Buffering Mechanism in Aortic Arch Artery Formation and Congenital Heart Disease.

BACKGROUND: The resiliency of embryonic development to genetic and environmental perturbations has been long appreciated; however, little is known about the mechanisms underlying the robustness of developmental processes. Aberrations resulting in neonatal lethality are exemplified by congenital heart disease arising from defective morphogenesis of pharyngeal arch arteries (PAAs) and their derivatives. METHODS: Mouse genetics, lineage tracing, confocal microscopy, and quantitative image analyses were used to investigate mechanisms of PAA formation and repair. RESULTS: The second heart field (SHF) gives rise to the PAA endothelium. Here, we show that the number of SHF-derived endothelial cells (ECs) is regulated by VEGFR2 (vascular endothelial growth factor receptor 2) and Tbx1. Remarkably, when the SHF-derived EC number is decreased, PAA development can be rescued by the compensatory endothelium. Blocking such compensatory response leads to embryonic demise. To determine the source of compensating ECs and mechanisms regulating their recruitment, we investigated 3-dimensional EC connectivity, EC fate, and gene expression. Our studies demonstrate that the expression of VEGFR2 by the SHF is required for the differentiation of SHF-derived cells into PAA ECs. The deletion of 1 VEGFR2 allele (VEGFR2SHF-HET) reduces SHF contribution to the PAA endothelium, while the deletion of both alleles (VEGFR2SHF-KO) abolishes it. The decrease in SHF-derived ECs in VEGFR2SHF-HET and VEGFR2SHF-KO embryos is complemented by the recruitment of ECs from the nearby veins. Compensatory ECs contribute to PAA derivatives, giving rise to the endothelium of the aortic arch and the ductus in VEGFR2SHF-KO mutants. Blocking the compensatory response in VEGFR2SHF-KO mutants results in embryonic lethality shortly after mid-gestation. The compensatory ECs are absent in Tbx1+/- embryos, a model for 22q11 deletion syndrome, leading to unpredictable arch artery morphogenesis and congenital heart disease. Tbx1 regulates the recruitment of the compensatory endothelium in an SHF-non-cell-autonomous manner. CONCLUSIONS: Our studies uncover a novel buffering mechanism underlying the resiliency of PAA development and remodeling.

Cell-Extracellular Matrix Interactions Play Multiple Essential Roles in Aortic Arch Development.

RATIONALE: Defects in the morphogenesis of the fourth pharyngeal arch arteries (PAAs) give rise to lethal birth defects. Understanding genes and mechanisms regulating PAA formation will provide important insights into the etiology and treatments for congenital heart disease. OBJECTIVE: Cell-ECM (extracellular matrix) interactions play essential roles in the morphogenesis of PAAs and their derivatives, the aortic arch artery and its major branches; however, their specific functions are not well-understood. Previously, we demonstrated that integrin α5ß1 and Fn1 (fibronectin) expressed in the Isl1 lineages regulate PAA formation. The objective of the current studies was to investigate cellular mechanisms by which integrin α5ß1 and Fn1 regulate aortic arch artery morphogenesis. METHODS AND RESULTS: Using temporal lineage tracing, whole-mount confocal imaging, and quantitative analysis of the second heart field (SHF) and endothelial cell (EC) dynamics, we show that the majority of PAA EC progenitors arise by E7.5 in the SHF and contribute to pharyngeal arch endothelium between E7.5 and E9.5. Consequently, SHF-derived ECs in the pharyngeal arches form a plexus of small blood vessels, which remodels into the PAAs by 35 somites. The remodeling of the vascular plexus is orchestrated by signals dependent on the pharyngeal ECM microenvironment, extrinsic to the endothelium. Conditional ablation of integrin α5ß1 or Fn1 in the Isl1 lineages showed that signaling by the ECM regulates aortic arch artery morphogenesis at multiple steps: (1) accumulation of SHF-derived ECs in the pharyngeal arches, (2) remodeling of the EC plexus in the fourth arches into the PAAs, and (3) differentiation of neural crest-derived cells adjacent to the PAA endothelium into vascular smooth muscle cells. CONCLUSIONS: PAA formation is a multistep process entailing dynamic contribution of SHF-derived ECs to pharyngeal arches, the remodeling of endothelial plexus into the PAAs, and the remodeling of the PAAs into the aortic arch artery and its major branches. Cell-ECM interactions regulated by integrin α5ß1 and Fn1 play essential roles at each of these developmental stages.

Relationship of Weight Outcomes, Co-Occurring Conditions, and Severity of Autism Spectrum Disorder in the Study to Explore Early Development.

OBJECTIVE: To assess contributing factors to increased obesity risk, by comparing children with autism spectrum disorder (ASD), developmental delays/disorders, and general population controls in weight status, and to examine associations between weight status and presence of co-occurring medical, behavioral, developmental, or psychiatric conditions across groups and ASD severity among children with ASD. STUDY DESIGN: The Study to Explore Early Development is a multisite cross-sectional study of children, 2-5 years of age, classified as children with ASD (n = 668), children with developmental delays/disorders (n = 914), or general population controls (n = 884). Using an observational cohort design, we compared the 3 groups. Children's heights and weights were measured during a clinical visit. Co-occurring conditions (medical, behavioral, developmental/psychiatric) were derived from medical records, interviews, and questionnaires. ASD severity was measured by the Ohio State University Global Severity Scale for Autism. RESULTS: The odds of overweight/obesity were 1.57 times (95% CI 1.24-2.00) higher in children with ASD than general population controls and 1.38 times (95% CI 1.10-1.72) higher in children with developmental delays/disorders than general population controls. The aORs were elevated for children with ASD after controlling for child co-occurring conditions (ASD vs general population controls: aOR = 1.51; 95% CI 1.14-2.00). Among children with ASD, those with severe ASD symptoms were 1.7 times (95% CI 1.1-2.8) more likely to be classified as overweight/obese compared with children with mild ASD symptoms. CONCLUSIONS: Prevention of excess weight gain in children with ASD, especially those with severe symptoms, and in children with developmental delays/disorders represents an important target for intervention.

Endothelium in the pharyngeal arches 3, 4 and 6 is derived from the second heart field.

Oxygenated blood from the heart is directed into the systemic circulation through the aortic arch arteries (AAAs). The AAAs arise by remodeling of three symmetrical pairs of pharyngeal arch arteries (PAAs), which connect the heart with the paired dorsal aortae at mid-gestation. Aberrant PAA formation results in defects frequently observed in patients with lethal congenital heart disease. How the PAAs form in mammals is not understood. The work presented in this manuscript shows that the second heart field (SHF) is the major source of progenitors giving rise to the endothelium of the pharyngeal arches 3 - 6, while the endothelium in the pharyngeal arches 1 and 2 is derived from a different source. During the formation of the PAAs 3 - 6, endothelial progenitors in the SHF extend cellular processes toward the pharyngeal endoderm, migrate from the SHF and assemble into a uniform vascular plexus. This plexus then undergoes remodeling, whereby plexus endothelial cells coalesce into a large PAA in each pharyngeal arch. Taken together, our studies establish a platform for investigating cellular and molecular mechanisms regulating PAA formation and alterations that lead to disease.

Identification of novel buffering mechanisms in aortic arch artery development and congenital heart disease.

Rationale: The resiliency of embryonic development to genetic and environmental perturbations has been long appreciated; however, little is known about the mechanisms underlying the robustness of developmental processes. Aberrations resulting in neonatal lethality are exemplified by congenital heart disease (CHD) arising from defective morphogenesis of pharyngeal arch arteries (PAA) and their derivatives. Objective: To uncover mechanisms underlying the robustness of PAA morphogenesis. Methods and Results: The second heart field (SHF) gives rise to the PAA endothelium. Here, we show that the number of SHF-derived ECs is regulated by VEGFR2 and Tbx1 . Remarkably, when SHF-derived EC number is decreased, PAA development can be rescued by the compensatory endothelium. Blocking such compensatory response leads to embryonic demise. To determine the source of compensating ECs and mechanisms regulating their recruitment, we investigated three-dimensional EC connectivity, EC fate, and gene expression. Our studies demonstrate that the expression of VEGFR2 by the SHF is required for the differentiation of SHF-derived cells into PAA ECs. The deletion of one VEGFR2 allele (VEGFR2 SHF-HET ) reduces SHF contribution to the PAA endothelium, while the deletion of both alleles (VEGFR2 SHF-KO ) abolishes it. The decrease in SHF-derived ECs in VEGFR2 SHF-HET and VEGFR2 SHF-KO embryos is complemented by the recruitment of ECs from the nearby veins. Compensatory ECs contribute to PAA derivatives, giving rise to the endothelium of the aortic arch and the ductus in VEGFR2 SHF-KO mutants. Blocking the compensatory response in VEGFR2 SHF-KO mutants results in embryonic lethality shortly after mid-gestation. The compensatory ECs are absent in Tbx1 +/- embryos, a model for 22q11 deletion syndrome, leading to unpredictable arch artery morphogenesis and CHD. Tbx1 regulates the recruitment of the compensatory endothelium in an SHF-non-cell-autonomous manner. Conclusions: Our studies uncover a novel buffering mechanism underlying the resiliency of PAA development and remodeling. Nonstandard Abbreviations and Acronyms in Alphabetical Order: CHD - congenital heart disease; ECs - endothelial cells; IAA-B - interrupted aortic arch type B; PAA - pharyngeal arch arteries; RERSA - retro-esophageal right subclavian artery; SHF - second heart field; VEGFR2 - Vascular endothelial growth factor receptor 2.

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction.

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Early life influences on child weight outcomes in the Study to Explore Early Development.

We examined associations between child body mass index at 2-5 years and maternal pre-pregnancy body mass index, gestational weight gain, and rapid weight gain during infancy in children with autism spectrum disorder, developmental delays, or population controls. The Study to Explore Early Development is a multi-site case-control study of children, aged 2-5 years, classified as autism spectrum disorder ( n = 668), developmental delays ( n = 914), or population controls ( n = 884). Maternal gestational weight gain was compared to the Institute of Medicine recommendations. Rapid weight gain was a change in weight-for-age z-scores from birth to 6 months > 0.67 standard deviations. After adjusting for case status, mothers with pre-pregnancy overweight/obesity were 2.38 times (95% confidence interval: 1.96-2.90) more likely, and mothers who exceeded gestational weight gain recommendations were 1.48 times (95% confidence interval: 1.17-1.87) more likely, to have an overweight/obese child than other mothers ( P < 0.001). Children with autism spectrum disorder showed the highest frequency of rapid weight gain (44%) and were 3.47 times (95% confidence interval: 1.85-6.51) more likely to be overweight/obese as children with autism spectrum disorder without rapid weight gain ( P < 0.001). Helping mothers achieve a healthy pre-pregnancy body mass index and gestational weight gain represent important targets for all children. Healthy infant growth patterns carry special importance for children at increased risk for an autism spectrum disorder diagnosis.

ASD Screening with the Child Behavior Checklist/1.5-5 in the Study to Explore Early Development.

We analyzed CBCL/1½-5 Pervasive Developmental Problems (DSM-PDP) scores in 3- to 5-year-olds from the Study to Explore Early Development (SEED), a multi-site case control study, with the objective to discriminate children with ASD (N = 656) from children with Developmental Delay (DD) (N = 646), children with Developmental Delay (DD) plus ASD features (DD-AF) (N = 284), and population controls (POP) (N = 827). ASD diagnosis was confirmed with the ADOS and ADI-R. With a cut-point of T ≥ 65, sensitivity was 80% for ASD, with specificity varying across groups: POP (0.93), DD-noAF (0.85), and DD-AF (0.50). One-way ANOVA yielded a large group effect (η2 = 0.50). Our results support the CBCL/1½-5's as a time-efficient ASD screener for identifying preschoolers needing further evaluation.

Poly (ADP) Ribose Glycohydrolase Can Be Effectively Targeted in Pancreatic Cancer.

Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have an average survival of less than 1 year, underscoring the importance of evaluating novel targets with matched targeted agents. We recently identified that poly (ADP) ribose glycohydrolase (PARG) is a strong candidate target due to its dependence on the pro-oncogenic mRNA stability factor HuR (ELAVL1). Here, we evaluated PARG as a target in PDAC models using both genetic silencing of PARG and established small-molecule PARG inhibitors (PARGi), PDDX-01/04. Homologous repair-deficient cells compared with homologous repair-proficient cells were more sensitive to PARGi in vitro. In vivo, silencing of PARG significantly decreased tumor growth. PARGi synergized with DNA-damaging agents (i.e., oxaliplatin and 5-fluorouracil), but not with PARPi therapy. Mechanistically, combined PARGi and oxaliplatin treatment led to persistence of detrimental PARylation, increased expression of cleaved caspase-3, and increased γH2AX foci. In summary, these data validate PARG as a relevant target in PDAC and establish current therapies that synergize with PARGi. SIGNIFICANCE: PARG is a potential target in pancreatic cancer as a single-agent anticancer therapy or in combination with current standard of care.