Xueping Zhou

Endothelial [Ca2+]i and caveolin-1 antagonistically regulate eNOS activity and microvessel permeability in rat venules

AIMS In this study, we investigated the mechanisms by which caveolin-1 (CAV) inhibits increases in permeability induced by platelet activating factor (PAF) and elucidated the relationship between the endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and CAV in regulating endothelial nitric oxide synthase (eNOS) activity and microvessel permeability in intact microvessels. METHODS AND RESULTS Experiments were conducted in individually perfused mesenteric venules in Sprague-Dawley rats. Permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca(2+)](i) and nitric oxide (NO) production were measured in fura-2- and DAF-2-loaded microvessels. Perfusion of the CAV scaffolding domain, AP-CAV, at 1 microM for 30 min did not affect PAF-induced increases in endothelial [Ca(2+)](i) but significantly attenuated PAF-induced NO production from 143 +/- 2 to 110 +/- 3% of control fluorescence intensity (FI). The PAF-induced Lp increase was correlatively reduced from a mean peak value of 7.5 +/- 0.9 to 1.9 +/- 0.5 times that of the control. Increasing extracellular [Ca(2+)] that potentiated PAF-induced peak [Ca(2+)](i) from 500 to 1225 nM augmented NO production to 193 +/- 13% and further increased Lp to 17.3 +/- 1.6 times the control value. More importantly, enhanced Ca(2+) influx restored the reduced NO production and Lp by AP-CAV with NO FI at 149% and Lp at 7.7 +/- 1.1 times the control value. CONCLUSION Our results indicate that eNOS inhibition and reduced NO production contribute to the inhibitory action of AP-CAV on PAF-induced increases in permeability. CAV and endothelial [Ca(2+)](i) antagonistically regulate eNOS activity in intact microvessels, and the level of produced NO is the key determinant of the degree of permeability increases during inflammation.

Improved measurements of intracellular nitric oxide in intact microvessels using 4,5-diaminofluorescein diacetate

4,5-Diaminofluorescein diacetate (DAF-2 DA) has been widely used for the measurement of nitric oxide (NO) in living cells and tissues. We previously established a method that demonstrated platelet activating factor (PAF)-induced endothelial NO production in intact venules using DAF-2 DA. In previous applications, the loading dye was removed from the extracellular space before NO measurements. However, in high permeability vessels, endothelial cells quickly released the accumulated intracellular DAF-2 after the washout, which compromises the NO measurement. The objective of this study was to investigate if the presence of DAF-2 DA during NO measurements could overcome the dye retention problem and enhance the sensitivity of NO detection. Experiments were conducted in individually perfused rat venules, and endothelial NO was measured using fluorescence imaging under basal and stimulated conditions with continuous perfusion of DAF-2 DA. Continuous dye perfusion was found to promote a relatively constant endothelial dye concentration in both normal and high permeability vessels throughout the experiment. With the use of this method, the basal and stimulated NO was quantified after endothelial DAF-2 concentrations reached a steady state. Our results showed enhanced sensitivity of detecting PAF-stimulated NO compared with a previous method. We also found that the hydrolyzed intracellular DAF-2, the precursor of DAF-2 triazole, contributed significantly to the measured fluorescence and that an appropriate subtraction of non-NO-dependent intracellular DAF-2 fluorescence is critical for the assessment of NO in living tissues. This method overcame the dye leakage problem, enhanced the sensitivity of NO detection, and improved NO quantification, demonstrating significant advantages over existing methodologies using DAF-2.

Interactions between A2A adenosine receptors, hydrogen peroxide, and KATP channels in coronary reactive hyperemia

Myocardial metabolites such as adenosine mediate reactive hyperemia, in part, by activating ATP-dependent K(+) (K(ATP)) channels in coronary smooth muscle. In this study, we investigated the role of adenosine A(2A) and A(2B) receptors and their signaling mechanisms in reactive hyperemia. We hypothesized that coronary reactive hyperemia involves A(2A) receptors, hydrogen peroxide (H(2)O(2)), and KATP channels. We used A(2A) and A(2B) knockout (KO) and A(2A/2B) double KO (DKO) mouse hearts for Langendorff experiments. Flow debt for a 15-s occlusion was repaid 128 ± 8% in hearts from wild-type (WT) mice; this was reduced in hearts from A(2A) KO and A(2A)/(2B) DKO mice (98 ± 9 and 105 ± 6%; P < 0.05), but not A(2B) KO mice (123 ± 13%). Patch-clamp experiments demonstrated that adenosine activated glibenclamide-sensitive KATP current in smooth muscle cells from WT and A(2B) KO mice (90 ± 23% of WT) but not A(2A) KO or A(2A)/A(2B) DKO mice (30 ± 4 and 35 ± 8% of WT; P < 0.05). Additionally, H(2)O(2) activated KATP current in smooth muscle cells (358 ± 99%; P < 0.05). Catalase, an enzyme that breaks down H(2)O(2), attenuated adenosine-induced coronary vasodilation, reducing the percent increase in flow from 284 ± 53 to 89 ± 13% (P < 0.05). Catalase reduced the repayment of flow debt in hearts from WT mice (84 ± 9%; P < 0.05) but had no effect on the already diminished repayment in hearts from A(2A) KO mice (98 ± 7%). Our findings suggest that adenosine A(2A) receptors are coupled to smooth muscle KATP channels in reactive hyperemia via the production of H(2)O(2) as a signaling intermediate.

Three-dimensional localization and quantification of PAF-induced gap formation in intact venular microvessels.

Combining single-vessel perfusion technique with confocal microscopy, this study presents a new approach that allows three-dimensional visualization and quantification of endothelial gaps under experimental conditions identical to those used to measure permeability coefficients, endothelial calcium concentration, and nitric oxide production in individually perfused intact microvessels. This approach provides an efficient means for defining the transport pathways and cellular mechanisms of increased microvascular permeability during inflammation. Platelet-activating factor (PAF) was used to increase the permeability of individually perfused rat mesenteric venules. Fluorescent microspheres (FMs, 100 nm) were used as leakage markers, and confocal images were acquired at successive focal planes through the perfused microvessel. Perfusion of FMs under control conditions produced a thin, uniform layer of FMs in the vessel lumen, but in PAF-stimulated microvessels significant amounts of FMs accumulated at endothelial junctions. Reconstructed confocal images three-dimensionally delineated the temporal and spatial development of endothelial gaps in PAF-stimulated microvessels. The FM accumulation, quantified as the total fluorescence intensity per square micrometer of vessel wall, was 8.4 +/- 1.8 times the control value within 10 min of PAF perfusion and declined to 5.0 +/- 0.6 and 1.4 +/- 0.2 times the control value when FMs were applied 30 and 60 min after PAF perfusion. The changes in the magnitude of FM accumulation closely correlated with the time course of PAF-induced increases in hydraulic conductivity (L(p)), indicating that the opening and closing of endothelial gaps contributed to the transient increase in L(p) in PAF-stimulated microvessels. Electron microscopic evaluations confirmed PAF-induced gap formation and FM accumulation at endothelial clefts.

Calcium influx-dependent differential actions of superoxide and hydrogen peroxide on microvessel permeability.

Our previous study demonstrated that reactive oxygen species (ROS) released from activated blood cells contribute significantly to the increased microvessel permeability during inflammation. This study aims to define the individual roles of hydrogen peroxide (H(2)O(2)) and superoxide in ROS-induced increases in permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (L(p)). Endothelial [Ca(2+)](i) was measured in fura-2 AM-loaded microvessels. Perfusing microvessels with superoxide generated by hypoxanthine and xanthine oxidase (HX/XO) induced immediate and transient increases in L(p). The mean peak value, which occurred within 5 min of HX/XO exposure, was 4.3 +/- 0.6 times that of the control. In contrast, the perfusion of H(2)O(2) (100 and 500 microM) caused no immediate increases in L(p). A significant L(p) increase, 3.6 +/- 0.6 times the control value, occurred 30 min after the perfusion of H(2)O(2) at 500 microM. The perfusion of H(2)O(2) at 100 or 500 microM for 1 h increased L(p) to 6.6 +/- 0.9 and 11.3 +/- 3.6 times the control value, respectively. The increased endothelial [Ca(2+)](i) in HX/XO or H(2)O(2) perfused vessels was correlated with the time course of the increases in L(p). Inhibiting Ca(2+) influx by LaCl(3) prevented the permeability increase induced by HX/XO or H(2)O(2). These results demonstrated differential actions of superoxide and H(2)O(2) on microvessel permeability and endothelial [Ca(2+)](i). Superoxide-induced permeability increases were immediate and transient, whereas H(2)O(2)-induced permeability increases were progressive, demonstrating concentration and time dependence. Ca(2+) influx plays an essential role in both superoxide and H(2)O(2)-induced permeability increases.

Caveolin-1 scaffolding domain promotes leukocyte adhesion by reduced basal endothelial nitric oxide-mediated ICAM-1 phosphorylation in rat mesenteric venules.

Exogenously applied caveolin-1 scaffolding domain (CAV) has been shown to inhibit inflammatory mediator-induced nitric oxide (NO) production and NO-mediated increases in microvessel permeability. However, the effect of CAV on endothelial basal NO that prevents leukocyte adhesion remains unknown. This study aims to investigate the roles of exogenously applied CAV in endothelial basal NO production, leukocyte adhesion, and adhesion-induced changes in microvessel permeability. Experiments were conducted in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). NO was quantified with fluorescence imaging in DAF-2-loaded vessels. Perfusing venules with CAV inhibited basal NO production without affecting basal Lp. Resuming blood flow in CAV-perfused vessels significantly increased leukocyte adhesion. The firmly adherent leukocytes altered neither basal Lp nor adherens junction integrity. Increases in Lp occurred only upon formyl-Met-Leu-Phe application that induces release of reactive oxygen species from the adherent leukocytes. The application of NO synthase inhibitor showed similar results to CAV, and NO donor abolished CAV-mediated leukocyte adhesion. Immunofluorescence staining showed increases in binding of ICAM-1 to an adhesion-blocking antibody concurrent with a Src-dependent ICAM-1 phosphorylation following CAV perfusion. Pre-perfusing vessels with anti-ICAM-1 blocking antibody or a Src kinase inhibitor attenuated CAV-induced leukocyte adhesion. These results indicate that the application of CAV, in addition to preventing excessive NO-mediated permeability increases, also causes reduction of basal NO and promotes ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.

H2O2-induced endothelial NO production contributes to vascular cell apoptosis and increased permeability in rat venules.

Although elevated levels of H(2)O(2) have been implicated to play important roles in the pathogenesis of various cardiovascular diseases, the underlying mechanisms remain unclear. This study aims to examine the effect of H(2)O(2) on endothelial nitric oxide (NO) production in intact venules, and elucidate the role and mechanisms of NO in H(2)O(2)-induced increases in microvessel permeability. Experiments were conducted on individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp), and endothelial [Ca(2+)](i) was measured on fura-2-loaded vessels. Perfusion of H(2)O(2) (10 μM) caused a delayed and progressively increased endothelial [Ca(2+)](i) and Lp, a pattern different from inflammatory mediator-induced immediate and transient response. Under the same experimental conditions, measuring endothelial NO via DAF-2 and the spatial detection of cell apoptosis by fluorescent markers revealed that H(2)O(2) induced two phases of NO production followed by caspase activation, intracellular Ca(2+) accumulation, and vascular cell apoptosis. The initial NO production was correlated with increased endothelial NO synthase (eNOS) Ser(1177) phosphorylation in the absence of elevated endothelial [Ca(2+)](i), whereas the second phase of NO depended on increased [Ca(2+)](i) and was associated with Thr(495) dephosphorylation without increased Ser(1177) phosphorylation. Inhibition of NOS prevented H(2)O(2)-induced caspase activation, cell apoptosis, and increases in endothelial [Ca(2+)](i) and Lp. Our results indicate that H(2)O(2) at micromolar concentration is able to induce a large magnitude of NO in intact venules, causing caspase activation-mediated endothelial Ca(2+) accumulation, cell apoptosis, and increases in permeability. The mechanisms revealed from intact microvessels may contribute to the pathogenesis of oxidant-related cardiovascular diseases.

A1 adenosine receptor negatively modulates coronary reactive hyperemia via counteracting A2A-mediated H2O2 production and KATP opening in isolated mouse hearts

We previously demonstrated that A2A, but not A2B, adenosine receptors (ARs) mediate coronary reactive hyperemia (RH), possibly by producing H2O2 and, subsequently, opening ATP-dependent K(+) (KATP) channels in coronary smooth muscle cells. In this study, A1 AR knockout (KO), A3 AR KO, and A1 and A3 AR double-KO (A1/A3 DKO) mice were used to investigate the roles and mechanisms of A1 and A3 ARs in modulation of coronary RH. Coronary flow of isolated hearts was measured using the Langendorff system. A1 KO and A1/A3 DKO, but not A3 KO, mice showed a higher flow debt repayment [~30% more than wild-type (WT) mice, P < 0.05] following a 15-s occlusion. SCH-58261 (a selective A2A AR antagonist, 1 μM) eliminated the augmented RH, suggesting the involvement of enhanced A2A AR-mediated signaling in A1 KO mice. In isolated coronary arteries, immunohistochemistry showed an upregulation of A2A AR (1.6 ± 0.2 times that of WT mice, P < 0.05) and a higher magnitude of adenosine-induced H2O2 production in A1 KO mice (1.8 ± 0.3 times that of WT mice, P < 0.05), which was blocked by SCH-58261. Catalase (2,500 U/ml) and glibenclamide (a KATP channel blocker, 5 μM), but not N(G)-nitro-l-arginine methyl ester, also abolished the enhanced RH in A1 KO mice. Our data suggest that A1, but not A3, AR counteracts the A2A AR-mediated CF increase and that deletion of A1 AR results in upregulation of A2A AR and/or removal of the negative modulatory effect of A1 AR, thus leading to an enhanced A2A AR-mediated H2O2 production, KATP channel opening, and coronary vasodilation during RH. This is the first report implying that A1 AR has a role in coronary RH.

Metabolic hyperemia requires ATP-sensitive K+ channels and H2O2 but not adenosine in isolated mouse hearts

We have previously demonstrated that adenosine-mediated H2O2 production and opening of ATP-sensitive K(+) (KATP) channels contributes to coronary reactive hyperemia. The present study aimed to investigate the roles of adenosine, H2O2, and KATP channels in coronary metabolic hyperemia (MH). Experiments were conducted on isolated Langendorff-perfused mouse hearts using combined pharmacological approaches with adenosine receptor (AR) knockout mice. MH was induced by electrical pacing at graded frequencies. Coronary flow increased linearly from 14.4 ± 1.2 to 20.6 ± 1.2 ml·min(-1)·g(-1) with an increase in heart rate from 400 to 650 beats/min in wild-type mice. Neither non-selective blockade of ARs by 8-(p-sulfophenyl)theophylline (8-SPT; 50 μM) nor selective A2AAR blockade by SCH-58261 (1 μM) or deletion affected MH, although resting flow and left ventricular developed pressure were reduced. Combined A2AAR and A2BAR blockade or deletion showed similar effects as 8-SPT. Inhibition of nitric oxide synthesis by N-nitro-l-arginine methyl ester (100 μM) or combined 8-SPT administration failed to reduce MH, although resting flows were reduced (by ∼20%). However, glibenclamide (KATP channel blocker, 5 μM) decreased not only resting flow (by ∼45%) and left ventricular developed pressure (by ∼36%) but also markedly reduced MH by ∼94%, resulting in cardiac contractile dysfunction. Scavenging of H2O2 by catalase (2,500 U/min) also decreased resting flow (by ∼16%) and MH (by ∼24%) but to a lesser extent than glibenclamide. Our results suggest that while adenosine modulates coronary flow under both resting and ischemic conditions, it is not required for MH. However, H2O2 and KATP channels are important local control mechanisms responsible for both coronary ischemic and metabolic vasodilation.

Spent Culture Medium from Virulent Borrelia burgdorferi Increases Permeability of Individually Perfused Microvessels of Rat Mesentery

Background Lyme disease is a common vector-borne disease caused by the spirochete Borrelia burgdorferi (Bb), which manifests as systemic and targeted tissue inflammation. Both in vitro and in vivo studies have shown that Bb-induced inflammation is primarily host-mediated, via cytokine or chemokine production that promotes leukocyte adhesion/migration. Whether Bb produces mediators that can directly alter the vascular permeability in vivo has not been investigated. The objective of the present study was to investigate if Bb produces a mediator(s) that can directly activate endothelial cells resulting in increases in permeability in intact microvessels in the absence of blood cells. Methodology/Principal Findings The effects of cell-free, spent culture medium from virulent (B31-A3) and avirulent (B31-A) B. burgdorferi on microvessel permeability and endothelial calcium concentration, [Ca2+]i, were examined in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca2+]i, a necessary signal initiating hyperpermeability, was measured in Fura-2 loaded microvessels. B31-A3 spent medium caused a rapid and transient increase in Lp and endothelial [Ca2+]i. Within 2–5 min, the mean peak Lp increased to 5.6±0.9 times the control, and endothelial [Ca2+]i increased from 113±11 nM to a mean peak value of 324±35 nM. In contrast, neither endothelial [Ca2+]i nor Lp was altered by B31-A spent medium. Conclusions/Significance A mediator(s) produced by virulent Bb under culture conditions directly activates endothelial cells, resulting in increases in microvessel permeability. Most importantly, the production of this mediator is associated with Bb virulence and is likely produced by one or more of the 8 plasmid(s) missing from strain B31-A.