Phloretin attenuates hyperuricemia‐induced endothelial dysfunction through co‐inhibiting inflammation and GLUT9‐mediated uric acid uptake

Hyperuricemia is an important risk factor for cardiovascular and renal diseases. Phloretin had shown antioxidant and anti‐inflammatory properties, but its role in endothelial injury is rarely reported. In this study, we aimed to investigate the protective effect of phloretin on UA‐induced injury in human umbilical vein endothelial cells. The effects of UA and phloretin on cell viability, inflammation, THP‐1 monocyte adhesion, endothelial cell tube formation, GLUT9 expression and UA uptake in human umbilical vein endothelial cells were evaluated. The changes of nuclear factor‐kappa B/extracellular regulated protein kinases signalling were also analysed. Our results showed that UA reduced cell viability and tube formation, and increased inflammation and monocytes adhesion in human umbilical vein endothelial cells in a dose‐dependent manner. In contrast, phloretin significantly attenuated pro‐inflammatory factors expression and endothelial injury induced by UA. Phloretin inhibited the activation of extracellular regulated protein kinases/nuclear factor‐kappa B pathway, and reduced GLUT9 and it mediated UA uptake in human umbilical vein endothelial cells. These results indicated that phloretin attenuated UA‐induced endothelial injury via a synergic mechanism including direct anti‐inflammatory effect and lowering cellular UA uptake. Our study suggested that phloretin might be a promising therapy for hyperuricemia‐related cardiovascular diseases.     

Exposure to polychlorinated biphenyls causes endothelial cell dysfunction

Environmental chemicals, such as polychlorinated biphenyls (PCBs), may be atherogenic by disrupting normal functions of the vascular endothelium. To investigate this hypothesis, porcine pulmonary artery-derived endothelial cells were exposed to 3,3′,4,4′-tetrachlorobiphenyl (PCB 77), 2,3,4,4′,5-pentachlorobiphenyl (PCB 114), or 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) for up to 24 hours. These PCBs were selected for their varying binding avidities with the aryl hydrocarbon (Ah) receptor and differences in their induction of cytochrome P450. PCB 77 and PCB 114 significantly disrupted, in a dose-dependent manner, endothelial barrier function by allowing an increase in albumin transfer across endothelial monolayers. These PCBs also contributed markedly to cellular oxidative stress, as measured by 2,7-dichlorofluorescin (DCF) fluorescence and lipid hydroperoxides, and caused a significant increase in intracellular calcium ([Ca²⁺]_i) levels. Enhanced oxidative stress and [Ca²⁺]_i in PCB 77- and PCB 114-treated cells were accompanied by increased activity and content of cytochrome P450 1A and by a decrease in the vitamin E content in the culture medium. In contrast to the effects of PCB 77 and PCB 114, cell exposure to PCB 153 had no effect on cellular oxidation, [Ca²⁺]_i, or endothelial barrier function. These results suggest that certain PCBs may play a role in the development of atherosclerosis by causing endothelial cell dysfunction and a decrease in the barrier function of the vascular endothelium. It is possible that interaction of PCBs with the Ah receptor and activation of the cytochrome P450 1A subfamily are involved in this pathology. © 1995 John Wiley & Sons, Inc.     

Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor‐mediated quorum sensing

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor‐mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis‐2‐decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.     

Hyperosmolarity‐mediated mitochondrial dysfunction requires Transglutaminase‐2 in human Corneal epithelial cells

Hyperosmolar‐induced ocular surface cell death is a key mitochondria‐mediated event in inflammatory eye diseases. Transglutaminase (TGM)‐2, a cross‐linking enzyme, is purported to mediate cell death, but its link to mitochondria is unclear. In the cornea, the integrity of the epithelial cells is important for maintaining transparency of the cornea and therefore functional vision. We evaluated the role of TGM‐2 and its involvement in hyperosmolarity‐stimulated mitochondrial cell death in human corneal epithelial (HCE‐T) cells. HCE‐T cell lines stably expressing either shRNA targeting TGM‐2 (shTG) or scrambled shRNA (shRNA) were constructed. Hyperosmolar conditions reduced viability and increased mitochondrial depolarization in shRNA cells. However, hyperosmolarity failed to induce mitochondrial depolarization to the same extent in shTG cells. Transient overexpression of TGM‐2 resulted in very high levels of TGM‐2 expression in shTG and shRNA cells. In the case of shTG cells after overexpression of TGM‐2, hyperosmolarity induced the same extent of mitochondrial depolarization as similarly treated shRNA cells. Overexpression of TGM‐2 also elevated transamidase activity and reduced viability. It also induced mitochondrial depolarization, increased caspase‐3/7 and ‐9 activity, and these increases were partially suppressed by pan‐caspase inhibitor Z‐VAD‐FMK. Corneal epithelial apoptosis via mitochondrial dysfunction after hyperosmolar stimulation is partially dependent on TGM‐2. This TGM‐2‐dependent mechanism occurs in part via caspase‐3/7 and ‐9. Protection against mitochondrial stress in the ocular surface targeting TGM‐2 may have important implications in the survival of cells in hyperosmolar stress. J. Cell. Physiol. 226: 693–699, 2011. © 2010 Wiley‐Liss, Inc.     

BDNF‐mediated migration of cardiac microvascular endothelial cells is impaired during ageing

This study indicates that brain‐derived neurotrophic factor (BDNF) can promote young cardiac microvascular endothelial cells (CMECs) to migrate via the activation of the BDNF‐TrkB‐FL‐PI3K/Akt pathway, which may benefit angiogenesis after myocardial infarction (MI). However, the ageing of CMECs led to changes in the expression of receptor Trk isoforms in that among the three isoforms (TrkB‐FL, TrkB‐T1 and TrkB‐T2), only one of its truncated isoforms, TrkB‐T1, continued to be expressed, which leads to the dysfunction of its ligand, a decrease in the migration of CMECs and increased injury in ageing hearts. This shift in receptor isoforms in aged CMECs, together with changes in the ageing microenvironment, might predispose ageing hearts to decreased angiogenic potential and increased cardiac pathology.     

Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta

Irradiation of the heart and vasculature can cause a spectrum of cardiovascular complications, including increased risk of myocardial infarction or coronary heart disease. Although irradiation is implicated in oxidant stress and chronic inflammation, the underlying molecular mechanisms have not been elucidated. We tested the hypothesis that irradiation-initiated upregulation of xanthine oxidase (XO), a primary source of cardiovascular reactive oxygen species, contributes to endothelial dysfunction and increased vascular stiffness. Twenty-two, 3-month-old Sprague–Dawley male rats were gamma-irradiated at the following doses: 0, 50, 160, and 500 cGy. Rats exposed to 500 cGy showed a significant increase in endothelial XO expression and a twofold increase in XO activity, compared to the 0 cGy controls. Endothelial function was investigated ex vivo through vascular tension dose–responses to the endothelial dependent vasodilator, acetylcholine. Endothelial-dependent relaxation in aorta of the 500 cGy exposed rats was significantly attenuated from the control group. Remarkably, specific inhibition of XO with oxypurinol restored the relaxation response to that of the control. Furthermore, these ex vivo results are reflected in vivo through alterations in vascular stiffness, as measured by pulse wave velocity (PWV). As early as 1-day post-exposure, rats exhibited a significant increase in PWV from pre-exposure. The PWV of irradiated rats (50, 160, and 500 cGy) were greater than those of 0 cGy control rats at 1 day, 1 and 2 weeks. The sham and irradiated rats possessed equivalent pre-exposure PWV, with sham showing no change over 2 weeks. Thus, these findings suggest that early upregulation of XO contributes to oxidative stress and endothelial nitro-redox imbalance with resultant endothelial dysfunction and altered vascular mechanics. Furthermore, these data identify XO as a potential molecular target for attenuating irradiation-induced cardiovascular injury.     

Lysosome-membrane fusion mediated superoxide production in hyperglycaemia-induced endothelial dysfunction

Lysosomal exocytosis and fusion to cellular membrane is critical in the oxidative stress formation of endothelium under apoptotic stimulus. We investigated the role therein of it in hyperglycaemia-induced endothelial dysfunction. The lysosome-membrane fusion was shown by the expression of lamp1, the lysosomal membrane marker, on cellular membrane and the transportation of lysosomal symbolic enzymes into cultural medium. We also examined the ceramide production, lipid rafts (LRs) clustering, colocalization of gp91^phox, a NADPH oxidase subunit (NOX) to LRs clusters, superoxide (O₂ ^{. -}) formation and nitric oxide (NO) content in human umbilical vein endothelial cells (HUVEC) and the endothelium-dependent NO-mediated vasodilation in isolated rat aorta. As compared to normal glucose (5.6 mmol/l, Ctrl) incubation, high glucose (22 mmol/l, HG) exposure facilitated the lysosome-membrane fusion in HUVEC shown by significantly increased quantity of lamp1 protein on cellular membrane and enhanced activity of lysosomal symbolized enzymes in cultural medium. HG incubation also elicited ceramide generation, LRs clustering and gp91^phox colocalization to LRs clusters which were proved to mediate the HG induced O₂ ^{. -} formation and NO depletion in HUVEC. Functionally, the endothelium-dependent NO-mediated vasodilation in aorta was blunted substantially after HG incubation. Moreover, the HG-induced effect including ceramide production, LRs clustering, gp91^phox colocalization to LRs clusters, O₂ ^{. -} formation and endothelial dysfunction could be blocked significantly by the inhibition of lysosome-membrane fusion. We propose that hyperglycaemia-induced endothelial impairment is closely related to the lysosome-membrane fusion and the following LRs clustering, LRs-NOX platforms formation and O₂ ^{. -} production.     

Epac1 increases migration of endothelial cells and melanoma cells via FGF2‐mediated paracrine signaling

Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N‐sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N‐sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2‐mediated cell–cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM‐induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N‐sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS–FGF2‐mediated cell–cell communication.     

Linoleic acid hydroperoxide increases the transfer of albumin across cultured endothelial monolayers

Exposure to albumin-bound linoleic acid (60 to 150 microM) for 24 h significantly increased the rate of albumin transfer across cultured endothelial monolayers. The increase was dependent on the linoleic acid (18:2) concentration to which the cultures were exposed. Linoleic acid hydroperoxide (18:2-OOH) further accelerated the rate of albumin transfer over that of 18:2. A near maximum albumin transfer was observed after a 2-h incubation with 90 microM 18:2-OOH. Cells exposed to 18:2-OOH caused a marked release of lactate dehydrogenase into the media. On the other hand, 18:2 at concentrations as high as 150 microM, did not significantly affect lactate dehydrogenase release. These results suggest that exposure to 18:2, and in particular to 18:2-OOH, reduces the ability of the endothelium to act as a selective permeability barrier to plasma components.     

Hypoxic trophoblast‐derived sFlt‐1 may contribute to endothelial dysfunction: implication for the mechanism of trophoblast—endothelial dysfunction in preeclampsia

The maternal systemic disorder of widespread endothelial dysfunction is a primary focus in understanding the development of preeclampsia. sFlt‐1 (soluble fms‐like tyrosine kinase receptor 1), an endogenous inhibitor of VEGF (vascular endothelial growth factor), may play important roles in endothelial dysfunction. The present study aimed to determine whether hypoxic trophoblast‐derived sFlt‐1 could lead to endothelial dysfunction by establishing a cocultured model of anoxic TEV‐1s (human first‐trimester extravillous trophoblasts) and HUVECs (human umbilical vein endothelial cells). The results showed that the hypoxic treatment significantly promoted sFlt‐1 mRNA and protein expression in TEV‐1s in a time‐dependent manner compared with the effect in HUVECs. When HUVECs were cocultured with anoxic TEV‐1s, the endothelial function, which was characterized by NO (nitric oxide) synthesis and monolayer barrier function of HUVECs, were notably decreased, accompanied by increasing sFlt‐1 and decreasing VEGF in cell‐conditioned medium. Moreover, the observed endothelial dysfunction described above was consistent with the dysfunction observed in VEGF siRNA‐treated cultures. The findings presented herein imply that chronically hypoxic trophoblasts may release sufficient sFlt‐1 to cause endothelial dysfunction by depriving cells of VEGF activity.     

Honokiol ameliorates angiotensin II‐induced hypertension and endothelial dysfunction by inhibiting HDAC6‐mediated cystathionine γ‐lyase degradation

Hypertension and endothelial dysfunction are associated with various cardiovascular diseases. Hydrogen sulphide (H₂S) produced by cystathionine γ‐lyase (CSE) promotes vascular relaxation and lowers hypertension. Honokiol (HNK), a natural compound in the Magnolia plant, has been shown to retain multifunctional properties such as anti‐oxidative and anti‐inflammatory activities. However, a potential role of HNK in regulating CSE and hypertension remains largely unknown. Here, we aimed to demonstrate that HNK co‐treatment attenuated the vasoconstriction, hypertension and H₂S reduction caused by angiotensin II (AngII), a well‐established inducer of hypertension. We previously found that histone deacetylase 6 (HDAC6) mediates AngII‐induced deacetylation of CSE, which facilitates its ubiquitination and proteasomal degradation. Our current results indicated that HNK increased endothelial CSE protein levels by enhancing its stability in a sirtuin‐3‐independent manner. Notably, HNK could increase CSE acetylation levels by inhibiting HDAC6 catalytic activity, thereby blocking the AngII‐induced degradative ubiquitination of CSE. CSE acetylation and ubiquitination occurred mainly on the lysine 73 (K73) residue. Conversely, its mutant (K73R) was resistant to both acetylation and ubiquitination, exhibiting higher protein stability than that of wild‐type CSE. Collectively, our findings suggested that HNK treatment protects CSE against HDAC6‐mediated degradation and may constitute an alternative for preventing endothelial dysfunction and hypertensive disorders.     

G protein‐coupled estrogen receptor‐mediated effects on cytosolic calcium and nanomechanics in brain microvascular endothelial cells

G protein‐coupled estrogen receptor (GPER) is a relatively recently identified non‐nuclear estrogen receptor, expressed in several tissues, including brain and blood vessels. The mechanisms elicited by GPER activation in brain microvascular endothelial cells are incompletely understood. The purpose of this work was to assess the effects of GPER activation on cytosolic Ca²⁺ concentration, [Ca²⁺]_i, nitric oxide production, membrane potential and cell nanomechanics in rat brain microvascular endothelial cells (RBMVEC). Extracellular but not intracellular administration of G‐1, a selective GPER agonist, or extracellular administration of 17‐β‐estradiol and tamoxifen, increased [Ca²⁺]_i in RBMVEC. The effect of G‐1 on [Ca²⁺]_i was abolished in Ca²⁺‐free saline or in the presence of a L‐type Ca²⁺ channel blocker. G‐1 increased nitric oxide production in RBMVEC; the effect was prevented by NG‐nitro‐l ‐arginine methyl ester. G‐1 elicited membrane hyperpolarization that was abolished by the antagonists of small and intermediate‐conductance Ca²⁺‐activated K⁺ channels, apamin, and charibdotoxin. GPER‐mediated responses were sensitive to G‐36, a GPER antagonist. In addition, atomic force microscopy studies revealed that G‐1 increased the modulus of elasticity, indicative of cytoskeletal changes and increase in RBMVEC stiffness. Our results unravel the mechanisms underlying GPER‐mediated effects in RBMVEC with implications for the effect of estrogen on cerebral microvasculature.

     

Oleic acid supplementation reduces oxidant-mediated dysfunction of cultured porcine pulmonary artery endothelial cells

We have previously shown that supplementing cultured porcine pulmonary artery endothelial cells (PAEC) with exogenous oleic acid (18:1ω9) alters the fatty acid composition of the cells and reduces oxidant-mediated cytotoxicity. Because the mechanisms by which lipid alterations modulate oxidant susceptibility have not been defined, the ability of 18:1 to reduce hydrogen peroxide (H₂O₂)-mediated PAEC dysfunction was evaluated. PAEC monolayers on polycarbonate filters were incubated for 3 h in maintenance medium supplemented with either 0.1 mM 18.1 in ethanol vehicle (ETOH) or with an equivalent volume of vehicle alone. Twenty-four hours later monolayers were treated for 30 min with 50 or 100 μM H₂O₂ in Hanks' balanced salt solution (HBSS) or with HBSS alone (nonoxidant control). As a functional index of PAEC monolayer integrity, the permeability of monolayers to albumin was then measured for 3 h. Treatment with 100 μM H₂O₂ caused cytotoxicity and progressive increases in PAEC monolayer permeability that were attenuated by 18:1 supplementation, whereas 50 μM H₂O₂ caused only a transient increase in permeability without cytotoxicity. Supplementation with 18:1 also attenuated H₂O₂-induced reductions in PAEC adenosine triphosphate (ATP) content and disruption of PAEC microfilament architecture. The ATP content of PAEC monolayers was reversibly reduced in the absence of oxidant stress by incubation with glucose-depleted medium containing deoxyglucose and antimycin A. Metabolic inhibitor-induced ATP depletion increased monolayer permeability and altered cytoskeletal architecture, alterations that resolved during recovery of PAEC ATP content. These results demonstrate that ATP depletion plays a critical role in barrier dysfunction and suggests that the ability of 18:1 to reduce oxidant-mediated PAEC dysfunction and injury may relate directly to its ability to preserve PAEC ATP content. © 1993 Wiley-Liss, Inc.