Perturbational phenotyping of human blood cells reveals genetically determined latent traits associated with subsets of common diseases.

Although genome-wide association studies (GWAS) have successfully linked genetic risk loci to various disorders, identifying underlying cellular biological mechanisms remains challenging due to the complex nature of common diseases. We established a framework using human peripheral blood cells, physical, chemical and pharmacological perturbations, and flow cytometry-based functional readouts to reveal latent cellular processes and performed GWAS based on these evoked traits in up to 2,600 individuals. We identified 119 genomic loci implicating 96 genes associated with these cellular responses and discovered associations between evoked blood phenotypes and subsets of common diseases. We found a population of pro-inflammatory anti-apoptotic neutrophils prevalent in individuals with specific subsets of cardiometabolic disease. Multigenic models based on this trait predicted the risk of developing chronic kidney disease in type 2 diabetes patients. By expanding the phenotypic space for human genetic studies, we could identify variants associated with large effect response differences, stratify patients and efficiently characterize the underlying biology.

Blood Pressure-Associated Genetic Variants in the Natriuretic Peptide Receptor 1 Gene Modulate Guanylate Cyclase Activity.

BACKGROUND: Human genetic variation in the NPR1 (natriuretic peptide receptor 1 gene, encoding NPR-A, atrial natriuretic peptide receptor 1) was recently shown to affect blood pressure (BP). NPR-A catalyzes the intracellular conversion of guanosine triphosphate to cGMP (cyclic 3',5'-guanosine monophosphate) on binding of ANP, BNP (atrial or brain natriuretic peptide). Increased levels of cGMP decrease BP by inducing natriuresis, diuresis, and vasodilation. METHODS: We performed a meta-analysis of low-frequency and rare NPR1 variants for BP association in up to 491 584 unrelated individuals. To examine whether the identified BP-associated variants affect NPR-A function, the cGMP response to ANP and BNP was measured in cells expressing wild-type NPR1 and cells expressing the NPR1 variants. RESULTS: In this study, we identified BP associations of 3 amino acid altering variants of NPR1. The minor alleles of rs35479618 (p.E967K, gnomAD non-Finnish European allele frequency 0.017) and rs116245325 (p.L1034F, allele frequency 0.0007) were associated with higher BP (P=4.0×10-25 and P=9.9×10-8, respectively), while the minor allele of rs61757359 (p.G541S, allele frequency 0.003) was associated with lower BP (P=1.8×10-9). Cells transiently expressing 967K or 1034F NPR-A displayed decreased cGMP production in response to ANP and BNP (all P<10-6), while cells expressing 541S NPR-A produced more cGMP compared with cells expressing wild-type NPR-A (P≤4.13×10-5 for ANP and P≤4.24×10-3 for BNP). CONCLUSIONS: In summary, the loss or gain of guanylate cyclase activity for these NPR1 allelic variants could explain the higher or lower BP observed for carriers in large population-based studies.

MicroRNA-425 and microRNA-155 cooperatively regulate atrial natriuretic peptide expression and cGMP production.

AIMS: Atrial natriuretic peptide (ANP), secreted primarily by atrial cardiomyocytes, decreases blood pressure by raising cyclic 3',5'-guanosine monophosphate (cGMP) levels and inducing vasorelaxation, natriuresis, and diuresis. Raising the level of ANP has been shown to be an effective treatment for hypertension. To advance the future development of an anti-microRNA (miR) approach to increasing expression of ANP, we investigated the regulation of NPPA expression by two miRs: miR-425 and miR-155. We examined whether miR-425 and miR-155 have an additive effect on the expression and function of ANP. METHODS AND RESULTS: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were transfected with miR-425, miR-155, or a combination of the two miRs. Two days later, NPPA expression was measured using real time qPCR. Each of the miRs decreased NPPA expression over a wide range of concentrations, with a significant reduction at concentrations as low as 1 nM. The combination of miR-425 and miR-155 reduced NPPA expression to a greater extent than either miR-425 or miR-155 alone. An in vitro assay was developed to study the potential biological significance of the miR-induced decrease in NPPA expression. The cooperative effect of miR-425 and miR-155 on NPPA expression was associated with a significant decrease in cGMP levels. CONCLUSIONS: These data demonstrate that miR-425 and miR-155 regulate NPPA expression in a cooperative manner. Targeting both miRNAs with anti-miRs (possibly at submaximal concentrations) might prove to be a more effective strategy to modulate ANP levels, and thus blood pressure, than targeting either miRNA alone.

Decreased Soluble Guanylate Cyclase Contributes to Cardiac Dysfunction Induced by Chronic Doxorubicin Treatment in Mice.

AIMS: The use of doxorubicin, a potent chemotherapeutic agent, is limited by cardiotoxicity. We tested the hypothesis that decreased soluble guanylate cyclase (sGC) enzyme activity contributes to the development of doxorubicin-induced cardiotoxicity. RESULTS: Doxorubicin administration (20 mg/kg, intraperitoneally [IP]) reduced cardiac sGC activity in wild-type (WT) mice. To investigate whether decreased sGC activity contributes to doxorubicin-induced cardiotoxicity, we studied mice with cardiomyocyte-specific deficiency of the sGC α1-subunit (mice with cardiomyocyte-specific deletion of exon 6 of the sGCα1 allele [sGCα1-/-CM]). After 12 weeks of doxorubicin administration (2 mg/kg/week IP), left ventricular (LV) systolic dysfunction was greater in sGCα1-/-CM than WT mice. To further assess whether reduced sGC activity plays a pathogenic role in doxorubicin-induced cardiotoxicity, we studied a mouse model in which decreased cardiac sGC activity was induced by cardiomyocyte-specific expression of a dominant negative sGCα1 mutant (DNsGCα1) upon doxycycline removal (Tet-off). After 8 weeks of doxorubicin administration, DNsGCα1tg/+, but not WT, mice displayed LV systolic dysfunction and dilatation. The difference in cardiac function and remodeling between DNsGCα1tg/+ and WT mice was even more pronounced after 12 weeks of treatment. Further impairment of cardiac function was attenuated when DNsGCα1 gene expression was inhibited (beginning at 8 weeks of doxorubicin treatment) by administering doxycycline. Furthermore, doxorubicin-associated reactive oxygen species generation was higher in sGCα1-deficient than WT hearts. Innovation and Conclusion: These data demonstrate that a reduction in cardiac sGC activity worsens doxorubicin-induced cardiotoxicity in mice and identify sGC as a potential therapeutic target. Various pharmacological sGC agonists are in clinical development or use and may represent a promising approach to limit doxorubicin-associated cardiotoxicity. Antioxid. Redox Signal. 26, 153-164.

Androgen-sensitive hypertension associated with soluble guanylate cyclase-α1 deficiency is mediated by 20-HETE.

Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1 (-/-) mice) display sex- and strain-specific hypertension: male but not female sGCα1 (-/-) mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex- and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGCα1 (-/-)S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGCα1 (-/-)S6 than of male sGCα1 (-/-)B6 mice. Furthermore, treating male sGCα1 (-/-)S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background- and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling.

Meox2/Tcf15 heterodimers program the heart capillary endothelium for cardiac fatty acid uptake.

BACKGROUND: Microvascular endothelium in different organs is specialized to fulfill the particular needs of parenchymal cells. However, specific information about heart capillary endothelial cells (ECs) is lacking. METHODS AND RESULTS: Using microarray profiling on freshly isolated ECs from heart, brain, and liver, we revealed a genetic signature for microvascular heart ECs and identified Meox2/Tcf15 heterodimers as novel transcriptional determinants. This signature was largely shared with skeletal muscle and adipose tissue endothelium and was enriched in genes encoding fatty acid (FA) transport-related proteins. Using gain- and loss-of-function approaches, we showed that Meox2/Tcf15 mediate FA uptake in heart ECs, in part, by driving endothelial CD36 and lipoprotein lipase expression and facilitate FA transport across heart ECs. Combined Meox2 and Tcf15 haplodeficiency impaired FA uptake in heart ECs and reduced FA transfer to cardiomyocytes. In the long term, this combined haplodeficiency resulted in impaired cardiac contractility. CONCLUSIONS: Our findings highlight a regulatory role for ECs in FA transfer to the heart parenchyma and unveil 2 of its intrinsic regulators. Our insights could be used to develop new strategies based on endothelial Meox2/Tcf15 targeting to modulate FA transfer to the heart and remedy cardiac dysfunction resulting from altered energy substrate usage.

Atrial natriuretic peptide is negatively regulated by microRNA-425.

Numerous common genetic variants have been linked to blood pressure, but no underlying mechanism has been elucidated. Population studies have revealed that the variant rs5068 (A/G) in the 3' untranslated region of NPPA, the gene encoding atrial natriuretic peptide (ANP), is associated with blood pressure. We selected individuals on the basis of rs5068 genotype (AG vs. AA) and fed them a low- or high-salt diet for 1 week, after which they were challenged with an intravenous saline infusion. On both diets, before and after saline administration, ANP levels were up to 50% higher in AG individuals than in AA individuals, a difference comparable to the changes induced by high-salt diet or saline infusion. In contrast, B-type natriuretic peptide levels did not differ by rs5068 genotype. We identified a microRNA, miR-425, that is expressed in human atria and ventricles and is predicted to bind the sequence spanning rs5068 for the A, but not the G, allele. miR-425 silenced NPPA mRNA in an allele-specific manner, with the G allele conferring resistance to miR-425. This study identifies miR-425 as a regulator of ANP production, raising the possibility that miR-425 antagonists could be used to treat disorders of salt overload, including hypertension and heart failure.

Increased cardiac myocyte PDE5 levels in human and murine pressure overload hypertrophy contribute to adverse LV remodeling.

BACKGROUND: The intracellular second messenger cGMP protects the heart under pathological conditions. We examined expression of phosphodiesterase 5 (PDE5), an enzyme that hydrolyzes cGMP, in human and mouse hearts subjected to sustained left ventricular (LV) pressure overload. We also determined the role of cardiac myocyte-specific PDE5 expression in adverse LV remodeling in mice after transverse aortic constriction (TAC). METHODOLOGY/PRINCIPAL FINDINGS: In patients with severe aortic stenosis (AS) undergoing valve replacement, we detected greater myocardial PDE5 expression than in control hearts. We observed robust expression in scattered cardiac myocytes of those AS patients with higher LV filling pressures and BNP serum levels. Following TAC, we detected similar, focal PDE5 expression in cardiac myocytes of C57BL/6NTac mice exhibiting the most pronounced LV remodeling. To examine the effect of cell-specific PDE5 expression, we subjected transgenic mice with cardiac myocyte-specific PDE5 overexpression (PDE5-TG) to TAC. LV hypertrophy and fibrosis were similar as in WT, but PDE5-TG had increased cardiac dimensions, and decreased dP/dtmax and dP/dtmin with prolonged tau (P<0.05 for all). Greater cardiac dysfunction in PDE5-TG was associated with reduced myocardial cGMP and SERCA2 levels, and higher passive force in cardiac myocytes in vitro. CONCLUSIONS/SIGNIFICANCE: Myocardial PDE5 expression is increased in the hearts of humans and mice with chronic pressure overload. Increased cardiac myocyte-specific PDE5 expression is a molecular hallmark in hypertrophic hearts with contractile failure, and represents an important therapeutic target.

Placental growth factor increases regional myocardial blood flow and contractile function in chronic myocardial ischemia.

Placental growth factor (PlGF) has a distinct biological phenotype with a predominant proangiogenic role in disease without affecting quiescent vessels in healthy organs. We tested whether systemic administration of recombinant human (rh)PlGF improves regional myocardial blood flow (MBF) and systolic function recovery in a porcine chronic myocardial ischemia model. We implanted a flow-limiting stent in the proximal left anterior descending coronary artery and measured systemic hemodynamics, regional myocardial function using MRI, and blood flow using colored microspheres 4 wk later. Animals were then randomized in a blinded way to receive an infusion of rhPlGF (15 µg·kg(-1)·day(-1), n = 9) or PBS (control; n = 10) for 2 wk. At 8 wk, myocardial perfusion and function were reassessed. Infusion of rhPlGF transiently increased PlGF serum levels >30-fold (1,153 ± 180 vs. 33 ± 18 pg/ml at baseline, P < 0.001) without affecting systemic hemodynamics. From 4 to 8 wk, rhPlGF increased regional MBF from 0.46 ± 0.11 to 0.85 ± 0.16 ml·min(-1)·g(-1), with a concomitant increase in systolic wall thickening from 11 ± 3% to 26 ± 5% in the ischemic area. In control animals, no significant changes from 4 to 8 wk were observed (MBF: 0.45 ± 0.07 to 0.49 ± 0.08 ml·min(-1)·g(-1) and systolic wall thickening: 14 ± 4% to 18 ± 1%). rhPlGF-induced functional improvement was accompanied by increased myocardial neovascularization, enhanced glycogen utilization, and reduced oxidative stress and cardiomyocyte apoptosis in the ischemic zone. In conclusion, systemic rhPlGF infusion significantly enhances regional blood flow and contractile function of the chronic ischemic myocardium without adverse effects. PlGF protein infusion may represent an attractive therapeutic strategy to increase myocardial perfusion and energetics in chronic ischemic cardiomyopathy.

Differential effects of progenitor cell populations on left ventricular remodeling and myocardial neovascularization after myocardial infarction.

OBJECTIVES: We compared biological repair after acute myocardial infarction (AMI) with selected porcine progenitor cell populations. BACKGROUND: Cell types and mechanisms responsible for myocardial repair after AMI remain uncertain. METHODS: In a blinded, randomized study, we infused autologous late-outgrowth endothelial progenitor cells (EPC) (n = 10, 34 +/- 22 x 10(6) CD29-31-positive, capable of tube formation), allogeneic green fluorescent peptide-labeled mesenchymal stem cells (MSC) (n = 11, 10 +/- 2 x 10(6) CD29-44-90-positive, capable of adipogenic and osteogenic differentiation), or vehicle (CON) (n = 12) in the circumflex artery 1 week after AMI. Systolic function (ejection fraction), left ventricular (LV) end-diastolic and end-systolic volumes, and infarct size were assessed with magnetic resonance imaging at 1 week and 7 weeks. Cell engraftment and vascular density were evaluated on postmortem sections. RESULTS: Recovery of LV ejection fraction from 1 to 7 weeks was similar between groups, but LV remodeling markedly differed with a greater increase of LV end-systolic volume in MSC and CON (+11 +/- 12 ml/m(2) and +7 +/- 8 ml/m(2) vs. -3 +/- 11 ml/m(2) in EPC, respectively, p = 0.04), and a similar trend was noted for LV end-diastolic volume (p = 0.09). After EPC, infarct size decreased more in segments with >50% infarct transmurality (p = 0.02 vs. MSC and CON) and was associated with a greater vascular density (p = 0.01). Late outgrowth EPCs secrete higher levels of the pro-angiogenic placental growth factor (733 [277 to 1,214] pg/10(6) vs. 59 [34 to 88] pg/10(6) cells in MSC, p = 0.03) and incorporate in neovessels in vivo. CONCLUSIONS: Infusion of late-outgrowth EPCs after AMI improves myocardial infarction remodeling via enhanced neovascularization but does not mediate cardiomyogenesis. Endothelial progenitor cell transfer might hold promise for heart failure prevention via pro-angiogenic or paracrine matrix-modulating effects.

Ventricular phosphodiesterase-5 expression is increased in patients with advanced heart failure and contributes to adverse ventricular remodeling after myocardial infarction in mice.

BACKGROUND: Ventricular expression of phosphodiesterase-5 (PDE5), an enzyme responsible for cGMP catabolism, is increased in human right ventricular hypertrophy, but its role in left ventricular (LV) failure remains incompletely understood. We therefore measured LV PDE5 expression in patients with advanced systolic heart failure and characterized LV remodeling after myocardial infarction in transgenic mice with cardiomyocyte-specific overexpression of PDE5 (PDE5-TG). METHODS AND RESULTS: Immunoblot and immunohistochemistry techniques revealed that PDE5 expression was greater in explanted LVs from patients with dilated and ischemic cardiomyopathy than in control hearts. To evaluate the impact of increased ventricular PDE5 levels on cardiac function, PDE5-TG mice were generated. Confocal and immunoelectron microscopy revealed increased PDE5 expression in cardiomyocytes, predominantly localized to Z-bands. At baseline, myocardial cGMP levels, cell shortening, and calcium handling in isolated cardiomyocytes and LV hemodynamic measurements were similar in PDE5-TG and wild-type littermates. Ten days after myocardial infarction, LV cGMP levels had increased to a greater extent in wild-type mice than in PDE5-TG mice (P<0.05). Ten weeks after myocardial infarction, LV end-systolic and end-diastolic volumes were larger in PDE5-TG than in wild-type mice (57+/-5 versus 39+/-4 and 65+/-6 versus 48+/-4 muL, respectively; P<0.01 for both). LV systolic dysfunction and diastolic dysfunction were more marked in PDE5-TG than in wild-type mice, associated with enhanced hypertrophy and reduced contractile function in isolated cardiomyocytes from remote myocardium. CONCLUSIONS: Increased PDE5 expression predisposes mice to adverse LV remodeling after myocardial infarction. Increased myocardial PDE5 expression in patients with advanced cardiomyopathy may contribute to the development of heart failure and represents an important therapeutic target.