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

Abstract

Transforming growth factor-␤ (TGF-␤) 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-␤ signaling via Smad1/5 in ECs, ALK5 knockout (KO) mice were not able to give us the precise mechanisms by which TGF-␤/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 Ncadherin 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, VEcadherin, and S1PR1 expressions in the vasculature.(Blood. 2012;119(22): 5320-5328) IntroductionAberrant vascularization leads to a number of diseases including atherosclerosis, tumorigenicity, and retinopathy, 1,2 whereas angiogenesis is essential during embryonic development as well as in adulthood. Angiogenesis is mediated by sprouting of new vessels from preexisting ones or by intussusceptive microvascular growth. In general, vascular formation is quiet in adulthood, although angiogenesis involved in wound healing, inflammation, ischemia, and the female reproductive cycle can be observed. Angiogenesis is divided into 2 phases: the activation phase and the resolution phase. The balance between physiologic stimulators (eg, vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), angiopoietins, and hypoxia) and inhibitors (eg, angiostatin, endostatin, and interferon-␣) is strategic to tuning of the angiogenic switch. Proliferation of endothelial cells (ECs), increase in vascular permeability, and degradation of extracellular matrix components can be observed during the activation phase. Consequently, ECs make new capillary sprouts. In the resolution phase, the proliferation and migration of ECs ceases and is followed by reconstitution of the basement membrane and maturation of the vessels. 3 Transforming growth factor-␤ (TGF-␤) is a pivotal cytokine that contributes to the behaviors and activities of most cells from the embryonic to the adult stage. The TGF-␤ signal is initiated when the ligand binds to its own TGF-␤ type II receptor (T␤RII); thereafter, the TGF-␤ type I receptor (T␤RI or activin receptor-like kinase [ALK]...

Excessive vascular sprouting underlies cerebral hemorrhage in mice lacking αVβ8-TGFβ signaling in the brain

“…Consistent with this, it was recently shown that endothelial cell-specific Smad2/Smad3 double mutants develop similar brain hemorrhage (Itoh et al, 2012). Although this report did not characterize brain vascular sprouting/branching, it is interesting to point out that S1pr1 mRNA expression was downregulated in Smad2/Smad3 mutants (Itoh et al, 2012), and endothelial cellspecific S1pr1 mutants (like Itgb8, Tgfb1, Tgfbr2 and Alk5 mutants) develop endothelial cell hypersprouting before hemorrhage (Gaengel et al, 2012). It is now important to determine how αVβ8-TGFβ signaling interacts with other pathways, including VEGF, Notch, BMP and S1PR1, known to regulate sprouting angiogenesis.…”

“…The effects of αVβ8-TGFβ1 appear to be directed to endothelial cells, not the neuroepithelium, because endothelial cell-specific Tgfbr2 and Alk5 mutants phenocopy Itgb8 mutants, and neuroepithelial cell-specific deletion of Tgfbr2 exhibited no vascular phenotype. Consistent with this, it was recently shown that endothelial cell-specific Smad2/Smad3 double mutants develop similar brain hemorrhage (Itoh et al, 2012). Although this report did not characterize brain vascular sprouting/branching, it is interesting to point out that S1pr1 mRNA expression was downregulated in Smad2/Smad3 mutants (Itoh et al, 2012), and endothelial cellspecific S1pr1 mutants (like Itgb8, Tgfb1, Tgfbr2 and Alk5 mutants) develop endothelial cell hypersprouting before hemorrhage (Gaengel et al, 2012).…”

“…As the αVβ8 integrin has been shown to mediate TGFβ activation in vitro (Cambier et al, 2005), this suggested that αVβ8 on neuroepithelial cells activates TGFβ, which subsequently promotes vascular integrity. The similarities in phenotype of neuroepithelialspecific Itgav and Itgb8 mutants with endothelial-specific mutations of Tgfbr2 (Robson et al, 2010), Alk5 (Nguyen et al, 2011) and Smad2/Smad3 (Itoh et al, 2012) support this proposal. In each mutant, vessels were 'stalled' in their growth from the pia mater towards the ventricle.…”

“…Glomeruloid formation and hemorrhage are relatively late phenotypes, probably secondary to elevated sprouting, branching and proliferation of endothelial cells. In addition to Itgav and Itgb8, brain hemorrhage during embryonic development is observed in other mutant mice, including Gpr124 (Anderson et al, 2011;Cullen et al, 2011;Kuhnert et al, 2010), Nrp1 (Gerhardt et al, 2004;Gu et al, 2003;Kawasaki et al, 1999), Wnt7a/ b-Bcat (Daneman et al, 2009;Liebner et al, 2008;Stenman et al, 2008), Tgfb1/3 (Mu et al, 2008), Tgfbr2 (Nguyen et al, 2011;Robson et al, 2010), Alk5 (Nguyen et al, 2011), Smad4 , Smad2/Smad3 (Itoh et al, 2012) and S1pr1 (Gaengel et al, 2012). Prior work either did not address the cause of hemorrhage (Nrp1, Tgfb1/3, Smad2/Smad3, S1pr1) or attributed it to a dysfunctional BBB [Gpr124 (Anderson et al, 2011), Bcat (Liebner et al, 2008), Smad4 , Itgb8 (Mobley et al, 2009), Itgav-Tgfbr2-Alk5 (Nguyen et al, 2011)].…”

FGF2-induced Ras/Erk MAPK signalling maintains lymphatic endothelial cell identity by up-regulating endothelial cell-specific gene expression and suppressing TGFβ signalling via Smad2

“…Our findings, together with the findings of Clavin and co-workers, imply that FGF2-meditated maintenance of LEC characteristics might be important for functional lymphatic regeneration during tissue repair in vivo. Recent studies have reported that endothelial-cell-specific knockout mice for Fgfr1 or Smad2 are viable (Itoh et al, 2012;Zhao et al, 2012), suggesting that both genes, and their potential roles in lymphatic EndMT are dispensable for developmental lymphangiogenesis and lymphatic vessel function under physiological conditions. However, it would be important to examine whether these mice exhibit defects in lymphatic vessel regeneration and function during wound healing, and whether and how FGFR, Ras and TGFb signalling contribute to lymphatic regeneration and function in vivo.…”