A rat fibroblastic cell line (rat-1/myc-ERtrade mark) was treated with different concentration of Antimycin A, a metabolic poison that affects mitochondrial respiratory chain complex III. The modes of cell death were analyzed by time-lapse videomicroscopy, in situ end-labeling (ISEL) technique, and ultrastructural analysis. Intracellular ATP levels were also measured in order to detect whether the energetic stores were determinant for the type of cell death. It was found that while apoptosis was the prevalent cell death in the fibroblasts treated with low doses, 100 or 200 microM Antimycin A, a new type of cell demise that shared dynamic, molecular, and morphological features with both apoptosis and necrosis represents the most common cell death when the cells were exposed to high doses, 300 or 400 microM, of the hypoxic stimulus. This new type of cell death has been chimerically termed aponecrosis. The inhibition of caspase 3, an enzyme critical for the apoptotic DNA degradation, caused a clear shift from aponecrosis to necrosis in the cell culture, suggesting that this new type of cell death could account for an incomplete execution of the apoptotic program and the following degeneration in necrosis. After being treated with higher doses, i.e., 1000 microM Antimycin A, almost all of the cells died by true necrosis. The analysis of the cellular energetic stores showed that the levels of ATP were a primary determinant in directing toward active cell death (apoptosis), aponecrosis, or necrosis. We conclude that chemically induced hypoxia produces different types of cell death depending on the intensity of the insult and on the ATP availability of the cell, and that the classic apoptosis and necrosis may represent only two extremes of a continuum of intermediate forms of cell demise.

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The antifibrotic potential of platelet-rich plasma (PRP) is controversial. This study examined the effects of PRP on in vitro transforming growth factor (TGF)-β1-induced differentiation of fibroblasts into myofibroblasts, the main drivers of fibrosis, and the involvement of vascular endothelial growth factor (VEGF)-A in mediating PRP-induced responses. The impact of PRP alone on fibroblast differentiation was also assessed. Myofibroblastic phenotype was evaluated by confocal fluorescence microscopy and western blotting analyses of α-smooth muscle actin (sma) and type-1 collagen expression, vinculin-rich focal adhesion clustering, and stress fiber assembly. Notch-1, connexin 43, and VEGF-A expression were also analyzed by RT-PCR. PRP negatively regulated fibroblast-myofibroblast transition via VEGF-A/VEGF receptor (VEGFR)-1-mediated inhibition of TGF-β1/Smad3 signaling. Indeed TGF-β1/PRP co-treated fibroblasts showed a robust attenuation of the myofibroblastic phenotype concomitant with a decrease of Smad3 expression levels. The VEGFR-1 inhibition by KRN633 or blocking antibodies, or VEGF-A neutralization in these cells prevented the PRP-promoted effects. Moreover PRP abrogated the TGF-β1-induced reduction of VEGF-A and VEGFR-1 cell expression. The role of VEGF-A signaling in counteracting myofibroblast generation was confirmed by cell treatment with soluble VEGF-A. PRP as single treatment did not induce fibroblast myodifferentiation. This study provides new insights into cellular and molecular mechanisms underpinning PRP antifibrotic action.

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