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Effect of adenosine on collagen production in cultured peritoneal mesothelial cells and fibroblasts. ( A ) Primary peritoneal mesothelial cells (PMC) were incubated with the A 2A R agonist CGS21680 (0.1–10 μ M). At 24 h supernatants were collected and analysed for collagen production by Sircol collagen dye-binding assay. ( B ) Primary peritoneal fibroblasts were isolated and cultured. The left panel shows a phase contrast picture of a representative fibroblast preparation. The right panel shows a representative fibroblast preparation stained by indirect immunofluorescence to vimentin. ( C ) Fibroblasts were incubated with adenosine (0.1–10 μ M), CGS21680 (0.1–10 μ M) in the presence or absence of the A 2A R antagonist (ZM241385, 10 nM) or ( D ) CGS21680 (0.1 μ M) in the presence of caffeine (100 μ M) or ZM241385 (10 nM). After 24 h supernatants were collected and analysed for collagen production by Sircol collagen dye-binding assay. The figure depicts a representative experiment out of three performed on different preparations of mesothelial cells or fibroblasts. Results are presented as mean ± SE of μ g/10 6 cells, n = 4 for each group, ∗ P < 0.05, ∗∗ P < 0.01 between untreated control and treatment.
Source publication
Long-term peritoneal dialysis (PD) is associated with peritoneal fibrosis and loss of function. It has been shown that activation of the adenosine A(2A) receptor (A(2A)R) promotes tissue repair, wound healing and extracellular matrix (ECM) production. We have previously shown that adenosine is a potent regulator of inflammation in the peritoneum. I...
Context in source publication
Context 1
... 2. Effect of caffeine on peritoneal fibrosis. CD1 mice were administered i.p. with CG or PDF for 2 or 4 weeks, respectively, and supplied with DW with or without caffeine (0.1% w/v). Sections of the peritoneal membrane were analysed for fibrosis intensity by ( A ) histological preparations stained with Masson’s trichrome (representative slides). Scale bar represents 200 μ m; the upper right panoramic figures were obtained by merging of three pictures. ( B ) Quantification of the mean submesothelial width and mean density of stained collagen analysed by the Image J software as described in Figure 1. ( C ) Determination of collagen content by the Sircol collagen dye-binding assay. ( D ) Lavage fluid collected and analysed for TGF- β by ELISA. ( E ) Extraction of total RNA and analysis of mRNA levels of FSP-1 and CTGF by quantitative real-time RT-PCR. mRNA levels were normalized to actin levels. Results are presented as mean ± SE, (B) + (D) fold of CT, n = 5 for each group. ∗ P < 0.05 and ∗∗ P < 0.01 between the marked groups. ˆ P < 0.05 and ˆˆ- P < 0.01 compared to the corresponding untreated control.
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Citations
... The CHX solution is usually injected intraperitoneally (ease of administration), but some researchers use a surgical procedure to insert the cannulas to avoid infection (transfer of skin microorganisms into the peritoneal cavity by repeated intraperitoneal injections) or the misconduct of intraperitoneal injection. The protocols used in the studies also differ in the number of CHX applications, which affect the duration of the study, extent of damage, inflammatory response, and pathological alterations [5][6][7][8][9][10][11][12]. ...
Understanding the tissue changes and molecular mechanisms of preclinical models is essential for creating an optimal experimental design for credible translation into clinics. In our study, a chlorhexidine (CHX)-induced mouse model of peritoneal fibrosis was used to analyze histological and molecular/cellular alterations induced by 1 and 3 weeks of intraperitoneal CHX application. CHX treatment for 1 week already caused injury, degradation, and loss of mesothelial cells, resulting in local inflammation, with the most severe structural changes occurring in the peritoneum around the ventral parts of the abdominal wall. The local inflammatory response in the abdominal wall showed no prominent differences between 1 and 3 weeks. We observed an increase in polymorphonuclear cells in the blood but no evidence of systemic inflammation as measured by serum levels of serum amyloid A and interleukin-6. CHX-induced fibrosis in the abdominal wall was more pronounced after 3 weeks, but the gene expression of fibrotic markers did not change over time. Complement system molecules were strongly expressed in the abdominal wall of CHX-treated mice. To conclude, both histological and molecular changes were already present in week 1, allowing examination at the onset of fibrosis. This is crucial information for refining further experiments and limiting the amount of unnecessary animal suffering.
... In contrast, we have shown that early desensitization of A 1 R can alter the balance towards an immunosuppressive A 2A R environment 11,12 . In previous studies, we characterized the immunosuppressive effects of A 1 R desensitization by pharmacological preconditioning (PPC) 11 . ...
Adenosine is widely known as a potent modulator of innate and acquired immunity. It is released during transplants, and acts on four subtype receptors. In previous studies, we demonstrated that pharmacological preconditioning (PPC), pre-administration of the selective A1 receptor (A1R) agonist led to A1R desensitization, is followed by upregulation of the adenosine A2A receptor. This immunosuppressive effect resulted in lymphopenia, and it reduced T-cell reactivity. The aim of the current study was to challenge the immunosuppressive effects of A1R-PPC in models of allogeneic grafts. PPC mice were treated by intraperitoneal injection using specific adenosine A1R agonist 24 h and 12 h before starting any procedure. We challenged our method in novel allogeneic muscle and skin grafts models. Mice and grafts were assessed by complete blood counts, MLR from PPC splenocytes, and pathological evaluation. We found a significant reduction in WBC and lymphocyte counts in PPC-treated mice. Two-way MLR with splenocytes from PPC grafted mice showed decreased proliferation and anergy. Histology of PPC allogeneic grafts revealed profoundly less infiltration and even less muscle necrosis compared to vehicle treated allografts. Similar results observed in PPC skin transplantation. To conclude, PPC moderated graft rejection in separate allogeneic challenges, and reduced lymphocytes infiltration and ischemic damage.
... Lower doses of CG or shorter time courses produced milder and more infrequent development of peritoneal fibrosis [99,100]. Recent studies showed that a standard peritoneal fibrosis model could be produced in mice following treatment with 0.1% CG every other day or three times a week for 1-3 weeks [101][102][103][104][105][106][107][108]. ...
Peritonitis is an important complication of peritoneal dialysis. Several animal peritonitis models have been described, including bacterial and fungal models that are useful for studying inflammation in peritonitis. However, these models have limitations for investigating peritoneal fibrosis induced by acute inflammation and present difficulties in handling the infected animals. Animal models of peritonitis which induced peritoneal fibrosis are important for establishing new therapies to improve peritoneal damage induced by peritonitis. Here, we present an overview of representative animal models of peritoneal dialysis-associated infectious and non-infectious peritonitis, including our novel animal models (scraping and zymosan models) that mimic peritoneal injury associated with fibrosis and neoangiogenesis caused by bacterial or fungal peritonitis.
... Caffeine has been associated with reduced collagen deposition and reduced fibrosis in the lung and liver (Feoktistov et al. 2009). This effect has been associated with an antagonist action of caffeine on adenosine receptors (Chan et al. 2006;Nakav et al. 2009). However, the inhibition of adenosine activity by caffeine is achieved by higher doses (25 mg/kg/ day) than the dose used in our experiment (approximately 2.5 mg/kg/day). ...
Coffee intake has been associated with a low risk of developing cancer, including prostate cancer, which is one of the most commonly diagnosed cancer in men. However, few studies have evaluated the chronic effects of caffeine, which is the most abundant methylxanthine in coffee, on prostate morphology and physiology. In the present study, we investigated the effects of chronic, low-dose caffeine intake on rat prostate morphology from puberty to adulthood. Five-week-old male Wistar rats were randomized into two experimental groups: caffeine-treated (20 ppm in drinking water, n = 12) and control (n = 12). The ventral and dorsolateral prostates were dissected, weighted and submitted to morphological, morphometrical and immunohistochemical analysis of cellular proliferation, apoptosis and androgen receptor (AR) tissue expression. The testosterone (T) and dihydrotestosterone (DHT) concentrations were measured in the plasma. Our results show that caffeine intake increased the concentrations of T and DHT, organ weight, epithelial cell proliferation and AR tissue expression in the ventral prostatic lobe. All the ventral prostates from the caffeine-treated animals presented various degrees of epithelial and stromal hyperplasia. Our results suggest that chronic caffeine intake from puberty increases androgenic signalling and cell proliferation in the rat prostate gland and can be related to the development of benign prostatic hyperplasia.
... A common complication of abdominal surgery or peritoneal dialysis is peritoneal fibrosis. Although peritoneal fibrosis is usually of little consequence, in some patients, peritoneal fibrosis can lead to bowel obstruction or other problems, so it is encouraging that recent studies in two different animal models of peritoneal fibrosis demonstrate that adenosine A 2A receptor blockade diminishes peritoneal fibrosis and adhesions [41]. ...
Adenosine-a purine nucleoside generated extracellularly from adenine nucleotides released by cells as a result of direct stimulation, hypoxia, trauma, or metabolic stress-is a well-known physiologic and pharmacologic agent. Recent studies demonstrate that adenosine, acting at its receptors, promotes wound healing by stimulating both angiogenesis and matrix production. Subsequently, adenosine and its receptors have also been found to promote fibrosis (excess matrix production) in the skin, lungs, and liver, but to diminish cardiac fibrosis. A commonly ingested adenosine receptor antagonist, caffeine, blocks the development of hepatic fibrosis, an effect that likely explains the epidemiologic finding that coffee drinking, in a dose-dependent fashion, reduces the likelihood of death from liver disease. Accordingly, adenosine may be a good target for therapies that prevent fibrosis of the lungs, liver, and skin.
... All animal experiments were approved by the Committee on Animal Experimentation of Sun Yat-sen University and performed in compliance with the university's Guidelines for the Care and Use of Laboratory Animals. Thirty male SD rats were randomly allocated into five groups: rats in group A (n ϭ 6) served as normal controls; rats in group B (n ϭ 6) and group C (n ϭ 6) received daily intraperitoneal injections of PDF named Dianeal ® PD-2 Peritoneal Dialysis Solution with 4.25% Dextrose (4.25% Dianeal; Baxter HealthCare, Deerfield, IL) at 100 ml/kg of body weight 36 ; rats in group D (n ϭ 6) were intraperitoneally injected with 10 mol/L DAPT together with 4.25% Dianeal; rats in group E (n ϭ 6) received the same amount of DMSO (the vehicle for DAPT) as group D together with 4.25% Dianeal. Rats of group B were sacrificed at 14 days and the rest of rats were sacrificed at 28 days after initial treatment. ...
Peritoneal fibrosis, a major complication of peritoneal dialysis, limits the effectiveness of peritoneal dialysis as a treatment of end-stage renal disease. Preventing this complication by identifying targets for therapy has recently received much attention. In the present study, we showed that Notch signaling was highly activated in rats in peritoneal dialysis fluid-induced fibrotic peritoneum, as indicated by increased expression of Jagged-1, Notch-1, and HES-1. Blocking Notch signaling activation by intraperitoneal injection of a gamma-secretase inhibitor, DAPT, significantly attenuated peritoneal fibrosis as indicated by the decreased expression of alpha-smooth muscle actin, collagen I, and vascular endothelial growth factor as well as increased expression of E-cadherin. Moreover, compared with control rats, DAPT-treated rats had a thinner peritoneum with less extracellular matrix accumulation, a lower mass transfer of glucose, and a higher ultrafiltration rate. In addition, transforming growth factor (TGF)-beta1 induced Notch signaling activation in primary rat peritoneal mesothelial cells. DAPT blocked this TGF-beta1-induced Notch signaling activation and therefore significantly inhibited TGF-beta1-induced expression of alpha-smooth muscle actin, collagen I, and vascular endothelial growth factor. Thus, a gamma-secretase inhibitor that interferes with Notch signaling prevents biochemical, histological, and functional consequences of peritoneal fibrosis through inhibiting epithelial to mesenchymal transition of rat peritoneal mesothelial cells. These results support the use of gamma-secretase inhibitors as a novel therapeutic approach for peritoneal fibrosis.
... However, as the overall pivotal role of CTGF in the fibrogenic process of the liver has been convincingly proven in experimental rat liver fibrosis with silenced CTGF [73,74], as strong overexpression of CTGF is found in both, fibrotic and tumor tissues [28,[65][66][67][68][69][70][71][72], and as caffeine is able to prevent D-GalN induced hepatic expression of CTGF in the rat in vivo, the molecular-biological mechanisms summarized above suggest a suppressive effect of caffeine, paraxanthine or cAMP analogs on human liver fibrosis. This suggestion is supported by very recent data showing that blocking adenosine A 2A receptors reduces peritoneal fibrosis in two independent experimental models in vivo [104]. Therefore, methylxanthines may eventually be proposed as a family of drugs useful in the treatment of chronic fibrogenic (or even carcinogenic) disorders. ...
Background:
Systemic sclerosis (SSc) is characterized by fibrosis, vascular disease and inflammation. Adenosine signaling plays a central role in fibroblast activation. The aim of the present study was to evaluate the therapeutic effects of adenosine depletion with pegylated adenosine deaminase (PEG-ADA) in preclinical models of SSc.
Methods:
The effects of PEG-ADA on inflammation, vascular remodeling and tissue fibrosis were analyzed in Fos-related antigen-2 transgenic (Fra2) mice and in the B10.D2→Balb/c(H-2d) model of sclerodermatous-chronic-graft-versus-host-disease (scl-cGvHD). The effects of PEG-ADA were confirmed in vitro in a human full-thickness-skin-model.
Results:
PEG-ADA effectively inhibited myofibroblast differentiation and reduced pulmonary (with decreased collagen expression by 34.3%, p=0.0079, n=6), dermal (51.8%, p=0.0006, n=6) and intestinal fibrosis (17.7%, p=0.0228, n=6) in Fra2 mice. Antifibrotic effects of PEG-ADA were also demonstrated in scl-cGvHD (38.4%, p=0.0063, n=8), and in a human full-thickness-skin model. PEG-ADA decreased inflammation and corrected the M2-Th2-ILC2-bias. Moreover, PEG-ADA inhibited proliferation of pulmonary vascular smooth muscle cells (40.5%, p<0.0001, n=6), prevented thickening of the vessel walls (39.6%, p=0.0028, n=6) and occlusions of pulmonary arteries (63.9%, p=0.0147, n=6). Treatment with PEG-ADA inhibited apoptosis of microvascular endothelial cells (65.4%, p=0.0001, n=6) and blunted the capillary rarefication (32.5%, p=0.0199, n=6). RNASeq demonstrated that treatment with PEG-ADA normalized multiple pathways related to fibrosis, vasculopathy and inflammation in Fra2 mice.
Conclusion:
Treatment with PEG-ADA ameliorates the three cardinal features of SSc in pharmacologically relevant and well-tolerated doses. These findings may have direct translational implications as PEG-ADA is already FDA-approved for the treatment of patients with ADA-deficient-SCID.
Background:
Connective tissue growth factor (CTGF/CCN2) regulates the signalling of other growth factors and promotes fibrosis. CTGF is increased in mice and humans with peritoneal fibrosis. Inhibition of CTGF has not been examined as a potential therapeutic target for peritoneal fibrosis because systemic CTGF knockout mice die at the perinatal stage.
Methods:
To study the role of CTGF in peritoneal fibrosis of adult mice, we generated CTGF conditional knockout (cKO) mice by crossing CTGF floxed mice with RosaCreERT2 mice. We administered tamoxifen to Rosa-CTGF cKO mice to delete the CTGF gene throughout the body. We induced peritoneal fibrosis by intraperitoneal injection of chlorhexidine gluconate (CG) in wild-type and Rosa-CTGF cKO mice.
Results:
Induction of peritoneal fibrosis in wild-type mice increased CTGF expression and produced severe thickening of the peritoneum. In contrast, CG-treated Rosa-CTGF cKO mice exhibited reduced thickening of the peritoneum. Peritoneal equilibration test revealed that the excessive peritoneal small-solute transport in CG-treated wild-type mice was normalized by CTGF deletion. CG-treated Rosa-CTGF cKO mice exhibited a reduced number of αSMA-, Ki67-, CD31- and MAC-2-positive cells in the peritoneum. Analyses of peritoneal mRNA showed that CG-treated Rosa-CTGF cKO mice exhibited reduced expression of Cd68, Acta2 (αSMA), Pecam1 (CD31) and Vegfa.
Conclusions:
These results indicate that a deficiency of CTGF can reduce peritoneal thickening and help to maintain peritoneal function by reducing angiogenesis and inflammation in peritoneal fibrosis. These results suggest that CTGF plays an important role in the progression of peritoneal fibrosis.
Adenosine, a nucleoside derived primarily from the extracellular hydrolysis of adenine nucleotides, is a potent regulator of inflammation. Adenosine mediates its effects on inflammatory cells by engaging one or more cell-surface receptors. The expression and function of adenosine receptors on different cell types change during the course of rheumatic diseases, such as rheumatoid arthritis (RA). Targeting adenosine receptors directly for the treatment of rheumatic diseases is currently under study; however, indirect targeting of adenosine receptors by enhancing adenosine levels at inflamed sites accounts for most of the anti-inflammatory effects of methotrexate, the anchor drug for the treatment of RA. In this Review, we discuss the regulation of extracellular adenosine levels and the role of adenosine in regulating the inflammatory and immune responses in rheumatic diseases such as RA, psoriasis and other types of inflammatory arthritis. In addition, adenosine and its receptors are involved in promoting fibrous matrix production in the skin and other organs, and the role of adenosine in fibrosis and fibrosing diseases is also discussed.
