Smad2/Smad3 in endothelium is indispensable for vascular stability via S1PR1 and N-cadherin expressions.

Transforming growth factor-ß (TGF-ß) is involved in vascular formation through activin receptor-like kinase (ALK)1 and ALK5. ALK5, which is expressed ubiquitously, phosphorylates Smad2 and Smad3, whereas endothelial cell (EC)-specific ALK1 activates Smad1 and Smad5. Because ALK5 kinase activity is required for ALK1 to transduce TGF-ß signaling via Smad1/5 in ECs, ALK5 knockout (KO) mice were not able to give us the precise mechanisms by which TGF-ß/ALK5/Smad2/3 signaling is implicated in angiogenesis. To delineate the role of Smad2/3 signaling in endothelium, the Smad2 gene in Smad3 KO mice was selectively deleted in ECs using Tie2-Cre transgenic mice, termed EC-specific Smad2/3 double KO (EC-Smad2/3KO) mice. EC-Smad2/3KO embryos revealed hemorrhage leading to embryonic lethality around E12.5. EC-Smad2/3KO embryos exhibited no abnormality of vasculogenesis and angiogenesis in both the yolk sac and the whole embryo, whereas vascular maturation was incomplete because of inadequate assembly of mural cells in the vasculature. Wide gaps between ECs and mural cells could be observed in the vasculature of EC-Smad2/3KO mice because of reduced expression of N-cadherin and sphingosine-1-phosphate receptor-1 (S1PR1) in ECs from those mice. These results indicated that Smad2/3 signaling in ECs is indispensable for maintenance of vascular integrity via the fine-tuning of N-cadherin, VE-cadherin, and S1PR1 expressions in the vasculature.

Generation of mouse conditional and null alleles of the type III sodium-dependent phosphate cotransporter PiT-1.

Accelerated vascular calcification occurs in several human diseases including diabetes and chronic kidney disease (CKD). In patients with CKD, vascular calcification is highly correlated with elevated serum phosphate levels. In vitro, elevated concentrations of phosphate induced vascular smooth muscle cell matrix mineralization, and the inorganic phosphate transporter-1 (PiT-1), was shown to be required. To determine the in vivo role of PiT-1, mouse conditional and null alleles were generated. Here we show that the conditional allele, PiT-1(flox), which has loxP sites flanking exons 3 and 4, is homozygous viable. Cre-mediated recombination resulted in a null allele that is homozygous lethal. Examination of early embryonic development revealed that the PiT-1(Deltae3,4/Deltae3,4) embryos displayed anemia, a defect in yolk sac vasculature, and arrested growth. Thus, conditional and null PiT-1 mouse alleles have been successfully generated and PiT-1 has a necessary, nonredundant role in embryonic development.

Phosphate and vascular calcification: Emerging role of the sodium-dependent phosphate co-transporter PiT-1.

Elevated serum phosphate is a risk factor for vascular calcification and cardiovascular events in kidney disease as well as in the general population. Elevated phosphate levels drive vascular calcification, in part, by regulating vascular smooth muscle cell (VSMC) gene expression, function, and fate. The type III sodium-dependent phosphate co-transporter, PiT-1, is necessary for phosphate-induced VSMC osteochondrogenic phenotype change and calcification, and has recently been shown to have unexpected functions in cell proliferation and embryonic development.

Generation of novel conditional and hypomorphic alleles of the Smad2 gene.

Smad2 is an intracellular mediator of the transforming growth factor beta signaling (TGFbeta) pathway. It has been previously shown that, in the mouse, ablation of functional Smad2 results in embryonic lethality due to gastrulation defects. To circumvent the early lethality and study the spatially and temporally specific functions of Smad2, we utilized the Cre-loxP system to generate a Smad2 conditional allele. Here we show that a conditional allele, Smad2(flox), was generated. In this allele, exons 9 and 10 are flanked by loxP sites and the gene is functionally wildtype. Cre-mediated recombination results in a deletion allele which phenocopies our previously reported Smad2(DeltaC) null mutation. To generate this conditional allele, we first made a targeted mutation which introduced a floxed neo cassette into intron 10. This allele (Smad2(3loxP)) functions hypomorphically when placed opposite a null allele, and unlike the other published Smad2 hypomorphic allele, can be maintained in the homozygous state.

Smad2 and Smad3 coordinately regulate craniofacial and endodermal development.

Ligands of the transforming growth factor-beta (TGF-beta) superfamily are involved in numerous developmental and disease processes. TGF-beta, activins, and nodal ligands operate through the highly homologous Smad2 and Smad3 intracellular mediators. Smad2 mutants exhibit early embryonic lethality, while Smad3 mutants are viable, but show a plethora of postnatal phenotypes, including immune dysfunction and skeletal abnormalities. Previously, we have shown that the Smad2 and Smad3 genes function cooperatively during liver morphogenesis. Here we show that Smad2 and Smad3 are required at a full dosage for normal embryonic development. Animals lacking one allele of each gene exhibit a variably penetrant phenotype in which structures in the anterior and ventral midline are reduced or lost; additionally, we demonstrate that this craniofacial defect and the previously reported hepatic phenotypes are both due to defects in the definitive endoderm. A reduction of endodermal gene expression as well as a failure to displace the visceral endoderm occurs despite the formation of a normal foregut pocket. This precedes any defects in anterior patterning and likely causes the abnormalities observed in craniofacial and midline development, as well as hepatogenesis.