Dumitru A. Iacobas

Gene expression alterations in connexin null mice extend beyond the gap junction

Connexin43 (Cx43) is the principal gap junction protein between astrocytes in the neonatal brain and also interconnects neural precursor cells during CNS development. In an attempt to understand global effects of expression of the Cx43 gap junction gene on development and function of the nervous system, we have compared gene expression patterns in cultured astrocytes and brains from wildtype mice with those in which Cx43 is deleted as well as in spinal cords of experimental autoimmune encepahlomyelitis (EAE) mice. One surprising result obtained from high densitity mouse cDNA studies was the large number of genes that were statistically altered in mice with decreased expression of Cx43. These altered genes encode proteins with a wide range of functions within cells, and thus deletion of normal gap junction expression appears to result in globally altered glial functions in addition to disruption of intercellular communication. Here we discuss those results in the context of the strategies and data analysis paradigms that we are using in such studies.

Reversion of gene expression alterations in hearts of mice with chronic chagasic cardiomyopathy after transplantation of bone marrow cells

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries, being associated with intense inflammatory response and fibrosis. We have previously shown that bone marrow mononuclear cell (BMC) transplantation improves inflammation, fibrosis, and ventricular diameter in hearts of mice with chronic Chagas disease. Here we investigated the transcriptomic recovery induced by BMC therapy by comparing the heart transcriptomes of control, chagasic, and BMC transplanted mice. Out of the 9390 unique genes quantified in all samples, 1702 had their expression altered in chronic chagasic hearts compared to those of normal mice. Major categories of significantly upregulated genes were related to inflammation, fibrosis and immune responses, while genes involved in mitochondrion function were downregulated. When BMC-treated chagasic hearts were compared to infected mice, 96% of the alterations detected in infected hearts were restored to normal levels, although an additional 109 genes were altered by treatment. Transcriptomic recovery, a new measure that considers both resotrative and side effects of treatment, was remarkably high (84%). Immunofluorescence and morphometric analyses confirmed the effects of BMC therapy in the pattern of inflammatory-immune response and expression of adhesion molecules. In conclusion, by using large-scale gene profiling for unbiased assessment of therapeutic efficacy we demonstrate immunomodulatory effects of BMC therapy in chronic chagasic cardiomyopathy and identify potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets.

Fluid shear stress upregulates vascular endothelial growth factor gene expression in osteoblasts

Abstract:  Fluid‐induced shear stress is widely recognized as an important biophysical signal in cell–cell mechanotransduction. To identify cellular signaling pathways that are regulated by fluid shear stress, we applied the unbiased approach of transcriptional profiling. Our cDNA array analysis detected that 1,165 of the 6,288 sampled unigenes were significantly affected by pulsatile fluid flow. GenMapp 2.1 analysis revealed pathways of genes regulated by shear stress: angiogenesis, blood vessel morphogenesis, regulation of endothelial cell proliferation, and prostaglandin biosynthesis. Individual genes significantly up‐/downregulated by shear stress included vascular endothelial growth factor A (Vegf a), cysteine‐rich protein 61 (Cyr61), platelet‐derived growth factor‐alpha (Pdgf a), connective tissue growth factor (Ctgf), Neuropilin 1 (Nrp1), angiotensin II receptor, type 1 a (Agtr1 a) and fibroblast growth factor 1 (Fgf1). Based on these findings, we hypothesize that fluid shear stress‐regulated Vegf most likely stimulates MC3T3‐E1 cells through autocrine/paracrine release and may provide a powerful recruitment signal for osteoclasts, endothelial cells, and/or stem cells during bone remodeling.

Gene Expression Changes Associated with Myocarditis and Fibrosis in Hearts of Mice with Chronic Chagasic Cardiomyopathy

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries. About 30% of Trypanosoma cruzi-infected individuals develop this severe symptomatic form of the disease, characterized by intense inflammatory response accompanied by fibrosis in the heart. We performed an extensive microarray analysis of hearts from a mouse model of this disease and identified significant alterations in expression of approximately 12% of the sampled genes. Extensive up-regulations were associated with immune-inflammatory responses (chemokines, adhesion molecules, cathepsins, and major histocompatibility complex molecules) and fibrosis (extracellular matrix components, lysyl oxidase, and tissue inhibitor of metalloproteinase 1). Our results indicate potentially relevant factors involved in the pathogenesis of the disease that may provide new therapeutic targets in chronic Chagas disease.

Organizational principles of the connexin-related brain transcriptome

We have found that deletion of genes encoding the gap junction proteins Cx43, Cx32 and Cx36 alter the expression levels of large numbers of genes in mouse brain located on all chromosomes and encoding proteins from all major functional categories. Gene regulation in Cx32 and Cx43 null brains was more similar than that in the Cx36 null brain, suggesting the possibility of transcriptomic controls exerted by both genes on both astrocytes and oligodendrocytes. In order to explore the nature of expression linkage among the genes, we examined coordinated expression patterns in wild-type and connexin null brains. Coordination with Cx43 in wild-type brain predicted regulation in Cx43 nulls with considerable accuracy. Moreover, interlinkage within gene networks was greatly perturbed in the Cx43 null brain. These findings suggest several principles regarding regulatory transcriptomic networks involving gap junction genes and raise the issue of the underlying cause of connexin null phenotypes as well as mechanisms of regulation.

Effect of microgravity on gene expression in mouse brain

Changes in gravitational force such as that experienced by astronauts during space flight induce a redistribution of fluids from the caudad to the cephalad portion of the body together with an elimination of normal head-to-foot hydrostatic pressure gradients. To assess brain gene profile changes associated with microgravity and fluid shift, a large-scale analysis of mRNA expression levels was performed in the brains of 2-week control and hindlimb-unloaded (HU) mice using cDNA microarrays. Although to different extents, all functional categories displayed significantly regulated genes indicating that considerable transcriptomic alterations are induced by HU. Interestingly, the TIC class (transport of small molecules and ions into the cells) had the highest percentage of up-regulated genes, while the most down-regulated genes were those of the JAE class (cell junction, adhesion, extracellular matrix). TIC genes comprised 16% of those whose expression was altered, including sodium channel, nonvoltage-gated 1 beta (Scnn1b), glutamate receptor (Grin1), voltage-dependent anion channel 1 (Vdac1), calcium channel beta 3 subunit (Cacnb3) and others. The analysis performed by GeneMAPP revealed several altered protein classes and functional pathways such as blood coagulation and immune response, learning and memory, ion channels and cell junction. In particular, data indicate that HU causes an alteration in hemostasis which resolves in a shift toward a more hyper-coagulative state with an increased risk of venous thrombosis. Furthermore, HU treatment seems to impact on key steps of synaptic plasticity and learning processes.

Similar Transcriptomic Alterations in Cx43 Knockdown and Knockout Astrocytes

Previous findings of widespread transcriptomic alteration in tissues from connexin null mice raise the issue of whether the transcriptomic changes are directly due to connexin down-regulation or to “compensatory” developmental alterations for the missing gene. To start addressing this question, the authors compared with wild-type control the gene expression profiles of connexin43 (Cx43) knockout and Cx43siRNA knockdown wild-type cortical astrocytes. Array analysis revealed remarkable parallelism of transcriptomic changes in knockout and knockdown astrocytes, with similarly altered genes being located on all chromosomes and encoding proteins involved in a wide diversity of cell functions. Moreover, gene expression variability was analogously higher in Cx43 null and siRNA-treated astrocytes, and expression interlinkages were similarly altered among a selected subset of genes. This highly significant overlap between transcriptomic alterations in Cx43 knockout and knockdown astrocytes suggests that the widespread changes more likely reflect connexin-dependent Gene Regulatory Networks rather than developmental compensation for the missing gene.

Gap Junction and Purinergic P2 Receptor Proteins as a Functional Unit: Insights from Transcriptomics

Gap junctions and purinergic P2 receptors (P2Rs) can be regarded as belonging to a common functional unit, given that they are involved in the transmission of calcium signals between cells. We have previously shown that deletion of the Gja1 gene alters expression levels of numerous genes encoding proteins with diverse functions, including purinergic receptors (P2Rs), and have found that genes synergistically or antagonistically expressed in wild-type tissues are more prone to be similarly or oppositely regulated in Cx43-nulls. We have now explored the use of coordination analysis of gene expression as a strategy to identify interlinked genes encoding functionally related proteins and pull-downs to evaluate their interlinkage. Our findings indicate that, in brain and in cultured astrocytes, several of these coexpressed genes encode proteins that are components of P2R signal-transduction pathways and/or directly interact with these receptors, including the gap junction protein connexin43 (Cx43) and Cx45 as well as pannexins. It is proposed that coordination analysis of gene expression may provide a novel unbiased strategy for the identification of proteins belonging to supramolecular complexes.

Transcriptomic alterations in Trypanosoma cruzi-infected cardiac myocytes.

Trypanosoma cruzi infection is a major cause of cardiomyopathy. Previous gene profiling studies of infected mouse hearts have revealed prominent changes in gene expression within many functional pathways. This variety of transcriptomic changes in infected mice raises the question of whether gene expression alterations in whole hearts are due to changes in infected cardiac myocytes or other cells or even to systemic effects of the infection on the heart. We employed microarrays to examine infected cardiac myocyte cultures 48 h post-infection. Statistical comparison of gene expression levels of 7624 well annotated unigenes in four independent cultures of infected and uninfected myocytes detected substantial (>or=1.5 absolute fold changes) in 420 (5.5%) of the sampled genes. Major categories of affected genes included those involved in immune response, extracellular matrix and cell adhesion. These findings on infected cardiac myocytes in culture reveal that alterations in cardiac gene expression described in Chagas disease are the consequence of both direct infection of the myocytes themselves as well as resulting from the presence of other cell types in the myocardium and systemic effects of infection.

Astrocyte proximity modulates the myelination gene fabric of oligodendrocytes.

Extensive literature documented that astrocytes release neurotransmitters, cytokines and other signaling molecules to modulate migration, maturation and myelin synthesis of oligodendrocytes through mechanisms primarily converging on cytosolic [Ca2+] transients. Considering the long-term effects, it is expected that astrocyte-conditioned medium is a major regulator of gene expression in oligodendrocytes even in the absence of cytosol-to-cytosol communication via astrocyte-oligodendrocyte gap junction channels. Indeed, by comparing the transcriptomes of immortalized precursor oligodendrocyte (Oli-neu) cells when cultured alone and co-cultured with non-touching astrocytes we found profound changes in the gene expression level, control and networking. Remarkably, the astrocyte proximity was more effective in remodeling the myelination (MYE) gene fabric and its control by cytokine receptor (CYR)-modulated intercellular Ca2+-signaling (ICS) transcriptomic network than the dibutyryl-cAMP (db-cAMP) treatment-induced transformation into myelin-associated glycoprotein-positive oligodendrocyte-like cells. Moreover, astrocyte proximity up-regulated 37 MYE genes and switched on another 14 MYE, 23 ICS and 4 CYR genes, enhancing the roles of the leukemia inhibitory factor receptor and connexins Cx29 and Cx47. The novel prominent gene analysis identified the enhancer of zeste homolog 2 as the most relevant MYE gene in the astrocyte proximity, notch gene homolog 1 in control and B-cell leukemia/lymphoma 2 in differentiated Oli-neu cells.