Here we describe in detail both the expression of Hoxa-11 in the wild-type mouse uterus and the defects resulting in maternal reproductive failure of Hoxa-11 null female mice. The Hoxa-11 gene is expressed at peak levels in uterine stromal cells during metestrus. Hoxa-11 transcripts were induced beginning on Day 2 of gestation in the stromal cells underlying the uterine epithelium and appeared in the secondary decidual zone between Days 6 and 8 of gestation. At early gestational stages, stromal, decidual, and glandular cell development were deficient in Hoxa-11 null uteri in comparison to wild-type as assessed by histology and immunohistochemical localization of the decidual cell marker epitope, stage-specific embryonic antigen-3 (SSEA-3). Both steroid-induced uterine stromal and glandular cell proliferation as well as oil-induced stromal decidualization after induction of pseudopregnancy were deficient in mutant uteri. Moreover, both Western blotting and immunohistochemistry demonstrated that the burst of glandular leukemia inhibitory factor (LIF) found in normal pregnant uteri at Day 4.5 of gestation was absent in Hoxa-11-deficient uteri. The LIF burst was also not observed in the uteri of bilaterally ovariectomized, hormonally stimulated Hoxa-11 mutants. These results demonstrate that the Hoxa-11 gene is required for normal uterine stromal cell and glandular differentiation during pregnancy, as is the presence of the steroid-induced glandular LIF burst initiating embryo implantation.

To examine the roles of TGFbeta isoforms on corneal morphogenesis, the eyes of mice that lack TGFbetas were analyzed at different developmental stages for cell proliferation, migration and apoptosis, and for expression patterns of keratin 12, lumican, keratocan and collagen I. Among the three Tgfb(-/-) mice, only Tgfb2(-/-) mice have abnormal ocular morphogenesis characterized by thin corneal stroma, absence of corneal endothelium, fusion of cornea to lens (a Peters'-like anomaly phenotype), and accumulation of hyaline cells in vitreous. In Tgfb2(-/-) mice, fewer keratocytes were found in stroma that has a decreased accumulation of ECM; for example, lumican, keratocan and collagen I were greatly diminished. The absence of TGFbeta2 did not compromise cell proliferation, nor enhance apoptosis. The thinner stroma resulting from decreased ECM synthesis may account for the decreased cell number in the stroma of Tgfb2 null mice. Keratin 12 expression was not altered in Tgfb2(-/-) mice, implicating normal corneal type epithelial differentiation. Delayed appearance of macrophages in ocular tissues was observed in Tgfb2(-/-) mice. Malfunctioning macrophages may account for accumulation of cell mass in vitreous of Tgfb2 null mice.

BackgroundPulmonary arterial hypertension (PAH) is thought to be driven by dysfunction of pulmonary vascular microendothelial cells (PMVEC). Most hereditary PAH is associated with BMPR2 mutations. However, the physiologic and molecular consequences of expression of BMPR2 mutations in PMVEC are unknown.MethodsIn vivo experiments were performed on adult mice with conditional endothelial-specific expression of the truncation mutation Bmpr2delx4+, with age-matched transactivator-only mice as controls. Phenotype was assessed by RVSP, counts of muscularized vessels and proliferating cells, and staining for thromboses, inflammatory cells, and apoptotic cells. The effects of BMPR2 knockdown in PMVEC by siRNA on rates of apoptosis were assessed. Affymetrix expression arrays were performed on PMVEC isolated and cultured from triple transgenic mice carrying the immortomouse gene, a transactivator, and either control, Bmpr2delx4+ or Bmpr2R899X mutation.ResultsTransgenic mice showed increased RVSP and corresponding muscularization of small vessels, with histologic alterations including thrombosis, increased inflammatory cells, increased proliferating cells, and a moderate increase in apoptotic cells. Expression arrays showed alterations in specific pathways consistent with the histologic changes. Bmpr2delx4+ and Bmpr2R899X mutations resulted in very similar alterations in proliferation, apoptosis, metabolism, and adhesion; Bmpr2delx4+ cells showed upregulation of platelet adhesion genes and cytokines not seen in Bmpr2R899X PMVEC. Bmpr2 mutation in PMVEC does not cause a loss of differentiation markers as was seen with Bmpr2 mutation in smooth muscle cells.ConclusionsBmpr2 mutation in PMVEC in vivo may drive PAH through multiple, potentially independent, downstream mechanisms, including proliferation, apoptosis, inflammation, and thrombosis.

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These results indicate that the maintenance of Tbdn-1 expression is important for retinal blood vessel homeostasis and for controlling retinal neovascularization in adults. Restoration of Tbdn-1 protein expression and/or activity may provide a novel approach for treating proliferative retinopathies.

Tubedown (Narg1, Tbdn), a member of the Nat1 family of proteins, associates with the acetyltransferase Ard1 and exerts an angiostatic function in adult retinal-blood-vessel homeostasis. The purpose of the present study was to gain a better understanding of the nature of the Tbdn protein complex and how it might exert a homeostatic influence on blood vessels. Immunoprecipitation of Tbdn from endothelial cells followed by gel electrophoresis and liquid-chromatography–tandem-mass-spectrometry identified the actin-cytoskeleton-binding protein cortactin as a co-immunopurifying species. Western blotting confirmed the association between Tbdn and cortactin. Immunofluorescence confocal microscopy revealed that Tbdn colocalizes with cortactin and F-actin in cytoplasmic regions and at the cortex of cultured endothelial cells. Because cortactin is known to regulate cellular permeability through its interaction with the actin cytoskeleton, a process that is crucial for endothelial cell homeostasis, the role of Tbdn on endothelial cell permeability was examined. Knockdown of Tbdn expression in endothelial cells led to the co-suppression of Ard1 protein expression and to a significant increase in cellular permeability measured by the transit of FITC-albumin across the cellular monolayer. Furthermore, the proliferative retinal neovascularization and thickening resulting from induction of Tbdn knockdown in endothelium in transgenic mice was associated with a significant increase in extravasation or leakage of albumin from abnormal retinal blood vessels in vivo. These results provide evidence that an association occurs between Tbdn and cortactin, and that Tbdn is involved in the regulation of retinal-endothelial-cell permeability to albumin. This work implicates a functional role for Tbdn in blood-vessel permeability dynamics that are crucial for vascular homeostasis.

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The de novo formation of blood vessels (vasculogenesis) is an integral part of embryogenesis. Elucidation of the role of cytokine cooperation in vasculogenesis may lead to a better understanding of organogenesis, blood vessel regulation during tumorigenesis, and tissue injury. We have used embryonic stem cells to derive an endothelial cell line, designated IEM, which expresses a range of endothelial markers, including Von Willibrand Factor VIII related antigen, vascular cell adhesion molecule, platelet-endothelial cell adhesion molecule (CD31), and receptors for acetylated low-density lipoprotein. More importantly, IEM cells can be induced upon exposure to combinations of basic fibroblast growth factor and leukemia inhibitory factor (LIF) to proliferate and undergo vasculogenesis in vitro, resulting in the formation of vascular tubes and microcapillary anastomoses. Moreover, exposure to both cytokines conditionally permits IEM cells to specifically chimerize microvascular endothelium in vivo following blastocyst injection. These results indicate that bFGF and LIF together contribute to the induction and support of embryonic vasculogenesis in an isolated endothelial cell line. Our results provide evidence that combined actions of bFGF/LIF may play a role in mechanisms controlling blood vessel development.