Yen Wanyi, Cai Bin, Yang Jinlin, Zhang Lin, Zeng Min, Tarbell John M, Fu Bingmei M
Department of Biomedical Engineering, The City College of the City University of New York, New York, New York, United States of America.
PLoS One. 2015 Jan 9;10(1):e0117133. doi: 10.1371/journal.pone.0117133. eCollection 2015.
Due to its unique location, the endothelial surface glycocalyx (ESG) at the luminal side of the microvessel wall may serve as a mechano-sensor and transducer of blood flow and thus regulate endothelial functions. To examine this role of the ESG, we used fluorescence microscopy to measure nitric oxide (NO) production in post-capillary venules and arterioles of rat mesentery under reduced (low) and normal (high) flow conditions, with and without enzyme pretreatment to remove heparan sulfate (HS) of the ESG and in the presence of an endothelial nitric oxide synthase (eNOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA). Rats (SD, 250-300 g) were anesthetized. The mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. An individual post-capillary venule or arteriole was cannulated and loaded for 45 min with 5 μM 4, 5-Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO production was measured for 10 min under a low flow (300 μm/s) and for 60 min under a high flow (1000 μm/s). In the 15 min after switching to the high flow, DAF-2-NO fluorescence intensity increased to 1.27-fold of its baseline, DAF-2-NO continuously increased under the high flow, to 1.53-fold of its baseline in 60 min. Inhibition of eNOS by 1 mM L-NMMA attenuated the flow-induced NO production to 1.13-fold in 15 min and 1.30-fold of its baseline in 60 min, respectively. In contrast, no significant increase in NO production was observed after switching to the high flow for 60 min when 1 h pretreatment with 50 mU/mL heparanase III to degrade the ESG was applied. Similar NO production was observed in arterioles under low and high flows and under eNOS inhibition. Our results suggest that ESG participates in endothelial cell mechanosensing and transduction through its heparan sulfate to activate eNOS.
由于其独特的位置，微血管壁管腔侧的内皮表面糖萼（ESG）可作为血流的机械传感器和转换器，从而调节内皮功能。为了研究ESG的这一作用，我们使用荧光显微镜在低流量（低）和正常流量（高）条件下，测量大鼠肠系膜毛细血管后微静脉和小动脉中一氧化氮（NO）的产生，有无酶预处理以去除ESG的硫酸乙酰肝素（HS），并在存在内皮型一氧化氮合酶（eNOS）抑制剂NG-单甲基-L-精氨酸（L-NMMA）的情况下进行测量。将大鼠（SD，250 - 300 g）麻醉。将肠系膜从腹腔中轻轻取出，放置在玻璃盖玻片表面进行测量。将单个毛细血管后微静脉或小动脉插管，并加载5 μM 4,5-二氨基荧光素二乙酸酯45分钟，这是一种用于NO的膜通透性荧光指示剂，然后在低流量（约300 μm/s）下测量约10分钟的NO产生，并在高流量（约1000 μm/s）下测量约60分钟。在切换到高流量后的15分钟内，DAF-2-NO荧光强度增加到其基线的1.27倍，在高流量下DAF-2-NO持续增加，在60分钟内达到其基线的1.53倍。1 mM L-NMMA抑制eNOS后，在15分钟内将流量诱导的NO产生减弱至基线的1.13倍，在60分钟内减弱至基线的1.30倍。相比之下，当用50 mU/mL硫酸乙酰肝素酶III预处理1小时以降解ESG后，在切换到高流量60分钟后未观察到NO产生有显著增加。在低流量和高流量以及eNOS抑制下，小动脉中观察到类似的NO产生。我们的结果表明，ESG通过其硫酸乙酰肝素参与内皮细胞的机械传感和转导，以激活eNOS。
