Male FVB Mice Research Articles

Cardiac interfibrillar mitoKATP channels are sensitive to diazoxide and have a regulatory subunit profile distinct from subsarcolemmal mitoKATP

Two distinct populations of mitochondria exist in cardiac muscle, subsarcolemmal and interfibrillar (SSM and IFM). SSM and IFM differ in location, respiratory capacity, and susceptibility to disease. Mitochondrial KATP channels (mitoKATP) play an important role in cardioprotective signaling; however, characterization of differences in mitoKATP between IFM and SSM is absent. The rationale that cellular demand during insult could affect SSM and IFM differently makes it plausible to hypothesize that differences exist in mitoKATP subunit composition and pharmacology. We tested isolated cardiac SSM and IFM of adult male FVB mice using Rhodamine123 fluorometry and JC‐1 flow cytometry. We measured membrane potential (ΔΨm) in response to the mitoKATP agonist diazoxide. We also compared immunoblots of putative mitoKATP subunits. Our results indicate that IFM mitoKATP are more sensitive to diazoxide, as ΔΨm depolarized more so than did SSM ΔΨm. Immunoblots showed differential expression of SUR subunits, with SSM containing SUR2A and SUR2B, while IFM appear to have a novel splice variant of SUR1. These data suggest that mitoKATP subunits are expressed differently in cardiac mitochondrial subpopulations and that IFM are more sensitive to diazoxide. These findings have implications for cardioprotective mechanisms, metabolic disease, and individual roles that IFM and SSM may have. T32‐HL090610

Shortened life span in mice generated by in vitro fertilization

In humans assisted reproductive technologies (ART) induce generalized vascular dysfunction in children. In line with this observation, ART induces vascular dysfunction and arterial hypertension in mice that is related to an epigenetic mechanism, but the long term consequences are unknown. To address this problem, we compared the long term survival of ART and control mice fed a normal chow diet (NC) or challenged with a high‐fat diet (HFD).80 male FVB mice generated by ART and 80 control mice born and raised in our animal facility were observed in these studies. Three to four animals were placed in a cage in the clean conventional mouse facility. 6 ART and 7 control mice had to be sacrificed because of injuries related to fighting. 32 ART and 20 control mice were fed NC, 42 ART mice and 53 control mice were fed a HFD (49.5% fat, 31.5% protein and 0% carbohydrate). Kaplan‐Meier survival curves were constructed using known birth and death dates and the log‐rank test was used to evaluate statistical differences between groups.We found that in mice fed with NC the life span was similar in the two groups. In contrast, HFD, while not having a detectable effect on life span in control mice, reduced the survival in ART‐mice by roughly 25%(P<0.0001).ART is associated with a marked reduction of the life span in normal mice challenged with a Western style high‐fat diet.

Cardiac-Specific Overexpression of Metallothionein Rescues against Cigarette Smoking Exposure-Induced Myocardial Contractile and Mitochondrial Damage

ObjectivesSecond hand cigarette smoke is an independent risk factor for cardiovascular disease. Although a tie between smoking and cardiovascular disease is well established, the underlying mechanisms still remains elusive due to the lack of adequate animal models. This study was designed to use a mouse model of exposure to cigarette smoke, a surrogate of environmental tobacco smoke, to evaluate the impact of cardiac overexpression of heavy metal scavenger metallothionein on myocardial geometry, contractile and intracellular Ca2+ properties and apoptosis following side-stream smoke exposure.MethodsAdult male wild-type FVB and metallothionein transgenic mice were placed in a chamber exposed to cigarette smoke for 1 hour daily for 40 days. Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties, fibrosis, apoptosis and mitochondrial damage were examined.ResultsOur data revealed that smoke exposure enlarged ventricular end systolic and diastolic diameters, reduced myocardial and cardiomyocyte contractile function, disrupted intracellular Ca2+ homeostasis, facilitated fibrosis, apoptosis and mitochondrial damage (cytochrome C release and aconitase activity), the effects of which were attenuated or mitigated by metallothionein. In addition, side-stream smoke expose enhanced phosphorylation of Akt and GSK3β without affecting pan protein expression in the heart, the effect of which was abolished or ameliorated by metallothionein. Cigarette smoke extract interrupted cardiomyocyte contractile function and intracellular Ca2+ properties, the effect of which was mitigated by wortmannin and NAC.ConclusionsThese data suggest that side-stream smoke exposure led to myocardial dysfunction, intracellular Ca2+ mishandling, apoptosis, fibrosis and mitochondrial damage, indicating the therapeutic potential of antioxidant against in second smoking-induced cardiac defects possibly via mitochondrial damage and apoptosis.

Gender and strain contributions to the variability of buprenorphine-related respiratory toxicity in mice

While most deaths from asphyxia related to buprenorphine (BUP) overdose have been reported in males, higher plasma concentrations of BUP and its toxic metabolite norbuprenorphine (NBUP) have been observed in females. We previously demonstrated that P-glycoprotein (P-gp) modulation at the blood–brain barrier (BBB) contributes highly to BUP-related respiratory toxicity, by limiting NBUP entrance into the brain. In this work, we sought to investigate the role of P-gp-mediated transport at the BBB in gender and strain-related variability of BUP and NBUP-induced respiratory effects in mice. Ventilation was studied using plethysmography, P-gp expression using western blot, and transport at the BBB using in situ cerebral perfusion. In male Fvb and Swiss mice, BUP was responsible for ceiling respiratory effects. NBUP-related reduction in minute volume was dose-dependent but more marked in Fvb (p<0.01 at 1mg/kg NBUP and p<0.001 at 3 and 9mg/kg NBUP) than in Swiss mice (p<0.001 at 9mg/kg NBUP). Female Fvb mice were more susceptible to BUP than males with significantly increased inspiratory time (p<0.05) and to NBUP with significantly increased expiratory time (p<0.01). Following BUP administration, plasma BUP concentrations were significantly higher (p<0.01) and plasma NBUP concentrations significantly lower (p<0.001) in Fvb mice compared to Swiss mice. Plasma BUP concentrations were significantly higher (p<0.05) and plasma NBUP concentrations significantly lower (p<0.01) in male compared to female Fvb mice. In contrast, following NBUP administration, comparable plasma NBUP concentrations were observed in both genders and strains. No differences in P-gp expression or BUP and NBUP transport across the BBB were observed between male and female Fvb mice as well as between Swiss and Fvb mice. Our results suggest that P-gp-mediated transport across the BBB does not play a key-role in gender and strain-related variability in BUP and NBUP-induced respiratory toxicity in mice. Both gender- and strain-related differences in respiratory effects of BUP could be attributed to BUP itself rather than to its metabolite, NBUP.

Abstract 180: Interleukin (IL) 16 Acts as a Regulator of Skin Electrolyte Metabolism and Blood Pressure

We showed that Na + is stored in the skin without commensurate water retention. In response to interstitial electrolyte load, mononuclear phagocyte system (MPS) cells secrete VEGF C, as regulated by tonicity enhancer binding protein (TonEBP) attaching to the VEGF C promoter. VEGF C induces hyperplasia of skin lymphcapillaries. As IL16 harbors a TonEBP binding site, we tested the hypothesis that IL16 participates in this process. We cultured bone marrow-derived macrophages in isotonic or hypertonic cell culture media and measured IL16 expression. We fed male FVB mice a low salt diet (LSD; &lt; 0.1% NaCl + tap) or a high salt diet (HSD; 4% NaCl + 0.9% saline) for two weeks. We treated the groups with either IL16 specific antibodies or no antibody. After two weeks we measured mean arterial blood pressure (MAP), skin Na + , Cl - , and water content (rSKNa, rSKCl, rSKW), quantified lymphcapillary density, VEGF C and TonEBP expression. IL16 was specifically secreted from macrophages in response to hypertonicity in vitro . HSD increased skin electrolytes, activated TonEBP/VEGF C, and resulted in lymphcapillary hyperplasia. Anti IL16 treatment under LSD increased skin electrolyte, water content and MAP. The IL16 antibody blunted the VEGF C driven lymphcapillary response despite salt load. No differences in skin electrolytes and MAP were found between IL16 antibody treated and untreated mice after HSD. We conclude that IL16 is a regulator of electrolyte and water metabolism and MAP, even in the absence of HSD. The finding that the IL16 promoter contains a TonEBP binding site suggests that IL16 could act as an osmoprotective gene that is involved in regulation of electrolyte homeostasis and MAP control.

Chronic intermittent hypoxia exposure improves left ventricular contractility in transgenic mice with heart failure

We previously reported the unexpected finding that 4 wk of exposure to intermittent hypoxia (IH), which simulates the hypoxic stress of obstructive sleep apnea, improved LV cardiac function in healthy, lean C57BL/6J mice. The purpose of the present study was to assess the impact of 4 wk of IH on cardiac function in a transgenic murine model that exhibits a natural history of heart failure. We hypothesized that IH exposure would exacerbate cardiac decompensation in heart failure. Adult male FVB (wild type) and transgenic mice with cardiac overexpression of tumor necrosis factor α (TNF-αTG) at 10-12 wk of age were exposed to 4 wk of IH (nadir inspired oxygen 5-6% at 60 cycles/h for 12 h during light period) or intermittent air (IA) as control. Cardiac function was assessed by echocardiography and pressure-volume loop analyses, and mRNA and protein expression were performed on ventricular homogenates. TNF-αTG mice exposed to IA exhibited impaired LV contractility and increased LV dilation associated with markedly elevated cardiac expression of atrial natriuretic peptide and brain natriuretic peptide compared with wild-type mice. When wild-type FVB mice were exposed to IH, they exhibited increases in arterial pressure and dP/dt(max), consistent with our previous report in C57BL/6J mice. Surprisingly, we found that TNF-αTG mice exposed to IH showed a reduction in end-diastolic volume (38.7 ± 3.8 to 22.2 ± 2.1 ul; P < 0.01) and an increase in ejection fraction (29.4 ± 2.5 to 41.9 ± 3.1%; P < 0.05). In contrast to our previous study in C56Bl/6J mice, neither FVB nor TNF-αTG mice exhibited an upregulation in β-adrenergic expression or cAMP in response to IH exposure. We conclude that 4 wk of exposure to IH in mice induces adaptive responses that improve cardiac function in not only healthy animals but also in animals with underlying heart failure.

Abstract 933: Peripheral neuropathy induced by chronic administration of Cisplatin, taxol and bortezomib in several murine models

Abstract Cisplatin (CDDP), Taxol (TAX) and Bortezomib (BZ) are chemotherapeutic drugs commonly employed in clinical practice for the treatment of solid and haematological tumors. Their clinical use is limited by the development of a peripheral neuropathy characterized by sensory alterations, pain and, in part, by motor and autonomic dysfunctions. Several rat models had been performed to study and describe the mechanisms of the peripheral neurotoxicity induced by these drugs. However, since only few cancer cell lines are able to induce the development of cancer in the rat, we focused our attention on mice models to allow the combined study of the antineoplastic activity and of the neurotoxic effects of the anticancer compounds. Moreover, different mice strains should be useful for different kind of studies. Here we investigated the interactions between the mice genotype and their drug response to determine the susceptibility to the development of chemotherapy-induced peripheral neuropathy. To this aim, we investigated the neurophysiological and neuropathological alterations induced by the chronic treatment with different chemotherapy drugs in female Balb/c, CD1 and c57 mice and in male c57 and FVB mice. Mice were injected with Cisplatin (2, 4 mg/Kg, ip, 2qw), or Taxol (50, 70, 80 mg/Kg, iv, 1qw) or Bortezomib (0.4, 0.8 mg/Kg, iv, 2qw) for a period of 4-6 weeks. At the end of the treatment the nerve conduction velocities (NCVs) were determined in the caudal and in the digital nerves and the dorsal root ganglia (DRG) and sciatic nerves collected for the neuropathological analysis; behavioural tests were performed to study if these drugs are able to induce allodynia or hyperalgesia. In all mice models we observed that TAX and CDDP induced a significant alteration in body weight at the end of the treatment. All the drugs induced a significant reduction in the caudal and digital NCV in Balb/c and CD1 mice. TAX and BZ caused the axonal degeneration of the sciatic nerves while CDDP and BZ morphological alterations in the DRG. In c57 female mice, only BZ induced a significant reduction in caudal NCV while in c57 male mice only CDDP caused neurophysiological impairments. By contrast, male FVB mice showed any neurophysiological alterations. All these models, except FVB mice, have shown the onset of allodynia and hyperalgesia at the end of the treatment. In conclusion we can affirm that, even if at different extent, Balb/c, CD1 and c57 mice were able to develop the different chemotherapy-induced peripheral neuropathies. Moreover, also the gender seemed to be involved in the severity of the neuropathy. By contrast, FVB mice were less susceptible to the neurotoxic damage. This study would give a useful guideline for the choice of mouse strains in the combined studies of the antineoplastic activity and of the neurotoxic effects of chemotherapy drugs. Partially supported by a grant from “Fondazione Banca del Monte di Lombardia”. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 933. doi:1538-7445.AM2012-933

Dietary isoflavones and supplemental selenium show interactive effects on blood‐glucose homeostasis in male FVB mice

Isoflavones and supplemental selenium have shown interactive effects on body weight, body fat, and blood‐glucose homeostasis. To further elucidate the effects of the interaction, FVB mice were fed either high or low‐isoflavone (HIF or LIF) diets, with or without supplemental selenium. Custom diets were designed to be equivalent in vitamin, mineral and amino acid profiles, but to differ in soy protein and specific isoflavone content. The HIF diet was tested to ensure 500mg/kg aglycone equivalents of genistein + daidzein. Selenium was administered by gavage daily (3mg/kg body wt/day) as Se‐methylselenocysteine, either from conception (Sel‐C), or weaning (Sel‐W). All groups were compared against a LIF‐no‐Sel control group. After 5 months HIF‐Sel‐W mice had elevated fasting glucose levels (HIF‐no sel 106.2 ± 2.76, HIF‐Sel‐W 121.6 ± 2.79 mg/dL, p=.008). LIF‐Sel‐W mice followed the same trend, but values were not significant (p=.109) Homeostatic Model Assessment of Insulin Resistance showed a 274% ± 36% increase of insulin resistance in the LIF‐Sel‐C group (p=.034), but not in HIF groups. Although blood‐glucose and insulin levels were affected by treatments, body weights and abdominal fat pads were not different, contrary to findings in other models. These results demonstrate an interactive effect of dietary isoflavone content and supplemental selenium on glucose regulation in the absence of changes in body fat.

Type 1 diabetes mellitus differentially regulates mitochondrially‐encoded proteins in cardiac mitochondrial subpopulations

Mitochondrial dysfunction is a contributing factor in diabetic cardiomyopathy. The mitochondrial genome encodes for thirteen proteins comprising essential constituents of the respiratory chain. Dysregulation of the mitochondrial genome has been shown to correlate with mitochondrial dysfunction. The purpose of this study was to determine whether the protein expression of mitochondrial genome‐encoded proteins were influenced during a type 1 diabetic insult in cardiac subsarcolemmal (SSM) and interfibrillar mitochondria (IFM). Male FVB mice were made diabetic through multiple low‐dose injections of streptozotocin and mitochondrial subpopulations were isolated from cardiac tissue five weeks post‐hyperglycemia. Western blot analyses revealed differential impact on mitochondrial genome encoded proteins from electron transport chain complexes I (ND2, ND3, ND4L, ND5, ND6), III, IV (CO1, CO2, CO3), and V (ATP6) (P&lt;0.05 for all). Of particular interest, was observed significant decreases to mitochondrially‐encoded electron transport chain complex IV proteins CO1 and CO3 within diabetic IFM (P&lt;0.05 for both). The results indicate that type 1 diabetes mellitus differentially regulates mitochondrially‐encoded electron transport chain complex constituents. Further, these effects appear specific for a given mitochondrial subpopulations.

Longitudinal assessment of type I diabetes mellitus using conventional echocardiography and speckle‐tracking based strain imaging

Cardiac complications, such as cardiomyopathy, are the leading cause of mortality among diabetic patients. Sensitive in vivo imaging is essential for assessment of cardiovascular function within small animal models. Echocardiographic speckle‐tracking based strain analysis has been suggested as a way to noninvasively evaluate cardiac performance in small animal models of acute pathological insult. The goal of this study was to provide a comparative longitudinal analysis of conventional echocardiographic and speckle‐tracking based strain imaging analyses in a small animal model of diabetes mellitus. Six week old male FVB mice were made diabetic through multiple low‐dose streptozotocin injections. M‐mode and B‐mode echocardiographic analyses revealed no differences in ejection fraction and fractional shortening in diabetic animals relative to control until approximately 5 weeks post diabetes onset (P&lt;0.05). Diabetic mice showed changes in average time‐to‐peak analysis using speckle‐tracking 1 week post diabetes onset and radial strain alterations at 4 weeks post diabetes onset relative to control (P&lt;0.05 for both). These findings indicate that assessment of myocardial strain may efficiently detect early contractile changes which may precede functional alterations observed with conventional M‐mode and B‐mode echocardiography. (Support: NIH DP2DK083095, NIH T32HL090610, AHA 10PRE3420006)

Differential expression of mitoKATP subunits in cardiac mitochondrial subpopulations and the influence of Type I diabetes

Mitochondrial dysfunction is an underlying risk for heart failure in Type I diabetes mellitus. MitoKATP channels are likely composed of an inwardly rectifying K+ channel subunit (Kir6.1) and a sulphonylurea receptor (SUR1). MitoKATP regulate mitochondrial volume and membrane potential: two functions disturbed by diabetes mellitus. In the heart, two mitochondrial populations exist: subsarcolemmal (SSM) and interfibrillar (IFM) which differ in their subcellular locations. It is not known whether: a) Kir6.1 or SUR1 expression is similar in IFM and SSM or whether b) diabetes mellitus alters the expression of Kir6.1 or SUR1 subunits in either SSM or IFM. Male FVB mice were made diabetic via multiple low‐dose injections of streptozoticin and examined after 5 weeks. Western blot analyses on cardiac mitochondrial subpopulations revealed Kir6.1 expression in non‐diabetic IFM was greater relative to SSM (p &lt; 0.05), but SUR1 expression was equivalent. Kir6.1 protein was reduced (p &lt; 0.05) in the IFM of diabetic mice. In contrast, diabetes mellitus had no effect on Kir6.1 protein expression in SSM or SUR1 expression in either IFM or SSM. These data show that expression Kir6.1 may be higher in IFM than SSM and that diabetes mellitus reduces Kir6.1 expression in IFM. This suggests that mitoKATP components may be differentially impacted by diabetes mellitus and that these effects may be specific to a subset of mitochondria.

Fiber‐type skeletal muscle response to dietary selenium and isoflavone supplementation in male mice

Previous research has shown selenium (Se) and isoflavone (IF) supplementation influences carbohydrate metabolism and insulin sensitivity. In response to different dietary IF contents and/or supplemental Se, we examined adaptations of mitochondrial proteins and FOXO1a in skeletal muscle. FOXO1a is a transcription factor that suppresses insulin sensitivity and alters glucose metabolism. Male FVB mice were fed diets for six months providing minimal IF or 500 mg/kg equivalents of genistein and daidzein. All other dietary components were equivalent. Selenium was administered by gavage daily (3mg/kg body wt/day as Se‐methylselenocysteine). Results show elevated dietary IF content decreased FOXO1a abundance in Tibialis Anterior (19±4.7%) and Red Quadriceps (RQ) (17±5%) vs. low IF. Supplemental Se decreased RQ FOXO1a levels in low IF mice (59±10% vs. control; p&lt;0.001). Our data show Se also decreases FOXO1a in White Quadriceps (12±4%) and RQ (17±9%) vs. no Se overall. As Se had the greatest effect by decreasing FOXO1a in the RQ, we investigated changes in mitochondrial content by measuring Uncoupling Protein 3 (UCP3) and Cytochrome C (CytC) in the same muscle. Both increased in response to supplemental Se (CytC 126±43%, UCP3 99±31.5% elevated vs. control, p&lt;0.05). These data suggest fiber‐type specific adaptation in response to supplemental Se and elevated dietary isoflavone content.

The effect of naringenin on the phosphorylation of AMPK in diet‐induced obese mice

Previously, we reported that naringenin, the predominant flavanone in grapefruit, acted like metformin to decrease glucose output from hepatocytes. The objective of this study is to investigate whether dietary naringenin modulates energy balance and metabolism through p‐AMPK, a central regulator of glucose and lipid metabolism in several tissues. Male FVB mice were fed a high fat diet for 19 days, and then switched to AIN‐93G diet with (N= 10) or without (N=8) naringenin for 13 days. Body weight and food intake of mice fed naringenin were not significantly different than values in mice fed diet without naringenin. Mice were fasted for 12 hr then necropsy performed. There was a trend of enhanced p‐AMPK:AMPK in quadriceps muscle in mice fed diet with naringenin but p‐AMPK:AMPK was not significantly different in liver or inguinal fat pad. The induction of p‐AMPK in skeletal muscle suggests that naringenin exerts effects on energy pathways regulated by AMPK. Currently, we are conducting a new study to investigate the effects of various doses of naringenin on p‐AMPK activity and energy balance and metabolism in a mouse model for diet‐induced obesity.

Fresh Green Tea and Gallic Acid Ameliorate Oxidative Stress in Kainic Acid-Induced Status Epilepticus

Green tea is one of the most-consumed beverages due to its taste and antioxidative polyphenols. However, the protective effects of green tea and its constituent, gallic acid (GA), against kainic acid (KA)-induced seizure have not been studied. We investigated the effect of fresh green tea leaf (GTL) and GA on KA-induced neuronal injury in vivo and in vitro. The results showed that GTL and GA reduced the maximal seizure classes, predominant behavioral seizure patterns, and lipid peroxidation in male FVB mice with status epilepticus (SE). GTL extract and GA provided effective protection against KA-stressed PC12 cells in a dose-dependent manner. In the protective mechanism study, GTL and GA decreased Ca(2+) release, ROS, and lipid peroxidation from KA-stressed PC12 cells. Western blot results revealed that mitogen-activated protein kinases (MAPKs), RhoA, and COX-2 expression were increased in PC12 cells under KA stress, and expression of COX-2 and p38 MAPK, but not RhoA, was significantly reduced by GTL and GA. Furthermore, GTL and GA were able to reduce PGE(2) production from KA-stressed PC12 cells. Taken together, the results showed that GTL and GA provided neuroprotective effects against excitotoxins and may have a clinical application in epilepsy.

N-nitroso-tris-chloroethylurea Induces Premalignant Squamous Dysplasia in Mice

Squamous cell carcinoma (SCC) and premalignant endobronchial lesions have been difficult to study in murine models. In this study, we evaluate the topical N-nitroso-tris-chloroethylurea (NTCU) murine SCC model, determine the extent to which resulting premalignant airway dysplasia develops, discuss clinicopathologic grading criteria in lesion progression, and confirm that immunohistochemical (IHC) staining patterns are consistent with those observed in human endobronchial dysplasia and SCC. Male and female FVB mice were treated biweekly with topical NTCU (4, 8, or 40 mmol/L) or vehicle for 32 weeks. Following sacrifice, squamous cell lesions were enumerated and categorized into the following groups: flat atypia, low-grade dysplasia, high-grade dysplasia, and invasive SCC. The 40 mmol/L NTCU concentration produced the entire spectrum of premalignant dysplasias and squamous cell carcinomas, but was associated with poor survival. Concentrations of 4 and 8 mmol/L NTCU were better tolerated and produced only significant levels of flat atypia. Squamous origin of the range of observed lesions was confirmed with IHC staining for cytokeratin 5/6, p63, thyroid transcription factor-1 (TTF-1), and Napsin-A. This study shows that topical application of high-dose NTCU produces endobronchial premalignant lesions with classic squamous characteristics and should allow for improved preclinical evaluation of potential chemopreventive agents.

Molecular Imaging of Bone Marrow Mononuclear Cell Survival and Homing in Murine Peripheral Artery Disease

This study aims to provide insight into cellular kinetics using molecular imaging after different transplantation methods of bone marrow-derived mononuclear cells (MNCs) in a mouse model of peripheral artery disease (PAD). MNC therapy is a promising treatment for PAD. Although clinical translation has already been established, there is a lack of knowledge about cell behavior after transplantation and about the mechanism whereby MNC therapy might ameliorate complaints of PAD. MNCs were isolated from F6 transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). Male FVB and C57Bl6 mice (n = 50) underwent femoral artery ligation and were randomized into 4 groups receiving the following: 1) single intramuscular (IM) injection of 2 × 10(6) MNCs; 2) 4 weekly IM injections of 5 × 10(5) MNCs; 3) 2 × 10(6) MNCs intravenously; and 4) phosphate-buffered saline as control. Cells were characterized by flow cytometry and in vitro bioluminescence imaging (BLI). Cell survival, proliferation, and migration were monitored by in vivo BLI, which was validated by ex vivo BLI, post-mortem immunohistochemistry, and flow cytometry. Paw perfusion and neovascularization was measured with laser Doppler perfusion imaging (LDPI) and histology, respectively. In vivo BLI revealed near-complete donor cell death 4 weeks after IM transplantation. After intravenous transplantation, BLI revealed that cells migrated to the injured area in the limb, as well as to the liver, spleen, and bone marrow. Ex vivo BLI showed presence of MNCs in the scar tissue and adductor muscle. However, no significant effects on neovascularization were observed, as monitored by LDPI and histology. This is one of the first studies to assess kinetics of transplanted MNCs in PAD using in vivo molecular imaging. MNC survival is short-lived, MNCs do not preferentially home to injured areas, and MNCs do not significantly stimulate perfusion in this particular model.

Safrole-2′,3′-oxide induces cytotoxic and genotoxic effects in HepG2 cells and in mice

Safrole-2′,3′-oxide (SAFO) is a reactive electrophilic metabolite of the hepatocarcinogen safrole, the main component of sassafras oil. Safrole occurs naturally in a variety of spices and herbs, including the commonly used Chinese medicine Xi xin (Asari Radix et Rhizoma) and Dong quai (Angelica sinensis). SAFO is the most mutagenic metabolite of safrole tested in the Ames test. However, little or no data are available on the genotoxicity of SAFO in mammalian systems. In this study, we investigated the cytotoxicity and genotoxicity of SAFO in human HepG2 cells and male FVB mice. Using MTT assay, SAFO exhibited a dose- and time-dependent cytotoxic effect in HepG2 cells with TC50 values of 361.9μM and 193.2μM after 24 and 48h exposure, respectively. In addition, treatment with SAFO at doses of 125μM and higher for 24h in HepG2 cells resulted in a 5.1–79.6-fold increase in mean Comet tail moment by the alkaline Comet assay and a 2.6–7.8-fold increase in the frequency of micronucleated binucleated cells by the cytokinesis-block micronucleus assay. Furthermore, repeated intraperitoneal administration of SAFO (15, 30, 45, and 60mg/kg) to mice every other day for a total of twelve doses caused a significant dose-dependent increase in mean Comet tail moment in peripheral blood leukocytes (13.3–43.4-fold) and in the frequency of micronucleated reticulocytes (1.5–5.8-fold). Repeated administration of SAFO (60mg/kg) to mice caused liver lesions manifested as a rim of ballooning degeneration of hepatocytes immediately surrounding the central vein. Our data clearly demonstrate that SAFO significantly induced cytotoxicity, DNA strand breaks, micronuclei formation both in human cells in vitro and in mice. More studies are needed to explore the role SAFO plays in safrole-induced genotoxicity.

Development of a castrate resistant transplant tumor model of prostate cancer

Currently, limited mouse models that mimic the clinical course of castrate resistant prostate development currently exist. Such mouse models are urgently required to conduct pre-clinical studies to assist in the understanding of disease progression and the development of rational therapeutic strategies to treat castrate resistant prostate cancer. Wild type intact FVB male mice were injected by subcutaneous injection with Myc-CaP cells to establish androgen sensitive Myc-CaP tumors. Tumor bearing mice were castrated and resulting tumors serially passaged in pre-castrated FVB male mice to produce a bone fide Myc-CaP castrate resistant tumor. Immunohistochemical analysis revealed that initial androgen sensitive Myc-CaP tumors had strong nuclear transcriptional active androgen receptor expression, as indicated by marked c-MYC staining and were highly proliferative. Castration of tumor bearing animals resulted in cytoplasmic relocation of androgen receptor concurrent with loss of transcriptional activity and tumor proliferation. Serial passaging of castrate refractory Myc-CaP in pre-castrated male FVB mice resulted in the development of a bona fide castrate resistant Myc-CaP tumor which pheno-copied the original androgen sensitive parental Myc-CaP tumor. Developing a murine castrate transplant resistant tumor model that mimics the clinical course of human castrate resistant prostate cancer will create better opportunities to understand the development of castrate resistant prostate cancer and also allow for more rapid pre-clinical studies to stratify rational novel therapies for this lethal form of prostate cancer.

Central nervous system exposure of next generation quinoline methanols is reduced relative to mefloquine after intravenous dosing in mice

BackgroundThe clinical use of mefloquine (MQ) has declined due to dose-related neurological events. Next generation quinoline methanols (NGQMs) that do not accumulate in the central nervous system (CNS) to the same extent may have utility. In this study, CNS levels of NGQMs relative to MQ were measured and an early lead chemotype was identified for further optimization.Experimental designThe plasma and brain levels of MQ and twenty five, 4-position modified NGQMs were determined using LCMS/MS at 5 min, 1, 6 and 24 h after IV administration (5 mg/kg) to male FVB mice. Fraction unbound in brain tissue homogenate was assessed in vitro using equilibrium dialysis and this was then used to calculate brain-unbound concentration from the measured brain total concentration. A five-fold reduction CNS levels relative to mefloquine was considered acceptable. Additional pharmacological properties such as permeability and potency were determined.ResultsThe maximum brain (whole/free) concentrations of MQ were 1807/4.9 ng/g. Maximum whole brain concentrations of NGQMs were 23 - 21546 ng/g. Maximum free brain concentrations were 0.5 to 267 ng/g. Seven (28%) and two (8%) compounds exhibited acceptable whole and free brain concentrations, respectively. Optimization of maximum free brain levels, IC90s (as a measure or potency) and residual plasma concentrations at 24 h (as a surrogate for half-life) in the same molecule may be feasible since they were not correlated. Diamine quinoline methanols were the most promising lead compounds.ConclusionReduction of CNS levels of NGQMs relative to mefloquine may be feasible. Optimization of this property together with potency and long half-life may be feasible amongst diamine quinoline methanols.

Adrenergic Regulation of AMP-activated Protein Kinase in Brown Adipose Tissue in Vivo

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK α2 protein and activity were reduced by 45%. In β(1,2,3)-adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr(172) phosphorylation. In contrast, acute α- or β-adrenergic blockade in WT mice resulted in increased AMPK α Thr(172) phosphorylation and AMPK α1 and α2 activity in BAT. But short term blockade of α-adrenergic signaling in β(1,2,3)-adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser(485/491), a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser(485/491) phosphorylation resulting from α-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of α-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr(172) phosphorylation and chronically through changes in the α catalytic subunit protein levels. Furthermore, AMPK α Ser(485/491) phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects.