Nitric oxide is a potent local vasodilator throughout the vasculature.  It is first produced by endothelial cells and then diffuses through the tissue to smooth muscle cells, which act to dilate the vessel.  The method by which nitric oxide (NO) is produced is of particular importance as failure to produce NO often results in arterial disease, such as hypertension.  Additionally, NO has atheroprotective effects, shielding the vessel from plaque build-up resulting in blockage of flow.  Our lab has conducted several studies to discover how the endothelial glycocalyx is the mechanotransducer for shear stress-induced nitric oxide production.  We first observed how critical glycosaminoglycans (GAGs) in the glycocalyx are involved in this process (Figure 1).

Figure 1. Enzymatic removal of critical GAGs in the glycocalyx (heparin sulfate, sialic acid, chondroitin sulfate, hyaluranon) and the resulting change in shear-induced NO2 levels. (Pahakis 2007)" width="600" height="412" /> Figure 1. Enzymatic removal of critical GAGs in the glycocalyx (heparin sulfate, sialic acid, chondroitin sulfate, hyaluranon) and the resulting change in shear-induced NO2 levels. (Pahakis 2007)

Figure 1. Enzymatic removal of critical GAGs in the glycocalyx (heparin sulfate, sialic acid, chondroitin sulfate, hyaluranon) and the resulting change in shear-induced NO2 levels. (Pahakis 2007)” width=”600″ height=”412″ /> Figure 1. Enzymatic removal of critical GAGs in the glycocalyx (heparin sulfate, sialic acid, chondroitin sulfate, hyaluranon) and the resulting change in shear-induced NO2 levels. (Pahakis 2007)

 

These GAGs are known to associate with particular proteoglycans in the glycocalyx; heparin sulfate and chondroitin sulfate bind to syndecan-1, heparin sulfate alone binds to glypican-1, and hyaluranon binds to CD44).  These associations as well as previous work modeling tip drag interactions spurred a recent study showing pertinent proteoglycans involved in shear-induced nitric oxide production (Figure 2).

Figure 2. Endothelial nitric oxide synthase (eNOS) is phosphorylated (peNOS) to activate increased nitric oxide production. Enzymatic removal of heparin sulfate decreases the shear-induced nitric oxide production (as measured by peNOS). Interestingly, siRNA knockdowns of glypican-1, but not syndecan-1, showed decreased nitric oxide production. (Ebong, et al. 2014)" width="550" height="481" /> Figure 2. Endothelial nitric oxide synthase (eNOS) is phosphorylated (peNOS) to activate increased nitric oxide production. Enzymatic removal of heparin sulfate decreases the shear-induced nitric oxide production (as measured by peNOS). Interestingly, siRNA knockdowns of glypican-1, but not syndecan-1, showed decreased nitric oxide production. (Ebong, et al. 2014)

Figure 2. Endothelial nitric oxide synthase (eNOS) is phosphorylated (peNOS) to activate increased nitric oxide production. Enzymatic removal of heparin sulfate decreases the shear-induced nitric oxide production (as measured by peNOS). Interestingly, siRNA knockdowns of glypican-1, but not syndecan-1, showed decreased nitric oxide production. (Ebong, et al. 2014)” width=”550″ height=”481″ /> Figure 2. Endothelial nitric oxide synthase (eNOS) is phosphorylated (peNOS) to activate increased nitric oxide production. Enzymatic removal of heparin sulfate decreases the shear-induced nitric oxide production (as measured by peNOS). Interestingly, siRNA knockdowns of glypican-1, but not syndecan-1, showed decreased nitric oxide production. (Ebong, et al. 2014)

 

Additionally, we are investigating the mechanism by which proteoglycans bind to the plasma membrane, which may influence shear-induced glycocalyx reorganization and signal transduction.  We recently focused on proteoglycans, their associated GAGs, and two different kinds of membrane rafts: caveolae and lipid rafts.  Our study shows how lipid rafts create mobility for the proteoglycan glypican-1 with shear stress (Figure 3).  Caveolae have been shown by other authors to colocalize with eNOS, and are thereby involved in nitric oxide production.

Figure 3. The mobility of proteoglycans (glypican-1 and syndecan-1) and membrane rafts (caveolae and lipid rafts) in response to varying timescales of shear stress exposure. (Zeng et al. 2014)

Figure 3. The mobility of proteoglycans (glypican-1 and syndecan-1) and membrane rafts (caveolae and lipid rafts) in response to varying timescales of shear stress exposure. (Zeng et al. 2014)