Purpose
To analyze if using a bioadhesive (sodium hyaluronate or HaNa) in combination with a blood derivative (s‐PRGF) improves the healing rate and quality of corneal epithelial ulcers.
Methods
...Rabbit corneas removed 7 and 30 days after an in vivo re‐epithelialization assay were include in paraffin and processed for haematoxylin–eosin staining or cryopreserved for immunofluorescent staining. In vitro proliferation and wound healing experiments were performed using the human corneal epithelial HCE cell line and rabbit primary corneal epithelial cultures. We studied the following treatments: 1) 90% s‐PRGF 2) 0.22% NaHa 3) s‐PRGF + NaHa 4) PBS as control treatment for the in vivo assay. All components in treatments 1, 2 and 3 were half the concentration for the in vitro assays, being 1% BSA the control treatment (4). To manufacture s‐PRGF whole blood was collected by venipuncture from healthy volunteers or New Zealand rabbits, respectively.
Results
Healing rate was higher in cultures and in eyes under s‐PRGF treatment than in those treated with HaNa or the combination of both. H‐E sections at 30 days showed more cells in the anterior stroma in eyes under HaNa treatments. In agreement with it, Ki67 staining as well as in vitro proliferation assays demonstrated that HaNa stimulated cell proliferation. All treatments produced stratified and mature epithelia (CK3 positiveness) with barrier function (ZO‐1 staining) and activation of limbal stem cells (CK15 staining), although the HaNa treated epithelia were the less organized. Moreover, eyes treated with only s‐PRGF showed the best integrin β4 staining.
Conclusions
HaNa bioadhesive promotes epithelial and stromal cell proliferation, but does not improve the corneal healing capacity of s‐PRGF. s‐PRGF shows a better healing quality in terms of epithelial‐stromal adhesion.
In our previous works, we have demonstrated that terfenadine (TEF) induces DNA damage and apoptosis in human melanoma cell lines. In this present work, we have studied the effect of histamine on ...viability of A375 human melanoma cells and the cell-signalling pathways through which TEF may induce its apoptotic effect. We have found that exogenous histamine stimulates A375 melanoma cell proliferation in a dose- and time-dependent manner. Moreover, TEF-induced apoptosis seems to occur via other cellular pathways independent of the histamine-signalling system since co-treatment of histamine with TEF did not protect melanoma cells from the cytotoxic effect of TEF, and alpha fluoromethylhistidine did not induce the same cytotoxic effect of TEF. In addition, we have observed that knocking down the H1 histamine receptor (HRH1) by small interference RNA approach protects melanoma cells only slightly from TEF-induced apoptosis. To explore the molecular mechanisms responsible for histamine and TEF effect on the cell growth, we analysed intracellular cyclic nucleotides and Ca2+ levels. TEF did not modify intracellular levels of cyclic adenosine 3′,5′-monophosphate and cyclic guanine 3′,5′-monophosphate; however, TEF induced a very sharp and sustained increase in cytosolic Ca2+ levels in A375 melanoma cells. On the contrary, histamine did not modulate intracellular Ca2+. TEF-induced Ca2+ rise and apoptosis appear to be phospholipase C (PLC) dependent since neomycin and U73122, two inhibitors of PLC, abolished cytosolic Ca2+ increase and protected the cells completely from cell death. Furthermore, inhibition of tyrosine kinase activity by genistein blocked cytosolic Ca2+ rise and TEF-induced apoptosis. These results suggest that TEF modulates Ca2+ homeostasis and induces apoptosis through other cellular pathways involving tyrosine kinase activity, independently of HRH1.
Retinoic acid-induced apoptosis of embryonic stem (ES) cells is an experimental system which resembles the physiological programmed cell death that occurs during differentiation in embryonic ...development. Our aim was to analyze the involvement of epigenetic modifications such as DNA methylation and chromatin structure in the apoptotic process and to investigate the metabolic activity of apoptotic bodies. We found a relationship between DNA methylation and apoptosis, shown by a dose-dependent induction of apoptosis after treatment with the inhibitor of DNA methylation 5-aza-2'-deoxycytidine. Interestingly, we found a slight demethylation of specific sequences of the U2afl-rs1 imprinted gene in those RA treated cells which were specifically undergoing apoptosis. In addition, apoptotic bodies exhibited an unexpected open chromatin conformation accessible to the endonuclease DNase-I. Furthermore, we observed a structural and functional preservation of specific DNA sequences and mRNA. These results suggest that biological activities, such as transcription or protein synthesis, could be maintained even towards the end of the apoptotic process.
In this work we have studied the behavior of some cell structures, such as actin, tubulin and chromatin during apoptosis induced in F9 cells after retinoic acid treatment. In this experimental model, ...all defined steps of morphological changes described for apoptosis are observed. The correlation between a partial maintenance of F-actin and microtubular structures and the spatial distribution of F-actin suggests a possible relationship between this molecule and the characteristic shape changes observed in apoptosis. Additionally, the disposition of monomeric G-actin suggests a possible relationship between the fragmentation of this molecule and the cleavage of DNA. The analysis of the U2af1-rs1 specific sequence shows that the internucleosomal fragmentation observed in this gene is randomly produced during apoptosis and is not dependent of demethylation status. The results obtained confirm that specific cleavage of these cell structures is inherent to the development of the apoptotic process and do not exclude the possibility that proteolysis of key actin and/or tubulin molecules or the cleavage of specific chromatin sequences other than the ones analyzed here, could control the different phases of the apoptotic process.
Epigenetic modifications such as DNA methylation and alterations to chromatin structure have been proposed as hallmarks of imprinting in somatic cells after fertilization. In the germ cell line, gene ...imprinting needs to be reset in order to transmit the correct sex-specific imprinting pattern to the next generation. The precise timing of imprint erasure and re-establishment for many genes remains to be determined and precise molecular mechanisms of genomic imprinting have not yet been fully characterized. Here, we have analysed the methylation state and DNase-I sensitivity of two genes with reciprocal genomic imprinting (U2af1-rs1 and H19 genes) in a male mouse primordial germ cell (PGC) derived cell line (EG-1), isolated post-natal spermatogonia and mature sperm cells. Our results show that establishment of imprinting of the U2af1-rs1 and H19 genes during male germ cell differentiation occurs at different stages of differentiation. Furthermore, the presence of DNase-I hypersensitive sites may constitute a molecular marker to identify alleles and subsequently acquire the appropriate methylation imprint. We propose that this molecular identifier may be present or absent for a specific gene according to the sex of the gamete.
Studies on biological development and cancer have pointed out the importance of specific epigenetic environments to maintain the equilibrium between repressed and activated genes. It has been ...possible to establish that this kind of environment induces chromatin structure modification and heritable changes in gene functions without altering primary DNA sequencing. We show here recent results of our laboratory on the expression of two imprinted genes, U2af1-rs1 and H19, in normal and pluripotent male germinal cells and in embryonic stem cell after induction of differentiation and apoptosis by retinoic acid treatments. These experimental observations can shed new light for a better understanding of testis embryonal carcinoma biology.
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