C-type natriuretic peptide (CNP) is synthesised and released by the endothelium and plays a vital role in the maintenance of vascular homeostasis (Moyes et al., 2014). However, a similar regulatory role of endogenous CNP in the heart has yet to be elucidated. Therefore, I have used three unique mouse strains with endothelium (Tie2-Cre), cardiomyocyte (αMHC-Cre) and fibroblast (Col1α2-Cre)-restricted deletion of CNP to investigate if the peptide modulates coronary vascular reactivity and cardiac function. Methods: Langendorff isolated hearts were used to investigate the effect of CNP deletion on coronary vascular reactivity in response to the endothelium-dependent vasodilators bradykinin (10nmol) and acetylcholine (0.1-1nmol). Vasodilatation associated with reperfusion was investigated by transient cessation of flow (20-80 seconds). Ischaemia reperfusion (IR) injury (35 minutes ischaemia followed by 60 minutes reperfusion) was also investigated in cell-specific knockout (KO) animals. Isoprenaline (ISO; 20mg/kg/day, 7days)- and pressure overload (abdominal aortic constriction [AAC]; 6 weeks)-induced heart failure were used to study the effect of CNP deletion during cardiac stress, with cardiac function assessed by echocardiography. Cardiac fibrosis and hypertrophy were determined by picro-sirius red and wheat-germ agglutinin fluorescence staining, respectively. A subset of experiments was repeated in mice with global deletion of natriuretic peptide receptor-C (NPR-C) to delineate the signalling pathway triggered by CNP. Real time qPCR was used to determine hypertrophic and fibrotic gene expression in left ventricles isolated from mice subjected to AAC or sham. Neonatal cardiomyocytes were isolated to investigate angiotensin (Ang)II-induced hypertrophy. Results: Coronary endothelial reactivity was reduced in endothelial CNP (ecCNP) KO mice compared to wild type (WT) in response to bradykinin, acetylcholine and reperfusion-induced vasodilatation. These observations were paralleled in NPR-C KO animals. ecCNP KO did not exacerbate IR injury, whilst mice with cardiomyocyte-restricted deletion of CNP (cmCNP KO) and NPR-C KO animals exhibited a larger infarct size compared to WT. cmCNP KO mice also displayed greater cardiac dysfunction and fibrosis after ISO infusion or AAC compared to WT; similar results were observed in fbCNP KO and NPR-C KO animals. Infusion of CNP (0.2mg/kg/day; osmotic mini-pump, s.c.) in WT, but not NPR-C KO, animals rescued the decline in cardiac function. CNP (1μM) administration in isolated cardiomyocyte also blunted Ang II-induced hypertrophy. Pro-hypertrophic and pro-fibrotic gene expression (ANP, β-MHC and MMP-2) was augmented in cmCNP KO and NPR-C KO mice compared to littermate controls following AAC. Conclusions: Endothelial, cardiomyocyte and fibroblast-derived CNP have distinct, complementary roles in the heart, modulating cardiac function by influencing coronary vascular tone and protecting against heart failure and IR injury. These protective effects of CNP are mediated, at least in part, via NPR-C activation. Developing CNP mimetics or selective NPR-C agonists could be a novel therapeutic intervention in cardiovascular disease.

C-type Natriuretic Peptide (CNP), a relatively new member of the natriuretic peptide family, is found throughout the central nervous system. Circumstantial evidence associates CNP with learning and memory, as its expression is highest in brain regions known to be involved in memory and associated with hippocampal physiology. Here, the first study housed rats in an enriched environment, regarded as providing an 'informal' learning experience, for either 14 or 28 days of housing in enrichment in six regions of interest, which was attributed to changes in the degradation of CNP. The second study examined a group of rats trained on object -recognition task – the bow-tie maze. A difference was found in CNP production in the limbic medial prefrontal cortex over repeated exposures to novel objects relative to controls that received 'yoked learning' an exposure only to the test room. CNP concentrations also tended to be lower in rats with better levels of discrimination between familiar objects. Together, these studies provide some initial evidence that CNP influences learning –induced plasticity in the intact brain.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22726号 / 医博第4644号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 柳田 素子, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20275号 / 医博第4234号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 小西 靖彦, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20979号 / 医博第4325号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 戸口田 淳也, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21660号 / 医博第4466号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 柳田 素子, 教授 滝田 順子, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21673号 / 医博第4479号 / 新制||医||1036(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 滝田 順子, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24787号 / 医博第4979号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一, 教授 森本 尚樹, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Calcific aortic valve disease (CAVD) occurs through multiple mutually non-exclusive mechanisms that are mediated by valvular interstitial cells (VICs). VICs undergo pathological differentiation during the progression of valve calcification; however the factors that regulate cellular differentiation are not well defined. Most commonly recognized are biochemical factors that induce pathological differentiation, but little is known regarding the biochemical factors that may suppress this process. Further, the contribution of matrix mechanics in valve pathology has been overlooked, despite increasing evidence of close relationships between changes in tissue mechanics, disease progression and the regulation of cellular response. In this thesis, the effect of matrix stiffness on the differentiation of and calcification by VICs in response to pro-calcific and anti-calcific biochemical factors was investigated. Matrix stiffness modulated the response of VICs to pro-calcific factors, leading to two distinct calcification processes. VICs cultured on the more compliant matrices underwent calcification via osteoblast differentiation, whereas those cultured on the stiffer matrices were prone to myofibroblast differentiation. The transition of fibroblastic VICs to myofibroblasts increased cellular contractility, which led to contraction-mediated, apoptosis-dependent calcification. In addition, C-type natriuretic peptide (CNP), a putative protective molecule against CAVD, was identified. CNP supressed myofibroblast and osteoblast differentiation of VICs, and thereby inhibited calcification in vitro. Matrix stiffness modulated the expression of CNP-regulated transcripts, with only a small number of CNP-regulated transcripts not being sensitive to matrix mechanics. These data demonstrate the combined effects of mechanical and biochemical cues in defining VIC phenotype and responses, with implications for the interpretation of in vitro models of VIC calcification and possibly disease devleopment. The findings from this thesis emphasize the necessity to consider both biochemical and mechanical factors in order to improve fundamental understanding of VIC biology.

Calcific aortic valve disease (CAVD) occurs through multiple mutually non-exclusive mechanisms that are mediated by valvular interstitial cells (VICs). VICs undergo pathological differentiation during the progression of valve calcification; however the factors that regulate cellular differentiation are not well defined. Most commonly recognized are biochemical factors that induce pathological differentiation, but little is known regarding the biochemical factors that may suppress this process. Further, the contribution of matrix mechanics in valve pathology has been overlooked, despite increasing evidence of close relationships between changes in tissue mechanics, disease progression and the regulation of cellular response. In this thesis, the effect of matrix stiffness on the differentiation of and calcification by VICs in response to pro-calcific and anti-calcific biochemical factors was investigated. Matrix stiffness modulated the response of VICs to pro-calcific factors, leading to two distinct calcification processes. VICs cultured on the more compliant matrices underwent calcification via osteoblast differentiation, whereas those cultured on the stiffer matrices were prone to myofibroblast differentiation. The transition of fibroblastic VICs to myofibroblasts increased cellular contractility, which led to contraction-mediated, apoptosis-dependent calcification. In addition, C-type natriuretic peptide (CNP), a putative protective molecule against CAVD, was identified. CNP supressed myofibroblast and osteoblast differentiation of VICs, and thereby inhibited calcification in vitro. Matrix stiffness modulated the expression of CNP-regulated transcripts, with only a small number of CNP-regulated transcripts not being sensitive to matrix mechanics. These data demonstrate the combined effects of mechanical and biochemical cues in defining VIC phenotype and responses, with implications for the interpretation of in vitro models of VIC calcification and possibly disease devleopment. The findings from this thesis emphasize the necessity to consider both biochemical and mechanical factors in order to improve fundamental understanding of VIC biology.