Cultured Artery Endothelial Cells Research Articles

SY 15-2 ENDOTHELIAL EPIGENETICS AND ITS ROLE IN MEDIATING BIOMECHANICAL STRESS OF HYPERTENSION

Abstract Hemodynamics creates a constantly changing physical and chemical environment to which the arterial endothelium is exquisitely sensitive. Biomechanical stresses are intrinsic to blood flow characteristics and blood pressure and therefore are important considerations in hypertension. Near branching anatomical sites in arteries, blood flow separates from the main flow to undergo complex multi-directional characteristics for a part of each cardiac cycle (collectively referred to as disturbed flow). Atherosclerosis and aneurysmal pathology develop preferentially at disturbed flow locations, particularly when an additional cardiovascular risk factor such as hypercholesterolemia or high blood pressure are present. In the pre-disease state, a pre-inflammatory endothelial phenotype is characteristically present in such regions in contrast to nearby endothelium outside of the complex disturbed flow fields. A regulatory hierarchy of mechanisms controls endothelial gene and protein expression (and function) in response to the flow environment. Gene expression regulation, the transcription of the DNA (genomic) code to mRNA, is well described by transcription factors, enhancers and repressors, many of which are flow responsive. However, emerging studies describe the additional role of localized disturbed blood flow upon epigenetic mechanisms of endothelial responses to biomechanical stress. Epigenetics and epigenomics (global analyses of epigenetic changes across the entire genome) encompass heritable and non-heritable changes in nuclear chromatin leading to gene expression changes that cannot be attributed to changes in the primary DNA sequence. Hemodynamic stimuli have recently been shown to induce epigenetic responses including transcriptional regulation by proximal promoter DNA methylation, and regulation of gene and protein expression by histone/chromatin remodeling and by noncoding RNA-based mechanisms.Dynamic epigenomic responses to flow that result in regulation of endothelial gene expression in vivo and in vitro include: (i) regions of stable differentially methylated DNA in swine and mouse endothelial genomes that are arterial site-specific and map to atherosusceptibility, (ii) disturbed flow (but not undisturbed flow) applied to endothelial cells in vitro that induces DNA methylation through DNA methyltransferase enrichment of gene promoter regions; the resulting hypermethylation suppresses gene expression, (iii) histone acetylation / deacetylation and methylation that create histone marks that enable or suppress gene expression by controlling access of transcription factors to chromatin DNA, and (iv) short microRNAs and long noncoding RNAs that interact with highly specific binding sites of newly synthesized mRNA to promote its degradation and thus suppress transcription. Flow-mediated epigenomic responses intersect with cis and trans factor regulation to maintain endothelial function in a shear-stressed or pressure-stressed environment and may contribute to localized endothelial dysfunctions that promote localized vascular pathology.The Encyclopedia of DNA Elements (ENCODE) and the International Human Epigenome Consortia (IHEC) have uncovered thousands of putative epigenetic regulatory sites in non-coding regions of the genome. Methylation of cytosine nucleosides in DNA both at/near gene promoter regions as well as at some distance upstream is a potent epigenetic suppressor of transcription. Recently, complete methylated DNA immunoprecipitation sequencing (MeDIP-seq) of regions of arterial endothelium associated with disturbed vs undisturbed flow in vivo in pigs has identified many differentially methylated DNA profiles enriched in exons and 5'UTR sequences of annotated genes, 60 of which are linked to cardiovascular disease. Furthermore, in human arterial endothelial cells in culture we have demonstrated DNA methylation plasticity to be regulated by disturbed flow resulting in suppression (hypermethylation) or stimulation (hypomethylation) of transcription. We propose dynamic DNA methylation to be a genome-wide epigenetic mechanism of transcriptional control that regulates adaptive endothelial phenotype plasticity in response to the local environment, including biomechanical stresses associated with disturbed flow characteristics and hypertension.The epigenomic code therefore has the potential to figure prominently in disease susceptibility and pathogenesis arising from environmental influences independent of, or in concert with, mutations and single nucleotide polymorphisms (SNP) linked to many complex diseases, including hypertension and cardiovascular disease. The role of disturbed flow as a hemodynamic regulator of epigenomic DNA methylation-mediated and histone-mediated gene expression may play a prominent role in endothelial vascular function and dysfunction in hypertension, atherogenesis and aneurysm development. The cellular mechanisms that link hemodynamics and biomechanical stress to the epigenomic code remain unexplored.

Cigarette smoke but not electronic cigarette aerosol activates a stress response in human coronary artery endothelial cells in culture

BackgroundIt is generally acknowledged that e-cigarettes are unlikely to be as harmful as conventional cigarettes, but there is little data that quantifies their relative harms. We investigated the biological response to e-cigarette aerosol exposure (versus conventional cigarette smoke exposure) at the cellular level, by exposing human coronary artery endothelial cells (HCAEC) to aqueous filtered extracts of e-cigarette aerosol or cigarette smoke and looking at gene expression changes consistent with a stress response. This included genes controlled by the oxidant-stress sensing transcription factor NFR2 (NFE2L2), and cytochrome P450 family members. MethodsCigarette smoke extract (CSE) was created using mainstream smoke from a single cigarette drawn through 10ml of endothelial cell growth media MV2. Electronic cigarette aerosol extract (eCAE) was created using the same apparatus, using a constant power output of 10.8w (4.2V) and 18mg/ml nicotine solution. eCAE was generated using 5 cycles of 5s heat with at least 10s in between each puff to allow the coil to cool, air being drawn through the device at 70ml/minute. ResultsHCAEC responded to the noxious components in CSE, resulting in activation of NRF2 and upregulation of cytochrome p450. However, eCAE did not induce NRF2 nuclear localisation, upregulation of NRF2-activated genes, or the upregulation of cytochrome p450. ConclusionsThe use of e-cigarettes as a substitute for conventional cigarettes is likely to reduce immediate tobacco-related harm, at least with respect to cardiovascular harms.

Non-nuclear Estrogen Receptor Signaling in the Endothelium

In addition to the classical function of estrogen receptors (ER) as transcription factors, evidence continues to accumulate that they mediate non-nuclear processes in numerous cell types, including the endothelium, in which they activate endothelial NO synthase. Non-nuclear ER signaling entails unique post-translational modifications and protein-protein interactions of the receptor with adaptor molecules, kinases, and G proteins. Recent in vitro and in vivo studies in mice using an estrogen-dendrimer conjugate that is excluded from the nucleus indicate that non-nuclear ER activation underlies the migration and growth responses of endothelial cells to estrogen but not the growth responses of endometrial or breast cancer cells to the hormone. In this minireview, the features of ERα and protein-protein interactions that enable it to invoke extranuclear signaling in the endothelium and the consequences of that signaling are discussed.

Characterization of the threshold for NAD(P)H:quinone oxidoreductase activity in intact sulforaphane-treated pulmonary arterial endothelial cells

Treatment of bovine pulmonary arterial endothelial cells in culture with the phase II enzyme inducer sulforaphane (5 μM, 24 h; sulf-treated) increased cell-lysate NAD(P)H:quinone oxidoreductase (NQO1) activity by 5.7 ± 0.6 (mean ± SEM)-fold, but intact-cell NQO1 activity by only 2.8 ± 0.1-fold compared to control cells. To evaluate the hypothesis that the threshold for sulforaphane-induced intact-cell NQO1 activity reflects a limitation in the capacity to supply NADPH at a sufficient rate to drive all the induced NQO1 to its maximum activity, total KOH-extractable pyridine nucleotides were measured in cells treated with duroquinone to stimulate maximal NQO1 activity. NQO1 activation increased NADP + in control and sulf-treated cells, with the effect more pronounced in the sulf-treated cells, in which the NADPH was also decreased. Glucose-6-phosphate dehydrogenase (G-6-PDH) inhibition partially blocked NQO1 activity in control and sulf-treated cells, but G-6-PDH overexpression via transient transfection with the human cDNA alleviated neither the restriction on intact sulf-treated cell NQO1 activity nor the impact on the NADPH/NADP + ratios. Intracellular ATP levels were not affected by NQO1 activation in control or sulf-treated cells. An increased dependence on extracellular glucose and a rightward shift in the K m for extracellular glucose were observed in NQO1-stimulated sulf-treated vs control cells. The data suggest that glucose transport in the sulf-treated cells may be insufficient to support the increased metabolic demand for pentose phosphate pathway-generated NADPH as an explanation for the NQO1 threshold.

(−)-Epicatechin Activation of Endothelial Cell Endothelial Nitric Oxide Synthase, Nitric Oxide, and Related Signaling Pathways

Recent reports indicate that (-)-epicatechin can exert cardioprotective actions, which may involve endothelial nitric oxide synthase (eNOS)-mediated nitric oxide production in endothelial cells. However, the mechanism by which (-)-epicatechin activates eNOS remains unclear. In this study, we proposed to identify the intracellular pathways involved in (-)-epicatechin-induced effects on eNOS, using human coronary artery endothelial cells in culture. Treatment of cells with (-)-epicatechin led to time- and dose-dependent effects that peaked at 10 minutes at 1 mumol/L. (-)-Epicatechin treatment activates eNOS via serine 633 and serine 1177 phosphorylation and threonine 495 dephosphorylation. Using specific inhibitors, we have established the participation of the phosphatidylinositol 3-kinase pathway in eNOS activation. (-)-Epicatechin induces eNOS uncoupling from caveolin-1 and its association with calmodulin-1, suggesting the involvement of intracellular calcium. These results allowed us to propose that (-)-epicatechin effects may be dependent on actions exerted at the cell membrane level. To test this hypothesis, cells were treated with the phospholipase C inhibitor U73122, which blocked (-)-epicatechin-induced eNOS activation. We also demonstrated inositol phosphate accumulation in (-)-epicatechin-treated cells. The inhibitory effects of the preincubation of cells with the calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 indicate that (-)-epicatechin-induced eNOS activation is at least partially mediated via the Ca(2+)/CaMKII pathway. The (-)-epicatechin stereoisomer catechin was only partially able to stimulate nitric oxide production in cells. Together, these results strongly suggest the presence of a cell surface acceptor-effector for the cacao flavanol (-)-epicatechin, which may mediate its cardiovascular effects.

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD + and NADPH/NADP + redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD + and NADPH/NADP + ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD +, but not NADPH/NADP + or NQO1 activity. Iodoacetate decreased NADH/NAD + but had no detectable effect on NADPH/NADP + or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP + or NADH/NAD + ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP + decreased by 84% with no impact on NADH/NAD +. Duroquinone alone also decreased NADPH/NADP + but not NADH/NAD +. The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP + than NADH/NAD + redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD + ratio.

Role of mitochondrial electron transport complex I in coenzyme Q1reduction by intact pulmonary arterial endothelial cells and the effect of hyperoxia

The objective was to determine the impact of intact normoxic and hyperoxia-exposed (95% O(2) for 48 h) bovine pulmonary arterial endothelial cells in culture on the redox status of the coenzyme Q(10) homolog coenzyme Q(1) (CoQ(1)). When CoQ(1) (50 microM) was incubated with the cells for 30 min, its concentration in the medium decreased over time, reaching a lower level for normoxic than hyperoxia-exposed cells. The decreases in CoQ(1) concentration were associated with generation of CoQ(1) hydroquinone (CoQ(1)H(2)), wherein 3.4 times more CoQ(1)H(2) was produced in the normoxic than hyperoxia-exposed cell medium (8.2 +/- 0.3 and 2.4 +/- 0.4 microM, means +/- SE, respectively) after 30 min. The maximum CoQ(1) reduction rate for the hyperoxia-exposed cells, measured using the cell membrane-impermeant redox indicator potassium ferricyanide, was about one-half that of normoxic cells (11.4 and 24.1 nmol x min(-1) x mg(-1) cell protein, respectively). The mitochondrial electron transport complex I inhibitor rotenone decreased the CoQ(1) reduction rate by 85% in the normoxic cells and 44% in the hyperoxia-exposed cells. There was little or no inhibitory effect of NAD(P)H:quinone oxidoreductase 1 (NQO1) inhibitors on CoQ(1) reduction. Intact cell oxygen consumption rates and complex I activities in mitochondria-enriched fractions were also lower for hyperoxia-exposed than normoxic cells. The implication is that intact pulmonary endothelial cells influence the redox status of CoQ(1) via complex I-mediated reduction to CoQ(1)H(2), which appears in the extracellular medium, and that the hyperoxic exposure decreases the overall CoQ(1) reduction capacity via a depression in complex I activity.

Quantitative analysis of sphingoid base-1-phosphates as bisacetylated derivatives by liquid chromatography–tandem mass spectrometry

Sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) are important signaling sphingolipids. The presence of nanomolar levels of S1P and DHS1P in tissues, cells, and biological fluids requires a highly sensitive and selective assay method for their reliable detection and quantitation. Preliminary findings employing positive ion electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis indicated significant sample carryover from previous injections of authentic standards of S1P and DHS1P. This article details a negative ion ESI LC–MS/MS technique following modification of the zwitterionic nature of S1P and DHS1P via derivatization. A highly selective and sensitive LC–MS/MS technique capable of reliable detection of less than 50 fmol of the derivatives of S1P and DHS1P without significant sample carryover was developed. Standard curves for S1P and DHS1P are linear over wide ranges (0–300 pmol) of analyte concentrations with correlation coefficients ( r 2) greater than 0.995. The levels of S1P and DHS1P in human platelet poor plasma were 590.8 ± 42.1 and 130.7 ± 20.7 pmol/ml, respectively. The levels of S1P and DHS1P in fetal bovine serum were 141.7 ± 4.6 and 0.6 ± 0.2 pmol/ml, respectively. The addition of sphingosine (1 μM) to human pulmonary artery endothelial cells in culture resulted in a more than 20-fold increase in the cellular level of S1P, whereas the level of DHS1P was unchanged.

Pulmonary arterial endothelial cells affect the redox status of coenzyme Q0.

The pulmonary endothelium is capable of reducing certain redox-active compounds as they pass from the systemic venous to the arterial circulation. This may have important consequences with regard to the pulmonary and systemic disposition and biochemistry of these compounds. Because quinones comprise an important class of redox-active compounds with a range of physiological, toxicological, and pharmacological activities, the objective of the present study was to determine the fate of a model quinone, coenzyme Q0 (Q), added to the extracellular medium surrounding pulmonary arterial endothelial cells in culture, with particular attention to the effect of the cells on the redox status of Q in the medium. Spectrophotometry, electron paramagnetic resonance (EPR), and high-performance liquid chromatography (HPLC) demonstrated that, when the oxidized form Q is added to the medium surrounding the cells, it is rapidly converted to its quinol form (QH2) with a small concentration of semiquinone (Q*-) also detectable. The isolation of cell plasma membrane proteins revealed an NADH-Q oxidoreductase located on the outer plasma membrane surface, which apparently participates in the reduction process. In addition, once formed the QH2 undergoes a cyanide-sensitive oxidation by the cells. Thus, the actual rate of Q reduction by the cells is greater than the net QH2 output from the cells.

Intracellular redox status affects transplasma membrane electron transport in pulmonary arterial endothelial cells.

Pulmonary arterial endothelial cells possess transplasma membrane electron transport (TPMET) systems that transfer intracellular reducing equivalents to extracellular electron acceptors. As one aspect of determining cellular mechanisms involved in one such TPMET system in pulmonary arterial endothelial cells in culture, glycolysis was inhibited by treatment with iodoacetate (IOA) or by replacing the glucose in the cell medium with 2-deoxy-D-glucose (2-DG). TPMET activity was measured as the rate of reduction of the extracellular electron acceptor polymer toluidine blue O polyacrylamide. Intracellular concentrations of NADH, NAD(+), NADPH, and NADP(+) were determined by high-performance liquid chromatography of KOH cell extracts. IOA decreased TPMET activity to 47% of control activity concomitant with a decrease in the NADH/NAD(+) ratio to 34% of the control level, without a significant change in the NADPH/NADP(+) ratio. 2-DG decreased TPMET activity to 53% of control and decreased both NADH/NAD(+) and NADPH/NADP(+) ratios to 51% and 55%, respectively, of control levels. When lactate was included in the medium along with the inhibitors, the effects of IOA and 2-DG on both TPMET activity and the NADPH/NADP(+) ratios were prevented. The results suggest that cellular redox status is a determinant of pulmonary arterial endothelial cell TPMET activity, with TPMET activity more highly correlated with the poise of the NADH/NAD(+) redox pair.

Ascorbate-mediated transplasma membrane electron transport in pulmonary arterial endothelial cells.

Pulmonary endothelial cells are capable of reducing certain electron acceptors at the luminal plasma membrane surface. Motivation for studying this phenomenon comes in part from the expectation that it may be important both as an endothelial antioxidant defense mechanism and in redox cycling of toxic free radicals. Pulmonary arterial endothelial cells in culture reduce the oxidized forms of thiazine compounds that have been used as electron acceptor probes for studying the mechanisms of transplasma membrane electron transport. However, they reduce another commonly studied electron acceptor, ferricyanide, only very slowly by comparison. In the present study, we examined the influence of ascorbate [ascorbic acid (AA)] and dehydroascorbate [dehydroascorbic acid (DHAA)] on the ferricyanide and thiazine reductase activities of the bovine pulmonary arterial endothelial cell surface. The endothelial cells were grown on microcarrier beads so that the reduction of ferricyanide and methylene blue could be studied colorimetrically in spectrophotometer cuvettes and in flow-through cell columns. The ferricyanide reductase activity could be increased 80-fold by adding DHAA to the medium, with virtually no effect on methylene blue reduction. The DHAA effect persisted after the DHAA was removed from the medium. AA also stimulated the ferricyanide reductase activity but was less potent, and the relative potencies of AA and DHAA correlated with their relative rates of uptake by the cells. The results are consistent with the hypothesis that AA is an intracellular electron donor for an endothelial plasma membrane ferricyanide reductase and that the stimulatory effect of DHAA is the result of increasing intracellular AA. Adding sufficient DHAA to markedly increase extracellular ferricyanide reduction had little effect on the plasma membrane methylene blue reductase activity, suggesting that pulmonary arterial endothelial cells have at least two separate transplasma membrane electron transport systems.

Western immunoblot analysis.

AbstractSodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1) combined with Western immunoblot analysis (2,3) is a widely used technique for quantitatively and qualitatively evaluating specific protein expression. This chapter describes a Western immunoblot technique routinely used in our laboratories for protein analysis with representative data shown in Fig. 1. The protocols for SDS-PAGE and electrophoretic transfer are based on using the Mini-PROTEAN II system (Bio-Rad, Hercules, CA). Protein detection is based on using the enhanced chemiluminescence (ECL) detection method (Amersham, Arlington Heights, IL). In this technique, mixtures of solubilized proteins under denaturing conditions (in the presence of detergent [SDS], and reducing agent [mercaptoenthanol]) are separated by electrophoresis based on their molecular weights, followed by transferring proteins to PVDF membrane and identifying a protein of interest using a specific antibody. It is highly recommended that, before beginning the protocol, the operator read Subheading 4. Western immunoblot analysis of protein expression of eNOS in ovine fetoplacental artery endothelial cells in culture: Proteins (2 μg protein/lane) were separated by electrophoresis (7.5% SDS-PAGE gels). After transfer to the membrane, immunoblotting was performed using a mouse monoclonal ecNOS antibody (1∶750 [0.33 μg/mL]; Transduction Laboratories, Lexington, KY) with ECL dectection at 5-min exposure, as described. Lanes 1–2, controls; lanes 3–8, cells treated with basic fibroblast growth factor (1 or 10 ng/mL) for 24 h. KeywordsGlass PlateTris BaseAmmonium PersulfateTransfer BufferElectrophoresis BufferThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Substrate Inhibition of Nitric Oxide Synthase in Pulmonary Artery Endothelial Cells in Culture

The effects of arginine on nitric oxide synthase (NOS) activity and NO production were studied in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 0–100 μM arginine increased NO production, detected as nitrite in the culture medium, in a dose-dependent manner. In contrast, incubation with concentrations of arginine in excess of 100 μM resulted in a reversible dose-dependent inhibition of NO production, even though intracellular arginine content increased in these cells. The NOS enzyme kinetics were studied in a total membrane preparation and in purified NOS protein and revealed that theKmof arginine as a substrate for NOS is 3–5 μM, theVmaxoccurred at 100 μM arginine, and substrate inhibition occurred at >100 μM arginine. Oxyhemoglobin, carboxy-PTIO, catalase, SOD, citrulline, hydroxyarginine, andd-arginine did not change NOS kinetics. These results indicate that substrate inhibition of eNOS exists in porcine PAEC in vitro.

Fibrin activation of vascular endothelial cells. Induction of IL-8 expression.

Fibrin deposition and leukocyte accumulation are classic histopathologic hallmarks of acute and chronic inflammation. Although both of these processes are common to inflammatory processes, surprisingly little is known about the interrelationship between fibrin deposition and leukocyte accumulation. We hypothesized that: 1) fibrin can regulate inflammation by directly inducing leukocyte chemotactic factor (LCF) expression from vascular endothelial cells (VEC), and 2) this fibrin-induced VEC expression of LCF would enhance leukocyte recruitment seen in acute and chronic inflammation. To test this hypothesis, we developed an in vitro model system of in situ polymerization of fibrin on isolated bovine pulmonary artery endothelial cells in culture. In the initial phase of our studies, we found that fibrin induced a time- and dose-dependent expression of leukocyte migration activity from VEC. Checkerboard analysis demonstrated that the leukocyte migration activity present in the fibrin-stimulated VEC culture supernatant was truly chemotactic in nature. In an effort to begin to characterize this chemotactic activity, we demonstrated that this fibrin-induced LCF activity was 1) protein dependent, 2) not derived from fibrin(ogen), and 3) had an apparent m.w. of 6 to 12 kDa. Our further studies demonstrated that fibrin was a potent stimulus for IL-8 Ag expression from the VEC. Thus, these studies clearly demonstrate that fibrin has the capability to induce leukocyte chemotactic activity in general, and IL-8 activity specifically, from VEC. These studies support our hypothesis that fibrin is an important activator of vascular endothelial cells in vivo.

Reduction of thiazine dyes by bovine pulmonary arterial endothelial cells in culture.

The uptake of methylene blue (MB), and toluidine blue O (TBO) by bovine pulmonary arterial endothelial cells grown on microcarrier beads was detected as a decrease in the concentration of dye in the medium after these thiazine dyes were added to the medium surrounding the cells. Because the reduced forms of these dyes are much more lipophilic than the oxidized forms, we considered the possibility that reduction of the dyes at the cell surface might have preceded the uptake by the cells. Therefore, we studied the ability of the cells to reduce a toluidine blue O-polyacrylamide polymer (TBOP), which was too large to enter the cells in either the oxidized or reduced form. The TBO moieties of the polymer were reduced by the cells, indicating that the dyes did not have to enter the cells to be reduced and that reduction can occur at, or near, the cell surface. The rate of TBOP reduction was about the same as the rate of uptake of the monomeric dyes, indicating that the cell surface reduction mechanism had a sufficient capacity to account for the monomer uptake by the cells. We also found that ferricyanide ion, which also did not permeate the cells, was reduced by the cells and that external ferricyanide inhibited the monomeric MB uptake. Thus the results with ferricyanide were also consistent with the concept that the monomeric thiazine dyes are reduced at the cell surface before the more lipophilic reduced forms are taken up by the endothelial cells.

Histamine degradative uptake by cultured human pulmonary vascular endothelial cells utilizes an inflammatory cell diamine oxidase

Neutrophil-endothelial interactions, altered clearance properties of the lung toward vasoactive mediators, and damaging effects of histamine that target the lung represent prominent elements of the inflammatory response at the systemic level. The pulmonary vasculature is unusual in that, unlike other tissues and vascular beds, it normally does not metabolize circulating histamine in vivo, although histamine-metabolizing enzyme activities have been detected in disrupted lung tissue. We have therefore explored the capability of human pulmonary artery endothelial cells in culture to express the receptor-mediated histamine degradative uptake system we previously defined in systemic endothelial cells (Haddock, R. C., Mack, P., Leal, S., and Baenziger, N. L. (1990) J. Biol. Chem. 265, 14395-14401). Pulmonary endothelial cells display all components of this system: histamine methyltransferase generating the proximal cell-associated metabolite tele-methylhistamine and receptors binding diamine oxidase which generates the distal product methylimidazoleacetic acid that is accumulated by the cells. A diamine oxidase released from human neutrophil granules by activation with Ca2+ ionophore binds pulmonary and systemic endothelial cell and fibroblast diamine oxidase receptors and, thereby, participates in histamine degradative uptake. This enzyme utilizes cell-associated tele-methylhistamine as a substrate, preferentially generating methylimidazoleacetic acid in addition to reactive oxygen species. Thus the enzymatic and interactive cellular machinery for histamine clearance is inherently present as a functional unit in two major human pulmonary cell types. It interacts with products of inflammatory host defense cells, and pulmonary endothelial-neutrophil interactions via this pathway may influence the progression of inflammation.

Isolation and maintenance of human pulmonary artery endothelial cells in culture isolated from transplant donors.

Even though endothelial cells from different locations have similarities, there are potential morphological and functional differences between cells from different vascular regions, as well as between species. Our laboratory is interested in studying the molecular regulation of vasoactive substances in pulmonary vasculature. Therefore, we have developed reproducible methodology to isolate and maintain cultures of human pulmonary artery endothelial cells. The major innovation has been the employment of sections of pulmonary artery from heart transplant donors, from which endothelial cells are isolated. Cell monolayers were identified as endothelial cells by phase-contrast microscopy. Representative dishes of cells were further characterized by indirect immunofluorescent staining for factor VIII antigen, uptake of acetylated low-density lipoprotein, and electron microscopy. These cells were also evaluated for the expression of endothelin-1 (ET-1), a vasoactive 21-amino acid peptide derived from endothelial cells. The cells expressed ET-1 peptide and mRNA as determined by radioimmunoassay and Northern analysis, respectively. We also demonstrated that these cells are useful in transient transfection experiments for potential evaluation of promoter elements. The availability and relevance of these cells provide an important investigative tool for studies on human pulmonary vascular disease.

Characteristics of Porcine Coronary Artery Endothelial Cells in Culture: Comparison with Aortic Endothelium

In the present study we compared the characteristics of porcine coronary artery endothelial cells in culture with the characteristics of aortic endothelium. Morphologically, coronary artery endothelial cells were larger than aortic endothelial cells. Under basal culture conditions, both coronary artery and aortic endothelial cells secreted significant amounts of prostacyclin and endothelin-1. The outstanding finding is that coronary artery endothelial cells secreted larger amounts of endothelin-1 than aortic endothelial cells. Immunofluorescence staining revealed that individual endothelial cells synthesize both substances. This comparative study provides important evidence indicating that the characteristics of coronary artery endothelial cells differ from those of aortic endothelial cells.

The effect of endothelins on nitric oxide and prostacyclin production from human umbilical vein, porcine aorta and bovine carotid artery endothelial cells in culture.

1. This study has investigated the effects of the endothelin isopeptides, endothelin-1 (ET-1), ET-2 and ET-3 on the production of the endothelium-derived relaxing factors, nitric oxide (NO) and prostacyclin (PGI2) from primary cultures of endothelial cells obtained from human umbilical vein (HUVECS), porcine aorta (PAECS) and bovine carotid artery (BCAECS). 2. NO generation was assessed indirectly by measuring production of cyclic GMP and PGI2 formation was measured by radioimmunoassay of 6-keto PGF1 alpha. 3. In HUVECS, histamine (1 microM) increased cyclic GMP and 6-keto PGF1 alpha production by 12.6 +/- 2.0 and 4.9 +/- 0.7 fold respectively over the corresponding basal values. Haemoglobin (10 microM) and the NO synthase inhibitor NG-monomethyl-L-arginine (10 microM) significantly inhibited the increase in cyclic GMP formation in response to histamine but had no effect on 6-keto PGF1 alpha production. In contrast to histamine, the endothelin isopeptides (ET-1, ET-2 and ET-3; 0.01-1000 nM) produced no significant change in either cyclic GMP or 6-keto PGF1 alpha production in HUVECS. 4. In a separate series of experiments, ET-3 (0.01-1000 nM) also failed to produce any significant change in cyclic GMP or 6-keto PGF1 alpha production from primary cultures of PAECS and BCAECS. In contrast, bradykinin (0.1 microM) and sodium nitroprusside (1 mM) were used as positive control agents and increased cyclic GMP production in these cells. 5. In conclusion, the endothelin isopeptides do not release NO and PGI2 from primary cultures of HUVECS, PAECS and BCAECS. This suggests that endothelin receptors are either absent from these cells or are not coupled to NO or PGI2 production.

An analysis of the time-dependent changes in intracellular calcium concentration in endothelial cells in culture induced by mechanical stimulation.

When bovine pulmonary artery endothelial cells in culture are subjected to mechanical strain, their physiology is altered. Experimentally, this mechanical strain is generated by increased tension in the substrate to which the cells are attached and results in altered levels of fibronectin. Studies of the structural response of the endothelial cell suggest that this stimulus is transmitted to the cell membrane, organelles, and cytoskeleton by natural cell attachments in a quantifiable and predictable manner. This report examines altered intracellular calcium homeostasis as a possible messenger for the observed strain-induced physiologic response. In particular, using the intracellular trapped calcium indicator dyes, Quin2 and Fura2, we observed changes in cytosolic free calcium ion concentration in response to biaxial strain of bovine pulmonary artery endothelial cells in culture. The magnitude and time course of this calcium transient resemble that produced by treatment with the calcium ionophore, Ionomycin, indicating that mechanical stimulation may alter cell membrane permeability to calcium. Additional experiments in the presence of EDTA indicated that calcium was also released from intracellular stores in response to strain. In order to explain the stretch-induced calcium transients, a first-order species conservation model is presented that takes into account both the cell's structural response and the calcium homeostatic mechanisms of the cell. It is hypothesized that the cell's calcium sequestering and pumping capabilities balanced with its mechanically induced changes in calcium ion permeability will determine the level and time course of calcium accumulation in the cytosol.