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.     

Genetic polymorphism of ganglioside expression in mouse organs

In previous studies it was demonstrated that there are three variations as to the expression of liver gangliosides in inbred strains of mice; the first group expresses GM3(NeuGc) as a major component, the second group, GM2(NeuGc), and the third group, GM2(NeuGc), GM1 (NeuGc), and GD1a(NeuGc). In the present study, we attempted to determine which organs, if any, exhibit the same polymorphic variations as those observed in the liver. Thus, the gangliosides in spleen, thymus, heart, lung, kidney, testis, and erythrocytes, as well as those in liver, were examined using a TLC-mapping technique or by one-dimensional TLC. WHT/Ht, BALB/c, and ICR mice, which are typical strains as to the polymorphic expression of liver gangliosides, were used for the analysis. The presence of GM1 was confirmed by not only chemical detection on TLC plates but also with a TLC-immunostaining procedure using choleragenoid. These comparative studies indicated that only erythrocytes exhibited the same polymorphic variations of ganglioside expression as those in the liver, but the other six organs showed specific patterns which were not polymorphic. In addition to this, there were the following two interesting findings. Firstly, WHT/Ht mice, in which GM2(NeuGc) and GM1(NeuGc) are not expressed in the liver and erythrocytes, did not express a detectable amount of GM2(NeuGc) but expressed GM1(NeuGc) in all the other organs. Secondly, marked polymorphic variation was found in the expression of GM4(NeuAc) in the erythrocytes.     

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.     

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.     

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.     

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40ng/ml in the culture medium, but decreased at 80ng/ml. Under CoCl(2)-induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or anti-angiogenic activities of BMSCs.