EC Exposure Research Articles

Direct-contact co-culture between smooth muscle and endothelial cells inhibits TNF-α-mediated endothelial cell activation

We used a direct-contact endothelial cell-smooth muscle cell (EC-SMC) co-culture to examine whether quiescent SMCs regulate the EC inflammatory response to tumor necrosis factor (TNF)-alpha. ECs were cultured under static and physiological flow conditions. Compared with TNF-alpha-treated ECs in monoculture, TNF-alpha-treated ECs in co-culture had less NF-kappaB nuclear translocation; less intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin surface protein expression; no change in TNF receptor expression, but greater Kruppel-like factor 2 (KLF2) gene expression. After flow preconditioning for 24 h at 15 dyne/cm(2), and exposure of ECs to flow and TNF-alpha for 4.5 h, ECs in co-culture had less ICAM-1, VCAM-1, and E-selectin surface protein expression. Exposure to flow greatly increased KLF2 gene expression levels in both EC cultures; as a result, ECs in co-culture and monoculture had similar levels of post-flow KLF2 gene expression. The reduced levels of TNF-alpha-induced adhesion molecule expression in co-culture required the presence of quiescent SMCs; adhesion to decellularized extracellular matrix (ECM) or co-culture with fibroblasts produced only a modest reduction in EC adhesion molecule expression. Furthermore, co-culture of quiescent SMCs and ECs on the opposite side of a 10-microm-thick porous membrane did not alter the TNF-alpha-mediated ICAM-1 surface protein expression. Although the ECM produced by SMCs plays some role in reducing TNF-alpha-mediated inflammation, these results suggest that the direct contact between ECs and quiescent SMCs is required to inhibit TNF-alpha-mediated activation.

Rapamycin Regulates Endothelial Cell Migration through Regulation of the Cyclin-dependent Kinase Inhibitor p27Kip1

Rapamycin is a macrolide antibiotic that inhibits vascular smooth muscle cell proliferation and migration and that is used clinically on drug-eluting stents to inhibit in-stent restenosis. Although inhibition of cell migration is an asset in preventing restenosis, it also leads to impaired stent endothelialization, a significant limitation of current drug-eluting stent technology that necessitates prolonged antiplatelet therapy. We measured the ability of rapamycin to inhibit the migration of human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAEC) toward the chemoattractant vascular endothelial cell growth factor. Although acute administration of rapamycin had no effect, exposure for 24 h inhibited HUVEC and HCAEC migration. Disruption of the mTORC2 via small interfering RNA was also effective in inhibiting HCAEC migration. Treatment of HCAECs for this period with rapamycin produced an increase in the cyclin-dependent kinase inhibitor p27(Kip), through a decrease in the targeting of the protein for degradation by phosphorylation at Thr(187). ECs isolated from a knock-in mouse expressing p27(Kip1) with a mutation of this residue to an alanine, blocking this phosphorylation, exhibited reduced migration compared with wild-type controls. Silencing of p27(Kip1) with small interfering RNA blocked the effects of rapamycin on migration and tube formation as well as RhoA activation and cytoskeletal reorganization. We conclude that prolonged exposure of ECs to rapamycin increases p27(Kip1) and in turn inhibits RhoA activation, blocking cell migration and differentiation. These data elucidate the molecular mechanism underlying regulation of p27(Kip1) protein and cell migration by rapamycin.

Realistic Temporal Variations of Shear Stress Modulate MMP-2 and MCP-1 Expression in Arteriovenous Vascular Access

Venous neointimal hyperplasia (VNH) lesions are prone to localized development within the vascular access junction (VAJ) and efferent vein of arteriovenous (AV) fistulae and grafts. The creation of VAJ dramatically alters the local venous hemodynamics with high pulsatile flow velocities enter the vein resulting in blood-flow separation, recirculation and flow reversal. This study conducted a computational hemodynamic investigation of an idealized AV graft and realistic AV fistula which demonstrated a complex hemodynamic environment within the VAJ, producing elevated wall shear stress (WSS) magnitudes and significant spatial and temporal WSS gradients in the VAJ. These hemodynamic patterns and non-physiological WSSs are postulated to initiate VNH development at the transcriptional level. Human umbilical vein endothelial cells (HUVEC) were exposed to elevated temporal WSS waveforms obtained from the aforementioned computational analysis, using a cone-and-plate bioreactor. Using real-time RT-PCR, early induction of MMP-2 and delayed transcriptional upregulation of MCP-1 was observed following EC exposure to VAJ high wall shear forces. These results indicate that MMP-2 and MCP-1 may be induced by high WSS present in the VAJ, suggesting a link between elevated WSS magnitudes and temporal gradients, extracellular matrix decomposition, smooth muscle cell migration and proliferation, and the subsequent VNH development in AV VAJs.

Source proximity and residential outdoor concentrations of PM2.5, OC, EC, and PAHs

We examined the effect of proximity to specific mobile, area, and point sources on the residential outdoor concentrations of fine particulate matter PM (PM(2.5)) and several of its particle components. Integrated (48-h) PM(2.5) samples were collected outside non-smoking residences in Elizabeth, NJ, between summer 1999 and spring 2001. Samples were analyzed for PM(2.5) mass, organic and elemental carbon (OC and EC, respectively), trace elements, particle-phase polycyclic aromatic hydrocarbons (p-PAHs), and other important particle species. Information about the proximity of the study homes to potential mobile and area sources of OC, EC, p-PAHs, sulfur (S), and selenium (Se) (including urban interstate highways, local roadways, the Newark International Airport, the Elizabeth seaport, and a nearby refinery in Linden, NJ) were retrieved from a database that included detailed emissions, meteorological, and geographical data for the study area. The dependence of residential outdoor concentrations on source proximity and on various meteorological parameters was then examined for each species by multiple linear regression analysis. As expected, the predicted ambient air concentrations of all particle species (except S, Se) decreased with increasing distance from the sources. Although the enhancement in PM(2.5) and OC levels outside the study homes closest to primary PM sources was modest (e.g., 1.6 and 2.5 times the background levels 37 m from interstate highways), the elevation of EC and p-PAH concentrations was substantial outside the closest study homes (i.e., about 20 times for p-PAHs 37 m from interstate highways and about 14 times for EC 192 m from the refinery in Linden, NJ). The predicted EC concentrations 192 and 500 m from the oil refinery were 22.8 and 3.0 microgC/m(3), compared with an urban background of 1 microgC/m(3). Thus, emissions from this source might dramatically affect EC exposure for residents living in its close proximity.

Candida albicans Triggers Activation of Distinct Signaling Pathways to Establish a Proinflammatory Gene Expression Program in Primary Human Endothelial Cells

Endothelial cells (EC) actively participate in the innate defense against microbial pathogens. Under unfavorable conditions, defense reactions can turn life threatening resulting in sepsis. We therefore studied the so far largely unknown EC reaction patterns to the fungal pathogen Candida albicans, which is a major cause of lethality in septic patients. Using oligonucleotide microarray analysis, we identified 56 genes that were transcriptionally up-regulated and 69 genes that were suppressed upon exposure of ECs to C. albicans. The most significantly up-regulated transcripts were found in gene ontology groups comprising the following categories: chemotaxis/migration; cell death and proliferation; signaling; transcriptional regulation; and cell-cell contacts/intercellular signaling. Further examination of candidate signaling cascades established a central role of the proinflammatory NF-kappaB pathway in the regulation of the Candida-modulated transcriptome of ECs. As a second major regulatory pathway we identified the stress-activated p38 MAPK pathway, which critically contributes to the regulation of selected Candida target genes such as CXCL8/IL-8. Depletion of MyD88 and IL-1R-associated kinase-1 by RNA interference demonstrates that Candida-induced NF-kappaB activation is mediated by pattern recognition receptor signaling. Additional experiments suggest that C. albicans-induced CXCL8/IL-8 expression is mediated by TLR3 rather than TLR2 and TLR4, which previously have been implicated with MyD88/IkappaB kinase-2/NF-kappaB activation by this fungus in other systems. Our study provides the first comprehensive analysis of endothelial gene responses to C. albicans and presents novel insights into the complex signaling patterns triggered by this important pathogen.

Evaluation of VEGF‐mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast‐mediated crosstalk to endothelial cells

Communication between endothelial and bone cells is crucial for controlling vascular supply during bone growth, remodeling, and repair but the molecular mechanisms coordinating this intercellular crosstalk remain ill-defined. We have used primary human and rat long bone-derived osteoblast-like cells (HOB and LOB) and human umbilical vein endothelial cells (HUVEC) to interrogate the potential autocrine/paracrine role of vascular endothelial cell growth factor (VEGF) in osteoblast:endothelial cell (OB:EC) communication and examined whether prostaglandins (PG), known modulators of both OB and EC behavior, modify VEGF production. We found that the stable metabolite of PGI2, 6-keto-PGF(1alpha) and PGE2, induced a concentration-dependent increase in VEGF release by HOBs but not ECs. In ECs, VEGF promoted early ERK1/2 activation, late cyclooxygenase-2 (COX-2) protein induction, and release of 6-keto-PGF1alpha. In marked contrast, no significant modulation of these events was observed in HOBs exposed to VEGF, but LOBs clearly exhibited COX-dependent prostanoid release (10-fold less than EC) following VEGF treatment. A low level of osteoblast-like cell responsiveness to exogenous VEGF was supported by VEGFR2/Flk-1 immunolabelling and by blockade of VEGF-mediated prostanoid generation by a VEGFR tyrosine kinase inhibitor (TKI). HOB alkaline phosphatase (ALP) activity was increased following long-term non-contact co-culture with ECs and exposure of ECs to VEGF in this system further increased OB-like cell differentiation and markedly enhanced prostanoid release. Our studies confirm a paracrine EC-mediated effect of VEGF on OB-like cell behavior and are the first supporting a model in which prostanoids may facilitate this unidirectional VEGF-driven OB:EC communication. These findings may offer novel regimes for modulating pathological bone remodeling anomalies through the control of the closely coupled vascular supply.

Circulating endothelial progenitor cells in multiple myeloma: implications and significance

AbstractAngiogenesis governs the progression of multiple myeloma (MM). Circulating endothelial cells (CECs) contribute to angiogenesis and comprise mature ECs and endothelial progenitor cells (EPCs). The present study sought to characterize CECs and their relation to disease activity and therapeutic response in 31 consecutive patients with MM. CECs, identified as CD34+/CD146+/CD105+/CD11b- cells, were 6-fold higher in patients compared to controls and correlated positively with serum M protein and β2-microglobulin. Circulating EPCs displayed late colony formation/outgrowth and capillary-like network formation on matrigel; these processes were inhibited after effective thalidomide treatment. Co-expression of vascular endothelial growth factor receptor-2 (KDR) and CD133 characterized EPCs in MM, and KDR mRNA elevations correlated with M protein levels. In vitro exposure of ECs to thalidomide or its derivative CC-5013 inhibited gene expression of the receptors for transforming growth factor-β and thrombin. Thus, elevated levels of CECs and EPCs covary with disease activity and response to thalidomide, underscoring the angiogenic aspect of MM and suggesting that angioblastlike EPCs are a pathogenic biomarker and a rational treatment target in MM. The results also highlight the anti-angiogenic properties of thalidomide and CC-5013 and further elucidate possible mechanisms of their effectiveness against MM. (Blood. 2005;105:3286-3294)

Fluid shear stress inhibits vascular inflammation by decreasing thioredoxin-interacting protein in endothelial cells

Regions in the vasculature that are exposed to steady laminar blood flow are protected from atherosclerosis as compared with regions where flow is disturbed. We found that flow decreased TNF-mediated VCAM1 expression by inhibiting JNK and p38. JNK inhibition correlated with inhibition of apoptosis signal–regulating kinase 1 (ASK1), a JNK and p38 activator. Thioredoxin-interacting protein (TXNIP) is a stress-responsive protein that inhibits thioredoxin (TRX) activity. Since thioredoxin inhibits ASK1, we hypothesized that changes in TXNIP-TRX-ASK1 interactions mediate the antiinflammatory effects of flow. To explore this, we used perfused vessels and cultured ECs. Exposure of rabbit aortae or ECs to normal flow (12 dyn/cm2, 24 hours) was associated with decreased TXNIP expression and increased TRX activity compared with exposure to low flow (0.4 dyn/cm2). Normal flow inhibited TNF activation of JNK/p38 and VCAM1 expression. In cultured ECs, reduction of TXNIP expression by small interfering RNA increased TRX binding to ASK1 and inhibited TNF activation of JNK/p38 and VCAM1 expression. Conversely, overexpression of TXNIP stimulated JNK and p38. In aortae from TXNIP-deficient mice, TNF-induced VCAM1 expression was inhibited. The data suggest that TXNIP and TRX are key components of biomechanical signal transduction and establish them as potentially novel regulators of TNF signaling and inflammation in ECs.

Fluid shear stress inhibits vascular inflammation by decreasing thioredoxin-interacting protein in endothelial cells

Regions in the vasculature that are exposed to steady laminar blood flow are protected from atherosclerosis as compared with regions where flow is disturbed. We found that flow decreased TNF-mediated VCAM1 expression by inhibiting JNK and p38. JNK inhibition correlated with inhibition of apoptosis signal-regulating kinase 1 (ASK1), a JNK and p38 activator. Thioredoxin-interacting protein (TXNIP) is a stress-responsive protein that inhibits thioredoxin (TRX) activity. Since thioredoxin inhibits ASK1, we hypothesized that changes in TXNIP-TRX-ASK1 interactions mediate the antiinflammatory effects of flow. To explore this, we used perfused vessels and cultured ECs. Exposure of rabbit aortae or ECs to normal flow (12 dyn/cm2, 24 hours) was associated with decreased TXNIP expression and increased TRX activity compared with exposure to low flow (0.4 dyn/cm2). Normal flow inhibited TNF activation of JNK/p38 and VCAM1 expression. In cultured ECs, reduction of TXNIP expression by small interfering RNA increased TRX binding to ASK1 and inhibited TNF activation of JNK/p38 and VCAM1 expression. Conversely, overexpression of TXNIP stimulated JNK and p38. In aortae from TXNIP-deficient mice, TNF-induced VCAM1 expression was inhibited. The data suggest that TXNIP and TRX are key components of biomechanical signal transduction and establish them as potentially novel regulators of TNF signaling and inflammation in ECs.

Fragile X mental retardation protein levels increase following complex environment exposure in rat brain regions undergoing active synaptogenesis

Fragile X mental retardation protein (FMRP), which is absent in fragile X syndrome, is synthesized in vitro in response to neurotransmitter activation. Humans and mice lacking FMRP exhibit abnormal dendritic spine development, suggesting that this protein plays an important role in synaptic plasticity. Previously, our laboratory demonstrated increased FMRP immunoreactivity in visual cortex of rats exposed to complex environments (EC) and in motor cortex of rats trained on motor-skill tasks compared with animals reared individually in standard laboratory housing (IC). Here, we use immunohistochemistry to extend those findings by investigating FMRP levels in visual cortex and hippocampal dentate gyrus of animals exposed to EC or IC. Rats exposed to EC for 20 days exhibited increased FMRP immunoreactivity in visual cortex compared with animals housed in standard laboratory caging. In the dentate gyrus, animals exposed to EC for 20 days had higher FMRP levels than animals exposed to EC for 5 or 10 days. In light of possible antibody crossreactivity with closely related proteins FXR1P and FXR2P, FMRP immunoreactivity in the posterior-dorsal one-third of cerebral cortex was also examined by Western blotting following 20 days of EC exposure. FMRP levels were greater in EC animals, whereas levels of FXR1P and FXR2P were unaffected by experience. These results provide further evidence for behaviorally induced alteration of FMRP expression in contrast to its homologues, extend previous findings suggesting regulation of its expression by synaptic activity, and support the theories associating FMRP expression with alteration of synaptic structure both in development and later in the life-cycle.

The Effect of a Shear Flow on the Uptake of LDL and Ac-LDL by Cultured Vascular Endothelial Cells

The effects of a shear flow on the uptake of fluorescence-labeled low-density lipoprotein (DiI-LDL), acetylated LDL (DiI-Ac-LDL), and lucifer yellow (LY; a tracer of fluid-phase endocytosis) by cultured bovine aortic ECs were studied using a rotating-disk shearing apparatus. It was found that 2hours’ exposure of ECs to a laminar shear flow that imposed ECs an area-mean shear stress of 10dynes/cm2 caused an increase in the uptake of DiI-LDL and LY. By contrast, the uptake of DiI-Ac-LDL was decreased by exposure of the ECs to a shear flow. Addition of dextran sulfate (DS), a competitive inhibitor of scavenger receptors, reversed the effect of a shear flow on the uptake of DiI-Ac-LDL, resulting in an increase by the imposition of a shear flow, while the uptake of DiI-LDL and LY remained unaffected. It was concluded that a shear flow promotes the endocytosis of DiI-LDL and LY by ECs, but suppresses the uptake of DiI-Ac-LDL by ECs by inhibiting scavenger receptor-mediated endocytosis.

Anatomical and Eco-Physiological Changes in Leaves of Couch-Grass (Elymus repens L.), a Temperate Loess Grassland Species, after 7 Years Growth Under Elevated CO<sub>2</sub> Concentration

Leaf anatomy and eco-physiology of Elymus repens, a temperate loess grassland species, were determined after seven years of exposure to 700 μmol (CO2) mol−1 (EC). EC treatment resulted in significant reduction of stomatal density on both surfaces of couch-grass leaves. Thickness of leaves and that of the sclerenchyma tissues between the vessels and the adaxial surfaces, the area of vascular bundle, and the volumes of phloem and tracheary increased at EC while abaxial epidermis and the sclerenchyma layer between the vessel and the abaxial surface were thicker at ambient CO2 concentration (AC). Stomatal conductance and transpiration rates were lower in EC, while net CO2 assimilation rate considerably increased at EC exposure. Contents of soluble sugars and starch were higher in EC-treated couch-grass leaves than in plants grown at AC.

Different Responses of Norway Spruce Needles from Shaded and Exposed Crown Layers to the Prolonged Exposure to Elevated CO<sub>2</sub> Studied by Various Chlorophyll a Fluorescence Techniques

The acclimation depression of capacity of photon utilisation in photochemical reactions of photosystem 2 (PS2) can develop already after three months of cultivation of the Norway spruces (Picea abies [L.] Karst.) under elevated concentrations of CO2 (i.e., ambient, AC, + 350 µmol(CO2) mol-1 = EC) in glass domes with adjustable windows. To examine the role that duration of EC plays in acclimation response, we determined pigment contents, rate of photosynthesis, and parameters of chlorophyll a fluorescence for sun and shade needles after three seasons of EC exposure. We found responses of shaded and exposed needles to EC. Whereas the shaded needles still profited from the EC and revealed stimulated electron transport, for the exposed needles the stimulation of both electron transport activity and irradiance saturated rate of CO2 assimilation (PNmax) under EC already disappeared. No signs of the PS2 impairment were observed as judged from high values of potential quantum yield of PS2 photochemistry (FV/FM) and uniform kinetics of QA reoxidation for all variants. Therefore, the long-term acclimation of the sun-exposed needles to EC is not necessarily accompanied with the damage to the PS2 reaction centres. The eco-physiological significance of the reported differentiation between the responses of shaded and sun exposed needles to prolonged EC may be in changed contribution of the upper and lower crown layers to the production activity of the tree. Whereas for the AC spruces, PNmax of shaded needles was only less than 25 % compared to exposed ones, for the EC spruces the PNmax of shaded needles reached nearly 40 % of that estimated for the exposed ones. Thus, the lower shaded part of the crown may become an effective consumer of CO2.

Shear-induced platelet activation and adhesion on human pulmonary artery endothelial cells seeded onto hydrophilic polymers.

We evaluated platelet activation and adhesion on two plasma polymerized surfaces, N-vinyl pyrrolidone (NVP) and gamma-butyro lactone (GBL), which have been shown previously to promote endothelial cell growth and adhesion as well as fibronectin-coated glass (1 microg/cm(2)) coverslips. Human pulmonary artery endothelial cells were seeded onto coverslips at a low density ( approximately 20,000 cells/cm(2)) and grown to confluence (3-5 days). The materials, both with and without ECs, were then exposed to a shear rate of 400 s(-1) in a closed loop recirculating flow system containing human platelet-rich plasma. Plasma samples were taken at 0, 5, 15, 30, and 60 min and analyzed for platelet and coagulation activation. The coverslips were examined for EC coverage and platelet adherence. EC retention over a 1-h period was approximately 75% for all three materials. All three materials without ECs were highly platelet activating having similar P-selectin expression, platelet factor 4 (PF4) release, mepacrine uptake, and microparticle production. Both microparticle production and platelet adhesion were significantly lower in EC-seeded materials. Dense granule and PF4 release were both slightly diminished in all three materials seeded with ECs. P-selectin expression was reduced slightly for GBL, but remained the same for the other two materials. The EC-seeded materials displayed favorable characteristics with respect to platelet activation and adhesion; however, they still demonstrated some thrombogenic tendencies due to EC loss and exposure of the underlying substrate. Therefore, both EC coverage and EC hemostatic function are important factors in determining the thromboresistance of an EC-seeded surface.

68 ENDOTHELIAL CELLS (EC) TOLERATE PROLONGED HYPOXIA AND GLUCOSE DEPRIVATION

ECs play a key role in organ damage after ischemia-reperfusion and oxygen toxicity. In vitro models have been described in which short-term exposure of ECs to hypoxia followed by hyperoxia causes cell damage. We cultured human ECs from umbilical veins, prelabeled them with 14C-adenine, and followed nucleotide catabolism as a sensitive indicator of cell damage during exposure to normoxia (N), 95% N2 + 5% CO2 (HYPO), or 95% O2 + 5% CO2 (HYPER), with either 0 or 5.5 mM glucose (G) in the medium. Up to 6h, adenine nucleotide turnover and hypoxanthine accumulation in the medium were similar under all incubation conditions. After 16h, cellular nucleotide levels, in comparison with N+G (=100+-9 %), Were:N-G 140+-17 %, HYPO+G 37+-9 %, HYPO-G 58+-2%, HYPER+G 16+-7 %, and HYPER-G 114+-35 %. When 16h of HYPO was followed by 8h of HYPER, depletion was total and cell death ensued in the presence and absence of G. We conclude that ECs tolerate both HYPO and G deprivation for several hours, and surprisingly they are more sensitive to both HYPO and HYPER in the presence than in the absence of G.

Impairment of environmental effects on brain weight by adrenergic drugs in rats

Injection of clonidine or methyldopa in rats immediately after daily two-hour exposure to a complex environment (EC) reduced the increase in occipital cortical weight normally associated with such exposure in comparison with animals kept in isolation. Drug injection four hours after EC exposure had no effect. Drug-induced suppression of REM sleep during the first four hours after EC exposure was suggested to be the critical variable.