Molecular organization and functional regulation of cell to cell junctions in the endothelium

Intercellular junctions are important structural determinants of endothelial permeability. These organelles are formed by a complex network of transmembrane proteins linked to a well developed plasmalemmal undercoat. One of the typical characteristics of endothelial junctions is their dynamic organization. Endothelial cells are able to rapidly change the architecture of the junctions to allow the passage of plasma constituents and circulating cells. This effect, in most of the cases, is quickly reversible and the endothelium is able to disorganize/reorganize the intercellular junctions within minutes. The mechanisms that regulate the opening and the closure of endothelial junctions are still obscure. It is conceivable that inflammatory agents increase permeability by binding to specific receptors generating intracellular signals which in turn cause cytoskeletal reorganization and opening of interendothelial gaps. Endothelial junctions also control leukocyte extravasation. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial clefts occurs. The mechanism by which this takes place is unknown, but it might present characteristics similar to that triggered by soluble mediators.     

Decreased protein kinase C activation mediates inhibitory effect of norathyriol on serotonin-mediated endothelial permeability

We examined the mechanisms of norathyriol on the serotonin-induced increased permeability of rat heart endothelial cell monolayers. The present study showed that the activation of rat heart endothelial cell protein kinase C by phorbol myristate acetate led to the dose-dependent increase in endothelial permeability to albumin, an effect that was inhibited by staurosporine (a protein kinase inhibitor). Staurosporine also attenuated the serotonin-induced increase in permeability. Norathyriol abolished both serotonin- and phorbol myristate acetate-induced permeability. We investigated whether norathyriol, by inhibiting protein kinase C activation, attenuated the serotonin-induced permeability. Immunofluorescence studies demonstrated that norathyriol prevented the redistribution of protein kinase C isozymes following stimulation with serotonin. Western blot analysis showed that norathyriol significantly inhibited the serotonin-induced translocation of the alpha protein kinase C isozyme from the cytosolic to the particulate fraction. In conclusion, norathyriol attenuates the serotonin-induced permeability of rat heart endothelial cells to macromolecules in association with inhibition of protein kinase C activation. This decrease in endothelial cell permeability may be one of the mechanisms for the protective effects of norathyriol against edema formation in response to inflammatory agonists in vivo.     

Catenin-dependent and -independent functions of vascular endothelial cadherin

Vascular endothelial cadherin (VE-cadherin, cadherin-5, or 7B4) is an endothelial specific cadherin that regulates cell to cell junction organization in this cell type. Cadherin linkage to intracellular catenins was found to be required for their adhesive properties and for localization at cell to cell junctions. We constructed a mutant form of VE-cadherin lacking the last 82 amino acids of the cytoplasmic domain. Surprisingly, despite any detectable association of this truncated VE-cadherin to catenin-cytoskeletal complex, the molecule was able to cluster at cell-cell contacts in a manner similar to wild type VE-cadherin. Truncated VE-cadherin was also able to promote calcium-dependent cell to cell aggregation and to partially inhibit cell detachment and migration from a confluent monolayer. In contrast, intercellular junction permeability to high molecular weight molecules was severely impaired by truncation of VE-cadherin cytoplasmic domain. These results suggest that the VE-cadherin extracellular domain is enough for early steps of cell adhesion and recognition. However, interaction of VE-cadherin with the cytoskeleton is necessary to provide strength and cohesion to the junction. The data also suggest that cadherin functional regulation might not be identical among the members of the family.     

Inhibitory effect of 2-phenyl-4-quinolone on serotonin-mediated changes in the morphology and permeability of endothelial monolayers

The integrity of endothelial cell monolayers, a critical requirement for barrier maintenance, is needed for the prevention of edema formation. To investigate the mechanisms by which 2-phenyl-4-quinolone (YT-1) provided protection against serotonin-induced exudation, rat heart endothelial cell cultures were used. In this study, serotonin and phorbol myristate acetate (PMA) caused endothelial cells to became permeable to macromolecules by causing cell contraction and intercellular gap formation. These responses were attenuated by staurosporine, a protein kinase C inhibitor. Further experiments showed that YT-1 (1) did not alter serotonin-mediated early signal events such as protein kinase C activation, (2) protected against serotonin-induced endothelial barrier dysfunction by increasing intracellular cAMP levels, (3) played a role in regulating adenylate cyclase activity, (4) reversed serotonin-induced permeability to macromolecules, an effect which did not correlate with intracellular cGMP concentrations. This study demonstrates a possible mechanism by which YT-1 protects endothelial function and preserves the microvasculature from pharmacologic injury by vasoactive agents.     

Characterization of the mechanical behaviour parameters of the costo-vertebral joint

Humic acid in the drinking water of blackfoot disease endemic areas in Taiwan has been implicated as one of the aetiological factors of the disease. For this report we examined the effects of humic acid on the expression of thrombomodulin (TM) cofactor activity by cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with humic acid (HA) isolated from the drinking water, as a synthetic humic acid polymer (SHA) or with commercial HA, resulted in a dose-dependent reduction of cell surface thrombomodulin activity. Characterization of the mechanism by which humic acid reduced the protein C activation indicated that inhibition was not caused by production or release of a protein C inhibitor. Kinetic analysis showed that binding affinities of TM to thrombin and of TM-thrombin complex to protein C was unchanged upon humic acid treatment. However, the cell surface TM activity was reduced by humic acid, which functions as an irreversible noncompetitive inhibitor of thrombin binding. Down-regulation of TM was inhibited by non-selective protein kinase C inhibitors and a selective inhibitor. These results suggest that protein kinase C is intricately involved in HA-induced TM down-regulation. Down-regulation of TM was also inhibited by free radical scavengers. All these changes occurred in the absence of significant cytotoxic effect. In conclusion, our results suggest that HA induces down-regulation of TM by directly increasing permeability of the cell membrane, thus causing elevation in [Ca2+]i. This species functions as a second messenger to activate protein kinase C, and/or Ca-dependent enzymes eventually inducing down-regulation of TM. Attenuation of vascular endothelial cell TM cofactor activity by humic acid may play a role in the humic acid-induced thrombotic vascular disorders of blackfoot disease.     

Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.     

Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells

The effect of thrombin on the fibrinolytic potential of human vascular smooth muscle cells (SMC) in culture was studied. SMC of different origin responded to thrombin treatment with a dose and time dependent increase in tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) levels in both cell lysates and conditioned media with maximum effects achieved at 10-20 IU/ml thrombin. PAI-1 antigen levels also increased in the extracellular matrix of thrombin treated SMC. PAI-2 levels in cell lysates of such SMC were not affected by thrombin. The effect was restricted to active thrombin, since DFP-thrombin and thrombin treated with hirudin showed no increasing effect on t-PA and PAI-1 levels in SMC. Enzymatically active thrombin also caused a four-fold increase in specific PAI-1 mRNA and a three-fold increase in t-PA mRNA. Furthermore we demonstrated the presence of high and low affinity binding sites for thrombin on the surface of SMC with a KD = 4.3 x 10(-10)M and 9.0 x 10(4) sites per cell and a KD = 0.6 x 10(-8) M and 5.8 x 10(5) sites per cell respectively. Thrombin could come in contact with SMC in case of vascular injury or following gap formation between endothelial cells. Our data support the idea that besides its known proliferative effect for SMC, thrombin could also modulate their fibrinolytic system.     

Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells

Polymorphonuclear leukocytes (PMN) traverse an endothelial cell (EC) barrier by crawling between neighboring EC. Whether EC regulate the integrity of their intercellular adhesive and junctional contacts in response to chemotaxing PMN is unresolved. EC respond to the binding of soluble mediators such as histamine by increasing their cytosolic free calcium concentration ([Ca++]i) (Rotrosen, D., and J.I. Gallin. 1986. J. Cell Biol. 103:2379-2387) and undergoing shape changes (Majno, G., S. M. Shea, and M. Leventhal. 1969. J. Cell Biol. 42:617-672). Substances such as leukotriene C4 (LTC4) and thrombin, which increased the permeability of EC monolayers to ions, as measured by the electrical resistance of the monolayers, transiently increased EC [Ca++]i. To determine whether chemotaxing PMN cause similar changes in EC [Ca++]i, human umbilical vein endothelial cells (HUVEC) maintained as monolayers were loaded with fura-2. [Ca++]i was measured in single EC during PMN adhesion to and migration across these monolayers. PMN-EC adhesion and transendothelial PMN migration in response to formyl-methionyl-leucyl-phenylalanine (fMLP) as well as to interleukin 1 (IL-1) treated EC induced a transient increase in EC [Ca++]i which temporally corresponded with the time course of PMN-EC interactions. When EC [Ca++]i was clamped at resting levels with a cell permeant calcium buffer, PMN migration across EC monolayers and PMN induced changes in EC monolayer permeability were inhibited. However, clamping of EC [Ca++]i did not inhibit PMN-EC adhesion. These studies provide evidence that EC respond to stimulated PMN by increasing their [Ca++]i and that this increase in [Ca++]i causes an increase in EC monolayer permeability. Such [Ca++]i increases are required for PMN transit across an EC barrier. We suggest EC [Ca++]i regulates transendothelial migration of PMN by participating in a signal cascade which stimulates EC to open their intercellular junctions to allow transendothelial passage of leukocytes.     

Cigarette smoking is associated with increased human monocyte adhesion to endothelial cells: reversibility with oral L-arginine but not vitamin C

OBJECTIVES: This study sought to assess the effect of cigarette smoking on adhesion of human monocytes to human endothelial cells and to measure the effect of L-arginine and vitamin C supplementation on this interaction. BACKGROUND: Cigarette smoking has been associated with abnormal endothelial function and increased leukocyte adhesion to endothelium, both key early events in atherogenesis. Supplementation with both oral L-arginine (the physiologic substrate for nitric oxide) and vitamin C (an aqueous phase antioxidant) may improve endothelial function; however, their benefit in cigarette smokers is not known. METHODS: Serum was collected from eight smokers (mean [+/-SD] age 33 +/- 5 years) with no other coronary risk factors and eight age- and gender-matched lifelong nonsmokers. The serum was added to confluent monolayers of human umbilical vein endothelial cells and incubated for 24 h. Human monocytes obtained by counterflow centrifugation elutriation were then added to these monolayers for 1 h, and adhesion then was measured by light microscopy. To assess reversibility, monocyte/ endothelial cell adhesion was then measured for each subject 2 h after 2 g of oral vitamin C and 2 h after 7 g of oral L-arginine. RESULTS: In smokers compared with control subjects, monocyte/ endothelial cell adhesion was increased (46.4 +/- 4.5% vs. 27.0 +/- 5.2%, p < 0.001), endothelial expression of intercellular adhesion molecule (ICAM)-1 was increased (0.31 +/- 0.02 vs. 0.22 +/- 0.03, p = 0.004), and vitamin C levels were reduced (33.7 +/- 24.1 vs. 53.4 +/- 11.5 mumol/liter, p = 0.028). After oral L-arginine, monocyte/ endothelial cell adhesion was reduced in smokers (from 46.4 +/- 4.5% to 35.1 +/- 4.0%, p = 0.002), as was endothelial cell expression of ICAM-1 (from 0.31 +/- 0.02 to 0.27 +/- 0.01, p = 0.001). After vitamin C, there was no significant change in monocyte/ endothelial cell adhesion or ICAM-1 expression from baseline in the smokers despite an increase in vitamin C levels (to 115 +/- 7 mumol/liter). CONCLUSIONS: Cigarette smoking is associated with increased monocyte-endothelial cell adhesion when endothelial cells are exposed to serum from healthy young adults. This abnormality is acutely reversible by oral L-arginine but not by vitamin C.     

Angiogenic activity of osteopontin-derived peptide SVVYGLR

Angiogenesis plays an important role in various pathological conditions as well as some physiological processes. Although a number of soluble angiogenic factors have been reported, extracellular matrix also has crucial effect on angiogenesis through interaction with endothelial cells. Since recent reports showed osteopontin had some angiogenic activity, the effect of the SVVYGLR peptide, novel binding motif in osteopontin molecule, on angiogenesis was examined in this study. Synthetic peptide SVVYGLR did not have proliferative effect on endothelial cells but adhesion and migration activity to endothelial cells. Furthermore, SVVYGLR had as potent activity for tube formation in three-dimensional collagen gel as vascular endothelial growth factor which is known to be the strongest angiogenic factor. Electron microscopical analysis showed a number of microvilli on the endothelial luminar surface and tight junction formation in the luminar intercellular border between endothelial cells, indicating SVVYGLR induced cell porarity and differentiation of endothelial cells. This small peptide might be expected to stimulate angiogenesis to improve some ischemic conditions in the future because of some advantages due to smaller molecular weight.     

High permeability of the intercellular contact

The mechanism of the intercellular contact (gap junction) high permeability is proposed on the following basis: 1) cell membranes are depolarized in the junction zone and 2) during depolarization a sharp increase of membrane permeability results from the formerly proposed [1--3] relationship between the trans-membrane potential and membrane surface potential (boundary jump), owing to the fact that the latter determines both the membranes conductance and conformation.     

Drinking in Atlantic salmon presmolts and smolts in response to growth hormone and salinity

Both thrombin and the synthetic tetracapeptide thrombin receptor-activating peptide (TRAP), recently described as a peptide mimicking the new amino terminus created by cleavage of the thrombin receptor, stimulated the proliferation of human umbilical vein endothelial cells (HUVEC) in culture. Although to a lesser extent, F-14, a tetradecapeptide representing the residues 365-378 of human prothrombin, also promoted HUVEC growth, thereby demonstrating that thrombin can stimulate HUVEC growth via both a proteolytic and a nonenzymatic pathway. Thrombin-TRAP, and F-14-induced HUVEC growth were inhibited by a thrombin receptor oligodeoxynucleotide antisense, showing that the growth-inducing effects of all 3 compounds were mediated through the same thrombin receptor. Thrombin and TRAP also stimulated intracellular Ca2+ increase, monolayer permeability increase, and prostacyclin release in HUVEC. None of these effects was observed with F-14 suggesting that thrombin-induced intracellular Ca2+ release, permeability increase, and prostacyclin release in HUVEC required catalytic cleavage of the receptor, whereas thrombin-induced growth might also be due to activation of the thrombin receptor through a nonproteolytic pathway.     

An in vitro model for toxin-mediated vascular leak syndrome: ricin toxin A chain increases the permeability of human endothelial cell monolayers

Vascular leak syndrome (VLS) is the dose-limiting toxicity observed in clinical trials of immunotoxins containing ricin toxin A chain (RTA). RTA itself is thought to cause VLS by damaging vascular endothelial cells, but the exact mechanism remains unclear. This is partially due to the paucity of appropriate models. To study VLS, we developed an in vitro model in which human umbilical vein-derived endothelial cells were first grown to confluence on microporous supports and then cultured under low pressure in the presence or absence of RTA. Endothelial cell barrier function was assessed by measuring the volume of fluid that passed through each monolayer per unit time. We found that RTA significantly increased monolayer permeability at times and concentrations consistent with the onset of VLS in patients treated with RTA-based immunotoxins. Scanning electron microscopy showed that intercellular gaps formed in endothelial monolayers exposed to RTA. Intercellular gap formation followed endothelial cell death caused by the enzymatic activity of RTA. We conclude that RTA is directly toxic to endothelial cells in vitro and speculate that this contributes to VLS in vivo.     

Mechanisms of edema formation after intracerebral hemorrhage: effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model

Recently, the authors showed that thrombin contributes to the formation of brain edema following intracerebral hemorrhage. The current study examines whether the action of thrombin is due to an effect on cerebral blood flow (CBF), vasoreactivity, blood-brain barrier (BBB) function, or cell viability. In vivo solutions of thrombin were infused stereotactically into the right basal ganglia of rats. The animals were sacrificed 24 hours later; CBF and BBB permeability were measured. The actions of thrombin on vasoreactivity were examined in vitro by superfusing thrombin on cortical brain slices while monitoring microvessel diameter with videomicroscopy. In separate experiments C6 glioma cells were exposed to various concentrations of thrombin, and lactate dehydrogenase release, a marker of cell death, was measured. The results indicate that thrombin induces BBB disruption as well as death of parenchymal cells, whereas CBF and vasoreactivity are not altered. The authors conclude that cell toxicity and BBB disruption by thrombin are triggering mechanisms for the edema formation that follows intracerebral hemorrhage.     

Dephosphorylation of the cadherin-associated p100/p120 proteins in response to activation of protein kinase C in epithelial cells

Protein kinase C signaling pathways have been implicated in the disruption of intercellular junctions, but mechanisms are not clear. p100 and p120 are members of the Armadillo family of proteins and are localized to cellular adherens junctions. In strain I Madin-Darby canine kidney cells, protein kinase C activation leads to disruption of tight junctions and an increase in permeability of cell monolayers. We show that this permeability increase is accompanied by dephosphorylation of p100/p120 on serine and threonine residues. The dephosphorylation of these proteins can also be induced by the kinase inhibitors staurosporine, KT5926, and G? 6976. Treatment of cells with phosphatase inhibitors induced hyperphosphorylation of p100 and p120. Thus, p100 and p120 participate in a regulatable cycle of serine/threonine phosphorylation and dephosphorylation. Protein kinase C must act, directly or indirectly, by perturbing this phosphorylation cycle, by inhibition of a p100/p120 kinase and/or activation of a phosphatase. These data clearly show that p100 and p120 are targets of a novel protein kinase C signaling pathway. Dephosphorylation of these proteins precedes the permeability increase across epithelial cell monolayers seen in response to phorbol esters, raising the possibility that this pathway may play a role in the modulation of intercellular junctions.     

Long-term visual outcome of choroidal neovascularization in pathologic myopia: natural history and laser treatment

Tumour-endothelial cell adhesion forms a key role in the establishment of distant metastases. This study examined the effect of gamma linolenic acid (GLA), an anti-cancer polyunsaturated fatty acid (PUFA), on both the gap junction communication of human vascular endothelial cells and tumour cell-endothelial interactions. By using scrape loading of Lucifer yellow dye, we showed that GLA at non-toxic levels increased Lucifer yellow transfer, indicating improved gap junction communication. The fatty acid also corrected the communication that was reduced by the mitogenic and motogenic factor HGF/SF. GLA inhibited the tyrosine phosphorylation of connexin-43, a protein that formed gap junction in this cell. When human tumour cells were added to quiescent or HGF/SF-activated endothelial cells, the presence of GLA reduced adhesion of tumour cells to the endothelium. It is concluded that GLA reduces tumour-endothelium adhesion, partly by improved gap junction communications of the endothelium.     

18F]fluoroethoxy-benzovesamicol, a PET radiotracer for the vesicular acetylcholine transporter and cholinergic synapses

CACO-2 BBE was used to determine the response of a gastrointestinal epithelium to tumor necrosis factor-alpha (TNF). Incubation of CACO-2 BBE with TNF did not produce any effect on transepithelial resistance (TER) within the first 6 hr but resulted in a 40-50% reduction in TER and a 30% decrease in 1SC (short circuit current) relative to time-matched control at 24 hr. The decrease in TER was sustained up to 1 week following treatment with TNF and was not associated with a significant increase in the transepithelial flux of [14C]-D-mannitol or the penetration of ruthenium red into the lateral intercellular space. Dilution potential and transepithelial 22Na+ flux studies demonstrated that TNF-treatment of CACO-2 BBE cell sheets increased the paracellular permeability of the epithelium to Na+ and Cl-. The increased transepithelial permeability did not associate with an increase in the incidence of apoptosis. However, there was a TNF-dependent increase in [3H]-thymidine labeling that was not accompanied by a change in DNA content of the cell sheet. The increase in transepithelial permeability was concluded to be across the tight junction because: (i) 1 mM apical amiloride reduced the basolateral to apical flux of 22Na+, and (ii) dilution potential studies revealed a bidirectionally increased permeability to both Na+ and Cl-. These data suggest that the increase in transepithelial permeability across TNF-treated CACO-2 BBE cell sheets arises from an alteration in the charge selectivity of the paracellular conductive pathway that is not accompanied by a change in its size selectivity.     

Macro allocation in health: an ethically based model

To investigate the role of 2-phenyl-4-quinolone in enhancing endothelial monolayer paracellular barrier function and preventing the disturbance of paracellular barrier function by vasoactive agents, the study examined the effect of 2-phenyl-4-quinolone on serotonin-mediated macromolecule transfer and microfilament changes in cultured rat heart endothelial cells. Serotonin-treated endothelial cells induced concentration-dependent increases in the passage of Evans blue dye-bound bovine serum albumin. Incubation of the endothelial monolayers with 2-phenyl-4-quinolone antagonized serotonin- and cytochalasin B-induced macromolecular permeability. 2-Phenyl-4-quinolone also opposed the effect of serotonin or cytochalasin B on the distribution and quantity of actin filaments in the endothelial cytoskeleton. Furthermore, 2-phenyl-4-quinolone alone led to an apparent quantitative increase in F actin fluorescence in endothelial cells. The addition of 10(-7) M 2-phenyl-4-quinolone had an effect on serotonin-induced changes in the myosin and distribution of myosin were comparable to that on serotonin monolayers. In conclusion, 2-phenyl-4-quinolone attenuated the serotonin-induced permeability of rat heart endothelial cells and this was associated with stabilization of F actin microfilaments and changes in the myosin organization. This result suggests that influences on cytoskeletal assembly may be involved in this process.     

Canine jugular vein endothelial cell monolayers in vitro: vasomediator-activated diffusive albumin pathway

Cultured canine jugular vein endothelial cells were seeded on polycarbonate filters to create an in vitro permeability assay. The calculated diffusive permeability coefficient for FITC-BSA across untreated monolayers was 1.1 +/- 0.4 x 10(-6) cm/s. After 15-min incubations with either histamine or bradykinin, the resistance to albumin flux across the monolayers was reduced significantly. Diffusive albumin permeability coefficients were 3.4 +/- 1.8 x 10(-6) and 4.1 +/- 2.0 x 10(-6) cm/s, respectively. Ultrastructural morphometric analyses of the endothelial cell monolayers served to define uniform dimensions of intercellular clefts and similar plasmalemmal vesicle densities in the untreated and the vasomediator-activated monolayers. These results are consistent with the interpretation that the vasomediator-activated pathway across the venous endothelial monolayers is not dependent on sustained intercellular gap formation or sustained expansion of the plasmalemmal vesicle population for the 15-min observation periods. Whether the increased albumin flux is dependent on transient gap formation or on physical changes within the venous endothelial cell glycocalyx remains to be tested.