Background: Endothelial-mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Results: Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin-dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up-regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively-active calcineurin A, suggesting dysregulation of myocardial calcineurin-VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium-derived growth factors, transforming growth factor b2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial-specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. Conclusions: The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium-related signaling events. Developmental Dynamics 244:31-42, 2015. V C 2014 Wiley Periodicals, Inc.

The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1(ko) /H2(ko) ) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1(ko) /H2(eko) ) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1(ko) /H2(ko) phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1(ko) /H2(eko) embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency.

Among members of the TGFβ superfamily, bone morphogenetic protein (BMP) 9 and BMP10 regulate vascular endothelium differentiation and morphogenesis by activating the specific receptor complex, which consists of ALK1 (or ACVRL1), BMPR2, and endoglin. Mutations in ACVRL1, BMPR2, or ENG are associated with hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension in humans [1, 2]. We previously identified TMEM100 as a downstream target gene of BMP9/BMP10-ALK1 signaling pathway [3]. TMEM100 is a novel intracellular protein with two putative transmembrane domains, and its amino acid sequence is highly conserved from fish to humans. To clarify the physiological significance of TMEM100, we generated Tmem100deficient mice and found that all mutant embryos died in utero around embryonic day 10.5 (E10.5). LacZ reporter driven by the Tmem100 locus was predominantly expressed in endothelial cells of developing arteries and endocardium. Tmem100 null embryos showed severe vascular dysmorphogenesis and cardiac enlargement at E9.5 and massive pericardial effusion and growth retardation at E10.5 (Fig. 21.1). These phenotypic abnormalities were virtually identical to those observed in Alk1/Acvrl1-deficient mice, suggesting that Tmem100 is an important downstream gene of BMP9/BMP10-ALK1 signaling during cardiovascular Note: This Chapter is also related to Part VI.

Background Pregnancy in dialysis patients is rare but challenging both for nephrologists and obstetricians. Although intensified hemodialysis is effective to achieve favorable clinical outcomes in this clinical setting, the evidence concerning reliable parameters for the optimal dialysis prescription is scarce. Herein, we report a case of a primigravida with advanced maternal age who underwent a normal spontaneous vaginal delivery at full term managed by intensified hemodialysis, and discuss reliable indicators of the appropriate dialysis prescription. Case presentation A 39-year-old primigravida with end-stage renal disease was referred to our department for hemodialysis management during pregnancy. She had a history of multiple gynecologic disorders, including cervical dysplasia, right tubal ectopic pregnancy and spontaneous abortion; consequently, she conceived through in vitro fertilization and embryo transfer. After pregnancy was confirmed, an intensified hemodialysis schedule with weekly duration of 36 hours was employed to prevent volume overload and the accumulation of hyperosmotic substances. Utilizing maternal blood pressure, plasma atrial natriuretic peptide levels and fetal development as indicators of volume status, we incremented her dry weight by 300–400 g per week in the first and second trimester and 100–200 g per week in the third trimester, respectively. Serum urea nitrogen levels were persistently maintained below 50 mg/dL. Throughout pregnancy, fetal growth and development were within normal limits. At 38 weeks and 3 days of gestation, she delivered a healthy child vaginally without complications. To date, the child’s development has been normal, both physically and mentally. Conclusions Pregnancy in dialysis patients with advanced age that leads to a normal childbirth is exceptionally rare. Our case is the hemodialysis patient with the most advanced age who achieves a normal spontaneous vaginal delivery at full term. Considering that cardiovascular system and endogenous metabolism dynamically alter during pregnancy, various clinical parameters should be closely monitored to modify dialysis settings accordingly. Of note, our case suggests that plasma atrial natriuretic peptide level is a useful parameter for the appropriate dry weight. Intensified hemodialysis is an effective and reliable measure that can achieve normal pregnancy in dialysis patients, even in a primigravida with advanced maternal age.

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