Ultrastructural observations on cell death by apoptosis in the “resting” human breast

Summary For the first time the process of epithelial cell deletion was studied within the parenchymal component of the resting human breast. The dying cells were initially recognised by specific nuclear changes involving peripheral condensation of the chromatin and nucleolar disintegration. At this stage the cells were retracted from the lumen and had lost desmosomal connections with their neighbours. Within the cytoplasm, there was evidence of ribosomal detachment from the endoplasmic reticulum with the formation of ribosome aggregates. The majority of dying cells were phagocytosed at this stage although a few underwent further morphological changes. These involved blebbing and fragmentation of the nucleus followed by cytoplasmic fragmentation. The dying cells and cell fragments were phagocytosed by epithelial or myoepithelial cells as well as mononuclear phagocytes and undergo lysosomal digestion within the phagosomes. These progressive morphological changes were consistent with cell deletion occurring by apoptosis.     

Developmental cell death: morphological diversity and multiple mechanisms

Summary Physiological cell death is a widespread phenomenon in the development of both vertebrates and invertebrates. This review concentrates on an aspect of developmental cell death that has tended to be neglected, the manner in which the cells are dismantled. It is emphasized that the dying cells may adopt one of at least three different morphological types: apoptotic, autophagic, and non-lysosomal vesiculate. These probably reflect a corresponding multiplicity of intracellular events. In particular, the destruction of the cytoplasm in these three types appears to be achieved primarily by heterophagy, by autophagy and by non-lysosomal degradation, respectively. The various mechanisms underlying both nuclear and cytoplasmic destruction are reviewed in detail. The multiplicity of destructive mechanisms needs to be born in mind in studies of other aspects of cell death such as the signals which trigger it, since different signals probably trigger different types of cell death.     

Combined effects of deafferentation and de-efferentation on isthmo-optic neurons during the period of their naturally occurring cell death

Summary We have studied the effects on the chick embryo's isthmo-optic nucleus of de-efferentation alone or in combination with deafferentation. De-efferentation was achieved by pharmacological destruction of the axonal target cells in the retina at E13, or by colchicine-blockade of axoplasmic transport in the intraocular parts of the isthmo-optic axons at E13; deafferentation was by a tectal lesion at E11 or E12. De-efferentation alone causes all the isthmo-optic neurons to die, and mostly by the endocytic-autophagic mode of cell death, which is characterized by pronounced endocytosis (of an intravascularly injected label) and by intense, clumped activity of two lysosomal enzymes (acid phosphatase and N-acetyl--glucosaminidase). Deafferentation plus de-efferentation caused there to be less endocytic-autophagic dying cells in the isthmo-optic nucleus than after de-efferentation alone, but all the neurons still died. Our interpretation is that deafferentation switched many of the isthmo-optic neurons to a completely different (nonendocytic, nonautophagic) mode of cell death.Abbreviations AcP acid phosphatase - E embryonic day - HRP horseradish peroxidase - ION isthmo-optic nucleus - KA kainate - NAG N-acetyl--glucosaminidase     

Autophagy Is a Renoprotective Mechanism During in Vitro Hypoxia and in Vivo Ischemia-Reperfusion Injury

Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. In response to stress, autophagy is induced and may either contribute to cell death or serve as a cell survival mechanism. Very little is known about autophagy in renal pathophysiology. This study examined autophagy and its pathological role in renal cell injury using in vitro and in vivo models of ischemia−reperfusion. We found that hypoxia (1% O₂) induced autophagy in cultured renal proximal tubular cells. Blockade of autophagy by 3-methyladenine or small-interfering RNA knockdown of Beclin-1 and ATG5 (two key autophagic genes) sensitized the tubular cells to hypoxia-induced apoptosis. In an in vitro model of ischemia−reperfusion, autophagy was not induced by anoxic (0% O₂) incubation in glucose-free buffer, but was induced during subsequent recovery/reperfusion period. In this model, suppression of autophagy also enhanced apoptosis. In vivo, autophagy was induced in kidney tissues during renal ischemia−reperfusion in mice. Autophagy was not obvious during the ischemia period, but was significantly enhanced during reperfusion. Inhibition of autophagy by chloroquine and 3-methyladenine worsened renal ischemia/reperfusion injury, as indicated by renal function, histology, and tubular apoptosis. Together, the results demonstrated autophagy induction during hypoxic and ischemic renal injury. Under these pathological conditions, autophagy may provide a protective mechanism for cell survival.Autophagy is a cellular process of “self-eating” wherein various cytoplasmic constituents are broken down and recycled through the lysosomal degradation pathway.¹ This process consists of several sequential steps, including sequestration of cytoplasmic portions by isolation membrane to form autophagosome, fusion of the autophagosome with lysosome to create an autolysosome, and degradation of the engulfed material to generate monomeric units such as amino acids.² Identification of the autophagy-related genes (ATG) in yeast and their orthologs in other organisms including mammals demonstrates that autophagy is evolutionarily conserved in all eukaryotic cells. The ATG genes constitute the core molecular machinery of autophagy and function at the different levels to regulate autophagy induction, progression, and completion.¹Autophagy occurs at basal level in most cells and contributes to the turnover of long-lived proteins and organelles to maintain intracellular homeostasis. In response to cellular stress, autophagy is up-regulated and can provide an adaptive strategy for cell survival, but may also directly or indirectly lead to cell demise.^3–6 With the dual role in life and death, autophagy is involved in various physiological processes, and more importantly, linked to the pathogenesis of a wide array of diseases, such as neurodegeneration, cancer, heart disease, aging, and infections.^1,2,6,7 However, it remains largely unknown how autophagy makes the life and death decisions of a stressed cell. Moreover, the conundrum is further complicated by the cross talk and coordinated regulation between autophagy and apoptosis.^4,5,8Despite rapid progress of autophagy research in other organ systems, the role of autophagy in the pathogenesis of renal diseases was not recognized until very recently. In 2007, Chien et al⁹ suggested the first evidence of autophagy during renal ischemia−reperfusion in rats. Subsequent work by Suzuki et al¹⁰ further showed autophagy in ischemic mouse kidneys and notably, in transplanted human kidneys. In nephrotoxic models of acute kidney injury, we and others have demonstrated autophagy during cisplatin nephrotoxicity and have suggested a role for autophagy in renoprotection.^11,12 A prosurvival role of autophagy was also shown in tubular cells during cyclosporine A nephrotoxicity.¹³ In contrast, Gozuacik et al¹⁴ suggested that autophagy may serve as a second cell killing mechanism that acts in concert with apoptosis to trigger kidney damage in tunicamycin-treated mice. A cell killing role for autophagy was also suggested by Suzuki et al¹⁰ during H₂O₂-induced renal tubular cell injury. As a result, whether autophagy is a mechanism of cell death or survival in renal pathology remains unclear.In this study, we have determined the role of autophagy in renal tubular cell injury using in vitro and in vivo models of renal ischemia−reperfusion. We show that autophagy is induced in these models. Importantly, blockade of autophagy sensitizes renal cells and tissues to injury by hypoxia and ischemia−reperfusion, suggesting a prosurvival role for autophagy.     

Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry

Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10ng/ml TNF- for 4h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2 ± 0.1 to 5.8s±0.1 and the lysosomal increased from 4.3±0.4 to 5.2±0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.     

Alterations of the retina in chick embryos induced by systemic alpha-bungarotoxin application

Summary The application of -bungarotoxin onto the chorio-allantoic membrane of chick embryos between the 11th and 18th day of incubation leads to alterations of retinal development. The most significant qualitative change is the appearance of retinal rosettes formed by receptor cells. These rosettes are infoldings of the receptor cell layer. Quantitatively, an enlargement in volume of the receptor and outer nuclear layer can be found together with a simultaneous decrease of the other retinal layers. The toxin seems to suspend the naturally occurring nerve cell death in the receptor cell population.In honour of the 90th birthday of Prof. Dr.Dr. h.c. hermann Voss     

Histochemical examination of lysosomal exzymes in necrotic proximal renal tubules of albino rats.

The lysosomal enzymatic activity of the necrotic proximal tubules was examined by light microscopy and electron microscopy in 24- and 48-h focal renal cortical necrosis induced by administration of oestrogen and posterior pituitary extract in rats. Organelles exhibiting acid phosphatase activity can also be seen in the necrotic cells but these differ in size and structure from the lysosomes of normal cells. The cytoplasmic nonspecific esterase and thioacetic acid hydrolase activities decrease considerably or disappear, although some morphologically damaged, but active, lysosomes can be observed. The role of thelysosomal enzymes is seen not in the development of the necrosis but rather in the breaking down of the already necrotic cell constituents.     

Fasciola hepatica: the effect of the microfilament inhibitor cytochalasin B on the ultrastructure of the adult fluke

The effect of the microfilament inhibitor cytochalasin B (10 and 100 g/ml) on the ultrastructure of adultFasciola hepatica was determined in vitro by scanning and transmission electron microscopy (SEM, TEM) using both intact flukes and tissue-slice material. SEM revealed that initial swelling of the tegument led to surface blebbing and limited areas of sloughing after 24 h treatment at 100 g/ml. In the tegumental syncytium, basal accumulations of secretory bodies (especially T2s) were evident in the earlier time periods but declined with longer incubations, until few secretory bodies remained in the syncytium overall. Blebbing of the apical plasma membrane and occasional areas of breakdown and sloughing of the tegument were observed over longer periods of treatment at 100 g/ml. In the tegumental cell bodies, the Golgi complexes gradually decreased in size and activity, and few secretory bodies were produced. In the later time periods, the cells assumed abnormal shapes, the cytoplasm shrinking in towards the nucleus. In the vitelline follicles, a random dispersion of shell protein globules was evident within the intermediate-type cells, rather than their being organized into distinct shell globule clusters. Disruption of this process was more severe at the higher concentration of 100 g/ml and again was more evident in tissue-slice material. In the latter, after prolonged (12 h) exposure to cytochalasin B, the intermediate and mature vitelline cells were filled with loosely packed and expanded shell globule clusters, containing few shell protein globules. The mature vitelline cells continued to lay down yolk globules and glycogen deposits. Disruption of the network of processes from the nurse cells was evident at the higher concentration of cytochalasin. Spaces began to appear between the vitelline cells and grew larger with progressively longer incubation periods, and the cells themselves assumed abnormal shapes. A number of binucleate stem cells were observed in tissue-slice material at the longest incubation period (12 h).     

The tissue dynamics of heart morphogenesis

Summary Zones of cell death in the chick embryo heart were demonstrated by a study of cardiac morphogenesis on the light microscopical level.Between the 2nd and 20th day of incubation 31 foci of cell degeneration were found.The greatest number of dying cells occured on the 4th day of incubation and was located in the heart bulbus and bulbar cushions. The largest number of different degenerative foci were present on the 6th incubation day. Starting on the 10th day of incubation, both the number of degeneration zones and their population of dying cells decreased.This work was in part supported by grants no. B70-12X-579 from the Swedish Medical Research Council and no. 108-K69 from the Swedish Cancer Society.     

Innate and adaptive immunity through autophagy

The two main proteolytic machineries of eukaryotic cells, lysosomes and proteasomes, receive substrates by different routes. Polyubiquitination targets proteins for proteasomal degradation, whereas autophagy delivers intracellular material for lysosomal hydrolysis. The importance of autophagy for cell survival has long been appreciated, but more recently, its essential role in both innate and adaptive immunity has been characterized. Autophagy is now recognized to restrict viral infections and replication of intracellular bacteria and parasites. Additionally, this pathway delivers cytoplasmic antigens for MHC class II presentation to the adaptive immune system, which then in turn is able to regulate autophagy. At the same time, autophagy plays a role in the survival and the cell death of T cells. Thus, the immune system utilizes autophagic degradation of cytoplasmic material, to both restrict intracellular pathogens and regulate adaptive immunity.     

Failure to complete apoptosis following neonatal hypoxia-ischemia manifests as "continuum" phenotype of cell death and occurs with multiple manifestations of mitochondrial dysfunction in rodent forebrain

Controversy surrounds proper classification of neurodegeneration occurring acutely following neonatal hypoxia-ischemia (HI). By ultrastructural classification, in the first 24 h after neonatal hypoxia-ischemia in the 7-day-old (p7) rat, the majority of striatal cells die having both apoptotic and necrotic features. There is formation of a functional apoptosome, and activation of caspases-9 and -3 occurring simultaneously with loss of structurally intact mitochondria to 34.7+/-25% and loss of mitochondrial cytochrome c oxidase activity to 34.7+/-12.7% of control levels by 3 h after hypoxia-ischemia. There is also loss of the mitochondrial motor protein, kinesin. This combination of activation of apoptosis pathways simultaneous with significant mitochondrial dysfunction may cause incomplete packaging of nuclear and cytoplasmic contents and a hybrid of necrotic and apoptotic features. Evidence for an intermediate biochemistry of cell death including expression of the 17 kDa isoform of caspase-3 in dying neurons lacking a classic apoptotic morphology and degradation of the neuronal cytoskeletal protein spectrin by caspase-3 and calcium-activated calpains yielding 120 kDa and 145/150 kDa fragments, respectively, is also found. In summary, neonatal hypoxia-ischemia triggers apoptotic cascades, and simultaneously causes mitochondrial structural and functional failure. The presence of a "continuum" phenotype of cell death that varies on a cell-by-cell basis suggests that the phenotype of cell death is dependent on the energy available to drive the apoptotic pathways to completion.     

The role of lysosomal rupture in neuronal death

Apoptosis research in the past two decades has provided an enormous insight into its role in regulating cell death. However, apoptosis is only part of the story, and inhibition of neuronal necrosis may have greater impact than apoptosis, on the treatment of stroke, traumatic brain injury, and neurodegenerative diseases. Since the “calpain–cathepsin hypothesis” was first formulated, the calpain- and cathepsin-mediated regulation of necrotic cascades observed in monkeys, has been demonstrated to be a common neuronal death mechanism occurring from simpler organisms to humans. However, the detailed mechanism inducing lysosomal destabilization still remains poorly understood. Heat-shock protein-70 (Hsp70) is known to stabilize lysosomal membrane and protect cells from oxidative stress and apoptotic stimuli in many cell death pathways. Recent proteomics approach comparing pre- and post-ischemic hippocampal CA1 neurons as well as normal and glaucoma-suffered retina of primates, suggested that the substrate protein upon which activated calpain acts at the lysosomal membrane of neurons might be Hsp70. Understanding the interaction between activated calpains and Hsp70 will help to unravel the mechanism that destabilizes the lysosomal membrane, and will provide new insights into clarifying the whole cascade of neuronal necrosis. Although available evidence is circumferential, it is hypothesized that activated calpain cleaves oxidative stress-induced carbonylated Hsp70.1 (a major human Hsp70) at the lysosomal membrane, which result in lysosomal rupture/permeabilization. This review aims at highlighting the possible mechanism of lysosomal rupture in neuronal death by a modified “calpain–cathepsin hypothesis”. As the autophagy–lysosomal degradation pathway is a target of oxidative stress, the implication of autophagy is also discussed.     

Nuclear bodies in the egg cells of aGyrodactylus species (Platyhelminthes,Monogenea)

The presence and ultrastructure of distinctive nuclear bodies in a mature egg cell of aGyrodactylus sp. are described. These electron-dense granular bodies, which appear in section as open or closed rings or as a solid mass are not membrane-bound and lie in clusters close to the nucleolus. The nuclear bodies are compared with the nuclear-inclusion bodies previously reported in platyhelminths. The possible origin, significance and role of the nuclear bodies in thisGyrodactylus sp. are discussed.This paper is dedicated to Prof. J.C. Pearson, Department of Parasitology, The University of Queensland, on the occasion of his 65th birthdayThis study was supported by a Macquarie University Research Grant (to M.K.J) and by a Queen Elizabeth II Fellowship awarded by the Australian Research Council (to I.D.W.)     

Autophagic death of adult hippocampal neural stem cells following insulin withdrawal

Novel therapeutic approaches using stem cell transplantation to treat neurodegenerative diseases have yielded promising results. However, survival of stem cells after transplantation has been very poor in animal models, and considerable efforts have been directed at increasing the viability of engrafted stem cells. Therefore, understanding the mechanisms that regulate survival and death of neural stem cells is critical to the development of stem cell-based therapies. Hippocampal neural (HCN) stem cells derived from the adult rat brain undergo cell death following insulin withdrawal, which is associated with downregulation of antiapoptotic Bcl-2 family members. To understand the type of cell death in HCN cells following insulin withdrawal, apoptosis markers were assessed. Of note, DNA fragmentation or caspase-3 activation was not observed, but rather dying cells displayed features of autophagy, including increased expression of Beclin 1 and the type II form of light chain 3. Electron micrographs showed the dramatically increased formation of autophagic vacuoles with cytoplasmic contents. Staurosporine induced robust activation of caspase-3 and nucleosomal DNA fragmentation, suggesting that the machinery of apoptosis is intact in HCN cells despite the apparent absence of apoptosis following insulin withdrawal. Autophagic cell death was suppressed by knockdown of autophagy-related gene 7, whereas promotion of autophagy by rapamycin increased cell death. Taken together, these data demonstrate that HCN cells undergo a caspase-independent, autophagic cell death following insulin withdrawal. Understanding the mechanisms governing autophagy of adult neural stem cells may provide novel strategies to improve the survival rate of transplanted stem cells for treatment of neurodegenerative diseases. Disclosure of potential conflicts of interest is found at the end of this article.     

Types of programmed cell death: two variants expressed by neonatal murine hepatocytes

Neonatal livers examined with the terminal transferase-mediated dUTP nick end labeling (TUNEL) method contained numerous positive cells. Although the majority of dying cells are either hematopoietic cells including erythroids and granulocytes or macrophages, a few hepatocytes were also positive. As for the ultrastructural features of these dying hepatocytes, two different types, type I and II, could be identified. The early features of type I appeared in the cytoplasm, which was characterized by dilated rough endoplasmic reticulum, and the cell fragments displayed a round, foamy appearance. Type II was characterized by nuclear compaction and margination of heterochromatin resulting in the formation of sharply circumscribed masses, followed by the condensation of the cytoplasm. The cell death of type I, characterized by the formation of massive vacuolization of the endoplasmic reticulum, corresponds to cytoplasmic type degeneration or nonapoptotic death, while that of type II corresponds to nuclear type cell death or classical apoptotic death. In the two types of programmed cell death, the incidence of nonapoptotic cell death was much higher than that of classical apoptosis in neonatal murine hepatocytes.     

Mammary "Comedo"-DCIS: Apoptosis,Oncosis, and Necrosis: An Electron Microscopic Examination of 8 Cases

The terms apoptosis and necrosis are commonly used to imply two distinct types of cell death. Apoptosis reflects a genetically mediated, ATP-dependent form of cell death. A passive form of cell death (oncosis) also occurs, often in response to someformof injury. Both pathways can lead to necrosis (postmortem autolytic cell changes). The nature of intraluminal necrosis in mammary ductal carcinoma in situ (DCIS) was evaluated using ultrastructural analysis on paraffin-embedded material of 8 cases with "comedo"-DCIS. In each case, intraepithelial proliferation zones and intraluminal zones (peripheral and central luminal zones) were examined. All cases with ``comedo''-DCIS revealed abundant apoptosis, characterized by apoptotic cells showing chromatin condensation and margination with sharply circumscribed, uniformly dense crescents, as well as cytoplasmic condensation. Numerous membrane-bound apoptotic bodies with condensed cytoplasm (with or without nuclear fragments) were also observed. The central luminal zones of "comedo"-DCIS, however, revealed necrotic debris characterized by severe degradative changes, largely devoid of recognizable cell structures. In addition, two cases displayed features of oncosis, characterized by nuclear and cytoplasmic swelling, vacuolization of cytoplasm,and mitochondrial swelling with occasional dense bodies. The results indicate that necrosis (postmortem, secondary degradative cell changes) in "comedo"-DCIS is the end result of either apoptosis (programmed cell death) alone or a combination of apoptosis and oncosis (passive or "accidental" cell death).     

7-Ketocholesterol Induces Autophagy in Vascular Smooth Muscle Cells through Nox4 and Atg4B

Oxidized lipoproteins stimulate autophagy in advanced atherosclerotic plaques. However, the mechanisms underlying autophagy induction and the role of autophagy in atherogenesis remain to be determined. This study was designed to investigate the mechanisms by which 7-ketocholesterol (7-KC), a major component of oxidized lipoproteins, induces autophagy. This study was also designed to determine the effect of autophagy induction on apoptosis, a central event in the development of atherosclerosis. Exposure of human aortic smooth muscle cells to 7-KC increased autophagic flux. Autophagy induction was suppressed by treating the cells with either a reactive oxygen species scavenger or an antioxidant. Administration of 7-KC concomitantly up-regulated Nox4 expression, increased intracellular hydrogen peroxide levels, and inhibited autophagy-related gene 4B activity. Catalase overexpression to remove hydrogen peroxide or Nox4 knockdown with siRNA reduced intracellular hydrogen peroxide levels, restored autophagy-related gene 4B activity, and consequently attenuated 7-KC–induced autophagy. Moreover, inhibition of autophagy aggravated both endoplasmic reticulum (ER) stress and cell death in response to 7-KC. In contrast, up-regulation of autophagic activity by rapamycin had opposite effects. Finally, activation of autophagy by chronic rapamycin treatment attenuated ER stress, apoptosis, and atherosclerosis in apolipoprotein E knockout (ApoE^−/−) mouse aortas. In conclusion, we demonstrate that up-regulation of autophagy is a cellular protective response that attenuates 7-KC–induced cell death in human aortic smooth muscle cells.Autophagy is a highly conserved cellular process for degradation of cytoplasmic components, such as long-lived proteins and damaged organelles in lysosomes.¹ The process is essential for the maintenance of cellular homeostasis and survival because the degradation of cytosolic components can provide amino acids and substrates for intermediary metabolism.² Although dysregulation of autophagy has been implicated in many human diseases, including neurodegeneration, cancer, and cardiomyopathy,^3–5 little information exists about the role of autophagy in the development of atherosclerosis. Recently, oxidized lipoproteins have been demonstrated to stimulate autophagy in advanced atherosclerotic plaques,⁶ leading us to hypothesize that oxidized lipoproteins may induce autophagy in vascular cells through increasing intracellular reactive oxygen species (ROS). In support of this model, a strong correlation between oxidative stress and the development of atherosclerosis has been established⁷ and starvation-induced ROS have been demonstrated to trigger autophagy by oxidizing a critical cysteine residue in autophagy-related gene 4 (Atg4) protein.⁸Atherosclerosis is characterized by the accumulation of oxidized lipoproteins in large arteries. Oxidation of lipoproteins leads to the formation of dozens of new lipids, such as oxysterols, aldehydes, and oxidized fatty acids.⁷ These oxidized lipoproteins could promote the progression of atherosclerosis by stimulating an inflammatory response, increasing foam cell formation, and inducing vascular cell apoptosis.⁹ Oxysterols, such as 7β-hydoxycholesterol and 7-ketocholesterol (7-KC), are major components of oxidized lipoproteins in human atherosclerotic plaques.¹⁰ The oxysterol 7-KC has been shown to accelerate ROS production and induce complex modes of cell death,¹¹ including necrosis,¹² apoptosis (type I cell death),¹³ and autophagic type II cell death. Martinet et al¹¹ demonstrated that 7-KC activates the ubiquitin proteasome system and induces the formation of a myelin figure and the processing of microtubule-associated protein light chain 3 (LC3). However, the molecular mechanisms by which 7-KC induces autophagy and the role of autophagy induction in the development of atherosclerosis remain undefined. Therefore, we investigated the mechanism by which 7-KC induces autophagy and the effect of autophagy on apoptosis, an important process in the development of atherosclerosis. We found that 7-KC increased Nox4-mediated hydrogen peroxide formation, which triggered autophagy through the inhibition of Atg4B activity. The induction of autophagy mitigated vascular smooth muscle cell (VSMC) death by suppressing the endoplasmic reticulum (ER) stress–apoptosis pathway.     

Mammary "comedo"-DCIS: apoptosis, oncosis, and necrosis: an electron microscopic examination of 8 cases

The terms apoptosis and necrosis are commonly used to imply two distinct types of cell death. Apoptosis reflects a genetically mediated, ATP-dependent form of cell death. A passive form of cell death (oncosis) also occurs, often in response to someformof injury. Both pathways can lead to necrosis (postmortem autolytic cell changes). The nature of intraluminal necrosis in mammary ductal carcinoma in situ (DCIS) was evaluated using ultrastructural analysis on paraffin-embedded material of 8 cases with "comedo"-DCIS. In each case, intraepithelial proliferation zones and intraluminal zones (peripheral and central luminal zones) were examined. All cases with 'comedo'-DCIS revealed abundant apoptosis, characterized by apoptotic cells showing chromatin condensation and margination with sharply circumscribed, uniformly dense crescents, as well as cytoplasmic condensation. Numerous membrane-bound apoptotic bodies with condensed cytoplasm (with or without nuclear fragments) were also observed. The central luminal zones of "comedo"-DCIS, however, revealed necrotic debris characterized by severe degradative changes, largely devoid of recognizable cell structures. In addition, two cases displayed features of oncosis, characterized by nuclear and cytoplasmic swelling, vacuolization of cytoplasm,and mitochondrial swelling with occasional dense bodies. The results indicate that necrosis (postmortem, secondary degradative cell changes) in "comedo"-DCIS is the end result of either apoptosis (programmed cell death) alone or a combination of apoptosis and oncosis (passive or "accidental" cell death).     

Autophagy Is a Component of Epithelial Cell Fate in Obstructive Uropathy

Epithelial cell fate and nephron loss in obstructive uropathy are not fully understood. We produced transgenic mice in which epithelial cells in the nephrons and collecting ducts were labeled with enhanced yellow fluorescent protein, and tracked the fate of these cells following unilateral ureteral obstruction (UUO). UUO led to a decrease in the number of enhanced yellow fluorescent protein-expressing cells and down-regulation of epithelial markers, E-cadherin, and hepatocyte nuclear factor-1β. Following UUO, enhanced yellow fluorescent protein-positive cells were confined within the tubular basement membrane, were not found in the renal interstitium, and did not express α-smooth muscle actin or S100A4, markers of myofibroblasts and fibroblasts. Moreover, when proximal tubules were labeled with dextran before UUO, dextran-retaining cells did not migrate into the interstitium or express α-smooth muscle actin. These results indicate that UUO leads to tubular epithelial loss but does not cause epithelial-to-mesenchymal transition that has been shown by others to be responsible for nephron loss and interstitial fibrosis. For the first time, we found evidence of enhanced autophagy in obstructed tubules, including accumulation of autophagosomes, increased expression of Beclin 1, and increased conversion of microtubular-associated protein 1 light chain 3-I to -II. Increased autophagy may represent a mechanism of tubular survival or may contribute to excessive cell death and tubular atrophy after obstructive injury.Obstructive uropathy and renal cystic dysplasia are the most common causes of end-stage renal disease in children. These disorders account for 16% of pediatric kidney transplantations in North America.¹ The pathogenesis of these diseases is not fully understood. Obstruction of the kidney during fetal development results in renal cystic dysplasia, which is likely due to a disturbance in epithelial differentiation and maturation.^2,3,4,5 In contrast, urinary tract obstruction in the postnatal kidney results in inflammation, tubular dilation, tubular atrophy, extracellular matrix accumulation, and renal fibrosis.⁶ Although the pathogenesis of obstructive uropathy is not identical in developing and mature kidneys, loss of normal renal tissue and increased interstitial fibrosis are common to both conditions.The mechanism of tubular atrophy and nephron loss in obstructive uropathy has not been fully elucidated. Previous studies have shown that epithelial cell apoptosis plays an important role.^2,7 Epithelial-mesenchymal transition (EMT) has also been proposed as a mechanism of interstitial fibrosis as well as nephron loss.^8,9 Lineage tracing studies in which proximal tubular cells were labeled with lacZ have shown that up to 36% of interstitial fibroblasts originate by EMT after unilateral ureteral obstruction (UUO).¹⁰ However, the extent to which EMT contributes to nephron loss and interstitial fibrosis remains controversial.^10,11,12 In a rat model of angiotensin II-induced renal fibrosis, fibroblasts originate from encroachment of interstitial myofibroblasts from the perivascular space rather than via EMT.¹¹ To further evaluate the role of EMT in nephron loss in obstructive uropathy, we performed lineage analysis using genetically modified mice in which epithelial cells of the proximal and distal nephron were labeled and cell fate was followed.Autophagy is another potential mechanism of nephron loss in obstructive uropathy. Autophagy is a lysosomal degradation pathway that is essential for cell survival, embryonic development, and tissue homeostasis.^13,14 Autophagy results in the degradation of cytoplasm by lysosomes in response to stress conditions, such as nutrient deprivation. The morphological hallmark of autophagy is the autophagosome, which is a double-membrane-bound vacuole that contains cytoplasmic contents and organelles. Fusion of autophagosomes with lysosomes results in the formation of autophagolysosomes in which the captured material is degraded. Autophagy protects cells against diverse pathologies, including infection, cancer, neurodegeneration, aging, and other diseases. However, under certain conditions, this self-cannibalistic function may be detrimental and could be associated with excessive cell death. Emerging evidence indicates that apoptosis (type I programmed cell death) and autophagy (type II programmed cell death) are coordinated processes. Bcl-2 family members have been shown to be dual regulators of apoptosis and autophagy.¹⁵ Increased cell death due to apoptosis has been observed in obstructive uropathy. Here, we explored the possibility that autophagy may also contribute to excessive cell death associated with nephron loss and tubular atrophy.     

Response of lung enzyme activities in rabbits following short-term exposure ton-hexane: Correlation between morphological and biochemical changes

The activity of lactatedehydrogenase, -glucuronidase, glucose-6-phosphate dehydrogenase, acid and alkaline phosphatase was studied in lung homogenate from New Zealand rabbits exposed to 3000 p.p.m. ofn-hexane 8 h per day for 8 days or filtered air.In hydrocarbon-treated animals all enzymes examined, except alkaline phosphatase, were markedly increased.The biochemical changes correlated well with the morphological changes and the results of cytological evaluation of bronchopulmonary lavage. It is suggested that high values in lung lysosomal enzymes from treated rabbits reflect the acute inflammation whilst the increase in lung glueose-6-phosphate dehydrogenase may depend upon reparative process subsequent ton-hexane-induced lung damage.