Department of Bioengineering, Northeastern University, Boston, MA, USA.
Department of Chemical Engineering, Northeastern University, Boston, MA, 02115, USA.
J Transl Med. 2018 Dec 18;16(1):364. doi: 10.1186/s12967-018-1721-2.
Endothelial-dependent atherosclerosis develops in a non-random pattern in regions of vessel bending and bifurcations, where blood flow exhibits disturbed flow (DF) patterns. In contrast, uniform flow (UF), normal endothelium, and healthy vessel walls co-exist within straight vessels. In clarifying how flow protectively or atherogenically regulates endothelial cell behavior, involvement of the endothelial surface glycocalyx has been suggested due to reduced expression in regions of atherosclerosis development. Here, we hypothesized that pro-atherosclerotic endothelial dysfunction occurs as a result of DF-induced reduction in glycocalyx expression and subsequently impairs endothelial sensitivity to flow. Specifically, we propose that glycocalyx degradation can induce pro-atherosclerotic endothelial dysfunction through decreased caveolin-1 and endothelial nitric oxide synthase expression and localization.
We studied endothelial cells in atherosclerotic-prone DF and atherosclerotic-resistant UF conditions in parallel plate flow culture and in C57Bl/6 mice. The effects of flow conditioning on endothelial cell behavior were quantified using immunocytochemistry. The glycocalyx was fluorescently labeled for wheat germ agglutinin, which serves as a general glycocalyx label, and heparan sulfate, a major glycocalyx component. Additionally, mechanosensitivity was assessed by immunocytochemical fluorescence expression and function of caveolin-1, the protein that forms the mechanosignaling caveolar invaginations on the endothelial surface, total endothelial-type nitric oxide synthase (eNOS), which synthesizes nitric oxide, and serine 1177 phosphorylated eNOS (eNOS-pS1177), which is the active form of eNOS. Caveolin function and eNOS expression and activation were correlated to glycocalyx expression. Heparinase III enzyme was used to degrade a major glycocalyx component, HS, to identify the role of the glycocalyx in caveoin-1 and eNOS-pS1177 regulation.
Results confirmed that DF reduces caveolin-1 expression and abolishes most of its subcellular localization preferences, when compared to the effect of UF. DF down-regulates caveolin-1 mechanosignaling, as indicated by its reduced colocalization with serine 1177 phosphorylated endothelial-type nitric oxide synthase (eNOS-pS1177), a vasoregulatory signaling molecule whose activity is regulated by its residence in caveolae. As expected, DF inhibited glycocalyx expression compared to UF. In the absence of heparan sulfate, a major glycocalyx component, UF-conditioned endothelial cells exhibited near DF-like caveolin-1 expression, localization, and colocalization with eNOS-pS1177.
This is the first demonstration of a flow-defined role of the glycocalyx in caveolae expression and function related to vasculoprotective endothelial mechanosensitivity that defends against atherosclerosis. The results suggest that a glycocalyx-based therapeutic targeted to areas of atherosclerosis development could prevent disease initiation and progression.
血管弯曲和分叉处的血管中，内皮依赖性动脉粥样硬化呈非随机模式发展，血流呈现出紊乱流（DF）模式。相比之下，直血管中存在均匀流（UF）、正常内皮和健康的血管壁。在阐明血流如何保护性或动脉粥样硬化性调节内皮细胞行为时，由于在动脉粥样硬化发展区域表达减少，已经提出了内皮表面糖萼的参与。在这里，我们假设，DF 诱导的糖萼表达减少导致促动脉粥样硬化性内皮功能障碍，进而损害内皮对血流的敏感性。具体而言，我们提出糖萼降解可以通过降低 caveolin-1 和内皮型一氧化氮合酶的表达和定位来诱导促动脉粥样硬化性内皮功能障碍。
我们在平行板流动培养和 C57Bl/6 小鼠中平行研究了易发生动脉粥样硬化的 DF 和抗动脉粥样硬化的 UF 条件下的内皮细胞。使用免疫细胞化学定量研究了流动条件对内皮细胞行为的影响。用荧光标记物标记糖萼，荧光标记物包括作为一般糖萼标记的麦胚凝集素和糖萼的主要成分硫酸乙酰肝素。此外，通过免疫细胞化学荧光表达和 caveolin-1 的功能、形成内皮表面机械信号 caveolar 内陷的蛋白、合成一氧化氮的内皮型一氧化氮合酶（eNOS）和丝氨酸 1177 磷酸化的 eNOS（eNOS-pS1177），即 eNOS 的活性形式，评估机械敏感性。caveolin 功能和 eNOS 的表达和激活与糖萼表达相关。肝素酶 III 酶用于降解主要的糖萼成分 HS，以确定糖萼在 caveoin-1 和 eNOS-pS1177 调节中的作用。
结果证实，与 UF 的作用相比，DF 降低了 caveolin-1 的表达并消除了其大部分亚细胞定位偏好。DF 下调 caveolin-1 机械信号转导，这表明其与丝氨酸 1177 磷酸化的内皮型一氧化氮合酶（eNOS-pS1177）的共定位减少，eNOS-pS1177 是一种血管调节信号分子，其活性受其在 caveolae 中的位置调节。如预期的那样，DF 与 UF 相比抑制了糖萼的表达。在缺乏硫酸乙酰肝素（一种主要的糖萼成分）的情况下，UF 条件下的内皮细胞表现出类似 DF 的 caveolin-1 表达、定位和与 eNOS-pS1177 的共定位。
这是首次证明糖萼在与血管保护内皮机械敏感性相关的 caveolae 表达和功能中具有明确的流动作用，该作用可抵抗动脉粥样硬化。结果表明，针对动脉粥样硬化发展区域的基于糖萼的治疗方法可能预防疾病的发生和进展。
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