Department of Pharmacology, Physiology & Neuroscience, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
Departamento de Ciencias Biológicas y Químicas, Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago, Chile.
Microcirculation. 2024 Jul;31(5):e12859. doi: 10.1111/micc.12859. Epub 2024 May 31.
The endothelium regulates crucial aspects of vascular function, including hemostasis, vasomotor tone, proliferation, immune cell adhesion, and microvascular permeability. Endothelial cells (ECs), especially in arterioles, are pivotal for flow distribution and peripheral resistance regulation. Investigating vascular endothelium physiology, particularly in microvascular ECs, demands precise isolation and culturing techniques.
Freshly isolated ECs are vital for examining protein expression, ion channel behavior, and calcium dynamics. Establishing primary endothelial cell cultures is crucial for unraveling vascular functions and understanding intact microvessel endothelium roles. Despite the significance, detailed protocols and comparisons with intact vessels are scarce in microvascular research. We developed a reproducible method to isolate microvascular ECs, assessing substrate influence by cultivating cells on fibronectin and gelatin matrix gels. This comparative approach enhances our understanding of microvascular endothelial cell biology.
Microvascular mesenteric ECs expressed key markers (VE-cadherin and eNOS) in both matrix gels, confirming cell culture purity. Under uncoated conditions, ECs were undetected, whereas proteins linked to smooth muscle cells and fibroblasts were evident. Examining endothelial cell (EC) physiological dynamics on distinct matrix substrates revealed comparable cell length, shape, and Ca elevations in both male and female ECs on gelatin and fibronectin matrix gels. Gelatin-cultured ECs exhibited analogous membrane potential responses to acetylcholine (ACh) or adenosine triphosphate (ATP), contrasting with their fibronectin-cultured counterparts. In the absence of stimulation, fibronectin-cultured ECs displayed a more depolarized resting membrane potential than gelatin-cultured ECs.
Gelatin-cultured ECs demonstrated electrical behaviors akin to intact endothelium from mouse mesenteric arteries, thus advancing our understanding of endothelial cell behavior within diverse microenvironments.
内皮细胞调节着血管功能的诸多关键方面，包括止血、血管舒缩张力、增殖、免疫细胞黏附以及微血管通透性。内皮细胞（endothelial cells，ECs），尤其是在小动脉中，对于血流分布和外周阻力调节至关重要。研究血管内皮细胞生理学，特别是在微血管 ECs 中，需要精确的分离和培养技术。
新鲜分离的 ECs 对于研究蛋白质表达、离子通道行为和钙动力学至关重要。建立原代内皮细胞培养对于揭示血管功能和理解完整微血管内皮细胞的作用至关重要。尽管这一点很重要，但在微血管研究中，详细的方案和与完整血管的比较仍然很少。我们开发了一种可重复的方法来分离微血管 ECs，通过在纤维连接蛋白和明胶基质凝胶上培养细胞来评估基质的影响。这种比较方法增强了我们对微血管内皮细胞生物学的理解。
肠系膜微血管 ECs 在两种基质凝胶中均表达关键标记物（VE-cadherin 和 eNOS），证实了细胞培养的纯度。在未包被的条件下，无法检测到 ECs，而与平滑肌细胞和成纤维细胞相关的蛋白则很明显。在不同的基质底物上研究内皮细胞（endothelial cell，EC）的生理动力学，发现在明胶和纤维连接蛋白基质凝胶上，雄性和雌性 ECs 的细胞长度、形状和 Ca 升高都相似。在缺乏刺激的情况下，与纤维连接蛋白培养的 ECs 相比，明胶培养的 ECs 对乙酰胆碱（acetylcholine，ACh）或三磷酸腺苷（adenosine triphosphate，ATP）的膜电位反应类似。与明胶培养的 ECs 相比，纤维连接蛋白培养的 ECs 的静息膜电位更为去极化。
明胶培养的 ECs 表现出与来自小鼠肠系膜动脉的完整内皮相似的电生理行为，从而加深了我们对内皮细胞在不同微环境中的行为的理解。
