Parul Gulati

Erk1 and Erk2 Regulate Endothelial Cell Proliferation and Migration during Mouse Embryonic Angiogenesis

Angiogenesis is a complex process orchestrated by both growth factors and cell adhesion and is initiated by focal degradation of the vascular basement membrane with subsequent migration and proliferation of endothelial cells. The Ras/Raf/MEK/ERK pathway is required for EC function during angiogenesis. Although in vitro studies implicate ERK1 and ERK2 in endothelial cell survival, their precise role in angiogenesis in vivo remains poorly defined. Cre/loxP technology was used to inactivate Erk1 and Erk2 in endothelial cells during murine development, resulting in embryonic lethality due to severely reduced angiogenesis. Deletion of Erk1 and Erk2 in primary endothelial cells resulted in decreased cell proliferation and migration, but not in increased apoptosis. Expression of key cell cycle regulators was diminished in the double knockout cells, and decreased DNA synthesis could be observed in endothelial cells during embryogenesis. Interestingly, both Paxillin and Focal Adhesion Kinase were expressed at lower levels in endothelial cells lacking Erk1 and Erk2 both in vivo and in vitro, leading to defects in the organization of the cytoskeleton and in cell motility. The regulation of Paxillin and Focal Adhesion Kinase expression occurred post-transcriptionally. These results demonstrate that ERK1 and ERK2 coordinate endothelial cell proliferation and migration during angiogenesis.

Effect of Postnatal Maturation on the Mechanisms of Esophageal Propulsion in Preterm Human Neonates: Primary and Secondary Peristalsis

OBJECTIVES:The changes in esophageal propulsive characteristics during maturation are not known. Our aim was to define the effects of postnatal maturation on esophageal peristaltic characteristics in preterm human neonates. We tested the hypotheses that: (i) maturation modifies esophageal bolus propulsion characteristics, and (ii) the mechanistic characteristics differ between primary and secondary peristalsis.METHODS:Esophageal motility in 10 premature neonates (mean 27.5 weeks gestational age) was evaluated twice at 33.8 weeks (time 1, earlier study) and 39.2 weeks (time 2, later study) mean postmenstrual age. Esophageal manometry waveform characteristics (amplitude and duration, peristaltic velocity, and intrabolus pressure domains) were analyzed during spontaneous primary peristalsis and infusion-induced secondary peristalsis. Repeated-measures and unstructured variance–covariance or compound symmetry matrixes were used for statistical comparison. Values stated as least squares means±s.e.m. or percent.RESULTS:A total of 200 primary peristalsis and 227 secondary peristalsis events were evaluated. Between time 1 and time 2: (i) proximal esophageal waveform amplitude increased (P<0.02), with primary peristalsis (38±6 vs. 48±7 mm Hg) and with secondary peristalsis (34±6 vs. 46±5 mm Hg); (ii) distal esophageal waveform amplitude was similar (P=NS), with primary peristalsis (42±4 vs. 43±4 mm Hg) and secondary peristalsis (29±3 vs. 32±4 mm Hg); (iii) proximal esophageal waveform onset to peak duration decreased (P=0.02) with primary (2.6±0.3 vs. 1.9±0.1 s, P<0.003) and with secondary peristalsis (2.2±0.2 vs. 1.8±0.1 s); (iv) distal esophageal waveform onset to peak duration decreased (P=0.01) with primary (2.4±0.3 vs. 1.8±0.1 s) and with secondary peristalsis (1.9±0.2 vs. 1.5±0.1 s); (v) effects of identical stimulus volume on intrabolus pressure were similar (P=NS); however, greater infusion volumes (2 vs. 1 ml) generated higher intrabolus pressure at both time 1 and time 2 (both Ps<0.05). Between primary and secondary peristalsis (mechanistic variable): (i) no differences were noted at either period, with proximal esophageal waveform amplitudes (P=NS); (ii) differences were noted with distal esophageal waveform amplitudes at each time period (P=0.0002); (iii) no differences were noted with both esophageal waveforms duration at either period (P=NS); (iv) peristaltic velocity was faster with secondary peristalsis than with primary peristalsis at either period (at earlier study, 7.9±1.4 vs. 2.5±1.4 cm/s and at later study 6.2±1.6 vs. 1.2±1.5 cm/s, both Ps<0.01).CONCLUSIONS:In preterm neonates, longitudinal maturation modulates the characteristics of primary and secondary peristalsis. Differences in proximal striated muscle and distal smooth muscle activity during peristalsis are evident. Peristaltic velocity is faster with secondary peristalsis. These findings may represent maturation of central and peripheral neuromotor properties of esophageal bolus propulsion in healthy preterm human neonates.

Indentation Modulus of the Alveolar Process in Dogs

One mechanism of bone adaptation is alteration in tissue level material properties. We hypothesized that alteration in the indentation modulus of the alveolar process is an adaptive response to the localized mechanical environment. Forty-eight specimens representing anterior and posterior regions of the maxilla and mandible were obtained from 6 mature male beagle dogs. The indentation properties of the alveolar bone proper and more distant osteonal cortical bone were estimated. The bone types were further divided into 3 regions (coronal, middle, and apical), with 27 indents being made in each region of tooth-supporting bone. There was a significant difference (p < 0.001) in the indentation moduli of the jaws (maxilla/mandible), location (anterior/posterior), and bone type (alveolar bone proper vs. cortical bone). However, statistical interactions exist which preclude the simple interpretation of results. The distribution of relative stiffness provides a better understanding of bone adaptations in the alveolar process.

Fully Moderated T-statistic for Small Sample Size Gene Expression Arrays

Gene expression microarray experiments with few replications lead to great variability in estimates of gene variances. Several Bayesian methods have been developed to reduce this variability and to increase power. Thus far, moderated t methods assumed a constant coefficient of variation (CV) for the gene variances. We provide evidence against this assumption, and extend the method by allowing the CV to vary with gene expression. Our CV varying method, which we refer to as the fully moderated t-statistic, was compared to three other methods (ordinary t, and two moderated t predecessors). A simulation study and a familiar spike-in data set were used to assess the performance of the testing methods. The results showed that our CV varying method had higher power than the other three methods, identified a greater number of true positives in spike-in data, fit simulated data under varying assumptions very well, and in a real data set better identified higher expressing genes that were consistent with functional pathways associated with the experiments.

Inflammatory Gene Regulatory Networks in Amnion Cells Following Cytokine Stimulation: Translational Systems Approach to Modeling Human Parturition

A majority of the studies examining the molecular regulation of human labor have been conducted using single gene approaches. While the technology to produce multi-dimensional datasets is readily available, the means for facile analysis of such data are limited. The objective of this study was to develop a systems approach to infer regulatory mechanisms governing global gene expression in cytokine-challenged cells in vitro, and to apply these methods to predict gene regulatory networks (GRNs) in intrauterine tissues during term parturition. To this end, microarray analysis was applied to human amnion mesenchymal cells (AMCs) stimulated with interleukin-1β, and differentially expressed transcripts were subjected to hierarchical clustering, temporal expression profiling, and motif enrichment analysis, from which a GRN was constructed. These methods were then applied to fetal membrane specimens collected in the absence or presence of spontaneous term labor. Analysis of cytokine-responsive genes in AMCs revealed a sterile immune response signature, with promoters enriched in response elements for several inflammation-associated transcription factors. In comparison to the fetal membrane dataset, there were 34 genes commonly upregulated, many of which were part of an acute inflammation gene expression signature. Binding motifs for nuclear factor-κB were prominent in the gene interaction and regulatory networks for both datasets; however, we found little evidence to support the utilization of pathogen-associated molecular pattern (PAMP) signaling. The tissue specimens were also enriched for transcripts governed by hypoxia-inducible factor. The approach presented here provides an uncomplicated means to infer global relationships among gene clusters involved in cellular responses to labor-associated signals.

Adaptations in the Mandible and Appendicular Skeleton of High and Low Bone Density Inbred Mice

The appendicular skeletons of high [C3H/HeJ (C3H)] and low [C57BL/6J (B6)] density inbred mice have been shown to differ in morphology, mechanical properties, and cellular activity. The focus of the current study was to (1) characterize the mandibular bone formation rate (BFR/BS), bone mass, indentation modulus (IM), and hardness of C3H and B6 mice and (2) investigate the relationship of the mechanical properties in three skeletal sites: mandible, femur, and tibia. Specimens from 17-week-old female C3H and B6 (n = 15/group) mice were obtained. Mandibular bone mass was estimated from the lateral-view area (LVA) and transverse cross sections. BFR/BS was measured in the mandibular section distal to the third molar. In addition, bone blocks from the distal surface of the third molar and the femoral and tibial midshaft were obtained for mechanical testing. BFR/BS, cortical area, and LVA were greater (P < 0.001) in C3H mandibles. IM was approximately 2 GPa higher in the C3H mandible (P > 0.05), femur (P < 0.001), and tibia (P < 0.01). Mandibular IM was lower (P < 0.05) than the femoral and tibial IM within each inbred mouse. IM was not significant between C3H and B6 mandibles. However, the magnitude of the difference (∼12%) in the mandible was similar to the difference in the appendicular skeleton. This mandibular bone phenotype is similar to that observed in the appendicular skeleton of these distinct inbred mice.

Impact of aprotinin and renal function on mortality: a retrospective single center analysis

BackgroundAn estimated up to 7% of high-risk cardiac surgery patients return to the operating room for bleeding. Aprotinin was used extensively as an antifibrinolytic agent in cardiac surgery patients for over 15 years and it showed efficacy in reducing bleeding. Aprotinin was removed from the market by the U.S. Food and Drug Administration after a large prospective, randomized clinical trial documented an increased mortality risk associated with the drug. Further debate arose when a meta-analysis of 211 randomized controlled trials showed no risk of renal failure or death associated with aprotinin. However, only patients with normal kidney function have been studied.MethodsIn this study, we look at a single center clinical trial using patients with varying degrees of baseline kidney function to answer the question: Does aprotinin increase odds of death given varying levels of preoperative kidney dysfunction?ResultsBased on our model, aprotinin use was associated with a 3.8-fold increase in odds of death one year later compared to no aprotinin use with p-value = 0.0018, regardless of level of preoperative kidney dysfunction after adjusting for other perioperative variables.ConclusionsLessons learned from our experience using aprotinin in the perioperative setting as an antifibrinolytic during open cardiac surgery should guide us in testing future antifibrinolytic drugs for not only efficacy of preventing bleeding, but for overall safety to the whole organism using long-term clinical outcome studies, including those with varying degree of baseline kidney function.

Abstract 110: A bioinformatics view of networking in the mouse mammary microenvironment

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Applying a novel bioinformatics strategy, the objective of this study was to identify signaling interactions that occur within cell types of the mouse mammary gland. Mining through typical microarray data is quite a challenge, but it is even more difficult to extract biological relevance within the mammary microenvironment. The approach here involves a combinatorial strategy that effectively integrates basic research design, statistics, and bioinformatics. We hypothesized that cells in the microenvironment potentiate tumorigenesis by signaling with each other through critical pathways sharing common ‘network hubs’. Mammary glands were harvested from 8-week-old mice that were wildtype (WT) or had fibroblast-specific Pten deletion (fPten-/-). Fibroblasts, macrophages, endothelial and epithelial cells were selected from the glands through cell sorting and selective cell culture. Replicate samples of their cDNA were applied onto Mouse Exon Arrays. The raw data were processed through Robust Mean Analysis (RMA), and then subjected to Empirical Bayes Arrays (EBArrays) analysis to generate lists of differentially expressed genes between the fPten-/- and WT mice for the four cell types. Each gene was given a probability value as a measure of its true differential expression, and only genes with a value more than or equal to 0.7 were considered for further analyses. Three bioinformatics tools- Database for Annotation, Visualization and Integrated Discovery (DAVID), Biometric Research Branch (BRB) ArrayTools, and Ingenuity Pathway Analysis® (IPA) - were used to analyze the four gene lists. Analysis by DAVID revealed that for the fibroblasts and the macrophages, the major biological machinery activated in the fPten-/- mice related to extracellular matrix remodeling and immune response. The endothelial cells displayed genes involved in complement activation pathway. Interestingly, the genes expressed in epithelial cells related to various aspects of epithelial-mesenchymal transition. Together, this suggests that even in the absence of tumor, the fPten-/- stromal signaling infuses a tumorigenic potential into the microenvironment. A filter on BRB ArrayTools selected genes that had a 2-fold change. Average hierarchical clustering based on Spearman correlation was done to generate heat maps. This refined the list of significant genes between the genotypes for each cell type. The four fPten-/- derived genelists were then uploaded into IPA. This confirmed the output from DAVID, and further revealed that ERBB2, MMP9, TNFα, TGFβ, and NFκB, β-catenin, and Ets, were key network hubs and transcription factors, respectively, through which signaling occurred in the mammary microenvironment. The top merged networks across cell types displayed shared nodes important for communication. In summary, this analytical approach gave an insight into the ‘network players’ and ‘cellular crosstalk’ critical for a tumorigenic environment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 110.