Microvascular Endothelial Cells Exhibit Optimal Aspect Ratio for Minimizing Flow Resistance

A recent analytical solution of the three-dimensional Stokes flow through a bumpy tube predicts that for a given bump area, there exists an optimal circumferential wavenumber which minimizes flow resistance. This study uses measurements of microvessel endothelial cell morphology to test whether this prediction holds in the microvasculature. Endothelial cell (EC) morphology was measured in blood perfused in situ microvessels in anesthetized mice using confocal intravital microscopy. EC borders were identified by immunofluorescently labeling the EC surface molecule ICAM-1 which is expressed on the surface but not in the EC border regions. Comparison of this theory with extensive in situ measurements of microvascular EC geometry in mouse cremaster muscle using intravital microscopy reveals that the spacing of EC nuclei in venules ranging from 27 to 106 μm in diameter indeed lies quite close to this predicted optimal configuration. Interestingly, arteriolar ECs are configured to minimize flow resistance not in the resting state, but at the dilated vessel diameter. These results raise the question of whether less organized circulatory systems, such as that found in newly formed solid tumors or in the developing embryo, may deviate from the optimal bump spacing predicted to minimize flow resistance

The endothelial glycocalyx: composition, functions, and visualization

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging

Mechanosignaling from extracellular matrix fibronectin mediates endothelial cell responses to flow

Thesis (Ph. D.)--University of Rochester. Department of Biomedical Engineering, 2015.The endothelium is constantly exposed to various hemodynamic forces including blood pressure and fluid shear stress which aid in regulating vascular development and physiology. Fluid shear stress acts in a tangential direction on the apical surface of endothelial cells (EC) and both in vivo and in vitro studies have demonstrated that EC are responsive to this mechanical stimulus. One approach to determine the mechanosensitivity of the endothelium is to measure changes in cell morphology and cytoskeletal realignment in the direction of blood flow. The existing paradigm holds that this realignment is signaled directly from changes in perceived wall shear stress by cellular mechanotransducers linked to the cytoskeleton. Components of the glycocalyx, cell-matrix adhesions and cell-cell junctions have all been implicated as potential candidates in the process of mechanotransduction. The studies presented in this thesis sought to establish whether non-cellular components such as ECM fibronectin contributed to the observed mechanoresponses. In response to mechanical force, type III repeats (FNIII) within fibronectin are predicted to unfold and expose cryptic binding sites. Previous studies have shown that the biological activity of ECM fibronectin is localized, in part, to a matricryptic, heparin-binding site located within its 1st type III repeat (FNIII1H). A series of engineered fibronectin matrix mimetics that mimic the effects of ECM fibronectin on cells have been developed using recombinant protein production and purification. These mimetics couple the matricryptic, heparin-binding site (FNIII1H) to variants of the cell-binding domain (FNIII8-10) and provide us with the ability to study the effects of cryptic site signaling and various fibronectin conformations on endothelial cell responses to flow. Therefore, the overall goal of this work was to determine the role of ECM fibronectin (FN) and specific matricryptic signaling elements within the 1st type III repeat in EC responses to flow. We subjected human umbilical vein endothelial cells seeded on various fibronectin matrix mimetics to flow in perfusable microslides and measured the short-term (t ≤ 8 hrs.) shear stress-induced changes in endothelial cell morphology and stress fiber and cell alignment. In the 1st part of the study, we show that ECM fibronectin via a matricryptic, heparin-binding site within its 1st type III repeat mediates endothelial cell mechanosensitive responses when flow is used as mechanical stimulus. Specifically, the matricryptic, heparin-binding site (FNIII1H) mediates stress fiber realignment when cells are adhered via the integrin α5β1 but is not necessary when endothelial cells are adhered via the integrin αvβ3. In the 2nd part of the study, we show that endothelial cell-cell junctions are not required for FNIII1H-mediated stress fiber realignment but they are necessary when endothelial cell adhesion is via the integrin αvβ3. In subconfluence, we also demonstrate that cell, but not stress fiber alignment is independent of matricryptic site signaling. Lastly, we show that subconfluent endothelial cells utilize the matricryptic site and the integrin αvβ3 to mediate different morphological responses to flow. In summary, the studies presented in this thesis identify a pivotal role for fibronectin conformation and cryptic site signaling in mediating endothelial cell mechanosensitive responses to mechanical stimuli