David K. Pritchard

A Hierarchical Network Controls Protein Translation during Murine Embryonic Stem Cell Self-Renewal and Differentiation

Stem cell differentiation involves changes in transcription, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of murine embryonic stem cells (ESCs) into embryoid bodies by integrating transcriptome analysis with global assessment of ribosome loading. While protein synthesis was parsimonious during self-renewal, differentiation induced an anabolic switch, with global increases in transcript abundance, polysome content, protein synthesis, and protein content. Furthermore, 78% of transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Transcripts under exclusive translational control included the transcription factor ATF5, the tumor suppressor DCC, and the beta-catenin agonist Wnt1. We show that a hierarchy of translational regulators, including mTOR, 4EBP1, and the RNA-binding proteins DAZL and GRSF1, control global and selective protein synthesis during ESC differentiation. Parsimonious translation in pluripotent state and hierarchical translational regulation during differentiation may be important quality controls for self-renewal and choice of fate in ESCs.

Capillary Regeneration in Scleroderma: Stem Cell Therapy Reverses Phenotype?

Background Scleroderma is an autoimmune disease with a characteristic vascular pathology. The vasculopathy associated with scleroderma is one of the major contributors to the clinical manifestations of the disease. Methodology/Principal Findings We used immunohistochemical and mRNA in situ hybridization techniques to characterize this vasculopathy and showed with morphometry that scleroderma has true capillary rarefaction. We compared skin biopsies from 23 scleroderma patients and 24 normal controls and 7 scleroderma patients who had undergone high dose immunosuppressive therapy followed by autologous hematopoietic cell transplant. Along with the loss of capillaries there was a dramatic change in endothelial phenotype in the residual vessels. The molecules defining this phenotype are: vascular endothelial cadherin, a supposedly universal endothelial marker required for tube formation (lost in the scleroderma tissue), antiangiogenic interferon α (overexpressed in the scleroderma dermis) and RGS5, a signaling molecule whose expression coincides with the end of branching morphogenesis during development and tumor angiogenesis (also overexpressed in scleroderma skin. Following high dose immunosuppressive therapy, patients experienced clinical improvement and 5 of the 7 patients with scleroderma had increased capillary counts. It was also observed in the same 5 patients, that the interferon α and vascular endothelial cadherin had returned to normal as other clinical signs in the skin regressed, and in all 7 patients, RGS5 had returned to normal. Conclusion/Significance These data provide the first objective evidence for loss of vessels in scleroderma and show that this phenomenon is reversible. Coordinate changes in expression of three molecules already implicated in angiogenesis or anti-angiogenesis suggest that control of expression of these three molecules may be the underlying mechanism for at least the vascular component of this disease. Since rarefaction has been little studied, these data may have implications for other diseases characterized by loss of capillaries including hypertension, congestive heart failure and scar formation.

Combined analysis of monocyte and lymphocyte messenger RNA expression with serum protein profiles in patients with scleroderma

OBJECTIVE We attempted to elucidate possible pathogenetic mechanisms in scleroderma by analysis of gene expression patterns of purified monocytes and lymphocytes, as well as protein profiles of cytokines and growth factors. METHODS Expression analysis was performed on messenger RNA (mRNA) from cells that had been purified with magnetic beads. Plasma samples from the same patients were used for multiplex cytokine analysis. Potential sources of proteins were also examined by in situ hybridization of skin specimens. RESULTS A total of 1,800 genes from monocytes and 863 genes from CD4+ T cells were differentially expressed in scleroderma patients. As observed by other investigators using unfractionated peripheral blood cells from patients with autoimmune connective tissue diseases, the cell type-specific analyses of our scleroderma samples showed expression of genes suggesting the presence of interferon-alpha (IFNalpha), despite the apparent absence of this cytokine in plasma. IFNalpha RNA was, however, expressed at enhanced levels in vascular and perivascular cells in scleroderma skin samples. While levels of interleukin-1alpha (IL-1alpha) and IL-16 were among 10 proteins found to be significantly elevated in scleroderma patients, none of the large panel of plasma cytokines we analyzed correlated with the expression levels of putative IFN response genes. CONCLUSION The pattern of up-regulation of mRNA in both the monocytes and CD4 lymphocytes of scleroderma patients, together with the detection of IFNalpha RNA in the microvasculature, suggests that leukocytes respond to this cytokine locally in the vessels. Detection of high levels of IL-1alpha and IL-16 in plasma and the independence of these protein levels from the IFN signature, implicates an independent contribution of other cytokines to immune activation and/or inflammation in scleroderma.

High-Density Lipoprotein Suppresses the Type I Interferon Response, a Family of Potent Antiviral Immunoregulators, in Macrophages Challenged With Lipopolysaccharide

Background— High-density lipoprotein (HDL) protects the artery wall by removing cholesterol from lipid-laden macrophages. However, recent evidence suggests that HDL might also inhibit atherogenesis by combating inflammation. Methods and Results— To identify potential antiinflammatory mechanisms, we challenged macrophages with lipopolysaccharide, an inflammatory microbial ligand for Toll-like receptor 4. HDL inhibited the expression of 30% (277 of 911) of the genes normally induced by lipopolysaccharide, microarray analysis revealed. One of its major targets was the type I interferon response pathway, a family of potent viral immunoregulators controlled by Toll-like receptor 4 and the TRAM/TRIF signaling pathway. Unexpectedly, the ability of HDL to inhibit gene expression was independent of macrophage cholesterol stores. Immunofluorescent studies suggested that HDL promoted TRAM translocation to intracellular compartments, which impaired subsequent signaling by Toll-like receptor 4 and TRIF. To examine the potential in vivo relevance of the pathway, we used mice deficient in apolipoprotein A-I, the major protein of HDL. After infection with Salmonella typhimurium, a Gram-negative bacterium that expresses lipopolysaccharide, apolipoprotein A-I–deficient mice had 6-fold higher plasma levels of interferon-&bgr;, a key regulator of the type I interferon response, than did wild-type mice. Conclusions— HDL inhibits a subset of lipopolysaccharide-stimulated macrophage genes that regulate the type I interferon response, and its action is independent of sterol metabolism. These findings raise the possibility that regulation of macrophage genes by HDL might link innate immunity and cardioprotection.

Epithelial phenotype confers resistance of ovarian cancer cells to oncolytic adenoviruses

We studied the susceptibility of primary ovarian cancer cells to oncolytic adenoviruses. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, we discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to coxsackie-adenovirus receptor or CD46. Specifically, we found that these adenovirus receptors were trapped in tight junctions and not accessible for virus binding. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-to-mesenchymal transition (EMT). We showed that specific, thus far little-explored adenovirus serotypes (Ad3, Ad7, Ad11, and Ad14) that use receptor(s) other than coxsackie-adenovirus receptor and CD46 were able to trigger EMT in epithelial ovarian cancer cells and cause efficient oncolysis. Our studies on ovarian cancer cultures and xenografts also revealed several interesting cancer cell biology features. Tumors in situ as well as tumor xenografts in mice mostly contained epithelial cells and cells that were in a hybrid stage where they expressed both epithelial and mesenchymal markers (epithelial/mesenchymal cells). These epithelial/mesenchymal cells are the only xenograft-derived cells that can be cultured and with passaging undergo EMT and differentiate into mesenchymal cells. Our study provides a venue for improved virotherapy of cancer as well as new insights into cancer cell biology.

cDNA microarray analysis reveals fundamental differences in the expression profiles of primary human monocytes, monocyte-derived macrophages, and alveolar macrophages

We report the systematic use of large‐scale cDNA microarrays to study the gene expression profiles of primary human peripheral blood monocytes (MONO) in comparison with in vitro‐differentiated, M‐CSF‐induced MONO‐derived macrophages (MAC) and primary human alveolar MAC (AM), obtained by bronchoalveolar lavage from the lungs of normal volunteers. These studies revealed large‐scale differences in the gene expression profile between both MAC types (MAC and AM) and MONO. In addition, large differences were observed in the gene expression profiles of the two MAC types. Specifically, 21% of genes on the array (2904 out of 13,582) were differentially expressed between AM and MONO, and 2229 out of 13,583 probes were differentially expressed between MAC and AM. Our expression data show remarkable differences in gene expression between different MAC subpopulations and emphasize the heterogeneity of different MAC populations. This study underscores the need to scrutinize models of MAC biology for relevance to specific disease processes.

TagSNP evaluation for the association of 42 inflammation loci and vascular disease: evidence of IL6, FGB, ALOX5, NFKBIA, and IL4R loci effects.

Inflammatory markers have consistently been associated with vascular disease. Evidence of genetic polymorphisms in inflammatory loci that predict severe carotid artery disease (CAAD) would suggest that this relationship is not secondary to other correlated factors, but related to inflammation itself. We examined the full common genetic variation in 42 inflammatory loci for prediction of severe CAAD versus ultrasound proven controls using a tagSNP approach. For selected loci, monocyte RNA levels were contrasted in subjects with and without CAAD. We confirm the association of IL6−174, FGB−455, and ALOX5 with CAAD and show that multiple ALOX5 SNPs independently predict CAAD. We provide evidence for previously unreported associations of SNPs in IL4R, NFKBIA, and PLG with CAAD, and weaker evidence for associations with CSF3, IL10RA, and VCAM1. The NFKBIA and IL10RA expression levels significantly differed between subjects with CAAD and controls. These results support a role for genetic variation related to inflammation in CAAD and a causal role for specific gene products.

Cholesterol Accumulation Regulates Expression of Macrophage Proteins Implicated in Proteolysis and Complement Activation

Objective—Cholesterol accumulation by macrophages plays a key role in atherogenesis. To begin to develop a global picture of this process, we used proteomics and transcriptomics to analyze foam cells generated with acetyl-low-density lipoprotein, a classic ligand for scavenger receptors. Methods and Results—Tandem mass spectrometry and stringent statistical analysis revealed that foam cells differentially expressed 15 of 542 proteins (2.8%) detected in macrophage-conditioned medium. Apolipoprotein E was one of the most upregulated proteins, confirming that proteins involved in lipid metabolism are important targets for regulation by sterol accumulation. However, levels of proteins linked to complement activation and lysosomal proteolysis also changed markedly. Transcriptional analysis demonstrated that 698 of 19 700 genes (3.5%) were regulated in foam cells, including many genes important in sterol metabolism. We also found that cholesterol accumulation regulated genes implicated in complement activation but failed to affect genes linked to proteolysis and macrophage polarization. Changes in protein levels in macrophage-conditioned medium were largely independent of changes in mRNA levels. Conclusion—Loading sterol into macrophages regulates levels of complement proteins and lysosomal proteases—key players in the immune system and plaque rupture. Posttranscriptional mechanisms are likely important for controlling levels of most of the proteins detected in macrophage medium.

Identification of Putative Immunologic Targets for Colon Cancer Prevention Based on Conserved Gene Upregulation from Preinvasive to Malignant Lesions

The length of time required for preinvasive adenoma to progress to carcinoma, the immunogenicity of colorectal cancer (CRC), and the identification of high-risk populations make development and testing of a prophylactic vaccine for the prevention of CRC possible. We hypothesized that genes upregulated in adenoma relative to normal tissue, which maintained increased expression in CRC, would encode proteins suitable as putative targets for immunoprevention. We evaluated existing adenoma and CRC microarray datasets and identified 160 genes that were ≥2-fold upregulated in both adenoma and CRC relative to normal colon tissue. We further identified 23 genes that showed protein overexpression in colon adenoma and CRC based on literature review. Silencing the most highly upregulated genes, CDH3, CLDN1, KRT23, and MMP7, in adenoma and CRC cell lines resulted in a significant decrease in viability (P < 0.0001) and proliferation (P < 0.0001) as compared to controls and an increase in cellular apoptosis (P < 0.05 for CDH3, KRT23). Results were duplicated across cell lines representing microsatellite instability, CpG island methylator, and chromosomal instability phenotypes, suggesting immunologic elimination of cells expressing these proteins could impact the progression of all CRC phenotypes. To determine whether these proteins were immunogens, we interrogated sera from early stage CRC patients and controls and found significantly elevated CDH3 (P = 0.006), KRT23 (P = 0.0007), and MMP7 (P < 0.0001) serum immunoglobulin G in cases as compared to controls. These data show a high throughput approach to the identification of biologically relevant putative immunologic targets for CRC and identified three candidates suitable for vaccine development. Cancer Prev Res; 6(7); 666–74. ©2013 AACR.

Microarray Analysis of Gene Expression Changes in Aging

Publisher Summary The most common application of microarray technology to the research of aging is the search for gene expression changes that correlates with organismal age. Microarrays can also be used to compare individuals or populations of similar age but with different aging potentials. In several research, these two types of experimental design have been combined in such a way that the microarray analysis is carried out on control and long-lived animals at two or more ages. Microarray analysis of tissues or cells derived from short-lived mutants offers the opportunity to identify gene expression changes correlated with short life span and, potentially, accelerated aging. Microarrays offer the opportunity to detect a group of gene expression biomarkers that more accurately reflect biological age than currently possible. Several potential biomarkers can be assayed simultaneously in a single array experiment. Appropriate experimental design for the identification of individual biomarkers of longevity using microarrays, however, is nontrivial. While considering the design of a microarray experiment, the sources of biological and technical variability must be considered as they affect the ability to detect true gene expression changes.