Monolayer Wound Research Articles

MiR-223 regulates migration and invasion by targeting Artemin in human esophageal carcinoma

BackgroundArtemin (ARTN) is a neurotrophic factor belonging to the glial cell-derived neurotrophic factor family of ligands. To develop potential therapy targeting ARTN, we studied the roles of miR-223 in the migration and invasion of human esophageal carcinoma.MethodsARTN expression levels were detected in esophageal carcinoma cell lines KYSE-150, KYSE-510, EC-9706, TE13, esophageal cancer tissues and paired non-cancerous tissues by Western blot. Artemin siRNA expression vectors were constructed to knockdown of artemin expression mitigated migration and invasiveness in KYSE150 cells. Monolayer wound healing assay and Transwell invasion assay were applied to observe cancer cell migration and invasion. The relative levels of expression were quantified by real-time quantitative PCR.ResultsARTN expression levels were higher in esophageal carcinoma tissue than in the adjacent tissue and was differentially expressed in various esophageal carcinoma cell lines. ARTN mRNA contains a binding site for miR-223 in the 3'UTR. Co-transfection of a mir-223 expression vector with pMIR-ARTN led to the reduced activity of luciferase in a dual-luciferase reporter gene assay, suggesting that ARTN is a target gene of miR-223. Overexpression of miR-223 decreased expression of ARTN in KYSE150 cells while silencing miR-223 increased expression of ARTN in EC9706 cells. Furthermore, overexpression of miR-223 in KYSE150 cells decreased cell migration and invasion. Silencing of miR-223 in EC9706 cells increased cell migration and invasiveness.ConclusionsThese results reveal that ARTN, a known tumor metastasis-related gene, is a direct target of miR-223 and that miR-223 may have a tumor suppressor function in esophageal carcinoma and could be used in anticancer therapies.

Psidin, a conserved protein that regulates protrusion dynamics and cell migration

Dynamic assembly and disassembly of actin filaments is a major driving force for cell movements. Border cells in the Drosophila ovary provide a simple and genetically tractable model to study the mechanisms regulating cell migration. To identify new genes that regulate cell movement in vivo, we screened lethal mutations on chromosome 3R for defects in border cell migration and identified two alleles of the gene psidin (psid). In vitro, purified Psid protein bound F-actin and inhibited the interaction of tropomyosin with F-actin. In vivo, psid mutations exhibited genetic interactions with the genes encoding tropomyosin and cofilin. Border cells overexpressing Psid together with GFP-actin exhibited altered protrusion/retraction dynamics. Psid knockdown in cultured S2 cells reduced, and Psid overexpression enhanced, lamellipodial dynamics. Knockdown of the human homolog of Psid reduced the speed and directionality of migration in wounded MCF10A breast epithelial monolayers, whereas overexpression of the protein increased migration speed and altered protrusion dynamics in EGF-stimulated cells. These results indicate that Psid is an actin regulatory protein that plays a conserved role in protrusion dynamics and cell migration.

Nitric oxide-induced collagen IV expression and angiogenesis: FAK or fiction? Focus on “Collagen IV contributes to nitric oxide-induced angiogenesis of lung endothelial cells”

<i>Nitric oxide-induced collagen IV expression and angiogenesis: FAK or fiction?</i> Focus on “Collagen IV contributes to nitric oxide-induced angiogenesis of lung endothelial cells”

Collagen IV contributes to nitric oxide-induced angiogenesis of lung endothelial cells

Nitric oxide (NO) mediates endothelial angiogenesis via inducing the expression of integrin α(v)β(3). During angiogenesis, endothelial cells adhere to and migrate into the extracellular matrix through integrins. Collagen IV binds to integrin α(v)β(3), leading to integrin activation, which affects a number of signaling processes in endothelial cells. In the present study, we evaluated the role of collagen IV in NO-induced angiogenesis. We found that NO donor 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18) causes increases in collagen IV mRNA and protein in lung endothelial cells and collagen IV release into the medium. Addition of collagen IV into the coating of endothelial culture increases endothelial monolayer wound repair, proliferation, and tube formation. Inhibition of collagen IV synthesis using gene silencing attenuates NOC-18-induced increases in monolayer wound repair, cell proliferation, and tube formation as well as in the phosphorylation of focal adhesion kinase (FAK). Integrin blocking antibody LM609 prevents NOC-18-induced increase in endothelial monolayer wound repair. Inhibition of protein kinase G (PKG) using the specific PKG inhibitor KT5823 or PKG small interfering RNA prevents NOC-18-induced increases in collagen IV protein and mRNA and endothelial angiogenesis. Together, these results indicate that NO promotes collagen IV synthesis via a PKG signaling pathway and that the increase in collagen IV synthesis contributes to NO-induced angiogenesis of lung endothelial cells through integrin-FAK signaling. Manipulation of collagen IV could be a novel approach for the prevention and treatment of diseases such as alveolar capillary dysplasia, severe pulmonary arterial hypertension, and tumor invasion.

A novel role for BRCA1 in regulating breast cancer cell spreading and motility.

BRCA1 C-terminal (BRCT) domains in BRCA1 are essential for tumor suppressor function, though the underlying mechanisms remain unclear. We identified ezrin, radixin, and moesin as BRCA1 BRCT domain-interacting proteins. Ezrin-radixin-moesin (ERM) and F-actin colocalized with BRCA1 at the plasma membrane (PM) of cancer cells, especially at leading edges and focal adhesion sites. In stably expressing cancer cells, high levels of enhanced green fluorescent protein (EGFP)-BRCA1(1634-1863) acted as a dominant-negative factor, displacing endogenous BRCA1 from the PM. This led to delayed cell spreading, increased spontaneous motility, and irregular monolayer wound healing. MCF-7 cells (intact BRCA1) showed lower motility than HCC1937 cells (truncated BRCA1), but expression of EGFP-BRCA1(1634-1863) in MCF-7 increased motility. Conversely, full-length BRCA1 expression in HCC1937 decreased motility but only if the protein retained ubiquitin ligase activity. We conclude that full-length BRCA1 is important for complete tumor suppressor activity via interaction of its BRCT domains with ERM at the PM, controlling spreading and motility of cancer cells via ubiquitin ligase activity.

Connective Tissue Growth Factor (CTGF/CCN2) Mediates Angiogenic Effect of S1P in Human Dermal Microvascular Endothelial Cells

The primary objective of this study was to examine the potential interaction between S1P, a pleiotropic lipid mediator, and CTGF/CCN2, a secreted multimodular protein, in the process of endothelial cell migration. The secondary objective was to determine whether C- and N-terminal domains of CTGF/CCN2 have a specific function in cell migration. Migration of HDMECs was examined in monolayer wound healing "scratch" assay, whereas capillary-like tube formation was examined in three-dimensional collagen co-culture assays. We observed that S1P stimulates migration of HDMECs concomitant with upregulation of CTGF/CCN2 expression. Furthermore, the blockade of endogenous CTGF/CCN2 via siRNA abrogated S1P-induced HDMEC migration and capillary-like tube formation. Full-length CTGF induced cell migration and capillary-like tube formation with a potency similar to that of S1P, while C-terminal domain of CTGF was slightly less effective. However, N-terminal domain had only a residual activity in inducing capillary-like tube formation. This study revealed that CTGF/CCN2 is required for the S1P-induced endothelial cell migration, which suggests that CTGF/CCN2 may be an important mediator of S1P-induced physiological and pathological angiogenesis. Moreover, this study shows that the pro-migratory activity of CTGF/CCN2 is located in the C-terminal domain.

S138 Clara cells inhibit alveolar epithelial wound repair through a TRAIL-dependent apoptosis mechanism

IntroductionAlveolar bronchiolization, a hyperproliferation of ciliated and non-ciliated (Clara) bronchiolar cells and their extension into the alveolar region, is a common feature of idiopathic pulmonary fibrosis. The role of this...

αB-Crystallin Is Elevated in Highly Infiltrative Apoptosis-Resistant Glioblastoma Cells

We have previously established two distinct glioma phenotypes by serial xenotransplantation of human glioblastoma (GBM) biopsies in nude rats. These tumors undergo a gradual transition from a highly invasive nonangiogenic to a less-invasive angiogenic phenotype. In a protein screen to identify molecular markers associated with the infiltrative phenotype, we identified α-basic-crystallin (αBc), a small heat-shock protein with cytoprotective properties. Its increased expression in the infiltrative phenotype was validated by immunohistochemistry and Western blots, confirming its identity to be tumor-derived and not from the host. Stereotactic human GBM biopsies taken from MRI-defined areas verified stronger αBc expression in the infiltrative edge compared to the tumor core. Cell migration assays and immunofluorescence staining showed αBc to be expressed by migrating cells in vitro. To determine αBc function, we altered its expression levels. αBc siRNA depletion caused a loss of migrating tumor cells from biopsy spheroids and delayed monolayer wound closure. In contrast, glioma cell migration in a Boyden chamber assay was unaffected by either αBc knockdown or overexpression, indicating that αBc is not functionally linked to the cell migration machinery. However, after siRNA αBc depletion, a significant sensitization of cells to various apoptotic inducers was observed (actinomycin, tumor necrosis factor α, and TNF-related apoptosis-inducing ligand [TRAIL]). In conclusion, αBc is overexpressed by highly migratory glioma cells where it plays a functional role in apoptosis resistance.

The nontoxic natural compound Curcumin exerts anti-proliferative, anti-migratory, and anti-invasive properties against malignant gliomas

BackgroundNew drugs are constantly sought after to improve the survival of patients with malignant gliomas. The ideal substance would selectively target tumor cells without eliciting toxic side effects. Here, we report on the anti-proliferative, anti-migratory, and anti-invasive properties of the natural, nontoxic compound Curcumin observed in five human glioblastoma (GBM) cell lines in vitro.MethodsWe used monolayer wound healing assays, modified Boyden chamber trans-well assays, and cell growth assays to quantify cell migration, invasion, and proliferation in the absence or presence of Curcumin at various concentrations. Levels of the transcription factor phospho-STAT3, a potential target of Curcumin, were determined by sandwich-ELISA. Subsequent effects on transcription of genes regulating the cell cycle were analyzed by quantitative real-time PCR. Effects on apoptosis were determined by caspase assays.ResultsCurcumin potently inhibited GBM cell proliferation as well as migration and invasion in all cell lines contingent on dose. Simultaneously, levels of the biologically active phospho-STAT3 were decreased and correlated with reduced transcription of the cell cycle regulating gene c-Myc and proliferation marking Ki-67, pointing to a potential mechanism by which Curcumin slows tumor growth.ConclusionsCurcumin is part of the diet of millions of people every day and is without known toxic side effects. Our data show that Curcumin bears anti-proliferative, anti-migratory, and anti-invasive properties against GBM cells in vitro. These results warrant further in vivo analyses and indicate a potential role of Curcumin in the treatment of malignant gliomas.

A mathematical model of intercellular signaling during epithelial wound healing

Recent experiments monitoring the healing process of wounded epithelial monolayers have demonstrated the necessity of MAPK activation for coordinated cell movement after damage. This MAPK activity is characterized by two wave-like phenomena. One MAPK “wave” that originates immediately after injury, propagates deep into the cell sheet, away from the edge, and then rebounds back to the wound interface. After this initial MAPK activity has largely disappeared, a second MAPK front propagates slowly from the wound interface and also continues into the cell sheet, maintaining a sustained level of MAPK activity throughout the cell sheet. It has been suggested that the first wave is initiated by Reactive Oxygen Species (ROS) generated at the time of injury. In this work, we develop a minimal mathematical model that reproduces the observed behavior. The main ingredients of our model are a competition between ligand (e.g., Epithelial Growth Factor) and ROS for the activation of Epithelial Growth Factor Receptor, and a feedback loop between receptor occupancy and MAPK activation. We explore the mathematical properties of the model and look for traveling wave solutions consistent with the experimentally observed MAPK activity patterns.

Coordinate Control of Host Centrosome Position, Organelle Distribution, and Migratory Response by Toxoplasma gondii via Host mTORC2

The invasion of host cells by Toxoplasma gondii is accompanied by a reorganization of host cell structure, in which the host centrosome and Golgi apparatus are localized to the vacuole, and mitochondria, microtubules, and endolysosomes are recruited to the vacuole perimeter. The mechanism and functional significance of this process have not been well defined. Here, we report that the centrosome-vacuole association was abolished in mammalian target of rapamycin complex 2 (mTORC2)-deficient cells, which also displayed a disordered distribution of perivacuolar host mitochondria and lysosomes. Infection of fibroblasts led to stable, mTORC2-dependent activation of Akt, and Akt inhibition mimicked the effect of mTORC2 ablation on centrosome, mitochondria, and lysosome localization. Mobilization of the centrosome by Akt inhibition was abrogated by inhibitors of glycogen synthase kinase 3 (GSK3), implying that the centrosome is constrained to the vacuole through an mTORC2-Akt-GSK3 pathway. Infected cells were incapable of migration in a wounded monolayer model, and this effect was associated with the inability of centrosomes to reorient in the direction of migration. Both migration and centrosome reorientation were fully restored upon ablation of mTORC2. These findings provide the first linkage of host signals to parasite-mediated host cell reorganization and demonstrate migratory suppression as a novel functional consequence of this process that is associated with mTORC2-mediated centrosome constraint.

Abstract LB-267: GRB7: A novel target in basal breast cancer

Abstract Basal breast cancers lack expression of estrogen and progesterone receptors and HER2, rendering them non-responsive to the most commonly used targeted therapies in breast cancer. These tumors are typically more aggressive, more locally invasive and more likely to metastasize to the brain than other breast cancer subtypes and, consequently, have a poor prognosis. GRB7 is a cytoplasmic adaptor protein that interacts with receptor tyrosine kinases, relaying information to their downstream signaling cascades. It also plays a role in integrin signaling and cell migration by binding focal adhesion kinase (FAK) and ephrin receptors. It is a part of the HER2 amplicon and is frequently overexpressed in HER2-amplified breast tumors. Breast tumors in which HER2 and GRB7 are both co-amplified and co-overexpressed have a poor prognosis. We and others have recently determined that high levels of GRB7 are associated with recurrence in women with triple-negative breast cancer treated with anthracyclines (Sparano et al. J Clin Oncol 27(15s) 500). In this study, we have tested the hypothesis that triple-negative breast cancer cell lines are dependent on GRB7 for proliferation, migration and invasion. We have utilized a highly specific cell-penetrating peptide inhibitor of GRB7 (G7-18NATE) and tested its effects on a panel of 5 basal breast cancer cell lines in a series of culture assays: monolayer wound healing assays (motility on 2D substrates), transwell invasion assays (in vitro invasiveness) and 3D Matrigel cultures (morphogenesis in 3D environment). In all cases evaluated, the Grb7 inhibitory peptide impaired the proliferation, migration and invasion of these cell lines, even though these cells express low levels of GRB7 compared to HER2-amplified cell lines. These data implicate GRB7 as a key mediator of several important phenotypes of basal breast cancer cells and suggest that GRB7 itself, or GRB7-dependent pathways, may prove to be important targets for the development of specific therapies for this disease. This work was supported in part by Susan G. Komen for the Cure (KG091136). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-267.

HMGB1 accelerates alveolar epithelial repair via an IL‐1β‐dependent activation of TGF‐β1 by the αvβ6 integrin.

High mobility group box 1 (HMGB1) protein, recently described as a proinflammatory mediator of acute lung injury, is a danger‐signaling molecule shown to be actively secreted or passively released from damaged cells. We hypothesized that HMGB1 would accelerate the re‐epithelialisation of the distal lung epithelium, a mechanism that is crucial for the recovery from acute lung injury. We found in the present study that HMGB1 is released in the extracellular space by alveolar epithelial cells that underwent a scratch wound. HMGB1 increased primary rat and human alveolar type II cell monolayer wound repair via an IL‐1β‐dependent activation of TGF‐β1. Specifically, HMGB1 induced the release of IL‐1β that caused the activation of TGF‐β1 via a p38 MAPK, RhoA and αvβ6 integrin ‐dependent mechanism. Furthermore, active TGF‐β1 accelerated the wound closure of primary rat epithelial cell monolayers via a PI3Kα‐dependent mechanism. In conclusion, this study demonstrates that HMGB1 accelerates wound closure in the distal lung epithelium via the IL‐1β‐induced αvβ6‐dependent activation of TGF‐β1, and could thus play a critical role in the resolution of acute lung injury by promoting repair of the injured alveolar epithelium.

Simulation of lung alveolar epithelial wound healing in vitro

The mechanisms that enable and regulate alveolar type II (AT II) epithelial cell wound healing in vitro and in vivo remain largely unknown and need further elucidation. We used an in silico AT II cell-mimetic analogue to explore and better understand plausible wound healing mechanisms for two conditions: cyst repair in three-dimensional cultures and monolayer wound healing. Starting with the analogue that validated for key features of AT II cystogenesis in vitro, we devised an additional cell rearrangement action enabling cyst repair. Monolayer repair was enabled by providing 'cells' a control mechanism to switch automatically to a repair mode in the presence of a distress signal. In cyst wound simulations, the revised analogue closed wounds by adhering to essentially the same axioms available for alveolar-like cystogenesis. In silico cell proliferation was not needed. The analogue recovered within a few simulation cycles but required a longer recovery time for larger or multiple wounds. In simulated monolayer wound repair, diffusive factor-mediated 'cell' migration led to repair patterns comparable to those of in vitro cultures exposed to different growth factors. Simulations predicted directional cell locomotion to be critical for successful in vitro wound repair. We anticipate that with further use and refinement, the methods used will develop as a rigorous, extensible means of unravelling mechanisms of lung alveolar repair and regeneration.

Wound Healing Assays in Well Plate—Coupled Microfluidic Devices with Controlled Parallel Flow

The migratory or proliferative responses mounted by wounded cell monolayers are important to drug discovery and drug safety testing, as well as to basic research across a number of disciplines, including stem-cell biology, cell biology, ophthalmology, endocrinology, microbiology, oncology, and developmental biology. Scratch wounding by mechanical means is the golden standard to achieve an appropriate model system in which to study these cellular reactions. The scratch wounding technique is plagued by wound size irregularity, release of cytosolic contents along the wound edge, and difficulty in scaling up to higher throughput screening. To address these issues, we developed a microfluidic device coupled to a well plate in which wounds were produced enzymatically using highly controlled laminar flow streams. Within the device, epithelial cells were cultivated and exposed to different compounds. Proliferation and migration were characterized by bright-field microscopy. Resulting wound size was highly uniform compared with reported variability of manual scratch wounding. The protocol for wounding was fully automated using customized software, and response to wounding was followed in real time by microscopy.

β-Actin Association with Endothelial Nitric-oxide Synthase Modulates Nitric Oxide and Superoxide Generation from the Enzyme

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that beta-actin is associated with eNOS oxygenase domain and that association of eNOS with beta-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that beta-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to beta-actin and prevented eNOS association with beta-actin in vitro. Peptide 326 also prevented beta-actin-induced decrease in superoxide formation and increase in NO and L-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-beta-actin binding and to prevent beta-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-beta-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-beta-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that beta-actin binding to eNOS through residues 326-333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by beta-actin plays an important role in endothelial function.

159. EXTRACELLULAR MATRIX DYNAMICS IN SCAR-FREE ENDOMETRIAL REPAIR

Tissue destruction and repair occur simultaneously within the menstruating uterus. Whilst the factors that control menstruation are increasingly understood (1) the endometrial milieu which governs repair remains elusive. The extracellular-matrix (ECM) plays a dynamic role within the repairing endometrium, with roles suggested for fibronectin and certain integrins (2). We here use both an in vivo mouse model of breakdown and repair (3) and an in vitro model of re-epithelialisation to determine factors involved in repair of this tissue. Murine uterine horns were subjected to laser-capture microdissection of the repairing luminal epithelium (LE) and immediate sub-luminal stroma. Extracted RNA was processed for pathway-focused array that revealed expression of epithelial and leukocyte expressed integrins along with their ligands, proteases and protease inhibitors and basement membrane proteins as elevated in repairing uterine horns. Immunohistochemistry confirmed expression and localisation of integrins a 5 and b 1 in addition to integrin ligands VCAM-1 and fibronectin. Additionally, we demonstrated intimate association of neutrophils with repairing/recently repaired luminal epithelium. To confirm the relevance of these ECM interactions in a human model, human endometrial luminal epithelial cell (ECC-1) monolayers were wounded and treated with inhibitors of MMPs (doxycycline, GM6001) or integrin-fibronectin interactions (RGDS). Significant inhibition of in vitro re-epithelialisation was seen following treatment of wounded ECC-1 monolayers with RGDS, doxycycline or GM6001, suggesting a requirement for these ECM components in endometrial re-epithelialisation. We subsequently demonstrated that menstrual blood derived factors may play a role in the initial endometrial re-epithelialisation. Menstrual blood and peripheral blood were collected on the same day from volunteers. Treatment of wounded ECC-1 monolayers with menstrual blood plasma enhanced the rate of repair compared with peripheral blood plasma from the same woman. Enhanced understanding of the mechanisms governing scar-free repair in the endometrium may help resolve pathological endometrial bleeding disorders and may be applied to aid healing in other tissues. (1) Jabbour et al, 2006. Endocr Rev. 27(1): 17–46.(2) Cao et al, 2007. Hum Reprod. 22(12): 3223–31(3) Brasted et al, 2003. Biol Reprod. 69(4): 1273–80.

Laminin-derived peptide AG73 regulates migration, invasion, and protease activity of human oral squamous cell carcinoma cells through syndecan-1 and β1 integrin

Squamous cell carcinoma is a prevalent head and neck tumor with high mortality. We studied the role played by laminin alpha1 chain peptide AG73 on migration, invasion, and protease activity of cells (OSCC) from human oral squamous cell carcinoma. Immunohistochemistry and immunofluorescence analyzed expression of laminin alpha1 chain and MMP9 in oral squamous cells carcinoma in vivo and in vitro. Migratory activity of AG73-treated OSCC cells was investigated by monolayer wound assays and in chemotaxis chambers. AG73-induced invasion was assessed in Boyden chambers. Invasion depends on MMPs. Conditioned media from cells grown on AG73 was subjected to zymography. We searched for AG73 receptors related to these activities in OSCC cells. Immunofluorescence analyzed AG73-induced colocalization of syndecan-1 and beta1 integrin. Cells had these receptors silenced by siRNA, followed by treatment with AG73 and analysis of migration, invasion, and protease activity. Oral squamous cell carcinoma expresses laminin alpha1 chain and MMP9. OSCC cells treated with AG73 showed increased migration, invasion, and protease activity. AG73 induced colocalization of syndecan-1 and beta1 integrin. Knockdown of these receptors decreased AG73-dependent migration, invasion, and protease activity. Syndecan-1 and beta1 integrin signaling downstream of AG73 regulate migration, invasion, and MMP production by OSCC cells.

Geniposide inhibits interleukin-6 and interleukin-8 production in lipopolysaccharide-induced human umbilical vein endothelial cells by blocking p38 and ERK1/2 signaling pathways

The aim of this study was to investigate the inhibitory effect of geniposide on lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and interleukin-8 (IL-8) production in human umbilical vein endothelial cells (HUVECs). Primary HUVECs were used. The mRNA/protein levels of IL-6 and IL-8 was determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). LPS-induced HUVEC migration and adhesion of monocytes to HUVECs were studied by monolayer wound healing experiments and monocytic cell adhesion assay, respectively. Expression of nuclear factor kappaB (NF-kappaB), inhibitory factor kappaB-alpha (IkappaB-alpha), p38 mitogen-activated protein kinase (MAPK) and ERK1/2 were determined by Western blot analysis. Geniposide effectively inhibited LPS-induced expression of IL-6 and IL-8 in HUVECs at the transcription and translation levels. Additionally, geniposide suppressed LPS-induced HUVEC migration and U937 monocyte adhesion to HUVECs. Signal transduction studies indicate that geniposide blocked the activation of NF-kappaB, degradation of IkappaB-alpha, and phosphorylation of p38 MAPK and ERK1/2 in HUVECs challenged by LPS. The results show that geniposide can inhibit LPS-induced IL-6 and IL-8 production in HUVECs by blocking p38 MAPK and ERK1/2 signaling pathways.

Fibroblast nemosis induces angiogenic responses of endothelial cells

Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-κB. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.