F-actin Stress Fiber Formation Research Articles

Cerebral Endothelial CXCR2 Promotes Neutrophil Transmigration into Central Nervous System in LPS-Induced Septic Encephalopathy.

Septic encephalopathy (SE) represents a severe inflammatory syndrome linked to elevated septic mortality rates, lacking specific therapeutic interventions, and often resulting in enduring neurological sequelae. The present investigation endeavors to elucidate the involvement of C-X-C Motif Chemokine Receptor 2 (CXCR2) in the pathogenesis of SE and to explore the potential of CXCR2 modulation as a therapeutic avenue for SE. Employing a murine SE model induced by lipopolysaccharide (LPS) administration, CXCR2 knockout mice and the CXCR2 inhibitor SB225002 were utilized to assess neutrophil recruitment, endothelial integrity, and transendothelial migration. Our findings substantiate that either CXCR2 deficiency or its inhibition curtails neutrophil recruitment without impacting their adhesion to cerebral endothelial cells. This phenomenon is contingent upon endothelial CXCR2 expression rather than CXCR2's presence on neutrophils. Furthermore, the CXCR2 blockade preserves the integrity of tight junction protein ZO-1 and mitigates F-actin stress fiber formation in cerebral endothelial cells following septic challenge. Mechanistically, CXCL1-mediated CXCR2 activation triggers cerebral endothelial actin contraction via Rho signaling, thereby facilitating neutrophil transmigration in SE. These observations advocate for the potential therapeutic efficacy of CXCR2 inhibition in managing SE.

Paeoniflorin alleviates TGF-β2-mediated extracellular matrix remodeling and oxidative stress in human trabecular meshwork cells.

The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-β2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-β2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment. Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-β2 (5ng/mL). PAE (300μM) was added to serum-starved confluent cultures of HTM cells for 2h, followed by incubation with TGF-β2 for 22h. SB-431542, a TGF-β receptor inhibitor (10μM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-β2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells. PAE attenuated TGF-β2-induced oxidative stress and suppressed TGF-β2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-β2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-β2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells. PAE alleviates TGF-β2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.

Antiviral Effect of Extracellular Matrix Protein ABI3BP on Vesicular Stomatitis Virus and Its Mechanism: A Preliminary Study In Vitro

Objective To explore the influence of extracellular matrix protein ABI-interactor 3-binding protein (ABI3BP) on vesicular stomatitis virus (VSV) genome replication and innate immune signaling pathway.Methods The small interfering RNA (siRNA) was transfected to knock down ABI3BP gene in human skin fibroblast BJ-5ta cells. VSV-green fluorescent protein (VSV-GFP)-infected cell model was established. The morphological changes and F-actin stress fiber formation were detected on ABI3BP knockdown cells by phalloidin immunofluorescence staining. The mRNA level of virus replication was detected by RT-qPCR in BJ-5ta cells after VSV-GFP infection; western blotting was performed to detect the changes in interferon regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK1) phosphorylation levels.Results The VSV-GFP-infected BJ-5ta cell model was successfully established. Efficient knockdown of ABI3BP in BJ-5ta cells was achieved. Phalloidin immunofluorescence staining revealed structural rearrangement of intracellular F-actin after ABI3BP gene knockdown. Compared with the control group, the gene copy number of VSV-GFP in ABI3BP knockdown cells increased by 2.2 - 3.5 times (P<0.01) and 2.2 - 4.0 times (P<0.01) respectively when infected with VSV of multiplicity of infection 0.1 and 1. The expression of viral protein significantly increased in ABI3BP knockdown cells after virus infection. The activation of type-I interferon pathway, as determined by phosphorylated IRF3 and phosphorylated TBK1, was significantly decreased in ABI3BP knockdown cells after VSV-GFP infection.Conclusions Extracellular matrix protein ABI3BP plays an important role in maintaining the formation and rearrangement of actin structure. ABI3BP gene deletion promotes RNA virus replication, and ABI3BP is an important molecule that maintains the integrity of type I interferon pathway.

Everolimus-induced hyperpermeability of endothelial cells causes lung injury.

The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the underlying mechanisms remain unclear. Endothelial cell barrier dysfunction plays a major role in the pathogenesis of the lung injury. This study hypothesizes that everolimus increases pulmonary endothelial permeability, which leads to lung injury. We tested the effects of everolimus on human pulmonary microvascular endothelial cell (HPMEC) permeability and a mouse model of intraperitoneal injection of everolimus was established to investigate the effect of everolimus on pulmonary vascular permeability. Our data showed that everolimus increased human pulmonary microvascular endothelial cell (HPMEC) permeability which was associated with MLC phosphorylation and F-actin stress fiber formation. Furthermore, everolimus induced an increasing concentration of intracellular calcium Ca2+ leakage in HPMECs and this was normalized with ryanodine pretreatment. In addition, ryanodine decreased everolimus-induced phosphorylation of PKCα and MLC, and barrier disruption in HPMECs. Consistent with in vitro data, everolimus treatment caused a visible lung-vascular barrier dysfunction, including an increase in protein in BALF and lung capillary-endothelial permeability, which was significantly attenuated by pretreatment with an inhibitor of PKCα, MLCK, and ryanodine. This study shows that everolimus induced pulmonary endothelial hyper-permeability, at least partly, in an MLC phosphorylation-mediated EC contraction which is influenced in a Ca2+-dependent manner and can lead to lung injury through mTOR-independent mechanisms.

Extracellular matrix viscoelasticity regulates TGFβ1-induced epithelial-mesenchymal transition and apoptosis via integrin linked kinase.

Transforming growth factor (TGF)-β1 is a multifunctional cytokine that plays important roles in health and disease. Previous studies have revealed that TGFβ1 activation, signaling, and downstream cell responses including epithelial-mesenchymal transition (EMT) and apoptosis are regulated by the elasticity or stiffness of the extracellular matrix. However, tissues within the body are not purely elastic, rather they are viscoelastic. How matrix viscoelasticity impacts cell fate decisions downstream of TGFβ1 remains unknown. Here, we synthesized polyacrylamide hydrogels that mimic the viscoelastic properties of breast tumor tissue. We found that increasing matrix viscous dissipation reduces TGFβ1-induced cell spreading, F-actin stress fiber formation, and EMT-associated gene expression changes, and promotes TGFβ1-induced apoptosis in mammary epithelial cells. Furthermore, TGFβ1-induced expression of integrin linked kinase (ILK) and colocalization of ILK with vinculin at cell adhesions is attenuated in mammary epithelial cells cultured on viscoelastic substrata in comparison to cells cultured on nearly elastic substrata. Overexpression of ILK promotes TGFβ1-induced EMT and reduces apoptosis in cells cultured on viscoelastic substrata, suggesting that ILK plays an important role in regulating cell fate downstream of TGFβ1 in response to matrix viscoelasticity.

RHOAL57V drives the development of diffuse gastric cancer through IGF1R-PAK1-YAP1 signaling.

Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr42-to-Cys (Y42C) and Leu57-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOAY42C exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOAL57V promotes DGC growth. In mouse gastric organoids with deletion of Cdh1, which encodes the cell adhesion protein E-cadherin, the expression of RHOAL57V, but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOAL57V also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOAL57V retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr42 and Leu57 in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOAL57V additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOAY42C. Our results reveal that RHOAL57V and RHOAY42C drive the development of DGC through distinct biochemical and signaling mechanisms.

Activation of cell adhesion and migration is an early event of platelet-rich plasma (PRP)-dependent stimulation of human adipose-derived stem/stromal cells.

Stem cell therapy is a promising treatment in regenerative medicine. Human adipose-derived stem/stromal cells (hASCs), a type of mesenchymal stem cell, are easy to harvest. In plastic and aesthetic surgery, hASC may be applied in the treatment of fat grafting, wound healing, and scar remodeling. Platelet-rich plasma (PRP) contains various growth factors, including platelet-derived growth factor (PDGF), which accelerates wound healing. We previously reported that PRP promotes the proliferation of hASC via multiple signaling pathways, and we evaluated the effect of PRP on the stimulation of hASC adhesion and migration, leading to the proliferation of these cells. When hASCs were treated with PRP, AKT, ERK1/2, paxillin and RhoA were rapidly activated. PRP treatment led to the formation of F-actin stress fibers. Strong signals for integrin β1, paxillin and RhoA at the cell periphery of RPR-treated cells indicated focal adhesion. PRP promoted cell adhesion and movement of hASC, compared with the control group. Imatinib, an inhibitor of the PDGF receptor tyrosine kinase, inhibited the promotion of PRP-dependent cell migration. PDGF treatment of hASCs also stimulated cell adhesion and migration but to a lesser extent than PRP treatment. PRP promoted the adhesion and the migration of hASC, mediated by the activation of AKT in the integrin signaling pathway. PRP treatment was more effective than PDGF treatment in enhancing cell migration. Thus, the ability of PRPs to promote migration of hASC to enhance cell growth is evident.

Evaluation of Tight Junction Integrity in Brain Endothelial Cells Using Confocal Microscopy.

The blood-brain barrier (BBB) is a highly complex and dynamic microvascular barrier that protects the brain parenchyma from the entry of pathogens, toxins, and other macromolecules and is a critical structure that helps to maintain brain homeostasis. The BBB is formed mainly by brain capillary endothelial cells and perivascular astrocytes and pericytes. One of the primary properties of the BBB is a tight regulation of paracellular permeability due to the presence of tight junctional complexes (also, adherens and gap junctions) between the neighboring microvascular endothelial cells. Alterations in the assembly of the tight junctions impair BBB properties, particularly influenced barrier integrity and permeability. The tight junctions of the BBB are mainly composed of proteins including claudins, occludin, and zonula occludens-1 (ZO-1). Zonula occludens-1 binds to the actin cytoskeleton, and its localization provides valuable information on the status of BBB integrity and permeability. Immunofluorescence localization of ZO-1 and/or other tight junction proteins is a reliable indicator of barrier integrity and permeability in microvascular endothelial cells. In microvascular endothelial cells, f-actin stress fiber formation significantly influences the rate and size of the inter-endothelial cell gap that form as cells retract from their borders. Rhodamine phalloidin is a popular conjugate used as a fluorescent label for f-actin. Herein, we describe the procedures for ZO-1 immunofluorescence and f-actin labeling followed by confocal microscopic imaging to determine barrier integrity and tight junction organization in brain microvascular endothelial cells in vitro.

A crucial stem cell plasticity regulation pathway: identification of key elements using the NCCIT human embryonic carcinoma cell line.

Upon removal of stemness factors, a small subpopulation of embryonic stem cells (ESCs) spontaneously extrudes the t-SNARE protein syntaxin-4, which upregulates the cell adhesion molecule P-cadherin and induces the onset of epithelial-mesenchymal transition (EMT)-like behaviors with loss of stemness in each cell. In this study, we identified a series of molecular elements responsible for this phenomenon using several small-molecule inhibitors and the human embryonic carcinoma cell line, NCCIT. We found that the syntaxin-4-triggered morphological changes and a decrease in stemness signatures were independently induced by the activation of Rho-associated kinase (ROCK) and the abrogation of PI3K/Akt signaling. We also found that the extracellular expression of syntaxin-4 inactivated focal adhesion kinase (FAK) in association with the augmented expression of P-cadherin, and comparable controls of either of these downstream elements of syntaxin-4 accelerated both ROCK-induced F-actin stress fiber formation and P13K/Akt-suppressed loss of stemness signatures. Cells expressing P-cadherin inactivated FAK but FAK inhibition did not affect P-cadherin expression, demonstrating a causal relationship between P-cadherin and FAK in the event of syntaxin-4 induction. These results reveal a novel signaling axis in stem cells and shed new light on the crucial elements for stem cell plasticity and the maintenance of stemness.

Long Noncoding RNA TPRG1-AS1 Suppresses Migration of Vascular Smooth Muscle Cells and Attenuates Atherogenesis via Interacting With MYH9 Protein.

Migration of human aortic smooth muscle cells (HASMCs) contributes to the pathogenesis of atherosclerosis. This study aims to functionally characterize long noncoding RNA TPRG1-AS1 (tumor protein p63 regulated 1, antisense 1) in HASMCs and reveal the underlying mechanism of TPRG1-AS1 in HASMCs migration, neointima formation, and subsequent atherosclerosis. The expression of TPRG1-AS1 in atherosclerotic plaques was verified a series of in silico analysis and quantitative real-time polymerase chain reaction analysis. Northern blot, rapid amplification of cDNA ends and Sanger sequencing were used to determine its full length. In vitro transcription-translation assay was used to investigate the protein-coding capacity of TPRG1-AS1. RNA fluorescent in situ hybridization was used to confirm its subcellular localization. Loss- and gain-of-function studies were used to investigate the function of TPRG1-AS1. Furthermore, the effect of TPRG1-AS1 on the pathological response was evaluated in carotid balloon injury model, wire injury model, and atherosclerosis model, respectively. TPRG1-AS1 was significantly increased in atherosclerotic plaques. TPRG1-AS1 did not encode any proteins and its full length was 1279nt, which was bona fide a long noncoding RNA. TPRG1-AS1 was mainly localized in cytoplasmic and perinuclear regions in HASMCs. TPRG1-AS1 directly interacted with MYH9 (myosin heavy chain 9) protein in HASMCs, promoted MYH9 protein degradation through the proteasome pathway, hindered F-actin stress fiber formation, and finally inhibited HASMCs migration. Vascular smooth muscle cell-specific transgenic overexpression of TPRG1-AS1 significantly reduced neointima formation, and attenuated atherosclerosis in apolipoprotein E knockout (Apoe-/-) mice. This study demonstrated that TPRG1-AS1 inhibited HASMCs migration through interacting with MYH9 protein and consequently suppressed neointima formation and atherosclerosis.

A Novel Mouse Model of TGFβ2-Induced Ocular Hypertension Using Lentiviral Gene Delivery.

Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) β2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFβ2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFβ2 in the TM. We developed an LV vector-encoding active hTGFβ2C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFβ2C226,228S. We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFβ2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFβ2C226,228S exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFβ2C226,228S that induces TM dysfunction and outflow resistance.

ETS-Related Gene Activation Preserves Adherens Junctions and Permeability in Microvascular Endothelial Cells.

ERG (ETS-related gene) is a member of the ETS (Erythroblast-transformation specific) family of transcription factors abundantly present in vascular endothelial cells. Recent studies demonstrate that ERG has important roles in blood vessel stability and angiogenesis. However, it is unclear how ERG is potentially involved in microvascular barrier functions and permeability. A wide variety of diseases and clinical conditions including trauma-hemorrhagic shock and burn injury are associated with microvascular dysfunctions, which causes excessive microvascular permeability, tissue edema and eventually, multiple organ dysfunction and death. The main purpose of this study was to determine the specific role of ERG in regulating microvascular permeability in human lung microvascular endothelial cells (HLMEC) and to evaluate if exogenous ERG will protect the barrier. The HLMECs were grown on Transwell inserts as monolayers and were transfected with ERG CRISPR/cas9 knockdown plasmid, ERG CRISPR activation plasmid, recombinant ERG protein or their respective controls. Recombinant vascular endothelial growth factor (VEGF) was used as an inducer of permeability for evaluating the effect of ERG activation on permeability. Changes in barrier integrity and permeability were studied using monolayer permeability assay and immunofluorescence of adherens junction proteins (VE-cadherin and β-catenin) respectively. CRISPR/cas9-based ERG knockdown as well as VEGF treatment induced monolayer hyperpermeability, VE-cadherin, and β-catenin junctional relocation and cytoskeletal F-actin stress fiber formation. CRISPR based ERG activation and recombinant ERG transfection attenuated VEGF-induced monolayer hyperpermeability. ERG activation preserved the adherens junctions and cytoskeleton. These results demonstrate that ERG is a potent regulator of barrier integrity and permeability in human lung microvascular endothelial cells and endogenously or exogenously enhancing ERG provides protection against barrier dysfunction and hyperpermeability.

Mechanosensitive channel inhibition attenuates TGFβ2-induced actin cytoskeletal remodeling and reactivity in mouse optic nerve head astrocytes

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFβ2) levels within astrocytes have been described in glaucoma, and TGFβ signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFβ2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFβ2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFβ2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining and immunoblotting for GFAP. Levels of fibronectin and collagen IV deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (F4/80) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFβ2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFβ2 treatment and the presence of f-actin stress fibers. TGFβ2 treatment significantly increased GFAP, fibronectin, and collagen IV levels, and GsMTx4 co-treatment ameliorated GFAP immunoreactivity. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

Depletion of Arg/Abl2 improves endothelial cell adhesion and prevents vascular leak during inflammation

Endothelial barrier disruption and vascular leak importantly contribute to organ dysfunction and mortality during inflammatory conditions like sepsis and acute respiratory distress syndrome. We identified the kinase Arg/Abl2 as a mediator of endothelial barrier disruption, but the role of Arg in endothelial monolayer regulation and its relevance in vivo remain poorly understood. Here we show that depletion of Arg in endothelial cells results in the activation of both RhoA and Rac1, increased cell spreading and elongation, redistribution of integrin-dependent cell-matrix adhesions to the cell periphery, and improved adhesion to the extracellular matrix. We further show that Arg is activated in the endothelium during inflammation, both in murine lungs exposed to barrier-disruptive agents, and in pulmonary microvessels of septic patients. Importantly, Arg-depleted endothelial cells were less sensitive to barrier-disruptive agents. Despite the formation of F-actin stress fibers and myosin light chain phosphorylation, Arg depletion diminished adherens junction disruption and intercellular gap formation, by reducing the disassembly of cell-matrix adhesions and cell retraction. In vivo, genetic deletion of Arg diminished vascular leak in the skin and lungs, in the presence of a normal immune response. Together, our data indicate that Arg is a central and non-redundant regulator of endothelial barrier integrity, which contributes to cell retraction and gap formation by increasing the dynamics of adherens junctions and cell-matrix adhesions in a Rho GTPase-dependent fashion. Therapeutic inhibition of Arg may provide a suitable strategy for the treatment of a variety of clinical conditions characterized by vascular leak.

ZnR/GPR39 controls cell migration by orchestrating recruitment of KCC3 into protrusions, re-organization of actin and activation of MMP

Actin re-organization and degradation of extracellular matrix by metalloproteases (MMPs) facilitate formation of cellular protrusions that are required for cell proliferation and migration. We find that Zn2+ activation of the Gq-coupled receptor ZnR/GPR39 controls these processes by regulating K+/Cl− co-transporter KCC3, which modulates cell volume. Silencing of KCC3 expression or activity reverses ZnR/GPR39 enhancement of cell proliferation, migration and invasion through Matrigel. Activation of ZnR/GPR39 recruits KCC3 into F-actin rich membrane protrusions, suggesting that it can locally control volume changes. Immunofluorescence analysis indicates that Zn2+ activation of ZnR/GPR39 and KCC3 are required to enhance formation of F-actin stress fibers and cellular protrusions. In addition, ZnR/GPR39 upregulation of KCC3-dependent transport increases the activity of matrix metalloproteases MMP2 and MMP9. Our study establishes a mechanism in which ZnR/GPR39 orchestrates localization and activation of KCC3, formation of F-actin rich cell protrusions and activation of MMPs, and thereby controls cell proliferation and migration.

Lanthanum chloride causes blood-brain barrier disruption through intracellular calcium-mediated RhoA/Rho kinase signaling and myosin light chain kinase.

Rare earth elements (REEs) have caused bioaccumulation and adverse health effects attributed to extensive application. The penetrability of REEs across the blood-brain barrier (BBB) contributes to their neurotoxicity process, but potential mechanisms affecting BBB integrity are still obscure. The present study was designed to investigate the effects of lanthanum on BBB adheren junctions and the actin cytoskeleton in vitro using bEnd.3 cells. After lanthanum chloride (LaCl3, 0.125, 0.25 and 0.5 mM) treatment, cytotoxicity against bEnd.3 cells was observed accompanied by increased intracellular Ca2+. Higher paracellular permeability presented as decreased TEER (transendothelial electrical resistance) and increased HRP (horse radish peroxidase) permeation, and simultaneously reduced VE-cadherin expression and F-actin stress fiber formation caused by LaCl3 were reversed by inhibition of ROCK (Rho-kinase) and MLCK (myosin light chain kinase) using inhibitor Y27632 (10 μM) and ML-7 (10 μM). Moreover, chelating overloaded intracellular Ca2+ by BAPTA-AM (25 μM) remarkably abrogated RhoA/ROCK and MLCK activation and downstream phosphorylation of MYPT1 (myosin phosphatase target subunit 1) and MLC2 (myosin light chain 2), therefore alleviating LaCl3-induced BBB disruption and dysfunction. In conclusion, this study indicated that lanthanum caused endothelial barrier hyperpermeability accompanied by loss of VE-cadherin and rearrangement of the actin cytoskeleton though intracellular Ca2+-mediated RhoA/ROCK and MLCK pathways.

Vascular Shutdown by Photodynamic Therapy Using Talaporfin Sodium.

Photodynamic therapy (PDT) is an attractive cancer treatment modality. Talaporfin sodium, a second-generation photosensitizer, results in lower systemic toxicity and relatively better selective tumor destruction than first-generation photosensitizers. However, the mechanism through which PDT induces vascular shutdown is unclear. In this study, the in vitro effects of talaporfin sodium-based PDT on human umbilical vein endothelial cells (HUVECs) were determined through cell viability and endothelial tube formation assays, and evaluation of the tubulin and F-actin dynamics and myosin light chain (MLC) phosphorylation. Additionally, the effects on tumor blood flow and tumor vessel destruction were assessed in vivo. In the HUVECs, talaporfin sodium-based PDT induced endothelial tube destruction and microtubule depolymerization, triggering the formation of F-actin stress fibers and a significant increase in MLC phosphorylation. However, pretreatment with the Rho-associated protein kinase (ROCK) inhibitor, Y27632, completely prevented PDT-induced stress fiber formation and MLC phosphorylation. The in vivo analysis and pathological examination revealed that the PDT had significantly decreased the tumor blood flow and the active area of the tumor vessel. We concluded that talaporfin sodium-based PDT induces the shutdown of existing tumor vessels via the RhoA/ROCK pathway by activating the Rho-GTP pathway and decreasing the tumor blood flow.

Cathepsin‐B is a Mediator of Barrier Dysfunction and Hyperpermeability in Blood‐Brain Barrier Endothelial Cells

Cathepsin B belongs to the papain‐like family of cysteine proteases and is an upstream mediator of the NLRP3 inflammasome pathway that leads to interleukin 1β (IL‐1β) secretion. Interleukin 1β is a potent inducer of tight junction disruption and hyperpermeability in blood‐brain barrier (BBB) endothelial cells. However, the involvement of cathepsin B in BBB dysfunction and hyperpermeability is not clearly known. Understanding this pathway is critical to preserving BBB integrity and protection against brain edema. In order to study this relationship, rat brain microvascular endothelial cells were transfected with active cathepsin B. Tight junction organization and changes in permeability were evaluated based on immunofluorescence, immunoblot analysis of zonula occudens‐1 (ZO‐1) and Transwell monolayer permeability assay using FITC‐dextran (10‐kDa). Cathepsin B activity and cell viability were evaluated fluorometrically. F‐actin stress fiber formation was studied using rhodamine phalloidin staining. Active cathepsin B induced ZO‐1 dislocation/tight junction disorganization, F‐actin stress fiber formation and monolayer hyperpermeability significantly. These effects were inhibited by pretreatment with NLRP3 inhibitors isoliquiritigenin and MCC950. Active cathepsin B had no significant effect on ZO‐1 protein expression or cell viability. Furthermore, cathepsin B inhibitor E‐64d attenuated hydrogen peroxide‐induced cathepsin B activity and monolayer hyperpermeability significantly. These results suggest that cathepsin B is a mediator of BBB dysfunction/hyperpermeability. Cathepsin B inhibition has protective effects against barrier dysfunction and hyperpermeability in BBB endothelial cells. One of the mechanisms by which cathepsin B induces barrier dysfunction is via the NLRP3 inflammasome pathway.

The Effect of Matrix Stiffness of Biomimetic Gelatin Nanofibrous Scaffolds on Human Cardiac Pericyte Behavior.

Congenital heart disease (CHD) is the most common and deadly congenital anomaly, accounting for up to 7.5% of all infant deaths. Survival in children born with CHD has improved dramatically over the past several decades (this positive trend being counterbalanced by the fact that more patients develop heart failure). Seminal data indicate an alteration of the extracellular matrix occurs with time in these hearts due to diffuse and abundant interstitial fibrosis. This results in an escalation in the stiffness of the local myocardial microenvironment. However, the influence of matrix stiffness in regulating the function of resident human stromal cells has not been reported. The objective of this study was to determine the impact of scaffold stiffness on the antigenic and functional profile of cardiac pericytes (CPs) isolated from patients with CHD. To this end, we have first manufactured gelatin nanofibrous scaffolds with varying degrees of stiffness using an in situ cross-linking electrospinning technique in a pure water solvent system. We assessed Young's modulus and performed a comprehensive physicochemical characterization of the scaffolds employing scanning electron microscopy and Fourier transform infrared spectroscopy. We next evaluated the changes induced by a different scaffold stiffness on CP morphology, antigenic profile, viability, proliferation, angiocrine activity, and induced differentiation. Results indicate that soft matrixes with a fiber diameter of ∼400 nm increase CP proliferation, secretion of angiopoietin 2, and F-actin stress fiber formation, without affecting the antigenic profile, viability, or differentiation. These data indicate for the first time that human CPs can be functionally influenced by slight changes in matrix stiffness. The study elucidates the importance of mechanical/morphological cues in modulating the behavior of stromal cells isolated from patients with CHD.

Abstract 67: MMP-7 decreases E-cadherin localization at cell-cell contacts resulting in F-actin cytoskeletal remodeling in prostate cancer micro-tumors formed by perlecan/HSPG2

Abstract Introduction: Polarized epithelium is maintained by cell-cell interactions via cadherins and cell adhesion molecules (CAMs). These interactions are further stabilized by cell-matrix interactions on the basement membrane. Perlecan/HSPG2 is a major component of the basement membrane, controlling signaling in resting tissues with unperturbed matrices. During epithelial to mesenchymal transition in prostate cancer (PCa), cell-cell adhesions decrease and the cells acquire an invasive phenotype. Proteolytic cleavage of perlecan decreases cell-matrix interactions and dysregulates cell signaling, permitting migration. In vivo, patients with bone metastases were found to have circulating DmIV fragments, with a positive correlation between MMP-7 staining and loss of perlecan in tissue samples from these same patients. Recent studies showed perlecan domain IV-3 (DmIV-3) drives cell cohesion and, when digested with matrix metalloproteinase-7 (MMP-7), drives cell dyscohesion. MMP-7 can cleave cadherins and other CAMs disrupting cell-cell adhesions. Additionally, DmIV-3 fragments generated by MMP-7 cleavage may further induce cell dyscohesion by disrupting interactions between CAMs and/or cadherins. Methods: To evaluate the impact of MMP-7 and domain IV-3 fragments, uniformly sized PCa cell clusters were pre-formed using a microwell system, enabling control of cluster size and cell number. These pre-formed cell clusters were transferred to DmIV-3 coated wells for 16-24 hours. Clusters then were treated with MMP-7 alone or MMP-7 plus DmIV-3 fragments and stained for E-cadherin and F-actin. Results: Consistent with previous data, PCa cell clusters maintained strong cell-cell contacts in the presence of DmIV-3. In contrast, we showed that in pre-formed PCa cell clusters cultured in the presence of DmIV-3 cleaved by MMP-7, E-cadherin localization at cell-cell interfaces was reduced. Pre-formed PCa cell clusters treated with MMP-7 had increased F-actin stress fibers present, with a decrease in F-actin organization at cell-cell contacts. Conclusion: Creating PCa micro-tumors using a microwell system provides a good model to study dynamic changes in cell-cell interactions. The purpose of this study was to identify which cell adhesion complexes are perturbed by MMP-7 in these PCa cell clusters. E-cadherin is known to regulate the cortical actin cytoskeleton; thus, decreased E-cadherin at cell-cell contacts results in actin reorganization and an increase in F-actin stress fiber formation, promoting a migratory cell phenotype. Perlecan fragments may augment this behavior by increasing MMP-7 access to the ectodomains of CAMs. This study will be novel in its identification of a DmIV fragment(s) that may provide a prediction marker for PCa metastasis as well as fragments that are positively associated with tumor dyscohesion. Citation Format: Lissette A. Cruz, Tristen V. Tellman, Mary C. Farach-Carson, Brian Grindel, Daniel D. Carson. MMP-7 decreases E-cadherin localization at cell-cell contacts resulting in F-actin cytoskeletal remodeling in prostate cancer micro-tumors formed by perlecan/HSPG2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 67.