Proteomic Profiles of Human Arterioles Isolated From Fresh Adipose Tissue or Following Overnight Storage.

Arterioles are key determinants of the total peripheral vascular resistance, which, in turn, is a key determinant of arterial blood pressure. However, the amount of protein available from one isolated human arteriole may be less than 5 µg, making proteomic analysis challenging. In addition, obtaining human arterioles requires manual dissection of unfrozen clinical specimens. This limits its feasibility, especially for powerful multicenter clinical studies in which clinical specimens need to be shipped overnight to a research laboratory for arteriole isolation. We performed a study to address low-input, test overnight tissue storage and develop a reference human arteriolar proteomic profile. In tandem mass tag proteomics, use of a booster channel consisting of human induced pluripotent stem cell-derived endothelial and vascular smooth muscle cells (1:5 ratio) increased the number of proteins detected in a human arteriole segment with a false discovery rate of <0.01 from 1051 to more than 3000. The correlation coefficient of proteomic profile was similar between replicate arterioles isolated freshly, following cold storage, or before and after the cold storage (1-way analysis of variance; P = .60). We built a human arteriolar proteomic profile consisting of 3832 proteins based on the analysis of 12 arteriole samples from 3 subjects. Of 1945 blood pressure-relevant proteins that we curated, 476 (12.5%) were detected in the arteriolar proteome, which was a significant overrepresentation (χ2 test; P < .05). These findings demonstrate that proteomic analysis is feasible with arterioles isolated from human adipose tissue following cold overnight storage and provide a reference human arteriolar proteome profile highly valuable for studies of arteriole-related traits.

Robustness of single-cell RNA-seq for identifying differentially expressed genes.

BACKGROUND: A common feature of single-cell RNA-seq (scRNA-seq) data is that the number of cells in a cell cluster may vary widely, ranging from a few dozen to several thousand. It is not clear whether scRNA-seq data from a small number of cells allow robust identification of differentially expressed genes (DEGs) with various characteristics. RESULTS: We addressed this question by performing scRNA-seq and poly(A)-dependent bulk RNA-seq in comparable aliquots of human induced pluripotent stem cells-derived, purified vascular endothelial and smooth muscle cells. We found that scRNA-seq data needed to have 2,000 or more cells in a cluster to identify the majority of DEGs that would show modest differences in a bulk RNA-seq analysis. On the other hand, clusters with as few as 50-100 cells may be sufficient for identifying the majority of DEGs that would have extremely small p values or transcript abundance greater than a few hundred transcripts per million in a bulk RNA-seq analysis. CONCLUSION: Findings of the current study provide a quantitative reference for designing studies that aim for identifying DEGs for specific cell clusters using scRNA-seq data and for interpreting results of such studies.

Isomer-specific effect of microRNA miR-29b on nuclear morphology.

Targeting mRNAs via seed region pairing is the canonical mechanism by which microRNAs (miRNAs) regulate cellular functions and disease processes. Emerging evidence suggests miRNAs might also act through other mechanisms. miRNA isomers that contain identical seed region sequences, such as miR-29a and miR-29b, provide naturally occurring, informative models for identifying those miRNA effects that are independent of seed region pairing. miR-29a and miR-29b are both expressed in HeLa cells, and miR-29b has been reported to localize to the nucleus in early mitosis because of unique nucleotide sequences on its 3' end. Here, we sought to better understand the mechanism of miR-29b nuclear localization and its function in cell division. We hypothesized that its nuclear localization may be facilitated by protein-miRNA interactions unique to miR-29b. Specific blockade of miR-29b resulted in striking nuclear irregularities not observed following miR-29a blockade. We also observed that miR-29b, but not miR-29a, is enriched in the nucleus and perinuclear clusters during mitosis. Targeted proteomic analysis of affinity-purified samples identified several proteins interacting with synthetic oligonucleotides mimicking miR-29b, but these proteins did not interact with miR-29a. One of these proteins, ADP/ATP translocase 2 (ANT2), known to be involved in mitotic spindle formation, colocalized with miR-29b in perinuclear clusters independently of Argonaute 2. Of note, ANT2 knockdown resulted in nuclear irregularities similar to those observed following miR-29b blockade and prevented nuclear uptake of endogenous miR-29b. Our findings reveal that miR-29 regulates nuclear morphology during mitosis and that this critical function is unique to the miR-29b isoform.

Genome-wide map of proximity linkage to renin proximal promoter in rat.

A challenge to understanding enhancer-gene relationships is that enhancers are not always sequentially close to the gene they regulate. Physical proximity mapping through sequencing can provide an unbiased view of the chromatin close to the proximal promoter of the renin gene ( Ren). Our objective was to determine genomic regions that physically interact with the renin proximal promoter, using two different genetic backgrounds, the Dahl salt sensitive and normotensive SS-13BN, which have been shown to have different regulation of plasma renin in vivo. The chromatin conformation capture method with sequencing focused at the Ren proximal promoter in rat-derived cardiac endothelial cells was used. Cells were fixed, chromatin close to the Ren promoter was captured, and fragments were sequenced. The clustering of mapped reads produced a genome-wide map of chromatin in contact with the Ren promoter. The largest number of contacts was found on chromosome 13, the chromosome with Ren, and contacts were found on all other chromosomes except chromosome X. These contacts were significantly enriched with genes positively correlated with Ren expression and with mapped quantitative trait loci associated with blood pressure, cardiovascular, and renal phenotypes. The results were reproducible in an independent biological replicate. The findings reported here represent the first map between a critical cardiovascular gene and physical interacting loci throughout the genome and will provide the basis for several new directions of research.

Mechanisms of Mas1 Receptor-Mediated Signaling in the Vascular Endothelium.

OBJECTIVE: Angiotensin II (AngII) has been shown to regulate angiogenesis and at high pathophysiological doses to cause vasoconstriction through the AngII receptor type 1. Angiotensin 1 to 7 (Ang-(1-7)) acting through the Mas1 receptor can act antagonistically to high pathophysiological levels of AngII by inducing vasodilation, whereas the effects of Ang-(1-7) signaling on angiogenesis are less defined. To complicate the matter, there is growing evidence that a subpressor dose of AngII produces phenotypes similar to Ang-(1-7). APPROACH AND RESULTS: This study shows that low-dose Ang-(1-7), acting through the Mas1 receptor, promotes angiogenesis and vasodilation similar to a low, subpressor dose of AngII acting through AngII receptor type 1. In addition, we show through in vitro tube formation that Ang-(1-7) augments the angiogenic response in rat microvascular endothelial cells. Using proteomic and genomic analyses, downstream components of Mas1 receptor signaling were identified, including Rho family of GTPases, phosphatidylinositol 3-kinase, protein kinase D1, mitogen-activated protein kinase, and extracellular signal-related kinase signaling. Further experimental antagonism of extracellular signal-related kinases 1/2 and p38 mitogen-activated protein kinase signaling inhibited endothelial tube formation and vasodilation when stimulated with equimolar, low doses of either AngII or Ang-(1-7). CONCLUSIONS: These results significantly expand the known Ang-(1-7)/Mas1 receptor signaling pathway and demonstrate an important distinction between the pathological effects of elevated and suppressed AngII compared with the beneficial effects of AngII normalization and Ang-(1-7) administration. The observed convergence of Ang-(1-7)/Mas1 and AngII/AngII receptor type 1 signaling at low ligand concentrations suggests a nuanced regulation in vasculature. These data also reinforce the importance of mitogen-activated protein kinase/extracellular signal-related kinase signaling in maintaining vascular function.

Mitochondria-regulated formation of endothelium-derived extracellular vesicles shifts the mediator of flow-induced vasodilation.

To examine the effect of endothelium-derived extracellular vesicles (eEVs) on the mediator of flow-induced dilation (FID), composition, formation, and functional effects on the mediator of FID were examined from two different eEV subtypes, one produced from ceramide, while the other was produced from plasminogen-activator inhibitor 1 (PAI-1). Using video microscopy, we measured internal-diameter changes in response to increases in flow in human adipose resistance arteries acutely exposed (30 min) to eEVs derived from cultured endothelial cells exposed to ceramide or PAI-1. FID was significantly impaired following exposure to 500K/ml (K = 1,000) of ceramide-induced eEVs (Cer-eEVs) but unaffected by 250K/ml. FID was reduced in the presence of PEG-catalase following administration of 250K/ml of Cer-eEVs and PAI-1 eEVs, whereas Nω-nitro-l-arginine methyl ester (l-NAME) had no effect. Pathway analysis following protein composition examination using liquid chromatography tandem mass spectrometry (LC-MS/MS) demonstrated that both subtypes were strongly linked to similar biological functions, primarily, mitochondrial dysfunction. Flow cytometry was used to quantify eEVs in the presence or absence of l-phenylalanine-4'-boronic acid (PBA) and mitochondria-targeted [93-boronophenyl)methyl]triphenyl-phosphonium (mito-PBA), cytosolic and mitochondrial-targeted antioxidants, respectively. eEV formation was significantly and dramatically reduced with mito-PBA treatment. In conclusion, eEVs have a biphasic effect, with higher doses impairing and lower doses shifting the mediator of FID from nitric oxide (NO) to hydrogen peroxide (H2O2). Despite differences in protein content, eEVs may alter vascular function in similar directions, regardless of the stimulus used for their formation. Furthermore, mitochondrial ROS production is required for the generation of these vesicles.NEW & NOTEWORTHY The vascular effect of endothelium-derived extracellular vesicles (eEVs) is biphasic, with higher doses decreasing the magnitude of flow-induced dilation (FID) compared with lower doses that shift the mediator of FID from nitric oxide to H2O2 eEVs may cause vascular dysfunction via similar pathways despite being formed from different stimuli, although both require mitochondrial reactive oxygen species for their formation.

Host genetic modifiers of nonproductive angiogenesis inhibit breast cancer.

PURPOSE: Multiple aspects of the tumor microenvironment (TME) impact breast cancer, yet the genetic modifiers of the TME are largely unknown, including those that modify tumor vascular formation and function. METHODS: To discover host TME modifiers, we developed a system called the Consomic/Congenic Xenograft Model (CXM). In CXM, human breast cancer cells are orthotopically implanted into genetically engineered consomic xenograft host strains that are derived from two parental strains with different susceptibilities to breast cancer. Because the genetic backgrounds of the xenograft host strains differ, whereas the inoculated tumor cells are the same, any phenotypic variation is due to TME-specific modifier(s) on the substituted chromosome (consomic) or subchromosomal region (congenic). Here, we assessed TME modifiers of growth, angiogenesis, and vascular function of tumors implanted in the SSIL2Rγ and SS.BN3IL2Rγ CXM strains. RESULTS: Breast cancer xenografts implanted in SS.BN3IL2Rγ (consomic) had significant tumor growth inhibition compared with SSIL2Rγ (parental control), despite a paradoxical increase in the density of blood vessels in the SS.BN3IL2Rγ tumors. We hypothesized that decreased growth of SS.BN3IL2Rγ tumors might be due to nonproductive angiogenesis. To test this possibility, SSIL2Rγ and SS.BN3IL2Rγ tumor vascular function was examined by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), micro-computed tomography (micro-CT), and ex vivo analysis of primary blood endothelial cells, all of which revealed altered vascular function in SS.BN3IL2Rγ tumors compared with SSIL2Rγ. Gene expression analysis also showed a dysregulated vascular signaling network in SS.BN3IL2Rγ tumors, among which DLL4 was differentially expressed and co-localized to a host TME modifier locus (Chr3: 95-131 Mb) that was identified by congenic mapping. CONCLUSIONS: Collectively, these data suggest that host genetic modifier(s) on RNO3 induce nonproductive angiogenesis that inhibits tumor growth through the DLL4 pathway.

Tumor Necrosis Factor α Regulates Endothelial Progenitor Cell Migration via CADM1 and NF-kB.

Shortly after the discovery of endothelial progenitor cells (EPCs) in 1997, many clinical trials were conducted using EPCs as a cellular based therapy with the goal of restoring damaged organ function by inducing growth of new blood vessels (angiogenesis). Results were disappointing, largely because the cellular and molecular mechanisms of EPC-induced angiogenesis were not clearly understood. Following injection, EPCs must migrate to the target tissue and engraft prior to induction of angiogenesis. In this study EPC migration was investigated in response to tumor necrosis factor α (TNFα), a pro-inflammatory cytokine, to test the hypothesis that organ damage observed in ischemic diseases induces an inflammatory signal that is important for EPC homing. In this study, EPC migration and incorporation were modeled in vitro using a coculture assay where TNFα treated EPCs were tracked while migrating toward vessel-like structures. It was found that TNFα treatment of EPCs increased migration and incorporation into vessel-like structures. Using a combination of genomic and proteomic approaches, NF-kB mediated upregulation of CADM1 was identified as a mechanism of TNFα induced migration. Inhibition of NF-kB or CADM1 significantly decreased migration of EPCs in vitro suggesting a role for TNFα signaling in EPC homing during tissue repair. Stem Cells 2016;34:1922-1933.

Improved rat genome gene prediction by integration of ESTs with RNA-Seq information.

MOTIVATION: RNA-Seq (also called whole-transcriptome sequencing) is an emerging technology that uses the capabilities of next-generation sequencing to detect and quantify entire transcripts. One of its important applications is the improvement of existing genome annotations. RNA-Seq provides rapid, comprehensive and cost-effective tools for the discovery of novel genes and transcripts compared with expressed sequence tag (EST), which is instrumental in gene discovery and gene sequence determination. The rat is widely used as a laboratory disease model, but has a less well-annotated genome as compared with humans and mice. In this study, we incorporated deep RNA-Seq data from three rat tissues-bone marrow, brain and kidney-with EST data to improve the annotation of the rat genome. RESULTS: Our analysis identified 32 197 transcripts, including 13 461 known transcripts, 13 934 novel isoforms and 4802 new genes, which almost doubled the numbers of transcripts in the current public rat genome database (rn5). Comparisons of our predicted protein-coding gene sets with those in public datasets suggest that RNA-Seq significantly improves genome annotation and identifies novel genes and isoforms in the rat. Importantly, the large majority of novel genes and isoforms are supported by direct evidence of RNA-Seq experiments. These predicted genes were integrated into the Rat Genome Database (RGD) and can serve as an important resource for functional studies in the research community. AVAILABILITY AND IMPLEMENTATION: The predicted genes are available at http://rgd.mcw.edu.

TNF-α increases endothelial progenitor cell adhesion to the endothelium by increasing bond expression and affinity.

Endothelial progenitor cells (EPCs) are a rare population of cells that participate in angiogenesis. To effectively use EPCs for regenerative therapy, the mechanisms by which they participate in tissue repair must be elucidated. This study focused on the process by which activated EPCs bind to a target tissue. It has been demonstrated that EPCs can bind to endothelial cells (ECs) through the tumore necrosis factor-α (TNF-α)-regulated vascular cell adhesion molecule 1/very-late antigen 4 (VLA4) interaction. VLA4 can bind in a high or low affinity state, a process that is difficult to experimentally isolate from bond expression upregulation. To separate these processes, a new parallel plate flow chamber was built, a detachment assay was developed, and a mathematical model was created that was designed to analyze the detachment assay results. The mathematical model was developed to predict the relative expression of EPC/EC bonds made for a given bond affinity distribution. EPCs treated with TNF-α/vehicle were allowed to bind to TNF-α/vehicle-treated ECs in vitro. Bound cells were subjected to laminar flow, and the cellular adherence was quantified as a function of shear stress. Experimental data were fit to the mathematical model using changes in bond expression or affinity as the only free parameter. It was found that TNF-α treatment of ECs increased adhesion through bond upregulation, whereas TNF-α treatment of EPCs increased adhesion by increasing bond affinity. These data suggest that injured tissue could potentially increase recruitment of EPCs for tissue regeneration via the secretion of TNF-α.

Physiogenomic resources for rat models of heart, lung and blood disorders.

Cardiovascular disorders are influenced by genetic and environmental factors. The TIGR rodent expression web-based resource (TREX) contains over 2,200 microarray hybridizations, involving over 800 animals from 18 different rat strains. These strains comprise genetically diverse parental animals and a panel of chromosomal substitution strains derived by introgressing individual chromosomes from normotensive Brown Norway (BN/NHsdMcwi) rats into the background of Dahl salt sensitive (SS/JrHsdMcwi) rats. The profiles document gene-expression changes in both genders, four tissues (heart, lung, liver, kidney) and two environmental conditions (normoxia, hypoxia). This translates into almost 400 high-quality direct comparisons (not including replicates) and over 100,000 pairwise comparisons. As each individual chromosomal substitution strain represents on average less than a 5% change from the parental genome, consomic strains provide a useful mechanism to dissect complex traits and identify causative genes. We performed a variety of data-mining manipulations on the profiles and used complementary physiological data from the PhysGen resource to demonstrate how TREX can be used by the cardiovascular community for hypothesis generation.

Bone marrow mononuclear cell angiogenic competency is suppressed by a high-salt diet.

Autologous bone marrow-derived mononuclear cell (BM-MNC) transplantation is a potential therapy for inducing revascularization in ischemic tissues providing the underlying disease process had not negatively affected BM-MNC function. Previously, we have shown that skeletal muscle angiogenesis induced by electrical stimulation is impaired by a high-salt diet (HSD; 4% NaCl) in Sprague-Dawley (SD) rats. In this study we tested the hypothesis that BM-MNC angiogenic function is impaired by an elevated dietary sodium intake. Following 1 wk on HSD, either vehicle or BM-MNCs derived from SD donor rats on HSD or normal salt diet (NSD; 0.4% NaCl) were injected into male SD rats undergoing hindlimb stimulation. Administration of BM-MNCs (intramuscular or intravenous) from NSD donors, but not HSD donors, restored the angiogenic response in HSD recipients. Angiotensin II (3 ng · kg(-1) · min(-1)) infusion of HSD donor rats restored angiogenic capacity of BM-MNCs, and treatment of NSD donor rats with losartan, an angiotensin II receptor-1 antagonist, inhibited BM-MNC angiogenic competency. HSD BM-MNCs and NSD losartan BM-MNCs exhibited increased apoptosis in vitro following an acute 6-h hypoxic stimulus. HSD BM-MNCs also had increased apoptosis following injection into skeletal muscle. This study suggests that BM-MNC transplantation can restore skeletal muscle angiogenesis and that HSD impairs the angiogenic competency of BM-MNCs due to suppression of the renin-angiotensin system causing increased apoptosis.

Histone Modifications and Their Contributions to Hypertension.

Essential hypertension, a multifaceted disorder, is a worldwide health problem. A complex network of genetic, epigenetic, physiological, and environmental components regulates blood pressure (BP), and any dysregulation of this network may result in hypertension. Growing evidence suggests a role for epigenetic factors in BP regulation. Any alterations in the expression or functions of these epigenetic regulators may dysregulate various determinants of BP, thereby promoting the development of hypertension. Histone posttranslational modifications are critical epigenetic regulators that have been implicated in hypertension. Several studies have demonstrated a clear association between the increased expression of some histone-modifying enzymes, especially HDACs (histone deacetylases), and hypertension. In addition, treatment with HDAC inhibitors lowers BP in hypertensive animal models, providing an excellent opportunity to design new drugs to treat hypertension. In this review, we discuss the potential contribution of different histone modifications to the regulation of BP.

Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress.

Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration.

Targeting the endothelial progenitor cell surface proteome to identify novel mechanisms that mediate angiogenic efficacy in a rodent model of vascular disease.

Endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials suggest autologous EPC-based therapy may be effective in treatment of vascular diseases. Albeit promising, variability in the efficacy of EPCs associated with underlying disease states has hindered the realization of EPC-based therapy. Here we first identify and characterize EPC dysfunction in a rodent model of vascular disease (SS/Mcwi rat) that exhibits impaired angiogenesis. To identify molecular candidates that mediate the angiogenic potential of these cells, we performed a broad analysis of cell surface protein expression using chemical labeling combined with mass spectrometry. Analysis revealed EPCs derived from SS/Mcwi rats express significantly more type 2 low-affinity immunoglobulin Fc-gamma (FCGR2) and natural killer 2B4 (CD244) receptors compared with controls. Genome-wide sequencing (RNA-seq) and qt-PCR confirmed isoforms of CD244 and FCGR2a transcripts were increased in SS/Mcwi EPCs. EPCs with elevated expression of FCGR2a and CD244 receptors are predicted to increase the probability of SS/Mcwi EPCs being targeted for death, providing a mechanistic explanation for their reduced angiogenic efficacy in vivo. Pathway analysis supported this contention, as "key" molecules annotated to cell death paths were differentially expressed in the SS/Mcwi EPCs. We speculate that screening and neutralization of cell surface proteins that "tag" and impair EPC function may provide an alternative approach to utilizing incompetent EPCs in greater numbers, as circulating EPCs are depleted in patients with vascular disease. Overall, novel methods to identify putative targets for repair of EPCs using discovery-based technologies will likely provide a major advance in the field of regenerative medicine.

Distinct roles of estrone and estradiol in endothelial colony-forming cells.

Our current understanding of the relationship between estrogen and human endothelial colony-forming cell (hECFC) function is based almost exclusively on studies investigating estradiol action at nuclear estrogen receptors. In the current study the hypothesis was tested that the less potent estrogen receptor agonist, estrone, affects hECFC proliferation, migration, secretion, and tube formation in a way that is unique from that of estradiol. The relationship between the estrogens, estradiol and estrone, is clinically important, particularly in postmenopausal women where estradiol levels wane and estrone becomes the predominant estrogen. Cultured hECFCs from peripheral blood mononuclear cell fractions were treated with concentrations of estradiol and estrone ranging from 1 nM to 1 µM separately and in combination. Following treatment, proliferation, migration, ability to attract other hECFCs (autocrine secretion), and ability to enhance endothelial cell tube formation (tubulogenesis) were tested. Functional assays revealed unique, concentration-dependent physiological effects of estrone and estradiol. Estradiol exposure resulted in increased hECFC proliferation, migration, secretion of chemoattractant, and enhancement of tube formation as expected. As with estradiol, hECFC secretion of chemoattractant increased significantly with each increase in estrone exposure. Estrone treatment produced a biphasic, concentration-dependent relationship with proliferation and tube formation and relatively no effect on hECFC migration at any concentration. The quantitative relationship between the effects of estrone and estradiol and each hECFC function was analyzed. The extent to which estrone was similar in effect to that of estradiol was dependent on both the concentrations of estradiol and estrone and the hECFC function measured. Interestingly, when the two estrogens were present, differing ratios resulted in unique functional responses. hECFCs that were treated with combinations of estrone and estradiol with high estrone to estradiol ratios showed decreased proliferative capacity. Conversely, hECFCs that were treated with combinations that were relatively high in estradiol, showed increased proliferative capacity. Cells that were treated with estrone and estradiol in equal concentrations showed an attenuated proliferative response that was decreased compared to the proliferation that either estrone or estradiol produced when they were present alone. This co-inhibitory relationship, which has not been previously reported, challenges the prevailing understanding of estrone as solely a weak agonist at estrogen receptors. This study provides evidence that estrone signaling is distinct from that of estradiol and that further investigation of estrone's mechanism of action and the biological effect may provide important insight into understanding the dysfunction and decreased number of hECFCs, and the resulting cardiovascular disease risk observed clinically in menopausal women and women undergoing hormone replacement therapy.

Metabolic responses of normal rat kidneys to a high salt intake.

In the present study, novel methods were developed which allowed continuous (24/7) measurement of blood pressure (BP) and renal blood flow (RBF) in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O 2 and metabolites. The study determined the effects of a high salt (HS) diet upon whole kidney O 2 consumption and the metabolomic profiles of normal Sprague Dawley (SD) rats. A separate group of rats was studied to determine changes in the cortex (Cx) and outer medulla (OM) tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to a 4.0% NaCl diet. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O 2 consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. Increased glycolysis was evident with the elevation of mRNA expression encoding key glycolytic enzymes and release of pyruvate and lactate from the kidney in the renal venous blood. Glycolytic production of NADH is used in either the production of lactate or oxidized via the malate aspartate shuttle. Aerobic glycolysis (e.g., Warburg-effect) may account for the needed increase in cellular energy. The study provides evidence that kidney metabolism responds to a HS diet enabling enhanced energy production while protecting from oxidate stress and injury.

Metabolic Responses of Normal Rat Kidneys to a High Salt Intake.

In this study, novel methods were developed, which allowed continuous (24/7) measurement of arterial blood pressure and renal blood flow in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2 and metabolites. Specifically, the study determined the effects of a high salt (HS; 4.0% NaCl) diet upon whole kidney O2 consumption and arterial and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats. A separate group of rats was studied to determine changes in the cortex and outer medulla tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to 4.0% NaCl diet. In addition, targeted mRNA expression analysis of cortical segments was performed. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O2 consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. A novel finding was the increased expression of glycolysis-related genes in Cx and isolated proximal tubular segments in response to an HS diet, consistent with increased release of pyruvate and lactate from the kidney to the renal venous blood. Data suggests that aerobic glycolysis (eg, Warburg effect) may contribute to energy production under these circumstances. The study provides evidence that kidney metabolism responds to an HS diet enabling enhanced energy production while protecting from oxidative stress and injury. Metabolomic and transcriptomic analysis of kidneys of Sprague-Dawley rats fed a high salt diet.

3D-bioprinting of patient-derived cardiac tissue models for studying congenital heart disease.

Introduction: Congenital heart disease is the leading cause of death related to birth defects and affects 1 out of every 100 live births. Induced pluripotent stem cell technology has allowed for patient-derived cardiomyocytes to be studied in vitro. An approach to bioengineer these cells into a physiologically accurate cardiac tissue model is needed in order to study the disease and evaluate potential treatment strategies. Methods: To accomplish this, we have developed a protocol to 3D-bioprint cardiac tissue constructs comprised of patient-derived cardiomyocytes within a hydrogel bioink based on laminin-521. Results: Cardiomyocytes remained viable and demonstrated appropriate phenotype and function including spontaneous contraction. Contraction remained consistent during 30 days of culture based on displacement measurements. Furthermore, tissue constructs demonstrated progressive maturation based on sarcomere structure and gene expression analysis. Gene expression analysis also revealed enhanced maturation in 3D constructs compared to 2D cell culture. Discussion: This combination of patient-derived cardiomyocytes and 3D-bioprinting represents a promising platform for studying congenital heart disease and evaluating individualized treatment strategies.

Mechanisms of cardioprotection resulting from Brown Norway chromosome 16 substitution in the salt-sensitive Dahl rat.

The SS-16(BN)/Mcwi consomic rat was produced by the introgression of chromosome 16 from the Brown Norway (BN/NHsdMcwi) rat onto the genetic background of the Dahl salt-sensitive (SS/Mcwi) rat by marker-assisted breeding. We have previously shown that the normotensive SS-16(BN)/Mcwi consomic strain is better protected from developing left ventricular dysfunction and fibrosis with aging than the hypertensive SS/Mcwi parental strain; however, the mechanism of this protection was not clear since the SS-16(BN)/Mcwi had both lowered blood pressure and an altered genetic background compared with SS/Mcwi. Microarray analysis of SS-16(BN)/Mcwi and SS/Mcwi left ventricle tissue and subsequent protein pathway analysis were used to identify alterations in gene expression in signaling pathways involved with the observed cardioprotection on the SS background. The SS-16(BN)/Mcwi rats exhibited much higher mRNA levels of expression of transcription factor JunD, a gene found on chromosome 16. Additionally, high levels of differential gene expression were found in pathways involved with angiogenesis, oxidative stress, and growth factor signaling. We tested the physiological relevance of these pathways by experimentally determining the responsiveness of neonatal cardiomyocytes to factors from identified pathways and found that cells isolated from SS-16(BN)/Mcwi rats had a greater growth response to epidermal growth factor and endothelin-1 than those from parental SS/Mcwi. We also demonstrate that the SS-16(BN)/Mcwi is better protected from developing fibrosis with surgically elevated afterload than other normotensive strains, indicating that gene-gene interactions resulting from BN chromosomal substitution confer specific cardioprotection. When combined with our previous findings, these data suggest that that SS-16(BN)/Mcwi may have an increased angiogenic potential and better protection from oxidative stress than the parental SS/Mcwi strain. Additionally, the early transient idiopathic left ventricular hypertrophy in the SS-16(BN)/Mcwi may be related to altered myocyte sensitivity to growth factors.