Genetic mouse models reveal key physiological functions of adrenomedullin signaling

Adrenomedullin (AM) is a potent 52-amino acid peptide vasodilator that is involved in a wide variety of physiological processes, including regulation of renal function, neurotransmission, apoptosis and growth. AM peptide levels are elevated in many cardiovascular conditions, including normal pregnancy, septic shock, hypertension, and renal failure. The multitude of conditions associated with elevated AM levels suggests that it serves to maintain physiological homeostasis during various stresses. AM exerts most of its biological functions by promoting increases in the intracellular messengers, cAMP and/or nitric oxide. Recent characterization of AM signaling has identified a unique mechanism of G-protein coupled receptor signaling, mediated by a class of single transmembrane proteins called receptor activity modifying proteins (RAMPs), that have been shown to dictate ligand binding specificity of the calcitonin receptor like receptor (CLR for protein, Calcrl for gene). To date, three RAMP proteins have been identified and it is the association of RAMP2 or 3 with CLR that designates an AM receptor. My research has utilized several lines of gene targeted mice to determine the in vivo role of AM and its signaling components in various physiologic contexts. I show here that mice lacking CLR suffer from extreme hydrops fetalis and die at mid-gestation with severe cardiovascular defects, including small overall heart sizes, thin vascular smooth muscle cell walls and defects in myocardial proliferation and apoptosis. To further examine the role of CLR in cardiac development and physiology, I crossed mice with a floxed Calcrl allele to two cardiomyocyte-specific Cre lines, [alpha]-MHC and cardiac troponin. These mice develop normally and are born at the expected Mendelian ratios. Additionally, echocardiography and histological examination revealed no significant differences in heart structure or function as late as 14 weeks of age. Cardiomyocyte-specific CLR knockouts and control littermates also responded similarly to cardiac challenge in two different disease models: transverse aortic constriction and angiotensin II infusion. A separate study revealed that RAMP2 and RAMP3 have distinct physiological functions from embryogenesis to old age, whereby genetic deletion of Ramp2 results in embryonic lethality while deletion of Ramp3 has no effect on embryonic development or survival. Finally, I used mice heterozygous for the AM gene to show that the AM peptide is required for the normal inflammatory response to LPS-induced septic shock. Collectively, the work presented here provides the first in vivo genetic characterization of several key genes involved in AM signaling during various physiological conditions

Loss of receptor activity-modifying protein 3 exacerbates cardiac hypertrophy and transition to heart failure in a sex-dependent manner

Sex differences exist in the hypertrophic response, cardiac remodeling, and transition to heart failure of hypertensive patients, and while some of these differences are likely influenced by estrogen, the genetic pathways downstream of estrogen that impact on cardioprotection have yet to be fully elucidated. We have previously shown that the cardioprotective effects of adrenomedullin (AM), an emerging clinical biomarker for cardiovascular disease severity, vary with sex in mouse models. AM signaling during cardiovascular stress is strongly modulated by receptor activity-modifying protein 3 (RAMP3) via its interaction with the G protein-coupled receptor calcitonin receptor-like receptor (CLR). Like AM, RAMP3 expression is potently regulated by estrogen, and so we sought to determine the consequences of genetic Ramp3 loss on cardiac adaptation to chronic hypertension, with a particular focus on characterizing potential sex differences. We generated and bred RAMP3−/− mice to RenTgMK mice that consistently display severe angiotensin II-mediated CV disease and compared CV disease progression in RenTgMK to that of RenTgMK:RAMP3−/− offspring. As expected, RAMP3 gene expression was higher in cardiovascular tissues of RenTgMK mice and more strongly up-regulated in female RenTgMK mice relative to wildtype controls. RAMP3 loss did not affect the development of hypertension or the presence and severity of perivascular and interstitial fibrosis in the left ventricle (LV). However, echocardiography revealed that while RenTgMK mice developed concentric cardiac hypertrophy with sustained systolic function, male RenTgMK:RAMP3−/− mice showed evidence of LV chamber dilatation and depressed systolic function, suggestive of cardiac decompensation. Consistent with these measures of heart failure, male RenTgMK:RAMP3−/− mice had increased cardiac apoptosis and elevated activation of Akt. These phenotypes were not present in female RenTgMK:RAMP3−/− mice. Collectively, these data demonstrate a sex-dependant, cardioprotective role of RAMP3 in the setting of chronic hypertension

Hydrops Fetalis, Cardiovascular Defects, and Embryonic Lethality in Mice Lacking the Calcitonin Receptor-Like Receptor Gene

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. To date, numerous in vitro studies have suggested that AM can mediate its biological effects through at least three different receptors. To determine the in vivo importance of the most likely candidate receptor, calcitonin receptor-like receptor, a gene-targeted knockout model of the gene was generated. Mice heterozygous for the targeted Calcrl allele appear normal, survive to adulthood, and reproduce. However, heterozygote matings fail to produce viable Calcrl(−/−) pups, demonstrating that Calcrl is essential for survival. Timed matings confirmed that Calcrl(−/−) embryos die between embryonic day 13.5 (E13.5) and E14.5 of gestation. The Calcrl(−/−) embryos exhibit extreme hydrops fetalis and cardiovascular defects, including thin vascular smooth muscle walls and small, disorganized hearts remarkably similar to the previously characterized AM(−/−) phenotype. In vivo assays of cellular proliferation and apoptosis in the hearts and vasculature of Calcrl(−/−) and AM(−/−) embryos support the concept that AM signaling is a crucial mediator of cardiovascular development. The Calcrl gene targeted mice provide the first in vivo genetic evidence that CLR functions as an AM receptor during embryonic development

Loss of receptor activity-modifying protein 3 exacerbates cardiac hypertrophy and transition to heart failure in a sex-dependent manner

Sex differences exist in the hypertrophic response, cardiac remodeling, and transition to heart failure of hypertensive patients, and while some of these differences are likely influenced by estrogen, the genetic pathways downstream of estrogen that impact on cardioprotection have yet to be fully elucidated. We have previously shown that the cardioprotective effects of adrenomedullin (AM), an emerging clinical biomarker for cardiovascular disease severity, vary with sex in mouse models. AM signaling during cardiovascular stress is strongly modulated by receptor activity-modifying protein 3 (RAMP3) via its interaction with the G protein-coupled receptor calcitonin receptor-like receptor (CLR). Like AM, RAMP3 expression is potently regulated by estrogen, and so we sought to determine the consequences of genetic Ramp3 loss on cardiac adaptation to chronic hypertension, with a particular focus on characterizing potential sex differences. We generated and bred RAMP3(−/−) mice to RenTgMK mice that consistently display severe angiotensin II-mediated CV disease and compared CV disease progression in RenTgMK to that of RenTgMK:RAMP3(−/−) offspring. As expected, RAMP3 gene expression was higher in cardiovascular tissues of RenTgMK mice and more strongly up-regulated in female RenTgMK mice relative to wildtype controls. RAMP3 loss did not affect the development of hypertension or the presence and severity of perivascular and interstitial fibrosis in the left ventricle (LV). However, echocardiography revealed that while RenTgMK mice developed concentric cardiac hypertrophy with sustained systolic function, male RenTgMK:RAMP3(−/−) mice showed evidence of LV chamber dilatation and depressed systolic function, suggestive of cardiac decompensation. Consistent with these measures of heart failure, male RenTgMK:RAMP3(−/−) mice had increased cardiac apoptosis and elevated activation of Akt. These phenotypes were not present in female RenTgMK:RAMP3(−/−) mice. Collectively, these data demonstrate a sex-dependant, cardioprotective role of RAMP3 in the setting of chronic hypertension

Cytochrome P450 2J2 Is Highly Expressed in Hematologic Malignant Diseases and Promotes Tumor Cell GrowthS⃞

Cytochrome P450 2J2 (CYP2J2) epoxygenase converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs) that exert multiple biological effects in the cardiovascular system and in various human solid cancers. However, it is unknown whether this enzyme is expressed or plays any role in malignant hematological diseases. In this study, we found strong and highly selective CYP2J2 expression in five human-derived malignant hematological cell lines and in leukemia cells from peripheral blood and bone marrow in 36 of 42 patients (86%) with malignant hematologic diseases. Furthermore, increased levels of EETs were detected in urine and blood samples from these patients. Addition of exogenous EET or CYP2J2 overexpression in cultured human-derived malignant hematologic cell lines markedly accelerated proliferation and attenuated apoptosis. Addition of the selective CYP2J2 inhibitor compound 26 (C26; 1-[4-(vinyl) phenyl]-4-[4-(diphenyl-hydroxymethyl)-piperidinyl]-butanone hydrochloride) inhibited cell proliferation and increased apoptosis, an effect that was significantly reversed by EET. CYP2J2 overexpression and exogenous EET activated AMP-activated protein kinase, c-Jun NH2-terminal kinase, and phosphatidylinositol 3-kinase/Akt signaling pathways, and increased epidermal growth factor receptor phosphorylation levels. CYP2J2 overexpression also enhanced malignant xenograft growth, which was efficiently inhibited by oral administration of C26 in Tie2-CYP2J2 transgenic mice and in severe combined immunodeficiency (SCID) xenograft mice. Together, these results suggest that CYP2J2 plays a key role in the pathogenesis of human hematologic malignant diseases. Selective inhibition of CYP2J2 may be a promising therapeutic strategy for these conditions

Cyclooxygenase-2 Regulates Th17 Cell Differentiation during Allergic Lung Inflammation

Rationale: Th17 cells comprise a distinct lineage of proinflammatory T helper cells that are major contributors to allergic responses. It is unknown whether cyclooxygenase (COX)-derived eicosanoids regulate Th17 cells during allergic lung inflammation