Faa1 membrane binding drives positive feedback in autophagosome biogenesis via fatty acid activation.

Autophagy serves as a stress response pathway by mediating the degradation of cellular material within lysosomes. In autophagy, this material is encapsulated in double-membrane vesicles termed autophagosomes, which form from precursors referred to as phagophores. Phagophores grow by lipid influx from the endoplasmic reticulum into Atg9-positive compartments and local lipid synthesis provides lipids for their expansion. How phagophore nucleation and expansion are coordinated with lipid synthesis is unclear. Here, we show that Faa1, an enzyme activating fatty acids, is recruited to Atg9 vesicles by directly binding to negatively charged membranes with a preference for phosphoinositides such as PI3P and PI4P. We define the membrane-binding surface of Faa1 and show that its direct interaction with the membrane is required for its recruitment to phagophores. Furthermore, the physiological localization of Faa1 is key for its efficient catalysis and promotes phagophore expansion. Our results suggest a positive feedback loop coupling phagophore nucleation and expansion to lipid synthesis.

Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH.

Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis.

Molecular Mechanism of Autophagosome-Lysosome Fusion in Mammalian Cells.

In eukaryotes, targeting intracellular components for lysosomal degradation by autophagy represents a catabolic process that evolutionarily regulates cellular homeostasis. The successful completion of autophagy initiates the engulfment of cytoplasmic materials within double-membrane autophagosomes and subsequent delivery to autolysosomes for degradation by acidic proteases. The formation of autolysosomes relies on the precise fusion of autophagosomes with lysosomes. In recent decades, numerous studies have provided insights into the molecular regulation of autophagosome-lysosome fusion. In this review, an overview of the molecules that function in the fusion of autophagosomes with lysosomes is provided. Moreover, the molecular mechanism underlying how these functional molecules regulate autophagosome-lysosome fusion is summarized.

mTORC1 hyperactivation and resultant suppression of macroautophagy contribute to the induction of cardiomyocyte necroptosis by catecholamine surges.

Previous studies revealed a controversial role of mechanistic target of rapamycin complex 1 (mTORC1) and mTORC1-regulated macroautophagy in isoproterenol (ISO)-induced cardiac injury. Here we investigated the role of mTORC1 and potential underlying mechanisms in ISO-induced cardiomyocyte necrosis. Two consecutive daily injections of ISO (85 mg/kg, s.c.) or vehicle control (CTL) were administered to C57BL/6J mice with or without rapamycin (RAP, 5 mg/kg, i.p.) pretreatment. Western blot analyses showed that myocardial mTORC1 signaling and the RIPK1-RIPK3-MLKL necroptotic pathway were activated, mRNA expression analyses revealed downregulation of representative TFEB target genes, and Evan's blue dye uptake assays detected increased cardiomyocyte necrosis in ISO-treated mice. However, RAP pretreatment prevented or significantly attenuated the ISO-induced cardiomyocyte necrosis, myocardial inflammation, downregulation of TFEB target genes, and activation of the RIPK1-RIPK3-MLKL pathway. LC3-II flux assays confirmed the impairment of myocardial autophagic flux in the ISO-treated mice. In cultured neonatal rat cardiomyocytes, mTORC1 signaling was also activated by ISO, and inhibition of mTORC1 by RAP attenuated ISO-induced cytotoxicity. These findings suggest that mTORC1 hyperactivation and resultant suppression of macroautophagy play a major role in the induction of cardiomyocyte necroptosis by catecholamine surges, identifying mTORC1 inhibition as a potential strategy to treat heart diseases with catecholamine surges.

Aggrephagy-related gene signature correlates with survival and tumor-associated macrophages in glioma: Insights from single-cell and bulk RNA sequencing.

BACKGROUND: Aggrephagy is a lysosome-dependent process that degrades misfolded protein condensates to maintain cancer cell homeostasis. Despite its importance in cellular protein quality control, the role of aggrephagy in glioma remains poorly understood. OBJECTIVE: To investigate the expression of aggrephagy-related genes (ARGs) in glioma and in different cell types of gliomas and to develop an ARGs-based prognostic signature to predict the prognosis, tumor microenvironment, and immunotherapy response of gliomas. METHODS: ARGs were identified by searching the Reactome database. We developed the ARGs-based prognostic signature (ARPS) using data from the Cancer Genome Atlas (TCGA, n = 669) by Lasso-Cox regression. We validated the robustness of the signature in clinical subgroups and CGGA cohorts (n = 970). Gene set enrichment analysis (GSEA) was used to identify the pathways enriched in ARPS subgroups. The correlations between ARGs and macrophages were also investigated at single cell level. RESULTS: A total of 44 ARGs showed heterogeneous expression among different cell types of gliomas. Five ARGs (HSF1, DYNC1H1, DYNLL2, TUBB6, TUBA1C) were identified to develop ARPS, an independent prognostic factor. GSEA showed gene sets of patients with high-ARPS were mostly enriched in cell cycle, DNA replication, and immune-related pathways. High-ARPS subgroup had higher immune cell infiltration states, particularly macrophages, Treg cells, and neutrophils. APRS had positive association with tumor mutation burden (TMB) and immunotherapy response predictors. At the single cell level, we found ARGs correlated with macrophage development and identified ARGs-mediated macrophage subtypes with distinct communication characteristics with tumor cells. VIM+ macrophages were identified as pro-inflammatory and had higher interactions with malignant cells. CONCLUSION: We identified a novel signature based on ARGs for predicting glioma prognosis, tumor microenvironment, and immunotherapy response. We highlight the ARGs-mediated macrophages in glioma exhibit classical features.

A metabolite sensor subunit of the Atg1/ULK complex regulates selective autophagy.

Cells convert complex metabolic information into stress-adapted autophagy responses. Canonically, multilayered protein kinase networks converge on the conserved Atg1/ULK kinase complex (AKC) to induce non-selective and selective forms of autophagy in response to metabolic changes. Here we show that, upon phosphate starvation, the metabolite sensor Pho81 interacts with the adaptor subunit Atg11 at the AKC via an Atg11/FIP200 interaction motif to modulate pexophagy by virtue of its conserved phospho-metabolite sensing SPX domain. Notably, core AKC components Atg13 and Atg17 are dispensable for phosphate starvation-induced autophagy revealing significant compositional and functional plasticity of the AKC. Our data indicate that, instead of functioning as a selective autophagy receptor, Pho81 compensates for partially inactive Atg13 by promoting Atg11 phosphorylation by Atg1 critical for pexophagy during phosphate starvation. Our work shows Atg11/FIP200 adaptor subunits bind not only selective autophagy receptors but also modulator subunits that convey metabolic information directly to the AKC for autophagy regulation.

ALS-related p97 R155H mutation disrupts lysophagy in iPSC-derived motor neurons.

Mutations in the AAA+ ATPase p97 cause multisystem proteinopathy 1, which includes amyotrophic lateral sclerosis; however, the pathogenic mechanisms that contribute to motor neuron loss remain obscure. Here, we use two induced pluripotent stem cell models differentiated into spinal motor neurons to investigate how p97 mutations perturb the motor neuron proteome. Using quantitative proteomics, we find that motor neurons harboring the p97 R155H mutation have deficits in the selective autophagy of lysosomes (lysophagy). p97 R155H motor neurons are unable to clear damaged lysosomes and have reduced viability. Lysosomes in mutant motor neurons have increased pH compared with wild-type cells. The clearance of damaged lysosomes involves UBXD1-p97 interaction, which is disrupted in mutant motor neurons. Finally, inhibition of the ATPase activity of p97 using the inhibitor CB-5083 rescues lysophagy defects in mutant motor neurons. These results add to the evidence that endo-lysosomal dysfunction is a key aspect of disease pathogenesis in p97-related disorders.

Human YKT6 forms priming complex with STX17 and SNAP29 to facilitate autophagosome-lysosome fusion.

Autophagy is crucial for degrading and recycling cellular components. Fusion between autophagosomes and lysosomes is pivotal, directing autophagic cargo to degradation. This process is driven by STX17-SNAP29-VAMP8 and STX7-SNAP29-YKT6 in mammalian cells. However, the interaction between STX17 and YKT6 and its significance remain to be revealed. In this study, we challenge the notion that STX17 and YKT6 function independently in autophagosome-lysosome fusion. YKT6, through its SNARE domain, forms a complex with STX17 and SNAP29 on autophagosomes, enhancing autophagy flux. VAMP8 displaces YKT6 from this complex, leading to the formation of the fusogenic complex STX17-SNAP29-VAMP8. We demonstrated that the YKT6-SNAP29-STX17 complex facilitates both lipid and content mixing driven by STX17-SNAP29-VAMP8, suggesting a priming role of YKT6 for efficient membrane fusion. Our results provide a potential regulation mechanism of autophagosome-lysosome fusion, highlighting the importance of YKT6 and its interactions with STX17 and SNAP29 in promoting autophagy flux.

TFEB activation triggers pexophagy for functional adaptation during oxidative stress under calcium deficient-conditions.

BACKGROUND: Calcium is a ubiquitous intracellular messenger that regulates the expression of various genes involved in cell proliferation, differentiation, and motility. The involvement of calcium in diverse metabolic pathways has been suggested. However, the effect of calcium in peroxisomes, which are involved in fatty acid oxidation and scavenges the result reactive oxygen species (ROS), remains elusive. In addition, impaired peroxisomal ROS inhibit the mammalian target of rapamycin complex 1 (mTORC1) and promote autophagy. Under stress, autophagy serves as a protective mechanism to avoid cell death. In response to oxidative stress, lysosomal calcium mediates transcription factor EB (TFEB) activation. However, the impact of calcium on peroxisome function and the mechanisms governing cellular homeostasis to prevent diseases caused by calcium deficiency are currently unknown. METHODS: To investigate the significance of calcium in peroxisomes and their roles in preserving cellular homeostasis, we established an in-vitro scenario of calcium depletion. RESULTS: This study demonstrated that calcium deficiency reduces catalase activity, resulting in increased ROS accumulation in peroxisomes. This, in turn, inhibits mTORC1 and induces pexophagy through TFEB activation. However, treatment with the antioxidant N-acetyl-l-cysteine (NAC) and the autophagy inhibitor chloroquine impeded the nuclear translocation of TFEB and attenuated peroxisome degradation. CONCLUSIONS: Collectively, our study revealed that ROS-mediated TFEB activation triggers pexophagy during calcium deficiency, primarily because of attenuated catalase activity. We posit that calcium plays a significant role in the proper functioning of peroxisomes, critical for fatty-acid oxidation and ROS scavenging in maintaining cellular homeostasis. These findings have important implications for signaling mechanisms in various pathologies, including Zellweger's syndrome and ageing.

Natural products targeting macroautophagy signaling in hepatocellular carcinoma therapy: Recent evidence and perspectives.

Hepatocellular carcinoma (HCC), presently the second leading cause of global cancer-related mortality, continues to pose significant challenges in the realm of medical oncology, impacting both clinical drug selection and mechanistic research. Recent investigations have unveiled autophagy-related signaling as a promising avenue for HCC treatment. A growing body of research has highlighted the pivotal role of autophagy-modulating natural products in inhibiting HCC progression. In this context, we provide a concise overview of the fundamental autophagy mechanism and delineate the involvement of autophagic signaling pathways in HCC development. Additionally, we review pertinent studies demonstrating how natural products regulate autophagy to mitigate HCC. Our findings indicate that natural products exhibit cytotoxic effects through the induction of excessive autophagy, simultaneously impeding HCC cell proliferation by autophagy inhibition, thereby depriving HCC cells of essential energy. These effects have been associated with various signaling pathways, including PI3K/AKT, MAPK, AMPK, Wnt/ß-catenin, Beclin-1, and ferroautophagy. These results underscore the considerable therapeutic potential of natural products in HCC treatment. However, it is important to note that the present study did not establish definitive thresholds for autophagy induction or inhibition by natural products. Further research in this domain is imperative to gain comprehensive insights into the dual role of autophagy, equipping us with a better understanding of this double-edged sword in HCC management.

Neuroactive steroid effects on autophagy in a human embryonic kidney 293 (HEK) cell model.

Neuropsychiatric and neurodegenerative disorders are correlated with cellular stress. Macroautophagy (autophagy) may represent an important protective pathway to maintain cellular homeostasis and functionality, as it targets cytoplasmic components to lysosomes for degradation and recycling. Given recent evidence that some novel psychiatric treatments, such as the neuroactive steroid (NAS) allopregnanolone (AlloP, brexanolone), may induce autophagy, we stably transfected human embryonic kidney 293 (HEK) cells with a ratiometric fluorescent probe to assay NAS effects on autophagy. We hypothesized that NAS may modulate autophagy in part by the ability of uncharged NAS to readily permeate membranes. Microscopy revealed a weak effect of AlloP on autophagic flux compared with the positive control treatment of Torin1. In high-throughput microplate experiments, we found that autophagy induction was more robust in early passages of HEK cells. Despite limiting studies to early passages for maximum sensitivity, a range of NAS structures failed to reliably induce autophagy or interact with Torin1 or starvation effects. To probe NAS in a system where AlloP effects have been shown previously, we surveyed astrocytes and again saw minimal autophagy induction by AlloP. Combined with other published results, our results suggest that NAS may modulate autophagy in a cell-specific or context-specific manner. Although there is merit to cell lines as a screening tool, future studies may require assaying NAS in cells from brain regions involved in neuropsychiatric disorders.

Upregulated pexophagy limits the capacity of selective autophagy.

Selective autophagy is an essential process to maintain cellular homeostasis through the constant recycling of damaged or superfluous components. Over a dozen selective autophagy pathways mediate the degradation of diverse cellular substrates, but whether these pathways can influence one another remains unknown. We address this question using pexophagy, the autophagic degradation of peroxisomes, as a model. We show in cells that upregulated pexophagy impairs the selective autophagy of both mitochondria and protein aggregates by exhausting the autophagy initiation factor, ULK1. We confirm this finding in cell models of the pexophagy-mediated form of Zellweger Spectrum Disorder, a disease characterized by peroxisome dysfunction. Further, we extend the generalizability of limited selective autophagy by determining that increased protein aggregate degradation reciprocally reduces pexophagy using cell models of Parkinson's Disease and Huntington's Disease. Our findings suggest that the degradative capacity of selective autophagy can become limited by an increase in one substrate.

Neuronal Autophagy: Regulations and Implications in Health and Disease.

Autophagy is a major degradative pathway that plays a key role in sustaining cell homeostasis, integrity, and physiological functions. Macroautophagy, which ensures the clearance of cytoplasmic components engulfed in a double-membrane autophagosome that fuses with lysosomes, is orchestrated by a complex cascade of events. Autophagy has a particularly strong impact on the nervous system, and mutations in core components cause numerous neurological diseases. We first review the regulation of autophagy, from autophagosome biogenesis to lysosomal degradation and associated neurodevelopmental/neurodegenerative disorders. We then describe how this process is specifically regulated in the axon and in the somatodendritic compartment and how it is altered in diseases. In particular, we present the neuronal specificities of autophagy, with the spatial control of autophagosome biogenesis, the close relationship of maturation with axonal transport, and the regulation by synaptic activity. Finally, we discuss the physiological functions of autophagy in the nervous system, during development and in adulthood.

Inhibition of VHL by VH298 Accelerates Pexophagy by Activation of HIF-1α in HeLa Cells.

Autophagy is a pivotal biological process responsible for maintaining the homeostasis of intracellular organelles. Yet the molecular intricacies of peroxisomal autophagy (pexophagy) remain largely elusive. From a ubiquitin-related chemical library for screening, we identified several inhibitors of the Von Hippel-Lindau (VHL) E3 ligase, including VH298, thereby serving as potent inducers of pexophagy. In this study, we observed that VH298 stimulates peroxisomal degradation by ATG5 dependently and escalates the ubiquitination of the peroxisomal membrane protein ABCD3. Interestingly, the ablation of NBR1 is similar to the curtailed peroxisomal degradation in VH298-treated cells. We also found that the pexophagy induced by VH298 is impeded upon the suppression of gene expression by the translation inhibitor cycloheximide. Beyond VHL inhibition, we discovered that roxadustat, a direct inhibitor of HIF-α prolyl hydroxylase, is also a potent inducer of pexophagy. Furthermore, we found that VH298-mediated pexophagy is blocked by silencing HIF-1α. In conclusion, our findings suggest that VH298 promotes pexophagy by modulating VHL-mediated HIF-α transcriptional activity.

Single-cell aggrephagy-related patterns facilitate tumor microenvironment intercellular communication, influencing osteosarcoma progression and prognosis.

Osteosarcoma, a common malignant tumor in children, has emerged as a major threat to the life and health of pediatric patients. Presently, there are certain limitations in the diagnosis and treatment methods for this disease, resulting in inferior therapeutic outcomes. Therefore, it is of great importance to study its pathogenesis and explore innovative approaches to diagnosis and treatment. In this study, a non-negative matrix decomposition method was employed to conduct a comprehensive investigation and analysis of aggregated autophagy-related genes within 331,394 single-cell samples of osteosarcoma. Through this study, we have elucidated the intricate communication patterns among various cells within the tumor microenvironment. Based on the classification of aggregated autophagy-related genes, we are not only able to more accurately predict patients' prognosis but also offer robust guidance for treatment strategies. The findings of this study hold promise for breakthroughs in the diagnosis and treatment of osteosarcoma, intervention of aggrephagy is expected to improve the survival rate and quality of life of osteosarcoma patients.

The hookup model of the HOPS complex in autophagosome-lysosome fusion.

Macroautophagy/autophagy is a highly conserved process that involves the degradation of proteins, damaged organelles, and other cytoplasmic macromolecules. Autophagosome-lysosome fusion is critical for successful substrate degradation and is mediated by SNARE proteins. The fusion process requires additional vesicle docking and tethering-regulating factors. Our recent work has uncovered a functional model of autophagosome-lysosome fusion. We demonstrated that the six-subunit homotypic fusion and vacuole protein sorting (HOPS) complex can be assembled by two subcomplexes, the VPS39-VPS11 subcomplex (HOPS-2) and the VPS41-VPS16-VPS18-VPS33A subcomplex (HOPS-4). VPS39 binds with RAB2 on the autophagosome and VPS41 binds with RAB39A on the lysosome, which then promotes membrane tethering and autophagic SNARE-mediated membrane fusion. Moreover, we have revealed that ALS- and FTD-related C9orf72 is a guanine exchange factor (GEF) for RAB39A. In this punctum, we discuss how the C9orf72-RAB39A-HOPS axis function regulates autophagosome-lysosome fusion.

The role of the N terminus of lipidated human Atg8-family proteins in cis-membrane association.

A multifunctional role of Atg8-family proteins (Atg8 from yeast and human LC3 and GABARAP subfamilies, all referred to here as ATG8s) in macroautophagy/autophagy is carried out by two protein domains, the N-terminal helical domain (NHD) and ubiquitin-like (UBL) domain. Previous studies showed that the NHD of PE-conjugated ATG8s mediates membrane hemifusion via a direct interaction with lipids in trans-membrane association, which would require the NHD in lipidated ATG8s to adopt a solvent-oriented, "open", conformation that unmasks a UBL domain surface needed for membrane tethering. A purpose of the "closed" conformation of the NHD masking the tethering surface on the UBL domain, a conformation seen in the most structures of non-lipidated ATG8s, remained elusive. A recent study by Zhang et al. discussed in this article, showed that the N terminus of lipidated human ATG8s adopts the "closed" conformation when it interacts with the membrane in cis-membrane association, i.e. with the same membrane ATG8 is anchored to. This finding suggests functions for two distinct conformations of the NHD in lipidated ATG8s and raises questions about determinants controlling cis- or trans-membrane associations of the NHD in ATG8-PE.Abbreviations: AIM, Atg8-family interacting motif; 3D-CLEM, three-dimensional correlative light and electron microscopy; FRET, Förster/fluorescence resonance energy transfer; LIR, LC3-interacting motif; MD, molecular dynamics; NHD, N-terminal helical domain; UBL, ubiquitin-like.

Flagella-mediated cytosolic motility of Salmonella enterica Paratyphi A aids in evasion of xenophagy but does not impact egress from host cells.

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Typhoidal serovars like Paratyphi A (SPA) are human restricted and cause severe systemic diseases, while many serovars like Typhimurium (STM) have a broad host range, and usually lead to self-limiting gastroenteritis. There are key differences between typhoidal and non-typhoidal Salmonella in pathogenesis, but underlying mechanisms remain largely unknown. Transcriptomes and phenotypes in epithelial cells revealed induction of motility, flagella and chemotaxis genes for SPA but not STM. SPA exhibited cytosolic motility mediated by flagella. In this study, we applied single-cell microscopy to analyze triggers and cellular consequences of cytosolic motility. Live-cell imaging (LCI) revealed that SPA invades host cells in a highly cooperative manner. Extensive membrane ruffling at invasion sites led to increased membrane damage in nascent Salmonella-containing vacuole, and subsequent cytosolic release. After release into the cytosol, motile bacteria showed the same velocity as under culture conditions in media. Reduced capture of SPA by autophagosomal membranes was observed by LCI and electron microscopy. Prior work showed that SPA does not use flagella-mediated motility for cell exit via the intercellular spread. However, cytosolic motile SPA was invasion-primed if released from host cells. Our results reveal flagella-mediated cytosolic motility as a possible xenophagy evasion mechanism that could drive disease progression and contributes to the dissemination of systemic infection.

Molecular Mechanisms of Macroautophagy, Microautophagy, and Chaperone-Mediated Autophagy.

Autophagy is a self-digestive process that is conserved in eukaryotic cells and responsible for maintaining cellular homeostasis through proteolysis. By this process, cells break down their own components in lysosomes. Autophagy can be classified into three categories: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Macroautophagy involves membrane elongation and microautophagy involves membrane internalization, and both pathways undergo selective or non-selective processes that transport cytoplasmic components into lysosomes to be degraded. CMA, however, involves selective incorporation of cytosolic materials into lysosomes without membrane deformation. All three categories of autophagy have attracted much attention due to their involvement in various biological phenomena and their relevance to human diseases, such as neurodegenerative diseases and cancer. Clarification of the molecular mechanisms behind these processes is key to understanding autophagy and recent studies have made major progress in this regard, especially for the mechanisms of initiation and membrane elongation in macroautophagy and substrate recognition in microautophagy and CMA. Furthermore, it is becoming evident that the three categories of autophagy are related to each other despite their implementation by different sets of proteins and the involvement of completely different membrane dynamics. In this review, recent progress in macroautophagy, microautophagy, and CMA are summarized.

Integrin ß5 subunit regulates hyperglycemia-induced vascular endothelial cell apoptosis through FoxO1-mediated macroautophagy.

BACKGROUND: Hyperglycemia frequently induces apoptosis in endothelial cells and ultimately contributes to microvascular dysfunction in patients with diabetes mellitus (DM). Previous research reported that the expression of integrins as well as their ligands was elevated in the diseased vessels of DM patients. However, the association between integrins and hyperglycemia-induced cell death is still unclear. This research was designed to investigate the role played by integrin subunit ß5 (ITGB5) in hyperglycemia-induced endothelial cell apoptosis. METHODS: We used leptin receptor knockout (Lepr-KO) ( db / db ) mice as spontaneous diabetes animal model. Selective deletion of ITGB5 in endothelial cell was achieved by injecting vascular targeted adeno-associated virus via tail vein. Besides, we also applied small interfering RNA in vitro to study the mechanism of ITGB5 in regulating high glucose-induced cell apoptosis. RESULTS: ITGB5 and its ligand, fibronectin, were both upregulated after exposure to high glucose in vivo and in vitro . ITGB5 knockdown alleviated hyperglycemia-induced vascular endothelial cell apoptosis and microvascular rarefaction in vivo.In vitro analysis revealed that knockdown of either ITGB5 or fibronectin ameliorated high glucose-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). In addition, knockdown of ITGB5 inhibited fibronectin-induced HUVEC apoptosis, which indicated that the fibronectin-ITGB5 interaction participated in high glucose-induced endothelial cell apoptosis. By using RNA-sequencing technology and bioinformatic analysis, we identified Forkhead Box Protein O1 (FoxO1) as an important downstream target regulated by ITGB5. Moreover, we demonstrated that the excessive macroautophagy induced by high glucose can contribute to HUVEC apoptosis, which was regulated by the ITGB5-FoxO1 axis. CONCLUSION: The study revealed that high glucose-induced endothelial cell apoptosis was positively regulated by ITGB5, which suggested that ITGB5 could potentially be used to predict and treat DM-related vascular complications.