Objective. No therapy for fibrotic disease is available. The proadhesive matricellular protein connective tissue growth factor CCN2 is a marker of fibrotic cells; however, the specific role of CCN2 in connective tissue biology in general and in fibrogenesis in particular is unclear. The aim of this study was to assess whether adult mice bearing a smooth muscle cell/fibroblastspecific deletion of CCN2 are resistant to bleomycininduced skin scleroderma.Methods. Cutaneous fibrosis was induced in mice by subcutaneous injection of bleomycin. Untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast/smooth muscle cellspecific deletion of CCN2 were investigated for changes in dermal thickness, collagen content, and the number of ␣-smooth muscle actin (␣-SMA)-positive cells. Dermal fibroblasts were isolated to assess whether the induction of collagen and ␣-SMA messenger RNA in response to transforming growth factor ␤ (TGF␤) was impaired.Results. The loss of CCN2 resulted in resistance to bleomycin-induced skin fibrosis. In response to bleomycin, wild-type mice possessed, but CCN2-deficient mice lacked, abundant ␣-SMA-expressing myofibroblasts within fibrotic lesions. Fibroblast responses to TGF␤, a potent inducer of myofibroblast differentiation, were not affected. Collectively, these results indicate that CCN2 is essential for bleomycin-induced skin fibrosis, likely due to a defect in myofibroblast recruitment.Conclusion. These data indicate that therapeutic strategies that involve blocking CCN2 in vivo may be of benefit in combating fibrotic skin disease.

SummaryIn tissue repair, fibroblasts migrate into the wound to produce and remodel extracellular matrix (ECM). Integrins are believed to be crucial for tissue repair, but their tissue-specific role in this process is poorly understood. Here, we show that mice containing a fibroblast-specific deletion of integrin 1 exhibit delayed cutaneous wound closure and less granulation tissue formation, including reduced production of new ECM and reduced expression of -smooth muscle actin (-SMA). Integrin-1-deficient fibroblasts showed reduced expression of type I collagen and connective tissue growth factor, and failed to differentiate into myofibroblasts as a result of reduced -SMA stress fiber formation. Loss of integrin 1 in adult fibroblasts reduced their ability to adhere to, to spread on and to contract ECM. Within stressed collagen matrices, integrin-1-deficient fibroblasts showed reduced activation of latent TGF. Addition of active TGF alleviated the phenotype of integrin-1-deficient mice. Thus integrin 1 is essential for normal wound healing, where it acts, at least in part, through a TGF-dependent mechanism in vivo.

Objective Enhanced adhesive signaling including activation of the focal adhesion kinase (FAK) is a hallmark of fibroblasts from lung fibrosis patients, and FAK has been therefore hypothesized to be a key mediator of this disease. This study was undertaken to characterize the contribution of FAK to the development of pulmonary fibrosis both in vivo and in vitro. Methods FAK expression and activity were analyzed in lung tissue samples from lung fibrosis patients by immunohistochemistry. Mice orally treated with the FAK inhibitor, PF-562,271, or with siRNA-mediated silencing of FAK, were exposed to intratracheally instilled bleomycin to induce lung fibrosis, and the lungs were harvested for histological and biochemical analysis. Using endothelin-1 (ET-1) as stimulus, cell adhesion and contraction, as well as profibrotic gene expression were studied in fibroblasts isolated from wild type and FAK-deficient mouse embryos. ET-1-mediated FAK activation and gene expression were studied in primary mouse lung fibroblasts, as well as in wild type and integrin β1-deficient fibroblasts. Results Increased FAK expression and activity are upregulated in fibroblast foci and remodeled vessels in lung fibrosis patients. Pharmacological or siRNA-mediated targeting of FAK resulted in marked abrogation of bleomycin-induced lung fibrosis. Loss of FAK impaired the acquisition of a profibrotic phenotype in response to ET-1. Profibrotic gene expression leading to myofibroblast differentiation required cell adhesion, and was driven by Jun N-terminal kinase activation through integrin β1/FAK signaling. Conclusion These results implicate FAK as a central mediator of fibrogenesis, and highlight this kinase as a potential therapeutic target in fibrotic diseases.

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It is well known that essential oil thymol exhibits antibacterial activity. The 13 protective effects of thymol on pig intestine during inflammation is yet to be investigated. In this 14 study, an in vitro lipopolysaccharide (LPS)-induced inflammation model using IPEC-J2 cells was 15 established. Cells were pre-treated with thymol for 1 h and then exposed to LPS for various assays. 16 Interleukin 8 (IL-8) secretion, the mRNA abundance of cytokines, reactive oxygen species (ROS), 17 nutrient transporters, and tight junction proteins was measured. The results showed that LPS 18 stimulation increased IL-8 secretion, ROS production, and tumor necrosis factor alpha (TNF-α) 19 mRNA abundance (P < 0.05), but the mRNA abundance of sodium-dependent glucose transporter 20 1 (SGLT1), excitatory amino acid transporter 1 (EAAC1) and H + /peptide cotransporter 1 (PepT1) 21 were decreased (P < 0.05). Thymol blocked ROS production (P < 0.05) and tended to decrease the 22 production of LPS-induced IL-8 secretion (P = 0.0766). The mRNA abundance of IL-8 and TNF-α 23 was reduced by thymol pre-treatment (P < 0.05), but thymol did not improve the gene expression 24 of nutrient transporters (P > 0.05). The transepithelial electrical resistance (TEER) was reduced 25 and cell permeability increased by LPS treatment (P < 0.05), but these effects were attenuated by 26 thymol (P < 0.05). Moreover, thymol increased zonula occludens-1 (ZO-1) and actin staining in 27 the cells. However, the mRNA abundance of ZO-1 and occludin-3 was not affected by either LPS 28 or thymol treatments. These results indicated that thymol enhances barrier function and reduce 29 ROS production and pro-inflammatory cytokine gene expression in the epithelial cells during 30 inflammation. The regulation of barrier function by thymol and LPS may be at post-transcriptional 31 or post-translational levels.32 Intestinal epithelial cells (IECs) are continuously lined monolayer cells, which play important 36 roles in the animal's physical defense. Normally, IECs function as the first line of defense against 37 the invasion of pathogenic agents in the external environment of gut lumen. 1 The maintenance of 38 the barrier function of IECs contributes to the gut homeostasis and health of animals. Gut disorder 39 and dysfunction might be harmful to the growth performance of food-producing animals, and may 40 induce gut diseases such as inflammatory bowel diseases and diarrhea, possibly due to complex 41 interactions among immunologic, genetic, microbial and environmental factors. 2 For instance, 42 diarrhea is a common gut disorders, which causes almost 5% mortality per year in weaned piglets. 3 43 Therefore, it is necessary to prevent gut disorder and diseases by maintaining proper barrier 44 function of IECs in animals. 45 In addition to the physical barrier, IECs also function as an extrinsic barrier. Under certain 46 circumstances, IECs secrete signaling molecules like mucins, cytokines, and chemokines to 47 prevent the invasion of harmful microorganisms in the gu...

Tissue repair requires that fibroblasts migrate into the wound to produce and remodel extracellular matrix, a process that requires adhesion. Failure to suppress the tissue repair program results in fibrotic disorders that are characterized by excessive adhesive signaling. The role of specific components of adhesive signaling in fibrogenic responses is unclear, but may involve small GTPases such as Rac1. To address the functions of Rac1 in fibroblasts, we generated mice containing a fibroblast-specific deletion of Rac1. These mice show delayed cutaneous wound closure, including reduced collagen production and myofibroblast formation. In cultured Rac1-deficient fibroblasts , adhesion , spreading , and migration were significantly inhibited. Rac1-deficient fibroblasts possessed impaired myofibroblast formation and function as visualized by reduced ␣-smooth muscle actin expression as well as matrix contraction. Both in vivo and in vitro, Rac1-deficient fibroblasts showed a reduced generation of reactive oxygen species; in vitro, hydrogen peroxide alleviated the phenotype of Rac1-deficient fibroblasts. Thus, Rac1 is an essential signaling integrator that is required for normal wound healing and dermal homeostasis. (Am J

Abstract-The availability of nitric oxide (NO), which is required for the normal regulation of vascular tone, may be decreased in preeclampsia, thus contributing to the vascular pathogenesis of this pregnancy disorder. Because ascorbate is essential for the decomposition of S-nitrothiols and the release of NO, we speculated that the ascorbate deficiency typical of preeclampsia plasma might result in decreased rates of decomposition of S-nitrosothiols. We tested the hypothesis that total S-nitrosothiol and S-nitrosoalbumin concentrations are increased in preeclampsia plasma, reflecting a decreased release of NO from these major reservoirs of NO. Gestationally matched plasma samples were obtained (before labor or intravenous MgSO 4 ) from 21 women with preeclampsia and 21 women with normal pregnancy, and plasma samples were also obtained from 12 nonpregnant women of similar age and body mass index during the follicular phase of the menstrual cycle. All were nonsmokers. The assay included ultraviolet-induced decomposition of S-nitrosothiols to liberate NO captured by a florigenic reagent, 4,5-diaminofluoresceine, to produce diaminofluoresceine-Triazole. Preeclampsia plasma contained significantly higher concentrations of total S-nitrosothiols (11.1Ϯ2.9 nmol/mL) than normal pregnancy samples (9.4Ϯ1.5 nmol/mL). Even greater differences were found between preeclampsia plasma and plasma samples from normal pregnancies and nonpregnant women (294Ϯ110, 186Ϯ25, and 151Ϯ25 pmol/mg protein, respectively) when S-nitrosothiol content was expressed per milligram protein. The albumin fraction contained 49.4% of total plasma S-nitrosothiols in the control samples and 53.7% and 56.8% of plasma S-nitrosothiols in normal pregnancy and preeclampsia, respectively. The level of S-nitrosoalbumin was significantly higher in preeclampsia than in normal pregnancy or nonpregnancy plasma (6.3Ϯ1.4, 5.1Ϯ0.7, and 4.2Ϯ1.0 nmol/mL, respectively). The increased concentration of S-nitrosoalbumin in preeclampsia almost completely accounted for the increased levels of S-nitrosothiols in total plasma. Due to combined increases in nitrosothiols and decreases in protein, the preeclampsia plasma concentration of S-nitrosoalbumin was greatly increased on a per milligram of protein basis (271% and 186% compared with normal nonpregnancy and normal pregnancy plasma, respectively). We conclude that S-nitrosoalbumin and total S-nitrosothiol concentrations are significantly increased in preeclampsia plasma and may reflect insufficient release of NO groups in this condition. Key Words: preeclampsia Ⅲ plasma Ⅲ nitrosothiols Ⅲ nitrosoalbumin Ⅲ ascorbate P reeclampsia, a pregnancy-specific disorder characterized by placental abnormalities and maternal vascular endothelial dysfunction, is the major cause of both maternal and fetal/neonatal morbidity and mortality. [1][2][3] Oxidative stress accompanied by a pronounced depletion of ascorbate 4 is thought to contribute to the endothelial dysfunction of preeclampsia. 4 -10 Although specific pathways through which a sh...