Incidence of microvascular dysfunction is increased in hyperlipidemic mice, reducing cerebral blood flow and impairing remote memory.

Introduction: The development of cognitive dysfunction is not necessarily associated with diet-induced obesity. We hypothesized that cognitive dysfunction might require additional vascular damage, for example, in atherosclerotic mice. Methods: We induced atherosclerosis in male C57BL/6N mice by injecting AAV-PCSK9DY (2x1011 VG) and feeding them a cholesterol-rich Western diet. After 3 months, mice were examined for cognition using Barnes maze procedure and for cerebral blood flow. Cerebral vascular morphology was examined by immunehistology. Results: In AAV-PCSK9DY-treated mice, plaque burden, plasma cholesterol, and triglycerides are elevated. RNAseq analyses followed by KEGG annotation show increased expression of genes linked to inflammatory processes in the aortas of these mice. In AAV-PCSK9DY-treated mice learning was delayed and long-term memory impaired. Blood flow was reduced in the cingulate cortex (-17%), caudate putamen (-15%), and hippocampus (-10%). Immunohistological studies also show an increased incidence of string vessels and pericytes (CD31/Col IV staining) in the hippocampus accompanied by patchy blood-brain barrier leaks (IgG staining) and increased macrophage infiltrations (CD68 staining). Discussion: We conclude that the hyperlipidemic PCSK9DY mouse model can serve as an appropriate approach to induce microvascular dysfunction that leads to reduced blood flow in the hippocampus, which could explain the cognitive dysfunction in these mice.

Secreted Protein Profiling of Human Aortic Smooth Muscle Cells Identifies Vascular Disease Associations.

BACKGROUND: Smooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular disease, the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a contractile to a synthetic phenotype characterized by an increased proliferation, migration, production of ECM (extracellular matrix) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of cardiovascular disease, including coronary artery disease, stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies. METHODS: Using human aortic SMCs from 123 multiancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted liquid chromatography-tandem mass spectrometry-based proteomic analysis of the conditioned media. RESULTS: We measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 (latent-transforming growth factor beta-binding protein 1) in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions. CONCLUSIONS: Our results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.

MRAS in coronary artery disease-Unchartered territory.

Genome-wide association studies (GWAS) have identified coronary artery disease (CAD) susceptibility locus on chromosome 3q22.3. This locus contains a cluster of several genes that includes muscle rat sarcoma virus (MRAS). Common MRAS variants are also associated with CAD causing risk factors such as hypertension, dyslipidemia, obesity, and type II diabetes. The MRAS gene is an oncogene that encodes a membrane-bound small GTPase. It is involved in a variety of signaling pathways, regulating cell differentiation and cell survival (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase) as well as acute phase response signaling (tumor necrosis factor [TNF] and interleukin 6 [IL6] signaling). In this review, we will summarize the role of genetic MRAS variants in the etiology of CAD and its comorbidities with the focus on tissue distribution of MRAS isoforms, cell type/tissue specificity, and mode of action of single nucleotide variants in MRAS associated complex traits. Finally, we postulate that CAD risk variants in the MRAS locus are specific to smooth muscle cells and lead to higher levels of MRAS, particularly in arterial and cardiac tissue, resulting in MAPK-dependent tissue hypertrophy or hyperplasia.

Beyond the Basics: Unraveling the Complexity of Coronary Artery Calcification.

Coronary artery calcification (CAC) is mainly associated with coronary atherosclerosis, which is an indicator of coronary artery disease (CAD). CAC refers to the accumulation of calcium phosphate deposits, classified as micro- or macrocalcifications, that lead to the hardening and narrowing of the coronary arteries. CAC is a strong predictor of future cardiovascular events, such as myocardial infarction and sudden death. Our narrative review focuses on the pathophysiology of CAC, exploring its link to plaque vulnerability, genetic factors, and how race and sex can affect the condition. We also examined the connection between the gut microbiome and CAC, and the impact of genetic variants on the cellular processes involved in vascular calcification and atherogenesis. We aimed to thoroughly analyze the existing literature to improve our understanding of CAC and its potential clinical and therapeutic implications.

Autoantibodies against the chemokine receptor 3 predict cardiovascular risk.

BACKGROUND AND AIMS: Chronic inflammation and autoimmunity contribute to cardiovascular (CV) disease. Recently, autoantibodies (aAbs) against the CXC-motif-chemokine receptor 3 (CXCR3), a G protein-coupled receptor with a key role in atherosclerosis, have been identified. The role of anti-CXCR3 aAbs for CV risk and disease is unclear. METHODS: Anti-CXCR3 aAbs were quantified by a commercially available enzyme-linked immunosorbent assay in 5000 participants (availability: 97.1%) of the population-based Gutenberg Health Study with extensive clinical phenotyping. Regression analyses were carried out to identify determinants of anti-CXCR3 aAbs and relevance for clinical outcome (i.e. all-cause mortality, cardiac death, heart failure, and major adverse cardiac events comprising incident coronary artery disease, myocardial infarction, and cardiac death). Last, immunization with CXCR3 and passive transfer of aAbs were performed in ApoE(-/-) mice for preclinical validation. RESULTS: The analysis sample included 4195 individuals (48% female, mean age 55.5 ± 11 years) after exclusion of individuals with autoimmune disease, immunomodulatory medication, acute infection, and history of cancer. Independent of age, sex, renal function, and traditional CV risk factors, increasing concentrations of anti-CXCR3 aAbs translated into higher intima-media thickness, left ventricular mass, and N-terminal pro-B-type natriuretic peptide. Adjusted for age and sex, anti-CXCR3 aAbs above the 75th percentile predicted all-cause death [hazard ratio (HR) (95% confidence interval) 1.25 (1.02, 1.52), P = .029], driven by excess cardiac mortality [HR 2.51 (1.21, 5.22), P = .014]. A trend towards a higher risk for major adverse cardiac events [HR 1.42 (1.0, 2.0), P = .05] along with increased risk of incident heart failure [HR per standard deviation increase of anti-CXCR3 aAbs: 1.26 (1.02, 1.56), P = .03] may contribute to this observation. Targeted proteomics revealed a molecular signature of anti-CXCR3 aAbs reflecting immune cell activation and cytokine-cytokine receptor interactions associated with an ongoing T helper cell 1 response. Finally, ApoE(-/-) mice immunized against CXCR3 displayed increased anti-CXCR3 aAbs and exhibited a higher burden of atherosclerosis compared to non-immunized controls, correlating with concentrations of anti-CXCR3 aAbs in the passive transfer model. CONCLUSIONS: In individuals free of autoimmune disease, anti-CXCR3 aAbs were abundant, related to CV end-organ damage, and predicted all-cause death as well as cardiac morbidity and mortality in conjunction with the acceleration of experimental atherosclerosis.

Secreted protein profiling of human aortic smooth muscle cells identifies vascular disease associations.

Background: Smooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular diseases (CVD), the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a "contractile" to a "synthetic" phenotype characterized by an increased proliferation, migration, production of extracellular matrix (ECM) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of CVD, including coronary artery disease (CAD), stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies (GWAS). Methods: Using human aortic SMCs from 123 multi-ancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of the conditioned media. Results: We measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping (pQTL) and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions. Conclusions: Our results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.

ADAMTS-7 Modulates Atherosclerotic Plaque Formation by Degradation of TIMP-1.

BACKGROUND: The ADAMTS7 locus was genome-wide significantly associated with coronary artery disease. Lack of the ECM (extracellular matrix) protease ADAMTS-7 (A disintegrin and metalloproteinase-7) was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating the risk of atherosclerosis. METHODS: Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, coimmunoprecipitation, and Förster resonance energy transfer-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. RESULTS: In humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of Timp-1 (tissue inhibitor of metalloprotease-1). In coimmunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target MMP-9 (matrix metalloprotease-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after a Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. To facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim of decreasing TIMP-1 degradation, we designed a Förster resonance energy transfer-based assay targeting the ADAMTS-7 catalytic site. CONCLUSIONS: ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with coronary artery disease. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for the reduction of atherosclerosis-related events.

pyHeart4Fish: Chamber-specific heart phenotype quantification of zebrafish in high-content screens.

Cardiovascular diseases (CVDs) are the leading cause of death. Of CVDs, congenital heart diseases are the most common congenital defects, with a prevalence of 1 in 100 live births. Despite the widespread knowledge that prenatal and postnatal drug exposure can lead to congenital abnormalities, the developmental toxicity of many FDA-approved drugs is rarely investigated. Therefore, to improve our understanding of drug side effects, we performed a high-content drug screen of 1,280 compounds using zebrafish as a model for cardiovascular analyses. Zebrafish are a well-established model for CVDs and developmental toxicity. However, flexible open-access tools to quantify cardiac phenotypes are lacking. Here, we provide pyHeart4Fish, a novel Python-based, platform-independent tool with a graphical user interface for automated quantification of cardiac chamber-specific parameters, such as heart rate (HR), contractility, arrhythmia score, and conduction score. In our study, about 10.5% of the tested drugs significantly affected HR at a concentration of 20 µM in zebrafish embryos at 2 days post-fertilization. Further, we provide insights into the effects of 13 compounds on the developing embryo, including the teratogenic effects of the steroid pregnenolone. In addition, analysis with pyHeart4Fish revealed multiple contractility defects induced by seven compounds. We also found implications for arrhythmias, such as atrioventricular block caused by chloropyramine HCl, as well as (R)-duloxetine HCl-induced atrial flutter. Taken together, our study presents a novel open-access tool for heart analysis and new data on potentially cardiotoxic compounds.

AFM-based nanoindentation indicates an impaired cortical stiffness in the AAV-PCSK9DY atherosclerosis mouse model.

Investigating atherosclerosis and endothelial dysfunction has mainly become established in genetically modified ApoE-/- or LDL-R-/- mice transgenic models. A new AAV-PCSK9DYDY mouse model with no genetic modification has now been reported as an alternative atherosclerosis model. Here, we aimed to employ this AAV-PCSK9DY mouse model to quantify the mechanical stiffness of the endothelial surface, an accepted hallmark for endothelial dysfunction and forerunner for atherosclerosis. Ten-week-old male C57BL/6 N mice were injected with AAV-PCSK9DY (0.5, 1 or 5 × 1011 VG) or saline as controls and fed with Western diet (1.25% cholesterol) for 3 months. Total cholesterol (TC) and triglycerides (TG) were measured after 6 and 12 weeks. Aortic sections were used for atomic force microscopy (AFM) measurements or histological analysis using Oil-Red-O staining. Mechanical properties of in situ endothelial cells derived from ex vivo aorta preparations were quantified using AFM-based nanoindentation. Compared to controls, an increase in plasma TC and TG and extent of atherosclerosis was demonstrated in all groups of mice in a viral load-dependent manner. Cortical stiffness of controls was 1.305 pN/nm and increased (10%) in response to viral load (≥ 0.5 × 1011 VG) and positively correlated with the aortic plaque content and plasma TC and TG. For the first time, we show changes in the mechanical properties of the endothelial surface and thus the development of endothelial dysfunction in the AAV-PCSK9DY mouse model. Our results demonstrate that this model is highly suitable and represents a good alternative to the commonly used transgenic mouse models for studying atherosclerosis and other vascular pathologies.

Apolipoprotein E derived from CD11c+ cells ameliorates atherosclerosis.

Atherosclerosis is studied in models with dysfunctional lipid homeostasis-predominantly the ApoE-/- mouse. The role of antigen-presenting cells (APCs) for lipid homeostasis is not clear. Using a LacZ reporter mouse, we showed that CD11c+ cells were enriched in aortae of ApoE-/- mice. Systemic long-term depletion of CD11c+ cells in ApoE-/- mice resulted in significantly increased plaque formation associated with reduced serum ApoE levels. In CD11ccre+ApoEfl/fl and Albumincre+ApoEfl/fl mice, we could show that ≈70% of ApoE is liver-derived and ≈25% originates from CD11c+ cells associated with significantly increased atherosclerotic plaque burden in both strains. Exposure to acLDL promoted cholesterol efflux from CD11c+ cells and cell-specific deletion of ApoE resulted in increased inflammation reflected by increased IL-1ß serum levels. Our results determined for the first time the level of ApoE originating from CD11c+ cells and demonstrated that CD11c+ cells ameliorate atherosclerosis by the secretion of ApoE.

Effect of Differences in the Microbiome of Cyp17a1-Deficient Mice on Atherosclerotic Background.

CYP17A1 is a cytochrome P450 enzyme that has 17-alpha-hydroxylase and C17,20-lyase activities. Cyp17a11 deficiency is associated with high body mass and visceral fat deposition in atherosclerotic female ApoE knockout (KO, d/d or -/-) mice. In the present study, we aimed to investigate the effects of diet and Cyp17a1 genotype on the gut microbiome. Female Cyp17a1 (d/d) × ApoE (d/d) (DKO) and ApoE (d/d) (controls) were fed either standard chow or a Western-type diet (WTD), and we demonstrated the effects of genetics and diet on the body mass of the mice and composition of their gut microbiome. We found a significantly lower alpha diversity after accounting for the ecological network structure in DKO mice and WTD-fed mice compared with chow-fed ApoE(d/d). Furthermore, we found a strong significant positive association of the Firmicutes vs. Bacteroidota ratio with body mass and the circulating total cholesterol and triglyceride concentrations of the mice when feeding the WTD, independent of the Cyp17a1 genotype. Further pathway enrichment and network analyses revealed a substantial effect of Cyp17a1 genotype on associated cardiovascular and obesity-related pathways involving aspartate and L-arginine. Future studies are required to validate these findings and further investigate the role of aspartate/L-arginine pathways in the obesity and body fat distribution in our mouse model.

sGC Activity and Regulation of Blood Flow in a Zebrafish Model System.

Soluble guanylyl cyclase (sGC) protein is a heterodimer formed by two subunits encoded by GUCY1A1 and GUCY1B1 genes. The chromosomal locus 4q32.1 harbors both of these genes, which has been previously significantly associated with coronary artery disease, myocardial infarction, and high blood pressure. Blood pressure is influenced by both the environment and genetics and is complemented by several biological pathways. The underlying mechanisms associated with this locus and its genes still need to be investigated. In the current study, we aimed to establish the zebrafish as a model organism to investigate the mechanisms surrounding sGC activity and blood pressure. A zebrafish mutant gucy1a1 line was generated using the CRISPR-Cas9 system by inducing a 4-bp deletion frameshift mutation. This mutation resulted in a reduction of gucy1a1 expression in both heterozygote and homozygote zebrafish. Blood flow parameters (blood flow, arterial pulse, linear velocity, and vessel diameter) investigated in the gucy1a1 mutants showed a significant increase in blood flow and linear velocity, which was augmented in the homozygotes. No significant differences were observed for the blood flow parameters measured from larvae with individual morpholino downregulation of gucy1a1 and gucy1b1, but an increase in blood flow and linear velocity was observed after co-morpholino downregulation of both genes. In addition, the pharmacological sGC stimulator BAY41-2272 rescued the impaired cGMP production in the zebrafish gucy1a1 ± mutant larvae. Downregulation of cct7 gene did not show any significant difference on the blood flow parameters in both wild-type and gucy1a1 ± background larvae. In summary, we successfully established a zebrafish platform for investigating sGC-associated pathways and underlying mechanisms in depth. This model system will have further applications, including for potential drug screening experiments.

In silico candidate variant and gene identification using inbred mouse strains.

Mice are the most widely used animal model to study genotype to phenotype relationships. Inbred mice are genetically identical, which eliminates genetic heterogeneity and makes them particularly useful for genetic studies. Many different strains have been bred over decades and a vast amount of phenotypic data has been generated. In addition, recently whole genome sequencing-based genome-wide genotype data for many widely used inbred strains has been released. Here, we present an approach for in silico fine-mapping that uses genotypic data of 37 inbred mouse strains together with phenotypic data provided by the user to propose candidate variants and genes for the phenotype under study. Public genome-wide genotype data covering more than 74 million variant sites is queried efficiently in real-time to provide those variants that are compatible with the observed phenotype differences between strains. Variants can be filtered by molecular consequences and by corresponding molecular impact. Candidate gene lists can be generated from variant lists on the fly. Fine-mapping together with annotation or filtering of results is provided in a Bioconductor package called MouseFM. In order to characterize candidate variant lists under various settings, MouseFM was applied to two expression data sets across 20 inbred mouse strains, one from neutrophils and one from CD4+ T cells. Fine-mapping was assessed for about 10,000 genes, respectively, and identified candidate variants and haplotypes for many expression quantitative trait loci (eQTLs) reported previously based on these data. For albinism, MouseFM reports only one variant allele of moderate or high molecular impact that only albino mice share: a missense variant in the Tyr gene, reported previously to be causal for this phenotype. Performing in silico fine-mapping for interfrontal bone formation in mice using four strains with and five strains without interfrontal bone results in 12 genes. Of these, three are related to skull shaping abnormality. Finally performing fine-mapping for dystrophic cardiac calcification by comparing 9 strains showing the phenotype with eight strains lacking it, we identify only one moderate impact variant in the known causal gene Abcc6. In summary, this illustrates the benefit of using MouseFM for candidate variant and gene identification.

The CAD risk locus 9p21 increases the risk of vascular calcification in an iPSC-derived VSMC model.

BACKGROUND: Coronary artery disease (CAD) is the leading cause of death worldwide. Chromosome locus 9p21 was the first to be associated with increased risk of CAD and coronary artery calcification (CAC). Vascular calcification increases the risk for CAD. Vascular smooth muscle cells (VSMCs) are one of the major cell types involved in the development of vascular calcification. METHODS: So far, mainly animal models or primary SMCs have been used to model human vascular calcification. In this study, a human in vitro assay using iPSC-derived VSMCs was developed to examine vascular calcification. Human iPSCs were derived from a healthy non-risk (NR) and risk (R) donor carrying SNPs in the 9p21 locus. Additionally, 9p21 locus knockouts of each donor iPSC line (NR and R) were used. Following differentiation, the iPSC-derived VSMCs were characterized based on cell type, proliferation, and migration rate, along with calcium phosphate (CaP) deposits. CaP deposits were confirmed using Calcein and Alizarin Red S staining and then quantified. RESULTS: The data demonstrated significantly more proliferation, migration, and CaP deposition in VSMCs derived from the R and both KO iPSC lines than in those derived from the NR line. Molecular analyses confirmed upregulation of calcification markers. These results are consistent with recent data demonstrating increased calcification when the 9p21 murine ortholog is knocked-out. CONCLUSION: Therefore, in conclusion, genetic variation or deletion of the CAD risk locus leads to an increased risk of vascular calcification. This in vitro human iPSC model of calcification could be used to develop new drug screening strategies to combat CAC.

Induced Pluripotent Stem Cells (iPSCs) in Vascular Research: from Two- to Three-Dimensional Organoids.

Stem cell technology has been around for almost 30 years and in that time has grown into an enormous field. The stem cell technique progressed from the first successful isolation of mammalian embryonic stem cells (ESCs) in the 1990s, to the production of human induced-pluripotent stem cells (iPSCs) in the early 2000s, to finally culminate in the differentiation of pluripotent cells into highly specialized cell types, such as neurons, endothelial cells (ECs), cardiomyocytes, fibroblasts, and lung and intestinal cells, in the last decades. In recent times, we have attained a new height in stem cell research whereby we can produce 3D organoids derived from stem cells that more accurately mimic the in vivo environment. This review summarizes the development of stem cell research in the context of vascular research ranging from differentiation techniques of ECs and smooth muscle cells (SMCs) to the generation of vascularized 3D organoids. Furthermore, the different techniques are critically reviewed, and future applications of current 3D models are reported.

Studies in Zebrafish Demonstrate That CNNM2 and NT5C2 Are Most Likely the Causal Genes at the Blood Pressure-Associated Locus on Human Chromosome 10q24.32.

Background: Globally, high blood pressure (BP) is the most important risk factor for cardiovascular disease. Several genome-wide association studies (GWAS) have identified variants associated with BP traits at more than 535 chromosomal loci with genome-wide significance. The post-GWAS challenge is to annotate the most likely causal gene(s) at each locus. Chromosome 10q24.32 is a locus associated with BP that encompasses five genes: CYP17A1, BORCS7, AS3MT, CNNM2, and NT5C2 and warrants investigation to determine the specific gene or genes responsible for the phenotype. Aim: To identify the most likely causal gene(s) associated with BP at the 10q24.32 locus using zebrafish as an animal model. Results: We report significantly higher blood flow, increased arterial pulse, and elevated linear velocity in zebrafish larvae with cnnm2 and nt5c2 knocked down using gene-specific splice modification transcriptional morpholinos, compared with controls. No differences in blood-flow parameters were observed after as3mt, borcs7, or cyp17a1 knockdown. There was no effect on vessel diameter in animals with any of the four genes knocked down. At the molecular level, expression of hypertension markers (crp and ace) was significantly increased in cnnm2 and nt5c2 knockdown larvae. Further, the results obtained by morpholino knockdown were validated using zebrafish knockout (KO) lines with cnnm2 and nt5c2 deficiency, again resulting in higher blood flow, increased arterial pulse, and elevated linear velocity. Analysis of nt5c2a KO larvae demonstrated that lack of this gene resulted in reduced expression of cnnm2a, with reciprocal downregulation of nt5c2a in cnnm2a KO larvae. Staining of whole-blood smears from nt5c2 mutants revealed that KO of this gene might be associated with an acute lymphoblastic leukemia phenotype, consistent with literature reports. Additional experiments were designed based on previous literature on cnnm2a mutant zebrafish revealed impaired renal function, high levels of renin, and significantly increased expression of the ren gene, leading us to hypothesize that the observed elevated blood-flow parameters may be attributable to triggering of the renin-angiotensin-aldosterone signaling pathway. Conclusion: Our zebrafish data establish CNNM2 and NT5C2 as the most likely causal genes at the 10q24.32 BP locus and indicate that they trigger separate downstream mechanistic pathways.