Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.

Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine release from the rat hippocampal formation

Insulin-like growth factors-I and -II (IGF-I and -II) are structurally related mitogenic polypeptides with potent growth promoting effects. These peptides and their corresponding IGF-I and -II receptors are selectively localized in the brain. To date, most of the effects of IGFs are believed to be mediated by IGF-I receptors whereas the significance of IGF-II receptor in mediating biological responses remains unclear. In the present study, we characterized the distribution of IGF-I and IGF-II receptor sites and investigated the effects of both factors on endogenous acetylcholine (ACh) release in adult rat hippocampus. [125I]IGF-I receptor binding sites are recognized by IGF-I> IGF-II> insulin, whereas [125I]IGF-II binding was competed potently by IGF-II> IGF-I but not by insulin. At the cellular level, IGF-I receptor sites were primarily noted in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon’s horn whereas IGF-II sites were localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus. IGF-I (10−14–10−8 M) and des(1–3) IGF-I (10−10–10−8 M) were found to inhibit whereas IGF-II (10−14–10−8 M) potentiated K+-evoked ACh release from hippocampal slices. Tetrodotoxin altered the effects of IGF-I but not those of IGF-II suggesting that IGF-I acts indirectly via the release of other modulators whereas IGF-II acts directly on or in close proximity to the cholinergic terminals. The inhibitory effects of IGF-I were also observed in the frontal cortex but not in the striatum. In contrast, the stimulatory effects of IGF-II were evident both in the frontal cortex and striatum. Taken together, these results reveal the differential localization of IGF-I and IGF-II receptor sites in the hippocampal formation and the opposite role for these growth factors in the acute regulation of ACh release likely via two distinct mechanisms. Additionally, these data provide the first evidence for a direct role for IGF-II and its receptors in the regulation of transmitter release in the central nervous system.

Altered gene expression in the host brain caused by a trematode parasite: Neuropeptide genes are preferentially affected during parasitosis

Schistosome parasites adjust the physiology and behavior of their intermediate molluscan hosts to their own benefit. Previous studies demonstrated effects of the avian-schistosome Trichobilharzia ocellata on peptidergic centers in the brain of the intermediate snail host Lymnaea stagnalis. In particular, electrophysiological properties and peptide release of growth- and reproduction-controlling neuroendocrine neurons were affected. We now have examined the possibility that the expression of genes that control physiology and behavior of the host might be altered during parasitosis. A cDNA library of the brain of parasitized Lymnaea was constructed and differentially screened by using mRNA from the brain of both parasitized and nonparasitized snails. This screening yielded a number of clones, including previously identified cDNAs as well as novel neuronal transcripts, which appear to be differentially regulated. The majority of these transcripts encode neuropeptides. Reverse Northern blot analysis confirmed that neuropeptide gene expression is indeed affected in parasitized animals. Moreover, the expression profiles of 10 transcripts tested showed a differential, parasitic stage-specific regulation. Changes in expression could in many cases already be observed between 1.5 and 5 hr postinfection, suggesting that changes in gene expression are a direct effect of parasitosis. We suggest that direct regulation of neuropeptide gene expression is a strategy of parasites to induce physiological and behavioral changes in the host.

Nontropic actions of neurotrophins: Subcortical nerve growth factor gene delivery reverses age-related degeneration of primate cortical cholinergic innervation

Normal aging is associated with a significant reduction in cognitive function across primate species. However, the structural and molecular basis for this age-related decline in neural function has yet to be defined clearly. Extensive cell loss does not occur as a consequence of normal aging in human and nonhuman primate species. More recent studies have demonstrated significant reductions in functional neuronal markers in subcortical brain regions in primates as a consequence of aging, including dopaminergic and cholinergic systems, although corresponding losses in cortical innervation from these neurons have not been investigated. In the present study, we report that aging is associated with a significant 25% reduction in cortical innervation by cholinergic systems in rhesus monkeys (P < 0.001). Further, these age-related reductions are ameliorated by cellular delivery of human nerve growth factor to cholinergic somata in the basal forebrain, restoring levels of cholinergic innervation in the cortex to those of young monkeys (P = 0.89). Thus, (i) aging is associated with a significant reduction in cortical cholinergic innervation; (ii) this reduction is reversible by growth-factor delivery; and (iii) growth factors can remodel axonal terminal fields at a distance, representing a nontropic action of growth factors in modulating adult neuronal structure and function (i.e., administration of growth factors to cholinergic somata significantly increases axon density in terminal fields). These findings are relevant to potential clinical uses of growth factors to treat neurological disorders.

Effect of rabies virus infection on gene expression in mouse brain

A variety of molecular genetic approaches were used to study the effect of rabies virus (RV) infection on host gene expression in mouse brain. The down-regulation of gene expression was found to be a major effect of RV infection by using subtraction hybridization. However, a combination of techniques identified approximately 39 genes activated by infection. These included genes involved in regulation of cell metabolism, protein synthesis, synaptic activity, and cell growth and differentiation. Northern blot analysis to monitor temporal activation of several of these genes following infection revealed essentially two patterns of activation: (i) an early response with up-regulation beginning within 3 days after infection and correlating with transcription of RV nuclear protein; and (ii) a late response with enhanced expression occurring at days 6–7 after infection and associated with peak RV replication. The gene activation patterns and the known functions of their products suggest that a number of host genes may be involved in the replication and spread of RV in the brain.

Ras Family GTPases Control Growth of Astrocyte Processes

Astrocytes in neuron-free cultures typically lack processes, although they are highly process-bearing in vivo. We show that basic fibroblast growth factor (bFGF) induces cultured astrocytes to grow processes and that Ras family GTPases mediate these morphological changes. Activated alleles of rac1 and rhoA blocked and reversed bFGF effects when introduced into astrocytes in dissociated culture and in brain slices using recombinant adenoviruses. By contrast, dominant negative (DN) alleles of both GTPases mimicked bFGF effects. A DN allele of Ha-ras blocked bFGF effects but not those of Rac1-DN or RhoA-DN. Our results show that bFGF acting through c-Ha-Ras inhibits endogenous Rac1 and RhoA GTPases thereby triggering astrocyte process growth, and they provide evidence for the regulation of this cascade in vivo by a yet undetermined neuron-derived factor.

FGF-2 regulation of neurogenesis in adult hippocampus after brain injury

Fibroblast growth factor-2 (FGF-2) promotes proliferation of neuroprogenitor cells in culture and is up-regulated within brain after injury. Using mice genetically deficient in FGF-2 (FGF-2−/− mice), we addressed the importance of endogenously generated FGF-2 on neurogenesis within the hippocampus, a structure involved in spatial, declarative, and contextual memory, after seizures or ischemic injury. BrdUrd incorporation was used to mark dividing neuroprogenitor cells and NeuN expression to monitor their differentiation into neurons. In the wild-type strain, hippocampal FGF-2 increased after either kainic acid injection or middle cerebral artery occlusion, and the numbers of BrdUrd/NeuN-positive cells significantly increased on days 9 and 16 as compared with the controls. In FGF-2−/− mice, BrdUrd labeling was attenuated after kainic acid or middle cerebral artery occlusion, as was the number of neural cells colabeled with both BrdUrd and NeuN. After FGF-2−/− mice were injected intraventricularly with a herpes simplex virus-1 amplicon vector carrying FGF-2 gene, the number of BrdUrd-labeled cells increased significantly to values equivalent to wild-type littermates after kainate seizures. These results indicate that endogenously synthesized FGF-2 is necessary and sufficient to stimulate proliferation and differentiation of neuroprogenitor cells in the adult hippocampus after brain insult.

Growth factor treatment enhances vestibular hair cell renewal and results in improved vestibular function

The vestibules of adult guinea pigs were lesioned with gentamicin and then treated with perilymphatic infusion of either of two growth factor mixtures (i.e., GF I or GF II). GF I contained transforming growth factor α (TGFα), insulin-like growth factor type one (IGF-1), and retinoic acid (RA), whereas GF II contained those three factors and brain-derived neurotrophic factor. Treatment with GF I significantly enhanced vestibular hair cell renewal in ototoxin-damaged utricles and the maturation of stereociliary bundle morphology. The addition of brain-derived neurotrophic factor to the GF II infusion mixture resulted in the return of type 1 vestibular hair cells in ototoxin-damaged cristae, and improved vestibular function. These results suggest that growth factor therapy may be an effective treatment for balance disorders that are the result of hair cell dysfunction and/or loss.

Brain-derived neurotrophic factor is an endogenous modulator of nociceptive responses in the spinal cord

The primary sensory neurons that respond to noxious stimulation and project to the spinal cord are known to fall into two distinct groups: one sensitive to nerve growth factor and the other sensitive to glial cell-line-derived neurotrophic factor. There is currently considerable interest in the ways in which these factors may regulate nociceptor properties. Recently, however, it has emerged that another trophic factor—brain-derived neurotrophic factor (BDNF)—may play an important neuromodulatory role in the dorsal horn of the spinal cord. BDNF meets many of the criteria necessary to establish it as a neurotransmitter/neuromodulator in small-diameter nociceptive neurons. It is synthesized by these neurons and packaged in dense core vesicles in nociceptor terminals in the superficial dorsal horn. It is markedly up-regulated in inflammatory conditions in a nerve growth factor-dependent fashion. Postsynaptic cells in this region express receptors for BDNF. Spinal neurons show increased excitability to nociceptive inputs after treatment with exogenous BDNF. There are both electrophysiological and behavioral data showing that antagonism of BDNF at least partially prevents some aspects of central sensitization. Together, these findings suggest that BDNF may be released from primary sensory nociceptors with activity, particularly in some persistent pain states, and may then increase the excitability of rostrally projecting second-order systems. BDNF released from nociceptive terminals may thus contribute to the sensory abnormalities associated with some pathophysiological states, notably inflammatory conditions.

Regulation of topographic projection in the brain: Elf-1 in the hippocamposeptal system.

The hippocampus and septum play central roles in one of the most important spheres of brain function: learning and memory. Although their topographic connections have been known for two decades and topography may be critical for cognitive functions, the basis for hippocamposeptal topographic projection is unknown. We now report for the first time that Elf-1, a membrane-bound eph family ligand, is a candidate molecular tag for the genesis of the hippocamposeptal topographic projection. Elf-1 is expressed in an increasing gradient from dorsal to ventral septum. Furthermore, Elf-1 selectively allows growth of neurites from topographically appropriate lateral hippocampal neurons, while inhibiting neurite outgrowth by medial hippocampal neurons. Complementary to the expression of Elf-1, an eph family receptor, Bsk, is expressed in the hippocampus in a lateral to medial gradient, consistent with a function as a receptor for Elf-1. Further, Elf-1 specifically bound Bsk, eliciting tyrosine kinase activity. We conclude that the Elf-1/Bsk ligand-receptor pair exhibits traits of a chemoaffinity system for the organization of hippocamposeptal topographic projections.

Low lead levels stunt neuronal growth in a reversible manner.

The developing brain is particularly susceptible to lead toxicity; however, the cellular effects of lead on neuronal development are not well understood. The effect of exposure to nanomolar concentrations of lead on several parameters of the developing retinotectal system of frog tadpoles was tested. Lead severely reduced the area and branchtip number of retinal ganglion cell axon arborizations within the optic tectum at submicromolar concentrations. These effects of lead on neuronal growth are more dramatic and occur at lower exposure levels than previously reported. Lead exposure did not interfere with the development of retinotectal topography. The deficient neuronal growth does not appear to be secondary to impaired synaptic transmission, because concentrations of lead that stunted neuronal growth were lower than those required to block synaptic transmission. Subsequent treatment of lead-exposed animals with the chelating agent 2,3-dimercaptosuccinic acid completely reversed the effect of lead on neuronal growth. These studies indicate that impaired neuronal growth may be responsible in part for lead-induced cognitive deficits and that chelator treatment counteracts this effect.

Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor.

The development of new capillary networks from the normal microvasculature of the host appears to be required for growth of solid tumors. Tumor cells influence this process by producing both inhibitors and positive effectors of angiogenesis. Among the latter, the vascular endothelial growth factor (VEGF) has assumed prime candidacy as a major positive physiological effector. Here, we have directly tested this hypothesis in the brain tumor, glioblastoma multiforme, one of the most highly vascularized human cancers. We introduced an antisense VEGF expression construct into glioblastoma cells and found that (i) VEGF mRNA and protein levels were markedly reduced, (ii) the modified cells did not secrete sufficient factors so as to be chemoattractive for primary human microvascular endothelial cells, (iii) the modified cells were not able to sustain tumor growth in immunodeficient animals, and (iv) the density of in vivo blood vessel formation was reduced in direct relation to the reduction of VEGF secretion and tumor formation. Moreover, revertant cells that recovered the ability to secrete VEGF regained each of these tumorigenic properties. These results suggest that VEGF plays a major angiogenic role in glioblastoma.

Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo.

Prostate carcinoma is the second leading cause of death from malignancy in men in the United States. Prostate cancer cells express type I insulin-like growth factor receptor (IGF-IR) and prostate cancer selectively metastazises to bone, which is an environment rich in insulin-like growth factors (IGFs), thereby supporting a paracrine action for cancer cell proliferation. We asked whether the IGF-IR is coupled to tumorigenicity and invasion of prostate cancer. When rat prostate adenocarcinoma cells (PA-III) were stably transfected with an antisense IGF-IR expression construct containing the ZnSO4-inducible metallothionein-1 transcriptional promoter, the transfectants expressed high levels of IGF-IR antisense RNA after induction with ZnSO4, which resulted in dramatically reduced levels of endogenous IGF-IR mRNA. A significant reduction in expression both of tissue-type plasminogen activator and of urokinase-type plasminogen activator occurred in PA-III cells accompanying inhibition of IGF-IR. Subcutaneous injection of either nontransfected PA-III or PA-III cells transfected with vector minus the IGF-IR insert into nude mice resulted in large tumors after 4 weeks. However, mice injected with IGF-IR antisense-transfected PA-III cells either developed tumors 90% smaller than controls or remained tumor-free after 60 days of observation. When control-transfected PA-III cells were inoculated over the abraded calvaria of nude mice, large tumors formed with invasion of tumor cells into the brain parenchyma. In contrast, IGF-IR antisense transfectants formed significantly smaller tumors with no infiltration into brain. These results indicate an important role for the IGF/IGF-IR pathway in metastasis and provide a basis for targeting IGF-IR as a potential treatment for prostate cancer.

Long-term functional recovery from age-induced spatial memory impairments by nerve growth factor gene transfer to the rat basal forebrain.

Nerve growth factor (NGF) stimulates functional recovery from cognitive impairments associated with aging, either when administered as a purified protein or by means of gene transfer to the basal forebrain. Because gene transfer procedures need to be tested in long-term experimental paradigms to assess their in vivo efficiency, we have used ex vivo experimental gene therapy to provide local delivery of NGF to the aged rat brain over a period of 2.5 months by transplanting immortalized central nervous system-derived neural stem cells genetically engineered to secrete NGF. By grafting them at two independent locations in the basal forebrain, medial septum and nucleus basalis magnocellularis, we show that functional recovery as assessed in the Morris water maze can be achieved by neurotrophic stimulation of any of these cholinergic cell groups. Moreover, the cholinergic neurons in the grafted regions showed a hypertrophic response resulting in a reversal of the age-associated atrophy seen in the learning-impaired aged control rats. Long-term expression of the transgene lead to an increased NGF tissue content (as determined by NGF-ELISA) in the transplanted regions up to at least 10 weeks after grafting. We conclude that the gene transfer procedure used here is efficient to provide the brain with a long-lasting local supply of exogenous NGF, induces long-term functional recovery of cognitive functions, and that independent trophic stimulation of the medial septum or nucleus basalis magnocellularis has similar consequences at the behavioral level.

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.