C-type natriuretic peptide signalling and cardiovascular disease Adrian Hobbs
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1 C-type natriuretic peptide signalling and cardiovascular disease Adrian Hobbs Professor of Cardiovascular Pharmacology William Harvey Research Institute Barts & The London School of Medicine & Dentistry Queen Mary University of London Charterhouse Square London EC1M 6BQ
2 Natriuretic peptide family L S R I D M R F C S R S R A Q S L C N S F R Y S P M K R I D M K R F C S Q V S S S S L C K V L R R H L R I D L K L F C S K S M S L C ANP 28aa peptide BNP 32aa peptide CNP 22aa peptide
3 Biological actions of natriuretic peptides ENDOCRINE PARACRINE Thirst Vasopressin Sympathetic ANP (atria) BNP (ventricles) CNP ANP BNP Na + /H 2 loss Aldosterone RELAXATION
4 Natriuretic peptide receptor (NPR) subtypes NPR-A NPR-B NPR-C ANP BNP CNP NP TP cmp TP NP Lysosome
5 Endothelium-dependent dilatation Blood vessel lumen PI 2 NO EDHF Endothelium AC s a IP b g s a Fe 2K + Na + /K + -ATPase 3Na + K + K IR sc K + ATP camp cmp TP HYPERPOLARISATION Smooth muscle cell RELAXATION RELAXATION
6 Vascular pharmacology of CNP Endothelial Cell ACh, Shear Stress, IL-1β, TNFα, TFβ CNP NPR-B NPR-C cmp TP Relaxation Smooth Muscle Cell
7 CNP EDHF CNP EDHF CNP EDHF % relaxation CNP and EDHF activate NPR-C! * * + HS NPR-A/B antagonist + M37249 NPR-C antagonist Rat isolated mesenteric arteries
8 Vascular pharmacology of CNP Endothelial Cell Leukocytes Platelets M37249 Proliferation CNP NPR-B IRK NPR-C β γ Hyperpolarisation K + i α γ β ERK 1/2 Proliferation Smooth Muscle Cell
9 ΔCPP (mmhg) Infarct size (% of total wet weight) A cytoprotective role for CNP in ischaemiareperfusion (I/R) injury -1-2 Control ** 5 25 * * -3 + Ba 2+ + ouabain CNP (nmol) Control + CNP (3nM) + CNP rep) Langendorff isolated heart ligature Non-ischaemic zone Ischaemic zone
10 A physiological role for CNP? Interesting pharmacodynamic profile! Presence of CNP in the endothelium suggests it has tissue localisation & functional remit to preserve vascular homeostasis Acts as an EDHF in mesenteric and coronary arteries Prevents the activation of leukocytes & platelets Maintains integrity of blood vessel wall (regulates EC and VSMC proliferation) Paucity of selective pharmacological interventions targeting natriuretic peptide signalling Difficult to define a physiological function
11 Development of an endothelium-specific CNP knockout Endogenous Nppc locus BamHI SacI AT Stop SspI NheI Exon 1 Exon 2 Exon 3
12 Development of an endothelium-specific CNP knockout Endogenous Nppc locus BamHI SacI AT Stop SspI NheI Exon 1 Exon 2 Exon 3 Targeting vector BamHI AT Stop FRT FRT NheI DTA LoxP Exon 1 Exon 2 Neo LoxP Homologous recombination Targeted Nppc locus BamHI LoxP AT Stop Exon 1 Exon 2 FRT Neo FRT LoxP NheI Exon 3 ES cell injection Tie 2 -Cre X Nppc flox/flox
13 Development of an endothelium-specific CNP knockout Endogenous Mouse CNP Locus AT Stop Exon 1 Exon 2 Exon 3 CNP +/+ Tie2 CNP - +/+ Tie2 - CNP CNP +/+ +/+ Tie2 + Tie2 + CNP +/flox CNP Tie2 +/flox - Tie2 - CNP +/flox CNP Tie2 +/flox + Tie2 + CNP flox/flox CNP flox/flox CNP flox/flox Tie2 - CNP Tie2 flox/flox + Tie2 - Tie2 + Floxed LoxP (Floxed) Mouse AT Stop 842 bp Tie2 LoxP Exon 1 Exon 2 LoxP Exon bp Mouse AT Stop Exons 1 & 2 Excised LoxP Exon 1 Exon 2 LoxP Exon 3
![Plasma [CNP] pg/ml Development of an endothelium-specific CNP knockout CNP mrna expression Plasma [CNP] EC KO](/docs-images/73/68356482/images/14-1.jpg "EC Liver KO Liver Lung KO Lung Kidney KO Kidney 1 CNP Exon 2 8 6 4 APDH 2 *** *** + LPS (12.5mg/kg; i.p.")
14 Plasma [CNP] pg/ml Development of an endothelium-specific CNP knockout CNP mrna expression Plasma [CNP] EC KO EC Liver KO Liver Lung KO Lung Kidney KO Kidney 1 CNP Exon APDH 2 *** *** + LPS (12.5mg/kg; i.p.; 14 hrs)
15 % Contraction % Contraction Vasoconstrictor responses are unaltered in vessels % Contraction % Contraction Aorta Mesentery pec 5 =6.91±.2 KO=6.87±.2 ] ns 5 pec 5 = 5.72±.6 KO= 5.71±.8 ] ns Log [Phenylephrine] M Log [Phenylephrine] M pec 5 =7.97 ±.3 KO= 7.87 ±.2 ] ns 5 pec 5 = 7.78±.1 KO =7.59 ±.16 ] ns Log [U46619] M Log [U46619] M
16 % Relaxation % Relaxation % Relaxation Endothelium-dependent relaxation is impaired in resistance arteries of mice Aorta Log [ACh] M pec 5 = 7.52±.4 KO= 7.54±.8 ] ns NO, PI 2 & EDHF-dependent relaxation Mesentery EDHF-dependent relaxation Log [ACh] M pec 5 8 =7.2 ±.2 KO=6.55 ± Log [ACh] M pec 5 ] * ] * =6.93±.22 KO=5.96 ±.29
17 MABP (mmhg) MABP (mmhg) Blood pressure is elevated in mice Circadian Rhythm MABP (24hr Mean) Light Light Dark Dark Light Light eccnp eccnp Het KO Time of Day *** *** *** eccnp Het eccnp KO enotype
18 Cell Rolling (cells/min) Cell Rolling (cells/min) Loss of endothelial CNP results in increased leukocyte rolling 3 25 Basal Rolling ** basal KO basal eccnp enotype IL-1β Treatment *** 2 1 IL-1β KO IL-1β eccnp enotype
19 Aggregation (AUC) % Platelet-leukocyte aggregates Loss of endothelial CNP: platelet function? Impedance aggregometry Platelet-leukocyte aggregates 3 2 * * * 4 3 ** 1 2 Collagen (3µg/ml) Collagen (1µg/ml) PAR4-AP (3µM) 1 (l)
20 CD62 CD62 Increased platelet & endothelial P-selectin expression in mice Endothelial staining intensity (a.u.) Platelet P-selectin expression CD41 CD62 * * * CD41 CD62 Endothelial P-selectin expression KO 8 ** 6 4 2
21 Lesion Size (% Aortic Arch) Lesion Size (% Thoracic Aorta) Lesion Size (% Entire Aorta) Lesion Size (% Abdominal Aorta) Accelerated atherogenesis in CNP KO mice Entire Aorta *** Abdominal Aorta * eccnp / Apoe KO / Apoe KO 2 2 eccnp / ApoE KO / ApoE KO eccnp / ApoE KO / ApoE KO enotype enotype Aortic Arch ** Thoracic Aorta eccnp / ApoE KO enotype / ApoE KO eccnp / ApoE KO enotype / ApoE KO Oil Red O Staining (Lipid) (12 week high-fat diet)
22 Intima/media thickness ratio Plaque Area (% of total lumen area) CNP/ApoE double knockout mice have larger atherosclerotic plaques 6 5 ** eccnp / Apoe KO / Apoe KO CNP / ApoE KO CNP KO/ ApoE KO H & E enotype * CNP / ApoE KO CNP KO/ ApoE KO α smooth muscle actin enotype (Brachiocephalic artery)
23 phenotyping summary Achieved selective deletion of CNP in endothelial cells Vascular phenotype Loss of endothelium-derived CNP results in impaired resistance artery function in vitro and hypertension in vivo CNP is involved in the regulation of inflammatory cell recruitment & platelet reactivity (increased endothelial & platelet P-selectin expression) CNP is important for the maintenance of vascular integrity and curbing the progression of atherosclerotic disease Tissue-specific CNP KO is an ideal model to further delineate the importance of CNP in cardiovascular health & disease Assessment of cardiac function in mice vs. cardiomyocyte (alphamhc-cre) CNP KO (EC telemetry; coronary vascular reactivity; ischaemia reperfusion (I/R) injury; heart failure) Pharmacology infers that the majority of the cytoprotective functions of CNP appear to be mediated via NPR-C Assessing the vascular importance of NPR-B and NPR-C (transgenic approach) Development of small molecule NPR-C agonists
24 Acknowledgements
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