Faculty Bibliography

Background/Purpose: Atherosclerosis is characterized by the accumulation of lipid-laden macrophages in the arterial walls. Patients with inflammatory arthritis and psoriasis are at greater risk of developing atherosclerotic plaques with their associated cardiovascular diseases. Recent studies indicate that effective therapy of inflammatory arthritis may reduce the risk of atherosclerotic cardiovascular disease although the basis for this risk reduction is not totally clear. Recently apremilast, an inhibitor of PDE4, has been introduced into the clinical armamentarium for the treatment of psoriasis and inflammatory arthritis. We and others have recently demonstrated that agents, such as adenosine A2A receptor stimuli, that stimulate cAMP accumulation in macrophages can reduce foam cell formation by a protein kinase A (PKA) - mediated mechanism involving enhanced efflux of cholesterol and lipids. We therefore asked whether apremilast might have a similar effect Methods: RAW264.7 murine macrophage cell line was infected with lentiviruses expressing shRNA to silence PKA, EPAC1 and EPAC2, or scrambled shRNA. Knockdown was confirmed by Western Blot. The effect of apremilast on foam cell formation was tested by examination of 70% confluent cells in 48-well plates following treatment with Interferon-gamma (IFNgamma, 0.5u/ul) for 24 hours and then treatment with acetylated LDL (50ug/ml) with/without apremilast (10uM) for another 48 hours. Cells were stained with oil-O-red and then cells containing lipid droplets were counted. To determine whether apremilast affected cholesterol efflux cells (70% confluent in 48-well plates) were treated with IFNgamma (0.5u/ul) for 24 hours and followed by treatment with acLDL (50ug/ml) for another 24 hours. After treatment with bodipy-cholesterol for 1 hour, cells were cultured in the equilibration buffer for 18 hours and treated with HDL (20ug/ml) and ApoA1(10ug/ml with/without apremilast (AP, 10uM) for 4 hours. 200ul of each supernant /cell lysate (lysed in 1% cholic acid) were analyzed by record fluorescence @482/515nm. Results: Apremilast treatment reduced foam cell formation in RAW264.7 cells stably expressing scrambled, EPAC1 and EPAC2, but not PKA shRNA (45+/-2%, 43+/-5%, 42+/-1% and -14+/-6% reduction, respectively, p<0.001, ANOVA). Apremilast treatment enhanced HDL-induced and apoA1-induced cholesterol efflux from RAW264.7 cells (2.345 +/- 0.03% vs. 1.733 +/- 0.08%, p<0.001, n=4 and 9.74 +/- 0.32% vs.3.96+/-0.77%, p<0.05, n=3). Furthermore, Apremilast treatment enhanced HDL-induced from Raw264.7 cells stably expressing scrambled, EPAC1, and PKA but not in EPAC2 shRNA (0.737+/-0.6% vs.1.19+/-0.04% of control, p<0.05, ANOVA); apremilast treatment enhanced ApoA1-induced cholesterol efflux from RAW264.7 cells stably expressing scrambled, but not EPAC1, EPAC2, and PKA shRNA (2.62+/-0.15%, 1.55+/-0.22%, 1.44+/-0.04% a vs. 0.95+/-0.15% of control, respectively, p<0.01, p<0.05 and p<0.01, ANOVA). Thus, these results suggested that 1)apremilast promotes HDL-induced cholesterol efflux through increasing intracellular cAMP with EPAC1dependent mechanism, and apoA1-induced cholesterol efflux through increasing intracellular cAMP with EPAC1, EPAC2, PKA dependent mechanisms, 2) apremilast inhibits foam cell formation through increasing intracellular cAMP by only PKA-dependent mechanism Conclusion: These results suggest that apremilast may be useful for the treatment/prevention of atherosclerosis in patients with psoriasis and inflammatory arthritis

Background/Purpose: Gout is an independent risk factor for cardiovascular disease (CVD). Investigators studying the relationship between gout and CVD have focused on acute coronary outcomes, with limited evidence available regarding peripheral arterial function. Using high-resolution ultrasound imaging of the brachial artery, we examined endothelial and smooth muscle arterial function in gout subjects versus healthy controls. Methods: 34 untreated male gout subjects and 64 healthy control males were included. By enrollment criteria some gout subjects, but no healthy controls, had coronary artery disease (CAD) or diabetes, or were current smokers. Demographics and medical history were recorded. Participants underwent brachial artery flow-mediated dilation (FMD; arterial response to blood flow after transient interruption using a distal blood pressure cuff) and nitroglycerine-mediated dilation (NMD) to assess endothelium-dependent and independent arterial smooth muscle responsiveness, respectively. Dynamic ultrasound images were assessed by two independent observers, with results reported as percentage change in arterial diameter from baseline. Results: Compared with healthy controls, gout subjects had a higher prevalence of CAD (21% vs 0%, p<0.05), chronic kidney disease (76% vs 0, p<0.05), hypertension (71% vs 22%, p<0.05) and hyperlipidemia (50% vs 18%, p<0.05), but a similar low prevalence of diabetes (6% vs 0%, p=0.12). 29% of gout patients were current smokers (p vs control<0.05). Gout subjects were slightly older (58.9 vs 53.2 years, p<0.05), and significantly more gout patients were African American (44% vs 8%). Both FMD (2.20+/-3.12 vs 3.56+/-2.50, p=0.021) and NMD (16.69+/-9.01 vs 24.51+/-7.18, p=0.00002) were significantly reduced in the gout group vs controls. Gout nonsmokers, white gout patients, and gout patients lacking specific co-morbidities persisted in having decreased FMD and NMD compared with controls. Gout patients with versus without specific co-morbidities had similar degrees of impaired FMD and NMD. Analysis of the gout group showed an inverse Pearson correlation between FMD and CRP (R=-0.42, p=0.017), a trend for inverse Pearson correlation between FMD and serum urate (R=-0.31, p= 0.08); but no correlation between NMD and CRP or serum urate. Conclusion: Compared with healthy controls, patients with gout have reduced arterial function as measured by FMD and NMD. While the increased prevalence of comorbidities among gout patients may contribute to diminished arterial function, it appears to be insufficient to explain the endothelial and smooth muscle dysfunction observed. Hyperuricemia and chronic inflammation may contribute to endothelial dysfunction among gout patients, but do not appear to contribute to smooth muscle dysfunction. Whether appropriate gout therapy may improve FMD and NMD in gout patients remains to be determined. (Figure Presented)

Background/Purpose: Human Immunodeficiency Virus (HIV) infection devastates the immune system but also affects tissues and organs such as kidney, liver, central nervous system, heart and bone. Bone alterations have been observed in HIV disease for nearly two decades, in particular a higher risk of low bone mineral density (BMD) and fragility fractures. Treatment of patients with tenofovir alone or in combination (as part of HAART), leads to changes in bone catabolism markers and significant reductions in BMD in children and young adults. Tenofovir is taken up by cells and phosphorylated; tenofovir-phosphate inhibits HIV-reverse transcriptase by mimicking AMP. We have recently found that tenofovir inhibits Pannexin-1/Connexin-43-mediated ATP release from cells and decreases extracellular adenosine levels and fibrosis in murine models. As adenosine and ATP are key regulators of bone homeostasis, we determined whether tenofovir directly affects bone by an adenosine- or ATP-dependent mechanism. Methods: M-CSF/RANKL-induced osteoclast (OC) and stimulated osteoblast (OB) differentiation were studied in primary murine bone marrow culture as the number of TRAP-positive or Alizarin Red-positive cells, respectively, after challenge with tenofovir (1nM-100mM) alone or in combination with dipyridamole (1nM-100mM), an agent that increases extracellular adenosine by blocking cellular adenosine uptake. Pannexin-1 and Connexin-43 expression were permanently knocked down by lentiviral infection with appropriate shRNA or scrambled shRNA and these cells were induced to differentiate into OC by RANKL. Male C57Bl/6 (WT), A2AKO and A2BKO mice received tenofovir 75mg/Kg/day for 4 weeks. Double labelling of bone with calcein (15mg/Kg)/Alizarin Red (30mg/Kg) was performed and long bones prepared for mCT and histology. Results: There was a dose-dependent increase in OC differentiation after treatment with Tenofovir (EC50=44.5nM), that was reversed by dipyridamole (IC50=0.3muM). Moreover, tenofovir inhibited OB differentiation (IC50=0.4muM) which was also reversed by dipyridamole (EC50=10nM). When both Pannexin-1 and Connexin-43 were absent, tenofovir did not increase OC number. Tenofovir treatment reduced bone formation in WT-mice (49+/-8mum vs 110+/-7mum untreated p<0.0005) but not in A2AKO (72+/-6mum vs 71+/-5mum untreated, p=ns) and A2BKO mice (64+/-8mum vs 86+/-9mum untreated, p=ns). mCT revealed decreased BMD and both cortical and trabecular bones were affected. TRAP-staining showed increased OCs in vivo in tenofovir-treated WT mice (21+/-1 vs 16+/-1 OC/hpf in untreated, p<0.005). There are increased osteoclasts in A2AKO mice and this was unaffected by tenofovir treatment (24+/-1 OC/hpf, vs 22+/-1 OC/hpf in untreated p=ns) and osteoclast number in A2BKO mice was unchanged (19+/-1 OC/hpf, vs 18+/-1 OC/hpf in untreated, p=ns). Similar results were obtained for Cathepsin K. Conclusion: These results indicate that tenofovir enhances osteoclast differentiation and inhibits osteoblast differentiation by an adenosine-dependent mechanism and suggests that treatment with agents that increase local adenosine concentrations, like dipyridamole, might prevent bone loss following tenofovir treatment

There is growing recognition that bone serves important endocrine and immunologic functions that are compromised in several disease states. While many factors are known to affect bone metabolism, recent attention has focused on investigating the role of purinergic signaling in bone formation and regulation. Adenosine is a purine nucleoside produced intracellularly and extracellularly in response to stimuli such as hypoxia and inflammation, which then interacts with P1 receptors. Numerous studies have suggested that these receptors play a pivotal role in osteoblast, osteoclast, and chondrocyte differentiation and function. This review discusses the various ways by which adenosine signaling contributes to bone and cartilage homeostasis, while incorporating potential therapeutic applications of these signaling pathways.

In humans low levels of growth hormone (GH) and its mediator, insulin-like growth factor-1 (IGF-1), associate with hepatic lipid accumulation. In mice, congenital liver-specific ablation of the GH receptor (GHR) results in reductions in circulating IGF-1 and hepatic steatosis, associated with systemic insulin-resistance. Due to the intricate relationship between GH and IGF-1, the relative contribution of each hormone to the development of hepatic steatosis is unclear. Our goal was to dissect the mechanisms by which hepatic GH resistance leads to steatosis and overall insulin resistance, independent of IGF-1. We have generated a combined mouse model with liver-specific ablation of GHR in which we restored liver IGF-1 expression via hepatic IGF-1 transgene. We found that liver-GHR ablation leads to increases in lipid uptake, de novo lipogenesis, hyperinsulinemia and hyperglycemia accompanied with severe insulin resistance, and increased body adiposity and serum lipids. Restoration of IGF-1 improved overall insulin sensitivity, lipid profile in serum, reduced body adiposity, but was insufficient to protect against steatosis-induced hepatic inflammation or oxidative stress. We conclude that the impaired metabolism in states of GH resistance results from direct actions of GH on lipid uptake and de novo lipogenesis, while its actions on extrahepatic tissues are mediated by IGF-1.