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Biomedical subjects

H J Federoff

Publications and source records attributed to H J Federoff.

At least 73 records · Page 4Linked to original sources
PubMed1995

Expression of vascular endothelial growth factor from a defective herpes simplex virus type 1 amplicon vector induces angiogenesis in mice.

Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific angiogenic growth factor. VEGF gene transfer strategies to stimulate focal angiogenesis could be used to ameliorate myocardial ischemia. To induce angiogenesis in vivo, we have constructed a replication-defective herpes simplex virus type 1 (HSV-1) amplicon vector that places the human VEGF-165 cDNA under the transcriptional control of the HSV immediate-early 4/5 promoter (HSVhvegf). Transduction of NIH 3T3 fibroblasts with HSVhvegf resulted in the secretion of high levels of biologically active VEGF, as assayed by microvascular endothelial mitogenesis. By use of an ex vivo protocol, BLK-CL4 fibroblasts were transduced with HSVhvegf or control HSVlac virus (expressing Escherichia coli beta-galactosidase), resuspended in basement membrane extract (matrigel), and coinjected subcutaneously into syngeneic C57BL/6 mice. One week later, the matrigel plugs with HSVhvegf showed a strong angiogenic response, in contrast to the plugs with HSVlac-transduced fibroblasts. These data indicate that transduction with HSVhvegf virus can induce an angiogenic response in vivo and suggest that this is a viable gene therapy approach for tissue ischemia.

3T3 Cells↗
PubMed1995

Expression of bcl-2 from a defective herpes simplex virus-1 vector limits neuronal death in focal cerebral ischemia.

BACKGROUND AND PURPOSE: A process resembling programmed cell death appears to contribute to postischemic neuronal loss in several models of stroke. Because the expression of the bcl-2 gene has been shown to rescue neurons from programmed cell death due to other causes, we determined whether it would be similarly neuroprotective in stroke. METHODS: Replication of defective herpes viral vectors that transduce bcl-2 (HSVbcl2) or Escherichia coli lacZ (HSVlac) were injected into two sites in the rat cerebral cortex 24 hours before induction of neocortical focal ischemia by tandem permanent occlusion of the right middle cerebral artery and ipsilateral common carotid artery. Local ischemic damage was determined 24 hours after occlusion by staining with 2% 2,3,5-triphenyltetrazolium chloride. RESULTS: Expression of bcl-2 in cerebral cortex was confirmed by immunohistochemistry in animals injected with the HSVbcl2 expression vector. Viable tissue was significantly increased at the injection sites in HSVbcl2- but not HSVlac-injected animals. The protection observed in the HSVbcl2 animals was localized to the injection sites. CONCLUSIONS: These data indicate that bcl-2 expression protects neurons in vivo from ischemic injury and suggest the feasibility of gene therapy for stroke and perhaps other neurological diseases in which programmed cell death is involved.

Animals↗
PubMed1995

Rapid and efficient gene transfer in Human hepatocytes by herpes viral vectors.

Retroviral vectors have been widely studied as vehicles for hepatocyte gene therapy, but they are limited by an inability to infect nondividing cells and the need for prolonged cell culture. Two replication deficient herpes simplex viral vectors (HSV) were constructed with the marker genes lac-Z/beta-galactosidase (HSVlac) or human-growth hormone (HSVhGH) to determine the efficiency of HSV gene transfer into adult human hepatocytes. Hepatocytes were isolated by collagenase perfusions and density centrifugation from liver wedge biopsy specimens obtained from six patients. After exposure to HSV (0, 50,000 and 500,000 viral particles/ 10(6) hepatocytes) for 20 minutes, 1 hour, or 2 hours, the hepatocytes were washed and placed in culture. Hepatocytes transduced with HSVlac were fixed at 24 hours and histochemically stained with X-gal, and media from HSVhgh-transduced cells were assayed at 48 hours by radioimmunoassay for hGH. After a 20-minute exposure at a multiplicity of infection of 0.5 (1 viral particle per 2 hepatocytes), greater than 35% of the hepatocytes expressed the lac-Z gene ( > 70% efficiency). hGH was also detected in the media from HSVhGH-transduced cells, showing that proteins coded for by foreign cells are not only expressed by transduced cells but are also secreted. Isolated liver perfusions using HSVlac were also performed in Fischer rats. A 20-minute isolated perfusion using 5 x 10(6) viral particles resulted in expression of the beta-galactosidase gene in the rodent livers 72 hours later without histological signs of tissue injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes.

BACKGROUND: Chronic diabetes causes structural changes in the retinal capillaries of nearly all patients with a disease duration of more than 15 years. Acellular occluded vessels cause hypoxia, which stimulates sight-threatening abnormal angiogenesis in 50% of all type I diabetic patients. The mechanism by which diabetes produces acellular retinal capillaries is unknown. MATERIALS AND METHODS: In this study, evidence of programmed cell death (PCD) was sought in the retinas of early diabetic rats, and the effect of nerve growth factor (NGF) on PCD and capillary morphology was evaluated. RESULTS: Diabetes induced PCD primarily in retinal ganglion cells (RGC) and Muller cells. This was associated with a transdifferentiation of Muller cells into an injury-associated glial fibrillary acidic protein (GFAP)-expressing phenotype, and an up-regulation of the low-affinity NGF receptor p75NGFR on both RGC and Muller cells. NGF treatment of diabetic rats prevented both early PCD in RGC and Muller cells, and the development of pericyte loss and acellular occluded capillaries. CONCLUSIONS: These data provide new insight into the mechanism of diabetic retinal vascular damage, and suggest that NGF or other neurotrophic factors may have potential as therapeutic agents for the prevention of human diabetic retinopathy.

Animals↗
PubMed1994

Functional characterization of the rat GAP-43 promoter.

GAP-43 is a highly conserved neuronal protein whose expression is spatially and temporally regulated. Because this regulation may occur, at least in part, at the level of transcription, we have begun to characterize the regions upstream of the GAP-43 transcription unit which direct its neuronal-specific expression. Functional analyses of GAP-43 promoter-reporter constructs have been performed in stably transfected cell lines, including PC12, C6 and RAT2. These data indicate that as little as 600 bp of GAP-43 5'-flanking DNA sequence directs the expression in a neuronal-specific manner. A shorter construct containing 230 bp of 5'-flanking DNA sequence defines a GAP-43 minimal promoter that is active in both neuronal and glial but not in non-neural cell lines. An upstream region, previously shown by other investigators to have promoter activity, was able to stimulate transcription when linked to the downstream minimal promoter. However, this upstream region was by itself unable to direct transcription of the reporting gene. In addition, we have demonstrated that two polypurine tracts within the 5'-flanking DNA sequence of the GAP-43 gene adopt a non-duplex configuration in plasmids, and, when studied in the context of chromosomal integration, these regions have a stimulatory effect on transcription.

Animals↗
PubMed1994

Cardiac and skeletal myopathy in beta myosin heavy-chain simian virus 40 tsA58 transgenic mice.

The mechanisms regulating cardiac muscle differentiation and development are incompletely understood. To examine the relationships between cardiocyte proliferation and differentiation, we tested the ability of a fragment from the rat beta myosin heavy-chain (MHC beta) gene to correctly target expression of a thermolabile simian virus 40 large tumor antigen allele (tsA58) in the developing mouse. Transgene expression in the heart was observed as early as 10 days postconception and was developmentally regulated in parallel with the endogenous MHC beta gene. Expression was also detected in developing skeletal muscle, although at low levels. Despite the temperature sensitivity of the mutant large tumor antigen protein, a subset of transgenic mice in several lineages developed marked cardiac and skeletal myopathies.

Animals↗
PubMed1994

Transfer of the nerve growth factor gene into cell lines and cultured neurons using a defective herpes simplex virus vector. Transfer of the NGF gene into cells by a HSV-1 vector.

Nerve growth factor (NGF) can be expressed in cells by gene transfer using a defective Herpes Simplex virus type 1 (HSV-1) vector. In this report, the defective HSV-1 vector, pHSVngf, is used to infect established cell lines and cultured neurons. Infection of cell lines with pHSVngf results in gene transcription, correct RNA processing, and production of biologically active NGF. Infection of the PC12 neuronal cell line results in the production of biologically active NGF and infection of NGF-dependent neonatal sympathetic neurons in primary culture with pHSVngf leads to neuronal survival in the absence of exogenously-added NGF. NGF expressed by pHSVngf-infected cells does not appear to work through an autocrine intracellular pathway since NGF antibody added to culture media of infected cells could block NGF action. Infection with pHSVngf of cholinergic striatal or septal neurons in dissociated cell culture resulted in an increase in choline acetyltransferase activity. These studies demonstrate the efficacy of defective HSV-1 vectors for delivery and expression of neurotrophin genes in cultured neural cells.

3T3 Cells↗
PubMed1993

Transsynaptic neuronal loss induced in hippocampal slice cultures by a herpes simplex virus vector expressing the GluR6 subunit of the kainate receptor.

Patients with severe temporal lobe epilepsy lose neurons within the CA3 and hilar regions of the hippocampus. Loss of CA3 and hilar neurons was also induced by transducing organotypic hippocampal slice cultures with a replication-defective herpes simplex virus (HSV) vector expressing the GluR6 kainate subtype of the glutamate receptor (HSVGluR6). In transduced fibroblasts, HSVGluR6 expressed a M(r) 115,000 protein that reacted with anti-GluR6 serum. After exposure of fibroblast to HSVGluR6, a kainate-dependent toxicity appeared in cells that were previously resistant to kainate. Microapplication of nanoliter amounts of recombinant HSV stocks into organotypic hippocampal slice cultures resulted in localized transduction and gene transfer at the site of microapplication. Microapplication of 100 HSVGluR6 virions into CA3 stratum pyramidale induced a large loss of CA3 pyramidal cells and hilar neurons, despite the small number of transduced neurons. This effect was not seen when 100 virions of HSVGluR6 were microapplied to CA1 stratum pyramidale. Tetrodotoxin or N-methyl-D-aspartate receptor antagonists inhibited the large loss of CA3 and hilar neurons, suggesting that the small cluster of HSVGluR6-transduced cells induced an N-methyl-D-aspartate-dependent transsynaptic loss of non-transduced neurons.

3T3 Cells↗
PubMed1993

Localized gene transfer into organotypic hippocampal slice cultures and acute hippocampal slices.

Viral vectors derived from herpes simplex virus, type-1 (HSV), can transfer and express genes into fully differentiated, post-mitotic neurons. These vectors also transduce cells effectively in organotypic hippocampal slice cultures. Nanoliter quantities of a virus stock of HSVlac, an HSV vector that directs expression of E. coli beta-galactosidase (beta-gal), were microapplied into stratum pyramidale or stratum granulosum of slice cultures. Twenty-four hours later, a cluster of transduced cells expressing beta-gal was observed at the microapplication site. Gene transfer by microapplication was both effective and rapid. The titer of the HSVlac stocks was determined on NIH3T3 cells. Eighty-three percent of the beta-gal forming units successfully transduced beta-gal after microapplication to slice cultures. beta-Gal expression was detected as rapidly as 4 h after transduction into cultures of fibroblasts or hippocampal slices. The rapid expression of beta-gal by HSVlac allowed efficient transduction of acute hippocampal slices. Many genes have been transduced and expressed using HSV vectors; therefore, this microapplication method can be applied to many neurobiological questions.

Animals↗
PubMed1993

Ontogeny, sex dimorphism, and neonatal sex hormone determination of synapse-associated messenger RNAs in rat brain.

Sex hormones influence neurite outgrowth and synaptogenesis in certain hormone-dependent areas of the rat brain during neonatal development. These alterations are thought to mediate changes in brain structure and function between the sexes. Growth-associated protein 43 kDa (GAP-43) gene expression is estrogen-regulated in the adult ventromedial hypothalamus (VMH) and sexually dimorphic (M:F = 1.8:1) in adult cortex (CTX). Such effects intimate hormonal regulation of synaptic plasticity. To investigate the nature of these dimorphisms, the present study examined the ontogeny of expression of mRNAs encoding 3 neural-specific proteins: GAP-43, SCG10, and synaptosomal-associated protein 25 kDa (SNAP-25); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in the VMH and CTX; and also the effects of altering the neonatal sex hormonal milieu on the development of these adult dimorphisms. Levels of specific mRNAs in VMH and CTX were quantitated by slot-blot hybridization in rats of both sexes at different postnatal ages. To determine the involvement of neonatal sex hormones on the levels of these mRNAs, male neonatal rat pups were treated with an estrogen receptor antagonist or an aromatase inhibitor, and neonatal female pups were treated with testosterone or estrogen prior to slot-blot evaluations in adulthood. In VMH, GAP-43 mRNA levels were high on days P1 and P4 with a 3-fold decrease by day P23; in CTX, GAP-43 mRNA first increased by day P11, then fell to baseline by day P23. In VMH, SCG10 mRNA showed only small increases with time; but in CTX, there was a 5-fold drop from days P4 to P23. In VMH, SNAP-25 mRNA was low and changed only slightly; but in CTX there was a 5-fold increase between days P4 and P60. At birth, there was no sex dimorphism in either VMH or CTX, but the levels of all 3 neural-specific mRNAs were sexually dimorphic in adult CTX (M:F = 1.76 for GAP-43, 1.46 for SCG10, 1.44 for SNAP-25). GAPDH mRNA levels were regulated developmentally in VMH and CTX, but there was no sex dimorphism in either area. In male rats who received either an estrogen antagonist or aromatase inhibitor at birth, the CTX GAP-43 and SNAP-25 mRNA levels fell by 30%, to levels similar to untreated females. Conversely, in female rats, neonatal treatment with either testosterone or estrogen increased GAP-43 and SNAP-25 mRNA levels by about 30%, to levels similar to the untreated adult male. SCG10 levels did not demonstrate neonatal hormonal dependence.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Nonenzymatic glycosylation of laminin and the laminin peptide CIKVAVS inhibits neurite outgrowth.

Nerve regeneration in diabetic animals is delayed and qualitatively impaired, but the mechanisms responsible for these defects have not been elucidated. The extracellular matrix protein laminin promotes the extension of neuronal processes, and recent studies have localized neurite-promoting activity to a lysine-containing sequence (IKVAV) within the laminin molecule. Because long-lived molecules such as laminin are likely to accumulate excessive amounts of nonenzymatic glycosylation products in diabetic subjects, we have investigated whether such adduct formation on laminin or the IKVAV peptide affects their neurite-promoting properties. These studies used the murine neuroblastoma cell line NB2a, which extends neurites on laminin when differentiated by cAMP. Neurite outgrowth in NB2a cells plated on glycosylated laminin was significantly decreased from that occurring on unmodified laminin. Similarly, neurite outgrowth in NB2a cells plated on glycosylated IKVAV peptide was inhibited compared with that observed on native IKVAV. These data suggest that nonenzymatic glycosylation of a biologically active domain within laminin may contribute to impaired nerve regeneration in diabetes.

Amino Acid Sequence↗
PubMed1992

Expression of nerve growth factor in vivo from a defective herpes simplex virus 1 vector prevents effects of axotomy on sympathetic ganglia.

Sympathetic neurons in the superior cervical ganglion (SCG) of adult rats depend on target-derived nerve growth factor (NGF) for maintenance of tyrosine hydroxylase (TH) levels and the noradrenergic neurotransmitter system. Axotomy of a SCG results in NGF deprivation, causing a decline in TH activity; continuous local application of NGF can prevent this decline in TH activity. We now report that injection of a defective herpes simplex virus 1 vector that expresses NGF (pHSVngf) into a SCG can prevent the decline in TH activity that follows axotomy. SCG of adult rats were injected with either pHSVngf virus or pNFlac virus, which expresses Escherichia coli beta-galactosidase. Analysis of RNA from pHSVngf-infected SCG indicated that the NGF gene was efficiently transcribed and processed. Furthermore, 4 days after pHSVngf injection animals underwent axotomy of the virus-injected SCG. After another 10 days, animals were sacrificed and both the injected-axotomized and contralateral control ganglia were assayed for TH activity. Axotomy of SCG injected with pNFlac virus produced a 50% decline in TH activity relative to control ganglia (P = 0.02). In contrast, SCG injected with pHSVngf virus did not show a decline in TH activity following axotomy; instead, these ganglia manifested an 18% increase in TH levels relative to control ganglia. These data demonstrate that herpes simplex virus 1 vectors can be used to modify neuronal physiology in vivo; specifically, expression of a critical gene product by neural cells that do not normally produce it has potential applications for gene therapy.

Base Sequence↗
PubMed1991

Estrogenic regulation and sex dimorphism of growth-associated protein 43 kDa (GAP-43) messenger RNA in the rat.

In addition to effects on brain protein synthesis, neurotransmitter release, and electrophysiology, estrogens alter neurite outgrowth and synaptogenesis. This study examined in the adult rat the effects of estrogen and sex on the expression of the GAP-43 gene; encoding a phosphoprotein structurally and physiologically linked to these two processes in the rat CNS. Ovariectomized (OVX) rats were injected with vehicle or estrogen, or male and female rats were either gonadectomized or left intact. Brains were dissected to obtain ventromedial hypothalamus (VMH), posterior hypothalamus (PH), or frontal cortex (CTX). Total RNA from these areas were extracted, and slot-blots of equal masses of total RNA were hybridized to 32P-labeled cDNAs for GAP-43 and beta-actin, and also to synthetic poly-dT. Resultant autoradiograms were scanned by laser densitometry, quantitated, and ratios of the gray scale generated by each probe were compared between experimental groups. GAP-43 mRNA expression, when compared to expression of either beta-actin mRNA or total poly(A)-containing RNA (poly(A) RNA), was higher in VMH and PH as compared to CTX. Estrogen treatment of OVX rats resulted in a 48-74% increase in GAP-43 mRNA levels in the VMH--in one experiment, this increase was noted after 2 h of estradiol treatment, and in another after 3 days of estradiol benzoate treatment; but PH and CTX were unaffected by either estrogen regimen. Conversely, ovariectomy of intact rats decreased GAP-43 mRNA expression by 45% in the VMH, but not in the CTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Abnormal cortisol secretion and responses to corticotropin-releasing hormone in women with hypothalamic amenorrhea.

Hypothalamic amenorrhea (HA) is a common disorder associated with hypoestrogenemia and has adverse effects. The mechanism of GnRH deficiency in these women is not yet known. To investigate the role of the hypothalamic-pituitary-adrenal axis in HA, we studied 10 women [mean age, 29 +/- 7 (+/- SD) yr] with 0.5-13 yr of amenorrhea (mean, 4.3 +/- 3.7 yr) related to simple weight loss or psychological stress. We investigated cortisol and ACTH responses to a bolus of ovine CRH, 24-h plasma cortisol levels obtained every 10 min, and urinary free cortisol levels in these patients. Results were compared with those obtained in normal women during all phases of the menstrual cycle. We found that mean basal concentrations of cortisol were significantly higher (P = 0.03) in the HA patients (mean, 210 +/- 130 nmol/L) than in the normal women (100 +/- 30 nmol/L). The delta (peak - basal) cortisol was significantly lower (P = 0.004) in the HA patients than in the normal women (320 +/- 100 vs. 440 +/- 90 nmol/L, respectively). ACTH responses to CRH did not differ between HA patients and normal women. The 24-h mean cortisol was significantly higher (P = 0.006) in the HA patients than in the normal controls (280 +/- 50 and 220 +/- 50 nmol/L, respectively), due to higher cortisol levels at night. The urinary free cortisol level was significantly higher (P = 0.005) in the HA patients (230 +/- 70 nmol/day) than in normal women (150 +/- 40 nmol/day). We conclude that women with HA have a blunted cortisol response to CRH administration. In addition, they have hypercortisolism, as demonstrated by elevated 24-h mean serum cortisol levels and urinary free cortisol values. This hypothalamic-pituitary-adrenal axis activation in patients with stress or weight loss may be a mechanism in the development of amenorrhea and may relate to other potential adverse effects of HA.

Adrenocorticotropic Hormone↗
PubMed1989

GAP-43 gene expression during development: persistence in a distinctive set of neurons in the mature central nervous system.

GAP-43 is a rapidly transported axonal protein most prominently expressed in regenerating and developing nerves. However, the low level persistence of GAP-43 in the adult CNS where growth and regenerative capacity are minimal may additionally indicate a role for this molecule in neuronal remodeling. Previous studies have revealed GAP-43 immunoreactivity in neurites throughout many regions of the CNS. To identify the CNS neurons that express GAP-43 at different stages of development, we utilized in situ hybridization and immunocytochemistry; the latter was performed with an antibody that recognizes GAP-43 immunoreactivity in both perikarya and neurites. In the perinatal period GAP-43 is expressed in all neurons. Subsequently its expression becomes progressively restricted such that by maturity most neurons no longer express detectable levels, although GAP-43 expression is still moderately high in the adult entorhinal cortex, and strikingly high in the adult hippocampus and olfactory bulb. In light of current notions about the function of GAP-43, it is tempting to speculate that this anatomy denotes neurons engaged in structural remodeling and functional plasticity.

Aging↗
PubMed1988

Dual regulation of GAP-43 gene expression by nerve growth factor and glucocorticoids.

GAP-43 is a neural-specific protein that is believed integral to neurite growth and to the plasticity of neuronal structure. Its gene expression is regulated in vivo and correlates with periods of axonal growth. We investigated the regulation of GAP-43 gene expression in PC12 cells, which are believed to resemble precursor cells of the adrenomedullary lineage. In these cells, nerve growth factor (NGF) increases GAP-43 expression, and corticosteroids decrease it. Corticosteroids diminish GAP-43 levels even in cells already differentiated by NGF, as well as in primary neurons of the superior cervical ganglion. Neither the NGF nor the steroid effect requires new protein synthesis. Nuclear run-on experiments show that the steroid repression is mediated at the level of gene transcription but that the NGF effect is likely to be posttranscriptional. NGF and corticosteroids are known to regulate bimodally the cell fate decision of sympathoadrenal precursors, with NGF promoting the neuronal phenotype and steroids promoting the chromaffin phenotype. The regulation of GAP-43 is consistent with the notion that this gene is bimodally regulated during these cell fate decisions.

Adrenal Gland Neoplasms↗
PubMed1983

Regulation of maltase synthesis in Saccharomyces carlsbergensis.

The induction of maltase (EC 3.2.1.20) by its inducer maltose in a strain of the yeast Saccharomyces carlsbergensis carrying a functional MAL locus is regulated at the level of transcription. Preceding the synthesis of increased levels of maltase is the de novo synthesis of maltase-specific RNA sequences. This was detected by determining the level of maltase mRNA by DNA-RNA hybridizations by using a maltase structural gene DNA sequence probe and by assaying functional maltase mRNA by in vitro RNA-directed synthesis of immunologically reactive maltase. Once maltase has accumulated, late in induction, further synthesis of the enzyme is inhibited, as reflected by reduced levels of the mRNA that encodes maltase.

Enzyme Induction↗