Rat Coronary Microvascular Endothelial Cells Research Articles

Effect of inflammation-mediated endothelial metabolic shift on endothelial barrier function

Abstract Background The integrity of the endothelial cell barrier of the microvasculature is compromised by inflammation. The increased vascular permeability leads to tissue injury and organ dysfunction. In recent years, considerable advances have been made in the understanding of signalling mechanisms regulating the endothelial barrier integrity. The role of endothelial metabolism as a modulator of endothelial barrier integrity is not yet well-studied. The aim of the present study was to investigate the effect of inflammation on endothelial metabolism and its role in the maintenance of endothelial barrier integrity. Methods The study was carried out on cultured human umbilical vein endothelial cells and rat coronary microvascular endothelial cells. Inflammatory condition was simulated by treating cells with low concentrations (1 ng/mL) of TNFα for 24h. Endothelial barrier function was analysed by measuring the flux of albumen through endothelial monolayers cultured on filter membranes. Gene expression was analysed by qPCR-based assays. The capacity of endothelial cells for maximal ATP synthesis rate was investigated by the real-time live-cell imaging using FRET-based ATP-biosensor (live cell FRET). Total cellular ATP concentration was measured using luminescence-based commercial kit (ATPLite, PerkinElmer). Mitochondrial mass was analysed by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). The cellular glucose uptake was measured by fluorescent microscopy using a fluorescent analogue of glucose (2-NBDG). Results Treatment of human endothelial cells with TNFα resulted in significant suppression of mitochondrial and upregulation of glycolytic ATP synthesis rate, suggesting a metabolic switch. This was accompanied by a reduction in mitochondrial content (mtDNA/nDNA), reduction in total cellular ATP levels, an enhanced expression of glycolytic enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), and enhanced glucose uptake by endothelial cells (n=5; p<0.05 for all parameters tested). Moreover, TNFα caused a 3-fold increase in endothelial permeability. Pharmacological inhibition of glycolysis either by partial replacement of glucose with 2-deoxy glucose (2DG) or an inhibition of PFKFB3 resulted in further worsening (a 5-fold increase in permeability) of TNFα-induced endothelial barrier failure. On the other hand pharmacological activation of AMPK, a potent inducer of mitochondrial biogenesis, could attenuate TNFα-induced but not 2DG-induced endothelial hyperpermeability. Conclusion The study demonstrates that TNFα induces metabolic switch towards glycolysis in endothelial cells. Moreover, the data suggest that upregulation of glycolysis may serve as an endogenous metabolic adaptation to the TNFα-induced suppression of mitochondrial ATP synthesis, which protects endothelial barrier integrity. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Justus-Liebig University GiessenDZHK (German Centre for Cardiovascular Research), partner site Rhein-Main, Bad Nauheim, Germany

NAD+ administration decreases microvascular damage following cardiac ischemia/reperfusion by restoring autophagic flux.

Microvascular damage is a key pathological change in myocardial ischemia/reperfusion (I/R) injury. Using a rat model of myocardial I/R, our current study has provided the first evidence that nicotinamide adenine dinucleotide (NAD+) administration can significantly attenuate myocardial I/R-induced microvascular damage, including reduced regional blood perfusion, decreased microvessel density and integrity, and coronary microvascular endothelial cells (CMECs) injury. In studies with primary cultured CMECs under hypoxia/reoxygenation (HR) and a rat model of I/R, our results suggested that the protective effect of NAD+ on CMECs exposed to HR or I/R is at least partially mediated by the NAD+-induced restoration of autophagic flux, especially lysosomal autophagy: NAD+ treatment markedly induced transcription factor EB (TFEB) activation and attenuated lysosomal dysfunction in the I/R or HR-exposed cells. Collectively, our study has provided the first in vivo and in vitro evidence that NAD+ significantly rescued the impaired autophagic flux and cell apoptosis that was induced by I/R in rat CMECs, which is mediated in part through the action of TFEB-mediated lysosomal autophagy.

Intermedin induces loss of coronary microvascular endothelial barrier via derangement of actin cytoskeleton: role of RhoA and Rac1

Intermedin (IMD) is a novel member of the calcitonin gene-related peptide family, which acts via calcitonin receptor-like receptors (CLRs), mediating activation of cAMP signalling. The main objective of the present study was to analyse the molecular mechanisms of the differential effects of IMD on the macromolecule permeability of endothelial cells of different vascular beds. Here we demonstrate that IMD increases permeability of rat coronary microvascular endothelial cells (RCECs) and reduces permeability of human umbilical vein endothelial cells (HUVECs) and rat aortic endothelial cells via CLRs and cAMP. Intermedin causes a derangement of the actin cytoskeleton accompanied by loss of vascular endothelial cadherin (VE-cadherin) in RCECs, while it causes a rearrangement of the actin cytoskeleton and VE-cadherin at cell-cell junctions in HUVECs. Intermedin inactivates the RhoA/Rho-kinase (Rock) pathway in both cell types; however, it inactivates Rac1 in RCECs but not in HUVECs. Inhibition and rescue experiments demonstrate that both RhoA and Rac1 are required for the RCEC barrier stability, while in HUVECs the inhibition of RhoA/Rock signalling does not interfere with basal permeability. The opposite effects of IMD on permeability of RCECs and HUVECs are due to differential regulation of actin cytoskeleton dynamics via RhoA and Rac1. Moreover, Rac1 activity is regulated by the RhoA/Rock pathway in RCECs but not in HUVECs.

Differential effects of cyclic and static stretch on coronary microvascular endothelial cell receptors and vasculogenic/angiogenic responses

Mechanical stretch, an important growth stimulus, results not only from pulsatile blood flow and diastolic stretch of the ventricles [cyclic stretch (CS)] but also from tissue expansion during growth [constant static stretch (SS)]. We compared growth factor receptor expression and vasculogenic/angiogenic responses of rat coronary microvascular endothelial cells (ECs) by exposing cells to CS (10% elongation at 30 cycles/min) and SS (constant 10% elongation). Both CS and SS increased VEGF receptor (VEGF-R)2 protein levels and the extent of tube formation and branching. Moreover, both CS and SS enhanced VEGF-induced cell proliferation and tube formation, indicating that both types of stretch increase the sensitivity of ECs to VEGF. Blockade of VEGF-R2 prevented the increases in EC proliferation and aggregate tube length. However, CS but not SS enhanced EC Tie-2 protein and migration. CS affected a greater increase in tube length and branch formation than did SS. A unique finding was that SS but not CS increased VEGFR-1 in ECs. Our study is the first to distinguish between the effects of CS and SS on growth factor receptor expression and rat coronary microvascular EC proliferation, migration, and tube formation. In conclusion, EC angiogenic responses to these two types of stretch display both differences and similarities, but both CS and SS are dependent on VEGF-R2 signaling for their vasculogenic/angiogenic effects.

Nitric Oxide Does Not Downregulate Rho-Kinase (ROCK-2) Expression in Rat Coronary Endothelial Cells

Rho kinase (ROCK) and nitric oxide (NO) are important targets in cardiovascular diseases. Therefore, we investigated the possible influence of NO on Rho kinase (ROCK-2 isoform) expressions in cultured rat coronary microvascular endothelial cells. The cells were isolated from Wistar rats on a Langendorff system, and were incubated overnight (approximately 16 h) with an NO generator, A-23187 (10 to 10 M), NO donors, such as sodium nitroprusside (10 to 10 M), glyceryl trinitrate (10 to 10 M), 2,2'-(hydroxynitrosohydrazono)bis-ethanimine (10 to 10 M), and NaNO2 (10 to 10 M) or a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methylester (2 x 10 M), or two ROCK inhibitors, (+)-(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10 M) and fasudil (10 M) in the absence or presence of thrombin (4 U/mL). ROCK-2 and endothelial NOS (eNOS) expressions were detected by Western blotting. Moreover, nitrite/nitrate levels were detected by Griess method in the presence of the ROCK inhibitors. The NO donors and the NO generator had no significant effects on ROCK-2 expression. Y-27632 and fasudil did not alter eNOS expression and NO production. Nitrite/nitrate levels were 4.4 +/- 0.32 microM in control and 4.0 +/- 0.93 microM and in Y-27632 group. These results demonstrate that prolong NO donation could not suppress the expression of ROCK-2 protein, and the ROCK inhibitor did not change e-NOS expression and NO production in the cultured rat coronary microvascular endothelial cells.

Therapeutic Concentrations of Tacrolimus Do Not Interfere With Endothelial Nitric Oxide Synthesis in Rat Thoracic Aortas and Coronary Arteries

This study aimed to investigate the potential effect of in vivo administration of immunosuppressive agent FK-506 (tacrolimus) on the endothelial function of rat thoracic aortas with respect to nitric oxide (NO) synthesis. In vitro effect of the drug on NO synthesis in cultured rat coronary microvascular endothelial cells (CMEC) was also studied.In vivo administration of tacrolimus (1 mg/kg/d, intramuscular) to rats for 14 days resulted in decreased relaxant responses to the higher concentrations (1 to 30 muM) of acetylcholine in the aortas; however, responses to calcium ionophore A23187, sodium nitroprusside, L-arginine, and L-NAME did not change significantly. No changes were observed in phenylephrine-induced contractions in endothelium-denuded or -intact preparations. Administration of the vehicle for 14 days did not affect these parameters. In order to evaluate the in vitro effect of tacrolimus on NO release, CMEC isolated from rat hearts were incubated with either tacrolimus (0.01, 0.1 microM) or the vehicle. Basal, calcium ionophore-stimulated, or interleukin-1 beta-induced NO synthesis was determined by measuring total nitrite in the media. Neither tacrolimus nor the vehicle changed nitrite accumulation. It has been concluded that therapeutic concentrations of tacrolimus do not alter NO production in rat thoracic aorta or cultured CMEC; however, it impairs relaxant responses of rat aorta induced by higher concentrations of acetylcholine, possibly through changes in the downstream of receptor activation or through an imbalance between endothelium-dependent relaxant and contracting factors within the endothelium in favor of the contracting factor(s).

Differential expression of α 1-adrenergic receptor subtypes in coronary microvascular endothelial cells in culture

It has been postulated that in blood vessels under α 1-related stimulation, the endothelial intracellular calcium concentration ([Ca 2+] i ) increases, which is necessary to induce nitric oxide synthesis, is the result of an increase in vascular smooth muscle, which subsequently, flows into the endothelial cells through gap junctions and it is not the result of a direct adrenergic stimulation of endothelial receptors. Others, however, postulate that endothelial α 1D-adrenoceptors, have a direct effect on nitric oxide synthesis. In order to clarify this phenomena, in this work we analyzed the presence of α 1receptor subtypes and their functional association with nitric oxide synthesis in rat coronary microvascular endothelial cells in culture, with pharmacological, immunological and reverse transcriptase polymerase chain reaction approaches. Our results show the presence and functional coupling with nitric oxide synthesis of α 1A and α 1D-adrenoceptor subtypes. α 1B-adrenoceptor subtype is not coupled with nitric oxide production.

Differential effects of cyclic and static stretch on expression of tyrosine kinase receptors in microvascular endothelial cells

Mechanical stretch, an important stimulus for vascular growth, results not only from pulsatile blood flow (cyclic stretch, CS), but also from tissue expansion during growth (constant static stretch, SS). In order to investigate whether both types of stretch provide similar or dissimilar stimuli for growth factor receptor up-regulation, rat coronary microvascular endothelial cells were cultured in Bioflex culture plates coated with collagen and exposed to CS or SS. The CS was generated by a computerized Flexercell Strain Unit with 10% elongation at 30 cycles/min, while the SS was induced at the same elongation but with constant stretch without relaxation. Controls consisted of cells seeded on the culture plates but not subjected to stretch. The protein level of tyrosine kinase receptor was assessed with Western blotting. The levels of Flk-1 protein increased in a time dependent manner following CS or SS. Maximal levels of Flk-1 reached 3.2 ± 0.54 fold at 6 hours after CS and 3.4 ± 0.75 fold at 18 hours after SS, respectively. SS enhanced protein level of Flt-1, peaked (2.1 ± 0.51 fold over the control value) by 18 hr. In contrast, CS did not alter the flt-1 expression. Both CS and SS caused up-regulation of Tie-2 protein after only 1 hour, however, CS affected relatively higher and longer expression of Tie-2, which remained elevated up to 18 hr of stretch (1.8 ± 0.23), while SS-induced up-regulation of Tie-2 returned to normal levels after 6 hr. Our study is the first to distinguish between the effects of the two types of stretch on key tyrosine kinase receptor expression. These results suggest that endothelial cell responses to two types of stretch display both differences and similarities with regard to receptor expression. Supported by NIH grant HL075446.

Coronary microvascular endothelial cell growth regulates expression of the gene encoding p22-phox

The mRNA expression of p22-phox, a membrane-bound component of NAD(P)H oxidase, is elevated in proliferating compared to quiescent rat coronary microvascular endothelial cells (CMEC). However, the underlying molecular mechanisms of this increase and its relevance to cell confluence remain unknown. This study shows greater levels of p22-phox mRNA/protein expression, NAD(P)H oxidase activity, and superoxide anion (O 2 −) production in proliferating versus fully confluent and growth-arrested 50% confluent CMEC. Both enhanced transcription of the p22-phox gene and enhanced mRNA half-life were shown to contribute to the increase in p22-phox mRNA levels as demonstrated by nuclear run-on studies and Northern analyses after actinomycin D transcriptional arrest, respectively. Suppression of O 2 − availability by a cell-permeable superoxide dismutase mimetic or inhibition of NAD(P)H oxidase activity via its specific inhibitors (apocynin, diphenyleneiodinium, phenylarsine oxide, and 4-(2-aminoethyl)bezenesulfonyl fluoride) or antisense p22-phox cDNA attenuated cell growth independent of changes in intracellular antioxidant glutathione levels. Inhibition of nascent protein synthesis by a translation inhibitor, cycloheximide, significantly reduced p22-phox mRNA stability in proliferating but not in confluent CMEC. These findings suggest that expression of p22-phox mRNA in CMEC is upregulated by growth through a mechanism that involves increases in transcriptional activity of the p22-phox gene and stability of the p22-phox message after its formation.

Stretch induces upregulation of key tyrosine kinase receptors in microvascular endothelial cells

We previously demonstrated that cyclic stretch of cardiac myocytes activates paracrine signaling via vascular endothelial growth factor (VEGF) leading to angiogenesis. The present study tested the hypothesis that cyclic stretch upregulates tyrosine kinase receptors in rat coronary microvascular endothelial cells (RCMEC) and human umbilical vein endothelial cells (HUVEC). VEGF receptor-2 (Flk-1) protein levels increased in HUVEC and RCMEC in a time-dependent manner, but the increase occurred much earlier in RCMEC than in HUVEC. The enhancement of Flk-1 protein level was not inhibited by addition of VEGF neutralizing antibodies, indicating that VEGF is not involved in stretch-induced Flk-1 expression. VEGF receptor-1 (Flt-1) protein and mRNA were not changed by stretch. However, Tie-2 and Tie-1 protein levels increased in RCMEC. Angiopoietin-1 and -2, the ligands for Tie-2, increased in cardiac myocytes subjected to cyclic stretch but were not affected by stretch in endothelial cells (EC). Stretch or incubation of RCMEC with VEGF increased cell proliferation moderately, whereas stretch + VEGF had an additive effect on proliferation. Mechanical stretch induces upregulation of the key tyrosine kinase receptors Flk-1, Tie-2, and Tie-1 in vascular EC, which underlies the increase in sensitivity of EC to growth factors and, therefore, facilitates angiogenesis. These in vitro findings support the concept that stretch of cardiac myocytes and EC plays a key role in coronary angiogenesis.

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells.

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E(rev)) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E(rev) of nicotine-induced current as a function of extracellular Na(+) concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K(+)/Na(+) permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca(2+) concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na(+), which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.

Effects of angiotensin II on nitric oxide generation in proliferating and quiescent rat coronary microvascular endothelial cells.

Nitric oxide (NO) and the mitogenic peptide angiotensin II (Ang II) have been implicated in endothelial cell growth. However, the putative relationship between these two opposing agents with respect to endothelial cell growth remains unknown. In this study, proliferating and confluent rat coronary microvascular endothelial cells (CMEC) were treated with different doses of Ang II, Ca2+ ionophore A23187, or valsartan (an Ang II type 1 (AT1) receptor inhibitor) alone or in combination for 24 h before measuring the nitrite levels as an index of NO generation. NO production and endothelial NO synthase (eNOS) mRNA/protein expression were found to be 3-fold greater in proliferating vs. quiescent CMEC. Treatments of CMEC with Ang II or Ca2+ ionophore A23187 equally increased NO production without altering the fold-difference in the basal release of NO from proliferating vs. confluent CMEC. Valsartan abolished NO production in CMEC treated with Ang II but not Ca2+ ionophore A23187. Treatments of endothelium-intact vascular rings with Ang II (1 nmol/l to 10 micromol/l) plus valsartan or PD-123319, an Ang II type 2 (AT2) receptor inhibitor, attenuated vascular responses to acetylcholine in an Ang II dose-dependent manner. In these rings, phenylephrine produced significant increases in contractile responses only at nmol/l concentrations of Ang II. In contrast, pharmacological and mechanical inactivation of endothelium enhanced contractile responses to phenylephrine at micromol/I concentrations of Ang II. These data demonstrate that Ang II stimulates NO production in CMEC in both an AT1- and an AT2 receptor-regulated manner, and that this stimulation of NO may be beneficial in counterbalancing the direct vasoconstrictor effect of Ang II on underlying smooth muscle cells.

Aprotinin impairs coronary endothelial function and down-regulates endothelial NOS in rat coronary microvascular endothelial cells.

The non-specific serine protease inhibitor aprotinin is currently used to reduce blood loss and the need for blood transfusion after cardiopulmonary bypass. We have recently reported that aprotinin impairs endothelium-dependent but not endothelium-independent relaxations in rat thoracic aortic rings due to its inhibitory effect on endothelial nitric oxide (NO) production. In light of these findings, the current study was designed to investigate the effects of aprotinin on coronary endothelial function in isolated rat hearts and on the expression of endothelial NO synthase (eNOS) in cultured rat coronary microvascular endothelial cells (CMEC). Hearts obtained from Sprague-Dawley rats were perfused on a constant flow Langendorff isolated heart system and coronary perfusion pressure and cardiac parameters were recorded. The coronary relaxant responses to bolus infusions of bradykinin (BK) and sodium nitroprusside (SNP) were recorded in the absence and presence of aprotinin. Total RNA and protein samples were extracted from CMEC incubated with aprotinin or the vehicle, 0.9% NaCl. RT-PCR-Southern blotting and Western analyses were carried out to assess eNOS mRNA and protein levels, respectively. Aprotinin (125 and 250 kIU/ml) increased coronary perfusion pressure without changing the heart rate and cardiac contractility. Aprotinin inhibited BK-induced coronary vasodilatation at 250 kIU/ml, but not at 125 kIU/ml concentrations. The relaxant response to SNP did not change in response to either concentration of the drug. Incubation of CMEC with aprotinin down-regulated eNOS mRNA and protein at 250 kIU/ml, but not at 125 kIU/ml concentration. These data suggest that aprotinin selectively inhibits NO synthesis at higher doses (> or = 250 kIU/ml) and therefore impairs endothelium-dependent coronary vascular tone. This effect of the drug may contribute to its 'blood-sparing' action, but may also account for the increase in the incidence of postoperative graft thrombosis observed in clinical practice during aprotinin therapy.

Aprotinin impairs endothelium-dependent relaxation in rat aorta and inhibits nitric oxide release from rat coronary endothelial cells.

Aprotinin, a non-specific serine protease inhibitor, reduces postoperative bleeding after coronary artery surgery. The mechanism of action for this 'blood-sparing' effect of aprotinin is only partially clarified. We therefore aimed to investigate the effect of aprotinin on the release of nitric oxide (NO), a vasodilator and antiaggregant factor, from rat coronary microvascular endothelial cells and on the NO-mediated endothelium-dependent relaxation of rat thoracic aorta. Endothelium-intact and endothelium-denuded thoracic aortic rings from Wistar rats (250-300 g) were suspended in organ chambers. Contractile and relaxant responses in the absence and presence of aprotinin (125, 250 and 500 KIU/ml) were recorded via a mechanotransducer. Coronary microvascular endothelial cells (CMEC) were isolated on a Langendorff system by collagenase perfusion of the hearts from the same rats. Calcium ionophore- (1 microM) induced release of NO from confluent cells was determined spectrophotometrically by measuring its stable metabolites, nitrite and nitrate, via Griess reaction. Aprotinin selectively enhanced phenylephrine-induced contractions in endothelium-intact rat thoracic aortic rings, but not in the endothelium-denuded rings. The use of a nitric oxide synthesis inhibitor Nomega-nitro-L-arginine methyl ester (100 microM) on endothelium-intact rings produced a similar increase in phenylephrine-induced contractions. KCl-induced contractions remained unaltered. Aprotinin inhibited acetylcholine-, calcium ionophore- and L-arginine-induced endothelium-dependent relaxations, but not sodium nitroprusside-induced endothelium-independent relaxation. Aprotinin had no significant effect on basal nitrite-nitrate release from CMEC, while it inhibited calcium ionophore-induced total nitrite accumulation in the supernatants. Aprotinin selectively impairs endothelium-dependent relaxation as well as basal NO availability in rat thoracic aortic rings and inhibits NO release from rat CMEC. This effect of the drug may contribute to its 'blood-sparing' action and may also account for the increase in perioperative restenosis risk observed in clinical practice during aprotinin therapy.

Presence of glutamine:fructose-6-phosphate amidotransferase for glucosamine-6-phosphate synthesis in endothelial cells: effects of hyperglycaemia and glutamine

Recent studies show that glucosamine infusion impairs endothelium-dependent blood flow in normoglycaemic rats. The pathophysiological relevance of this finding, however, depends on whether de novo glucosamine synthesis occurs in endothelial cells. The aim of this study was to test the hypothesis of whether glutamine:fructose-6-phosphate amidotransferase (the first and key regulatory enzyme in hexosamine synthesis) is present for endothelial glucosamine synthesis. Bovine venular, bovine aortic, human microvascular, human umbilical vein, and rat coronary microvascular endothelial cells were used to measure glutamine:fructose-6-phosphate amidotransferase activity. To determine glucosamine-6-phosphate synthesis in intact cells, they were incubated for 1 h in Krebs bicarbonate buffer containing 5, 15 or 30 mmol/l [U-14C]glucose and 0.5, 2 or 4 mmol/l glutamine. The [14C]Glucosamine-6-phosphate and its end products ([14C]UDP-N-acetylglucosamine and [14C]UDP-Nacetylgalactosamine) were separated by HPLC. There were high glutamine:fructose-6-phosphate amidotransferase activities in all endothelial cells studied. Exposure of cells to 15 to 30 mmol/l glucose or 2 to 4 mmol/l glutamine increased enzyme activity. Glucosamine-6-phosphate, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine syntheses increased with increasing extracellular concentrations of glucose from 5 to 30 mmol/l or of glutamine from 0.5 to 4 mmol/l. Our results show the presence of glutamine:fructose-6-phosphate amidotransferase for de novo glucosamine synthesis in endothelial cells and the modulation of this pathway by hyperglycaemia and glutamine. As glucosamine inhibits endothelial nitric oxide synthesis, these findings could have important implications for impaired endothelium-dependent relaxation and vascular dysfunction in diabetes mellitus.

Phenotypic changes in rat and guinea pig coronary microvascular endothelium after culture: loss of nitric oxide synthase activity.

Coronary microvascular endothelial cells (CMVEs) can modulate the contractile performance of the adjacent myocardium by the release of agents such as nitric oxide (NO). Most previous studies using CMVEs have been done in situ, in the intact organ. We set out to study possible differences in NO synthase (NOS) regulation between freshly isolated and cultured rat and guinea pig CMVEs. CMVEs were isolated from Wistar rats and Dunkin Hartley guinea pigs and then grown in culture for varying times. Fura-2 fluorescence was used to measure agonist-induced changes in CMVE intracellular calcium levels. Agonist-induced changes in CMVE cGMP levels were measured by commercial radioimmunoassay kit. Western blot analysis was used to measure endothelial, constitutive NOS (ecNOS) and soluble guanylate cyclase (sGC) protein levels. Reverse transcription, polymerase chain reactions and Southern blotting were used to measure ecNOS mRNA transcripts. In both fresh (1 h post-isolation) and cultured (14 days with one passage) CMVEs of the rat and guinea pig, bradykinin (BK) and the calcium ionophore A23187 (both 1 microM) elicited significant (P < 0.01) increases in the fura-2 340/380 fluorescence ratio. In cultured CMVEs, basal cGMP levels were unaffected by exposure to BK or A23187. Exposure to sodium nitroprusside (SNP) or atrial natriuretic peptide (ANP) (both 1 microM) induced significant (P < 0.01) increases in cGMP in guinea pig cells, whereas in rat cells only ANP produced a significant (P < 0.01) response. By contrast, freshly isolated CMVEs of both species had higher basal cGMP levels than cultured cells, and on exposure to BK and A23187, responded with significant (P < 0.01) increases in cGMP. Moreover, exposure of both fresh rat and guinea pig CMVEs to SNP or ANP also resulted in significant (P < 0.01) increases in cGMP. Western blot analysis demonstrated that ecNOS and sGC protein were lost from the rat CMVEs following culture. Furthermore, there was also a significant loss of ecNOS mRNA from the rat cells following culture. These data demonstrate that freshly isolated rat and guinea pig CMVEs possess ecNOS activity, and that this activity is downregulated following culture. At least for the rat, this effect would seem to lie at both the transcriptional and translational level. Furthermore, rat CMVEs have reduced activity of sGC following culture.

Expression of functional neutrophil-type NADPH oxidase in cultured rat coronary microvascular endothelial cells.

The production of reactive oxygen species (e.g., superoxide) by endothelial cells is relevant to tissue injury during ischemia-reperfusion, and may also play a role in intracellular signaling pathways. However, the molecular identities of the enzymes responsible for endothelial superoxide production are poorly defined, although xanthine oxidase, NADH/NADPH oxidoreductases and nitric oxide synthase are among proteins suggested to contribute. Recent studies suggest that an NADH/NADPH oxidase similar to that found in neutrophils is an important source of superoxide in vascular smooth muscle. We investigated whether a phagocyte-type NADH/NADPH oxidase complex is present in rat cultured coronary microvascular endothelial cells. The expression of NADPH oxidase components was studied by RT-PCR and Western blot analyses, while functional activity was assessed by measurement of superoxide production by lucigenin-enhanced chemiluminescence. The major component of the phagocyte-type NADH/NADPH oxidase complex, a cytochrome b558 heterodimer, was shown to be present both at mRNA and protein levels, using oligonucleotide primers designed from published neutrophil and vascular smooth muscle sequences and anti-neutrophil antibodies respectively. Functional activity of the enzyme was also confirmed by NADPH-evoked superoxide production in cell homogenates, which was inhibited either by the superoxide chelator Tiron or by diphenyleneiodonium, an inhibitor of the oxidase. A functional phagocyte-type NADPH oxidase is expressed in coronary microvascular endothelial cells, where it may contribute to the physiological and/or pathophysiological effects of reactive oxygen species. These data, together with reports of the presence of a similar oxidase in other non-phagocytic cell types, suggest that this enzyme complex is widely expressed in many tissues where it may subserve signaling and other functions.

Hematologically Important Mutations: X-Linked Chronic Granulomatous Disease

The mRNA expression of p22-phox, a membrane-bound component of NAD(P)H oxidase, is elevated in proliferating compared to quiescent rat coronary microvascular endothelial cells (CMEC). However, the underlying molecular mechanisms of this increase and its relevance to cell confluence remain unknown. This study shows greater levels of p22-phox mRNA/protein expression, NAD(P)H oxidase activity, and superoxide anion (O2−) production in proliferating versus fully confluent and growth-arrested 50% confluent CMEC. Both enhanced transcription of the p22-phox gene and enhanced mRNA half-life were shown to contribute to the increase in p22-phox mRNA levels as demonstrated by nuclear run-on studies and Northern analyses after actinomycin D transcriptional arrest, respectively. Suppression of O2− availability by a cell-permeable superoxide dismutase mimetic or inhibition of NAD(P)H oxidase activity via its specific inhibitors (apocynin, diphenyleneiodinium, phenylarsine oxide, and 4-(2-aminoethyl)bezenesulfonyl fluoride) or antisense p22-phox cDNA attenuated cell growth independent of changes in intracellular antioxidant glutathione levels. Inhibition of nascent protein synthesis by a translation inhibitor, cycloheximide, significantly reduced p22-phox mRNA stability in proliferating but not in confluent CMEC. These findings suggest that expression of p22-phox mRNA in CMEC is upregulated by growth through a mechanism that involves increases in transcriptional activity of the p22-phox gene and stability of the p22-phox message after its formation.

A2-adenosine receptor stimulation increases macromolecule permeability of coronary endothelial cells.

The effect of the A2-adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) on macromolecule permeability (PM; indicator fluorescein isothiocyanate-labeled albumin) of endothelial cells was investigated using confluent monolayers of rat coronary microvascular endothelial cells (CEC) and porcine aortic macrovascular endothelial cells (AEC). In CEC, NECA (10(-7) M) increased PM by 39%. Similar results were obtained by isoproterenol (10(-6) M) and forskolin (10(-5) M). The effect of NECA could be antagonized by 8-phenyltheophylline (8-PT; 10(-5) M). In AEC, NECA (10(-7) M) caused an opposite effect in that it decreased PM by 26% as did isoproterenol (10(-6) M) and forskolin (10(-5) M). The response to NECA was abolished in the presence of 8-PT (10(-5) M). In AEC but not CEC, NECA could reduce the rise in PM caused by endothelial energy depletion (in the presence of 5 mM KCN and 5 mM 2-deoxy-D-glucose). It was common to AEC and CEC that NECA (10(-7) M), isoproterenol (10(-6) M), and forskolin (10(-5) M) stimulated production of adenosine 3',5'-cyclic monophosphate (cAMP). The stimulatory effect of NECA on production of cAMP could be antagonized by 8-PT (10(-5) M). In summary, the results indicate that in AEC and CEC PM is modulated by an A2-adenosine receptor-mediated stimulation of adenylate cyclase. The secondary effects of stimulation of adenylate cyclase are different in CEC and AEC, however, since it caused a reduction of PM in AEC, but an increase in CEC.

Macromolecule permeability of coronary and aortic endothelial monolayers under energy depletion

The dependence of macromolecule permeability (MP; indicator fluorescein isothiocyanate-labeled albumin) of endothelial cells on their energetic state was investigated using confluent monolayers of rat coronary microvascular endothelial cells and porcine aortic macrovascular endothelial cells. When oxidative and glycolytic energy productions were inhibited (5 mM KCN plus 5 mM 2-deoxy-D-glucose) 90% of the endothelial ATP contents were lost within 15 min, followed by a progressive increase of MP, disintegration of the actin cytoskeleton, and the opening of intercellular gaps. Elution of the blocker and a subsequent 3-h incubation in complete culture medium reversed the effects of 2-h metabolic blockade, completely for MP and partially for ATP levels. Coronary microvascular and aortic macrovascular endothelial cells responded similarly to energy depletion and repletion, the microvascular cells being more sensitive. The results demonstrate that 1) energetic inhibition augments endothelial macromolecule permeability when both oxidative and glycolytic energy production are inhibited, 2) increased macromolecule permeability in energy-depleted endothelial monolayers is caused by the opening of intercellular gaps, and 3) endothelial cells reversibly tolerate up to 2 h of almost complete ATP depletion.