Expression study of the atherosclerosis mouse aorta reveals significant disturbance of calcium signaling pathway

Atherosclerosis is a complex disease resulting from interactions of genetic and environmental risk factors leading to heart failure and stroke. Using an atherosclerotic mouse model (ldlr-/-, apobec1-/- designated as LDb), we performed microarray analysis to identify candidate genes and pathways, which are most perturbed in changes in the following risk factors: genetics (control C57BL/6 vs. LDb mice), shearstress (lesion-prone vs. lesion-resistant regions in LDb mice), diet (chow vs. high fat fed LDb mice) and age (2-month-old vs. 8-month old LDb mice). ^ Atherosclerotic lesion quantification and lipid profile studies were performed to assess the disease phenotype. A microarray study was performed on lesion-prone and lesion-resistant regions of each aorta. Briefly, 32 male C57BL/6 and LDb mice (n =16/each) were fed on either chow or high fat diet, sacrificed at 2- and 8-months old, and RNA isolated from the aortic lesion-prone and aortic lesion-resistant segments. Using 64 Affymetrix Murine 430 2.0 chips, we profiled differentially expressed genes with the cut off value of FDR ≤ 0.15 for t-test, and q <0.0001 for the ANOVA. The data were normalized using two normalization methods---invariant probe sets (Loess) and Quantile normalization, the statistical analysis was performed using t-tests and ANOVA, and pathway characterization was done using Pathway Express (Wayne State). The result identified the calcium signaling pathway as the most significant overrepresented pathway, followed by focal adhesion. In the calcium signaling pathway, 56 genes were found to be significantly differentially expressed out of 180 genes listed in the KEGG calcium signaling pathway. Nineteen of these genes were consistently identified by both statistical tests, 11 of which were unique to the test, and 26 were unique to the ANOVA test, using the cutoffs noted above. ^ In conclusion, this finding suggested that hypercholesterolemia drives the disease progression by altering the expression of calcium channels and regulators which subsequently results in cell differentiation, growth, adhesion, cytoskeletal change and death. Clinically, this pathway may serve as an important target for future therapeutic intervention, and thus the calcium signaling pathway may serve as an important target for future diagnostic and therapeutic intervention. ^

Adenosine induced pulmonary fibrosis and the contribution of the adenosine A2B receptor to the induction of osteopontin in a mouse model of pulmonary fibrosis

Pulmonary fibrosis is a devastating and lethal lung disease with no current cure. Research into cellular signaling pathways able to modulate aspects of pulmonary inflammation and fibrosis will aid in the development of effective therapies for its treatment. Our laboratory has generated a transgenic/knockout mouse with systemic elevations in adenosine due to the partial lack of its metabolic enzyme, adenosine deaminase (ADA). These mice spontaneously develop progressive lung inflammation and severe pulmonary fibrosis suggesting that aberrant adenosine signaling is influencing the development and/or progression of the disease in these animals. These mice also show marked increases in the pro-fibrotic mediator, osteopontin (OPN), which are reversed through ADA therapy that serves to lower lung adenosine levels and ameliorate aspects of the disease. OPN is known to be regulated by intracellular signaling pathways that can be accessed through adenosine receptors, particularly the low affinity A2BR receptor, suggesting that adenosine receptor signaling may be responsible for the induction of OPN in our model. In-vitro, adenosine and the broad spectrum adenosine receptor agonist, NECA, were able to induce a 2.5-fold increase in OPN transcripts in primary alveolar macrophages. This induction was blocked through antagonism of the A2BR receptor pharmacologically, and through the deletion of the receptor subtype in these cells genetically, supporting the hypothesis that the A2BR receptor was responsible for the induction of OPN in our model. These findings demonstrate for the first time that adenosine signaling is an important modulator of pulmonary fibrosis in ADA-deficient mice and that this is in part due to signaling through the A2BR receptor which leads to the induction of the pro-fibrotic molecule, otseopontin. ^