Dexamethasone reduces vascular density and plasminogen activator activity in 9L rat brain tumors.

Angiogenesis, a process dependent upon perivascular proteolysis, is required for solid tumor growth and is inhibited by certain steroids including glucocorticoids. We examined the relationship between tumor growth and vessel density in experimental rat brain 9L glial tumors following chronic treatment with the glucocorticoid dexamethasone. Tumor growth was inhibited by intraperitoneal administration of 3 mg/kg/day dexamethasone. Maximal cross-sectional areas of post-implantation day 9 tumors were 4.6 +/- 1.0 mm2 in dexamethasone-treated animals and 17.0 +/- 3.4 mm2 in controls (P < 0.01). Microvessel density assessed by laminin immunohistochemistry was 59% lower in dexamethasone-treated tumors (P < 0.01). Plasminogen activator (PA) activity, a proteolytic enzyme related to endothelial migration and vessel growth, was 4.2 +/- 0.9 IU/micrograms protein in dexamethasone-treated tumors and 9.0 +/- 1.0 IU/micrograms protein in control tumors (P < 0.01). Exposure of cultured 9L and central nervous system microvessel endothelial cells to dexamethasone concentrations comparable to those achieved in vivo had no effect on cell growth, but reduced the PA activity of culture supernatant fractions by 78% and 99%, respectively. These findings suggest that inhibition of proteolytic steps involved in vessel growth may underlie, in part, the mechanism by which glucocorticoids decrease brain tumor growth.

Regulation of in vitro glia-induced microvessel morphogenesis by urokinase.

Plasminogen activators (PAs) regulate a variety of processes involved in tissue morphogenesis and differentiation. We used a coculture system in which microvascular endothelial cells are induced by glial cells to form capillary-like structures in order to examine the role of urokinase-type PA (uPA) during microvessel morphogenesis within the central nervous system (CNS). Endothelia-derived uPA activity decreased sevenfold within glial-endothelial cocultures when capillary-like structures were formed. Incubation of cocultures with concentrations of phorbol 12-myristate 13-acetate (0.1 and 1.0 nM) that induced endothelial uPA activity (45-210%) inhibited endothelial differentiation (25-70%). Furthermore, incubation of cocultures with proteolytically active low molecular weight uPA (5-500 IU/ml) inhibited endothelial differentiation (37-75%), whereas the amino terminal cell-binding fragment of uPA had minimal effect. Inhibition of plasminogen activation in cocultures with the serine protease/plasmin inhibitors aprotinin and soybean trypsin inhibitor increased glia-induced capillary-like structure formation (96-98%). These findings establish a paracrine/autocrine function for urokinase and its inhibitors in regulating endothelial responses to perivascular glia and provide insight into mechanisms of microvascular reactions to CNS pathology.

Inhibition of astroglia-induced endothelial differentiation by inorganic lead: a role for protein kinase C.

Microvascular endothelial function in developing brain is particularly sensitive to lead toxicity, and it has been hypothesized that this results from the modulation of protein kinase C (PKC) by lead. We examined the effects of inorganic lead on an in vitro model of central nervous system endothelial differentiation in which astroglial cells induce central nervous system endothelial cells to form capillary-like structures. Capillary-like structure formation within C6 astroglial-endothelial cocultures was inhibited by lead acetate with 50% maximal inhibition at 0.5 microM total lead. Inhibition was independent of effects on cell viability or growth. Under conditions that inhibited capillary-like structure formation, we found that lead increased membrane-associated PKC in both C6 astroglial and endothelial cells. Prolonged exposure of C6 cells to 5 microM lead for up to 16 h resulted in a time-dependent increase in membranous PKC as determined by immunoblot analysis. Membranous PKC increased after 5-h exposures to as little as 50 nM lead and was maximal at approximately 1 microM. Phorbol esters were used to determine whether PKC modulation was causally related to the inhibition of endothelial differentiation by lead. Phorbol 12-myristate 13-acetate (10 nM) inhibited capillary-like structure formation by 65 +/- 5%, whereas 4 alpha-phorbol 12,13-didecanoate was without effect. These findings suggest that inorganic lead induces cerebral microvessel dysfunction by interfering with PKC modulation in microvascular endothelial or perivascular astroglial cells.

Endothelial cell implantation and survival within experimental gliomas.

The delivery of therapeutic genes to primary brain neoplasms opens new opportunities for treating these frequently fatal tumors. Efficient gene delivery to tissues remains an important obstacle to therapy, and this problem has unique characteristics in brain tumors due to the blood-brain and blood-tumor barriers. The presence of endothelial mitogens and vessel proliferation within solid tumors suggests that genetically modified endothelial cells might efficiently transplant to brain tumors. Rat brain endothelial cells immortalized with the adenovirus E1A gene and further modified to express the beta-galactosidase reporter were examined for their ability to survive implantation to experimental rat gliomas. Rats received 9L, F98, or C6 glioma cells in combination with endothelial cells intracranially to caudate/putamen or subcutaneously to flank. Implanted endothelial cells were identified by beta-galactosidase histochemistry or by polymerase chain reaction in all tumors up to 35 days postimplantation, the latest time examined. Implanted endothelial cells appeared to cooperate in tumor vessel formation and expressed the brain-specific endothelial glucose transporter type 1 as identified by immunohistochemistry. The proliferation of implanted endothelial cells was supported by their increased number within tumors between postimplantation days 14 and 21 (P = 0.015) and by their expression of the proliferation antigen Ki67. These findings establish that genetically modified endothelial cells can be stably engrafted to growing gliomas and suggest that endothelial cell implantation may provide a means of delivering therapeutic genes to brain neoplasms and other solid tumors. In addition, endothelial implantation to brain may be useful for defining mechanisms of brain-specific endothelial differentiation.

Delivery of human fibroblast growth factor-1 gene to brain by modified rat brain endothelial cells.

Fibroblast growth factor (FGF) is an endothelial cell mitogen and serves as a mitogen and/or differentiating factor that can be neuroprotective for other cell types within the CNS. We established brain microvascular endothelial cell lines that secrete FGF-1 with the ultimate goal of examining their usefulness as a cellular platform for FGF gene delivery to brain. A chimeric gene consisting of the secretory sequence of FGF-4 linked at the 5' end of human FGF-1 (sp-hst/KS3:FGF-1) was transfected into rat microvascular endothelial cells previously altered to express the lacZ reporter gene (RBEZ), and numerous clones were found to secrete FGF-1 (RBEZ-FGF). Immunoblotting of conditioned medium demonstrated an 18-kDa protein corresponding to FGF-1. Conditioned medium from RBEZ-FGF cells enhanced [3H]thymidine incorporation in BALB/c3T3 fibroblasts by up to sevenfold when compared with conditioned medium of control cell lines, corresponding to as much as 110 ng of active FGF-1/mg of cell protein/24 h. RBEZ-FGF cell lines remained contact-inhibited and proliferated independent of exogenous endothelial mitogens, in contrast to control lines that are mitogen-dependent. Incubation of PC12 cells with RBEZ-FGF cells or their conditioned medium induced neurite outgrowth by PC12 cells. RBEZ-FGF cells survived following implantation to neonatal and adult rat caudate-putamen for at least 21 days based on 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) histochemistry, and FGF-1 gene expression by these cells in vivo was demonstrated by in situ hybridization and reverse transcriptase-PCR. These findings suggest that endothelial cells may be useful for FGF gene delivery to the CNS.