Nilima Biswas

We hypothesized that a strategy employing tissue-specific endothelial cells (EC) might facilitate the identification of tissue- or organ-specific vascular functions of ubiquitous metabolites. An unbiased approach was employed to identify water-soluble small molecules with mitogenic activity on choroidal EC. We identified adenosine diphosphate (ADP) as a candidate, following biochemical purification from mouse EL4 lymphoma extracts. ADP stimulated the growth of bovine choroidal EC (BCEC) and other bovine or human eye-derived EC. ADP induced rapid phosphorylation of extracellular signal-regulated kinase in a dose- and time-dependent manner. ADP-induced BCEC proliferation could be blocked by pretreatment with specific antagonists of the purinergic receptor P2Y1 but not with a vascular endothelial growth factor (VEGF) inhibitor, indicating that the EC mitogenic effects of ADP are not mediated by stimulation of the VEGF pathway. Intravitreal administration of ADP expanded the neovascular area in a mouse model of choroidal neovascularization. Single-cell transcriptomics from human choroidal datasets show the expression of P2RY1, but not other ADP receptors, in EC with a pattern similar to VEGFR2. Although ADP has been reported to be a growth inhibitor for vascular EC, here we describe its growth-stimulating effects for BCEC and other eye-derived EC.

Hypertension, a major cause of cardiomyopathy, is one of the most critical risk factors for heart failure and mortality worldwide. Loss of metabolic flexibility of cardiomyocytes is one of the major causes of heart failure. Although Catestatin (CST) treatment is known to be both hypotensive and cardioprotective, its effect on cardiac metabolism is unknown. In this study, we undertook a transcriptomic approach to identify differentially expressed genes that were filtered using Boolean implication relationships to develop a model of gene regulation in saline or CST-supplemented CST knockout (CST-KO) mice. The analysis revealed a set of gene signatures (fibroblast, cardiomyocyte, and macrophage) rescued after CST supplemented CST-KO mice compared to wild-type. Furthermore, we independently validated these gene signature models using publicly available patient datasets. Since the gene signature includes genes related to glucose, fatty acid metabolism, and mitochondrial function, we assessed the glucose and fatty acid uptake after CST treatment. We found that CST treatment can restore the cardiac metabolic inflexibility in CST-KO heart due to the metabolic shift of glucose utilization to fatty acid as energy source. Binding studies after immunoprecipitation and mass spectrometry revealed CST binding with ATP synthase, supported by molecular simulation and computational modeling that predicted CST binding to α/β subunit of ATP synthase. Colocalization of CST with mitochondria and increased mitochondrial membrane potential and ATP production upon CST treatment in neonatal cardiomyocytes further exhibit CST as a key regulator of cardiac metabolism and mitochondrial function.

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.

Chromogranins are pro-hormone secretory proteins released from neuroendocrine cells, with effects on control of blood pressure. We conducted a genome-wide association study for plasma catestatin, the catecholamine release inhibitory peptide derived from chromogranin A (CHGA), and other CHGA- or chromogranin B (CHGB)-related peptides, in 545 US and 1252 Australian subjects. This identified loci on chromosomes 4q35 and 5q34 affecting catestatin concentration (P = 3.40 × 10⁻³⁰ for rs4253311 and 1.85 × 10⁻¹⁹ for rs2731672, respectively). Genes in these regions include the proteolytic enzymes kallikrein (KLKB1) and Factor XII (F12). In chromaffin cells, CHGA and KLKB1 proteins co-localized in catecholamine storage granules. In vitro, kallikrein cleaved recombinant human CHGA to catestatin, verified by mass spectrometry. The peptide identified from this digestion (CHGA_360–373) selectively inhibited nicotinic cholinergic stimulated catecholamine release from chromaffin cells. A proteolytic cascade involving kallikrein and Factor XII cleaves chromogranins to active compounds both in vivo and in vitro.

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 ∼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.