In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

https://biblio.ugent.be/publication/8555786/file/8555788.pdf

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

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Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

Cells constantly adapt their metabolism to meet their energy needs and respond to nutrient availability. Eukaryotes have evolved a very sophisticated system to sense low cellular ATP levels via the serine/threonine kinase AMP-activated protein kinase (AMPK) complex. Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP generation and decrease ATP consumption. In the past decade, the discovery of numerous new AMPK substrates has led to a more complete understanding of the minimal number of steps required to reprogramme cellular metabolism from anabolism to catabolism. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and autophagy. Recent studies have revealed that one ancestral function of AMPK is to promote mitochondrial health, and multiple newly discovered targets of AMPK are involved in various aspects of mitochondrial homeostasis, including mitophagy. This Review discusses how AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology.

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AMPK: guardian of metabolism and mitochondrial homeostasis.

Autophagy and apoptosis control the turnover of organelles and proteins within cells, and of cells within organisms, respectively, and many stress pathways sequentially elicit autophagy, and apoptosis within the same cell. Generally autophagy blocks the induction of apoptosis, and apoptosis-associated caspase activation shuts off the autophagic process. However, in special cases, autophagy or autophagy-relevant proteins may help to induce apoptosis or necrosis, and autophagy has been shown to degrade the cytoplasm excessively, leading to 'autophagic cell death'. The dialogue between autophagy and cell death pathways influences the normal clearance of dying cells, as well as immune recognition of dead cell antigens. Therefore, the disruption of the relationship between autophagy and apoptosis has important pathophysiological consequences.

Self-consumption: the interplay of autophagy and apoptosis

Resolvin D1 reduces expression and secretion of cytokines and monocyte adhesion triggered by Angiotensin II, in rat cardiac fibroblasts.

Objective The purpose of this investigation was to determine whether the aminopeptidase inhibitor with broad specificity, bestatin, affects angiotensin I (Ang I)-, angiotensin II (Ang II)- or angiotensin III (Ang III)stimulated collagen gel contraction in cardiac fibroblasts. Design and methods Cardiac fibroblasts (from normal male adult rats) were cultured to confluency in Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum (FBS). These fibroblasts (100,000 cells) were then further incubated in a floating collagen gel lattice with the test products Ang I (1 µmol/L), Ang II (100 nmol/L), Ang III (100 nmol/L) and bestatin (100 µmol/L) for three days in DMEM without FBS. The area of the collagen gels embedded with cardiac fibroblasts was determined by a densitometric analysis. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide. Results

Effect of bestatin on angiotensin I-, II- and III-induced collagen gel contraction in cardiac fibroblasts.

The purpose of this investigation was to determine whether aminopeptidase inhibition could affect the angiotensin II-stimulated collagen gel contraction in basal (control) and TGF-beta1-treated cardiac fibroblasts (or myofibroblasts). The tested aminopeptidase inhibitors were the broad range aminopeptidase inhibitor bestatin, the specific inhibitor of alanine aminopeptidase leuhistin, and the specific inhibitor of arginine aminopeptidase arphamenine A. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and incubated with(out) 400 pmol/L TGF-beta1 in Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). These fibroblasts were then further incubated in a floating collagen gel lattice with the tested products (angiotensin II, bestatin, leuhistin, or arphamenine A) for 3 days in DMEM without FBS. The contraction of the collagen gel lattice by cardiac fibroblasts was determined by measuring the gel volume with tritiated water. Aminopeptidase activity was estimated by spectrophotometric determination of the liberation of p-nitroaniline from alanine- or arginine-p-nitroanilide. Angiotensin II (100 nmol/L) reduced the gel volume in control and TGF-beta1-treated cardiac fibroblasts. The angiotensin II-stimulated collagen gel contraction in control and TGF-beta1-treated fibroblasts was almost completely reversed by leuhistin and arphamenine A (100 micromol/L). Bestatin (100 micromol/L) only partially inhibited the angiotensin II-stimulated collagen gel contraction in control fibroblasts, although it did not affect the angiotensin II-induced contraction in TGF-beta1-treated fibroblasts. In control and TGF-beta1-treated cardiac fibroblasts, 100 micromol/L leuhistin or arphamenine A only partially inhibited alanine aminopeptidase activity, whereas bestatin (100 micromol/L) completely inhibited the alanine aminopeptidase activity. Arginine aminopeptidase activity was only partially inhibited by leuhistin and arphamenine A at 100 micromol/L in control and TGF-beta1-treated fibroblasts. Bestatin, however, completely blocked the arginine aminopeptidase activity in control fibroblasts and only partially in TGF-beta1-treated fibroblasts at 100 micromol/L. Our data suggest that both alanine and arginine aminopeptidases are involved in the reversal of the angiotensin II-stimulated collagen gel contraction in control and TGF-beta1-treated cardiac fibroblasts or myofibroblasts.

Reversal of angiotensin II-stimulated collagen gel contraction in cardiac fibroblasts by aminopeptidase inhibition.

Effect of angiotensin (1–7) on collagen production in cardiac fibroblasts

Cardiac fibroblasts are a cell population that controls the homeostasis of the extracellular matrix and orchestrates a damage response to maintain cardiac architecture and performance. Due to these functions, fibroblasts play a central role in cardiac fibrosis development, and there are large differences in matrix protein secretion profiles between fibroblasts from aged versus young animals. Senescence is a multifactorial and complex process that has been associated with inflammatory and fibrotic responses. After damage, transient cellular senescence is usually beneficial, as these cells promote tissue repair. However, the persistent presence of senescent cells within a tissue is linked with fibrosis development and organ dysfunction, leading to aging-related diseases such as cardiovascular pathologies. In the heart, early cardiac fibroblast senescence after myocardial infarction seems to be protective to avoid excessive fibrosis; however, in non-infarcted models of cardiac fibrosis, cardiac fibroblast senescence has been shown to be deleterious. Today, two new classes of drugs, termed senolytics and senostatics, which eliminate senescent cells or modify senescence-associated secretory phenotype, respectively, arise as novel therapeutical strategies to treat aging-related pathologies. However, further studies will be needed to evaluate the extent of the utility of senotherapeutic drugs in cardiac diseases, in which pathological context and temporality of the intervention must be considered. 

Guillermo Díaz-Araya

Papers

Resolvin D1 reduces expression and secretion of cytokines and monocyte adhesion triggered by Angiotensin II, in rat cardiac fibroblasts.

Effect of bestatin on angiotensin I-, II- and III-induced collagen gel contraction in cardiac fibroblasts.

Reversal of angiotensin II-stimulated collagen gel contraction in cardiac fibroblasts by aminopeptidase inhibition.

Effect of angiotensin (1–7) on collagen production in cardiac fibroblasts

Senescent cardiac fibroblasts: A key role in cardiac fibrosis.