In vitro chondrotoxicity of bupivacaine, levobupivacaine and ropivacaine and their effects on caspase activity in cultured canine articular chondrocytes.

Bupivacaine, levobupivacaine and ropivacaine are potent, long acting, amide-type local anesthetics that have several clinical applications including intra-articular administration. The objectives of this study were to evaluate their in vitro effects on cell viability and caspase activity to elucidate whether they activate the extrinsic or intrinsic pathways of apoptosis in canine articular chondrocytes. Chondrocytes in monolayer culture were treated with culture medium as the control, or with 0.062% (0.62 mg/mL) bupivacaine, 0.062% levobupivacaine, and 0.062% ropivacaine for 24 hr. Cell viability was evaluated using the live/dead, 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), and Cell Counting Kit-8 (CCK-8) assays. Evaluation of caspase-3, caspase-8, and caspase-9 activity was performed using colorimetric assays. The MTT and CCK-8 assays were used to evaluate the effect of caspase inhibitors on local anesthetic chondrotoxicity. All three local anesthetics decreased chondrocyte viability after 24 hr (P<0.001). Apoptosis was induced through both the extrinsic and intrinsic pathways. Bupivacaine increased caspase-3, caspase-8, and caspase-9 activity (P<0.001). Levobupivacaine increased caspase-3 (P=0.03) while ropivacaine did not significantly upregulate activity for all three caspases. Caspase inhibition did not suppress bupivacaine chondrotoxicity whereas inhibition of caspase-8 and caspase-9 decreased ropivacaine chondrotoxicity and mildly attenuated levobupivacaine chondrotoxicity. In summary, the level of chondrotoxicity, the type of caspase activated, the level of caspase activation, and the response to caspase inhibitors was dependent on the type of local anesthetic. Therefore, ropivacaine may be a safer choice for intra-articular administration compared to levobupivacaine and bupivacaine.

An IBD-associated pathobiont synergises with NSAID to promote colitis which is blocked by NLRP3 inflammasome and Caspase-8 inhibitors.

Conflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesized that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli (AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10-/- mice, we found that NSAID aggravated colitis in AIEC-colonized animals. This was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers, activated T-cells and macrophages, improved histology, and increased abundance of Clostridium cluster XIVa species. Our findings provide new insights into how NSAIDs and an opportunistic gut-pathobiont can synergize to worsen IBD symptoms. Targeting the NLRP3 inflammasome or Caspase-8 could be a potential therapeutic strategy in IBD patients with gut inflammation, which is worsened by NSAIDs.

VX-765 attenuates silica-induced lung inflammatory injury and fibrosis by modulating alveolar macrophages pyroptosis in mice.

Silicosis is a diffuse fibrotic lung disease in which excessive inflammatory responses are triggered by silica exposure. Pyroptosis, a pro-inflammatory mode of programmed cell death, is mediated by gasdermin and may play a pivotal role in the development of silicosis. The caspase-1 inhibitor, VX-765, was used in vivo and in vitro to investigate the effects of silica-induced early inflammatory injury and later lung fibrosis. Our findings show that VX-765 reduces inflammatory lung injury by inhibiting silica-induced pyroptosis of alveolar macrophages in a silicosis mouse model. VX-765 limits the infiltration of inflammatory M1 alveolar macrophages, decreasing expression of inflammatory cytokines, including IL-1ß, TNF-α, IL-6, CCL2, and CCL3, and down-regulating endogenous DAMPs and inflammatory immune-related cell pattern recognition receptors TLR4 and NLRP3. Furthermore, VX-765 alleviates fibrosis by down-regulating α-smooth muscle actin (α-SMA), collagen, and fibronectin. In this study, we illustrate that Alveolar macrophages pyroptosis occur in the early stages of silicosis, and VX-765 can alleviate the development of silicosis by inhibiting the pyroptosis signaling pathway. These results may provide new insight into the prevention and treatment of early-stage silicosis.

Natural killer cells induce HIV-1 latency reversal after treatment with pan-caspase inhibitors.

The establishment of a latency reservoir is the major obstacle for a cure of HIV-1. The shock-and-kill strategy aims to reactivate HIV-1 replication in HIV -1 latently infected cells, exposing the HIV-1-infected cells to cytotoxic lymphocytes. However, none of the latency reversal agents (LRAs) tested so far have shown the desired effect in people living with HIV-1. We observed that NK cells stimulated with a pan-caspase inhibitor induced latency reversal in co-cultures with HIV-1 latently infected cells. Synergy in HIV-1 reactivation was observed with LRAs prostratin and JQ1. The supernatants of the pan-caspase inhibitor-treated NK cells activated the HIV-1 LTR promoter, indicating that a secreted factor by NK cells was responsible for the HIV-1 reactivation. Assessing changes in the secreted cytokine profile of pan-caspase inhibitor-treated NK cells revealed increased levels of the HIV-1 suppressor chemokines MIP1α (CCL3), MIP1ß (CCL4) and RANTES (CCL5). However, these cytokines individually or together did not induce LTR promoter activation, suggesting that CCL3-5 were not responsible for the observed HIV-1 reactivation. The cytokine profile did indicate that pan-caspase inhibitors induce NK cell activation. Altogether, our approach might be-in combination with other shock-and-kill strategies or LRAs-a strategy for reducing viral latency reservoirs and a step forward towards eradication of functionally active HIV-1 in infected individuals.

Caspase-8 is involved in pyroptosis, necroptosis and the maturation and release of IL-1ß in Aspergillus fumigatus keratitis.

PURPOSE: To explore the role of caspase-8 in mediating the transition between different death modes in fungal keratitis. METHODS: The expression of caspase-8 in Aspergillus fumigatus (A. fumigatus) keratitis was detected using western blotting and immunofluorescence. After subconjunctival injection of Z-IETD-FMK (caspase-8 inhibitor) or VX765 (caspase-1 inhibitor), the mice corneas of A. fumigatus keratitis were observed and scored under a slit lamp. Colony plate count, immunofluorescence staining, western blotting and qRT-PCR experiments were used to detect fungal load, inflammatory cells, and the production of related mRNAs and proteins. In vitro experiments, the LDH release test, Cell Count Kit-8(CCK-8) assay, ELISA, qRT-PCR and western blotting were used to detect cell viability, related mRNAs and proteins. RESULTS: The caspase-8 protein was upregulated following fungal infection. Compared with the A. fumigatus keratitis group, the mice treated with Z-IETD-FMK had heavier corneal turbidity, higher clinical scores, more fungal load and fewer inflammatory cells. The expression of NLRP3, cleaved-caspase-1, N-GSDMD, and IL-1ß in the fungal infection group after Z-IETD-FMK pretreatment were downregulated, while RIPK3 and p-MLKL were upregulated. In the fungal infection group after VX765 pretreatment, the expression of cleaved-caspase-8 was up-regulated, while N-GSDMD was downregulated. CONCLUSIONS: Caspase-8 is involved in the early immune defense response of A. fumigatus keratitis. It is essential for the recruitment of inflammatory cells and the clearance of the fungus. In A. fumigatus keratitis, activated caspase-8 promoted the caspase-1/GSDMD signaling pathway to participate in pyroptosis, inhibited RIPK3/MLKL signaling pathway-mediated necroptosis, and promoted IL-1ß maturation and release by activating the NLRP3 inflammasomes.

Isorhamnetin Suppresses Human Gastric Cancer Cell Proliferation through Mitochondria-Dependent Apoptosis.

Derivates of natural products have been wildly utilized in the treatment of malignant tumors. Isorhamnetin (ISO), a most important active ingredient derived from flavonoids, shows great potential in tumor therapy. However, the therapeutic effects of ISO on gastric cancer (GC) remain unclear. Here, we demonstrate that ISO treatment dramatically inhibited the proliferation of two types of GC cells (AGS-1 and HGC-27) both in vitro and in vivo in time- and dose-dependent manners. These results are consistent with the transcriptomic analysis of ISO-treated GC cells, which yielded hundreds of differentially expressed genes that were enriched with cell growth and apoptosis. Mechanically, ISO treatment initiated the activation of caspase-3 cascade and elevated the expression of mitochondria-associated Bax/Bcl-2, cytosolic cytochrome c, followed by the activation of the cleavage of caspase-3 as well as poly ADP-ribose polymerase (PARP), resulting in the severe reduction of the mitochondrial potential and the accumulation of reactive oxygen species (ROS), while pre-treatment of the caspase-3 inhibitor could block the anti-tumor effect. Therefore, these results indicate that ISO treatment induces the apoptosis of GC cells through the mitochondria-dependent apoptotic pathway, providing a potential strategy for clinical GC therapy.

The Anti-Apoptotic Effects of Caspase Inhibitors in Propyl Gallate-Treated Lung Cancer Cells Are Related to Changes in Reactive Oxygen Species and Glutathione Levels.

Propyl gallate [3,4,5-trihydroxybenzoic acid propyl ester; PG] exhibits an anti-growth effect in various cells. In this study, the anti-apoptotic effects of various caspase inhibitors were evaluated in PG-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. Treatment with 800 µM PG inhibited the proliferation and induced the cell death of both Calu-6 and A549 cells at 24 h. Each inhibitor of pan-caspase, caspase-3, caspase-8, and caspase-9 reduced the number of dead and sub-G1 cells in both PG-treated cells at 24 h. PG increased ROS levels, including O2∙-, in both lung cancer cell lines at 24 h. Generally, caspase inhibitors appeared to decrease ROS levels in PG-treated lung cancer cells at 24 h and somewhat reduced O2∙- levels. PG augmented the number of GSH-depleted Calu-6 and A549 cells at 24 h. Caspase inhibitors did not affect the level of GSH depletion in PG-treated A549 cells but differently and partially altered the depletion level in PG-treated Calu-6 cells. In conclusion, PG exhibits an anti-proliferative effect in Calu-6 and A549 lung cancer cells and induced their cell death. PG-induced lung cancer death was accompanied by increases in ROS levels and GSH depletion. Therefore, the anti-apoptotic effects of caspase inhibitors were, at least in part, related to changes in ROS and GSH levels.

Characterization of caspase-2 inhibitors based on specific sites of caspase-2-mediated proteolysis.

Since the discovery of the caspase-2 (Casp2)-mediated ∆tau314 cleavage product and its associated impact on tauopathies such as Alzheimer's disease, the design of selective Casp2 inhibitors has become a focus in medicinal chemistry research. In the search for new lead structures with respect to Casp2 selectivity and drug-likeness, we have taken an approach by looking more closely at the specific sites of Casp2-mediated proteolysis. Using seven selected protein cleavage sequences, we synthesized a peptide series of 53 novel molecules and studied them using in vitro pharmacology, molecular modeling, and crystallography. Regarding Casp2 selectivity, AcITV(Dab)D-CHO (23) and AcITV(Dap)D-CHO (26) demonstrated the best selectivity (1-6-fold), although these trends were only moderate. However, some analogous tetrapeptides, most notably AcDKVD-CHO (45), showed significantly increased Casp3 selectivities (>100-fold). Tetra- and tripeptides display decreased or no Casp2 affinity, supporting the assumption that a motif of five amino acids is required for efficient Casp2 inhibition. Overall, the results provide a reasonable basis for the development of both selective Casp2 and Casp3 inhibitors.

Caspase-mediated regulation of the distinct signaling pathways and mechanisms in neuronal survival.

Caspases are intimately associated with altering various signaling pathways, resulting in programmed cell death or apoptosis. Apoptosis is necessary for the normal homeostasis of cells and their development. The untoward activation of apoptotic pathways indirectly or directly results in pathologies of various diseases. Identifying different caspases in apoptotic pathways directed the research to develop caspase inhibitors as therapeutic agents. However, no drug is available in the market that targets caspase inhibition and produces a therapeutic effect. Here, we will shed light on the role of caspases in the number of neuronal disorders and neurodegenerative diseases. The article reviews the findings about the activation of various upstream mechanisms associated with caspases in neurodegenerative disorders along with the recent progress in the generation of caspase inhibitors and the challenge faced in their development as therapeutic agents for neurological indications.

Novel role for caspase 1 inhibitor VX765 in suppressing NLRP3 inflammasome assembly and atherosclerosis via promoting mitophagy and efferocytosis.

Atherosclerosis is a maladaptive chronic inflammatory disease, which remains the leading cause of death worldwide. The NLRP3 inflammasome constitutes a major driver of atherosclerosis, yet the mechanism of action is poorly understood. Mitochondrial dysfunction is essential for NLRP3 inflammasome activation. However, whether activated NLRP3 inflammasome exacerbates mitochondrial dysfunction remains to be further elucidated. Herein, we sought to address these issues applying VX765, a well-established inhibitor of caspase 1. VX765 robustly restrains caspase 1-mediated interleukin-1ß production and gasdermin D processing. Our study assigned VX765 a novel role in antagonizing NLRP3 inflammasome assembly and activation. VX765 mitigates mitochondrial damage induced by activated NLRP3 inflammasome, as evidenced by decreased mitochondrial ROS production and cytosolic release of mitochondrial DNA. VX765 blunts caspase 1-dependent cleavage and promotes mitochondrial recruitment and phosphorylation of Parkin, a key mitophagy regulator. Functionally, VX765 facilitates mitophagy, efferocytosis and M2 polarization of macrophages. It also impedes foam cell formation, migration and pyroptosis of macrophages. VX765 boosts autophagy, promotes efferocytosis, and alleviates vascular inflammation and atherosclerosis in both ApoE-/- and Ldlr-/- mice. However, these effects of VX765 were abrogated upon ablation of Nlrp3 in ApoE-/- mice. This work provides mechanistic insights into NLRP3 inflammasome assembly and this inflammasome in dictating atherosclerosis. This study highlights that manipulation of caspase 1 paves a new avenue to treatment of atherosclerotic cardiovascular disease.

A Fluorescence-Polarization-Based Lipopolysaccharide-Caspase-4 Interaction Assay for the Development of Inhibitors.

Recognition of intracellular lipopolysaccharide (LPS) by Caspase-4 (Casp-4) is critical for host defense against Gram-negative pathogens. LPS binds to the N-terminal caspase activation and recruitment domain (CARD) of procaspase-4, leading to auto-proteolytic activation followed by pro-inflammatory cytokine release and pyroptotic cell death. Aberrant hyper-activation of Casp-4 leads to amplification of the inflammatory response linked to sepsis. While the active site of a caspase has been targeted with peptide inhibitors, inhibition of LPS-Casp-4 interaction is an emerging strategy for the development of selective inhibitors with a new mode of action for treating infectious diseases and sepsis induced by LPS. In this study, a high-throughput screening (HTS) system based on fluorescence polarization (FP) was devised to identify inhibitors of the LPS and Casp-4 interaction. Using HTS and IC50 determination and subsequently showing inhibited Casp-4 activity, we demonstrated that the LPS-Casp-4 interaction is a druggable target for Casp-4 inhibition and possibly a non-canonical inflammatory pathway.

Caspase Inhibition Restores NEP Expression and Rescues Olfactory Deficit in Rats Caused by Prenatal Hypoxia.

Development of the olfactory system begins early in embryogenesis and is important for the survival of new-borns in postnatal life. Olfactory malfunction in early life disrupts development of behavioural patterns while with ageing manifests development of neurodegenerative disorders. Previously, we have shown that prenatal hypoxia in rats leads to impaired olfaction in the offspring and correlates with reduced expression of a neuropeptidase neprilysin (NEP) in the brain structures involved in processing of the olfactory stimuli. Prenatal hypoxia also resulted in an increased activity of caspases in rat brain and its inhibition restored NEP content in the brain tissue and improved rat memory. In this study, we have analysed effects of intraventricular administration of a caspase inhibitor Ac-DEVD-CHO on NEP mRNA expression, the number of dendritic spines and olfactory function of rats subjected to prenatal hypoxia on E14. The data obtained demonstrated that a single injection of the inhibitor on P20 restored NEP mRNA levels and number of dendritic spines in the entorhinal and parietal cortices, hippocampus and rescued rat olfactory function in food search and odour preference tests. The data obtained suggest that caspase activation caused by prenatal hypoxia contributes to the olfactory dysfunction in developing animals and that caspase inhibition restores the olfactory deficit via upregulating NEP expression and neuronal networking. Because NEP is a major amyloid-degrading enzyme, any decrease in its expression and activity not only impairs brain functions but also predisposes to accumulation of the amyloid-ß peptide and development of neurodegeneration characteristic of Alzheimer's disease.

Reprogramming monocyte-derived macrophages through caspase inhibition.

Macrophages are widely distributed innate immune cells that play an indispensable role in a variety of physiologic and pathologic processes, including organ development, host defense, acute and chronic inflammation, solid and hematopoietic cancers. Beyond their inextricable role as conveyors of programmed cell death, we have previously highlighted that caspases exert non-apoptotic functions, especially during the differentiation of monocyte-derived cells in response to CSF-1. Here, we found that non-canonic cleavages of caspases, reflecting their activation, are maintained during IL-4-induced monocyte-derived macrophages polarization. Moreover, Emricasan, a pan-caspase inhibitor that demonstrated promising preclinical activity in various diseases and safely entered clinical testing for the treatment of liver failure, prevents the generation and the anti-inflammatory polarization of monocyte-derived macrophages ex vivo. Interestingly, caspase inhibition also triggered the reprogramming of monocyte-derived cells evidenced by RNA sequencing. Taken together, our findings position Emricasan as a potential alternative to current therapies for reprogramming macrophages in diseases driven by monocyte-derived macrophages.

Mechanism of Caspase-1 Inhibition by Four Anti-inflammatory Drugs Used in COVID-19 Treatment.

The inflammatory protease caspase-1 is associated with the release of cytokines. An excessive number of cytokines (a "cytokine storm") is a dangerous consequence of COVID-19 infection and has been indicated as being among the causes of death by COVID-19. The anti-inflammatory drug colchicine (which is reported in the literature to be a caspase-1 inhibitor) and the corticosteroid drugs, dexamethasone and methylprednisolone, are among the most effective active compounds for COVID-19 treatment. The SERM raloxifene has also been used as a repurposed drug in COVID-19 therapy. In this study, inhibition of caspase-1 by these four compounds was analyzed using computational methods. Our aim was to see if the inhibition of caspase-1, an important biomolecule in the inflammatory response that triggers cytokine release, could shed light on how these drugs help to alleviate excessive cytokine production. We also measured the antioxidant activities of dexamethasone and colchicine when scavenging the superoxide radical using cyclic voltammetry methods. The experimental findings are associated with caspase-1 active site affinity towards these compounds. In evaluating our computational and experimental results, we here formulate a mechanism for caspase-1 inhibition by these drugs, which involves the active site amino acid Cys285 residue and is mediated by a transfer of protons, involving His237 and Ser339. It is proposed that the molecular moiety targeted by all of these drugs is a carbonyl group which establishes a S(Cys285)-C(carbonyl) covalent bond.

Computational research of Belnacasan and new Caspase-1 inhibitor on cerebral ischemia reperfusion injury.

Cerebral ischemia-reperfusion injury is one of the most severe diseases in terms of mortality and disability, which seriously threatens human life and health. In clinical treatment, drug thrombolysis or mechanical interventional thrombolysis are used to quickly restore the blood supply of ischemic brain tissue. But with the rapid recovery of blood flow, complex pathophysiological processes such as oxidative stress and inflammation will further aggravate brain tissue damage, namely cerebral ischemia-reperfusion injury, for which there is no effective treatment. Recent studies have shown that the medical community has paid the role of inflammation and pyroptosis in cerebral ischemia-reperfusion injury more and more attention. And Caspase-1 was found to play a vital role in regulating inflammation pathways and pyroptosis in many inflammation-associated diseases, especially in cerebral ischemia-reperfusion injury. Not only that, Caspase-1 inhibitors have been shown to reduce the damage of cerebral ischemia-reperfusion injury by inhibiting inflammation and pyroptosis. And the Caspase-1 inhibitor, Belnacasan, has been proved to modify the active site of Caspase-1 and lead to the blocking of Caspase-1, thus correlating with tissue protection of inflammatory diseases in animal models. Therefore, it's essential to screen and design potential Caspase-1 inhibitors to reduce cerebral ischemia-reperfusion injury and protect brain function by reducing inflammation and pyroptosis, which provides a new idea for clinical treatment of the cerebral ischemia-reperfusion injury. This study applied a group of computer-aided technology, such as Discovery Studio 4.5, Schrodinger, and PyMol, to screen and assess potential Caspase-1 inhibitors. Moreover, the ADME (absorption, distribution, metabolism, excretion) and TOPKAT (Toxicity Prediction by Computer Assisted Technology) molecules of Discovery Studio 4.5 were conducted to evaluate molecules' pharmacological and toxicological features. Then, precise molecular docking was applied to assess the binding mechanism and affinity between Caspase-1 and selected compounds. Besides, molecular dynamics simulations were performed to determine the stability of ligand-receptor complexes in the natural environment. In summary, this study lists promising drug candidates and their pharmacological properties, promoting the development of Caspase-1 inhibitors and deepening the understanding of the interaction between inhibitors and Caspase-1.

Caspase-9 inhibition decreases expression of Mmp9 during chondrogenesis.

Besides cell death, caspase-9 participates in non-apoptotic events, including cell differentiation. To evaluate a possible impact on the expression of chondrogenic/osteogenic factors, a caspase-9 inhibitor was tested in vitro. For this purpose, mouse forelimb-derived micromass cultures, the most common chondrogenic in vitro model, were used. The following analyses were performed based on polymerase chain reaction (PCR) arrays and real-time PCR. The expression of several chondrogenesis-related genes was shown to be altered, some of which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodelling of the extracellular matrix (Col1a2, Mmp9) related to endochondral ossification. From the cluster of genes with altered expression, Mmp9 showed the most significant decrease in expression, of more than 50-fold. Additionally, we determined the possible impact of caspase-9 downregulation on the expression of other Mmp genes. A mild increase in Mmp14 was observed, but there was no change in the expression of other studied Mmp genes (-2, -3, -8, -10, -12, -13). Interestingly, inhibition of Mmp9 in micromasses led to decreased expression of some chondrogenic markers related to caspase-9. These samples also showed a decreased expression of caspase-9 itself, suggesting a bidirectional regulation of these two enzymes. These results indicate a specific impact of caspase-9 inhibition on the expression of Mmp9. The localisation of these two enzymes overlaps in resting, proliferative and pre-hypertrophic chondrocytes during in vivo development, which supports their multiple functions, either apoptotic or non-apoptotic. Notably, a coincidental expression pattern was identified in Pik3cg, a possible candidate for Mmp9 regulation.

Cholinergic anti-inflammatory pathway ameliorates murine experimental Th2-type colitis by suppressing the migration of plasmacytoid dendritic cells.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease. Several studies have demonstrated that α7 nicotinic acetylcholine receptors (α7nAChRs) exert anti-inflammatory effects on immune cells and nicotine suppress UC onset and relapse. Plasmacytoid dendritic cells (pDCs) reportedly accumulate in the colon of UC patients. Therefore, we investigated the pathophysiological roles of α7nAChRs on pDCs in the pathology of UC using oxazolone (OXZ)-induced Th2-type colitis with BALB/c mice. 2-deoxy-D-glucose, a central vagal stimulant suppressed OXZ colitis, and nicotine also ameliorated OXZ colitis with suppressing Th2 cytokines, which was reversed by α7nAChR antagonist methyllycaconitine. Additionally, α7nAChRs were expressed on pDCs, which were located very close to cholinergic nerve fibers in the colon of OXZ mice. Furthermore, nicotine suppressed CCL21-induced bone marrow-derived pDC migration due to Rac 1 inactivation, which was reversed by methyllycaconitine, a JAK2 inhibitor AG490 or caspase-3 inhibitor AZ-10417808. CCL21 was mainly expressed in the isolated lymphoid follicles (ILFs) of the colon during OXZ colitis. The therapeutic effect of cholinergic pathway on OXZ colitis probably through α7nAChRs on pDCs were attributed to the suppression of pDC migration toward the ILFs. Therefore, the activation of α7nAChRs has innovative therapeutic potential for the treatment of UC.

Arsenic trioxide activates yes-associated protein by lysophosphatidic acid metabolism to selectively induce apoptosis of vascular smooth muscle cells.

Inhibition of vascular smooth muscle cells (VSMCs) proliferation without dysregulating endothelial cells (ECs) may provide an ideal therapy for in-stent restenosis. Due to its anti-proliferation effect on VSMCs and pro-endothelium effect, arsenic trioxide (ATO) has been used in a drug-eluting stent in a recent clinical trial. However, the underlying mechanism by which ATO achieves this effect has not been determined. In the present work, we showed that ATO induced apoptosis in VSMCs but not in ECs. Mechanistically, ATO achieved this through modulation of cellular metabolism to increase lysophosphatidic acid (LPA) in VSMCs, while LPA concentration was stable in ECs. The elevated LPA facilitated the nuclear accumulation and initiated the transcriptional function of Yes-associated protein (YAP) in VSMCs. YAP regulated the transcription of N6-Methyladenosine (m6A) modulators (Mettl14 and Wtap) to increase the m6A methylation levels of apoptosis-related genes to induce their high expression and exacerbate VSMCs apoptosis. On the other hand, YAP nuclear accumulation in ECs was not observed. Collectively, our data exhibited the molecular process involved in selective apoptosis of VSMCs induced by ATO.

Cellular autophagy, an unbidden effect of caspase inhibition by zVAD-fmk.

zVAD-fmk is a widely used pan-caspase inhibitor that blocks apoptosis but has undesirable side effects, including autophagy. In this issue, Needs et al. propose that zVAD-fmk induces autophagy by inhibiting the N-glycanase NGLY1 rather than caspases. NGLY1 is essential for the ERAD response and patients with inactivating mutations in NGLY1 present with neurodevelopmental defects and organ dysfunction. The ability of NGLY1 to inhibit basal levels of autophagy may contribute to this pathology. This study demonstrates possible crosstalk between protein turnover and autophagy while also underscoring the importance of specificity when using chemical tools to interrogate these pathways. Comment on https://doi.org/10.1111/febs.16345.