Platelet rich plasma and growth factors cocktails for diabetic foot ulcers treatment: State of art developments and future prospects.

Current advances in diabetic foot ulcers (DFU) treatment are discussed. Normal and pathological wound healing process are observed and the role of growth factors (GFs) is elucidated. Current techniques involving GFs and platelet rich plasma (PRP) are compared. Up-to-date research suggests that treatment with single growth factor (GF) could be insufficient and not encompassing all pathological changes in DFU bed. Efficiency of PRP is rather controversial and lacks evidence. Thus the use of cocktail of particular GFs is suggested. Pro et contra of each approach are discussed.

[Energy corrective and antioxidative actions of cytoflavin during postischemic period of human dermal fibroblasts in vitro].

The influence of metabolic drug Cytoflavin (CF) with antihypoxic and antioxidative properties on human dermal fibroblasts in a model of ischemia-reoxygenation in vitro was studied. It was revealed that the restoration of ATP synthesis in fibroblasts in the postischemic period was considerably accelerated (in 2.1 times) by the addition of CF to the culture medium. The drug had a cell protective effect of reducing cell mortality during the reoxygenation after ischemia by 2-2.7 times. CF effectively reduced the level of reactive oxygen species (ROS) in fibroblasts after H2O2 treatment which allowed maintaining their survival at the level of control cells. Pretreatment of the cells with CF for one day ensured the maintenance of normal levels of ROS during the investigated time period in the fibroblasts subjected to H2O2 treatment, and reduced H2O2-induced cell death by almost a third compared to control cells. The introduction of CF in culture medium after ischemia showed no influence on Hsp70 synthesis, but led to decrease in GRP78 synthesis, raised after ischemia, to the control level, indicating a resolve of the endoplasmic reticulum (ER) stress and functional normalization of ER.

[Metalloprotease-mediated HB-EGF release regulates EGF receptor transactivation in A431 cells under oxidative stress].

We have shown earlier that H2O2 induces EGF receptor transactivation in different cells overexpressing EGF receptor. Mechanism of H2O2-induced EGF receptor transactivation in A431 human epidermoid carcinoma cells was examined in this work. We have demonstrated autophosphorylation of Tyr1045, 1068, 1148, 1173 as well as phosphorylation of Tyr845 of EGF receptor in response to H2O2, as assessed by autophosphorylation specific antibody. Tyrosine phosphorylation of EGF receptor by H2O2 did not involve receptor autophosphorylation at Tyr992. Blocking functions of metalloproteases by broad-spectrum inhibitor GM6001 suppressed H2O2-induced phosphorylation of EGF receptor, suggesting dependence of the transactivation on metalloproteases activity. To elucidate the possible role of EGF receptor agonists in its activation we used HB-EGF and TGF-alpha neutralizing antibody. H2O2-induced EGF receptor phosphorylation was inhibited by HB-EGF, but not TGF-alpha, neutralizing antibody. Taken together, our data suggest that, in human epidermoid carcinoma A431 cells, H2O2 stimulates EGF receptor transactivation via metalloprotease-dependent HB-EGF release and autophosphorylation.

[Interferon gamma-mediated growth regulation of epithelial cells].

Interferon gamma (IFNgamma) is known to inhibit proliferation of certain transformed cell lines. Recently, we have demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to IFNgamma (Burova et al., 2007) and provided direct evidence for the dependence of IFNgamma-induced EGFR transactivation upon EGFR expression level in epithelial cells (Gonchar et al., 2008). This study examines an antiproliferative effect of IFNgamma on human epithelial cells lines A431 and HeLa which express high levels of EGFR, as well as HEK293, which expresses low levels of EGFR. We characterized the IFNgamma-induced changes in these cells by studying cell growth, the cell cycle and induction of apoptosis. The response to IFNgamma differed in the tested cell lines: cell growth was inhibited in both A431 and HeLa cells, but not in HEK293 cells, as shown by cell counts and MTT. The cell cycle phases analyzed by flow cytometry were disturbed in A431 and HeLa cells in response to IFNgamma. In contrast, IFNgamma treatment did not alter distribution by cell cycle phases in HEK293. Our results indicate that IFNgamma exhibit an antiproliferative effect depending on the increased expression of EGFR in A431 and HeLa cells. Further, it was demonstrated that IFNgamma induced the caspase 3 activation in A431 cells, suggesting an involvement of active caspase 3 in IFNgamma-induced apoptosis.

[The level of EGF receptor expression effects its transactivation by IFN gamma in epithelial cells].

Earlier, we demonstrated transactivation of the epidermal growth factor receptor (EGFR) in response to interferon gamma (IFNgamma) in epidermal carcinoma A431 cells. It was shown that IFNgamma-induced EGFR transactivation is impossible in some cancer epithelial cells. Here, we hypothesize that IFNgamma-dependent EGFR transactivation in these cells correlates with EGFR quantity on the cell surface. To test this suggestion, a line of stably transfected HEK293 cells (HEK293delta99 cells) expressing high level of mutant EGFR lacking 99 C-terminal residues has been established. HEK293delta99 cells demonstrated EGFR transactivation in response to IFNgamma unlike the parent HEK293 cells, in which transactivation lacked. In HEK293delta99 and A431 cells, the time courses of EGFR activation induced by IFNgamma have the same pattern. In HEK293delta99 cells like A431, IFNgamma-induced EGFR transactivation requires EGFR kinase activity and occurs via autophosphorylation mechanism. Taken together, these data provide direct evidence of the dependence of IFNgamma-induced EGFR transactivation upon EGFR expression level in epithelial cells.

[STAT1 and STAT3 activation by oxidative stress in A431 cells involves Src-dependent EGF receptor transactivation]. / Aktivatsiia transkriptsionnykh faktorov STAT1 i STAT3 pri okislitel'nom stresse v kletkakh A431 vkliuchaet Src-zavisimuiu transaktivatsiiu retseptora EGF.

Different cellular signal transduction cascades are affected by environmental stressors (UV-radiation, gamma-irradiation, hyperosmotic conditions, oxidants). In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of STATs in A431 carcinoma cells. Oxidative stress, initiated by addition of H2O2 (1-2 mM) to A431 cells, activates STAT3 and, to a lesser extent, STAT1 in dose- and time-dependent manner. Maximum phosphorylation levels were observed after a 2 minutes stimulation at 1-2 mM H2O2. Phosphorylation was blocked by AG1478, a pharmacological inhibitor of the epidermal growth factor receptor tyrosine kinase, implicating intrinsic EGF receptor tyrosine kinase in this process. Consistent with this observation, H2O2-stimulated EGFR tyrosine phosphorylation was abolished by specific Src kinase family inhibitor CGP77675, implicating Src in H2O2-induced EGFR activation. An essential role for Src and JAK2 in STATs activation was suggested by three findings. 1. Src kinase family inhibitor CGP77675 blocked STAT3 and STAT1 activation by H2O2 in a concentration-dependent manner. 2. In Src-/-fibroblasts, activation of both STAT3 and STAT1 by H2O2 was significantly attenuated. 3. Inhibiting JAK2 activity with the specific inhibitor AG490 reduced the level of H2O2-induced STAT3 phosphorylation, but not STAT1 in A431 cells. These data show essential roles for Src and JAK2 inactivation of STAT3. In contrast, H2O2-mediated activation of STAT1 requires only Src kinase activity. Herein, we postulate also that H2O2-induced STAT activation in carcinoma cells involves Src-dependent EGFR transactivation.

[Dependence of EGF receptor and STAT factor activation on redox of A431 cells]. / Aktivatsiia retseptora EGF i faktorov STAT v zavisimosti ot okislitel'no-vosstanovitel'nogo statusa kletok A431.

Reactive oxygen species (ROS) were established to play an important role in cellular signaling as second messengers by integrating different pathways. Recently, we showed that EGF initiated a rapid tyrosine phosphorylation of both EGF-receptor and STAT factors with simultaneous increase in the intracellular ROS level. Now, we have investigated the effect of intracellular red-ox state on EGF- and H2O2-induced activation of EGF receptor, STAT1 and STAT3. We demonstrated that the pretreatment of A431 cells with antioxidant N-acetyl-L-cysteine (NAC) partly reduced the level of EGF-induced phosphorylation of proteins under investigation. Besides, H2O2-induced activation of EGF receptor, and STAT factors was fully prevented by NAC pretreatment. The inhibition of ROS generation by DPI declined EGF-dependent activation of EGF receptor and STAT factors to basal level. Our results demonstrate the essential role of cellular red-ox status in the modulation of EGF-mediated activation of receptor and STAT factors. We have postulated that EGF-induced ROS generation is a very important initial event promoting physiological activation of EGF receptor and subsequent STAT factor activation.