Polymorphisms at the F12 and KLKB1 loci have significant trait association with activation of the renin-angiotensin system.

BACKGROUND: Plasma coagulation Factor XIIa (Hageman factor; encoded by F12) and kallikrein (KAL or Fletcher factor; encoded by KLKB1) are proteases of the kallikerin-kinin system involved in converting the inactive circulating prorenin to renin. Renin is a key enzyme in the formation of angiotensin II, which regulates blood pressure, fluid and electrolyte balance and is a biomarker for cardiovascular, metabolic and renal function. The renin-angiotensin system is implicated in extinction learning in posttraumatic stress disorder. METHODS & RESULTS: Active plasma renin was measured from two independent cohorts- civilian twins and siblings, as well as U.S. Marines, for a total of 1,180 subjects. Genotyping these subjects revealed that the carriers of the minor alleles at the two loci- F12 and KLKB1 had a significant association with reduced levels of active plasma renin. Meta-analyses confirmed the association across cohorts. In vitro studies verified digestion of human recombinant pro-renin by kallikrein (KAL) to generate active renin. Subsequently, the active renin was able to digest the synthetic substrate angiotensinogen to angiotensin-I. Examination of mouse juxtaglomerular cell line and mouse kidney sections showed co-localization of KAL with renin. Expression of either REN or KLKB1 was regulated in cell line and rodent models of hypertension in response to oxidative stress, interleukin or arterial blood pressure changes. CONCLUSIONS: The functional variants of KLKB1 (rs3733402) and F12 (rs1801020) disrupted the cascade of enzymatic events, resulting in diminished formation of active renin. Using genetic, cellular and molecular approaches we found that conversion of zymogen prorenin to renin was influenced by these polymorphisms. The study suggests that the variant version of protease factor XIIa due to the amino acid substitution had reduced ability to activate prekallikrein to KAL. As a result KAL has reduced efficacy in converting prorenin to renin and this step of the pathway leading to activation of renin affords a potential therapeutic target.

Molecular Mechanism for Hypertensive Renal Disease: Differential Regulation of Chromogranin A Expression at 3'-Untranslated Region Polymorphism C+87T by MicroRNA-107.

Chromogranin A (CHGA) is coreleased with catecholamines from secretory vesicles in adrenal medulla and sympathetic axons. Genetic variation in the CHGA 3'-region has been associated with autonomic control of circulation, hypertension, and hypertensive nephropathy, and the CHGA 3'-untranslated region (3'-UTR) variant C+87T (rs7610) displayed peak associations with these traits in humans. Here, we explored the molecular mechanisms underlying these associations. C+87T occurred in a microRNA-107 (miR-107) motif (match: T>C), and CHGA mRNA expression varied inversely with miR-107 abundance. In cells transfected with chimeric luciferase/CHGA 3'-UTR reporters encoding either the T allele or the C allele, changes in miR-107 expression levels had much greater effects on expression of the T allele. Cotransfection experiments with hsa-miR-107 oligonucleotides and eukaryotic CHGA plasmids produced similar results. Notably, an in vitro CHGA transcription/translation experiment revealed that changes in hsa-miR-107 expression altered expression of the T allele variant only. Mice with targeted ablation of Chga exhibited greater eGFR. Using BAC transgenesis, we created a mouse model with a humanized CHGA locus (T/T genotype at C+87T), in which treatment with a hsa-miR-107 inhibitor yielded prolonged falls in SBP/DBP compared with wild-type mice. We conclude that the CHGA 3'-UTR C+87T disrupts an miR-107 motif, with differential effects on CHGA expression, and that a cis:trans (mRNA:miR) interaction regulates the association of CHGA with BP and hypertensive nephropathy. These results indicate new strategies for probing autonomic circulatory control and ultimately, susceptibility to hypertensive renal sequelae.

MicroRNA-22 and promoter motif polymorphisms at the Chga locus in genetic hypertension: functional and therapeutic implications for gene expression and the pathogenesis of hypertension.

Hypertension is a common hereditary syndrome with unclear pathogenesis. Chromogranin A (Chga), which catalyzes formation and cargo storage of regulated secretory granules in neuroendocrine cells, contributes to blood pressure homeostasis centrally and peripherally. Elevated Chga occurs in spontaneously hypertensive rat (SHR) adrenal glands and plasma, but central expression is unexplored. In this report, we measured SHR and Wistar-Kyoto rat (control) Chga expression in central and peripheral nervous systems, and found Chga protein to be decreased in the SHR brainstem, yet increased in the adrenal and the plasma. By re-sequencing, we systematically identified five promoter, two coding and one 3'-untranslated region (3'-UTR) polymorphism at the SHR (versus WKY or BN) Chga locus. Using HXB/BXH recombinant inbred (RI) strain linkage and correlations, we demonstrated genetic determination of Chga expression in SHR, including a cis-quantitative trait loci (QTLs) (i.e. at the Chga locus), and such expression influenced biochemical determinants of blood pressure, including a cascade of catecholamine biosynthetic enzymes, catecholamines themselves and steroids. Luciferase reporter assays demonstrated that the 3'-UTR polymorphism (which disrupts a microRNA miR-22 motif) and promoter polymorphisms altered gene expression consistent with the decline in SHR central Chga expression. Coding region polymorphisms did not account for changes in Chga expression or function. Thus, we hypothesized that the 3'-UTR and promoter mutations lead to dysregulation (diminution) of Chga in brainstem cardiovascular control nuclei, ultimately contributing to the pathogenesis of hypertension in SHR. Accordingly, we demonstrated that in vivo administration of miR-22 antagomir to SHR causes substantial (∼18 mmHg) reductions in blood pressure, opening a novel therapeutic avenue for hypertension.

Long human CHGA flanking chromosome 14 sequence required for optimal BAC transgenic "rescue" of disease phenotypes in the mouse Chga knockout.

Chromogranin A (CHGA) plays a catalytic role in formation of catecholamine storage vesicles and also serves as precursor to the peptide fragment catestatin, a catecholamine secretory inhibitor whose expression is diminished in the hypertensive individuals. We previously reported the hypertensive, hyperadrenergic phenotype of Chga-/- knockout (KO) mice and rescue by the human ortholog. In the present study, we compare two humanized CHGA mouse models. Into the Chga null background, by bacterial artificial chromosome transgenesis human CHGA transgene has been introduced. Both lines have the complete approximately 12 kbp CHGA gene integrated stably in the genome but have substantial differences in CHGA expression, as well as consequent sympathochromaffin biochemistry and physiology. A mouse model with longer-insert HumCHGA31 displays integration encompassing not only CHGA but also long human flanking sequences. This is in contrast to mouse model HumCHGA19 with limited flanking human sequence co-integrated. As a consequence, HumCHGA19 mice have normal though diminished pattern of spatial expression of CHGA, and 14-fold lower circulating CHGA, with failure to rescue KO phenotypes to normalcy. In the longer-insert HumCHGA31 mice, catecholamine secretion, exaggerated responses to environmental stress, and hypertension were all alleviated. Promoter regions of the transgenes in both HumCHGA19 and HumCHGA31 display minimal CpG methylation, weighing against differential "position effects" of integration, and thus suggesting that lack of cis elements required for optimal CHGA expression occurs in HumCHGA19 mice. Such "humanized" CHGA mouse models may be useful in probing the physiological consequences of variation in CHGA expression found in humans, with consequences for susceptibility to hypertension and cardiovascular disease.

A novel pathway of insulin sensitivity in chromogranin A null mice: a crucial role for pancreastatin in glucose homeostasis.

Chromogranin A (CHGA/Chga), a proprotein, widely distributed in endocrine and neuroendocrine tissues (not expressed in muscle, liver, and adipose tissues), generates at least four bioactive peptides. One of those peptides, pancreastatin (PST), has been reported to interfere with insulin action. We generated a Chga knock-out (KO) mouse by the targeted deletion of the Chga gene in neuroendocrine tissues. KO mice displayed hypertension, higher plasma catecholamine, and adipokine levels and lower IL-6 and lipid levels compared with wild type mice. Liver glycogen content was elevated, but the nitric oxide (NO) level was diminished. Glucose, insulin, and pyruvate tolerance tests and hyperinsulinemic-euglycemic clamp studies established increased insulin sensitivity in liver but decreased glucose disposal in muscle. Despite higher catecholamine and ketone body levels and muscle insulin resistance, KO mice maintained euglycemia due to increased liver insulin sensitivity. Suppressed mRNA abundance of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase) in KO mice further support this conclusion. PST administration in KO mice stimulated phosphoenolpyruvate carboxykinase and G6Pase mRNA abundance and raised the blood glucose level. In liver cells transfected with G6Pase promoter, PST caused transcriptional activation in a protein kinase C (PKC)- and NO synthase-dependent manner. Thus, PST action may be mediated by suppressing IRS1/2-phosphatidylinositol 3-kinase-Akt-FOXO-1 signaling and insulin-induced maturation of SREBP1c by PKC and a high level of NO. The combined effects of conventional PKC and endothelial NO synthase activation by PST can suppress insulin signaling. The rise in blood PST level with age and in diabetes suggests that PST is a negative regulator of insulin sensitivity and glucose homeostasis.

Discovery of common human genetic variants of GTP cyclohydrolase 1 (GCH1) governing nitric oxide, autonomic activity, and cardiovascular risk.

GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variation at GCH1 alters transmitter synthesis and predisposes to disease. Here we undertook a systematic search for polymorphisms in GCH1, then tested variants' contributions to NO and catecholamine release as well as autonomic function in twin pairs. Renal NO and neopterin excretions were significantly heritable, as were baroreceptor coupling (heart rate response to BP fluctuation) and pulse interval (1/heart rate). Common GCH1 variant C+243T in the 3'-untranslated region (3'-UTRs) predicted NO excretion, as well as autonomic traits: baroreceptor coupling, maximum pulse interval, and pulse interval variability, though not catecholamine secretion. In individuals with the most extreme BP values in the population, C+243T affected both diastolic and systolic BP, principally in females. In functional studies, C+243T decreased reporter expression in transfected 3'-UTRs plasmids. We conclude that human NO secretion traits are heritable, displaying joint genetic determination with autonomic activity by functional polymorphism at GCH1. Our results document novel pathophysiological links between a key biosynthetic locus and NO metabolism and suggest new strategies for approaching the mechanism, diagnosis, and treatment of risk predictors for cardiovascular diseases such as hypertension.

Naturally occurring genetic variants in human chromogranin A (CHGA) associated with hypertension as well as hypertensive renal disease.

Chromogranin A (CHGA) plays a fundamental role in the biogenesis of catecholamine secretory granules. Changes in storage and release of CHGA in clinical and experimental hypertension prompted us to study whether genetic variation at the CHGA locus might contribute to alterations in autonomic function, and hence hypertension and its target organ consequences such as hypertensive renal disease (nephrosclerosis). Systematic polymorphism discovery across the human CHGA locus revealed both common and unusual variants in both the open reading frame and such regulatory regions as the proximal promoter and 30-UTR. In chromaffin cell-transfected CHGA 30-UTR and promoter/luciferase reporter plasmids, the functional consequences of the regulatory/non-coding allelic variants were documented. Variants in both the proximal promoter and the 30-UTR displayed statistical associations with hypertension. Genetic variation in the proximal CHGA promoter predicted glomerular filtration rate in healthy twins. However, for hypertensive renal damage, both end-stage renal disease and rate of progression of earlier disease were best predicted by variants in the 30-UTR. Finally, mechanistic studies were undertaken initiated by the clue that CHGA promoter variation predicted circulating endothelin-1. In cultured endothelial cells, CHGA triggered co-release of not only the vasoconstrictor and pro-fibrotic endothelin-1, but also the pro-coagulant von Willebrand Factor and the pro-angiogenic angiopoietin-2. These findings, coupled with stimulation of endothelin-1 release from glomerular capillary endothelial cells by CHGA, suggest a plausible mechanism whereby genetic variation at the CHGA locus eventuates in alterations in human renal function. These results document the consequences of genetic variation at the CHGA locus for cardiorenal disease and suggest mechanisms whereby such variation achieves functional effects.

Chromogranin A pathway: from pathogenic molecule to renal disease.

BACKGROUND: Chromogranin A (CHGA) is an index granin protein critical for biogenesis and exocytotic release of catecholamine storage granules. It is elevated in plasma of patients with sympathetic over-activity and kidney dysfunction. Several CHGA polymorphisms are associated with hypertensive kidney disease. Previously, we unraveled the molecular mechanism by which CHGA expression is regulated in African Americans carrying a genetic variation associated with hypertensive chronic kidney disease (CKD). METHOD: Experimental CKD mouse model were created by 5/6th nephrectomy (Npx) using wild-type and Chga-/- knockout mouse strains to delineate the role of CHGA in CKD. RESULT: Wild-type-Npx mice expressing Chga developed exacerbated azotemia and fibrosis as compared with their knockout-Npx counterparts. Gene expression profiling revealed downregulation of mitochondrial respiratory complexes genes consistent with maladaptive mitochondria in wild-type-Npx mice, contrasted to knockout-Npx. In healthy individuals, an inverse relationship between circulating CHGA levels and glomerular function was observed. In vitro, mesangial cells treated with CHGA-triggered nitric oxide release by a signaling mechanism involving scavenger receptor SR-A. The CHGA-treated and untreated mesangial cells displayed differential expression of cytokine, chemokine, complement, acute phase inflammatory and apoptotic pathway genes. Thus, build-up of plasma CHGA because of kidney injury served as an insult to the mesangial cells resulting in expression of genes promoting inflammation, fibrosis, and progression of CKD. CONCLUSION: These findings improve understanding of the role of elevated CHGA in the progression of CKD and reveal novel pathways that could be exploited for therapeutic strategies in hypertensive kidney disease.

Uric Acid and Acute Kidney Injury in the Critically Ill.

RATIONALE & OBJECTIVE: Uric acid is excreted by the kidney and accumulates in acute kidney injury (AKI). Whether higher plasma uric acid level predisposes to AKI or its complications is not known. STUDY DESIGN: Prospective observational cohort study. SETTING & PARTICIPANTS: 2 independent cohorts of critically ill patients: (1) 208 patients without AKI admitted to the intensive care unit (ICU) at Brigham & Women's Hospital between October 2008 and December 2016; and (2) 250 participants with AKI requiring renal replacement therapy (RRT) who had not yet initiated RRT enrolled in the Acute Renal Failure Trial Network (ATN) Study. EXPOSURE: Plasma uric acid level upon ICU admission and before RRT initiation in the ICU and ATN Study cohorts, respectively. OUTCOMES: Incident AKI and 60-day mortality in the ICU and ATN Study cohorts, respectively. ANALYTICAL APPROACH: Logistic regression models were used to test the association of plasma uric acid level with incident AKI and 60-day mortality. RESULTS: In the ICU cohort, median plasma uric acid level was 4.7 (interquartile range [IQR], 3.6-6.4) mg/dL, and 40 patients (19.2%) developed AKI. Higher plasma uric acid levels associated with incident AKI, but this association was confounded by serum creatinine level and was not significant after multivariable adjustment (adjusted OR per doubling of uric acid, 1.50; 95% CI, 0.80-2.81). In the ATN Study cohort, median plasma uric acid level was 11.1 (IQR, 8.6-14.2) mg/dL, and 125 participants (50.0%) died within 60 days. There was no statistically significant association between plasma uric acid levels and 60-day mortality in either unadjusted models or after multivariable adjustment for demographic, severity-of-illness, and kidney-specific covariates (adjusted OR per doubling of uric acid, 1.15; 95% CI, 0.71-1.86). LIMITATIONS: Heterogeneity of ICU patients. CONCLUSIONS: Plasma uric acid levels upon ICU admission or before RRT initiation are not independently associated with adverse clinical outcomes in critically ill patients.

Catecholamine release-inhibitory peptide catestatin (chromogranin A(352-372)): naturally occurring amino acid variant Gly364Ser causes profound changes in human autonomic activity and alters risk for hypertension.

BACKGROUND: Chromogranin A, coreleased with catecholamines by exocytosis, is cleaved to the catecholamine release-inhibitory fragment catestatin. We identified a natural nonsynonymous variant of catestatin, Gly364Ser, that alters human autonomic function and blood pressure. METHODS AND RESULTS: Gly364Ser heterozygotes and controls underwent physiological and biochemical phenotyping, including catecholamine production, chromogranin A precursor, and its catestatin product. Case-control studies replicated effects of the gene on blood pressure in the population. Gly364Ser displayed diminished inhibition of catecholamine secretion from cultured neurons. Gly/Ser heterozygotes displayed increased baroreceptor slope during upward deflections (by approximately 47%) and downward deflections (by approximately 44%), increased cardiac parasympathetic index (by approximately 2.4-fold), and decreased cardiac sympathetic index (by approximately 26%). Renal norepinephrine excretion was diminished by approximately 26% and epinephrine excretion by approximately 34% in Gly/Ser heterozygotes. The coalescent dated emergence of the variant to approximately 70,000 years ago. Gly364Ser was in linkage disequilibrium with 1 major Chromogranin A promoter haplotype, although promoter haplotypes did not predict autonomic phenotypes. The 364Ser variant was associated with lower diastolic blood pressure in 2 independent/confirmatory groups of patients with hypertension; genotype groups differed by approximately 5 to 6 mm Hg, and the polymorphism accounted for approximately 1.8% of population diastolic blood pressure variance, although a significant gene-by-sex interaction existed, with an enhanced effect in men. CONCLUSIONS: The catestatin Gly364Ser variant causes profound changes in human autonomic activity, both parasympathetic and sympathetic, and seems to reduce risk of developing hypertension, especially in men. A model for catestatin action in the baroreceptor center of the nucleus of the tractus solitarius accounts for these actions.

Proteolytic cleavage of human chromogranin a containing naturally occurring catestatin variants: differential processing at catestatin region by plasmin.

The plasma level of chromogranin A (CgA) is elevated in genetic hypertension. Conversely, the plasma level of the CgA peptide catestatin is diminished in individuals with established hypertension and those with a genetic risk of this disease. Resequencing of the human CHGA gene identified three naturally occurring variants of catestatin (Gly364Ser, Pro370Leu, and Arg374Gln) that exhibit different potencies in inhibiting catecholamine secretion. Here, we have examined whether there is any differential processing of the three CHGA variants to catestatin by the endoproteolytic enzyme plasmin. Plasmin digestion of the purified CgA proteins generated a stable biologically active 14-amino acid peptide (human CgA(360-373)) from the wild-type, Gly364Ser, and Arg374Gln proteins despite the disruption of the dibasic site (Arg(373)Arg(374)) in the Arg374Gln variant. Unexpectedly, the action of plasmin in generating the catestatin peptide from the Pro370Leu protein was less efficient. The efficiency of cleavage at the dibasic Arg(373) downward arrowArg(374) site in synthetic human CgA(360-380) was 3- to 4-fold less in Pro370Leu CgA, compared with the wild type. Circular dichroism of the synthetic CgA(352-372) suggested a difference in the amount of alpha-helix and beta-sheet between the wild-type and Pro370Leu CgA peptides. Because the Pro(370) residue is in the P4 position, the local secondary structure in the vicinity of the cleavage site may enforce the specificity or accessibility to plasmin. The less efficient proteolytic processing of the Pro370Leu protein by plasmin, coupled with the strong association of this variant with ethnicity, suggests that the Pro370Leu CHGA gene variant may contribute to the differential prevalence of cardiovascular disease across ethnic groups.

Hypertension from targeted ablation of chromogranin A can be rescued by the human ortholog.

The secretory prohormone chromogranin A (CHGA) is overexpressed in essential hypertension, a complex trait with genetic predisposition, while its catecholamine release-inhibitory fragment catestatin is diminished, and low catestatin predicts augmented adrenergic pressor responses. These findings from studies on humans suggest a mechanism whereby diminished catestatin might increase the risk for hypertension. We generated Chga and humanized mice through transgenic insertion of a human CHGA haplotype in order to probe CHGA and catestatin in vivo. Chga mice displayed extreme phenotypic changes, including: (a) decreased chromaffin granule size and number; (b) elevated BP; (c) loss of diurnal BP variation; (d) increased left ventricular mass and cavity dimensions; (e) decreased adrenal catecholamine, neuropeptide Y (Npy), and ATP contents; (f) increased catecholamine/ATP ratio in the chromaffin granule; and (g) increased plasma catecholamine and Npy levels. Rescue of elevated BP to normalcy was achieved by either exogenous catestatin replacement or humanization of Chga mice. Loss of the physiological "brake" catestatin in Chga mice coupled with dysregulation of transmitter storage and release may act in concert to alter autonomic control of the circulation in vivo, eventuating in hypertension.

Mice overexpressing chromogranin A display hypergranulogenic adrenal glands with attenuated ATP levels contributing to the hypertensive phenotype.

OBJECTIVE: Elevated circulating chromogranin A (CHGA) is observed in human hypertension. CHGA is critical for granulogenesis and exocytosis of catecholamine stores from secretory large dense core vesicles (LDCV). This study aims to understand the morphological, molecular and phenotypic changes because of excess CHGA and the mechanistic link eventuating in hyper-adrenergic hypertension. METHODS: Blood pressure and heart rate was monitored in mouse models expressing normal and elevated level of CHGA by telemetry. Catecholamine and oxidative stress radicals were measured. Adrenal ultrastructure, LDCV content and mitochondrial abundance were compared and respiration analyzed by Seahorse assay. Effect of CHGA dosage on adrenal ATP content, electron transport chain components and uncoupling protein 2 (UCP-2) were compared in vivo and in vitro. RESULTS: Mice with excess-CHGA displayed hypertensive phenotype, higher heart rate and increased sympathetic tone. They had elevated plasma catecholamine and adrenal ROS levels. Excess-CHGA caused an increase in size and abundance of LDCV and adrenal mitochondria. Nonetheless, they had attenuated levels of ATP. Isolated adrenal mitochondria from mice with elevated CHGA showed higher maximal respiration rates in the presence of protonophore, which uncouples oxidative phosphorylation. Elevated CHGA resulted in overexpression of UCP2 and diminished ATP. In vitro in chromaffin cells overexpressing CHGA, concomitant increase in UCP2 protein and decreased ATP was detected. CONCLUSION: Elevated CHGA expression resulted in underlying bioenergetic dysfunction in ATP production despite higher mitochondrial mass. The outcome was unregulated negative feedback of LDCV exocytosis and secretion, resulting in elevated levels of circulating catecholamine and consequently the hypertensive phenotype.

Stability of Fibroblast Growth Factor 23 in Human Plasma.

BACKGROUND: Given the important emerging field of fibroblast growth factor 23 (FGF23) biology, there is a growing need for reliable data on FGF23 assay characteristics. We therefore evaluated the effects of different processing and storage conditions on FGF23 stability, as well as the assay precision of FGF23 measurements using 2 commercially available FGF23 ELISA kits. METHODS: We measured plasma concentrations of intact FGF23 (iFGF23) and C-terminal FGF23 (cFGF23) in duplicate in 12 patients with a wide range of kidney function. We used blinded replicate samples to calculate the interassay CV for both assays. We processed the samples immediately after collection, after 6 h at 22 °C, or after 24 h at 4 °C. We also exposed samples to 0, 1, 2, or 3 freeze-thaw cycles. RESULTS: The interassay CVs for iFGF23 and cFGF23 were 5.2% and 7.2%, respectively. Delayed processing for either 6 h at 22 °C or 24 h at 4 °C had no significant effect on either iFGF23 or cFGF23, although a nonsignificant trend toward decreased iFGF23 concentrations was observed compared with immediate processing (23% relative decline in concentrations under both delayed processing conditions). Three freeze-thaw cycles had no effect on either iFGF23 or cFGF23 concentrations. CONCLUSIONS: FGF23 measurements in human plasma are stable with delayed processing or after undergoing multiple freeze-thaw cycles.

Analysis and validation of traits associated with a single nucleotide polymorphism Gly364Ser in catestatin using humanized chromogranin A mouse models.

OBJECTIVE: The human prohormone chromogranin A (CHGA), an index member of the granin family is processed to generate catestatin, a peptide that is hypotensive in action and modulates catecholamine release within the sympathoadrenal system. Hypertensive patients with excess sympathetic activity have diminished catestatin. Often the study of physiological consequences of human genetic variation is confounded by elements such as other variations in obligatory linkage disequilibrium with the variant being studied. Also the phenotype of the variant may be influenced by genetic background that varies amongst individuals. This study addresses the effects of a human catestatin polymorphism (rs9658667) using humanized CHGA mouse models. METHODS: We created pertinent humanized mouse models wherein the mouse Chga gene locus was replaced by the human ortholog wild-type and the variant versions. This allowed for probing of the effects of catestatin variation in vivo with controls for other variations and global genetic background. RESULTS: Both the wild-type and variant human catestatin expressing mouse models were normotensive. The variant catestatin mouse model recapitulated physiological influence of the polymorphism on autonomic traits. These mice had diminished catecholamine, attenuated stress response and increased baroreceptor slopes that would suggest reduced risk of developing hypertension. Elevated plasma glucose, a trait observed in humans was not observed in mice expressing the variant catestatin. CONCLUSION: This functional genomics approach of creating humanized mouse models to study rs9658667 polymorphism recapitulated and validated many of the human trait associations. This approach can also be applied in the study of other human gene polymorphisms.

Cardiac electrical activity in a genomically "humanized" chromogranin a monogenic mouse model with hyperadrenergic hypertension.

The prohormone chromogranin A (CHGA) is ubiquitously found in vesicles of adrenal chromaffin cells and adrenergic neurons, and it is processed to the hypotensive hormone peptide catestatin (CST). Both CHGA and CST regulate blood pressure and cardiac function. This study addresses their role in cardiac electrical activity. We have generated two genomically "humanized" transgenic mouse strains (Tg31CHGA+/+; Chga-/- (HumCHGA31) and Tg19CHGA+/+; Chga-/- (HumCHGA19)) with varied CHGA expression and the ability to rescue the Chga-/- phenotype (hypertensive, hyperadrenergic with dilated cardiomyopathy). The normotensive HumCHGA31 mice express CHGA at levels comparable to wild-type. In contrast, the hypertensive HumCHGA19 mice have low levels of CHGA. EKG recordings revealed that the QT interval, R-amplitude, and QRS time-voltage integral are markedly longer in HumCHGA19 compared to wild-type and HumCHGA31 mice. These differences are accompanied by increased heart rate and QT variability, indicating that ventricular assault happens in a status of low levels of circulating CST.

Effects of chromogranin A deficiency and excess in vivo: biphasic blood pressure and catecholamine responses.

OBJECTIVE: The phenotype of the chromogranin A (Chga) null (knockout) mouse is hypertensive. However, hypertensive humans and spontaneously hypertensive rats display elevated CHGA expression. This study addresses the paradox that both ablation and elevation of CHGA result in hypertension. METHODS: Mice with varying copy number of the CHGA gene were generated. In these mice CHGA, catecholamine and blood pressure (BP) were measured. Also a cohort of healthy human individuals was stratified into tertiles based on plasma CHGA expression and phenotyped for characteristics including their BP response to environmental (cold) stress. RESULTS: The mice displayed a direct CHGA gene dose-dependent (0-4 copies/genome) activation of CHGA expression in both plasma and adrenal gland, yet the BP dependence of CHGA gene dose was U-shaped, maximal at 0 and four copies of the gene, whereas minimal at two copies (i.e., the wild-type gene dosage). Plasma catecholamine showed a parallel U-shaped dose/response in mice, whereas adrenal epinephrine exhibited a reciprocal (inverted) U-shaped response, suggesting dysregulated neurotransmission at both extremes of CHGA expression. The human individuals also showed a nonlinear relationship between CHGA expression and pressor responses to environmental (cold) stress, that were maximal in the highest and lowest tertiles, though basal BPs did not differ among the groups. The human CHGA tertiles also differed in epinephrine secretion as well as degree of CHGA processing to catestatin (catecholamine release-inhibitory peptide derived from CHGA processing). CONCLUSION: Thus, across mammalian species, an optimal amount of CHGA may be required to establish appropriate catecholamine storage and release, and hence BP homeostasis.

Cathepsin L colocalizes with chromogranin a in chromaffin vesicles to generate active peptides.

Chromogranin A (CgA), the major soluble protein in chromaffin granules, is proteolytically processed to generate biologically active peptides including the catecholamine release inhibitory peptide catestatin. Here we sought to determine whether cysteine protease cathepsin L (CTSL), a novel enzyme for proteolytic processing of neuropeptides, acts like the well-established serine proteases [prohormone convertase (PC)1/3 or PC2] to generate catestatin by proteolytic processing of CgA. We found that endogenous CTSL colocalizes with CgA in the secretory vesicles of primary rat chromaffin cells. Transfection of PC12 cells with an expression plasmid encoding CTSL directed expression of CTSL toward secretory vesicles. Deconvolution fluorescence microscopy suggested greater colocalization of CTSL with CgA than the lysosomal marker LGP110. The overexpression of CTSL in PC12 cells caused cleavage of full-length CgA. CTSL also cleaved CgA in vitro, in time- and dose-dependent fashion, and specificity of the process was documented through E64 (thiol reagent) inhibition. Mass spectrometry on CTSL-digested recombinant CgA identified a catestatin-region peptide, corresponding to CgA(360-373). The pool of peptides generated from the CTSL cleavage of CgA inhibited nicotine-induced catecholamine secretion from PC12 cells. CTSL processing in the catestatin region was diminished by naturally occurring catestatin variants, especially Pro370Leu and Gly364Ser. Among the CTSL-generated peptides, a subset matched those found in the catestatin region in vivo. These findings indicate that CgA can be a substrate for the cysteine protease CTSL both in vitro and in cella, and their colocalization within chromaffin granules in cella suggests the likelihood of an enzyme/substrate relationship in vivo.

Naturally occurring human genetic variation in the 3'-untranslated region of the secretory protein chromogranin A is associated with autonomic blood pressure regulation and hypertension in a sex-dependent fashion.

OBJECTIVES: We aimed to determine whether the common variation at the chromogranin A (CHGA) locus increases susceptibility to hypertension. BACKGROUND: CHGA regulates catecholamine storage and release. Previously we systematically identified genetic variants across CHGA. METHODS: We carried out dense genotyping across the CHGA locus in >1,000 individuals with the most extreme blood pressures (BPs) in the population, as well as twin pairs with autonomic phenotypes. We also characterized the function of a trait-associated 3'-untranslated region (3'-UTR) variant with transfected CHGA 3'-UTR/luciferase reporter plasmids. RESULTS: CHGA was overexpressed in patients with hypertension, especially hypertensive men, and CHGA predicted catecholamines. In individuals with extreme BPs, CHGA genetic variants predicted BP, especially in men, with a peak association occurring in the 3'-UTR at C+87T, accounting for up to approximately 12/ approximately 9 mm Hg. The C+87T genotype predicted CHGA secretion in vivo, with the +87T allele (associated with lower BP) also diminishing plasma CHGA by approximately 10%. The C+87T 3'-UTR variant also predicted the BP response to environmental (cold) stress; the same allele (+87T) that diminished basal BP in the population also decreased the systolic BP response to stress by approximately 12 mm Hg, and the response was smaller in women (by approximately 6 mm Hg). In a chromaffin cell-transfected CHGA 3'-UTR/luciferase reporter plasmid, the +87T allele associated with lower BP also decreased reporter expression by approximately 30%. In cultured chromaffin cells, reducing endogenous CHGA expression by small interfering ribonucleic acid caused approximately two-thirds depletion of catecholamine storage vesicles. CONCLUSIONS: Common variant C+87T in the CHGA 3'-UTR is a functional polymorphism causally associated with hypertension especially in men of the population, and we propose steps ("intermediate phenotypes") whereby in a sex-dependent fashion this genetic variant influences the ultimate disease trait. These observations suggest new molecular strategies to probe the pathophysiology, risk, and rational treatment of hypertension.

Biogenesis of the secretory granule: chromogranin A coiled-coil structure results in unusual physical properties and suggests a mechanism for granule core condensation.

The secretory pro-hormone chromogranin A (CHGA) is densely packed into storage granules along with catecholamines, playing a catalytic role in granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, or a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein "packing" in the secretory granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disrupted regulated secretory protein traffic by approximately 65%, while targeted ablation of the CHGA gene in the mouse reduced chromaffin granule cotransmitter concentrations by approximately 40-80%. These results suggest new roles for secretory protein tertiary structure in hormone and transmitter storage, with implications for secretory cargo condensation (or dense core "packing" structure) within the regulated pathway.