Girão-Silva Thaís, Fonseca-Alaniz Miriam Helena, Oliveira Dallan Luís Alberto, Valãdao Iuri Cordeiro, Oliveira da Rocha Gustavo Henrique, Krieger José Eduardo, Miyakawa Ayumi Aurea
Instituto do Coraçao (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo; Cardiology Research Group, Faculty VI Medicine and Health Sciences, Carl von Ossietzky University of Oldenburg.
Instituto do Coraçao (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo.
J Vis Exp. 2023 Apr 21(194). doi: 10.3791/65122.
Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.
冠状动脉旁路移植术（CABG）是一种使缺血心肌血管再通的手术。尽管与动脉血管相比，大隐静脉作为CABG血管桥的长期通畅率较低，但仍被用作CABG血管桥。与血管桥动脉化相关的血流动力学应激的突然增加会导致血管损伤，尤其是内皮损伤，这可能会影响大隐静脉桥（SVG）的低通畅率。在此，我们描述了人隐静脉内皮细胞（hSVECs）的分离、表征和扩增。通过胶原酶消化分离的细胞呈现典型的鹅卵石形态，并表达内皮细胞标志物CD31和血管内皮钙黏蛋白。为了评估机械应力的影响，本研究采用了相关方案来研究动脉化SVG上的两种主要物理刺激，即剪切应力和拉伸。hSVECs在平行板流动腔中培养以产生剪切应力，细胞沿流动方向排列，且KLF2、KLF4和NOS3的表达增加。hSVECs也可以在硅膜中培养，该硅膜允许模拟静脉（低）和动脉（高）拉伸的可控细胞拉伸。动脉拉伸相应地调节内皮细胞的F-肌动蛋白模式和一氧化氮（NO）分泌。总之，我们提出了一种详细的方法来分离hSVECs，以研究血流动力学机械应力对内皮细胞表型的影响。
