Alterações estruturais e moleculares (cDNA) precoces em veias safenas humanas cultivadas sob regime pressórico arterial Precocious structural and molecular (cDNA) changes in the human saphenous veins cultivated under arterial hemodynamic conditions

Abstract

OBJETIVO: A veia safena (VS) empregada na revascularização do miocárdio (RM) fica submetida a estresse tênsil elevado e contínuo. Sua resposta adaptativa à nova condição hemodinâmica pode predispor à oclusão do enxerto. Este trabalho visou verificar as alterações estruturais precoces e moleculares (cDNA) entre VS humanas submetidas a baixo regime pressórico versus condições hemodinâmicas sistêmicas. MÉTODO: Quarenta segmentos de VS foram cultivados "ex-vivo" sob condição hemodinâmica venosa (CHV) (sem pressão, fluxo:5 ml/min) e sob condição hemodinâmica arterial (CHA) (pressão: 80mmHg, fluxo:50mL/min). Foram analisadas: viabilidade celular (coloração MTT), densidade celular (coloração hoechst 33258) e apoptose (ensaio TUNEL), antes e um, dois e quatro dias após o procedimento. Determinamos alvos moleculares alterados precocemente nas veias cultivadas sob condição arterial, através da análise "cDNA microarray" de segmentos das VS. A busca desses alvos foi realizada através de pool homogeneizado do RNA desses segmentos venosos, interagindo por homologia em lâmina contendo 16000 genes humanos pré-determinados (Agilent Technologies slide). Os genes com expressão alterada foram certificados por PCR em tempo real, em veias de 16 diferentes indivíduos. RESULTADOS: Houve diminuição gradual da densidade celular e da viabilidade tecidual nas VS cultivadas mediante CHA, enquanto nenhuma alteração ocorreu quando a veia foi cultivada até quatro dias na CHV. No grupo sob CHA houve sinais de processo apoptótico celular (TUNEL-positivo) já a partir do 1º dia de cultivo, o que não ocorreu no outro grupo. A densidade celular das veias sob regime arterial, decorridas 24h de cultivo, era similar à das amostras frescas das mesmas, mas inúmeras células já apresentavam indícios de processo apoptótico. Os alvos moleculares mais alterados(de acordo com o PCR em tempo real) e selecionados para pesquisa foram o Oncogene 3 e a Interleucina 1ß. A expressão do Oncogene 3 estava elevada em 11 (68,7%) das veias cultivadas sob regime arterial, enquanto observou-se aumento da expressão da Interleucina 1ß em nove (56,2%) desses segmentos venosos (pOBJECTIVE: The saphenous vein (SV) used in coronary artery bypass grafting is submitted to elevated and continuous shear stress. Occlusion of the grafts can occur in response to the new hemodynamic conditions. The aim of this study is to compare the precocious structural and molecular (cDNA) changes in saphenous veins grafts submitted to low pressure hemodynamic conditions versus systemic hemodynamic conditions. METHOD: Forty sections of SV were cultivated "ex-vivo" under venous hemodynamic conditions (VHC) (without pressure, flow: 5 mL/min) and under arterial hemodynamic conditions (AHC) (pressure: 80 mmHg, flow: 50 mL/min). The following variables were analyzed: cellular viability (MTT assay) cellular density (hoeschst 33258 staining) and apoptosis (TUNEL assay), before and 1, 2 and 4 days after the procedure. "cDNA microarray" analysis of the SV sections was used to determine the precociously changed molecular targets in the veins cultivated under arterial conditions. The identification of these targets was achieved using a RNA homogenized pool of these vein sections, interacting on slides with 16,000 pre-determined human genes (Agilent Technologies slide). The genes with changed expressions were verified by real time PCR in the veins of 16 patients. RESULTS: There was a gradual reduction in the cellular density and in the tissue viability in the saphenous veins cultivated under AHC, whereas no alterations were observed in the saphenous veins cultivated under VHC of up to 4 days. In the AHC group there were signs of a cellular apoptotic process (positive - TUNEL) from the first day after cultivation. In the VHC group these alterations were not observed. Although the cellular density was the same in the veins submitted to arterial conditions, after 24 hours of cultivation, many cells already showed signs of the apoptotic process. The Oncogene 3 and the Interleucin 1ß were the most common sites with alterations identified in this research. The Oncogene 3 expression was elevated in 11 (68.7%) of the veins cultivated under AHC, and the Interleucin 1ß expression was elevated in 9 (56.2%) of these vein sections (p<0.05). CONCLUSION: The "ex vivo" study model was able to mimic events that occur "in vivo" by SVs utilized in the coronary artery bypass grafting. In the AHC group precocious loss of cellular viability (apoptosis) and significant elevation in the Oncogene 3 and Interleucin 1ß genic expressions were observed. The long-term follow up of these patients is important to determine the real effect of these immediate changes in the patency of the vein grafts.

Keywords

• Revascularização miocárdica
• Veia safena
• Expressão gênica
• Myocardial revascularization
• Saphenous Vein
• Gene Expression