Development Of Cardiovascular System Research Articles

Effects of assisted reproductive technology on gene expression in heart and spleen tissues of adult offspring mouse.

Assisted reproductive technology (ART) is an important part of reproductive medicine, whose possible effects on offspring's health have drawn widespread attention in recent years. However, relevant studies are limited to postnatal short-term follow-up and lack of diverse sample sources analysis other than blood. In this study, a mouse model was used to explore the effects of ART on fetal development and gene expression in the organs of offspring in the adulthood using next-generation sequencing. The sequencing results were then analyzed. The results showed that it caused abnormal expression in 1060 genes and 179 genes in the heart and spleen, respectively. Differentially expressed genes (DEGs) in the heart are mainly enriched in RNA synthesis and processing, and the cardiovascular system development also shows enrichment. STRING analysis identified Ccl2, Ptgs2, Rock1, Mapk14, Agt, and Wnt5a as the core interacting factors. DEGs in the spleen are significantly enriched in anti-infection and immune responses, which include the core factors Fos, Jun and Il1r2. Further exploration revealed the abnormal expression of 42 and 5 epigenetic modifiers in the heart and spleen, respectively. The expression of the imprinted genes Dhcr7, Igf2, Mest and Smoc1 decreased in the hearts of ART offspring, and the DNA methylation levels of Igf2- and Mest-imprinting control regions (ICRs) increased abnormally. In the mouse model, ART can interfere with the gene expression pattern in the heart and spleen of the adult offspring and that these changes are related to the aberrant expression of epigenetic regulators.

Optical transparency and label-free vessel imaging of zebrafish larvae in shortwave infrared range as a tool for prolonged studying of cardiovascular system development

Optical techniques are utilized for the non-invasive analysis of the zebrafish cardiovascular system at early developmental stages. Being based mainly on conventional optical microscopy components and image sensors, the wavelength range of the collected and analyzed light is not out of the scope of 400–900 nm. In this paper, we compared the non-invasive optical approaches utilizing visible and near infrared range (VISNIR) 400–1000 and the shortwave infrared range (SWIR) 900–1700 nm. The transmittance spectra of zebrafish tissues were measured in these wavelength ranges, then vessel maps, heart rates, and blood flow velocities were calculated from data in VISNIR and SWIR. An increased pigment pattern transparency was registered in SWIR, while the heart and vessel detection quality in this range is not inferior to VISNIR. Obtained results indicate an increased efficiency of SWIR imaging for monitoring heart function and hemodynamic analysis of zebrafish embryos and larvae and suggest a prolonged registration period in this range compared to other optical techniques that are limited by pigment pattern development.

Generation and functional evaluation of novel monoclonal antibodies targeting glycosylated human stem cell factor.

Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses. KEY POINTS: • Soluble glycosylated human SCF exerted improved proliferative activity on UT-7 cells. • Three mAbs with high specificity targeting glycosylated human SCF were obtained. • mAbs applications comprise sandwich ELISA, western blot, and immunofluorescence assays.

Abstract 14969: Transcriptome Signature of Cardiac Fibroblasts in HCM Patients Identifies Novel Drivers Of ECM Remodeling and Pro-Inflammatory Signaling

Introduction: Cardiac fibroblasts (CFs) have a crucial role in ECM remodeling as well as influencing contractility through crosstalk with the cardiomyocytes. There is accumulating evidence that the phenotype of CFs is disease specific. We here investigate the transcriptome signature of CFs in HCM patients and their functional significance. Methods: Primary CFs were isolated from the myectomy specimens of 12 clinically phenotyped HCM patients and 3 controls. RNA library was prepared from the cell isolates, and whole transcriptome sequencing was performed. Differential expression analysis was carried out using the Tuxedo pipeline. Pathway, Gene Ontology (GO) enrichment, and gene network analyses were performed using Ingenuity Pathways Analysis (IPA) and Gprofiler, respectively. Expression of candidate genes was validated using real time PCR, ICC, immunohistochemistry, cytokines profiling and western blotting. Results: Whole transcriptome analysis identified 292 significant differentially expressed genes (DEGs) (pvalue <0.05 threshold) in HCM fibroblasts compared to controls. The DEGs identified with the highest significance were associated with the “Cardiovascular System development and Function” network (1.55e-27 <pvalue< 3.10e-08). The most significant GO terms identified were associated with ECM remodeling, collagen binding, signal receptor binding, circulatory system development and biological adhesion and other relevant processes. The DEGs encompassed gene families such as collagens ( COL7A1, COL17A1, COL21A1, COL15A1 and COL5A3 ), proteases ( MMP9, MMP12, ABI13BP and ADAM19 ), fibulins (FBLN1 and FBLN5), inflammatory and signaling cytokines ( IL6, CCL2, CCL4, CCL11, IGF2, CSF1, FGF16 and FGF2 ), integrins and signaling receptors ( ITGA4, ITGA7, NRP2, SULF2, ADORA2B, CXCR4, FOLR2, PLXNA4 and TREM2 ) and kinases ( DDR1, EFNB2, RGCC, MAPK11, CDKN2B, PTK7, MARCKSL1, ROCK2, MET, TYROBP and KSR1 ). Assessment of candidate genes was further performed by studying their expression patterns in patients’ samples (CFs extracts and tissue sections). Conclusion: Transcriptome signature of CFs derived from HCM identifies new candidate genes and pathways for ECM remodeling, collagen binding and pro-inflammatory signaling.

Abstract 12552: Causal Effect of Exposure to Elevated Lipoprotein(a) Levels on the Blood Transcriptome : A Mendelian Randomization Study

INTRODUCTION: Lipoprotein(a) [Lp(a)] is one of the strongest genetic risk factors for atherosclerotic cardiovascular diseases (ASCVD). Several ex vivo studies have revealed a pro-inflammatory and pro-atherogenic role of Lp(a) in the etiology of ASCVD, mainly through induction of inflammation, metabolic processes, and apoptosis. However, whether Lp(a) causally influences the human transcriptome is unknown. Here, we aimed at identifying whether genetically-predicted Lp(a) levels could also affect the whole blood expression of genes involved in metabolic- and inflammatory-related pathways. Methods: We performed a series of inverse-variance weighted mendelian randomization analyses using single-nucleotide polymorphisms located in the LPA locus (±500kb; p<5x10 -8 ) from the UK Biobank GWAS on Lipoprotein(a) quantiles as exposure (n=377,590 and whole blood expression levels of 14,288 coding gene from eQTLGen as outcomes (n=31,684 from 37 studies). We then used the Functional Mapping and Annotation of Genome-Wide Association Studies tool (FUMA) to identify gene sets for cardiometabolic- and inflammatory-related pathways from gene ontology biological processes. Results: The MR analyses identified 1363 genes that may be causally influenced by exposure to high Lp(a) levels (with p<0.05). The 10 most strongly associated genes with genetically-predicted Lp(a) levels are FSTL3 , PRKC1 , PDE6C , PLXNB3 , SESN1 , SASH1 , CD1D , CCDC102A , SS18L1 and TCF12 . These genes are also differentially expressed in the heart. We identified 16 cardiometabolic-related pathways, mostly related to immune response, signaling, apoptosis, cardiovascular and vasculature development, cell proliferation, metabolic process, and catalytic activity. The 5 most significant pathways are regulation of cell population proliferation, regulation of intracellular signal transduction, positive regulation of cell population proliferation, cardiovascular system development and TOR signaling. Conclusions: Results of this study highlight a potentially causal effect of exposure to elevated Lp(a) levels on several key genes involved in metabolic processes and inflammation and provide mechanistic insight on the pathobiological role of Lp(a) in the etiology of ASCVD.

SORBS2 upregulation may contribute to dysfunction in LVNC via the Notch pathway.

Background: LVNC is congenital cardiomyopathy characterized by hypertrabeculation of the left ventricle. It was reported that SORBS2, which is expressed most highly in the heart, functions in cytoskeletal organization, cell adhesion, and signaling pathways. The Notch pathway plays an important role in the development of the normal cardiovascular system, dysfunction of which can lead to cardiovascular disease. In view of the localization and function of SORBS2, we hypothesized that elevated SORBS2 might combine with the Notch pathway and be involved in the molecular mechanism of LVNC. Methods: Comparative microarray analysis was used to screen differentially expressed genes in cardiomyocytes. The high expression of SORBS2 in hESC-CMs was further confirmed by western blotting and immunohistology. Recombinant AAV9 vectors carrying SORBS2 cDNA with a cardiac-specific promoter cTNT were constructed to study the distribution and function of SORBS2 in cardiomyocytes in vivo and in vitro. Results: In our study, changes in the Notch pathway were observed in cells overexpressing SORBS2. We tested and verified the consistent trend of Tbx20, Hey2, Anf, Bmp10, and Cx40 in the Notch pathway through microarray and western blotting. In view of the localization and function of SORBS2 in the heart, we hypothesized that elevated SORBS2 might be involved in the Notch pathway and thereby participate in the molecular mechanism behind LVNC. Conclusion: Myocardial SORBS2 interacted with Cx40 and participated in the Notch pathway, resulting in myocardial fibrosis and heart function impairment. Our findings suggest that elevated myocardial SORBS2 might be involved in the molecular mechanism behind LVNC.

Reconstruction and analysis of potential biomarkers for hypertrophic cardiomyopathy based on a competing endogenous RNA network

Hypertrophic cardiomyopathy (HCM) is a common heritable cardiomyopath. Although considerable effort has been made to understand the pathogenesis of HCM, the mechanism of how long noncoding RNA (lncRNA)-associated competing endogenous RNA (ceRNA) network result in HCM remains unknown. In this study, we acquired a total of 520 different expression profiles of lncRNAs (DElncRNAs) and 371 messenger RNAs (mRNA, DEGs) by microarray and 33 microRNAs (DEmiRNAs) by sequencing in plasma of patients with HCM and healthy controls. Then lncRNA–miRNA pairs were predicted using miRcode and starBase and crossed with DEmiRNAs. MiRNA–mRNA pairs were retrieved from miRanda and TargetScan and crossed with DEGs. Combined with these pairs, the ceRNA network with eight lncRNAs, three miRNAs, and 22 mRNAs was constructed. lncRNA RP11-66N24.4 and LINC00310 were among the top 10% nodes. The hub nodes were analyzed to reconstruct a subnetwork. Furthermore, quantitative real-time polymerase chain reaction results showed that LINC00310 was significantly decreased in patients with HCM. For LINC00310, GO analysis revealed that biological processes were enriched in cardiovascular system development, sprouting angiogenesis, circulatory system development, and pathway analysis in the cGMP-PKG signaling pathway. These results indicate that the novel lncRNA-related ceRNA network in HCM and LINC00310 may play a role in the mechanism of HCM pathogenesis, which could provide insight into the pathogenesis of HCM.

Analyzing integrated network of methylation and gene expression profiles in lung squamous cell carcinoma

Gene expression, DNA methylation, and their organizational relationships are commonly altered in lung squamous cell carcinoma (LUSC). To elucidate these complex interactions, we reconstructed a differentially expressed gene network and a differentially methylated cytosine (DMC) network by partial information decomposition and an inverse correlation algorithm, respectively. Then, we performed graph union to integrate the networks. Community detection and enrichment analysis of the integrated network revealed close interactions between the cell cycle, keratinization, immune system, and xenobiotic metabolism gene sets in LUSC. DMC analysis showed that hypomethylation targeted the gene sets responsible for cell cycle, keratinization, and NRF2 pathways. On the other hand, hypermethylated genes affected circulatory system development, the immune system, extracellular matrix organization, and cilium organization. By centrality measurement, we identified NCAPG2, PSMG3, and FADD as hub genes that were highly connected to other nodes and might play important roles in LUSC gene dysregulation. We also found that the genes with high betweenness centrality are more likely to affect patients’ survival than those with low betweenness centrality. These results showed that the integrated network analysis enabled us to obtain a global view of the interactions and regulations in LUSC.

Maternal and zygotic ZFP57 regulate coronary vascular formation and myocardium maturation in mouse embryo.

Genomic and epigenomic dynamics both play critical roles during embryogenesis. Zfp57 maintains genomic imprinting with both maternal and zygotic functions. In our previous study, we found that maternal and zygotic Zfp57 modulate NOTCH signaling during cardiac development. In this study, we investigated Zfp57 mutants from E11.5 to E13.5 to delineate its function during cardiac development. Here, we describe novel roles of maternal and zygotic Zfp57 during cardiovascular system development. We found that maternal and zygotic Zfp57 was required for coronary vascular development. Maternal and zygotic loss of Zfp57 perturbed the sprouting of the sinus venosus-derived endothelial cells and led to underdeveloped coronary vasculature, meanwhile, there was an ectopic overproduction of blood islands over the ventricles. Furthermore, loss of Zfp57 and failed vasculature disturbed myocardium maturation. Loss of maternal and zygotic Zfp57 resulted in hyper trabeculation and failed myocardium compaction. Zfp57 zygotic mutant (M+ Z- ) hearts displayed noncompaction cardiomyopathy at E18.5. Our results suggest that maternal and zygotic ZFP57 are essential for coronary vascular formation and myocardium maturation in mice. Our research provides evidence for the role of genomic imprinting during embryogenesis.

Human-derived decellularized extracellular matrix scaffold incorporating autologous bone marrow stem cells from patients with congenital heart disease for cardiac tissue engineering.

Stem cells are used as an alternative treatment option for patients with congenital heart disease (CHD) due to their regenerative potential, but they are subject to low retention rate in the injured myocardium. Also, the diseased microenvironment in the injured myocardium may not provide healthy cues for optimal stem cell function. In this study, we prepared a novel human-derived cardiac scaffold to improve the functional behaviors of stem cells. Decellularized extracellular matrix (ECM) scaffolds were fabricated by removing cells of human-derived cardiac appendage tissues. Then, bone marrow c-kit+ progenitor cells from patients with congenital heart disease were seeded on the cardiac ECM scaffolds. Cell adhesion, survival, proliferation and cardiac differentiation on human cardiac decellularized ECM scaffold were evaluated invitro. Label-free mass spectrometry was applied to analyze cardiac ECM proteins regulating cell behaviors. It was shown that cardiac ECM scaffolds promoted stem cell adhesion and proliferation. Importantly, bone marrow c-kit+ progenitor cells cultured on cardiac ECM scaffold for 14 days differentiated into cardiomyocyte-like cells without supplement with any inducible factors, as confirmed by the increased protein level of Gata4 and upregulated gene levels of Gata4, Nkx2.5, and cTnT. Proteomic analysis showed the proteins in cardiac ECM functioned in multiple biological activities, including regulation of cell proliferation, regulation of cell differentiation, and cardiovascular system development. The human-derived cardiac scaffold constructed in this study may help repair the damaged myocardium and hold great potential for tissue engineering application in pediatric patients with CHD.

Long-term changes in plasma proteomic profiles in premenopausal and postmenopausal Black and White women: the Atherosclerosis Risk in Communities study.

The activity, localization, and turnover of proteins within cells and plasma may contribute to physiologic changes during menopause and may influence disease occurrence. We examined cross-sectional differences and long-term changes in plasma proteins between premenopausal and naturally postmenopausal women. We used data from 4,508 (19% Black) women enrolled in the Atherosclerosis Risk in Communities study. SOMAscan multiplexed aptamer technology was used to measure 4,697 plasma proteins. Linear regression models were used to compare differences in proteins at baseline (1993-1995) and 18-year change in proteins from baseline to 2011-2013. At baseline, 472 women reported being premenopausal and 4,036 women reported being postmenopausal, with average ages of 52.3 and 61.4 years, respectively. A greater proportion of postmenopausal women had diabetes (15 vs 9%), used hypertension (38 vs 27%) and lipid-lowering medications (10 vs 3%), and had elevated total cholesterol and waist girth. In multivariable adjusted models, 38 proteins differed significantly between premenopausal and postmenopausal women at baseline, with 29 of the proteins also showing significantly different changes between groups over the 18-year follow-up as the premenopausal women also reached menopause. These proteins were associated with various molecular/cellular functions (cellular development, growth, proliferation and maintenance), physiological system development (skeletal and muscular system development, and cardiovascular system development and function), and diseases/disorders (hematological and metabolic diseases and developmental disorders). We observed significantly different changes between premenopausal and postmenopausal women in several plasma proteins that reflect many biological processes. These processes may help to understand disease development during the postmenopausal period.

High-throughput transcriptome sequencing reveals the key stages of cardiovascular development in zebrafish embryos

BackgroundThe cardiovascular developmental process is a tightly regulated network involving multiple genes. The current understanding of the molecular mechanism behind cardiovascular development is insufficient and requires further research.ResultsTranscriptome sequencing of three developmental stages in zebrafish embryos was performed and revealed three key cardiovascular developmental stages. Then, the differentially expressed genes (DEGs) involved in cardiovascular development were screened out. The three developmental stages were 18 (T1), 24 (T2), and 42 h post fertilization (hpf) (T3), and the three stages were confirmed by detecting differences in expression between cardiomyocyte and endothelial marker genes (cmlc2, fli1) using in situ hybridization, which represents the characteristics of cardiovascular development. Thousands of DEGs were identified using transcriptome analysis. Of them, 2605 DEGs were in T1-vs-T2, including 2003 up-regulated and 602 down-regulated genes, 6446 DEGs were in T1-vs-T3, consisting of 4608 up-regulated and 1838 down-regulated genes, and 3275 DEGs were in T2-vs-T3, including 2420 up-regulated and 855 down-regulated genes. There were 644 common DEGs and 167 common five-fold higher differentially expressed genes (HDEGs) identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Significant differences was observed in the levels of gene expression among different developmental stages in multiple GO terms and KEGG pathways, such as cell migration to the midline involved in heart development, cardiovascular system development, circulatory system process for biological processes of GO terms; and cardiac muscle contraction, adrenergic signaling in cardiomyocytes for KEGG pathways. These results demonstrated that these three stages were important period for the development of the cardiovascular system. Lastly, we used quantitative real-time PCR (qPCR) to validate the reliability of RNA-sequencing by selecting 21 DEGs.ConclusionsThese results demonstrated that these three stages represented the important periods for cardiovascular system development of zebrafish and some candidate genes was obtained and provided a solid foundation for additional functional studies of the DEGs.

Cell-free DNA in maternal blood and artificial intelligence: accurate prenatal detection of fetal congenital heart defects

DNA cytosine nucleotide methylation (epigenomics and epigenetics) is an important mechanism for controlling gene expression in cardiac development. Combined artificial intelligence and whole-genome epigenomic analysis of circulating cell-free DNA in maternal blood has the potential for the detection of fetal congenital heart defects. This study aimed to use genome-wide DNA cytosine methylation and artificial intelligence analyses of circulating cell-free DNA for the minimally invasive detection of fetal congenital heart defects. In this prospective study, whole-genome cytosine nucleotide methylation analysis was performed on circulating cell-free DNA using the Illumina Infinium MethylationEPIC BeadChip array. Multiple artificial intelligence approaches were evaluated for the detection of congenital hearts. The Ingenuity Pathway Analysis program was used to identify gene pathways that were epigenetically altered and important in congenital heart defect pathogenesis to further elucidate the pathogenesis of isolated congenital heart defects. There were 12 cases of isolated nonsyndromic congenital heart defects and 26 matched controls. A total of 5918 cytosine nucleotides involving 4976 genes had significantly altered methylation, that is, a P value of <.05 along with ≥5% whole-genome cytosine nucleotide methylation difference, in congenital heart defect cases vs controls. Artificial intelligence analysis of the methylation data achieved excellent congenital heart defect predictive accuracy (areas under the receiver operating characteristic curve, ≥0.92). For example, an artificial intelligence model using a combination of 5 whole-genome cytosine nucleotide markers achieved an area under the receiver operating characteristic curve of 0.97 (95% confidence interval, 0.87-1.0) with 98% sensitivity and 94% specificity. We found epigenetic changes in genes and gene pathways involved in the following important cardiac developmental processes: "cardiovascular system development and function," "cardiac hypertrophy," "congenital heart anomaly," and "cardiovascular disease." This lends biologic plausibility to our findings. This study reported the feasibility of minimally invasive detection of fetal congenital heart defect using artificial intelligence and DNA methylation analysis of circulating cell-free DNA for the prediction of fetal congenital heart defect. Furthermore, the findings supported an important role of epigenetic changes in congenital heart defect development.

A review of heat stress in chickens. Part I: Insights into physiology and gut health.

Heat stress (HS) compromises the yield and quality of poultry products and endangers the sustainability of the poultry industry. Despite being homeothermic, chickens, especially fast-growing broiler lines, are particularly sensitive to HS due to the phylogenetic absence of sweat glands, along with the artificial selection-caused increase in metabolic rates and limited development of cardiovascular and respiratory systems. Clinical signs and consequences of HS are multifaceted and include alterations in behavior (e.g., lethargy, decreased feed intake, and panting), metabolism (e.g., catabolic state, fat accumulation, and reduced skeletal muscle accretion), general homeostasis (e.g., alkalosis, hormonal imbalance, immunodeficiency, inflammation, and oxidative stress), and gastrointestinal tract function (e.g., digestive and absorptive disorders, enteritis, paracellular barrier failure, and dysbiosis). Poultry scientists and companies have made great efforts to develop effective solutions to counteract the detrimental effects of HS on health and performance of chickens. Feeding and nutrition have been shown to play a key role in combating HS in chicken husbandry. Nutritional strategies that enhance protein and energy utilization as well as dietary interventions intended to restore intestinal eubiosis are of increasing interest because of the marked effects of HS on feed intake, nutrient metabolism, and gut health. Hence, the present review series, divided into Part I and Part II, seeks to synthesize information on the effects of HS on physiology, gut health, and performance of chickens, with emphasis on potential solutions adopted in broiler chicken nutrition to alleviate these effects. Part I provides introductory knowledge on HS physiology to make good use of the nutritional themes covered by Part II.

Transmembrane Protein 100 Inhibits the Progression of Colorectal Cancer by Promoting the Ubiquitin/Proteasome Degradation of HIF-1α.

Transmembrane protein 100 (TMEM100) is involved in embryonic cardiovascular system development. However, the biological role of TMEM100 in human cancers, particularly colorectal cancer (CRC), is unclear. In this study, tissue microarrays were stained using immunohistochemistry methods to evaluate the association between TMEM100 levels and clinic-pathological features for CRC. Kaplan–Meier and log-rank tests revealed that decreased levels of TMEM100 correlated with shorter overall survival. Cox regression revealed that reduced levels of TMEM100 was an independent prognostic factor for detrimental survival in CRC. A lentiviral vector was used to overexpress TMEM100 in HCT116 cells, and small interfering RNA was used to knockdown TMEM100 in SW480 cells. The CCK-8 assay, colony formation analysis, cell cycle analysis, cell migration assay, mouse xenograft model and mouse lung metastasis model showed that TMEM100 suppressed CRC cell proliferation and migration in vitro and in vivo. IHC scores of TMEM100 and HIF-1α were significantly negatively correlated. A half-time determination analysis in which cells were treated with cycloheximide revealed that TMEM100 shortened the HIF-1α half-life. Further immunoprecipitation experimental results showed that TMEM100 promoted the ubiquitination of HIF-1α, which caused HIF-1α degradation via the 26S proteasome pathway. Angiogenesis assay and migration assay results revealed that TMEM100 suppressed the migration and angiogenesis induction capacities of HCT116 cells, but this inhibitory effect was abolished when HIF-1α degradation was blocked by MG132 treatment. These results indicated that TMEM100 inhibited the migration and the angiogenesis induction capacities of CRC cells by enhancing HIF-1α degradation via ubiquitination/proteasome pathway.

Environmental Hypoxia is a Driver of Sonic Hedgehog-dependent Troglomorphic Evolution in Astyanax Cavefish

Astyanax mexicanus cavefish (CF) have evolved numerous troglomorphic traits since their divergence from surface fish (SF) ancestors about 20,000 years ago. A large number of these traits are under the control of Sonic Hedgehog (Shh), a signaling morphogen that is overexpressed along the midline in CF relative to SF embryos. The Shh-dependent traits include eye degeneration and increases in the olfactory lobes, hypothalamus, jaws, and oral taste buds. Eye degeneration and tastebud enhancement are antagonistic tradeoffs governed by the pleiotropic shh gene. CF have also evolved multiple changes in circulatory system development, highlighted by larger hematopoietic domains, more erythrocytes, and reversals in normal heart asymmetry, which are also controlled by Shh signaling. Although Shh signaling is an important factor in CF troglomorphism, the shh gene is not associated with any of the many QTL identified by genetic analysis, suggesting that upstream processes, rather than mutation of the shh locus itself, may be important in Shh regulation. Due to the absence of primary productivity, cave pools harboring Astyanax CF are deficient in dissolved oxygen. Accordingly, the Hypoxia Inducing Factor (HIF) transcription factors and their downstream targets are constitutively active during CF development. To determine if oxygen deficiency is an upstream regulator of Shh signaling, we subjected early SF embryos to 8 or 16 hours of hypoxia in the laboratory and assayed the effects on shh expression, erythrocyte formation, heart asymmetry, and eye development. The results indicated that hypoxia can cause shh overexpression, increase erythropoiesis, reverse heart asymmetry, and induce eye degeneration in SF embryos. We conclude that environmental hypoxia may be responsible for the evolution of troglomorphic traits in Astyanax CF through hyperactivation of the Shh signaling pathway.

Transcriptional regulation of transcriptional Mediator complexes in cardiovascular development and disease.

During the development of the mammalian cardiovascular system, the formation of a mature and fully functional cardiovascular system needs the fine coordination of the morphogenesis of various molecules, cells, tissues, and organs. Abnormalities in these processes usually lead to serious congenital heart defects. The determination and maintenance of cell fate in multicellular organisms depend to a large extent on the precise timing and control of RNA polymerase II (Pol II) transcription, and the transcription Mediator complex plays an irreplaceable role in the Pol II transcription process. Mediator is an evolutionarily conserved multi-subunit protein complex, including four parts: head, middle, tail, and kinase. It is a functional bridge between transcription factors and basic transcription machines. In recent years, due to the key role of Mediator in the transcriptional regulation of gene expression, many of human heart diseases have been confirmed to be related to specific Mediator gene mutations, such as heart valve defects, translocation of the great arteries, DiGeorge syndrome and some cardiovascular diseases related to energy homeostasis. In this review, we summarize the role of Mediator in cardiovascular development and disease, focusing on the role of Mediator in the development of cardiovascular disease, and provides a broad idea for the research on Mediator-related cardiovascular system development and diseases.

A Combined Human in Silico and CRISPR/Cas9-Mediated in Vivo Zebrafish Based Approach to Provide Phenotypic Data for Supporting Early Target Validation.

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts as part of the process for validating potential new drug targets. Traditional approaches (e.g., genetically-modified mice) are optimal for assessing small numbers of genes, but less practical when multiple genes are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation, by providing more confidence in target relevance and facilitating gene selection for definitive loss of function studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including changes in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports further investigation of the potential roles of API5, HSPB7, and LMO2 in human cardiovascular disease. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.

Impact of airborne total suspended particles (TSP) and fine particulate matter (PM2.5 )-induced developmental toxicity in zebrafish (Danio rerio) embryos.

Airborne total suspended particles (TSP) and particulate matter (PM2.5 ) threaten global health and their potential impact on cardiovascular and respiratory diseases are extensively studied. Recent studies attest premature deaths, low birth weight, and congenital anomalies in the fetus of pregnant women exposed to air pollution. In this regard, only few studies have explored the effects of TSP and PM2.5 on cardiovascular and cerebrovascular development. As both TSP and PM2.5 differ in size and composition, this study is attempted to assess the variability in toxicity effects between TSP and PM2.5 on the development of cardiovascular and cerebrovascular systems and the underlying mechanisms in a zebrafish model. To explore the potential toxic effects of TSP and PM2.5 , zebrafish embryos/larvae were exposed to 25, 50, 100, 200, and 400 μg/ml of TSP and PM2.5 from 24 to 120 hpf (hours post-fertilization). Both TSP and PM2.5 exposure increased the rate of mortality, malformations, and oxidative stress, whereas locomotor behavior, heart rate, blood flow velocity, development of cardiovasculature and neurovasculature, and dopaminergic neurons were reduced. The expression of genes involved in endoplasmic reticulum stress (ERS), Wnt signaling, and central nervous system (CNS) development were altered in a dose- and time-dependent manner. This study provides evidence for acute exposure to TSP and PM2.5 -induced cardiovascular and neurodevelopmental toxicity, attributed to enhanced oxidative stress and aberrant gene expression. Comparatively, the effects of PM2.5 were more pronounced than TSP.

Mechanism of Peptide Agonist Binding in CXCR4 Chemokine Receptor.

Chemokine receptors are key G-protein-coupled receptors (GPCRs) that control cell migration in immune system responses, development of cardiovascular and central nervous systems, and numerous diseases. In particular, the CXCR4 chemokine receptor promotes metastasis, tumor growth and angiogenesis in cancers. CXCR4 is also used as one of the two co-receptors for T-tropic HIV-1 entry into host cells. Therefore, CXCR4 serves as an important therapeutic target for treating cancers and HIV infection. Apart from the CXCL12 endogenous peptide agonist, previous studies suggested that the first 17 amino acids of CXCL12 are sufficient to activate CXCR4. Two 17-residue peptides with positions 1–4 mutated to RSVM and ASLW functioned as super and partial agonists of CXCR4, respectively. However, the mechanism of peptide agonist binding in CXCR4 remains unclear. Here, we have investigated this mechanism through all-atom simulations using a novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method. The Pep-GaMD simulations have allowed us to explore representative binding conformations of each peptide and identify critical low-energy states of CXCR4 activated by the super versus partial peptide agonists. Our simulations have provided important mechanistic insights into peptide agonist binding in CXCR4, which are expected to facilitate rational design of new peptide modulators of CXCR4 and other chemokine receptors.