Peptide-Stimulated Angiogenesis: Role of Lung Endothelial Caveolar Signaling and Nitric Oxide

Abstract

Endothelial nitric oxide (NO) synthase (eNOS)-derived NO plays a critical role in the modulation of angiogenesis in the pulmonary vasculature. We recently reported that an eleven amino acid (SSWRRKRKESS) cell penetrating synthetic peptide (P1) activates caveolar signaling, caveloae/eNOS dissociation, and enhance NO production in lung endothelial cells (EC). This study examines whether P1 promote angiogenesis via modulation of caveolar signaling and the level of NO generation in EC and pulmonary artery (PA) segments. P1-enhanced tube formation and cell sprouting were abolished by caveolae disruptor Filipin (FIL) in EC and PA, respectively. P1 enhanced eNOS activity and angiogenesis were attenuated by inhibition of eNOS as well as PLCγ-1, PKC-α but not PI3K-mediated caveolar signaling in intact EC and/or PA. P1 failed to enhance the catalytic activity of eNOS and angiogenesis in caveolae disrupted EC by FIL. Lower (0.01 mM) concentration of NOC-18 enhanced angiogenesis without inhibition of eNOS activity whereas higher concentration of NOC-18 (1.0 mM) inhibited eNOS activity and angiogenesis in EC. Inhibition of eNOS by L-NAME in the presence of P1 resulted in near total loss of tube formation in EC. Although P1 enhanced angiogenesis mimicked only by lower concentrations of NO generated by NOC-18, this response is independent of caveolar signaling/integrity. These results suggest that P1-enhanced angiogenesis is regulated by dynamic process involving caveolar signaling-mediated increased eNOS/NO activity or by the direct exposure to NOC-18 generating only physiologic range of NO independent of caveolae in lung EC and PA segments.

1. Introduction

Angiogenesis is the complex process of formation of new vessels from pre-existing vessels by multiple stimulatory processes including generation of nitric oxide (NO) in the vasculature [1-4]. Vascular endothelium generates NO from the metabolism of L-arginine via an oxidative catabolic reaction mediated by endothelial cell (EC) nitric oxide synthase (eNOS). The catalytic activity of eNOS is regulated by multiple mechanisms including protein:protein interaction with the caveolae marker protein caveolin-1 (Cav-1) and by caveolar signaling leading to increased phosphorylation of eNOS Ser, Tyr, or Thr residues [5-7]. Caveolae are cholesterol/sphingolipid-enriched microdomain invaginations of the plasma membrane in EC [8]. The putative functions of caveolae include transmembrane transport by endocytosis, potocytosis, and signal transduction [8-10]. The loss and/or modulation of caveolae integrity can result in impaired NO production, calcium signaling, and associated pathophysiologic responses including angiogenesis [10-13].

A number of studies have reported that EC caveolae localizes multiple receptors and signaling modules including PI3K, PLC-γ, and PKC-α [8-10]. An agonist-stimulated Ca²⁺ wave in EC has been shown to originate in the caveole-rich region and to propagate throughout the cell [14]. Exogenous stimuli activate eNOS and enhance physiologic levels of NO production by the induction of calcium flux that activates caveolar signaling modules leading to modulation of Cav-1/eNOS association/dissociation processes in EC [14-17]. The regulatory role of Cav-1/eNOS promoting angiogenesis in EC has been reported using Cav-1 and eNOS deficient in vitro and in vivo models [1, 2, 11-13, 18-20]. However, the selective role of extracellular stimulus on modulation of caveolae or caveolar-specific signaling modules in lung EC/PA angiogenesis was not examined.

The catalytic activity of eNOS is also dependent on the rate of electron transfer from the reductase to the oxygenase domain of eNOS protein regulated by a 40 amino acid (604-RPEQHKSYKIRFNSISCSDPLVSSWRRKESSNDTSAGA-643) electron transfer control element or autoinhibitory domain [21, 22]. Our previous reports demonstrated that an 11-amino acid (626-SSWRRKRKESS-636) peptide (P1) derived from 40 amino acid autoinhibitory domain of eNOS protein modulated EC and lung function. For example, P1-stimulation of EC and/or PA resulted in : i) activation of eNOS independent of increased expression or phosphorylation of eNOS leading to NO-dependent vasorelaxation of contracted PA [23] and ii) enhanced compartmentalization and activation of eNOS in lung EC [24]. More recently we reported that P1-stimulation modulates caveolar PKC-α signaling that enhances eNOS activation, NO production, intracellular Ca²⁺ release, and phosphorylation of PKC-α and regulates internalization of P1 in lung EC [10]. P1-mediated activation of eNOS is associated with increased phosphorylation of Cav-1 and Cav-1/eNOS dissociation in caveolae-enriched fractions of EC and in an intact EC [24]. In addition, P1-mediated time-dependent increased phosphorylation of PKC-α and Cav-1 was independent of the basal level of these proteins (24). As such, this cell penetrating peptide can significantly impact EC function in the lung vasculature. Our objectives of the present study were to assess the role of P1-stimulated modulation of caveolar PI3K, PLC-γ, and PKC-α signaling modules, eNOS activity, and NO-mediated angiogenesis in lung EC and PA segments. Since P1 is known to generate physiologic levels of NO, we also examined the comparative and direct impact of varying levels of NO donor NOC-18 on angiogenesis in EC and PA segments.

2. Materials and methods

2.1 Reagents and antibodies

Primary antibodies for western blotting, immunoprecipitation, and immunofluorescence were commercially obtained as rabbit polyclonal anti-Cav-1, and mouse-anti-p-Cav-1(Tyr14) antibodies (BD Transduction Laboratories, San Jose, CA), and rabbit polyclonal anti-p-PKC-α (Ser 657) antibody, rabbit polyclonal anti-p-PLCγ-1(Tyr 783), and rabbit polyclonal anti-VWF (Von Willebrand factor) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies (peroxidase conjugated-donkey anti-mouse, donkey anti-rabbit IgG and Rhodamine Red labeled donkey anti-rabbit) were obtained from Jackson Immuno Research Laboratories (West Grove, PA). Western blotting reagents were obtained from Bio-Rad Laboratories (Hercules, CA). PI3K inhibitor Wortmannin (WT), eNOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME), PKC-α inhibitor GO6976, and NO donor NOC-18 were obtained from Calbiochem (Gibbstown. USA). Geltrex™ Reduced Growth Factor Basement Membrane 3D Matrix (RGFBM) from Invitrogen (Grand Island, NY), caveolae disruptor Filipin (FIL) [25], PLC inhibitor U73122 (IN), PLC non active analog U73433 (AI), and all other lab chemicals/reagents/buffers were obtained from Sigma Chemicals (St. Louis, MO), Santa Cruz Biotechnology (Santa Cruz, CA), and Fisher Scientific (Orlando, FL).

2.2 Tissue cultures and treatments

Pulmonary artery ECs were obtained from the main PA of 6 to 7 month-old pigs and propagated in monolayer culture as previously described [23]. Cells grown to confluence in 100-mm dishes were used for preparation of caveolae-enriched membrane fractions, immunoprecipitation, eNOS activity, western blot analysis, and to seed in 24 well plates on RGFBM for tube formation assay. In each experiment, cells were studied 1 or 2 days after confluence at passages 3-5 and were matched for cell line, passage number, and days after confluence. Cell monolayers were incubated with P1 (100 μM) in RPMI 1640 for 1 h at 37° C. Controls were incubated in RPMI 1640 only under identical conditions. The cells were pretreated with either PI3K inhibitor WT (1 μM) or caveolae disruptor FIL (0.05 μg/mL) for 1 h at 37° C followed by incubation with or without P1 (100 μM) for 1 h at 37° C or indicated with each individual experiment and figure. The cells were also pretreated individually with PLC inhibitor IN (1 μM); PLC non active analog AI (1 μM); eNOS inhibitor L-NAME (1 mM); PKC-α inhibitor GO6976 (10 μM); and NO donor NOC-18 (0.01mM-1mM) for 1h at 37° C, followed by co-incubation with or without P1 (100 μM) in RPMI 1640 for 1 h at 37° C. After treatment the cells were used: i.) to perform western blot analysis on isolated caveolar enriched fractions and immunoprecipitated proteins of interest (Cav-1, p-PLCγ-1, p-PKC-α, and p-Cav-1) from the treated cells, ii) to determine eNOS activity, and iii) to perform angiogenesis assays by tube formation and cell sprouting.

2.3 Animals

Male Sprague-Dawley rats (250-300 g) (Harlan Laboratories) were used to assess EC sprouting in isolated PA segments. Rats were housed in specific pathogen free, 12/12 light/dark cycle conditions with free access to standard food and water. Experimental protocol was approved by the Institutional Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals. The main PA was surgically removed and transversely cut into 0.5mm-1mm thick sections in the form of rings and embedded radially in 35 mm glass bottom dish coated with fresh RGFBM (150 μL), maintained at 4° C, followed by polymerization for 30 minutes incubation at 37° C, and then used for treatment with or without P1 (100 μM), FIL (0.05 μg/mL), and NOC-18 (0.01, 0.1, and 1.0 mM). For isolation of EC, main pulmonary arteries of 6- to 7-month old pigs were obtained from USDA inspected local slaughterhouse and processed as previously described [23].

2.4 Preparation of caveolae-enriched membrane fractions

The caveolae enriched membrane fractions were isolated using detergent-free method as we previously described [10, 24]. The isolated membrane fractions were analyzed by western blots using 50 μg of protein to determine the level of the caveolae marker protein Cav-1. Protein contents were measured with Bicinchoninic acid (BCA) using commercial kits supplied by Pierce in keeping with the manufacturer's instructions. The caveloae-enriched fraction #5 and representative non-caveolae enriched fraction #9 were identified and used in the study.

2.5 Immunoprecipitation assay

The immunoprecipitation of caveolar proteins by anti Cav-1 antibody were carried out as previously described [24]. In brief, 10 μg of Cav-1 antibody was added to the tube containing cold pre-cleared cell lysate obtained from EC treated with or without P1 or PKC/PLC inhibitors with or without P1 and incubated at 4°C overnight. After incubation, Protein A slurry (50 μl) was washed thrice in 450 μl lysis buffer and centrifuged at 10,000 rpm for 10 min. The pellets were suspended in pre-chilled lysis buffer containing caveolae-enriched immunoprecipitate fraction, incubated at 4°C for 1 h, and centrifuged at 10,000 rpm for 10 min. Supernatants were discarded, protein A bead pellets were saved, washed thrice in cold lysis buffer, and immunoprecipitated Cav-1 proteins were solubilized in Laemmli sample buffer for western blots.

2.6 Measurement of eNOS activity

The catalytic activity of eNOS was measured by monitoring the formation of L-[³H] citrulline from L-[³H] arginine in cell lysates obtained from EC treated with or without P1 or in combination with PI3K/PLC/eNOS/PKC inhibitors with or without P1 for 1 h at 37°C. The cell lysate (100–200 μg of protein) were incubated in 0.4 ml of Tris HCl buffer containing 1 mM NADPH, 100 nM calmodulin, 10 μM tetrahydrobiopterin, and 5 μM combined L-arginine and purified L-[³H] arginine for 30 min at 37°C [24]. Purification of L-[³H] arginine and measurement of L-[³H] citrulline formation were carried out as previously described [26].

2.7 Determination of angiogenesis

To determine P1-induced angiogenesis, tube formation in EC and sprouting of EC in PA segments were performed using Geltrex™ Reduced Growth Factor Basement Membrane 3D Matrix (RGFBM) to mimic the in vivo conditions as previously described [27]. In brief, porcine lung EC monolayers propagated in 24 well cell culture dishes coated with RGFBM (100 μL) or rat lung PA segments propagated in glass bottom 35 mm cell culture dishes coated with RGFBM (150 μL), were incubated with PI3K inhibitor (WT, 1μM), PLCγ-1 inhibitor (IN, 1μM), eNOS inhibitor (L-NAME, 1mM ), PKC-α inhibitor (GO6976, 1μM), NO donor (NOC-18; 0.01-1.0 mM), or caveolae disruptor FIL (0.05 μg/mL) with and without P1(100 M) in RPMI 1640 medium containing 1% fetal bovine serum for 12 h at 37°C in EC tube formation assay or 7 days at 37°C for PA segments sprouting. In PA sprouting assay the media was replaced with fresh media containing NOC-18 or P1 after every 24 h. It has been reported that 0.01 mM NOC-18 steady-state of 1-3 μM NO in medium which is comparable to endogenous production of NO by eNOS [28]. EC seeded on coated plates at a density of 3 × 10⁴ cells per well in RPMI 1640 medium containing 1% FBS at 37°C. Tube formation is optimal after 12 h and begins to fade after 16 h. Phase contrast microscopic images of tube formation in EC and sprouting of EC in PA segments obtained and compared with respective controls.

2.8 Western blot analysis

The western blots developed as previously described [3 and 10]. In brief, cell lysate, immunoprecipitates, or caveolar enriched fraction obtained as described in Methods. Western blots were developed for p-PKC-α, p-PLCγ-1, p-Cav-1, and Cav-1 using standard sandwich technique [10, 24]. The blots were developed using primary antibodies for p-PKC-α, p-PLC-γ1, p-Cav, and Cav (1:2000 dilutions or as directed by vendor) and appropriate secondary horse radish peroxidase antibody. Antigen antibody sandwich was detected by chemiluminescence method. The optical densities of the blots were measured by Quantity 1-D analysis software of Bio-Rad gel modular imager Gel Documentation system.

2.9 Immunofluorescence studies

To identify EC sprouting from PA segments, the sections of PA segments were placed in 35 mm glass bottom dishes coated with fresh RGFBM and maintained at 4° C, followed by 30 minutes incubation at 37° C and then further used for treatment with P1 induced sprouting of cells in RPMI with 1% serum at 37° C for 7 days. The PA segments were observed regularly each day under phase contrast microscope for P1 induced sprouting of cells. After detection of cell sprouting, the PA segments were detached from RGFBM and gently placed in another 35 mm glass bottom dishes. To confirm whether sprouted cells are EC, VWF (Von Willebrand Factor) protein was detected by immunofluorescence assay using VWF primary antibody (dilution 1:100) and Rhodamine red labeled secondary antibody (dilution 1:100). The fluorescence micrographs of sprouted cells were obtained by confocal microscopy. The cells were observed and analyzed at wavelength 543 nm for Rhodamine red labeling using a Zeiss LSM 510 inverted 2-photon confocal microscope and images were captured through 20 X objective, keeping all the conditions of microscope and settings of software identical for each experiment.

3.0 Statistical analysis

The data were subjected to statistical analysis and expressed as mean ± SE for n experiments. Significance for the effect of eNOS, PKC, PI3K, or PLC inhibitor and caveolae disruptor (FIL) on P1 mediated eNOS activity in EC or effect of different concentrations of NOC-18 vs control on eNOS activity in EC were determined with ANOVA and a Student's paired t-test. A value of p <0.05 was considered statistically significant.

3. Results

3.1 Caveolae integrity is critical for P1 stimulated angiogenesis in lung EC and PA

To determine whether P1 stimulation enhances angiogenesis in lung EC and PA, tube formation and EC sprouting assays were performed as described in Methods. As shown in Fig. 1A, P1 stimulation for 12 h enhanced tube formation in EC (panel ii) and progressive EC sprouting in PA from 4 to 7 days post P1 stimulation (panels iv and vi) as compared to respective controls. Since P1 internalization is known to be regulated by EC caveolae [10], we determine whether P1-mediated EC and PA angiogenesis is caveolae-dependent. EC and PA were pretreated with caveolae disruptor FIL prior to P1 stimulation as described in Methods. As shown in Fig. 1B, FIL-mediated disruption of caveolae attenuated P1-mediated tube formation in EC (panel ii) and 4 and 7 days EC sprouting in PA (panels iv and vi), respectively. Immunofluorescence detection using EC marker VWF (Von Willebrand factor) indicated that the P1-stimulated sprouted cells in PA are EC (Fig. 1C). These results suggest that P1stimulation enhances EC and PA angiogenesis via caveolae-dependent processes.

Figure 1.

Assessment of P1 induced angiogenesis in lung EC and PA segments. EC tube formation assay were performed in 24 well plates for 12 h with or without P1 (100 μM) treatment (panels A-i, ii). PA segment EC sprouting assay were performed in 35 mm glass bottom dishes for 4-7 days with and without P1(100 μM) treatment using Reduced Growth Factor Basement Membrane 3D Matrix (RGFBM) containing RPMI with 1% serum (panels A-iii-vi). To examine the effects of caveolae disruptor FIL on P1-mediated angiogenesis, ECs were pre-incubated with medium containing FIL (0.05 g/mL) for 90 min in 100 mm dishes and tube formation assay were performed in 24 well plates for 12 h with (panel B- ii) or without (control, CON) (panel B- i) the presence of P1(100 μM). For PA segments EC sprouting assay, PA were co-incubated with filipin in 35 mm glass bottom dishes for 4-7 days with or without P1(100 μM) using RGFBM containing RPMI with 1% serum (panels B-iii-vi). ECs were examined after each six hours for tube formation or PA segments were examined each day up to 7 days for EC sprouting. To Confirm whether P1 induced sprouted cells are EC, PA segments were incubated with P1 (100 μM) in 35 mm glass bottom dishes for 2 days were subjected to immunofluorescence assay against EC marker VWF(Von Willebrand Factor ) protein using VWF primary antibody and rhodamine red labeled secondary antibody and the 2-photon confocal microscopy as described in methods Panels C-i, fluorescence; ii, light, and iii, merged (fluorescence + light) micrograph of sprouted cells in the same microscopic field. All micrographs in panels A, B, and C are the representative of 3 to 6 independent experiments.

3.2 P1-Stimulated eNOS activity and angiogenesis is regulated via caveolar PLCγ-, PKC-α, and Cav-1 phosphorylation in lung EC

Since P1 stimulation increases eNOS activity, intracellular Ca²⁺ release, and caveolar PKC-α phosphorylation but not basal levels of PKC-α in cell lysate or in caveolae-enriched fractions in EC [10, 24], we examined whether caveolae-selective signaling module involving Cav-1, PLCγ-1, PKC-α, and PI3K is associated with P1-mediated increased eNOS activity and angiogenesis in EC. Fig. 2 shows a representative Western blots of the basal levels of PKC-α and PLCγ in cell lysate (panel A-i) and caveolae-enriched fractions (panel A-ii) in P1-stimulated EC for 60 min, respectively. Basal levels of PKC-α and PLC-γ were comparable on control and P1-stimulated cell lysates and caveolae-enriched fractions as we previously reported (24). Fig. 2, Panel B shows time-dependent effect of P1 on p-PLCγ-1, p-PKC-α, p-Cav-1, and Cav-1expression (panel-i), and quantitative analysis of corresponding blots performed by relative density ratio of p-PLCγ-1, p-PKC-α, and p-Cav-1 over Cav-1 (panel-ii) in intact EC. These results demonstrate that P1 mediated increased p- PLCγ-1 was maximum at 10 min that was gradually diminished to near control level in 60 min. Whereas, P1-mediated increased p-PKC-α, and p-Cav-1 was observed only at 60 min. This suggests that P1-stimulated increased phosphorylation of caveolar PLCγ-1, PKC-α, and Cav-1 is time-dependent. As shown in panel C, P1-stimulation increased p-PKC-α only in caveolae-enriched fraction number 5 of control and WT treated but not in IN treated and FIL-mediated caveolae disrupted cells. Fig. 2 (panel D), shows the effects P1-stimulation on the catalytic activity of eNOS with or without pretreatment with WT, AI, IN or FIL in EC. The catalytic activity of eNOS was significantly increased in P1, WT+P1, and AI+P1 but not in IN+P1 treated and in FIL-mediated caveolae disrupted EC. To determine whether P1 induced angiogenesis in lung ECs is regulated via PI3K or PLC signaling or required to maintain caveolae integrity, EC tube formation assay was performed with or without the pretreatment with PI3K/PLC inhibitor WT/IN or caveolae disruptor FIL. Fig. 2, panel E shows the representative micrographs of three to six experiments of P1, AI+ 1, and WT+P1 stimulated angiogenesis. However, IN+P1 treatment and caveolae disruption by FIL resulted in total loss of tube formation in EC as compared to respective controls. These data suggest that P1-mediated eNOS activation via caveolae PLCγ-1 but not by PI3K signaling pathway is critical for enhanced angiogenesis in lung EC.

Figure 2.

P1-stimulated angiogenesis is regulated via eNOS and caveolar signaling in lung EC. ECs were treated with or without P1 (100 μM) for 10 to 60 min at 37° C, and immunoprecipitated against Cav-1 antibody. Cell lysates and/or the immunoprecipitated proteins were used to develop immuno-blots for PKC-α, PLC-γ-1, and Cav-1 as well as p-PLCγ-1 (Tyr 783), p-Cav-1 (Tyr 14), p-PKC-α (Ser 657), and Cav-1 as described in Methods. Panel A-i and ii shows representative of three separate immuno-blots for PKC-α, PLC-γ-1, and Cav-1 in cell lysate and in caveolae enriched fraction (#5) and non-caveolae enriched fraction (#9) of P1-stimulated EC for 60 min, respectively. Panel B-i shows representative Western blots of three separate immune-blots for p-PLCγ-1, p-Cav-1, p-PKC-α and Cav-1 from immunoprecipitated protein by Cav-1 antibody of EC treated without and with P1 for 10 min, 30 min and 60 min. Panel B-ii shows quantitative relative ratio of Optical Densities unit (ODu) mm-² of p-PLCγ-1, p-cav-1, p-PKC-α to the Cav-1 of three blots. EC monolayers were also pre-incubated with or without (control, CON) the presence of inhibitors of PI3 kinase (WT; 1 μM) and PLCγ-1 (IN; 1 μM) or (AI, inactive analog of PLC inhibitor; 1 μM) for 1 h or caveolae disruptor (FIL; 0.05 μg.mL⁻¹) for 90 min at 37°C, followed by incubation with or without the presence of P1 (100 μM) in RPMI 1640 for 1 h (for caveolar enriched fraction isolation, immuno-blot and eNOS assay) or 12 h (for tube formation assay) at 37° C or as indicated in the methods for each experiment. After treatment, the levels of p-PKC-α and Cav-1 were determined by immuno-blot analysis in caveolae enriched fraction (#5) and non-caveolae enriched fraction (#9), the catalytic activity of eNOS was detected in intact EC and tube formation was examined as described in Methods. Representative immuno-blots of p-PKC-α and Cav-1 are shown in panel C. The eNOS activity and tube formation are shown in panels D and E, respectively. Data in panels A, C, and E are representative of 3 separate experiments. Data in panels B and D represent mean ± SE, n=6 for each treatment. *p < 0.05 vs CON, # p < 0.05 and **p < 0.05 vs respective CON, 10, and 30 min in panel B. *p < 0.05 vs respective CON, AI, and WT in panel D.

Since FIL-mediated caveolar disruption abolishes tube formation in EC, we determined whether this response is associated with modulation eNOS activity and/or caveolae PKC-α signaling. As shown in Fig. 2 C (iv), FIL treatment inhibited, P1-mediated enhanced phosphorylation of PKC-α in caveolae enriched fraction 5 as well as failed to enhance the P1-stimulated catalytic activity of eNOS in intact EC (Fig. 2 D) and tube formation in EC (Fig. 2 E i-x).

3.3 P1 failed to enhance eNOS activity and angiogenesis in the presence of L-NAME or GO7976

Since P1-stimulation increases the catalytic activity of eNOS via Cav-1/eNOS dissociation associated with increased phosphorylation of caveolar PKC-α [10, 22], we examined the effect of eNOS inhibitor L-NAME or PKC-α inhibitor GO6976 (GO) on eNOS activity, PKC-α phosphorylation and/or angiogenesis in EC. As shown in Fig. 3, P1-stimulation enhanced the catalytic activity of eNOS that was abolished or blocked by pretreatment of cells with L-NAME or GO, respectively (panels A-i and B-i). Pretreatment of EC with L-NAME and GO failed to enhance P1-mediated phosphorylation of PKC-α (panels A-ii and B-ii). Increased phosphorylation of PKC-α is required for Cav-1/eNOS dissociation and enhanced eNOS activity (10, 22). Similarly, P1-mediated tube formation in EC was abolished by L-NAME and GO pretreatment (panel C). These results suggest that P-1-mediated angiogenesis is regulated via activation of eNOS in EC.

Figure 3.

P1 failed to enhance eNOS activity and angiogenesis in the presence of eNOS inhibitor L-NAME or PKC-α inhibitor GO6979 (GO). ECs were pretreated overnight with L-NAME (1 mM) or GO (10 μM) in RPMI 1640 followed by 60 min at 37°C with or without (control, CON) the presence of P1 (100 μM) as described in methods. After treatment cells were used to monitor eNOS activity, immuno-blot detection of p-PKC-α, and tube formation. Panels A-i and B-i shows the effects P1 on eNOS activity with or without the presence of L-NAME or GO. Representative Western blots of three separate experiments shows the effects of L-NAME and GO on P1-mediated p-PKC-α in panels A-ii and B-ii, respectively. Panel C shows representative phase contrast micrographs of tube formation of 3-6 independent experiments. Data in panels A-i and B-i represent means ± SE, n=6 for each treatment. *p <0.05 vs CON in panels A-i and B-i.

3.4 Lower but not higher levels of NO released by NOC-18 induced angiogenesis in lung EC and PA

Since P1-mediated activation of caveolar signaling and eNOS is associated with enhanced angiogenesis in EC, we examined whether varying levels of NO released by NO donor mimic the effects of P1 on angiogenesis in EC and PA. As shown in Fig. 4, the catalytic activity of eNOS in EC treated with lower concentration of NOC-18 (0.01 mM) was comparable to controls. The higher concentrations of NOC-18 (0.1 and 1.0 mM) resulted in significant loss of the catalytic activity of eNOS (panel A). Lower concentration (0.01 mM) of NOC increased the tube formation in EC and progressive cell sprouting in PA segments from 4 to 7 days post treatment. In contrast, higher concentrations (0.1 and 1.0 mM) of NOC-18 resulted in near total loss of tube formation and cell sprouting in EC and PA, respectively (panel B). To determine whether this NOC-18-mediated angiogenic response is associated with activation of eNOS, PKC-α or caveolar integrity, we examined the effects of L-NAME, GO, and FIL. As shown in panel C, NOC-18-mediated tube formation in EC remained unchanged with or without the presence of L-NAME, GO, and FIL.

Figure 4.

NO donor induced angiogenesis in EC and PA is dose dependent and independent of caveolar integrity. ECs and PAs were incubated with or without (control. CON) presence of varying concentrations (0.01, 0.1, and 1.0 mM) of NOC-18 and 100 μM P1 for 12 hr or 4-7 days, respectively (panels A and B). In some experiments, ECs were treated with eNOS inhibitor L-NAME (1 mM), PKC-α inhibitor (GO, 10 μM) and Filipin (FIL, 0.05 μg/mL) in the presence of NOC-18 (0.01 mM) (panel C) for 12 h. with or without P1 (100 μM). For PA treatment, media was replaced daily containing NOC-18 and/or P1. After treatment eNOS activity in EC (panel A) and cell sprouting and tube formation (panels B and C, respectively) were determined as described in methods. Data in panel A represent mean ± SE, n=6 for each treatment. *p < 0.05 vs CON. The micrographs in panels B and C are representative of 3-6 independent experiments.

4. Discussion

This study demonstrated that synthetic peptide P1 promotes angiogenesis via modulation of caveolar signaling and activation of eNOS in EC and PA segments. Activation of caveolar PLCγ-1 and PKC-α but not PI3K are critical for P1-stimulated angiogenesis in EC. Disruption of caveolar integrity and/or inhibition of eNOS activity abolished P1-medeated angiogenesis in EC and PA. Since P1-mediated activation of eNOS leads to elevated NO release and NO/cGMP-stimulated vasorelaxation in the lung vasculature [23, 29], we also demonstrated that lower concentration of NO donor NOC-18 enhanced angiogenesis was independent of caveolar signaling/integrity in EC and PA. These results indicate that P1-stimulated but not NOC-18 mimicked angiogenesis is regulated by activation of caveolar signaling and eNOS.

Caveolae are dynamic structures defined as flask-shaped invaginations also known as lipid rafts of plasma membranes with multiple functions including signal transduction and endocytosis [9, 10]. A number of studies have reported that EC caveolae localizes multiple receptors and signaling modules including Ca²⁺-dependent enzymes PI3K, PLC-γ, and PKC-α that are associated with activation of eNOS via increased phosphorylation of Cav-1 leading Cav-1/eNOS dissociation [8-10]. However, the selective role of extracellular stimulus-mediated activation of caveolar signaling events leading to modulation EC functions including angiogenesis remained largely elusive. An agonist-stimulated Ca²⁺ wave in EC has been shown to originate in the caveolae-rich region and to propagate throughout the cell [14]. We reported that P1-stimulated increased phosphorylation of caveolar PKCα without change in the basal level of PKC-α was associated with the regulation of P1 internalization, intracellular Ca²⁺ release, as well as eNOS dissociation, activation, and elevated NO production in EC [10, 24]. The results of this study indicate that the caveolar proteins are the primary targets in P1-mediated responses. For example, P1-stimulated angiogenesis is selectively regulated by activation of caveolar PLC-γ but not PI3K as confirmed by using IN and WT, respectively. However, P1-mediated enhanced phosphorylation of PKC-α and the catalytic activity of eNOS was significantly increased in P1, WT+P1 but not in IN+P1 treated cells indicating selective regulatory roles of PKC-α and PLC-γ in activation of eNOS and enhanced angiogenesis, respectively. P1-mediated activation of caveolar PKC-α is also shown to be associated with limited internalization of P1 in EC [10]. Moreover, time course study indicates that P1-stimulated activation of PLC-γ was observed as early as 10 minutes whereas activation of PKC-α was a delayed event requiring 60 minutes. Thus, activation of caveolar PLCγ and PKC-α appears to be associated with P1-stimulated time-dependent intracellular Ca²⁺ release in EC [10]. This response is consistent with our previous report demonstrating that phorbol ester-mediated activation of Ca²⁺-dependent PKCs mimicked P1-stimulated PKC-α phosphorylation (24). Similarly, the catalytic activity of eNOS is regulated by multiple mechanisms including increased intracellular Ca²⁺ release, phosphorylation of Cav-1, and by eNOS/Cav-1 dissociation leading to intracellular translocation of eNOS and elevated NO production in EC [10, 16, 17, 24]. Our results support the fact that P1-mediated enhanced angiogenesis is regulated via eNOS activation since inhibition of eNOS by L-NAME abolished P1-stimulated angiogenesis. Similarly, inhibition of PKC-α phosphorylation by GO6976 also abolished P1-stimulated angiogenesis indicating that P1-stimulated eNOS activation appears to be regulated by two distinct but interrelated mechanisms i.e., direct activation via increased Ca²⁺ release and increased phosphorylation of PKC-α critical for activation of eNOS via Cav-1/eNOS dissociation. This P1-stimulated activation of caveolae-specific signaling events, eNOS activation/dissociation, and enhanced angiogenesis was markedly abolished by caveolae disrupting agent FIL. Therefore, maintaining the caveolar integrity is critical for P1-stimulated angiogenesis in EC and PA.

We also demonstrated that NO generated by 0.01 mM NOC-18 treatment can mimic P1-stimulated angiogenesis in EC and PA. This suggests that the lower concentration of NOC-18-mediated and P1-stimulated NO releases in EC and PA segments are in comparable range. Higher concentrations of NO (1 and 1.0 mM) resulted loss of angiogenic response appears to be associated with higher/toxic levels of NO production. In addition, the lower concentration of NOC-18-mediated angiogenic response was independent of caveolar integrity as FIL treatment failed to block NOC-18-stimulated agniogenesis. Moreover, NOC-18-mediaded angiogenic response remains unchanged with or without the modulation of eNOS activity, PKC-α phosphorylation or disruption of caveolae. The molecular events involved in promoting direct angiogenic response by NOC-18 in EC remained to be determined but may be associated with indirect involvement of increased expression of cyclic nucleotide-gated channel isoform A2 (CNGA2). We previously reported that exposure to NO gas or NOC-18 generated NO upregulates CNGA2 expression and elevation of intracellular Ca²⁺ in EC [28]. Since elevated Ca²⁺ release can activated calcium-dependent protein kinases including PLC-γ and PKC-α as well as eNOS, it is possible that the effect of NOC-18 on promoting angiogenesis may be regulated via indirect pathway such as increased expression of CNGA2. Similar indirect effect of NO donor modulated thrombospondin-1 was reported on the regulation of angiogenesis in human vascular endothelial cells [30]. Although P1-stimulared angiogenic response in EC and PA segments was mimicked by lower concentrations of NOC-18, the dynamics of the molecular events involved in these processes appears distinctly different. Irrespective of the specific mechanisms involved in promoting P1 or NOC-18-mediated angiogenesis, it is critical to maintain NO level within the physiologic range. This is specifically important if P1 or NOC-18 to be utilized for tissue repair therapies such as ischemic injury, chronic inflammation, and wound healing that require angiogenesis.

5. Conclusion

P1-stimulated angiogenesis is regulated via activation of caveolar signaling and eNOS in EC and PA. Although EC are known to generate low levels of NO via eNOS, P1 elevated NO via activation of eNOS is critical for enhanced angiogenesis in EC. Our findings also suggest that maintaining caveolae integrity is critical as caveolae impaired cell signaling and/or disruption of caveolae abolished P1-stimulated angiogenesis in EC and PA. We also demonstrated that physiological but not toxic levels of NO released by NOC-18 mimicked P1-stimulated angiogenesis in EC and PA via caveolae independent pathway. Thus, therapeutic utility of this synthetic peptide can be of significant relevance for modulation of selective physiological and pathological processes such as chemical or ischemic tissue injury, chronic inflammation, and wound healing that require angiogenesis.

Highlights.

• Cell penetrating synthetic peptide P1 promotes angiogenesis in lung.

• P1-mediated angiogenesis is regulated via caveolae signaling and eNOS activation.

• Disruption of caveolae abolished P1-stimulated angiogenesis.

• Exogenous NO donor NOC-18 mimicked P1-stimulated angiogenesis.

• P1 can be utilized to modulate physiologic processes in biology and medicine.