Inhibition of fibroblast growth factor/fibroblast growth factor receptor activity in glioma cells impedes tumor growth by both angiogenesis-dependent and -independent mechanisms.

We undertook a series of systematic studies to address the role of fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) activity in tumor growth and angiogenesis. We expressed dominant-negative FGFR2 (FGFR2-DN) or FGFR1 (FGFR1-DN) in glioma C6 cells by using constitutive or tetracycline-regulated expression systems. Anchorage-dependent or independent growth was inhibited in FGFR-DN-expressing cells. Tumor development after xenografting FGFR-DN-expressing cells in immunodeficient mice or after transplantation in rat brain was strongly inhibited. Quantification of microvessels demonstrated a significant decrease in vessel density in tumors derived from FGFR-DN-expressing cells. Furthermore, in a rabbit corneal assay, the angiogenic response after implantation of FGFR-DN-expressing cells was decreased. In tumors expressing FGFR-DN, vascular endothelial growth factor expression was strongly inhibited as compared with control tumor. These results indicate that inhibition of FGF activity may constitute a dominant therapeutic strategy in the treatment of FGF-producing cerebral malignancies and may disrupt both angiogenesis-dependent and -independent signals required for glioma growth and invasion.

In vitro and in vivo characterization of neural stem cells.

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.

Loss of basic fibroblast growth factor in the subcommissural organ of old spontaneously hypertensive rats.

The immunocytochemical localization of basic fibroblast growth factor (bFGF) was studied in subcommissural organ (SCO) of aged-matched normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats at 10, 14 and 18 months of age using a polyclonal antibody against bFGF. The bFGF-like immunoreactivity (bFGF-ir) was observed in SCO ependymal cells of young and old normotensive rats. However, a progressive loss of bFGF-immunopositive ependymal SCO cells was observed with age in SH rats (27.24, 57.5 and 96.9% in 10, 14 and 18 months old respectively) compared with aged-matched WKY normotensive rats. Considering the potential role of the SCO in sleep regulation and sodium homeostasis, which are altered in essential hypertension, these data show a new neuroendocrine anomaly to be added to the many others previously observed in this rat strain, when they develop hypertension as they get old.

Apoptosis of glioma cells induced by the fibroblast growth factor inhibitor 1,3,6-naphthalenetrisulfonate.

Fibroblast growth factors (FGFs) are powerful angiogenic polypeptides that are involved in the autocrine growth stimulation of gliomas. We report here that addition to glioma cell cultures of 1,3,6-naphthalenetrisulfonate (NTS), an inhibitor of the mitogenic activity of FGFs, significantly enhanced apoptosis, as assessed by terminal deoxynucleotidyl transferase (TdT) assay. The pro-apoptotic effect of NTS was time-dependent. These findings suggest that FGF may play a pivotal role in the survival of glioma cells, and support a clinical interest of NTS as a leading compound for the development of new antitumorals.

Abolished angiogenicity and tumorigenicity of rat glioma by 1-naphthalenemonosulfonate.

Suramins and suradistas, an important group of potential anti-cancer agents, inhibit fibroblast growth factor (FGF) mitogenic activity. It has been shown that naphthalenesulfonates, with a common chemical function to the family of suramins and suradistas, mimic their inhibitory activity, abolishing FGF-induced angiogenesis in vivo, and inducing apoptosis of C6 glioma cells in culture. In the present report, we show that intratumoral administration of 1-naphthalenemonosulfonate induces a considerable regression of gliomas in rats, significantly enhances apoptosis, and attenuates tumor angiogenesis. These findings may lead to new approaches for the treatment of glioblastoma, a most common primary malignant brain tumor of very poor prognosis, as well as of other angiogenesis-dependent malignancies.

Intramembranous cytochemistry: a new morphological technique for studying cholesterol in the astrocyte plasma membrane of ischemic brain cells.

The distribution of cholesterol in the astrocytic plasma membranes of normal and ischemic caudate nucleus in the cat was investigated with the polyene antibiotic, filipin, a morphological probe for membrane cholesterol domains in freeze-fracture tissue replicas. After filipin incubation of prefixed vibratome slices, filipin-cholesterol complexes appeared as 20 to 30 nm protuberances and pits on P- and E-faces of the replicas. Distinct patterns of filipin-cholesterol complexes were found on nonischemic and ischemic astrocyte membranes. The filipin-treated specimens showed an 80% decrease in astroglial plasmalemma of cholesterol 1 hour after cerebral ischemia.

Fibroblast growth factor-1 inhibits medial smooth muscle cells apoptosis after balloon injury.

We performed balloon injury in the rat common carotid artery and identified apoptosis of vascular smooth muscle cells (VSMCs) by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, 2 h after injury. Balloon injury induces apoptosis and loss of bcl-X protein in the innermost layers of the media of the common carotid artery. Treatment with acidic fibroblast growth factor (aFGF, FGF-1) attenuated by 56% the balloon angioplasty-induced apoptosis. In addition, FGF-1 treatment also induces expression of the bcl-X anti-apoptotic protein in the same site of the media showing VSMC apoptosis. These data suggest that the anti-apoptotic effect of FGF in injured vascular wall was mediated by a bcl-X pathway and identified FGF as an important factor in vascular remodeling.

Immunohistochemical localization of basic fibroblast growth factor in choroid plexus of the rat.

In the present study, we report that an intense bFGF-immunoreactivity has been detected in the choroid plexus of the brain ventricles of adult rats. These results suggest that epithelial choroid plexus cells may be the source of the cerebrospinal fluid bFGF.

Acidic fibroblast growth factor inhibits junctional communication of Schwann cells in culture.

The effect of acidic fibroblast growth factor (aFGF) was investigated on junctional communication of rat Schwann cells (SC) in culture. As measured by dye transfer, the incidence of coupling between SC was very low during the phase of proliferation and increased slowly and progressively with time under culture conditions that induced the myelinating phenotype. Treatment with aFGF alone or in combination with heparin decreased markedly coupling between SC in both culture stages. The coupling inhibition was rapid, the earliest effects being apparent 5-15 min after addition of growth factor, and was transient with a slower recovery of coupling at 1-3 h. The uncoupling effect of aFGF could be prevented by an inhibitor of protein-tyrosine kinase. Addition of heparin to cultures decreased the most effective aFGF concentration by 100-fold, from 100 ng ml-1 to 1 ng ml-1. The dose-response curves exhibited a characteristic window-shape. The results suggest that FGF might be involved in the regulation of the junctional communication between rat SC via tyrosine kinases.

Inhibition of intra-tumoral angiogenesis and glioma growth by the fibroblast growth factor inhibitor 1,3,6-naphthalenetrisulfonate.

1,3,6-naphthalenetrisulfonate (NTS) can inhibit the proliferation in vitro of cells of various origin including glioma. We have studied the effects of NTS on intra-tumoral angiogenesis and tumor growth in the rabbit cornea after implantation of C6 rat glioma cells. It was found that neovascularization and glioma growth were abolished by topical administration of NTS. This effect could be mediated by both induction of programmed cell death and inhibition of growth, in endothelium and in tumor cells, most likely as a consequence of the disruption of the autocrine and paracrine effects of FGF released from endothelial and tumor cells. The results suggest that NTS is a promising candidate to lead the development of new angiogenesis inhibitors for the treatment of cancer and other diseases whose progression is dependent upon the development of new blood vessels.

Acidic fibroblast growth factor rescues gerbil hippocampal neurons from ischemic apoptotic death.

Neurotrophic factors have been shown to support the survival of injured neurons and promote their recovery. Here, we investigated whether acidic fibroblast growth factor (aFGF) could modify programmed cell death caused by transient forebrain ischemia in the gerbil. The data show that systemic administration of 2.6 microg aFGF after 5 min ischemia followed by 7 days of brain reperfusion significantly (p < 0.05) reduced the occurrence of apoptotic cell death in CA1 neurons. These data suggest that aFGF would contribute to brain protection after acute stroke.

Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment.

Neural stem cells (NSCs) are self-renewable, multipotential cells capable of differentiating into the three major neural cell types, but the mechanisms which regulate their development are not fully understood. Both basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote the proliferation of NSCs. However, studies on the role of FGFs in the differentiation of EGF-expanded NSCs are still incomplete. We have studied the expression of distinct FGF receptors (FGFRs) in the progeny of EGF-expanded NSCs isolated from E15 rat striatum. In situ hybridization analysis and immunocytochemistry showed a developmentally related expression pattern and a cell lineage-specific distribution of these receptors. FGFR1 and FGFR2 were identified in many early precursors and in the oligodendrocyte lineage. The latter receptor was also present in a subpopulation of astrocytes. FGFR3 was detected in a restricted population of early precursors, in oligodendroglial progenitors, and in neurons and protoplasmic astrocytes of late-term cultures. Basic FGF treatment of the progeny of NSCs increased the proliferative rate of precursors and the number of oligodendrocytes generated, whereas the number of differentiating neurons was significantly reduced. Together these data provide evidence that FGFs modulate the development of EGF-expanded NSCs, and that this is at least partly determined by a cell lineage-specific expression of multiple FGFRs.

Administration of bovine superoxide dismutase prevents sequelae of spinal cord ischemia in the rabbit.

The contribution of free radical-mediated reperfusion injury to the ischemic damage caused by total arterial occlusion has been investigated in a model of transient spinal cord ischemia in the rabbit. Spinal cord ischemia was produced in 20 anaesthetized rabbits by temporary luminal occlusion (20 min) of the abdominal aorta below the renal arteries. Superoxide dismutase (5 mg/kg) (10 animals) was infused before and during reperfusion below aortic occlusion using an infusion pump that infused the enzyme through the contralateral femoral artery. Control (10 animals) received sterile saline with the same procedure. In this later group, 4 animals developed paraplegia, 4 were paretic and only 2 were normal. However, in the treated group, 6 animals were normal while 3 were paretic and only one appeared paralyzed. We conclude that: a) oxygen free radicals generated during reperfusion are involved in producing the ischemic injury, and b) the ischemic spinal cord injury is prevented by superoxide dismutase.

Pericyte endothelial gap junctions in human cerebral capillaries.

Human cerebral tissue has been ultrastructurally studied and gap junctions have been visualized between endothelial cells and pericytes that permit ion exchange. We propose that the functional interrelationship between endothelium and pericytes may play a role in the alteration of capillary diameter for the control of local cerebral blood flow.

Fibroblast growth factor-1 prevents myocardial apoptosis triggered by ischemia reperfusion injury.

BACKGROUND: Apoptosis is a constant feature of reperfusion injury in ischemic cardiac myocytes, leading to late cell death. Since fibroblast growth factors (FGFs) inhibit apoptosis in differentiated cells, we hypothesized that FGF-1 (acidic FGF), in its native form, and a non-mitogenic isoform would attenuate myocardial ischemia-reperfusion- induced apoptosis. METHODS AND RESULTS: The effect of native and non-mitogenic fibroblast growth factor-1 mutein (FGF-1 and m-FGF-1) on apoptosis assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method was tested in a rat model of 20 min regional myocardial ischemia and 24h reperfusion. Myocardial ischemia followed by reperfusion resulted in a high myocardial apoptosis rate in the area at risk. When given as a systemic bolus inmediately after myocardial ischemia, both FGF-1 and m-FGF-1 significantly reduced apoptosis (by 60 and 61.2, respectively; p<0.0001). CONCLUSIONS: The programed myocyte cell death triggered by ischemia-reperfusion injury is attenuated by FGF-1 in its native or non mitogenic isoforms, suggesting that this effect does not depend on the mitogenic properties of this protein. FGF-1 would contribute to the functional preservation of the myocardium after acute myocardial infarction.

Fibroblast growth factor protects the kidney against ischemia-reperfusion injury.

Ischemia-reperfusion injury, a common source of renal dysfunction in adults, is associated with tubular epithelial cell damage. Since fibroblast growth factors (FGF) attenuated tissue injury after transient myocardial ischemia, we hypothesized that acidic fibroblast growth factor (aFGF; FGF-1) would attenuate renal ischemia-reperfusion injury. We studied the effects of FGF-1 in a rat model of acute renal failure induced by bilateral renal ischemia (60 min) and 1, 2 or 7 days reperfusion. After FGF-1 administration at the onset of renal reperfusion, there was less functional impairment of the kidneys. The histological changes were not as severe as in controls. Increases in serum creatinine and blood urea nitrogen 24 h after reperfusion were attenuated by 35% (p< 0.01) and by 53% (p< 0.001), respectively, in FGF-1-treated animals compared to vehicle-treated rats. The ischemia/reperfusion-induced increase in tissue myeloperoxidase, a marker of neutrophil infiltration, was mitigated (67% reduction, p< 0.05) with FGF-1 treatment. As shown by histology, neutrophil infiltration and tubular cell necrosis in medulla were less pronounced (p< 0.0001 and p< 0.05, respectively) in animals receiving FGF-1. Furthermore, ischemia-induced apoptosis, prevalent in tubular cells of the cortex, was also attenuated by FGF-1-treatment (83% reduction, p< 0.0001). Pretreatment of animals with Nw-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, abolished the attenuating effects of FGF-1 on neutrophil infiltration, suggesting that nitric oxide might participate in the anti-inflammatory effects of FGF-1 in this experimental design. Our data support a role for FGF-1 in attenuation of renal damage or failure after ischemia-reperfusion injury of the kidney, in part at least by inhibition of neutrophil infiltration.

Cardioprotection from ischemia by fibroblast growth factor: role of inducible nitric oxide synthase.

Growing evidence from both animal experiments and clinical observations indicates that fibroblast growth factor (FGF) plays a protective role in myocardial reperfusion injury. The molecular and cellular mechanisms that lead to this postischemic myocardial protection, however, remain largely unexplored. We studied the cardioprotective effects of human recombinant acidic fibroblast growth factor (aFGF, FGF-1) in a rat model of myocardial reperfusion injury, induced by 20 minutes of left coronary artery occlusion followed by 24 hours of reperfusion. Intravenous FGF-1 administration at the onset of heart reperfusion attenuated both the functional impairment and the histological changes of ischemia/reperfusion injury. FGF-1 increases more than twice the left ventricular contractile function (p <0.005) compared to vehicle-treated rats. As shown by histology, myocardial tissue is better preserved with FGF-1 treatment. The infarct size, normalized for the area at risk, was significantly smaller in the FGF-1 group (p <0.01) than in the vehicle group. Furthermore, FGF-1 administration resulted in expression of inducible nitric oxide synthase (iNOS) in the area at risk. Since increased expression of iNOS could potentiate cardioprotection against myocardial reperfusion injury, our findings support a new non-mitogenic role for FGF and add a clinical interest for this protein in increasing myocardial ischemic tolerance.

Quantitative electron microscopic study of calcium accumulation in cerebral ischemia mitochondria.

Formation of calcium deposits in mitochondria is a consistent feature of irreversible injury in ischemic myocardium. We studied calcium accumulation in nerve cell mitochondria in a cat model 30 and 60 minutes after cerebral ischemia localized in the anterior part of the caudate nucleus and adjacent internal capsule. In control animals, calcium deposits were visible in synaptic vesicles, Golgi apparatus, mitochondria, lysosomes, and in glial and neuronal nuclei. After cerebral ischemia, findings included astrocytic swelling and degeneration of neurons, with an increase in calcium pyroantimoniate mitochondrial deposits. Content of intramitochondrial calcium deposits is related to duration of ischemia as well as the amount of cellular lesions.

Adrenergic innervation of human middle cerebral artery. Ultrastructural observations.

The innervation of the human middle cerebral artery is studied with transmission electron microscopy using the chromaffin reaction technique. Adrenergic nerve fibers and related terminals, with granular electrodense neurotransmitter vesicles, are confined to the tunica adventitia-tunica media transitional zone and the outer layers of the media. These findings may indicate the presence of an adrenergic vasoconstrictor system for circulation control in the human middle cerebral artery.

Intraluminal thrombus and neointimal hyperplasia after microvascular surgery.

Despite advances in instrumentation, suture materials, and techniques, thrombosis and intimal hyperplasia due to myointimal cell proliferation are still problems in microvascular anastomosis. Platelet factors stimulate smooth-muscle cell proliferation and migration, but little is known about platelet-vessel wall interaction in microvascular surgery. This study evaluates the effect of the disposition of platelet or luminal thrombus, or both, in intimal evolution in autologous venous micrografts interposed in the common carotid artery of 30 rats. Three hours postoperatively, venous graft and suture line endothelium was completely denuded, and the host artery exhibited focal de-endothelialization. Thrombus deposition was observed on suture line and venous graft luminal surfaces, whereas host artery-denuded areas exhibited platelet adhesion. Two weeks postoperatively, intimal thickenings developed in venous graft and suture line, becoming narrower toward the venous graft. The distribution and size of intimal thickening did not change significantly at later observation periods (3-8 months). Intimal thrombus deposition and intimal hyperplasia, appearing in later stages of evolution, show a similar pattern of distribution.