Mechanisms Of Inflammation On Primary Baboon Femoral Artery Endothelial Cells And Intervention Studies

BackgroundIt is well recognized that endothelial cell cytotoxicity induced by inflammatory factors plays a key role in the pathogenesis of cardiovascular disease. TNF-alpha, an endothelial cell-derived cytokine commonly found in atherosclerotic lesions, can promote apoptosis and inflammation[1], which subsequently contribute to endothelial cell injury and cellular dysfunction[3], So attenuating TNF-alpha induced endothelial cell cytotoxicity is important in preventing cardiovascular disease.Epidemiological studies have shown that Mediteranean diets are associated with reduced risk of cardiovasular diseases [4,5]. Resveratrol (RSV), a polyphenol found in grapes and red wine, is the most important constituent involved in mediteranean dietary. Resveratrol has been shown to have direct cardiovascular protective effect by improving myocardial perfusion, enhancing angiogenesis, reducing oxidant stress or inhibiting platelet aggregation [6-9]. However, the precise mechanism of its action is not completely understood.In the present study, we examined whether resveratrol could attenuate endothelial cell injury generated by TNF-alpha in baboon femoral arterial endothelial cells (BAECs) and investigated the molecular mechanisms involved in the process. Methods1. Cell cultureBaboon Femoral Arterial Endothelial Cells (BAEC) were isolated from baboon femoral arteries. Baboons were immobilized and a2-cm segment of femoral artery in the upper part of the thigh was obtained under sterile surgical procedure. The artery was digested with0.1%collagenase I at37℃for15min. The released cells were seeded immediately on1.0%gelatin-coated culture plates. Primary BAEC were cultured in F-12K growth medium supplemented with20%FCS, and treated with tumor necrosis factor (TNF lOng/mL) with and without resveratrol at various concentrations and for the different time periods indicated in the text.2. CD31+magnetic beads selectionBAECs were labeled by CD31+immunomagnetic beads for endothelial selection, purification and cell culture.3. Endothelial function and cytoskeleton determinationImmunocytochemistry method was simplified as fixiation, blocking, primary antibody incubation, washing, secondary antibody incubation, DAPI staining, mounted with slowfade reagents and observed under fluorescence microscopy. Dil-LDL/UEA-1, vWF and VE-cadherin are specific markers of endothelial cells, F-actin and β-tubulin are non-specific cytoskeleton staining of endothelial cells, a-actin is relatively specific staining of the fiberblast cells.4. Endothelial proliferation assayMTT assay was used to test proliferation of BAECs with different treatments by resveratrol (RSV,0.1-100μmol/L) and/or TNF-α(10ng/mL). According to the protocol of MTT assay, BAECs were incubated with20μl (1mg/ml) MTT for4hours at37℃to allow MTT to form formazan crystals and disolved by150μl of DMSO. The absorbance of each well was measured by a microplate reader at490nm wavelength (A490). 5. Endothelial cellular adhension moleculars expressionFlowcytometry was used to test the expression of endothelial cellular adhension moleculars (CAMs) under different treatment with resveratrol (RSV,0.1-100μmol/L) and/or TNF-α (10ng/mL).Following antibodies were used in this study:anti-human CD62E (clone BBIG-E5; R&D Systems, Inc), anti-human CD54(clone HA58; BD Biosciences, San Jose, CA), and anti-human CD106(clone5K267; US Biological, Swampscott, MA).6. Cytokine expressionELISA was used to quantification of, test the expression of cytokine production induced by TNF-α(10ng/mL).Commercial enzyme-linked immunosorbent assay kits from R&D Systems were used for the measurement of MCP-1and Human Cytokine Lincoplex kit (LINCO Research Inc, MO) for the measurement of measurement of IL-1β.7. Determination of inflammatory injury of endothelial cellsWound-Healing Assay was used to test endothelial migration with treatments by resveratrol (RSV,0.1-100μmol/L) and/or TNF-α (10ng/mL) incubation. Tube formation was used to test in vitro angiogenesis ability of BAECs with RSV and TNF-α (10ng/mL) incubation.Results1. Isolation and Identification of BAECsBaboon arterial endothelial cells have a typical cobblestone shape (Fig.l A, B), take3-7days to reach confluence according to cell seeding density and passages. We identified BAEC by staining the cells with VE-Cadherin, vWF, CD31+magnetic beads selection and Dil-LDL/UEA uptake and expression of cellular adhension molecules. 2. BAECs can be activated by TNF-α(10ng/ml)BAEC can be activated by TNF-α and highly express ICAM-1, VCAM-1and E-selectin, by41.38,3-5,12.2folds when compared with normal control。As well as increased MCP-1and IL-1β secretion。 Proliferation and migration of BAEC can be impared by TNF-α(10ng/ml).3. RSV can attenuate impairment of BAECs induced by TNF-alphaRSV can improve proliferation and migration ability of BAECs impaired by TNF-alpha, especially at10,50μmol/L. RSV (10,50μmol/L) can inhibit TNF-alpha induced expression of VCAM (CD106) and ICAM (CD54) in BAECs, when BAECs were incubated by RSV and TNF-a together. While RSV pre-treatment and after-treatment has no effects on activated BAECs. This anti-inflammation effect can be attributed to the inhibition of NF-K B singnal pathway activated by TNF-aConclusions1. Primary femeral arterial endothelial cells which can be constantly abtained from baboon with typical cobblestone morphology and was identified by vWF/VE-Cadherin immunocytochemistry staining and CD31+magnetic beads selection.2. RSV improves BAEC proliferation and attenuates BAECs migration impaired by TNF-alpha.3. RSV can inhibit TNF alpha induced expression of VCAM (CD106) and ICAM (CD54) in BAECs, which may be attributed to inhibition of NF-K B activated by TNF-alpha in BAECs, while no effects can be seen in pretreatment and aftertreatment incubation. BackgroundSince its discovery30years ago, oxidative modification of LDL is considered an important mechanism in atherogenesis. Results from animal models and human subjects suggest that LDL and its derivatives are involved in endothelial dysfunction creating a procoagulatory and proinflammatory local environment, which is a key step in the genesis of both stable and unstable atherosclerosis. However, the mechanisms by which LDL mediates these effects have been debated for decades; oxidized LDL preparations by individual investigators have shown numerous pro-and antiatherogenic properties. While neither LDL particle concentration nor the fatty acid composition of the particles has been proven to promote atherosclerosis directly, a strong relationship between LDL and risk of coronary vascular disease has been well documented.The endothelium, a monolayer tissue lining on the inner surface of blood vessels, is the first target of circulating LDL. However, assessment of the effect of LDL on endothelial cells has produced conflicting results. For example, while some have reported that LDL or oxidized LDL causes endothelial apoptosis, others have claimed that these substances have growth-promoting effects. Because oxidative LDL preparations have neither been well defined nor thoroughly characterized, the heterogeneous nature of the preparations due to their sources, methods of preparation and treatment, and conditions of storage and use has been attributed to be responsible for the variable pathophysiological effects of LDL on the endothelium. In addition, there are few data demonstrating whether LDL damages the endothelium directly by itself or indirectly in concert with other cell types. To better understand LDL-mediated effects, we investigated both the effects of defined LDL preparations on endothelial cells and the potential involvement of monocytes in mediating the impact of LDL on endothelial cells.This study was conducted using an Old World nonhuman primate, the baboon. There is increasing awareness that animal models play a critical role in providing relevant information about mechanistic questions. Different animal models have provided insights into mechanisms of atherogenesis, but large animals, especially nonhuman primates, are better suited for translation to humans. This is particularly important in regard to investigations involving LDL because the oxidation of LDL is a complex process during which both the proteins and the lipids undergo oxidative changes and form complex products. Since baboon and human LDL are remarkably similar, the baboon provides us a unique opportunity to address this question. At the Southwest National Primate Research Center, we are able to obtain a large and steady supply of blood and arteries that are from healthy baboons for experimental use and that give consistent and reproducible results. Our results have a direct impact on understanding the relationship of LDL to atherosclerosis in human beings.MethodsIsolation and oxidation of LDLPlasma was ultracentrifuged at105,000x g for24h at4℃. LDL in the density range of1.019-1.063g/mL was collected.0.1mM antioxidant butylated hydroxytoluene (BHT) was added to the plasma that was used for obtaining native LDL. The LDL preparation was then sterilized by passing through a0.2-μM filter. Extensively oxidized LDL was prepared by adding freshly prepared CuSO4solution at the final concentration of5μM at37℃for6hours. Minimally oxidized LDL was obtained by incubation of freshly isolated LDL at37℃for6hours without CuSO4. Endothelial cell and monocyte isolation and cultureBaboons were anesthetized under10mg/kg ketamine hydrochloride, and a2-4cm segment of femoral artery in the upper part of the thigh was obtained under sterile surgical procedure. The artery was gently digested with0.1%collagenase at37℃for20min. The released cells were seeded immediately on1.0%gelatin-coated culture plates. Cells were allowed to reach70-90%confluence until treatments.Peripheral blood mononuclear cells (PBMNCs) were obtained from buffy coats using a Ficoll gradient and centrifuged for10min at1300rpm to pellet cells. And incubated overnight at37℃in5%CO2. Attached cells were detached by add-ing10mL PBS/0.53mM EDTA and incubated for10min at37℃in a5%CO2incubator. The cells were identified by flowcytometry with anti-CD14-FITC and anti-CD36-FITC incubation. And monocyte functionalities were determined by measuring uptake of Dil-LDL and UEA-1using fluorescence microscopy.Monocyte activation by LDLWe exposed cells to50and100μg/ml native LDL for0,0.5,1,4,8,12and24hours in replicate wells.500ng/mL LPS was used as a positive control. We measured TNF-a, IL-1β and MCP-1with ELISA kits.Endothelial activation by LDL and monocyte co-cultureWe added native LDL, minimally oxidized LDL, and extensively oxidized LDL at indicated concentrations for24hours. In regard to co-culture, monocytes were seeded into the co-culture insert (8.0μm pore) on top of the endothelial layer at a density of0.5-1.0×106/well in the12-well plates in0.5mL F-12K medium with2%FCS. We added50μg/ml or100μg/ml native LDL in2%FCS medium for24hours; equal volume of PBS and10ng/mL TNF-a were added as vehicle and positive controls. Triplicate wells were evaluated for each treatment. Endothelial activation was determined by examining either cellular adhesion molecule expression or MCP-1release.Roles of TNF-a and IL-1β in monocyte conditioned media on endothelial activationWe added100μg/mL native LDL to monocyte cultures and collected conditioned media24hours later and divided the conditioned media into two parts; we added10μg/mL goat anti-human TNF-a and10μg/mL anti-human IL-1β IgG to the neutralization groups and an equal amount of nonspecific goat IgG to the control groups. After incubation for4hours at37℃in a humidified CO2incubator, the control and neutralized groups were separately added to endothelial cell cultures and incubated for an additional4hours. Endothelial activation was monitored by expression of E-selectin, ICAM-1and VCAM-1using flow cytometry.ResultsCharacteristics of LDL preparationsNative LDL had TBARS content ranging from zero to2nmol/mg protein, and extensively oxidized LDL had TBARS content from160to200nmol/mg protein. Extensively oxidized LDL had a higher electrophoretic mobility than minimally oxidized LDL and native LDL. Minimally oxidized LDL often showed a diffuse band of electrophoresis. Native LDL prepared using the current protocol retained maximally the chemical and physical features of freshly isolated LDL.Effects of LDL preparations on endothelial cell growthNative LDL at a lower dosage (50μg/mL) had no effect on cell growth, but a higher dosage of100μg/ml led to cell loss after24hours of incubation.Minimally oxidized LDL at concentrations of50and100μg/mL had no significant changes in baboon endothelial cells or human arterial endothelial cells (HAEC) after24hours of incubation.Comprised to native LDL and minimally oxidized LDL. Extensively oxidized LDL caused significant cell loss, which can be confirmed by EC morphology and by Annexin V detection, whereas native or minimally oxidized LDL caused minimal damage.Cytokine expressionCompared with TNF-a, various LDL preparations were not potent stimuli for endothelial CAM expression, even at24h incubation.Extensively oxidized LDL tremendously decreased MCP-1expression, while native LDL and minimally oxidized LDL tends to maintain endothelial cells and activate them to secrete chemokine MCP-1.Native LDL increased the expression of TNF-a and IL-1β in dosage-and time-dependent manners, and significant decreased CD36levels of monocyte.Interactive effects of LDL and monocytes on endothelial cellsNative LDL greately increased E-Selectin, VCAM and ICAM expression of endothelial cells when co-cultured with monocyte, which can be inhibited by neutralized media with anti-humans TNF-a and10μg/mL anti-human IL-1β IgG.Conclusions1. We set up standard method to define three LDL preparations with varying extents of oxidation from the same source for the first time.2. We demonstrated in this study that LDL-mediated effects were largely dependent on the extent of LDL oxidation.3. We explored the synergistic effects of monocytes and native LDL in endothelial cell activation under co-culture system, which can be blocked by anti-humans TNF-a and10μg/mL anti-human IL-1β IgG.4. Our results also suggest that genetic variation controlling endothelial and monocyte response to circulating LDL may have an important role in determining risk of atherosclerosis.