Theodor K. Bammler

Recombinant erythropoietin is neuroprotective in a novel mouse oxidative injury model

To identify neuroprotective changes in gene expression, we developed a neonatal mouse model of moderate to severe oxidative brain injury and hypothesized that recombinant erythropoietin (rEpo) would decrease the expression of proapoptotic and proinflammatory genes 24 and 48 h, respectively, after injury and increase the expression of neurogenic and angiogenic genes 168 h after injury. Postnatal day 10 BALB-c mice underwent sham surgery or right common carotid artery occlusion followed by alternating hypoxia and hyperoxia and were then treated with rEpo (5,000 U/kg s.c.) or saline (vehicle) daily for up to three doses. At death, gross brain injury was assessed, then hippocampus, cortex, and thalamus were isolated for RNA or protein extraction. Microarray analysis, real-time polymerase chain reaction, and Bio-Plex® suspension array system validation were performed. rEpo decreased both incidence and severity of brain injury (median injury score 3 vs. 0, p < 0.0001) and reduced the injury-induced increases in interleukin-1α and interleukin-6 gene expression (p < 0.001), with corresponding effects on protein translation. Similarly, the expression of caspase-1, caspase-4, and caspase-6 and of p53 was increased by brain injury at 24 h, but mitigated by rEpo (p < 0.01). The interleukin-10 expression was higher in the rEpo-treated animals. Apoptotic and proinflammatory gene expression persisted for 168 h. There was no increase in angiogenic gene expression at the time points studied.

Expression levels of DNA replication and repair genes predict regional somatic repeat instability in the brain but are not altered by polyglutamine disease protein expression or age

Expansion of CAG/CTG trinucleotide repeats causes numerous inherited neurological disorders, including Huntingtons disease (HD), several spinocerebellar ataxias and myotonic dystrophy type 1. Expanded repeats are genetically unstable with a propensity to further expand when transmitted from parents to offspring. For many alleles with expanded repeats, extensive somatic mosaicism has been documented. For CAG repeat diseases, dramatic instability has been documented in the striatum, with larger expansions noted with advancing age. In contrast, only modest instability occurs in the cerebellum. Using microarray expression analysis, we sought to identify the genetic basis of these regional instability differences by comparing gene expression in the striatum and cerebellum of aged wild-type C57BL/6J mice. We identified eight candidate genes enriched in cerebellum, and validated four--Pcna, Rpa1, Msh6 and Fen1--along with a highly associated interactor, Lig1. We also explored whether expression levels of mismatch repair (MMR) proteins are altered in a line of HD transgenic mice, R6/2, that is known to show pronounced regional repeat instability. Compared with wild-type littermates, MMR expression levels were not significantly altered in R6/2 mice regardless of age. Interestingly, expression levels of these candidates were significantly increased in the cerebellum of control and HD human samples in comparison to striatum. Together, our data suggest that elevated expression levels of DNA replication and repair proteins in cerebellum may act as a safeguard against repeat instability, and may account for the dramatically reduced somatic instability present in this brain region, compared with the marked instability observed in the striatum.

The Cycad Genotoxin MAM Modulates Brain Cellular Pathways Involved in Neurodegenerative Disease and Cancer in a DNA Damage-Linked Manner

Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O 6-methyldeoxyguanosine lesions, O 6-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O 6-mG DNA methyltransferase (MGMT) showed elevated O 6-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimers disease.

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: evidence for stromal induction of hepatocellular carcinoma.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.

Genetic polymorphisms of catechol-O-methyltransferase modify the neurobehavioral effects of mercury in children.

Mercury (Hg) is neurotoxic and children may be particularly susceptible to this effect. A current major challenge is identification of children who may be uniquely susceptible to Hg toxicity because of genetic disposition. This study examined the hypothesis that genetic variants of catechol-O-methyltransferase (COMT) that are reported to alter neurobehavioral functions that are also affected by Hg in adults might modify the adverse neurobehavioral effects of Hg exposure in children. Five hundred and seven children, 8–12 yr of age at baseline, participated in a clinical trial to evaluate the neurobehavioral effects of Hg from dental amalgam tooth fillings. Subjects were evaluated at baseline and at seven subsequent annual intervals for neurobehavioral performance and urinary Hg levels. Following the clinical trial, genotyping assays were performed for single-nucleotide polymorphisms (SNPs) of COMT rs4680, rs4633, rs4818, and rs6269 on biological samples provided by 330 of the trial participants. Regression-modeling strategies were employed to evaluate associations between allelic status, Hg exposure, and neurobehavioral test outcomes. Similar analysis was performed using haplotypes of COMT SNPs. Among girls, few interactions for Hg exposure and COMT variants were found. In contrast, among boys, numerous gene–Hg interactions were observed between individual COMT SNPs, as well as with a common COMT haplotype affecting multiple domains of neurobehavioral function. These findings suggest increased susceptibility to the adverse neurobehavioral effects of Hg among children with common genetic variants of COMT, and may have important implications for strategies aimed at protecting children from the potential health risks associated with Hg exposure.

CHF1/Hey2 Promotes physiological hypertrophy in response to pressure overload through selective repression and activation of specific transcriptional pathways

We have previously found that CHF1/Hey2 prevents the development of phenylephrine-induced cardiac hypertrophy. To determine the role of CHF1/Hey2 in pressure overload hypertrophy, we performed ascending aortic banding on wild-type and transgenic mice overexpressing CHF1/Hey2 in the myocardium. We found that both wild-type and transgenic mice developed increased ventricular weight to body weight ratios 1 week after aortic banding. Wild-type mice also developed decreased fractional shortening after 1 week when compared to preoperative echocardiograms and sham-operated controls. Transgenic mice, in comparison, demonstrated preserved fractional shortening. Histological examination of explanted heart tissue demonstrated extensive fibrosis in wild-type hearts, but minimal fibrosis in transgenic hearts. TUNEL staining demonstrated increased apoptosis in the wild-type hearts but not in the transgenic hearts. Exposure of cultured neonatal myocytes from wild-type and transgenic animals to hydrogen peroxide, a potent inducer of apoptosis, demonstrated increased apoptosis in the wild-type cells. Gene Set Analysis of microarray data from wild-type and transgenic hearts 1 week after banding revealed suppression and activation of multiple pathways involving apoptosis, cell signaling, and biosynthesis. These findings demonstrate that CHF1/Hey2 promotes physiological over pathological hypertrophy through suppression of apoptosis and regulation of multiple transcriptional pathways. These findings also suggest that CHF1/Hey2 and its downstream pathways provide a variety of targets for novel heart failure drug discovery, and that genetic polymorphisms in CHF1/Hey2 may affect susceptibility to hypertrophy and heart failure.

Transcriptome correlation analysis identifies two unique craniosynostosis subtypes associated with IRS1 activation.

The discovery of causal mechanisms associated with nonsyndromic craniosynostosis has proven to be a difficult task due to the complex nature of the disease. In this study, differential transcriptome correlation analysis was used to identify two molecularly distinct subtypes of nonsyndromic craniosynostosis, termed subtype A and subtype B. In addition to unique correlation structure, subtype A was also associated with high IGF pathway expression, whereas subtype B was associated with high integrin expression. To identify a pathologic link between altered gene correlation/expression and the disease state, phosphorylation assays were performed on primary osteoblast cell lines derived from cases within subtype A or subtype B, as well as on primary osteoblast cell lines with novel IGF1R variants previously reported by our lab (Cunningham ML, Horst JA, Rieder MJ, Hing AV, Stanaway IB, Park SS, Samudrala R, Speltz ML. Am J Med Genet A 155A: 91-97, 2011). Elevated IRS1 (pan-tyr) and GSK3β (ser-9) phosphorylation were observed in two novel IGF1R variants with receptor L domain mutations. In subtype A, a hypomineralization phenotype coupled with decreased phosphorylation of IRS1 (ser-312), p38 (thr-180/tyr-182), and p70S6K (thr-412) was observed. In subtype B, decreased phosphorylation of IRS1 (ser-312) as well as increased phosphorylation of Akt (ser-473), GSK3β (ser-9), IGF1R (tyr-1135/tyr-1136), JNK (thr-183/tyr-187), p70S6K (thr-412), and pRPS6 (ser-235/ser-236) was observed, thus implicating the activation of IRS1-mediated Akt signaling in potentiating craniosynostosis in this subtype. Taken together, these results suggest that despite the stimulation of different pathways, activating phosphorylation patterns for IRS1 were consistent in cell lines from both subtypes and the IGF1R variants, thus implicating a key role for IRS1 in the pathogenesis of nonsyndromic craniosynostosis.

Transcriptional analysis of human cranial compartments with different embryonic origins

OBJECTIVE Previous investigations suggest that the embryonic origins of the calvarial tissues (neural crest or mesoderm) may account for the molecular mechanisms underlying sutural development. The aim of this study was to evaluate the differences in the gene expression of human cranial tissues and assess the presence of an expression signature reflecting their embryonic origins. METHODS Using microarray technology, we investigated global gene expression of cells from the frontal and parietal bones and the metopic and sagittal intrasutural mesenchyme (ISM) of four human foetal calvaria. qRT-PCR of a selected group of genes was done to validate the microarray analysis. Paired comparison and correlation analyses were performed on microarray results. RESULTS Of six paired comparisons, frontal and parietal compartments (distinct tissue types of calvaria, either bone or intrasutural mesenchyme) had the most different gene expression profiles despite being composed of the same tissue type (bone). Correlation analysis revealed two distinct gene expression profiles that separate frontal and metopic compartments from parietal and sagittal compartments. TFAP2A, TFAP2B, ICAM1, SULF1, TNC and FOXF2 were among differentially expressed genes. CONCLUSION Transcriptional profiles of two groups of tissues, frontal and metopic compartments vs. parietal and sagittal compartments, suggest differences in proliferation, differentiation and extracellular matrix production. Our data suggest that in the second trimester of human foetal development, a gene expression signature of neural crest origin still exists in frontal and metopic compartments while gene expression of parietal and sagittal compartments is more similar to mesoderm.

The pulmonary inflammatory response to multiwalled carbon nanotubes is influenced by gender and glutathione synthesis

Inhalation of multiwalled carbon nanotubes (MWCNTs) during their manufacture or incorporation into various commercial products may cause lung inflammation, fibrosis, and oxidative stress in exposed workers. Some workers may be more susceptible to these effects because of differences in their ability to synthesize the major antioxidant and immune system modulator glutathione (GSH). Accordingly, in this study we examined the influence of GSH synthesis and gender on MWCNT-induced lung inflammation in C57BL/6 mice. GSH synthesis was impaired through genetic manipulation of Gclm, the modifier subunit of glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis. Twenty-four hours after aspirating 25 µg of MWCNTs, all male mice developed neutrophilia in their lungs, regardless of Gclm genotype. However, female mice with moderate (Gclm heterozygous) and severe (Gclm null) GSH deficiencies developed significantly less neutrophilia. We found no indications of MWCNT-induced oxidative stress as reflected in the GSH content of lung tissue and epithelial lining fluid, 3-nitrotyrosine formation, or altered mRNA or protein expression of several redox-responsive enzymes. Our results indicate that GSH-deficient female mice are rendered uniquely susceptible to an attenuated neutrophil response. If the same effects occur in humans, GSH-deficient women manufacturing MWCNTs may be at greater risk for impaired neutrophil-dependent clearance of MWCNTs from the lung. In contrast, men may have effective neutrophil-dependent clearance, but may be at risk for lung neutrophilia regardless of their GSH levels.

Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates

BACKGROUND: Most early preterm births are associated with intraamniotic infection and inflammation, which can lead to systemic inflammation in the fetus. The fetal inflammatory response syndrome describes elevations in the fetal interleukin‐6 level, which is a marker for inflammation and fetal organ injury. An understanding of the effects of inflammation on fetal cardiac development may lead to insight into the fetal origins of adult cardiovascular disease. OBJECTIVE: The purpose of this study was to determine whether the fetal inflammatory response syndrome is associated with disruptions in gene networks that program fetal cardiac development. STUDY DESIGN: We obtained fetal cardiac tissue after necropsy from a well‐described pregnant nonhuman primate model (pigtail macaque, Macaca nemestrina) of intrauterine infection (n=5) and controls (n=5). Cases with the fetal inflammatory response syndrome (fetal plasma interleukin‐6 >11 pg/mL) were induced by either choriodecidual inoculation of a hypervirulent group B streptococcus strain (n=4) or intraamniotic inoculation of Escherichia coli (n=1). RNA and protein were extracted from fetal hearts and profiled by microarray and Luminex (Millipore, Billerica, MA) for cytokine analysis, respectively. Results were validated by quantitative reverse transcriptase polymerase chain reaction. Statistical and bioinformatics analyses included single gene analysis, gene set analysis, Ingenuity Pathway Analysis (Qiagen, Valencia, CA), and Wilcoxon rank sum. RESULTS: Severe fetal inflammation developed in the context of intraamniotic infection and a disseminated bacterial infection in the fetus. Interleukin‐6 and ‐8 in fetal cardiac tissues were elevated significantly in fetal inflammatory response syndrome cases vs controls (P<.05). A total of 609 probe sets were expressed differentially (>1.5‐fold change, P<.05) in the fetal heart (analysis of variance). Altered expression of select genes was validated by quantitative reverse transcriptase polymerase chain reaction that included several with known functions in cardiac injury, morphogenesis, angiogenesis, and tissue remodeling (eg, angiotensin I converting enzyme 2, STEAP family member 4, natriuretic peptide A, and secreted frizzled‐related protein 4; all P<.05). Multiple gene sets and pathways that are involved in cardiac morphogenesis and vasculogenesis were downregulated significantly by gene set and Ingenuity Pathway Analysis (hallmark transforming growth factor beta signaling, cellular morphogenesis during differentiation, morphology of cardiovascular system; all P<.05). CONCLUSION: Disruption of gene networks for cardiac morphogenesis and vasculogenesis occurred in the preterm fetal heart of nonhuman primates with preterm labor, intraamniotic infection, and severe fetal inflammation. Inflammatory injury to the fetal heart in utero may contribute to the development of heart disease later in life. Development of preterm labor therapeutics must also target fetal inflammation to lessen organ injury and potential long‐term effects on cardiac function.