The endothelial cell-specific vascular endothelial growth factor (VEGF) and its cellular receptors Flt-1 and Flk-1 have been implicated in the formation of the embryonic vasculature. This is suggested by their colocalized expression during embryogenesis and the impaired vessel formation in Flk-1 and Flt-1 deficient embryos. However, because Flt-1 also binds placental growth factor, a VEGF homologue, the precise role of VEGF was unknown. Here we report that formation of blood vessels was abnormal, but not abolished, in heterozygous VEGF-deficient (VEGF+/-) embryos, generated by aggregation of embryonic stem (ES) cells with tetraploid embryos (T-ES) and even more impaired in homozygous VEGF-deficient (VEGF-/-) T-ES embryos, resulting in death at mid-gestation. Similar phenotypes were observed in F1-VEGF+/- embryos, generated by germline transmission. We believe that this heterozygous lethal phenotype, which differs from the homozygous lethality in VEGF-receptor-deficient embryos, is unprecedented for a targeted autosomal gene inactivation, and is indicative of a tight dose-dependent regulation of embryonic vessel development by VEGF.
Indirect evidence suggests a crucial role for the fibrinolytic system and its physiological triggers, tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator, in many proteolytic processes. Inactivation of the t-PA gene impairs clot lysis and inactivation of the u-PA gene results in occasional fibrin deposition. Mice with combined t-PA and u-PA deficiency suffer extensive spontaneous fibrin deposition, with its associated effects on growth, fertility and survival.
Homozygous plasminogen activator inhibitor-i (PAI-1 )-deficient (PAI-1 /-I) mice were generated by homologous recombination in D3 embryonic stem cells. Deletion of the genomic sequences encompassing the transcription initiation site and the entire coding regions of murine PAI-i was demonstrated by Southern blot analysis. A 3.0-kb PAI-i-specific mRNA was identified by Northern blot analysis in liver from PAI-1 wild type (PAI-i +1+) but not from PAI-1i--mice. Plasma PAI-i levels, measured 2-4 h after endotoxin (2.0 mg/kg) injection were 63±2 ng/ml, 30±10 ng/ml, and undetectable (< 2 ng/ ml) in PAl-i ++, heterozygous (PA-i +/-) and PAMl-/-mice, respectively (mean±SEM, n = 4-11). PAI-1-specific immunoreactivity was demonstrable in kidneys of PAI-1 +/+ but not of PAl-i--mice. SDS-gel electrophoresis of plasma incubated with '25I-labeled recombinant human tissue-type plasminogen activator revealed an 115,000-M, component with plasma from endotoxin-stimulated (0.5 mg/kg) PA-i +/+ but not from PAM--/-mice, which could be precipitated with a polyclonal anti-PAI-1 antiserum. PAM-i-mice were viable, produced similar sizes of litters as PA-+/+ mice, and showed no apparent macroscopic or microscopic histological abnormalities. (J. Clin. Invest. 1993. 92:2746-2755
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.