doi: 10.1161/CIRCRESAHA.108.174839
© 2008 American Heart Association, Inc.
Editorials |
Dragging Along
The Glycocalyx and Vascular Endothelial Cell Mechanotransduction
From the Department of Mechanical and Aeronautical Engineering, University of California, Davis.
Correspondence to Abdul I. Barakat, Mechanical and Aeronautical Engineering, University of California, Davis, One Shields Ave, Davis, CA 95616. E-mail abarakat{at}ucdavis.edu
See related article, pages 770–776
Key Words: mechanotransduction • glycocalyx
The glycocalyx is a complex and highly dynamic polymeric meshwork that coats the surfaces of most cells and consists of proteoglycans, glycosaminoglycans (GAGs), and glycoproteins, as well as adherent plasma proteins.1,2 In the microvasculature, the glycocalyx layer on the endothelial cell surface plays a critical role in regulating vessel permeability, modulating the dynamics of near-wall movement of red blood cells, and coordinating interactions between leukocytes and the vascular wall during inflammation. In large-vessel endothelium, perturbation of the glycocalyx appears to be associated with vascular damage, as well as increased vulnerability to the development of atherosclerosis.3–5 A number of studies have demonstrated that the presence of an intact glycocalyx is essential for endothelial cell sensitivity and responsiveness to fluid mechanical stimulation, thereby supporting the idea of a central role for the glycocalyx in endothelial cell flow-mediated mechanotransduction.6–12
In this issue of Circulation Research, Potter and Damiano13 use fluorescent microparticle image velocimetry (µ-PIV) to demonstrate that the hydrodynamically relevant endothelial cell glycocalyx surface layer observed in vivo in mouse cremaster muscle venules is absent from the surfaces of human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) grown in circular collagen microchannels and maintained in vitro under standard cell culture conditions. The µ-PIV technique allows the measurement of the velocity profile of fluid flow within the venule or microchannel. A linear regression analysis of the near-wall velocity is performed, and the resulting fit is extrapolated to the venule or microchannel wall. If this extrapolation leads to a negative velocity at the wall, then the distance from the wall to the position where the linear fit yields 0 velocity is taken to be the thickness of the hydrodynamically relevant glycocalyx. If, on the other hand, the fit does not extrapolate to a negative velocity at the wall, then a hydrodynamically relevant glycocalyx is assumed to be absent.
Previous studies have demonstrated via immunostaining that the various components of the glycocalyx are indeed present on the surfaces of cultured endothelial cells.6,11,12 These and other studies have also shown that the glycocalyx plays an integral role in mechanosensing in endothelial cells both in vivo and in vitro and that the individual components of the glycocalyx may, to a certain extent, play specialized roles in coordinating responsiveness to mechanical stimulation. For instance, enzymatic digestion of heparan sulfate, sialic acid, or hyaluronan abolishes flow-induced release of nitric oxide (NO) from endothelial cells,7,9 whereas removal of chondroitin sulfate fails to inhibit the NO response.9 Removal of heparan sulfate also attenuates or abolishes a number of other flow-induced endothelial responses, including remodeling of peripheral actin and the actin-binding protein vinculin11; cellular elongation and alignment in the direction of flow12; and suppression of cell proliferation.12
A question that arises is whether data demonstrating that an intact glycocalyx layer is present on the surfaces of endothelial cells in vitro and that glycocalyx removal modulates various flow responses are inconsistent with the results of Potter and Damiano.13 This is not necessarily the case. The findings of Potter and Damiano do not make a determination as to whether or not the HUVEC/BAEC glycocalyx is physically absent in cultured cells and provide no information on the thickness, composition, or overall state of the glycocalyx layer if it is present. Rather, the findings establish that whatever glycocalyx is present on the surfaces of HUVECs and BAECs in vitro exhibits hydrodynamic drag characteristics that are functionally different from those present in microvessels in vivo.
The implications of the results of Potter and Damiano13 for in vitro studies of endothelial cell mechanotransduction remain to be determined. Many endothelial cell mechanotransduction studies are motivated by a desire to elucidate the role of flow in the etiology and pathogenesis of atherosclerosis, a disease of medium and large arteries. Potter and Damiano13 performed their in vivo measurements on mouse cremaster muscle venules. Therefore, it is important to establish whether the in vivo drag characteristics of the glycocalyx in large vessel endothelium are similar to those in the microvasculature and if there are variations in these characteristics among vascular beds and species. Such studies are certainly technically challenging. Potter and Damiano13 obtained their in vitro results on cells cultured in collagen microchannels, a culture system that is different from that used in most flow studies in which cells are grown on flat surfaces and exposed to flow in parallel plate flow chambers or cone-and-plate viscometers. Therefore, an important question is whether or not the hydrodynamic behavior of the glycocalyx in cultured endothelial cells depends on the geometry of the flow system and/or the identity of the substrate. Another important prerequisite for understanding the full implications of the findings of Potter and Damiano13 is the need to determine which subset of the numerous endothelial cell responses to flow is sensitive to the hydrodynamic behavior of the glycocalyx. A long-standing controversy in this field is whether flow modulates endothelial cell function via a direct effect of the flow force on the cells (a shear stress effect), an indirect effect of altered transport of agonists to which the cells respond (a shear rate effect), or a combination of both of these pathways.14 Because Potter and Damiano13 focus on the hydrodynamic drag behavior of the glycocalyx, their results are more likely to be applicable to endothelial responses that are shear stress- rather than shear rate-dependent.
Although the glycocalyx appears to function as a mechanosensor and to regulate certain responses to flow in endothelial cells, there are reports of glycocalyx-independent responses. For example, glycocalyx removal appears to have no effect on flow-induced production of prostacyclin (also known as prostaglandin I2) in endothelial cells.7 It remains unclear whether the prostacyclin response is truly glycocalyx-independent or whether its suppression would simply require more complete removal of the glycocalyx (the enzymes used in the experiments often do not fully digest the glycocalyx). The presence of glycocalyx-independent flow responses in endothelium would suggest the existence of multiple and independent flow-sensing pathways. Other candidate flow sensors that have been proposed include flow-sensitive ion channels,15–17 integrins,18 the platelet endothelial cell adhesion molecule-1/cadherin/vascular endothelial growth factor receptor-2 complex,19 GTP-binding proteins (G proteins), and G protein–coupled receptors.20,21 In support of the existence of glycocalyx-independent mechanosensing pathways, fluid flow has been shown to activate ion channels and to mobilize intracellular calcium in endothelial cells, even in the absence of protein in the perfusate.15–17,22 The presence of protein in the fluid is generally considered necessary for imparting structure to the glycocalyx surface layer.23,24
The composition of the endothelial glycocalyx in vivo remains to be accurately defined. Potter and Damiano13 report that hyaluronidase treatment abolishes the hydrodynamically relevant glycocalyx layer present in vivo. This may indicate that hyaluronan constitutes the major component of the glycocalyx in vivo, although this remains to be determined in light of the imperfect specificity of hyaluronidase to hyaluronan. In vitro, heparan sulfate appears to be the most prevalent GAG constituent on the endothelial cell surface.2 Alternatively, the results of Potter and Damiano13 may indicate that hyaluronan is the primary determinant of the hydrodynamic drag provided by the glycocalyx, even if other GAGs and proteoglycans are present at high concentrations.
Over the past 2 decades, in vitro models have provided an invaluable tool for understanding mechanosensing and mechanotransduction in vascular endothelial cells. The results of Potter and Damiano13 generally underscore the need for carefully assessing the direct applicability of results derived from these models to the in vivo environment and highlight the need for the development of capabilities for studying vascular mechanotransduction in vivo.
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Acknowledgments |
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Sources of Funding
Research performed by the author is supported by NIH grants HL087078 and HL068035.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Circ. Res. 2008 102: 770-776.
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