Geetha A. Shetty

Stem/progenitor cell proliferation factors FGF‐2, IGF‐1, and VEGF exhibit early decline during the course of aging in the hippocampus: Role of astrocytes

Dentate neurogenesis, important for learning and memory, declines dramatically by middle age. Although studies have shown that this age‐related decrease can be reversed to some extent by exogenous applications of mitogenic factors, it is unclear whether one or more of these factors exhibits decline by middle age. We hypothesize that multiple stem/progenitor cell proliferation factors exhibit early decline during the course of aging in the hippocampus, and some of these declines are linked to age‐related alterations in hippocampal astrocytes. We measured the concentrations of fibroblast growth factor‐2 (FGF‐2), insulin‐like growth factor‐1 (IGF‐1), and vascular endothelial growth factor (VEGF) in the hippocampus of young, middle‐aged, and aged F344 rats, using enzyme‐linked immunosorbent assay (ELISA). In addition, we quantified the total number of FGF‐2 immunopositive (FGF‐2+) and glial fibrillary acidic protein immunopositive (GFAP+) cells in the dentate gyrus and the entire hippocampus. Our results provide new evidence that the concentrations of FGF‐2, IGF‐1, and VEGF decline considerably by middle age but remain steady between middle age and old age. Further, decreased concentrations of FGF‐2 during aging are associated with decreased numbers of FGF‐2+ astrocytes. Quantification of GFAP+ cells, and GFAP and FGF‐2 dual immunostaining analyses, reveal that aging does not decrease the total number of astrocytes but fractions of astrocytes that express FGF‐2 decline considerably by middle age. Thus, dramatically decreased dentate neurogenesis by middle age is likely linked to reduced concentrations of FGF‐2, IGF‐1, and VEGF in the hippocampus, as each of these factors can individually influence the proliferation of stem/progenitor cells in the dentate gyrus. Additionally, the results demonstrate that decreased FGF‐2 concentration during aging is a consequence of age‐related impairment in FGF‐2 synthesis by astrocytes.

Brain-derived neurotrophic factor, phosphorylated cyclic AMP response element binding protein and neuropeptide Y decline as early as middle age in the dentate gyrus and CA1 and CA3 subfields of the hippocampus

The hippocampus is very susceptible to aging. Severely diminished dentate neurogenesis at middle age is one of the most conspicuous early changes in the aging hippocampus, which is likely linked to an early decline in the concentration of neurotrophic factors and signaling proteins that influence neurogenesis. We analyzed three proteins that are well-known to promote dentate neurogenesis and learning and memory function in the dentate gyrus and the hippocampal CA1 and CA3 subfields of young, middle-aged and aged F344 rats. These include the brain-derived neurotrophic factor (BDNF), the transcription factor phosphorylated cyclic AMP response element binding protein (p-CREB) and the neuropeptide neuropeptide Y (NPY). The BDNF was analyzed via ELISA and BDNF immunohistochemistry, the p-CREB through densitometric analysis of p-CREB immunopositive cells, and the NPY via stereological counting of NPY-immunopositive interneurons. We provide new evidence that the BDNF concentration, the p-CREB immunoreactivity and the number of NPY immunopositive interneurons decline considerably by middle age in both dentate gyrus and CA1 and CA3 subfields of the hippocampus. However, both BDNF concentration and NPY immunopositive interneuron numbers exhibit no significant decrease between middle age and old age. In contrast, the p-CREB immunoreactivity diminishes further during this period, which is also associated with reduced BDNF immunoreaction within the soma of dentate granule cells and hippocampal pyramidal neurons. Collectively, these results suggest that severely dampened dentate neurogenesis observed at middle age is linked at least partially to reduced concentrations of BDNF, p-CREB and NPY, as each of these proteins is a positive regulator of dentate neurogenesis. Dramatically diminished CREB phosphorylation (and persistently reduced levels of BDNF and NPY) at old age may underlie the learning and memory impairments observed during senescence.

Nitric Oxide Synthase Gene Therapy Rapidly Reduces Adhesion Molecule Expression and Inflammatory Cell Infiltration in Carotid Arteries of Cholesterol-Fed Rabbits

BACKGROUND Hypercholesterolemia reduces nitric oxide bioavailability, manifested by reduced endothelium-dependent vascular relaxation, and also induces vascular adhesion molecule expression and inflammatory cell infiltration. We have previously shown that gene therapy with NO synthase in hypercholesterolemic rabbits substantially reverses the deficit in vascular relaxation. In the present study, we show that NO synthase gene therapy rapidly and substantially reduces vascular adhesion molecule expression, lipid deposition, and inflammatory cell infiltration. METHODS AND RESULTS Thirty male New Zealand White rabbits were maintained on a 1% cholesterol diet for 11 to 13 weeks, then underwent carotid artery gene transfer with Ad.nNOS or Ad.betaGal (recombinant adenoviruses expressing neuronal NO synthase or beta-galactosidase, respectively), or received medium alone in a sham procedure. Arteries were harvested at 1 and 3 days after gene transfer, and the following parameters were determined by immunohistochemical and image-analysis techniques: intercellular adhesion molecule-1, vascular cell adhesion molecule-1, lipid deposition by oil red O staining, lymphocyte infiltration (CD43-positive cells), and monocyte infiltration (RAM-11-positive cells). In Ad.nNOS-treated arteries, all markers were significantly decreased relative to Ad. betaGal or sham-treated arteries within 3 days after gene transfer. Ad.nNOS had a particularly striking impact on monocyte infiltration; as early as 24 hours after gene transfer, Ad.nNOS-treated arteries had >3-fold fewer monocytes than Ad.betaGal- or sham-treated arteries. CONCLUSIONS NO synthase gene therapy rapidly ameliorates several markers of atherosclerosis in the cholesterol-fed rabbit.

Adenoviral gene transfer of nitric oxide synthase : High level expression in human vascular cells

OBJECTIVES Nitric oxide synthases (NOS) generate nitric oxide (NO), a second messenger with key regulatory roles. In the cardiovascular system, deficient endothelial NO production is an early, persistent feature of atherosclerosis and vascular injury. Accordingly, the NOS isoforms represent attractive targets for vascular gene therapy. We aimed to generate and evaluate an adenoviral vector for gene transfer of an NOS isoform to vascular cells. METHODS We constructed a recombinant adenovirus, Ad.nNOS, for gene transfer of the neuronal isoform of NOS (nNOS) and characterized its expression in 293 cells, human vascular smooth muscle cells (hVSMC) and human umbilical vein endothelial cells (HUVEC). NOS expression was analyzed by Western immunoblotting, and NOS enzyme activity in response to receptor-dependent and receptor-independent agonists was determined by Griess assay or by NO chemiluminescence. RESULTS Ad.nNOS-infected 293 cells expressed high levels of functional nNOS enzyme, even higher than in 293.NOS cells (a cell line that expresses supraphysiologic levels of nNOS). In hVSMC, nNOS activity reached levels 50% of those seen in 293.NOS cells. nNOS expression and activity in hVSMC increased linearly with titer of Ad.nNOS. NO production in hVSMC was stimulated both by calcium ionophore and by physiologic agonists such as acetylcholine or bradykinin. In HUVEC, endogenous NOS activity was significantly augmented by Ad.nNOS infection. Supplementation with the tetrahydrobiopterin precursor sepiapterin enhanced NOS activity in all cells. CONCLUSIONS Ad.nNOS, a novel adenoviral vector for gene transfer of NOS, generates high-level nNOS expression in a variety of vascular cell types. nNOS activity in hVSMC is physiologically regulated and of a magnitude comparable to native eNOS activity in HUVEC. Our findings demonstrate Ad.nNOS to be a versatile and efficient tool for nNOS gene transfer, with widespread potential applications in cell culture and for gene therapy.

Hippocampal neurotrophin levels after injury: Relationship to the age of the hippocampus at the time of injury.

Aging impairs the competence of the hippocampus for synaptic reorganization after injury. This potentially is due to the inability of the aging hippocampus to up‐regulate the critical neurotrophic factors for prolonged periods after injury to levels at which they can stimulate neurite outgrowth and facilitate synaptic reorganization. We hypothesize that the concentrations of neurotrophins in the hippocampus after injury depend on the age at the time of injury. We quantified the concentrations of brain‐derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin‐3 (NT‐3) in the hippocampus of young, middle‐aged, and aged Fischer 344 rats at 4 days after kainic acid (KA)‐induced injury. In comparison with the age‐matched intact hippocampus, the KA‐lesioned hippocampus exhibited increased levels of BDNF and NGF in all three age groups. In contrast, the NT‐3 concentration was unaltered after KA lesion. Notwithstanding similar percentage increases in BDNF after injury, the lesioned middle‐aged and aged hippocampus contained 45–52% less BDNF than the lesioned young hippocampus. NGF and NT‐3 levels after injury were comparable across the three age groups, however. Furthermore, lower BDNF concentration in the injured aging hippocampus was associated with normal astrocytic response but significantly diminished microglial reaction. Thus, in comparison with the injured young hippocampus, the injured aging hippocampus contains considerably less BDNF but similar levels of NGF and NT‐3. Lower BDNF levels in the injured aging hippocampus might underlie the diminished spontaneous healing response observed in the aging hippocampus after injury, particularly in terms of synaptic reorganization and dentate neurogenesis.

Acute Host-Mediated Endothelial Injury After Adenoviral Gene Transfer in Normal Rabbit Arteries: Impact on Transgene Expression and Endothelial Function

Acute injury after adenoviral vascular gene transfer remains incompletely characterized. Here, we describe the early response (< or =days) in 52 New Zealand White rabbits undergoing gene transfer (beta-galactosidase or empty vector) or sham procedures to both carotid arteries. After gene transfer, arteries were either left in vivo for 1 hour to 3 days (in vivo arteries) or were excised immediately after gene transfer and cultured (ex vivo arteries). Within 1 hour, in vivo arteries receiving infectious titers of > or = 4X10(9) plaque-forming units (pfu)/mL showed endothelial activation, with an acute inflammatory infiltrate developing by 6 hours. Ex vivo arteries showed endothelial activation but no inflammatory infiltrate. There were also significant differences in transgene expression between in vivo and ex vivo arteries. Ex vivo arteries showed titer-dependent increases in beta-galactosidase expression through 2X10(10) pfu/mL, whereas in in vivo arteries, titers above 4X10(9) pfu/mL merely increased acute inflammatory response, without increasing transgene expression. In vivo arteries showed significant time- and titer-dependent impairment in endothelium-dependent relaxation, with no effect on contraction or nitroprusside-induced relaxation. Interestingly, however, if rabbits were made neutropenic with vinblastine, their arteries maintained full endothelium-dependent relaxation, even after very high titer vascular infection (up to 1X10(11) pfu/mL). These findings show that recombinant adenovirus triggers an early inflammatory response, and it is the inflammatory response that in turn causes functional endothelial injury. This occurs at much lower titers than previously appreciated (though the precise threshold will undoubtedly vary between laboratories). However, titers below the inflammatory threshold produce excellent transgene expression without inflammation or vascular injury.

Hippocampal neurotrophin levels in a kainate model of temporal lobe epilepsy: a lack of correlation between brain‐derived neurotrophic factor content and progression of aberrant dentate mossy fiber sprouting

A significant upregulation of neurotrophins particularly brain‐derived neurotrophic factor (BDNF) is believed to be involved in the initiation of epileptogenic changes such as the aberrant axonal sprouting and synaptic reorganization in the injured hippocampus. However, it is unknown which of the neurotrophins are upregulated during the peak period of aberrant mossy fiber sprouting in the chronically injured hippocampus. We measured chronic changes in the levels of BDNF, nerve growth factor (NGF) and neurotrophin‐3 (NT‐3) in the adult hippocampus using enzyme‐linked immunosorbent assay (ELISA) after a unilateral intracerebroventricular administration of kainic acid (KA), a model of temporal lobe epilepsy. For comparison, neurotrophins were also measured from the control intact hippocampus. Further, to see the association between changes in neurotrophin levels and the progression of mossy fiber sprouting, chronic changes in the mossy fiber distribution within the dentate supragranular layer (DSGL) were quantified. In the KA‐lesioned hippocampus, the neurotrophins BDNF and NGF were upregulated at 4 days post‐lesion, in comparison to their levels in the intact hippocampus. However, the concentration of BDNF reached the baseline level at 45 days post‐lesion and dramatically diminished at 120 days post‐lesion. In contrast, the upregulation of NGF observed at 4 days post‐lesion was sustained at both 45 days and 120 days post‐lesion. The concentration of NT‐3 was upregulated at 45 days post‐lesion but remained comparable to baseline levels at 4 days and 120 days post‐lesion. Interestingly, analysis of mossy fiber sprouting revealed that most of the aberrant sprouting in the lesioned hippocampus occurs between 45 days and 120 days post‐lesion. Taken together, these results suggest that the period of robust mossy fiber sprouting does not correlate with the phase of post‐lesion BDNF upregulation. Rather, it shows a relationship with the time of upregulation of neurotrophins NGF and NT‐3.

In Vivo Gene Transfer of Nitric Oxide Synthase Enhances Vasomotor Function in Carotid Arteries From Normal and Cholesterol-Fed Rabbits

BACKGROUND The vascular endothelium is anatomically intact but functionally abnormal in preatherosclerotic states, and an early deficit in the bioavailability of nitric oxide (NO) or related molecules has been described in both humans and animal models. We hypothesized that the targeted gene transfer of NO synthase (NOS) isoforms might ameliorate or reverse the deficit. METHODS AND RESULTS We constructed a recombinant adenovirus, Ad.nNOS, that expresses the neuronal isoform of NOS (nNOS) and used it for in vivo endovascular gene transfer to carotid arteries (CA) from normal and cholesterol-fed rabbits. Vessels were harvested 3 days after gene transfer. In CA from normal rabbits, Ad.nNOS generated high levels of functional nNOS protein predominantly in endothelial cells and increased vascular NOS activity by 3.4-fold relative to sham-infected control CA. Ad.nNOS gene transfer also significantly enhanced endothelium-dependent vascular relaxation to acetylcholine; at 3 micromol/L acetylcholine, Ad.nNOS-treated arteries showed an 86+/-4% reduction in precontracted tension, whereas control CA showed a 47+/-6% reduction in tension. Contraction in response to phenylephrine and relaxation in response to nitroprusside were unaffected in both control and Ad.nNOS-treated CA. To determine the effect of Ad.nNOS in atherosclerotic arteries, 10 male New Zealand White rabbits maintained on a 1% cholesterol diet for 10 to 12 weeks underwent gene transfer according to the same protocol used in normal rabbits. Ad.nNOS-treated arteries showed a 2-fold increase in NADPH-diaphorase staining intensity relative to sham-infected and Ad. betaGal-treated arteries. The CA from cholesterol-fed rabbits showed impaired acetylcholine-induced relaxation, but this abnormality was almost entirely corrected by Ad.nNOS gene transfer. CONCLUSIONS In vivo adenovirus-mediated endovascular delivery of nNOS markedly enhances vascular NOS activity and can favorably influence endothelial physiology in the intact and atherosclerotic vessel wall.

Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels.

OBJECTIVES Gene therapy may provide new approaches to reduce vein graft failure following coronary or peripheral bypass surgery. The aim of this study was to investigate the relative efficacy of intraoperative adenoviral gene transfer to vein grafts, comparing transgene expression in vein grafts with that in matched native vessels in the same animal. In addition, we assessed the impact of bypass grafting on the cellular targets of gene transfer. METHODS New Zealand White rabbits underwent interposition bypass grafting of the carotid artery, using the ipsilateral external jugular vein, which was infected with an adenovirus expressing beta-galactosidase immediately prior to bypass grafting (n = 16). The contralateral native jugular vein (n = 16) and carotid artery (n = 8) were infected concurrently with the same adenoviral preparation. After 3, 7 or 14 days, beta-galactosidase protein expression was quantified by ELISA, and specific cell types expressing beta-galactosidase were identified by X-Gal staining and by immunohistochemistry. RESULTS After 3 days, endothelial cells were efficiently transduced in all vessels; medial smooth muscle cells were transduced infrequently. In contrast to jugular veins after gene transfer, endothelium in vein grafts showed expression of VCAM-1 and ICAM-1, and intense inflammation with CD18+ leukocytes. Transgene expression in vein grafts at day 3 was maintained at levels approximately 50% of that in ungrafted jugular veins, but continued to decrease through day 7. CONCLUSIONS Although vascular injury in early venous bypass grafts reduces gene transfer efficacy, significant transgene expression is maintained for at least 7 days. These findings have important implications for intraoperative gene transfer strategies in vein grafts.

An autonomously self‐assembling dendritic DNA nanostructure for target DNA detection

There is a growing need for sensitive and reliable nucleic acid detection methods that are convenient and inexpensive. Responsive and programmable DNA nanostructures have shown great promise as chemical detection systems. Here, we describe a DNA detection system employing the triggered self-assembly of a novel DNA dendritic nanostructure. The detection protocol is executed autonomously without external intervention. Detection begins when a specific, single-stranded target DNA strand (T) triggers a hybridization chain reaction (HCR) between two, distinct DNA hairpins (α and β). Each hairpin opens and hybridizes up to two copies of the other. In the absence of T, α and β are stable and remain in their poised, closed-hairpin form. In the presence of T, α hairpins are opened by toe-hold mediated strand-displacement, each of which then opens and hybridizes two β hairpins. Likewise, each opened β hairpin can open and hybridize two α hairpins. Hence, each layer of the growing dendritic nanostructure can in principle accommodate an exponentially increasing number of cognate molecules, generating a high molecular weight nanostructure. This HCR system has minimal sequence constraints, allowing reconfiguration for the detection of arbitrary target sequences. Here, we demonstrate detection of unique sequence identifiers of HIV and Chlamydia pathogens.