Resveratrol inhibits proteinase-activated receptor-2-induced release of soluble vascular endothelial growth factor receptor-1 from human endothelial cells

We recently reported that (i) activation of the proinflammatory receptor, proteinase-activated receptor-2 (PAR-2) caused the release of an important biomarker in preeclampsia, soluble vascular endothelial growth factor receptor-1 (sVEGFR-1, also known as sFlt-1) from human umbilical vein endothelial cells (HUVECs), and (ii) that the anti-oxidant and anti-inflammatory agent, resveratrol, is capable of inhibiting the proinflammatory cytokine-induced sVEGFR-1 release from human placenta. Based on these findings and because PAR-2 is up-regulated by proinflammatory cytokines, we sought to determine whether resveratrol can inhibit PAR-2-induced sVEGFR-1 release. PAR-2 expressing cells, HUVECs and human embryonic kidney cells (HEK-293) transfected with a human VEGFR-1 promoter-luciferase reporter construct were incubated with PAR-2-activating peptide and/or resveratrol. Cell supernatants were assayed for sVEGFR-1 by enzyme-linked immunosorbent assay (ELISA), and VEGFR-1 promoter-luciferase assay was performed on the harvested cell lysates. Preincubation of HEK-293 cells with resveratrol significantly inhibited PAR-2-induced VEGFR-1 promoter activity without affecting cell viability as assessed by MTT assay. The addition of resveratrol also blocked PAR-2-mediated sVEGFR-1 release from HUVECs. The present study demonstrates that resveratrol suppressed both VEGFR-1 promoter activity and sVEGFR-1 protein release induced by PAR-2 activation, which further endorses our recent findings of a potential therapeutic role for resveratrol in preeclampsia

Exhaustive exercise and vitamins C and E modulate thyroid hormone levels at low and high altitudes

Thyroid hormones play an important role in cell growth and differentiation and regulation of oxygen consumption and thermogenesis. The effect of altitude and vitamin supplementation on thyroid hormone levels in animals or humans performing acute exhaustive exercise have not been investigated before. Therefore, we thought to test whether exhaustive exerciseinduced stress with antioxidant supplementation was capable of modulating the level of thyroid hormones at different altitudes. Serum levels of T4 (Thyroxin), T3 (Triiodothyronine), and TSH (Thyroid Stimulating Hormone) were measured in rats (N=36) born and bred in low altitude (600 m above sea level) and high altitude (2200 m above sea level) following forced swimming with or without vitamins C and E (25 mg/kg) pre-treatments. Thyroid levels were significantly decreased in resting rats at high altitude compared to low altitude, and swimming exercise moderately increased T3 and TSH at both high and low altitudes, whereas T4 was markedly increased (62 %) at low altitude compared to a moderate high altitude increase (28 %). Co-administration of vitamins C and E augmented the observed forced swimminginduced thyroid release. However, the conversion of T4 to T3 was reduced in both altitude areas following swimming exercise and vitamin pre-treatment had no effect. We conclude that acute stress induced thyroidal hormones in rats, which was augmented by antioxidant drugs in both high and low altitude areas. These findings may play an important role in the human pathophysiology of thyroid gland at different altitudes

The Release of Nitric Oxide from S-Nitrosothiols Promotes Angiogenesis

BACKGROUND: Free nitric oxide (NO) reacts with sulphydryl residues to form S-nitrosothiols, which act as NO reservoirs. We sought to determine whether thiol-preserving agents and antioxidants, such as dithiothreitol (DTT) and vitamin C, induce NO release from S-nitrosylated proteins in endothelial cell cultures to promote angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: NO release was measured directly in cell supernatants using a Sievers NO Analyser, and in vitro angiogenesis was assessed by quantifying capillary-like tube network formation of porcine aortic endothelial cells (PAEC) on growth factor-reduced Matrigel. Incubation of PAEC with DTT or vitamin C significantly increased NO release in a concentration-dependent manner. However, the nitric oxide synthase (NOS) inhibitors, L-NNA and L-NIO, had no effect on DTT- or vitamin C-induced NO release, and there was no concomitant increase in the phosphorylation of endothelial NOS at serine-1177 following DTT or vitamin C treatment. DTT and vitamin C increased capillary-like tube network formation by nine- and two-fold, respectively, and the addition of copper ions doubled the effect of vitamin C. Surprisingly, DTT maintained endothelial tube networks for up to one month under serum-free conditions, and selective inhibitors of guanylyl cyclase (ODQ) and PKG (KT-5823) blocked this, demonstrating the requirement of cyclic GMP and PKG in this process. CONCLUSIONS/SIGNIFICANCE: Both DTT and vitamin C are capable of releasing sufficient NO from S-nitrosothiols to induce capillary morphogenesis. This study provides the first evidence that increased denitrosylation leads to increased bioavailability of NO, independent of NOS activity, to promote sustained angiogenesis

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

Angiopoietin-2 confers Atheroprotection in apoE-/- mice by inhibiting LDL oxidation via nitric oxide

Atherosclerosis is promoted by a combination of hypercholesterolemia and vascular inflammation. The function of Angiopoietin (Ang)-2, a key regulator of angiogenesis, in the maintenance of large vessels is unknown. A single systemic administration of Ang-2 adenovirus (AdAng-2) to apoE-/- mice fed a Western diet significantly reduced atherosclerotic lesion size 8 40%) and oxidized LDL and macrophage content of the plaques. These beneficial effects were abolished by the inhibition of nitric oxide synthase (NOS). In endothelial cells, endothelial NOS activation per se inhibited LDL oxidation and Ang-2 stimulated NO release in a Tie2-dependent manner to decrease LDL oxidation. These findings demonstrate a novel atheroprotective role for Ang-2 when endothelial cell function is compromised and suggest that growth factors, which stimulate NO release without inducing inflammation, could offer atheroprotection

Proteinase-activated receptor 2: differential activation of the receptor by tethered ligand and soluble peptide analogs

ABSTRACT Activation of rat proteinase-activated receptor 2 (PAR2) by trypsin involves the unmasking of the tethered sequence S 37 LIGRL 42 that either tethered or on its own as a free peptide, activates PAR2. We aimed to determine whether different peptide sequences acting either as trypsin-revealed tethered ligands or as soluble peptides had the same relative activities for triggering the receptor. A comparison was also made between the different soluble and tethered receptor activating sequences in receptor constructs with extracellular loop 2 (ECL2) residues E 232 E 233 (PAR2SR/EE) mutated to R 232 R 233 (PAR2SR/ RR). Using site-directed mutagenesis, we prepared PAR2 constructs with trypsin-revealed tethered ligand sequences corresponding to the synthetic receptor-activating peptides (PAR2APs): SLIGRL-NH 2 (SR-NH 2 ), SLIGAL-NH 2 (SA-NH 2 ), and SLIGEL-NH 2 (SE-NH 2 ). Kirsten virus-transformed rat kidney cells stably expressing 1) wild-type PAR2 with site-mutated tethered ligands (PAR2SA/EE and PAR2SE/EE); 2) wild-type PAR2 with ECL2 mutated to R 232 R 233 (PAR2SR/RR); and 3) PAR2 constructs with both the RR mutation in ECL2 and a mutation in the tethered ligand (PAR2SA/RR and PAR2SE/RR) were assessed for receptor-mediated calcium signaling and cell growth inhibition, upon activation either by trypsin or the above-mentioned PAR2APs. Trypsin exerted equivalent and full agonist activity on the PAR2 constructs, causing a maximum response between 20 to 80 nM. In contrast, the PAR2APs as free peptide agonists showed marked potency differences in all wild-type receptors with mutated tethered ligands (SR-NH 2 Ͼ Ͼ SA-NH 2 Ͼ Ͼ SE-NH 2 ) and in all ECL2 RR mutated constructs (SE-NH 2 Ͼ SR-NH 2 Ͼ Ͼ SA-NH 2 ). We conclude that for receptor activation, the trypsin-revealed PAR2 tethered ligand sequence interacts differently for receptor activation than does the same peptide sequence as a free peptide. PAR2 activation by trypsin involves the proteolytic unmasking of an amino terminal receptor sequence that acts as a tethered ligan

Nano particles as new disinfectan In Moringa oleifera

ABSTRACT. Contamination is a big problem in in vitro cultures, especially in woody plants. In this paper, different sterilization material been used to compare their efficiency in sterilization with Nanoparticles. Silver and copper Nanoparticles had been widely used in many fields. Plants in vitro rarely get used with nanoparticles. Silver &Cupper nanoparticles had used in many concentrations. It was clear that using these particles have a good benefit to get clean cultures, even with high concentrations. This paper gives us clear idea to use silver Nanoparticles as alternative cleaning solution