HHS Public Access Author manuscript Author Manuscript
Hypertension. Author manuscript; available in PMC 2016 October 01. Published in final edited form as: Hypertension. 2015 October ; 66(4): 830–835. doi:10.1161/HYPERTENSIONAHA.115.05428.
AT2R AUTOANTIBODIES BLOCK ANGIOTENSIN II AND AT1R AUTOANTIBODY-INDUCED VASOCONSTRICTION
Author Manuscript
Campbell Liles*, Hongliang Li*, Vineet Veitla, Jonathan T. Liles, Taylor A. Murphy, Madeleine W. Cunningham, Xichun Yu†, and David C. Kem† Endocrinology & Heart Rhythm Institute, Department of Medicine (C.L., H. L., V.V., J.T.L., X.Y., D.C.K.); and Department of Microbiology and Immunology (M.W.C.), University of Oklahoma Health Sciences Center and Veterans Affairs Medical Center, Oklahoma City, OK
Abstract
Author Manuscript
Activating autoantibodies to the angiotensin type 1 receptor (AT1R) are associated with hypertensive disorders. The angiotensin type 2 receptor (AT2R) is known to counter-regulate the actions of AT1R. We investigated whether AT2R autoantibodies produced in immunized rabbits will activate AT2R and suppress the vasopressor responses to angiotensin II (Ang II) and AT1Ractivating autoantibodies. Five rabbits immunized with a peptide corresponding to the second extracellular loop of AT2R developed high AT2R antibody titers. Rabbit anti-AT2R sera failed to directly dilate isolated rat cremaster arterioles; however, when co-perfused with Ang II or AT1Ractivating autoantibodies, the anti-AT2R sera significantly inhibited their contractile effects. Rabbit anti-AT2R sera recognized a predominant sequence near the N-terminus of the AT2R second extracellular loop. A decoy peptide based on this sequence effectively reversed the opposing effect of the anti-AT2R sera on Ang II-induced contraction of rat cremaster arterioles. A similar blockade of the anti-AT2R sera effect was observed with the AT2R antagonist PD 123319 and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Rabbit antiAT2R sera reacted specifically with AT2R. No cross-reactivity with AT1R was observed. Blood pressure did not change in immunized animals. However, the pressor responses to incremental Ang II infusions were blunted in immunized animals. Thirteen subjects with primary aldosteronism demonstrated increased AT2R autoantibody levels compared to normal controls. In conclusion, AT2R autoantibodies produced in immunized rabbits have the ability to activate AT2R and counteract the AT1R-mediated vasoconstriction. These autoantibodies provide useful and selective tools for study of their roles in blood pressure regulation and possible therapeutic intervention.
Author Manuscript
Correspondence to: David C. Kem, MD, Endocrinology and Heart Rhythm Institute, University of Oklahoma Health Sciences Center, TCH 6E103, 1200 Everett Dr, Oklahoma City, OK 73104. Tel: 405-271-5896, Fax: 405-271-7455, [email protected]. *C. Liles and H. Li contributed equally to this work. †X. Yu and D. Kem served as co-senior authors. Presented in part at the 40th International Aldosterone Conference, June 2014, Chicago, IL. Disclosures: None.
Liles et al.
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Author Manuscript
Keywords activating autoantibodies; angiotensin II type 1 receptor; angiotensin II type 2 receptor; hypertension; vasoconstriction; rabbit
Introduction
Author Manuscript
The renin angiotensin system plays a key role in the regulation of blood pressure and cardiovascular function. Angiotensin II (Ang II) binds to two major subtypes of receptor, the angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R). The majority of the actions of Ang II are mediated by AT1R; however, recent evidence suggests that AT2R opposes AT1R by promoting vasodilatation rather than vasoconstriction.1–4 There has been an increasing awareness of the presence of autoantibodies directed toward the extracellular loops of several G protein-coupled receptors and these often express remarkable specificity for a given receptor.5, 6 Autoantibodies that activate the AT1R have been described and appear to be highly selective for their target domain on the second extracellular loop (ECL2) of AT1R.7, 8 These autoantibodies were initially identified in a limited number of subjects, but this list has expanded rapidly in the last few years in several hypertensive-related conditions such as preeclampsia,9 malignant and refractory hypertension,10, 11 renal allograft rejection,12 and more recently with primary aldosteronism.13–15 There is no currently published information as to whether activating autoantibodies to the AT2R exist; and whether they might alter the complex role of Ang II in vascular homeostasis.
Author Manuscript
We have recently developed a rabbit autoimmune model of AT1R-activating autoantibody (AT1R-AAb)-mediated hypertension.16 These animals were immunized with a peptide containing the known functional AT1R-AAb epitope in the ECL2 of AT1R. After immunization, all animals developed elevated blood pressure. The anti-AT1R sera induced significant AT1R activation and vasoconstriction in vitro. In the present study, we describe our successful efforts to develop AT2R-activating autoantibodies (AT2R-AAbs) in immunized rabbits and to demonstrate their potential impact on Ang II-mediated hypertension and AT1R-AAb-mediated vasoconstriction. We have discovered AT1R-AAbs and AT2R-AAbs maintain a high degree of specificity for their target receptors and produce opposing effects, thus providing a potentially valuable tool for studies of their roles in hypertension.
Methods Author Manuscript
This study protocol was approved by the Institutional Animal Care and Use Committee of the Oklahoma City Veterans Affairs Medical Center and Oklahoma University Health Sciences Center, and conforms to international standards for animal safety and comfort. Animal Model Five New Zealand white rabbits (2.5–3 kg) were fed on standard rabbit chow and were immunized with 1 mg of a synthetic peptide corresponding to the AT2R ECL2
Hypertension. Author manuscript; available in PMC 2016 October 01.
Liles et al.
Page 3
Author Manuscript
(YFRDVRTIEYLGVNACIMAFPPEKYAQWS) (GenScript, Piscataway, NJ) in 0.5 ml of complete Freund’s adjuvant. The animals were boosted with the same peptide plus incomplete Freund’s adjuvant (1 mg/0.5 ml) at 2 and 4 weeks. Arterial blood pressure was measured at preimmune and 6 weeks after immunization. Under anesthesia (ketamine/ xylazine 35 mg/5 mg/kg), the rabbit central ear artery was cannulated and the catheter connected to a pressure transducer. To determine the acute effect of Ang II on blood pressure before and after immunization, increasing dosages of Ang II (10, 100, and 500 ng/kg) were injected intravenously at 5-minute intervals using an infusion pump, and the blood pressure response at each dose was recorded. Each rabbit served as its own control. Preimmune and postimmune sera were obtained from all animals for ELISA and functional assays of the expected antibodies generated during immunization. AT1R-AAbs were obtained from the rabbits previously immunized with the AT1R ECL2 peptide.16
Author Manuscript
Human Studies Sera were available for testing autoantibodies from a group of 13 hypertensive subjects who previously had been diagnosed with primary aldosteronism using an IRB-approved protocol. These subjects for the most part had not had lateralization studies since their hypertension was generally under good control with medications, and the patients had declined to consider surgery irrespective of the testing outcome or there were evident contraindications from the physician’s viewpoint. Each fulfilled criteria for establishing the diagnosis using one of the established methods for confirmation of an abnormal plasma aldosterone/plasma renin activity (PA/PRA) ratio of over 30.17 The two defining tests used included a captopril suppression study (n=9) and saline infusion study (n=1), while others had a PA/PRA ratio of >50 and an absolute PA value over 25 ng/dl (n=3) negating the need for further diagnostic tests.
Author Manuscript
ELISA Antibodies produced in the sera were detected by ELISA. Briefly, microtiter plates were coated with the AT2R ECL2 peptide at 10 μg/ml. To determine antibody titer, rabbit sera were diluted 1:10000 and thereafter serially diluted 2-fold. Goat anti-rabbit IgG conjugated with alkaline phosphatase and its substrate para-nitrophenyl-phosphate 104 were used to detect antibody binding. Titers were determined as the highest dilution with an optical density (OD) value of 0.10 at 60 minutes. Human sera were diluted 1:100, and goat antihuman IgG conjugated with alkaline phosphatase was used for the secondary antibody. Their OD values were read at 60 min. Epitope Mapping
Author Manuscript
Epitope mapping for rabbit AT2R autoantibodies was performed with multipin peptides (constructed in Dr. Judith James’s laboratory, Oklahoma Medical Research Foundation), which comprised a set of 22 octapeptides, overlapping by 7 amino acids spanning the ECL2 of AT2R. Rabbit anti-AT2R sera (1:200) was added to the wells of 96-well plate along with the multipin peptides and incubated for 2 hours at room temperature. The multipin peptides were washed with PBS-Tween and incubated with anti-rabbit IgG antibody conjugated with horseradish peroxidase. Reactivity of rabbit anti-AT2R sera to the pins was measured by
Hypertension. Author manuscript; available in PMC 2016 October 01.
Liles et al.
Page 4
Author Manuscript
adding tetramethyl benzidine substrate solution in the developing plate. The OD values were read at 405 nm. Isolated Arteriole Assay
Author Manuscript
Autoantibody contractile activity was assayed using an isolated rat cremaster arteriole assay as previously described.16, 18 Briefly, arteriolar segments were microdissected from the cremaster muscle of anesthetized rats and cannulated with glass micropipettes. After equilibration and development of steady-state myogenic tone, the arterioles were perifused with rabbit sera. Dosage responses for Ang II (10−10 ~ 10−7 M) and rabbit anti-AT1R sera (1:500 - 1:50) in the absence and presence of rabbit anti-AT2R sera (1:50) were performed to examine the impact of AT2R-AAbs on Ang II and AT1R-AAb-induced contractile response. The AT2R antagonist PD 123319 (10 nM) was used to block AT2R-mediated effect. The potent soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ, 1 μM) was also tested for neutralization of the vasodilatory effect of AT2R-AAbs. Measurements of vessel diameter were made using a video edge detector. Data are reported as percentage of baseline diameter to normalize each response. Statistical Analysis Data are presented as mean ± SD. Comparison between two groups and multiple group comparisons were performed using the nonparametric Mann-Whitney test and KruskalWallis test followed by Dunn’s multiple comparison test, respectively. Statistical significance was set at P
Hypertension. Author manuscript; available in PMC 2016 October 01. Published in final edited form as: Hypertension. 2015 October ; 66(4): 830–835. doi:10.1161/HYPERTENSIONAHA.115.05428.
AT2R AUTOANTIBODIES BLOCK ANGIOTENSIN II AND AT1R AUTOANTIBODY-INDUCED VASOCONSTRICTION
Author Manuscript
Campbell Liles*, Hongliang Li*, Vineet Veitla, Jonathan T. Liles, Taylor A. Murphy, Madeleine W. Cunningham, Xichun Yu†, and David C. Kem† Endocrinology & Heart Rhythm Institute, Department of Medicine (C.L., H. L., V.V., J.T.L., X.Y., D.C.K.); and Department of Microbiology and Immunology (M.W.C.), University of Oklahoma Health Sciences Center and Veterans Affairs Medical Center, Oklahoma City, OK
Abstract
Author Manuscript
Activating autoantibodies to the angiotensin type 1 receptor (AT1R) are associated with hypertensive disorders. The angiotensin type 2 receptor (AT2R) is known to counter-regulate the actions of AT1R. We investigated whether AT2R autoantibodies produced in immunized rabbits will activate AT2R and suppress the vasopressor responses to angiotensin II (Ang II) and AT1Ractivating autoantibodies. Five rabbits immunized with a peptide corresponding to the second extracellular loop of AT2R developed high AT2R antibody titers. Rabbit anti-AT2R sera failed to directly dilate isolated rat cremaster arterioles; however, when co-perfused with Ang II or AT1Ractivating autoantibodies, the anti-AT2R sera significantly inhibited their contractile effects. Rabbit anti-AT2R sera recognized a predominant sequence near the N-terminus of the AT2R second extracellular loop. A decoy peptide based on this sequence effectively reversed the opposing effect of the anti-AT2R sera on Ang II-induced contraction of rat cremaster arterioles. A similar blockade of the anti-AT2R sera effect was observed with the AT2R antagonist PD 123319 and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Rabbit antiAT2R sera reacted specifically with AT2R. No cross-reactivity with AT1R was observed. Blood pressure did not change in immunized animals. However, the pressor responses to incremental Ang II infusions were blunted in immunized animals. Thirteen subjects with primary aldosteronism demonstrated increased AT2R autoantibody levels compared to normal controls. In conclusion, AT2R autoantibodies produced in immunized rabbits have the ability to activate AT2R and counteract the AT1R-mediated vasoconstriction. These autoantibodies provide useful and selective tools for study of their roles in blood pressure regulation and possible therapeutic intervention.
Author Manuscript
Correspondence to: David C. Kem, MD, Endocrinology and Heart Rhythm Institute, University of Oklahoma Health Sciences Center, TCH 6E103, 1200 Everett Dr, Oklahoma City, OK 73104. Tel: 405-271-5896, Fax: 405-271-7455, [email protected]. *C. Liles and H. Li contributed equally to this work. †X. Yu and D. Kem served as co-senior authors. Presented in part at the 40th International Aldosterone Conference, June 2014, Chicago, IL. Disclosures: None.
Liles et al.
Page 2
Author Manuscript
Keywords activating autoantibodies; angiotensin II type 1 receptor; angiotensin II type 2 receptor; hypertension; vasoconstriction; rabbit
Introduction
Author Manuscript
The renin angiotensin system plays a key role in the regulation of blood pressure and cardiovascular function. Angiotensin II (Ang II) binds to two major subtypes of receptor, the angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R). The majority of the actions of Ang II are mediated by AT1R; however, recent evidence suggests that AT2R opposes AT1R by promoting vasodilatation rather than vasoconstriction.1–4 There has been an increasing awareness of the presence of autoantibodies directed toward the extracellular loops of several G protein-coupled receptors and these often express remarkable specificity for a given receptor.5, 6 Autoantibodies that activate the AT1R have been described and appear to be highly selective for their target domain on the second extracellular loop (ECL2) of AT1R.7, 8 These autoantibodies were initially identified in a limited number of subjects, but this list has expanded rapidly in the last few years in several hypertensive-related conditions such as preeclampsia,9 malignant and refractory hypertension,10, 11 renal allograft rejection,12 and more recently with primary aldosteronism.13–15 There is no currently published information as to whether activating autoantibodies to the AT2R exist; and whether they might alter the complex role of Ang II in vascular homeostasis.
Author Manuscript
We have recently developed a rabbit autoimmune model of AT1R-activating autoantibody (AT1R-AAb)-mediated hypertension.16 These animals were immunized with a peptide containing the known functional AT1R-AAb epitope in the ECL2 of AT1R. After immunization, all animals developed elevated blood pressure. The anti-AT1R sera induced significant AT1R activation and vasoconstriction in vitro. In the present study, we describe our successful efforts to develop AT2R-activating autoantibodies (AT2R-AAbs) in immunized rabbits and to demonstrate their potential impact on Ang II-mediated hypertension and AT1R-AAb-mediated vasoconstriction. We have discovered AT1R-AAbs and AT2R-AAbs maintain a high degree of specificity for their target receptors and produce opposing effects, thus providing a potentially valuable tool for studies of their roles in hypertension.
Methods Author Manuscript
This study protocol was approved by the Institutional Animal Care and Use Committee of the Oklahoma City Veterans Affairs Medical Center and Oklahoma University Health Sciences Center, and conforms to international standards for animal safety and comfort. Animal Model Five New Zealand white rabbits (2.5–3 kg) were fed on standard rabbit chow and were immunized with 1 mg of a synthetic peptide corresponding to the AT2R ECL2
Hypertension. Author manuscript; available in PMC 2016 October 01.
Liles et al.
Page 3
Author Manuscript
(YFRDVRTIEYLGVNACIMAFPPEKYAQWS) (GenScript, Piscataway, NJ) in 0.5 ml of complete Freund’s adjuvant. The animals were boosted with the same peptide plus incomplete Freund’s adjuvant (1 mg/0.5 ml) at 2 and 4 weeks. Arterial blood pressure was measured at preimmune and 6 weeks after immunization. Under anesthesia (ketamine/ xylazine 35 mg/5 mg/kg), the rabbit central ear artery was cannulated and the catheter connected to a pressure transducer. To determine the acute effect of Ang II on blood pressure before and after immunization, increasing dosages of Ang II (10, 100, and 500 ng/kg) were injected intravenously at 5-minute intervals using an infusion pump, and the blood pressure response at each dose was recorded. Each rabbit served as its own control. Preimmune and postimmune sera were obtained from all animals for ELISA and functional assays of the expected antibodies generated during immunization. AT1R-AAbs were obtained from the rabbits previously immunized with the AT1R ECL2 peptide.16
Author Manuscript
Human Studies Sera were available for testing autoantibodies from a group of 13 hypertensive subjects who previously had been diagnosed with primary aldosteronism using an IRB-approved protocol. These subjects for the most part had not had lateralization studies since their hypertension was generally under good control with medications, and the patients had declined to consider surgery irrespective of the testing outcome or there were evident contraindications from the physician’s viewpoint. Each fulfilled criteria for establishing the diagnosis using one of the established methods for confirmation of an abnormal plasma aldosterone/plasma renin activity (PA/PRA) ratio of over 30.17 The two defining tests used included a captopril suppression study (n=9) and saline infusion study (n=1), while others had a PA/PRA ratio of >50 and an absolute PA value over 25 ng/dl (n=3) negating the need for further diagnostic tests.
Author Manuscript
ELISA Antibodies produced in the sera were detected by ELISA. Briefly, microtiter plates were coated with the AT2R ECL2 peptide at 10 μg/ml. To determine antibody titer, rabbit sera were diluted 1:10000 and thereafter serially diluted 2-fold. Goat anti-rabbit IgG conjugated with alkaline phosphatase and its substrate para-nitrophenyl-phosphate 104 were used to detect antibody binding. Titers were determined as the highest dilution with an optical density (OD) value of 0.10 at 60 minutes. Human sera were diluted 1:100, and goat antihuman IgG conjugated with alkaline phosphatase was used for the secondary antibody. Their OD values were read at 60 min. Epitope Mapping
Author Manuscript
Epitope mapping for rabbit AT2R autoantibodies was performed with multipin peptides (constructed in Dr. Judith James’s laboratory, Oklahoma Medical Research Foundation), which comprised a set of 22 octapeptides, overlapping by 7 amino acids spanning the ECL2 of AT2R. Rabbit anti-AT2R sera (1:200) was added to the wells of 96-well plate along with the multipin peptides and incubated for 2 hours at room temperature. The multipin peptides were washed with PBS-Tween and incubated with anti-rabbit IgG antibody conjugated with horseradish peroxidase. Reactivity of rabbit anti-AT2R sera to the pins was measured by
Hypertension. Author manuscript; available in PMC 2016 October 01.
Liles et al.
Page 4
Author Manuscript
adding tetramethyl benzidine substrate solution in the developing plate. The OD values were read at 405 nm. Isolated Arteriole Assay
Author Manuscript
Autoantibody contractile activity was assayed using an isolated rat cremaster arteriole assay as previously described.16, 18 Briefly, arteriolar segments were microdissected from the cremaster muscle of anesthetized rats and cannulated with glass micropipettes. After equilibration and development of steady-state myogenic tone, the arterioles were perifused with rabbit sera. Dosage responses for Ang II (10−10 ~ 10−7 M) and rabbit anti-AT1R sera (1:500 - 1:50) in the absence and presence of rabbit anti-AT2R sera (1:50) were performed to examine the impact of AT2R-AAbs on Ang II and AT1R-AAb-induced contractile response. The AT2R antagonist PD 123319 (10 nM) was used to block AT2R-mediated effect. The potent soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ, 1 μM) was also tested for neutralization of the vasodilatory effect of AT2R-AAbs. Measurements of vessel diameter were made using a video edge detector. Data are reported as percentage of baseline diameter to normalize each response. Statistical Analysis Data are presented as mean ± SD. Comparison between two groups and multiple group comparisons were performed using the nonparametric Mann-Whitney test and KruskalWallis test followed by Dunn’s multiple comparison test, respectively. Statistical significance was set at P
