76 research outputs found

    Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function

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    The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17–3.02μm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078±0.016μm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same vessels in a timedependent manner, with changes in all three true vessel wall permeability coefficients (hydraulic conductivity, reflection coefficient, and diffusive solute permeability). These novel technologies expand the range of techniques that permit direct studies of the structure of the endothelial glycocalyx and dependent microvascular functions in vivo, and demonstrate that sialic acid residues within the endothelial glycocalyx are critical regulators of microvascular permeability to both water and albumin

    Shear stress-induced angiogenesis in mouse muscle is independent of the vasodilator mechanism and quickly reversible.

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    Aim: Is modulation of skeletal muscle capillary supply by altering blood flow due to a presumptive shear stress response per se, or dependent on the vasodilator mechanism? Methods: The response to four different vasodilators, and cotreatment with blockers of NO and prostaglandin synthesis, was compared. Femoral artery blood flow was correlated with capillary-to-fibre ratio (C:F) and protein levels of putative angiogenic compounds. Results: All vasodilators induced a similar increase in blood flow after 14 days, with a similar effect on C:F (1.62 ± 0.05, 1.60 ± 0.01, 1.57 ± 0.06, 1.57 ± 0.07, respectively, all P < 0.05 vs. control 1.20 ± 0.01). Concomitant inhibitors revealed differential effects on blood flow and angiogenesis, demonstrating that a similar response may have different signalling origins. The time course of this response with the most commonly used vasodilator, prazosin, showed that blood flow increased from 0.40 mL min−1 to 0.61 mL min−1 by 28 days (P < 0.05), dropped within 1 week after the cessation of treatment (0.54 mL min−1; P < 0.05) and returned to control levels by 6 weeks. In parallel with FBF, capillary rarefaction began within 1 week (P < 0.05), giving C:F values similar to control by 2 weeks. Of the dominant signalling pathways, prazosin decreased muscle VEGF, but increased its cognate receptor Flk-1 (both P < 0.01); levels of eNOS varied with blood flow (P < 0.05), and Ang-1 initially increased, while its receptor Tie-2 was unchanged, with only modest changes in the antiangiogenic factor TSP-1. Conclusion: Hyperaemia-induced angiogenesis, likely in response to elevated shear stress, is independent of the vasodilator involved, with a rapid induction and quick regression following the stimulus withdrawal

    Heparan Sulfate Regrowth Profiles Under Laminar Shear Flow Following Enzymatic Degradation

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    The local hemodynamic shear stress waveforms present in an artery dictate the endothelial cell phenotype. The observed decrease of the apical glycocalyx layer on the endothelium in atheroprone regions of the circulation suggests that the glycocalyx may have a central role in determining atherosclerotic plaque formation. However, the kinetics for the cells’ ability to adapt its glycocalyx to the environment have not been quantitatively resolved. Here we report that the heparan sulfate component of the glycocalyx of HUVECs increases by 1.4-fold following the onset of high shear stress, compared to static cultured cells, with a time constant of 19 h. Cell morphology experiments show that 12 h are required for the cells to elongate, but only after 36 h have the cells reached maximal alignment to the flow vector. Our findings demonstrate that following enzymatic degradation, heparan sulfate is restored to the cell surface within 12 h under flow whereas the time required is 20 h under static conditions. We also propose a model describing the contribution of endocytosis and exocytosis to apical heparan sulfate expression. The change in HS regrowth kinetics from static to high-shear EC phenotype implies a differential in the rate of endocytic and exocytic membrane turnover.National Heart, Lung, and Blood Institute (Grant HL090856-01)Singapore-MIT Allianc

    Endothelial Surface Layer Degradation by Chronic Hyaluronidase Infusion Induces Proteinuria in Apolipoprotein E-Deficient Mice

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    Functional studies show that disruption of endothelial surface layer (ESL) is accompanied by enhanced sensitivity of the vasculature towards atherogenic stimuli. However, relevance of ESL disruption as causal mechanism for vascular dysfunction remains to be demonstrated. We examined if loss of ESL through enzymatic degradation would affect vascular barrier properties in an atherogenic model. Eight week old male apolipoprotein E deficient mice on Western-type diet for 10 weeks received continuous active or heat-inactivated hyaluronidase (10 U/hr, i.v.) through an osmotic minipump during 4 weeks. Blood chemistry and anatomic changes in both macrovasculature and kidneys were examined. Infusion with active hyaluronidase resulted in decreased ESL (0.32±0.22 mL) and plasma volume (1.03±0.18 mL) compared to inactivated hyaluronidase (0.52±0.29 mL and 1.28±0.08 mL, p<0.05 respectively).Active hyaluronidase increased proteinuria compared to inactive hyaluronidase (0.27±0.02 vs. 0.15±0.01 µg/µg protein/creatinin, p<0.05) without changes in glomerular morphology or development of tubulo-interstitial inflammation. Atherosclerotic lesions in the aortic branches showed increased matrix production (collagen, 32±5 vs. 18±3%; glycosaminoglycans, 11±5 vs. 0.1±0.01%, active vs. inactive hyaluronidase, p<0.05). ESL degradation in apoE deficient mice contributes to reduced increased urinary protein excretion without significant changes in renal morphology. Second, the induction of compositional changes in atherogenic plaques by hyaluronidase point towards increased plaque vulnerability. These findings support further efforts to evaluate whether ESL restoration is a valuable target to prevent (micro) vascular disease progressio

    Mechanical Strain Stabilizes Reconstituted Collagen Fibrils against Enzymatic Degradation by Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8)

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    Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain.The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded.In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen

    The endothelial glycocalyx mediates shear-induced changes in hydraulic conductivity

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    Recent in vitro and in vivo studies have reported fluid shear stress-induced increases in endothelial layer hydraulic conductivity (Lp) that are mediated by an increased production of nitric oxide (NO). Other recent studies have shown that NO induction by shear stress is mediated by the glycocalyx that decorates the surface of endothelial cells. Here we find that a selective depletion of the major components of the glycocalyx with enzymes can block the shear stress-induced response of Lp. Heparinase and hyaluronidase block shear-induced increases in Lp, which is consistent with their effects on NO production. But chondroitinase, which does not suppress shear-induced NO production, also inhibits shear-induced Lp. A further surprise is that treatment with the general proteolytic enzyme pronase does not suppress the shear Lp response. We also find that heparinase does not alter baseline Lp significantly, whereas chondroitinase, hyaluronidase, and pronase increase it significantly

    Combined effects of shear stress and glucose on the morphology, actin filaments, and VE-cadherin of endothelial cells in vitro

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    Objective: The purpose of the present study was to analyze the effects of glucose and shear stress on the morphology and density of endothelial cells, actin filamen, and the expression of VE-cadherin. Methods: After confluency of endothelial cells (3–4 days), 22 mM of d-glucose was administered for 7 days. Endothelial cells were exposed to shear stress of 10 dyne/cm2 for varied durations of 5, 8, 12, and 15 min. Morphology of ECs was observed using an inverted microscope before and after shear stress exposure. VE-cadherin and actin filament were analyzed immunohistochemically. Results: Exposure to high glucose induces more shrinkage and the cell density decreased at 15 min. High glucose reduced actin filaments and the more globular ones, especially around the nuclei. There was a decline in VE-cadherin scores with significant differences between treatments with 5 mM and 22 mM of glucose. Conclusion: Combination exposure of shear stress and high glucose changes morphology, reduces actin filament and VE-cadherin, of endothelial cells
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