Niclosamide Prevents Systemic Sclerosis in a Reactive Oxygen Species-Induced Mouse Model.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibrosis of the skin and inner organs, vasculopathy, and immunological abnormalities. Recent insights on the implication of STAT3, AKT, and Wnt/ß-catenin in fibrosis have prompted us to investigate, in a mouse model of ROS-induced SSc, the effects of niclosamide, an antihelmintic drug that inhibits both of these signaling pathways. SSc was induced in BALB/c mice by daily s.c. injections of hypochlorous acid (HOCl). Mice were treated or not every other day, 5 d a week, for 6 wk, by niclosamide. Skin and lung fibrosis as well as immunological features were studied. Mice exposed to HOCl developed a diffuse cutaneous SSc with pulmonary fibrosis and anti-DNA topoisomerase 1 autoantibodies. STAT3, AKT, and Wnt/ß-catenin pathways were hyperactivated in the skin and the lungs of diseased mice. Niclosamide reversed fibrosis of the skin and the lungs. Beneficial immunological effects were also observed because niclosamide decreased the activation of CD4+ and CD8+ T cells, autoimmune B cell activation, as well as IL-4 and IL-13 production in the skin. The improvement permitted by niclosamide in the mouse model of HOCl-induced SSc as well as the well-documented safety profile of this drug provide a rationale for the evaluation of niclosamide in the management of patients affected by this disease.

Imbalance of the Vanin-1 Pathway in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and visceral organs and vascular alterations. SSc pathophysiology involves systemic inflammation and oxidative stress. Because the vanin-1 gene (vnn1) encodes an enzyme with pantetheinase activity that converts vasculoprotective pantethine into profibrotic pantothenic acid and pro-oxidant cystamine, we tested this pathway in the pathophysiology of SSc. Activation of the vanin-1/pantetheinase pathway was investigated in wild-type BALB/c mice with hypochlorous acid (HOCl)-induced SSc by ELISA and Western blotting. We then evaluated the effects of the inactivation of vnn1 on the development of fibrosis, endothelial alterations, and immunological activation in mice with HOCl- and bleomycin-induced SSc. We then explored the vanin-1/pantetheinase pathway in a cohort of patients with SSc and in controls. In wild-type mice with HOCl-induced SSc, the vanin-1/pantetheinase pathway was dysregulated, with elevation of vanin-1 activity in skin and high levels of serum pantothenic acid. Inactivation of the vnn1 gene in vnn1-/- mice with HOCl-induced SSc prevented the development of characteristic features of the disease, including fibrosis, immunologic abnormalities, and endothelial dysfunction. Remarkably, patients with diffuse SSc also had increased expression of vanin-1 in skin and blood and elevated levels of serum pantothenic acid that correlated with the severity of the disease. Our data demonstrate that vanin-1/pantetheinase controls fibrosis, vasculopathy, autoimmunity, and oxidative stress in SSc. The levels of vanin-1 expression and pantothenic acid determine SSc severity and can be used as markers of disease severity. More importantly, inhibition of vanin-1 can open new therapeutic approaches in SSc.

ADAM9 expression promotes an aggressive lung adenocarcinoma phenotype.

A disintegrin and metalloproteinase 9 (ADAM9) possesses potent metastasis-inducing capacities and is highly expressed in several cancer cells. Previous work has shown that ADAM9 participates in the adhesive-invasive phenotype in lung cancer cells in vitro. In this study, we evaluated whether ADAM9 expression plays a critical role in metastatic processes in vivo and in angiogenesis. We first found that high ADAM9 expression was correlated with poor lung adenocarcinoma patient prognosis on Prognoscan data base. In vivo model based on intravenous injection in nude mice showed that a stable downregulation of ADAM9 in A549 (TrA549 A9-) cells was associated with a lower number of nodules in the lung, suggesting lower potentials for extravasation and metastasis. On a subcutaneous xenograft we showed that TrA549 A9- produced significantly smaller tumours and exhibited fewer neovessels. In addition, in vitro human umbilical vein endothelial cells exposed to supernatant from TrA549 A9- could reduce the formation of more vessel-like structures. To further understand the mechanism, a human antibody array analysis confirmed that five cytokines were downregulated in TrA549 A9- cells. Interleukin 8 was the most significantly downregulated, and its interaction with CXCR2 was implicated in angiogenesis on an in vitro model. These results emphasize the critical influence of ADAM9 on lung cancer progression and aggressiveness. ADAM9 should at least be a marker of cancer aggressiveness and a potential therapeutic target for cancer treatment.

RhoA/Rho-kinase activation promotes lung fibrosis in an animal model of systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a connective-tissue disease characterized by vascular injury, immune-system disorders, and excessive fibrosis of the skin and multiple internal organs. Recent reports found that RhoA/Rho-kinase (ROCK) pathway is implicated in various fibrogenic diseases. Intradermal injection of hypochlorous acid (HOCl)-generating solution induced inflammation, autoimmune activation, and fibrosis, mimicking the cutaneous diffuse form of SSc in humans. Our study aimed firstly to describe pulmonary inflammation and fibrosis induced by HOCl in mice, and secondly to determine whether fasudil, a selective inhibitor of ROCK, could prevent lung and skin fibroses in HOCl-injected mice. METHODS: Female C57BL/6 mice received daily intradermal injection of hypochlorous acid (HOCl) for 6 weeks to induce SSc, with and without daily treatment with fasudil (30 mg·kg(-1)·day(-1)) by oral gavage. RESULTS: HOCl intoxication induced significant lung inflammation (macrophages and neutrophils infiltration), and fibrosis. These modifications were prevented by fasudil treatment. Simultaneously, HOCl enhanced ROCK activity in lung and skin tissues. Inhibition of ROCK reduced skin fibrosis, expression of α-smooth-muscle actin and 3-nitrotyrosine, as well as the activity of ROCK in the fibrotic skin of HOCl-treated mice, through inhibition of phosphorylation of Smad2/3 and ERK1/2. Fasudil significantly decreased the serum levels of anti-DNA-topoisomerase-1 antibodies in mice with HOCl-induced SSc. CONCLUSIONS: Our findings confirm HOCl-induced pulmonary inflammation and fibrosis in mice, and provide further evidence for a key role of RhoA/ROCK pathway in several pathological processes of experimental SSc. Fasudil could be a promising therapeutic approach for the treatment of SSc.

The level of H2O2 type oxidative stress regulates virulence of Theileria-transformed leukocytes.

Theileria annulata infects predominantly macrophages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macrophages are aggressively invasive, but this virulence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their characterization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of monocytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α activates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes maintain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H2 O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels consequently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence.

Amelioration of systemic fibrosis in mice by angiotensin II receptor blockade.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by microvascular damage, fibrosis of skin and visceral organs, and autoimmunity. Previous studies have shown that angiotensin II is involved in the synthesis of type I collagen. We investigated whether the blockade of angiotensin II receptor type I (AT1 ) by irbesartan reduces skin and lung fibrosis in 2 murine models of SSc. METHODS: SSc was induced by daily intradermal injection of HOCl into the backs of BALB/c mice (HOCl-induced SSc). Mice were treated daily with irbesartan by oral gavage. RESULTS: Irbesartan reduced dermal thickness, collagen concentration, Smad2/3, and α-smooth muscle actin expression, as well as fibroblast proliferation and H-Ras expression in the skin of mice with HOCl-induced SSc. Mice treated with irbesartan also displayed less lung fibrosis, less inflammation, and a lower concentration of collagen in the lungs than untreated mice. Exhaled nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine expression in the lungs were decreased following irbesartan treatment. Moreover, irbesartan reduced the number and the proliferation of splenic B and T cells and the serum levels of anti-DNA topoisomerase I autoantibodies. CONCLUSION: Irbesartan, an AT1 antagonist, prevents fibrosis and inflammation and inhibits nitric oxide production in HOCl-induced models of systemic fibrosis. Our findings extend the indication of an AT1 antagonist to SSc patients with diffuse fibrosis, especially those with lung involvement.

Arsenic trioxide prevents murine sclerodermatous graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) follows allogeneic hematopoietic stem cell transplantation. It results from alloreactive processes induced by minor MHC incompatibilities triggered by activated APCs, such as plasmacytoid dendritic cells (pDCs), and leading to the activation of CD4 T cells. Therefore, we tested whether CD4(+) and pDCs, activated cells that produce high levels of reactive oxygen species, could be killed by arsenic trioxide (As(2)O(3)), a chemotherapeutic drug used in the treatment of acute promyelocytic leukemia. Indeed, As(2)O(3) exerts its cytotoxic effects by inducing a powerful oxidative stress that exceeds the lethal threshold. Sclerodermatous GVHD was induced in BALB/c mice by body irradiation, followed by B10.D2 bone marrow and spleen cell transplantation. Mice were simultaneously treated with daily i.p. injections of As(2)O(3). Transplanted mice displayed severe clinical symptoms, including diarrhea, alopecia, vasculitis, and fibrosis of the skin and visceral organs. The symptoms were dramatically abrogated in mice treated with As(2)O(3). These beneficial effects were mediated through the depletion of glutathione and the overproduction of H(2)O(2) that killed activated CD4(+) T cells and pDCs. The dramatic improvement provided by As(2)O(3) in the model of sclerodermatous GVHD that associates fibrosis with immune activation provides a rationale for the evaluation of As(2)O(3) in the management of patients affected by chronic GVHD.

Arsenic trioxide exerts antitumor activity through regulatory T cell depletion mediated by oxidative stress in a murine model of colon cancer.

Immunotherapy is a promising antitumor strategy that can successfully be combined with current anticancer treatment. In this study, arsenic trioxide (As(2)O(3)) was shown to increase the antitumor immune response in CT26 colon tumor-bearing mice through the modulation of regulatory T cell (T(reg)) numbers. As(2)O(3) induced T(reg)-selective depletion in vitro. In vivo, tumor-bearing mice injected with 1 mg/kg As(2)O(3) showed a significant decrease in the T(reg)/CD4 cell ratio and in absolute T(reg) count versus controls. As(2)O(3) exerted antitumor effects only in immunocompetent mice and enhanced adoptive immunotherapy effects. Inhibition of As(2)O(3)-induced T(reg) depletion by the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester and the superoxide dismutase mimic manganese [III] tetrakis-(5, 10, 15, 20)-benzoic acid porphyrin suggested that it was mediated by oxidative and nitrosative stress. The differential effect of As(2)O(3) on T(reg) versus other CD4 cells may be related to differences in the cells' redox status, as indicated by significant differences in 2'7'dichlorodihydrofluorescein diacetate and 4,5-diaminofluorescein diacetate fluorescence levels. In conclusion, these results show for the first time, to our knowledge, that low doses As(2)O(3) can delay solid tumor growth by depleting T(regs) through oxidative and nitrosative bursts, and suggest that As(2)O(3) could be used to enhance the antitumor activity of adoptive immunotherapy strategies in human cancer.

Sunitinib inhibits the phosphorylation of platelet-derived growth factor receptor ß in the skin of mice with scleroderma-like features and prevents the development of the disease.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by fibrosis of the skin and visceral organs, vascular dysfunction, and immunologic dysregulation. Platelet-derived growth factors (PDGFs) have been implicated in the development of fibrosis and dysregulation of vascular function. We investigated the effects of sunitinib and sorafenib, two tyrosine kinase inhibitors that interfere with PDGF signaling, in a mouse model of diffuse SSc. METHODS: SSc was induced in BALB/c mice by subcutaneous injections of HOCl daily for 6 weeks. Mice were randomized to treatment with sunitinib, sorafenib, or vehicle. The levels of native and phosphorylated PDGF receptor ß (PDGFRß) and vascular endothelial growth factor receptor (VEGFR) in the skin were assessed by Western blot and immunohistochemical analyses. Skin and lung fibrosis were evaluated by histologic and biochemical methods. Autoantibodies were detected by enzyme-linked immunosorbent assay, and spleen cell populations were analyzed by flow cytometry. RESULTS: Phosphorylation of PDGFRß and VEGFR was higher in fibrotic skin from HOCl-injected mice with SSc than from PBS-injected mice. Injections of HOCl induced cutaneous and lung fibrosis, increased the proliferation rate of fibroblasts in areas of fibrotic skin, increased splenic B cell and T cell counts, and increased anti-DNA topoisomerase I autoantibody levels in BALB/c mice. All of these features were reduced by sunitinib but not by sorafenib. Sunitinib significantly reduced the phosphorylation of both PDGF and VEGF receptors. CONCLUSION: Inhibition of the hyperactivated PDGF and VEGF pathways by sunitinib prevented the development of fibrosis in HOCl-induced murine SSc and may represent a new SSc treatment for testing in clinical trials.

Reactive oxygen species-mediated killing of activated fibroblasts by arsenic trioxide ameliorates fibrosis in a murine model of systemic sclerosis.

OBJECTIVE: In patients with systemic sclerosis (SSc), activated fibroblasts produce reactive oxygen species (ROS) that stimulate their proliferation and collagen synthesis. By analogy with tumor cells that undergo apoptosis upon cytotoxic treatment that increases ROS levels beyond a lethal threshold, we tested whether activated fibroblasts could be selectively killed by the cytotoxic molecule arsenic trioxide (As(2) O(3) ) in a murine model of SSc. METHODS: SSc was induced in BALB/c mice by daily intradermal injections of HOCl. Mice were simultaneously treated with daily intraperitoneal injections of As(2) O(3) . RESULTS: As(2) O(3) limited dermal thickness and inhibited collagen deposition, as assessed by histologic examination and measurement of mouse skin and lung collagen contents. As(2) O(3) abrogated vascular damage, as shown by serum vascular cell adhesion molecule 1 level, and inhibited the production of autoantibodies, interleukin-4 (IL-4), and IL-13 by activated T cells. These beneficial effects were mediated through ROS generation that selectively killed activated fibroblasts containing low levels of glutathione. CONCLUSION: Our findings indicate that treatment with As(2) O(3) dramatically improves skin and lung fibrosis in a mouse model of SSc, providing a rationale for the evaluation of As(2) O(3) treatment in patients with SSc.

The mTOR/AKT inhibitor temsirolimus prevents deep infiltrating endometriosis in mice.

Deep infiltrating endometriosis (DIE) is a particular clinical and histological entity of endometriosis responsible for chronic pelvic pain and infertility. Here we characterize the proliferative phenotype of DIE cells, to explore the cellular and molecular mechanisms that could explain their aggressive potential. In addition, the inhibition of mTOR/AKT pathway was tested, as a potential treatment of DIE. Included were 22 patients with DIE and 12 control patients without endometriosis. Epithelial and stromal cells were extracted from biopsies of eutopic endometrium and deep infiltrating endometriotic nodules from patients with DIE. Cell proliferation was determined by thymidine incorporation. Oxidative stress was assayed by spectrofluorometry. The ERK and mTOR/AKT pathways were analyzed in vitro by Western blot and for AKT in vivo in a mouse model of DIE. The proliferation rate of eutopic endometrial cells and of deep infiltrating endometriotic cells from DIE patients was higher than that of endometrial cells from controls. The hyperproliferative phenotype of endometriotic cells was associated with an increase in endogenous oxidative stress, and with activation of the ERK and mTOR/AKT pathways. mTOR/AKT inhibition by temsirolimus decreased endometriotic cell proliferation both in vitro and in vivo in a mouse model of DIE. Blocking the mTOR/AKT pathway offers new prospects for the treatment of DIE.

Clopidogrel protects from cell apoptosis and oxidative damage in a mouse model of renal ischaemia-reperfusion injury.

Renal ischaemia-reperfusion injury (IRI) is consecutive to tissue oxidative damage and cell apoptosis that lead to acute renal failure (ARF) in renal allografts. The aim of this study was to investigate the beneficial effects of a pretreatment by clopidogrel on renal IRI in mice. IRI was induced by bilateral renal ischaemia for 45 min followed by reperfusion. Sixty-two healthy male BALB/c mice were randomly assigned to one of the following groups: PBS + ischaemia-reperfusion (IR); clopidogrel + IR; PBS + sham IR; clopidogrel + sham IR. Clopidogrel (25 mg/kg) or PBS was administered per os to the animals via a gastric cannula 24 h before operation. All mice were given a single dose of clopidogrel or PBS. Renal function histological damage, renal cell apoptosis, renal antioxidant activities, and CD41 expression were determined 24 h after reperfusion. The survival rates were evaluated over 7 days. Animals pretreated with clopidogrel had lower plasma levels of blood urea nitrogen (BUN) and creatinine, lower histopathological scores, and improved survival rates following IR. Renal cell apoptosis induced by IR was decreased in kidneys of mice pretreated by clopidogrel, with an increase in Bcl-2 and Bcl-xL expression and a decrease in caspase-3, caspase-8, and Bax expression. Renal reduced glutathione, superoxide dismutase, and catalase activities were unmodified by the pretreatment with clopidogrel. However, clopidogrel resulted in an increased total antioxidant capacity of the kidney. Furthermore, pretreatment by clopidogrel decreased the number of CD41-positive cells. Thus, clopidogrel exerts protective effects on renal IRI in mice by abrogating renal cell apoptosis as a consequence of improved renal antioxidant capacity and could be tried as a novel therapeutic tool in renal IRI.

Sialylation levels of anti-proteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis (Wegener's).

OBJECTIVE: To investigate whether the glycosylation and sialylation levels of anti-proteinase 3 (anti-PR3) antibodies could affect their pathogenicity, and whether these levels could be correlated with the activity of granulomatosis with polyangiitis (Wegener's) (GPA). METHODS: Forty-two serum samples positive for anti-PR3 antibodies from 42 patients with active or weakly active/inactive GPA were included. Anti-PR3 antibodies were assayed by enzyme-linked immunosorbent assay, and their levels of glycosylation and sialylation were assessed by enzyme-linked lectin assay. The glycosylation and sialylation levels of IgG purified from the serum of healthy donors and patients with active, remitted, or weakly active disease were assessed by permethylation and mass spectrometry analysis of glycans, following neuraminidase digestion. The neutrophil oxidative burst induced by purified IgG was assayed by spectrofluorimetry. RESULTS: The mean sialylation ratio of anti-PR3 antibodies was significantly lower in patients with active disease than in patients with weakly active or inactive disease, and this was inversely correlated with the Birmingham Vasculitis Activity Score (BVAS) (P < 0.0001). Similar results were obtained using the BVAS/GPA. The area under the receiver operating characteristic curve for the sialylation ratio of anti-PR3 antibodies, as a test to determine the activity of GPA, was 0.82 (P = 0.0006). The characterization of N-glycans showed a decrease in 2,6-linked sialylated N-glycans and an increase in dHex1 Hex3 HexNAc4 (mass/charge 1,836) agalactosylated structures in purified IgG from patients with active disease compared with controls. The anti-PR3 antibody-induced oxidative burst of neutrophils was inversely correlated with the sialylation levels of anti-PR3 IgG. CONCLUSION: The sialylation level of anti-PR3 antibodies contributes to the clinical activity of GPA, by modulating the oxidative burst of neutrophils induced by these autoantibodies.

Increased NOS coupling by the metabolite tetrahydrobiopterin (BH4) reduces preeclampsia/IUGR consequences.

Preeclampsia (PE) is a high-prevalence pregnancy disease characterized by placental insufficiency, gestational hypertension, and proteinuria. Overexpression of the A isoform of the STOX1 transcription factor (STOX1A) recapitulates PE in mice, and STOX1A overexpressing trophoblasts recapitulate PE patients hallmarks in terms of gene expression and pathophysiology. STOX1 overexpression induces nitroso-redox imbalance and mitochondrial hyper-activation. Here, by a thorough analysis on cell models, we show that STOX1 overexpression in trophoblasts alters inducible nitric oxide synthase (iNOS), nitric oxide (NO) content, the nitroso-redox balance, the antioxidant defense, and mitochondrial function. This is accompanied by specific alterations of the Krebs cycle leading to reduced l-malate content. By increasing NOS coupling using the metabolite tetrahydrobiopterin (BH4) we restore this multi-step pathway in vitro. Moving in vivo on two different rodent models (STOX1 mice and RUPP rats, alike early onset and late onset preeclampsia, respectively), we show by transcriptomics that BH4 directly reverts STOX1-deregulated gene expression including glutathione metabolism, oxidative phosphorylation, cholesterol metabolism, inflammation, lipoprotein metabolism and platelet activation, successfully treating placental hypotrophy, gestational hypertension, proteinuria and heart hypertrophy. In the RUPP rats we show that the major fetal issue of preeclampsia, Intra Uterine Growth Restriction (IUGR), is efficiently corrected. Our work posits on solid bases BH4 as a novel potential therapy for preeclampsia.

Bystander effect of vinorelbine alters antitumor immune response.

The bystander effect (BE) is the ability of malignant cells affected by an anticancer agent to induce damage in neighboring cancer cells. In this study, we showed that it could also affect immune cells surrounding the tumor and interfere with the antitumor immune response. We observed that the exposure of human lung cancer cells A549 to vinorelbine induced a BE on neighboring human peripheral blood mononuclear cells (PBMCs) in vitro and on mice splenocytes in vivo. In vitro, the number of PBMCs killed because of their coculture with vinorelbine-pretreated A549 cells was 33% higher than those killed by A549 control cells (p = 0.003). In addition, we showed that when vinorelbine-pretreated A549 cells were injected into immunocompetent mice, splenocyte proliferation ex vivo toward tumor cells decreased by 27% compared with that seen in mice injected with untreated A549 cells (p = 0.03). Finally, in vivo experiment in A549 tumor bearing nude mice showed that adoptive transfer of A549 immune splenocytes was not able to delay tumor growth when vinorelbine-pretreated A549 cells were used for immunization. Inhibition of the BE by the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester, and the superoxide dismutase mimic, mangafodipir, suggested that it was mediated by oxidative and nitrosative stress. In conclusion, exposure of cancer cells to vinorelbine alters the antitumor immune response through a BE mediated by cellular oxidative and nitrosative stress. Our results offer new prospects for using oxidative stress modulators to restore the antitumor immune response in patients treated with anticancer agents.

Postresuscitation syndrome: potential role of hydroxyl radical-induced endothelial cell damage.

OBJECTIVE: After out of hospital cardiac arrest, it has been reported that endothelium dysfunction may occur during the postresuscitation syndrome. However, the consequences of the reperfusion phase on endothelial reactive oxygen species production and redox homeostasis have not been explored in out of hospital cardiac arrest patients. DESIGN: Prospective, observational study. SETTING: Medical intensive care unit in a university hospital. PATIENTS: Twenty successfully resuscitated out of hospital cardiac arrest patients, seven septic shock patients, and ten healthy volunteers. INTERVENTION: Plasma was collected from patients at admission and 12, 24, 36, 48, and 72 hrs after cardiac arrest. We studied the production of reactive oxygen species and cell survival during plasma perfusion using perfused endothelial cells (human umbilical vein endothelial cells) as a model. Cell antioxidant response was studied by measuring superoxide dismutase, glutathione peroxidase, and glutathione reductase activities and reduced and oxidized glutathione levels. Mitochondrial respiratory chain activity was assessed by measuring complex I, II, III, and IV activities and anaerobic glycolysis by measuring glucose-6-phosphate dehydrogenase activity. MEASUREMENTS AND MAIN RESULTS: Using perfused endothelial cells as a model, we demonstrate that plasma from out of hospital cardiac arrest patients induced on naive human umbilical vein endothelial cells a significant and massive cell death compared to plasma from septic shock patients and healthy volunteers. An increase of reactive oxygen species production with a decrease in antioxidant defenses (superoxide dismutase, glutathione peroxidase, and glutathione reductase activities, reduced and oxidized glutathione levels) was observed. The metabolic consequence of plasma exposure showed that mitochondrial respiratory chain activity was significantly impaired and anaerobic glycolysis was significantly increased. Inhibiting hydroxyl radical production significantly decreased cell death, suggesting that plasma from out of hospital cardiac arrest induced significant cell death by triggering the Fenton reaction. CONCLUSION: Plasma from out of hospital cardiac arrest induces major endothelial toxicity with an acute pro-oxidant state in the cells and impairment of mitochondrial respiratory chain activity. This toxicity could be due to hydroxyl radical production by activation of the Fenton reaction.

Production of superoxide anions by keratinocytes initiates P. acnes-induced inflammation of the skin.

Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes), a gram-positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at determining whether reactive oxygen species (ROS) are produced by keratinocytes upon P. acnes infection, dissecting the mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response induced by P. acnes. In our hands, ROS, and especially superoxide anions (O2(*-)), were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In P. acnes-stimulated keratinocytes, O2(*-) was produced by NAD(P)H oxidase through activation of the scavenger receptor CD36. O2(*-) was dismuted by superoxide dismutase to form hydrogen peroxide which was further detoxified into water by the GSH/GPx system. In addition, P. acnes-induced O2(*-) abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O2(*-) with nitric oxide to form peroxynitrites. Finally, retinoic acid derivates, the most efficient anti-acneic drugs, prevent O2(*-) production, IL-8 release and keratinocyte apoptosis, suggesting the relevance of this pathway in humans.

Antiproliferative effects of cannabinoid agonists on deep infiltrating endometriosis.

Deep infiltrating endometriosis (DIE) is characterized by chronic pain, hyperproliferation of endometriotic cells and fibrosis. Since cannabinoids are endowed with antiproliferative and antifibrotic properties, in addition to their psychogenic and analgesic effects, cannabinoid agonists have been evaluated in DIE both in vitro and in vivo. The in vitro effects of the cannabinoid agonist WIN 55212-2 were evaluated on primary endometriotic and endometrial stromal and epithelial cell lines extracted from patients with or without DIE. Cell proliferation was determined by thymidine incorporation and production of reactive oxygen species by spectrofluorometry. ERK and Akt pathways were studied by immunoblotting. Immunoblotting of α-smooth muscle actin was studied as evidence of myofibroblastic transformation. The in vivo effects of WIN 55212-2 were evaluated on Nude mice implanted with human deep infiltrating endometriotic nodules. The in vitro treatment of stromal endometriotic cells by WIN 55212-2 decreased cell proliferation, reactive oxygen species production, and α-smooth muscle actin expression. The decrease in cell proliferation induced by WIN 55212-2 was not associated with a decrease in ERK activation, but was associated with the inhibition of Akt activation. WIN 55212-2 abrogated the growth of endometriotic tissue implanted in Nude mice. Cannabinoid agonists exert anti-proliferative effects on stromal endometriotic cells linked to the inhibition of the Akt pathway. These beneficial effects of cannabinoid agonists on DIE have been confirmed in vivo.

Targeting the cannabinoid pathway limits the development of fibrosis and autoimmunity in a mouse model of systemic sclerosis.

Our aim was to evaluate the roles of the cannabinoid pathway in the induction and propagation of systemic sclerosis (SSc) in a mouse model of diffuse SSc induced by hypochlorite injections. BALB/c mice injected subcutaneously every day for 6 weeks with PBS or hypochlorite were treated intraperitoneally with either WIN-55,212, an agonist of the cannabinoid receptors 1 (CB1) and receptors 2 (CB2), with JWH-133, a selective agonist of CB2, or with PBS. Skin and lung fibrosis were then assessed by histological and biochemical methods, and the proliferation of fibroblasts purified from diseased skin was assessed by thymidine incorporation. Autoantibodies were detected by ELISA, and spleen cell populations were analyzed by flow cytometry. Experiments were also performed in mice deficient for CB2 receptors (Cnr2(-/-)). Injections of hypochlorite induced cutaneous and lung fibrosis as well as increased the proliferation rate of fibroblasts isolated from fibrotic skin, splenic B cell counts, and levels of anti-DNA topoisomerase-1 autoantibodies. Treatment with WIN-55,212 or with the selective CB2 agonist JWH-133 prevented the development of skin and lung fibrosis as well as reduced fibroblast proliferation and the development of autoantibodies. Experiments performed in CB2-deficient mice confirmed the influence of CB2 in the development of systemic fibrosis and autoimmunity. Therefore, we demonstrate that the CB2 receptor is a potential target for the treatment of SSc because it controls both skin fibroblast proliferation and the autoimmune reaction.

Protein kinase inhibitors can control the progression of endometriosis in vitro and in vivo.

Endometriosis affects 6-10% of women in their reproductive years, causing chronic pelvic pain and infertility. Its pathogenesis remains poorly understood and current treatments, based on hormonal therapy or surgery, are often insufficient. The purpose of our study was to investigate the role of the ERK pathway in the development of endometriosis and to test the effects of protein kinase inhibitors on the proliferation of endometriotic cells in vitro and in vivo. We studied ex vivo human endometrial and endometriotic cells in culture. Stromal and epithelial cells were extracted from endometrial and endometriotic biopsies from patients with endometriosis and from patients without endometriosis. The ERK pathway was explored by western blot on cell lysates and by ELISA on total crushed specimens of endometrium. Cells in culture were treated with A771726, PD98059, and U0126. Human endometriotic lesions were implanted in nude mice. Mice were treated with A771726, leflunomide, PD98059, U0126 or PBS during 2 weeks before sacrifice and extraction of the endometriotic implants for histological examination. We found that the ERK pathway was significantly activated in endometriotic cells and in endometrial cells from patients with endometriosis compared to endometrial cells of control patients, both by ELISA and by western blot. This phenomenon was associated with an increased proliferation of endometriotic cells compared to endometrial cells. Treating endometriotic cells with A771726, PD98059 or U0126 abrogated the phosphorylation of ERK and significantly decreased the cellular proliferation in vitro. In vivo, A771726, leflunomide, PD98059, and U0126 controlled the growth of endometriotic implants in the mouse model of endometriosis. Our study shows that protein kinase inhibitors could be new candidates to treat endometriosis. However, further studies are needed to evaluate their effects and tolerability in humans.