Ashish Kurundkar

Reversal of Persistent Fibrosis in Aging by Targeting Nox4-Nrf2 Redox Imbalance

Fibrosis resolution is impaired by aging and is mediated by altered cellular redox homeostasis because of a Nox4-Nrf2 imbalance that promotes an apoptosis-resistant myofibroblast phenotype. Scarred for Life? Fibrosis or “scarring” of vital internal organs is an increasing cause of debilitation and death worldwide. The risk of organ fibrosis increases with age, accounting for a growing “epidemic” of fibrotic disorders in aging populations such as in the United States. A study by Hecker et al. provides new insights into how the aging process may lead to a predisposition to fibrosis. In a mouse model of injury-induced lung fibrosis, these investigators found that the ability to resolve fibrosis was impaired in aged mice compared to young cohorts. Resolution of fibrosis is normally dependent on a process known as “apoptosis” (or programmed cell death) of myofibroblasts in injured tissues; this normal wound-healing response was found to be less efficient in aged mice. Myofibroblasts from aged mice acquired a prolonged senescent and apoptosis-resistant phenotype, which was attributed to an imbalance between the oxidant-generating enzyme Nox4 [reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4] and the antioxidant response factor Nrf2 (NFE2-related factor 2). Genetic or pharmacologic approaches to suppress the expression or activation of Nox4 in aged mice with persistent fibrosis enhanced the capacity for fibrosis resolution. There was evidence for Nox4-Nrf2 imbalance and apoptosis-resistant behavior of myofibroblasts in the lungs of human subjects with the progressive and fatal fibrotic disorder idiopathic pulmonary fibrosis. The results of these studies improve our understanding of how and why elderly patients become susceptible to progressive fibrotic disorders, such as idiopathic pulmonary fibrosis. Additionally, this study uncovers new approaches for treating fibrotic disorders by targeting the “stubborn” and apoptosis-resistant myofibroblast. The incidence and prevalence of pathological fibrosis increase with advancing age, although mechanisms for this association are unclear. We assessed the capacity for repair of lung injury in young (2 months) and aged (18 months) mice. Whereas the severity of fibrosis was not different between these groups, aged mice demonstrated an impaired capacity for fibrosis resolution. Persistent fibrosis in lungs of aged mice was characterized by the accumulation of senescent and apoptosis-resistant myofibroblasts. These cellular phenotypes were sustained by alterations in cellular redox homeostasis resulting from elevated expression of the reactive oxygen species–generating enzyme Nox4 [NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-4] and an impaired capacity to induce the Nrf2 (NFE2-related factor 2) antioxidant response. Lung tissues from human subjects with idiopathic pulmonary fibrosis (IPF), a progressive and fatal lung disease, also demonstrated this Nox4-Nrf2 imbalance. Nox4 mediated senescence and apoptosis resistance in IPF fibroblasts. Genetic and pharmacological targeting of Nox4 in aged mice with established fibrosis attenuated the senescent, antiapoptotic myofibroblast phenotype and led to a reversal of persistent fibrosis. These studies suggest that loss of cellular redox homeostasis promotes profibrotic myofibroblast phenotypes that result in persistent fibrosis associated with aging. Our studies suggest that restoration of Nox4-Nrf2 redox balance in myofibroblasts may be a therapeutic strategy in age-associated fibrotic disorders, potentially able to resolve persistent fibrosis or even reverse its progression.

Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis

Matrix stiffening and myofibroblast resistance to apoptosis are cardinal features of chronic fibrotic diseases involving diverse organ systems. The interactions between altered tissue biomechanics and cellular signaling that sustain progressive fibrosis are not well defined. In this study, we used ex vivo and in vivo approaches to define a mechanotransduction pathway involving Rho/Rho kinase (Rho/ROCK), actin cytoskeletal remodeling, and a mechanosensitive transcription factor, megakaryoblastic leukemia 1 (MKL1), that coordinately regulate myofibroblast differentiation and survival. Both in an experimental mouse model of lung fibrosis and in human subjects with idiopathic pulmonary fibrosis (IPF), we observed activation of the Rho/ROCK pathway, enhanced actin cytoskeletal polymerization, and MKL1 cytoplasmic-nuclear shuttling. Pharmacologic disruption of this mechanotransduction pathway with the ROCK inhibitor fasudil induced myofibroblast apoptosis through a mechanism involving downregulation of BCL-2 and activation of the intrinsic mitochondrial apoptotic pathway. Treatment with fasudil during the postinflammatory fibrotic phase of lung injury or genetic ablation of Mkl1 protected mice from experimental lung fibrosis. These studies indicate that targeting mechanosensitive signaling in myofibroblasts to trigger the intrinsic apoptosis pathway may be an effective approach for treatment of fibrotic disorders.

Plasma concentrations of inflammatory cytokines rise rapidly during ECMO-related SIRS due to the release of preformed stores in the intestine

Extracorporeal membrane oxygenation (ECMO) is a life-saving support system used in neonates and young children with severe cardiorespiratory failure. Although ECMO has reduced mortality in these critically ill patients, almost all patients treated with ECMO develop a systemic inflammatory response syndrome (SIRS) characterized by a ‘cytokine storm’, leukocyte activation, and multisystem organ dysfunction. We used a neonatal porcine model of ECMO to investigate whether rising plasma concentrations of inflammatory cytokines during ECMO reflect de novo synthesis of these mediators in inflamed tissues, and therefore, can be used to assess the severity of ECMO-related SIRS. Previously healthy piglets (3-week-old) were subjected to venoarterial ECMO for up to 8 h. SIRS was assessed by histopathological analysis, measurement of neutrophil activation (flow cytometry), plasma cytokine concentrations (enzyme immunoassays), and tissue expression of inflammatory genes (PCR/western blots). Mast cell degranulation was investigated by measurement of plasma tryptase activity. Porcine neonatal ECMO was associated with systemic inflammatory changes similar to those seen in human neonates. Tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) concentrations rose rapidly during the first 2 h of ECMO, faster than the tissue expression of these cytokines. ECMO was associated with increased plasma mast cell tryptase activity, indicating that increased plasma concentrations of inflammatory cytokines during ECMO may result from mast cell degranulation and associated release of preformed cytokines stored in mast cells. TNF-α and IL-8 concentrations rose faster in plasma than in the peripheral tissues during ECMO, indicating that rising plasma levels of these cytokines immediately after the initiation of ECMO may not reflect increasing tissue synthesis of these cytokines. Mobilization of preformed cellular stores of inflammatory cytokines such as in mucosal mast cells may have an important pathophysiological role in ECMO-related SIRS.

Apolipoprotein A-I mimetic 4F alters the function of human monocyte-derived macrophages

HDL and its major protein component apolipoprotein A-I (apoA-I) exert anti-inflammatory effects, inhibit monocyte chemotaxis/adhesion, and reduce vascular macrophage content in inflammatory conditions. In this study, we tested the hypothesis that the apoA-I mimetic 4F modulates the function of monocyte-derived macrophages (MDMs) by regulating the expression of key cell surface receptors on MDMs. Primary human monocytes and THP-1 cells were treated with 4F, apoA-I, or vehicle for 7 days and analyzed for expression of cell surface markers, adhesion to human endothelial cells, phagocytic function, cholesterol efflux capacity, and lipid raft organization. 4F and apoA-I treatment decreased the expression of HLA-DR, CD86, CD11b, CD11c, CD14, and Toll-like receptor-4 (TLR-4) compared with control cells, suggesting the induction of monocyte differentiation. Both treatments abolished LPS-induced mRNA for monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1), regulated on activation, normal T-expressed and presumably secreted (RANTES), IL-6, and TNF-alpha but significantly upregulated LPS-induced IL-10 expression. Moreover, 4F and apoA-I induced a 90% reduction in the expression of CD49d, a ligand for the VCAM-1 receptor, with a concurrent decrease in monocyte adhesion (55% reduction) to human endothelial cells and transendothelial migration (34 and 27% for 4F and apoA-I treatments) compared with vehicle treatment. In addition, phagocytosis of dextran-FITC beads was inhibited by 4F and apoA-I, a response associated with reduced expression of CD32. Finally, 4F and apoA-I stimulated cholesterol efflux from MDMs, leading to cholesterol depletion and disruption of lipid rafts. These data provide evidence that 4F, similar to apoA-I, induces profound functional changes in MDMs, possibly due to differentiation to an anti-inflammatory phenotype.

Epidermal growth factor reduces autophagy in intestinal epithelium and in the rat model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Epidermal growth factor (EGF) is one of the most promising candidates in NEC prophylaxis. Autophagy regulates cell homeostasis, but uncontrolled activation of autophagy may lead to cellular injury. The aim was to evaluate the effects of EGF on intestinal autophagy in epithelial cells and in the rat NEC model and measure autophagy in NEC patients. Intestinal epithelial cells (IEC-6) and the rat NEC model were used to study the effect of EGF on intestinal autophagy. Protein levels of Beclin 1 and LC3II were measured in the intestinal epithelium in both in vivo and in vitro models. Ultrastructural changes in intestinal epithelium were studied by electron microscopy. Expression of Beclin 1, LC3II, and p62 protein was evaluated in biopsies from NEC patients. Autophagy was induced in IEC-6 cells and inhibited by adding EGF into the culture. In the rat NEC model, EGF treatment of NEC reduced expression of Beclin 1 and LC3II in ileal epithelium. Morphologically, typical signs of autophagy were observed in the epithelium of the NEC group, but not in the EGF group. A strong signal for Beclin 1 and LC3II was detected in the intestine from patients with NEC. Autophagy is activated in the intestinal epithelium of NEC patients and in the ileum of NEC rats. Supplementation of EGF blocks intestinal autophagy in both in vivo and in vitro conditions. Results from this study indicate that EGF-mediated protection against NEC injury is associated with regulation of intestinal autophagy.

Novel Mechanisms for the Antifibrotic Action of Nintedanib

Idiopathic pulmonary fibrosis (IPF) is a disease with relentless course and limited therapeutic options. Nintedanib (BIBF-1120) is a multiple tyrosine kinase inhibitor recently approved by the U.S. Food and Drug Administration for the treatment of IPF. The precise antifibrotic mechanism(s) of action of nintedanib, however, is not known. Therefore, we studied the effects of nintedanib on fibroblasts isolated from the lungs of patients with IPF. Protein and gene expression of profibrotic markers were assessed by Western immunoblotting and real-time PCR. Autophagy markers and signaling events were monitored by biochemical assays, Western immunoblotting, microscopy, and immunofluorescence staining. Silencing of autophagy effector proteins was achieved with small interfering RNAs. Nintedanib down-regulated protein and mRNA expression of extracellular matrix (ECM) proteins, fibronectin, and collagen 1a1 while inhibiting transforming growth factor (TGF)-β1-induced myofibroblast differentiation. Nintedanib also induced beclin-1-dependent, ATG7-independent autophagy. Nintedanibs ECM-suppressive actions were not mediated by canonical autophagy. Nintedanib inhibited early events in TGF-β signaling, specifically tyrosine phosphorylation of the type II TGF-β receptor, activation of SMAD3, and p38 mitogen-activated protein kinase. Nintedanib down-regulates ECM production and induces noncanonical autophagy in IPF fibroblasts while inhibiting TGF-β signaling. These mechanisms appear to be uncoupled and function independently to mediate its putative antifibrotic effects.

Epithelial cells in fetal intestine produce chemerin to recruit macrophages

Macrophages are first seen in the fetal intestine at 11-12 wk and rapidly increase in number during the 12- to 22-wk period of gestation. The development of macrophage populations in the fetal intestine precedes the appearance of lymphocytes and neutrophils and does not require the presence of dietary or microbial antigens. In this study, we investigated the role of chemerin, a recently discovered, relatively selective chemoattractant for macrophages, in the recruitment of macrophage precursors to the fetal intestine. Chemerin mRNA/protein expression was measured in jejunoileal tissue from 10- to 24-wk human fetuses, neonates operated for intestinal obstruction, and adults undergoing bariatric surgery. The expression of chemerin in intestinal epithelial cells (IECs) was confirmed by using cultured primary IECs and IEC-like cell lines in vitro. The regulatory mechanisms involved in chemerin expression were investigated by in silico and immunolocalization techniques. IECs in the fetal, but not mature, intestine express chemerin. Chemerin expression peaked in the fetal intestine at 20-24 wk and then decreased to original low levels by full term. During the 10- to 24-wk period, chemerin accounted for most of the macrophage chemotactic activity of cultured fetal IECs. The maturational changes in chemerin expression correlated with the expression of retinoic acid receptor-beta in the intestine. Chemerin is an important mediator of epithelial-macrophage cross talk in the fetal/premature, but not in the mature, intestine. Understanding the regulation of the gut macrophage pool is an important step in development of novel strategies to boost mucosal immunity in premature infants and other patient populations at risk of microbial translocation.

Chronic Parenteral Nutrition Induces Hepatic Inflammation, Steatosis, and Insulin Resistance in Neonatal Pigs

Prematurity and overfeeding in infants are associated with insulin resistance in childhood and may increase the risk of adult disease. Total parenteral nutrition (TPN) is a major source of infant nutritional support and may influence neonatal metabolic function. Our aim was to test the hypothesis that TPN induces increased adiposity and insulin resistance compared with enteral nutrition (EN) in neonatal pigs. Neonatal pigs were either fed enteral formula orally or i.v. administered a TPN mixture for 17 d; macronutrient intake was similar in both groups. During the 17-d period, we measured body composition by dual-energy X-ray absorptiometry scanning; fasting i.v. glucose tolerance tests (IVGTT) and hyperinsulinemic-euglycemic clamps (CLAMP) were performed to quantify insulin resistance. On d 17, tissue was collected after 1-h, low-dose CLAMP for tissue insulin signaling assays. TPN pigs gained less lean and more body fat and developed hepatic steatosis compared with EN pigs. After 7 and 13 d, IVGTT showed evidence of insulin resistance in the TPN compared with the EN group. Fasting plasma glucose and insulin also were higher in TPN pigs. CLAMP showed that insulin sensitivity was markedly lower in TPN pigs than in EN pigs. TPN also reduced the abundance of the insulin receptor, insulin receptor substrate 1, and phosphatidylinositol 3 kinase in skeletal muscle and liver and the proliferation of total pancreatic cells and β-cells. Hepatic proinflammatory genes as well as c-Jun-N-terminal kinase 1 phosphorylation, plasma interleukin 6, and tumor necrosis factor-α were all higher in TPN pigs than in EN pigs. The results demonstrate that chronic TPN induces a hepatic inflammatory response that is associated with significant insulin resistance, hepatic steatosis, and fat deposition compared with EN in neonatal pigs. Further studies are warranted to establish the mechanism of TPN-induced insulin resistance and hepatic metabolic dysfunction and whether there are persistent metabolic consequences of this lifesaving form of infant nutritional support.

Gut mucosal injury in neonates is marked by macrophage infiltration in contrast to pleomorphic infiltrates in adult: evidence from an animal model

Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. In tissue samples of NEC, we identified numerous macrophages and a few neutrophils but not many lymphocytes. We hypothesized that these pathoanatomic characteristics of NEC represent a common tissue injury response of the gastrointestinal tract to a variety of insults at a specific stage of gut development. To evaluate developmental changes in mucosal inflammatory response, we used trinitrobenzene sulfonic acid (TNBS)-induced inflammation as a nonspecific insult and compared mucosal injury in newborn vs. adult mice. Enterocolitis was induced in 10-day-old pups and adult mice (n = 25 animals per group) by administering TNBS by gavage and enema. Leukocyte populations were enumerated in human NEC and in murine TNBS-enterocolitis using quantitative immunofluorescence. Chemokine expression was measured using quantitative polymerase chain reaction, immunoblots, and immunohistochemistry. Macrophage recruitment was investigated ex vivo using intestinal tissue-conditioned media and bone marrow-derived macrophages in a microchemotaxis assay. Similar to human NEC, TNBS enterocolitis in pups was marked by a macrophage-rich leukocyte infiltrate in affected tissue. In contrast, TNBS-enterocolitis in adult mice was associated with pleomorphic leukocyte infiltrates. Macrophage precursors were recruited to murine neonatal gastrointestinal tract by the chemokine CXCL5, a known chemoattractant for myeloid cells. We also demonstrated increased expression of CXCL5 in surgically resected tissue samples of human NEC, indicating that a similar pathway was active in NEC. We concluded that gut mucosal injury in the murine neonate is marked by a macrophage-rich leukocyte infiltrate, which contrasts with the pleomorphic leukocyte infiltrates in adult mice. In murine neonatal enterocolitis, macrophages were recruited to the inflamed gut mucosa by the chemokine CXCL5, indicating that CXCL5 and its cognate receptor CXCR2 merit further investigation as potential therapeutic targets in NEC.

Mechanosensing by the α6-integrin confers an invasive fibroblast phenotype and mediates lung fibrosis.

Matrix stiffening is a prominent feature of pulmonary fibrosis. In this study, we demonstrate that matrix stiffness regulates the ability of fibrotic lung myofibroblasts to invade the basement membrane (BM). We identify α6-integrin as a mechanosensing integrin subunit that mediates matrix stiffness-regulated myofibroblast invasion. Increasing α6-expression, specifically the B isoform (α6B), couples β1-integrin to mediate MMP-2-dependent pericellular proteolysis of BM collagen IV, leading to myofibroblast invasion. Human idiopathic pulmonary fibrosis lung myofibroblasts express high levels of α6-integrin in vitro and in vivo. Genetic ablation of α6 in collagen-expressing mesenchymal cells or pharmacological blockade of matrix stiffness-regulated α6-expression protects mice against bleomycin injury-induced experimental lung fibrosis. These findings suggest that α6-integrin is a matrix stiffness-regulated mechanosensitive molecule which confers an invasive fibroblast phenotype and mediates experimental lung fibrosis. Targeting this mechanosensing α6(β1)-integrin offers a novel anti-fibrotic strategy against lung fibrosis.