CircItgb5 promotes synthetic phenotype of pulmonary artery smooth muscle cells via interacting with miR-96-5p and Uba1 in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare but fatal cardiopulmonary disease mainly characterized by pulmonary vascular remodeling. Aberrant expression of circRNAs has been reported to play a crucial role in pulmonary vascular remodeling. The existing literature predominantly centers on studies that examined the sponge mechanism of circRNAs. However, the mechanism of circRNAs in regulating PAH-related protein remains largely unknown. This study aimed to investigate the effect of circItgb5 on pulmonary vascular remodeling and the underlying functional mechanism. MATERIALS AND METHODS: High-throughput circRNAs sequencing was used to detect circItgb5 expression in control and PDGF-BB-treated pulmonary arterial smooth muscle cells (PASMCs). Localization of circItgb5 in PASMCs was determined via the fluorescence in situ hybridization assay. Sanger sequencing was applied to analyze the circularization of Itgb5. The identification of proteins interacting with circItgb5 was achieved through a RNA pull-down assay. To assess the impact of circItgb5 on PASMCs proliferation, an EdU assay was employed. Additionally, the cell cycle of PASMCs was examined using a flow cytometry assay. Western blotting was used to detect biomarkers associated with the phenotypic switch of PASMCs. Furthermore, a monocrotaline (MCT)-induced PAH rat model was established to explore the effect of silencing circItgb5 on pulmonary vascular remodeling. RESULTS: CircItgb5 was significantly upregulated in PDGF-BB-treated PASMCs and was predominately localized in the cytoplasm of PASMCs. In vivo experiments revealed that the knockdown of circItgb5 attenuated MCT-induced pulmonary vascular remodeling and right ventricular hypertrophy. In vitro experiments revealed that circItgb5 promoted the transition of PASMCs to synthetic phenotype. Mechanistically, circItgb5 sponged miR-96-5p to increase mTOR level and interacted with Uba1 protein to activate the Ube2n/Mdm2/ACE2 pathway. CONCLUSIONS: CircItgb5 promoted the transition of PASMCs to synthetic phenotype by interacting with miR-96-5p and Uba1 protein. Knockdown of circItgb5 mitigated pulmonary arterial pressure, pulmonary vascular remodeling and right ventricular hypertrophy. Overall, circItgb5 has the potential for application as a therapeutic target for PAH.

miR-142-3p is associated with aberrant WNT signaling during airway remodeling in asthma.

Asthma is characterized by a chronic inflammation and remodeling of the airways. Although inflammation can be controlled, therapeutic options to revert remodeling do not exist. Thus, there is a large and unmet need to understand the underlying molecular mechanisms to develop novel therapies. We previously identified a pivotal role for miR-142-3p in regulating airway smooth muscle (ASM) precursor cell proliferation during lung development by fine-tuning the Wingless/Integrase I (WNT) signaling. Thus, we here aimed to investigate the relevance of this interaction in asthma. We performed quantitative RT-PCR and immune staining in a murine model for ovalbumin-induced allergic airway inflammation and in bronchial biopsies from patients with asthma and isolated primary fibroblasts thereof. miR-142-3p was increased in hyperproliferative regions of lung in murine and human asthma, whereas this microRNA (miRNA) was excluded from regions with differentiated ASM cells. Increases in miR-142-3p were associated with a decrease of its known target Adenomatous polyposis coli. Furthermore, we observed a differential expression of miR-142-3p in bronchial biopsies from patients with early or late onset severe asthma, which coincided with a differential WNT signature. Our data suggest that miR-142-3p is involved in regulating the balance between proliferation and differentiation of ASM cells in asthma, possibly via controlling WNT signaling. Thus, this miRNA might be an interesting target to prevent ASM hyperproliferation in asthma.

Vascular wall progenitor cells in health and disease.

The vasculature plays an indispensible role in organ development and maintenance of tissue homeostasis, such that disturbances to it impact greatly on developmental and postnatal health. Although cell turnover in healthy blood vessels is low, it increases considerably under pathological conditions. The principle sources for this phenomenon have long been considered to be the recruitment of cells from the peripheral circulation and the re-entry of mature cells in the vessel wall back into cell cycle. However, recent discoveries have also uncovered the presence of a range of multipotent and lineage-restricted progenitor cells in the mural layers of postnatal blood vessels, possessing high proliferative capacity and potential to generate endothelial, smooth muscle, hematopoietic or mesenchymal cell progeny. In particular, the tunica adventitia has emerged as a progenitor-rich compartment with niche-like characteristics that support and regulate vascular wall progenitor cells. Preliminary data indicate the involvement of some of these vascular wall progenitor cells in vascular disease states, adding weight to the notion that the adventitia is integral to vascular wall pathogenesis, and raising potential implications for clinical therapies. This review discusses the current body of evidence for the existence of vascular wall progenitor cell subpopulations from development to adulthood and addresses the gains made and significant challenges that lie ahead in trying to accurately delineate their identities, origins, regulatory pathways, and relevance to normal vascular structure and function, as well as disease.

Genetically modified human myoblasts with eNOS may improve regenerative ability of myogenic stem cells to infarcted heart.

BACKGROUND: Modern therapies of post infarcted heart failure are focused on perfusion improvement of the injured myocardium. This effect can be achieved by, among other means, implanting stem cells which could be genetically modified with factors inducing the formation of new blood vessels in the post infarction scar area. Combined stem cell and gene therapy seems to be a promising strategy to heal an impaired myocardium. The creation of new blood vessels can be indirectly stimulated via factors inducing vascular endothelial growth factor synthesis, for example endothelial nitric oxide synthase (eNOS). The product of this enzyme, nitric oxide, is a molecule that can influence numerous physiological activities; it can contribute to vasodilation, stimulation of endothelial cell growth, prevention of platelet aggregation and leukocyte adhesion to the endothelium. AIM: To verify the pro-angiogenic and regenerative potential of human primary myoblasts and murine myoblast cell line C2C12 transiently transfected with eNOS gene. METHODS: Stem cells (either human or murine) were maintained in standard in vitro conditions. Next, both types of myoblasts were modified using electroporation and lipofection (human and murine cells), respectively. The efficacy of the transfection method was evaluated using flow cytometry. The concentration of eNOS protein was measured by ELISA immunoassay. The biological properties of modified cells were assessed using an MTT proliferation test and DAPI cell cycle analysis. To verify the influence of oxidative stress on myoblasts, cytometric tests using Annexin V and propidium iodide were applied. To check possible alterations in myogenic gene expression of stem cells transduced by genetic modification, the myogenic regulatory factors were evaluated by real-time PCR. The function of genetic modification was confirmed by a HUVEC capillary sprouting test using myoblasts supernatants. RESULTS: Electroporation turned out to be an efficient transfection method. High amounts of secreted protein were obtained (in the range 2,000 pg/mL) in both cell types studied. Moreover, the functionality of gene overexpression product was confirmed in capillary development assay. Human myoblasts did not exhibit any changes in cell cycle; however, eNOS transfected murine myoblasts revealed a statistically significant reduction in cell cycle ratio compared to controls (p < 0.001). In the case of myogenic gene expression, a decrease in Myogenin level was only detected in the human transfected myoblast population (p < 0.05). CONCLUSIONS: The results of our study may suggest that transplantation of myoblasts overexpressing eNOS could be promising for cell therapy in regenerating the post infarction heart.

Tissue engineered esophagus by mesenchymal stem cell seeding for esophageal repair in a canine model.

PURPOSE: Acellular porcine small intestinal submucosa (SIS) has been successfully used for esophagoplasty in dogs. However, this has not led to complete epithelialization and muscular regeneration. We undertook the present study to assess the effect of tissue-engineered esophagus generated by seeding bone marrow mesenchymal stem cells (BMSCs) onto an SIS scaffold (BMSCs-SIS) in a canine model. METHODS: We cultured, passaged, and measured autologous BMSCs and myoblasts with cell proliferation and immunohistochemical assays. We labeled the third passage of BMSCs with PKH-26, a fluorescent dye, before seeded it onto the SIS. We resected canine cervical esophagus to generate a defect 5 cm in length and 50% in circumference, which we repaired with BMSCs-SIS or SIS alone. RESULTS: Four weeks later, barium esophagram demonstrated that esophageal lumen surface of the patch graft was smoother in the BMSCs-SIS group compared with the SIS group. Histological examination suggested a strong similarity between BMSCs and esophageal myoblasts in terms of morphology and function. Although both BMSCs-SIS and SIS repaired the esophageal defects, we noted complete re-epithelialization with almost no inflammation only in the former group. By 12 wk after the surgery, we observed long bundles of skeletal muscles only in the BMSCs-SIS group, where the microvessel density was also much greater. CONCLUSIONS: Bone marrow mesenchymal stem cells on an SIS scaffold can promote re-epithelialization, revascularization, and muscular regeneration. This approach may provide an attractive option for esophageal regeneration.

Canonical Wnt signaling regulates smooth muscle precursor development in the mouse ureter.

Smooth muscle cells (SMCs) are a key component of many visceral organs, including the ureter, yet the molecular pathways that regulate their development from mesenchymal precursors are insufficiently understood. Here, we identified epithelial Wnt7b and Wnt9b as possible ligands of Fzd1-mediated ß-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated ureteric mesenchyme. Mice with a conditional deletion of Ctnnb1 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional obstruction of the ureter. Histological analysis revealed that the layer of undifferentiated mesenchymal cells directly adjacent to the ureteric epithelium did not undergo characteristic cell shape changes, exhibited reduced proliferation and failed to differentiate into SMCs. Molecular markers for prospective SMCs were lost, whereas markers of the outer layer of the ureteric mesenchyme fated to become adventitial fibroblasts were expanded to the inner layer. Conditional misexpression of a stabilized form of Ctnnb1 in the prospective ureteric mesenchyme resulted in the formation of a large domain of cells that exhibited histological and molecular features of prospective SMCs and differentiated along this lineage. Our analysis suggests that Wnt signals from the ureteric epithelium pattern the ureteric mesenchyme in a radial fashion by suppressing adventitial fibroblast differentiation and initiating smooth muscle precursor development in the innermost layer of mesenchymal cells.

Proteomic characterization of human early pro-angiogenic cells.

Early pro-angiogenic cells (EPCs) have been shown to be involved in neovascularization, angiogenesis and re-endothelialization and cathepsin L inhibition blunted their pro-angiogenic effect. In the present study, we have analysed and mapped the proteome and secretome of human EPCs, utilizing a combination of difference in-gel electrophoresis (DIGE) and shotgun proteomics. A population of 206 protein spots were analysed, with 171 being identified in the cellular proteome of EPCs. 82 proteins were identified in their conditioned medium, including the alternative macrophage markers C-C motif chemokine 18 (CCL18) and the hemoglobin scavenger receptor CD163 as well as platelet factor 4 (CXCL4) and platelet basic protein (CXCL7) with "platelet alpha granule" being returned as the top category according to the Gene Ontology Annotation. Apart from cathepsin L, the cathepsin L inhibitor also attenuated the release of a wide range of other cathepsins and lysosomal proteins such as legumain, but stimulated the secretion of members of the S100 protein family. The data presented here are the most comprehensive characterization of protein expression and secretion in human EPCs to date and highlight the potential importance of cysteine proteases in the processing of platelet factors for their pro-angiogenic potential. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Circulating smooth muscle progenitor cells in arterial remodeling.

The proliferation and migration of vascular smooth muscle cells (SMCs) from the media toward the intimal layer are key components in vascular proliferative diseases. In addition, the differentiation of circulating bone marrow-derived mononuclear cells (BMMCs) into SMCs has been described to contribute to lesion progression in experimental models of atherosclerosis, transplant arteriosclerosis, and neointima formation. In vitro, CD14(+) BMMCs from peripheral blood acquire a spindle-shaped phenotype and express specific SMC markers in response to platelet-derived growth factor-BB. However, the 'trans-differentiation' capacity of BMMCs into definitive SMCs in vivo remains a highly controversial issue. Whereas SMCs within atherosclerotic plaques have been demonstrated to be exclusively of local origin, more severe injury models have shown a wide diversity of SMCs or smooth muscle-like cells derived from BMMCs. In hindsight, these discrepancies may be attributed to methodological differences, e.g., the use of high-resolution microscopy or the specificity of the SMC marker proteins. In fact, the analysis of mouse strains that express marker genes under the control of a highly specific smooth muscle-myosin heavy chain (SM-MHC) promoter and a time-course analysis on the dynamic process of neointima formation have recently shown that BMMCs temporarily express α-smooth muscle actin, not SM-MHC. Additionally, BM-derived cells disappear from the neointimal lesion after the inflammatory response to the injury has subsided. Although CD14(+)/CD68(+) have important paracrine effects on arterial lesion progression, BMMCs account for more of the 'SMC-like macrophages' than the highly 'trans-differentiated' and definitive SMCs in vivo. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

Bone marrow-derived CX3CR1 progenitors contribute to neointimal smooth muscle cells via fractalkine CX3CR1 interaction.

Smooth muscle cells play a major role in numerous vascular diseases that contribute to remodeling, repair after injury, and arteriogenesis, and the source of these cells is thought to lie within the vessel wall and the circulating blood. Currently, the precise origin and mechanism of differentiation of extravascular smooth muscle progenitor cells (SPCs) is unclear. We show here that the CX(3)CR1 mononuclear cell population of murine bone marrow provides a source of SPCs that contributes to smooth muscle cells within the neointimal plaque after vascular injury. Moreover, CX(3)CR1-fractalkine (FKN) interaction in vivo is essential for smooth muscle cell differentiation of bone marrow-derived progenitor cells at the vessel wall level. Functional competence of bone marrow-derived CX(3)CR1 positive cells to interact with FKN is also crucial in part for neointima formation following vascular injury. Finally, in a pure preparation of bone marrow-derived CX(3)CR1 positive cells, we show that in vitro smooth muscle cell differentiation increases markedly in the presence of FKN. Our data highlight a novel functional relationship between the myeloid and vascular systems and in the context of vascular injury and repair underscores a key chemokine-receptor pathway that may regulate cell fate when smooth muscle cell differentiation is required.

The role of a human hematopoietic mesenchymal progenitor in wound healing and fibrotic diseases and implications for therapy.

The human peripheral blood contains a multipotent precursor that shows hematopoietic stem cell features and transiently expresses markers of the myeloid lineage. Under permissive conditions, this precursor gives rise to committed progenitors of various lineages, including a mesenchymal progenitor cell known by the name of fibrocyte. The fibrocytes still express some hematopoietic and myeloid antigens together with fibroblast markers. They constitutively release pro-fibrotic and angiogenic factors and can modulate ongoing inflammatory reactions through the release of a number of chemokines. Under appropriate stimulation, fibrocytes produce increased amounts of extracellular matrix components and acquire a contractile phenotype similar to that of activated fibroblasts (myofibroblasts). Fibrocytes synthesizing new collagen or acquiring myofibroblast markers have been detected in pulmonary diseases characterized by an extensive remodeling of the bronchial wall or progressive fibrosis, in the skin of patients affected by nephrogenic systemic fibrosis, in human hypertrophic scars, in proliferative vitreoretinopathies and atherosclerotic lesions. Similar cells also participate in the stromal reaction to tumor development. Prevention of detrimental tissue remodeling in fibrotic diseases may be achieved by inhibiting the accumulation of fibrocytes. In-vitro expanded fibrocytes may be used to improve ineffective tissue repair or may be engineered for the delivery of gene constructs in anti-cancer therapy.

Cellular phenotypes in human stenotic lesions from haemodialysis vascular access.

BACKGROUND: Haemodialysis vascular access dysfunction (due to venous stenosis and thrombosis) is a leading cause of hospitalization and morbidity. The aim of the current study was to identify the specific cell types present within stenotic tissue samples from patients with AV fistula and graft failure. METHODS: Discarded tissue segments were collected from the stenotic portions (usually near the graft-vein anastomosis or the AV anastomosis) of 23 dialysis grafts and 20 AV fistulae, and examined for expression of smooth muscle alpha actin, desmin, vimentin and a macrophage marker. RESULTS: The majority of cells within the venous neointima (both grafts and fistulae) were myofibroblasts, with a smaller number of desmin positive smooth muscle cells. The graft neointima had a similar cellular phenotype, albeit without any desmin positive contractile smooth muscle cells. The majority of cells within the PTFE graft material were macrophages. Analysis of sequential sections revealed the presence of fibroblasts within the venous neointima and intragraft region. CONCLUSIONS: Our results demonstrate that contractile smooth muscle cells, myofibroblasts, fibroblasts and macrophages all play a role in the pathogenesis of dialysis access dysfunction (grafts and fistulae). Targeting these specific cell types might result in the development of novel therapeutic paradigms for haemodialysis vascular access dysfunction.

TGF-beta1 enhances the expression of alpha-smooth muscle actin in cultured human pulpal fibroblasts: immunochemical and ultrastructural analyses.

Transforming growth factor-beta 1 (TGF-beta1) has been related to induce the expression of alpha-smooth muscle actin (alpha-SMA) in fibroblasts during repair. Because pulpal fibroblasts seem to be somewhat different from other fibroblasts, the present study investigated in vitro whether TGF-beta1 enhances the expression of alpha-SMA in human pulpal fibroblasts. TGF-beta1 was added in doses between 5-10 ng/mL to cultures of both dental pulp and gingival human fibroblasts. The expression of alpha-SMA was analyzed by immunofluorescence and Western blotting, whereas the ultrastructure was evaluated by electron microscopy. In addition, the expression of tenascin, osteonectin, and vimentin was also investigated. Both cell types were immunoreactive for alpha-SMA even without TGF-beta1. When TGF-beta1 was added to cell cultures, the expression of alpha-SMA increased dramatically in pulpal fibroblasts, independent of the concentration used. It was confirmed by the Western blotting analysis. Ultrastructure revealed myofilaments and indented nuclei in both fibroblasts treated with TGF-beta1. Tenascin and osteonectin were only immunolabeled in pulpal fibroblasts treated or not with TGF-beta1. Both fibroblast types were positive for vimentin. The present findings showed that TGF-beta1 up-regulated the expression of alpha-SMA, thus inducing pulpal fibroblasts to acquire the myofibroblast phenotype.

Co-regulation by Notch and Fos is required for cell fate specification of intermediate precursors during C. elegans uterine development.

The Notch pathway is the key signal for many cell fate decisions in the nematode Caenorhabditis elegans including the uterine pi cell fate, crucial for a proper uterine-vulval connection and egg laying. Expression of the egl-13 SOX domain transcription factor is specifically upregulated upon induction of the pi lineage and not in response to other LIN-12/Notch-mediated decisions. We determined that dual regulation by LIN-12 and FOS-1 is required for egl-13 expression at specification and for complete rescue of egl-13 mutants. We found that fos-1 mutants exhibit uterine defects and fail to express pi markers. We show that FOS-1 is expressed at pi cell specification and can bind in vitro to egl-13 upstream regulatory sequence (URS) as a heterodimer with C. elegans Jun.

Interleukin-31 stimulates production of inflammatory mediators from human colonic subepithelial myofibroblasts.

Interleukin (IL)-31 is mainly produced by CD4+ T cells, in particular T cells skewed toward a Th2 phenotype. Here we report for the first time that IL-31 stimulates secretion of proinflammatory cytokines, chemokines and matrix metalloproteinases (MMPs) from human colonic subepithelial myofibroblasts (SEMFs). The effects of IL-31 were investigated by cDNA microarrays, enzyme-linked immunosorbent assay, and real-time PCR. IL-31 effectively induced chemokines [IL-8, GRO-alpha (growth-related oncogene-alpha), MCP-3 (monocyte chemoattractant protein-3), CXCL3, CCL13 and CCL15], proinflammatory cytokines (IL-6, IL-16 and IL-32) and matrix metalloproteinases (MMP-1, MMP-3, MMP-25 and MMP-7). IL-31 dose-dependently induced secretion of IL-6, IL-8, GRO-alpha, MCP-3, MMP-1 and MMP-3. The effects of IL-31 were comparable to the effects of IL-17A. IL-31 and IL-17A showed additive effects on IL-6, IL-8, GRO-alpha, MCP-3, MMP-1 and MMP-3 secretion. In conclusion, we demonstrated that IL-31 is a potent inducer of proinflammatory mediators in human colonic SEMFs. IL-31 may function as a proinflammatory cytokine derived from Th2 cells.

Selective clearance of macrophages in atherosclerotic plaques by the protein synthesis inhibitor cycloheximide.

Macrophages are an essential component of unstable atherosclerotic plaques and play a pivotal role in the destabilization process. We have demonstrated previously that local delivery of the mammalian target of rapamycin (mTOR) inhibitor everolimus selectively clears macrophages in rabbit plaques. Because mTOR controls mRNA translation, inhibition of protein synthesis might induce selective macrophage cell death. We therefore investigated in the present study the effect of the protein synthesis inhibitor cycloheximide on macrophage and smooth muscle cell (SMC) viability. In vitro studies with cultured macrophages and SMCs showed that cycloheximide induced selective apoptosis of macrophages in a concentration- and time-dependent manner. Moreover, macrophages could be selectively depleted in rabbit carotid artery rings with collar-induced atherosclerotic plaques after in vitro treatment with cycloheximide. Local in vivo administration of cycloheximide via osmotic minipumps to rabbit carotid arteries with collar-induced atherosclerotic plaques significantly reduced the macrophage but not the SMC content. Cycloheximide-treated plaques showed signs of apoptosis (increased terminal deoxynucleotidyl transferase end labeling and fluorescein isothiocyanate-Val-Ala-dl-Asp(O-methyl)-fluoromethylketone labeling) that did not colocalize with SMCs. Organ chamber studies demonstrated that the functionality of SMCs and the endothelium were not influenced by cycloheximide treatment. All together, these findings demonstrate that cycloheximide decreases the macrophage load in atherosclerotic plaques by induction of apoptosis without changing SMC content or contractility.

Regulation of amphiregulin and epiregulin expression in human colonic subepithelial myofibroblasts.

Amphiregulin and epiregulin belong to the epidermal growth factor (EGF) family, and act as mitogenic stimulators via binding to EGF receptors (EGFRs). Amphiregulin and epiregulin are thought to play a role in regenerative responses in the gastrointestinal tract. In this study, we investigated secretion of amphiregulin and epiregulin in human colonic subepithelial myofibroblasts (SEMFs). The mRNA expression and protein secretion of amphiregulin and epiregulin were evaluated by Northern blotting and Western blotting, respectively. The trophic effects of amphiregulin and epiregulin on SEMFs were analyzed by MTT assays. Amphiregulin and epiregulin mRNAs were not detected in unstimulated SEMFs. Among the various cytokines and growth factors, interleukin-1beta, tumor necrosis factor-alpha, and EGF strongly induced amphiregulin and epiregulin mRNA expression. These responses were markedly reduced by AG1478, a specific inhibitor of EGF receptor tyrosine kinases. Amphiregulin and epiregulin secretion were also detected at the protein level. MTT assays demonstrated that amphiregulin and epiregulin stimulate the proliferation of SEMFs. We demonstrated expression of amphiregulin and epiregulin in SEMFs. Amphiregulin and epiregulin may play an important role in the mechanism underlying wound healing in damaged colonic mucosa.

Reversal of renal fibrosis, inflammation, and glomerular hypertrophy by kallikrein gene delivery.

Evidence suggests that the progression of renal fibrosis is a reversible process. Because inflammation plays a crucial role in the development of renal injury, we examined the effect of kallikrein and activation of the kinin B2 receptor on the reversal of salt-induced inflammation and renal fibrosis in Dahl salt-sensitive (DSS) rats. Four weeks after high salt loading, when renal injury was apparent, adenovirus harboring the human tissue kallikrein gene was injected into DSS rats. To determine the role of the B2 receptor in mediating the actions of kallikrein, icatibant, a kinin B2 receptor antagonist, was infused with kallikrein gene delivery. Two weeks after adenovirus injection, salt-induced glomerular sclerosis, tubular protein cast formation, and monocyte/ macrophage accumulation in the kidney were notably reversed by kallikrein. Decreased intercellular adhesion molecule-1 expression paralleled this observation. Kallikrein gene delivery also dramatically reduced collagens I, III, and IV and reticulin deposition, accompanied by a decline in myofibroblast accumulation and transforming growth factor-beta(1) expression. Moreover, kallikrein reversed salt-induced glomerular hypertrophy and inhibited the increase in levels of the cell cycle-inhibitory proteins p21 and p27. These protective actions of kallikrein were abolished by icatibant, indicating a B2 receptor-mediated event. In addition, kallikrein protected against salt-induced renal injury by diminishing urinary protein and blood urea nitrogen levels. Furthermore, kallikrein gene delivery restored nitric oxide production and suppressed NADH oxidase activity and superoxide generation. These results indicate that tissue kallikrein, through the kinin B2 receptor, reverses salt-induced inflammation, renal fibrosis, and glomerular hypertrophy via suppression of oxidative stress.

Lung myofibroblasts as targets of salmeterol and fluticasone propionate: inhibition of alpha-SMA and NF-kappaB.

Lung myofibroblasts play a major role in the pathophysiology of asthma, contributing not only to tissue remodelling but also to airway inflammation. Nevertheless, only recently, attention has been focused on these cells as potential targets for anti-allergic drugs. Herein, we analysed the pharmacological response of lung myofibroblasts to beta2-agonists associated or not to inhaled corticosteroids, investigating their effects on (i) the constitutive and transforming growth factor-beta (TGF-beta)-induced expression of alpha-smooth muscle actin (alpha-SMA), the main functional marker of myofibroblastic differentiation and contractility; (ii) isometric contraction and (iii) tumour necrosis factor-alpha (TNF-alpha)-induced nuclear translocation of the pro-inflammatory transcription factor nuclear factor-kappaB (NF-kappaB). The beta2-agonist salmeterol (SMl) has on human lung myofibroblasts new direct anti-contractile/anti-inflammatory effects that are amplified by the combined use of low concentrations of the glucocorticoid fluticasone propionate (FP). First, SMl and/or FP (10(-12) M) inhibits the constitutive and TGF-beta-induced expression of alpha-SMA. Second, the two drugs block the TNF-alpha-induced nuclear translocation of the pro-inflammatory transcription factor NF-kappaB. Finally, SMl decreases TNF- alpha-induced production of the inflammatory cytokine IL-6. The complementary anti-inflammatory/ anti-contractile effects displayed by SMl and FP on lung myofibroblasts in vitro may be related to the improvement in lung function and symptom control obtained in vivo by the early use of low doses of glucocorticoids in combination with long-acting beta2-agonists.

Nicotine inhibits myofibroblast differentiation in human gingival fibroblasts.

Cigarette smoking has been suggested as a risk factor for several periodontal diseases. It has also been found that smokers respond less favorably than non-smokers to periodontal therapy. Previous work in our lab has shown that nicotine inhibits human gingival cell migration. Since myofibroblasts play an important role in wound closure, we asked if nicotine affects gingival wound healing process by regulating myofibroblast differentiation. Human gingival fibroblasts (HGFs) from two patients were cultured in 10% fetal bovine serum cell culture medium. Cells were pretreated with different doses of nicotine (0, 0.01, 0.1, and 1 mM) for 2 h, and then incubated with transforming growth factor beta (TGF-beta1) (0, 0.25, 0.5, and 1 ng/ml) with or without nicotine for 30 h. The expression level of alpha-smooth muscle actin (alpha-SMA), a specific marker for myofibroblasts, was analyzed by Western blots, immunocytochemistry, and real-time polymerase chain reaction (real-time PCR). Phosphorylated p38 mitogen-activated protein kinase (Phospho-p38 MAPK) activity was analyzed by Western blots. TGF-beta1 induced an increase of alpha-SMA protein and mRNA expression, while nicotine (1 mM) inhibited the TGF-beta1-induced expression of alpha-SMA but not beta-actin. Nicotine treatment down-regulated TGF-beta1-induced p38 MAPK phosphorylation. Our results demonstrated for the first time that nicotine inhibits myofibroblast differentiation in human gingival fibroblasts in vitro; supporting the hypothesis that delayed wound healing in smokers may be due to decreased wound contraction by myofibroblasts.

Butyrate blocks interferon-gamma-inducible protein-10 release in human intestinal subepithelial myofibroblasts.

BACKGROUND: Interferon (IFN)-gamma-inducible protein (IP)-10 is a chemoattractant for CXCR 3-expressing T lymphocytes and monocytes. IP-10 has been reported to mediate chronic inflammation such as that in inflammatory bowel disease (IBD). However, the local secretion of IP-10 in the intestine remains unclear. In this study, we investigated IP-10 secretion in human colonic subepithelial myofibroblasts (SEMFs). METHODS: IP-10 secretion was determined by enzyme-linked immunosorbent assay (ELISA), and IP-10 mRNA expression was evaluated by Northern blotting. RESULTS: Interleukin (IL)-10 mRNA was not detected in unstimulated SEMFs. Interferon (IFN)-gamma strongly induced IP-10 mRNA expression. Tumor necrosis factor (TNF)-alpha also stimulated IP-10 mRNA expression, but this was much weaker than that induced by IFN-gamma. The effects of IFN-gamma and TNF-alpha were detected in a dose- and time-dependent manner. These responses were also observed at the protein levels. The IFN-gamma-induced IP-10 secretion was not affected by acetate or propionate, but was significantly reduced by butyrate. Trichostatin A, a specific inhibitor of histone deacetylase, also blocked the IFN-gamma- and TNF-alpha-induced IP-10 mRNA expression, but the effects of trichostatin A were weaker than those of butyrate. The inhibitory effect of butyrate on IFN-gamma-induced IP-10 release was not associated with STAT (signaling transducer and activator of transcription)-1alpha activation. CONCLUSIONS: We demonstrated that human colonic SEMFs are the local site for the secretion IP-10. The regulation of IP-10 release by IFN-gamma and butyrate may play an important role in controlling chronic mucosal inflammation in pathological entities such as IBD.