Microelastic mapping of living endothelial cells exposed to shear stress in relation to three-dimensional distribution of actin filaments

The surface topography and local elastic moduli of endothelial cells exposed to shear stress were measured using atomic force microscopy. Bovine aortic endothelial cells were exposed to shear stress of 2Pa for 6, 12 or 24h. In addition, a confocal laser-scanning microscope used in conjunction with the atomic force microscope was used to observe the actin filament structure of these endothelial cells to elucidate the relationship between mechanical properties and cytoskeletal structure. The elastic modulus, calculated using the Hertz model, was measured at 50x50 points at 1mum intervals within 40min. For endothelial cells sheared for 6h and 12h, the elastic modulus at the upstream region was found to be higher than that at the downstream region. For endothelial cells sheared for 24h, the elastic modulus at both the upstream and downstream regions increased. Fluorescent images showed thick, elongated actin filaments oriented in the direction of flow at the ventral surface of the cells. In the middle plane of the cells, actin filaments developed around the nucleus, while in the upper plane, short, thick actin filaments were observed but thick stress fibers were not present. The high elastic modulus came from the stress fibers. These results indicate that the higher elastic modulus observed in the upstream and downstream regions of sheared endothelial cells is mainly due to the development of stress fibers at the ventral surface and middle plane of the cell.     

Flow and High Affinity Binding Affect the Elastic Modulus of the Nucleus,Cell Body and the Stress Fibers of Endothelial Cells

Cell mechanical properties are important in the adhesion of endothelial cells to synthetic vascular grafts exposed to shear flow. We hypothesized that the local apparent elastic modulus of the nucleus and the cell body would increase to a greater extent for cells adherent via the dual ligand (integrin-fibronectin/avidin-biotin) and exposed to flow, than for cells treated with either ligand alone. High affinity avidin-biotin bonds and in vitro flow exposure were used to improve adhesion to grafts thereby altering the mechanical properties of endothelial cells. Introduction of the dual ligand chemistry at the cell-substrate interface increased the apparent elastic modulus of the cells as compared to cells adherent with the fibronectin-integrin bonds only. Cells cultured on the dual ligand surface exhibited higher elastic moduli of the nucleus and cell body relative to cells cultured on fibronectin alone. Exposure of cells to flow increased the apparent elastic modulus of the cell body, nucleus, and stress fibers of cells adherent to the fibronectin surface. A similar effect was seen for cells adherent to the dual ligand surface, although there was little effect on the elastic modulus of the nucleus. While the dual ligand surface produces an increase in adhesion strength, focal contact area and elastic modulus, the change in elastic modulus after exposure to flow is due only to an increase in stress fibers and not an increase in contact area.     

Softening of the actin cytoskeleton by inhibition of myosin II

We have investigated the mechanical properties of fibroblast cells after adding the myosin inhibitor blebbistatin and the Rho-kinase inhibitor Y-27632 by atomic force microscopy (AFM). We have observed a decrease in the elastic modulus from a value of around 20 kPa down to a value around 8 kPa on a time scale of around 30–60 min when applying the myosin inhibitor blebbistatin, whereas the Y-27632 did not show any prominent mechanical effects. From topographic images, we can conclude that, after adding blebbistatin, actin filaments are not visible any more, whereas Y-27632 did not show any prominent effects in cell morphology. This study shows that tension generated by myosin contributes to the cellular stiffness and thus can be observed by measuring the elastic modulus of cells.     

A simple microindentation technique for mapping the microscale compliance of soft hydrated materials and tissues

Several recent studies have shown that cells respond to the elastic modulus and elasticity gradients on soft substrates. However, traditional macroscale methods for measuring elastic modulus cannot resolve elastic gradients or differences between the macroscale and microscale elastic modulus of layered tissues. Here, we present a technique for measurement of the microscale elastic modulus of soft, hydrated gels and tissues. This technique requires less equipment than equivalent atomic force microscopy (AFM) and can easily measure larger samples with high adhesiveness. We validate this technique by measuring the microscale modulus of a hydrogel with elasticity that does not depend on measurement scale. We show that the elastic modulus measured using microindentation correlates with measurements using AFM and the macroscale tensile modulus. We verified the ability of this technique to characterize a hydrogel with an elastic gradient of 2.2 kPa/mm across 19 mm and to measure the microscale elastic modulus of the endothelial side of human greater saphenous vein, which is an order of magnitude less than the whole vein macroscale modulus. This simple, inexpensive system allows the measurement of the spatial organization of microscale elastic properties of fully hydrated, soft gels and tissues as a routine laboratory technique.     

Characterization of cellular elastic modulus using structure based double layer model

The mechanical characterization of cells is important for understanding cellular behavior and physiological functions. We used atomic force microscopy (AFM) to obtain a force-displacement curve and estimate the elastic modulus of hepatocellular carcinoma cells (HEP-G2) utilizing both linear Hertz-Sneddon (HS) and non-linear elastic models. In order to overcome the limitations of HS model, which assumes a linear homogeneous cell body, a cell is modeled as a double-layered body with an outer cytoplasmic layer made mostly of interconnected fibers of cytoskeleton proteins and a nucleus. By disrupting all cytoskeletal protein networks, we estimate the elastic modulus of the core nucleus using FEM for a single ellipsoid. Based on the nucleic modulus and cellular dimensions found by 3D confocal imaging, we develop a novel double-layered cellular (DLC) finite element model. The DLC model provides a more reliable estimate of the elastic modulus of the cell than conventionally used HS model and correlates closely with experimental results.     

大腿假肢支撑期有限元分析

目的分析大腿截肢患者的硅胶套材料属性,对支撑期残肢与接受腔之间接触面的力学分布的影响,为大腿假肢适配方案中硅胶套的选取提供参考。方法利用计算机断层扫描技术获取大腿截肢患者残端与接受腔的断层图像,通过影像学信息和工程学方法,分别获取接受腔、硅胶套、残肢、骨骼等结构的三维模型;根据角度变化调整模型,获得初始接触期、负荷反应期、站立中期、站立末期、摆动前期5个时相的组装模型;根据三维动作捕捉系统Motion和Kistler三维测力平台测得的地面反作用力及髋关节角度变化的结果,对5个时相下大腿假肢模型分别进行有限元非线性接触分析;在此基础上,模拟分析了硅胶套不同弹性模量的变化对残肢表面等效应力以及剪切应力分布的影响。结果穿戴不同弹性模量的硅胶套时,残肢所受最大等效应力以及最大剪切应力在初始接触期、负荷反应期、站立中期、站立末期时相时出现在残肢内侧和接受腔口型边缘对应的残肢位置,在摆动前期时相时则出现在残肢内侧、接受腔口型边缘对应的残肢位置和坐骨周围等位置。当硅胶套弹性模量在0.98~2.70 MPa范围内变化时,在摆动前期残肢所受等效应力变化范围为13.85~23.55 k Pa,最大剪切应力变化范围为7.82~13.46 k Pa,而其他时相基本一致。结论硅胶套的力学特性影响大腿假肢残肢与接受腔之间接触面的受力分布,摆动前期残肢所受最大等效应力与最大剪切应力随硅胶套弹性模量变化大,在实际适配过程中需注意。     

温度对单个人红细胞膜力学特性的即时作用

目的：探讨温度对单个红细胞膜力学性质的即时影响。 方法： 利用静态显微图像分析技术和动态显微图像分析技术，在无损、实时、在位的情况下，观察和测量不同温度下单个人红细胞的形态、大小、膜弯曲弹性模量和剪切弹性模量的变化。 结果： 红细胞的接触面积和直径随温度的升高而减小，胞膜的弯曲弹性模量和剪切弹性模量都在生理温度37 ℃时最小，而温度低于或高于37 ℃时红细胞膜的弯曲弹性模量和剪切弹性模量都增大。 结论： 红细胞在生理温度37 ℃时有最好的形态和力学变形性，便于发挥其生理功能。     

流体切应力对内皮细胞粘附分子表达的影响

在动脉粥样硬化(Atherosclerosis,As)的发生发展中各种白细胞,包括单核细胞对内皮细胞的粘附可能起着较为重要的作用。在体内,血流切应力对内皮细胞的形态和功能有重要影响。为了阐明流体切应力对内皮细胞表面粘附分子表达的影响,本文研究了流体切应力(2.23～6.08dyne/cm     

Superparamagnetic iron oxide nanoparticles change endothelial cell morphology and mechanics via reactive oxygen species formation

Superparamagnetic iron oxide nanoparticles are used in various medical applications including magnetic resonance imaging, magnetic hyperthermia, and targeted drug and gene delivery. When used in vivo, these nanoparticles interact with endothelial cells lining all blood vessels, therefore it is crucial to understand endothelial cell functional changes and toxicity upon nanoparticle exposure. We incubated porcine aortic endothelial cells with varying concentrations of bare iron oxide nanoparticles (20-40 nm), and measured cellular reactive oxygen species (ROS) formation, morphology and cytoskeletal organization, death, and elastic modulus. Intracellular ROS increased more than 800% after 3 h of nanoparticle exposure (0.5 mg mL(-1)). Endothelial cells elongated to more than twice their initial length by 12 h, and actin stress fibers formed within the cells. This change in the actin cytoskeleton increased cell elastic modulus by 50%. When ROS formation was blocked using scavengers, initial cell morphology and the actin cytoskeleton remained intact, and cell viability increased. These studies suggest that iron oxide nanoparticles induce ROS formation, which disrupts the actin cytoskeleton and alters endothelial cell morphology and mechanics. If ROS formation is decreased using ROS inhibitors, either as a component of the nanoparticle coating or by systemic administration, higher nanoparticle concentrations might be used with greater efficacy and diminished side effects.     

Tetronic®-based composite hydrogel scaffolds seeded with rat bladder smooth muscle cells for urinary bladder tissue engineering applications

Natural hydrogels such as collagen offer desirable properties for tissue engineering, including cell adhesion sites, but their low mechanical strength is not suitable for bladder tissue regeneration. In contrast, synthetic hydrogels such as poly (ethylene glycol) allow tuning of mechanical properties, but do not elicit protein adsorption or cell adhesion. For this reason, we explored the use of composite hydrogel blends composed of Tetronic (BASF) 1107-acrylate (T1107A) in combination with extracellular matrix moieties collagen and hyaluronic acid seeded with bladder smooth muscle cells (BSMC). This composite hydrogel supported BSMC growth and distribution throughout the construct. When compared to the control (acellular) hydrogels, mechanical properties (peak stress, peak strain, and elastic modulus) of the cellular hydrogels were significantly greater. When compared to the 7-day time point after BSMC seeding, results of mechanical testing at the 14-day time point indicated a significant increase in both ultimate tensile stress (4.1–11.6 kPa) and elastic modulus (11.8–42.7 kPa) in cellular hydrogels. The time-dependent improvement in stiffness and strength of the cellular constructs can be attributed to the continuous collagen deposition and reconstruction by BSMC seeded in the matrix. The composite hydrogel provided a biocompatible scaffold for BSMC to thrive and strengthen the matrix; further, this trend could lead to strengthening the construct to match the mechanical properties of the bladder.     

Three-dimensional analysis of shear wave propagation observed by in vivo magnetic resonance elastography of the brain

Dynamic magnetic resonance elastography (MRE) is a non-invasive method for the quantitative determination of the mechanical properties of soft tissues in vivo. In MRE, shear waves are generated in the tissue and visualized using phase-sensitive MR imaging methods. The resulting two-dimensional (2-D) wave images can reveal in-plane elastic properties when possible geometrical biases of the wave patterns are taken into account. In this study, 3-D MRE experiments of in vivo human brain are analyzed to gain knowledge about the direction of wave propagation and to deduce in-plane elastic properties. The direction of wave propagation was determined using a new algorithm which identifies minimal wave velocities along rays from the surface into the brain. It was possible to quantify biases of the elastic parameters due to projections onto coronal, sagittal and transversal image planes in 2-D MRE. It was found that the in-plane shear modulus is increasingly overestimated when the image slice is displaced from narrow slabs of 2-5cm through the center of the brain. The mean shear modulus of the brain was deduced from 4-D wave data with about 3.5kPa. Using the proposed slice positions in 2-D MRE, this shear modulus can be reproduced with an acceptable error within a fraction of the full 3-D examination time.     

Monocyte adhesion and changes in endothelial cell number, morphology, and F-actin distribution elicited by low shear stress in vivo.

Left common carotid arteries of New Zealand white rabbits were ligated rostral to origin of the thyroid artery to reduce flow in the carotid upstream of this branch, and the vessels were examined 5 days later. Estimates of mean shear stress in the upstream carotid artery indicated a decrease of 73% (from 12.1 +/- 1.6 dynes/cm2 to 3.26 +/- 0.58 dynes/cm2). The contralateral common carotid artery carried collateral flow and experienced a 170% increase in shear stress (from 11.3 +/- 1.6 dynes/cm2 to 30.5 +/- 4.6 dynes/cm2). There was an adaptive reduction in the diameter in the left common carotid artery (low shear) from 2.07 +/- 0.06 mm to 1.75 +/- 0.12 mm, but the diameter of the right carotid was unchanged. Fluorescence microscopy and scanning electron microscopy of endothelium exposed to low shear revealed attachment of leukocytes (5.02 +/- 1.59 cells/mm2, mean +/- SE) that were identified as monocytes using the monoclonal antibody HAM 56. Laser confocal microscopy demonstrated that they were migrating across the endothelial cell monolayer. Fluorescence microscopy and scanning electron microscopy of left common carotid artery (low shear) also revealed cell morphology suggestive of endothelial cell desquamation. Endothelial cell loss was confirmed by morphometric determination of cell number (1.29 +/- 0.13 x 10(4) cells/mm length in experimental animals versus 1.71 +/- 0.08 x 10(4) cells/mm length in sham-operated animals). This endothelial cell loss may be an adaptation to a narrowing of carotid arteries exposed to low shear, which reduces luminal surface area of the vessel. Staining of F-actin with rhodamine phalloidin showed that endothelial cells exposed to low shear were less elongated and had fewer stress fibers than normal cells. By contrast, increasing shear stress by two- to threefold caused an increase in the number of stress fibers and a reduction in peripheral actin staining. Distal carotid ligation provided a consistent and well-defined in vivo technique for manipulating shear stresses imposed on a large population of endothelial cells.     

Intermittent short-duration exposure to low wall shear stress induces intimal thickening in arteries exposed to chronic high shear stress

We sought to determine whether intermittent short-duration exposure to low wall shear stress could induce intimal thickening in arteries chronically exposed to high shear stress. An arteriovenous fistula (AVF) was created between the left common carotid artery and the corresponding external jugular vein in 20 Japanese white male rabbits. After 4 weeks, blood flow was increased 10-fold to 182 +/- 39 ml/min and shear stress was increased to 33.4 +/- 13 dyn/cm(2). The AVF was then occluded for 1 h by finger compression with an 85% reduction in carotid artery blood flow (27 +/- 7 ml/min) and a reduction in wall shear stress to 4.9 +/- 1.7 dyn/cm(2) (P < 0.0001). Release of finger compression restored flow to the AVF and high shear stress to the carotid artery. This procedure was repeated at weekly intervals with a cumulative total of 4 h of low shear stress exposure. Arteries exposed to intermittent low shear stress developed a layer of intimal thickening which consisted of 3-4 layers of smooth muscle cells lined with thin elastic fibers and medial hyperplasia. Control arteries exposed to 8 weeks of continuous high shear had no intimal thickening. Transient exposure to low shear stress upregulated TGF-beta1, MMP-2, -14, and TIMP-2 gene expression while MMP-9 expression was downregulated. We conclude that repeated, intermittent short-duration exposure to low shear stress in the setting of high flow and high shear stress can induce arterial intimal thickening. Short-duration alterations in hemodynamic forces can induce rapid vascular cell message expression, which may effect arterial remodeling. This experiment suggests that a threshold value of 5 dyn/cm(2) may be needed in order to initiate and sustain the intimal thickening response.     

Development of a mechanical testing assay for fibrotic murine liver

In this article, a novel protocol for mechanical testing, combined with finite element modeling, is presented that allows the determination of the elastic modulus of normal and fibrotic murine livers and is compared to an independent mechanical testing method. The novel protocol employs suspending a portion of murine liver tissue in a cylindrical polyacrylamide gel, imaging with a microCT, conducting mechanical testing, and concluding with a mechanical property determination via a finite element method analysis. More specifically, the finite element model is built from the computerized tomography (CT) images, and boundary conditions are imposed in order to simulate the mechanical testing conditions. The resulting model surface stress is compared to that obtained during mechanical testing, which subsequently allows for direct evaluation of the liver modulus. The second comparison method involves a mechanical indentation test performed on a remaining liver lobe for comparison. In addition, this lobe is used for histological analysis to determine relationships between elasticity measurements and tissue health. This complete system was used to study 14 fibrotic livers displaying advanced fibrosis (injections with irritant), three control livers (injections without irritant), and three normal livers (no injections). The moduli evaluations for nondiseased livers were estimated as 0.62 +/- 0.09 kPa and 0.59 +/- 0.1 kPa for indenter and model-gel-tissue (MGT) assay tests, respectively. Moduli estimates for diseased liver ranged from 0.6-1.64 kPa and 0.96-1.88 kPa for indenter and MGT assay tests, respectively. The MGT modulus, though not equivalent to the modulus determined by indentation, demonstrates a high correlation, thus indicating a relationship between the two testing methods. The results also showed a clear difference between nondiseased and diseased livers. The developed MGT assay system is quite compact and could easily be utilized for controlled evaluation of soft-tissue moduli as shown here. In addition, future work will add the correlative method of elastography such that direct controlled validation of measurement on tissue can be determined.     

Photocrosslinked tyramine-substituted hyaluronate hydrogels with tunable mechanical properties improve immediate tissue-hydrogel interfacial strength in articular cartilage

Articular cartilage lacks the ability to self-repair and a permanent solution for cartilage repair remains elusive. Hydrogel implantation is a promising technique for cartilage repair; however for the technique to be successful hydrogels must interface with the surrounding tissue. The objective of this study was to investigate the tunability of mechanical properties in a hydrogel system using a phenol-substituted polymer, tyramine-substituted hyaluronate (TA-HA), and to determine if the hydrogels could form an interface with cartilage. We hypothesized that tyramine moieties on hyaluronate could crosslink to aromatic amino acids in the cartilage extracellular matrix. Ultraviolet (UV) light and a riboflavin photosensitizer were used to create a hydrogel by tyramine self-crosslinking. The gel mechanical properties were tuned by varying riboflavin concentration, TA-HA concentration, and UV exposure time. Hydrogels formed with a minimum of 2.5 min of UV exposure. The compressive modulus varied from 5 to 16 kPa. Fluorescence spectroscopy analysis found differences in dityramine content. Cyanine-3 labelled tyramide reactivity at the surface of cartilage was dependent on the presence of riboflavin and UV exposure time. Hydrogels fabricated within articular cartilage defects had increasing peak interfacial shear stress at the cartilage-hydrogel interface with increasing UV exposure time, reaching a maximum shear stress 3.5× greater than a press-fit control. Our results found that phenol-substituted polymer/riboflavin systems can be used to fabricate hydrogels with tunable mechanical properties and can interface with the surface tissue, such as articular cartilage.     

Mechanical properties of collagen gels derived from rats of different ages

In a previous study, we found that different collagen gels produced using collagen fibrils extracted from 1-, 4- and 8-month-old rat tails essentially influenced the morphogenesis of epithelial cells. More importantly, the youngest collagen gel induces the highest level of cell apoptosis. The objective of this study was to investigate mechanical properties of various collagen gels correlated to the rat ages. A rheometer and dynamic mechanical analyzer were used to measure shear and compressive properties of hydrated collagen gels. Experimental results obtained from both testing modes showed that older age-related collagen gels possessed a larger elastic modulus, possibly due to the enhanced cross-linking degree. The moduli obtained in shear mode were 1.4-2.7-times greater than those in compression. The results of shear test and compressive test consistently indicated the age of rats did have a statistically significant effect on mechanical properties of hydrated collagen gels.     

Subcutaneous tissue mechanical behavior is linear and viscoelastic under uniaxial tension

Subcutaneous tissue is part of a bodywide network of "loose" connective tissue including interstitial connective tissues separating muscles and surrounding all nerves and blood vessels. Despite its ubiquitous presence in the body and its potential importance in a variety of therapies utilizing mechanical stretch, as well as normal movement and exercise, very little is known about loose connective tissue's biomechanical behavior. This study aimed to determine elastic and viscoelastic mechanical properties of ex-vivo rat subcutaneous tissue in uniaxial tension with incremental stress relaxation experiments. The elastic response of the tissue was linear, with instantaneous and equilibrium tensile moduli of 4.77 kPa and 2.75 kPa, respectively. Using a 5 parameter Maxwell solid model, material parameters micro(1) = 0.95 +/- 0.24 Ns/m and micro(2) = 8.49 +/- 2.42 Ns/m defined coefficients of viscosity related to time constants tau(1M) = 3.83 +/- 0.15 sec and tau(2M) = 30.15 +/- 3.16 sec, respectively. Using a continuous relaxation function, parameters C = 0.25 +/- 0.12, tau(1C) = 1.86 +/- 0.34 sec, and tau(2C) = 110.40 +/- 25.59 sec defined the magnitude and frequency limits of the relaxation spectrum. This study provides baseline information for the stress-strain behaviors of subcutaneous connective tissue. Our results underscore the differences in mechanical behaviors between loose and high-load bearing connective tissues and suggest that loose connective tissues may function to transmit mechanical signals to and from the abundant fibroblasts, immune, vascular, and neural cells present within these tissues.     

Mechanical properties of collagen gels derived from rats of different ages

In a previous study, we found that different collagen gels produced using collagen fibrils extracted from 1-, 4- and 8-month-old rat tails essentially influenced the morphogenesis of epithelial cells. More importantly, the youngest collagen gel induces the highest level of cell apoptosis. The objective of this study was to investigate mechanical properties of various collagen gels correlated to the rat ages. A rheometer and dynamic mechanical analyzer were used to measure shear and compressive properties of hydrated collagen gels. Experimental results obtained from both testing modes showed that older age-related collagen gels possessed a larger elastic modulus, possibly due to the enhanced cross-linking degree. The moduli obtained in shear mode were 1.4–2.7-times greater than those in compression. The results of shear test and compressive test consistently indicated the age of rats did have a statistically significant effect on mechanical properties of hydrated collagen gels.     

Zonal Uniformity in Mechanical Properties of the Chondrocyte Pericellular Matrix: Micropipette Aspiration of Canine Chondrons Isolated by Cartilage Homogenization

The pericellular matrix (PCM) is a region of tissue that surrounds chondrocytes in articular cartilage and together with the enclosed cells is termed the chondron. Previous studies suggest that the mechanical properties of the PCM, relative to those of the chondrocyte and the extracellular matrix (ECM), may significantly influence the stress–strain, physicochemical, and fluid-flow environments of the cell. The aim of this study was to measure the biomechanical properties of the PCM of mechanically isolated chondrons and to test the hypothesis that the Young's modulus of the PCM varies with zone of origin in articular cartilage (surface vs. middle/deep). Chondrons were extracted from articular cartilage of the canine knee using mechanical homogenization, and the elastic properties of the PCM were determined using micropipette aspiration in combination with theoretical models of the chondron as an elastic incompressible half-space, an elastic compressible bilayer, or an elastic compressible shell. The Young's modulus of the PCM was significantly higher than that reported for isolated chondrocytes but over an order of magnitude lower than that of the cartilage ECM. No significant differences were observed in the Young's modulus of the PCM between surface zone (24.0 ± 8.9 kPa) and middle/deep zone cartilage (23.2 ± 7.1 kPa). In combination with previous theoretical biomechanical models of the chondron, these findings suggest that the PCM significantly influences the mechanical environment of the chondrocyte in articular cartilage and therefore may play a role in modulating cellular responses to micromechanical factors.     

Elevated glucose attenuates agonist- and flow-stimulated endothelial nitric oxide synthase activity in microvascular retinal endothelial cells.

Impaired vasoactive release of opposing vasodilator and vasoconstrictor mediators due to endothelial dysfunction is integral to the pathogenesis of diabetic retinopathy. The aim of this study was to determine the effect of hyperglycemia on the expression of endothelial nitric oxide synthase (eNOS) and the release of nitric oxide (NO) in bovine microvascular retinal endothelial cells (BRECs) under both static (basal and acetylcholine stimulated) and flow (laminar shear stress [10 dynes/cm2 and pulsatile flow 0.3 to 23 dynes/cm2) conditions using a laminar shear apparatus and an in vitro perfused transcapillary culture system. The activity and expression of eNOS, measured by nitrate levels and immunoblot, respectively, were determined following exposure of BRECs to varying concentrations of glucose and mannitol (0 to 25 mM). Under static conditions the expression of eNOS decreased significantly following exposure to increasing concentrations of glucose when compared to osmotic mannitol controls and was accompanied by a significant dose-dependent decrease in nitrate levels in conditioned medium. The acetylcholine stimulated increase in NO release (2.0 +/- 0.3-fold) was significantly reduced by 55% +/- 5% and 65% +/- 4.5% following exposure to 16 and 25 mM glucose, respectively, when compared to osmotic controls. In parallel studies, glucose significantly inhibited both laminar shear stress and pulsatile flow-induced activity when compared to mannitol. We conclude that hyperglycemia impairs agonist- and flow-dependent release of NO in retinal microvascular endothelial cells and may thus contribute to the vascular endothelial dysfunction and impaired autoregulation of diabetic retinopathy.