Distribution and relative expression of vasoactive receptors on arteries

Arterial tone is regulated by multiple ligand-receptor interactions, and its dysregulation is involved in ischemic conditions such as acute coronary spasm or syndrome. Understanding the distribution of vasoactive receptors on different arteries may help guide the development of tissue-specific vasoactive treatments against arterial dysfunction. Tissues were harvested from coronary, mesenteric, pulmonary, renal and peripheral human artery (n = 6 samples of each) and examined using a human antibody array to determine the expression of 29 vasoactive receptors and 3 endothelin ligands. Across all types of arteries, outer diameter ranged from 2.24 ± 0.63 to 3.65 ± 0.40 mm, and AVPR1A was the most abundant receptor. The expression level of AVPR1A in pulmonary artery was similar to that in renal artery, 2.2 times that in mesenteric artery, 1.9 times that in peripheral artery, and 2.2 times that in coronary artery. Endothelin-1 was expressed at significantly higher levels in pulmonary artery than peripheral artery (8.8 times), mesenteric artery (5.3 times), renal artery (7.9 times), and coronary artery (2.4 times). Expression of ADRA2B was significantly higher in coronary artery than peripheral artery. Immunohistochemistry revealed abundant ADRA2B in coronary artery, especially vessels with diameters below 50 μm, but not in myocardium. ADRA2C, in contrast, was expressed in both myocardium and blood vessels. The high expression of ADRA2B in coronary artery but not myocardium highlights the need to further characterize its function. Our results help establish the distribution and relative levels of tone-related receptors in different types of arteries, which may guide artery-specific treatments.


Distribution and relative expression of vasoactive receptors on arteries
Xinhao Liu 1,3 , Dan Luo 1,3 , Jie Zhang 2 & Lei Du 1* Arterial tone is regulated by multiple ligand-receptor interactions, and its dysregulation is involved in ischemic conditions such as acute coronary spasm or syndrome. Understanding the distribution of vasoactive receptors on different arteries may help guide the development of tissue-specific vasoactive treatments against arterial dysfunction. Tissues were harvested from coronary, mesenteric, pulmonary, renal and peripheral human artery (n = 6 samples of each) and examined using a human antibody array to determine the expression of 29 vasoactive receptors and 3 endothelin ligands. Across all types of arteries, outer diameter ranged from 2.24 ± 0.63 to 3.65 ± 0.40 mm, and AVPR1A was the most abundant receptor. The expression level of AVPR1A in pulmonary artery was similar to that in renal artery, 2.2 times that in mesenteric artery, 1.9 times that in peripheral artery, and 2.2 times that in coronary artery. Endothelin-1 was expressed at significantly higher levels in pulmonary artery than peripheral artery (8.8 times), mesenteric artery (5.3 times), renal artery (7.9 times), and coronary artery (2.4 times). Expression of ADRA2B was significantly higher in coronary artery than peripheral artery. Immunohistochemistry revealed abundant ADRA2B in coronary artery, especially vessels with diameters below 50 μm, but not in myocardium. ADRA2C, in contrast, was expressed in both myocardium and blood vessels. The high expression of ADRA2B in coronary artery but not myocardium highlights the need to further characterize its function. Our results help establish the distribution and relative levels of tone-related receptors in different types of arteries, which may guide artery-specific treatments.
Arterial tone (extent of constriction) modulates the perfusion of vital organs and is well regulated by interactions between neurohumoral ligands and their receptors on vascular smooth muscle or endothelial cells 1,2 . Disorder of arterial tone may impair blood supply and lead to acute, potentially life-threatening organ dysfunction, such as coronary artery spasm 3 or acute coronary syndrome 4 . A powerful treatment for such dysfunction would be vasodilators that act locally at the ischemic organ without affecting other vasculature, thereby maintaining pressure to push more blood to the target organ. Developing such specific vasodilators requires understanding the distribution of vasoactive receptors and their ligands across different artery types. For example, the cardiovascular system, especially the coronary artery, shows extensive sympathetic innervation and may be associated with cardiovascular diseases 6,7 . Investigating the expression pattern of various subtypes of adrenoceptors may help in treating ischemic heart disease. Endothelin-1 and its receptors are positively associated with cardiovascular diseases 8 , pulmonary arterial hypertension 9 , resistant hypertension 10,11 and chronic kidney diseases 12 . Investigation of endothelin expression in different arteries may help develop treatments for different target organs.
As a step in this direction, the present study investigated the distribution of 29 types of receptors and 3 types of endothelin ligands related to vascular smooth muscle cells and endothelial cells in small and medium-sized arteries, which are believed to be the primary determinants of organ perfusion 13 . In particular, we focused on adrenoceptors in coronary arteries because the sympathetic nerve is involved in the pathophysiology of ischemic heart disease 14 , a leading cause of death worldwide 15  www.nature.com/scientificreports/ condition that might compromise ability to regulate vascular tone, including history of smoking, daily alcohol use, diabetes mellitus, or long-term use of corticosteroids. Patients or family members provided written informed consent for their tissues to be taken and used for biomedical research. This study was approved by the Ethics Committee of West China Hospital and registered in the Chinese Clinical Trial Registry (https ://www.chict r.org.cn/showp rojen .aspx?proj=10753 ) as ChiCTR-OPC-15006229.
All samples were processed within 2 h after collection. Connective tissue surrounding the blood vessels was removed on ice, and samples were stored at − 80 °C until further analyses, for which they were thawed only once.

Human antibody array.
A portion of all artery samples was homogenized for 30 min with a high-speed dispersion cutter in cell lysis buffer containing protease inhibitor cocktail. Homogenates were centrifuged for 20 min at 4 °C, and the supernatant was processed using a membrane-based array of biotinylated human antibodies (catalog no. AAH-BLG-CUST, RayBiotech, Guangzhou, China) according to the manufacturer's instructions. This kit detects three subtypes of endothelin and 29 receptors of seven types: nine adreno, nine cholinergic, two angiotensin, three vasopressin, four 5-hydroxytryptamine, one prostaglandin I2 and one thromboxane A2 receptor ( Supplementary Fig. 1). The abbreviations of all detected ligands and receptors are listed in Table 1.
Samples were processed according to the manufacturer's instructions. After dialysis of samples, total protein concentration was determined, and primary amines were biotinylated at room temperature for 30 min. The sample was incubated in blocking buffer to remove unbound biotin, loaded onto the membrane array, covered with a lid to prevent drying out, and incubated at 4 °C overnight. The membrane was washed four times, incubated with horseradish peroxidase-conjugated streptavidin, then incubated in detection buffer. Signal on the membrane was detected using a chemiluminescence imaging analysis system (ImageQuant LAS4000 Scanner, GE Healthcare, USA), and signal intensity was quantified by densitometry. The array contained positive control spots of different concentrations of biotinylated bovine serum albumin (Pos1a, Pos2a, Pos3a and Pos1b, Pos2b, Pos3b), which served as reference points for orienting the arrays and normalizing results from different arrays.
Western blotting of all artery types. A portion of all artery samples was initially pulverized in liquid nitrogen, lysed in RIPA buffer containing protease inhibitor cocktail (Roche) for 1 h at 4 °C, and centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was transferred to a fresh centrifuge tube, protein concentration was determined, and 30 μg of protein was separated by SDS-PAGE in 12% acrylamide gels and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against ADRA2B (1:2000; AP17872PU-N, Origene) or GAPDH (1:5000; ab8245, Abcam). Membranes were washed in phosphate-buffered saline three times and incubated with goat anti-rabbit secondary antibodies for 1 h at room temperature. Finally, membranes were covered with ECL reagent (Thermo Fisher Scientific), then developed using a chemiluminescence imaging analysis system (ImageQuant LAS4000 Scanner).
Statistical analysis. Data were analyzed using SPSS 19.0 (IBM, Armonk, NY USA). Continuous variables were reported as mean and standard deviation. Inter-group differences were assessed for significance using ANOVA, and multiple comparisons were performed using the Tukey method. Differences were considered significant if associated with a two-tailed p < 0.05.

Ethics approval and Consent to participate. This study was approved by the Ethics Committee of West
China Hospital. All procedures involving human participants were performed in accordance with the ethical standards of our institutions as well as the 1964 Helsinki Declaration and its later amendments. Patients or their families gave written informed consent for their tissues to be donated for this research.
Consent for publication. Patients or their families gave written informed consent for the analyses of their tissues to be published.

Results
Patient characteristics. Characteristics of tissue donors and blood vessels are shown in Table 2. Tissues analyzed by human antibody array and Western blot came from four patients receiving a transplanted heart because of dilated cardiomyopathy, one patient receiving a transplanted heart because of coronary artery disease, and one patient who died in a traffic accident and donated his heart for heart transplantation, but the heart   www.nature.com/scientificreports/ In coronary artery, AVPR1A and ET1 were again the most abundant, followed by ADRA2B. CHRM and CHRNA were expressed at relatively low levels. Prostaglandin I2 receptor (PTGIR) was expressed at relatively high levels in coronary artery, but not significantly more than in other artery types ( Supplementary Fig. 2).
In pulmonary artery, ET1 was expressed most abundantly, followed by AVPR1A and then by the four receptors ADRA2B, ADRA2C, AHRM and CHRNA, which were expressed at similar levels.

Alpha-adrenoceptors.
In all types of artery, ADRA2B and ADRA2C were expressed at higher levels than ADRA1A, ADRA1B, ADRA1D or ADRA2A (Fig. 1a). In renal, mesenteric and peripheral arteries, ADRA2B and ADRA2C were expressed at similarly high levels, but ADRA2B in coronary artery (gray value 1403 ± 802) was expressed at a significantly higher level than ADRA2C (530 ± 505, p = 0.006; Fig. 1b) and a slightly higher level than ADRA2B in peripheral artery (634 ± 214, p = 0.063; Fig. 1c). These results were confirmed by Western blot (Fig. 1d) and immunohistochemistry (Fig. 1e). The latter technique further showed that ADRA2C was expressed in both coronary artery and myocardium, and that ADRA2B expression was higher in arteries with diameters smaller than 50 μm than in larger arteries (Fig. 1e).
ADRA1A and ADRA1B were observed in myocardium at lower levels than ADRA2C, but they were barely detectable in arteries. ADRA1D and ADRA2A were scarcely expressed in either myocardium or blood vessels.
AVPR1B and AVPR2 were expressed at much lower levels than AVPR1A (Supplementary Fig. 4b).

Discussion
Various mediators and their receptors regulate the balance between vasoconstriction and vasodilation, and perturbation of this equilibrium can cause life-threatening disease. Here we provide what appears to be the first description of the distribution of 29 vasoactive receptors and 3 ligands in various types of human arteries. Our results suggest that AVPR1A is expressed at higher levels than any receptor that we examined, regardless of the artery type, and that it is expressed at lower levels in coronary artery than in renal, pulmonary, mesenteric or systemic arteries. ADRA2B was expressed at higher levels in coronary artery than other types of alphaadrenoceptors, while ET1 was expressed at higher levels in pulmonary artery. These insights into tissue-specific distribution of vasoactive receptors and ligands may help guide the clinical use of certain vasoactive agents that limit drug activity to specific artery types.
We focused on receptors in the sympathetic nerve system because of their potential association with the development of coronary artery atherosclerosis and acute myocardial ischemia. These receptors can activate platelets and thereby promote atherosclerosis, and they can constrict the coronary artery, triggering acute ischemic events 16 . Sudden activation of sympathetic nerves, such as during severe emotional disturbance, can induce acute cardiovascular events in individuals with coronary artery atherosclerosis 17 . This surge in sympathetic nerve activity can increase the myocardial infarction area 18 and lead to poor prognosis after ischemia 19,20 , including sudden cardiac death. Blocking the thoracic sympathetic preganglionic fibers inhibits release of sympathetic neurotransmitters acting on coronary arteries, which can substantially increase coronary blood flow 21 . Similarly, high epidural block can relieve angina and improve the prognosis of patients with coronary heart disease 22 .
Our results identify ADRA2B as a novel therapeutic target in ischemic heart disease. This receptor was expressed more strongly in coronary artery, especially in vessels with diameters below 50 μm, than in other artery types. Specific agonists of this and other alpha 2-adrenorceptors significantly constrict the coronary artery and reduce coronary blood flow 23,24 . The D/D genotype of the ADRA2B gene leads to a receptor that hyper-responds artery with diameter < 50 μm. Bottom right corner: artery with diameter ≥ 50 μm. As a negative control, sections were incubated with phosphate-buffered saline instead of primary antibody. Data are mean ± SD, and P < 0.05 for Student's t test was set as the significance threshold.  www.nature.com/scientificreports/ to adrenaline, decreasing coronary blood flow 25 and increasing risk of acute myocardial infarction and sudden cardiac death [26][27][28] . We found the most abundant vasoactive receptors in myocardium to be ADRA1A, ADRA2A, and ADRA2C. It is possible that during stimulation of the sympathetic nervous system, these receptors are activated by norepinephrine to increase contractility. This may, in turn, increase oxygen consumption and lead to myocardial ischemia.

Scientific RepoRtS
In our samples of systemic artery, AVPR1A was more strongly expressed than any other assayed receptor. Interestingly, blood pressure is sensitive to arginine vasopressin 29 . The high expression of AVPR1A may contribute to the sensitive response of blood pressure to arginine vasopressin. Using vasopressin to treat hypotension can induce mesenteric and digital ischemia 30-32 as well as skin necrosis 33 , and vasopressin treatment is associated with higher risk of digital ischemia than norepinephrine treatment 34 . Whether these side effects are associated with high expression of AVPR1A need further functional studies.
AVPR1A was expressed at a moderate level in coronary artery. This supports the use of low-dose arginine vasopressin to treat hypotension while minimizing interference with the coronary artery. High vasopressin concentration leads to coronary vasoconstriction and myocardial depression in isolated animal hearts 35,36 , and the AVPR1A agonist selepressin increases coronary vascular resistance in a canine model 37 . High-dose arginine vasopressin infusion significantly reduces left ventricular ejection fraction and increases mortality in a mouse model of ischemia/reperfusion 38 . Delivering vasopressin to patients at < 0.03 U/min improves cardiac output 34,39 and reduces risk of renal failure and mortality 40 , but delivering it at > 0.05 U/min increases risk of cardiac arrest 39 and other poor outcomes 41 .
We found that ET1 was abundant in pulmonary artery, consistent with its potent vasoconstrictive activity 42 and its involvement in pulmonary arterial hypertension 43 . Indeed, the ET receptor antagonist bosentan can significantly improve hemodynamics, reduce severity of pulmonary hypertension and strengthen motor ability of patients with pulmonary hypertension 44,45 . The ET receptor antagonists maxitetan and ambesantan can also increase arterial oxygen saturation and the exercise ability of patients with pulmonary hypertension 46,47 . Our results suggest that another strategy to treat pulmonary hypertension may be to block ET1 synthesis or its activity using a specific antagonist.
In our study, pulmonary artery expressed abundant cholinergic receptors, where they regulate not only vasodilation and vasoconstriction, but also inflammation, airway remodeling, and airway mucus secretion. Indeed, activation of CHRM3 has been implicated in pulmonary artery contraction 48 . Muscarinic receptor antagonists are widely used to treat asthma 49 , chronic obstructive pulmonary disorder 50 and pulmonary hypertension 51 . The acetylcholinesterase inhibitor pyridostigmine is used to reduce pulmonary vascular resistance and remodeling, as well as right ventricle afterload 51 . Our results showed that CHRM3 has a relatively abundant expression compared to other muscarinic receptors across all type of arteries. This may suggest that CHRM3 plays important physiological roles in various arteries. Future studies should examine whether CHRM3 agonists or antagonists can be used to treat disorders of other organs.
Our results should be interpreted carefully because of several limitations. First, tissues came from older and younger patients, and receptor expression patterns may differ with age. Second, it is not always safe to assume that the relative abundance of a given receptor reflects its vasoconstrictive effects. For example, AVPR1A is highly expressed in systemic and pulmonary arteries, but it induces vasoconstriction only in the former 52 . Deoxyepinephrine induces vasoconstriction 10 times more strongly in systemic than pulmonary artery, suggesting that alpha-adrenoceptors may respond differently depending on the tissue. Third, receptors can behave differently under normal or pathological conditions. For example, ADRA2 triggers vasospasm and contraction of coronary artery in patients with coronary atherosclerosis but not in individuals with normal coronary artery 24 . Such differences may reflect differences in available levels of receptor ligand. For example, patients with hypertension or symptomatic atherosclerosis have elevated plasma levels of ET1 53 . Therefore, the distribution and effects of receptors in various vascular diseases need further study. Fourth, we found that patients varied substantially in expression of certain receptors in coronary artery (e.g. AVPR1A, ET1 and PTGIR). While this variability may make our results less generalizable, it implies that these receptors may be targets for precision medicine. Fifth, we were unable to sample all types of arteries because some types, such as cerebral artery, are difficult to sample; moreover, our protein array did not contain certain vasoactive substances synthesized by endothelial cells (e.g. NO, PGI2 and TXA2) because they are unstable. Lastly, we did not analyze receptor functionality in our study, which should be the subject of future work.
Despite these limitations, our results may provide an important reference for developing tissue-specific vasoactive drugs against hypotension, ischemic heart disease, and pulmonary hypertension.

Data availability
The original data on which this work is based are available from the corresponding author on reasonable request, depending on national laws and international regulations regarding sharing of confidential patient data.