Among the first mechanisms proposed to relate wall shear stress patterns to the localization of atherosclerosis in the 1970s was one in which the fluid (blood)- phase resistance to transport of lipid (LDL) or other atherogens was controlled by the local wall shear rate. This provided the impetus for our studies of shear dependent permeability of endothelial layers and the mechanisms of LDL transport across the arterial wall. Other studies suggested that direct effects of blood flow on the endothelium must be important. Such direct effects were demonstrated most graphically by the observation that endothelial cells change their morphology to elongate and align in the direction of flow. Subsequently investigators began to examine how fluid shear stress on endothelial cells might influence the production of physiologically important biochemicals by the cells. In the mid 1980s it was observed that shear stress applied to static cells in culture that had not been exposed to shear stress previously, induced the rapid (within minutes) and sustained (over a period of hours) production of prostacyclin, an important anti-thrombotic, anti-proliferative, vasodilatory agent. These studies were followed by observations that shear stress on static endothelial cells induced the transient release of intra-cellular calcium (Ca++), an important intra-cellular signaling molecule and the sustained release (over hours) of the anti-proliferative vasodilator, nitric oxide. Concurrently, during the early 1990’s, the effects of shear stress on the expression of individual endothelial genes came into focus.
In the early 1990s the idea of single gene analysis was advanced by the discovery of shear stress response elements (SSREs), or common cis-acting elements of gene promoters that are regulated by a single shear-controlled transcription factor. Endothelial cells’ keen ability to sense and convert mechanical stimuli into biochemical signaling responsesWith the advent of high throughput genome-wide analysis technology such as the microarray it became possible to study the expression of thousands of genes throughout the genome simultaneously. This greatly impacted the field of vascular mechanobiology by enabling researchers to track a large host of genes in a controlled, well-defined environment. The resulting studies provided clear evidence of the endothelial cells’ keen ability to sense and convert mechanical stimuli into biochemical signaling responses. Specific intracellular signaling pathways that become prevalent under low/oscillatory, atheroprone shear stress are displayed along with pathways that dominate under high, atheroprotective, laminar shear areas, cyclic stretch, and appropriate hydrostatic pressure in Figure 1.
Mechanotransduction is the process by which a cell senses a specific mechanical force and transduces it into an intracellular biomolecular response. Several mechanosensors/transducers on endothelial cells have been proposed (Figure 2). The intact mechanosensory complex at endothelial cell junctions consisting of PECAM-1, VE Cadherin, and VEGFR2, is crucial for the endothelial cell to be able to activate anti-atherosclerotic gene programs in response to laminar flow. Several other important mechanotransduction pathways begin at the endothelial surface proteoglycan layer (glycocalyx) and the cell membrane, including integrins (FAK), cell membrane proteins (RTKs, GPCRs), ion channels (Ca++), and intercellular junctions, are propagated biochemically through the cytosol or mechanically via the cytoskeleton to ultimately affect gene expression in the nucleus. The glycocalyx, integrins and the intercellular junction are of particular interest in the Coulter Lab.
A surface proteoglycan / glycoprotein layer – the glycocalyx (GCX) covers all mammalian cells. The concept that a thin endocapillary layer might cover the entire endothelial surface was first proposed in the 1940s. However, this layer evaded observation by light and electron microscopy (EM) until 1966, when ruthenium red staining and EM were used to clearly detect a thin layer (∼ 20 nm thick) in rat intestinal mucosa. More recent techniques have been used to overcome the dehydration artifacts if EM with the result that endothelial GCX is believed to be on the order of 0.5 – 5.0 µm thick as shown in Figure 3 for capillaries in the rat mesentery.
A cartoon that integrates all of the components of the GCX is shown in Figure 4. Major components of the GCX are glycoproteins bearing acidic oligosaccharides and terminal sialic acids (SA), and proteoglycans (PGs) with their associated glycosaminoglycan (GAG) side chains. GAGs are characterized by distinct disaccharide unit repeats that give rise to different components such as heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) commonly associated with ECs. Proteoglycans (PGs) are proteins that contain specific sites where sulfated GAGs are covalently attached. The transmembrane syndecans and membrane bound glypicans are among the three major protein core families of heparan sulfate proteoglycans (HSPGs) found on ECs (along with the basement matrix associated perlecans). From the syndecan family of core proteins, syndecans -1 (33 kDa), -2 (22 kDa), and -4 (22 kDa) are expressed on ECs.
Glypican-1 (64 kDa) is the only glypican expressed on ECs. Glypican-1 is bound directly to the plasma membrane through a C-terminal glycosylphosphatidylinositol (GPI) anchor. The GPI anchor localizes this proteoglycan to lipid rafts, which are cholesterol and sphingolipid rich membranous domains involved in vesicular transport and cell signaling. Caveolae can be considered a subset of lipid rafts, which arise from the incorporation of the protein caveolin-1, a cholesterol carrier, into the membrane, where they may form characteristic cave-like structures (~100 nm) that are supported by the cytoskeleton. In contrast with CS and HS, HA is a much longer disaccharide polymer, on the order of 1000 kDa, which is synthesized on the cell surface and is not covalently attached to a core protein. It is not sulfated, but obtains its negative charge from carboxyl groups that endow it with exceptional hydration properties. HA associates with the GCX through its interaction with surface HA receptors, such as the transmembrane CD44, and CS chains.
The primary evidence that supports a central role for the GCX in mechanotransduction comes from experiments involving use of enzymes to selectively degrade specific components of the GCX, followed by a reassessment of function. Our group used the enzyme heparinase III to selectively degrade the HS component of endothelial GAGs in vitro and observed that normal production of nitric oxide (NO) induced over 3 h by steady or oscillatory shear stress could be completely inhibited (Figure 5). In another study, the enzymes hyaluronidase, chondroitinase and neuraminidase were employed and all but chondroitinase blocked shear-induced NO production. These studies and many others support a role for the GCX in sensing and transducing fluid shear stress.
There are several possible mechanisms relating the GCX to mechanotransduction. The simplest is based on the idea that GAGs are the shear sensors that transmit force to the cell by way of the core proteins. The hypothesis is that deflections of the fibers due to exposure to shear stress cause molecular displacement of signaling proteins in the endothelial cytoskeleton.
A schematic of this model is shown in Figure 6. A prediction from the model is that fluid flow within the EC surface layer due to the applied fluid shear stress is restricted to the outer 10% of the surface layer. Structures below this surface region are not exposed to fluid shear forces. Thus, in an intact glycocalyx layer, not all components of an inner layer such as ion channels and surface receptors or caveolae are exposed directly to FSS. They are likely to respond directly to applied fluid shear only in the absence of the GCX. In the presence of an intact GCX, the applied fluid shear stress is transmitted to the cell as a solid mechanical force via the core proteins that interface with the cell membrane or cytoskeleton. This is somewhat analogous to the force of the wind blowing through the leaves of a tree being transmitted to the ground through the tree trunk.