The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a -/-). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac.

Rationale The density of native (pre-existing) collaterals varies widely and is a significant determinant of variation in severity of stroke, myocardial infarction and peripheral artery disease. However, little is known about mechanisms responsible for formation of the collateral circulation in healthy tissues. Objective We previously found that variation in VEGF expression causes differences in collateral density of newborn and adult mice. Herein, we sought to determine mechanisms of collaterogenesis in the embryo and the role of VEGF in this process. Methods and Results Pial collaterals begin forming between embryonic day (E) 13.5 and 14.5 as sprout-like extensions from arterioles of existing cerebral artery trees. Global VEGF-A overexpressing mice (Vegf hi/+) formed more—and Vegf lo/+ formed fewer—collaterals during embryogenesis, in association with differences in vascular patterning. Conditional global reduction of Vegf or Flk1 only during collaterogenesis significantly reduced collateral formation, but now without affecting vascular patterning, and the effects remained in adulthood. Endothelial-specific Vegf reduction had no effect on collaterogenesis. Endothelial-specific reduction of a disintegrin-and-metalloprotease-domain-10 (Adam10) and inhibition of γ-secretase increased collateral formation, consistent with their roles in VEGF-induced Notch1 activation and suppression of “pro-sprouting” signals. Endothelial-specific knockdown of Adam17 reduced collateral formation, consistent with its roles in endothelial cell migration and embryonic vascular stabilization, but not in activation of ligand-bound Notch1. These effects also remained in adulthood. Conclusions Formation of pial collaterals occurs during a narrow developmental window via a sprouting angiogenesis-like mechanism, requires paracrine VEGF-stimulation of Flk1-Notch signaling, and adult collateral number is dependent on embryonic collaterogenesis.

During developmental hematopoiesis, multilineage hematopoietic progenitors are thought to derive from a subset of vascular endothelium. Herein, we define the phenotype of such hemogenic endothelial cells and demonstrate, on a clonal level, that they exhibit multilineage hematopoietic potential. Furthermore, we have begun to define the molecular signals that regulate their development. We found that the formation of yolk sac hemogenic endothelium and its hematopoietic potential were significantly impaired in the absence of retinoic acid (RA) signaling, and could be restored in RA-deficient (Raldh2−/−) embryos by provision of exogenous RA in utero. Thus, we identify a novel, critical role for RA signaling in the development of hemogenic endothelium that contributes to definitive hematopoiesis.

Rationale Severity of tissue injury in occlusive disease is dependent on the extent (number and diameter) of collateral vessels, which varies widely among healthy mice and humans. However, the causative genetic elements are unknown. Recently, much of the variation among different mouse strains, including C57Bl/6J (B6, high extent) and BALB/cByJ (Bc, low), was linked to a QTL on chromosome 7 (Candq1). Objective We used congenic mapping to refine Candq1 and its candidate genes and create an “isogenic” strain-set with large differences in collateral extent to assess their and Candq1’s impact, alone, on ischemic injury. Methods and Results Six congenic strains possessing portions of Candq1 introgressed from B6 into Bc were generated and phenotyped. Candq1 was refined from 27 to 0.737 Mb with full retention of effect, ie, return/rescue of phenotypes from the poor values in Bc to nearly those of wildtype B6 in the B6/B6 congenic mice: 83% rescue of low pial collateral extent, and 4.5-fold increase in blood flow and 85% reduction of infarct volume after middle cerebral artery occlusion; 54% rescue of low skeletal muscle collaterals, and augmented recovery of perfusion (83%) and function after femoral artery ligation. Gene deletion and in-silico analysis further delineated the candidate genes. Conclusion We have significantly refined Candq1 (now designated Determinant of collateral extent-1, Dce1), demonstrated that genetic background-dependent variation in collaterals is a major factor underlying differences in ischemic tissue injury, and generated a congenic strain-set with wide, allele-dose-dependent variation in collateral extent for use in investigations of the collateral circulation.

Background and Purpose The extent (number and diameter) of collateral vessels varies widely and is a major determinant, along with arteriogenesis (collateral remodeling), of variation in severity of tissue injury following large artery occlusion. Differences in genetic background underlie the majority of the variation in collateral extent in mice, through alterations in collaterogenesis (embryonic collateral formation). In brain and other tissues, ~80% of the variation in collateral extent among different mouse strains has been linked to a region on chromosome 7. We recently used congenic (CNG) fine-mapping of C57BL/6 (B6, high extent) and BALB/cBy (BC, low extent) mice to narrow the region to a 737 Kb locus, Dce1. Herein, we report the causal gene. Methods We used additional CNG mapping and knockout mice to narrow the number of candidate genes. Subsequent inspection identified a non-synonymous SNP between B6 and BC within Rabep2 (rs33080487). We then created B6 mice with the BC SNP at this locus plus three other lines for predicted alteration or knockout of Rabep2 using gene editing. Results The single amino acid change caused by rs33080487 accounted for the difference in collateral extent and infarct volume between B6 and BC mice attributable to Dce1. Mechanistically, variants of Rabep2 altered collaterogenesis during embryogenesis but had no effect on angiogenesis examined in vivo and in vitro. Rabep2 deficiency altered endosome trafficking known to be involved in VEGF-A→VEGFR2 signaling required for collaterogenesis. Conclusions Naturally occurring variants of Rabep2 are major determinants of variation in collateral extent and stroke severity in mice.

Altered blood pressure and flow impact cardiac function during morphogenesis. How the arterial system supports cardiac morphogenesis after circulatory disruptions is not well characterized. We manipulated arterial flow via left atrial ligation (LAL) or arterial load via right vitelline artery ligation (VAL) in HamburgerHamilton (HH) stage 21 chick embryos. Embryos were reincubated for 1·h (HH21), 14·h (HH24) or 30·h (HH27). At each stage we measured simultaneous dorsal aortic blood pressure and flow, and calculated arterial compliance, impedance and hydraulic power. LAL acutely reduced stroke volume (VS), cardiac output (Q) and hydraulic power. Arterial pressure was preserved by a compensatory increase in characteristic impedance and decrease in compliance. Impedance parameters and compliance normalized by HH24 and all parameters normalized by HH27. VAL acutely increased arterial resistance. Embryos maintained arterial pressure by decreasing VS and Q. These parameters remained altered through HH27. In summary, despite the intervention, compensatory alterations in VS and arterial resistance maintained arterial pressure and fraction of oscillatory power within a narrow range. These results suggest that the maintenance of arterial pressure and circulatory energy efficiency, but not arterial flow, is critical to embryogenesis.

Premenopausal women and intact female rodents sustain smaller cerebral infarctions than males. Several sex-dependent differences have been identified as potential contributors, but many questions remain unanswered. Mice exhibit wide variation in native collateral number and diameter (collateral extent) that is dependent on differences in genetic background, aging, and other comorbidities and that contributes to their also-wide differences in infarct volume. Likewise, variation in infarct volume correlates with differences in collateral-dependent blood flow in patients with acute ischemic stroke. We examined whether extent of pial collateral arterioles and posterior communicating collateral arteries (PComAs) differ depending on sex in young, aged, obese, hypertensive, and genetically different mice. We combined new data with meta-analysis of our previously published data. Females of C57BL/6J (B6) and BALB/cByJ (BC) strains sustained smaller infarctions than males after permanent MCA occlusion. This protection was unchanged in BC mice after introgression of the B6 allele of Dce1, the major genetic determinant of variation in pial collaterals among mouse strains. Consistent with this, collateral extent in these and other strains did not differ with sex. Extent of PComAs and primary cerebral arteries also did not vary with sex. No dimorphism was evident for loss of pial collateral number and/or diameter (collateral rarefaction) caused by aging, obesity, and hypertension, nor for collateral remodeling after pMCAO. However, rarefaction was greater in females with long-standing hypertension. We conclude that smaller infarct volume in female mice is not due to greater collateral extent, greater remodeling, or less rarefaction caused by aging, obesity, or hypertension.

Oxygen tension is a critical factor for appropriate embryonic and fetal development. Chronic hypoxia exposure alters cardiovascular (CV) function and structure in the late fetus and newborn, yet the immature myocardium is considered to be less sensitive to hypoxia than the mature heart. We tested the hypothesis that hypoxia during the period of primary CV morphogenesis impairs immature embryonic CV function and embryo growth. We incubated fertile white Leghorn chick embryos in 15% oxygen (hypoxia) or 21% oxygen (control) until Hamburger-Hamilton stage 21 (3.5 d). We assessed in ovo viability and dysmorphic features and then measured ventricular pressure and dimensions and dorsal aortic arterial impedance at stage 21. Chronic hypoxia decreased viability and embryonic wet weight. Chronic hypoxia did not alter heart rate or the ventricular diastolic indices of end-diastolic pressure, maximum ventricular -dP/dt, or tau. Chronic hypoxia decreased maximum ventricular ϩdP/dt and peak pressure, increased ventricular end-systolic volume, and decreased ventricular ejection fraction, consistent with depressed systolic function. Arterial afterload (peripheral resistance) increased and both dorsal aortic SV and steady-state hydraulic power decreased in response to hypoxia. Thus, reduced oxygen tension during early cardiac development depresses ventricular function, increases ventricular impedance (afterload), delays growth, and decreases embryo survival, suggesting that a critical threshold of oxygen tension is required to support morphogenesis and cardiovascular function in the early embryo. (Pediatr Res 59: 116-120, 2006)