The present study demonstrates two fundamentally different situations of cell death in cardiac tissue. In embryonic mice, cardiomyocytes undergo caspase-dependent cell death. However, cardiomyocytes in heart failure show caspase-independent autophagic cell death rather than apoptotic cell death.
Objective-Oxysterols such as 7-ketocholesterol (7-KC) are important mediators of cell death in atherosclerosis. Therefore, in vitro studies of human smooth muscle cell (SMC) death in response to 7-KC were undertaken to investigate the potential mechanisms. Methods and Results-Human aortic SMCs treated with 7-KC showed enhanced immunoreactivity for the oxidative stress marker 4-hydroxy-2-nonenal and upregulated several stress genes (70-kDa heat shock protein 1, heme oxygenase 1, and growth arrest and DNA damage-inducible protein 153) at the mRNA but not at the protein level. 7-KC-treated SMCs rapidly underwent cell death as determined by neutral red, counting of adherent cells, and depolarization of the mitochondrial inner membrane. Cell death was associated with upregulation of ubiquitin mRNA and ubiquitination of cellular proteins. Inhibition of the proteasome by lactacystin potentiated considerably the toxicity of 7-KC. Transmission electron microscopy revealed formation of myelin figures, extensive vacuolization, and depletion of organelles. Formation of autophagosomes was suggested by labeling cells with LysoTracker and monitoring processing of microtubule-associated protein 1 light chain 3 (LC3). Analogous to our in vitro studies, human atherosclerotic plaques showed signs of ubiquitination in SMCs. Key Words: 7-ketocholesterol Ⅲ ubiquitination Ⅲ smooth muscle cells Ⅲ myelin figures Ⅲ LC3 Ⅲ autophagy Ⅲ atherosclerosis C ell death is a major event in the progression of atherosclerosis. Indeed, a large body of evidence suggests that apoptosis or type I programmed cell death 1 frequently occurs in advanced human plaques (1% to 2% TUNEL-positive nuclei). 2-5 However, results from electron microscopy studies showed that the majority of dying cells have an ultrastructure typical of cells undergoing "accidental" cell death or oncosis 6 (type III programmed cell death). 1 Furthermore, it is important to note that there are multiple pathways leading to cell death. Cells sometimes die with a morphology that is intermediate between apoptosis and oncosis (eg, aponecrosis and paraptosis), or they undergo cell death with a less clear morphology or mechanism such as lysosome-mediated cell death or autophagy (type II programmed cell death). 1 It is presently unknown whether cells in human atherosclerotic plaques die by mechanisms distinct from apoptosis or oncosis, as shown recently for myocytes in failing human hearts. 7,8 Oxidative processes, particularly oxidation of low-density lipoprotein (LDL), are thought to play a pivotal role in atherogenesis. 9 Oxidized LDL (oxLDL) exerts its proatherogenic effects in several ways, including stimulation of inflammatory responses, foam cell formation, and induction of cell death. 9 The mechanism of oxLDL-induced cell death is unclear, as oncosis and apoptosis have been reported, 10 and largely depends on the oxidation degree, exposure time, and concentration of oxLDL. 3 In macrophages, oxLDL induces mitochondrial dysfunction and lysosomal damage, 11,12 indicating that oxLDL upt...
BackgroundIn this study we explored the possibility of automating the PGP9.5 immunofluorescence staining assay for the diagnosis of small fiber neuropathy using skin punch biopsies. The laboratory developed test (LDT) was subjected to a validation strategy as required by good laboratory practice guidelines and compared to the well-established gold standard method approved by the European Federation of Neurological Societies (EFNS). To facilitate automation, the use of thinner sections. (16 µm) was evaluated. Biopsies from previously published studies were used. The aim was to evaluate the diagnostic performance of the LDT compared to the gold standard. We focused on technical aspects to reach high-quality standardization of the PGP9.5 assay and finally evaluate its potential for use in large scale batch testing.ResultsWe first studied linear nerve fiber densities in skin of healthy volunteers to establish reference ranges, and compared our LDT using the modifications to the EFNS counting rule to the gold standard in visualizing and quantifying the epidermal nerve fiber network. As the LDT requires the use of 16 µm tissue sections, a higher incidence of intra-epidermal nerve fiber fragments and a lower incidence of secondary branches were detected. Nevertheless, the LDT showed excellent concordance with the gold standard method. Next, the diagnostic performance and yield of the LDT were explored and challenged to the gold standard using skin punch biopsies of capsaicin treated subjects, and patients with diabetic polyneuropathy. The LDT reached good agreement with the gold standard in identifying small fiber neuropathy. The reduction of section thickness from 50 to 16 µm resulted in a significantly lower visualization of the three-dimensional epidermal nerve fiber network, as expected. However, the diagnostic performance of the LDT was adequate as characterized by a sensitivity and specificity of 80 and 64 %, respectively.ConclusionsThis study, designed as a proof of principle, indicated that the LDT is an accurate, robust and automated assay, which adequately and reliably identifies patients presenting with small fiber neuropathy, and therefore has potential for use in large scale clinical studies.
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