RESUMO
Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng+/- and Eng+/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p<0.05) and embryonic studies (p<0.001); MBE binding was approximately twofold higher in Eng+/+ cells and embryos compared with their Eng+/- counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.
Assuntos
Embrião de Mamíferos/diagnóstico por imagem , Células Endoteliais/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Animais , Western Blotting , Adesão Celular/fisiologia , Endoglina , Células Endoteliais/diagnóstico por imagem , Genótipo , Linfócitos Nulos , Camundongos , Camundongos Knockout , Microbolhas , Imagem Molecular , Ratos , UltrassonografiaRESUMO
Endoglin is a TGF-beta superfamily receptor critical for endothelial cell function. Mutations in this gene are associated with hereditary hemorrhagic telangiectasia type I (HHT1), and clinical signs of disease are generally more evident later in life. We previously showed that systemic vessels of adult Eng heterozygous (Eng(+/-)) mice exhibit increased vasorelaxation due to uncoupling of endothelial nitric oxide synthase (eNOS). We postulated that these changes may develop with age and evaluated pulmonary arteries from newborn and adult Eng(+/-) mice for eNOS-dependent, acetylcholine (ACh-induced) vasorelaxation, compared with that of age-matched littermate controls. While ACh-induced vasorelaxation was similar in all newborn mice, it was significantly increased in the adult Eng(+/-) vs. control vessels. The vasodilatory responses were inhibited by l-NAME suggesting eNOS dependence. eNOS uncoupling was observed in lung tissues of adult, but not newborn, heterozygous mice and was associated with increased production of reactive O(2) species (ROS) in adult Eng(+/-) vs. control lungs. Interestingly, ROS generation was higher in adult than newborn mice and so were the levels of NADPH oxidase 4 and SOD 1, 2, 3 isoforms. However, enzyme protein levels and NADPH activity were normal in adult Eng(+/-) lungs indicating that the developmental maturation of ROS generation and scavenging cannot account for the increased vasodilatation observed in adult Eng(+/-) mice. Our data suggest that eNOS-dependent H(2)O(2) generation in Eng(+/-) lungs accounts for the heightened pulmonary vasorelaxation. To the extent that these mice mimic human HHT1, age-associated pulmonary vascular eNOS uncoupling may explain the late childhood and adult onset of clinical lung manifestations.
Assuntos
Envelhecimento/metabolismo , Heterozigoto , Peptídeos e Proteínas de Sinalização Intracelular/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Artéria Pulmonar/enzimologia , Envelhecimento/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos/efeitos dos fármacos , Endoglina , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Proteínas de Choque Térmico HSP90/metabolismo , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/enzimologia , Camundongos , NADPH Oxidases/metabolismo , Artéria Pulmonar/diagnóstico por imagem , Artéria Pulmonar/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Tomografia Computadorizada por Raios X , Vasodilatação/efeitos dos fármacosRESUMO
Spontaneously hypertensive rats (SH) with an increased number of red blood cells (RBC), microcytosis, and normal hemoglobin (Hb) concentration were used to study the effect of different manipulations of the erythron on erythropoietin production and on erythroid progenitor proliferation by bone marrow cells in order to gain insight regarding the regulation of erythropoiesis. The serum erythropoietin (Ep) level was increased in untreated SH rats. After stimulation by either bleeding, hemolysis, or acute hypoxia, both the erythropoietin level and erythroid colony-forming unit (CFU-E) proliferation by bone marrow cells increased in SH rats to levels that were similar to those of normotensive Wistar (W) rats. Exposure to chronic hypoxia induced an increase in Hb concentration in SH rats concomitantly with the increase in RBC. The results obtained in SH rats raise the possibility of a defect in nonEp stimulators of erythropoiesis that may alter Hb synthesis.
Assuntos
Hemoglobina A/metabolismo , Policitemia/sangue , Ratos Endogâmicos SHR/sangue , Ratos Endogâmicos/sangue , Animais , Medula Óssea/patologia , Ensaio de Unidades Formadoras de Colônias , Eritroblastos/metabolismo , Índices de Eritrócitos , Feminino , Masculino , RatosRESUMO
Mesenchymal stem/stromal cells (MSCs) offer considerable promise as a novel therapeutic strategy for acute respiratory distress syndrome (ARDS). MSCs may be able to "reprogramme" the immune response to reduce destructive inflammatory elements while preserving the host response to pathogens. In addition, MSCs may be able to enhance the repair and resolution of lung injury. Resolution of ARDS is impeded by destruction of the integrity of the epithelial barrier, which inhibits alveolar fluid clearance and depletes surfactant. MSCs appear to restore epithelial and endothelial function, via both paracrine and cell contact dependent effects. ARDS is frequently a component of a generalized process resulting in dysfunction and failure of multiple organs. MSCs have been demonstrated to decrease injury and/or restore function in other organs, including the kidney, liver and heart. MSCs may directly attenuate bacterial sepsis, the commonest and most severe cause of ALI/ARDS. The fact that MSCs are in clinical studies for a wide range of disease processes is a clear advantage for translating MSCs to clinical testing in patients with ARDS. However, some important knowledge gaps exist that may impede clinical translation. The ultimate success of MSCs as a therapy for patients with ARDS will likely be dependent on a greater knowledge of their mechanisms of action and the determination of the optimal strategies for their use in the clinical setting.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais , Síndrome do Desconforto Respiratório/terapia , Lesão Pulmonar Aguda/terapia , Humanos , Síndrome do Desconforto Respiratório/epidemiologiaRESUMO
Bone marrow megakaryocytes and their progenitors were studied in SHR in order to obtain more information about megakaryocytopoiesis in hypertension since it is known that various anomalies of platelet function occur in hypertension. Megakaryocytopoiesis under steady state conditions and following stimulated erythropoiesis and thrombocytopenia was not found to be significantly different in SHR from that in normotensive Wistar controls.
Assuntos
Medula Óssea/patologia , Hipertensão/patologia , Megacariócitos/citologia , Animais , Contagem de Células , Eritropoese , Eritropoetina/análise , Feminino , Contagem de Plaquetas , Ratos , Ratos Endogâmicos SHRRESUMO
BACKGROUND: Inhibition of nitric oxide (NO) synthase by L-arginine analogs is associated with elevation of blood pressure in rats. Because endothelium-dependent vasomotion in different vascular beds is not homogenous, the aim of this study was to characterize and compare regional hemodynamic responses in carotid, femoral, and renal vascular beds after chronic NO inhibition in spontaneously hypertensive rats. The possible role of circulating endothelin and renin angiotensin systems in mediating the effects of chronic NO inhibition was also studied. METHODS: Systemic and regional hemodynamics, left ventricular mass, plasma renin activity, and plasma endothelin-1 were determined in control and Nomega-nitro-Larginine methyl ester (L-NAME)-treated (10 mg/kg/day, 4 weeks) spontaneously hypertensive rats. RESULTS: L-NAME treatment increased arterial pressure and total peripheral and regional vascular resistance and decreased cardiac output, stroke volume, and regional blood flow. An increase in blood flow ratio and a decrease in vascular resistance ratio between carotid and renal as well as femoral and renal vascular beds in rats treated with L-NAME was found. Blood flow and vascular resistance ratios between femoral and carotid vascular beds remained unchanged. L-NAME increased plasma renin activity and left ventricular weight/body weight ratio, whereas plasma endothelin-1 was not modified. CONCLUSIONS: The results of this study showed that the renal circulation seemed to be more sensitive to the effects of chronic NO inhibition than carotid and femoral vascular beds. Simultaneous activation of the renin angiotensin system may further potentiate cardiovascular effects of chronic NO inhibition. No evidence that circulating endothelin-1 plays a role in this model of hypertension was found.
Assuntos
Hemodinâmica/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/antagonistas & inibidores , Circulação Renal/efeitos dos fármacos , Angiotensinas/sangue , Animais , Pressão Sanguínea/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiologia , Endotelina-1/sangue , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/fisiologia , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Fluxo Sanguíneo Regional/efeitos dos fármacos , Artéria Renal/efeitos dos fármacos , Artéria Renal/fisiologia , Renina/sangue , Volume Sistólico/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacosRESUMO
To examine whether changes in renomedullary osmolality and the activity of the renin-angiotensin system may influence the ability of the renal medulla to exert an antihypertensive function, rats were exposed to several manoeuvers. These affected either the medullary osmolality or the renin-angiotensin system (salt or saccharose load, salt depletion, treatment with captopril alone or in combination with salt depletion). A comparison of the antihypertensive capacity of the renal medulla was studied by transplanting renal medullae from the various groups into one-kidney one-clip hypertensive rats. A significant and quantitatively similar reduction in blood pressure was observed in hypertensive rats that received transplants of the medullae from control, salt or saccharose loaded rats and captopril treated rats. In contrast, medullae from salt depleted rats did not affect blood pressure when transplanted into hypertensive animals. The addition of captopril restored the antihypertensive function of renal medulla in salt depleted rats. The results do not support the view that osmolality of the renal medulla regulates its antihypertensive capacity, and suggest that angiotensin II may restrain renomedullary antihypertensive function.
Assuntos
Pressão Sanguínea/fisiologia , Hipertensão Renovascular/fisiopatologia , Medula Renal/fisiologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Captopril/farmacologia , Feminino , Medula Renal/transplante , Masculino , Ratos , Ratos Endogâmicos , Sistema Renina-AngiotensinaRESUMO
The mechanisms of increased susceptibility to nephrotoxins in aging are complex and incompletely understood. It is very important to try to increase our knowledge of them because adults become increasingly vulnerable to nephrotoxic substances, as they grow older. In addition, the percentage of elderly people will increase markedly in the near future, at least in the developed countries. Drugs such as diuretics, laxatives, NSAIDs, aminoglycosides and other nephrotoxic antibiotics, and converting enzyme inhibitors are used a lot by aging people and can produce severe renal problems. Beside drugs, the clinical use of radiocontrast agents also rises in older patients. It seems that the main mechanism of the increased renal susceptibility to toxic substances in the elderly is a disbalance between vasoconstrictor and vasodilator factors (in favor of vasoconstrictor ones). Increased propensity to vasoconstriction (to Ang II, ET and PAF), as well as increased levels of oxidatively modified biomolecules in the elderly, may enhance susceptibility of old kidney to toxic substances. In addition, all mechanisms that influence both mesangial and fibroblast cell proliferation and over-production of extracellular matrix might also be involved in the processes that make the old kidney prone to drug-induced chronic toxic injury.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Envelhecimento/fisiologia , Rim/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Injúria Renal Aguda/fisiopatologia , Idoso , Anti-Inflamatórios não Esteroides/efeitos adversos , Endotelinas/fisiologia , Produtos Finais de Glicação Avançada/fisiologia , Humanos , Rim/fisiologia , Fator de Ativação de Plaquetas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Renina/sangueRESUMO
Endoglin is a component of the TGF-beta receptor complex present in the kidney at the human glomerular mesangium. Since the cellular origin of the glomerular endoglin is unknown, in the present study we investigated the expression of endoglin in mesangial cells in culture, as well as their response to TGF-beta1. Western and Northern blot analysis identified the expression of endoglin protein and mRNA transcript in both human and rat mesangial cells. Flow cytometry and immunocytochemistry analyses revealed that endoglin is present on the cell membrane. Exogenous TGF-beta1 stimulated not only the expression of collagen alpha1 (I) I and TGF-beta1, but also that of endoglin. These data provide the first evidence for the expression of endoglin in mesangial cells, as well as its upregulation by TGF-beta1, thus suggesting that endoglin may have a role in modulating the effects of TGF-beta1 on the glomerular mesangium.
Assuntos
Mesângio Glomerular/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Antígenos CD , Células Cultivadas , Colágeno/genética , Endoglina , Humanos , Imuno-Histoquímica , RNA Mensageiro/genética , Ratos , Receptores de Superfície Celular , Fator de Crescimento Transformador beta/genética , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
BACKGROUND: The pathophysiology of renal ischaemia, resulting in tubular cell injury and leading to acute renal failure (ARF), remains unclear. An ever-increasing number of investigations focus on a possible role of nitric oxide (NO) in regulating circulation during ARF. In this context, we investigated the influence of chronic stimulation or inhibition of NO synthesis, or both, on haemodynamic parameters, histology and plasma renin activity (PRA) after ischaemia-reperfusion injury of rat kidneys. METHODS: Experiments were performed on adult, male Wistar rats. Before induction of ARF, a group of animals was treated with a NO synthesis inhibitor (L-NAME) and another group was treated with a precursor of NO synthesis (L-arginine). The animals received those substances for 4 weeks. Control groups received the same amount of tap water for 4 or 8 weeks and were divided into groups with ARF (4 weeks--ARF group and 8 weeks ARF group) and a sham-operated group. Another group of rats was treated first with L-NAME and then with L-arginine in their drinking water, for 4 weeks for each of these two substances. All parameters were evaluated 24 h after the induction of ischaemic ARF or the sham operation. RESULTS: Our results show that such long-term stimulation of NO release by L-arginine improved renal haemodynamics in the ischaemic form of ARF. Renal blood flow (RBF) increased by 96% in the L-arginine-treated rats with ARF compared with the group with ARF alone. Inhibition of NO synthesis worsens renal haemodynamics after ARF. However, this aggravation can be reversed by L-arginine. The rate of water reabsorption was reduced in all groups with ARF, but this reduction was least in the group treated with L-arginine. The rate of Na+ reabsorption was reduced in all groups 24 h after renal ischaemia, but a significant decrease was observed after the inhibition of NO synthesis. Histological examination of the kidney specimens showed that morphological changes were least in the rats treated with L-arginine, when compared with all other groups with ARF. Nevertheless, the lesions were most prominent in the L-NAME+ARF group. In this group, the areas of corticomedullar necrosis were more widespread in comparison with other groups, especially the L-arginine group where only swelling of the proximal tubular cells was observed. Treatment with L-NAME was not accompanied by any significant alteration in the plasma concentration of angiotensin I (ANG I), while in the group treated with L-arginine ANG I had a tendency to decrease. CONCLUSIONS: Acute post-ischaemic renal failure may be alleviated by administering the NO substrate (L-arginine). NO acts cytoprotectively on tubular epithelial cells in ischaemia--reperfusion injury of rat kidney. Evidence of this comes from both histopathological findings and increased tubular water and sodium reabsorption. However, inhibition of NO synthesis (provoked by L-NAME) worsens renal haemodynamics and aggravates morphological changes after ARF. These aggravations can, however, be reversed by L-arginine.