Intrastromal versus subconjunctival injection of mesenchymal stem/stromal cells for promoting corneal repair.

PURPOSE: Different approaches to delivery of mesenchymal stem/stromal cells (MSCs) for ameliorating corneal injuries have been investigated. This study was aimed to compare the efficacy of intrastromal and subconjunctival injection of human bone marrow-derived MSCs (hBM-MSCs) in a corneal epithelial injury model. METHODS: Twenty-four C57BL/6J mice underwent total corneal and limbal epithelial debridement. Then, the mice were divided into three different groups: (1) intrastromal hBM-MSCs injection, (2) subconjunctival hBM-MSCs injection, and (3) injection of frozen medium as a control. Mice were monitored by slit lamp and underwent anterior segment optical coherence tomography (ASOCT). Following euthanasia, the corneas were further evaluated by histology and immunostaining. RESULTS: hBM-MSC injection successfully healed epithelial defects regardless of the delivery route (P < 0.001). However, intrastromal injection was superior to subconjunctival injection in reducing defect area (P = 0.001). Intrastromal injection of hBM-MSCs also significantly reduced corneal opacity and neovascularization and improved ASOCT parameters compared to subconjunctival injection or no treatment (P < 0.001, P = 0.003, and P < 0.001, respectively). Although both of the treatment groups were positive for CK12 and had reduced levels of MUC5AC compared to the control, CK12 staining was stronger in the intrastromal group compared to the subconjunctival group. Also, persistency of MSCs was confirmed by in vivo (up to 2 weeks) and in vitro assessments (up to 4 weeks). CONCLUSIONS: Although the injection of hBM-MSC using both intrastromal and subconjunctival methods improve wound healing and reduce neovascularization and opacity, the intrastromal approach is superior in terms of corneal healing.

The effect of decellularization protocols on characterizations of thermoresponsive and light-curable corneal extracellular matrix hydrogels.

To compare the effects of two decellularization protocols on the characteristics of fabricated COrnea Matrix (COMatrix) hydrogels. Porcine corneas were decellularized with Detergent (De) or Freeze-Thaw (FT)-based protocols. DNA remnant, tissue composition and α-Gal epitope content were measured. The effect of α-galactosidase on α-Gal epitope residue was assessed. Thermoresponsive and light-curable (LC) hydrogels were fabricated from decellularized corneas and characterized with turbidimetric, light-transmission and rheological experiments. The cytocompatibility and cell-mediated contraction of the fabricated COMatrices were assessed. Both protocols reduced the DNA content to < 0.1 µg/mg (native, > 0.5 µg/mg), and preserved the collagens and glycosaminoglycans. The α-Gal epitope remnant decreased by > 50% following both decellularization methods. We observed more than 90% attenuation in α-Gal epitope after treatment with α-galactosidase. The thermogelation half-time of thermoresponsive COMatrices derived from De-Based protocol (De-COMatrix) was 18 min, similar to that of FT-COMatrix (21 min). The rheological characterizations revealed significantly higher shear moduli of thermoresponsive FT-COMatrix (300.8 ± 22.5 Pa) versus De-COMatrix 178.7 ± 31.3 Pa, p < 0.01); while, this significant difference in shear moduli was preserved after fabrication of FT-LC-COMatrix and De-LC-COMatrix (18.3 ± 1.7 vs 2.8 ± 2.6 kPa, respectively, p < 0.0001). All thermoresponsive and light-curable hydrogels have similar light-transmission to human corneas. Lastly, the obtained products from both decellularization methods showed excellent in vitro cytocompatibility. We found that FT-LC-COMatrix was the only fabricated hydrogel with no significant cell-mediated contraction while seeded with corneal mesenchymal stem cells (p < 0.0001). The significant effect of decellularization protocols on biomechanical properties of hydrogels derived from porcine corneal ECM should be considered for further applications.

Bone marrow mesenchymal stromal cells in a 3D system produce higher concentration of extracellular vesicles (EVs) with increased complexity and enhanced neuronal growth properties.

PURPOSE: Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to possess great potential in preclinical models. An efficient biomanufacturing platform is necessary for scale up production for clinical therapeutic applications. The aim of this study is to investigate the potential differences in neuro-regenerative properties of MSC-derived EVs generated in 2D versus 3D culture systems. METHOD: Human bone marrow MSCs (BM-MSCs) were cultured in 2D monolayer and 3D bioreactor systems. EVs were isolated using ultracentrifugation followed by size and concentration measurements utilizing dynamic light scattering (NanoSight) and by fluorescence staining (ExoView). Mouse trigeminal ganglia (TG) neurons were isolated from BALB/c mice and cultured in the presence or absence of EVs derived from 2D or 3D culture systems. Neuronal growth and morphology were monitored over 5 days followed by immunostaining for ß3 tubulin. Confocal images were analyzed by Neurolucida software to obtain the density and length of the neurites. RESULTS: The NanoSight tracking analysis revealed a remarkable increase (24-fold change) in the concentration of EVs obtained from the 3D versus 2D culture condition. ExoView analysis showed a significantly higher concentration of CD63, CD81, and CD9 markers in the EVs derived from 3D versus 2D conditions. Furthermore, a notable shift toward a more heterogeneous phenotype was observed in the 3D-derived EVs compared to those from 2D culture systems. EVs derived from both culture conditions remarkably induced neurite growth and elongation after 5 days in culture compared to untreated control. Neurolucida analysis of the immunostaining images (ß3 tubulin) showed a significant increase in neurite length in TG neurons treated with 3D- versus 2D-derived EVs (3301.5 µm vs. 1860.5 µm, P < 0.05). Finally, Sholl analysis demonstrated a significant increase in complexity of the neuronal growth in neurons treated with 3D- versus 2D-derived EVs (P < 0.05). CONCLUSION: This study highlights considerable differences in EVs obtained from different culture microenvironments, which could have implications for their therapeutic effects and potency. The 3D culture system seems to provide a preferred environment that modulates the paracrine function of the cells and the release of a higher number of EVs with enhanced biophysical properties and functions in the context of neurite elongation and growth.

A Light-Curable and Tunable Extracellular Matrix Hydrogel for In Situ Suture-Free Corneal Repair.

Corneal injuries are a major cause of blindness worldwide. To restore corneal integrity and clarity, there is a need for regenerative bio-integrating materials for in-situ repair and replacement of corneal tissue. Here, we introduce Light-curable COrnea Matrix (LC-COMatrix), a tunable material derived from decellularized porcine cornea extracellular matrix containing un-denatured collagen and sulfated glycosaminoglycans. It is a functionalized hydrogel with proper swelling behavior, biodegradation, and viscosity that can be cross-linked in situ with visible light, providing significantly enhanced biomechanical strength, stability, and adhesiveness. Cross-linked LC-COMatrix strongly adheres to human corneas ex vivo and effectively closes full-thickness corneal perforations with tissue loss. Likewise, in vivo, LC-COMatrix seals large corneal perforations, replaces partial-corneal stromal defects and bio-integrates into the tissue in rabbit models. LC-COMatrix is a natural ready-to-apply bio-integrating adhesive that is representative of native corneal matrix with potential applications in corneal and ocular surgeries.

Preclinical Evaluation of the Safety and Efficacy of Cryopreserved Bone Marrow Mesenchymal Stromal Cells for Corneal Repair.

Purpose: Mesenchymal stromal cells (MSCs) have been shown to enhance tissue repair as a cell-based therapy. In preparation for a phase I clinical study, we evaluated the safety, dosing, and efficacy of bone marrow-derived MSCs after subconjunctival injection in preclinical animal models of mice, rats, and rabbits. Methods: Human bone marrow-derived MSCs were expanded to passage 4 and cryopreserved. Viability of MSCs after thawing and injection through small-gauge needles was evaluated by vital dye staining. The in vivo safety of human and rabbit MSCs was studied by subconjunctivally injecting MSCs in rabbits with follow-up to 90 days. The potency of MSCs on accelerating wound healing was evaluated in vitro using a scratch assay and in vivo using 2-mm corneal epithelial debridement wounds in mice. Human MSCs were tracked after subconjunctival injection in rat and rabbit eyes. Results: The viability of MSCs after thawing and immediate injection through 27- and 30-gauge needles was 93.1% ± 2.1% and 94.9% ± 1.3%, respectively. Rabbit eyes demonstrated mild self-limiting conjunctival inflammation at the site of injection with human but not rabbit MSCs. In scratch assay, the mean wound healing area was 93.5% ± 12.1% in epithelial cells co-cultured with MSCs compared with 40.8% ± 23.1% in controls. At 24 hours after wounding, all MSC-injected murine eyes had 100% corneal wound closure compared with 79.9% ± 5.5% in controls. Human MSCs were detectable in the subconjunctival area and peripheral cornea at 14 days after injection. Conclusions: Subconjunctival administration of MSCs is safe and effective in promoting corneal epithelial wound healing in animal models. Translational Relevance: These results provide preclinical data to support a phase I clinical study.

Dose-dependent therapeutic effects of topical 1,25 OH-vitamin D3 on corneal wound healing.

Conflicting results have been reported regarding the effects of 1,25 OH-vitamin D3 on corneal wound healing. Therefore, we undertook this study to determine whether the observed differences are dose related. The dose-dependent effects of 1,25 OH-vitamin D3 on corneal wound healing were evaluated using scratch assays on human corneal limbal-epithelial cells (HCLEs) and in vivo mouse corneal epithelial debridement. To evaluate the anti-inflammatory effects of 1,25 OH-vitamin D3, macrophages were stimulated by a Toll-Like Receptor (TLR) ligand followed by treatment with the 10-6 M, 10-7 M and 10-8 M 1,25 OH-vitamin D3. 10-7 M 1,25 OH-vitamin D3 induced faster scratch wound closure compared with the other concentrations of 1,25 OH-vitamin D3 tested (10-6 M and 10-8 M), and 0.02% ethanol as a control (85.8 ± 2.6%, 33.9 ± 6.74%, 32.6 ± 3.35%, and 31.6 ± 3.99%, respectively, P < 0.0001). Single-time treatment with 10-7 M 1,25 OH-vitamin D3 also significantly improved the healing of mouse corneal epithelial wound compared to multiple treatments and control (74.1 ± 17.3% vs. 52.4 ± 11.6% and 45.8 ± 13.4%, respectively). Polyinosinic: polycytidylic acid (poly [I:C])-stimulated macrophage cells and 10-7 M 1,25 OH-vitamin D3 significantly decreased gene expression of ICAM1, TLR3, IL6, IL8, and TNFα (P < 0.0001). Our results suggest the dose-dependent therapeutic effect of 1,25 OH-vitamin D3 in corneal wound healing which can be potentially used as a non-invasive option in the treatment of corneal wounds.

In-situ porcine corneal matrix hydrogel as ocular surface bandage.

PURPOSE: Bioactive substrates can be used therapeutically to enhance wound healing. Here, we evaluated the effect of an in-situ thermoresponsive hydrogel from decellularized porcine cornea ECM, COMatrix (COrnea Matrix), for application as an ocular surface bandage for corneal epithelial defects. METHODS: COMatrix hydrogel was fabricated from decellularized porcine corneas. The effects of COMatrix hydrogel on attachment and proliferation of human corneal epithelial cells (HCECs) were evaluated in vitro. The effect of COMatrix on the expressions of the inflammatory genes, IL-1ß, TNF-α, and IL-6 was assessed by RT-PCR. The in-situ application and also repairing effects of COMatrix hydrogel as an ocular bandage was studied in a murine model of corneal epithelial wound. The eyes were examined by optical coherence tomography (OCT) and slit-lamp microscopy in vivo and by histology and immunofluorescence post-mortem. RESULTS: In vitro, COMatrix hydrogel significantly enhanced the attachment and proliferation of HCECs relative to control. HCECs exposed to COMatrix had less induced expression of TNF-α (P < 0.05). In vivo, COMatrix formed a uniform hydrogel that adhered to the murine ocular surface after in-situ curing. Corneal epithelial wound closure was significantly accelerated by COMatrix hydrogel compared to control (P < 0.01). There was significant increase in the expression of proliferation marker Ki-67 in wounded corneal epithelium by COMatrix hydrogel compared to control (P < 0.05). CONCLUSIONS: COMatrix hydrogel is a naturally derived bioactive material with potential application as an ocular surface bandage to enhance epithelial wound healing.

Gene dosage manipulation alleviates manifestations of hereditary PAX6 haploinsufficiency in mice.

In autosomal dominant conditions with haploinsufficiency, a single functional allele cannot maintain sufficient dosage for normal function. We hypothesized that pharmacologic induction of the wild-type allele could lead to gene dosage compensation and mitigation of the disease manifestations. The paired box 6 (PAX6) gene is crucial in tissue development and maintenance particularly in eye, brain, and pancreas. Aniridia is a panocular condition with impaired eye development and limited vision due to PAX6 haploinsufficiency. To test our hypothesis, we performed a chemical screen and found mitogen-activated protein kinase kinase (MEK) inhibitors to induce PAX6 expression in normal and mutant corneal cells. Treatment of newborn Pax6-deficient mice (Pax6Sey-Neu/+ ) with topical or systemic MEK inhibitor PD0325901 led to increased corneal PAX6 expression, improved corneal morphology, reduced corneal opacity, and enhanced ocular function. These results suggest that induction of the wild-type allele by drug repurposing is a potential therapeutic strategy for haploinsufficiencies, which is not limited to specific mutations.

Reproducible Derivation and Expansion of Corneal Mesenchymal Stromal Cells for Therapeutic Applications.

Purpose: A reproducible protocol for the production of corneal mesenchymal stem/stromal cells (cMSCs) is necessary for potential clinical applications. We aimed to describe successful generation and expansion of cMSCs using an explant method. Methods: Corneoscleral rims of human cadaveric eyes were divided into four pieces and used as explants to allow outgrowth of cMSCs (passage 0, or P0). The cells were subcultured at a 1:10 ratio until passage 5 (P5). The characteristics as well as therapeutic effects of expanded cMSCs were evaluated both in vitro, using a scratch assay, and in vivo using epithelial debridement and chemical injury mouse models. Results: All explants demonstrated outgrowth of cells by 7 days. Although the initial outgrowth included mixed mesenchymal and epithelial cells, by P1 only cMSCs remained. By subculturing each flask at a ratio of 1:10, the potential yield from each cornea was approximately 12 to 16 × 1010 P5 cells. P5 cMSCs demonstrated the cell surface markers of MSCs. The secretome of P5 cMSCs induced faster closure of wounds in an in vitro scratch assay. Subconjunctival injection of P5 cMSCs in mouse models of mechanical corneal epithelial debridement or ethanol injury led to significantly faster wound healing and decreased inflammation, relative to control. Conclusions: cMSCs can be reproducibly derived from human cadaveric corneas using an explant method and expanded with preservation of characteristics and corneal wound healing effects. Translational Relevance: The results of our study showed that cMSCs produced using this scheme can be potentially used for clinical applications.

Therapeutic Effects of Lyophilized Conditioned-Medium Derived from Corneal Mesenchymal Stromal Cells on Corneal Epithelial Wound Healing.

Objectives: The conditioned-medium derived from corneal mesenchymal stromal cells (cMSCs) has been shown to have wound healing and immunomodulatory effects in corneal injury models. Here, the therapeutic effects of lyophilized cMSC conditioned-medium were compared with fresh conditioned-medium. Methods: The epithelial wound healing effects of fresh and lyophilized cMSC conditioned-medium were compared with conditioned-medium from non-MSC cells (corneal epithelial cells) using scratch assay. To evaluate the anti-inflammatory effects of fresh and lyophilized cMSC conditioned-media, macrophages were stimulated by a Toll-Like Receptor (TLR) ligand followed by treatment with the conditioned-media and measuring the expression of inflammatory genes. In vivo wound healing effects of fresh and lyophilized cMSC conditioned-media were assessed in a murine model of cornea epithelial injury. Results: Both fresh and lyophilized cMSCs-derived conditioned-medium induced significantly faster closure of in vitro epithelial wounds compared to conditioned-medium from non-MSC cells (P < .0001). Treating stimulated macrophages with fresh or lyophilized cMSCs-derived conditioned-media significantly decreased the expression of inflammatory genes compared to control (P < .0001). Murine corneal epithelial wounds were healed by 87.6 ± 2.7% and 86.2 ± 4.6% following treatment with fresh and lyophilized cMSC conditioned-media, respectively, while the control was healed by 64.7 ± 16.8% (P < .05). Conclusion: Lyophilized cMSC-derived conditioned-medium is as effective as fresh conditioned-medium in promoting wound healing and modulating inflammation. The results of this study support the application of lyophilized cMSCs-derived conditioned-medium, which allows for more extended storage, as a promising non-invasive option in the treatment of corneal wounds.

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion.

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.

Cornea-Derived Mesenchymal Stromal Cells Therapeutically Modulate Macrophage Immunophenotype and Angiogenic Function.

Macrophages are crucial drivers of inflammatory corneal neovascularization and thus are potential targets for immunomodulatory therapies. We hypothesized that therapeutic use of cornea-derived mesenchymal stromal cells (cMSCs) may alter the function of macrophages. We found that cMSCs can modulate the phenotype and angiogenic function of macrophages. In vitro, cMSCs induce apoptosis of macrophages while preferentially promoting a distinct CD14hi CD16hi CD163hi CD206hi immunophenotype that has significantly reduced angiogenic effects based on in vitro angiogenesis assays. In vivo, application of cMSCs to murine corneas after injury leads to reduced macrophage infiltration and higher expression of CD206 in macrophages. Macrophages cocultured ("educated") by cMSCs express significantly higher levels of anti-angiogenic and anti-inflammatory factors compared with control macrophages. In vivo, injured corneas treated with cMSC-educated macrophages demonstrate significantly less neovascularization compared with corneas treated with control macrophages. Knocking down the expression of pigment epithelial derived factor (PEDF) in cMSCs significantly abrogates its modulating effects on macrophages, as shown by the reduced rate of apoptosis, decreased expression of sFLT-1/PEDF, and increased expression of vascular endothelial growth factor-A in the cocultured macrophages. Similarly, cMSCs isolated from PEDF knockout mice are less effective compared with wild-type cMSCs at inhibiting macrophage infiltration when applied to wild-type corneas after injury. Overall, these results demonstrate that cMSCs therapeutically suppress the angiogenic capacity of macrophages and highlight the role of cMSC secreted PEDF in the modulation of macrophage phenotype and function. Stem Cells 2018;36:775-784.

In vitro antiretroviral activity and in vivo toxicity of the potential topical microbicide copper phthalocyanine sulfate.

BACKGROUND: Copper has antimicrobial properties and has been studied for its activity against viruses, including HIV. Copper complexed within a phthalocyanine ring, forming copper (II) phthalocyanine sulfate (CuPcS), may have a role in microbicide development when used intravaginally. METHODS: CuPcS toxicity was tested against cervical epithelial cells, TZM-BL cells, peripheral blood mononuclear cells (PBMC), and cervical explant tissues using cell viability assays. In vivo toxicity was assessed following intravaginal administration of CuPcS in female BALB/C mice and measured using a standardized histology grading system on reproductive tract tissues. Efficacy studies for preventing infection with HIV in the presence of various non-toxic concentrations of CuPcS were carried out in TZM-BL, PBMC, and cervical explant cultures using HIV-1BAL and various pseudovirus subtypes. Non-linear regression was applied to the data to determine the EC50/90 and CC50/90. RESULTS: CuPcS demonstrated inhibition of HIV infection in PBMCs at concentrations that were non-toxic in cervical epithelial cells and PBMCs with EC50 values of approximately 50 µg/mL. Reproductive tract tissue analysis revealed no toxicity at 100 mg/mL. Human cervical explant tissues challenged with HIV in the presence of CuPcS also revealed a dose-response effect at preventing HIV infection at non-toxic concentrations with an EC50 value of 65 µg/mL. CONCLUSION: These results suggest that CuPcS may be useful as a topical microbicide in concentrations that can be achieved in the female genital tract.

Critical role of non-muscle myosin light chain kinase in thrombin-induced endothelial cell inflammation and lung PMN infiltration.

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.

LPS activation of Toll-like receptor 4 signals CD11b/CD18 expression in neutrophils.

We identify herein a novel signaling function of the Toll-like receptor-4 (TLR4), the lipopolysaccharide (LPS) receptor mediating the innate immune response, in inducing the expression of CD11b/CD18 integrin in polymorphonuclear leukocytes (PMNs). Studies were made in PMNs isolated from TLR4-deficient (TLR4(-/-)) and C57BL/6 [wild-type (WT)] mice. We observed increased CD11b expression in WT PMNs within 3 h after LPS challenge, whereas CD11b was not expressed in TLR4(-/-) PMNs above basal levels. TLR4-activated CD11b expression was cycloheximide sensitive and involved the activation of transcription factors, NF-kappaB and c-Jun/PU.1. TLR4(-/-) PMNs challenged with LPS were functionally defective as the result of the impaired CD11b expression in that they failed to adhere and did not migrate across endothelial cells in response to N-formylmethionyl-leucyl-phenylalanine. TLR4 also promoted increased binding of LPS to PMNs on the basis of expression of CD11b. Thus TLR4 signaling activates synthesis and upregulation of CD11b and is essential for PMN adhesion and transmigration. Our data suggest an important role of TLR4-activated CD11b expression in the mechanism of the PMN host-defense response to LPS.

RhoA/Rho-associated kinase pathway selectively regulates thrombin-induced intercellular adhesion molecule-1 expression in endothelial cells via activation of I kappa B kinase beta and phosphorylation of RelA/p65.

We investigated the involvement of the RhoA/Rho-associated kinase (ROCK) pathway in regulating ICAM-1 expression in endothelial cells by the procoagulant, thrombin. Exposure of HUVECs to C3 exoenzyme, a selective inhibitor of Rho, markedly reduced thrombin-induced ICAM-1 expression. Inhibition of ROCK, the downstream effector of Rho, also prevented thrombin-induced ICAM-1 expression. Blockade of thrombin-induced ICAM-1 expression was secondary to inhibition of NF-kappaB activity, the key regulator of ICAM-1 expression in endothelial cells. In parallel studies we observed that inhibition of the RhoA/ROCK pathway by the same pharmacological and genetic approaches failed to inhibit TNF-alpha-induced NF-kappaB activation and ICAM-1 expression. The effect of RhoA/ROCK inhibition on thrombin-induced NF-kappaB activation was secondary to inhibition of IkappaB kinase activation and subsequent IkappaBalpha degradation and nuclear uptake and the DNA binding of NF-kappaB. Inhibition of the RhoA/ROCK pathway also prevented phosphorylation of Ser(536) within the transactivation domain 1 of NF-kappaB p65/RelA, a critical event conferring transcriptional competency to the bound NF-kappaB. Thus, the RhoA/ROCK pathway signals thrombin-induced ICAM-1 expression through the activation of IkappaB kinase, which promotes NF-kappaB binding to ICAM-1 promoter and phosphorylation of RelA/p65, thus mediating the transcriptional activation of bound NF-kappaB.

Synergistic anti-inflammatory activity of omega-3 lipid and rofecoxib pretreatment on macrophage proinflammatory cytokine production occurs via divergent NF-kappaB activation.

UNLABELLED: Omega-3 lipid pretreatment significantly decreases TNF-alpha production in LPS-stimulated Mphis; however, this response is only a partial inhibition, suggesting that other nonsubstrate- (lipid) dependent mechanisms are involved. The cyclooxygenase (COX)-2 enzyme is principally responsible for lipid metabolism; thus, a selective COX-2 inhibitor (Rofecoxib) would clarify if it is an omega-3 lipid direct effect or a COX-2 enzyme-associated modulated reduction in TNF-alpha. Moreover, potential synergy between omega-3 lipids and selective COX-2 inhibition is postulated. HYPOTHESIS: Through divergent regulatory mechanisms, omega-3 lipids in combination with Rofecoxib will synergistically decrease the LPS-stimulated Mphi inflammatory response. METHODS: RAW 264.7 cells were pretreated with omega-3 lipids, Rofecoxib, or combination treatment and then washed and exposed to LPS. Supernatants were collected for ELISA, total proteins were obtained to determine COX-2 protein expression by Western blot, and nuclear extracts were isolated to determine NF-kappaB activation by electromobility shift assay. RESULTS: TNF-alpha and PGE2 production was significantly decreased with omega-3 and Rofecoxib pretreatment, and with combination treatment a further decrease in TNF-alpha production was observed. COX-2 protein expression was demonstrated to increase in omega-3, Rofecoxib, and combination groups stimulated with LPS. No alteration in NF-kappaB activation was observed with Rofecoxib or combination pretreatment compared with LPS-stimulated control cells. Repletion of prostaglandin (PGE2) in the Mphi model significantly decreased TNF-alpha in all groups. CONCLUSIONS: Omega-3 lipids and Rofecoxib independently decrease TNF-alpha and PGE2 production in LPS-stimulated Mphi, yet in combination a synergistic reduction in TNF-alpha production is observed. Although the anti-inflammatory effects observed from omega-3 lipids are known to occur partially through decreasing NF-kappaB activation, we demonstrated that Rofecoxib or even a combination of omega-3 and Rofecoxib does not alter NF-kappaB activation, as seen with omega-3 lipids alone. These data support that combination treatment may result in decreased Mphi inflammation, yet this occurs via divergent mechanisms.

cAMP targeting of p38 MAP kinase inhibits thrombin-induced NF-kappaB activation and ICAM-1 expression in endothelial cells.

We investigated the mechanisms by which elevated intracellular cAMP concentration inhibits the thrombin-induced ICAM-1 expression in endothelial cells. Exposure of human umbilical vein endothelial cells to forskolin or dibutyryl cAMP, which increase intracellular cAMP by separate mechanisms, inhibited the thrombin-induced ICAM-1 expression. This effect of cAMP was secondary to inhibition of NF-kappaB activity, the key regulator of thrombin-induced ICAM-1 expression in endothelial cells. The action of cAMP occurred downstream of IkappaBalpha degradation and was independent of NF-kappaB binding to the ICAM-1 promoter. We observed that cAMP interfered with thrombin-induced phosphorylation of NF-kappaB p65 (RelA) subunit, a crucial event promoting the activation of the DNA-bound NF-kappaB. Because p38 MAPK can induce transcriptional activity of RelA/p65 without altering the DNA binding function of NF-kappaB, we addressed the possibility that cAMP antagonizes thrombin-induced NF-kappaB activity and ICAM-1 expression by preventing the activation of p38 MAPK. We observed that treating cells with forskolin blocked the activation of p38 MAPK, and inhibition of p38 MAPK interfered with phosphorylation of RelA/p65 induced by thrombin. Our data demonstrate that increased intracellular cAMP concentration in endothelial cells prevents thrombin-induced ICAM-1 expression by inhibiting p38 MAPK activation, which in turn prevents phosphorylation of RelA/p65 and transcriptional activity of the bound NF-kappaB.

Inhibition of activator protein-1 transcription factor activation by omega-3 fatty acid modulation of mitogen-activated protein kinase signaling kinases.

BACKGROUND: Lipopolysaccharide (LPS)-stimulated macrophages (Mphi) produce excess tumor necrosis factor (TNF), and the direct inhibition of IkappaB phosphorylation and its subsequent separation from the nuclear factor kappaB (NFkappaB)-IkappaB complex has been experimentally supported as a mechanism for omega-3 fatty acid (FA) inhibition of this TNF response. However, TNF production is a "late" event in the LPS-induced Mpsi inflammatory cascade, and in addition to NFkappaB-associated pathways, a separate transcription factor, activator protein-1 (AP-1) is an important pathway for Mpsi proinflammatory cytokine production. The mitogen-activated protein kinase (MAPK) cascade regulates both NFkappaB-IkappaB--and AP-1-associated gene transcription through several cross-amplifying phosphorylation kinases, specifically p44/42 [ie, extracellular signal-regulated kinase (ERK) 1/2], p38, and c//jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The activation of these kinases occurs in the proximal MAPK cascade and activation modulates AP-1 activation. In this set of experiments, it was hypothesized that inhibition of MAPK signaling phosphorylation kinases by omega-3 fatty acids in a model of LPS-stimulated Mphi(s) would alter the activation of the proinflammatory cytokine transcription factor AP-1. METHODS: RAW 264.7 cells were pretreated with a sterile, commercially available, pharmaceutical grade omega-3 FA emulsion, equivalent grade omega-6 FA emulsion, or Dulbecco's modified eagles medium (media alone) for 4 hours. Cells were washed twice and exposed to LPS for 15 minutes. Total cell lysates were collected, and both total and phosphorylated portions of the p44/42, p38, and JNK/SAPK proteins were determined by Western blotting. AP-1 nuclear translocation was determined by electromobility shift assay. RESULTS: Phosphorylation of p44/42 and JNK/SAPK proteins of the MAPK pathways in LPS-stimulated Mpsi(s) was significantly reduced by omega-3 FA treatment compared with Mphi treated with omega-6 FA or media alone. In contrast, phosphorylation of p38 was not inhibited in the presence of omega-3 or (omega-6 FA treatment compared with media alone. Omega-3 FA pretreatment inhibited AP-1 activation. CONCLUSIONS: omega-3 FA inhibited p44/42 and JNK/SAPK phosphorylation; however, p38 remained unchanged. Phosphorylation of p44/42 and JNK/SAPK are the immediate prior steps in AP-1 activation. Attenuated AP-1 activation and subsequent attenuated gene-level proinflammatory cytokine elaboration is anticipated after inhibition of these MAPK intermediates and is confirmed by the reduction in AP-1 activity. These results provide further evidence for the transcriptional level regulation in the elaboration of proinflammatory cytokines by omega-3 FA in this Mphi model.

Tumor necrosis factor-alpha induces early-onset endothelial adhesivity by protein kinase Czeta-dependent activation of intercellular adhesion molecule-1.

We tested the hypothesis that TNF-alpha induces early-onset endothelial adhesivity toward PMN by activating the constitutive endothelial cell surface ICAM-1, the beta2-integrin (CD11/CD18) counter-receptor. Stimulation of human pulmonary artery endothelial cells with TNF-alpha resulted in phosphorylation of ICAM-1 within 1 minute, a response that was sustained up to 15 minutes after TNF-alpha challenge. We observed that TNF-alpha induced 10-fold increase in PMN adhesion to endothelial cells in an ICAM-1-dependent manner and that this response paralleled the rapid time course of ICAM-1 phosphorylation. We also observed that the early-onset TNF-alpha-induced endothelial adhesivity was protein synthesis-independent and associated with cell surface ICAM-1 clustering. Pretreatment of cells with the pan-PKC inhibitor, chelerythrine, prevented the activation of endothelial adhesivity. As PKCzeta, an atypical PKC isoform abundantly expressed in endothelial cells, is implicated in signaling TNF-alpha-induced ICAM-1 gene transcription, we determined the possibility that PKCzeta was involved in mediating endothelial adhesivity through ICAM-1 expression. We observed that TNF-alpha stimulation of endothelial cells induced PKCzeta activation and its association with ICAM-1. Inhibition of PKCzeta by pharmacological and genetic approaches prevented the TNF-alpha-induced phosphorylation and the clustering of the cell surface ICAM-1 as well as activation of endothelial adhesivity. Thus, TNF-alpha induces early-onset, protein synthesis-independent expression of endothelial adhesivity by PKCzeta-dependent phosphorylation of cell surface ICAM-1 that precedes the de novo ICAM-1 synthesis. The rapid ICAM-1 expression represents a novel mechanism for promoting the stable adhesion of PMN to endothelial cells that is needed to facilitate the early-onset transendothelial migration of PMN.