Abstract Aims Arterial stiffness is an underlying risk factor and a hallmark of cardiovascular diseases. The endothelial cell (EC) glycocalyx is a glycan rich surface layer that plays a key role in protecting against EC dysfunction and vascular disease. However, the mechanisms by which arterial stiffness promotes endothelial cell dysfunction and vascular disease are not fully understood, and whether the mechanism involves the protective endothelial glycocalyx is yet to be determined. We hypothesized that endothelial glycocalyx protects the endothelial cells lining the vascular wall from dysfunction and disease in response to arterial stiffness. Methods and Results Cells cultured on polyacrylamide (PA) gels of substrate stiffness 10 kPa (mimicking the subendothelial stiffness of aged, unhealthy arteries) showed a significant inhibition of glycocalyx expression compared to cells cultured on softer PA gels (2.5 kPa, mimicking the subendothelial stiffness of young, healthy arteries). Specifically, gene and protein analyses revealed that a glycocalyx core protein Glypican 1 was inhibited in cells cultured on stiff PA gels. These cells had enhanced endothelial cell dysfunction as determined by enhanced cell inflammation (enhanced inflammatory gene expression, monocyte adhesion and inhibited nitric oxide expression), proliferation and EndMT. Removal of Glypican 1 using gene-specific silencing with siRNA or gene overexpression using a plasmid revealed that Glypican 1 is required to protect against stiffness-mediated endothelial cell dysfunction. Consistent with this, using a model of age-mediated stiffness, older mice exhibited a reduced expression of Glypican 1 and enhanced endothelial cell dysfunction compared to young mice. Glypican1 gene deletion in knockout mice (GPC1 -/-) exacerbated endothelial dysfunction in young mice, which normally had high endothelial expression, but not in old mice that normally expressed low levels. Endothelial cell dysfunction was exacerbated in young, but not aged, Glypican 1 knockout mice (GPC1 -/-). Conclusions Arterial stiffness promotes EC dysfunction and vascular disease at least partly through the suppression of the glycocalyx protein Glypican 1. Glypican 1 contributes to the protection against endothelial cell dysfunction and vascular disease in endothelial cells. Translational Perspective This work reveals for the first time the effect of arterial stiffness on the glycocalyx protein Glypican 1 in endothelial cell function. Stiffness promotes endothelial cell dysfunction and subsequently vascular disease at least partly through the suppression of Glypican 1. Thus, Glypican 1 could be a novel therapeutic candidate for the treatment/reversal of arterial stiffness-mediated vascular disease.

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The yellow color of 9,IO-phenanthrenequinone in alcohol solution is bleached under the influence of exciting radiation in a band of wavelengths extending from the ultraviolet to the visible green. The bleaching is due to a photoreduction of the quinone yielding 9,lOdihydroxyphenanthrene. In the presence of certain divalent metal ions, irradiation of the quinoid material results in a blue-green long lived intermediate which may be further photoreduced to the colorless 9.1 0-diol. The blue-green photo-intermediate is a cation-semiquinone complex.

The surface proteoglycan/glycoprotein layer (glycocalyx) on tumor cells has been associated with cellular functions that can potentially enable invasion and metastasis. In addition, aggressive renal carcinoma cells (SN12L1) with high metastatic potential have enhanced invasion rates compared to low metastatic (SN12C) cells in response to interstitial flow stimuli in vitro. Our previous studies suggest that heparan sulfate (HS) and hyaluronic acid (HA) in the glycocalyx play an important role in this flow mediated mechanotransduction and upregulation of invasive and metastatic potential. In our recent study, SN12L1 cells were genetically modified to suppress HS production by knocking down its synthetic enzyme NDST1. Using modified Boyden chambers with defined interstitial flow, we showed that flow‐enhanced invasion is suppressed in HS deficient cells. We also examined two prominent HSPGs on renal carcinoma cells – glypican‐1 and syndecan‐1 and one prominent HA receptor – CD44. We observed higher glypican‐1 levels in flow dependent SN12L1 cells when compared to SN12C cells. Caki‐1 (highly metastatic) cells did not display flow‐dependent invasion in vitro and did not display elevated glypican‐1 compared to low metastatic Caki‐2 cells. However we did observe significantly increased HS, HA, syndecan‐1 and CD44 in Caki‐1 compared to Caki‐2 cells suggesting an alternative mechanism for reported higher metastatic rates in these cells. All of our data are consistent with the hypothesis that glypican‐1 is the core protein responsible for flow sensing in metastatic cancer cells. This is also consistent with observations in endothelial cells. To assess the ability of tumor cells to metastasize in vivo, parental or HS knockdown SN12L1 cells expressing fluorescent reporters were injected into kidney capsules in SCID mice. Histological analysis confirmed that there was a large reduction (95%) in metastasis to distant organs by tumors formed from knockdown cells compared to control cells with intact HS. The reduction was even greater (98%) in lungs where most of the metastases from the control cells were observed. The ability of these knockdown cells to invade surrounding tissue was also impaired. The substantial inhibition of metastasis and invasion upon reduction of HS suggests an active role for the tumor cell glycocalyx and glypican‐1 in tumor progression.Support or Funding InformationGrant: RO1CA204949 Targeting glycocalyx mediated mechanisms of tumor metastsisThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.