Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro.

Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.     

Introduction to the blood coagulation cascade and cloning of blood coagulation factors

The coagulation cascade that occurs in mammalian plasma involves a large number of plasma proteins that participate in a stepwise manner and eventually give rise to the formation of thrombin. This enzyme then converts fibrinogen to an insoluble fibrin clot. This series of reactions involves a number of glycoproteins that particupate as enzymes as well as cofactors. These proteins that circulate in the blood in a precursor or zymogen form are multifunctional proteins that share many common segments or domains. One group includes the vitamin K-dependent glycoproteins (prothrombin, factor IX, factor X, and protein C) that show considerable homology in both their amino acid sequences and their gene structures. The proteins that participate in the contact or early phase of the blood coagulation cascade include plasma prekallikrein, factor XII, and factor IX. The amino-terminal regions of both factor XI and plasma prekallikrein contain four tandem repeats of about 90 amino acids, and these tandem repeats show considerable amino acid sequence homology. Factor XII contains four different domains in the amino-terminai region of the protein, including a kringle structure, two growth factor domains, and type I and type II finger domains. The finger domains were first identified in fibronectin. The carboxyl-terminal portion of plasma prekallikrein, factor XII, and factor XI contains the serine or protease portion of the molecule. These various plasma proteins that share common domains appear to have evolved by gene shuffling that may have, in some cases, involved introns.     

VEGF regulates cell behavior during vasculogenesis

Prominent among molecules that control neovascular processes is vascular endothelial growth factor (VEGF). The VEGF ligands comprise a family of well-studied mitogens/permeability factors that bind cell surface receptor tyrosine kinases. Targets include VEGF receptor-1/Flt1 and VEGF receptor-2/Flk1. Mice lacking genes for VEGF ligand or VEGF receptor-2 die early in gestation, making it difficult to determine the precise nature of underlying endothelial cellular behavior(s). To examine the effect(s) of VEGF signaling on cell behavior in detail, we conducted loss-of-function studies using avian embryos. Injection of soluble VEGFR-1 results in malformed vascular networks and the absence of large vessels. In the most severe cases embryos exhibited vascular atresia. Closely associated with the altered phenotype was a clear endothelial cell response-a marked decrease in cell protrusive activity. Further, we demonstrate that VEGF gain of function strikingly increased cell protrusive activity. Together, our data show that VEGF/VEGF receptor signaling regulates endothelial cell protrusive activity, a key determinant of blood vessel morphogenesis. We propose that VEGF functions as an instructive molecule during de novo blood vessel morphogenesis.     

The ubiquitin-like protein MNSFbeta regulates ERK-MAPK cascade

MNSFbeta is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta covalently binds to intracellular proapoptotic protein Bcl-G in mitogen-activated murine T cells. In this study, we further investigated the intracellular mechanism of action of MNSFbeta in macrophage cell line, Raw 264.7 cells. We present evidence that MNSFbeta.Bcl-G complex associates with ERKs in non-stimulated Raw 264.7. We found that MNSFbeta.Bcl-G directly bound to ERKs and inhibited ERK activation by MEK1. In Raw 264.7 cells treated with MNSFbeta small interfering RNA (siRNA) lipopolysaccharide (LPS)-induced ERK1/2 activation was enhanced and LPS-induced JNK and p38 activation was unaffected. SiRNA-mediated knockdown of MNSFbeta increased tumor necrosis factor alpha (TNFalpha) expression at mRNA and protein levels in LPS-stimulated Raw 264.7 cells. Finally, we found that transfection with MNSFbeta expression construct resulted in a significant inhibition of LPS-induced ERK activation and TNFalpha production. Co-transfection experiments with MNSFbeta and Bcl-G greatly enhanced this inhibition. Collectively, these findings indicate that MNSFbeta might be implicated in the macrophage response to LPS.     

Estrogen regulates tyrosine hydroxylase expression in the neonate mouse midbrain

Estrogen plays an important role during differentiation of midbrain dopaminergic neurons. This is indicated by the presence of estrogen receptors and the transient expression of the estrogen-forming enzyme aromatase within the dopaminergic cell groups. We have previously shown that estrogen regulates the plasticity of dopamine cells through the stimulation of neurite growth/arborization. In this study, we have analyzed the capability of estrogen to influence the activity of developing mouse dopamine neurons. The expression of tyrosine hydroxylase (TH) was assessed by competitive RT-PCR and Western blotting. The developmental expression of TH in the ventral midbrain was studied from embryonic day 15 until postnatal day 15 and revealed highest TH levels early postnatally. This profile coincides with the transient aromatase expression in this brain area. Using cultured midbrain cells, we found that estrogen increased TH mRNA/protein levels. The application of the estrogen receptor antagonist ICI 182,780 resulted in a complete inhibition of estrogen effects. To verify these data in vivo, fetuses were exposed in utero from E15 until birth to the aromatase inhibitor CGS 16949A or to CGS supplemented with estrogen. CGS caused a robust reduction in TH mRNA/protein levels in the midbrain, which could be restored by estrogen substitution. Taken together, our data strongly suggest that estrogen controls dopamine synthesis in the developing nigrostriatal dopaminergic system and support the concept that estrogen is implicated in the regulation of ontogenetic steps but also in the function of midbrain dopamine neurons.     

Heparan sulfate regulates VEGF165- and VEGF121-mediated vascular hyperpermeability

VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF(165) and VEGF(121). Because VEGF(121) does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF(165) and VEGF(121). Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF(165) and VEGF(121), suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.     

Bacillus subtilis rapD, a direct target of transcription repression by RghR, negatively regulates srfA expression

The Bacillus subtilis genome encodes eleven Rap proteins, which are conserved tetratricopeptide-containing regulatory proteins. Of those characterized to date, all except RapI negatively regulate response regulators, including Spo0F, ComA and DegU, via protein-protein interactions. RapD has not yet been fully characterized. It was examined whether RapD inhibits the expression of spoIIE, srfA and aprE, which are Spo0F-, ComA- and DegU-regulated genes, respectively. It was observed that multicopy rapD inhibited srfA expression, which suggests that RapD inhibits ComA. This was reinforced by the fact that multicopy rapD also blocked the expression of rapC and rapF, which belong to the ComA regulon. The expression of rapD was reported to depend on the extracytoplasmic function sigma factor SigX. DNA microarray analysis and gel retardation assays revealed that rapD expression is directly repressed by RghR. Thus, the ComA regulon is regulated by rapD in a SigX- and RghR-dependent manner.     

Assessment of coagulation cascade during air microembolization of the lung

Experiments were performed to determine whether activation of the coagulation cascade was required for pulmonary vascular permeability to increase during microembolization of the lung. For 30-45 min air microemboli were intravenously infused (0.05-0.10 ml X kg-1 X min-1) into awake sheep with chronic lung-lymph fistulas and anesthetized mongrel dogs. During embolization the pulmonary arterial pressure increased, and O2 partial pressure (PaO2) fell by more than 20 Torr (P less than 0.01). Subsequently lymph flow nearly tripled without a change in the lymph-to-plasma protein concentration ratio. Partial thromboplastin and prothrombin times, biological activity of antithrombin III, and circulating concentration of 125I-labeled dog or sheep fibrinogen did not change during or following air infusion. In two additional sheep an intravenous infusion of thrombin at 0.6 U X kg-1 X min-1 for 15 min resulted in a 20% decrease in 125I-labeled sheep fibrinogen concentration without a change in pulmonary arterial pressure or PaO2. We conclude that air microembolization can increase permeability to water and protein without a detectable activation of the coagulation cascade in the sheep or dog.     

A cAMP-responsive element regulates expression of the mouse steroid 11 beta-hydroxylase gene

In Y1 mouse adrenocortical tumor cells, expression of steroid 11 beta-hydroxylase (11 beta-OHase) is stimulated by cAMP following a delay of 4-6 h. Our results demonstrate that a cAMP-responsive element (CRE) within the 11 beta-OHase promoter region is a major determinant of this induction. The 5'-flanking sequences from the mouse 11 beta-OHase gene were placed in front of a human growth hormone reporter gene and transfected into Y1 cells. Treatment of transfected cells with 8-bromo-cAMP increased expression directed by the 11 beta-OHase 5'-flanking region by 3.8-fold. In 5'-deletion analyses, 123 base pairs of 5'-flanking sequences were sufficient for cAMP induction, whereas cAMP treatment did not affect expression of a plasmid with only 40 base pairs of 5'-flanking sequence. Within these 123 base pairs, a region from -56 to -49 matched 7 of 8 bases comprising the consensus sequence for the CRE. 11 beta-OHase 5'-flanking sequences from -65 to -42, including the CRE-like sequence, conferred cAMP inducibility to promoters from the thymidine kinase and chorionic gonadotropin alpha-subunit genes. DNase I footprinting and Southwestern blotting analyses demonstrated that the protein which interacted with the CRE in the 11 beta-OHase promoter region was similar to the CRE-binding protein associated with other cAMP-regulated genes. Together, these results suggest that an interaction between the 11 beta-OHase CRE and CRE-binding protein mediates cAMP induction of the 11 beta-OHase gene.