150

Intravital Imaging Window For Visualization Of An Orthotopic Tumor Microenvironment

Dmitriy Kedrin, MS, Albert Einstein College of Medicine, Bronx, NY, USA; Jacco van Rheenen, PhD, Hubrecht Institute, Utrecht, NA, Netherlands; Bojana Gligorijevic, PhD, Albert Einstein College of Medicine, Bronx, NY, USA; Jeffrey Wyckoff, MFA, Albert Einstein College of Medicine, Bronx, NY, USA; John Condeelis, PhD, Albert Einstein College of Medicine, Bronx, NY, USA; Jeffrey Segall, PhD, Albert Einstein College of Medicine, Bronx, NY, USA

The various aspects of in vivo tumor cell behavior have been widely studied using a number of well established techniques. Tumor cell invasion, motility and intravasation have been linked with metastatic propensity of a variety of tumor models. Intravital imaging approaches allow for qualitative and quantitative direct observation of tumor cell behavior and interaction with various stromal components, including macrophages and extracellular matrix fibers. However, due to a number of technical difficulties, current approaches lack spatial and temporal resolution. Here, we present a technique for in vivo imaging of mammary gland tumors which allows for high resolution imaging of tumor cell behavior, including protrusion, invasion and movement towards and into the blood vessels that are part of the tumor microenvironment, as well as interaction with macrophages and ECM components. Not only does this technique allows us to obtain subcellular resolution temporal images of the tumor microenvironment, but we are able to image the same tumor using different microscope modalities, including epifluorescence, confocal and multiphoton microscopy. The use of this technique demonstrated the existance of heterogenious microenvironments in the primary tumor,which support invasion and intravasation of tumor cells.

151

Molecular Analyses of Deafferentation-Induced Cell Death in Chick Cochlear Nucleus Neurons

Hope Nicely, BS, University of Kansas Medical Center, Kansas City, KS, USA; Dianne Durham, PhD, University of Kansas Medical Center, Kansas City, KS, USA

By age 75, more than 30% of Americans will suffer from hearing loss. Hearing loss is a significant and costly public health concern, which is growing in magnitude as the population ages. Hearing loss occurs from damage to inner ear or brainstem structures of the auditory system. Current technologies to restore hearing, like hearing aids and cochlear implants, have advanced the therapeutic options to treat the disorder, but their focus is limited to inner ear structures. The central nervous system (CNS) component is equally as important as the ear component in preserving auditory function, but little is known about the process of auditory neuronal damage and recovery. Here, we evaluate the CNS response to disruptions in auditory signaling. Specifically, we use the chick auditory system to study changes in auditory neurons after loss of afferent support. Neurons in the avian cochlear nucleus, nucleus magnocellularis (NM) receive exclusive excitatory input from the ipsilateral cochlea. Cochlea removal permanently abolishes this input, and NM neurons undergo rapid ultrastructural, metabolic, and functional changes; 30-50% of cells ultimately die. NM neurons destined to die exhibit early indications of cell compromise, including disassembly of ribosomes and degradation of rough endoplasmic reticulum. As a consequence, these "ghost" neurons stain poorly with Thionin and are readily distinguishable from the NM neurons destined to survive deafferentation. Demonstration of caspase up-regulation suggested that the abrupt cell death that occurs following deafferentation is apoptotic in nature. Paradoxically, caspase-9 and caspase-3 are observed throughout deafferented NM, in both degrading and surviving neurons. To better characterize the molecular determinants of cell fate in deafferented NM, we investigated a late marker of apoptosis, loss of nuclear DNA integrity, using a TUNEL assay. Young broiler birds underwent unilateral cochlea removal and were sacrificed 12 hours to 7 days later. Nuclear TUNEL labeling appears at 12 hours, consistent with apoptosis-mediated DNA fragmentation. Interestingly, an unusual pattern of diffuse, cytoplasmic TUNEL labeling emerges 24 hours after deafferentation. Cytoplasmic TUNEL labeling appears to coincide with the number and pattern of "ghost" neurons that also contain abnormal mitochondria. To examine genetic contributions to cell fate, we performed laser capture microdissection (LCM) on Thionin-stained sections and microdissected three populations for differential gene expression analyses: 1) deafferented NM "ghost" neurons that stained poorly with Thionin; 2) deafferented, surviving NM neurons that stained intensely for Thionin; 3) and healthy control neurons from contralateral NM. From each population, RNA was extracted, analyzed for integrity, and hybridized to PCR expression arrays containing probes for apoptosis-related genes. Differential gene expression was compared across populations.

152

Molecular Dependence Of Estrogen Receptor Negative Breast Cancer To A Notch-Survivin Signaling Axis

Connie Lee, MS, University of Massachusetts Medical School, Worcester, MA, USA; Igor Prudovsky, PhD, Maine Medical Center Research Institute, Scarborough, ME, USA; Dario Altieri, MD, University of Massachusetts Medical School, Worcester, MA, USA

Despite progress in the management of breast cancer, the molecular underpinnings of clinically aggressive subtypes of the disease are not well understood.  Here, activation of Notch developmental signaling in estrogen receptor (ER) negative breast cancer cells resulted in transcriptional upregulation of the apoptosis inhibitor and cell cycle regulator, survivin.  This was associated with increased expression of survivin at mitosis, enhanced cell proliferation and heightened viability at cell division.  Conversely, targeting Notch signaling with peptidyl -secretase inhibitors suppressed survivin levels, induced apoptosis, abolished colony formation in soft agar, and inhibited localized and metastatic tumor growth in mice, without organ or systemic toxicity.  In contrast, ER+ breast cancer subtypes or normal cells were insensitive to Notch inhibition.  Therefore, ER- breast cancer cells become dependent on Notch-survivin signaling for their maintenance, in vivo.  Therapeutic targeting of this pathway may be explored for individualized treatment of clinically aggressive, ER- breast cancer patients.

153

Identification and Characterization of Potential Inhibitors of hsFATP2-mediated Fatty Acid Uptake

Aalap Chokshi, BS, Albany Medical College, Albany, NY, USA; Angel Sandoval, PhD, Ordway Research Institute, Albany, NY, USA; Concetta DiRusso, PhD, Ordway Research Institute, Albany, NY, USA

Obesity is a complex syndrome that results from a disturbance in energy balance: an increased caloric intake and decreased physical activity. Obesity correlates with the prevalence of health conditions and diseases such as metabolic syndrome, dyslipidemias, type 2 diabetes, heart disease, stroke, respiratory diseases, and cancer. Current drugs suppress appetite or decrease fat digestion by limiting absorption in the intestine. However, several side effects have been reported and neither drug has resulted in sustained weight reductions. In order to address this need for safe pharmaceuticals targeted to fat accumulation, a library of 100,000 small compounds were screened to find potential inhibitors of hsFATP2, the primary fatty acid transporter in humans. 224 compounds were selected and retested for the transport of the fluorescent fatty acid analogue C1-BODIPY-C12 into yeast humanized with cloned hsFATP2. Of these, 8 compounds were eliminated due to quenching, 14 compounds were eliminated due to permeabilization, and 32 were grouped by structure-activity relationships. 21 compounds of diverse structure were chosen for generation of graded log-dose-response curves for the transport of C1-BODIPY-C12 into the humanized yeast and Caco-2 cells, a human intestinal epithelial cell line. These studies resulted in the identification of 8 compounds, which specifically inhibit uptake of the fatty acid analogue without toxic side effects. These compounds will now be used as the basis to develop an expanded set of rationally designed compound derivatives to improve efficacy and potency and to further evaluate specificity of the compound toward the transporter.

154

The Role of Pin1 in NF-?B Regulated Gene Expression

George Atkinson, BS, Department of Cell Biology, Birmingham, AL, USA; Etty Benveniste, PhD, Department of Cell Biology, Birmingham, AL, USA; Susan Nozell, PhD, Department of Cell Biology, Birmingham, AL, USA; Daniel Harrison, MD, Department of Pathology, Birmingham, AL, USA; Dongquan Chen, PhD, Department of Medicine, Birmingham, AL, USA; Mark Stonecypher, MD/PhD, Department of Cell Biology, Birmingham, AL, USA

Glioblastoma (GBM) is the most frequent and malignant primary brain tumor in adults.  While new therapeutic regimens have marginally improved overall patient lifespans, the lethality and aggressive nature of GBM warrants further investigation into its underlying biology.  The mammalian NF-κB family includes five members: p65 (RelA), RelB, c-Rel, p50/p105 and p52/p100.  The NF-κB pathway, particularly p65, is aberrantly activated in GBMs.  A protein that has been shown to play a regulatory role in the NF-κB pathway is peptidyl-prolyl isomerase (PPIase) Pin1.  Pin1 is the only member of the PPIase family that has been shown to preferentially bind phosphorylated serine or threonine residues immediately preceding proline residues (pSer/Thr-Pro) and promote isomerization of this bond in target proteins.  It has also been demonstrated previously that Pin1 directly interacts with p65.  We hypothesize that Pin1 retains activated p65 in the nucleus, fostering increases in NF-κB signaling and corresponding increases in the expression of NF-κB-regulated genes.

Our primary model for this study is a modified human glioma U251-MG cell line capable of producing Tet-inducible shRNA that specifically targets Pin1 mRNA.  In support of our hypothesis, we have determined that Pin1 knockdown reduces the amount of active p65 in the nucleus after TNFα stimulation.  However, in contrast to a previous report, total levels of nuclear p65 appear unaffected by the absence or presence of Pin1.  Through microarray and qRT-PCR analysis, we have identified a subset of NF-κB regulated genes whose expression is inhibited in Pin1 knockdown cells, including IL-8 and IP-10.  Interestingly, we have also found a number of NF-κB target genes that show enhanced expression in Pin1 knockdown cells.  Preliminary chromatin immunoprecipitation (ChIP) experiments suggest that Pin1 is binding at the promoters of some of these genes, suggesting a possible role as a transcriptional cofactor.  Future studies will include the full characterization of the IL-8 promoter, using ChIP to determine how Pin1 affects the transcriptional program of this gene.  We also plan on studying the role of Pin1 in apoptosis and migration in our system.

155

Ceramide Activates Volume-sensitive Chloride Current in Rabbit Ventricular Myocytes

Frank Raucci, MS, Virginia Commonwealth University, Richmond, VA, USA; Clive Baumgarten, PhD, Virginia Commonwealth University, Richmond, VA, USA

A volume-sensitive chloride current (ICl,swell) is present in cardiac myocytes of several species, including humans.  This current is due to activation of a class of ion channels that responds to increases in cell volume or mechanical stretch.  Activation of ICl,swell leads to action potential duration shortening, depolarization of resting membrane potential, and ultimately modulation of cell volume.  In models of congestive heart failure (CHF), ICl,swell is chronically activated.   Sphingolipids are known to accumulate in many forms of cardiovascular disease, including CHF and acute myocardial infarction, and are involved in numerous processes including apoptosis and mitogenesis.  The sphingolipid ceramide recently was implicated in the regulation of ion channels, including the cardiac isoform of the cystic fibrosis transmembrane regulator (CFTR) chloride current. Using whole-cell patch clamp techniques, we explored the hypothesis that ceramide is involved in signaling pathways that modulate the activity of ICl,swell in rabbit ventricular myocytes.  Isosmotic bathing solutions designed to isolate anion currents were used.  Using each myocyte as its own control, addition of 2 μM C2-ceramide to the bathing solution increased the chloride current density 181 ± 17% (n = 10, p <0.01) after 7-15 min of exposure.  Such activation does not appear to occur with the inactive analogue dihydro-C2-ceramide (n = 2).  DCPIB (4-(2-butyl-6,7-dichloro-2-cyclo -pentylindan-1- on-5-yl) oxybutyric acid) is a specific blocker of ICl,swell.  When 10 μM DCPIB was added in the continuous presence of C2-ceramide, activation of ICl,swell was diminished by 76 ± 8% after 7-12 min (n = 6, p <0.01)  The current density in the presence of DCPIB plus C2-ceramide was not statistically different from that under control conditions.   These data indicate that there is a ceramide-sensitive component of ICl,swell.  Given that ceramide accumulation has been observed in CHF patients and that ICl,swell is persistently activated in similar patients, it is reasonable to postulate that ceramide may play a role in the regulation of ICl,swell in cardiovascular disease states.

156

KATP-Mediated Vasodilation is Impaired in Obese Zucker Rats

Benjamin Hodnett, BS, University of Mississippi Medical Center, Jackson, MS, USA; Lusha Xiang, MD, University of Mississippi Medical Center, Jackson, MS, USA; Jennifer Dearman, BS, University of Mississippi Medical Center, Jackson, MS, USA; Cory Carter, BS, University of Mississippi Medical Center, Jackson, MS, USA; Robert Hester, Ph.D., University of Mississippi Medical Center, Jackson, MS, USA

Background and Objective: One of the predominant mechanisms of skeletal muscle blood flow control under both resting and active states is the release of endothelium-derived factors to dilate arterioles, leading to an overall increase in blood flow (functional hyperemia). The arachidonic acid metabolite prostacyclin (PGI2) is a vasodilatory prostaglandin released by endothelial cells and is considered to be a regulator of skeletal muscle blood flow control. PGI2 results in a vasodilation through stimulation of the PGI2 receptor (IP) and activation of potassium channels including ATP-sensitive potassium channels (KATP). Stimulation of the IP receptor using the PGI2 analog iloprost in the obese Zucker rat (OZR) has been shown to be attenuated both in isolated arterioles from gracilis muscles and in vivo preparations of the spinotrapezius muscle. The mechanisms underlying the impaired IP receptor-mediated vasodilation in the OZR are unclear. One possible mechanism underlying this impaired vasodilation is an alteration in the downstream effector mechanisms activated through IP receptor signaling mechanisms, i.e. KATP channels. The objective of this study was to test the hypothesis that the attenuated vasodilation in skeletal muscle arterioles of OZR is due to altered KATP channel-mediated vasodilation.

Methods: The KATP channel function was determined using isolated skeletal muscle (gracilis) arterioles in response to the KATP channel opener cromakalim (0.1 – 10 μM), both under normal myogenic tone and enhanced α-adrenergic-mediated tone (0.1 μM phenylephrine). The right spinotrapezius muscle was prepared and the vasodilatory responses to muscle stimulation or iloprost (0.028 μM – 2.8 μM) were observed before and after the application of the KATP channel inhibitor glibenclamide (10 μM). KATP channel subunit expression was determined using western blot analyses.

Results: Cromakalim concentration response curves were shifted right in OZR as compared to lean controls (P < 0.001 for both myogenic and enhanced tone). OZR exhibited an impaired functional (22 ± 1 μm vs. 26 ± 1 μm) and iloprost-induced vasodilation (32 ± 4 μm vs. 41 ± 3 μm; max conc.) as compared to the lean controls. Glibenclamide inhibited the functional and iloprost-induced dilation in the lean rats (P = 0.013; P < 0.001, respectively) with no effects in the obese animals. KATP channel subunit expression was similar in the femoral arteries.

Conclusion: These results suggest that the impaired functional hyperemia in the OZR may be due to alterations in the function of the KATP channels that mediate PGI2-mediated vasodilation. Furthermore, the mechanism underlying the impaired KATP channel response is apparently not due to a decrease in channel number but rather due to decreased channel sensitivity.

157

In Vitro Expanded Living-Skin Matrix: A Potential Source of Autologous Tissue for Reconstructive Procedures

Mitchell Ladd, BS, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USA; Sang Jin Lee, PhD, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USA; Anthony Atala, MD, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USA; James Yoo, MD, PhD, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, USA

     Numerous reconstructive procedures result in wounds that require skin grafting. However, the amount of autologous tissue available from donor sites is often limited and large wounds remain difficult to manage [1]. In vivo tissue expanders have been used clinically to generate larger autologous skin. However, this method requires an additional surgical procedure for expander implantation and a long waiting time to obtain sufficient tissue for reconstruction. Moreover, discomfort associated with the increasing expander volume and frequent tissue fibrosis are among the limitations [2]. In this study we investigated whether these limitations could be solved by increasing the surface area of skin tissue in vitro while maintaining tissue viability.  

     Human foreskin was incrementally expanded over 6 days to increase its surface dimensions in a computer-controlled bioreactor system under tissue culture conditions. Morphological, structural and mechanical properties of the foreskin were evaluated before and after expansion using various analytical methods. Tensile testing was performed at a strain rate of 10.6 mm/min[3] as cited in the literature. 

The surface area of the tissue was increased by 83% with the maintenance of cell viability and proliferative potential. Histomorphological and ultrastructural analyses showed that dermal structural integrity was preserved. The mechanical properties of the expanded skin tissue remained similar to those of the initial state.

     These findings show that expansion of living skin can be achieved using a bioreactor system. This technique provides an opportunity to generate large amounts of skin for reconstructive procedures and may overcome the current limitations of the in vivo tissue expander system.

References:

Atiyeh, BS et. al. New technologies for burn wound closure and healing-review of the literature. Burns. 2005. 31(8): p. 944-956.

LoGiudice J, Gosain AK. Pediatric tissue expansion: indications and complications. J Craniofac Surg. 2003 Nov;14(6):866-72.

Edsberg, LE et. al. Mechanical characteristics of human skin subjected to static versus cyclic normal pressures. J Rehabil Res Dev. 1999. 36(2): p. 133-141.

158

Multiple Plasma Biomarkers of Oxidative Stress Predict Risk of Cardiovascular Mortality

Claire Heslop, HBMSc, University of British Columbia, Vancouver, BC, Canada; Jiri Frohlich, MD, University of British Columbia, vancouver, BC, Canada; John Hill, PhD, University of British Columbia, vancouver, BC, Canada

Background: Reactive oxygen species mediate the signalling of inflammatory pathways throughout all stages of atherosclerosis, from endothelial dysfunction to plaque rupture. Activated leukocytes within atherosclerotic lesions produce reactive oxygen species, causing local and systemic oxidative injury; however, contributions of oxidative stress to the clinical features of atherosclerosis have not yet been clearly established. Biomarkers of oxidative stress associated with presence of coronary artery disease (CAD) include myeloperoxidase (MPO), nitrotyrosine (N–tyr), and oxidized LDL (oxLDL). Although these markers have been found within atherosclerotic plaques—where they directly contribute to atherogenesis—their relative or additive abilities for predicting outcomes in stable CAD have not yet been determined.

Study Design: We tested the hypothesis that plasma levels of oxidative stress biomarkers MPO, N–tyr, and oxLDL, and plasma total antioxidant potential (AOP), predict cardiovascular mortality in patients with CAD. Biomarkers were measured using commercial enzyme–linked immunoassays in a cohort of 1117 coronary angiography patients. After a mean follow–up time of 8.5 years, 233 deaths were observed, of which 109 were cardiovascular disease (CVD)–deaths. The relative value of oxidative stress biomarkers for prediction of CVD mortality was determined with adjustment for: age, sex, smoking status, hypertension, diabetes, BMI, LDL–cholesterol, total:HDL–cholesterol, C-reactive protein, and family history of CAD.

Interpretation: Patients whose plasma MPO levels were in the highest tertile had a higher risk of CVD mortality [Adjusted HR(95%CI):2.04(1.02–4.10) p=0.04]. Plasma AOP also predicted CAD presence and risk of CVD mortality. Non–significant trends were observed between elevated N-tyr and oxLDL and CAD, and between N-tyr and risk of CVD–death. Individuals with ≥2 biomarkers in the highest tertile had a 2–fold higher risk of CVD–death [2.03(1.03–4.41) p=0.04], and patients with 3 biomarkers in the highest tertile had a 3–fold higher risk [2.97(1.06–8.34) p=0.04]. While oxidative stress biomarkers associate with long-term risk of CVD mortality, further studies are warranted to establish the clinical prognostic value of these biomarkers.

159

ACE2 Gene Therapy Improves Glycemic Control in Diabetic Mice.

Sharell Bindom, BS, LSU Health Sciences Center, New Orleans, New Orleans , LA, USA; Eric Lazartigues, PhD, LSU Health Sciences Center, New Orleans, New Orleans , LA, USA; Huijing Xia, PhD, LSU Health Sciences Center, New Orleans, New Orleans , LA, USA; Eric Lazartigues, PhD, LSU Health Sciences Center, New Orleans, New Orleans , LA, USA

Angiotensin converting enzyme (ACE) inhibitor therapy has been shown to prevent new onset diabetes and pancreatic beta cell dysfunction. ACE2, a new member of the renin-angiotensin system (RAS), counteracts Angiotensin (Ang) II signaling cascades by cleaving Ang-II to generate Ang-(1-7). Thus, we hypothesized that ACE2 would ameliorate glycemia in diabetic mice. Diabetic obese mice (db/db) and control lean littermates (db/m) were infected with human ACE2 (hACE2)or control (GFP) adenovirus, (2x106 pfu, 100μL) in the pancreas. After 7d, fasting blood glucose (12hrs) and glucose tolerance (GT, 2g/kg IP) were measured as indicies of glycemic control. ACE2 immunostaining (arbitrary units) revealed high expression of hACE2 in the pancreas of infected ice with Ad-hACe2 vs uninfected (16.7+/- 5 vs. 0.55 +/-1, n=11, p<0.01). Body weight g in db/db mice was not affected by Ad-ACE2 (35+/-1, n=5) or Ad-GRP (36+/-1, n=5) delivery. Baseline fasting blood glucose was significantly higher in db/db vs db/m mice (246+/-18 vs 102+/-6 p<0.01) and was dramatically reduced in Ad-ACE2 vs Ad-GFP treated db/db mice (157+/-12 vs. 203+/-6, n=5, p<0.05). Ad-ACE2 had no effect on fasting blood glucose in db/m mice (98.2+/-8, n=10) compared to Ad-GFP treated mice (75+/-10). In addition, Ad-ACE2 infection improved IPGT in db/db mice compared to GFP treated mice (area under the curve 51.1+/-7 vs. 73.3+/-2, n=5, p<0.05). These data confirm the role of ACE2 in diabetes and suggest that ACE2 gene therapay can ameliorate glucose homeostasis in diabetic mice. (NIH NS052479 and RR018766)

160

Cystic Fibrosis: In Utero Gene Replacement Therapy Using Chitosan-DNA Nanoparticles and C31 Integrase

Patrick Yang, Bachelor of Science, University of British Columbia, Vancouver, BC, Canada; Erik Skarsgard, MD, University of British Columbia, Vancouver, BC, Canada; William Jia, PhD, University of British Columbia, Vancouver, BC, Canada

In utero gene replacement is a novel treatment paradigm for Cystic Fibrosis and other monogenic disorders diagnosed in the fetus. Traditional viral vector-mediated gene delivery carries the risks of endogenous virus recombination and random insertion into the genome, resulting in insertional oncogenesis. To increase therapeutic safety, non-viral gene delivery systems using complex polymers combined with phage recombinase genes (integrase) which lead to site-specific transgene insertion are currently under investigation.

The purposes of our studies are: 1) to use a non-viral vector (chitosan nanoparticles) containing reporter gene Green Fluorescent Protein (GFP) to perform gene transfer to HEK 293T cells in vitro;   2) to perform in utero GFP gene delivery via intra-amniotic injection of chitosan-pEGFP construct combined with ?C31 phage integrase (p?C31); and 3) to analyze post-natal pup tissues and maternal tissues for transgene presence and expression.

Methods: 5´105 HEK293T cells were transfected with either chitosan-pEGFP or naked pEGFP with a total of 6.5mg DNA per well (using chitosan and saline controls). GFP positive cells were visualized after 48 hours using direct fluorescence microscopy.

Time-mated CD-1 pregnant dams underwent laparotomy on gestational day 17 (term=21d), and individual amniotic sacs were injected with chitosan-pEGFP-p?C31 (12.5ug DNA). Following spontaneous delivery, pups and dams were sacrificed on post-natal day 1. All tissues were examined for GFP transgene presence and expression by DNA PCR, qRT-PCR, and whole mount fluorescent microscopy.

Results: Transfection of HEK293T cells was seen with the chitosan-pEGFP constructs, suggesting that chitosan is functional as a vector to deliver genes into cells in culture. In vivo transfection of fetal mice via intra-amniotic injections of chitosan-GFP-integrase resulted in GFP integration and expression in lungs and intestines (target organs for Cystic Fibrosis) as confirmed by DNA PCR, qRT-PCR, and direct fluorescent microscopy. Maternal mice were negative for DNA transfection by PCR.

Conclusions: Chitosan appears to be an effective vector for gene delivery both in vitro and to fetal mice in utero.  The non-viral Chitosan-?C31 integrase vector shows promise as a safe and effective transgene delivery system for the purpose of in utero gene replacement therapy for Cystic Fibrosis. 

161

Growth Hormone-Releasing Peptide-2 Administration to Dyslipidemic Mice

Jane Titterington, BA, Tulane University School of Medicine, New Orleans, LA, USA; Cyril Bowers, MD, Tulane University School of Medicine, New Orleans, LA, USA; Patrick Delafontaine, MD, Tulane University School of Medicine, New Orleans, LA, USA

Purpose of Study:  Growth Hormone-Releasing Peptide-2 (GHRP-2) is a synthetic peptide that increases circulating Growth Hormone and Insulin-Like Growth Factor-1 (IGF-1) levels.  It also binds to CD36, a scavenger receptor for oxidized low-density lipoprotein (OxLDL), and may prevent cellular uptake of this pro-atherogenic lipoprotein complex.  This study was conducted to determine whether GHRP-2 would be a suitable anti-atherogenic therapy in vivo. 

Methods: GHRP-2 was given subcutaneously to Apolipoprotein-E knockout mice, a hypercholesterolemic animal model of atherosclerosis. Male ApoE knockout mice were fed a high fat, high cholesterol diet beginning at 9 weeks of age. During this time and throughout the 12 week duration of the study, they were assigned to one of four treatment groups: 10 mcg GHRP-2 once per day, Phosphate Buffered Saline (PBS) once per day, 20 mcg GHRP-2 twice per day, or PBS twice per day.

Summary of Results: GHRP-2 administration lead to an increase in weight gain without increased food intake. GHRP-2 treated mice also had higher circulating IGF-1 levels. GHRP-2 treated mice had a small but statistically insignificant reduction in urine 8-isoprostane levels, a marker for systemic oxidative stress. GHRP-2 treated mice did not have any significant changes in blood pressure or heart rate. GHRP-2 did not change the atherosclerotic plaque area in either aortas stained en face with Oil Red O or in cross sections at the aortic sinus.

Conclusions: Subcutaneous delivery of GHRP-2 did not decrease atherosclerosis in ApoE knockout mice. This lack of efficacy in vivo may be due to drug delivery issues and unfavorable pharmacokinetics or a pharmacodynamic inability to adequately increase IGF-1 levels and/or block CD36-mediated OxLDL uptake. Future experiments will determine if GHRP-2 administration altered inflammatory or oxidative mediators of atherogenesis in ApoE KO mice.

162

Neurotoxicity of Nigrostriatal Dopamine Neurons is Attenuated in Cannabinoid Receptor Deficient Mice

Kelly Janis, BS, Michigan State University, East Lansing, MI, USA; Keith Lookingland, PHD, Michigan State University, East Lansing, MI, USA; John Goudreau, DO, PHD, Michigan State University, East Lansing, MI, USA

Parkinson’s disease (PD) results from the progressive loss of nigrostriatal dopamine (NSDA) neurons.  Cannabinoids are endogenous bioactive metabolites of arachidonic acid, synthesized and released from post-synaptic neurons into the synapse, where they act in a retrograde negative feedback fashion to inhibit pre-synaptic release of neurotransmitters.  Cannabinoid 1 (CB1) receptor activation increases NSDA neuronal activity and DA release, likely due inhibition of GABA release from pre-synaptic neurons.  MPTP is a selective DA neurotoxin which induces the loss of NSDA neurons and a compensatory increase in DA release from surviving, unlesioned neurons.  The purpose of this study was to determine if endogenous cannabinoids acting at CB1 or CB2 receptors mediate the compensatory activation of NSDA neurons.  In wild type C57BL/6 control mice, MPTP caused a significant decrease in DA and tyrosine hydroxylase (TH) in the striatum (consistent with a loss of NSDA axon terminals).  This loss was associated with a compensatory increase in the activity of surviving NSDA neurons as indicated by the ratio of the DA metabolite DOPAC to DA.  In CB1/CB2 receptor knockout mice, MPTP caused a similar decrease in striatal TH and an attenuated decrease in DA, but no increase in the DOPAC/DA ratio.  These results suggest that endogenous cannabinoids may play a role in DA receptor-mediated neuronal feedback regulation of NSDA neurons.  Lack of the CB1 receptor may provide a partial neuroprotective effect by decreasing the activity of remaining NSDA neurons slowing or halting their loss.  CB2 receptors have only recently been found on neurons in the central nervous system, suggesting that lack of these receptors may or may not contribute to the partial resistance of DA depletion in the striatum.  Thus resistance to MPTP induced DA depletion in the striatum of CB1/CB2 receptor knockout mice compared to C57BL/6 wildtype mice may be attributed to the lack of one or both CB receptors.

163

Human Airway Epithelial Cell Cultures of Respiratory Secretions from Patients with Acute Kawasaki Disease

Bridget Banach, BS, Loyola University, Maywood, IL, USA; Anne Rowley, MD, Northwestern University, Chicago, IL, USA; Stanford Shulman, MD, Northwestern University, Chicago, IL, USA; Jan Orenstein, MD PhD, George Washington University, Washington DC, DC, USA; Susan Baker, PhD, Loyola University, Maywood, IL, USA

BACKGROUND: Although the etiology of Kawasaki disease (KD) is unknown, inflamed tissues from acute KD patients are infiltrated by IgA plasma cells and CD8 T lymphocytes, suggesting an immune response to an intracellular pathogen with a mucosal portal of entry. Furthermore, intracytoplasmic inclusion bodies have been identified in ciliated bronchial epithelium of acute KD but not infant control patients using synthetic KD antibodies. Propagation of the etiologic agent in cell culture would greatly enhance efforts to develop a diagnostic test for KD. We hypothesize that the KD agent replicates in human bronchial epithelial cells.   Therefore, we established human airway epithelial (HAE) cell cultures, and are inoculating the cultures with KD respiratory samples in an attempt to propagate and analyze the KD agent. 

METHODS: Primary human bronchial epithelial cells are cultured for 4 to 6 weeks to produce well-differentiated, pseudo-stratified columnar epithelial cells, and mucus-producing Goblet cells. HAE cultures are then inoculated with respiratory secretions from acute KD patients or a control virus. Cultures are analyzed by immunofluorescence assay using synthetic KD antibodies or control antibodies, and by transmission electron microscopy (TEM) to visualize viral infection. Cell-free supernatants are harvested and isolated RNA is amplified by whole transcriptome amplification (WTA). WTA reaction products are cloned and sequenced to identify novel sequences. 

RESULTS: Experiments using HAE cultures and a control respiratory virus show we can propagate and identify virus-infected HAE cells using immunofluorescence, TEM and WTA assays. We have inoculated HAE cultures with respiratory secretions from 3 patients with acute KD, and to date we have detected a small number of antigen positive cells in one of the three KD cultures. Passage experiments are in progress and respiratory secretions from additional KD patients will also be cultured.

CONCLUSIONS: Inoculation of KD respiratory secretions on human airway epithelial cell cultures followed by immunofluorescence, TEM, and highly sensitive molecular amplification of culture supernatants are promising new approaches for the propagation of an agent associated with KD.

164

Pyrophosphate As A Dynamic Probe Of The Human RNA Polymerase II Mechanism

Woo Moon, BS, Michigan State University College of Osteopathic Medicine, East Lansing, MI, USA; Anthony Nazione, Mr, Department of Biochemistry & Molecular Biology, East Lansing, MI, USA; Rita Grantner, Ms, Department of Biochemistry & Molecular Biology, East Lansing, MI, USA; Zachary Burton, PhD, Department of Biochemistry & Molecular Biology, East Lansing, MI, USA

Human RNA polymerase II is an enzyme that synthesizes pre-messenger RNA, which is important for protein synthesis and gene regulation in development, cancer and viral infection. Our laboratory utilized pyrophosphate as a dynamic probe to study the intricate elongation mechanism of this enzyme. Pyrophosphorolysis is the reverse of the RNA synthesis reaction, in which pyrophosphate interacts with the pre-translocated elongation complex to remove the 3’-NMP (nucleoside monophosphate) from a nascent RNA chain to release NTP (nucleoside triphosphate). Although mononucleotide products are expected, we find that exposing human RNA polymerase II elongation complexes to pyrophosphate can result in apparent cleavage of a dinucleotide, in a reaction that mimics dinucleotide cleavage stimulated by the transcription elongation factor TFIIS. In addition to facilitating dinucleotide cleavage, pyrophosphate suppresses transcriptional pausing when escaping from a stall position, presumably by retaining the elongation complex on the active synthesis pathway. When added to stalled complexes together with TFIIS, pyrophosphate does not inhibit TFIIS-mediated dinucleotide cleavage, but, when added prior to stalling, pyrophosphate does inhibit TFIIS-mediated RNA cleavage, presumably by blocking formation of off-pathway complexes that are sensitive to TFIIS. When added together with NTP substrates, pyrophosphate inhibits elongation, particularly at low NTP concentrations. Approaching a stall position, conditions can be obtained in which reverse synthesis through pyrophosphorolysis appears to balance forward synthesis. Addition of the next templated substrate NTP to these complexes results in generation of longer products, showing that delayed complexes are not arrested but, rather, remain elongation competent. This result confirms that pre-translocated and post-translocated elongation complexes co-exist at the stall position. Translocation, therefore, appears to progress via a thermal ratchet, in which pre-translocated elongation complexes, which bind pyrophosphate, are in equilibrium with post-translocated elongation complexes, which bind NTPs.

165

The SIV-Infected Rhesus Macaque is an Ideal Model to Examine the Effects of Chronic Alcohol Consumption on HIV Pathogenesis

Jessica Thomas, MPH, LSUHSC- New Orleans, New Orleans, LA, USA; Nedra Lacour, BS, LSUHSC- New Orleans, New Orleans, LA, USA; Gregory Bagby, PhD, LSUHSC- New Orleans, New Orleans, LA, USA; Angela Amedee, PhD, LSUHSC- New Orleans, New Orleans, LA, USA

Most HIV-1 infections are acquired across mucosal surfaces; however, oral transmission rarely occurs in adults due to high levels of expression of protective innate immune factors in saliva. Chronic alcohol abuse has been shown to impair multiple aspects of immune system function and has been associated with an increased risk of HIV infection and accelerated disease progression. The combined effect of chronic alcohol abuse and HIV infection on immune function and how this correlates with disease progression is not well understood. The SIV-infected rhesus macaque provides an excellent model to evaluate HIV pathogenesis and host immune defenses, as well as determining the effects of chronic alcohol consumption on these factors. We have adapted a widely used in vitro HIV neutralization assay for use with the SIV-macaque model. Plasma and saliva samples were collected from rhesus macaques treated with a chronic-binge alcohol protocol and from sucrose-treated controls over a 48-week period post-SIVmac251 infection (w.p.i.). Because effective humoral immune responses are essential for the control of HIV/SIV replication and disease progression, we evaluated the development of neutralizing antibody (Nab) titers in the plasma of all animals at 1, 8, 12, 30 and 48 w.p.i. Most animals have developed a significant Nab titer against a neutralization-sensitive SIV reference isolate by 12 w.p.i. Animals that progressed quickly to end-stage disease failed to develop a robust Nab response. There was little difference in Nab titer between alcohol and sucrose-treated monkeys; however, alcohol-treated animals were slower to develop Nab responses and titers generally peaked later than control animals. Using this in vitro HIV neutralization assay, we also examined the ability of whole saliva from normal rhesus macaques to inhibit infection of SIV. Macaque saliva inhibited in vitro infection of SIV, similar to previous observations with HIV and human saliva. The degree of inhibition varied by individual animal and SIV isolate used. To determine the impact of alcohol consumption on the inhibitory capacity of saliva, we evaluated saliva from our alcohol-treated and sucrose-treated cohorts. Saliva from alcohol-treated animals showed a significantly reduced ability to inhibit SIV infection in vitro as compared to controls (p= 0.0001), potentially indicating an alcohol-mediated impairment to the innate immune factors present in saliva. The SIV-infected rhesus macaque provides an ideal model to characterize the innate and specific factors that limit progression and transmission of HIV. These studies suggest that chronic alcohol use may delay development of critical humoral immune responses in the plasma and may increase susceptibility to viral transmission through impairment of protective factors in the oral cavity and, potentially, at other mucosal sites. Supported by NIH grants AA09803 and AA07577.

166

Caveolin-1 Up-Regulation During Epithelial to Mesenchymal Transition (EMT) is Mediated by Focal Adhesion Kinase.

Kelly Bailey, MD/PhD student, West Virginia University, Morgantown, WV, USA

Emerging evidence has shown that caveolin-1 is up-regulated in a number of metastatic cancers and can influence various aspects of cell migration. However, in general, the role of caveolin-1 in cancer progression is poorly understood. In the present study, we examined alterations in caveolin-1 expression during epithelial-to-mesenchymal transition (EMT) and the ability of caveolin-1 to alter cancer cell adhesion, an aspect of cell motility. We employed two EMT cell models, the human embryonic carcinoma cell line NT2/D1 and TGF-?1 treated NMuMG cells, which are derived from normal mouse mammary epithelia. Caveolin-1 expression was substantially up-regulated in both cell lines following the induction of EMT and was preceded by increased activation of focal adhesion kinase (FAK) and Src, two known tyrosine kinases involved in EMT. We hypothesized that caveolin-1 expression could be influenced by increased FAK phosphorylation, to which Src is a known contributor. Examination of FAK+/+ and FAK-/- mouse embryonic fibroblasts revealed that in cells devoid of FAK, caveolin-1 expression is strikingly diminished. Using FAK and superFAK constructs and the novel FAK inhibitor PF-228, we were able to demonstrate that indeed, FAK can regulate caveolin-1 expression. We also found that Src can contribute to increases in caveolin-1 expression, however, only in the presence of FAK. From the culmination of this data and our functional analyses, we conclude that caveolin-1 expression can be up-regulated during EMT, and further, once expressed, caveolin-1 can greatly influence cancer cell adhesion.

167

Magnetic Protein Microspheres as Dynamic Contrast Agents for Targeting Integrin Receptors

Freddy Nguyen, BS, BA, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Freddy Nguyen, BS BA, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Elizabeth Dibbern, PhD, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Eric Chaney, BS, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Amy Oldenburg, PhD, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Kenneth Suslick, PhD, University of Illinois at Urbana-Champaign, Urbana, IL, USA; Stephen Boppart, MD PhD, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Optical coherence tomography (OCT) is an emerging biomedical optical imaging modality that has been developed over the last 15 years in a number of clinical applications. Recently, OCT has been used for the intraoperative imaging of tumor margins and axillary lymph nodes in breast cancer providing a real time in-vivo assessment of the tissue morphology for the staging of breast cancer. OCT has been extensively studied in the field of cardiology. In comparison to intravascular ultrasound, OCT provides much higher resolution by at least an order of magnitude. The strengths of structural OCT as an imaging modality lie in its high-resolution (μm and sub-μm) capabilities, deep depth penetrations in highly-scattering tissue (2-3 mm), and its high acquisition rate.

As diagnostic medicine continues to push for earlier detection, the development of functional imaging modalities to probe molecular information in-vivo continues to increase. This information can be combined with the morphological information (observed under OCT) allowing for a real-time microscopic analysis of the tissue specimen. A novel functional modality of OCT called magnetomotive-OCT (MM-OCT) uses a magnetic field that is modulated at half the axial scan frequency. This modulates the OCT image between an “on” and “off” state generating an MM-OCT image that is overlaid on top of the structural OCT image. The contrast agent itself does not necessarily need to exhibit significant scattering or absorbing properties (the contrast mechanism needed for OCT) in their optical profiles in order to be detected in MM-OCT, but simply need to be able to displace nearby tissue scatterers adding dimensionality to the contrast mechanisms needed for OCT.

The contrast agent in this study designed to work with MM-OCT is a protein microsphere with an oil core and a BSA protein shell functionalized with RGD peptide sequences for targeting integrin receptors. Magnetic nanoparticles (Fe3O4) and Nile Red fluorescent dye have been encapsulated into its oil core. These microspheres have been functionalized with RGD using an LBL electrostatic adhesion process. Results show that these magnetic microspheres (2.0-5.0μm), are readily detectable under MM-OCT when embedded in a different number of environments including 5% agarose gel, a 3-D scaffold of macrophage cells previously incubated with the microspheres, and when injected in-vivo into a tumor from an NMU-carcinogen rat animal model. Current studies also confirm the increased binding affinity and specificity of these microspheres to the integrin receptors in cancer cell lines. Initial ex-vivo studies in an atherosclerotic rat model have confirmed their binding affinity to atherosclerotic lesions found in the aortas using histology. Ongoing studies are underway to further study the sensitivity and specificity of the RGD targeting to the α(v)β(3) receptor.

168

Strategies To Discover New Therapies That Target Microsatellite Instable (MSI) Colorectal Cancer (CRC).

Eduardo Vilar, MD, MS, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA; Bhramar Mukherjee, PhD, Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA; Gad Rennert, MD, PhD, Clalit Health Services, National Cancer Control Center, Haifa, NA, Israel; Stephen Gruber, MD, PhD, MPH, Departments of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

BACKGROUND. Sporadic MSI tumors represent approximately 15% of CRC cases. Several preclinical and clinical studies have identified differences in response to 5-Fluorouracil and Irinotecan between Microsatellite Stable (MSS) and MSI tumors. CMap is a systems biology publicly available which enables researchers to match a gene expression data defining a tumor subgroup with a database containing expression data obtained from different cell lines after being exposed to a panel of 164 small molecules. Our objective was to identify new compounds to target MSI tumors using CMap and validate them in the preclinical setting. METHODS. We have used gene expression data generated from sporadic MSI tumors included in the Molecular Epidemiology of Colorectal Cancer study and from two signatures published previously (Watanabe et al and Koinuma et al). In addition, two aggregated signature were generated, one containing common genes found to be up and downregulated through the three signatures, and other integrating all the probes from the three signatures. The most active compounds selected for cytotoxicity assays were those fulfilling each of the following criteria: >2 negative scores in the global analysis using the CMap; >50% negative scores; and compounds highly ranked in terms of a summary measure obtained from aggregating p-values across the different signatures. CRC cell lines showing MSI due to mutations in mismatch repair (MMR) genes, MSI due to hypermethylation of the promoter of MLH1, and MSS were selected to perform cytotoxicity and flow cytometer experiments. RESULTS. Four candidate compounds arose from comparison of CRC expression profiles and the CMap: Trichostatin, which acts through HDAC inhibition; 17-Allyamino-Geldamycin (17-AAG) which targets hsp90; LY-294002, a PI3K inhibitor; and Sirolimus which acts through the mTOR pathway. These experiments show that Sirolimus and 17-AAG are preferentially targeting cell lines with microsatellite instability due to hypermetylation in the MLH1 promoter. MSS cell lines showed 4-fold higher and 2-fold and IC50 with respect to MSI cells due to hypermethylation of MLH1 treated with Sirolimus (18.3 vs 4.4 µM) and 17-AAG (98 vs 48 nM), respectively. Apoptosis analysis were performed using Annexin V and PI staining revealing a high increase in the percentage of apoptotic cells under treatment with 17-AAG, although no changes were observed with Sirolimus. However cell cycle analysis by flow cytometry demonstrates an arrest in G1 phase under treatment with both Sirolimus and 17-AAG consistent with previous studies. CONCLUSIONS. We report the application of CMap systems biology tools for discovering of new small molecules to specifically target MSI CRC. Further investigation of Sirolimus and 17-AAG in combination with standard chemotherapeutic agents is warranted for MSI colorectal cancers.

169

Age- And Time- Dependent Effects Of Seizures On Proliferation In The Juvenile Period

Yasmine Khalil, BS (DO/MS in Progres, New York College of Osteopathic Medicine/New York Institute of Technology, Hicksville, NY, USA; Henry Hong, BS, New York College of Osteopathic Medicine/New York Institute of Technology, old westbury, NY, USA; Toni Webster, BS (DO/MS in Progres, New Yor College of Osteopathic Medicine, old westbury, NY, USA; Edward Lopez, BS (DO in Progress), New York College of Osteopathic Medicine/New York Institute of Technology, old westbury, NY, USA; Imeh Sampson, BS (DO in Progress), New York College of Osteopathic Medicine/New York Institute of Technology, old westbury, NY, USA; Linda Friedman, PhD, New York College of Osteopathic Medicine/New York Institute of Technology, old westbury, NY, USA

There is an age-dependent switch in neuronal vulnerability following a single episode of kainic acid (KA)-induced status epilepticus from almost no damage in the first two weeks of life to selective injury in CA1/CA2 regions at prepubescent ages (P20-P30) to preferential CA3 injury in adulthood (P60). In rat pups neurogenesis of the hippocampus is constrained by seizures whereas in adult rats it is provoked.  We hypothesized that inhibition or stimulation of hippocampal progenitors in the juvenile period may depend upon the injured region affected, number of juvenile seizures, and time point examined.  Seizures were induced once with KA (1x KA) either on postnatal (P) day 30 or 60 or twice (2x KA) on P25 and P30.  In control rats, (P30 or P60) immunohistochemistry with Ki67 and bromo-deoxyuridine (BrdU) antibodies showed scattered newly dividing cells were few in number and located at the hilar border.  Few or no progenitors were counted in other hippocampal layers.  At P30, following 1x KA or 2x KA, the hippocampus rapidly expressed moderate numbers of Ki67 and BrdU labeled cells in the CA3 and dentate gyrus (DG) and high numbers in the CA1 within 48 hrs of the KA injection. Histological analysis revealed robust CA1 injury after 1x KA but spared CA1 neurons after 2x KA. After 9 days, progenitor counts returned to control or lower levels in the DG and vulnerable CA1 subregions.  Interestingly, CA1 sparing was observed after 1x KA and 2x KA at this time.  In contrast, P60 rats with 1x KA, expressed high numbers of progenitors throughout the hippocampal subfields particularly in regions of injury (e.g. CA3).  Increased proliferation was also noted in the molecular cell layer (MCL) of the DG in all groups. Co-labeling with neuronal and non-neuronal markers showed progenitor types were differentially stimulated and predominantly of neural origin. Results also indicate that stimulated proliferation and injury in P30 rats is transient after 1x KA and even more so after 2x KA. It is possible progenitors replaced the dying pyramidal neurons whereas in older animals the injury persists due to poor survival of progenitors with maturation.

170

SDF-1/CXCL12 Modulates Serotonergic Neuronal Excitability In The Rat Dorsal Raphe Nucleus

Silke Heinisch, BS, Temple University School of Medicine, Philadelphia, PA, USA; Lynn Kirby, PhD, Temple Universiy School of Medicine, Philadelphia, PA, USA

The serotonin (5-hydroxytryptamine; 5-HT) system is typically altered in major depression, and recent reports describe increasing comorbidities of mood-immune disorders, suggesting an immunological component. Chemokines (chemotactic cytokines), immune proteins which mediate leukocyte trafficking in the periphery, and their G-protein coupled receptors are widely distributed on cells within the central nervous system (CNS), are critical for proper neuronal patterning, and are implicated in numerous neuropathologies such as multiple sclerosis, meningitis, and neuroAIDS. “Sickness behavior,” a mental state symptomatically identical to depression, can be induced by these immune proteins, implying that chemokine-serotonin interactions in the CNS may mediate this clinical condition. The purpose of this study is to investigate the neuroanatomical relationship between and functional impact of the chemokine stromal cell-derived factor-1α (SDF-1α/CXCL12) and its receptor CXCR4 with the 5-HT system. Both anatomical and electrophysiological techniques are used to determine chemokine-serotonin associations in rat midbrain dorsal and median raphe nuclei (DRN, MRN). Immunohistochemical analysis in raphe nuclei reveals that greater than 70% of 5-HT neurons colocalize with SDF-1α/CXCL12 and CXCR4. SDF-1α/CXCL12 localizes as discrete puncta throughout the cytoplasm and processes of 5-HT neurons, whereas CXCR4 primarily concentrates to the outer plasma membrane and processes of these neurons. SDF-1α/CXCL12 and CXCR4 are also colocalized to cells within the raphe nuclei. Functionally, SDF-1α/CXCL12 depolarizes 5-HT neurons, thus increasing the excitability of these neurons. Furthermore, SDF-1α/CXCL12 increases the frequency of spontaneous inhibitory postsynaptic currents (IPSC) selectively in 5-HT neurons. SDF-1α/CXCL12 also dose-dependently increases evoked IPSC (eIPSC) amplitude while decreasing eIPSC paired-pulse ratio (PPR) selectively in 5-HT neurons. These data indicate that SDF-1α/CXCL12 increases presynaptic GABA release at these neurons. The presynaptic effects of SDF-1α/CXCL12 on evoked GABA release are mediated by the CXCR4 receptor since the SDF-1α/CXCL12-induced effects on eIPSC amplitude and PPR are blocked by pretreatment with the CXCR4 receptor antagonist AMD3100. Using immunohistochemistry, CXCR4 was also detected on GABAergic neurons within the raphe nuclei, suggesting a mechanism by which SDF-1α/CXCL12 is able to interact with GABAergic neurons and modulate GABA release. SDF-1α/CXCL12’s direct effects on 5-HT neurons and indirect effects on GABA synaptic activity provide multiple mechanisms whereby this chemokine can impact 5-HT neurotransmission. Future therapies targeting SDF-1α/CXCL12, as well as other immune proteins, may be developed to treat serotonin related mood disorders, particularly depression experienced by immune-compromised individuals.

171

Electrical Stimulation of the Hippocampal Fimbria Facilitates Nitric Oxide Efflux in the Nucleus Accumbens via a Neuronal NOS-dependent Mechanism.

Kristina Hoque, B.S. , RFUMS, North Chicago, IL, USA; Anthony West, PhD, RFUMS, North Chicago, IL, USA

The nucleus accumbens (NAc) receives motivational and locomotor information from limbic system afferents originating in the hippocampus, amygdala and ventral tegmental area. Dysregulation of NAc neurotransmission is observed in neuropsychiatric disorders such as addiction and schizophrenia. Anatomical studies have demonstrated that nitric oxide (NO)-producing interneurons localized to the core and shell of the NAc give rise to a dense plexus of NO synthase (NOS)-immunoreactive fibers. It is postulated that these interneurons play an important role in the feed-forward regulation of projection neurons in the NAc. In this study, NO efflux was measured in anesthetized rats using NO-selective, amperometric microsensors implanted into the shell of the NAc. NO oxidation current was recorded during electrical stimulation of the ipsilateral fimbria.  Electrical stimulation of the fimbria at low frequencies (0.5 Hz) had no effect on NO efflux. Electrical stimulation of the fimbria at intermediate frequencies (5 Hz) induced a modest increase in NAc NO efflux. The magnitude of NO efflux evoked during the 5Hz stimulation was not dependent on the intensity of the stimulus (500 – 1000 µA). Interestingly, train stimulation (50 Hz) of the fimbria elicited a robust stimulus-intensity dependent increase in NAc NO efflux. The facilitatory effect of train stimulation on NAc NO efflux was transient and attenuated by systemic administration of the neuronal NOS inhibitor N-propyl-L-arginine. These observations demonstrate that NO synthesis in the NAc is facilitated by robust activation of hippocampal afferents. Furthermore, evoked NO efflux in the NAc is at least partially dependent on activation of the neuronal isoform of NOS. Further characterization of the role of NO in the regulation of NAc function may facilitate the development of pharmacotherapies designed to treat disorders of motivation and cognition.

172

Diet and Risk of Acute Myocardial Infarction in a Multiethnic Cohort

Nitzan Roth, B.S., University of Southern California, Pasadena, CA, USA; Wendy Mack, PhD, University of Southern California, Los Angeles, CA, USA; Kristine Monroe, PhD, University of Southern California, Los Angeles, CA, USA; Laurence Kolonel, MD PhD MPH, University of Hawaii, Honolulu, HI, USA; Brian Henderson, MD, University of Southern California, Los Angeles, CA, USA

Background: There is substantial evidence that diet influences risk of acute myocardial infarction (AMI) but the optimal low-risk diet is not established. In the Multiethnic Cohort (MEC), an inverse association between alcohol consumption and risk of death due to AMI was previously reported. Methods: We examined associations between diet and risk of AMI in 44,137 African-American and Latino men and postmenopausal women, aged 45-75, residing in Los Angeles County and who enrolled in the MEC between 1993 and 1996 and did not report a history of angina or heart attack at cohort entry. We identified incident cases of AMI through 2003 by linkage of the cohort with death files and records from the California Office of Statewide Health Planning and Development's hospitalization discharge database. Relative risks (RRs) of AMI were calculated according to relative consumption (as percentage of daily caloric intake) of saturated fat, monounsaturated fat, omega-3 polyunsaturated fat (PUFA), omega-6 PUFA, and alcohol. We adjusted for age, ethnicity, education, body mass index, smoking, physical activity, hypertension, diabetes, stroke, cardiac revascularization, use of aspirin or blood pressure medications, caloric intake, and consumption of dietary fiber, cholesterol, sodium, and caffeine. For women, we also adjusted for age at menopause, type of menopause (natural or surgical), and use of hormone therapy. Results: There were 1,851 incident AMI cases during an average of 9.2 years of follow-up. Alcohol consumption was inversely associated with AMI risk in men [RR for the highest quartile of alcohol consumption versus nondrinkers = 0.75, 95% confidence interval (CI) = 0.62-0.91; p for trend

173

The Role of Receptor Dynamics in High Affinity IgE Receptor Signaling

Nicholas Andrews, Bachelor of Science, University of New Mexico, Albuquerque, NM, USA; Keith Lidke, PhD, University of New Mexico, Albuquerque, NM, USA; Janet Pfeiffer, Bachelor of Science, University of New Mexico, Albuquerque, NM, USA; Alan Burns, PhD, Sandia National Laboratories, Albuquerque, NM, USA; Bridget Wilson, PhD, University of New Mexico, Albuquerque, NM, USA; Janet Oliver, PhD, University of New Mexico, Albuquerque, NM, USA; Diane Lidke, PhD, University of New Mexico, Albuquerque, NM, USA

The high affinity IgE receptor, FcεRI, is the principal multi-subunit immunoreceptor on the surface of human mast cells and basophils.  Crosslinking of IgE-bound FcεRI by multivalent allergen triggers a signaling cascade which leads to a rise in intracellular calcium and subsequent release of pre-formed mediators of allergic inflammation from mast cell granules.  Biochemical studies have provided a wealth of knowledge regarding the sequence of events occuring downstream of FcεRI crosslinking; however, the precise mechanism by which crosslinking initiates these events remains unclear.  To study the dynamic events that induce FcεRI signaling, we generated monovalent quantum dot (QD)-IgE that binds FcεRI without crosslinking.  As receptor immobilization has been shown to be a consequence of crosslink-induced activation, we developed a real-time assay for this process.  Employing this assay, we found that immobilization occurs within seconds of crosslinking (at 1 µg/ml of crosslinking agent) but the kinetics of this response are largely dose-dependent.  Treatment with the actin-disrupting agent latrunculin B delayed the kinetics of this response and also significantly increased the diffusion coefficient of crosslinked FcεRI.  Furthermore, inhibition of src-family kinases did not have a significant effect on crosslink-induced immobilization.  Finally, we show that direct crosslinking by multivalent antigen is required for immobilization since crosslinking of a subset of dansyl-specific-IgE bound receptors does not induce immobilization of DNP-specific QD-IgE-FcεRI complexes.  These results indicate that receptor immobilization lies upstream of FcεRI phosphorylation and is dependent upon an intact actin cytoskeleton.

174

The Effect Angiotensin 1-7 on Sympathetic Neurotransmitter Overflow in Spontaneously Hypertensive Rats and the Mechanisms Involved

Mirnela Byku, BA, SAint Louis University, Saint Louis, MO, USA; Heather Macarthur, PhD, Saint Louis University School of Medicine, Saint Louis, MO, USA; Thomas Westfall, PhD, Saint Louis University School of Medicine, Saint Louis, MO, USA

Neuropeptide Y (NPY) is a cotransmitter with Norepinephrine (NE) and ATP in sympathetic nerves. There is evidence for increased activity of the sympathetic nervous system and the renin-angiotensin system (RAS) as well as a role for NPY in the development of hypertension in experimental animal models and in humans. Angiotensin II (Ang II) is known to facilitate sympathetic neurotransmission; an effect greater in Spontaneously Hypertensive Rats (SHR) than normotensive Wistar-Kyoto (WKY) rats. A newly discovered product of the RAS is the peptide Angiotensin 1-7 (Ang 1-7). There is evidence suggesting that Ang 1-7 opposes the actions of Ang II resulting in hypotensive effects. The objective of this study was to investigate the role of Ang 1-7 on the nerve stimulated overflow of NE and NPY from the mesenteric arterial bed of SHR and the mechanisms involved in mediating any effects produced. 

Ang 1-7 (0.001, 0.01, 0.1μM) decreased nerve stimulated NE and NPY overflow, as well as perfusion pressure in preparations obtained from SHR. This effect was greater in preparations of SHR than WKY controls. In addition, Ang 1-7 decreased NE overflow to a greater extent than NPY overflow. Administration of the Mas Receptor Antagonist (MRA), D-Ala Ang 1-7 attenuated the decrease in both NE and NPY overflow due to Ang 1-7 administration. However, Angiotensin Type 2 Receptor Antagonist (AT2A), PD123391 attenuated the effect of Ang 1-7 on NE overflow without affecting the decrease in NPY overflow. Moreover, in the presence of L-Name, Ang 1-7 decreased NPY overflow but not NE overflow.

Ang 1-7 decreases the nerve stimulated overflow of NE and NPY in preparations of SHR, while Ang II enhances it. Therefore, Ang 1-7 may counteract the effects of Ang II by acting on AT2 and Mas Receptors. In addition, our data suggests that Ang 1-7 modulates sympathetic neurotransmission through a nitric oxide dependent mechanism. (Sponsored by NIH HL60260 and AHA)

175

An Investigation of Genetic Variability at the Aggrecan Locus in African American Osteoarthritis Patients.

Dawn Franks, PhD, Rush University Medical Center, Chicago, IL, USA; Cristina Fheid , MS, Rush University Medical Center, Chicago, IL, USA; Rediet Kokebie, MD, Stroger County Hospital, Chicago, IL, USA; Augustine Manadan, MD, Stroger County Hospital, Chicago, IL, USA; Joel Block, MD, Rush University Medical Center, Chicago, IL, USA; Theodore Oegema, PhD, Rush Universtiy Medical Center, Chicago, IL, USA

INTRODUCTION: Future treatment of disease is likely to involve analysis of the genetic background of the patient for gene variants that have been shown to affect response to medication or predisposition to rapid progression of the condition. The genetic polymorphisms that constitute our genetic diversity often vary between ethnic groups and the NCBI Single Nucleotide Polymorphism Database reports this variation for 5 groups, when available. Through a collaboration with a local organ donor network, we have recently found that a variable number tandem repeat (VNTR) polymorphism in aggrecan, which encodes a major structural protein in cartilage, is present in the Caucasian donors at a frequency similar to that reported by others for this ethnic group. There are 13 alleles of the aggrecan VNTR, with 87% of the Caucasians tested exhibiting 26, 27, or 28 repeats. The shortest allele has 13 repeats and the longest 33 and all are rare. However, our small number of African American (AA) donors show a drastically different allele distribution compared to the Caucasian. In general, we are finding many more of the so-called short alleles in the AA donors. Since there are no published reports about the aggrecan polymorphism in the AA or African populations we are unable to assess whether the allelic distribution we have found is representative of this particular group. This study was designed to 1) determine the aggrecan allele distribution in the AA population and 2) determine if the AA donors in our study exhibit genotypes that can be associated with osteoarthritis (OA). The relevance of the aggrecan polymorphism to the study of OA in general pertains to the fact that the VNTR occurs in the portion of the gene coding for chondroitin sulfate chain attachment sites the water retaining portion of the molecule. The shortest and longest of the 13 alleles documented have the potential for a difference of up to 40 CS chains. The potential lower fixed charge density proposed to be present in carriers of the shorter alleles has been suggested to be a predisposition to loss of cartilage due to a decreased ability to resist compression. If we can document that the shorter VNTR alleles are present at high frequency in the AA population, then the short allele hypothesis can be tested. METHODS: We are collecting DNA from AA-OA patients at a Chicago-area hospital and genotyping them for the VNTR by PCR. We are comparing the frequency of each allele in the case group to that of a general population group consisting of 100 unrelated AA individuals whose DNA is available through the Coriell Institute. RESULTS: As collection of samples proceeds, we have found the short aggrecan alleles at a much higher frequency in both AA groups compared to the Caucasian. We have also identified an allele hitherto not documented in other ethnic groups. We hope to soon have samples numbers sufficient for statistical significance.