Effects of oral vitamin C on monocyte:Endothelial cell adhesion in healthy subjects

Monocyte recruitment and retention in the vasculature is influenced by oxidative stress and is involved in cardiovascular disease (CVD). Individuals with low plasma ascorbate are at elevated risk of CVD. It is unknown whether vitamin C supplementation affects monocyte adhesion to endothelial cells (ECs) in healthy non-smokers. In a randomised double-blind crossover study the effect of vitamin C supplementation (six weeks, 250 mg/day) was determined in subjects with normal (HIC) and below average (LOC) plasma vitamin C concentration at baseline (mean = 67μM, n = 20, mean = 32μM, n = 20, respectively). LOC subjects showed 30% greater monocyte adhesion to ECs. This was significantly reduced by 37% (P < 0.02) following vitamin C supplementation to levels of HIC monocyte adhesion. No differences in plasma malondialdehyde concentrations were observed between groups or after supplementation. In conclusion, vitamin C supplementation normalises monocyte adhesion in subjects with low plasma vitamin C (LOC). This process may be related to a direct effect on monocytes, independent of lipid peroxidation. © 2002 Elsevier Science (USA). All rights reserved.

The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis

VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.

Autocrine activity of soluble Flt-1 controls endothelial cell function and angiogenesis

Background - The negative feedback system is an important physiological regulatory mechanism controlling angiogenesis. Soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), acts as a potent endogenous soluble inhibitor of VEGF- and placenta growth factor (PlGF)-mediated biological function and can also form dominant-negative complexes with competent full-length VEGF receptors. Methods and results - Systemic overexpression of VEGF-A in mice resulted in significantly elevated circulating sFlt-1. In addition, stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF-A, induced a five-fold increase in sFlt-1 mRNA, a time-dependent significant increase in the release of sFlt-1 into the culture medium and activation of the flt-1 gene promoter. This response was dependent on VEGF receptor-2 (VEGFR-2) and phosphoinositide-3'-kinase signalling. siRNA-mediated knockdown of sFlt-1 in HUVEC stimulated the activation of endothelial nitric oxide synthase, increased basal and VEGF-induced cell migration and enhanced endothelial tube formation on growth factor reduced Matrigel. In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation. Preeclampsia is associated with elevated placental and systemic sFlt-1. Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta. Conclusion - These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.

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.

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.

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.

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.

Vascular endothelial growth factor-induced endothelial cell proliferation is regulated by interaction between VEGFR-2, SH-PTP1 and eNOS

VEGF receptor-2 plays a critical role in endothelial cell proliferation during angiogenesis. However, regulation of receptor activity remains incompletely explained. Here, we demonstrate that VEGF stimulates microvascular endothelial cell proliferation in a dose-dependent manner with VEGF-induced proliferation being greatest at 5 and 100 ng/ml and significantly reduced at intermediate concentrations (>50% at 20 ng/ml). Neutralization studies confirmed that signaling occurs via VEGFR-2. In a similar fashion, ERK/MAPK is strongly activated in response to VEGF stimulation as demonstrated by its phosphorylation, but with a decrease in phosphoryation at 20 ng/ml VEGF. Immunoblotting analysis revealed that VEGF did not cause a dose-dependent change in expression of VEGFR-2 but instead resulted in reduced phosphorylation of VEGFR-2 when cells were exposed to 10 and 20 ng/ml of VEGF. VEGFR-2 dephosphorylation was associated with an increase in the protein tyrosine phosphatase, SH-PTP1, and endothelial nitric oxide synthase (eNOS). Immunoprecipitation and selective immunoblotting confirmed the association between VEGFR-2 dephosphorylation and the upregulation of SH-PTP1 and eNOS. Transfection of endothelial cells with antisense oligonucleotide against VEGFR-2 completely abolished VEGF-induced proliferation, whereas anti SH-PTP1 dramatically increased VEGF-induced proliferation by 1 and 5-fold at 10 and 200 ng/ml VEGF, respectively. Suppression of eNOS expression only abolished endothelial cell proliferation at VEGF concentrations above 20 ng/ml. Taken together, these results indicate that activation of VEGFR-2 by VEGF enhances SH-PTP1 activity and eNOS expression, which in turn lead to two diverse events: one is that SH-PTP1 dephosphorylates VEGFR-2 and ERK/MAPK, which weaken VEGF mitogenic activity, and the other is that eNOS increases nitric oxide production which in turn lowers SH-PTP1 activity via S-nitrosylation.

Human mesenchymal stem cells stimulate EaHy926 endothelial cell migration:combined proteomic and in vitro analysis of the influence of donor-donor variability

Mesenchymal stem cells (MSCs) stimulate angiogenesis within a wound environment and this effect is mediated through paracrine interactions with the endothelial cells present. Here we report that human MSC-conditioned medium (n=3 donors) significantly increased EaHy-926 endothelial cell adhesion and cell migration, but that this stimulatory effect was markedly donor-dependent. MALDI-TOF/TOF mass spectrometry demonstrated that whilst collagen type I and fibronectin were secreted by all of the MSC cultures, the small leucine rich proteoglycan, decorin was secreted only by the MSC culture that was least effective upon EaHy-926 cells. These individual extracellular matrix components were then tested as culture substrata. EaHy-926 cell adherence was greatest on fibronectin-coated surfaces with least adherence on decorin-coated surfaces. Scratch wound assays were used to examine cell migration. EaHy-926 cell scratch wound closure was quickest on substrates of fibronectin and slowest on decorin. However, EaHy-926 cell migration was stimulated by the addition of MSC-conditioned medium irrespective of the types of culture substrates. These data suggest that whilst the MSC secretome may generally be considered angiogenic, the composition of the secretome is variable and this variation probably contributes to donor-donor differences in activity. Hence, screening and optimizing MSC secretomes will improve the clinical effectiveness of pro-angiogenic MSC-based therapies.

Endothelial cell redox regulation of ischemic angiogenesis

The endothelium produces and responds to reactive oxygen and nitrogen species (RONS), providing important redox regulation to the cardiovascular system in physiology and disease. In no other situation are RONS more critical than in the response to tissue ischemia. Here, tissue healing requires growth factor-mediated angiogenesis that is in part dependent on low levels of RONS, which paradoxically must overcome the damaging effects of high levels of RONS generated as a result of ischemia. While generation of endothelial cell RONS in hypoxia/reoxygenation is acknowledged, the mechanism for their role in angiogenesis is still poorly understood. During ischemia, the major low molecular weight thiol glutathione (GSH) reacts with RONS and protein cysteines, producing GSH-protein adducts. Recent data indicate that GSH adducts on certain proteins are essential to growth factor responses in endothelial cells. Genetic deletion of the enzyme glutaredoxin-1, which selectively removes GSH protein adducts, improves, while its overexpression impairs, revascularization of the ischemic hindlimb of mice. Ischemia-induced GSH adducts on specific cysteine residues of several proteins, including p65 NFkB and the sarcoplasmic reticulum calcium ATPase-2 (SERCA2), appear to promote ischemic angiogenesis. Identifying the specific proteins in the redox response to ischemia has provided therapeutic opportunities to improve clinical outcomes of ischemia.

Glucolipotoxicity and the control of pancreatic ℓ-cell apoptosis

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Glucolipotoxicity and the control of pancreatic ℓ-cell apoptosis

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Endothelial cell-derived Pentraxin 3 limits the vasoreparative therapeutic potential of Circulating Angiogenic Cells

AIMS: Circulating Angiogenic Cells (CACs) promote revascularization of ischemic tissues although their underlying mechanism of action and the consequences of delivering varying numbers of these cells for therapy remain unknown. This study investigates molecular mechanisms underpinning CAC modulation of blood vessel formation.

METHODS & RESULTS: CACs at low (2x10(5)cells/ml) and mid (2x10(6)cells/ml) cellular densities significantly enhanced endothelial cell (EC) tube formation in vitro, while high density CACs (2x10(7)cells/ml) significantly inhibited this angiogenic process. In vivo, Matrigel-based angiogenesis assays confirmed mid-density CACs as pro-angiogenic and high density CACs as anti-angiogenic. Secretome characterization of CAC-EC conditioned media identified pentraxin 3 (PTX3) as only present in the high density CAC-EC co-culture. Recombinant PTX3 inhibited endothelial tube formation in vitro and in vivo Importantly, our data revealed that the anti-angiogenic effect observed in high density CAC-EC co-cultures was significantly abrogated when PTX3 bioactivity was blocked using neutralizing antibodies or PTX3 siRNA in endothelial cells. We show evidence for an endothelial source of PTX3, triggered by exposure to high density CACs. In addition, we confirmed that PTX3 inhibits FGF2-mediated angiogenesis, and that the PTX3 N-terminus, containing the FGF-binding site, is responsible for such anti-angiogenic effects.

CONCLUSIONS: Endothelium, when exposed to high density CACs, releases PTX3 which markedly impairs the vascular regenerative response in an autocrine manner. Therefore, CAC density and accompanying release of angiocrine PTX3 are critical considerations when using these cells as a cell therapy for ischemic disease.