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Master's Dissertation
DOI
https://doi.org/10.11606/D.17.2020.tde-23082020-143746
Document
Author
Full name
Maynara Santana Gonçalves
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2020
Supervisor
Committee
Rodrigues, Maria Carolina de Oliveira (President)
Kayser, Cristiane
Passaglia, Rita de Cassia Aleixo Tostes
Title in Portuguese
Avaliação da integridade endotelial e fibrose na pele de pacientes com esclerose sistêmica submetidos a transplante autólogo de células-tronco hematopoéticas
Keywords in Portuguese
Dano vascular
Esclerose sistêmica
Fibrose
Microvasculatura
Transplante autólogo de células-tronco hematopoéticas
Abstract in Portuguese
A esclerose sistêmica (ES) é uma doença autoimune caracterizada por dano vascular, desregulação do sistema imunológico e fibrose da pele e órgãos internos. A lesão e a apoptose das células endoteliais são indicadas como eventos iniciais da doença, levando a alterações fisiológicas no endotélio e vasculopatia. O transplante de células-tronco hematopoéticas autólogas (TACTH) promove o resetting (do inglês, reprogramação) do sistema imunológico e emergiu como uma opção de tratamento para pacientes com ES grave e progressiva. Embora o TACTH leve à melhora clínica, os mecanismos ainda não são completamente compreendidos. Neste estudo, objetivamos avaliar a expressão de marcadores de integridade, dano e ativação endotelial e aspectos de fibrose na pele de pacientes com esclerose sistêmica tratados com TACTH e correlacioná-los com dados clínicos. Dados clínicos e amostras de pele do antebraço de 35 pacientes com ES foram avaliados por imuno-histoquímica (IHC) antes e aos 6 e 12 meses após o TACTH. Amostras de soro de 15 pacientes encontravam-se disponíveis para análise por técnica de multiplex para marcadores endoteliais (E-SELECTINA, ENDOTELINA-1 e VEGFA). As biópsias foram marcadas com anticorpos anti-CD31, -VEGF, -VEGFR2, - ANGIOPOIETINA 1, -ANGIOPOIETINA 2, -TIE, -ENDOTELINA-1, E-SELECTINA, VECADERINA e coradas com Hematoxilina e Eosina (HE) e Picrosirius red. As imagens foram analisadas pelo programa ImageJ. A maioria dos participantes era do sexo feminino (80%), com idade média (desvio padrão, DP) de 36 (9,4) anos. O tempo médio (DP) entre o diagnóstico da doença e o transplante foi de 3,2 (3,1) anos. A avaliação clínica da fibrose, avaliada pelo escore de Rodnan (mRSS), melhorou aos 6 e 12 meses em comparação ao período PRÉ. (p <0,0001). A análise histológica da fibrose, avaliada por HE e picrosirius red, mostrou redução da espessura da pele (p <0,0001) e da densidade de colágeno (p<0.0001) após o TACTH. Na microvasculatura, observamos, através da capilaroscopia, alterações morfológicas, com aumento do número de capilares e uma redução nos mega-capilares. Os únicos marcadores endoteliais histológicos que mudaram após o TATCH foram ANG1 (p <0,0001) e E-selectina (p <0,0001), que diminuíram. Todos os demais marcadores avaliados não apresentaram alterações (p> 0,05). A expressão de e-selectina se associou com o comprometimento da pele (mRSS) e se correlacionou com a expressão de VEGF. Não houve alterações dos marcadores séricos endoteliais antes e após o TACTH. Nossos dados sugerem que a reversibilidade do dano endotelial associado à doença não é alcançada integralmente após o TACTH. Entretanto, pontuais melhoras são observadas clínica e histologicamente. Acreditamos que o efeito terapêutico do TACTH possa estar mais associado à melhora da fibrose do que a efeitos sobre o compartimento microvascular.
Title in English
Assessment of endothelial integrity and fibrosis in the skin of systemic sclerosis patients treated with autologous hematopoietic stem cell transplantation
Keywords in English
Autologous hematopoietic stem cell transplantation
Fibrosis
Microvasculature
Systemic sclerosis
Vascular damage
Abstract in English
Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular damage, deregulation of the immune system and fibrosis of the skin and internal organs. Injury and apoptosis of endothelial cells are indicated as initial events of the disease, leading to physiological changes in the endothelium and subsequent vasculopathy. Autologous Hematopoietic Stem Cell Transplantation (AHSCT) promotes resetting of the immune system and has emerged as a treatment option for patients with severe and progressive SSc. Although AHSCT leads to clinical improvement, the mechanistic effects are still not completely understood. In this study, we aimed to evaluate the expression of markers of integrity, damage and endothelial activation, as well as aspects of fibrosis in the skin of SSc patients treated with AHSCT and correlate them with clinical data. Skin samples from the forearms of 35 SSc patients were evaluated by immunohistochemistry (IHC) before and at 6 and 12 months after AHSCT. Serum samples from 15 patients were available for analysis using the multiplex technique for endothelial markers (E-SELECTIN, ENDOTHELIN-1, and VEGFA). Biopsies were marked with anti-CD31, -VEGF, -VEGFR2, -ANGIOPOIETIN 1, -ANGIOPOIETIN 2, - TIE, -ENDOTHELIN-1, E-SELECTIN and -VE-CADHERIN antibodies and stained with Hematoxylin and Eosin (HE) and Picrosirius Red. Images were analyzed by ImageJ software. Most participants were female (80%) with a mean (standard deviation, SD) age of 36 (9.4) years. The mean (SD) time between diagnosis of disease and transplantation was 3.2 (3.1) years. Clinical evaluation of fibrosis, assessed by the modified Rodnan's Skin Score (mRSS) improved at 6 and 12 months, compared to baseline (p<0.0001). Histological analysis of fibrosis evaluated by HE and picrosirius red showed reduction of skin thickness (p<0.0001) and collagen density (p=0.0193) after AHSCT. Capillaroscopic analyses of the microvasculature showed morphological changes, with an increased number of capillaries and a reduction in mega-capillaries after AHSCT. The only histological endothelial markers that decreased after AHSCT were ANG1 (p<0.0001) and E-selectin (p<0.0001). All the other evaluated markers remained stable (p>0.05) after AHSCT, when compared to baseline. E-selectin expression in the skin was associated with skin involvement (mRSS) and correlated with VEGF expression. Our data suggest that the reversibility of endothelial damage associated with the disease is not fully achieved after AHSCT. However, occasional improvements are observed clinically and histologically. We believe that the therapeutic effects of AHSCT may be more associated with the improvement of fibrosis than with effects on the microvascular compartment.
 
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Publishing Date
2020-10-20
 
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