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  • br Methods br Results br Discussion First the cells

    2023-01-29


    Methods
    Results
    Discussion First, the Erastin isolated from non-PD TA tissue and PD plaque tissue were characterized. The results showed that both cell groups were positive for vimentin and negative for desmin suggesting that these cells were fibroblasts. It was also observed that treatment with TGF-β1 did not alter the expression of these specific markers, but significantly increased the expression of α-SMA confirming myofibroblast phenotype. Myofibroblasts have a crucial role in pathophysiology of fibrosis, as these cells are not only responsible for contraction but also for production of several cytokines and abundant ECM proteins. Myofibroblast transformation is well established in the literature as being an important contributor in the pathophysiology of several fibrotic disorders including PD. A key feature of myofibroblasts is their α-SMA expression, which can be targeted to investigate the myofibroblast differentiation. Both mRNA and protein levels of α-SMA were assessed by exposing cells derived from non-PD TA tissue and PD plaque tissue to TGF-β1. In both cell groups, a significant increase of α-SMA mRNA levels was observed when treated with TGF-β1. The effect of TGF-β1 on mRNA levels was mirrored by α-SMA protein expression, that is, both cell populations showed an increase in α-SMA-positive cells in the presence of TGF-β1. These results are supported by previously published reports that have shown that both α-SMA mRNA and protein levels increase in the presence of TGF-β1 in fibroblasts obtained from PD tissue.10, 11 Adenosine receptors have been associated with the progression of chronic tissue injuries when the levels of adenosine are increased, suggesting that adenosine may promote fibrosis. To the best of the author’s knowledge, this is the first time that the expression of adenosine receptors has been investigated in cells derived from non-PD TA tissue and PD plaque tissue. In order to demonstrate the involvement of adenosine receptors in myofibroblast transformation in PD, the expression of these receptors was investigated in cells isolated from non-PD TA tissue and PD plaque tissue. Among the 4 receptors, the expression of ADORA1 and ADORA2B was significantly higher in PD plaque-derived cells than in non-PD TA cells; however, this increase was dramatically higher in ADORA1 transcripts levels. The expression of ADORA2A and ADORA3 was below the detection limits, therefore these 2 receptors were not studied further. Zhong et al (2005) found that primary human lung fibroblasts expressed high mRNA levels of ADORA2B and the expression of this receptor at protein levels was confirmed by immuno-fluorescence. These authors also showed that the activation of the ADORA2B by adenosine promoted myofibroblast transformation. In our study, the effect of TGF-β1 on mRNA levels of both adenosine receptors was also assessed, leading to a significant decrease of ADORA1 and ADORA2B mRNA levels in both cell groups. Even though the mRNA levels were significantly decreased, it may not represent a biological change in the transcript levels, as according to MIQE guidelines, to show biological changes a 2-fold (recommended cut-off value) increase or decrease should be observed. The protein levels of ADORA1 and ADORA2B in both cell populations was assessed using the ICE assay, showing that a significant increase of ADORA1 was observed in cells treated with TGF-β1; however, no difference was observed between non-PD TA cells and PD plaque-derived cells. In addition, Wen et al (2010) showed that primary corpus cavernosal fibroblasts from mice expressed ADORA2B, which was suggested to be responsible for adenosine-mediated penile fibrosis. Furthermore, 2 other studies have also shown that deaminase-deficient mice had an increment of adenosine levels and ADORA2B activation in the penis, suggesting an essential mechanism for the progression of priapism in these mice.23, 32 The role of ADORA1 and ADORA2B was assessed in TGF-β1-induced myofibroblast transformation using ADORA1 and ADORA2B agonists and antagonists. CPA and BAY 60-6583 were utilized as ADORA1 and ADORA2B agonists, respectively, while SLV 320 and MRS 1754 were used as ADORA1 and ADORA2B antagonists, respectively. ADORA1 agonist, CPA, showed an inhibitory effect on TGF-β1-induced myofibroblast transformation only at high concentrations while affecting the cell viability; therefore, we conclude that the inhibitory effect is most probably due to cytotoxicity at the high concentrations. ADORA2B agonist BAY 60-6583 significantly inhibited the transformation in a concentration-dependent manner with an IC50 value of 30 μmol/L and had no cytotoxicity either on fibroblasts or myofibroblasts. No inhibition of TGF-β1-induced myofibroblast transformation was observed with the ADORA1 antagonist (SLV 320) and ADORA2B antagonist (MRS 1754). We are particularly surprised to see no effect by ADORA2B antagonist since we would have expected inhibition of myofibroblast transformation with this compound. However, we have observed an inhibition by ADORA2B agonist suggesting that endogenous adenosine may have anti-fibrotic effect under these conditions, which obviously warrants further research.