The review draws a sharp line between two phenomena that have often been lumped together under the vague heading of lysosomal damage. The first, lysosomal membrane permeabilization, or LMP, was defined between 1972 and 1999 through cytochemical experiments by Brunk and colleagues using cultured cells exposed to various stresses. In LMP, the lysosomal limiting membrane remains ultrastructurally intact when viewed under the electron microscope, yet tiny pores—far too small to be resolved—allow a gradual and selective leakage of lysosomal contents into the cytoplasm. The second phenomenon, lysosomal membrane rupture, or LMR, was first identified in 1996 by Yamashima and coworkers using electron microscopic analysis of hippocampal CA1 neurons in Japanese macaque monkeys after transient global brain ischemia. In LMR, the limiting membrane shows an apparent, visible disruption: larger holes that permit an acute and massive outpouring of hydrolytic cathepsin enzymes, leading to the rapid, almost explosive vanishing of the lysosomes themselves.

The distinction matters because the two modes of membrane failure appear to steer cells toward entirely different fates. According to the review, LMP acts as an amplifier of cell death signaling and participates in apoptosis, the orderly, programmed form of cell suicide. The selective release of lysosomal enzymes through submicroscopic pores triggers the proteolytic activation of caspases, the executioner enzymes of apoptosis. LMR, by contrast, unleashes a catastrophic flood of cathepsins that drives necrosis, the messy, inflammatory form of cell death in which cells essentially dissolve. Yamashima proposes that the calpain-cathepsin cascade—a sequence in which the calcium-activated protease calpain and the lysosomal cathepsins work in concert—executes necrotic death, whereas caspase activation through LMP executes apoptosis. Which of the two outcomes prevails depends on the size and number of membrane perforations, the amount of enzyme leakage, and the cellular context.

The historical thread runs back to 1966, when de Duve and Wattiaux first proposed that cell degeneration and necrosis occur through the leakage of hydrolytic enzymes from damaged lysosomes. In 1972, Brunk and Ericsson observed an extensive diffusion of the lysosomal enzyme acid phosphatase through membranes that looked structurally intact, although the improper fixation used in those experiments may itself have created artificial pores. It was not until 1999 that Brunk and Svensson, working with human Jurkat leukemia cells exposed to oxidative stress, confirmed LMP directly by tracking the leakage of the lysosome-accumulating dye acridine orange. Because the electron microscope showed nothing wrong with the membrane, the researchers concluded that pores too small to image must be responsible. For decades afterward, lysosomes were widely regarded as sturdy organelles whose membranes might become leaky but would not visibly rupture until the cell was already dead—a view that caused interest in lysosomal cell death to fade.

That view changed with the 1996 primate study. In hippocampal CA1 neurons three to five days after transient ischemia—neurons that were degenerating but still alive—Yamashima’s group captured electron micrographs showing lysosomes with unmistakable holes in their limiting membranes, spilling their contents before necrosis had run its course. Similar ruptures were later documented in cultured macrophages and in mouse alveolar macrophages exposed to nanomaterials in 2012 and 2016. In 2016, Li and colleagues reported that loss of the scav-3 gene in the roundworm Caenorhabditis elegans—the worm counterpart of the human lysosomal membrane protein LIMP-2, a key guardian of membrane integrity—also produced LMR. Yamashima’s team subsequently observed comparable membrane disruptions, often appearing as double contours or irregular lysosomal shapes near autophagolysosomes and damaged mitochondria, in degenerating cortical neurons from patients with Alzheimer’s disease.

Recent live-cell imaging has brought the two phenomena into even sharper contrast. In experiments on a human hepatocellular carcinoma cell line, HepG2, the researchers compared the effects of epirubicin, a chemotherapeutic agent, with those of 4-hydroxy-2-nonenal, or 4-HNE, a lipid peroxidation product. Time-lapse imaging with LysoTracker, a fluorescent dye retained in acidic lysosomes, showed that epirubicin caused cell shrinkage and membrane blebbing while most lysosomes remained visible until apoptosis was complete—consistent with gradual, selective leakage through invisible pores. The 4-HNE treatment, however, produced bursting cell death in which every lysosome vanished before the cytoplasm and nuclear chromatin disintegrated, reflecting rapid, massive leakage through ruptured membranes. Notably, the intensity of lysosomal staining proved to be a poor proxy for the degree of membrane damage, and lysosomes could still be detected after apoptosis but not after necrosis had been executed.

Why are lysosomes so vulnerable in the first place? Unlike mitochondria, they lack antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. They are also rich in iron released from degraded iron-containing proteins. When reactive oxygen species flood the cell, ferrous iron inside the lysosome reacts with hydrogen peroxide in the Fenton reaction, generating hydroxyl radicals that attack the membrane’s lipids. Experiments with silica particles illustrate the point: macrophages that swallowed iron-laden particles suffered extensive lysosomal damage, while those given particles pretreated with an iron chelator showed only modest leakage. The review identifies 4-HNE as a central culprit. Generated both from dietary linoleic acid in vegetable oils and from cardiolipin in the inner mitochondrial membrane, 4-HNE forms adducts with the amino acids cysteine, histidine, lysine and arginine in proteins, including the heat-shock protein Hsp70.1, which normally stabilizes the lysosomal membrane.

The molecular story that emerges is intricate. Hsp70.1 performs a dual role: it ferries damaged proteins to lysosomes for degradation via chaperone-mediated autophagy, and it preserves membrane integrity by promoting the interaction between the lipid BMP and acid sphingomyelinase, an enzyme whose product, ceramide, reinforces the membrane against rupture. In the ischemic monkey hippocampus, two-dimensional oxyblot analysis revealed a three- to nine-fold upregulation of Hsp70.1 together with a drop in its molecular weight, indicating that oxidative carbonylation had struck the arginine at position 469 of the protein during reperfusion. Once carbonylated, Hsp70.1 becomes susceptible to cleavage by the calcium-activated protease μ-calpain. In vitro experiments showed that calpain alone could not cut intact Hsp70.1, but cleavage proceeded in a time-dependent manner once 4-HNE had modified the protein. In living monkeys injected with 4-HNE over 24 weeks, Western blotting of liver tissue confirmed the same calpain-mediated cleavage, and immunofluorescence revealed extensive overlap of Hsp70.1 and activated calpain in hepatocytes and CD68-positive Kupffer cells, whose lysosomes largely faded away through rupture.

The review also reaches beyond apoptosis and necrosis. Pyroptosis, a highly inflammatory, lytic form of programmed cell death first named in 2001, defends the body against intracellular pathogens by rupturing the plasma membrane and releasing pro-inflammatory cytokines. Ferroptosis, described in 2012, is an iron-dependent death driven by lipid peroxidation of membranes. Because the acidic lysosomal lumen, packed with reactive iron and hydrogen peroxide, is an ideal chemical reactor for generating reactive oxygen species, recent work by Saimoto and colleagues suggests that lipid peroxidation at the lysosomal membrane triggers LMP during ferroptosis, with intralysosomal iron leakage then fanning cell-wide lipid peroxidation. Yamashima argues that the overlap runs deeper: given the shared involvement of the Fenton reaction, lipid peroxidation, calcium signaling and lysosomal membrane failure, ferroptosis may well employ the same calpain-cathepsin cascade that drives necrosis.

The practical upshot is methodological as much as conceptual. Because LMP involves pores that electron microscopy cannot resolve, it must be detected cytochemically—for instance with the lysosomal galectin puncta assay, which registers the translocation of galectins 1 and 3 to damaged membranes earlier than cathepsin-release measurements can. LMR, by contrast, is fleeting: ruptures occur within minutes or hours and the lysosomes then disappear, so researchers have only a narrow window to catch them in degenerating tissue. The review’s summary point is unambiguous: distinguishing LMP from LMR by careful electron microscopy is indispensable for elucidating the mechanisms of cell death in organs stressed by ischemia, toxins or disease. If the calpain-cathepsin hypothesis holds across neurons, hepatocytes and immune cells alike, then protecting the lysosomal membrane—and shielding Hsp70.1 from carbonylation—may become a unified therapeutic strategy against some of medicine’s most stubborn degenerative conditions.

Subject of Research: Distinct roles of lysosomal membrane permeabilization and rupture in determining apoptotic versus necrotic cell death

Article Title: Lysosomal membrane-permeabilization (LMP) and -rupture (LMR) are distinct for cell death

Article References: Yamashima, T. (2025). Lysosomal membrane-permeabilization (LMP) and -rupture (LMR) are distinct for cell death. Frontiers in Cell Death, 4, Article 1669955. https://doi.org/10.3389/fceld.2025.1669955

Image Credits: AI Generated

DOI: 10.3389/fceld.2025.1669955

Keywords: lysosome, lysosomal membrane permeabilization, lysosomal membrane rupture, apoptosis, necrosis, cathepsins, calpain, Hsp70.1, 4-hydroxy-2-nonenal, ferroptosis, pyroptosis, cell death

The system in question is autophagy, the cellular recycling program that engulfs damaged proteins and organelles in double-membraned vesicles and delivers them to lysosomes for degradation. Within mitochondria, a specialized form of this process, known as mitophagy, selectively removes dysfunctional mitochondria before they leak reactive oxygen species and trigger cell death. When autophagic flux runs smoothly, the endothelium stays clean and energetic. When it stalls, debris accumulates, mitochondria fragment into dysfunctional shards, and the cell’s metabolic machinery begins to misfire. The French-led research team, working across institutions in Paris and Angers with collaborators in Japan, set out to determine whether calpains, enzymes best known for degrading structural proteins under stress, might be the hidden hand jamming this recycling machinery in diabetes.

The investigators began with human umbilical vein endothelial cells and primary lung endothelial cells exposed to high glucose concentrations in the laboratory. Under these diabetic-mimicking conditions, they observed a clear activation of calpains, detectable through the characteristic cleavage of their substrates. More importantly, they documented a full-blown autophagic flux blockade. The cargo receptor protein SQSTM1, also known as p62, accumulated to abnormal levels because it was no longer being efficiently cleared, while expression of LC3B, the lipidated protein that decorates autophagosomal membranes, was reduced. In practical terms, the conveyor belt carrying cellular waste to the lysosome had ground to a halt, and the mitochondria within these cells responded by fragmenting, losing the elongated, interconnected network architecture that healthy endothelial cells depend upon for efficient energy production.

To test whether calpains were truly the drivers of this collapse rather than innocent bystanders, the team deployed two complementary strategies. The first was pharmacological: small-molecule inhibitors that block calpain enzymatic activity. The second was genetic: overexpression of calpastatin, the endogenous protein that naturally inhibits calpains and serves as the body’s built-in brake on these proteases. Remarkably, both approaches produced the same result. Blocking calpain activity restored autophagic flux, allowing SQSTM1 to be cleared and LC3B-mediated autophagosome formation to resume, and it preserved the mitochondrial network, preventing the pathological fragmentation that hyperglycemia would otherwise induce. The convergence of drug-based and gene-based evidence strengthens the causal argument considerably, ruling out the possibility that the inhibitor was working through some unrelated off-target pathway.

To understand the broader consequences of calpain inhibition at the level of gene expression, the researchers performed transcriptomic profiling of endothelial cells subjected to high glucose with and without calpain blockade. The results revealed that calpain inhibition counteracted several major hyperglycemia-driven transcriptional programs. Among the most prominent were pathways governing oxidative phosphorylation, the mitochondrial process that generates most of the cell’s ATP; reactive oxygen species signaling, which spirals dangerously upward when mitochondria fail; and the unfolded protein response, a stress program activated when the endoplasmic reticulum is overwhelmed. In other words, restraining calpains did not merely patch one leak in the diabetic endothelium but appeared to rebalance an entire constellation of stress responses that high glucose had pushed out of alignment.

The mechanistic picture that emerges is elegant in its simplicity. Hyperglycemia floods endothelial cells with calcium signals and oxidative stress, which overactivate calpains. These proteases then disrupt the autophagy-mitophagy axis, either by directly degrading components of the autophagic machinery or by damaging the lysosomal system that completes the degradation process. With mitophagy impaired, damaged mitochondria escape removal, fragment, and pour out reactive oxygen species, which in turn inflict further damage on proteins and organelles, creating a self-reinforcing cycle of dysfunction. Calpain inhibition breaks this vicious cycle at its upstream point, restoring the cell’s capacity for self-renewal before the downstream damage becomes irreversible. This positions calpains not as one among many secondary casualties of diabetes but as a genuine regulatory node in the pathogenesis of vascular disease.

Crucially, the team did not confine their work to cell culture dishes. They extended their findings into living animals, using mouse models of diabetes to ask whether calpain inhibition could preserve vascular function in the intact organism. The answer was affirmative: treatment that restrained calpain activity preserved vascular reactivity in diabetic mice, the ability of blood vessels to dilate and constrict appropriately in response to physiological cues. This functional rescue is the metric that matters most for translational relevance, because endothelial dysfunction in patients is measured precisely by the loss of this dynamic responsiveness, whether in the coronary arteries feeding the heart or the glomerular capillaries sustaining the kidneys. The study also made use of mitoQC reporter mice, a sophisticated genetic tool developed in Ian Ganley’s laboratory at the University of Dundee, which allows mitophagy to be visualized directly in tissues, providing in vivo confirmation of the cellular findings.

The implications for diabetic patients are significant but must be weighed with appropriate caution. Calpains have been implicated in a wide range of physiological processes, including platelet activation, muscle remodeling, and neuronal function, which means that systemic calpain inhibition carries potential risks that will need careful evaluation. The study’s use of both pharmacological inhibitors and calpastatin overexpression suggests that even partial or tissue-targeted modulation of calpain activity might be sufficient to confer vascular protection, which could open the door to therapeutic strategies that maximize benefit while limiting collateral effects. It is also worth noting that the work was supported by the Société francophone du diabète, Laboratoire Servier, and the EFSD/Novo Nordisk Programme for Diabetes Research in Europe, reflecting a serious institutional commitment to translating basic endothelial biology into clinical applications.

What makes this research resonate beyond the diabetes field is its contribution to a growing appreciation of autophagy as a master regulator of vascular health. Endothelial cells are among the longest-lived cells in the body, exposed continuously to mechanical forces, metabolic fluctuations, and inflammatory insults over decades. Their survival depends on relentless intracellular housekeeping, and any mechanism that compromises that housekeeping, whether it is a protease gone rogue or a lysosome gone sluggish, accelerates vascular aging. By identifying calpain overactivation as a specific and druggable point of failure in the diabetic endothelium, Salemkour, Lenoir, and their colleagues have added a precise new entry to the molecular map of diabetic vascular disease, and they have demonstrated that the map can be redrawn toward health with the right molecular intervention.

The road from a mouse study to a bedside therapy is long, and the authors themselves frame calpain inhibition as a promising strategy rather than a finished treatment. Nevertheless, the convergence of cellular mechanistics, transcriptomic breadth, and intact-animal functional data makes this one of the more complete preclinical demonstrations in the endothelial autophagy literature. For the hundreds of millions of people worldwide living with diabetes, whose risk of cardiovascular death is roughly doubled compared with the general population, the finding that a single enzymatic family stands between high glucose and the collapse of mitochondrial quality control offers something genuinely new: a target that, if safely inhibited, could preserve the vessels that diabetes slowly destroys. The next chapter will be written in the clinic, but the molecular groundwork has now been firmly laid.

Subject of Research: The role of calpain-mediated autophagy and mitophagy blockade in diabetic endothelial dysfunction

Article Title: Calpain inhibition prevents high-glucose-induced autophagy blockade and mitochondrial fragmentation in endothelial cells and preserves vascular functions in diabetic mice

Article References: Salemkour, Y., Dionet, L., Vessieres, E., Saito, R., Le Gall, M., Letavernier, E., Henrion, D., Tharaux, P.-L., & Lenoir, O. (2026). Calpain inhibition prevents high-glucose-induced autophagy blockade and mitochondrial fragmentation in endothelial cells and preserves vascular functions in diabetic mice. Cellular and Molecular Life Sciences. https://doi.org/10.1007/s00018-026-06433-5

Image Credits: AI Generated

DOI: 10.1007/s00018-026-06433-5

Keywords: diabetes, endothelial dysfunction, calpain, autophagy, mitophagy, mitochondria, hyperglycemia, vascular function, calpastatin, oxidative stress, endothelium, diabetic complications