RAPHAEL DOS SANTOS COUTINHO E SILVA
Projetos de Pesquisa
Unidades Organizacionais
Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina
LIM/11 - Laboratório de Cirurgia Cardiovascular e Fisiopatologia da Circulação, Hospital das Clínicas, Faculdade de Medicina
LIM/11 - Laboratório de Cirurgia Cardiovascular e Fisiopatologia da Circulação, Hospital das Clínicas, Faculdade de Medicina
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- Long-term lung inflammation is reduced by estradiol treatment in brain dead female rats(2021) RICARDO-DA-SILVA, Fernanda Yamamoto; ARMSTRONG-JR, Roberto; VIDAL-DOS-SANTOS, Marina; CORREIA, Cristiano de Jesus; SILVA, Raphael dos Santos Coutinho e; ANUNCIACAO, Lucas Ferreira da; MOREIRA, Luiz Felipe Pinho; LEUVENINK, Henri Gerrit Derk; BREITHAUPT-FALOPPA, Ana CristinaOBJECTIVES: Lung transplantation is limited by the systemic repercussions of brain death (BD). Studies have shown the potential protective role of 17 beta-estradiol on the lungs. Here, we aimed to investigate the effect of estradiol on the long-lasting lung inflammatory state to understand a possible therapeutic application in lung donors with BD. METHODS: Female Wistar rats were separated into 3 groups: BD, subjected to brain death (6h); E2-T0, treated with 17 beta-estradiol (50 mu g/mL, 2 mL/h) immediately after brain death; and E2-T3, treated with 17 beta-estradiol (50 mu g/ml, 2 ml/h) after 3h of BD. Complement system activity and macrophage presence were analyzed. TNF-alpha, IL-1 beta, IL-10, and IL-6 gene expression (RT-PCR) and levels in 24h lung culture medium were quantified. Finally, analysis of caspase-3 gene and protein expression in the lung was performed. RESULTS: Estradiol reduced complement C3 protein and gene expression. The presence of lung macrophages was not modified by estradiol, but the release of inflammatory mediators was reduced and TNF-alpha and IL-1 beta gene expression were reduced in the E2-T3 group. In addition, caspase-3 protein expression was reduced by estradiol in the same group. CONCLUSIONS: Brain death-induced lung inflammation in females is modulated by estradiol treatment. Study data suggest that estradiol can control the inflammatory response by modulating the release of mediators after brain death in the long term. These results strengthen the idea of estradiol as a therapy for donor lungs and improving transplant outcomes.
conferenceObject SEX-DIFFERENCES ON PLATELET AGGREGATION AND MICROVASCULAR PERFUSION AFTER BRAIN DEATH IN RATS(2019) CORREIA, Cristiano de Jesus; BREITHAUPT-FALOPPA, Ana Cristina; SILVA, Raphael dos Santos Coutinho e; SANTOS, Marina Vidal dos; ANUNCIACAO, Lucas Ferreira da; LEUVENINK, Hendrik Gerrit Derk; MOREIRA, Luiz Felipe Pinho- Bilateral sympathectomy improves postinfarction left ventricular remodeling and function(2017) ZANONI, Fernando Luiz; SIMAS, Rafael; SILVA, Raphael Grillo da; BREITHAUPT-FALOPPA, Ana Cristina; SILVA, Raphael dos Santos Coutinho e; JATENE, Fabio Biscegli; MOREIRA, Luiz Felipe P.Objectives: To evaluate the influence of bilateral or left sympathectomy on left ventricular remodeling and function after myocardial infarction in rats. Methods: Myocardial infarction was induced in rats by ligation of the left anterior descending coronary. Seven days later, rats were divided into 4 groups: the myocardial infarction, myocardial infarction with left sympathectomy, myocardial infarction with bilateral sympathectomy, and sham groups. After 8 weeks, left ventricular function was evaluated with the use of a pressure-volume conductance catheter under steady-state conditions and pharmacological stress. Infarct size and extracellular matrix fibrosis were evaluated, and cardiac matrix metalloproteinases and myocardial inflammatory markers were analyzed. Results: The myocardial infarction and left sympathectomy group had an increased end diastolic volume, whereas the bilateral sympathectomy group had a mean end-diastolic volume similar to that of the sham group (P <. 002). Significant reduction in ejection fraction was observed in the myocardial infarction and left sympathectomy group, whereas it was preserved after bilateral sympathectomy (P < .001). In response to dobutamine, left ventricular contractility increased in sham rats, rising stroke work, cardiac output, systolic volume, end-diastolic volume, ejection fraction, and dP/dt max. Only bilateral sympathectomy rats had significant increases in ejection fraction (P <. 001) with dobutamine. Fibrotic tissue and matrix metalloproteinase expression decreased in the bilateral sympathectomy group compared to that in the myocardial infarction group (P <. 001) and was associated with left ventricular wall thickness maintenance and better apoptotic markers in noninfarcted myocardium. Conclusions: Bilateral sympathectomy effectively attenuated left ventricular remodeling and preserved systolic function after myocardial infarction induction in rats.
- Protective role of 17 beta-estradiol treatment in renal injury on female rats submitted to brain death(2021) ARMSTRONG-JR, Roberto; RICARDO-DA-SILVA, Fernanda Yamamoto; VIDAL-DOS-SANTOS, Marina; CORREIA, Cristiano de Jesus; ANUNCIACAO, Lucas Ferreira; SILVA, Raphael dos Santos Coutinho e; MOREIRA, Luiz Felipe Pinho; LEUVENINK, Henri Gerrit Derk; BREITHAUPT-FALOPPA, Ana CristinaBackground: Clinical and experimental data highlight the consequences of brain death on the quality of organs and demonstrate the importance of donor state to the results of transplantation. Female rats show higher cardio-pulmonary injury linked to decreased concentrations of female sex hormones after brain-dead (BD). This study evaluated the effect of 17 beta-estradiol on brain death induced renal injury in female rats. Methods: Female Wistar rats were randomically allocated into 4 groups: false-operation (Sham), BD, treatment with 17 beta-estradiol (50 mu g/mL, 2 mL/h) 3 h after brain death (E2-T3), or immediately after brain death confirmation (E2-T0). Creatinine, urea, cytokines, and complement system components were quantified. Renal injury markers, such as KIM-1, Caspase-3, BCL-2 and MMP2/9 were evaluated. Results: Brain death leads to increased kidney KIM-1 expression and longer 17 beta-estradiol treatment resulted in downregulation (P<0.0001). There was increase of neutrophil numbers in kidney from BD rats and E2 treatment was able to reduce it (P=0.018). Regarding complement elements, E2-T3 group evidenced E2 therapeutic effects, reducing C5b-9 (P=0.0004), C3aR (P=0.054) and C5aR (P=0.019). In parallel, there were 17 beta-estradiol effects in reducing MMP2 (P=0.0043), MMP9 (P=0.011), and IL-6 (P=0.024). Moreover, E2-T3 group improved renal function in comparison to BD group (P=0.0938). Conclusions: 17 beta-estradiol treatment was able to reduce acute kidney damage in BD female rats owing to its ability to prevent tissue damage, formation of C5b-9, and local synthesis of inflammatory mediators.
- Protective effects of 17 beta-oestradiol on coagulation and systemic inflammation after total occlusion of the descending aorta in male rats(2022) SOBRAL, Marcelo Luiz Peixoto; DIAS, Ricardo Ribeiro; CORREIA, Cristiano de Jesus; SILVA, Raphael dos Santos Coutinho e; ANUNCIACAO, Lucas Ferreira da; BREITHAUPT-FALOPPA, Ana Cristina; MOREIRA, Luiz Felipe PinhoOBJECTIVES: The surgical treatment for diseases of the descending aorta is related to a high mortality rate because of the activation of a systemic inflammatory process due to ischaemia and reperfusion (I/R) injury. Activation of coagulation can contribute to the inflammatory process, resulting in microcirculatory damage and multiple organ failure. Our goal was to evaluate the role of prophylactic intravenous 17 beta-oestradiol (E2) in coagulation, the inflammatory response and hepatic injury after occlusion of the descendent proximal aorta in male rats. METHODS: Wistar male rats were randomized and allocated to 3 groups (n = 8 per group): sham, surgically manipulated; IR, animals subjected to I/R; and E2, animals treated with E2 (280 mu g/kg, intravenously) before I/R. I/R was induced by insertion of a 2-Fr Fogarty arterial embolectomy catheter in the descending aorta, which was occluded for 20 min, followed by a reperfusion period of 2 h. Serological markers, platelet aggregation, hepatic vascular flow, systemic and liver inflammatory response and apoptosis were analysed. The coagulation process was evaluated by thromboelastometry. RESULTS: The aortic occlusion led to a reduction in plasma fibrinogen concentration in parallel with increased clotting time, greater clot firmness and reduced lysis. E2 treatment was able to increase fibrinogen, prevent the increase in clotting time and normalize clot firmness, but it exerted only a mild effect on clot lysis. Platelet aggregation was increased by IR, and E2 treatment was able to reduce it. There was a reduction in flow percentage in the IR group that was not prevented by E2. In parallel, higher aggregate formation was observed in the vessels of the IR group of animals. There was increased systemic release of interleukin-1-beta, interleukin-6 and interleukin-10 in the IR group, which was reduced in the treated animals. CONCLUSIONS: The current results suggest that pretreatment with E2 before an ischaemic period induced by occlusion of the proximal descending aorta is effective in preventing alterations in coagulation and systemic inflammation due to I/R injury.
- Thoracic bilateral sympathectomy attenuates oxidative stress and prevents ventricular remodelling in experimental pulmonary hypertension(2022) SILVA, Raphael dos Santos Coutinho e; WIGGENHAUSER, Lucas Moritz; SIMAS, Rafael; ZANONI, Fernando Luiz; MEDEIROS, Geisla; SILVA, Fernanda Beatriz da; OGATA, Daniel Cury; BREITHAUPT-FALOPPA, Ana Cristina; KRENNING, Guido; MOREIRA, Luiz Felipe PinhoOBJECTIVES: Pulmonary arterial hypertension (PAH) is a cardiopulmonary disease that affects the pulmonary vasculature, leading to increased afterload and eventually right ventricular (RV) remodelling and failure. Bilateral sympathectomy (BS) has shown promising results in dampening cardiac remodelling and dysfunction in several heart failure models. In the present study, we investigated whether BS reduces pulmonary arterial remodelling and mitigates RV remodelling and failure. METHODS: PAH was induced in male Wistar rats by intraperitoneal injection of monocrotaline. Rats were divided into 3 groups, involving untreated PAH (n = 15), BS-treated PAH (n = 13) and non-manipulated control rats (n = 13). Three weeks after PAH induction, the rats were anaesthetized and RV function was assessed via the pressure-volume loop catheter approach. Upon completion of the experiment, the lungs and heart were harvested for further analyses. RESULTS: BS was found to prevent pulmonary artery remodelling, with a clear reduction in alpha-smooth muscle actin and endothelin-1 expression. RV end-systolic pressure was reduced in the BS group, and preload recruitable stroke work was preserved. BS, therefore, mitigated RV remodelling and cardiomyocyte hypertrophy and diminished oxidative stress. CONCLUSIONS: We showed that thoracic BS may be an important treatment option for PAH patients. Blockade of the sympathetic pathway can prevent pulmonary remodelling and protect the RV from oxidative stress, myocardial remodelling and function decay. Pulmonary arterial hypertension (PAH) is a cardiopulmonary disease that affects both the pulmonary vasculature and the right ventricle (RV).
- Sex differences in the coagulation process and microvascular perfusion induced by brain death in rats(2020) CORREIA, Cristiano de Jesus; SILVA, Fernanda Yamamoto Ricardo da; ARMSTRONG, Roberto Junior; SANTOS, Marina Vidal dos; ANUNCIACAO, Lucas Ferreira da; SOBRAL, Marcelo Luiz Peixoto; SILVA, Raphael dos Santos Coutinho e; LEUVENINK, Hendrik Gerrit Derk; BREITHAUPT-FALOPPA, Ana Cristina; MOREIRA, Luiz Felipe PinhoBrain death (BD) leads to a systemic inflammation associated with the activation of coagulation, which could be related to decreased microcirculatory perfusion. Evidence shows that females exhibit higher platelet aggregability than males. Thus, we investigated sex differences in platelets, coagulation and microcirculatory compromise after BD. BD was induced in male and female (proestrus) Wistar rats. After 3 h, we evaluated: (i) intravital microscopy to evaluate mesenteric perfusion and leucocyte infiltration; (ii) platelet aggregation assay; (iii) rotational thromboelastometry; and (iv) SerumNOx-. Female rats maintained the mesenteric perfusion, whereas male reduced percentage of perfused vessels. Male BD presented higher platelet aggregation than the controls. In contrast, female BD had lower platelet aggregation than the control. Thromboelastometry indicated a reduction in clot firmness with increased clotting time in the female group compared with the male group. SerumNOx-level in female BD was higher than that in the male BD and female control. There is sex dimorphism in platelet function and clotting process, which are altered in different ways by BD. Thus, it is possible to connect the reduction in microcirculatory perfusion in males to intravascular microthrombi formation and the maintenance of perfusion in females to a higher inflammatory response and NO synthesis.
conferenceObject ESTRADIOL TREATMENT REDUCES LUNG LEUKOCYTE INFILTRATION AFTER BRAIN DEATH IN FEMALE RATS(2019) RICARDO-DA-SILVA, Fernanda Yamamoto; ARMSTRONG- JR., Roberto; VIDAL-DOS-SANTOS, Marina; SILVA, Raphael dos Santos Coutinho e; CORREIA, Cristiano de Jesus; MOREIRA, Luiz Felipe Pinho; LEUVENINK, Henri; BREITHAUPT-FALOPPA, Ana Cristina- 17 beta-Estradiol Treatment Protects Lungs Against Brain Death Effects in Female Rat Donor(2021) RICARDO-DA-SILVA, Fernanda Yamamoto; JR, Roberto Armstrong; VIDAL-DOS-SANTOS, Marina; CORREIA, Cristiano de Jesus; SILVA, Raphael dos Santos Coutinho e; ANUNCIACAO, Lucas Ferreira da; MOREIRA, Luiz Felipe Pinho; LEUVENINK, Hendrik Gerrit Derk; BREITHAUPT-FALOPPA, Ana CristinaBackground. Brain death (BD) affects the viability of lungs for transplantation. A correlation exists between high-lung inflammation after BD and the decrease in female sex hormones, especially estradiol. Therefore, we investigated the effects of 17 beta-estradiol (E2) treatment on the lungs of female brain dead rats. Methods. Female Wistar rats were divided into 4 groups: BD (submitted to BD for 6 h), sham (false operated), E2-T0 (treated with E2 immediately after BD; 50 mu g/mL, 2 mL/h), and E2-T3 (treated with E2 after 3 h of BD; 50 mu g/mL, 2 mL/h). Lung edema, hemorrhage, and leukocyte infiltration were analyzed. Adhesion molecules were evaluated, and analysis of NO synthase gene and protein expression was performed using real-time PCR and immunohistochemistry, respectively. Release of chemokines and matrix degradation in the lungs was analyzed. Results. BD increased leukocyte infiltration, as shown by intravital microscopy (P = 0.017), bronchoalveolar lavage cell count (P = 0.016), the release of inflammatory mediators (P = 0.02), and expression of adhesion molecules. BD also increased microvascular permeability and the expression and activity of matrix metalloproteinase-9 in the lungs. E2 treatment reduced leukocyte infiltration, especially in the E2-T3 group, release of inflammatory mediators, adhesion molecules, and matrix metalloproteinase activity in the lungs. Conclusions. E2 treatment was successful in controlling the lung inflammatory response in females submitted to BD. Our results suggest that E2 directly decreases the release of chemokines, restraining cell traffic into the lungs. Thus, E2 has a therapeutic potential, and its role in improving donor lung quality should be explored further.
conferenceObject ESTRADIOL REDUCES HEART INJURY AND APOPTOSIS AFTER BRAIN DEATH IN FEMALE RATS(2019) JR, Roberto Armstrong; RICARDO-DA-SILVA, Fernanda Yamamoto; VIDAL-DOS-SANTOS, Marina; SILVA, Raphael dos Santos Coutinho e; CORREIA, Cristiano de Jesus; MOREIRA, Luiz Felipe Pinho; LEUVENINK, Henri Gerrit Derk; BREITHAUPT-FALOPPA, Ana Cristina