Talaignair N. Venkatraman

Hedgehog Signaling Antagonist Promotes Regression of Both Liver Fibrosis and Hepatocellular Carcinoma in a Murine Model of Primary Liver Cancer

Objective Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50–60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2−/− mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC. Methods Hepatic injury and fibrosis, Hh pathway activation, and liver progenitor populations were compared in Mdr2−/− mice and age-matched wild type controls. A dose finding experiment with the Hh signaling antagonist GDC-0449 was performed to optimize Hh pathway inhibition. Mice were then treated with GDC-0449 or vehicle for 9 days, and effects on liver fibrosis and tumor burden were assessed by immunohistochemistry, qRT-PCR, Western blot, and magnetic resonance imaging. Results Unlike controls, Mdr2−/− mice consistently expressed Hh ligands and progressively accumulated Hh-responsive liver myofibroblasts and progenitors with age. Treatment of aged Mdr2-deficient mice with GDC-0449 significantly inhibited hepatic Hh activity, decreased liver myofibroblasts and progenitors, reduced liver fibrosis, promoted regression of intra-hepatic HCCs, and decreased the number of metastatic HCC without increasing mortality. Conclusions Hh pathway activation promotes liver fibrosis and hepatocarcinogenesis, and inhibiting Hh signaling safely reverses both processes even when fibrosis and HCC are advanced.

Xenon Neuroprotection in Experimental Stroke Interactions with Hypothermia and Intracerebral Hemorrhage

Background:Xenon has been proven to be neuroprotective in experimental brain injury. The authors hypothesized that xenon would improve outcome from focal cerebral ischemia with a delayed treatment onset and prolonged recovery interval. Methods:Rats were subjected to 70 min temporary focal ischemia. Ninety minutes later, rats were treated with 0, 15, 30, or 45% Xe for 20 h or 0 or 30% Xe for 8, 20, or 44 h. Outcome was measured after 7 days. In another experiment, after ischemia, rats were maintained at 37.5° or 36.0°C for 20 h with or without 30% Xe. Outcome was assessed 28 days later. Finally, mice were subjected to intracerebral hemorrhage with or without 30% Xe for 20 h. Brain water content, hematoma volume, rotarod function, and microglial activation were measured. Results:Cerebral infarct sizes (mean ± SD) for 0, 15, 30, and 45% Xe were 212 ± 27, 176 ± 55, 160 ± 32, and 198 ± 54 mm3, respectively (P = 0.023). Neurologic scores (median ± interquartile range) followed a similar pattern (P = 0.002). Infarct size did not vary with treatment duration, but neurologic score improved (P = 0.002) at all xenon exposure durations (8, 20, and 44 h). Postischemic treatment with either 30% Xe or subtherapeutic hypothermia (36°C) had no effect on 28-day outcome. Combination of these interventions provided long-term benefit. Xenon improved intracerebral hemorrhage outcome measures. Conclusion:Xenon improved focal ischemic outcome at 7, but not 28 days postischemia. Xenon combined with subtherapeutic hypothermia produced sustained recovery benefit. Xenon improved intracerebral hemorrhage outcome. Xenon may have potential for clinical stroke therapy under carefully defined conditions.

Brain Natriuretic Peptide Improves Long-Term Functional Recovery after Acute CNS Injury in Mice

There is emerging evidence to suggest that brain natriuretic peptide (BNP) is elevated after acute brain injury, and that it may play an adaptive role in recovery through augmentation of cerebral blood flow (CBF). Through a series of experiments, we tested the hypothesis that the administration of BNP after different acute mechanisms of central nervous system (CNS) injury could improve functional recovery by improving CBF. C57 wild-type mice were exposed to either pneumatic-induced closed traumatic brain injury (TBI) or collagenase-induced intracerebral hemorrhage (ICH). After injury, either nesiritide (hBNP) (8 microg/kg) or normal saline were administered via tail vein injection at 30 min and 4 h. The mice then underwent functional neurological testing via rotorod latency over the following 5 days and neurocognitive testing via Morris water maze testing on days 24-28. Cerebral blood flow (CBF) was assessed by laser Doppler from 25 to 90 min after injury. After ICH, mRNA polymerase chain reaction (PCR) and histochemical staining were performed during the acute injury phase (<24 h) to determine the effects on inflammation. Following TBI and ICH, administration of hBNP was associated with improved functional performance as assessed by rotorod and Morris water maze latencies (p < 0.01). CBF was increased (p < 0.05), and inflammatory markers (TNF-alpha and IL-6; p < 0.05), activated microglial (F4/80; p < 0.05), and neuronal degeneration (Fluoro-Jade B; p < 0.05) were reduced in mice receiving hBNP. hBNP improves neurological function in murine models of TBI and ICH, and was associated with enhanced CBF and downregulation of neuroinflammatory responses. hBNP may represent a novel therapeutic strategy after acute CNS injury.

BIOCHEMICAL ABNORMALITIES OF THE MEDIAL TEMPORAL LOBE AND MEDIAL PREFRONTAL CORTEX IN LATE-LIFE DEPRESSION

We utilized single-voxel (1)H magnetic resonance spectroscopy (MRS) to investigate biochemical abnormalities related to late-life depression in the medial prefrontal cortex and medial temporal lobe. Fourteen elderly subjects whose depression responded to treatment and 12 nondepressed subjects were enrolled. Subjects were scanned using a GE 3.0 Tesla whole body MR scanner. Metabolite concentrations were quantified using the LC Model software and adjusted for CSF and ratio of gray to white matter. ANCOVA models tested for group differences while controlling for age and sex. Older previously depressed individuals showed significantly reduced concentrations of total N-acetyl aspartate (NAA), choline, and creatine in the prefrontal cortex and significantly elevated left medial temporal lobe concentrations of NAA and myo-inositol. There were no significant group differences in right temporal metabolite concentrations. The prefrontal cortex observations suggest that reduced neuronal, phospolipid, and energy metabolism is present even in clinically improved depression. In contrast, elevated NAA and myo-inositol concentrations in the left medial temporal lobe could be associated with neuronal and glial cell changes in the amygdala.

Anticancer therapeutic potential of Mn porphyrin/ascorbate system

Ascorbate (Asc) as a single agent suppressed growth of several tumor cell lines in a mouse model. It has been tested in a Phase I Clinical Trial on pancreatic cancer patients where it exhibited no toxicity to normal tissue yet was of only marginal efficacy. The mechanism of its anticancer effect was attributed to the production of tumoricidal hydrogen peroxide (H2O2) during ascorbate oxidation catalyzed by endogenous metalloproteins. The amount of H2O2 could be maximized with exogenous catalyst that has optimized properties for such function and is localized within tumor. Herein we studied 14 Mn porphyrins (MnPs) which differ vastly with regards to their redox properties, charge, size/bulkiness and lipophilicity. Such properties affect the in vitro and in vivo ability of MnPs (i) to catalyze ascorbate oxidation resulting in the production of H2O2; (ii) to subsequently employ H2O2 in the catalysis of signaling proteins oxidations affecting cellular survival pathways; and (iii) to accumulate at site(s) of interest. The metal-centered reduction potential of MnPs studied, E1/2 of Mn(III)P/Mn(II)P redox couple, ranged from -200 to +350 mV vs NHE. Anionic and cationic, hydrophilic and lipophilic as well as short- and long-chained and bulky compounds were explored. Their ability to catalyze ascorbate oxidation, and in turn cytotoxic H2O2 production, was explored via spectrophotometric and electrochemical means. Bell-shape structure-activity relationship (SAR) was found between the initial rate for the catalysis of ascorbate oxidation, vo(Asc)ox and E1/2, identifying cationic Mn(III) N-substituted pyridylporphyrins with E1/2>0 mV vs NHE as efficient catalysts for ascorbate oxidation. The anticancer potential of MnPs/Asc system was subsequently tested in cellular (human MCF-7, MDA-MB-231 and mouse 4T1) and animal models of breast cancer. At the concentrations where ascorbate (1mM) and MnPs (1 or 5 µM) alone did not trigger any alteration in cell viability, combined treatment suppressed cell viability up to 95%. No toxicity was observed with normal human breast epithelial HBL-100 cells. Bell-shape relationship, essentially identical to vo(Asc)oxvs E1/2, was also demonstrated between MnP/Asc-controlled cytotoxicity and E1/2-controlled vo(Asc)ox. Magnetic resonance imaging studies were conducted to explore the impact of ascorbate on T1-relaxivity. The impact of MnP/Asc on intracellular thiols and on GSH/GSSG and Cys/CySS ratios in 4T1 cells was assessed and cellular reduction potentials were calculated. The data indicate a significant increase in cellular oxidative stress induced by MnP/Asc. Based on vo(Asc)oxvs E1/2 relationships and cellular toxicity, MnTE-2-PyP(5+) was identified as the best catalyst among MnPs studied. Asc and MnTE-2-PyP(5+) were thus tested in a 4T1 mammary mouse flank tumor model. The combination of ascorbate (4 g/kg) and MnTE-2-PyP(5+) (0.2mg/kg) showed significant suppression of tumor growth relative to either MnTE-2-PyP(5+) or ascorbate alone. About 7-fold higher accumulation of MnTE-2-PyP(5+) in tumor vs normal tissue was found to contribute largely to the anticancer effect.

ApolipoproteinE mimetic peptides improve outcome after focal ischemia.

Growing clinical evidence implicates isoform-specific effects of apolipoprotein E (apoE) in reducing neuroinflammation and mediating adaptive responses following ischemic and traumatic brain injury. However, the intact apoE holoprotein does not cross the blood-brain barrier and thus has limited therapeutic potential. We have created a small peptide, COG1410 (acetyl-AS-Aib-LRKL-Aib-KRLL-amide), derived from the apoE receptor-binding region. COG1410 retains the anti-inflammatory and neuroprotective biological properties of the intact holoprotein and penetrates the blood-brain barrier. In the current study, we utilized a murine model of transient focal cerebral ischemia and reperfusion to demonstrate that intravenous (IV) administration of COG1410 reduces infarct volume and radiographic progression of infarct, and improves functional outcome as assessed by rotarod when delivered up to 4h after ischemia onset.

Effects of Deep Hypothermic Circulatory Arrest on the Blood Brain Barrier in a Cardiopulmonary Bypass Model – A Pilot Study

BACKGROUND Neurologic injury is common after cardiac surgery and disruption of the blood brain barrier (BBB) has been proposed as a contributing factor. We sought to study BBB characteristics in a rodent model of cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). METHODS Adult rats were subjected to CPB/DHCA or to sham surgery. Analysis included Western blotting of relevant BBB proteins in addition to in vivo brain magnetic resonance imaging (MRI) with a clinically used low-molecular contrast agent. RESULTS While quantitative analysis of BBB proteins revealed similar expression levels, MRI showed evidence of BBB disruption after CPB/DHCA compared to sham surgery. CONCLUSIONS Combining molecular BBB analysis and MRI technology in a rodent model is a highly translatable approach to study adverse neurologic outcomes following CPB/DHCA.

Mn Porphyrins as Novel Molecular Magnetic Resonance Imaging Contrast Agents

BACKGROUND AND PURPOSE In this study, we investigated the potential of a new class of therapeutic Mn porphyrins as molecular MRI probes for prostate cancer imaging. Two compounds of different bioavailibility were investigated: Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)) and Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP(5+)). These compounds have previously been shown to have adjunctive antineoplastic activity through their actions as powerful superoxide dismutase mimics, peroxynitrite scavengers, and modulators of cellular redox-based signaling pathways. Strong paramagnetic MRI contrast properties and affinity for cancer cells suggest their potential application as novel diagnostic imaging agents. MATERIALS AND METHODS MRI experiments were performed at 7.0T on a Bruker Biospec horizontal bore scanner. All in-vivo experiments were performed on 12 C57 black mice implanted with RM-9 prostate cancer cells on the hind limb. Two mg/kg of MnTnHex-2-PyP(5+) (n=6) and 8 mg/kg MnTE-2-PyP(5+) (n=6) were administered intraperitoneally 90 minutes before imaging. All the images were collected using a volume coil and processed using Paravision 4.0. RESULTS Phantom studies reveal remarkably high T1 relaxivity changes for both metalloporphyrins, which are twofold to threefold higher than commercially available gadolinium chelates. Observable detection limits using conventional T1-weighted MRI are in the low micromolar range for both compounds. In vivo, MR relaxation changes in prostate tumor xenografts were readily observed after a single injection of either MnTE-2-PyP(5+)or MnTnHex-2-PyP(5+), with tumor contrast to background ratio greatest after MnTE-2-PyP(5+) administration. CONCLUSION After a single dose of MnTE-2-PyP(5+), contrast changes in prostate tumors are up to sixfold greater than in surrounding, noncancerous tissues, suggesting the potential use of this metalloporphyrin as a novel diagnostic probe for detecting prostate malignancy using MRI.

The role of angiogenesis in Group 3 medulloblastoma pathogenesis and survival

Background Of the 4 medulloblastoma subgroups, Group 3 is the most aggressive but the importance of angiogenesis is unknown. This study sought to determine the role of angiogenesis and identify clinically relevant biomarkers of tumor vascularity and survival in Group 3 medulloblastoma. Methods VEGFA mRNA expression and survival from several patient cohorts were analyzed. Group 3 xenografts were implanted intracranially in nude rats. Dynamic susceptibility weighted (DSC) MRI and susceptibility weighted imaging (SWI) were obtained. DSC MRI was used to calculate relative cerebral blood volume (rCBV) and flow (rCBF). Tumor vessel density and rat vascular endothelial growth factor alpha (VEGFA) expression were determined. Results Patient VEGFA mRNA levels were significantly elevated in Group 3 compared with the other subgroups (P < 0.001) and associated with survival. Xenografts D283, D341, and D425 were identified as Group 3 by RNA hierarchical clustering and MYC amplification. The D283 group had the lowest rCBV and rCBF, followed by D341 and D425 (P < 0.05). These values corresponded to histological vessel density (P < 0.05), rat VEGFA expression (P < 0.05), and survival (P = 0.002). Gene set enrichment analysis identified 5 putative genes with expression profiles corresponding with these findings: RNH1, SCG2, VEGFA, AGGF1, and PROK2. SWI identified 3 xenograft-independent categories of intratumoral vascular architecture with distinct survival (P = 0.004): organized, diffuse microvascular, and heterogeneous. Conclusions Angiogenesis plays an important role in Group 3 medulloblastoma pathogenesis and survival. DSC MRI and SWI are clinically relevant biomarkers for tumor vascularity and overall survival and can be used to direct the use of antivascular therapies for patients with Group 3 medulloblastoma.

Neuroprotective pentapeptide CN-105 improves functional and histological outcomes in a murine model of intracerebral hemorrhage.

Presently, no pharmacological treatments have been demonstrated to improve long-term functional outcomes following intracerebral hemorrhage (ICH). Clinical evidence associates apolipoprotein E (apoE) genotype with ICH incidence and outcome. While apoE modifies neuroinflammatory responses through its adaptive role in glial downregulation, intact apoE holoprotein is too large to cross the blood-brain barrier (BBB). Therefore, we developed a 5-amino acid peptide – CN-105 – that mimics the polar face of the apoE helical domain involved in receptor interactions. In the current study, we investigated the therapeutic potential of CN-105 in a mouse model of ICH. Three doses of CN-105 (0.05 mg/kg) was administered by tail vein injection within 24 hours after ICH induction. Functional assessment showed durable improvement in vestibulomotor performance after CN-105 treatment, as quantified by increased Rotarod latencies on Days 1–5 post-ICH, and long-term improvement in neurocognitive performance, as quantified by reduced Morris water maze latencies on Days 29–32 post-ICH. Further, brain water content was significantly reduced, neuroinflammation was decreased and hippocampal CA3 neuronal survival was increased, although hemorrhage volume was not affected by CN-105. We concluded, therefore, that pentapeptide CN-105 improved short- and long-term neurobehavioral outcomes in a murine model of ICH, suggesting therapeutic potential for patients with acute ICH.