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

Activation of Nrf2/HO-1 signaling: An important molecular mechanism of herbal medicine in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress

Heart failure (HF) remains the most common cause of death and disability, and a major economic burden, in industrialized nations. Physiological, pharmacological, and clinical studies have demonstrated that activation of the renin-angiotensin system is a key mediator of HF progression. Angiotensin-converting enzyme 2 (ACE2), a homolog of ACE, is a monocarboxypeptidase that converts angiotensin II into angiotensin 1-7 (Ang 1-7) which, by virtue of its actions on the Mas receptor, opposes the molecular and cellular effects of angiotensin II. ACE2 is widely expressed in cardiomyocytes, cardiofibroblasts, and coronary endothelial cells. Recent preclinical translational studies confirmed a critical counter-regulatory role of ACE2/Ang 1-7 axis on the activated renin-angiotensin system that results in HF with preserved ejection fraction. Although loss of ACE2 enhances susceptibility to HF, increasing ACE2 level prevents and reverses the HF phenotype. ACE2 and Ang 1-7 have emerged as a key protective pathway against HF with reduced and preserved ejection fraction. Recombinant human ACE2 has been tested in phase I and II clinical trials without adverse effects while lowering and increasing plasma angiotensin II and Ang 1-7 levels, respectively. This review discusses the transcriptional and post-transcriptional regulation of ACE2 and the role of the ACE2/Ang 1-7 axis in cardiac physiology and in the pathophysiology of HF. The pharmacological and therapeutic potential of enhancing ACE2/Ang 1-7 action as a novel therapy for HF is highlighted.

Role of the ACE2/Angiotensin 1-7 Axis of the Renin-Angiotensin System in Heart Failure

Targeting autophagy-related protein kinases for potential therapeutic purpose.

Inhibiting cell survival and inducing cell death are the main approaches of tumor therapy. Autophagy plays an important role on intracellular metabolic homeostasis by eliminating dysfunctional or unnecessary proteins and damaged or aged cellular organelles to recycle their constituent metabolites that enable the maintenance of cell survival and genetic stability and even promotes the drug resistance, which severely limits the efficacy of chemotherapeutic drugs. Currently, targeting autophagy has a seemingly contradictory effect to suppress and promote tumor survival, which makes the effect of targeting autophagy on drug resistance more confusing and fuzzier. In the review, we summarize the regulation of autophagy by emerging ways, the action of targeting autophagy on drug resistance and some of the new therapeutic approaches to treat tumor drug resistance by interfering with autophagy-related pathways. The full-scale understanding of the tumor-associated signaling pathways and physiological functions of autophagy will hopefully open new possibilities for the treatment of tumor drug resistance and the improvement in clinical outcomes.

/pdf/targeting-autophagy-to-overcome-drug-resistance-further-34yrrbxm2m.pdf

Targeting autophagy to overcome drug resistance: further developments.

Studies regarding macroautophagic/autophagic regulation in endothelial cells (ECs) under diabetic conditions are very limited. Clinical evidence establishes an endothelial protective effect...

https://www.tandfonline.com/doi/pdf/10.1080/15548627.2019.1569913

Metformin alleviates hyperglycemia-induced endothelial impairment by downregulating autophagy via the Hedgehog pathway

Fibroblast growth factor 21 protects the heart from angiotensin II-induced cardiac hypertrophy and dysfunction via SIRT1.

Baicalin is the major component found in Scutellaria baicalensis root, a widely used herb in traditional Chinese medicine, which exhibits strong anti-inflammatory, anti-viral and anti-tumor activities. The present work was devoted to elucidate the molecular and cellular mechanisms underlying the protective effects of Baicalin against diabetes-induced oxidative damage, inﬂammation and endothelial dysfunction. Diabetic mice, induced by streptozotocin (STZ), were treated with intraperitoneal Baicalin injections. Human umbilical vein endothelial cells (HUVECs) were cultured either in normal glucose (NG, 5.5 mM) or high glucose (HG, 33 mM) medium in the presence or absence of Baicalin for 72 h. We observed an obvious inhibition of hyperglycemia-triggered oxidative damage and inﬂammation in HUVECs and diabetic aortal vasculature by Baicalin, along with restoration of hyperglycemia-impaired nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway activity. However, the protective effects of Baicalin almost completely abolished in HUVECs transduced with shRNA against Nrf2, but not with nonsense shRNA. Mechanistic studies demonstrated that HG decreased Akt and GSK3B phosphorylation, restrained nuclear export of Fyn and nuclear localization of Nrf2, blunted Nrf2 downstream target genes, and subsequently induced oxidative stress in HUVECs. However, those destructive cascade, were well prevented by Baicalin in HUVECs. Furthermore, LY294002 and ML385 (inhibitor of PI3K and Nrf2) attenuated Baicalin mediated Nrf2 activation and the ability of facilitates angiogenesis in vivo and ex vivo. Taken together, the endothelial protective effect of Baicalin under hyperglycemia condition could be partly attributed to its role in downregulating reactive oxygen species (ROS) and inﬂammation via the Akt/GSK3B/Fyn-mediated Nrf2 activation.

/pdf/baicalin-alleviates-hyperglycemia-induced-endothelial-4frobr483q.pdf

Baicalin alleviates hyperglycemia-induced endothelial impairment 1 via Nrf2.

c-Met up-regulates the expression of PD-L1 through MAPK/NF-κBp65 pathway

Multidrug resistance is the main obstacle to curing hepatocellular carcinoma (HCC). Acid‐sensing ion channel 1a (ASIC1a) has critical roles in all stages of cancer progression, especially invasion and metastasis, and in resistance to therapy. Epithelial to mesenchymal transition (EMT) transforms epithelial cells into mesenchymal cells after being stimulated by extracellular factors and is closely related to tumour infiltration and resistance. We used Western blotting, immunofluorescence, qRT‐PCR, immunohistochemical staining, MTT, colony formation and scratch healing assay to determine ASIC1a levels and its relationship to cell proliferation, migration and invasion. ASIC1a is overexpressed in HCC tissues, and the amount increased in resistant HCC cells. EMT occurred more frequently in drug‐resistant cells than in parental cells. Inactivation of ASIC1a inhibited cell migration and invasion and increased the chemosensitivity of cells through EMT. Overexpression of ASIC1a upregulated EMT and increased the cells’ proliferation, migration and invasion and induced drug resistance; knocking down ASIC1a with shRNA had the opposite effects. ASIC1a increased cell migration and invasion through EMT by regulating α and β‐catenin, vimentin and fibronectin expression via the AKT/GSK‐3β/Snail pathway driven by TGFβ/Smad signals. ASIC1a mediates drug resistance of HCC through EMT via the AKT/GSK‐3β/Snail pathway.

Jiaojiao Liang

Papers

Metformin alleviates hyperglycemia-induced endothelial impairment by downregulating autophagy via the Hedgehog pathway

Fibroblast growth factor 21 protects the heart from angiotensin II-induced cardiac hypertrophy and dysfunction via SIRT1.

Baicalin alleviates hyperglycemia-induced endothelial impairment 1 via Nrf2.

c-Met up-regulates the expression of PD-L1 through MAPK/NF-κBp65 pathway

ASIC1a stimulates the resistance of human hepatocellular carcinoma by promoting EMT via the AKT/GSK3β/Snail pathway driven by TGFβ/Smad signals