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Endostatin uncouples NO and Ca²⁺ response to bradykinin through enhanced O₂^–· production in the intact coronary endothelium

Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 3 March 2004 ; accepted in final form 1 October 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study tested the hypothesis that endostatin stimulates superoxide (O₂^–·) production through a ceramide-mediating signaling pathway and thereby results in an uncoupling of bradykinin (BK)-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) from nitric oxide (NO) production in coronary endothelial cells. With the use of high-speed, wavelength-switching, fluorescence-imaging techniques, the [Ca²⁺]_i and NO levels were simultaneously monitored in the intact endothelium of freshly isolated bovine coronary arteries. Under control conditions, BK was found to increase NO production and [Ca²⁺]_i in parallel. When the arteries were pretreated with 100 nM human recombinant endostatin for 1 h, this BK-induced NO production was reduced by 89%, whereas [Ca²⁺]_i was unchanged. With the conversion rate of L-[³H]arginine to L-[³H]citrulline measured, endostatin had no effect on endothelial NO synthase (NOS) activity, but it stimulated ceramide by activation of sphingomyelinase (SMase), whereby O₂^–· production was enhanced in endothelial cells. O₂^–· scavenging by tiron and inhibition of NAD(P)H oxidase by apocynin markedly reversed the effect of endostatin on the NO response to BK. These results indicate that endostatin increases intracellular ceramide levels, which enhances O₂^–· production through activation of NAD(P)H oxidase. This ceramide-O₂^–· signaling pathway may contribute importantly to endostatin-induced endothelial dysfunction.

collagen; sphingolipid; free radicals; signal transduction; coronary artery

Despite extensive studies on the action of endostatin on endothelial cell growth or apoptosis and related mechanisms, there are only a few studies done that addresses whether endostatin alters the function of cultured endothelial cells. So far little is known about the action of this angiostatic peptide on the intact endothelium on arterial wall and in vivo vasculature (29). In this regard, a recent study reported that endostatin decreased vascular endothelium growth factor (VEGF)-induced activation of nitric oxide (NO) synthase (NOS) in cultured umbilical vein endothelial cells (34). It has been suggested that endostatin may inhibit NO synthesis and thereby results in endothelial dysfunction (34). On the other hand, in cultured aortic endothelial cells, endostatin has been reported to increase intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Considering the stimulatory role of Ca²⁺ in NO production in endothelial cells, these results raised two important questions: 1) Does endostatin decrease NO production but increase intracellular Ca²⁺ concentrations? 2) What is the mechanism by which endostatin decreases NO in endothelial cells? Those previous studies did not answer these questions because they did not simultaneously measure intracellular Ca²⁺ and NO concentrations. We recently developed a novel method that uses high-speed, wavelength-switching, fluorescence-imaging techniques to simultaneously in situ monitor intracellular NO and Ca²⁺ concentrations in the intact coronary endothelium (37). This assay system may be used to answer the questions listed above and to further explore the mechanisms mediating endostatin-induced decrease in NO levels in arterial endothelial cells.

The present study hypothesized that endostatin uncouples endothelial NO production from increases in [Ca²⁺]_i in endothelial cells and that this uncoupling of Ca²⁺ and NO response may be associated with enhanced O₂^–· production. To test this hypothesis, we simultaneously monitored intracellular NO and Ca²⁺ levels in the intact endothelium of freshly dissected bovine coronary arteries and examined the effect of endostatin on bradykinin (BK)-induced increase in [Ca²⁺]_i and NO levels. We then determined the effects of endostatin on the activity of endothelial NOS and on the production of ceramide, an endothelial O₂^–·-stimulating signaling lipid. Finally, we examined the effects of endostatin on NAD(P)H-dependent O₂^–· production and NO-O₂^–· interaction in the intact endothelium of these coronary arteries.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fluorescent imaging analysis of NO levels and [Ca²⁺]_i in the intact endothelium of coronary arteries. Recently, we developed a fluorescence imaging analysis to simultaneously monitor [Ca²⁺]_i with fura 2 as indicator and intracellular NO levels with 4,5-diaminofluorescein as a probe in the intact endothelium of freshly dissected small bovine coronary arteries (37). It has been demonstrated that there is no interference between fura 2 and 4,5-diaminofluoroscein (DAF-2) signals in this assay system; therefore, DAF-2 signal is attributable to NO, which is not related with Ca²⁺ signal (37). This technique allows us to directly determine the relationship between [Ca²⁺]_i and NO levels in the endothelium, and the results obtained from these experiments more precisely represent the behavior of NOS and Ca²⁺ in endothelial cells under physiological conditions. In brief, the arterial segment was cut open along its longitudinal axis and put into a recording chamber with the vessel lumen side downward facing the objective of an inverted microscope. Care was taken not to disrupt the endothelium. The chamber was filled with Hanks’ buffered saline solution containing (in mM) 137 NaCl, 5.4 KCl, 4.2 NaHCO₃, 3 Na₂HPO₄, 0.4 KH₂PO4, 1.5 CaCl₂, 0.5 MgCl₂, 0.8 MgSO₄, 10 glucose, and 10 HEPES; pH 7.4. After a 30-min equilibration period, the artery was loaded with DAF-2 diacetate (10 µM) and fura 2-AM (10 µM) in Hanks buffer for 40 min at room temperature.

After dye loading was completed, temperature of the chamber solution was adjusted to 37°C by a temperature control system (Warner Instruments; Hamden, CT). An inverted microscope with epifluorescence attachments (Diaphot 200; Nikon, Tokyo, Japan) with a x20 phase/fluor objective (Nikon Diaphot) was used to visualize individual endothelial cells on the endothelium of coronary arteries. The excitation light from a xenon lamp was filtered to provide wavelengths of 340 ± 10, 380 ± 10 (for fura 2), and 480 ± 20 (for DAF-2) nm with a high-speed wavelength switcher (Lambda DG-4; Sutter, Novato, CA). Emission light from endothelial cells was passed through a dichroic mirror (500 nm) and through an emission filter of 510 ± 20 nm for fura 2 or 535 ± 25 nm for DAF-2 with a high-speed rotating filter wheel (Lambda 10–2; Sutter). The fluorescence images were captured by a digital camera (SPOT RT Monochrome; Diagnostic Instruments). Metafluor imaging and analysis software (Universal Imaging) was used to acquire, digitize, and store the images and for off-line processing and statistical analysis. To reduce photobleaching of these fluorescent dyes, images with excitation of 340 and 380 nm for Ca²⁺ were acquired at 2-s intervals, and images at excitation of 480 nm for NO were acquired at 10-s intervals. F₃₄₀/F₃₈₀, a fluorescence ratio of excitation at 340 nm to that at 380 nm, was determined after background subtraction, and [Ca²⁺]_i was calculated by using the equation: [Ca²⁺]_i = K_d[(R – R_min)/(R_min – R)], where K_d for the fura 2-Ca²⁺ complex is 224 nM; R is the fluorescence ratio (F₃₄₀/F₃₈₀); R_max and R_min are the maximal and minimal fluorescence ratios measured by addition of 10 µM of Ca²⁺ ionophore ionomycin to the Ca²⁺-replete (2.5 mM CaCl₂) solution and Ca²⁺-free (5 mM EGTA) solution, respectively; and is the fluorescence ratio at 380-nm excitation determined at R_min and R_max, respectively. The peak of Ca²⁺ transient was used as the maximal Ca²⁺ response. Intracellular NO production was expressed as relative fluorescence (f), which is the net increment of DAF-2 fluorescence at excitation/emission of 480/535 nm relative to its basal value (f = F/F₀ x 1,000), where F is DAF-2 fluorescence intensity obtained during experiments and F₀ is its basal fluorescence intensity. Because NO does not dissociate from DAF-2 once this dye reacts with NO, the detected NO-sensitive fluorescence with DAF-2 primarily represents a cumulative amount of NO within the cells. To more representatively show the features of NO-DAF-2 fluorescence and to accurately present the relationship between NO production and Ca²⁺ concentration in the cells, we performed a differential conversion of time-dependent NO-DAF-2 fluorescence curve to calculate NO production rate, df/dt using the following equation as described previously (37):

where t is time, t₀ is time when BK is added, and a, b, and c are constants that define the shape for a specific-fitting curve of BK response and can be calculated with the curve-fitting program of SigmaPlot 8.0. We plotted the converted df/dt against the reaction time and presented this curve with [Ca²⁺] changes in parallel. The area under the df/dt curve (AUC) was calculated to represent the cumulative amount of NO in the cells.

NOS activity assay. NOS activity was determined by measuring the conversion of L-[³H]arginine to L-[³H]citrulline using an isotopic NOS detection kit (Calbiochem) as we described previously (38, 39). Briefly, the homogenates prepared from cultured bovine coronary endothelial cells (25 µg protein) were incubated in 50-µl reaction mixture containing the following (in mM): 25 Tris·HCl (pH 7.4), 0.6 CaCl₂, 1 -NADPH, 0.003 tetrahydrobiopterin, 0.001 flavin adenine dinucleotide, 0.001 flavin mononucleotide, and 0.005 cold L-arginine, along with 1.0 µCi L-[³H]arginine in the absence or presence of endostatin. After incubation for 15 min at 37°C, the reaction was terminated by the addition of 400 µl of ice-cold stop buffer containing the following (in mM): 50 HEPES (pH 5.5) and 5 EDTA. Equilibrated cation exchange resin was added to the samples, and they were then applied to spin columns. After centrifugation, the eluate (containing [³H]citrulline) was collected, and the radioactivity was determined with a liquid scintillation counter. To determine the effect of endostatin on NOS activity in intact endothelial cells, the confluent endothelial cell cultures in 150-mm dishes were treated with endostatin for 60 min, followed by harvesting and homogenization. The formation of [³H]citrulline was then assayed as described above. In these experiments, the formation rate of citrulline represented NOS activity, which was expressed as picomoles per minute per milligram protein.

Ceramide assay. Lipids from endothelial cells were extracted as we described previously (38), and the lower chloroform phase was dried under N₂ and analyzed for ceramide concentration within 72 h. The dried lipids were solubilized into a detergent solution containing 7.5% n-octyl-D-glucopyranoside, and 5 mM cardiolipin in 1 mM diethylenetriaminepentaacetic acid solution, and then mixed with diacylglycerol kinase (Calbiochem) and 4 µCi [-³²P]ATP to a final volume of 100 µl. After incubation at 25°C for 3 h, the reaction was stopped by extracting the lipids with 600 µl chloroform-methanol (1:1 vol/vol), 20 µl 1% perchloric acid, and 150 µl 1 M NaCl. The lower organic phase was recovered and dried with N₂. The ³²P-labeled ceramide (ceramide-1-P) was separated from other lipids by thin layer chromatography (TLC) with a solvent consisting of chloroform-acetone-methanol-acetic acid-water (10:4:3:2:1, vol/vol/vol/vol/vol). After visualization by autoradiography, the ceramide-1-P band was recovered by scraping and counted in a scintillation counter. The phosphorylation of C₆ ceramide as an internal control was determined in parallel. The identity of ceramide was confirmed by HPLC analysis as we reported previously (39).

To determine the effect of endostatin on intracellular ceramide levels in endothelial cells, the confluent cell cultures in 150-mm dishes were treated with endostatin (100 nM) for 2, 5, and 30 min, followed by harvesting and homogenization. Fas ligand (Fas L, 100 ng/ml) was used as a positive control to stimulate ceramide production in all the experiments (11).

SMase activity assay. To explore the mechanism for endostatin-stimulated ceramide production, the activities of SMases, the major enzyme accounting for ceramide production, were determined as we reported previously (38). Briefly, N-[methyl-¹⁴C]sphingomyelin was incubated with endothelial cell homogenates, and the metabolites of sphingomyelin, [¹⁴C]choline phosphate, and ceramide were quantified. For acidic SMase (A-SMase), an aliquot of homogenates (20 µg) was mixed with 0.02 µCi of N-[methyl-¹⁴C]sphingomyelin in 100 µl acidic reaction buffer containing 100 mM sodium acetate and 0.1% Triton X-100, pH 5.0, and incubated at 37°C for 15 min. The reaction was terminated by adding 1.5 ml chloroform-methanol (2:1) and 0.2 ml double-distilled water. The samples were then vortexed and centrifuged at 1,000 g for 5 min to separate into two phases. A portion of the upper aqueous phase was transferred to scintillation vials and counted for the formation of [¹⁴C]choline phosphate in a Beckman liquid scintillation counter. For magnesium-dependent neutral SMase (N-SMase), the activity was determined using the neutral reaction buffer containing 100 mM Tris·HCl, 5 mM MgCl₂, and 0.1% Triton X-100; pH 7.5. The [¹⁴C]choline phosphate (another product metabolized by SMase from sphingosine) formation rate (pmol·min^–1·mg protein^–1) was calculated to represent the enzyme activity. To determine the effect of endostatin on SMase activity, the cells were treated with vehicle, endostatin (100 nM) for 2, 5, and 30 min, or Fas L (100 ng/ml) for 5 min, respectively.

Measurement of intracellular O₂^–· within the endothelium of small bovine coronary arteries. Intracellular O₂^–· was monitored by detecting dihydroethidium (DHE) fluorescence using the fluorescence imaging system described above. DHE can enter the cell and is fluorescent with excitation-emission of 380/435 nm in cell cytoplasm. DHE is oxidized specifically by O₂^–· to yield ethidium bromide (EtBr), which binds to DNA and has fluorescence at 480/610 nm excitation-emission. The assays were performed on the endothelium of isolated small bovine coronary arteries as described above and previously (39). Briefly, bovine arteries with the endothelium side up were incubated with 50 µM DHE in Hanks’ buffer for 30–60 min at room temperature. Fluorescent images for EtBr and DHE were then captured and analyzed as described above. Previous studies in our laboratory have demonstrated that O₂^–· detected by DHE in this preparation was located in the endothelium (39). O₂^–· fluorescence was measured every 1 min in a single area of the endothelial layer for 60 min. The ratio of EtBr and DHE fluorescence was recorded and calculated as the percent change relative to basal fluorescence ratio before stimulation. This ratiometric measurement of O₂^–· in the endothelial cell of intact arteries prevented the influence of differences in DHE loading levels on the sensitivity and specificity of the O₂^–· fluorescence assay (16, 25).

Fluorescence spectrometric assay of O₂^–· production. A DHE-based fluorescence assay was used to assess O₂^–· production from NAD(P)H oxidase in endothelial cells (40). Briefly, homogenates (20 µg) freshly prepared from endothelial cells were incubated with DHE (100 µM) and salmon testes DNA (0.5 mg/ml) in 200 µl phosphate-buffered saline. Immediately before fluorescence was recorded, NADPH (final concentration 1 mM) was added, and ethidium-DNA fluorescence was measured using a fluorescence microplate reader (Series 4000, Applied Biosystems). Salmon test DNA was added to the reaction mixture to bind ethidium and amplify fluorescence signal. The ethidium fluorescence increase (arbitrary unit) was used to represent NAD(P)H oxidase activity as described previously (40).

Statistics. Data are presented as means ± SE. Significant differences between and within multiple groups were examined using ANOVA for repeated measures, followed by Duncan’s multiple-range test. Student’s t-test was used to evaluate the significant differences between two groups of observations. P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of endostatin on BK-induced NO production and Ca²⁺ increase in bovine coronary arterial endothelium. Figure 1A presents some typical fluorescence images for endothelial Ca²⁺ and NO response in coronary endothelium preparation. Under resting condition (without endostatin treatment), BK (1 µM) significantly increased fura 2 fluorescence ratio (images taken before and 2 min after BK) in the intact endothelium, indicating a BK-induced increase in [Ca²⁺]_i. In parallel, BK increased DAF-2 fluorescence, which represented an increase in intracellular NO (images taken before and 5 min after BK). However, treatment of the endothelium with endostatin (100 nM) for 1 h had no significant effect on BK-induced changes in fura 2 fluorescence, but it blocked BK-induced increase in DAF-2 fluorescence (Fig. 1A).

View larger version (40K): [in this window] [in a new window]   Fig. 1. Simultaneous in situ measurements of intracellular Ca2+ concentrations ([Ca2+]i) and nitric oxide (NO) levels in the bovine coronary arterial endothelium. A: top, representative fura 2 fluorescence ratio images [taken at excitation wavelengths of 340 and 380 nm (F340/F380) and an emission wavelength of 510 nm]; bottom, representative 4,5-diaminofluorescein (DAF-2) fluorescence images (taken at excitation wavelength of 480 nm and an emission wavelength of 535 nm). Images were taken under resting conditions before (control) and after addition of bradykinin (BK) in the intact arterial endothelium or in the endothelium pretreated with endostatin (EST + BK). B: relationship of BK-induced increase in [Ca2+]i and NO production rate shown by change in relative fluorescence (f) with time (df/dt) of DAF-2 fluorescence. C: relationship of BK-induced increase in [Ca2+]i and NO production rate in coronary arteries pretreated with endostatin. A 5-min baseline recording before BK treatment is presented in both B and C.

Next, we observed the effect of endostatin on this endothelial response to BK. Endostatin (10–1,000 nM) did not acutely alter basal fura 2 fluorescence ratio or DAF-2 fluorescence in the endothelium or over a 60-min recording duration when added into the bath solution. As shown in Fig. 1C, a continuous recording of this endostatin-induced uncoupling of BK-induced NO production from increase in [Ca²⁺]_i was observed. It is clear that the pattern and extent of fura 2 fluorescence increase was similar to that observed before endostatin treatment (Fig. 1, C vs. B). However, the increase in NO level under endostatin treatment was markedly attenuated. The endostatin concentration used in the present study is in the range of concentrations that has been reported to inhibit endothelial cell proliferation and migration and induce endothelial cell apoptosis (4, 27).

The maximal responses of endothelial [Ca²⁺]_i and NO to BK and another stimulator of NO production with or without pretreatment of endostatin are presented in Fig. 2. As summarized in Fig. 2A, BK increased [Ca²⁺]_i by fourfold and DAF fluorescence by eightfold (control vs. BK) in the absence of endostatin. When the arterial endothelium was pretreated with endostatin, BK no longer increased NO but still stimulated an increase in [Ca²⁺]_i (endostatin + BK). Another receptor-independent endothelial NO stimulatory agonist A23187 [GenBank] (1 µM) increased [Ca²⁺]_i by 5.3-fold and DAF fluorescence by 25-fold. Similar to BK, pretreatment of vessels with endostatin inhibited A23187 [GenBank] -induced NO production, but it had no significant effect on the Ca²⁺ response. These results were summarized in Fig. 2B.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effect of endostatin on BK (A) or A23187 [GenBank] (B)-induced maximal increase in [Ca2+]i and NO production in intact endothelium of small bovine coronary arteries (n = 7 hearts). Peak of Ca2+ transient represented the maximal Ca2+ response. The accumulated NO production induced by BK was represented as arbitrary units (AUC) under the curve of NO production rate within 15 min. Control, basal [Ca2+]i and NO level in the arterial endothelium under resting condition. BK or A23187 [GenBank] , maximal [Ca2+]i and NO production in response to BK or A23187 [GenBank] in the absence of endostatin. EST + BK or EST + A23187 [GenBank] , maximal [Ca2+]i and NO production in response to BK or A23187 [GenBank] in the endothelium pretreated with endostatin (100 nM). *P < 0.05 vs. control (C). #P < 0.05 vs. BK or A23187 [GenBank] .

View larger version (9K): [in this window] [in a new window]   Fig. 3. Effect of EST on the activity of endothelial NO synthase (NOS). The homogenate of coronary endothelial cells (n = 6) were incubated with EST (100 nM), and the formation of [3H]citrulline from [3H]arginine was measured. NG-nitro-L-arginine methyl ester (L-NAME) was used as a negative control. *P < 0.05 vs. control.

View larger version (29K): [in this window] [in a new window]   Fig. 4. EST-induced increase in intracellular ceramide (Cer) levels in endothelial cells. A: representative autoradiograph of phospholipids fractionated by thin-layer chromatography (TLC) after diacylglycerol (DG) kinase assay. The bands represented ceramide-1-phosphate (Cer-1-P), which were from sample ceramide, and C6-ceramide-1-P (C6-Cer-1-P) from internal standard, respectively. B: summarized quantitative changes in Cer-1-P normalized to C6-Cer-1-P. The Cer-1-P and C6-Cer-1-P bands were scraped, and the radioactivity was counted using a liquid scintillation counter. *P < 0.05 vs. control (n = 6).

View larger version (19K): [in this window] [in a new window]   Fig. 5. A: effect of EST on sphinomyelinase (SMase) activity in endothelial cells. The cells were treated with vehicle, EST (100 nM) for 2, 5, 30 min, or Fas ligand (Fas L, 100 ng/l, 5 min). Both acid SMase (A-SMase) and neutral SMase (N-SMase) were determined by measuring [14C]choline formation rate. *P < 0.05 vs. control (n = 6). B: effect of desipramine on EST-induced ceramide production in endothelial cells. The cells were treated with desipramine (0.01–1 mM) for 15 min followed by EST (100 nM) for 30 min. *P < 0.05 vs. control; #P < 0.05 vs. EST (n = 6).

View larger version (30K): [in this window] [in a new window]   Fig. 6. Measurement of intracellular O2–· levels in the endothelium. A: representative ethidium bromide (EtBr) fluorescence images in the endothelium taken under control conditions or after 60 min treatment with EST (100 nM). B: time course for the change in EtBr-to-DHE fluorescence ratio in the bovine arterial endothelium under control or incubation of EST in the absence or presence of a cell-membrane permeable superoxide dismutase (SOD) mimetic tiron (1 mM). C: summarized data showing the maximal ratio of EtBr-to-DHE fluorescence ratio at 60 min in the intact endothelium. Apocynin (100 µM), a NAD(P)H oxidase inhibitor, was used to block NAD(P)H oxidase-mediated O2–· production in the presence of EST. Menadione (MEN, 1 mM) was used as a positive control in these experiments. *P < 0.05 vs. control. #P < 0.05 vs. EST.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Effect of EST on NAD(P)H oxidase activity in endothelial cells. The NADPH-dependent O2–· production in endothelial cells treated with vehicle or EST (100 nM) for 2, 5, 15, 30 min or desipramine (Desipr: 100 µM) or apocynin (100 µM), followed by EST for 30 min (n = 7–10). *P < 0.05 vs. control; #P < 0.05 vs. EST (30 min).

View larger version (21K): [in this window] [in a new window]   Fig. 8. Effect of tiron (A) or apocynin (B) on BK-induced NO production in the intact bovine arterial endothelium pretreated with EST. A: data were summarized to show the effect of tiron on [Ca2+]i and NO production before (Tiron + EST) and after addition of BK (Tiron + EST + BK) in the intact endothelium treated with EST (n = 7 hearts). *P < 0.05 vs. Tiron + EST (n = 7 hearts). B: effect of NAD(P)H oxidase inhibitor apocynin on [Ca2+]i and NO production before (Apocynin + EST) and after addition of BK (Apocynin + EST + BK) in the intact endothelium treated with EST. *P < 0.05 vs. Apocynin + EST. (n = 5 hearts).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Endostatin had no effect on basal [Ca²⁺]_i or NO levels in the intact endothelium. However, pretreatment of the arteries with endostatin for 1 h substantially reduced BK-induced NO increases. This inhibitory effect of endostatin was observed while [Ca²⁺]_i increased. It appears that an uncoupling of BK-induced [Ca²⁺]_i increase from NO production occurs in this bovine coronary arterial endothelium treated by endostatin. To our knowledge, these results provide the first evidence that endostatin blunts NO response to BK in the intact mature arterial endothelial cells, and this blunted NO response is not associated with inhibition of its Ca²⁺ response. Similar to BK, A23187 [GenBank] -induced NO production was also blocked by endostatin with no changes in the Ca²⁺ response. These results suggest that the effect of endostatin is not only specific for BK-induced response, but it as a general mechanism may affect all agonist-induced NO responses in the endothelium.

With regard to the effect of endostatin on [Ca²⁺]_i, a previous study in cultured aortic endothelial cells has shown that endostatin acutely increases [Ca²⁺]_i through D-myo-inositol-1,4,5-trisphosphate-sensitive Ca²⁺ release and extracellular Ca²⁺ entry, whereas overnight treatment with endostatin attenuated the increase in [Ca²⁺]_i induced by VEGF or FGF-2 (13). Although the cultured endothelial cells used in that study may behave differently to the intact in situ endothelial cells used in the present studies, we did not demonstrate any effect of endostatin on basal Ca²⁺ level in cultured coronary arterial endothelial cells. Therefore, the difference in preparations used in our study (intact endothelium) and those previous studies seemed not to be the determinant for different action of endostatin on basal Ca²⁺ levels in the endothelium. Another possible explanation for this difference may be related to different vascular beds used in both studies (coronary vs. aorta).

One of the important findings of the present study was that endostatin attenuated BK-induced NO increase but it had no effect on the BK-induced Ca²⁺ transient in endothelial cells. BK activates its receptors on vascular endothelial cells and subsequently causes intracellular Ca²⁺ mobilization and stimulation of endothelial NOS activity through a calmodulin-dependent mechanism (35). This Ca²⁺-dependent activation of NOS in endothelial cells importantly contributes to NO production in response to different agonists. In the present study, the uncoupling of BK-induced Ca²⁺ increase from NO accumulation by endostatin indicates that this angiostatic peptide alters the NO levels in the endothelium through a Ca²⁺-independent mechanism. By measuring the conversion rate of [³H]arginine to [³H]citrulline, endostatin was found to have no effect on NOS activity in bovine coronary arterial endothelial cells. This suggests that this peptide does not directly inhibit NOS enzyme activity to decrease NO levels.

Recently, the mechanism mediating the actions of endostatin on cell apoptosis or angiogenesis has been extensively studied (29). Several cell surface proteins, such as glypicans and integrins, may participate in the mediation or regulation of endostatin-induced actions (15, 31, 32). Glypicans, which are classified as a family of cell surface glycosyl-phosphatidylinositol anchored heparan sulfate proteoglycans, act as low-affinity endostatin receptors and cointeract with yet unidentified high-affinity receptors through protein-protein interaction to transmit endostatin signaling (15). Moreover, cell surface integrins, especially with _v1, a major fibronectin receptor in endothelial cells, have been implicated in the action of endostatin (31). Binding of the integrins to extracellular ligands may initiate a series of events, including integrins clustering, cytoskeletal reorganization, and other downstream signaling events (29, 36). In addition, endostatin exerts its antimigratory or antiangiogenetic action by inhibition of matrix metalloproteinase-2 activity, direct interaction with cell surface VEGF receptors, protein phosphorylation related to phosphatase PP2A activity or tyrosine kinase activity, and downregulation of growth- and apoptosis-related genes and antiapoptotic protein levels (7, 17, 21, 33, 34). However, most of these mechanisms are thought to contribute to inhibition of cell growth or apoptosis induced by endostatin. There is no evidence indicating that these mechanisms contribute to the NO decrease induced by endostatin in endothelial cells.

Given the potent apoptotic action of endostatin in endothelial cells, it is possible that its action is related to the signaling of death receptors. Recent studies have indicated that various death receptor ligands or stimulators such as TNF-, Fas L, and angiostatin may induce apoptosis by activating ceramide production in addition to caspase activation (12, 28). These stimuli could stimulate SMase activity to produce ceramide and thereby induce cell apoptosis (28). More recently, work in our laboratory and by others has demonstrated that ceramide increase by TNF- activated NAD(P)H oxidase to produce O₂^–·, thereby producing endothelial dysfunction and reducing NO-mediated vasodilation in isolated small bovine coronary arteries (38, 40). Based on these previous results, we hypothesized that endostatin may activate SMases and in this way stimulate ceramide production, which activates NAD(P)H oxidase for O₂^–· generation, thereby decreasing NO levels and producing endothelial dysfunction. To test this hypothesis, we first examined whether endostatin increases the ceramide level in endothelial cells. With the use of TLC analysis, it was found that endostatin produced a time-dependent increase in endothelial ceramide levels, suggesting that the ceramide-mediated signaling is involved in the actions of endostatin. Interestingly, a recent study has shown that another angiogenesis inhibitor angiostatin acutely increased ceramide in the endothelial cells, which is associated with free radical production (12). Taken together, it appears that ceramide serves as a general signaling molecule mediating the acute response of angiogenesis inhibitory peptides such as endostatin and angiostatin, especially during the early stage of their actions.

To explore the mechanism by which ceramide level is increased by endostatin, we examined the activities of SMases in these endothelial cells. Endostatin was found to produce a rapid activation of endothelial A-SMase but not N-SMase. Desipramine, a selective A-SMase inhibitor, concentration dependently decreased endostatin-stimulated ceramide production, suggesting that an A-SMase-ceramide pathway mediates the early response to endostatin. In previous studies, A-SMase was found to translocate from cytosol to sphingomylin-enriched membrane in response to various stimuli such as Fas L, whereby it is activated to produce ceramide (11). Ceramide then serves as a signaling molecule to stimulate the activities of a variety of enzymes such as NAD(P)H oxidase and increase intracellular oxidative stress, resulting in impairment of many cellular functions. Previous studies in our laboratory have demonstrated that ceramide can activate NAD(P)H oxidase and thereby increase O₂^–· production, leading to endothelial dysfunction in the coronary circulation (40). Therefore, it is possible that endostatin increases intracellular O₂^–· through this ceramide-NAD(P)H oxidase pathway in the endothelium.

With the use of DHE as a fluorescence indicator for O₂^–·, endostatin was found to induce a time-dependent increase in DHE-O₂^–· fluorescence in the intact endothelium of coronary arteries. This endostatin-induced O₂^–· production was attenuated by a SOD mimetic tiron. To our knowledge, these results provide the first direct evidence that intracellular O₂^–· levels increased in the intact endothelium with endostatin. In our previous studies, ceramide activated NAD(P)H oxidase to increase O₂^–· production without an effect on other O₂^–·-generating systems such as mitochondrial electron transport chain enzymes (10, 40). Therefore, we wondered whether the action to produce O₂^–· is associated with ceramide-mediated activation of NAD(P)H oxidase. With the use of an A-SMase inhibitor desipramine or a NAD(P)H oxidase inhibitor apocynin, endostatin-induced increase in O₂^–· production in the intact endothelium could be blocked. By biochemical analysis, NAD(P)H oxidase activity was also found significantly increased in endothelial cells pretreated with endostatin in a time-dependent manner, which was markedly inhibited by desipramine and apocynin. Taken together, these results suggest that endostatin is a potent stimulus for the production of O₂^–· in coronary endothelial cells through ceramide-mediated activation of NAD(P)H oxidase.

It is well documented that O₂^–· reduces NO bioavailability through a rapid formation of peroxynitrite and thereby results in the impairment of endothelial function (2). To further address whether endostatin decreases NO by enhanced intracellular O₂^–· production, we explored the possibility that O₂^–· scavenging restores BK-induced NO production in the endostatin-treated endothelium. The SOD mimetic tiron reversed the inhibitory effect of endostatin on BK-induced NO production without affecting the Ca²⁺ response. Tiron completely blocked endostatin-induced production of O₂^–·. Similar to tiron, the NAD(P)H oxidase inhibitor apocynin also recovered the NO response of the endothelium to BK. These results further confirm that NAD(P)H oxidase-derived O₂^–· is involved in endostatin-induced decrease in NO response to agonists in coronary arterial endothelium. This reduced endothelial NO response may result in impairment of endothelial-dependent vasodilation (20).

In summary, the present study demonstrated that 1) a relatively short-time treatment of endostatin blocked BK-induced NO increase in the intact endothelium; 2) endostatin increased A-SMase activity and intracellular ceramide levels; and 3) endostatin stimulated production of intracellular O₂^–· level through activation of NAD(P)H oxidase in coronary arterial endothelial cells. It is concluded that endostatin activates A-SMase to produce ceramide, which enhances intracellular O₂^–· production and consequently impairs endothelial NO response to BK in coronary arteries.

	ACKNOWLEDGMENTS

 
This study was supported by National Institute of Health Grants HL-57244, HL-70726, HL-51055, and DK-54927, American Heart Association Established Investigator Grant 9940167N (to P.-L. Li), and Predoctoral Fellowship 0410061Z (to A. Y. Zhang).

	FOOTNOTES

 

Address for reprint requests and other correspondence: P.-L. Li, Dept. of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: pli{at}mcw.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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