Svitlana Danchuk

Human multipotent stromal cells attenuate lipopolysaccharide-induced acute lung injury in mice via secretion of tumor necrosis factor-α-induced protein 6

IntroductionMultipotent stromal cells (MSCs) are currently in clinical trials for a number of inflammatory diseases. Recent studies have demonstrated the ability of MSCs to attenuate inflammation in rodent models of acute lung injury (ALI) suggesting that MSCs may also be beneficial in treating ALI.MethodsTo better understand how human MSCs (hMSCs) may act in ALI, the lungs of immunocompetent mice were exposed to lipopolysaccharide (LPS) and four hours later bone marrow derived hMSCs were delivered by oropharyngeal aspiration (OA). The effect of hMSCs on lung injury was assessed by measuring the lung wet/dry weight ratio and total protein in bronchoalveolar lavage (BAL) fluid 24 or 48 h after LPS. BAL fluid was also analyzed for the presence of inflammatory cells and cytokine expression by multiplex immunoassay. Microarray analysis of total RNA isolated from treated and untreated lungs was performed to elucidate the mechanism(s) involved in hMSC modulation of lung inflammation.ResultsAdministration of hMSCs significantly reduced the expression of pro-inflammatory cytokines, neutrophil counts and total protein in bronchoalveolar lavage. There was a concomitant reduction in pulmonary edema. The anti-inflammatory effects of hMSCs were not dependent on localization to the lung, as intraperitoneal administration of hMSCs also attenuated LPS-induced inflammation in the lung. Microarray analysis revealed significant induction of tumor necrosis factor (TNF)-α-induced protein 6 (TNFAIP6/ TSG-6) expression by hMSCs 12 h after OA delivery to LPS-exposed lungs. Knockdown of TSG-6 expression in hMSCs by RNA interference abrogated most of their anti-inflammatory effects. In addition, intra-pulmonary delivery of recombinant human TSG-6 reduced LPS-induced inflammation in the lung.ConclusionsThese results show that hMSCs recapitulate the observed beneficial effects of rodent MSCs in animal models of ALI and suggest that the anti-inflammatory properties of hMSCs in the lung are explained, at least in part, by activation of hMSCs to secrete TSG-6.

Deregulation of selective autophagy during aging and pulmonary fibrosis: the role of TGFβ1.

Aging constitutes a significant risk factor for fibrosis, and idiopathic pulmonary fibrosis (IPF) is characteristically associated with advancing age. We propose that age‐dependent defects in the quality of protein and cellular organelle catabolism may be causally related to pulmonary fibrosis. Our research found that autophagy diminished with corresponding elevated levels of oxidized proteins and lipofuscin in response to lung injury in old mice and middle‐aged mice compared to younger animals. More importantly, older mice expose to lung injury are characterized by deficient autophagic response and reduced selective targeting of mitochondria for autophagy (mitophagy). Fibroblast to myofibroblast differentiation (FMD) is an important feature of pulmonary fibrosis in which the profibrotic cytokine TGFβ1 plays a pivotal role. Promotion of autophagy is necessary and sufficient to maintain normal lung fibroblasts’ fate. On the contrary, FMD mediated by TGFβ1 is characterized by reduced autophagy flux, altered mitophagy, and defects in mitochondrial function. In accord with these findings, PINK1 expression appeared to be reduced in fibrotic lung tissue from bleomycin and a TGFβ1‐adenoviral model of lung fibrosis. PINK1 expression is also reduced in the aging murine lung and biopsies from IPF patients compared to controls. Furthermore, deficient PINK1 promotes a profibrotic environment. Collectively, this study indicates that an age‐related decline in autophagy and mitophagy responses to lung injury may contribute to the promotion and/or perpetuation of pulmonary fibrosis. We propose that promotion of autophagy and mitochondrial quality control may offer an intervention against age‐related fibrotic diseases.

Comparison of the therapeutic effects of human and mouse adipose-derived stem cells in a murine model of lipopolysaccharide-induced acute lung injury.

IntroductionAdipose-derived stem cells (ASCs) have emerged as important regulators of inflammatory/immune responses in vitro and in vivo and represent attractive candidates for cell-based therapies for diseases that involve excessive inflammation. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive due to lack of effective therapies. In this study, the therapeutic effects of ASC-based therapy were assessed in vivo by comparison of the anti-inflammatory properties of both human and murine ASCs in a mouse model of lipopolysaccharide (LPS)-induced ALI.MethodsHuman ASCs (hASCs) or mouse ASCs (mASCs) were delivered to C57Bl/6 mice (7.5 × 105 total cells/mouse) by oropharyngeal aspiration (OA) four hours after the animals were challenged with lipopolysaccharide (15 mg/kg). Mice were sacrificed 24 and 72 hours after LPS exposure, and lung histology examined for evaluation of inflammation and injury. Bronchoalveolar lavage fluid (BALF) was analyzed to determine total and differential cell counts, total protein and albumin concentrations, and myeloperoxidase (MPO) activity. Cytokine expression in the injured lungs was measured at the steady-state mRNA levels and protein levels for assessment of the degree of lung inflammation.ResultsBoth human and mouse ASC treatments provided protective anti-inflammatory responses. There were decreased levels of leukocyte (for example neutrophil) migration into the alveoli, total protein and albumin concentrations in BALF, and MPO activity after the induction of ALI following both therapies. Additionally, cell therapy with both cell types effectively suppressed the expression of proinflammatory cytokines and increased the anti-inflammatory cytokine interleukin 10 (IL-10). Overall, the syngeneic mASC therapy had a more potent therapeutic effect than the xenogeneic hASC therapy in this model.ConclusionsTreatment with hASCs or mASCs significantly attenuated LPS-induced acute lung injury in mice. These results suggest a potential benefit for using an ASC-based therapy to treat clinical ALI and may possibly prevent the development of acute respiratory distress syndrome (ARDS).

Marinobufagenin Inhibits Proliferation and Migration of Cytotrophoblast and CHO Cells

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid that is involved in the inhibition of the sodium pump Na(+)/K(+)-ATPase. Increased plasma levels of MBG have been reported in patients with volume expansion-mediated hypertension and preeclampsia. We have recently demonstrated that MBG impairs both the proliferation and growth factor-induced migration of human first trimester cytotrophoblast (CTB) cells, crucial for proper placental development. However, the intracellular signaling mechanisms regulating the MBG-induced impairment of CTB differentiation, migration and invasion are unknown. The human extravillous CTB cell line SGHPL-4 was utilized for this study. The phosphorylation of MAP kinase protein ERK1/2 was evaluated by Cellular Activation of Signaling ELISA (CASE) in control CTB cells and those treated with MBG. MBG at concentrations of 10 and 100nM inhibited CTB cell proliferation, migration and invasion (60%, 50% and 50%, respectively). MBG also caused a significant decrease in the phosphorylation of ERK1/2. In addition, MBG decreased proliferation, migration, and ERK1/2 activity in another motile cell line, CHO cells. Another sodium pump inhibitor, ouabain, similarly decreased proliferation and ERK1/2 activity in CTB and CHO cells. These data suggest that the changes observed in cell function may be mediated by inhibition of Na(+)/K(+)-ATPase. We demonstrate that the MBG-induced impairment of CTB cell proliferation, migration and invasion is associated with decreased ERK1/2 activity which may be mediated by inhibition of Na(+)/K(+)-ATPase.

Interleukin 6 Mediates the Therapeutic Effects of Adipose-Derived Stromal/Stem Cells in Lipopolysaccharide-Induced Acute Lung Injury

Adipose‐derived stromal/stem cells (ASCs) have anti‐inflammatory as well as immunosuppressive activities and are currently the focus of clinical trials for a number of inflammatory diseases. Acute lung injury (ALI) is an inflammatory condition of the lung for which standard treatment is mainly supportive due to lack of effective therapies. Our recent studies have demonstrated the ability of both human ASCs (hASCs) and mouse ASCs (mASCs) to attenuate lung damage and inflammation in a rodent model of lipopolysaccharide‐induced ALI, suggesting that ASCs may also be beneficial in treating ALI. To better understand how ASCs may act in ALI and to elucidate the mechanism(s) involved in ASC modulation of lung inflammation, gene expression analysis was performed in ASC‐treated (hASCs or mASCs) and control sham‐treated lungs. The results revealed a dramatic difference between the expression of anti‐inflammatory molecules by hASCs and mASCs. These data show that the beneficial effects of hASCs and mASCs in ALI may result from the production of different paracrine factors. Interleukin 6 (IL‐6) expression in the mASC‐treated lungs was significantly elevated as compared to sham‐treated controls 20 hours after delivery of the cells by oropharyngeal aspiration. Knockdown of IL‐6 expression in mASCs by RNA interference abrogated most of their therapeutic effects, suggesting that the anti‐inflammatory properties of mASCs in ALI are explained, at least in part, by activation of IL‐6 secretion. Stem Cells 2014;32:1616–1628

Resibufogenin corrects hypertension in a rat model of human preeclampsia

The study of the pathogenesis of preeclampsia has been hampered by a relative dearth of animal models. We developed a rat model of preeclampsia in which the excretion of a circulating inhibitor of Na/K ATPase, marinobufagenin (MBG), is elevated. These animals develop hypertension, proteinuria, and intrauterine growth restriction. The administration of a congener of MBG, resibufogenin (RBG), reduces blood pressure to normal in these animals, as is the case when given to pregnant animals rendered hypertensive by the administration of MBG. Studies of Na/K ATPase inhibition by MBG and RBG reveal that these agents are equally effective as inhibitors of the enzyme.

Pharmacological inhibition of HDAC6 attenuates endothelial barrier dysfunction induced by thrombin

BACKGROUND Endothelial barrier dysfunction (EBD) involves microtubule disassembly and enhanced cell contractility. Histone deacetylase 6 (HDAC6) deacetylates α-tubulin, and thereby destabilizes microtubules. This study investigates a role for HDAC6 in EBD. METHODS EBD was induced with thrombin±HDAC6 inhibitors (tubacin and MC1575), and assessed by transendothelial electrical resistance (TEER). Markers for microtubule disassembly (α-tubulin and acetylated α-tubulin) and contraction (phosphorylated myosin light chain 2, P-MLC2) were measured using immunoblots and immunofluorescence. RESULTS AND CONCLUSION Thrombin induced a ∼50% decrease in TEER that was abrogated by the HDAC6 inhibitors. Moreover, inhibition of HDAC6 diminished edema in the lung injured by lipopolysaccharide. Lastly, inhibition of HDAC6 attenuated thrombin-induced microtubule disassembly and P-MLC2. Our results suggest that HDAC6 can be targeted to limit EBD.

Effects of Resibufogenin in Experimental Hypertension

Background/Aims: There are two major pathophysiologic processes involved in the development of hypertension: (1) expanded extracellular fluid volume and (2) vasoconstriction. We have developed a model of preeclampsia in the rat, in which excessive volume expansion (VE) plays a role. These animals excrete increased amounts of the bufodienolide, marinobufagenin (MBG), even before their hypertension and proteinuria become established. Furthermore, their hypertension is corrected by administration of resibufogenin (RBG), a compound structurally similar to MBG. Method: We studied two models of experimental hypertension in the nonpregnant animal, produced either by deoxycorticosterone acetate (DOCA)-salt administration or by angiotensin infusion. Results: RBG administered to the DOCA-salt rats lowered blood pressure and reduced proteinuria in the VE animals, but had no affect on the rats infused with angiotensin. Furthermore, although the production of superoxide anion in the aortas of both groups of hypertensive rats was increased over control, RBG reduced these levels to normal in the VE (DOCA-salt) animals only. RBG had no effect in the angiotensin-infused rats. The urinary excretion of angiotensinogen did not rise in VE-mediated hypertension, but did increase in the angiotensin-infused rats. Conclusions: MBG plays an important role in the causation of hypertension in the VE rats, but not in the vasoconstrictive model. RBG is effective only in VE-mediated hypertension.

Cigarette smoke represses the innate immune response to asbestos.

Both cigarette smoke (CS) and asbestos cause lung inflammation and lung cancer, and at high asbestos exposure levels, populations exposed to both of these carcinogens display a synergistic increase in the development of lung cancer. The mechanisms through which these two toxic agents interact to promote lung tumorigenesis are poorly understood. Here, we begin to dissect the inflammatory signals induced by asbestos in combination with CS using a rodent inhalation model and in vitro cell culture. Wild‐type C57BL/6 mice were exposed to room air as a control, CS, and/or asbestos (4 days per week to CS and 1 day per week to asbestos for 5 weeks). Bronchoalveolar lavage (BAL) fluid was collected following exposure and analyzed for inflammatory mediators. Asbestos‐exposed mice displayed an increased innate immune response consistent with NLRP3 inflammasome activation. Compared to mice exposed only to asbestos, animals coexposed to CS + asbestos displayed attenuated levels of innate immune mediators and altered inflammatory cell recruitment. Histopathological changes in CS + asbestos‐exposed mice correlated with attenuated fibroproliferative lesion development relative to their counterparts exposed only to asbestos. In vitro experiments using a human monocyte cell line (THP‐1 cells) supported the in vivo results in that coexposure to cigarette smoke extract repressed NLRP3 inflammasome markers in cells treated with asbestos. These observations indicate that CS represses central components of the innate immune response to inhaled asbestos.

Battling Inflammation in Acute Lung Injury and Acute Respiratory Distress Syndrome: Stem Cell-Based Therapy Targeting the Root Cause of Acute Lung Injury

Acute lung injury (ALI) describes one or more initiating assaults either directly to the lungs or systemically that, if not treated in a timely manner, ultimately progresses to the development of acute respiratory distress syndrome (ARDS) a condition which is characterized by atelectasis, pulmonary hypertension, and an intense, overwhelming inflammatory response that leads to obliterating pulmonary fibrosis and ultimately respiratory failure. The long-term complications associated with ALI/ARDS can be subverted if the fibrotic phase of the disease is suppressed; therefore, it is essential to control inflammation in such a way that endogenous self-protection mechanisms are maintained while not allowing escalating inflammatory damage to occur in the local environment of the lungs. Current treatment strategies focus on optimal ventilator management and treatment of the underlying condition. Cell-based approaches are an attractive option for directed therapeutic intervention for ALI/ARDS. In particular, mesenchymal stem cells (MSCs) from bone marrow, adipose, umbilical cord, and lung tissue as well as induced pluripotent stem (iPS) cells have been shown to facilitate lung repair in several animal models of ALI. The exact mechanism(s) by which these cells accomplish this feat are as yet unknown; however, mounting evidence suggests that they possess potent immunomodulatory and antimicrobial capabilities which diminish the injury-induced inflammatory responses and reduce infection-mediated ALI, respectively, in these various models. Both direct delivery of stem cells to the lung and systemic administration have been somewhat effective, suggesting that stem cells utilize paracrine mechanisms, at least in part, to perform these functions. Aside from their endogenous ability to suppress inflammation and infection, gene-modified MSCs and iPS cells have recently been used as vehicles for carrying anti-inflammatory agents to the lung. Taken together, stem cell therapy is a promising alternative to current therapeutic intervention for ALI/ARDS.