Majid Katebi

Adenosine A2A receptors play an active role in mouse bone marrow-derived mesenchymal stem cell development.

Bone marrow‐derived mesenchymal stem cells (BM‐MSCs) play a role in wound healing and tissue repair and may also be useful for organ regeneration. As we have demonstrated previously that A2A adenosine receptors (A2AR) promote tissue repair and wound healing by stimulating local repair mechanisms and enhancing accumulation of endothelial progenitor cells, we investigated whether A2AR activation modulates BM‐MSC proliferation and differentiation. BM‐MSCs were isolated and cultured from A2A‐deficient and ecto‐5′nucleotidase (CD73)‐deficient female mice; the MSCs were identified and quantified by a CFU‐fibroblast (CFU‐F) assay. Procollagen α2 type I expression was determined by Western blotting and immunocytochemistry. MSC‐specific markers were examined in primary cells and third‐passage cells by cytofluorography. PCR and real time‐PCR were used to quantitate adenosine receptor and CD73 expression. There were significantly fewer CFU‐Fs in cultures of BM‐MSCs from A2AR knockout (KO) mice or BM‐MSCs treated with the A2AR antagonist ZM241385, 1 μM. Similarly, there were significantly fewer procollagen α2 type I‐positive MSCs in cultures from A2AR KO and antagonist‐treated cultures as well. In late passage cells, there were significantly fewer MSCs from A2A KO mice expressing CD90, CD105, and procollagen type I (P<0.05 for all; n=3). These findings indicate that adenosine and adenosine A2AR play a critical role in promoting the proliferation and differentiation of mouse BM‐MSCs.

Adenosine A2A Receptor Blockade or Deletion Diminishes Fibrocyte Accumulation in the Skin in a Murine Model of Scleroderma, Bleomycin-induced Fibrosis

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 A2A receptor-deficient and A2A antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis, thus the aim of this study was to determine whether the adenosine A2A 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, A2A knockout and A2A antagonist-treated mice were stained for Procollagen α2 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 A2A receptors. Because fibrocytes play a central role in tissue fibrosis these results suggest that diminished adenosine A2A 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 A2A receptors may represent a new target for the treatment of such fibrosing diseases as scleroderma or nephrogenic fibrosing dermopathy.

Neuroprotective Effects of Diazoxide and Its Antagonism by Glibenclamide in Pyramidal Neurons of Rat Hippocampus Subjected to Ischemia-Reperfusion-Induced Injury

Mitochondrial ATP-sensitive potassium channel opener, diazoxide, is shown to have protective effect on the heart and brain following ischemia-reperfusion-induced injury (IR/II). However, the detailed effect of diazoxide and its antagonist on neuronal death, mitochondrial changes, and apoptosis in cerebral IR/II has not fully studied. IR/II was induced in rats by the 4-vessel occlusion model. Neuronal cell death and mitochondrial changes in CA1–CA4 pyramidal cells of the hippocampus were studied by light and electron microscopy, respectively. Apoptosis was assessed by measuring the amount of protein expressed by Bax and Bcl-2 genes. In light microscopy studies, the number of total and normal cells were increased only following 18 mg/kg of diazoxide. Lower doses (2 and 6 mg/kg) failed to change the cell numbers. All three doses of glibenclamide (1, 5, and 25 mg/kg) decreased the number of total and normal cell populations. In electron microscopy studies, different doses of diazoxide and glibenclamide prevented and aggravated the IR-induced morphological changes, respectively. Western blot analysis showed that diazoxide and glibenclamide inhibited and enhanced Bax protein expression respectively. Regarding Bcl-2 expression, only diazoxide showed a significant enhancement of gene expression. In conclusion, the results show that diazoxide can exhibit neuroprotective effects against IR/II in hippocampal regions, possibly through the opening of mitochondrial ATP-sensitive K+ channels.

TGF- β1–mediated Apoptosis Associated With SMAD-dependent MitochondrialBcl-2 Expression

BACKGROUND Transforming growth factor (TGF) β1 can elicit various cellular responses, including inhibition of cell growth, migration, differentiation, and apoptosis. In addition, TGF-β1 is able to induce apoptosis in certain lymphomas. METHODS In the present study, the role of SMADs, Bax, Bcl-xl, and Bcl2 was characterized in 2 B-lymphoma cell lines, Burkitt and pre-B cell. RESULTS Apoptosis was detected after exposure of TGF-β on Raji and Nalm 6 cell lines and was evaluated by flow cytometry by using annexin V, reverse transcriptase-polymerase chain reaction, and Western blot analysis. Flow Cytometry With Cell Sorting analysis showed that apoptosis could be observed after 24 hours of TGF-β treatment and was continued after 48 hours. TGF-β downregulated the Bcl-xl and Bcl-2, whereas the Bax was upregulated. Furthermore, messenger RNA of SMAD6 and SMAD7 showed the significant upregulation. CONCLUSION The results indicated that alteration in gene expression and protein level may determine the induction of apoptosis pathway in these lymphoma cell lines exposed to TGF-β.

Brain Derived Neurotrophic Factor Modification of Epileptiform Burst Discharges in a Temporal Lobe Epilepsy Model

Introduction: Transforming Growth Factor-Beta 1 (TGF-β1) is a pleiotropic cytokine with potent anti-inflammatory property, which has been considered as an essential risk factor in the inflammatory process of Ischemic Stroke (IS), by involving in the pathophysiological progression of hypertension, atherosclerosis, and lipid metabolisms. -509C/T TGF-β1 gene polymorphism has been found to be associated with the risk of IS. The aim of this meta-analysis was to provide a relatively comprehensive account of the relation between -509C/T gene polymorphisms of TGF-β1 and susceptibility to IS. Methods: Male Wistar rats were divided into sham (receiving phosphate buffered saline within dorsal hippocampus), pilocarpine (epileptic model of TLE), single injection BDNF (epileptic rats which received single high dose of BDBF within dorsal hippocampus), and multiple injections BDNF (epileptic rats which received BDNF in days 10, 11, 12, and 13 after induction of TLE) groups. Their electrocorticogram was recorded and amplitude, frequency, and duration of spikes were evaluated. Results: Amplitude and frequency of epileptiform burst discharges were significantly decreased in animals treated with BDNF compared to pilocarpine group. Conclusion: Our findings suggested that BDNF may modulate the epileptic activity in the animal model of TLE. In addition, it may have therapeutic effect for epilepsy. More studies are necessary to clarify the exact mechanisms of BDNF effects.