Author + information
- Received October 19, 2018
- Revision received January 29, 2019
- Accepted February 18, 2019
- Published online May 20, 2019.
- Yong Dai, PhDa,b,c,∗,
- Xiao Chen, PhDa,b,c,∗,
- Xiaoxiao Song, MSca,b,c,∗,
- Xijun Chen, MSca,b,c,
- Wenrui Ma, MSca,b,c,
- Jibin Lin, PhDa,b,c,
- Hailang Wu, PhDa,b,c,
- Xiajun Hu, PhDa,b,c,
- Yanzhao Zhou, PhDa,b,c,
- Hongrong Zhang, PhDa,b,c,
- Yuhua Liao, PhDa,b,c,
- Zhihua Qiu, PhDa,b,c,∗∗ (, )@Qiuzhihua3 and
- Zihua Zhou, PhDa,b,c,∗ ()
- aDepartment of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- bInstitute of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- cKey Lab of Molecular Biological Targeted Therapies of the Ministry of Education, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- ↵∗Address for correspondence:
Dr. Zihua Zhou, Department of Cardiology, Institute of Cardiology, Key Lab of Molecular Biological Targeted Therapies of the Ministry of Education, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China.
- ↵∗∗Dr. Zhihua Qiu, Department of Cardiology, Institute of Cardiology, Key Lab of Molecular Biological Targeted Therapies of the Ministry of Education, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China.
Background Pulmonary arterial hypertension (PAH) is a chronic fatal disease. The treatment of PAH is less than ideal and the control is far from satisfactory worldwide. Vaccination provides a promising approach for treatment of PAH.
Objectives This study sought to find a vaccine against endothelin-1 (ET-1) receptor type A (ETAR) for treating PAH.
Methods The ETRQβ-002 vaccine was screened and the specific antibodies against epitope ETR-002 belonging to the second extracellular loop of ETAR (including the polyclonal and monoclonal antibody) were produced. The effect of the antibodies on Ca2+-dependent signal transduction events was investigated. In vivo, ETRQβ-002 vaccine was used to vaccinate monocrotaline (MCT)- and Sugen/hypoxia–induced pulmonary hypertension animals. The monoclonal antibody (mAb) against ETR-002 was also injected into the PAH animals. The effect of ETRQβ-002 vaccine on pulmonary hypertension and remodeling of pulmonary arterioles and right ventricle (RV) was carefully evaluated. Further, the possible immune-mediated damage was detected in normal vaccinated animals.
Results ETR-002 peptide has perfect immunogenicity and ETRQβ-002 vaccine could induce strong antibody production. In vitro, the anti–ETR-002 antibody bound to ETAR and inhibited Ca2+-dependent signal transduction events, including extracellular signal-regulated kinase phosphorylation and elevation of intracellular Ca2+ concentration induced by ET-1. In vivo, both ETRQβ-002 vaccine and the mAb significantly decreased the RV systolic pressure up to 20 mm Hg and 10 mm Hg in MCT-exposed rats and Sugen/hypoxia–exposed mice, respectively. Also, ETRQβ-002 vaccine/mAb obviously ameliorated pathological remodeling of pulmonary arterioles and hypertrophy of the RV in PAH animals. Additionally, no significant immune-mediated damage was detected in vaccinated animals.
Conclusions ETRQβ-002 vaccine/mAb attenuated remodeling of pulmonary arterioles and RV in MCT- and Sugen/hypoxia–induced PAH animals and decreased RV systolic pressure effectively through diminishing the pressure response and inhibiting signal transduction initiated by ET-1. ETRQβ-002 vaccine/mAb may provide a novel and promising method for PAH treatment.
Pulmonary arterial hypertension (PAH) is a fatal disease caused by irreversible functional and structural changes in the pulmonary vasculature, leading to a progressive increasing in pulmonary vascular resistance and subsequent right ventricular (RV) failure and ultimately death (1). A number of agents have recently been introduced for primary and secondary PAH, mainly including high-dose calcium-channel blockers, endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, and prostacyclin derivatives (1,2). Except for monotherapy, combination therapy (using 2 or more classes of drugs simultaneously) has been used successfully in the treatment of patients with moderate-to-severe pulmonary hypertension (3). Although these drugs have obviously improved the outcome of PAH patients, therapies against PAH remain palliative, and poor treatment compliance and quite high price have limited the application of these drugs, especially in developing countries. Most PAH patients still die from the disease, with a 5-year survival rate of 59% (2,4). To improve the poor treatment compliance and reduce the high cost, it is necessary to find a new therapy for PAH treatment.
Therapeutic vaccine is a new approach for neoplastic, cardiovascular, and cerebrovascular diseases (5). Vaccines can produce specific antibodies against related target molecules, may provide a new effective and safe way for PAH treatment. Based on virus-like particle (VLP) technology platform, our team invented an antihypertensive vaccine (ATRQβ-001) against angiotensin II receptor type 1 (AT1R) and a cholesterol-lowering vaccine that targets proprotein convertase subtilisin/kexin type 9 (PCSK9Qβ-003) (6,7). The ATRQβ-001 vaccine could significantly reduce the blood pressure and protect target organs of hypertensive animals (6), and even ameliorate atherosclerosis (8) and nephropathy in animal models (9). The PCSK9Qβ-003 vaccine obviously decreased plasma total cholesterol and regulated lipid homeostasis in both Balb/c mice and LDLR+/– mice (7).
Endothelin-1 (ET-1) receptor type A (ETAR) is a classical G protein–coupled receptor, which has a second extracellular loop (ECL2) that plays an important role in activation of the receptor (10). Activation of the endothelin system has been demonstrated in both plasma and lung tissue of PAH patients (11). ETAR is an ideal target for peptide vaccine design against PAH. Here, a linear epitope ETR-002, derived from the ECL2 of human ETAR, was designed. To improve the low immunogenicity of the ETR-002 peptide, it was conjugated with a VLP carrier protein, whose remarkable feature is the highly repetitive and ordered surface structure (12). In this study, the ETR-002 peptide was covalently conjugated with a Qβ bacteriophage VLP as a vaccine (designated ETRQβ-002). ETRQβ-002 vaccine and the monoclonal antibody (mAb) against ETR-002 was used to inject monocrotaline (MCT)-induced rats and Sugen/hypoxia–induced mice to evaluate the effect of lowering pulmonary hypertension. The effect of the ETRQβ-002 vaccine/mAb on attenuating remodeling of pulmonary arterioles and the RV was also investigated.
The study was carried out in strict accordance with the Guidelines for the Care and Use of Laboratory Animals (Science & Technology Department of Hubei Province, China, 2005). The protocol was approved by Animal Care and Use Committee of Hubei Province (Nos. 00009367 and 00021468). Animals were housed under specific pathogen-free conditions with 12-h light/dark cycle and 22 ± 2°C and 60 ± 5°C humidity. Sterile water and chow were available ad libitum. All efforts were made to minimize suffering and the procedure was performed under sodium pentobarbital anesthesia if necessary.
Vaccine preparation and screening
Epitope E-Y-R-G-E-Q-H-K-T-C (termed ETR-002), which belongs to ECL2 of ETAR, was selected and covalently conjugated with Qβ VLP (termed ETRQβ-002 vaccine) by cross-linker Sulfo-SMCC (Thermo, Rockford, Illinois) according to the manufacturer’s instruction. Male Sprague Dawley rats (Vital River, Beijing, China) aged 6 weeks were randomly divided into 2 groups: 1) a saline-treated control group (Con) (n = 6); and 2) a ETRQβ-002 vaccine group (ETRQβ-002) (n = 6). Rats were injected with ETRQβ-002 vaccine (200 μg/rat) on days 0 and 14. ETRQβ-002 vaccine specific peptide antibody titer was detected on days 0, 14, 35, 49, and 56 by enzyme-linked immunosorbent assay (ELISA).
The polyclonal antibody against ETR-002 peptide was from rabbits vaccinated with ETR-002-KLH conjugation vaccine. The antibody was purified using protein A affinity chromatography and epitope-linked gel affinity chromatography (GL Ltd., Shanghai, China). The mAb (A1C5) against ETR-002 peptide was also from GL Ltd. through hybridoma development and purification.
Cell culture and in vitro test
Primary pulmonary arterial smooth muscle cells (PASMCs) were isolated and cultured as previously described (13). PASMCs from passages 3 to 8 were used in this study. Chinese hamster ovary (CHO) (ATCC, Manassas, Virginia) cells were stably transfected with a pcDNA3.1 (+) vector expressing ETAR (CHO-ETAR), and cultured in Dulbecco's modified Eagle's medium–Ham’s F12 (Gibco, Carlsbad, California). CHO-ETAR cells from passages 4 to 10 were used in this study. The phosphorylation level of extracellular signal-related kinase 1/2 (ERK1/2) was detected by Western blotting. The cross-reactivity of ETR-002 antibody with ETBR was examined by immunofluorescent staining assay and ELISA. PASMCs proliferation was accessed by Cell Counting Kit-8 assay (Dojindo Molecular Technologies, Kumamoto Prefecture, Japan). The messenger RNA expression of vascular proliferation factors Skp2 and p27 and fibrosis factors including transforming growth factor β1, collagen I, fibronectin, and tenascin C was measured by quantitative real-time polymerase chain reaction (Applied Biosystems, Bio-Rad, Hercules, California). The primer sequence is shown in Online Table 1.
Monocrotaline-induced pulmonary hypertension
Male Sprague Dawley rats (Vital River) 4 weeks of age were randomly divided into 6 groups: 1) a saline-treated control group (Con) (n = 8); 2) an MCT-exposed group (MCT) (n = 18); 3) an MCT and Bosentan group (MCT+Bosentan) (n = 16); 4) an MCT and ETRQβ-002 vaccine group (MCT+ETRQβ-002) (n = 20); 5) an MCT and ETRQβ-002 monoclonal antibody A1C5 group (MCT+ ETRQβ-002[A1C5]) (n = 14); and 6) an MCT and Qβ VLP group (MCT+VLP) (n = 10). The ETRQβ-002 vaccine and VLP groups were immunized subcutaneously on days 0, 14, and 28, with 400 μg ETRQβ-002 vaccine or Qβ VLP formulated in aluminum hydroxide gel, respectively. On day 29, all rats except the control group received a single subcutaneous injection of MCT (60 mg/kg [cayman, Ann Arbor, Michigan]). The Bosentan group was administered with peroral bosentan (100 mg/kg/day; Actelion, Allschwil, Switzerland) from days 29 to 60. Rats in the A1C5 group experienced tail vein injection of A1C5 mAb (350 μg) once a week from day 29 to day 60. ETRQβ-002 vaccine specific peptide antibody titer was detected on days 0, 10, 21, 41, and 58 by ELISA. The blood pressure of animals was measured using the tail-cuff method (BP-2010A, Softron, Tokyo, Japan). All rats were sacrificed on day 60. The process map is shown in Online Figure 1.
Sugen/hypoxia–induced pulmonary hypertension
Male C57BL/6 mice (Vital River) 3 weeks of age were randomly divided into 6 groups: 1) a saline-treated control group (Con) (n = 8); 2) a hypoxia- and Su5416-exposed group (SuHx) (n = 17); 3) an SuHx+ETRQβ-002(a) group (n = 16); 4) an SuHx+ETRQβ-002(s) group (n = 13); 5) an SuHx+ETRQβ-002(A1C5) group (n = 13); and 6) an SuHx+VLP group (n = 11). All mice except the control group experienced a weekly subcutaneous injection of Su5416 (20 mg/kg; TargetMol, Boston, Massachusetts) and were exposed to chronic normobaric hypoxia in a hypoxic chamber flushed with a mixture of N2 and room air (10% O2). The mice in the SuHx+ETRQβ-002(a) group were immunized with ETRQβ-002 vaccine (100 μg/mouse) on days 0, 14, and 28 before being exposed to chronic hypoxia. However, the mice in the SuHx+ETRQβ-002(s) group were immunized with ETRQβ-002 vaccine (100 μg/mouse) on days 28, 42, and 56 when initially exposed to chronic hypoxia. The mice in the SuHx+ETRQβ-002(A1C5) group were treated weekly with A1C5 mAb (250 μg) for 7 weeks. After being exposed to chronic normobaric hypoxia for 3 weeks, the animals were followed by re-exposure to normoxia (21% O2) for 4 additional weeks. ETRQβ-002 vaccine specific peptide antibody titer in group 3 was detected on days 14, 28, 42, 56, 84, and 91, while the titer in group 4 was measured on days 42, 70, 84, and 91 by ELISA. All mice were sacrificed on day 91. The process map is shown in Online Figure 2.
Another 2 groups of normal SD rats aged 6 weeks were designed to evaluate the safety of ETRQβ-002 vaccine: 1) a saline-treated control group (Con) (n = 4); and 2) a ETRQβ-002 vaccine group (ETRQβ-002) (n = 8). The rats were immunized subcutaneously on days 0, 14, and 28 with ETRQβ-002 vaccine (400 μg/rat). ETRQβ-002 vaccine specific peptide antibody titer was detected on days 0, 14, 35, 49 56 and 74 by ELISA. The sera content including alanine aminotransferase, aspartate aminotransferase, albumin, serum creatinine, and blood urea nitrogen was measured using biochemical kits (Jiancheng, Nanjing, China).
Animals were anesthetized with 1% pentobarbital (4 ml/kg). RV systolic pressure (RVSP) was measured with a polygraph system (LabChart 7.3.7; AD Instruments, New South Wales, Australia) using polyethylene catheters (Scientific Commodities Inc., Lake Havasu City, Arizona).
Cardiomyocyte diameter and the fibrotic area (%) of the heart were determined through hematoxylin and eosin (HE) and Masson’s trichrome staining. Pulmonary arterioles remodeling was calculated by HE and α-smooth muscle actin (anti–α-smooth muscle actin; Sigma-Aldrich, St. Louis, Missouri). The percentage of medial wall thickness (MT%) and wall area (WA%) of vessels (diameter <200 μm) was measured according to the previous method (14). All of the analyses were performed in a blinded manner. Parts of fresh renal cortex were immediately fixed in 0.25% glutaraldehyde for transmission electron microscopy (TEM).
Data are expressed as mean ± SEM. Student's t-test (for comparison between 2 groups) and a 1-way analysis of variance using Bonferroni’s method (for comparison of more than 2 groups) were used for the statistical analyses. The differences in some observations (cross-sectional area of cardiomyocytes, MT%, and WA%) were analyzed using a mixed-effects model. The variables with heterogeneity of variance were performed using nonparametric testing (Kruskal-Wallis test). The calculation was carried out using the statistical program SPSS version 19.0 (IBM Corporation, Armonk, New York). A p value <0.05 was accepted as significant.
Target peptides screening and preliminary functional testing
The ETR-002 peptide derived from the ECL2 of human ETAR was first screened after peptides antibody production verification. Epitope ETR-002 was covalently conjugated with Qβ VLP (ETRQβ-002 vaccine), and the sodium dodecyl sulfate polyacrylamide gel electrophoresis manifested that 1 monomer of VLP coupling 1 to 4 ETR-002 epitopes (2 ETR-002 epitope per 1 VLP monomer averagely) (Figure 1A). Then, normal Sprague Dawley rats were vaccinated with ETRQβ-002 vaccine. ELISA testing confirmed that the anti–ETR-002 antibody titer was 1:50,000 to 1:80,000 in the ETRQβ-002 group (Figure 1B), which indicated the perfect immunogenicity of the ETR-002 peptide.
Second, the binding capacity of the ETR-002 peptide with ETAR was determined. Results of the immunofluorescence assays and Western blotting demonstrated that the polyclonal anti–ETR-002 antibody specifically bound to ETAR in rat PASMCs and CHO-ETAR (Online Figure 3). When the antibody was neutralized by the ETR-002 peptide, the binding with ETAR was significantly blocked (Online Figure 3). Further, our results showed that ET-1–induced ERK1/2 phosphorylation in the anti–ETR-002 antibody preincubated group was obviously lower than that of the control antibody or neutralized ETAR group in CHO-ETAR cells (65.4% decrease, p < 0.001) (Figure 1C). Also, the anti–ETR-002 antibody obviously inhibited the increase of intracellular Ca2+ concentration ([Ca2+]cyt) induced by ET-1 (45.4% decrease, p < 0.0001) (Figure 1D), similar to ETAR inhibitor BQ123. No agonistic or antagonistic role was detected when stimulating the cells using the anti–ETR-002 antibody only.
To facilitate animal experiments, the specific mAb (A1C5) against ETR-002 was also produced. The immunofluorescence assays demonstrated that A1C5 specifically bound to ETAR in rat PASMCs and CHO-ETAR (Online Figure 4). And, A1C5 has no cross-reactivity with ETBR (Online Figure 4). Further test confirmed that A1C5 not only inhibited ET-1–induced ERK1/2 phosphorylation in CHO-ETAR cells, but also decreased the expression of proliferation and fibrosis factors in rat PASMCs (Online Figure 5).
ETRQβ-002 vaccine/mAb ameliorated MCT-induced PAH and RV remodeling
ETRQβ-002 vaccine/mAb were injected into MCT-induced rats. ELISA confirmed that the anti–ETR-002 antibody titer was 1:100,000 to 1:150,000 in the ETRQβ-002 group (Online Figure 6). All the groups except the control group had significantly reduced body weight (Online Table 2). The blood pressure and heart rate had no meaningful difference (Online Table 2). Further, the survival rate had no obvious difference among groups except for the control group (Online Table 3).
To evaluate pulmonary arterial pressure accurately, right heart catheterization was carried out. The results showed that RVSP of all groups was significantly increased compared with the control group (Figures 2A and 2E). However, RVSP in the ETRQβ-002, A1C5, and Bos groups was decreased compared with the MCT group (ETRQβ-002, 34.7 ± 2.9 mm Hg, p < 0.001; A1C5, 39.6 ± 2.7 mm Hg, p = 0.015; Bosentan, 39.2 ± 1.8 mm Hg, p = 0.02; MCT, 53.3 ± 2.8 mm Hg) (Figures 2A and 2E). No obvious difference was found among the Bos, ETRQβ-002, and A1C5 groups. Representative images of PAH in the 6 groups are shown in Figure 2E.
To determine the pathological changes of the RV, the weight ratio of RV/(left ventricle + interventricular septum) and cardiomyocytes CSA were measured. Results showed that the weight ratio of RV/(left ventricle + interventricular septum) in the MCT group was more increased than in the ETRQβ-002, A1C5, and Bos groups (ETRQβ-002, 0.489 ± 0.037, p = 0.041; A1C5, 0.463 ± 0.056, p = 0.021; Bos, 0.485 ± 0.021, p = 0.024; MCT, 0.642 ± 0.042) (Figure 2B). Compared with those in the ETRQβ-002, A1C5, or Bos groups, cardiomyocytes in the MCT group were obviously hypertrophied. The CSA of RV myocardium in the ETRQβ-002, A1C5, and Bos groups was significantly decreased compared with the MCT group, but no difference was observed among the ETRQβ-002, A1C5, and Bos groups (ETRQβ-002, 291.8 ± 23.1 μm2, p < 0.001; A1C5, 320.3 ± 24.4 μm2, p = 0.012; Bos, 313.4 ± 22.6 μm2, p = 0.035; MCT, 405.9 ± 22.8 μm2) (Figures 2C and 2F). Furthermore, Masson’s trichrome staining of RV showed that ETRQβ-002 vaccine and A1C5 significantly decreased the fibrosis induced by MCT exposure, similar to the Bos group. In the MCT group, the fibrotic area of RV was 21.8 ± 2.5%, whereas it was only 8.1 ± 1.1% in ETRQβ-002 (p = 0.039), 8.3 ± 0.9% in A1C5 (p = 0.03), and 8.0 ± 0.7% in Bos (p = 0.05) groups, as shown in Figures 2D and 2G.
ETRQβ-002 vaccine/mAb prevented MCT-induced pathological remodeling of pulmonary arterioles
Human PAH is characterized by significant medial hyperplasia or hypertrophy in small pulmonary arterioles. Representative images of the pulmonary vascular remodeling (<50 μm, 50 to 100 μm, and 100 to 200 μm) in the 6 different groups are shown in Figure 3A. As illustrated in Figure 3B, compared with the control group, the MT% and WA% of pulmonary arterioles were remarkably increased in rats treated with MCT, but ETRQβ-002 vaccine, A1C5, and Bosentan were able to reverse the increase in values of WA% and MT% induced by MCT (Figure 3). Furthermore, the full muscularization of pulmonary arterioles was attenuated in the ETRQβ-002 vaccine, A1C5, and Bosentan groups compared with the MCT group (ETRQβ-002, 19.0 ± 5.6%, p = 0.058; A1C5, 9.2 ± 3.1%, p < 0.001; Bos, 19.0 ± 5.9%, p = 0.057; MCT, 38.3 ± 3.3%) (Figure 3C).
ETRQβ-002 vaccine/mAb alleviated sugen/hypoxia–induced PAH and RV remodeling
ETRQβ-002 vaccine/mAb were injected into Sugen/hypoxia–induced PAH mice. ELISA confirmed that the anti–ETR-002 antibody titer was 1:1,000,000 to 1:1,500,000 and 1:500,000 to 1:1,000,000 in the SuHx+ETRQβ-002(a) and SuHx+ETRQβ-002(s) groups, respectively (Online Figure 7). All groups had no significantly reduced body weight (Online Table 4). The blood pressure had no meaningful difference (Online Table 4). In all groups, no mice died.
The results of right heart catheterization showed, RVSP of all groups was significantly increased except the control group (Figures 4A and 4E). However, RVSP in the ETRQβ-002(a), ETRQβ-002(s), and A1C5 groups was decreased compared with the SuHx group (ETRQβ-002(a), 26.9 ± 1.2 mm Hg, p < 0.001; ETRQβ-002(s), 25.8 ± 1.4 mm Hg, p < 0.0001; A1C5, 27.4 ± 1.6 mm Hg, p = 0.009; SuHx, 35.7 ± 2.0 mm Hg) (Figure 4A). No obvious difference was found among the ETRQβ-002(a), ETRQβ-002(s), and A1C5 groups, which indicated the preventive and curable effect of ETRQβ-002 vaccine on hypoxia-induced PAH. Representative images of PAH in the 6 groups are shown in Figure 4E.
To determine the pathological changes of RV, the weight ratio of RV/(left ventricle + interventricular septum) and cardiomyocytes CSA were determined. Results showed that the weight ratio of RV/(left ventricle + interventricular septum) in the SuHx group was more increased than the SuHx+ETRQβ-002(a), SuHx+ETRQβ-002(s), and A1C5 groups (ETRQβ-002(a), 0.240 ± 0.006, p = 0.016; ETRQβ-002(s), 0.233 ± 0.008, p = 0.004; A1C5, 0.242 ± 0.005, p = 0.03; SuHx, 0.276 ± 0.004) (Figure 4B). Compared with those in the SuHx+ETRQβ-002(a), SuHx+ETRQβ-002(s), and A1C5 groups, cardiomyocytes in the SuHx group were obviously hypertrophied. The CSA of RV myocardium in the SuHx+ETRQβ-002(a), SuHx+ETRQβ-002(s), and A1C5 groups was significantly decreased compared with the SuHx group, but no difference was observed among the SuHx+ETRQβ-002(a), SuHx+ETRQβ-002(s), and A1C5 groups (ETRQβ-002(a), 233.3 ± 11.6 μm2, p = 0.005; ETRQβ-002(s), 235.3 ± 13.4 μm2, p = 0.003; A1C5, 231.0 ± 12.9 μm2, p = 0.004; SuHx, 288.6 ± 13.6 μm2) (Figures 4C and 4F). However, no obvious difference was observed among 6 groups in RV fibrosis because of unobvious myocardium fibrosis (Figures 4D and 4G).
ETRQβ-002 vaccine/mAb decreased sugen/hypoxia–induced pathological remodeling of pulmonary arterioles
Representative images of the pulmonary vascular remodeling (100× and 400×, HE and SMA) in the 6 groups are shown in Figure 5A. As illustrated in Figure 5B, compared with the control group, the MT% and WA% of pulmonary arterioles were remarkably increased in mice treated with hypoxia and SU5416, but the SuHx+ETRQβ-002(a), SuHx+ETRQβ-002(s), and A1C5 groups were able to reverse the increase in values of WA% and MT% (Figure 5). Furthermore, the partial muscularization of pulmonary arterioles was reversed in the SuHx+ETRQβ-002(a), SuHx+ETRQβ-002(s), and A1C5 groups compared with the SuHx group (ETRQβ-002(a), 38.7 ± 1.4%, p = 0.025; ETRQβ-002(s), 38.1 ± 2.7%, p = 0.032; A1C5, 36.8 ± 5.3%, p = 0.018; SuHx, 57.4 ± 3.3%) (Figure 5C).
No immune-mediated injury was observed in vaccinated animals
For safety consideration, normal Sprague Dawley rats were immunized with ETRQβ-002 vaccine. No evidence of skin damage at the site of subcutaneous injection was noted in the vaccine-treated animals. Blood pressure, heart rate, and body weight had no difference between the control and ETRQβ-002 groups (Online Table 5). The anti–ETR-002 antibody titer could reach to 1:600,000 in the ETRQβ-002 group after the third immunization, and then decreased gradually (Figure 6A). The hepatic and renal function (including ALT, AST, Alb, Scr, and BUN) had no difference between the control group and the ETRQβ-002 vaccine group (Figures 6B and 6C). Light microscopy showed that no significant immune-mediated damage was detected in critical organs (heart, liver, spleen, lung, and kidney) of the vaccinated animals (Figure 6D). TEM showed that no obvious injury or immune complex was observed in kidney between the 2 groups (Figure 6E). In addition, the pathological results of our experimental animals (MCT-treated and SuHx-induced PAH animals) also showed that no significant immune damage was detected in lung or off-target organs from the vaccinated animals (Online Figures 8 and 9).
This study demonstrates for the first time that immunotherapy against ETAR (ETRQβ-002 vaccine/mAb) effectively prevented and treated pulmonary arterioles remodeling, the elevation of RV pressure and RV hypertrophy in MCT-treated and Sugen/hypoxia–induced PAH animals (Central Illustration). The duration of the ETRQβ-002 antibody was longer than general chemical drugs. Compared with chemical drugs, immunotherapy without taking medication every day may have potentially superior advantages, including the ability to steadily lower PAH, greatly improve patient compliance, and lower the possible cost. The ETRQβ-002 vaccine/mAb may provide a new and effective therapeutic approach for PAH, a devastating disease with no cure.
Autoantibodies against ETAR have been reported in autoimmune diseases such as systemic sclerosis (15,16), cystic fibrosis (17), and Sjogren’s syndrome (18). Although the antibodies against ETAR discovered thus far show agonistic effects, we cannot rule out the possible existence of blocking antibodies to ETAR. Researchers have attempted to identify a G protein–coupled receptor antibody that can act as an agonist or an antagonist. A classic example is the detection of the autoantibodies in patients with Graves’ disease that display various functions, including activation, inhibition, or antigen-antibody binding without signal transduction activity (19–21). In autoimmune diseases, preeclampsia, and malignant hypertension (22,23), classical agonistic autoantibodies against AT1R were found, leading to elevated blood pressure and impaired target organs. To explore the possible existence of blocking antibodies to AT1R, we developed the peptide vaccine against AT1R (ATRQβ-001) successfully, which could significantly reduce the blood pressure and protect target organs (6).
Based on the success of AT1R vaccine and the structure characteristics of ETAR, we further developed the ETRQβ-002 vaccine against ETAR. The target peptide ETR-002 epitope was from ECL2 of ETAR, which plays a key role in ligand-induced ETAR activation (10,24,25). ELISA testing confirmed the perfect immunogenicity of ETR-002 peptide. In vitro testing showed that the anti–ETR-002 antibody specifically bound to ETAR in rat PASMCs and CHO-ETAR but not to ETBR. And, the anti–ETR-002 antibody not only inhibited phosphorylation of extracellular signal-regulated kinase and elevation of [Ca2+]cyt, but also decreased the expression of proliferation and fibrosis factors in rat PASMCs induced by ET-1.
MCT-induced PAH is a classical animal model, involving rapid medial remodeling in pulmonary arterioles, which leads to increased pulmonary artery pressure, RV dysfunction, and death in animals a few weeks after insult (26). The ETRQβ-002 vaccine was produced and injected into MCT-induced rats to evaluate the treatment effect of PAH. Results showed that the ETRQβ-002 vaccine induced prominent antibody production, significantly decreased RVSP up to 25 mm Hg, and prevented RV hypertrophy and fibrosis of MCT-induced PAH rats. Also, the ETRQβ-002 vaccine obviously inhibited pathological remodeling of pulmonary arterioles. To further confirm the effect of the ETRQβ-002 vaccine, it was injected into Sugen/hypoxia–induced PAH mice (27). As the results showed, the ETRQβ-002 vaccine significantly decreased Sugen/hypoxia–induced PAH. The ETRQβ-002 vaccine obviously attenuated Sugen/hypoxia–induced pathological RV hypertrophy and remodeling of pulmonary arterioles.
To evaluate the curable effect of the ETRQβ-002 vaccine on PAH, the animals were synchronously immunized with the vaccine when exposed to chronic Sugen/hypoxia. Further, mAb (A1C5) against ETR-002 was also produced and was injected into established PAH animals. Results demonstrated that the SuHx+ETRQβ-002(s) and A1C5 groups both experienced significantly decreased PAH, RV hypertrophy, and pathological remodeling of pulmonary arterioles, similar to the SuHx+ETRQβ-002(a) and Bosentan groups.
The safety of the ETRQβ-002 vaccine is an important concern. As the target peptide of the vaccine was only 10 amino acids in length, it was smaller than the minimal size of a T cell epitope and therefore should not be able to induce a T cell response (28). Peptide-VLP hypertension vaccine (CYT-006-AngQβ) showed good safety in clinical trials (29). Similar peptide-VLP hypertension vaccine (ATRQβ-001 and PCSK9Qβ-003) invented by our group also showed no obvious inflammation lesions in series of studies (6–9), which indicates that this kind of vaccine is safe for the immunized animals. Further, the immune response mechanism of the peptide-VLP vaccine has been elucidated in our previous work, which indicated that the peptide-VLP vaccine has satisfactory safety (30). Similarly, ETRQβ-002 vaccination did not cause immune injury in important organs and tissues of rats. The hepatic and renal function (including ALT, AST, Alb, Scr, and BUN) had no difference between the control group and the ETRQβ-002 vaccine group. TEM showed that no obvious injury was observed in kidney between the 2 groups. All of these indicated the safety of ETRQβ-002 vaccine. Nevertheless, the potential toxicity of ETRQβ-002 vaccine needs to be further investigated.
The study was performed with restricted observation (2 and 3 months after vaccine injection) that may have limited power and limit the advantage of improving survival rate in the ETRQβ-002 vaccine. Therefore, future studies will focus on long-term efficacy and safety endpoints of the ETRQβ-002 vaccine.
The ETRQβ-002 vaccine antibody inhibited Ca2+-dependent signal transduction events, including extracellular signal-regulated kinase phosphorylation and elevation of intracellular Ca2+ concentration ([Ca2+]cyt) induced by ET-1. However, the exact mechanism of ETRQβ-002 vaccine antibody on ETAR needs future investigation, especially the G protein signal pathway.
Immunotherapy against ETAR (ETRQβ-002 vaccine/mAb) is a novel therapeutic method for PAH, which may become a promising treatment method for PAH in humans.
COMPETENCY IN MEDICAL KNOWLEDGE: PAH is a fatal disease caused by irreversible functional and structural changes in the pulmonary vascular, leading to progressively increasing pulmonary vascular resistance and RV failure. In an animal model of PAH, ETRQβ-002 vaccine/mAb attenuated remodeling of pulmonary arterioles decreased RVSP.
TRANSLATIONAL OUTLOOK: Further studies are needed to determine whether this therapy can be translated to patients with PAH.
↵∗ Drs. Yong Dai, Xiao Chen, and Xiaoxiao Song contributed equally to this work as joint first authors.
This work was supported by National Natural Science Funds of China (No. 81470494, No. 81770366, No. 81400314, No. 81270331) and the Major Research Plan of the National Natural Science Foundation of China (No. 91439207). The authors have reported that they have no relationships relevant to the contents of this paper to disclose.
Listen to this manuscript's audio summary by Editor-in-Chief Dr. Valentin Fuster on JACC.org.
- Abbreviations and Acronyms
- angiotensin II receptor type 1
- intracellular Ca2+concentration
- Chinese hamster ovary
- second extracellular loop
- enzyme-linked immunosorbent assay
- extracellular signal-related kinase 1/2
- endothelin-1 receptor type A
- hematoxylin and eosin
- monoclonal antibody
- medial wall thickness
- pulmonary arterial hypertension
- primary pulmonary arterial smooth muscle cell
- right ventricular
- right ventricular systolic pressure
- transmission electron microscopy
- virus-like particle
- wall area
- Received October 19, 2018.
- Revision received January 29, 2019.
- Accepted February 18, 2019.
- 2019 American College of Cardiology Foundation
- Galiè N.,
- Humbert M.,
- Vachiery J.L.,
- et al.
- Humbert M.,
- Lau E.M.,
- Montani D.,
- Jais X.,
- Sitbon O.,
- Simonneau G.
- Boucly A.,
- Weatherald J.,
- Savale L.,
- et al.
- Oparil S.,
- Schmieder R.E.
- Zhou Y.,
- Wang S.,
- Qiu Z.,
- et al.
- Ding D.,
- Du Y.,
- Qiu Z.,
- et al.
- Davenport A.P.,
- Hyndman K.A.,
- Dhaun N.,
- et al.
- Zhou L.,
- Chen Z.,
- Vanderslice P.,
- et al.
- Riemekasten G.,
- Philippe A.,
- Nather M.,
- et al.
- Budding K.,
- van de Graaf E.A.,
- Hoefnagel T.,
- et al.
- Sugiura T.,
- Yamanaka S.,
- Takeuchi H.,
- Morimoto N.,
- Kamiokan M.,
- Matsumura Y.
- Abe K.,
- Toban M.,
- Alzoubi A.,
- et al.
- Hu X.,
- Deng Y.,
- Chen X.,
- et al.