The concomitant prevalence of toxic cyanobacteria blooms and plastic pollution in aquatic ecosystems is emerging as a pressing global water pollution dilemma. While toxic cyanobacteria and microplastics (MPs) can each independently exert significant impacts on aquatic biota, the magnitude and trajectory of the combined interactions remains rudimentary. In this study, we evaluated how MPs influences cyanobacterial stress on keystone grazer Daphnia, focusing on population, individual, biochemical and toxicogenomic signatures. We found that toxic Microcystis (TM) adversely affected the fitness of Daphnia populations (intrinsic rate of population increase), and these adverse effects were amplified in the presence of MPs. Through detailed observation, it was ascertained that MPs promoted the ingestion of TM, culminating in enhanced microcystin bioaccumulation. Using the Eco-Evo model, we found that there was potential absence of correlation between the MPs toxicity and the effect size of MPs on the TM. Utilizing gene set enrichment analysis (GSEA), we further identified a marked suppression of molecular pathways and entities crucial to individual growth and development in the TM-MPs consortium compared to exposure to TM alone. The present study provides important insights about the influence of MPs on cyanobacteria toxicity and the prediction the risk of harmful algal blooms in aquatic ecosystems.

C1ORF106 has previously been associated with inflammatory bowel diseases (IBD) via large-scale genetic studies. Increased intestinal permeability is a hallmark of IBD and is observed in at-risk individuals prior to the appearance of clinical symptoms. C1ORF106 was previously shown to regulate intestinal barrier permeability through the regulation of adherens junction stability and through the formation of tight junctions, which impacted actin assembly. However, the downstream impact and molecular mechanisms involved in actin regulation by C1ORF106 haven't been explored. Our study aimed at identifying which pathways involved in intestinal epithelial barrier regulation and F-actin regulation are impacted by C1ORF106 and its IBD-associated variant.

We knocked down (KD) the expression of C1ORF106 in human colonic epithelial cells and characterized the function of the 333F variant in intestinal epithelial spheroid cultures obtained from patient-derived human induced pluripotent stem cell (hiPSC). We measured barrier permeability and characterized spheroid formation, actin regulation and cell migration though immunofluorescence, western blots and permeability assays.

C1ORF106 KD leads to impaired cortical actin belt dynamics and regulation of stress fiber formation, resulting in increased cell constriction, impaired barrier permeability, cell polarity and cell migration. Moreover, we demonstrated that an inhibition of ROCK rescues the actin belt and cell polarity phenotypes in C1ORF106 KD cells, demonstrating that C1ORF106 regulates these phenotypes through a ROCK-dependent mechanism. We also observed an altered nmMYO2-P localization in C1ORF106 KD cells associated with the formation of Vacuolar Apical Compartments (VACs), which are important for 3D epithelial spheroid formation. We observed a similar impact on cell polarity in intestinal epithelial spheroids obtained from hiPSC carrying the 333F variant, providing additional support that this pathway is involved in disease development.

We provide insights into the molecular mechanisms by which C1ORF106 controls actin dynamics to regulate intestinal epithelial integrity.

The small GTPases RhoA and Cdc42 and their effector proteins play crucial roles in neutrophil chemotaxis. However, endogenous localization and regulation of these proteins have remained largely unknown. Here, we show, using a trichloroacetic acid fixation method, that endogenous RhoA and Cdc42 are preferentially accumulated at the F-actin-rich leading edge (pseudopod) during chemotaxis of human neutrophil-like PLB-985 cells in response to the chemoattractant C5a. Interestingly, the enrichment of RhoA is impaired by knockdown of Cdc42, indicating a positive regulation by Cdc42. Depletion of Cdc42 or RhoA each induces the formation of multiple pseudopods, confirming their significance in cell polarization with an organized actin network at the front. The Rho-associated kinase ROCK is also recruited to the leading edge during chemotaxis in a manner dependent on not only RhoA and Cdc42 but also aPKC, a Cdc42-interacting kinase that can also bind to ROCK. ROCK promotes phosphorylation of the myosin light chain at the front, possibly regulating pseudopod contractility. Knockdown of aPKC suppresses neutrophil chemotaxis by disturbing pseudopod orientation without forming multiple protrusions. An incorrectly oriented pseudopod is also observed in ROCK-depleted cells. Thus, aPKC, as well as RhoA and Cdc42, likely regulates neutrophil chemotaxis partly by recruiting ROCK to the leading edge for correct directionality.

Rho-associated protein kinases (ROCKs) are a key regulator of T cell function, influencing a wide range of processes from activation to differentiation. Experimental autoimmune uveoretinitis (EAU) is an animal model of human non-infectious uveitis. This study aimed to evaluate the suppressive effects of ripasudil, a ROCK inhibitor, on ocular inflammation when administered from the onset of EAU and to elucidate the underlying mechanisms of its inhibitory effects.

EAU was induced in wild-type C57BL/6 mice by immunisation with IRBP peptide. Ripasudil or its vehicle, PBS, was intraperitoneally administered daily starting from 8 days post-immunisation. Clinical and histopathological examinations and analysis of T cell activation state were conducted. In addition, T cell gene expression profiles in the relevant immune functions were identified using single-cell RNA sequencing (scRNA-seq).

The development of EAU was significantly attenuated and T cell activation and Th1 cell differentiation were significantly inhibited in mice with ripasudil (RIP-EAU) compared with mice with PBS (PBS-EAU), scRNA-seq using splenic T cells indicated that genes involved in the ROCK signalling pathway were highly expressed in low-differentiated Th1/Th17 cells, intermediate Th1 cells and differentiated Th1 cells. In addition, although differentiated Th1 and Th17 cells constituted similar proportions between PBS-EAU and RIP-EAU mice, RIP-EAU mice exhibited fewer low-differentiated Th1/Th17 cells and intermediate Th1 cells compared with PBS-EAU mice.

Ripasudil suppressed EAU when administered from the onset of the disease by inhibiting cells that strongly express genes involved in the ROCK signalling pathway and differentiate into Th1 cells.

Metastatic dissemination is driven by genetic, biochemical, and biophysical cues that favor the distant colonization of organs and the formation of life-threatening secondary tumors. We have previously demonstrated that endothelial cells (ECs) actively remodel during extravasation by enwrapping arrested tumor cells (TCs) and extruding them from the vascular lumen while maintaining perfusion. In this work, we dissect the cellular and molecular mechanisms driving endothelial remodeling. Using high-resolution intravital imaging in zebrafish embryos, we demonstrate that the actomyosin network of ECs controls tissue remodeling and subsequent TC extravasation. Furthermore, we uncovered that this cytoskeletal remodeling is driven by altered endothelial-calcium (Ca2+) signaling caused by arrested TCs. Accordingly, we demonstrated that the inhibition of voltage-dependent calcium L-type channels impairs extravasation. Lastly, we identified P2X4, TRP, and Piezo1 mechano-gated Ca2+ channels as key mediators of the process. These results further highlight the central role of endothelial remodeling during the extravasation of TCs and open avenues for successful therapeutic targeting.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline and loss of neuronal integrity. Emerging evidence suggests that RhoA, Rho-associated coiled-coil kinase (ROCK), and their downstream effector molecule glycogen synthase 3β (GSK3β) interact within a complex signaling pathway (RhoA/ROCK/GSK3β) that plays a crucial role in the pathogenesis of AD. RhoA, a small GTPase, along with its downstream effector, ROCK, regulates various cellular processes, including actin cytoskeleton dynamics, apoptosis, and synaptic plasticity. GSK3β, a serine/threonine kinase, plays a key role in neuronal function and AD pathology, including the regulation of tau phosphorylation and amyloid-beta cleavage. Overactive GSK3β has been closely linked to tau hyperphosphorylation, neurodegeneration, and the progression of AD. Thus, GSK3β has been considered as a promising therapeutic target for treating AD and mitigating cognitive impairment. However, clinical trials of GSK3β in AD have faced considerable challenges due to the complexity of the specific neuronal inhibition of GSK3β. In this review, we summarize the literature regarding the relationship of RhoA/ROCK and GSK3β signaling pathways in AD pathogenesis. We further discuss recent findings of the sTREM2-transgelin-2 (TG2) axis as a potential mediator of this complex pathway and provide our review on a novel targeting strategy for AD.

Innate immune responses triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection play pivotal roles in the pathogenesis of COVID-19, while host factors including proinflammatory cytokines are critical for viral containment. By utilizing quantitative and qualitative models, we discovered that soluble factors secreted by human monocytes potently inhibit SARS-CoV-2-induced cell-cell fusion in viral-infected cells. Through cytokine screening, we identified that interleukin-1β (IL-1β), a key mediator of inflammation, inhibits syncytia formation mediated by various SARS-CoV-2 strains. Mechanistically, IL-1β activates RhoA/ROCK signaling through a non-canonical IL-1 receptor-dependent pathway, which drives the enrichment of actin bundles at the cell-cell junctions, thus prevents syncytia formation. Notably, in vivo infection experiments in mice confirmed that IL-1β significantly restricted SARS-CoV-2 spread in the lung epithelium. Together, by revealing the function and underlying mechanism of IL-1β on SARS-CoV-2-induced cell-cell fusion, our study highlights an unprecedented antiviral function for cytokines during viral infection.

Fluid viscosity and osmolarity are among some of the underappreciated mechanical stimuli that cells can detect. Abnormal changes of multiple fluidic factors such as viscosity and osmolarity have been linked with diseases such as cystic fibrosis, cancer, and coronary heart disease. Changes in viscosity have been recently suggested as a regulator of cell locomotion. These novel studies focus on cell migration and spreading on glass substrates and through microchannels, and it remains a question whether viscosity impacts the cellular remodeling of extracellular matrices (ECMs). Here, we demonstrate that elevated viscosity induces cellular remodeling of collagen substrates and enhances cell spreading on ECM-mimetic substrates. Our results expand on recent work showing that viscosity induces increased cellular forces and demonstrates that viscosity can drive local ECM densification. Our data further show that microtubules, Ras-related C3 botulinum toxin substrate 1 (Rac1), actin-related protein 2/3 (Arp2/3) complex, Rho-associated protein kinase 1 (ROCK), and myosin are important regulators of viscosity-induced ECM remodeling. In the context of viscosity-induced cell spreading, cells cultured on glass and collagen substrates exhibit markedly different responses to pharmacological treatments, indicating that microtubules, Rac1, and Arp2/3 play distinct roles in regulating cellular spreading depending on the substrate. In addition, our results demonstrate that high osmotic pressures override viscosity-induced cell spreading by suppressing membrane ruffling. Our results demonstrate viscosity as a regulator of ECM remodeling and cell spreading in a fibrillar microenvironment. We also reveal a complex interplay between viscosity and osmolarity. We anticipate that our research can pave the way for future investigations into the crucial roles played by viscosity in both physiological and pathological conditions.

Reperfusion therapy inevitably leads to brain-blood barrier (BBB) disruption and promotes damage despite its benefits for acute ischaemic stroke (AIS). An effective brain cytoprotective treatment is still needed as an adjunct to reperfusion therapy. Here, we explore the potential benefits of therapeutic hypothermia (HT) in attenuating early BBB leakage and improving neurological outcomes. Mild HT was induced during the early and peri-recanalization stages in a mouse model of transient middle cerebral artery occlusion and reperfusion (tMCAO/R). The results showed that mild HT attenuated early BBB leakage in AIS, decreased the infarction volume, and improved functional outcomes. RNA sequencing data of the microvessels indicated that HT decreased the transcription of the actin polymerization-related pathway. We further discovered that HT attenuated the ROCK1/MLC pathway, leading to a decrease in the polymerization of G-actin to F-actin. Arachidonic acid (AA), a known structural ROCK agonist, partially counteracted the protective effects of HT in the tMCAO/R model. Our study highlights the importance of early vascular protection during reperfusion and provides a new strategy for attenuating early BBB leakage by HT treatment for ischaemic stroke.

Bronchopulmonary dysplasia (BPD) frequently affects extremely preterm and low birth weight infants, with current treatments lacking specificity. Enhancing extra-uterine preterm alveoli development and repairing damage are crucial for BPD management. Here we show that exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-Exos) can enhance fetal lung development in mice by delivering specific contents. Briefly, hucMSCs-Exos were extracted using ultracentrifugation and identified by transmission electron microscopy (TEM), flow cytometry, Western blot (WB), and nanoparticle tracking analysis (NTA). These exosomes were then administered to pregnant mice via tail vein injection. Embryonic lung tissues were collected at E13.5 and E18.5 via cesarean section and analyzed using hematoxylin-eosin (HE) staining, immunofluorescence, and TEM. Proteomic analysis was conducted to identify protein components in the exosomes, and WB was used to assess protein expression changes. hucMSCs-Exos from full-term infants were more effective in promoting cell proliferation than those from preterm infants. In vivo, full-term hucMSCs-Exos significantly enhanced alveolarization in fetal lung tissues. Proteomic analysis revealed higher Wnt5a expression in full-term hucMSCs-Exos, and further experiments confirmed a direct interaction between Wnt5a and ROCK1. WB also showed increased expression of the autophagy marker LC3B in the lung tissues of mice treated with full-term exosomes. In conclusion, term hucMSCs-Exos may directly regulate the phosphorylation of ROCK1 in mouse lung tissue through naturally enriched Wnt5a, thus promoting autophagy of AT2 cells and lamellar body development, and ultimately enhance the alveolarization and reducing the incidence of BPD in premature infants.

Immune checkpoint therapies have spearheaded drug innovation over the last decade, propelling cancer treatments toward a new era of precision therapies. Nonetheless, the challenges of low response rates and prevalent drug resistance underscore the imperative for a deeper understanding of the tumor microenvironment (TME) and the pursuit of novel targets. Recent findings have revealed the profound impacts of biomechanical forces within the tumor microenvironment on immune surveillance and tumor progression in both murine models and clinical settings. Furthermore, the pharmacological or genetic manipulation of mechanical checkpoints, such as PIEZO1, DDR1, YAP/TAZ, and TRPV4, has shown remarkable potential in immune activation and eradication of tumors. In this review, we delved into the underlying biomechanical mechanisms and the resulting intricate biological meaning in the TME, focusing mainly on the extracellular matrix, the stiffness of cancer cells, and immune synapses. We also summarized the methodologies employed for biomechanical research and the potential clinical translation derived from current evidence. This comprehensive review of biomechanics will enhance the understanding of the functional role of biomechanical forces and provide basic knowledge for the discovery of novel therapeutic targets.

Rho-associated protein kinase (ROCK) inhibitors are therapeutic candidates in ischemic stroke and subarachnoid hemorrhage. However, their efficacy in intracerebral hemorrhage (ICH) is unknown. Here, we tested the efficacy of fasudil (10 mg/kg), an isoform-nonselective ROCK inhibitor, and NRL-1049 (10 mg/kg), a novel inhibitor with 43-fold higher selectivity for ROCK2 isoform compared with ROCK1, in a collagenase-induced ICH model in mice. Both short (1-3 days) and prolonged (14 days) therapeutic paradigms were tested using robust sample sizes in both males and females and in active and inactive circadian stages. Outcome readouts included weight loss, mortality, hematoma volume, hemispheric swelling, brain water content, BBB permeability to large molecules, and sensorimotor and cognitive function. We found the treatments safe but not efficacious in improving the hematoma volume, BBB disruption, or neurological deficits in this collagenase-induced ICH model. Intriguingly, however, induction of ICH during the active circadian stage was associated with worse tissue and behavioral outcomes compared with the inactive stage.

Cerebral cavernous malformation (CCM) is a type of cerebrovascular abnormality in the central nervous system linked to both germline and somatic genetic mutations. Recent preclinical and clinical studies have shown that various drugs can effectively reduce the burden of CCM lesions. Despite significant progress, the mechanisms driving CCM remain incompletely understood, and to date, no drugs have been developed that can cure or prevent CCM. This review aims to explore the genetic mutations, molecular mechanisms, and pharmacological interventions related to CCM.

Literatures on the genetic mechanisms and pharmacological treatments of CCM can be searched in PubMed and Web of Science.

Germline and somatic mutations mediate the onset and development of CCM through several molecular pathways. Medications such as statins, fasudil, rapamycin, and propranolol can alleviate CCM symptoms or hinder its progression by specifically modulating the corresponding targets.

Understanding the molecular mechanisms underlying CCM offers potential for targeted therapies. Further research into novel mutations and treatment strategies is essential for improving patient outcomes.

Belumosudil mesylate (REZUROCK® Tablets hereafter belumosudil) is a novel selective rho-associated, coiled-coil containing protein kinase 2 (ROCK2) inhibitor. ROCK2 is a kinase involved in immune cell differentiation and tissue fibrosis. Belumosudil exerts its effect by decreasing the inflammation and fibrosis in various organs which are the two key features of cGVHD. In the phase III clinical study in Japan, the primary endpoint was met, best overall response rate (best ORR), defined as the percentage of patients who achieved complete response (CR) or partial response (PR), was 85.7%. Belumosudil received manufacturing and marketing approval for the treatment of chronic graft-versus-host disease (cGVHD) in patients who have insufficient response to steroid therapy in March 2024 and launched in May 2024. The Japanese MHLW has also granted orphan drug designation in May 2023 for the treatment of cGVHD.

Nicotinic α7 receptors (α7 nAChRs) present in perisynaptic Schwann cells (PSCs) control acetylcholine (ACh) spillover from the neuromuscular synapse by transiently increasing intracellular Ca2+, which fosters adenosine release via type 1 equilibrative nucleoside transporters (ENT1) and retrograde activation of presynaptic A1 inhibitory receptors. The putative Ca2+-dependent pathways downstream α7 nAChRs involved in the sensing inhibitory drive operated by PSCs is unknown. Herein, we used phrenic nerve-hemidiaphragm preparations from Wistar rats. Time-lapse video-microscopy was instrumental to assess nerve-evoked (50-Hz bursts) transmitter exocytosis and intracellular NO oscillations in nerve terminals and PSCs loaded with FM4-64 and DAF-FM diacetate fluorescent dyes, respectively. Selective activation of α7 nAChRs with PNU 282987 reduced transmitter exocytosis (FM4-64 dye unloading) during 50-Hz bursts. Inhibition of calmodulin activity (with W-7), Ca2+/calmodulin-dependent protein kinase II (CaMKII; with KN-62) and Rho-kinase (ROCK; with H1152) all prevented the release inhibitory effect of PNU 282987. The α7 nAChR agonist transiently increased NO inside PSCs; the same occurred during phrenic nerve stimulation with 50-Hz bursts in the presence of the cholinesterase inhibitor, neostigmine. The nitric oxide synthase (NOS) inhibitor, L-NOARG, but not with the guanylylcyclase (GC) inhibitor, ODQ, prevented inhibition of transmitter exocytosis by PNU 282987. Inhibition of adenosine kinase with ABT 702 favors the intracellular accumulation and translocation of the nucleoside to the synaptic cleft, thus overcoming prevention of the PNU 282987 effect caused by H1152, but not by L-NOARG. In conclusion, the α7nAChR-mediated cholinergic inhibitory drive operated by PSCs involves two distinct Ca2+-dependent intracellular pathways: a CaMKII/ROCK cascade along with a GC-independent NO pathway with divergent end-effects concerning ADK inhibition.

RHOA, RHOB, and RHOC comprise a subfamily of RHO GTPase proteins famed for controlling cytoskeletal dynamics. RHO proteins operate downstream of multiple signals emerging from the microenvironment, leading to diverse cell responses, such as proliferation, adhesion, and migration. Therefore, RHO signaling has been centrally placed in the regulation of blood cells. Despite their high homology, unique roles of RHOA, RHOB, and RHOC have been described in hematopoietic cells. In this article, we overview the contribution of RHO proteins in the development and function of each blood cell lineage. Additionally, we highlight the aberrations of the RHO signaling pathways found in hematological malignancies, providing clues for the identification of new therapeutic targets.

Since the late 20th century, researchers have known that caspases are a pillar of cell death, particularly apoptosis. However, recent advances in cell biology have unraveled the multiple roles of caspases. These enzymes have an unconventional role in cell proliferation, differentiation, and invasion. As a result, caspase deregulation can fuel the fire of cancer, incite flames of inflammation, flare neurodegenerative disorders, and exacerbate skin pathologies. Several therapeutic approaches toward caspase inhibition have been investigated, but can caspase inhibitors harness the maladaptive effect of these proteases without causing significant side effects? A few studies have exploited caspase induction for cancer or adoptive cell therapies. Here, we provide a compelling picture of caspases, starting with their evolution, their polytomous roles beyond cell death, the flaws of their deregulation, and the merits of targeting them for therapeutic implications. Furthermore, we provide a deeper understanding of the evolution of caspase-related research up to the current era, pinpointing the role of caspases in cell survival and aiding in the development of effective caspase-targeted therapies.

This study aimed to investigate the effects of Y-27632 on the long-term maintainence of mouse submandibular epithelial cells (SG-Epis) in vitro and to elucidate the underlying mechanisms.

The role of the Rho-associated kinase (ROCK) inhibitor Y-27632 in maintaining SG-Epis and its underlying mechanisms were evaluated by examining the in vitro expansion of mouse SG-Epis. Changes in key cellular characteristics, such as proliferation, long-term expansion, and mRNA and protein expression, were assessed in the presence or absence of Y-27632.

Treatment with Y-27632 significantly enhanced the proliferative potential of SG-Epis, preserving Krt8 and Krt14 expression over 17 passages. In the absence of Y-27632, SG-Epis lost their epithelial morphology. However, Y-27632 treatment maintained the epithelial morphology and downregulated mRNA levels of Tgf-β1, Ctgf, and Rock2. Treatment with TGF-β1 indicated that TGF-β/CTGF/p38 signaling is responsible for the maintenance of SG-Epis, while RNA interference studies revealed that ROCK2/c-Jun N-terminal kinase (JNK) signaling is also crucial for SG-Epis proliferation and maintenance.

The TGF-β1/CTGF/p38 and ROCK2/JNK signaling pathways are responsible for SG-Epis proliferation, and Y-27632 treatment effectively inactivates these pathways, enabling long-term in vitro maintenance of SG-Epis. The culture method utilizing Y-27632 provides an effective approach for the in vitro expansion of SG-Epis.

Regenerative medicine using human induced pluripotent stem cells (hiPSCs) is available for treating type 1 diabetes; however, the efficiency and maturation of hiPSC differentiation into pancreatic beta cells requires improvement. Various protocols, including three-dimensional (3D) culture, have been developed to improve differentiation efficiency and maturation. Several methods for 3D culture have been reported; however, they require costly and complicated equipment, special materials, and complicated operations. To solve these problems, we developed a simple 3D culture method under static conditions using a cyclo-olefin polymer (COP) characterized by high moisture barrier properties, low surface energy, and hydrophobicity. Using this 3D method and our simple and low-cost protocol, we found that differentiation into the definitive endoderm (DE) was better when the spheroids were attached. Therefore, upon the addition of Y-27632, attached spheroids with unique shapes and cavities were formed, and the differentiation efficiency into DE increased. During DE differentiation, the attachment of spheroids to the substrate and their subsequent floating improved differentiation efficiency. We found that the amount of C-peptide in spheroids differentiated using COP dishes was greater than that in rotary culture. Furthermore, INSULIN was highly expressed in areas with low cell density, suggesting that the unique shape of the spheroids made from COP dishes improved differentiation efficiency. Our study suggests that a device-free, simple 3D culture method that controls spheroid attachment improves the efficiency of hiPSC differentiation into pancreatic beta cells.

The maintenance of corneal transparency and normal vision is dependent on preservation of epithelial and endothelial cell layer homeostases. Different types of corneal injury can induce swelling and losses in transparency. Fuchs endothelial corneal dystrophy (FECD) is one type of injury that is commonly treated with rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors. While their clinical benefit is apparent, certain aspects of their mechanism of action require clarification. Specifically, although topical eye drops containing ROCK inhibitors have been employed to treat corneal endothelial dysfunction-associated corneal edema, it remains unclear whether interactions between both corneal epithelial and endothelial cell contribute to mitigating clinical signs that compromise normal vision. To address this question, we first review the intricate ROCK signaling pathways and their role in modulating a variety of functions that are related to the maintenance of corneal transparency and normal vision. We also review the results of ongoing clinical trials employing current FDA-approved ROCK inhibitors, highlighting the prominent role of Y-27632 in the treatment of a variety of ocular conditions, particularly FECD, and its promising results in reversing losses in normal vision through facilitating cell proliferation and suppressing apoptosis. This review shows that the ROCK inhibitor clinical benefit is affected by their interactions between the epithelium and the endothelium. This realization makes it likely that ROCK inhibitors will be approved for use in a clinical setting to treat FECD.

Formation of fluid-filled lumina by epithelial tissues is essential for organ development. How cells control the hydraulic and cortical forces to control lumen morphology is not well understood. Here, we quantified the mechanical role of tight junctions in lumen formation using MDCK-II cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for the hydraulic inflation of a lumen. However, the depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements of hydrostatic lumen pressure and junctional tension with modeling enabled us to explain lumen morphologies from the pressure-tension force balance. Tight junctions promote lumen inflation by decreasing cortical tension via the inhibition of myosin. In addition, our results suggest that excess apical area contributes to lumen opening. Overall, we provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

Ras homolog family member A (RhoA) acts as a signaling hub in many cellular processes, including cytoskeletal dynamics, division, migration, and adhesion. RhoA activity is tightly spatiotemporally controlled, but whether downstream effectors share these activation dynamics is unknown. We developed a novel single-color FRET biosensor to measure Rho-associated kinase (ROCK) activity with high spatiotemporal resolution in live cells. We report the validation of the Rho-Kinase Activity Reporter (RhoKAR) biosensor. RhoKAR activation was specific to ROCK activity and was insensitive to PKA activity. We then assessed the mechanisms of ROCK activation in mouse fibroblasts. Increasing intracellular calcium with ionomycin increased RhoKAR activity and depleting intracellular calcium with EGTA decreased RhoKAR activity. We also investigated the signaling intermediates in this process. Blocking calmodulin or CaMKII prevented calcium-dependent activation of ROCK. These results indicate that ROCK activity is increased by calcium in fibroblasts and that this activation occurs downstream of CaM/CaMKII.

The efficacy of cell implantation via 3D-spheroids to treat basal tone in fecal incontinence remains unclear. To address this, in this study, we aimed to identify cell differentiation and assess the development of a contractile phenotype corresponding to smooth muscle cells (SMCs) following implantation of 3D-spheroid and 2D-cultured human adipose stem cells (hASCs) in an in vivo internal anal sphincter (IAS)-targeted mouse model.

We developed an IAS-targeted in vivo model via rapid freezing (at - 196 °C) of the dorsal layers of the region of interest (ROI) of the IAS ring posterior quarter, between the submucosal and muscular layers, following submucosal dissection (n = 60 rats). After implantation of tetramethylindocarbocyanine perchlorate (Dil)-stained 3D and 2D-cells into randomly allocated cryoinjured rats, the entire sphincter ring or only the cryoinjured ROI was harvested. Expression of SMC markers, RhoA/ROCKII and its downstream molecules, and fibrosis markers was analyzed. Dil, α-smooth muscle actin (α-SMA), and RhoA signals were used for cell tracking.

In vitro, 3D-spheroids exhibited higher levels of SMC markers and RhoA/ROCKII-downstream molecules than 2D-hASCs. The IAS-targeted cryoinjured model exhibited substantial loss of SMC layers of the squamous epithelium lining of the anal canal, as well as reduced expression of SMC markers and RhoA-related downstream molecules. In vivo, 3D-spheroid implantation induced SMC markers and contractile molecules weakly at 1 week. At 2 weeks, the mRNA expression of aSma, Sm22a, Smoothelin, RhoA, Mypt1, Mlc20, Cpi17, and Pp1cd increased, whereas that of fibrosis markers reduced significantly in the 3D-spheroid implanted group compared to those in the sham, non-implanted, and 2D-hASC implanted groups. Protein levels of RhoA, p-MYPT1, and p-MLC20 were higher in the 3D-spheroid-implanted group than in the other groups. At 2 weeks, in the implanted groups, the cryoinjured tissues (which exhibited Dil, α-SMA, and RhoA signals) were restored, while they remained defective in the sham and non-implanted groups.

These findings demonstrate that, compared to 2D-cultured hASCs, 3D-spheroids more effectively induce a contractile phenotype that is initially weak but subsequently improves, inducing expression of RhoA/ROCKII-downstream molecules and SMC differentiation associated with IAS basal tone.

Immunoglobulin E (IgE)-mediated immediate hypersensitivity reactions are the most concerning adverse events after penicillin antibiotics (PENs) administration because of their rapid progression and potential for fatal outcome. However, the diagnosis of allergic death is a forensic challenge because it mainly depends on nonspecific characteristic morphological changes, as well as exclusion and circumstantial evidence. In this study, an untargeted metabolomics approach based on liquid chromatography-mass spectrometry (LC-MS) was used to screen potential forensic biomarkers of fatal anaphylactic shock induced by four PENs (benzylpenicillin (BP), amoxicillin (AMX), oxacillin (OXA), and mezlocillin (MEZ)), and analyzed the metabolites, metabolic pathway and the mechanism which were closely related to the allergic reactions. The metabolomics results discovered that a total of 24 different metabolites in all four anaphylactic death (AD) groups, seven of which were common metabolites. A biomarker model consisting of six common metabolites (linoleic acid, prostaglandin D2, lysophosphatidylcholine (18:0), N-acetylhistamine, citric acid and indolelactic acid) AUC value of Receiver Operating Characteristic (ROC) curve was 0.978. Metabolism pathway analysis revealed that the pathogenesis of PENs-induced AD is closely related to linoleic acid metabolism. Our results revealed that the metabolomic profiling has potential in PENs-induced AD post-mortem diagnosis and metabolic mechanism investigations.

Blocking the RhoA/ROCK II/MLC 2 (Ras homolog gene family member A/Rho kinase II/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the RhoA/ROCK II/MLC 2 signaling pathway changes the pathogenic processes of the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH.

Scalp acupuncture (SA) therapy was performed on rats with ICH at the acupuncture point "Baihui"-penetrating "Qubin," and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the RhoA/ROCK II/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging.

We found that ROCK II acts as a promoter of the RhoA/ROCK II/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the pre-intervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK II, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at "Baihui"-penetrating "Qubin" and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the RhoA/ROCK II/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats.

This study found that these experimental data indicated that SA at "Baihui"-penetrating "Qubin" could preserve BBB integrity and neurological function recovery after ICH by inhibiting RhoA/ROCK II/MLC 2 signaling pathway activation and by regulating endothelial cell-related proteins.

Genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A in non-small cell lung cancer. Previously, we and others have identified that SMARCA4-mutant lung cancers are highly dependent on oxidative phosphorylation (OXPHOS). Despite initial excitements, therapeutics targeting metabolic pathways such as OXPHOS have largely been disappointing due to rapid adaptation of cancer cells to inhibition of single metabolic enzymes or pathways, suggesting novel combination strategies to overcome adaptive responses are urgently needed. Here, we performed a functional genomics screen using CRISPR-Cas9 library targeting genes with available FDA approved therapeutics and identified ROCK1/2 as a top hit that sensitizes cancer cells to OXPHOS inhibition. We validate these results by orthogonal genetic and pharmacologic approaches by demonstrating that KD025 (Belumosudil), an FDA approved ROCK inhibitor, has highly synergistic anti-cancer activity in vitro and in vivo in combination with OXPHOS inhibition. Mechanistically, we showed that this combination induced a rapid, profound energetic stress and cell cycle arrest that was in part due to ROCK inhibition-mediated suppression of the adaptive increase in glycolysis normally seen by OXPHOS inhibition. Furthermore, we applied global phosphoproteomics and kinase-motif enrichment analysis to uncover a dynamic regulatory kinome upon combination of OXPHOS and ROCK inhibition. Importantly, we found converging phosphorylation-dependent regulatory cross-talk by AMPK and ROCK kinases on key RHO GTPase signaling/ROCK-dependent substrates such as PPP1R12A, NUMA1 and PKMYT1 that are known regulators of cell cycle progression. Taken together, our study identified ROCK kinases as critical mediators of metabolic adaptation of cancer cells to OXPHOS inhibition and provides a strong rationale for pursuing ROCK inhibitors as novel combination partners to OXPHOS inhibitors in cancer treatment.

It is currently not known whether mRNAs fulfill structural roles in the cytoplasm. Here, we report the fragile X-related protein 1 (FXR1) network, an mRNA-protein (mRNP) network present throughout the cytoplasm, formed by FXR1-mediated packaging of exceptionally long mRNAs. These mRNAs serve as an underlying condensate scaffold and concentrate FXR1 molecules. The FXR1 network contains multiple protein binding sites and functions as a signaling scaffold for interacting proteins. We show that it is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. Point mutations in FXR1, found in its homolog FMR1, where they cause fragile X syndrome, disrupt the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. Our findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as an organizer of signaling reactions.

Glaucoma is identified by the loss of retinal ganglion cells (RGCs). The primary approach to managing glaucoma is to control intraocular pressure (IOP). Lately, there has been an increasing focus on neuroprotective therapies for glaucoma because of the limited effectiveness of standard methods in reducing IOP and preventing ongoing vision deterioration in certain glaucoma patients. Various drug-based techniques with neuroprotective properties have demonstrated the ability to decrease the mortality of retinal ganglion cells. This study will analyze the currently recommended drug-based techniques for neuroprotection in the prospective treatment of glaucoma.

Acupuncture has been proven effective for various types of pain, and peripheral molecular signals around acupuncture-treated areas have been suggested to contribute to the analgesic effects of acupuncture. However, the underlying mechanism from these peripheral molecular signals to central ones remains unclear. The purpose of this study was to investigate whether peripheral Rho-associated protein kinase (ROCK) activation induced by acupuncture treatment mediates acupuncture analgesia, and also to investigate the relationship between ROCK activation and extracellular signal-regulated kinase (ERK), which has previously been proven to mediate acupuncture analgesia and other related molecular changes during acupuncture.

Acupuncture was treated at the bilateral GB34 acupoints of C57BL/6 mice, after which changes in ROCK activation and the location of its expression in the skin were analyzed. To verify the role of ROCK in acupuncture analgesia, we administrated ROCK inhibitor Y-27632 (0.3 μg/ul) into the skin before acupuncture treatment with formalin and complete Freund adjuvant (CFA) induced pain models, then the nociceptive responses were analyzed.

Acupuncture treatment produced ROCK2 activation in the skin after 30 and 60 min, and the histological analyses revealed that ROCK2 was activated in the fibroblast of the dermis. The acupuncture-induced ROCK2 expression was significantly attenuated by the ERK inhibitor, whereas phospho-ERK expression was not inhibited by ROCK inhibitor. In both the formalin- and CFA-induced mouse pain models, acupuncture analgesia was blocked by ROCK inhibitor administration.

Acupuncture treatment-induced ROCK2 expression is a downstream effector of phospho-ERK in the skin and plays a crucial role in acupuncture analgesia.

The discipline of structure-based drug design (SBDD) is several decades old and it is tempting to think that the proliferation of experimental structures for many drug targets might make computer-aided drug design (CADD) straightforward. However, this is far from true. In this review, we illustrate some of the challenges that CADD scientists face every day in their work, even now. We use Rho-associated protein kinase (ROCK), and public domain structures and data, as an example to illustrate some of the challenges we have experienced during our project targeting this protein. We hope that this will help to prevent unrealistic expectations of what CADD can accomplish and to educate non-CADD scientists regarding the challenges still facing their CADD colleagues.

Despite of yet unknown mechanism, microvascular deposition of oligomeric Tau (oTau) has been implicated in alteration of the Blood-Brain Barrier (BBB) function in Alzheimer's disease (AD) brains. In this study, we employed an in vitro BBB model using primary mouse cerebral endothelial cells (CECs) to investigate the mechanism underlying the effects of oTau on BBB function. We found that exposing CECs to oTau induced oxidative stress through NADPH oxidase, increased oxidative damage to proteins, decreased proteasome activity, and expressions of tight junction (TJ) proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5. These effects were suppressed by the pretreatment with Fasudil, a RhoA/ROCK signaling inhibitor. Consistent with the biochemical alterations, we found that exposing the basolateral side of CECs to oTau in the BBB model disrupted the integrity of the BBB, as indicated by an increase in FITC-dextran transport across the model, and a decrease in trans endothelial electrical resistance (TEER). oTau also increased the transmigration of peripheral blood mononuclear cells (PBMCs) in the BBB model. These functional alterations in the BBB induced by oTau were also suppressed by Fasudil. Taken together, our findings suggest that targeting the RhoA/ROCK pathway can be a potential therapeutic strategy to maintain BBB function in AD.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors encoded in the human genome, and they initiate cellular responses triggered by a plethora of extracellular stimuli ranging from neurotransmitters and hormones to photons. Upon stimulation, GPCRs activate heterotrimeric G proteins (Gαβγ) in the cytoplasm, which then convey signals to their effectors to elicit cellular responses. Given the broad biological and biomedical relevance of GPCRs and G proteins in physiology and disease, there is great interest in developing and optimizing approaches to measure their signaling activity with high accuracy and across experimental systems pertinent to their functions in cellular communication. This review provides a historical perspective on approaches to measure GPCR-G protein signaling, from quantification of second messengers and other indirect readouts of activity to biosensors that directly detect the activity of G proteins. The latter is the focus of a more detailed overview of the evolution of design principles for various optical biosensors of G protein activity with different experimental capabilities. We will highlight advantages and limitations of biosensors that detect different G protein activation hallmarks, like dissociation of Gα and Gβγ or nucleotide exchange on Gα, as well as their suitability to detect signaling mediated by endogenous versus exogenous signaling components or in physiologically relevant systems like primary cells. Overall, this review intends to provide an assessment of the state-of-the-art for biosensors that directly measure G protein activity to allow readers to make informed decisions on the selection and implementation of currently available tools. SIGNIFICANCE STATEMENT: G protein activity biosensors have become essential and widespread tools to assess GPCR signaling and pharmacology. Yet, investigators face the challenge of choosing from a growing list of G protein activity biosensors. This review provides an overview of the features and capabilities of different optical biosensor designs for the direct detection of G protein activity in cells, with the aim of facilitating the rational selection of systems that align with the specific scientific questions and needs of investigators.

Control of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis.

We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs.

The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis.

Cassiae semen, commonly consumed as roasted tea, has been widely used for both medicinal purposes and dietary supplements. In this study, we investigated the nephrotoxic effects and underlying mechanisms of Cassiae semen aqueous extracts (CSAEs) using computational and animal models. Both male and female Sprague Dawley rats were treated with 4.73-47.30 g/kg (body weight) of CSAEs by oral gavage twice a day for 7-28 days. We found that serum and urinary biomarkers of kidney injury and kidney coefficients were increased in a dose-dependent manner, and were accompanied by morphological alterations in the kidneys of CSAEs-treated rats. Computational and molecular docking approaches predicted that the three most abundant components of CSAEs-obtusifolin, aurantio-obtusin, and obtusin-exhibited strong affinity for the binding of F-actin, ROCK1, and Rac1, and the RhoA-ROCK pathway was identified as the most likely regulatory mechanism mediating the nephrotoxicity of CSAEs. Consistently, immunofluorescence staining revealed F-actin and cytoskeleton were frequently disturbed in renal cells and brush borders at high doses of CSAEs. Results from gene expression analyses confirmed that CSAEs suppressed the key proteins in the RhoA-ROCK signaling pathway and consequently the expression of F-actin and its stabilization genes. In summary, our findings suggest that Cassiae semen can depolymerize and destabilize actin cytoskeleton by inhibition of the RhoA-ROCK pathway and/or direct binding to F-actin, leading to nephrotoxicity. The consumption of Cassiae semen as a supplement and medicine warrants attention.

Proper hydration and the clarity of the cornea are maintained through the crucial function of the corneal endothelium. Inflammation of the corneal endothelium, known as endotheliitis, can disrupt endothelial function, resulting in alterations to vision. Corneal endotheliitis is characterised by corneal oedema, the presence of keratic precipitates, inflammation within the anterior chamber, and occasionally, limbal injection, neovascularisation, and the concurrent or overlapping presence of uveitis. The aetiology of this condition is diverse, predominantly viral, but it may also be drug-induced, result from bacterial or fungal infections, be associated with systemic diseases and procedures, or remain idiopathic with no identifiable cause. To date, no standardised protocol for the treatment of this ocular disease exists, and in severe cases, corneal transplantation may be required. A 31-year-old male was transferred to our hospital for the management of corneal endothelial decompensation resulting from corneal endotheliitis. Hormonal therapy and antiviral medications proved ineffective, rendering the patient a candidate for corneal transplantation. As a final measure, treatment with the ROCK inhibitor netarsudil was initiated. The patient demonstrated significant improvement in symptoms, and the inflammation was successfully managed after nine months. In this study, a novel approach employing ROCK inhibitor therapy was utilised for the treatment of corneal endotheliitis, leading to marked recovery during patient follow-up. This case report represents the inaugural application of the ROCK inhibitor netarsudil in managing corneal endothelial decompensation attributed to corneal endotheliitis. These findings suggest that this method warrants consideration as a potential novel treatment option for similar conditions.

The mechanical phenotype of a cell determines its ability to deform under force and is therefore relevant to cellular functions that require changes in cell shape, such as migration or circulation through the microvasculature. On the practical level, the mechanical phenotype can be used as a global readout of the cell's functional state, a marker for disease diagnostics, or an input for tissue modeling. We focus our review on the current knowledge of structural components that contribute to the determination of the cellular mechanical properties and highlight the physiological processes in which the mechanical phenotype of the cells is of critical relevance. The ongoing efforts to understand how to efficiently measure and control the mechanical properties of cells will define the progress in the field and drive mechanical phenotyping toward clinical applications.

MEASURE2 (Multisite Evaluation Study on Analytical Methods for Non-clinical Safety Assessment of HUman-derived REgenerative Medical Products 2) is a Japanese experimental public-private partnership initiative that aims to standardize testing methods for tumorigenicity evaluation of human pluripotent stem cell (hPSC)-derived cell therapy products (CTPs). MEASURE2 organized multisite studies to optimize the methodology of the highly efficient culture (HEC) assay, a sensitive culture-based in vitro assay for detecting residual undifferentiated hPSCs in CTPs.

In these multisite studies, 1) the efficiency of colony formation by human induced pluripotent stem cells (hiPSCs) under two different culture conditions and 2) the sorting efficiency of microbeads conjugated to various anti-hPSC markers during hiPSC enrichment were evaluated using samples in which hiPSCs were spiked into hiPSC-derived mesenchymal stem cells.

The efficiency of colony formation was significantly higher under culture conditions with the combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) than with Y-27632, which is widely used for the survival of hPSCs. Between-laboratory variance was also smaller under the condition with CEPT than with Y-27632. The sorting efficiency of microbeads conjugated with the anti-Tra-1-60 antibody was sufficiently higher (>80%) than those of the other various microbeads investigated.

Results of these multisite studies are expected to contribute to improvements in the sensitivity and robustness of the HEC assay, as well as to the future standardization of the tumorigenicity risk assessment of hPSC-derived CTPs.

It is currently not known whether mRNAs fulfill structural roles in the cytoplasm. Here, we report the FXR1 network, an mRNA-protein (mRNP) network present throughout the cytoplasm, formed by FXR1-mediated packaging of exceptionally long mRNAs. These mRNAs serve as underlying condensate scaffold and concentrate FXR1 molecules. The FXR1 network contains multiple protein binding sites and functions as a signaling scaffold for interacting proteins. We show that it is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. Point mutations in FXR1, found in its homolog FMR1, where they cause Fragile X syndrome, disrupt the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. These findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as organizer of signaling reactions.

To investigate the biological significance of Rho-associated coiled-coil-containing protein kinase (ROCK) 2 in the human trabecular meshwork (HTM), changes in both metabolic phenotype and gene expression patterns against a specific ROCK2 inhibitor KD025 were assessed in planar-cultured HTM cells. A seahorse real-time ATP rate assay revealed that administration of KD025 significantly suppressed glycolytic ATP production rate and increased mitochondrial ATP production rate in HTM cells. RNA sequencing analysis revealed that 380 down-regulated and 602 up-regulated differentially expressed genes (DEGs) were identified in HTM cells treated with KD025 compared with those that were untreated. Gene ontology analysis revealed that DEGs were more frequently related to the plasma membrane, extracellular components and integral cellular components among cellular components, and related to signaling receptor binding and activity and protein heterodimerization activity among molecular functions. Ingenuity Pathway Analysis (IPA) revealed that the detected DEGs were associated with basic cellular biological and physiological properties, including cellular movement, development, growth, proliferation, signaling and interaction, all of which are associated with cellular metabolism. Furthermore, the upstream regulator analysis and causal network analysis estimated IL-6, STAT3, CSTA and S1PR3 as possible regulators. Current findings herein indicate that ROCK2 mediates the IL-6/STAT3-, CSTA- and S1PR3-linked signaling related to basic biological activities such as glycolysis in HTM cells.

Efferocytosis, the process of engulfing and removing apoptotic cells, plays an essential role in preserving tissue health and averting undue inflammation. While macrophages are primarily known for this task, dendritic cells (DCs) also play a significant role. This review delves into the unique contributions of various DC subsets to efferocytosis, highlighting the distinctions in how DCs and macrophages recognize and handle apoptotic cells. It further explores how efferocytosis influences DC maturation, thereby affecting immune tolerance. This underscores the pivotal role of DCs in orchestrating immune responses and sustaining immune equilibrium, providing new insights into their function in immune regulation.