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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretatoin of assays for monitoring autophagy

A link between oxidative stress and hypertension has been firmly established in multiple animal models of hypertension but remains elusive in humans While initial studies focused on inactivation of nitric oxide by superoxide, our understanding of relevant reactive oxygen species (superoxide, hydrogen peroxide, and peroxynitrite) and how they modify complex signaling pathways to promote hypertension has expanded significantly In this review, we summarize recent advances in delineating the primary and secondary sources of reactive oxygen species (nicotinamide adenine dinucleotide phosphate oxidases, uncoupled endothelial nitric oxide synthase, endoplasmic reticulum, and mitochondria), the posttranslational oxidative modifications they induce on protein targets important for redox signaling, their interplay with endogenous antioxidant systems, and the role of inflammasome activation and endoplasmic reticular stress in the development of hypertension We highlight how oxidative stress in different organ systems contributes to hypertension, describe new animal models that have clarified the importance of specific proteins, and discuss clinical studies that shed light on how these processes and pathways are altered in human hypertension Finally, we focus on the promise of redox proteomics and systems biology to help us fully understand the relationship between ROS and hypertension and their potential for designing and evaluating novel antihypertensive therapies

Oxidative Stress and Hypertension

Dihydromyricetin alleviates doxorubicin-induced cardiotoxicity by inhibiting NLRP3 inflammasome through activation of SIRT1.

As a potent chemotherapeutic agent, doxorubicin (DOX) is widely used for the treatment of a variety of cancers However, its clinical utility is limited by dose-dependent cardiotoxicity, and pathogenesis has traditionally been attributed to the formation of reactive oxygen species (ROS). Accordingly, the prevention of DOX-induced cardiotoxicity is an indispensable goal to optimize therapeutic regimens and reduce morbidity. Acetylation is an emerging and important epigenetic modification regulated by histone deacetylases (HDACs) and histone acetyltransferases (HATs). Despite extensive studies of the molecular basis and biological functions of acetylation, the application of acetylation as a therapeutic target for cardiotoxicity is in the initial stage, and further studies are required to clarify the complex acetylation network and improve the clinical management of cardiotoxicity. In this review, we summarize the pivotal functions of HDACs and HATs in DOX-induced oxidative stress, the underlying mechanisms, the contributions of noncoding RNAs (ncRNAs) and exercise-mediated deacetylases to cardiotoxicity. Furthermore, we describe research progress related to several important SIRT activators and HDAC inhibitors with potential clinical value for chemotherapy and cardiotoxicity. Collectively, a comprehensive understanding of specific roles and recent developments of acetylation in doxorubicin-induced cardiotoxicity will provide a basis for improved treatment outcomes in cancer and cardiovascular diseases.

Role of acetylation in doxorubicin-induced cardiotoxicity.

Targeted inhibition of ULK1 enhances daunorubicin sensitivity in acute myeloid leukemia.

Oxidative stress and apoptosis play a vital role in the pathogenesis of contrast-induced acute kidney injury (CI-AKI). The purpose of our study was to investigate the protective effects and mechanisms of melatonin against CI-AKI in a CI-AKI mouse model and NRK-52E cells. We established the CI-AKI model in mice, and the animals were pretreated with melatonin (20 mg/kg). Our results demonstrated that melatonin treatment exerted a renoprotective effect by decreasing the level of serum creatinine (SCr) and blood urea nitrogen (BUN), lessening the histological changes of renal tubular injuries, and reducing the expression of neutrophil gelatinase-associated lipid (NGAL), a marker of kidney injury. We also found that pretreatment with melatonin remarkably increased the expression of Sirt3 and decreased the ac-SOD2 K68 level. Consequently, melatonin treatment significantly decreased the oxidative stress by reducing the Nox4, ROS, and malondialdehyde (MDA) content and by increasing the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity levels. The antiapoptotic effect of melatonin on CI-AKI was revealed by decreasing the ratio of Bax/Bcl2 and the cleaved caspase3 level and by reducing the number of apoptosis-positive tubular cells. In addition, melatonin treatment remarkably reduced the inflammatory cytokines of interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), and transforming growth factor β (TGFβ) in vivo and in vitro. Sirt3 deletion and specific Sirt3 siRNA abolished the above renoprotective effects of melatonin in mice with iohexol-induced acute kidney injury and in NRK-52E cells. Thus, our results demonstrated that melatonin exhibited the renoprotective effects of antioxidative stress, antiapoptosis, and anti-inflammation by the activation of Sirt3 in the CI-AKI model in vivo and in vitro. Melatonin may be a potential drug to ameliorate CI-AKI in clinical practice.

/pdf/melatonin-alleviates-contrast-induced-acute-kidney-injury-by-4bhuusu2t7.pdf

Melatonin Alleviates Contrast-Induced Acute Kidney Injury by Activation of Sirt3

Sunitinib malate is a multi-targeted tyrosine kinase inhibitor used extensively for treatment of human tumors. However, cardiovascular adverse effects of sunitinib limit its clinical use. It is pivotal to elucidate molecular targets that mediate sunitinib-induced cardiotoxicity. Sirtuin 3 (Sirt3) is an effective mitochondrial deacetylase that has been reported to regulate sensitivity of different types of cells to chemotherapies, but roles of Sirt3 in sunitinib-induced cardiotoxicity have not been investigated. In the present study, we established wild type, Sirt3-knockout, and Sirt3-overexpressing mouse models of sunitinib (40 mg kg−1 day−1 for 28 days)-induced cardiotoxicity and examined cardiovascular functions and pathological changes. We further cultured wild type, Sirt3-knockout, and Sirt3-overexpressing primary mouse cardiac pericytes and analyzed sunitinib (10 μMol for 48 h)-induced alterations in cellular viability, cell death processes, and molecular pathways. Our results show that sunitinib predominantly induced hypertension, left ventricular systolic dysfunction, and cardiac pericyte death accompanied with upregulation of Sirt3 in cardiac pericytes, and these cardiotoxicities were significantly attenuated in Sirt3-knockout mice, but aggravated in Sirt3-overexpressing mice. Mechanistically, sunitinib induced cardiac pericyte death through inhibition of GSTP1/JNK/autophagy pathway and Sirt3 interacted with and inhibited GSTP1, further inhibiting the pathway and aggravating sunitinib-induced pericyte death. Conclusively, we demonstrate that Sirt3 promotes sensitivity to sunitinib-induced cardiotoxicity via GSTP1/JNK/autophagy pathway. Our results suggest Sirt3 might be a potential target for developing cardioprotective therapies for sunitinib-receiving patients.

Sirt3 promotes sensitivity to sunitinib-induced cardiotoxicity via inhibition of GTSP1/JNK/autophagy pathway in vivo and in vitro.

Ranolazine alleviates contrast-associated acute kidney injury through modulation of calcium independent oxidative stress and apoptosis.

Lymphangiogenesis is possibly capable of attenuating hypertension-induced cardiac injury. Sirtuin 3 (SIRT3) is an effective mitochondrial deacetylase that has the potential to modulate this process; however, its role in hypertension-induced cardiac lymphangiogenesis to date has not been investigated. Our experiments were performed on 8-week-old wild-type (WT), SIRT3 knockout (SIRT3-KO) and SIRT3 overexpression (SIRT3-LV) mice infused with angiotensin II (Ang II) (1000 ng/kg per minute) or saline for 28 days. After Ang II infusion, SIRT3-KO mice developed a more severe cardiac remodelling, less lymphatic capillaries and lower expression of lymphatic marker when compared to wild-type mice. In comparison, SIRT3-LV restored lymphangiogenesis and attenuated cardiac injury. Furthermore, lymphatic endothelial cells (LECs) exposed to Ang II in vitro exhibited decreased migration and proliferation. Silencing SIRT3 induced functional decrease in LECs, while SIRT3 overexpression LECs facilitated. Moreover, SIRT3 may up-regulate lymphangiogenesis by affecting vascular endothelial growth factor receptor 3 (VEGFR3) and ERK pathway. These findings suggest that SIRT3 could promote lymphangiogenesis and attenuate hypertensive cardiac injury.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jcmm.16661

Lingxin Liu

Papers

Melatonin Alleviates Contrast-Induced Acute Kidney Injury by Activation of Sirt3

Sirt3 promotes sensitivity to sunitinib-induced cardiotoxicity via inhibition of GTSP1/JNK/autophagy pathway in vivo and in vitro.

Ranolazine alleviates contrast-associated acute kidney injury through modulation of calcium independent oxidative stress and apoptosis.

Sirtuin 3 deficiency aggravates angiotensin II-induced hypertensive cardiac injury by the impairment of lymphangiogenesis.