Fluoride-induced hypertension by regulating RhoA/ROCK pathway and phenotypic transformation of vascular smooth muscle cells: In vitro and in vivo evidence.

Fluoride exposure has been implicated as a potential risk factor for hypertension, but the underlying mechanisms remain unclear. This study investigated the role of the RhoA/ROCK signaling pathway in fluoride-induced hypertension. Male Wistar rats were divided into different groups and exposed to varying concentrations of sodium fluoride (NaF) or sodium chloride (NaCl) via drinking water. The rats' blood pressure was measured, and their aortic tissue was utilized for high-throughput sequencing analysis. Additionally, rat and A7r5 cell models were established using NaF and/or Fasudil. The study evaluated the effects of fluoride exposure on blood pressure, pathological changes in the aorta, as well as the protein/mRNA expression levels of phenotypic transformation indicators (a-SMA, calp, OPN) in vascular smooth muscle cells (VSMCs), along with the RhoA/ROCK signaling pathway (RhoA, ROCK1, ROCK2, MLC/p-MLC). The results demonstrated that fluoride exposure in rats led to increased blood pressure. High-throughput sequencing analysis revealed differential gene expression associated with vascular smooth muscle contraction, with the RhoA/ROCK signaling pathway emerging as a key regulator. Pathological changes in the rat aorta, such as elastic membrane rupture and collagen fiber deposition, were observed following NaF exposure. However, fasudil, a ROCK inhibitor, mitigated these pathological changes. Both in vitro and in vivo models confirmed the activation of the RhoA/ROCK signaling pathway and the phenotypic transformation of VSMCs from a contractile to a synthetic state upon fluoride exposure. Fasudil effectively inhibited the activities of ROCK1 and ROCK2 and attenuated the phenotypic transformation of VSMCs. In conclusion, fluoride has the potential to induce hypertension through the activation of the RhoA/ROCK signaling pathway and phenotypic changes in vascular smooth muscle cells. These results provide new insights into the mechanism of fluoride-induced hypertension.

Plakophilin 4 controls the spatio-temporal activity of RhoA at adherens junctions to promote cortical actin ring formation and tissue tension.

Plakophilin 4 (PKP4) is a component of cell-cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs was required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization.

Effects of fasudil on glial cell activation induced by tooth movement.

BACKGROUND: Orthodontic pain affects the physical and mental health of patients. The spinal trigeminal subnucleus caudalis (SPVC) contributes to the transmission of pain information and serves as a relay station for integrating orofacial damage information. Recently, glial cells have been found to be crucial for both acute and maintenance phases of pain. It has also been demonstrated that rho kinase (ROCK) inhibitors can manage different pain models by inhibiting glial cell activation. Here, we hypothesized that orthodontic pain is related to glial cells in the SPVC, and Fasudil, a representative rho/rock kinase inhibitor, can relieve orthodontic pain by regulating the function of glial cells and the related inflammatory factors. In this study, we constructed a rat model of tooth movement pain and used immunofluorescence staining to evaluate the activation of microglia and astrocytes. Quantitative real-time PCR was used to detect the release of related cytokines and the expression of pain-related genes in the SPVC. Simultaneously, we investigated the effect of Fasudil on the aforementioned indicators. RESULTS: In the SPVC, the expression of c-Fos peaked on day 1 along with the expression of OX42 (related to microglial activation), CD16 (a pro-inflammatory factor), and CD206 (an anti-inflammatory factor) on day 3 after tooth movement, followed by a gradual decrease. GFAP-staining showed that the number of activated astrocytes was the highest on day 5 and that cell morphology became complex. After Fasudil treatment, the expression of these proteins showed a downward trend. The mRNA levels of pro-inflammatory factors (IL-1ß and TNF-α) peaked on day 3, and the mRNA expression of the anti-inflammatory factor TGF-ß was the lowest 3 days after tooth movement. Fasudil inhibited the mRNA expression of pain-related genes encoding CSF-1, t-PA, CTSS, and BDNF. CONCLUSION: This study shows that tooth movement can cause the activation of glial cells in SPVC, and ROCK inhibitor Fasudil can inhibit the activation of glial cells and reduce the expression of the related inflammatory factors. This study presents for the first time the potential application of Fasudil in othodontic pain.

Exercise Attenuate Diaphragm Atrophy in COPD Mice via Inhibiting the RhoA/ROCK Signaling.

Background: Exercise is an indispensable component of pulmonary rehabilitation with strong anti-inflammatory effects. However, the mechanisms by which exercise prevents diaphragmatic atrophy in COPD (chronic obstructive pulmonary disease) remain unclear. Methods: Forty male C57BL/6 mice were assigned to the control (n=16) and smoke (n=24) groups. Mice in the smoke group were exposed to the cigarette smoke (CS) for six months. They were then divided into model and exercise training groups for 2 months. Histological changes were observed in lung and diaphragms. Subsequently, agonist U46639 and antagonist Y27632 of RhoA/ROCK were subjected to mechanical stretching in LPS-treated C2C12 myoblasts. The expression levels of Atrogin-1, MuRF-1, MyoD, Myf5, IL-1ß, TNF-α, and RhoA/ROCK were determined by Western blotting. Results: Diaphragmatic atrophy and increased RhoA/ROCK expression were observed in COPD mice. Exercise training attenuated diaphragmatic atrophy, decreased the expression of MuRF-1, and increased MyoD expression in COPD diaphragms. Exercise also affects the upregulation of RhoA/ROCK and inflammation-related proteins. In in vitro experiments with C2C12 myoblasts, LPS remarkably increased the level of inflammation and protein degradation, whereas Y27632 or combined with mechanical stretching prevented this phenomenon considerably. Conclusion: RhoA/ROCK plays an important role in the prevention of diaphragmatic atrophy in COPD.

The Toxoplasma secreted effector TgWIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2.

The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a 'Trojan Horse' mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro.

Enhanced Migration of Fuchs Corneal Endothelial Cells by Rho Kinase Inhibition: A Novel Ex Vivo Descemet's Stripping Only Model.

Descemet's Stripping Only (DSO) is a surgical technique that utilizes the peripheral corneal endothelial cell (CEnC) migration for wound closure. Ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, has shown potential in DSO treatment; however, its mechanism in promoting CEnC migration remains unclear. We observed that ripasudil-treated immortalized normal and Fuchs endothelial corneal dystrophy (FECD) cells exhibited significantly enhanced migration and wound healing, particularly effective in FECD cells. Ripasudil upregulated mRNA expression of Snail Family Transcriptional Repressor (SNAI1/2) and Vimentin (VIM) while decreasing Cadherin (CDH1), indicating endothelial-to-mesenchymal transition (EMT) activation. Ripasudil activated Rac1, driving the actin-related protein complex (ARPC2) to the leading edge, facilitating enhanced migration. Ex vivo studies on cadaveric and FECD Descemet's membrane (DM) showed increased migration and proliferation of CEnCs after ripasudil treatment. An ex vivo DSO model demonstrated enhanced migration from the DM to the stroma with ripasudil. Coating small incision lenticule extraction (SMILE) tissues with an FNC coating mix and treating the cells in conjunction with ripasudil further improved migration and resulted in a monolayer formation, as detected by the ZO-1 junctional marker, thereby leading to the reduction in EMT. In conclusion, ripasudil effectively enhanced cellular migration, particularly in a novel ex vivo DSO model, when the stromal microenvironment was modulated. This suggests ripasudil as a promising adjuvant for DSO treatment, highlighting its potential clinical significance.

Gnao1 is a molecular switch that regulates the Rho signaling pathway in differentiating neurons.

GNAO1 encodes G protein subunit alpha O1 (Gαo). Pathogenic variations in GNAO1 cause developmental delay, intractable seizures, and progressive involuntary movements from early infancy. Because the functional role of GNAO1 in the developing brain remains unclear, therapeutic strategies are still unestablished for patients presenting with GNAO1-associated encephalopathy. We herein report that siRNA-mediated depletion of Gnao1 perturbs the expression of transcripts associated with Rho GTPase signaling in Neuro2a cells. Consistently, siRNA treatment hampered neurite outgrowth and extension. Growth cone formation was markedly disrupted in monolayer neurons differentiated from iPSCs from a patient with a pathogenic variant of Gαo (p.G203R). This variant disabled neuro-spherical assembly, acquisition of the organized structure, and polarized signals of phospho-MLC2 in cortical organoids from the patient's iPSCs. We confirmed that the Rho kinase inhibitor Y27632 restored these morphological phenotypes. Thus, Gαo determines the self-organizing process of the developing brain by regulating the Rho-associated pathway. These data suggest that Rho GTPase pathway might be an alternative target of therapy for patients with GNAO1-associated encephalopathy.

S1PR2 participates in intestinal injury in severe acute pancreatitis by regulating macrophage pyroptosis.

Background: Severe acute pancreatitis (SAP) is an inflammatory disorder affecting the gastrointestinal system. Intestinal injury plays an important role in the treatment of severe acute pancreatitis. In this study, we mainly investigated the role of S1PR2 in regulating macrophage pyroptosis in the intestinal injury of severe acute pancreatitis. Methods: The SAP model was constructed using cerulein and lipopolysaccharide, and the expression of S1PR2 was inhibited by JTE-013 to detect the degree of pancreatitis and intestinal tissue damage in mice. Meanwhile, the level of pyroptosis-related protein was detected by western blot, the level of related mRNA was detected by PCR, and the level of serum inflammatory factors was detected by ELISA. In vitro experiments, LPS+ATP was used to construct the pyroptosis model of THP-1. After knockdown and overexpression of S1PR2, the pyroptosis proteins level was detected by western blot, the related mRNA level was detected by PCR, and the level of cell supernatant inflammatory factors were detected by ELISA. A rescue experiment was used to verify the sufficient necessity of the RhoA/ROCK pathway in S1PR2-induced pyroptosis. Meanwhile, THP-1 and FHC were co-cultured to verify that cytokines released by THP-1 after damage could regulate FHC damage. Results: Our results demonstrated that JTE-013 effectively attenuated intestinal injury and inflammation in mice with SAP. Furthermore, we observed a significant reduction in the expression of pyroptosis-related proteins within the intestinal tissue of SAP mice upon treatment with JTE-013. We confirmed the involvement of S1PR2 in THP-1 cell pyroptosis in vitro. Specifically, activation of S1PR2 triggered pyroptosis in THP-1 cells through the RhoA/ROCK signaling pathway. Moreover, it was observed that inflammatory factors released during THP-1 cell pyroptosis exerted an impact on cohesin expression in FHC cells. Conclusion: The involvement of S1PR2 in SAP-induced intestinal mucosal injury may be attributed to its regulation of macrophage pyroptosis.

Rear cortex contraction aids in nuclear transit during confined migration by increasing pressure in the cell posterior.

As cells migrate through biological tissues, they must frequently squeeze through micron-sized constrictions in the form of interstitial pores between extracellular matrix fibers and/or other cells. Although it is now well recognized that such confined migration is limited by the nucleus, which is the largest and stiffest organelle, it remains incompletely understood how cells apply sufficient force to move their nucleus through small constrictions. Here, we report a mechanism by which contraction of the cell rear cortex pushes the nucleus forward to mediate nuclear transit through constrictions. Laser ablation of the rear cortex reveals that pushing forces behind the nucleus are the result of increased intracellular pressure in the rear compartment of the cell. The pushing forces behind the nucleus depend on accumulation of actomyosin in the rear cortex and require Rho kinase (ROCK) activity. Collectively, our results suggest a mechanism by which cells generate elevated intracellular pressure in the posterior compartment to facilitate nuclear transit through three-dimensional (3D) constrictions. This mechanism might supplement or even substitute for other mechanisms supporting nuclear transit, ensuring robust cell migrations in confined 3D environments.

Protective effects of Y-27632 against cisplatin-induced ototoxicity: A zebrafish model Y-27632 and cisplatin-induced ototoxicity.

Cisplatin is an effective chemotherapy agent against various solid malignancies; however, it is associated with irreversible bilateral sensorineural hearing loss, emphasizing the need for drug development to prevent this complication, with the current options being very limited. Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine protein kinase involved in various cellular processes, including apoptosis regulation. In this study, we used a transgenic zebrafish model (Brn3C: EGFP) in which hair cells within neuromasts are observed in green under fluorescent microscopy without the need for staining. Zebrafish larvae were exposed to cisplatin alone or in combination with various concentrations of Y-27632, a potent ROCK inhibitor. Hair cell counts, apoptosis assessments using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay, FM1-43FX labeling assay and behavioral analyses (startle response and rheotaxis) were performed to evaluate the protective effects of Y-27632 against cisplatin-induced ototoxicity. Cisplatin treatment reduced the number of hair cells in neuromasts, induced apoptosis, and impaired zebrafish larval behaviors. Y-27632 demonstrated a dose-dependent protective effect against cisplatin-induced hair cell loss and apoptosis. These findings suggest that Y-27632, as a ROCK inhibitor, mitigates cisplatin-induced hair cell loss and associated ototoxicity in zebrafish.

Low-intensity pulsed ultrasound combined with ST36 modulate gastric smooth muscle contractile marker expression via RhoA/Rock and MALAT1/miR-449a/DLL1 signaling in diabetic rats.

BACKGROUND: Low-intensity pulsed ultrasound (LIPUS) combined with acupoint can promote gastric motility of diabetic rats. The switch of gastric smooth muscle cell (GSMCs) phenotype was related to the diabetes-induced gastric dysfunction, but the mechanism is not clearly elucidated. This study was aimed at exploring the underlying mechanism of LIPUS stimulation application in diabetic gastroparesis rats. METHODS: In this study, Sprague-Dawley male rats were divided into three groups: control group (CON), diabetic gastroparesis group (DGP), and LIPUS-treated group (LIPUS). LIPUS irradiation was performed bilaterally at ST36 for 20 min per day for 4 weeks. The gastric emptying rate was measured by ultrasound examination. Contraction ability of GSMCs was assessed by muscle strip experiment. The expression of related proteins or mRNAs including α-SMA, SM22α, MHC, RhoA, Rock2, p-MYPT1, MYPT1, p-MLC, MLC, MALAT1, miR-449a, and DLL1 was detected by different methods such as western blotting, RT-qPCR, immunohistochemistry, and immunofluorescence staining, as appropriate. KEY RESULTS: (a) LIPUS stimulation at ST36 could improve the gastric motility dysfunction of diabetic rats. (b) LIPUS increased RhoA, Rock2, p-MYPT1, and p-MLC expression level. (c) MALAT1 and DLL1 contents were decreased, but the level of miR-449a was increased in the LIPUS group. CONCLUSIONS & INFERENCES: LIPUS may affect the contractile marker expression of gastric smooth muscle through the RhoA/Rock and MALAT1/miR-449a/DLL1 pathway to ameliorate DGP.

The Application of Rho Kinase Inhibitors in the Management of Glaucoma.

Glaucoma is a chronic neurodegenerative disease that poses a significant threat of irreversible blindness worldwide. Current treatments for glaucoma focus on reducing intraocular pressure (IOP), which is the only modifiable risk factor. Traditional anti-glaucomatous agents, including carbonic anhydrase inhibitors, beta-blockers, alpha-2 agonists, and prostaglandin analogs, work by either improving uveoscleral outflow or reducing aqueous humor production. Rho kinase (ROCK) inhibitors represent a novel class of anti-glaucomatous drugs that have emerged from bench to bedside in the past decade, offering multifunctional characteristics. Unlike conventional medications, ROCK inhibitors directly target the trabecular meshwork outflow pathway. This review aims to discuss the mechanism of ROCK inhibitors in reducing IOP, providing neuroprotection, and preventing fibrosis. We also highlight recent studies and clinical trials evaluating the efficacy and safety of ROCK inhibitors, compare them with other clinical anti-glaucomatous medications, and outline future prospects for ROCK inhibitors in glaucoma treatment.

Connecting GSK-3ß Inhibitory Activity with IKK-ß or ROCK-1 Inhibition to Target Tau Aggregation and Neuroinflammation in Alzheimer's Disease-Discovery, In Vitro and In Cellulo Activity of Thiazole-Based Inhibitors.

GSK-3ß, IKK-ß, and ROCK-1 kinases are implicated in the pathomechanism of Alzheimer's disease due to their involvement in the misfolding and accumulation of amyloid ß (Aß) and tau proteins, as well as inflammatory processes. Among these kinases, GSK-3ß plays the most crucial role. In this study, we present compound 62, a novel, remarkably potent, competitive GSK-3ß inhibitor (IC50 = 8 nM, Ki = 2 nM) that also exhibits additional ROCK-1 inhibitory activity (IC50 = 2.3 µM) and demonstrates anti-inflammatory and neuroprotective properties. Compound 62 effectively suppresses the production of nitric oxide (NO) and pro-inflammatory cytokines in the lipopolysaccharide-induced model of inflammation in the microglial BV-2 cell line. Furthermore, it shows neuroprotective effects in an okadaic-acid-induced tau hyperphosphorylation cell model of neurodegeneration. The compound also demonstrates the potential for further development, characterized by its chemical and metabolic stability in mouse microsomes and fair solubility.

SMP30 Interacts with ROCK1 to Inhibit HCC Invasiveness by Effecting the Epithelial-Mesenchymal Transition.

BACKGROUND: The senescence marker protein 30 (SMP30) is a calcium-binding protein whose expression decreases with age, and is closely associated with hepatocellular carcinoma (HCC) development. The primary goal of this study was to examine the mechanistic effect of SMP30 on HCC migration and invasion. METHODS: Bioinformatic and immunohistochemical approaches were used to examine the expression of SMP30 in HCC tissues and its relationship to patient survival. We investigated the effects of SMP30 expression on HCC cell proliferation, migration, invasion, and cell cycle dynamics. cDNA microarray technology was used to determine the gene expression profile of SK-Hep-1 cells following recombinant SMP30 overexpression to identify genes downstream of SMP30 that regulate HCC cell migration and invasion. We identified SMP30 interacting proteins by affinity purification-mass spectrometry (AP-MS) and co-immunoprecipitation/western blotting (COIP-WB). RESULTS: SMP30 expression was lower in HCC tissues compared with normal liver tissues, and its expression positively correlated with overall survival in HCC patients. Additionally, SMP30 overexpression effectively blocked the migratory and invasive properties of SK-Hep-1 cells, but did not affect either proliferation rates or cell cycle. cDNA microarray results confirmed that many of the differentially expressed genes identified are involved in the process of epithelial-mesenchymal transition (EMT). AP-MS and COIP-WB experiments confirmed that Rho-associated protein kinase 1 (ROCK1) interacts with SMP30 in SK-Hep-1 cells, and ROCK1 is known to intimately regulate the EMT process. CONCLUSION: SMP30 inhibits HCC metastasis by influencing the expression of EMT-related proteins after interacting with ROCK1.

FAK regulates tension transmission to the nucleus and endothelial transcriptome independent of kinase activity.

The mechanical environment generated through the adhesive interaction of endothelial cells (ECs) with the matrix controls nuclear tension, preventing aberrant gene synthesis and the transition from restrictive to leaky endothelium, a hallmark of acute lung injury (ALI). However, the mechanisms controlling tension transmission to the nucleus and EC-restrictive fate remain elusive. Here, we demonstrate that, in a kinase-independent manner, focal adhesion kinase (FAK) safeguards tension transmission to the nucleus to maintain EC-restrictive fate. In FAK-depleted ECs, robust activation of the RhoA-Rho-kinase pathway increased EC tension and phosphorylation of the nuclear envelope protein, emerin, activating DNMT3a. Activated DNMT3a methylates the KLF2 promoter, impairing the synthesis of KLF2 and its target S1PR1 to induce the leaky EC transcriptome. Repleting FAK (wild type or kinase dead) or inhibiting RhoA-emerin-DNMT3a activities in damaged lung ECs restored KLF2 transcription of the restrictive EC transcriptome. Thus, FAK sensing and control of tension transmission to the nucleus govern restrictive endothelium to maintain lung homeostasis.

Paeoniflorin regulates RhoA/ROCK1 and Nrf2 pathways in PDLIM1-dependent or independent manners in oxidative stressed melanocytes.

BACKGROUND: The adhesive properties of vitiligo melanocytes have decreased under oxidative stress., cytoskeleton proteins can control cell adhesion. Paeoniflorin (PF) was proved to resist hydrogen peroxide (H2O2)-induced oxidative stress in melanocytes via nuclear factorE2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. OBJECTIVES: This study was to investigate whether PF exerts anti-oxidative effect through influencing cytoskeleton markers or potential signaling pathway. METHODS: Human Oxidative Stress Plus array was used to identify the differentially expressed genes between H2O2 + PF group and H2O2 only group, in PIG1 and PIG3V melanocyte cell lines respectively. Western blotting was used to verify the PCR array results and to test the protein expression levels of cytoskeleton markers including Ras homolog family member A (RhoA), Rho-associated kinase 1 (ROCK1) and antioxidative marker Nrf2. Small interfering RNA was used to knock down PDZ and LIM domain 1 (PDLIM1). RESULTS: PF increased the expressions of PDLIM1, RhoA and ROCK1 in H2O2-induced PIG1, in contrast, decreased the expressions of PDLIM1 and ROCK1 in H2O2-induced PIG3V. Knockdown of PDLIM1 increased the expressions of RhoA and Nrf2 in PF-pretreated H2O2-induced PIG1, and ROCK1 and Nrf2 in PF-pretreated H2O2-induced PIG3V. CONCLUSIONS: PF regulates RhoA/ROCK1 and Nrf2 pathways in PDLIM1-dependent or independent manners in H2O2-induced melanocytes. In PIG1, PF promotes PDLIM1 to inhibit RhoA/ROCK1 pathway or activates Nrf2/HO-1 pathway, separately. In PIG3V, PF directly downregulates ROCK1 in PDLIM1-independent manner or upregulates Nrf2 dependent of PDLIM1.

Sema4D Knockout Attenuates Choroidal Neovascularization by Inhibiting M2 Macrophage Polarization Via Regulation of the RhoA/ROCK Pathway.

Purpose: The aim of this study was to elucidate the role of Sema4D in the pathogenesis of senescence-associated choroidal neovascularization (CNV) and to explore its underlying mechanisms. Methods: In this study, we utilized a model of laser-induced CNV in both young (3 months old) and old (18 months old) mice, including those with or without Sema4D knockout. The expression and localization of Sema4D in CNV were assessed using PCR, Western blot, and immunostaining. Subsequently, the morphological and imaging examinations were used to evaluate the size of CNV and vascular leakage. Finally, the expression of M2 markers, senescence-related markers, and molecules involved in the RhoA/ROCK pathway was detected. Results: We found that Sema4D was predominantly expressed in macrophages within CNV lesions, and both the mRNA and protein levels of Sema4D progressively increased following laser photocoagulation, a trend more pronounced in old mice. Moreover, Sema4D knockout markedly inhibited M2 polarization in senescent macrophages and reduced the size and leakage of CNV, particularly in aged mice. Mechanistically, aging was found to upregulate RhoA/ROCK signaling, and knockout of Sema4D effectively suppressed the activation of this pathway, with more significant effects observed in aged mice. Conclusions: Our findings revealed that the deletion of Sema4D markedly inhibited M2 macrophage polarization through the suppression of the RhoA/ROCK pathway, ultimately leading to the attenuation of senescence-associated CNV. These data indicate that targeting Sema4D could offer a promising approach for gene editing therapy in patients with neovascular age-related macular degeneration.

Hypericin-mediated photodynamic therapy inhibits metastasis and EMT of colorectal cancer cells by regulating RhoA-ROCK1 signaling pathway.

Colorectal cancer (CRC) is significantly contributed to global cancer mortality rates. Treating CRC is particularly challenging due to metastasis and drug resistance. There is a pressing need for new treatment strategies against metastatic CRC. Photodynamic therapy (PDT) offers a well-established, minimally invasive treatment option for cancer with limited side effects. Hypericin (HYP), a potent photosensitizer for PDT, has been documented to induce cytotoxicity and apoptosis in various types of cancers. However, there are few reports on the inhibitory effects of HYP-mediated PDT on the metastatic ability of CRC cells. Here, we evaluate the inhibitory effects of HYP-mediated PDT against metastatic CRC cells and define its underlying mechanisms. Wound-healing and Transwell assays show that HYP-mediated PDT suppresses migration and invasion of CRC cells. F-actin visualization assays indicate HYP-mediated PDT decreases F-actin formation in CRC cells. TEM assays reveal HYP-mediated PDT disrupts pseudopodia formation of CRC cells. Mechanistically, immunofluorescence and western blotting results show that HYP-mediated PDT upregulates E-cadherin and downregulates N-cadherin and Vimentin. HYP-mediated PDT also suppresses key EMT regulators, including Snail, MMP9, ZEB1 and α-SMA. Additionally, the expressions of RhoA and ROCK1 are downregulated by HYP-mediated PDT. Together, these findings suggest that HYP-mediated PDT inhibits the migration and invasion of HCT116 and SW620 cells by modulating EMT and RhoA-ROCK1 signaling pathway. Thus, HYP-mediated PDT presents a potential therapeutic option for CRC.

Oligomeric Tau-induced oxidative damage and functional alterations in cerebral endothelial cells: Role of RhoA/ROCK signaling pathway.

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.