Department of Anesthesiology, College of Medicine-Tucson, The University of Arizona, Tucson, AZ 85724, USA.
Center for Translational Science, Florida International University, Port St. Lucie, FL 34987, USA.
Int J Mol Sci. 2023 Sep 26;24(19):14568. doi: 10.3390/ijms241914568.
The endothelial glycocalyx is a dynamic signaling surface layer that is involved in the maintenance of cellular homeostasis. The glycocalyx has a very diverse composition, with glycoproteins, proteoglycans, and glycosaminoglycans interacting with each other to form a mesh-like structure. Due to its highly interactive nature, little is known about the relative contribution of each glycocalyx constituent to its overall function. Investigating the individual roles of the glycocalyx components to cellular functions and system physiology is challenging, as the genetic manipulation of animals that target specific glycocalyx components may result in the development of a modified glycocalyx. Thus, it is crucial that genetically modified animal models for glycocalyx components are characterized and validated before the development of mechanistic studies. Among the glycocalyx components, glypican 1, which acts through eNOS-dependent mechanisms, has recently emerged as a player in cardiovascular diseases. Whether glypican 1 regulates eNOS in physiological conditions is unclear. Herein, we assessed how the deletion of glypican 1 affects the development of the pulmonary endothelial glycocalyx and the impact on eNOS activity and endothelial function. Male and female 5-9-week-old wild-type and glypican 1 knockout mice were used. Transmission electron microscopy, immunofluorescence, and immunoblotting assessed the glycocalyx structure and composition. eNOS activation and content were assessed by immunoblotting; nitric oxide production was assessed by the Griess reaction. The pulmonary phenotype was evaluated by histological signs of lung injury, in vivo measurement of lung mechanics, and pulmonary ventilation. Glypican 1 knockout mice showed a modified glycocalyx with increased glycocalyx thickness and heparan sulfate content and decreased expression of syndecan 4. These alterations were associated with decreased phosphorylation of eNOS at S1177. The production of nitric oxides was not affected by the deletion of glypican 1, and the endothelial barrier was preserved in glypican 1 knockout mice. Pulmonary compliance was decreased, and pulmonary ventilation was unaltered in glypican 1 knockout mice. Collectively, these data indicate that the deletion of glypican 1 may result in the modification of the glycocalyx without affecting basal lung endothelial function, validating this mouse model as a tool for mechanistic studies that investigate the role of glypican 1 in lung endothelial function.
内皮糖萼是一种动态的信号表面层，参与细胞内稳态的维持。糖萼的组成非常多样化，糖蛋白、蛋白聚糖和糖胺聚糖相互作用，形成网状结构。由于其高度交互的性质，对于糖萼各组成部分对其整体功能的相对贡献知之甚少。研究糖萼成分对细胞功能和系统生理学的个别作用具有挑战性，因为针对特定糖萼成分的动物基因操作可能导致糖萼发生改变。因此，在进行机制研究之前，对糖萼成分的基因修饰动物模型进行特征描述和验证至关重要。在糖萼成分中，通过 eNOS 依赖机制发挥作用的糖蛋白聚糖 1 最近成为心血管疾病的一个参与者。糖蛋白聚糖 1 是否在生理条件下调节 eNOS 尚不清楚。在此，我们评估了糖蛋白聚糖 1 的缺失如何影响肺内皮糖萼的发育及其对 eNOS 活性和内皮功能的影响。使用 5-9 周龄雄性和雌性野生型和糖蛋白聚糖 1 敲除小鼠。透射电子显微镜、免疫荧光和免疫印迹评估糖萼结构和组成。通过免疫印迹评估 eNOS 激活和含量；通过格里斯反应评估一氧化氮产生。通过肺损伤的组织学迹象、体内肺力学测量和肺通气评估肺表型。糖蛋白聚糖 1 敲除小鼠的糖萼发生改变，糖萼厚度和肝素硫酸盐含量增加，黏附素 4 表达减少。这些改变与 eNOS 在 S1177 处的磷酸化减少有关。糖蛋白聚糖 1 的缺失并不影响一氧化氮的产生，并且糖蛋白聚糖 1 敲除小鼠的内皮屏障得以保留。糖蛋白聚糖 1 敲除小鼠的肺顺应性降低，肺通气不变。总之，这些数据表明，糖蛋白聚糖 1 的缺失可能导致糖萼发生改变，而不影响基础肺内皮功能，验证了这种小鼠模型作为研究糖蛋白聚糖 1 在肺内皮功能中的作用的机制研究工具。
