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Background —These studies were initiated to confirm that high-level thrombomodulin overexpression is sufficient to limit neointima formation after mechanical overdilation injury. Methods and Results —An adenoviral construct expressing thrombomodulin (Adv/RSV-THM) was created and functionally characterized in vitro and in vivo. The impact of local overexpression of thrombomodulin on neointima formation 28 days after mechanical overdilation injury was evaluated. New Zealand White rabbit common femoral arteries were treated with buffer, viral control, or Adv/RSV-THM and subjected to mechanical overdilation injury. The treated vessels (n=4 per treatment) were harvested after 28 days and evaluated to determine intima-to-media (I/M) ratios. Additional experiments were performed to determine early (7-day) changes in extracellular elastin and collagen content; local macrophage, T-cell, and neutrophil infiltration; and local thrombus formation as potential contributors to the observed impact on 28-day neointima formation. The construct significantly decreased neointima formation after mechanical dilation injury in this model. By histological analysis, buffer controls exhibited mean I/M ratios of 0.76±0.06%, whereas viral controls reached 0.77±0.08%; in contrast, Adv/RSV-THM reduced I/M ratios to 0.47±0.06%. Local inflammatory infiltrate decreased in the Adv/RSV-THM group relative to controls, whereas matrix remained relatively preserved. Rates of early thrombus formation also decreased in Adv/RSV-THM animals. Conclusions —This construct thus offers a viable technique for promoting a locally neointima-resistant small-caliber artery via decreased thrombus bulk, normal matrix preservation, and decreased local inflammation without the inflammatory damage that has limited many other adenoviral applications.

Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that suppresses metastasis in multiple human and murine carcinoma cell lines. BRMS1 interacts with several nuclear proteins including SIN3:HDAC chromatin remodeling complexes that are involved in repressing transcription. However, recent reports suggest BRMS1 may function in the cytoplasm. BRMS1 has two predicted nuclear localization sequences (NLS) that are located near the C-terminus (amino acids 198-205 and 238-244, NLS1 and NLS2 respectively). We hypothesized that nuclear localization sequences of BRMS1 were essential for BRMS1 mediated metastasis suppression. Replacement of NLS2 with NLS1 (BRMS1(NLS1,1)), truncation at 238 (BRMS1(ΔNLS2)), or switching the location of NLS1 and NLS2 (BRMS1(NLS2,1)) did not affect nuclear localization; but, replacement of NLS1 with NLS2 (BRMS1(NLS2,2)) or truncation at 197 (BRMS1(ΔNLS) which removes both NLS) promoted cytoplasmic localization. MDA-MB-231 human metastatic breast cancer cells transduced with BRMS1(NLS1,1), BRMS1(NLS2,2) or BRMS1(NLS2,1) were evaluated for metastasis suppression in an experimental xenograft mouse model. Interestingly, while NLS2 was not necessary for nuclear localization, it was found to be important for metastasis suppression since BRMS1(NLS2,2) suppressed metastasis by 85%. In contrast, BRMS1(NLS2,1) and BRMS1(NLS1,1) did not significantly suppress metastasis. Both BRMS1 and BRMS1(NLS2,2) co-immunoprecipitated with SIN3A in the nucleus and cytoplasm; however, BRMS1(NLS1,1) and BRMS1(NLS2,1) were associated with SIN3A in the nucleus only. Moreover, BRMS1 and BRMS1(NLS2,2), but not BRMS1(NLS1,1) and BRMS1(NLS2,1), down-regulated the pro-metastatic microRNA, miR-10b. Together, these data demonstrate an important role for NLS2 in the cytoplasm that is critical for metastasis suppression and is distinct from nuclear localization.

To investigate transrenal arteriovenous graft creation in an animal model and to report survival and patency.Arteriovenous grafts were created from the renal artery to the renal vein in 10 swine. Renal access was accomplished with a combined percutaneous and transvascular approach. A reinforced 6-mm polytetrafluoroethylene conduit was tunneled between both flank access points, and stent-grafts were deployed from each of the renal origins into the conduit. Clopidogrel (10 mg per kilogram of body weight) was administered intravenously during the procedure, followed by a daily oral dose (75 mg) for up to 2 weeks. Animals were monitored with auscultation and angiography for up to 1 month; necropsy was performed in all animals.Rapid arteriovenous flow at completion angiography was achieved in eight of 10 animals. Shunts were patent in five of six animals that were followed for 1 month. Diffuse pseudointimal hyperplasia was mild in three of six shunts and moderate in two (focal stenoses). Immediate thrombosis occurred in the first two animals when the clopidogrel bolus was administered after stent-graft deployment. These shunts were recanalized mechanically. Shunts were immediately patent in five of the six remaining shunts when the clopidogrel bolus was administered prior to stent-graft deployment. Complications occurred in four of 10 animals, three of which were euthanized within 1 week. The bowel was traversed in two animals, and renal vein laceration occurred during two procedures because of failure of the stent-graft delivery system.Transrenal arteriovenous graft creation in swine is technically feasible, and long-term patency is possible with antiplatelet therapy.

PURPOSE Alpha-1-antitrypsin (AAT) is the major circulating elastase inhibitor. Deficiency of elastase inhibition leads to emphysema and vascular abnormalities including accelerated neointima. Because recent evidence suggests that tissue AAT levels determine inhibitory function, the authors hypothesize that local tissue-based expression of AAT limits elastase activity sufficiently to guide arterial response to injury. MATERIALS AND METHODS Rabbit common femoral arteries were injured by mechanical overdilation and treated with buffer, viral control, or an adenovirus expressing AAT (Ad/AAT). After 3 and 28 days, intima-to-media (I/M) ratios were evaluated. Additionally, early changes in elastase inhibition potential (3 d), extracellular elastin and collagen content (3 d), and local macrophage and neutrophil infiltration (7 d) were determined. RESULTS Ad/AAT significantly decreased neointima formation after mechanical dilation injury after 28 days: buffer controls exhibited mean I/M ratios of 0.76 ± 0.06, whereas viral controls reached 0.77 ± 0.09; in contrast, Ad/AAT reduced I/M ratios to 0.44 ± 0.06. Both early elastin and collagen content were preserved in the Ad/AAT group relative to controls. The Ad/AAT group also reversed the local inflammation that characterized viral controls. CONCLUSIONS This strategy demonstrates that local increases in elastase inhibition potential promote a neointimaresistant small-caliber artery, which may offer new promise in management of patients undergoing angioplasty.

Purpose: Indirect evidence suggests that tissue plasminogen activator (tPA) either limits or does not alter restenosis. However, tPA enhances tumor invasiveness through matrix remodeling, and several elements of degraded matrix enhance smooth muscle cell mitogenesis. We use either local adenoviral-mediated overexpression of tPA or systemic infusion of recombinant tPA combined with mechanical overdilation of rabbit common femoral arteries to evaluate the impact of tPA on neointima formation. Methods: Left common femoral arteries of New Zealand white rabbits were transfected in situ either with an adenoviral-construct-expressing tPA or a viral control (adenoviral-construct-expressing β-galactosidase) or nonviral (buffer) control after balloon angioplasty injury. At 7 and 28 days, left common femoral artery segments were harvested (n = 4 for each group and time point). Vessel segments were examined for intimato-media ratio, smooth muscle cell proliferation, extracellular matrix, and inflammatory response. Thrombus formation was evaluated after 3 days (n = 3 for each group). In a second experiment, New Zealand white rabbits (n = 3 per group, per time point) underwent mechanical dilation followed by buffer treatment or systemic tPA infusion according to a widely clinically used accelerated infusion protocol. Treated artery segments were harvested after 7 or 28 days and processed for intima-to-media ratio determination and class-wide histochemical determination of collagenous extracellular matrix and collagen content. Results: Both rate and degree of neointima formation increase dramatically with overexpression (250%-461% relative to controls at 7 and 28 days). Substantial early matrix degradation is observed in vessels treated with local overexpression of tPA, although no increases in local inflammation or in smooth muscle proliferation occur. Late enhancement of smooth muscle proliferation emerges, consistent with secondary impact of perturbed matrix components. Systemic infusion of tPA according to clinical protocols also results in early and late enhancement of neointima formation in this model (34%-52% relative to controls at at 7 and 28 days), with significant early collagenous matrix degradation. Systemic infusion, although significant, did not attain the degree of neointima formation present with overexpression. Conclusion: With some evidence of dose-dependence, tissue plasminogen activator enhances neointima formation after angioplasty in a rabbit model. Early matrix degradation precedes change in rates of proliferation and underlies this effect in spite of several antirestenotic actions including decreased thrombus and decreased macrophage recruitment in this model. (J Vasc Surg 2001;33:821-8.)

The main focus of the present study was to define the rotational response of 6-hydroxydopamine-lesioned rats to dopaminergic agonists to separate the partially lesioned rats from those having complete substantia nigra (SN) and ventral tegmental area (VTA) lesions. Animals were challenged by amphetamine and L-DOPA for 10 consecutive weeks. There was a correlation between rotational behavior and extent of midbrain cell loss. Rats with complete SN and

Guanine nucleotides have been examined for their effect on [ 3 H]-[1-(2-thienyl)-cyclohexyl]-piperidine ([ 3 H]TCP) binding to rat forebrain synaptic plasma membranes (SPM). We report that of the series of guanine nucleotides tested, GTP, GDP, 5′-guanylylimidodiphosphate (Gpp(NH)p) and 5′-guanylylmethylenediphosphate (Gpp(CH 2 )p) are significantly more potent at decreasing [ 3 H]TCA binding than GMP, cyclic GMP, and guanosine. GTP, the most potent compound tested, inhibited basal [ 3 H]TCP binding with an IC 50 of 38.7 μM. Stimulation of [ 3 H]TCP binding with either the N-methyl-D-aspartate (NMDA) agonist, L-glutamate, or Mg 2+ was also inhibited by GTP. Addition of GTP resulted in a rightward shift in the glutamate dose-response curve and a decrease in the maximum level of stimulation. The Mg 2+ stimulation of [ 3 H]TCP binding was completely blocked by the addition of GTP. These results, coupled with the previous findings that guanine nucleotides inhibit the binding of L-[ 3 H]glutamate to the NMDA recognition site (Monahan et al., 1988), indicate that guanine nucleotides antagonize NMDA receptor-mediated neurotransmission, at least in part, through their action (direct or indirect) on the NMDA recognition site and thus may be endogenous negative modulators of the NMDA receptor.

A single phage was isolated from a λ gt11 rat brain cDNA library by screening with antibodies prepared against rat renal glutaminase. Partial proteolysis of the fusion protein produced by a lysogen of the isolated phage generated a series of immunoreactive peptides that co-migrated with those derived from the purified brain glutaminase. The cDNA has a single open reading frame which encodes 326 amino acids that are in frame with β-galactosidase. A 72-kDa protein, corresponding in size to the precursor of mitochondrial glutaminase, was immunoprecipitated from the translation products of rat renal mRNA that selectively hybridized to the cDNA. A probe made from the glutaminase cDNA detected an mRNA about 6 kb in length. This mRNA was present in rat brain and normal kidney RNA, increased 6-fold in acidotic kidney RNA, but was not detectable in liver RNA.

Radiation inactivation was used to estimate the molecular weight of the benzodiazepine (BZ), γ-amino-butyric acid (GABA), and associated chloride ionophore (picrotoxinin/barbiturate) binding sites in frozen membranes prepared from rat forebrain. The target size of the BZ recognition site (as defined by the binding of the agonists [3H]diazepam and [3H]flunitrazepam, the antagonists [3H]Ro 15–1788 and [3H]CGS 8216, and the inverse agonist [3H]ethyl-β-carboline-3-carboxylate) averaged 51,000 × 2,000 daltons. The presence or absence of GABA during irradiation had no effect on the target size of the BZ recognition site. The apparent molecular weight of the GABA binding site labelled with [3H]muscimol was identical to the BZ receptor when determined under identical assay conditions. However the target size of the picrotoxinin/barbiturate binding site labelled with the cage convulsant [35S]t-butylbicyclophosphorothionate was about threefold larger (138,000 daltons). The effects of lyophilization on BZ receptor binding activity and target size analysis were also determined. A decrease in the number of BZ binding sites (Bmax) was observed in the nonirradiated, lyophilized membranes compared with frozen membranes. Lyophilization of membranes prior to irradiation at – 135°C or 30°C resulted in a 53 and 151% increase, respectively, in the molecular weight (target size) estimates of the BZ recognition site when compared with frozen membrane preparations. Two enzymes were also added to the membrane preparations for subsequent target size analysis. In lyophilized preparations irradiated at 30°C, the target size for (i-galactosidase was also increased 71% when compared with frozen membrane preparations. In contrast, the target size for glucose-6-phosphate dehydrogenase was not altered by lyophilization. Thus, in some cases lyophilization may lead to an increase in protein target size, possibly due to aggregation of protein subunits. We conclude that in frozen membrane preparations the functional target size of the BZ and GABA binding sites reflects the subunit molecular weights for the BZ/GABA/chloride ionophore receptor complex.

Plasma and brain levels of the dopamine metabolite, homovanillic acid, were measured in rats after acute and chronic treatment with fluphenazine. After acute administration, homovanillic acid levels were increased in the whole frontal cortex, caudate nucleus, nucleus accumbens, anterior cingulate (supragenual) cortex, prefrontal cortex and plasma. Following chronic administration, the effects of single doses of fluphenazine in increasing homovanillic acid were markedly attenuated in the caudate nucleus, nucleus accumbens, the whole frontal cortex, anterior cigulate cortex and plasma, but not in the prefrontal (anteromedial) cortex. Following fluphenazine administration plasma homovanillic acid levels were correlated with homovanillic acid concentrations in whole frontal cortex (r = 0.63, p less than 0.01) and caudate nucleus (r = 0.51, p less than 0.05). Further, prior intracerebroventricular administration of 6-hydroxydopamine prevented the fluphenazine-induced increase in plasma homovanillic acid. These data suggest that in the rat, plasma and brain (caudate nucleus and whole frontal cortex) homovanillic acid levels change in a similar fashion during acute and chronic neuroleptic administration.

Pretreatment of rats in vivo with γ-aminobutyric acid (GABA) agonists and drugs which increase GABA levels in brain leads to an increased affinity of [3H]diazepam binding sites in rat cerebral cortex. These drugs include amino-oxyacetic acid, muscimol, γ-butyrolactone and β-(p-chlorophenyl)-GABA; pentobarbital and thiosemicarbazide are without major effect. Bicuculline and picrotoxin (at high concentrations) reverse this increase in [3H]diazepam binding site affinity.

A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N-methyl-D-aspartate receptor complex. Incubation of SPM with [3H]glycine for 10 min at 2 degrees C results in saturable, reversible binding with a KD of 0.234 microM and a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D-serine (Ki = 0.27 microM), D-alanine (Ki = 1.02 microM), and D-cycloserine (Ki = 2.33 microM) are potent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki = 25.4 microM, 15.9 microM, and greater than 100 microM, respectively). Inactive at concentrations of up to 100 microM were strychnine, L-valine, N,N-dimethylglycine, aminomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to Triton X-100-washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate [3H]TCP binding.

Glutaminase mRNA levels increased over 3-fold relative to total RNA, poly(A)+ RNA, and beta-actin mRNA in neonatal rat cerebellar granule cells as the cells differentiated between days 3 and 8 in culture. In contrast, mRNA levels of another glutamate cycle enzyme, glutamine synthetase, remained constant. Glutaminase protein levels increased per cell more than 2-fold between days 3 and 8, and at least 3-fold by day 10 in these cells. The total amount of glutamate per cell increased about 40% during this period. Glutaminase induction paralleled the development of Ca2+-dependent glutamate release, and the formation of neurites, synaptic vesicles, and synapses. The induction of glutaminase in developing granule cells is consistent with a special role for glutaminase in the synthesis of neurotransmitter glutamate.

A cDNA has been isolated from a human brain expression library using anti-bovine glutamate dehydrogenase (GDH) antibodies. The cDNA has an open reading frame of 774 nucleotides, which codes for 258 amino acids. The 258-amino-acid sequence is 95% homologous to the carboxy terminus of human liver GDH. This high degree of homology indicates that the cDNA codes for brain GDH. Fourteen differences between the amino acid sequence deduced from this cDNA and the sequence reported for human liver GDH suggest that there may be two active human GDH genes. A cRNA probe synthesized from the cDNA detects a 3.7-kilobase (kb) mRNA from human brain. Rat liver and kidney each contain two GDH mRNAs, 3.5 and 2.8 kb, respectively. The 3.5-kb transcript is prominent in rat brain, whereas the 2.8-kb transcript is barely detectable, a result suggesting that GDH gene expression is differentially controlled in rat brain.

The pathway of breakdown of membrane-bound benzodiazepine binding sites has been examined with proteolytic enzymes. Photoaffinity labeled benzodiazepine receptors were degraded for varying amounts of time and at varying enzyme concentrations. The properties of fractions both remaining in the membrane and released into the supernatant were examined for their apparent molecular weight by SDS gel electrophoresis. Trypsin treatment converted the 46K subunits of the GABA/BDZ complex which bind 3H-flunitrazepam into 40K and 27.5K fragments which remained in the membrane and finally a small fragment which was released into the supernatant. An endogenous trypsin-like activity in the membrane fractions has similar proteolytic effects on the membrane bound receptor.

ALTHOUGH neurophysiological, behavioural and biochemical evidence suggests that benzodiazepines (BZ) may affect several neuronal systems within the central nervous system (CNS), recent studies indicate that some of these effects result from a specific interaction with GABAergic transmission1. The precise mechanism of this interaction remains in doubt, as facilitation of GABAergic transmission2, potentiation of GABAergic inhibition3,4, direct activation of γ-aminobutyric acid (GABA) receptors5, and antagonism of GABA-mediated inhibition have all been attributed to the benzodiazepines6,7. In studies from this laboratory8, both the systemic and iontophoretic administration of BZs potentiated an inhibitory response produced by GABA in the dorsal raphe nucleus—suggesting that the potentiation was mediated through a change in the post-synaptic receptor. In addition, direct binding studies using 3H-diazepam have indicated a specific high affinity binding site which may be relevant to the pharmacological actions of BZ in brain9. In this study, we show that GABA can modulate the responsiveness of this BZ binding site since the addition of GABA to cortical membranes in vitro results in an increased affinity of the 3H-diazepam binding site for its ligand. This effect is mimicked by the GABA analogue, muscimol10, and antagonised by the GABA antagonist, (+)bicuculline.

Neurophysiological studies have shown that glycine potentiates the NMDA response in cultured neurons by a strychnine-insensitive mechanism. Autoradiographic data have demonstrated a correspondence between strychnine-insensitive [3H]glycine binding sites and NMDA-sensitive [3H]glutamate binding sites. Here we report that in synaptic plasma membranes from rat brain, the binding of a PCP analog, [3H]TCP, was enhanced more than 5-fold by 1 microM glycine. This glycine stimulation of binding of [3H]TCP was blocked by the competitive NMDA-receptor antagonist, D-AP7. These data provide support for the hypothesis that a unique amino acid recognition site is associated with the proposed NMDA/PCP receptor complex in brain.

An in vivo method for labeling specific benzodiazepine (BDZ) binding sites in brain was developed using intravenously injected [3H]diazepam. Labeling of these sites is blocked by pretreatment of animals with high doses of pharmacologically active BDZs (but not by an inactive BDZ). Using this in vivo binding technique, specific BDZ binding is enhanced by pretreatment of rats with the GABA agonist muscimol or with amino-oxyacetic acid, which increases GABA levels in brain.

The high-affinity binding site for [3H] diazepam has been solubilized from rat brain using 0.5% Lubrol-PX. Using a polyethylene glycol (PEG)-γ-globulin assay, it has been possible to demonstrate solubilization of about 60% of the binding sites in a single step. The solubilized binding site possesses a KD of 11 nM for [3H] diazepam compared to approximately 4 nM for the membrane-bound form, and binding is to a single class of sites. The order of potency of benzodiazepines is identical for the solubilized receptor and the membrane-bound form. Binding of [3H] diazepam is temperature dependent and higher at 4° than 37°C. Both urea and guanidine-HC1 were capable of totally inhibiting binding, and this inhibition was partly reversible; neither sulfhydryl groups nor carbohydrate moieties seem to be important for binding. γ-Aminobutyric acid which enhanced [3H] diazepam binding to membrane fractions was without effect on the solubilized binding site.

We studied the regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486 [11 beta-(4-dimethylaminophenyl)17 beta-hydroxy-17 alpha-(prop-1-ynyl)estra-4,9-dien-3-one], a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Increased enzyme activity was specific for glucocorticoids; other steroid hormones were essentially without effect. The induction of glutamine synthetase was selective, in that glutaminase activity was not induced by dexamethasone treatment of L6 cells. Thus, glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

Glutamic acid decarboxylase (GAD) mRNA was quantified in different regions of rat brain using an antisense RNA probe (ribo-probe) prepared from a cloned feline cDNA. In all brain regions studied a single band of GAD mRNA of approximately 3.7 kb was detected. The level of GAD mRNA was highest in the cerebellum, followed by the hypothalamus greater than thalamus greater than striatum greater than hippocampus greater than frontal cortex = parietal cortex greater than or equal to medulla = pons. Since GAD has been previously localized to intrinsic neurons of the striatum, we examined the effects of intrastriatal kainic acid administration on striatal GAD mRNA. The level of GAD mRNA in the kainic acid-lesioned striatum was reduced by 70-75% when compared to the contralateral (unlesioned) striatum. In contrast, the level of glutamine synthetase (an enzyme localized to glia) mRNA was increased approximately 290% in the kainic acid-lesioned striatum. There were no significant differences in GAD mRNA levels between the ipsilateral and contralateral cerebral cortices and hippocampi of rats injected with intrastriatal kainic acid.

IT HAS been a common clinical practice to "reperitonealize" areas of injury to the visceral peritoneum in order to prevent the formation of adhesions between such areas and omentum, intestine or other intraabdominal structures. The usual method of doing this for serosal injuries to the bowel is to draw adjacent uninjured serosa together over the area of injury with interrupted sutures of a fine nonabsorbable material, such as 0000 silk, taking delicate bites of tissue with a small round-pointed needle. Experiments carried out by Chandy in this laboratory have indicated that silk itself commonly becomes encapsulated by omentum and that the usual seromuscular stitch tied in place would, of itself, produce an adhesion in the experimental animal (rat). EXPERIMENTAL STUDIES The following experiments were carried out to see if the incidence of adhesions resulting from areas of bowel denuded of serosa was reduced or increased by oversewing. The animals used