The glycocalyx and its significance in human medicine.

Cells are covered by a surface layer of glycans that is referred to as the 'glycocalyx'. In this review, we focus on the role of the glycocalyx in vascular diseases (atherosclerosis, stroke, hypertension, kidney disease and sepsis) and cancer. The glycocalyx and its principal glycosaminoglycans [heparan sulphate (HS) and hyaluronic acid (HA)] and core proteins (syndecans and glypicans) are degraded in vascular diseases, leading to a breakdown of the vascular permeability barrier, enhanced access of leucocytes to the arterial intima that propagate inflammation and alteration of endothelial mechanotransduction mechanisms that protect against disease. By contrast, the glycocalyx on cancer cells is generally robust, promoting integrin clustering and growth factor signalling, and mechanotransduction of interstitial flow shear stress that is elevated in tumours to upregulate matrix metalloproteinase release which enhances cell motility and metastasis. HS and HA are consistently elevated on cancer cells and are associated with tumour growth and metastasis. Later, we will review the agents that might be used to enhance or protect the glycocalyx to combat vascular disease, as well as a different set of compounds that can degrade the cancer cell glycocalyx to suppress cell growth and metastasis. It is clear that what is beneficial for either vascular disease or cancer will not be so for the other. The overarching conclusions are that (i) the importance of the glycocalyx in human medicine is only beginning to be recognized, and (ii) more detailed studies of glycocalyx involvement in vascular diseases and cancer will lead to novel treatment modalities.

Computational model of collagen turnover in carotid arteries during hypertension.

It is well known that biological tissues adapt their properties because of different mechanical and chemical stimuli. The goal of this work is to study the collagen turnover in the arterial tissue of hypertensive patients through a coupled computational mechano-chemical model. Although it has been widely studied experimentally, computational models dealing with the mechano-chemical approach are not. The present approach can be extended easily to study other aspects of bone remodeling or collagen degradation in heart diseases. The model can be divided into three different stages. First, we study the smooth muscle cell synthesis of different biological substances due to over-stretching during hypertension. Next, we study the mass-transport of these substances along the arterial wall. The last step is to compute the turnover of collagen based on the amount of these substances in the arterial wall which interact with each other to modify the turnover rate of collagen. We simulate this process in a finite element model of a real human carotid artery. The final results show the well-known stiffening of the arterial wall due to the increase in the collagen content.

DBS-relevant electric fields increase hydraulic conductivity of in vitro endothelial monolayers.

Deep brain stimulation (DBS) achieves therapeutic outcome through generation of electric fields (EF) in the vicinity of energized electrodes. Targeted brain regions are highly vascularized, and it remains unknown if DBS electric fields modulate blood-brain barrier (BBB) function, either through electroporation of individual endothelial cells or electro-permeation of barrier tight junctions. In our study, we calculated the intensities of EF generated around energized Medtronic 3387 and 3389 DBS leads by using a finite element model. Then we designed a novel stimulation system to study the effects of such fields with DBS-relevant waveforms and intensities on bovine aortic endothelial cell (BAEC) monolayers, which were used as a basic analog for the blood-brain barrier endothelium. Following 5 min of stimulation, we observed a transient increase in endothelial hydraulic conductivity (Lp) that could be related to the disruption of the tight junctions (TJ) between cells, as suggested by zonula occludens-1 (ZO-1) protein staining. This 'electro-permeation' occurred in the absence of cell death or single cell electroporation, as indicated by propidium iodide staining and cytosolic calcein uptake. Our in vitro results, using uniform fields and BAEC monolayers, thus suggest that electro-permeation of the BBB may occur at electric field intensities below those inducing electroporation and within intensities generated near DBS electrodes. Further studies are necessary to address potential BBB disruption during clinical studies, with safety and efficacy implications.

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.

A computational study of flow in a compliant carotid bifurcation-stress phase angle correlation with shear stress.

The present study presents a three-dimensional, unsteady supercomputer simulation of the coupled fluid-solid interaction problem associated with flow through a compliant model of the bifurcation of the common carotid artery into the internal and external carotid arteries. The fluid wall shear stress (WSS) and solid circumferential stress/strain (CS) are computed and analyzed for the first time using the complex ratio of CS to WSS (CS/WSS). This analysis reveals a large negative phase angle between CS and WSS (stress phase angle--SPA) on the outer wall of the carotid sinus where atherosclerotic plaques are localized. This finding is consistent with other measurements and computations of the SPA in coronary arteries and the aortic bifurcation that show large negative SPA correlating with sites of plaque location and in vitro studies of endothelial cells showing that large negative SPA induces pro-atherogenic gene expression and metabolite release profiles.

Fenestral pore size in the internal elastic lamina affects transmural flow distribution in the artery wall.

Interstitial flow through the subendothelial intima and media of an artery wall was simulated numerically to investigate the water flow distribution through fenestral pores which affects the wall shear stress on smooth muscle cells right beneath the internal elastic lamina (IEL). A two-dimensional analysis using the Brinkman model of porous media flow was performed. It was observed that the hydraulic permeability of the intimal layer should be much greater than that of the media in order to predict a reasonable magnitude for the pressure drop across the subendothelial intima and IEL (about 23 mostly at a 70 mm Hg luminal pressure). When Ki was set equal to the value in the media, this pressure drop was unrealistically high. Furthermore, the higher value of Ki produced a nearly uniform distribution of water flow through a simple array of fenestral pores all having the same diameters (1.2 microm), whereas when Ki was set at the value in the media, the flow distribution through fenestral pores was highly nonuniform and nonphysiologic. A deformable intima model predicted a nonuniform flow distribution at high pressure (180 mm Hg). Damage to the IEL was simulated by introducing a large fenestral pore (up to 17.8 microm) into the array. A dramatic increase in flow through the large pore was observed implying an altered fluid mechanical environment on the smooth muscle cells near the large pore which has implications for intimal hyperplasia and atherosclerosis. The model also predicted that the fluid shear stress on the bottom surface of an endothelial cell is on the order of 10 dyne/cm2, a level which can affect cell function.

Shear stress regulates occludin content and phosphorylation.

Previous studies determined that shear stress imposed on bovine aortic endothelial cell (BAEC) monolayers increased the hydraulic conductivity (L(P)); however, the mechanism by which shear stress increases L(P) remains unknown. This study tested the hypothesis that shear stress regulates paracellular transport by altering the expression and phosphorylation state of the tight junction protein occludin. The effect of shear stress on occludin content was examined by Western blot analysis. Ten dyn/cm(2) significantly reduced occludin content in a time-dependent manner such that after a 3 h exposure to shear, occludin content decreased to 44% of control. Twenty dyn/cm(2) decreased occludin content to 50% of control and increased L(P) by 4.7-fold after 3 h. Occludin expression and L(P) depend on tyrosine kinase activity because erbstatin A (10 microM) attenuated both the shear-induced decrease in occludin content and increase in L(P). Shear stress increased occludin phosphorylation after 5 min, 15 min, and 3 h exposures. The shear-induced increase in occludin phosphorylation was attenuated with dibutyryl (DB) cAMP (1 mM), a reagent previously shown to reverse the shear-induced increase in L(P). We conclude that shear stress rapidly (< or = 5 min) increases occludin phosphorylation and significantly decreases the expression of occludin over 1-4 h. Alterations in the occludin phosphorylation state and occludin total content are potential mechanisms by which shear stress increases L(P).

Laser Doppler velocimetry and flow visualization studies in the regurgitant leakage flow region of three mechanical mitral valves.

Streak line flow visualization and laser Doppler velocimetry (LDV) were conducted in the regurgitant leakage flow region of 3 mechanical heart valve types: CarboMedics, Medtronic Hall, and St. Jude Medical. Streak line flow visualization identified regions of high regurgitant flow, and LDV measurements were focused on those locations. Maximum regurgitant flow velocities after valve closure ranged from 0.7 to 2.6 m/s, and maximum Reynolds shear stress after valve closure ranged from 450 to 3,600 dyne/cm2. These data indicate that leakage flows can generate turbulent jets with elevated Reynolds stresses even in bileaflet valves.

Integrating particle image velocimetry and laser Doppler velocimetry measurements of the regurgitant flow field past mechanical heart valves.

This study investigates the transient regurgitant flow downstream of a prosthetic heart valve using both laser Doppler velocimetry (LDV) and particle image velocimetry (PIV). Until now, LDV has been the more commonly used tool in investigating the flow characteristics associated with mechanical heart valves. The LDV technique allows point-by-point velocity measurements and provides enough information about the temporal variations in the flow. The main drawback of this technique is the time consuming nature of the data acquisition process in order to assess an entire flow field area. The PIV technique, on the other hand, allows measurement of the entire flow field in space in a plane at a given instant. In this study, PIV with spatial resolution of 0 (1 mm) and LDV with a temporal resolution of 0 (1 ms) were used to measure the regurgitant flow proximal to the Björk-Shiley monostrut (BSM) valve in the mitral position. With PIV, the ability to measure 2 velocity components over an entire plane simultaneously provides a very different insight into the flow field compared to a more traditional point-to-point technique like LDV. In this study, a picture of the effects of occluder motion on the fluid flow in the atrial chamber is interpreted using an integration of PIV and LDV measurements. Specifically, fluid velocities in excess of 3.0 m/s were recorded in the pressure-driven jet during valve closure, and a 1.5 m/s sustained regurgitant jet was observed on the minor orifice side. Additionally, the effects of the impact and subsequent rebound of the occluder on the flow also were clearly recorded in spatial and temporal detail by the PIV and LDV measurements, respectively. The PIV results provide a visually intuitive way of interpreting the flow while the LDV data explore the temporal variations and trends in detail. This analysis is an integrated flow description of the effects of valve closure and leakage on the pulsatile regurgitation flow field past a tilting-disc mechanical heart valve (MHV). It further reinforces the hypothesis that the planar flow visualization techniques, when integrated with traditional point-to point techniques, provide significantly more insight into the complex pulsatile flow past MHVs.

Kinetics of placenta growth factor/vascular endothelial growth factor synergy in endothelial hydraulic conductivity and proliferation.

Vascular endothelial growth factor (VEGF) was originally discovered as vascular permeability factor because of its ability to increase microvascular permeability to plasma proteins. Since then, it has been shown to induce proliferation and migration in endothelial cells. Placenta growth factor (PlGF) is a member of the VEGF family of growth factors, but has little or undetectable mitogenic activity on endothelial cells. Intriguingly, however, PlGF was able to potentiate the action of low concentrations of VEGF on endothelial cell growth and macromolecule permeability in vitro. Here we show that PlGF can potentiate the effects of VEGF on the hydraulic conductivity of certain endothelial cells and that the duration of pretreatment with PlGF determines the resulting response. Hydraulic conductivity (Lp) was calculated from the water flux across the monolayer of human umbilical vein endothelial cells (HUVECs) or bovine aortic endothelial cells (BAECs). After 2 h of exposure to VEGF(165), the Lp of BAEC monolayers increased threefold, but the Lp of HUVEC monolayers did not increase. PlGF alone induced a small (63%) increase in Lp in BAECs, but not in HUVECs. BAEC, but not HUVEC, monolayers exposed first to PlGF and then to VEGF exhibited a seven- to eightfold increase in Lp. This enhancement in BAEC Lp could be observed for 4 h after the administration of PlGF. PlGF also potentiated the effect of VEGF on BAEC proliferation. Thus, augmentation of VEGF action by PlGF depends on the duration of PlGF exposure and on the origin of endothelial cells.

A biphasic, anisotropic model of the aortic wall.

A biphasic, anisotropic elastic model of the aortict wall is developed and compared to literature values of experimental measurements of vessel wall radii, thickness, and hvdraulic conductivity as a function of intraluminal pressure. The model gives good predictions using a constant wall modulus for pressures less than 60 mmHg, but requires a strain-dependent modulus for pressures greater than this. In both bovine and rabbit aorta, the tangential modulus is found to be approximately 20 times greater than the radial modulus. These moduli lead to predictions that, when perfused in a cylindrical geometry, the aortic volume and its specific hydraulic coonductivity are relatively independent of perfusion pressure, in agreement with experimental measurements. M, the parameter that relates specific hydraulic conductivy, to tissue dilation, is found to be a positive quantity correcting a previous error in the literature.

Effect of VEGF on retinal microvascular endothelial hydraulic conductivity: the role of NO.

PURPOSE: Vascular endothelial growth factor (VEGF) increases microvascular permeability in vivo and has been hypothesized to play a role in plasma leakage in diabetic retinopathy. Few controlled studies have been conducted to determine the mechanism underlying the effect of VEGF on transport properties (e.g., hydraulic conductivity [Lp]). This study was conducted to determine the effect of VEGF on bovine retinal microvascular endothelial LP and the role of nitric oxide (NO) and the guanylate cyclase/guanosine 3', 5'-cyclic monophosphate/protein kinase G (GC/cGMP/PKG) pathway downstream of NO in mediating the VEGF response. METHODS: Bovine retinal microvascular endothelial cells (BRECs) were grown on porous polycarbonate filters, and water flux across BREC monolayers in response to a pressure differential was measured to determine endothelial LP RESULTS: VEGF (100 ng/ml) increased endothelial LP: within 30 minutes of addition and by 13.8-fold at the end of 3 hours of exposure. VEGF stimulated endothelial monolayers to release NO and incubation of the BRECs with the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA; 100 microM) significantly attenuated the VEGF-induced LP increase. It was observed that incubation of the monolayers with the GC inhibitor LY-83583 (10 microM) did not alter the VEGF-mediated LP: response. Addition of the cGMP analogue 8-br-cGMP (1 mM) did not change the baseline LP over 4 hours. Also, the PKG inhibitor KT5823 (1 microM) did not inhibit the response of BREC LP to VEGF. CONCLUSIONS: These experiments indicate that VEGF elevates hydraulic conductivity in BRECs through a signaling mechanism that involves NO but not the GC/cGMP/PKG pathway.

Observation and quantification of gas bubble formation on a mechanical heart valve.

Clinical studies using transcranial Doppler ultrasonography in patients with mechanical heart valves (MHV) have detected gaseous emboli. The relationship of gaseous emboli release and cavitation on MHV has been a subject of debate in the literature. To study the influence of cavitation and gas content on the formation and growth of stable gas bubbles, a mock circulatory loop, which employed a Medtronic-Hall pyrolytic carbon disk valve in the mitral position, was used. A high-speed video camera allowed observation of cavitation and gas bubble release on the inflow valve surfaces as a function of cavitation intensity and carbon dioxide (CO2) concentration, while an ultrasonic monitoring system scanned the aortic outflow tract to quantify gas bubble production by calculating the gray scale levels of the images. In the absence of cavitation, no stable gas bubbles were formed. When gas bubbles were formed, they were first seen a few milliseconds after and in the vicinity of cavitation collapse. The volume of the gas bubbles detected in the aortic track increased with both increased CO2 and increased cavitation intensity. No correlation was observed between O2 concentration and bubble volume. We conclude that cavitation is an essential precursor to stable gas bubble formation, and CO2, the most soluble blood gas, is the major component of stable gas bubbles.

The osmotic swelling characteristics of cardiac valve prostheses.

Several types of mechanical cardiac prostheses have been constructed with Delrin occluders, a material that is subject to osmotic swelling. The leaftets are designed to expand to specific tolerances when immersed in blood. The synthetic blood analogs commonly used in vitro contain hydrophilic compounds that can alter the osmotic expansion of the Delrin occluders. A static leak test chamber was employed to illustrate the effects of various test fluids on the sustained regurgitation phase of Delrin valves.

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 H(2)O differential pressure over a 6-hour period without significant compaction. Up to 1 dyne/cm(2) shear stress on the suspended SMCs could be induced by the pressure-driven interstitial flow. The suspended SMCs were globular, had a diameter of approximately 10 microm, 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 approximately 100 nm, and the concentration of collagen was 2.5 mg/mL. Using these parameters, fiber matrix theory predicted a Darcy permeability coefficient (K:(p)) of 1.22x10(-)(8) cm(2), which was close to the measured value of K:(p). The production rates of prostaglandin (PG) I(2) and PGE(2) were used as markers of biochemical responsiveness of SMCs to fluid shear stress. Both PGI(2) and PGE(2) production rates under 1 dyne/cm(2) shear stress were significantly elevated relative to static (no-flow) controls. The production rates, however, were approximately 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.

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/cm(2)), oscillatory shear (10 +/- 10 dyn/cm(2), rigid tube), and oscillatory shear (10 +/- 10 dyn/cm(2)) 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.

Fluid dynamics of a pediatric ventricular assist device.

The number of pediatric patients requiring some form of mechanical circulatory assistance is growing throughout the world because of new surgical procedures and the success of pediatric cardiac transplantation. However, the salvage rate for those patients requiring circulatory support may be as low as 25%. Despite the fact that Penn State's 70 cc pneumatic ventricular assist device has been used with a success rate of over 90% in more than 250 patients worldwide, efforts to scale down the pump have encountered difficulties. Animal experiments with a 15 cc version were unsuccessful, with explanted pumps showing extensive thrombus deposition within the pumping chamber. The materials used to fabricate the smaller pump as well as the basic operating principles are identical to the successful adult-sized version. It is therefore believed that reducing the size of the pump altered the internal flow field, and that fluid dynamic factors were responsible for the high degree of thrombus observed with the implanted devices. A dimensional analysis was conducted that revealed significant differences in both Reynolds (Re) and Strouhal (St) numbers between the successful and unsuccessful pumps. Two component laser Doppler velocimetry was then used to characterize the internal flow field quantitatively. Comparison with data from the 70 cc pump showed a reduction in wall shear stress and turbulence levels in the 15 cc pump that would yield an environment conducive to clot formation.

Flow visualization in mechanical heart valves: occluder rebound and cavitation potential.

High density particle image velocimetry, with spatial resolution of O(1 mm), was used to measure the effect of occluder rebound on the flow field near a Bjork-Shiley Monostrut tilting-disk mitral valve. The ability to measure two velocity components over an entire plane simultaneously provides a very different insight into the flow compared to the more traditional point to point techniques (like Laser Doppler Velocimetry) that were utilized in previous investigations of the regurgitant flow. A picture of the effects of occluder rebound on the fluid flow in the atrial chamber is presented. Specifically, fluid velocities in excess of 1.5 m/s traveling away from the atrial side were detected 3 mm away from the valve seat in the local low pressure region created by the occluder rebound on the major orifice side where cavitation has been observed. This analysis is the first spatially detailed flow description of the effects of occluder rebound on the flow field past a tilting-disk mechanical heart valve and further reinforces the hypothesis that the rebound effect plays a significant role in the formation of cavitation, which has been implicated in the hemolysis and wear associated with tilting-disk valves in vivo.

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.

Interstitial flow through the internal elastic lamina affects shear stress on arterial smooth muscle cells.

Interstitial flow through the tunica media of an artery wall in the presence of the internal elastic lamina (IEL), which separates it from the subendothelial intima, has been studied numerically. A two-dimensional analysis applying the Brinkman model as the governing equation for the porous media flow field was performed. In the numerical simulation, the IEL was modeled as an impermeable barrier to water flux, except for the fenestral pores, which were uniformly distributed over the IEL. The tunica media was modeled as a heterogeneous medium composed of a periodic array of cylindrical smooth muscle cells (SMCs) embedded in a fiber matrix simulating the interstitial proteoglycan and collagen fibers. A series of calculations was conducted by varying the physical parameters describing the problem: the area fraction of the fenestral pore (0. 001-0.036), the diameter of the fenestral pore (0.4-4.0 microm), and the distance between the IEL and the nearest SMC (0.2-0.8 microm). The results indicate that the value of the average shear stress around the circumference of the SMC in the immediate vicinity of the fenestral pore could be as much as 100 times greater than that around an SMC in the fully developed interstitial flow region away from the IEL. These high shear stresses can affect SMC physiological function.