Inhibition of endogenous VEGF impedes revascularization and astroglial proliferation: roles for VEGF in brain repair

Vascular endothelial growth factor (VEGF) is upregulated following injury to the CNS. Our previous work has shown that exogenous application of VEGF promotes angiogenesis, blood-brain barrier permeability, and astroglial mitogenicity in the traumatized brain. To develop a model that could link endogenously secreted VEGF to brain tissue repair, a specific neutralizing antibody to VEGF was infused by osmotic minipump directly into the neocortex and striatum for up to 1 week. Tissues adjacent to the infusion/wound site were analyzed for specific vascular and astroglial protein markers and proliferation, necrosis/apoptosis (via TUNEL staining), VEGF, the VEGF receptors flt-1 and flk-1, and bFGF expression using immunohistochemistry and semi-quantitative RT-PCR. Neutralization of native VEGF caused significant decreases in angiogenic activity, astroglial proliferation, and nestin immunoexpression, while vascular and astroglial degeneration was substantially increased, resulting in much larger wound cavities when compared to controls. The hindrance of brain tissue repair occurred despite an increase in bFGF expression at the wound sites. VEGF appears to be an integral factor in CNS wound healing that is essential for vascular endothelial proliferation and survival and may also be necessary for astroglial proliferation and maintenance during the repair of brain injury.     

Growth and heat resistance of rats subjected to repeated exposure to heat in early ontogeny

The effect of 10 daily exposures to heat from the 1st to the 10th, 10th to the 20th, and 20th to the 30th days of life, respectively, on subsequent growth and resistance of the animals to high temperatures was studied. Growth and survival of the rats were found to depend on the conditions of heat exposure. Animals aged 10–20 days were the most susceptible to the action of high ambient tempertures. In these rats, heat resistance was increased to the greatest degree during the period of sexual maturity, whereas the rate of growth was slower than in animals exposed to heat at the other age periods.Laboratory of Age Physiology, Institute of Physiology, Academy of Sciences of the Uzbek SSR, Tashkent. (Presented by Academician V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 5, pp. 537–539, May, 1977.     

Synthesis and immunotropic activity of derivatives of thiazolo[3,4-a]benzimidazole

Translated from Khimiko-farmatsevticheskii Zhurnal, Vol. 25, No. 1, pp. 40–42, January, 1991.     

Pulsed high-intensity focused ultrasound enhances uptake of radiolabeled monoclonal antibody to human epidermoid tumor in nude mice.

The aim of this study was to determine if pulsed high-intensity focused ultrasound (HIFU) exposures could enhance tumor uptake of (111)In-MX-B3, a murine IgG1kappa monoclonal antibody directed against the Le(y) antigen. METHODS: MX-B3 was labeled with (111)In, purified, and confirmed for its binding to the antigen-positive A431 cell line. Groups of nude mice were inoculated subcutaneously with A431 tumor cells on both hind flanks. A tumor on one flank was treated with pulsed-HIFU; the other tumor was used as an untreated control. Within 10 min after the HIFU exposure, the mice received intravenous (111)In-MX-B3 for imaging and biodistribution studies. Mice were euthanized at 1, 24, 48, and 120 h after injection for biodistribution studies. RESULTS: The HIFU exposure shortened the peak tumor uptake time (24 vs. 48 h for the control) and increased the peak tumor uptake value (38 vs. 25 %ID/g [percentage injected dose per gram] for the control). The HIFU effect on enhancing tumor uptake was greater at earlier times up to 24 h, but the effect was gradually diminished thereafter. The HIFU effect on enhancing tumor uptake was substantiated by nuclear imaging studies. HIFU also increased the uptake of the antibody in surrounding tissues, but the net increase was marginal compared with the increase in tumor uptake. CONCLUSION: This study demonstrates that pulsed-HIFU significantly enhances the delivery of (111)In-MX-B3 in human epidermoid tumors xenografted in nude mice. The results of this pilot study warrant further evaluation of other treatment regimens, such as repeated HIFU exposures for greater delivery enhancement of antibodies labeled with cytotoxic radioisotopes or pulsed-HIFU exposure in addition to a combined therapy of (90)Y-B3 and taxol to enhance the synergistic effect.     

Activation of receptor-mediated angiogenesis and signaling pathways after VEGF administration in fetal rat CNS explants.

SUMMARY: The angiogenic role of vascular endothelial growth factor (VEGF) receptors, flk-1 and flt-1, and their downstream signaling pathways, MAPK/ERK and PI-3 kinase, were examined in a fetal rat cortical explant model after exposure to exogenous VEGF. Treatment with VEGF resulted in substantial neovascularization characterized by increased vascular flk-1 receptor expression, whereas flt-1 receptor protein expression was absent. The specific role of flk-1 receptors in the angiogenic process was confirmed by the addition of antisense oligonucleotides (AS-ODNs) to flk-1, which blocked angiogenesis, whereas AS-ODNs to flt-1 had no effect. These results were further supported by the finding that specific chemical inhibition of flk-1 receptors caused disruption of the angiogenic response, whereas inhibition of the flt-1 receptors had no effect. Application of either MAPK/ERK or PI-3 kinase pathway inhibitors disrupted VEGF-induced angiogenesis, thereby indicating that both signaling pathways mediate this process. Thus VEGF binding to the endothelial flk-1 receptor activates the MAPK/ERK and PI-3 kinase pathways, resulting in neoangiogenic events. Of interest is the fact that although VEGF is regarded as a vascular permeability factor, its application to nascent cortical tissue caused an increase in a key physiologic protein of the blood-brain barrier function, glucose transporter-1, suggesting that the cytokine may have a role in blood-brain barrier development.     

Expression of adrenomedullin mRNA is altered with gestation and labour in human placenta and fetal membranes

The aim of the present study was to investigate whether placental and fetal membrane AdM (adrenomedullin) mRNA expression changes with gestation and human labour, as we have previously found labour-associated changes in AdM content in fetal membranes [Al-Ghafra, Gude, Brennecke and King (2003) Clin. Sci. 105, 419-423]. Placentas and fetal membranes were collected either at term or pre-term from women either in-labour or not-in-labour, and AdM mRNA abundance was measured in tissue extracts by Northern blot analysis. Increases were found in the relative abundance of amniotic tissue AdM mRNA in both in-labour and not-in-labour groups at term compared with those at pre-term, and there were positive correlations with gestational age. Relative abundance of choriodecidual tissue AdM mRNA was also significantly elevated in the not-in-labour groups between pre-term and term tissues, although there was no significant correlation with gestational age. However, placental AdM mRNA expression was neither significantly increased at term (compared with pre-term) nor correlated with gestational age. In addition, there were significant increases in AdM mRNA in amnion and choriodecidua in the in-labour group compared with the not-in-labour group for both pre-term and term gestations. There was no difference in AdM mRNA in placental tissues between labour groups. In conclusion, the present study provides evidence that AdM production by fetal membranes is increased in amniotic and choriodecidual tissues at term, compared with pre-term, and in response to labour.     

Astrocyte growth effects of vascular endothelial growth factor (VEGF) application to perinatal neocortical explants: receptor mediation and signal transduction pathways

The non-angiogenic role of vascular endothelial growth factor (VEGF), and its receptors flt-1 and flk-1, together with downstream signaling pathways were examined in fetal and postnatal rat cerebral cortical organotypic explants. VEGF application in both paradigms caused a significant increase in astroglial proliferation and a dose-dependent increase in GFAP and nestin immunoreactivity. The VEGF receptor flt-1 was observed on most, though not all astrocytes, while flk-1 receptor immunoexpression was absent. Treatment with antisense oligonucleotides (AS-ODNs) to flt-1 resulted in a dramatic decrease in GFAP and nestin immunoreactivity, which further confirmed the role of flt-1 in mediating VEGF's gliotrophic effects, while AS-ODNs to flk-1 had no effect. VEGF-induced gliotrophic effects were found to be mediated by the MAPK/ERK and PI-3 kinase signaling pathways, since the both the MEK1 inhibitor, PD98059 and the PI-3 kinase inhibitor, Wortmannin abolished VEGF-induced astrocytic GFAP(+) expression. Although high dose VEGF application resulted in strong upregulation of both GFAP and nestin immunoreactivity in astrocytes, overlap of the two proteins was not observed in all cells, suggesting that some of the nestin(+) cells might be neural progenitors. Exposure to VEGF resulted in upregulation of both VEGF and bFGF mRNA at the one-day time point, and bFGF protein by 3 days; VEGF activated astrocytes expressed bFGF to a much greater degree than those in untreated explants. The increased expression of bFGF induced by VEGF, may serve in the proliferation of multipotential neural stem/progenitor cells in vitro. VEGF, an established angiogenic factor, appears to play a significant role in the growth and differentiation of astrocytes in the CNS.     

Vascular endothelial growth factor promotes neurite maturation in primary CNS neuronal cultures

Recent studies have demonstrated that vascular endothelial growth factor (VEGF) and its receptor VEGFR2 (flk-1) are expressed by neurons during development and following hypoxic-ischemic events. Moreover, fetal CNS tissue explants exposed to exogenous VEGF exhibit increased neuronal Map-2 expression, suggesting that VEGF could have an effect on neuronal maturation. To determine whether this effect is of a direct nature, we examined the expression of Map-2 in the presence of VEGF in primary CNS neuronal cultures. After 3 days in culture, a statistically significant dose-dependent increase in the length of Map-2(+) processes was observed, with the peak occurring at 10 ng/ml of VEGF. Immunohistochemical analysis of the cultures demonstrated the presence of VEGFR2 after VEGF treatment, as well as the expression of the VEGF receptor VEGFR1 (flt-1). Treatment of the cultures with antisense oligonucleotides against VEGFR2, but not against VEGFR1, abolished the effect of VEGF on the length of Map-2(+) processes. RT-PCR analyses of Map-2 and VEGFR1 indicated that mRNAs of these two genes are upregulated in the presence of VEGF. The addition of wortmannin, an inhibitor of PI3K/Akt signal-transduction pathway, to the media did not affect the VEGF-dependent increase in Map-2(+) length. In contrast PD98059, which inhibits the MAPK pathway, partially abolished this effect of VEGF. These experiments suggest that VEGF has a direct effect on neuronal growth and maturation under normoxic conditions during CNS development, which is mediated by the VEGFR2 receptor via the MAPK pathway.