The Role of eNOS Phosphorylation in Causing Drug-induced Vascular Injury

Abstract

Previously we found that regulation of eNOS is an important part of the pathogenic process of Drug-induced vascular injury (DIVI) for PDE4i. The aims of the current study were to examine the phosphorylation of eNOS in mesentery versus aorta at known regulatory sites across DIVI-inducing drug classes and to compare changes across species. We found that phosphorylation at S615 in rats was elevated 35-fold 2 hr after the last dose of CI-1044 in mesentery versus 3-fold in aorta. Immunoprecipitation studies revealed that many of the upstream regulators of eNOS activation were associated with eNOS in 1 or more signalosome complexes. Next rats were treated with drugs from 4 other classes known to cause DIVI. Each drug was given alone and in combination with SIN-1 (NO donor) or L-NAME (eNOS inhibitor), and the level of eNOS phosphorylation in mesentery and aorta tissue was correlated with the extent of vascular injury and measured serum nitrite. Drugs or combinations produced altered serum nitrite levels as well as vascular injury score in the mesentery. The results suggested that phosphorylation of S615 may be associated with DIVI activity. Studies with the species-specific A2A adenosine agonist CI-947 in rats versus primates showed a similar pattern.

Keywords

animal models vascular system drug development risk identification.

Introduction

The upregulation of cyclic adenosine monophosphate (cAMP) by phosphodiesterase-4 inhibitors (PDE-4i) causing relaxation of vascular smooth muscle cells and suppression of pro-inflammatory cytokines clearly has the potential to treat diseases such as asthma and chronic obstructive pulmonary disease (Burnouf et al. 2000; Heaslip et al. 1994). However, preclinical safety evaluations have indicated that PDE-4i induce vascular lesions, primarily in the mesenteric arteries of rats and in the coronary arteries of dogs, pigs, and monkeys (Hanton et al. 2008; Heaslip et al. 1994; Losco et al. 2004; Zhang et al. 2008). It is not known whether similar effects would be observed in humans. A study where tissues were obtained at autopsy from patients taking minoxidil as an antihypertensive drug did not find similar lesions (Sobota 1989). Drug-induced vascular injury (DIVI) has become a major cause of attrition in the development of this class of drugs, as well as a number of other classes of pharmaceuticals.

The mechanism by which PDE-4i causes DIVI has not been clearly elucidated. Although the location of the injury among species varies greatly, pathological evaluation indicates that similar etiologies are observed, namely endothelial cell damage with medial necrosis and hemorrhage along with perivascular inflammation (Hanton et al. 2008; Zhang et al. 2008). Treatment of rats with PDE4i CI-1018 (Slim et al. 2003) or SCH 351591 (Zhang et al. 2002) resulted in DIVI histopathology and deposition of nitrotyrosine adducts in the vascular smooth muscle and endothelial cell layers. These results suggest that inflammation is a major component of PDE-inhibitor-related vascular injury and that nitrative stress may be important for inducing the observed necrosis.

Nitric oxide (NO) is a short-lived second messenger that causes relaxation of the medial smooth muscle cells and thereby maintains vascular tone. However, excessively elevated levels of NO can cause inflammation by reacting with reactive oxygen/nitrogen species (ROS/RNS) and superoxide dismutase (SOD; Pacher, Beckman, and Liaudet 2007). Serum nitrite is a more stable breakdown product of NO and has also been shown to be elevated in association with DIVI (Sheth et al. 2011; Weaver et al. 2008; Weaver et al. 2010), suggesting a causative role for NO in DIVI formation. The addition of a NO donor stimulated the effect, while a nitric oxide synthase (NOS) inhibitor ameliorated the response. Furthermore, fenoldopam, a dopaminergic-1 agonist, induced NO-mediated mesenteric injury in rats that was modulated by a NO-donor and NOS inhibitor in a similar manner as CI-1044 (Brott, Richardson, and Louden 2012). This suggests the possibility of a common mechanism for DIVI-inducing drugs possibly involving nitrative stress.

NO is made by 3 isoforms of NOS: inducible NOS (iNOS), endothelial cell-specific NOS (eNOS), and neuronal NOS (nNOS; Förstermann and Sessa 2012; Sessa 2004). Because eNOS is constitutively expressed, the ongoing regulation of its activity is essential for normal function. A number of factors regulate eNOS activity, including calmodulin-mediated dimerization, heat shock protein 90, tetrahydrobiopterin (BH₄), and caveolin (Desjardins, Delisle, and Gratton 2012; Fleming 2010). Kinases and phosphatases also play pivotal roles in the regulation of eNOS activity (Mount, Kemp, and Power 2007). There are 7 known phosphorylation sites on eNOS: Y81, S114, T495, S615, S633, Y657, and S1177 (numbering based on the amino acid sequence of human eNOS; Hornbeck et al. 2012). Phosphorylation of eNOS by PKA or AKT at S1177 leads to increased activity of eNOS (Chen et al. 1999; Fulton et al. 1999). In contrast, phosphorylation at T495, which is in the calmodulin binding site, prevents dimerization, and hence reduces eNOS activity (Chen et al. 1999). Phosphorylation at S615, Y81, and S633 promotes activity, while Y657 and S114 phosphorylation are thought to be inhibitory (Bauer et al. 2003; Boo et al. 2002; Fulton et al. 2008). The action of phosphatases such as PP1 and PP2A are also important for regulating eNOS activity (Zhang and Hintze 2006). It is likely that there is an interplay among a number of sites that regulate the activity of eNOS.

The aims of our present study were (1) to examine the factors regulating the phosphorylation status of eNOS in response to CI-1044, (2) to determine the relationship of eNOS phosphorylation status with serum nitrite and vascular lesions observed, (3) to examine whether other drugs that cause vascular injury operated in a similar fashion, and (4) to compare the response to CI-947, an adenosine 2A receptor antagonist, in rats versus nonhuman primates to see if the presence or absence of DIVI observed between these species were reflected in the pattern of activation of eNOS in different tissues.

Materials and Methods

Animals

Male Sprague-Dawley (SD) rats (10–12 weeks old) were purchased from Taconic (Hudson, NY) and were doubly housed. Rats were fed Certified Purina Chow #5002 (Ralston Purina Co., St Louis, MO) and water ad libitum. Rats were anesthetized by inhalation of isofluorane and serum and tissue harvested using experimental procedures approved by the Institutional Animal Care and Use Committee, Center for Drug Evaluation and Research, FDA, and carried out in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility. Group size for rat studies was 5 animals, consistent with previous studies (Sheth et al. 2011).

Cynomologous monkeys (less than 2.5 yr) were purchased from Covance Primates (Princeton, NJ) and were doubly housed. Animals were fed a standard diet of pelleted food (certified Primate diet 5K91, PMI Feeds, Inc.) supplemented with vegetables and/ or fruit twice daily and water ad libitum. Monkeys were anesthetized by intravenous barbiturate administration followed by exsanguination and mesentery, aorta and coronary artery tissue harvested. All primate experimental procedures were approved by Institutional Animal Care and Use Committee of Pfizer, Inc.

Chemicals

CI-1044 [N-[9-Amino-3,4,6,7-tetrahydro-4-oxo-1-phenylpyrrolo[3,2,1-j,k][1,4] benzodiazepin-3-yl]-3-pyridinecarboxamide], a PDE-4 inhibitor (Burnouf et al. 2000); SK&F 95654, a PDE-3 inhibitor; and CI-947, an A2A adenosine agonist were supplied by Pfizer (Groton, CT). Midodrine hydrochloride, an α-1 adrenergic vasopressor, was purchased from Sigma (St. Louis, MO). Nicorandil, a vasodilatory K-channel agonist and NO donor, was purchased from TCI America (Portland, OR). Fenoldopam mesylate, a dopaminergic DA₁ agonist, was purchased from LKT Laboratories (St. Paul, MN). SIN-1 and L-NAME were purchased from Sigma (St. Louis, MO). CI-1044, CI-947 and nicorandil were resuspended in 0.5% (wt/vol) carboxymethylcellulose (CMC), while fenoldopam mesylate, midodrine, SIN-1, and L-NAME were resuspended in saline and SK&F 95654 was resuspended in dimethyl sulfoxide (DMSO).

Experimental Procedures

Time course

Rats were dosed with CI-1044 (80 mg/kg) or carboxymethylcellulose vehicle daily for 3 days by oral gavage. The animals were dosed and sacrificed in the same sequence order to maintain consistent timing across the group. Rats were anesthetized with isofluorane and blood was collected for processing to serum. Then the animals were sacrificed and mesentery tissues for histopathology were harvested as described previously (Sheth et al. 2011). Sections of mesentery and aorta tissue were flash frozen in liquid N₂ and stored at −80°C for Western blot analysis.

Effect of Different Classes of Drugs on DIVI

Rats were dosed with the following: midodrine (25 mg/kg) or nicorandil (500 mg/kg) or CMC vehicle for 4 days by oral gavage, SK&F 95654 (50 mg/kg) or DMSO vehicle, for 3 days or fenoldopam (100 mg/kg) or PBS vehicle, for 1 day, respectively, by subcutaneous injection. These drugs were selected to provide the widest diversity of intended pharmacological action among drugs known to cause DIVI. Rats were also dosed with either SIN-1 (30 mg/kg) or L-NAME (60 mg/kg) by oral gavage as described previously (Sheth et al. 2011). The combination of midodrine and L-NAME was particularly lethal and therefore could not be investigated as rats died or were sacrificed in a moribund state in less than 8 hr with L-NAME doses as low as 10 mg/kg. Serum and mesentery tissue for histopathology were harvested as described previously (Sheth et al. 2011). Sections of mesentery and aorta tissue were flash frozen in liquid N₂ and stored at −80°C for Western blot analysis.

Effect of CI-947 on Cross-species Variation of DIVI

Rats were dosed with CI-947 (20 mg/kg) and/or SIN-1 and L-NAME for 3 days and serum and mesentery, aorta and coronary artery tissue harvested as described previously. Monkeys were dosed with CI-947 (50 mg/kg) for 3 or 7 days and serum and mesentery, aorta and coronary artery tissue harvested as described previously.

Histopathology

The mesentery tissue was harvested for histopathological evaluation as described previously (Sheth et al. 2011). Briefly, the gastrointestinal tract was removed and laid out flat to enable removal of the intestines and mesenteric lymph node. The mesentery tissue was then rolled onto the plunger of a 1-cc syringe, placed in a tissue cassette and fixed in 10% formalin prior to embedding in paraffin. Sections were prepared and stained with hematoxylin and eosin for light microscopy examination. The extent of vascular injury was scored on a scale of 0 (no injury) to 5 (severe injury) as detailed in Zhang et al. (2008).

Serum Nitrite Concentration Determination

The level of NO in the blood, as reflected by the level of serum nitrite, was measured using the Total Nitric Oxide Assay kit (R&D Systems, Inc., Minneapolis, MN) as previously described (Sheth et al. 2011).

Protein Extraction and Western Blot Analysis

Approximately 30 mg of mesentery tissue was sonicated in cell lysis buffer (25 mM HEPES, 300 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.1% TX-100, containing 1 mM DTT, 1 mM NaVO₃, 20 mM NaF, Protease Inhibitor Cocktail (Sigma), 10 μg/ml aprotinin, 12.5 μM leupeptin, 1 mM PMSF, and 20 mM β-glycerophosphate), and samples were left on ice for 20 min prior to centrifugation to remove cellular debris. Approximately 1 cm of the aorta or coronary artery was cut into 8 equal sized pieces. The pieces were then placed in cell lysis buffer with 0.2 g of 0.5 mm Zirconia/Silica beads (Biospec Products, Bartlesville, OK) and placed in a mini bead beater (Biospec Products, Bartlesville, OK) on high for 60 sec. The samples were then clarified by centrifugation, and the lysate was collected from below the fatty layer and above the beads. All lysates were quantified for total protein concentration using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific Inc., Rockford, IL) and stored at −80°C.

Cell lysates (25–30 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes using the Criterion Cell apparatus (BioRad, Hercules, CA). Specifically, proteins were separated on 4 to 12% Bis-Tris Criterion XT gels using XT Mops running buffer (BioRad). Proteins were then transferred to PVDF membranes using the Criterion blotter and a Tris/Glycine/20% MeOH transfer buffer (BioRad). Western blot analysis for phosphorylated eNOS, eNOS, and upstream regulators was carried out using the following antibodies: eNOS, #9572 (Cell Signaling, Danvers, MA), phospho-Y81/Y657, p-Tyr-100, #9411 (Cell Signaling), phospho-S114, ab78004 (Abcam, Cambridge, MA), phospho-T495, sc-19827 (Santa Cruz Biotechnology, Inc., Dallas, TX), phospho-S615, #07-561 (Upstate, Billerica, MA), phospho-S633, ab76199 (Abcam), phospho-S1177, ab75639 (Abcam), and actin, A5441 (Sigma, St Louis, MO) as detailed in the manufacturer’s instructions. Due to the lack of specific antibodies for Y81 and Y657, we used the anti-phosphotyrosine antibody, p-Tyr-100, and results for the Y81/Y657 phosphorylation sites are denoted as p-Tyr. Bands were visualized using an ImmunStar Western C Substrate kit (BioRad) with a ChemiDoc XRS chemiluminescent detection system. The density of bands was measured using the Quantity One 1-D Analysis Software (BioRad) supplied with the instrument. Band density was normalized relative to actin and was expressed as fold change relative to vehicle control.

Immunoprecipitation

Lysates of mesentery tissue (500 μg) prepared as mentioned earlier were incubated with 2μg anti eNOS antibody, ab66127 (Abcam) overnight at 4°C with gentle shaking. Immunocomplexes were harvested by incubating with Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Inc.) for 4 hr at 4°C and examined for the presence of the following proteins by Western blot analysis: eNOS, #9572 (Cell Signaling), β-arrestin-2, ab31294 (Abcam), PDE-4A, NB300-677 (Novus Biologicals, Inc.), PDE-4B, NB300-679 (Novus Biologicals, Inc.), PDE-4D, NB300-637 (Novus Biologicals, Inc.), exchange protein directly activated by cAMP (EPAC-1), ab21236 (Abcam), ras-related protein (RAP-1), ab25980 (Abcam), AKT, #4691 (Cell Signaling), pAKT (Ser473), #4060 (Cell Signaling), and PKAα/β/γ cat, sc-28892 (Santa Cruz Biotechnology).

Statistical Analysis

Statistical significance was calculated using the Student’s t-test. A p value < 0.05 was considered statistically significant.

Results

We have previously demonstrated that the production of NO through eNOS activation has a causative link with DIVI, since treatment of rats with CI-1044 in combination with a NO donor, SIN-1, exacerbated vascular injury, while the combination of CI-1044 and a NO synthase inhibitor, L-NAME, ameliorated vascular injury (Sheth et al. 2011). To extend these studies, and to help elucidate the mechanism by which eNOS activation leads to vascular injury, SD rats were dosed with 80 mg/kg CI-1044 for 3 days and serum, mesentery, and aorta tissues were harvested over the subsequent 24-hr time period to enable evaluation of serum nitrite and the extent of vascular injury.

The serum nitrite levels remained around baseline levels in the first 8 hr, but rose to double the vehicle control by 24 hr, although this increase was not statistically significant (Figure 1A). Interestingly, the histopathology evaluation of mesentery tissue showed a similar trend, with the greatest number of lesions observed 24 hr after the final dose of CI-1044 (Figure 1B). These results further confirm the link between production of NO and vascular injury.

Figure 1.

CI-1044 treatment causes an increased production of total serum nitrite and incidence of vascular lesions with time in SD rats with an increased eNOS phosphorylation at S615. (A) Measurement of total nitrite in serum from SD rats harvested 1 to 24 hr after the last dose of CI-1044. (B) Histopathological evaluation of mesentery tissue collected from SD rats 1 to 24 hr after the last treatment of CI-1044. (C) Densitometry quantitation of Western blot lysates from mesentery and aorta tissue for phosphorylated eNOS at S615, 1 to 24 hr after the last dose of CI-1044. *Indicates p < 0.05 relative to Vehicle (24 hr). Note. SD = Sprague-Dawley; eNOS = endothelial nitric oxide synthase.

To determine what role phosphorylation of eNOS may play in the production of NO over time and to discern differences between physiological and toxicological effects, we next examined lysates of mesentery and aorta tissue collected 1 to 24 hr after the last dose of CI-1044 for differences in phosphorylation at Y81/Y657, S114, T495, S615, S633, and S1177 (Figures 1C and 2). Studies of factors governing eNOS activation previously demonstrated that an increase in phosphorylation of S1177 and inhibition of phosphorylation of T495 correlate with eNOS activity (Mount, Kemp, and Power 2007). Interestingly, a comparison of the density of phosphorylated bands between mesentery and aorta tissue demonstrated that the most striking effect was observed at S615, where phosphorylation was increased 35-fold in the mesentery by 2 hr while in the aorta the increase was only 3-fold at the same time point (Figure 1C). The level of phosphorylated S615 decreased in the mesentery with time, although it remained at a higher level than in the aorta at all time points. In comparison, phosphorylation at S1177 was decreased at all time points while phosphorylation at S633 was elevated 8 hr after the last dose in the mesentery. Phosphorylation at the other sites was not elevated more than 4-fold at any time (Figure 2). Our results suggest that an early event in the activation of eNOS is phosphorylation at S615, followed by a prolongation of the signal through S633 phosphorylation. This is then followed by an elevation in NO production as measured by serum nitrite production and by a further increase in vascular injury.

Figure 2.

Time course of changes in eNOS phosphorylation levels over time following the third daily dose of the PDE4i CI-1044. Columns—Mesentery or aorta, rows—specific phosphorylation sites. Note: eNOS = endothelial nitric oxide synthase.

This suggests that the regulation of eNOS activity is a dynamic process and requires upstream regulators to be in close proximity to enable phosphorylation at multiple phosphorylation sites. To determine if eNOS was part of a rat endothelial cell signalosome complex in response to CI-1044, mesentery lysates from SD rats treated with CI-1044 were immunoprecipitated with eNOS antibodies and immunocomplexes examined by Western blot analysis (Figure 3). eNOS coprecipitated with β-arrestin-2, AKT, PDE-4, EPAC-1, RAP-1, and PKA. This provides evidence for the presence of multiple upstream regulators in close proximity to eNOS and allows for the coordinated regulation of eNOS activity. The data show strong staining for these proteins; however, there was no detectable change in the composition of the complex or complexes as a result of drug treatment. It is plausible that the overall phosphorylation status of eNOS is more important than phosphorylation at one particular site, for regulating activity.

Figure 3.

Interaction of eNOS with upstream regulators and β-arrestin-2. Lysates of mesentery tissue from SD rats were treated with CI-1044, and immunoprecipitated with total eNOS antibodies. Complexes were analyzed by Western blot for eNOS, β-arrestin-2, PDE-4, EPAC-1, RAP-1, AKT, pAKT, and PKAα/β/γ cat. IP Control corresponds to immunoprecipitates of CI-1044-treated mesentery lysates where the total eNOS antibody was omitted. (A) Western blot analysis, (B) quantitation of individual Western blots. *p < 0.05. Note. eNOS = endothelial nitric oxide synthase; PDE-4i = phosphodiesterase-4 inhibitors; SD = Sprague-Dawley; VH = vehicle control.

To examine the factors governing the mechanism of DIVI in other classes of drugs, rats were treated with either a phosphodiesterase-3 inhibitor, SK&F 95654; an α-1 adrenergic vasopressor, midodrine; a vasodilatory K-channel agonist and NO donor, nicorandil; and a dopaminergic D1 receptor agonist, fenoldopam, as described in the Materials and Methods section; and the extent of vascular injury was evaluated by histological evaluation. Typical lesions consistent with previous descriptions of DIVI with these compounds were observed (Figure 4). To determine whether eNOS activation played a role in the extent of vascular injury observed, SD rats were treated with the drugs of interest, as well as the NO donor, SIN-1, or the NO synthase inhibitor, L-NAME. Quantitation of the extent of vascular injury by histopathologic evaluation demonstrated that SK&F 95654 treatment increased vascular injury by 1.5-fold (Figure 5A). Addition of SIN-1 further increased the vascular injury score, while the combination of SK&F 95654 and L-NAME led to a complete inhibition of vascular injury. Treatment with the vasoconstrictor midodrine increased the vascular injury score 4-fold relative to vehicle control, and the addition of SIN-1 decreased the vascular injury score to 2-fold (Figure 5B). The combination of midodrine and L-NAME was extremely lethal even at 1/6 of the usual dose of L-NAME, and therefore this combination could not be evaluated. Of the drugs in this group, nicorandil caused the most severe vascular injury with a histopathology score of 5 (Figure 5C). As we had some unexpected deaths in the SIN-1 and L-NAME combinations with nicorandil, these arms of the study had to be terminated early so we could not assess the effect of SIN-1 and L-NAME for this drug. The effect of fenoldopam in inducing vascular injury has been well documented (Brott, Richardson, and Louden 2012; Kerns et al. 1989); however, in our hands, fenoldopam alone induced mild vascular injury, and these differences may reflect difference in the dosing regimens used between the studies. Interestingly, the combination of fenoldopam and SIN-1 produced a more than additive effect than fenoldopam or SIN-1 alone (Figure 5D). L-NAME, as expected, in combination with fenoldopam inhibited the production of any vascular injury. These results show that modulation of eNOS activity through the addition of SIN-1 or L-NAME appears to be important for regulating the extent of the mesenteric vascular injury observed.

Figure 4.

Representative photomicrographs of DIVI in the mesenteric artery of SD rats. (A) A small-sized mesenteric artery showing normal appearance in control rats. (B) SK&F 95654-induced arterial hemorrhage and acute inflammatory response in the perivascular space. (C) Fenoldopam-induced acute inflammatory response in the perivascular space. (D) Midodrine-induced severe inflammatory cell infiltration in the perivascular space, with rare hemorrhage in the arterial wall. (E & F) Nicorandil-induced arterial hemorrhage, with severe chronic inflammatory response and severe fibrosis in the perivascular space in mesenteric arteries. Note. Arterial endothelial cells become activated and degenerated. A-F, X200, H&E stain. Note. SD = Sprague-Dawley; DIVI = drug-induced vascular injury.

Figure 5.

Mesenteric histopathology score changes in response to several DIVI-inducing drugs. (A) PDE3i SKF 95654, (B) α₁-adrenergic agonist midodrine, (C) NO donor and Kir channel activator nicorandil, (D) dopaminergic (DA₁) receptor agonist fenoldopam. Note. DIVI = drug-induced vascular injury; NO = nitric oxide; VH = vehicle control.

As a measure of eNOS activation, we measured the serum nitrite levels at the termination of the experiment from rats treated with the drugs of interest. SK&F 95654 showed a small decrease in serum nitrite 24 hr after the drug treatment (Figure 6A); however, a peak in serum nitrite between 2 hr and 4 hr after the last dose was observed (Figure 6B). Interestingly, the combination of SK&F 95654 and SIN-1 led to an increase in serum nitrite, while the addition of L-NAME inhibited serum nitrite production. This suggests that the PDE-3i, SK&F 95654 induces vascular injury in a similar fashion as the PDE-4i, CI-1044. In contrast, midodrine showed a nonsignificant increase in serum nitrite with drug alone, while midodrine and SIN-1 combined normalized the measured serum nitrite levels, mirroring the effect seen with the vascular injury score (Figure 6C). Since nicorandil is an NO donor as well as a vasodilatory K-channel agonist, the serum nitrite levels were extremely high with this drug treatment (Figure 6D). The pattern of serum nitrite production upon treatment with fenoldopam/SIN-1/L-NAME also mirrored that observed for vascular injury, with fenoldopam alone not having much of an effect on serum nitrite production, while fenoldopam and SIN-1 increased and fenoldopam and L-NAME decreased serum nitrite production respectively (Figure 6E).

Figure 6.

Serum nitrite level changes in response to several DIVI-inducing drugs. (A) PDE3i SK&F 95654, (B) Time course with SK&F 95654, (C) α₁-adrenergic agonist midodrine, (D) NO donor and Kir channel activator nicorandil, (E) dopaminergic (DA₁) receptor agonist fenoldopam. Note. DIVI = drug-induced vascular injury; VH = vehicle control.

Phosphorylation at Y81-657 was increased in response to nicorandil in the mesentery (Figure 7). There was also a slight increase in phosphorylation with fenoldopam treatment at this site; however, there was no correlation with the SIN-1 or L-NAME treatment, suggesting the effects observed were not specifically due to any of the drugs regulating eNOS activity. Phosphorylation at S114 was increased in response to SK&F 95654 and nicorandil, but again there was no correlation with SIN-1 or L-NAME treatment. Phosphorylation at T495 was increased in response to fenoldopam and showed a typical SIN-1 and L-NAME response. SK&F 95654 increased phosphorylation at S615, and there was a concomitant increase in phosphorylation with the addition of SIN-1 and a further decrease with L-NAME. Nicorandil increased phosphorylation at S615 by 4-fold. Fenoldopam alone did not affect S615 phosphorylation in the mesentery, while the combination of fenoldopam and SIN-1 increased phosphorylation, and L-NAME addition to fenoldopam inhibited. Midodrine treatment did not affect S615 phosphorylation. These general patterns of phosphorylation with these 4 drugs resembled those seen for vascular injury and serum nitrite production with CI-1044.

Figure 7.

Changes in phosphorylation at various sites on eNOS in response to several DIVI-inducing drugs. Columns = DIVI drug; rows = eNOS phosphorylation site. Note: DIVI = drug-induced vascular injury; eNOS = endothelial Nitric Oxide Synthase; VH = vehicle control.

The results with CI-1044 suggested that S633 may be important for prolonging the activation of eNOS for CI-1044 (Figure 2). However, this did not seem to be a common feature for the other drugs tested, as only the combination of fenoldopam and SIN-1 produced an increase in S633 phosphorylation in the mesentery, which decreased to basal levels with L-NAME. Phosphorylation at S1177 is the most commonly reported effect of eNOS activation; however, consistent with our observations for CI-1044 (Figure 2), phosphorylation at S1177 was decreased by midodrine, nicorandil, and fenoldopam treatments in the mesentery. SK&F 95654 treatment showed an increase in S1177 phosphorylation in both the mesentery and aorta. Drug treatment did not affect the total eNOS levels. A summary view of the results of this section of the research is given in Table 1.

Table 1.

Summary of effects across drug classes.

Drug	Class	Vascular activity	SN for drug alone	SN drug + L-NAME	SN drug + SIN-1	Histology drug alone	Histology drug + L-NAME	Histology drug + SIN-1
CI-1044	PDE4i	Weak dilation	Up 2×	No change	Up 4.5×	DIVI	No DIVI	More DIVI
SKF 95654	PDE3i	Dilation	No change	No change	Up 1.7×	DIVI	No DIVI	More DIVI
Midodrine	α-1 Adrenergic	Contract	Up 1.4×	Death	No change	DIVI	Death	Less DIVI
Nicorandil	K+ channel and sGC, NO donor	Dilation	Up 23×	Not evaluated	Not evaluated	DIVI	Not evaluated	Not evaluated
Fenoldopam	Dopaminergic agonist	Dilation	No change	No change	Up 1.15×	Small DIVI	No DIVI	More DIVI
CI-947 in rats	Adenosine agonist	Dilation	No change	No change	Up 2.0×	No DIVI	No DIVI	No DIVI
CI-947 in cyno	Adenosine agonist	Probable dilation	Up 1.8× on day 3	Not measured	Not measured	DIVI	n/a	n/a

Note: L-NAME = eNOS inhibitor, SIN-1 = NO donor, PDE#i = phosphodiesterase 3 or 4 inhibitor, SN = serum nitrite, sGC = soluble guanylate cyclase agonist, DIVI = drug-induced vascular injury.

One of the most striking features of vascular injury in the different animal species is that although the etiology of disease is similar, the location of the injury varies, with the most common site of the injury being located in the mesentery of rats and in the coronary arteries of dogs and nonhuman primates, while this type of vascular injury has not been reported in mice. In an attempt to identify differences in disease progression and whether they correlated with eNOS activation, we examined the effect of CI-947, an adenosine 2A receptor antagonist, on eNOS activation and vascular injury in the rat and nonhuman primate. Interestingly, previous internal Pfizer studies have demonstrated that this drug causes injury in the nonhuman primate, but not in the rat. Treatment of SD rats with CI-947 did not affect the production of serum nitrite in the rats (Figure 8A), while in the nonhuman primate, there was an increase in serum nitrite 3 days after CI-947 treatment, which returned to baseline by day 7 (Figure 8B). We examined the effect of SIN-1 and L-NAME on serum nitrite production in the rat and found that the CI-947/SIN-1 combination increased serum nitrite levels, while the CI-947/L-NAME combination had baseline levels of serum nitrite (Figure 8A).

Figure 8.

Serum nitrite levels changes in response to CI-947 (A) rat with CI-947 and SIN-1 or L-NAME. (B) Primate at day 3 or day 7 ± CI-947. VH = vehicle control.

Our results suggest that CI-947 treatment leads to increased eNOS activity in the nonhuman primate, but not the rat. To determine whether drug treatment led to differences in vascular injury, mesentery, aorta, and coronary artery tissue sections from both species were assessed for vascular injury. CI-947 did not cause any vascular injury in the rat in mesentery, aorta, or heart (data not shown). In contrast, CI-947 treatment produced inflammation, necrosis, and hemorrhage in the coronary arteries and heart of the nonhuman primate (Table 2). There was no injury noted in the mesentery of these primates, further confirming the localized effect of the drug across species.

Table 2.

Summary of histopathology findings in CI-947-treated nonhuman primates.

	Control						CI-947-Treated
	Day 3			Day 7			Day 3			Day 7
Animal number	7	8	9	1	2	3	10	11	12	4	5	6
Mesenteric arteries
No significant lesions	×	×	×	×	×	×	×	×	×	×	×	×
Heart
No significant lesions	×
Mononuclear cell infiltrate	1+	1	1	1	1	1		2		2
Necrosis, myocyte with associated inflammation			1		1				2	2	2	2
Hemorrhage, subendocardial							2		2	1	2	2
Hemorrhage, subepicardial									2
Inflammation, adventitia, artery, atrium				1
Coronary arteries and/or branches
No significant lesion	×	×	×	×	×	×		×
Cellular infiltrate, adventitia, intramural arteries										1,1	1,2	1,1
Inflammation adventitia							2,3*		1,2
Fibrinoid necrosis, smooth muscle							2,3		1,2
Hemorrhage, smooth muscle							1,2		1,1
Testes
No significant findings	×	×	×	×	×	×	×	×	×	×	×	×
Skin
Hemorrhage, subcutaneous	×		×							×	×	×
Ulceration, epidermis			×									×
Dermatitis			×									×
Folliculitis												×

Note. × = indicates lesion is present but not scored for severity. Numbers indicate severity of the lesion. + = indicates increased severity. * = indicates increased severity and increased distribution.

Examination of eNOS phosphorylation in the rat showed there was very little effect of CI-947 treatment in the mesentery except at S1177 where there was a 5-fold increase in phosphorylation with drug treatment (Figure 9). SIN-1 also increased phosphorylation although the effect was not additive. There was no particular effect noted for phosphorylation at S615. Phosphorylation at T495 was increased in both the mesentery and aorta, and for the aorta, the addition of either SIN-1 or L-NAME did not affect the pattern of phosphorylation. Overall, the phosphorylation pattern is quite different from that observed with drugs causing DIVI.

Figure 9.

Changes in phosphorylation at various sites on eNOS in response to CI-947 in rats versus primate. Columns = species, rows = eNOS phosphorylation site. Note. eNOS = endothelial nitric oxide synthase; VH = vehicle control.

To determine whether the difference in etiology was due to differences in eNOS phosphorylation status, we also examined the phosphorylation pattern of mesentery, aorta, and coronary arteries from nonhuman primates treated with CI-947 for either 3 or 7 days. There was very little change in phosphorylation levels for any of the sites in the mesentery (Figure 9). In the aorta and coronary artery, the only significant increases in phosphorylation at day 3 were observed at S114. In general, there was an increase in basal phosphorylation in the coronary artery between day 3 and day 7 for most sites examined. Surprisingly, CI-947 treatment led to an inhibition of phosphorylation at day 7. CI-947 treatment led to an increase in phosphorylation at Y81-657, S114, and T495, with phosphorylation at T495 being increased 3-fold in the aorta. This suggests that it is the early effect on eNOS phosphorylation that is associated with the vascular injury observed in the primate.

Discussion

The present work provides further information on the critical role of eNOS phosphorylation in the activation process leading to the excessive production of NO in the vascular system in response to several drugs known to cause DIVI. We have used 3 complementary experimental approaches to this problem. First, we have investigated the signaling around eNOS in considerable detail using the PDE4i CI-1044 as our model. Second, we have then extended this work using 4 additional compounds known to cause DIVI in the rat. Finally, we have compared eNOS signaling across species, using a compound that causes DIVI in the nonhuman primate (NHP) but not in the rat.

Signaling around eNOS is quite complex and only partially understood. A simplified classical signaling pathway diagram is shown in Figure 10A. This shows published signaling linkages around eNOS and should be interpreted with some caution as our research implies additional currently unknown influences on eNOS activity.

Figure 10.

Simplified views of signaling around eNOS. (A) Classical signaling pathway view; (B) Signalosome view. Drugs causing DIVI are highlighted in yellow, green shapes are signaling proteins shown to be involved in eNOS signaling pathways. Light gray shapes are cell surface receptor molecules. Note: DIVI = drug-induced vascular injury; eNOS = endothelial nitric oxide synthase.

Results from the first segment of our studies demonstrates that one of the most noteworthy events in eNOS activation in response to CI-1044 treatment is the elevation in phosphorylation at S615, accompanied by overproduction of NO and vascular injury. These elevations are notably higher in the mesentery as compared to the aorta which is not damaged in response to treatment with PDE-4i in rats. This group of findings does not appear to be specific to PDE-4i, since each of the 4 classes of drugs we also examined produced a similar response, in that alteration of vascular injury correlated with serum nitrite production and eNOS phosphorylation. This association was significant only at 24 hr after the last dose. The late arrival of the serum nitrate signal may be due to late over production or it may be that the rise in serum is secondary to leakage from damaged vascular tissue. Furthermore, phosphorylation of S615 to activate eNOS appears to be a signal specific for mesenteric activation of eNOS since in rats and nonhuman primates treated with CI-947, no vascular injury was observed in the mesentery, and altered phosphorylation at S615 was absent. Vascular injury was present in the aorta and coronary arteries in the NHP and interestingly, the phosphorylation pattern of eNOS was different from that which we had seen before, suggesting differential activation of eNOS by upstream regulators ultimately leads to a similar product, although the means of producing the effect may vary. From the results of these and other studies, it is clear that typical lesions of DIVI can be caused by both vasodilators and by vasoconstrictors and that both can alter regulation of eNOS. Our results with eNOS modulators, although complex, are consistent with the hypothesis t+hat reversing the deviation from normal eNOS activity also tends to reverse DIVI (Table 1).

Since eNOS is constitutively expressed, posttranslational modifications are critically important for the regulation of normal function. The major regulator of eNOS activity is Ca²⁺-dependent dimerization induced by binding to calmodulin (Bredt and Snyder 1990). Other binding proteins, such as caveolin and BH₄, are also important for activity, as are the actions of multiple kinases and phosphatases. Of the 7 phosphorylation sites on eNOS, the most widely studied is S1177; a number of stimuli such as shear stress, bradykinin, insulin, statins, ischemia/reperfusion, and vascular endothelial growth factor (VEGF) have been reported to cause activation of eNOS through phosphorylation at this site (Mount, Kemp, and Power 2007). In contrast, phosphorylation at T495 leads to inactivation of eNOS as phosphorylation at this site blocks the calmodulin binding site, preventing dimerization (Sessa 2004). Phosphorylation of S615 is increased in response to shear stress in a model using treadmill running mice (Zhang et al. 2009). Phosphorylation of S633 has also been shown to activate eNOS, although with delayed kinetics relative to S1177 phosphorylation, suggesting that S1177 phosphorylation is an early event in eNOS activation. Phosphorylation at S633 prolongs the signal (Mount, Kemp, and Power 2007). S114 phosphorylation is postulated to cause inhibition of eNOS activity (Mount, Kemp, and Power 2007), while phosphorylation at the tyrosine sites, Y81 and Y657, have been reported to either stimulate or inhibit activity (Fulton et al. 2008).

We were interested in the factors that regulate eNOS activity in the whole animal, and to determine whether modulation of eNOS activation would lead to alterations in the extent of downstream vascular injury. It was possible that changes in phosphorylation of eNOS would not necessarily correlate with changes in eNOS activity as had previously been observed in uterine artery endothelial cells from pregnant sheep (Cale and Bird 2006). It was remarkable that in the rat the major response to CI-1044 treatment was primarily through early phosphorylation of S615, with a 30-fold increase in phosphorylation observed within 2 hr of the last dose. Surprisingly, phosphorylation of S1177 was lower than baseline. Phosphorylation of S1177 has been shown to be an early event in eNOS activation (Bauer et al. 2003; Zhang and Hintze 2006), so it is possible that the S1177 response may have peaked prior to the first sample collection. The excessive phosphorylation of S615 in the mesentery appears to be contributing to the eNOS production of NO resulting in the injurious event.

A number of upstream pathways regulate the phosphorylation of eNOS (Figure 10). PKA regulates the phosphorylation of S1177 and S633 (Boo et al. 2002; Sessa 2004), while PI3K/AKT coordinates the phosphorylation of S615 and S1177 (Fulton et al. 1999; Li et al. 2005). Phosphorylation of T495 and Y657 is regulated by PKC (Chen et al. 1999; Mount, Kemp, and Power 2007) and Y81 is regulated by cSRC (Fulton et al. 2007). These findings suggest that a number of upstream regulators can play a role in regulating phosphorylation at each site to produce the desired response.

It was striking that phosphorylation at one key site was induced by several compounds binding to different ligands and led to the deregulation of eNOS activity in the rat mesentery associated with DIVI. Increased eNOS phosphorylation at a number of sites, in particular S615, as well as increased phosphorylation of AKT has been demonstrated in a shear stress-related model of exercised mice (Zhang 2009). However, in a more long-term conditioning experiment, no effect on eNOS or AKT phosphorylation was observed (Pellegrin 2011). Therefore, there may be a more temporal aspect to activation of eNOS activity where, in response to upstream regulators, subtle variations in phosphorylation of one site over another produces the response to the stimulus, rather than the effect at one particular phosphorylation site.

It was noteworthy that immunoprecipitation using eNOS antibodies pulled down a number of upstream regulators, suggesting that eNOS is held in a signalosome complex with a number of upstream regulators, enabling crosstalk between the different pathways. Crosstalk between the PKA and AKT pathways has been identified previously; cAMP-mediated eNOS activation via signaling through the AKT pathway was observed in canine coronary microvessels (Zhang and Hintze 2006), while cAMP treatment increased phosphorylation of a 125-kDa PI3K-binding protein (Yamanaka et al. 2012). Furthermore, complexes between eNOS and PKA/AKT held in close proximity by the scaffolding protein β-arrestin-2 have been identified in human primary T cells (Bjørgo et al. 2010). Large signalosome complexes composed of PKA, AKT, PI3K, ERK1/2, and MEK have been identified in HEK293 cells (Shen et al. 2011). A direct connection between cAMP and PI3K occurs through EPAC-1 and RAP-1 (Tawa et al. 2010). This pathway is involved in multiple endothelial functions, including angiogenesis as well as exocytosis of Weibel-Palade bodies (van Hooren et al. 2012). EPAC-1 is also involved in additional signalosomes mediating connexin stability with PDE-4D and integrin function with PDE-3B (Maurice 2011). This suggests that it is the interplay among many upstream regulators in a large signaling complex that coordinates the phosphorylation state of eNOS leading to its activation. This type of physical interaction allows apparently common mediators such as cAMP to have compartmentalized responses even within the same cell (Dekkers et al. 2012). Our finding of eNOS in a complex with a number of upstream regulators provides further evidence that the communal interplay among the different upstream regulators leads to the coordinated phosphorylation of eNOS (Figure 10B). Hence, the overall phosphorylation state of the molecule may be more critical than the phosphorylation state of one particular amino acid. Until the basic biology of the regulation of eNOS is more clearly understood, it will be difficult to dissect the root causes of the disregulation leading to DIVI.

In conclusion, our work further demonstrates that in response to CI-1044, activation of eNOS produces excessive levels of NO and vascular injury, and modulation of upstream activators of eNOS leads to regulation of eNOS activity. Our findings are that 4 other classes of drugs appear to cause vascular injury in a manner similar to CI-1044 and cause similar changes in eNOS phosphorylation patterns. These findings support the hypothesis that a common mechanism may underlie DIVI in rats and this concept deserves further scrutiny.

Footnotes

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a Cooperative Research and Development Agreement between the U.S. FDA and Pfizer Inc. This project was supported in part by an appointment to the Oak Ridge Institute for Science and Education (ORISE) Research Participation Program at the Center for Drug Evaluation and Research administered by the ORISE through an agreement between the U.S. Department of Energy and Center for Drug Evaluation and Research (CDER).

Authors’ Note

The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be considered to represent any agency determination or policy.

Abbreviations

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