Dynamics of serum-induced endothelial cell apoptosis in patients with myocardial infarction.

BACKGROUND: In patients with ST-segment elevation myocardial infarction (STEMI) reperfused with primary coronary intervention (PCI), the dynamics of endothelial cell (EC) viability, apoptosis and necrosis and its relationship with the structural consequences on the left ventricle have not been addressed so far. DESIGN: In 20 STEMI patients, we incubated human umbilical vein endothelial cells (HUVECs) with serum drawn before reperfusion and subsequently afterwards (24, 96 h, 30 days). Viability, apoptosis and necrosis percentages were evaluated by flow cytometry. Values were compared with 12 age- and sex-matched control subjects with normal coronary arteries. Cardiac magnetic resonance (CMR) was performed during the first week after infarction. RESULTS: Serum from STEMI patients induced a progressive loss of EC viability, with a nadir of 67.7 ± 10.2% at 96 h (baseline: 75 ± 6% and controls: 80.2 ± 3.9%, P < 0.001 in both cases). This is due to an increase in apoptosis that peaked at 96 h after reperfusion (15.2 ± 7.1% vs. 11 ± 6 at baseline and 5.8 ± 1.6% in controls, P < 0.001 in both cases). However, no significant dynamic changes in EC necrosis were detected. Extensive myocardial oedema (> 30%, median of left ventricular mass) was the only CMR variable significantly associated with a higher percentage of EC apoptosis at 96 h (extensive vs. nonextensive oedema: 18.3 ± 6.8% vs. 12.1 ± 6.3%, P < 0.05). CONCLUSIONS: Dynamic changes in EC viability occur in the setting of STEMI patients reperfused with PCI, these changes peak late after reperfusion, they are mainly the result of an increase of apoptosis and are associated with the presence of extensive myocardial oedema.

Development of a multiparametric cell-based protocol to screen and classify the hepatotoxicity potential of drugs.

Hepatotoxicity is a major reason for drug nonapprovals and withdrawals. The multiparametric analysis of xenobiotic toxicity at the single cells level using flow cytometry and cellular imaging-based approaches, such as high-content screening (HCS) technology, could play a key role in the detection of toxicity and the classification of compounds based on patterns of cellular injury. This study aimed to develop and validate a practical, reproducible, in vitro multiparametric cell-based protocol to assess those drugs that are potentially hepatotoxic to humans and to suggest their mechanisms of action. The assay was applied to HepG2 human cell line cultured in 96-well plates and exposed to 78 different compounds for 3 and 24 h at a range of concentrations (1-1000µM). After treatments, cells were simultaneously loaded with five fluorescent dyes showing optical compatibility and were then analyzed with the High-Content Screening Station Scan^R (Olympus). By using the new technology of HCS cell parameters associated with nuclear morphology, plasma membrane integrity, mitochondrial function, intracellular calcium concentration, and oxidative stress, indicative of prelethal cytotoxic effects and representative of different mechanisms of toxicity, were measured at the single cells level, which allows high-throughput screening. This strategy appears to identify early and late events in the hepatotoxic process and also suggests the mechanism(s) implicated in the toxicity of compounds to thereby classify them according to their degree of injury (no injury, low, moderate, and high injury).

The significance of sperm DNA oxidation in embryo development and reproductive outcome in an oocyte donation program: a new model to study a male infertility prognostic factor.

OBJECTIVE: One byproduct resulting from free radical damage is the DNA hydroxylation also known as DNA oxidation. Our aim with this work was to determine the relevance of sperm DNA oxidation on embryo quality in oocyte donation cycles. DESIGN: We prospectively studied pairs of oocyte donation cycles, i.e., the same oocyte donors, donating to two recipients, where the only difference between the two treatments was the use of a different sperm sample. SETTING: University-affiliated private IVF setting. PATIENT(S): Infertile male partners from couples undergoing oocyte donation cycles (n = 38): 76 semen aliquots analyzed before and after semen processing by swim up. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): We measured sperm DNA oxidation by flow cytometry using the OxiDNA assay and correlated it with embryo quality parameters, implantation, and pregnancy outcome. RESULT(S): A positive correlation was seen between embryo fragmentation and DNA oxidation of capacitated samples at 48 hours and 72 hours after fertilization. However, when we analyzed the differences in the IVF outcome parameters of the couples who shared the oocyte cohort (same donor) with the differences in the OxiDNA values, we observed increased and further relationships with cell embryo division 48 hours after fertilization. A negative association with blastocyst formation was also detected. CONCLUSION(S): Oxidative damage in the DNA is clearly increased in samples with lower sperm motility. An association between early and late embryo quality and sperm DNA oxidation supports the relevance of the hydroxylation of 8-oxoguanine as a biomarker of sperm quality reflecting the free radical damage in human sperm.