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, 250–300g) 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.

Tumor cell adhesion to the microvessel wall is a critical step during tumor metastasis. Vascular endothelial growth factor (VEGF), a secretion of tumor cells, can increase microvessel permeability and tumor cell adhesion in the microvessel. To test the hypothesis that inhibiting permeability increase can reduce tumor cell adhesion, we used in vivo fluorescence microscopy to measure both microvessel permeability and adhesion rates of human mammary carcinoma MDA-MB-231 cells in post-capillary venules of rat mesentery under the treatment of VEGF and a cAMP analog, 8-bromo-cAMP, which can decrease microvessel permeability. By immunostaining adherens junction proteins between endothelial cells forming the microvessel wall, we further investigated the structural mechanism by which cAMP abolishes VEGF-induced increase in microvessel permeability and tumor cell adhesion. Our results demonstrate that 1) Pretreatment of microvessels with cAMP can abolish VEGF-enhanced microvessel permeability and tumor cell adhesion; 2) Tumor cells prefer to adhere to the endothelial cell junctions instead of cell bodies; 3) VEGF increases microvessel permeability and tumor cell adhesion by compromising endothelial junctions while cAMP abolishes these effects of VEGF by reinforcing the junctions. These results suggest that strengthening the microvessel wall integrity can be a potential approach to inhibiting hematogenous tumor metastasis.

To test the hypothesis that endothelial surface glycocalyx (ESG) plays a role in mechanosensing and transduction of the microvessel wall, we used fluorescence microscopy to measure the NO levels in the post‐capillary venules of rat mesentery under low and high flow conditions and with/out enzyme treatment for removing heparan sulfate (HS) of the ESG. Rats (SD, 250–300g) were anesthetized with pentobarbital sodium given subcutaneously, the mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. After perfusion for 1 hr with 1% BSA Ringer for the control or with 50 mU/mL heparanase III for the treatment, an individual post‐capillary venule (35–50 μm) was loaded for 45 min with 5 μM 4, 5‐Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO was measured for ~10min under a low flow (< 300 μm/s) as the baseline and for ~60min under a high flow (1500–2000 μm/s mean velocity). In 1 min after switching to the high low, NO increased by 1.14 ± 0.05‐fold (n=3) under control and 1.15 ± 0.04‐fold (n=3) under enzyme treatment. NO continuously increased after 1 min under high flow, reached a plateau of 1.42 ± 0.02‐fold in ~40 min under control and of 1.22 ± 0.03 in ~15min under treatment. The results suggest that ESG participate in EC mechanosensing and transduction through its HS and other components. Supported by NIH R01HL094889–01.