulto , Animais , Ouro , Voluntários Saudáveis , Humanos , Pulmão/patologia , Masculino , Camundongos , Nanopartículas , Nanoestruturas/análise , Tamanho da Partícula , Doenças Vasculares/terapia
13.
Sci Rep ; 7: 44233, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28287126

RESUMO

Assessing the retention of cell therapies following implantation is vital and often achieved by labelling cells with 2'-[18F]-fluoro-2'-deoxy-D-glucose (18F-FDG). However, this approach is limited by local retention of cell-effluxed radiotracer. Here, in a preclinical model of critical limb ischemia, we assessed a novel method of cell tracking using 3'-deoxy-3'-L-[18F]-fluorothymidine (18F-FLT); a clinically available radiotracer which we hypothesise will result in minimal local radiotracer reuptake and allow a more accurate estimation of cell retention. Human endothelial cells (HUVECs) were incubated with 18F-FDG or 18F-FLT and cell characteristics were evaluated. Dynamic positron emission tomography (PET) images were acquired post-injection of free 18F-FDG/18F-FLT or 18F-FDG/18F-FLT-labelled HUVECs, following the surgical induction of mouse hind-limb ischemia. In vitro, radiotracer incorporation and efflux was similar with no effect on cell viability, function or proliferation under optimised conditions (5 MBq/mL, 60 min). Injection of free radiotracer demonstrated a faster clearance of 18F-FLT from the injection site vs. 18F-FDG (p ≤ 0.001), indicating local cellular uptake. Using 18F-FLT-labelling, estimation of HUVEC retention within the engraftment site 4 hr post-administration was 24.5 ± 3.2%. PET cell tracking using 18F-FLT labelling is an improved approach vs. 18F-FDG as it is not susceptible to local host cell reuptake, resulting in a more accurate estimation of cell retention.


Assuntos
Rastreamento de Células/métodos , Didesoxinucleosídeos , Células Endoteliais da Veia Umbilical Humana/citologia , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Didesoxinucleosídeos/farmacocinética , Didesoxinucleosídeos/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Traçadores Radioativos
14.
PLoS Med ; 14(2): e1002248, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28245243

RESUMO

BACKGROUND: Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension. METHODS AND FINDINGS: To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 µg/kg/d and then 60 min at 30 µg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow. Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; p < 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study's main limitations were the relatively small sample size and stable, well-compensated population. CONCLUSIONS: Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required. TRIAL REGISTRATION: ClinicalTrials.gov NCT01640964.


Assuntos
Nefropatias/tratamento farmacológico , Rim/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Relaxina/farmacologia , Relaxina/uso terapêutico , Adolescente , Adulto , Idoso , Animais , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Humanos , Rim/irrigação sanguínea , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Fluxo Sanguíneo Regional/efeitos dos fármacos , Escócia , Adulto Jovem
15.
Biochem Pharmacol ; 129: 73-84, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28131845

RESUMO

Use of topical glucocorticoid for inflammatory skin conditions is limited by systemic and local side-effects. This investigation addressed the hypothesis that topical 5α-tetrahydrocorticosterone (5αTHB, a corticosterone metabolite) inhibits dermal inflammation without affecting processes responsible for skin thinning and impaired wound healing. The topical anti-inflammatory properties of 5αTHB were compared with those of corticosterone in C57Bl/6 male mice with irritant dermatitis induced by croton oil, whereas its effects on angiogenesis, inflammation, and collagen deposition were investigated by subcutaneous sponge implantation. 5αTHB decreased dermal swelling and total cell infiltration associated with dermatitis similarly to corticosterone after 24h, although at a five fold higher dose, but in contrast did not have any effects after 6h. Pre-treatment with the glucocorticoid receptor antagonist RU486 attenuated the effect of corticosterone on swelling at 24h, but not that of 5αTHB. After 24h 5αTHB reduced myeloperoxidase activity (representative of neutrophil infiltration) to a greater extent than corticosterone. At equipotent anti-inflammatory doses 5αTHB suppressed angiogenesis to a limited extent, unlike corticosterone which substantially decreased angiogenesis compared to vehicle. Furthermore, 5αTHB reduced only endothelial cell recruitment in sponges whereas corticosterone also inhibited smooth muscle cell recruitment and decreased transcripts of angiogenic and inflammatory genes. Strikingly, corticosterone, but not 5αTHB, reduced collagen deposition. However, both 5αTHB and corticosterone attenuated macrophage infiltration into sponges. In conclusion, 5αTHB displays the profile of a safer topical anti-inflammatory compound. With limited effects on angiogenesis and extracellular matrix, it is less likely to impair wound healing or cause skin thinning.


Assuntos
Corticosterona/análogos & derivados , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Administração Tópica , Animais , Corticosterona/administração & dosagem , Corticosterona/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL
16.
Vascul Pharmacol ; 89: 19-30, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27717848

RESUMO

Angiogenesis is important in cancer progression and can be influenced by tumor-associated myofibroblasts. We addressed the hypothesis that glucocorticoids indirectly affect angiogenesis by altering the release of pro-angiogenic factors from colon cancer-derived myofibroblasts. Our study shows that glucocorticoids reduced prostanoids, urokinase-type plasminogen activator (uPA) and angiopoietin-like protein-2 (ANGPTL2) levels, but increased angiogenin (ANG) in supernatant from human CT5.3hTERT colon cancer-derived myofibroblasts. Conditioned medium from solvent- (CMS) and dexamethasone (Dex)-treated (CMD) myofibroblasts increased human umbilical vein endothelial cell (HUVEC) proliferation, but did not affect expression of pro-angiogenic factors or tube-like structure formation (by HUVECs or human aortic ECs). In a HUVEC scratch assay CMS-induced acceleration of wound healing was blunted by CMD treatment. Moreover, CMS-induced neovessel growth in mouse aortic rings ex vivo was also blunted using CMD. The latter effect could be ascribed to both Dex-driven reduction of secreted factors and potential residual Dex present in CMD (indicated using a dexamethasone-spiked CMS control). A similar control in the scratch assay, however, revealed that altered levels of factors in the CMD, and not potential residual Dex, were responsible for decreased wound closure. In conclusion, our results suggest that glucocorticoids indirectly alter endothelial cell function during tumor development in vivo.


Assuntos
Inibidores da Angiogênese/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Dexametasona/farmacologia , Células Endoteliais/efeitos dos fármacos , Glucocorticoides/farmacologia , Miofibroblastos/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Angiopoietinas/metabolismo , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia , Fatores de Tempo , Técnicas de Cultura de Tecidos , Microambiente Tumoral , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
17.
Hypertension ; 69(2): 275-285, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28028193

RESUMO

The role of smooth muscle endothelinB (ETB) receptors in regulating vascular function, blood pressure (BP), and neointimal remodeling has not been established. Selective knockout mice were generated to address the hypothesis that loss of smooth muscle ETB receptors would reduce BP, alter vascular contractility, and inhibit neointimal remodeling. ETB receptors were selectively deleted from smooth muscle by crossing floxed ETB mice with those expressing cre-recombinase controlled by the transgelin promoter. Functional consequences of ETB deletion were assessed using myography. BP was measured by telemetry, and neointimal lesion formation induced by femoral artery injury. Lesion size and composition (day 28) were analyzed using optical projection tomography, histology, and immunohistochemistry. Selective deletion of ETB was confirmed by genotyping, autoradiography, polymerase chain reaction, and immunohistochemistry. ETB-mediated contraction was reduced in trachea, but abolished from mesenteric veins, of knockout mice. Induction of ETB-mediated contraction in mesenteric arteries was also abolished in these mice. Femoral artery function was unaltered, and baseline BP modestly elevated in smooth muscle ETB knockout compared with controls (+4.2±0.2 mm Hg; P<0.0001), but salt-induced and ETB blockade-mediated hypertension were unaltered. Circulating endothelin-1 was not altered in knockout mice. ETB-mediated contraction was not induced in femoral arteries by incubation in culture medium or lesion formation, and lesion size was not altered in smooth muscle ETB knockout mice. In the absence of other pathology, ETB receptors in vascular smooth muscle make a small but significant contribution to ETB-dependent regulation of BP. These ETB receptors have no effect on vascular contraction or neointimal remodeling.


Assuntos
Pressão Sanguínea/fisiologia , Regulação da Expressão Gênica , Hipertensão/genética , Músculo Liso Vascular/metabolismo , RNA/genética , Receptor de Endotelina B/genética , Vasoconstrição/fisiologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Neointima , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Endotelina B/biossíntese , Remodelação Vascular
18.
PLoS One ; 11(9): e0163815, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27684374

RESUMO

Endocrinopathic laminitis (EL) is a vascular condition of the equine hoof resulting in severe lameness with both welfare and economic implications. EL occurs in association with equine metabolic syndrome and equine Cushing's disease. Vascular dysfunction, most commonly due to endothelial dysfunction, is associated with cardiovascular risk in people with metabolic syndrome and Cushing's syndrome. We tested the hypothesis that horses with EL have vascular, specifically endothelial, dysfunction. Healthy horses (n = 6) and horses with EL (n = 6) destined for euthanasia were recruited. We studied vessels from the hooves (laminar artery, laminar vein) and the facial skin (facial skin arteries) by small vessel wire myography. The response to vasoconstrictors phenylephrine (10-9-10-5M) and 5-hydroxytryptamine (5HT; 10-9-10-5M) and the vasodilator acetylcholine (10-9-10-5M) was determined. In comparison with healthy controls, acetylcholine-induced relaxation was dramatically reduced in all intact vessels from horses with EL (% relaxation of healthy laminar arteries 323.5 ± 94.1% v EL 90.8 ± 4.4%, P = 0.01, laminar veins 129.4 ± 14.8% v EL 71.2 ± 4.1%, P = 0.005 and facial skin arteries 182.0 ± 40.7% v EL 91.4 ± 4.5%, P = 0.01). In addition, contractile responses to phenylephrine and 5HT were increased in intact laminar veins from horses with EL compared with healthy horses; these differences were endothelium-independent. Sensitivity to phenylephrine was reduced in intact laminar arteries (P = 0.006) and veins (P = 0.009) from horses with EL. Horses with EL exhibit significant vascular dysfunction in laminar vessels and in facial skin arteries. The systemic nature of the abnormalities suggest this dysfunction is associated with the underlying endocrinopathy and not local changes to the hoof.

19.
Endocrinology ; 157(6): 2479-88, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27145015

RESUMO

The testicular vasculature forms a complex network, providing oxygenation, micronutrients, and waste clearance from the testis. The vasculature is also instrumental to testis function because it is both the route by which gonadotropins are delivered to the testis and by which T is transported away to target organs. Whether Sertoli cells play a role in regulating the testicular vasculature in postnatal life has never been unequivocally demonstrated. In this study we used models of acute Sertoli cell ablation and acute germ cell ablation to address whether Sertoli cells actively influence vascular structure and function in the adult testis. Our findings suggest that Sertoli cells play a key role in supporting the structure of the testicular vasculature. Ablating Sertoli cells (and germ cells) or germ cells alone results in a similar reduction in testis size, yet only the specific loss of Sertoli cells leads to a reduction in total intratesticular vascular volume, the number of vascular branches, and the numbers of small microvessels; loss of germ cells alone has no effect on the testicular vasculature. These perturbations to the testicular vasculature leads to a reduction in fluid exchange between the vasculature and testicular interstitium, which reduces gonadotropin-stimulated circulating T concentrations, indicative of reduced Leydig cell stimulation and/or reduced secretion of T into the vasculature. These findings describe a new paradigm by which the transport of hormones and other factors into and out of the testis may be influenced by Sertoli cells and highlights these cells as potential targets for enhancing this endocrine relationship.


Assuntos
Células de Sertoli/metabolismo , Testículo/metabolismo , Testosterona/sangue , Testosterona/metabolismo , Animais , Células Cultivadas , Células Germinativas/citologia , Células Germinativas/metabolismo , Gonadotropinas/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Camundongos , Microvasos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Células de Sertoli/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatogênese/fisiologia , Testículo/citologia , Testículo/efeitos dos fármacos
20.
PLoS One ; 11(5): e0154987, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27159530

RESUMO

AIMS: Studies in global androgen receptor knockout (G-ARKO) and orchidectomised mice suggest that androgen accelerates reperfusion of the ischaemic hindlimb by stimulating angiogenesis. This investigation used novel, vascular cell-specific ARKO mice to address the hypothesis that the impaired hindlimb reperfusion in G-ARKO mice was due to loss of AR from cells in the vascular wall. METHODS AND RESULTS: Mice with selective deletion of AR (ARKO) from vascular smooth muscle cells (SM-ARKO), endothelial cells (VE-ARKO), or both (SM/VE-ARKO) were compared with wild type (WT) controls. Hindlimb ischaemia was induced in these mice by ligation and removal of the femoral artery. Post-operative reperfusion was reduced in SM-ARKO and SM/VE-ARKO mice. Immunohistochemistry indicated that this was accompanied by a reduced density of smooth muscle actin-positive vessels but no change in the density of isolectin B4-positive vessels in the gastrocnemius muscle. Deletion of AR from the endothelium (VE-ARKO) did not alter post-operative reperfusion or vessel density. In an ex vivo (aortic ring culture) model of angiogenesis, AR was not detected in vascular outgrowths and angiogenesis was not altered by vascular ARKO or by exposure to dihydrotestosterone (DHT 10(-10)-10(-7)M; 6 days). CONCLUSION: These results suggest that loss of AR from vascular smooth muscle, but not from the endothelium, contributes to impaired reperfusion in the ischaemic hindlimb of G-ARKO. Impaired reperfusion was associated with reduced collateral formation rather than reduced angiogenesis.


Assuntos
Vasos Sanguíneos/metabolismo , Membro Posterior/irrigação sanguínea , Receptores Androgênicos/metabolismo , Traumatismo por Reperfusão/metabolismo , Androgênios/farmacologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Receptores Androgênicos/genética
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