John M. Tarbell

Heparan sulfate proteoglycan is a mechanosensor on endothelial cells.

Abstract— The objective of this study was to test whether a glycosaminoglycan component of the surface glycocalyx layer is a fluid shear stress sensor on endothelial cells (ECs). Because enhanced nitric oxide (NO) production in response to fluid shear stress is a characteristic and physiologically important response of ECs, we evaluated NOx (NO2− and NO3−) production in response to fluid shear stress after enzymatic removal of heparan sulfate, the dominant glycosaminoglycan of the EC glycocalyx, from cultured ECs. The significant NOx production induced by steady shear stress (20 dyne/cm2) was inhibited completely by pretreatment with 15 mU/mL heparinase III (E.C.4.2.2.8) for 2 hours. Oscillatory shear stress (10±15 dyne/cm2) induced an even greater NOx production than steady shear stress that was completely inhibited by pretreatment with heparinase III. Addition of bradykinin (BK) induced significant NOx production that was not inhibited by heparinase pretreatment, demonstrating that the cells were still able to produce abundant NO after heparinase treatment. Fluorescent imaging with a heparan sulfate antibody revealed that heparinase III treatments removed a substantial fraction of the heparan sulfate bound to the surfaces of ECs. In summary, these experiments demonstrate that a heparan sulfate component of the EC glycocalyx participates in mechanosensing that mediates NO production in response to shear stress. The full text of this article is available online at http://www.circresaha.org.

Mechanotransduction and the glycocalyx

Endothelial cells (ECs) line all blood vessel walls and are exposed to the mechanical forces of blood flow which modulate their function and play a role in vascular regulation, remodelling and disease. The principal mechanical forces sensed by ECs are the shear stress of flowing blood on their apical surface, and the circumferential stress resisting blood pressure, which induces stretch in the cell body. ‘Mechanotransduction’ refers to the mechanisms by which these forces are transduced into biomolecular responses of the cells. Given the importance of endothelial mechanotransduction in cardiovascular physiology and pathology, numerous research efforts have been dedicated to identifying the mechanosensory component(s) of ECs. This review focuses on mechanotransduction of shear stress by ECs and considers the evidence in support of the surface glycocalyx acting as a mechanotransducer.

Shear stress and the endothelial transport barrier.

The shear stress of flowing blood on the surfaces of endothelial cells that provide the barrier to transport of solutes and water between blood and the underlying tissue modulates the permeability to solutes and the hydraulic conductivity. This review begins with a discussion of transport pathways across the endothelium and then considers the experimental evidence from both in vivo and in vitro studies that shows an influence of shear stress on endothelial transport properties after both acute (minutes to hours) and chronic (hours to days) changes in shear stress. Next, the effects of shear stress on individual transport pathways (tight junctions, adherens junctions, vesicles and leaky junctions) are described, and this information is integrated with the transport experiments to suggest mechanisms controlling both acute and chronic responses of transport properties to shear stress. The review ends with a summary of future research challenges.

Interaction between Wall Shear Stress and Circumferential Strain Affects Endothelial Cell Biochemical Production

The wall shear stress (WSS) of flowing blood and the circumferential strain (CS) driven by the pressure pulse interact to impose a dynamic force pattern on endothelial cells (ECs) which can be characterized by the temporal phase angle between WSS and CS, a quantity which varies significantly throughout the circulation. To study the interaction of WSS and CS on endothelial production of vasodilators (prostacyclin and nitric oxide) and a vasoconstrictor (endothelin-1), bovine aortic ECs were cultured on the inner surface of compliant tubes and subjected to various flow conditions: steady shear (10 dyn/cm2), oscillatory shear (10 ± 10 dyn/cm2, rigid tube), and oscillatory shear (10 ± 10 dyn/cm2) with CS (8%) either in or out of phase with shear. The 4-hour production rates of vasoactive agents show that steady shear stimulates the highest production of vasodilators whereas oscillatory shear stimulates the highest vasoconstrictor production. The addition of CS in concert with oscillatory shear enhances the production of vasodilators and inhibits the production of vasoconstrictors, and this effect is modulated by the phase angle between WSS and CS. These data suggest that the interactions of WSS and CS are important in vascular regulation and remodeling.

Permeability of Endothelial and Astrocyte Cocultures: In Vitro Blood–Brain Barrier Models for Drug Delivery Studies

The blood–brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (Lp), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that Lp and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2–4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.

Fluid Flow Mechanotransduction in Vascular Smooth Muscle Cells and Fibroblasts

Understanding how vascular wall endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts (FBs) sense and transduce the stimuli of hemodynamic forces (shear stress, cyclic strain, and hydrostatic pressure) into intracellular biochemical signals is critical to prevent vascular disease development and progression. ECs lining the vessel lumen directly sense alterations in blood flow shear stress and then communicate with medial SMCs and adventitial FBs to regulate vessel function and disease. Shear stress mechanotransduction in ECs has been extensively studied and reviewed. In the case of endothelial damage, blood flow shear stress may directly act on the superficial layer of SMCs and transmural interstitial flow may be elevated on medial SMCs and adventitial FBs. Therefore, it is also important to investigate direct shear effects on vascular SMCs as well as FBs. The work published in the last two decades has shown that shear stress and interstitial flow have significant influences on vascular SMCs and FBs. This review summarizes work that considered direct shear effects on SMCs and FBs and provides the first comprehensive overview of the underlying mechanisms that modulate SMC secretion, alignment, contraction, proliferation, apoptosis, differentiation, and migration in response to 2-dimensional (2D) laminar, pulsatile, and oscillating flow shear stresses and 3D interstitial flow. A mechanistic model of flow sensing by SMCs is also provided to elucidate possible mechanotransduction pathways through surface glycocalyx, integrins, membrane receptors, ion channels, and primary cilia. Understanding flow-mediated mechanotransduction in SMCs and FBs and the interplay with ECs should be helpful in exploring strategies to prevent flow-initiated atherosclerosis and neointima formation and has implications in vascular tissue engineering.

Cellular Fluid Mechanics and Mechanotransduction

Mechanotransduction, the transformation of an applied mechanical force into a cellular biomolecular response, is briefly reviewed focusing on fluid shear stress and endothelial cells. Particular emphasis is placed on recent studies of the surface proteoglycan layer (glycocalyx) as a primary sensor of fluid shear stress that can transmit force to apical structures such as the plasma membrane or the actin cortical web where transduction can take place or to more remote regions of the cell such as intercellular junctions and basal adhesion plaques where transduction can also occur. All of these possibilities are reviewed from an integrated perspective.

Numerical simulation of pulsatile flow in a compliant curved tube model of a coronary artery.

The endothelial cells (ECs) lining a blood vessel wall are exposed to both the wall shear stress (WSS) of blood flow and the circumferential strain (CS) of pulsing artery wall motion. These two forces and their interaction are believed to play a role in determining remodeling of the vessel wall and development of arterial disease (atherosclerosis). This study focused on the WSS and CS dynamic behavior in a compliant model of a coronary artery taking into account the curvature of the bending artery and physiological radial wall motion. A three-dimensional finite element model with transient flow and moving boundaries was set up to simulate pulsatile flow with physiological pressure and flow wave forms characteristic of the coronary arteries. The characteristic coronary artery curvature and flow conditions applied to the simulation were: aspect ratio (lambda) = 10, diameter variation (DV) = 6 percent, mean Reynolds number (Re) = 150, and unsteadiness parameter (alpha) = 3. The results show that mean WSS is about 50 percent lower on the inside wall than the outside wall while WSS oscillation is stronger on the inside wall. The stress phase angle (SPA) between CS and WSS, which characterizes the dynamics of the mechanical force pattern applied to the endothelial cell layer, shows that CS and WSS are more out of phase in the coronaries than in any other region of the circulation (-220 deg on the outside wall, -250 deg on the inside wall). This suggests that in addition to WSS, SPA may play a role in localization of coronary atherosclerosis.

Effect of Fluid Flow on Smooth Muscle Cells in a 3-Dimensional Collagen Gel Model

A 3D collagen gel model was developed to simulate interstitial fluid flow and to assess the importance of this flow on the biochemical production rates of vascular smooth muscle cells (SMCs). Rat aortic SMCs were suspended in type I collagen, and the gel was supported by nylon fibers that allowed a 9-cm length of the SMC-gel model to withstand 90 cm H2O differential pressure over a 6-hour period without significant compaction. Up to 1 dyne/cm2 shear stress on the suspended SMCs could be induced by the pressure-driven interstitial flow. The suspended SMCs were globular, had a diameter of ≈10 &mgr;m, and were distributed uniformly throughout the gel. The collagen fibers formed a network that was connected randomly with the surface of SMCs and nylon fibers. The diameter of the collagen fibers was ≈100 nm, and the concentration of collagen was 2.5 mg/mL. Using these parameters, fiber matrix theory predicted a Darcy permeability coefficient (Kp) of 1.22×10−8 cm2, which was close to the measured value of Kp. The production rates of prostaglandin (PG) I2 and PGE2 were used as markers of biochemical responsiveness of SMCs to fluid shear stress. Both PGI2 and PGE2 production rates under 1 dyne/cm2 shear stress were significantly elevated relative to static (no-flow) controls. The production rates, however, were ≈10 times lower than observed when the same cells were plated on collagen-treated glass slides (2D model) and exposed to the same level of shear stress by use of a rotating disk apparatus. The results indicate that interstitial flow can affect SMC biology and that SMCs are more quiescent in 3D cultures than in 2D cultures. The 3D collagen gel model should be useful for future studies of interstitial flow effects on SMC function.

Compliance and diameter mismatch affect the wall shear rate distribution near an end-to-end anastomosis

The development of intimal hyperplasia near the anastomosis of a vascular graft to an artery may be related to changes in the wall shear rate distribution. Mismatches in compliance and diameter at the end-to-end anastomosis of a compliant artery and a rigid graft cause shear rate disturbances that may induce intimal hyperplasia and ultimately graft failure. The goal of this study is to determine how compliance mismatch, diameter mismatch, and impedance phase angle affect the wall shear rate distribution in end-to-end anastomosis models under sinusoidal flow conditions. Wall shear rates are obtained through flow visualization using a photochromic dye. In a model with a well-matched graft diameter (6% undersized), the compliance mismatch causes low mean wall shear rates near the distal anastomosis. Considering diameter mismatch, the wall shear rate distributions in 6% undersized, 16% undersized, and 13% oversized graft models are markedly different at similar phase angles. In the two undersized graft models, the minimum mean shear rate occurs near the distal anastomosis, and this minimum is lower in the model with greater diameter mismatch. The oversized graft model has a minimum mean shear rate near the proximal anastomosis. Thus in all three models, the minimum mean wall shear rate is observed at the site of the divergent geometry. The impedance phase angle, which can be altered by disease states and vasoactive drugs, has a minor effect on the wall shear rate amplitude far from the anastomosis but a more pronounced effect closer to the anastomosis. Mean wall shear rates under sinusoidal flow conditions are significantly lower than under steady flow conditions at the same mean flow rate, but they are fairly insensitive to phase angle changes. In order to avoid the divergent geometry that may cause lower wall shear rates, we recommend that compliance mismatch be minimized whenever possible and that graft diameter be chosen to match the arterial diameter at the relevant physiologic pressure, not at the reduced pressure present when the graft is implanted.