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Abstract(s)
A doença venosa crónica (DVeC) é muito prevalente a nível mundial e o seu tratamento, bem
como o das suas complicações, acarretam elevados custos socioeconómicos. A sua
fisiopatologia ainda não é completamente conhecida, facto que tem contribuído para o atraso
do desenvolvimento de terapias eficazes e que suspendam, efetivamente, a evolução da
doença. Estudos prévios demonstraram que a desregulação de metaloproteinases (MMP) e dos
seus inibidores (TIMP) contribuem, muito provavelmente, para o processo patológico na
parede venosa. Também são conhecidos os resultados de estudos que sugerem que o fator
transformador beta 1 (TGF-β1) e os seus recetores (TGFβR) podem interferir na expressão e
na atividade de MMP e TIMP, podendo assim estar implicados na fisiopatologia da DVeC. Não
obstante, os dados disponíveis na literatura não são conclusivos e não têm em consideração o
fator estádio de evolução da doença. Por esta razão, no presente trabalho, foram realizados
dois estudos complementares com o objetivo de contribuir para o conhecimento científico
nesta área.
O primeiro estudo, de natureza observacional, comparou amostras de veias dos membros
inferiores colhidas em 13 indivíduos sem doença venosa e 31 indivíduos com DVeC (19 em
estádio inicial e 12 em estádio avançado da doença) para analisar as diferenças na expressão
génica de MMP1, MMP2, MMP8, MMP9, MMP12, MMP13, TIMP1, TIMP2, TIMP3, TIMP4, TGFβR1,
TGFβR2, e TGFβR3 e na imunomarcação de MMP2, TIMP2 e TGFβR2 na parede venosa. As
amostras foram agrupadas segundo a localização anatómica da amostra venosa e o estádio
evolutivo da doença, tendo sido sujeitas a polymerase chain reaction convencional e
quantitativa e depois a imuno-histoquímica. O segundo estudo, de natureza intervencional,
comparou amostras de veia grande safena saudável de oito sujeitos, que foram divididas em
dois segmentos e cultivadas in vitro, tendo sido um dos segmentos tratado com TGF-β1 e o
outro não, com o propósito de analisar as diferenças na expressão de MMP2, MMP9, MMP12,
TIMP1, TIMP2, TIMP3, TIMP4, TGFβR2 e TGFβR3 entre os grupos de amostras e demonstrar um
efeito direto do TGF-β1 na expressão de MMP e TIMP na parede venosa.
Os resultados do primeiro estudo revelaram uma diminuição da expressão génica de MMP12,
TIMP2, TIMP3, TIMP4, e TGFβR2 na parede de veias insuficientes, quando comparadas com
veias saudáveis. Quando o fator estádio evolutivo da doença foi considerado, os resultados
demonstraram uma expressão diminuída de MMP9 e de TGFβR3, assim como um aumento da
expressão de MMP2 e TIMP3 em veias insuficientes no estádio avançado da doença. Os
resultados imuno-histoquímicos, sobretudo para a túnica íntima, foram coerentes com os
resultados da expressão génica. Já no segundo estudo foi demonstrado o aumento da
expressão génica de MMP9, MMP12, TIMP1 e TIMP2 em amostras de veia tratadas com TGF-β1,
quando comparadas com as amostras não tratadas. Em conclusão, poderá afirmar-se que existem, efetivamente, diferenças de expressão de
MMP/TIMP em veias insuficientes, quando comparadas com veias saudáveis, e que o TGF-β1
influencia diretamente a expressão de MMP/TIMP na parede venosa. Acresce ainda o facto da
diminuição de expressão de TGFβR na parede venosa poder ter um papel importante na
redução da influência do TGF-β1 na desregulação da expressão local de MMP/TIMP em
estádios avançados da DVeC.
Chronic venous disease (CVeD) is a worldwide prevalent condition with a significant socioeconomic burden. Its pathophysiology is only just beginning to be understood, which has been delaying the development of efficient therapies able to suspend the disease progression. Previous studies showed that the dysregulation of matrix metalloproteinases (MMP) and their inhibitors (TIMP), within the varicose vein wall, may be implicated in the pathologic process. Moreover, it is highly plausible that MMP and TIMP expression and activity are affected by transforming growth factor beta-1 (TGF-β1) and its signalling receptors (TGFβR), which can also be implicated in CVeD pathophysiology. Nevertheless available data are inconsistent and disregard clinical progression. Hence, two studies were performed in order to contribute to scientific knowledge within this field. The first study, an observational study, was undertaken to analyse genetic and immunohistochemical differences between lower limb vein samples harvested from 13 individuals without venous disease and from 31 individuals with CVeD (19 on early stages and 12 on advanced stages of the disease). Samples were grouped based on anatomic harvest site and clinical progression and were subjected to gene expression analysis by conventional and quantitative polymerase chain reaction for MMP1, MMP2, MMP8, MMP9, MMP12, MMP13, TIMP1, TIMP2, TIMP3, TIMP4, TGFβR1, TGFβR2, and TGFβR3, and then to immunohistochemistry for immunolocalization of MMP2, TIMP2, and TGFβR2. The second study, an interventional one, was performed with eight subjects from whom healthy great saphenous vein samples were harvested. Each vein sample was divided into two segments, which were then cultivated separately in vitro, being one of the segments treated with TGF- β1, as opposed to the other, in order to analyse the gene expression for MMP2, MMP9, MMP12, TIMP1, TIMP2, TIMP3, TIMP4, TGFβR2 e TGFβR3 and to demonstrate a direct effect of TGF-β1 on MMP and TIMP gene expressions in the vein wall. The results of the first study results showed a decreased gene expression of MMP12, TIMP2, TIMP3, TIMP4, and TGFβR2 in varicose veins when compared to controls. A decreased gene expression of MMP9 and TGFβR3 and an increased gene expression of MMP2 and TIMP3 were found in advanced clinical stages. Most immunohistochemistry results, mainly for tunica intimae, were coherent with quantitative real time-polymerase chain reaction results. The second study showed a general increase of MMP9, MMP12, TIMP1 and TIMP2 gene expressions in TGF-β1 treated samples when compared to non treated samples. In conclusion, there are in fact MMP/TIMP gene expression differences between varicose and healthy veins and TGF-β1 has a direct effect on MMP/TIMP expression in the vein wall. Moreover, the decreased expression of TGFβRs in the vein wall might suggest a reduction of TGF-β1 participation in the MMP/TIMP imbalance in advanced stages of CVeD.
Chronic venous disease (CVeD) is a worldwide prevalent condition with a significant socioeconomic burden. Its pathophysiology is only just beginning to be understood, which has been delaying the development of efficient therapies able to suspend the disease progression. Previous studies showed that the dysregulation of matrix metalloproteinases (MMP) and their inhibitors (TIMP), within the varicose vein wall, may be implicated in the pathologic process. Moreover, it is highly plausible that MMP and TIMP expression and activity are affected by transforming growth factor beta-1 (TGF-β1) and its signalling receptors (TGFβR), which can also be implicated in CVeD pathophysiology. Nevertheless available data are inconsistent and disregard clinical progression. Hence, two studies were performed in order to contribute to scientific knowledge within this field. The first study, an observational study, was undertaken to analyse genetic and immunohistochemical differences between lower limb vein samples harvested from 13 individuals without venous disease and from 31 individuals with CVeD (19 on early stages and 12 on advanced stages of the disease). Samples were grouped based on anatomic harvest site and clinical progression and were subjected to gene expression analysis by conventional and quantitative polymerase chain reaction for MMP1, MMP2, MMP8, MMP9, MMP12, MMP13, TIMP1, TIMP2, TIMP3, TIMP4, TGFβR1, TGFβR2, and TGFβR3, and then to immunohistochemistry for immunolocalization of MMP2, TIMP2, and TGFβR2. The second study, an interventional one, was performed with eight subjects from whom healthy great saphenous vein samples were harvested. Each vein sample was divided into two segments, which were then cultivated separately in vitro, being one of the segments treated with TGF- β1, as opposed to the other, in order to analyse the gene expression for MMP2, MMP9, MMP12, TIMP1, TIMP2, TIMP3, TIMP4, TGFβR2 e TGFβR3 and to demonstrate a direct effect of TGF-β1 on MMP and TIMP gene expressions in the vein wall. The results of the first study results showed a decreased gene expression of MMP12, TIMP2, TIMP3, TIMP4, and TGFβR2 in varicose veins when compared to controls. A decreased gene expression of MMP9 and TGFβR3 and an increased gene expression of MMP2 and TIMP3 were found in advanced clinical stages. Most immunohistochemistry results, mainly for tunica intimae, were coherent with quantitative real time-polymerase chain reaction results. The second study showed a general increase of MMP9, MMP12, TIMP1 and TIMP2 gene expressions in TGF-β1 treated samples when compared to non treated samples. In conclusion, there are in fact MMP/TIMP gene expression differences between varicose and healthy veins and TGF-β1 has a direct effect on MMP/TIMP expression in the vein wall. Moreover, the decreased expression of TGFβRs in the vein wall might suggest a reduction of TGF-β1 participation in the MMP/TIMP imbalance in advanced stages of CVeD.
Description
Keywords
Doença venosa crónica - Membros inferiores - Fisiopatologia Doença venosa crónica - Membros inferiores - Terapia - Metaloproteínases Doença venosa crónica - Membros inferiores - Estudo epidemiológico Insuficiência venosa - Membros inferiores - Fisiopatologia Insuficiência venosa - Mebros inferiores - Metaloproteínases Insuficiência venosa - Membros inferiores - Estudo epidemiológico