Charge effects on the hindered transport of macromolecules across the endothelial surface glycocalyx layer

A fluid mechanical and electrostatic model for the transport of solute molecules across the vascular endothelial surface glycocalyx layer (EGL) was developed to study the charge effect on the diffusive and convective transport of the solutes. The solute was assumed to be a spherical particle with a constant surface charge density, and the EGL was represented as an array of periodically arranged circular cylinders of like charge, with a constant surface charge density. By combining the fluid mechanical analyses for the flow around a solute suspended in an electrolyte solution and the electrostatic analyses for the free energy of the interaction between the solute and cylinders based on a mean field theory, we estimated the transport coefficients of the solute across the EGL. Both of diffusive and convective transports are reduced compared to those for an uncharged system, due to the stronger exclusion of the solute that results from the repulsive electrostatic interaction. The model prediction for the reflection coefficient for serum albumin agreed well with experimental observations if the charge density in the EGL is ranged from approximately -10 to -30 mEq/l.

Rotational co-culture of clonal β-cells with endothelial cells:effect of PPAR-γ agonism in vitro on insulin and VEGF secretion

Aim: Delayed graft revascularization impedes the success of human islet transplantation. This study utilized rotational co-culture of insulin secreting ß-cells with human umbilical vein endothelial cells (HUVECs) and a peroxisome proliferator-activated receptor gamma (PPAR-?) agonist to promote insulin and vascular endothelial growth factor (VEGF) secretory function. Methods: Clonal BRIN-BD11 (D11) cells were maintained in static culture (SC) and rotational culture (RC) ± HUVEC and ± the TZD (thiazolidinedione) rosiglitazone (10 mmol/l) as a specific PPAR-? agonist. HUVECs were cultured in SC and RC ± D11 and ± TZD. D11 insulin secretion was induced by static incubation with low glucose (1.67 mmol/l), high glucose (16.7 mmol/l) and high glucose with 10 mmol/l theophylline (G+T) and assessed by enzyme-linked immunosorbent assay (ELISA). HUVEC proliferation was determined by ATP luminescence, whereas VEGF secretion was quantified by ELISA. Co-cultured cells were characterized by immunostaining for insulin and CD31. Results: D11 SC and RC showed enhanced insulin secretion in response to 16.7 mmol/l and G+T (p <0.01); without significant alteration by the TZD. Co-culture with HUVEC in SC and RC also increased D11 insulin secretion when challenged with 16.7 mmol/l and G+T (p <0.01), and this was slightly enhanced by the TZD. The presence of HUVEC increased D11 SC and RC insulin secretion in response to high glucose and G+T, respectively (p <0.01). Addition of the TZD increased SC and RC HUVEC ATP content (p <0.01) and VEGF production (p <0.01) in the presence and absence of D11 cells. Conclusions: Rotational co-culture of insulin secreting cells with endothelial cells, and exposure to a PPAR-? agonist may improve the prospects for graft revascularization and function after implantation. © 2011 Blackwell Publishing Ltd.

Flow across microvessel walls through the endothelial surface glycocalyx and the interendothelial cleft

A mathematical model is presented for steady fluid flow across microvessel walls through a serial pathway consisting of the endothelial surface glycocalyx and the intercellular cleft between adjacent endothelial cells, with junction strands and their discontinuous gaps. The three-dimensional flow through the pathway from the vessel lumen to the tissue space has been computed numerically based on a Brinkman equation with appropriate values of the Darcy permeability. The predicted values of the hydraulic conductivity Lp, defined as the ratio of the flow rate per unit surface area of the vessel wall to the pressure drop across it, are close to experimental measurements for rat mesentery microvessels. If the values of the Darcy permeability for the surface glycocalyx are determined based on the regular arrangements of fibres with 6nm radius and 8nm spacing proposed recently from the detailed structural measurements, then the present study suggests that the surface glycocalyx could be much less resistant to flow compared to previous estimates by the one-dimensional flow analyses, and the intercellular cleft could be a major determinant of the hydraulic conductivity of the microvessel wall.

Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial Nox2 in vivo. To address this question, we generated transgenic mice with endothelial-specific overexpression of Nox2 (Tg) and studied the effects on endothelial function and blood pressure. Tg had an about twofold increase in endothelial Nox2 levels which was accompanied by an increase in p22phox levels but no change in levels of other Nox isoforms or endothelial nitric oxide synthase (eNOS). Basal NADPH oxidase activity, endothelial function and blood pressure were unaltered in Tg compared to wild-type littermates. Angiotensin II caused a greater increase in ROS production in Tg compared to wild-type aorta and attenuated acetylcholine-induced vasorelaxation. Both low and high dose chronic angiotensin II infusion increased telemetric ambulatory blood pressure more in Tg compared to wild-type, but with different patterns of BP change and aortic remodeling depending upon the dose of angiotensin II dose. These results indicate that an increase in endothelial Nox2 levels contributes to angiotensin II-induced endothelial dysfunction, vascular remodeling and hypertension. © 2011 The Author(s).

Celiac disease-specific TG2-targeted autoantibodies inhibit angiogenesis ex vivo and in vivo in mice by interfering with endothelial cell dynamics

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility. © 2013 Kalliokoski et al.

Celiac disease patient IgA antibodies induce endothelial adhesion and cell polarization defects via extracellular transglutaminase 2

We have recently found that celiac disease patient serum-derived autoantibodies targeted against transglutaminase 2 interfere with several steps of angiogenesis, including endothelial sprouting and migration, though the mechanism involved remained to be fully characterized. This study now investigated the processes underlying the antiangiogenic effects exerted by celiac disease patient antibodies on endothelial cells, with particular regard to the adhesion, migration, and polarization signaling pathway. We observed that celiac IgA reduced endothelial cell numbers by affecting adhesion without increasing apoptosis. Endothelial cells in the presence of celiac IgA showed weak attachment, a high susceptibility to detach from fibronectin, and a disorganized extracellular matrix due to a reduction of protein cross-links. Furthermore, celiac patient IgA led to secretion of active transglutaminase 2 from endothelial cells into the culture supernatants. Additionally, cell surface transglutaminase 2 mediated integrin clustering in the presence of celiac IgA was coupled to augmented expression of ß1-integrin. We also observed that celiac patient IgA-treated endothelial cells had migratory defects and a less polarized phenotype when compared to control groups, and this was associated with the RhoA signaling pathway. These biological effects mediated by celiac IgA on endothelial cells were partially influenced but not completely abolished by R281, an irreversible extracellular transglutaminase 2 enzymatic activity inhibitor. Taken together, our results imply that celiac patient IgA antibodies disturb the extracellular protein cross-linking function of transglutaminase 2, thus altering cell-extracellular matrix interactions and thereby affecting endothelial cell adhesion, polarization, and motility. © 2013 Springer Basel.

Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA

Purpose: To investigate the role of thioredoxin (TRX), a novel regulator of extracellular transglutaminase 2 (TG2), in celiac patients IgA (CD IgA) mediated TG2 enzymatic activation. Methods: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs) under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA) were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. Results: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. Conclusions: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX. © 2013 Nadalutti et al.

Experiences of patients with age-related macular degeneration receiving anti-vascular endothelial growth factor therapy:a qualitative study

The societal cost for the average health authority in the United Kingdom for the care of wet age-related macular degeneration (AMD) has been suggested to be around £7.4 million. It is vital that the best possible care based on the best available evidence is provided to reduce the impact of AMD on patients' lives and the financial cost to the health-care system. This study explored the experiences of AMD patients treated with intravitreal ranibizumab injections. Three semistructured interviews were conducted with seven participants over the course of 18 months. Transcripts were analysed using interpretative phenomenological analysis. Analysis identified four themes: preparing for treatment, the treatment process, patient-provider communication, and results of treatment. Patient experiences highlighted the need to move away from the reliance on letters for information provision, and the need for clearer guidelines about when to cease AMD treatment. Interviews highlighted the need for the inclusion of rigorous qualitative evidence with experiential data in future good clinical practice guideline development for AMD. © The Author(s) 2013.

Ceramide and insulin resistance in muscle and endothelial cells

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Age-related macular degeneration is associated with increased vascular endothelial growth factor, hemorheology and endothelial dysfunction

OBJECTIVE: To investigate laboratory evidence of abnormal angiogenesis, hemorheologic factors, endothelial damage/dysfunction, and age-related macular degeneration (ARMD). DESIGN: Comparative cross-sectional study. PARTICIPANTS: We studied 78 subjects (26 men and 52 women; mean age 74 years; standard deviation [SD] 9.0) with ARMD attending a specialist referral clinic. Subjects were compared with 25 healthy controls (mean age, 71 years; SD, 11). INTERVENTION AND OUTCOME MEASURES: Levels of vascular endothelial growth factor (VEGF, an index of angiogenesis), hemorheologic factors (plasma viscosity, hematocrit, white cell count, hemoglobin, platelets), fibrinogen (an index of rheology and hemostasis), and von Willebrand factor (a marker of endothelial dysfunction) were measured. RESULTS: Median plasma VEGF (225 vs. 195 pg/ml, P = 0.019) and mean von Willebrand factor (124 vs. 99 IU/dl, P = 0.0004) were greater in ARMD subjects than the controls. Mean plasma fibrinogen and plasma viscosity levels were also higher in the subjects (both P < 0.0001). There were no significant differences in other indices between cases and controls. When "dry" (drusen, atrophy, n = 28) and "exudative" (n = 50) ARMD subjects were compared, there was no significant differences in VEGF, fibrinogen, viscosity, or von Willebrand factor levels. There were no significant correlations between the measured parameters. Stepwise multiple regression analysis did not demonstrate any significant clinical predictors (age, gender, smoking, body mass index, history of vascular disease, or hypertension) for plasma VEGF or fibrinogen levels, although smoking status was a predictor of plasma von Willebrand factor levels (P < 0.05). CONCLUSIONS: This study suggests an association between markers of angiogenesis (VEGF), hemorheologic factors, hemostasis, endothelial dysfunction, and ARMD. The interaction between abnormal angiogenesis and the components of Virchow's triad for thrombogenesis may in part contribute to the pathogenesis of ARMD.

Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via toll-like receptor 2

To determine whether non-enterobacterial endotoxins, which are likely to constitute the majority of the circulating endotoxin pool, may stimulate coronary artery endothelial cell activation. Interleukin-8 secretion, monocyte adhesion, and E-selectin expression were measured in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) challenged in vitro with highly purified endotoxins of common host colonisers Escherichia coli, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Bacteroides fragilis. HCAECs but not HUVECs expressed Toll-like receptor (TLR)-2 and were responsive to non-enterobacterial endotoxins. Transfection of TLR-deficient HEK-293 cells with TLR2 or TLR4/MD2 revealed that while E. coli endotoxin utilised solely TLR4 to signal, the endotoxins, deglycosylated endotoxins (lipid-A), and whole heat-killed bacteria of the other species stimulated TLR2-but not TLR4-dependent cell-signalling. Blockade of TLR2 with neutralizing antibody prevented HCAEC activation by non-enterobacterial endotoxins. Comparison of each endotoxin with E. coli endotoxin in limulus amoebocyte lysate assay revealed that the non-enterobacterial endotoxins are greatly underestimated by this assay, which has been used in all previous studies to estimate plasma endotoxin concentrations. Circulating non-enterobacterial endotoxins may be an underestimated contributor to endothelial activation and atherosclerosis in individuals at risk of increased plasma endotoxin burden.

Fatty acid and phospholipid chlorohydrins cause cell stress and endothelial adhesion

The oxidation of low-density lipoprotein (LDL) is thought to contribute to atherogenesis, which is an inflammatory disease involving activation of phagocytic cells. Myeloperoxidase, an enzyme which is able to produce hypochlorous acid (HOCl), is released from these phagocytic cells, and has been found in an active form in atherosclerotic plaques. HOCl can oxidize both the lipid and protein moiety of LDL, and HOCl-modified LDL has been found to be pro-inflammatory, although it is not known which component is responsible for this effect. As HOCl can oxidize lipids to give chlorohydrins, we hypothesized that phospholipid chlorohydrins might have toxic and pro-inflammatory effects. We have formed chlorohydrins from fatty acids (oleic, linoleic and arachidonic acids) and from phospholipids (stearoyl-oleoyl phosphatidylcholine, stearoyl-linoleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine), and investigated various biological effects of these oxidation products. Fatty acid and phospholipid chlorohydrins were found to deplete ATP levels in U937 cells in a concentration-dependent manner, with significant effects observed at concentrations of 25 µM and above. Low concentrations (25 µM) of stearoyl-oleoyl phosphatidylcholine and stearoyl-arachidonoyl phosphatidylcholine chlorohydrins were also found to increase caspase-3 activity. Finally, stearoyl-oleoyl phosphatidylcholine chlorohydrin increased leukocyte adhesion to artery segments isolated from C57Bl/6 mice. These results demonstrate potentially harmful effects of lipid chlorohydrins, and suggest that they may contribute to some of the pro-inflammatory effects that HOCl-modified low density lipoprotein has been found to induce.

Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro

STUDY DESIGN: The effect of human intervertebral disc aggrecan on endothelial cell growth was examined using cell culture assays. OBJECTIVE: To determine the response of endothelial cells to human intervertebral disc aggrecan, and whether the amount and type of aggrecan present in the intervertebral disc may be implicated in disc vascularization. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration has been associated with a loss of proteoglycan, and the ingrowth of blood vessels and nerves. Neovascularization is a common feature also of disc herniation. Intervertebral disc aggrecan is inhibitory to sensory nerve growth, but the effects of disc aggrecan on endothelial cell growth are not known. METHODS: Aggrecan monomers were isolated separately from the anulus fibrosus and nucleus pulposus of human lumbar intervertebral discs, and characterized to determine the amount and type of sulfated glycosaminoglycan side chains present. The effects of these aggrecan isolates on the cellular adhesion and migration of the human endothelial cell lines, HMEC-1 and EAhy-926, were examined in vitro. RESULTS: Homogenous substrata of disc aggrecan inhibited endothelial cell adhesion and cell spreading in a concentration dependent manner. In substrata choice assays, endothelial cells seeded onto collagen type I migrated over the collagen until they encountered substrata of disc aggrecan, where they either stopped migrating, retreated onto the collagen, or, more commonly, changed direction to align along the collagen-aggrecan border. The inhibitory effect of aggrecan on endothelial cell migration was concentration dependent, and reduced by enzymatic treatment of the aggrecan monomers with a combination of chondroitinase ABC and keratinase/keratinase II. Anulus fibrosus aggrecan was more inhibitory to endothelial cell adhesion than nucleus pulposus aggrecan. However, this difference did not relate to the extent to which the different aggrecan isolates were charged, as determined by colorimetric assay with 1,9-dimethylmethylene blue, or to marked differences in the distribution of chondroitin sulfated and keratan sulfated side chains. CONCLUSIONS: Human intervertebral disc aggrecan is inhibitory to endothelial cell migration, and this inhibitory effect appears to depend, in part, on the presence of glycosaminoglycan side chains on the aggrecan monomer.

Plasma vascular endothelial growth factor, angiopoietin-2, and soluble angiopoietin receptor tie-2 in diabetic retinopathy:effects of laser photocoagulation and angiotensin receptor blockade

Background: Proliferative diabetic retinopathy (PDR) may be a response to abnormal angiogenic growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), and the soluble angiopoietin receptor tie-2. The authors hypothesised the following: (a) there are differences in plasma levels of these growth factors in different grades of diabetic retinopathy; and (b) that the effects of intervention with panretinal laser photocoagulation (PRP) for PDR, and angiotensin receptor blockade (using eprosartan) for patients with other grades of diabetic retinopathy will be to reduce levels of the growth factors. Methods: Cross sectional and interventional study (using PRP and eprosartan) in diabetic patients. VEGF, Ang-2, and tie-2 were measured by ELISA. Results: VEGF (p<0.001) and Ang-2 levels (p<0.001) were significantly higher in 93 diabetic patients compared to 20 healthy controls, with the highest levels in grade 2 and grade 3 diabetic retinopathy (p<0.05). Tie-2 was lower in diabetics compared to controls (p = 0.008), with no significant differences between the diabetic subgroups. Overall, VEGF significantly correlated with Ang-2 (p<0.001) and tie-2 (p = 0.004) but the correlation between Ang-2 and tie-2 levels was not significant (p = 0.065). Among diabetic patients only, VEGF levels were significantly correlated with Ang-2 (p<0.001) and tie-2 (p<0.001); the correlation between Ang-2 and tie-2 levels was also significant (p<0.001). There were no statistically significant effects of laser photocoagulation on plasma VEGF, Ang-2, and tie-2 in the 19 patients with PDR, or any effects of eprosartan in the 28 patients with non-proliferative diabetic retinopathy. Conclusion: Increased plasma levels of VEGF and Ang-2, as well as lower soluble tie-2, were found in diabetic patients. The highest VEGF and Ang-2 levels were seen among patients with pre-proliferative and proliferative retinopathy, but there was no relation of tie-2 to the severity of retinopathy. As the majority of previous research into Ang-2 and tie-2 has been in relation to angiogenesis and malignancy, the present study would suggest that Ang-2 and tie-2 may be used as potential indices of angiogenesis in diabetes mellitus (in addition to VEGF) and may help elucidate the role of the angiopoietin/tie-2 system in this condition.

Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis

Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase Cgamma1. Consistent with these findings, the VEGFR-1-specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2-activated phospholipase Cgamma1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.