Faculty Bibliography

Methotrexate remains a cornerstone in the treatment of rheumatoid arthritis and other rheumatic diseases. Folate antagonism is known to contribute to the antiproliferative effects that are important in the action of methotrexate against malignant diseases, but concomitant administration of folic or folinic acid does not diminish the anti-inflammatory potential of this agent, which suggests that other mechanisms of action might be operative. Although no single mechanism is sufficient to account for all the anti-inflammatory activities of methotrexate, the release of adenosine from cells has been demonstrated both in vitro and in vivo. Methotrexate might also confer anti-inflammatory properties through the inhibition of polyamines. The biological effects on inflammation associated with adenosine release have provided insight into how methotrexate exerts its effects against inflammatory diseases and at the same time causes some of its well-known adverse effects. These activities contribute to the complex and multifaceted mechanisms that make methotrexate efficacious in the treatment of inflammatory disorders

Atherosclerotic cardiovascular disease (ASCVD) contributes to morbidity and mortality in systemic lupus erythematosus (SLE). Immunologic derangements may disrupt cholesterol balance in vessel wall monocytes/macrophages and endothelium. We determined whether lupus plasma impacts expression of cholesterol 27-hydroxylase, an anti-atherogenic cholesterol-degrading enzyme that promotes cellular cholesterol efflux, in THP-1 human monocytes and primary human aortic endothelial cells (HAEC). THP-1 monocytes and HAEC were incubated in medium containing SLE patient plasma or apparently healthy control human plasma (CHP). SLE plasma decreased 27-hydroxylase message in THP-1 monocytes by 47 +/- 8% (p < 0.008) and in HAEC by 51 +/- 5.5% (n = 5, p < 0.001). THP-1 macrophages were incubated in 25% lupus plasma or CHP and cholesterol-loaded (50 microg ml(-1) acetylated low density lipoprotein). Lupus plasma more than doubled macrophage foam cell transformation (74 +/- 3% vs. 35 +/- 3% for CHP, n = 3, p < 0.001). Impaired cholesterol homeostasis in SLE provides further evidence of immune involvement in atherogenesis. Strategies to inhibit or reverse arterial cholesterol accumulation may benefit SLE patients

OBJECTIVE: Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Because identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and developing new treatments, we studied the effect of adenosine A(1) receptor blockade or deletion on bone density. METHODS: The bone mineral density (BMD) in adenosine A(1) receptor-knockout (A(1)R-knockout) mice was analyzed by dual x-ray absorptiometry (DXA) scanning, and the trabecular and cortical bone volume was determined by microfocal computed tomography (micro-CT). The mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analyzed by DXA scanning and micro-CT. A histologic examination of bones obtained from A(1)R-knockout and wild-type mice was carried out. Visualization of osteoblast function (bone formation) after tetracycline double-labeling was performed by fluorescence microscopy. RESULTS: Micro-CT analysis of bones from A(1)R-knockout mice showed significantly increased bone volume. Electron microscopy of bones from A(1)R-knockout mice showed the absence of ruffled borders of osteoclasts and osteoclast bone resorption. Immunohistologic analysis demonstrated that although osteoclasts were present in the A(1)R-knockout mice, they were smaller and often not associated with bone. No morphologic changes in osteoblasts were observed, and bone-labeling studies revealed no change in the bone formation rates in A(1)R-knockout mice. CONCLUSION: These results suggest that the adenosine A(1) receptor may be a useful target in treating diseases characterized by excessive bone turnover, such as osteoporosis and prosthetic joint loosening

Predicting complications of diseases such as rheumatoid arthritis (RA) as well as the efficacy and toxicity of drugs used to treat the disease based on an understanding of genetic differences is leading to the development of highly individualized, personal medicine. The prevention of cardiovascular complications of RA has assumed greater importance as our ability to treat the underlying joint disease has improved and it may be possible to predict which patients with RA are at greatest risk of developing cardiovascular disease

Purpose: Adenosine is a purine molecule necessary for normal cell metabolism and growth. Recent work from our laboratory using adenosine A₁-deficient mice has demonstrated that adenosine A₁receptors play a critical role in regulating bone turnover, raising the intriguing possibility of targeting A₁receptors for therapeutic advancement in osteoporosis and other bone diseases. In the present study, we investigated the mechanism by which A₁receptors regulate mouse osteoclast differentiation induced by macrophagecolony stimulating factor (M-CSF) and the receptor activator of NF-kappaB ligand (RANKL) from monocyte/macrophage cell lineage of bone marrow cells. Methods: Osteoclast differentiation was studied in vitro as the M-CSF/RANKL stimulated formation of multinucleated (>3 nuclei), TRAP-positive cells from primary murine (C57Bl/6) bone marrow-derived precursors. Signaling events were studied by Western Blot for activated (phosphorylated) signaling molecules and changes in message were determined by RT-PCR. Results: As we have previously demonstrated, the A₁-receptor specific antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) dose-dependently inhibited RANKL-stimulated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation from bone marrow cells and the bone-resorptive activity of mature osteoclasts (p<0.001, n=3, IC₅₀= 0.1muM). This inhibition is accompanied by reduction of osteoclast-specific target genes including MMP9, Integrin av, Integrin beta3, Cathepsin K and TRAP (p<0.05, n=4). Furthermore, DPCPX inhibited accumulation of the RANKL-stimulated c-fos mRNA at day 3 in bone marrow cells (p<0.05, n=3); c-fos is a key transcription factor for the differentiation of osteoclasts. DPCPX also dose-dependently suppressed RANKL-induced gene expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) at day 4 in bone marrow cells (p<0.05, n=3). Among the RANK signaling MAPK pathways, DPCPX (1muM) inhibited phosphorylation of JNK (p<0.001, n=4), which is upregulated in response to RANKL in bone marrow macrophages. Conclusion: Collectively, these data suggest that Adenosine A₁receptor blockade inhibits RANKL-induced osteoclast formation by inhibiting activation of JNK/c-Jun pathway, thereby suppressing the gene expression of c-fos and NFATc1 in osteoclast precursors. These results are consistent with the hypothesis that endogenously released adenosine, acting at A₁receptors, is critical for the expression and activation of critical signaling intermediates required for osteoclastogenesis. Moreover, these results further support the notion that blockade of adenosine A₁receptors may be useful in the treatment and prevention of osteoporosis and premature bone loss

Purpose: Adenosine is a nucleoside that is generated at sites of injury and hypoxia and which mediates its physiologic and pharmacologic effects via activation of one or more G protein coupled receptors (A1, A_2A, A_2Band A₃). We have previously reported that adenosine A₁receptor ligation is required for osteoclast formation and that blockade or deletion of adenosine A₁receptors leads to increased bone density. Because all four adenosine receptors are expressed on osteoclasts and osteoclast precursors we determined whether adenosine A_2Areceptors also regulate osteoclast formation in vivo and in vitro. Methods: Osteoclast differentiation was studied in vitro as the GM-CSF/RANKL stimulated formation of multinucleated (>=3 nuclei), TRAP-positive cells from primary murine (C57Bl/6) bone marrow-derived precursors. Signaling events were studied by Western Blot for activated (phosphorylated) signaling molecules and changes in message were determined by RT-PCR. Histological examination of bones from A_2Aknockout and wild type mice was carried out with TRAP and alcian blue stains. Murine bone was further analyzed by micro CT and electron microscopy. Results: The highly selective A_2Areceptor agonist CGS21680 inhibited osteoclast differentiation as much as 38 +/-1% with an IC₅₀of approximately 50nM (p<0.05), reversed by the selective high affinity antagonist ZM241385. CGS21680 (1uM) stimulated phosphorylation of ERK1/2 (122+/-3 and 134+/-3% of control at day 3 and 7 of differentiation, p<0.001 for both) and JAK1 (107+/-1% of control phosphorylation at day 7 of differentiation, p<0.01), activation that was reversed by ZM241385 (not shown). Moreover, CGS21680 inhibited NFkB translocation to the nucleus (nuclear NFkB decreased by 44+/-5% at day 3, p<0.001). There was also a marked increase in cellular IkB levels (maximal IkB increased by 19+/-1% at day 7, p<0.001) whereas pretreatment with ZM241385 increased phosphorylation of IkB (maximal pIkB increased by 13+/-0.4% at day 6, p <0.001) and enhanced nuclear translocation of NFkB (by 14+/-2% at day 3, p<0.01). MicroCT analysis of femurs from A_2AKO mice showed significantly decreased bone volume/total volume ratio (17.49+/-0.95 for A_2AKO vs. 22.61+/-2.15 for wild type, p<0.05), trabecular number (4.4+/-0.2 for A_2AKO vs 5.8+/-0.07 for wild type, p<0.001) and increased trabecular space (0.19 +/-0.006 for A_2AKO vs. 0.13+/-0.0004 for wild type, p<0.001). There were more osteoclasts in the TRAP-stained femurs of A_2AKO (50+/-13/lpf) than wild type mice (35+/-1/lpf, 6 fields each from femurs of 2 different mice each). Electron microscopy of osteoclasts in femurs from A_2AKO mice showed marked osteoclast membrane folding and increased osteoclast bone resorption. Conclusions: These results indicate that adenosine A_2Areceptors inhibit GM-CSF/RANKL-stimulated osteoclast differentiation and thereby regulate bone turnover. Because adenosine mediates the anti-inflammatory effects of methotrexate we further speculate that the capacity of methotrexate to inhibit bone erosion in patients with Rheumatoid Arthritis may be mediated by methotrexate-stimulated increases in adenosine concentration

Background: Increased production of extra cellular matrix in the skin is the hallmark of Scleroderma. We have previously reported that adenosine, a purine nucleoside produced in ischemic tissue, acting at A2A receptors, enhances dermal collagen production both in vitro and in a murine model of dermal fibrosis, although the mechanism by which adenosine receptor stimulation promotes collagen production is not clear. Fli1 is a known transcriptional repressor of fibrillar collagen genes and connective tissue growth factor (CTGF/CCN2) in dermal fibroblasts. To further clarify the mechanism by which A2A receptor stimulation induces dermal collagen accumulation, we explored the effects of A2A receptor occupancy on Fli1 and downstream mediators of fibroblast matrix production. Methods: Primary human dermal fibroblasts were stimulated with the selective adenosine A2AR agonist CGS21680 (1mM) for varying time periods and message levels (real time-RTPCR) for fli1 and CTGF were quantitated. In addition we measured cell associated and supernatant levels of collagen I and nuclear levels of fli1 by Western Blot. Results: Adenosine A2A receptor stimulation for 4 hours reduced Fli1 mRNA expression by 47+/-18% (4 hrs, p<0.05 vs. control, n=4) and reduced nuclear protein levels of Fli1 by 32+/-13% (24hrs, p<0.05, n=4). Because diminished nuclear fli1 should increase CTGF levels we next examined the effect of A2A receptor stimulation on CTGF mRNA and protein secretion. CTGF mRNA level was increased following A2A receptor stimulation for 8 hours by 3.7 fold. Consistent with the change in CTGF mRNA, CGS21680 stimulated a 4.7 fold increase in CTGF protein secretion at (24 hours) as well (p<0.03, n=4). As expected A2A receptor stimulation increased collagen I secretion (1.8-fold vs. control, p<0.05, n=3) and the increase in collagen production was completely abrogated by an antibody to CTGF (1.1+0.04 fold of control, p<0.05 vs.CGS, n=3) but not by control antiserum (1.6 +0.03fold of control, n=3). Conclusion: A2AR occupancy promotes dermal matrix production by suppressing expression of the transcriptional repressor fli1 leading to an increase in CTGF expression which acts in an autocrine fashion to stimulate collagen production. These findings further suggest that modulation of A2AR function may be a novel therapeutic target for limiting fibrosis in such conditions as scleroderma

ABSTRACT : Genome-wide association studies are the most comprehensive and straightforward approach to teasing out the identity of genetic polymorphisms associated with any given disease or characteristic. With the availability of DNA banks from large cohorts of ethnically matched patients and healthy individuals it is now possible to define even marginal genetic associations between genetic polymorphisms and diseases. As increasing numbers of these studies are carried out and as associations with smaller and smaller risks are identified, there is the growing concern that the findings will be of increasingly marginal value. Thus, the glut of new genetic associations is rapidly overwhelming our interest in the results, a situation that could be described as TMI (too much information). Recent genetic association studies in rheumatoid arthritis suggest that we may be approaching the TMI stage of genome-wide association studies