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. Author manuscript; available in PMC: 2023 Dec 1.
Published in final edited form as: J Cardiovasc Pharmacol. 2022 Dec 1;80(6):842–851. doi: 10.1097/FJC.0000000000001354

Apelin-Induced Relaxation of Coronary Arteries is Impaired in a Model of Second-Hand Cigarette Smoke Exposure

Santo Anto 1, Venkatachalem Sathish 1, Chengwen Sun 1, Stephen T O’Rourke 1
PMCID: PMC9729429  NIHMSID: NIHMS1829255  PMID: 35976142

Abstract

Apelin, an endogenous ligand for APJ receptors, causes nitric oxide (NO)-dependent relaxation of coronary arteries. Little is known about the effects of apelin/APJ receptor signaling in the coronary circulation under pathological conditions. Here, we tested the hypothesis that the vasorelaxing effect of apelin is impaired by cigarette smoke extract (CSE), an established model for secondhand smoke exposure. Isolated rat coronary arteries were treated with 2% CSE for four hours. Apelin-induced relaxation of coronary arteries was abolished by CSE exposure, while relaxations to acetylcholine (ACh) (endothelium-dependent relaxation) and to DEA NONOate (NO donor) were similar in control and CSE-treated arteries. Immunoblot analysis demonstrated that apelin increased eNOSser1177 phosphorylation under control conditions but had no effect after exposure to CSE. Moreover, GRK2 expression was increased in CSE-exposed coronary endothelial cells. Pretreatment with CMPD101, a GRK2 inhibitor, improved the relaxation response to apelin in CSE-exposed coronary arteries. CSE treatment failed to inhibit relaxations evoked by CMF-019, an APJ receptor biased agonist that has little effect on GRK2. In arteries exposed to CSE, apelin impaired the response to ACh but not to DEA NONOate. ACh-induced relaxation was unaffected by CMF-019 in either control or CSE-treated coronary arteries. Results suggest that APJ receptor signaling via the GRK2 pathway contributes to both the loss of relaxation to apelin itself as well as the ability of apelin to inhibit endothelium-dependent relaxation to ACh in CSE-exposed coronary arteries, likely due to impaired production of NO from endothelial cells. These changes in apelin/APJ receptor signaling under pathologic conditions (e.g., exposure to second-hand smoke) could create an environment that favors increased vasomotor tone in coronary arteries.

Keywords: apelin, APJ receptors, cigarette smoke extract, coronary arteries, GRK2, nitric oxide

INTRODUCTION

The peptide hormone, apelin, dilates coronary arteries and thereby increases coronary blood flow(1). Apelin-induced coronary vasodilation results from activation of APJ receptors located on endothelial cells in the coronary arterial wall (2). Binding of apelin to APJ receptors stimulates the release of endothelium-derived nitric oxide (NO) (2,3), which diffuses to the underlying smooth muscle to cause relaxation.

APJ receptors are G-protein-coupled receptors that signal via the PI3-kinase/Akt pathway to activate eNOS and stimulate NO production(4). A substantial body of evidence indicates that APJ receptors can also transduce extracellular signals in a G-protein-independent manner via activation of the GRK/β-arrestin pathway(5–7). Several GRK isoforms have been identified(8,9), with native vascular endothelial cells primarily expressing the GRK2 isoform(10,11). Activation of this G-protein-independent pathway may be particularly relevant in disease states. Indeed, activation of GRK2 inhibits eNOS activity and NO production in endothelial cells(11,12), and increased GRK2 expression and activity in the vasculature is associated with the pathogenesis of cardiovascular disease(13,14).

Based on the putative beneficial effects of apelin on the heart and coronary circulation, apelin and apelin-like analogs are being investigated for cardiovascular disorders such as heart failure and pulmonary hypertension(1,15). Surprisingly, the effects of apelin on vasomotor tone in diseased coronary arteries are largely unknown. Cigarette smoking, both active and passive (i.e. second-hand smoke), is a known risk factor for the development of coronary artery disease(16,17). Second-hand smoke exposure has been reported to increase GRK2 expression in isolated trophoblasts(18); however, the effects, if any, of cigarette smoke on GRK2 expression in blood vessels have not been determined. Therefore, we tested the hypothesis that the vasodilator effect of apelin is impaired in coronary arteries exposed to cigarette smoke extract, a well-established model of second-hand smoke(19,20).

MATERIALS AND METHODS

Animals and Tissue Preparation

Experiments were performed on tissues obtained from 12-week-old male Sprague-Dawley rats purchased from Envigo RMS (Indianapolis, IN). Rats were housed on a 12-hr/12-hr light/dark cycle at 22 ± 2°C and were provided with food and water ad libitum. All animal protocols used in this study were approved by the North Dakota State University Institutional Animal Care and Use Committee. The animals were anesthetized with isoflurane and hearts were isolated and placed into ice-cold physiologic salt solution (PSS) of the following composition: 118.9 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4.7H2O, 2.5 mM CaCl2, 1.2 mM KH2PO4, 0.03 mM EDTA, 5.5 mM glucose, and 25.0 mM NaHCO3. Epicardial coronary arteries were dissected free and cleaned of surrounding tissues.

Cigarette Smoke Extract Preparation

Cigarette smoke extract (CSE) was freshly prepared from research grade cigarettes (3R4F) purchased from the Tobacco Research Institute (University of Kentucky, Lexington, KY), using a modified method of Blue and Janoff(21). Briefly, CSE was prepared freshly for each experiment using a 60 ml syringe controlled by a three-way stopcock, which mimics the gas fluid pathway in the human lung during smoking(22). Cigarette smoke was drawn into the syringe and then slowly bubbled through 10 ml of phosphate buffered saline. The obtained CSE solution was pH corrected (7.4), and then filtered (0.2 μm) to remove bacteria and large particles. The concentration of the CSE solution obtained (stock) was considered as 100%(22). The CSE concentration used (i.e. 2%), as well as the incubation period (4 h), were selected based on previous reports of altered protein expression within that time frame in pulmonary endothelial cells(23).

Vascular Function Studies

Rat coronary arterial rings (120–150 μm; 1.2 mm in length) were mounted in wire myographs (DMT, Aarhus, Denmark) for isometric tension recording. The myograph chambers were filled with PSS (5 ml), which was maintained at 37°C and continuously aerated with 95% O2/5% CO2 throughout the experiment. The arterial rings were stretched up to a resting force of 6 mN by sequential stretching and then allowed to stabilize for 30 minutes with intermittent washings. Vascular reactivity was established by evoking a contractile response to KCl (60 mM). In some rings, the endothelium was removed by gently rubbing the intimal surface with a human hair. The absence or presence of endothelium was verified with the endothelium-dependent vasodilator, acetylcholine (ACh; 10−6 M). Responses to vasodilators used in this study were obtained in arterial rings contracted with 5-hydroxytryptamine (5-HT; 10−7 M). Inhibitors were added to the myograph chamber 20 min prior to contraction with 5-HT. When apelin (10−7 M) or CMF-019 (10−7 M) was used as an inhibitor, it was added to the tissues for 5 minutes, after the contraction to 5-HT had stabilized, in order to minimize desensitization of APJ receptors. All inhibitors remained in the myograph solution for the remainder of the experiment. Experiments with untreated control rings were conducted in parallel with rings treated with inhibitors from the same animal.

Isolation of Coronary Endothelial Cells

Coronary artery endothelial cells were isolated using previously established procedures(24), with minor modifications. Briefly, rat coronary arteries were digested in dissociation solution (55 mM NaCl, 6 mM KCl, 80 mM Na-glutamate, 2 mM MgCl2, 0.1 mM CaCl2, 10 mM HEPES, and 10 mM glucose, pH 7.3) containing elastase (0.5 mg/ml) (Worthington, Lakewood, NJ) and neutral protease (0.5 mg/ml) (Worthington) for 60 minutes at 37°C, followed by collagenase type II (0.5 mg/ml) (Worthington) in the same solution for 2 minutes. The enzyme solution was removed, and arteries were treated with dissociation solution without enzyme for 10 min followed by trituration with a polished Pasteur pipette to produce a suspension of single endothelial cells. Cells were re-suspended in endothelial cell growth medium MV (EGM MV) (Promo cell, Heidelberg, Germany) with 1% penicillin-streptomycin solution and maintained at 37°C (5% CO2, 95% air) until 80% confluent. Cells from 2nd to 4th passage were used for experiments. Phenotype stability was confirmed periodically using the endothelial cell marker, platelet endothelial cell adhesion molecule (PECAM 1), and smooth muscle cell marker, smooth muscle α-actin.

Western Immunoblotting

Rat coronary arteries were collected and frozen immediately using liquid nitrogen. For eNOS phosphorylation studies at serine-1177, freshly isolated coronary arteries were exposed to CSE (4 hrs) followed by treatment with apelin (10−7 M) for 10 minutes at 37OC, and were then snap frozen. Frozen tissues were crushed using a mortar and pestle and the powdered tissue was homogenized at 4°C using an IKA Ultra-Turrax T8 homogenizer (IKA Works Inc., Wilmington, NC) in lysis buffer containing protease and phosphatase inhibitor cocktail (ThermoFisher Scientific, Waltham, MA). Standard procedures were followed for coronary endothelial cell lysis. Protein estimation was performed using a Pierce BCA protein estimation kit (ThermoFisher Scientific, Waltham, MA). Proteins were separated by SDS–polyacrylamide gel electrophoresis using Mini-PROTEAN precast gels (4–15% gradient; Bio-Rad, Hercules, CA) and transferred onto a polyvinylidene difluoride membrane. After blocking with 5% bovine serum albumin in Tris buffered saline (TBS, pH 7.4), blots were incubated overnight at 4°C with appropriate primary antibodies specific for APJ receptors (sc-517300, Santa Cruz Biotechnology Inc.), GRK2 (sc-13143, Santa Cruz Biotechnology Inc.), phospho-eNOS (9570S, Cell Signaling Technology) or eNOS (sc-376751, Santa Cruz Biotechnology Inc.) using a dilution of 1:100 1:1000 (phospho-eNOS), followed by incubation with a horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology Inc.). To ensure equal loading, the blots were analyzed for β-actin protein expression using an anti-actin antibody with a dilution of 1:500 (sc-47778, Santa Cruz Biotechnology Inc.) Protein bands and relative densities were measured using an enhanced chemiluminescence light detection kit (Thermo Fisher Scientific, Waltham, MA).

Drugs

The following drugs were used: acetylcholine, diethyl amine (DEA) NONOate, diltiazem, and 5-HT (Sigma Chemical, St. Louis, MO); apelin-13 and F13A trifluoro-acetate salt (Bachem, Torrance, CA); CMPD 101 (Tocris, Ellisville, MO); and CMF 019 (Aobious, Gloucester, MA). Drug solutions were freshly prepared in double-distilled water with the exception of CMF-019 and CMPD 101, which were dissolved initially in DMSO and followed by further dilutions in double-distilled water.

Data Analysis

Relaxation responses are shown as a percent of initial tension induced by 5-HT (10−7). The EC50 values (concentration of drug that gives half-maximal response) were determined, converted to their negative logarithms, and expressed as -log molar EC50 (pD2). The data from all sets of experiments are presented as mean ± standard error of mean and n indicates the number of animals from which blood vessels were taken. The statistical significance was evaluated by paired t-test or two-way ANOVA followed by Bonferroni post hoc analysis, as appropriate. Differences were considered significant when P < 0.05.

RESULTS

CSE exposure inhibits apelin-induced relaxation of isolated coronary arteries

Apelin (10−8 – 3 × 10−6 M) caused concentration-dependent relaxation of control coronary arteries (i.e., not previously exposed to CSE) contracted with 5-HT (10−7 M) (Figure 1A). The pD2 value for apelin was 7.25 ± 0.16 and the maximal relaxation (Emax) was 43 ± 5%. Coronary arteries that had been exposed to 2% CSE for four hours failed to relax in response to apelin (Figure 1A). In contrast, the relaxation response to the classic endothelium-dependent vasodilator, acetylcholine (ACh; 10−9 – 10−5 M), was similar in control and CSE-treated arteries (pD2 = 6.97 ± 0.11 vs 6.83 ± 0.21 in control and 2% CSE-exposed arteries, respectively; P > 0.05, n = 6) (Figure 1B). Moreover, relaxations to the NO donor, DEA NONOate (10−9 – 10−5 M), were also unaltered by exposure of coronary arteries to 2% CSE (pD2 = 6.12 ± 0.26 vs 6.05 ± 0.21 in control and 2% CSE-exposed arteries, respectively; P > 0.05, n = 6) (Figure 1C). Immunoblot analysis indicated that basal levels of p-eNOS/eNOS were similar in control and CSE-treated arteries (Figure 2). Apelin (10−7 M) caused a nearly 3-fold increase in p-eNOS/eNOS under control conditions but had no effect after exposure to 2% CSE (Figure 2).

FIGURE 1.

FIGURE 1.

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Effect of cigarette smoke extract (CSE) exposure on apelin-induced relaxation of isolated rat coronary arteries. (A) Mean data demonstrating apelin-induced relaxation in control, which was abolished in the CSE-treated arteries; (B) Log concentration-response curves for acetylcholine (ACh) in control and CSE-treated arteries; (C) Log concentration-response curves for DEA in control and CSE-treated arteries. Data are expressed as percent relaxation of 5-HT-induced contractions, which averaged 3.98 ± 0.54 vs 4.39 ± 0.46 mN in control and CSE-treated rings, respectively (P > 0.05). Each point represents the mean ± S.E.M. (n = 6). *P < 0.05 indicates a significant difference from the corresponding control value.

FIGURE 2.

FIGURE 2.

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Effects of apelin on eNOS-Ser1177 phosphorylation in CSE exposed coronary arteries. (A) Representative western blot showing eNOS-Ser1177 phosphorylation in CSE exposed (4 hr) coronary arteries treated with apelin (10−7 M) for 10 minutes. β-actin was used as loading control. (B) Bar graph summarizing the effect of apelin on eNOS activity expressed as p-eNOS/eNOS protein levels in coronary arteries (n=3). Each bar represents mean ± SEM. *P<0.05 as compared with untreated control; #P<0.05 as compared with apelin alone.

GRK2 expression is increased in coronary endothelial cells exposed to CSE

Expression of eNOS, APJ receptor, and GRK2 proteins was identified by Western immunoblot analysis in cultured rat coronary endothelial cells. GRK2 protein expression was increased in coronary endothelial cells exposed to 2% CSE for four hours, whereas APJ receptor and eNOS protein expression were largely unaffected by CSE exposure (Figure 3).

FIGURE 3.

FIGURE 3.

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Effect of CSE on GRK2 expression in coronary endothelial cells. Representative immunoblots and bar graphs showing expression of: (A) GRK2 protein; (B) APJ receptors (APJR); and (C) eNOS in control (lane 1) and CSE-treated (lane 2) cultured coronary endothelial cells; β-actin was used as a loading control. Data are represented as mean ± S.E.M (n = 4–5). *P < 0.05 indicates a significant difference from the corresponding control value.

GRK2 mediates the inhibitory effect of CSE on apelin-induced relaxation

Based on the observation that GRK2 expression was increased in coronary endothelial cells exposed to 2% CSE (Figure 3A), we tested whether the GRK2 inhibitor, CMPD101(25,26), could rescue apelin-induced relaxation in coronary arteries exposed to CSE. CMPD101 (3×10−5 M) had no effect on the concentration-response curve to apelin in control arteries not exposed to CSE (pD2 = 6.89 ± 0.26 vs 6.88 ± 0.07 in absence and presence of CMPD101, respectively; P > 0.05, n = 6) (Figure 4A); however, treatment of coronary arteries with the GRK2 inhibitor restored apelin-induced relaxation in arteries previously exposed to CSE (pD2 = 7.01 ± 0.17 and Emax = 32% ± 4% in the presence of CMPD101) (Figure 4B).

FIGURE 4.

FIGURE 4.

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Effect of CSE on apelin-induced relaxation in the presence of the GRK2 inhibitor, CMPD101. Log concentration-response curves for apelin-induced relaxation in the absence or presence of CMPD101 (3 × 10−5 M) in: (A) control; and (B) CSE-treated coronary arteries. 5-HT-induced contractions averaged 5.50 ± 0.76 vs 7.07 ± 0.93 mN in control and CSE-treated rings, respectively (P > 0.05). Each point represents the mean ± S.E.M. (n = 6). *P < 0.05 indicates a significant difference from the corresponding control value.

We also tested the ability of CMF-019, an APJ receptor biased agonist that acts selectively through the G-protein-dependent pathway with little effect on GRK2(27), to cause relaxation of coronary arteries. CMF-019 (10−9 - 10−5 M) caused concentration-dependent relaxation of untreated (control) coronary arteries with intact endothelium (Figure 5A). The pD2 value for CMF-019 was 7.27 ± 0.36 and the Emax was 40% ± 3%. CMF-019 induced relaxation was abolished by removal of the endothelium and by the APJ receptor antagonist, F13A (10−7 M) (Figure 5B), consistent with activation of endothelial APJ receptors(2). In contrast to our findings above with apelin (Figure 1A), exposure of coronary arteries to 2% CSE had no effect on CMF-019-induced relaxation (pD2 = 7.17 ± 0.24 vs 6.90 ± 0.22 and Emax = 40% ± 6% vs 41% ± 7% in control and CSE-treated arteries, respectively; P > 0.05, n = 6) (Figure 5C).

FIGURE 5.

FIGURE 5.

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Endothelium-dependent relaxation of CMF-019 is mediated through APJ receptors. (A-B) Log concentration-response curves for CMF-019-induced relaxation in coronary arteries with endothelium (E+), which was abolished in endothelium denuded (E-) segments; or (B) in the presence of F13A (10−7 M). (C) Log concentration-response curves for CMF-019-induced relaxation in control and CSE-treated coronary arteries. 5-HT-induced contractions averaged 4.41 ± 0.51 mN in control rings and did not differ significantly in rings treated with F13A or CSE. Each point represents the mean ± S.E.M. (n = 6). *P < 0.05 indicates a significant difference from the corresponding control value.

Apelin, but not CMF-019, inhibits ACh-induced relaxation in coronary arteries exposed to CSE

Since GRK2 activation can cause inhibition of eNOS and decreased NO production in endothelial cells(11), we investigated whether apelin could inhibit coronary arterial relaxations mediated by ACh (an endothelium- and NO-dependent vasodilator). In coronary arteries exposed to 2% CSE, apelin (10−7 M) caused a rightward shift in the concentration-response curve to ACh (pD2 = 7.43 ± 0.24 vs 6.78 ± 0.20 without and with apelin, respectively; P < 0.05, n=6) (Figure 6A); however, apelin alone had no effect on ACh-induced relaxation in control coronary arteries (pD2 = 7.16 ± 0.17 vs 7.38 ± 0.20 without and with apelin, respectively; P < 0.05, n=6). Moreover, the concentration-response curve to DEA NONOate (10−9 – 10−5 M) was unaffected by the presence of apelin (10−7 M) in coronary arteries treated with 2% CSE (pD2 = 6.21 ± 0.35 vs 6.22 ± 0.25 without and with apelin, respectively; P > 0.05, n=6) (Figure 6B). In contrast to apelin, CMF-019 (10−7 M) had no effect on ACh-induced relaxation in CSE-treated arteries (pD2 = 7.04 ± 0.21 vs 6.94 ± 0.18 in control and 2% CSE, respectively; P > 0.05, n = 6) (Figure 6C).

FIGURE 6.

FIGURE 6.

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Effect of CSE on ACh-induced endothelium-dependent relaxation and DEA-induced endothelium-independent relaxation in the presence and absence of apelin. Log concentration-response curves for: (A) ACh in CSE-treated arteries in the absence and presence of apelin (10−7 M); (B) DEA in CSE-treated arteries in the absence and presence of apelin (10−7 M); and (C) Log concentration-response curves for ACh-induced relaxation in the absence and presence of CMF-019 (10−7 M) in CSE-treated arteries. 5-HT-induced contractions averaged 3.72 ± 0.44 mN in control rings and did not differ significantly in rings treated with apelin or CMF-019. Each point represents the mean ± S.E.M. (n = 6). *P < 0.05 indicates a significant difference from the corresponding control value.

DISCUSSION

Data from this study demonstrate that apelin-induced, endothelium-dependent relaxation of isolated coronary arteries is markedly impaired following exposure to CSE for 4 hours. Moreover, the function of apelin changed after exposure to CSE; i.e, rather than causing relaxation of coronary arteries, the peptide instead inhibited relaxation evoked by ACh, an endothelium-dependent vasodilator that acts via the release of NO from endothelial cells in rat coronary arteries(2). Results from mechanistic studies provide evidence that GRK2 may be a pivotal link in the phenotypic switch in the coronary artery response to apelin that occurs in CSE-treated arteries. The findings are significant inasmuch as they suggest that changes in apelin/APJ receptor signaling under pathologic conditions (e.g., exposure to second-hand smoke) could create an environment that favors increased vasomotor tone in coronary arteries, which would predictably lead to a reduction in coronary blood flow and myocardial ischemia.

Under normal physiologic conditions, apelin causes relaxation of coronary arteries by releasing NO from vascular endothelial cells(2). In coronary arteries exposed to CSE, apelin strikingly failed to cause relaxation of the tissues, whereas the response to acetylcholine (ACh), the prototypical endothelium-dependent vasodilator that releases NO from endothelial cells, was retained. CSE exposure also had no effect on relaxation to the NO donor, DEA NONOate. These findings with ACh and DEA indicate that the impaired response to apelin is unlikely due to a defect in the intrinsic ability of the endothelial cells to produce bioavailable NO, or in the responsiveness of the underlying smooth muscle cells to NO. Taken together, the data suggest that coupling between APJ receptor activation by apelin and NO synthesis by eNOS is selectively impaired in endothelial cells from coronary arteries exposed to CSE. This premise was evaluated by measuring phosphorylation of eNOS at serine-1177, a widely used marker of eNOS activation(28,29). We found that apelin increased p-eNOS in control arteries and, importantly, that this response to apelin was abolished in arteries exposed to CSE. These data indicate that apelin/APJ receptor signaling fails to increase eNOS activity in CSE-treated arteries, which would predictably lead to impaired NO production and relaxation.

Impaired coupling between apelin/APJ receptor activation and NO synthesis by eNOS could be explained, at least in part, by changes in expression of proteins involved in intracellular signaling. Relevant to this possibility, exposure to CSE has been reported to cause increases as well as decreases in protein expression(23,30). Nonetheless, it is not likely that the impaired response to apelin is due a reduction in either APJ receptors or eNOS since expression of these proteins was not altered by CSE exposure. In contrast, GRK2 expression was significantly elevated in CSE-treated coronary endothelial cells. GRK2 is a multidomain protein that interacts with a complex array of cellular effectors and is a key component of the G-protein independent intracellular signaling cascade(31). Since increased GRK2 is known to inhibit eNOS activity and NO production in endothelial cells(11,32), our finding that GRK2 expression is increased in coronary endothelial cells after CSE exposure suggests a potential mechanism for the impaired response to apelin. Indeed, the observation that CMPD101, a potent and selective GRK2 inhibitor(33,34), restored the response to apelin in CSE-treated coronary arteries strongly supports a major role for GRK2 in response to binding of apelin to APJ receptors in coronary arteries exposed to CSE.

In order to further explore the role of the G-protein-independent signaling pathway in the response to apelin in CSE-treated coronary arteries we compared the effects of apelin with CMF-019, a G-protein biased agonist at APJ receptors(35). Our data show that, like apelin, CMF-019 causes endothelium-dependent relaxation of isolated rat coronary arteries and that this response is inhibited by the APJ receptor antagonist, F13A. In contrast to apelin, however, CMF-019-induced coronary artery relaxation was unaffected by exposure to CSE. That the effects of the G-protein biased APJ receptor agonist did not differ in control and CSE-treated coronary arteries is consistent with a role for G-protein independent signaling via GRK2 in the impaired response to apelin in CSE-treated arteries.

Although the endothelium-dependent relaxation response to apelin is lost in coronary arteries exposed to CSE, it is noteworthy that the apelin/APJ receptor signaling system takes on a completely different function in these blood vessels. Rather than cause relaxation, apelin inhibited endothelium-dependent relaxation in response to ACh. These data suggest that APJ receptors retain their affinity for apelin in CSE-treated coronary arteries but process the apelin signal in a manner that differs from that in control arteries. That CMF-019 had no effect on ACh-induced relaxation further implicates a role for G-protein-independent signaling via APJ receptors under these conditions. In addition, apelin had no effect on relaxation of CSE-treated coronary arteries in response to DEA NONOate. These findings with the NO-donor indicate that apelin does not interfere with the smooth muscle response to NO and are consistent with a phenotypic switch in apelin/APJ receptor pharmacology occurring most likely at the level of the endothelial cells, resulting in impaired signaling between APJ receptor activation and NO production.

CONCLUSION

In conclusion, the present study demonstrates that apelin-induced relaxation of coronary arteries is impaired in a model of second-hand smoke exposure. Apelin/APJ receptor signaling via the GRK2 pathway may contribute to both the loss of relaxation to apelin itself as well as the ability of apelin to inhibit endothelium-dependent relaxation to ACh in CSE-treated coronary arteries. The results with CMF-019 suggest that, at least under certain pathologic conditions, APJ receptor biased agonists have the potential to be more effective than apelin itself as therapeutic agents for treating cardiovascular disorders.

Acknowledgements:

National Institutes of Health National Heart, Lung, and Blood Institute (HL124338)

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