Faculty Bibliography

Wound healing and tissue repair are critical processes, and adenosine, released from injured or ischemic tissues, plays an important role in promoting wound healing and tissue repair. Recent studies in genetically manipulated mice demonstrate that adenosine receptors are required for appropriate granulation tissue formation and in adequate wound healing. A(2A) and A(2B) adenosine receptors stimulate both of the critical functions in granulation tissue formation (i.e., new matrix production and angiogenesis), and the A(1) adenosine receptor (AR) may also contribute to new vessel formation. The effects of adenosine acting on these receptors is both direct and indirect, as AR activation suppresses antiangiogenic factor production by endothelial cells, promotes endothelial cell proliferation, and stimulates angiogenic factor production by endothelial cells and other cells present in the wound. Similarly, adenosine, acting at its receptors, stimulates collagen matrix formation directly. Like many other biological processes, AR-mediated promotion of tissue repair is critical for appropriate wound healing but may also contribute to pathogenic processes. Excessive tissue repair can lead to problems such as scarring and organ fibrosis and adenosine, and its receptors play a role in pathologic fibrosis as well. Here we review the evidence for the involvement of adenosine and its receptors in wound healing, tissue repair and fibrosis

Purpose: Unlike other DMARDs methotrexate diminishes the risk of Atherosclerotic Cardiovascular Disease (ASCVD) in patients with Rheumatoid Arthritis and adenosine, acting at adenosine A2A receptors, has been shown to mediate the anti-inflammatory effects of methotrexate. Adenosine inhibits the first step in formation of atherosclerotic plaque, foam cell formation in macrophages and this effect appears to be mediated by enhanced expression of cholesterol 27-hydroxylase, an enzyme involved in reverse cholesterol transport. We therefore asked whether the effect of adenosine A2A receptors on foam cell formation in vitro are mediated by apoE or 27-hydroxylase (27OH'ase), proteins involved in reverse cholesterol transport. Method: THP-1 cells, a human monocytoid cell line, were infected with lentiviral vectors expressing siRNA for either apoE or 27OH'ase or scrambled RNA and infected cell lines were selected by incubation with puromycin. Foam cell formation was induced in THP-1 cells by incubation with interferon- (500U/ml) and % foam cells enumerated in 5 high power fields. 3H-Cholesterol efflux was measured after loading with label. Results: Specific lentiviral siRNA infection markedly reduces apoE (p< 0.0001, apoE siRNA vs. control, n=3) or 27OH'ase mRNA (p< 0.0001, 27-hydroxylase siRNA vs. control, n=3) and protein (p< 0.0107, 27-hydroxylase siRNA vs. control n= 3) in THP-1 cells. Despite diminished apoE expression CGS-21680 (1muM), an adenosine A2A receptor agonist, inhibits IFN-induced foam cell formation (p< 0.0002, IFN CGS vs. IFN alone, n= 4) but has no effect on foam cell formation in 27OH'ase KD cells. CGS21680 increases cholesterol efflux in wild type and apoE1 KD cells (from 9.5% to 17.5+2.5% and from 10.0+2% to 17.5 +2%, respectively) but not 27OH'ase KD cells. Conclusion: Adenosine A2A receptor-mediated increases in reverse cholesterol transport leading to diminished foam cell formation explains the anti-atherosclerotic effects of methotrexate

Purpose: Recent studies indicate that increased fructose ingestion is a risk factor for development of hyperuricemia and gout. Hyperuricemia is also more common in obese individuals and often accompanies the metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). It has long been known that fructose induces ATP hydrolysis in the liver and the hydrolyzed ATP is further metabolized to uric acid in the intracellular and extracellular space (by ecto-5'nucleotidase, CD73). An intermediate in ATP hydrolysis to uric acid, adenosine, acts at one or more of four receptors (A1, A2A, A2B and A3) to effect a variety of physiologic and pharmacologic effects. Because we have recently demonstrated that A1 and A2B adenosine receptors play a critical role in the development of fatty liver due to ethanol ingestion, another risk factor for hyperuricemia (Peng, Z. J Clin Invest, 2009), we therefore determined whether adenosine and its receptors play a role in fructose-induced NAFL as well. Method: Fatty liver was induced by feeding a high-fructose diet (protein 20%, fat 13%, fructose 60%, and complex carbohydrates 7% of total kcal) for six weeks. Mice (all on C57Bl6 background) were treated with either the selective A1 receptor antagonist DPCPX or the selective A2B receptor antagonist enprofylline for the final four weeks of the experiment. Hepatic steatosis was graded semiquantitatively and hepatic triglyceride was measured photometrically. Adenosine concentration in the supernatant of cultured hepatic slices was measured by HPLC. Results: Livers from fructose-fed mice released significantly more adenosine than chow-fed mice (643+/-53 vs 91+/-2nM, n=6, p<0.01) and this increase depended, in part, on the presence of ecto-5'nucleotidase since the increase was blunted in livers of CD73KO mice (384+/-18 vs 26+/-3 nM, n=6, p<0.01). WT and A3KO mice developed severe hepatic steatosis after chronic fructose ingestion with obvious lipid droplets present in nearly 100% of hepatocytes in both periportal and pericentral areas, but A1KO mice and DPCPX-treated WT mice suffered only minimal fatty change (Steatosis grades: WT 3.9+/-0.6 or A3KO 3.7+/-0.4 vs A1KO 2.7+/-0.5 or DPCPX: 2.8+/-0.5, p<0.01, respectively). Similarly hepatic triglyceride levels increased following fructose ingestion in WT and A3KO mice (WT: from 30+/-3 to 131+/-11mg/g; A3KO from 32+/-2 to 138+/-11mg/g, n=6, p<0.01, respectively) but not so much in A1KO mice or mice treated with DPCPX (A1KO: from 29+/-3 to 76+/-6mg/g; DPCPX: 74+/-11mg/g vs untreated, n=6, p<0.01, respectively). Mice treated with enprofylline were similarly protected from developing hepatic steatosis as reflected by steatosis grade (2.5+/-0.3, n=6, p<0.01, vs untreated) and hepatic triglyceride content (72+/-8mg/g, n=6, p<0.01, vs untreated). Conclusion: These results indicate that chronic fructose ingestion leads to increased release of adenine nucleotides resulting in higher uric acid levels and fatty liver

OBJECTIVE: To determine whether methotrexate (MTX) can overcome the atherogenic effects of cyclooxygenase 2 (COX-2) inhibitors and interferon-gamma (IFNgamma), both of which suppress cholesterol efflux protein and promote foam cell transformation in human THP-1 monocyte/macrophages. METHODS: Message and protein levels of the reverse cholesterol transport proteins cholesterol 27-hydroxylase and ATP-binding cassette transporter A1 (ABCA1) in THP-1 cells were evaluated by real-time polymerase chain reaction and immunoblot, respectively. Expression was evaluated in cells incubated in the presence or absence of the COX-2 inhibitor NS398 or IFNgamma, with and without MTX. Foam cell transformation of lipid-laden THP-1 macrophages was detected with oil red O staining and light microscopy. RESULTS: MTX increased 27-hydroxylase message and completely blocked NS398-induced down-regulation of 27-hydroxylase (mean +/- SEM 112.8 +/- 13.1% for NS398 plus MTX versus 71.1 +/- 4.3% for NS398 alone; P < 0.01). MTX also negated COX-2 inhibitor-mediated down-regulation of ABCA1. The ability of MTX to reverse inhibitory effects on 27-hydroxylase and ABCA1 was blocked by the adenosine A2A receptor-specific antagonist ZM241385. MTX also prevented NS398 and IFNgamma from increasing transformation of lipid-laden THP-1 macrophages into foam cells. CONCLUSION: This study provides evidence supporting the notion of an atheroprotective effect of MTX. Through adenosine A2A receptor activation, MTX promotes reverse cholesterol transport and limits foam cell formation in THP-1 macrophages. This is the first reported evidence that any commonly used medication can increase expression of antiatherogenic reverse cholesterol transport proteins and can counteract the effects of COX-2 inhibition. Our results suggest that one mechanism by which MTX protects against cardiovascular disease in rheumatoid arthritis patients is through facilitation of cholesterol outflow from cells of the artery wall

Peripheral blood fibrocytes are a newly identified circulating leukocyte subpopulation that migrates into injured tissue where it may display fibroblast-like properties and participate in wound healing and fibrosis of skin and other organs. Previous studies in our lab demonstrated that A(2A) receptor-deficient and A(2A) antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis, thus the aim of this study was to determine whether the adenosine A(2A) receptor regulates recruitment of fibrocytes to the dermis in this bleomycin-induced model of dermal fibrosis. Sections of skin from normal mice and bleomycin-treated wild type, A(2A) knockout and A(2A) antagonist-treated mice were stained for Procollagen alpha2 Type I and CD34 and the double stained cells, fibrocytes, were counted in the tissue sections. There were more fibrocytes in the dermis of bleomycin-treated mice than normal mice and the increase was abrogated by deletion or blockade of adenosine A(2A) receptors. Because fibrocytes play a central role in tissue fibrosis these results suggest that diminished adenosine A(2A) receptor-mediated recruitment of fibrocytes into tissue may play a role in the pathogenesis of fibrosing diseases of the skin. Moreover, these results provide further evidence that adenosine A(2A) receptors may represent a new target for the treatment of such fibrosing diseases as scleroderma or nephrogenic fibrosing dermopathy