Hongmei Gu

Sca-1+ cardiac stem cells mediate acute cardioprotection via paracrine factor SDF-1 following myocardial ischemia/reperfusion

Background Cardiac stem cells (CSCs) promote myocardial recovery following ischemia through their regenerative properties. However, little is known regarding the implication of paracrine action by CSCs in the setting of myocardial ischemia/reperfusion (I/R) injury although it is well documented that non-cardiac stem cells mediate cardioprotection via the production of paracrine protective factors. Here, we studied whether CSCs could initiate acute protection following global myocardial I/R via paracrine effect and what component from CSCs is critical to this protection. Methodology/Principal Findings A murine model of global myocardial I/R was utilized to investigate paracrine effect of Sca-1+ CSCs on cardiac function. Intracoronary delivery of CSCs or CSC conditioned medium (CSC CM) prior to ischemia significantly improved myocardial function following I/R. siRNA targeting of VEGF in CSCs did not affect CSC-preserved myocardial function in response to I/R injury. However, differentiation of CSCs to cardiomyocytes (DCSCs) abolished this protection. Through direct comparison of the protein expression profiles of CSCs and DCSCs, SDF-1 was identified as one of the dominant paracrine factors secreted by CSCs. Blockade of the SDF-1 receptor by AMD3100 or downregulated SDF-1 expression in CSCs by specific SDF-1 siRNA dramatically impaired CSC-induced improvement in cardiac function and increased myocardial damage following I/R. Of note, CSC treatment increased myocardial STAT3 activation after I/R, whereas downregulation of SDF-1 action by blockade of the SDF-1 receptor or SDF-1 siRNA transfection abolished CSC-induced STAT3 activation. In addition, inhibition of STAT3 activation attenuated CSC-mediated cardioprotection following I/R. Finally, post-ischemic infusion of CSC CM was shown to significantly protect I/R-caused myocardial dysfunction. Conclusions/Significance This study suggests that CSCs acutely improve post-ischemic myocardial function through paracrine factor SDF-1 and up-regulated myocardial STAT3 activation.

Type V collagen induced tolerance suppresses collagen deposition, TGF-β and associated transcripts in pulmonary fibrosis.

Rationale Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by progressive scarring and matrix deposition. Recent reports highlight an autoimmune component in IPF pathogenesis. We have reported anti-col(V) immunity in IPF patients. The objective of our study was to determine the specificity of col(V) expression profile and anti-col(V) immunity relative to col(I) in clinical IPF and the efficacy of nebulized col(V) in pre-clinical IPF models. Methods Col(V) and col(I) expression profile was analyzed in normal human and IPF tissues. C57-BL6 mice were intratracheally instilled with bleomycin (0.025 U) followed by col(V) nebulization at pre-/post-fibrotic stage and analyzed for systemic and local responses. Results Compared to normal lungs, IPF lungs had higher protein and transcript expression of the alpha 1 chain of col(V) and col(I). Systemic anti-col(V) antibody concentrations, but not of anti-col(I), were higher in IPF patients. Nebulized col(V), but not col(I), prevented bleomycin-induced fibrosis, collagen deposition, and myofibroblast differentiation. Col(V) treatment suppressed systemic levels of anti-col(V) antibodies, IL-6 and TNF-α; and local Il-17a transcripts. Compared to controls, nebulized col(V)-induced tolerance abrogated antigen-specific proliferation in mediastinal lymphocytes and production of IL-17A, IL-6, TNF-α and IFN-γ. In a clinically relevant established fibrosis model, nebulized col(V) decreased collagen deposition. mRNA array revealed downregulation of genes specific to fibrosis (Tgf-β, Il-1β, Pdgfb), matrix (Acta2, Col1a2, Col3a1, Lox, Itgb1/6, Itga2/3) and members of the TGF-β superfamily (Tgfbr1/2, Smad2/3, Ltbp1, Serpine1, Nfkb/Sp1/Cebpb). Conclusions Anti-col(V) immunity is pathogenic in IPF, and col(V)-induced tolerance abrogates bleomycin-induced fibrogenesis and down regulates TGF- β-related signaling pathways.

Role of Complement Activation in Obliterative Bronchiolitis Post–Lung Transplantation

Obliterative bronchiolitis (OB) post-lung transplantation involves IL-17–regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB is unknown. The current study examines the role of complement activation in OB. Complement-regulatory protein (CRP) (CD55, CD46, complement receptor 1–related protein y/CD46) expression was downregulated in human and murine OB; and C3a, a marker of complement activation, was upregulated locally. IL-17 differentially suppressed complement receptor 1–related protein y expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen- or autoantigen (type V collagen)-reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17–mediated downregulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed-forward loop that may enhance CRP downregulation, suggesting that complement blockade could be a therapeutic strategy for OB.

IL-17 Induces Type V Collagen Overexpression and EMT via TGF-β dependent Pathways in Obliterative Bronchiolitis

Obliterative bronchiolitis (OB), a fibrotic airway lesion, is the leading cause of death after lung transplantation. Type V collagen [col(V)] overexpression and IL-17-mediated anti-col(V) immunity are key contributors to OB pathogenesis. Here, we report a previously undefined role of IL-17 in inducing col(V) overexpression, leading to epithelial mesenchymal transition (EMT) and subsequent OB. We observed IL-17-mediated induction of col(V) α1 chains [α1 (V)] in normal airway epithelial cells in vitro and detected α1 (V)-specific antibodies in bronchoalveolar lavage fluid of lung transplant patients. Overexpression of IL-17 and col(V) was detected in OB lesions in patient lung biopsies and in a murine OB model. IL-17 is shown to induce EMT, TGF-β mRNA expression, and SMAD3 activation, whereas downregulating SMAD7 expression in vitro. Pharmacological inhibition of TGF-βRI tyrosine kinase, p38 MAPK, or focal adhesion kinase prevented col(V) overexpression and EMT. In murine orthotopic lung transplants, neutralizing IL-17 significantly decreased TGF-β mRNA and protein expression and prevented epithelial repair/OB. Our findings highlight a feed-forward loop between IL-17 and TGF-β, leading to induction of col(V) and associated epithelial repair, thus providing one possible link between autoimmunity and OB after lung transplantation.

Peptide-Mediated Inhibition of Mitogen-Activated Protein Kinase–Activated Protein Kinase–2 Ameliorates Bleomycin-Induced Pulmonary Fibrosis

Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2, or MK2), a serine/threonine kinase downstream of p38 mitogen-activated protein kinase, has been implicated in inflammation and fibrosis. Compared with pathologically normal lung tissue, significantly higher concentrations of activated MK2 are evident in lung biopsies of patients with idiopathic pulmonary fibrosis (IPF). Expression is localized to fibroblasts and epithelial cells. In the murine bleomycin model of pulmonary fibrosis, we observed robust, activated MK2 expression on Day 7 (prefibrotic stage) and Day 14 (postfibrotic stage). To determine the effects of MK2 inhibition during the postinflammatory/prefibrotic and postfibrotic stages, C57BL/6 mice received intratracheal bleomycin instillation (0.025 U; Day 0), followed by PBS or the MK2 inhibitor (MK2i; 37.5 μg/kg), administered via either local (nebulized) or systemic (intraperitoneal) routes. MK2i or PBS was dosed daily for 14 days subsequent to bleomycin injury, beginning on either Day 7 or Day 14. Regardless of mode of administration or stage of intervention, MK2i significantly abrogated collagen deposition, myofibroblast differentiation and activated MK2 expression. MK2i also decreased circulating TNF-α and IL-6 concentrations, and modulated the local mRNA expression of profibrotic cytokine il-1β, matrix-related genes col1a2, col3a1, and lox, and transforming growth factor-β family members, including smad3, serpine1 (pai1), and smad6/7. In vitro, MK2i dose-dependently attenuated total MK2, myofibroblast differentiation, the secretion of collagen Type I, fibronectin, and the activation of focal adhesion kinase, whereas activated MK2 was attenuated at optimal doses. The peptide-mediated inhibition of MK2 affects both inflammatory and fibrotic responses, and thus may offer a promising therapeutic target for IPF.

Oxidative stress-mediated NFeκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

Estradiol treatment promotes cardiac stem cell (CSC)-derived growth factors, thus improving CSC-mediated cardioprotection after acute ischemia/reperfusion

INTRODUCTION Studies from our group and others have indicated that paracrine function is one of major mechanisms underlying stem cell-mediated cardioprotection. To improve therapeutic efficacy of cardiac stem cells (CSCs), modification of CSCs to enhance their paracrine actions is of great interest. We have shown previously that stem cells from female sex produced greater levels of protective growth factors compared with male stem cells. In addition, 17β-estradiol (E2)-treated mesenchymal stem cells provided better protection in the ischemia/reperfusion (I/R)-injured myocardium compared with untreated cells. In this study, therefore, we hypothesized that (1) treatment with E2 would improve CSC-mediated acute protection of cardiac function after global I/R; and (2) this greater protection in E2-treated CSCs would be attributable to the beneficial effect of E2 on paracrine actions of CSCs. METHOD CSCs were harvested from C57BL mouse hearts. Myocardial I/R was performed in isolated mouse hearts via a Langendorff model. A total of 0.1 × 10(6)/mL of untreated CSCs or E2-treated CSCs was infused into mouse hearts before ischemia or during the initiation of reperfusion. Heart tissue was used for analysis of activation of caspase-3 and STAT3. Secretion of vascular endothelial growth factor and stromal cell-derived factor 1α by CSCs and E2-treated CSCs was determined. In addition, the conditioned medium from the cultivation of CSCs and E2-modified CSCs was used to treat cardiomyocytes during hypoxia. RESULTS E2-treated CSCs produced greater levels of vascular endothelial growth factor and stromal cell-derived factor 1α compared with untreated CSCs. Preischemic infusion of CSCs and E2-treated CSCs improved myocardial function, increased activation of myocardial STAT3 (a prosurvival signaling), and reduced active caspase-3 after acute I/R compared with the vehicle group. The greater protection was observed in E2-treated CSC group than in CSC group. Additionally, infusion of E2-treated CSCs, but not untreated CSCs, during the initiation of reperfusion protected cardiac function after I/R, further indicating the beneficial effect of E2 on CSC protective function. CONCLUSION Treatment with E2 enhanced CSC-derived protective factor production and improved CSC-mediated protection of cardiac function and myocyte survival after acute I/R, suggesting that in vitro modification of CSCs may improve their therapeutic outcome.

Distinct patterns of histone modifications at cardiac-specific gene promoters between cardiac stem cells and mesenchymal stem cells

Mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs) possess different potential to develop into cardiomyocytes. The mechanism underlying cardiomyogenic capacity of MSCs and CSCs remains elusive. It is well established that histone modifications correlate with gene expression and contribute to cell fate commitment. Here we hypothesize that specific histone modifications accompany cardiac-specific gene expression, thus determining the differentiation capacity of MSCs and CSCs toward heart cells. Our results indicate that, at the promoter regions of cardiac-specific genes (Myh6, Myl2, Actc1, Tnni3, and Tnnt2), the levels of histone acetylation of H3 (acH3) and H4 (acH4), as a mark indicative of gene activation, were higher in CSCs (Sca-1(+)CD29(+)) than MSCs. Additionally, lower binding levels of histone deacetylase (HDAC) 1 and HDAC2 at promoter regions of cardiac-specific genes were noticed in CSCs than MSCs. Treatment with trichostatin A, an HDAC inhibitor, upregulated cardiac-specific gene expression in MSCs. Suppression of HDAC1 or HDAC2 expression by small interfering RNAs led to increased cardiac gene expression and was accompanied by enhanced acH3 and acH4 levels at gene loci. We conclude that greater levels of histone acetylation at cardiac-specific gene loci in CSCs than MSCs reflect a stronger potential for CSCs to develop into cardiomyocytes. These lineage-differential histone modifications are likely due to less HDAC recruitment at cardiac-specific gene promoters in CSCs than MSCs.

IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis

Interleukin 17A (IL‐17A) and complement (C′) activation have each been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have reported that IL‐17A induces epithelial injury via TGF‐β in murine bronchiolitis obliterans; that TGF‐β and the C′ cascade present signaling interactions in mediating epithelial injury; and that the blockade of C′ receptors mitigates lung fibrosis. In the present study, we investigated the role of IL‐17A in regulating C′ in lung fibrosis. Microarray analyses of mRNA isolated from primary normal human small airway epithelial cells indicated that IL‐17A (100 ng/ml; 24 h; n = 5 donor lungs) induces C′ components (C′ factor B, C3, and GPCR kinase isoform 5), cytokines (IL8, ‐6, and ‐1B), and cytokine ligands (CXCL1, ‐2, ‐3, ‐5, ‐6, and ‐16). IL‐17A induces protein and mRNA regulation of C′ components and the synthesis of active C′ 3a (C3a) in normal primary human alveolar type II epithelial cells (AECs). Wild‐type mice subjected to IL‐17A neutralization and IL‐17A knockout (i717a−/−) mice were protected against bleomycin (BLEO)‐induced fibrosis and collagen deposition. Further, BLEO‐injured i17a−/− mice had diminished levels of circulating Krebs Von Den Lungen 6 (alveolar epithelial injury marker), local caspase‐3/7, and local endoplasmic reticular stress‐related genes. BLEO‐induced local C′ activation [C3a, C5a, and terminal C′ complex (C5b‐9)] was attenuated in il17a−/− mice, and IL‐17A neutralization prevented the loss of epithelial C′ inhibitors (C′ receptor‐1 related isoform Y and decay accelerating factor), and an increase in local TUNEL levels. RNAi‐mediated gene silencing of il17a in fibrotic mice arrested the progression of lung fibrosis, attenuated cellular apoptosis (caspase‐3/7) and lung deposition of collagen and C′ (C5b‐9). Compared to normals, plasma from IPF patients showed significantly higher hemolytic activity. Our findings demonstrate that limiting complement activation by neutralizing IL‐17A is a potential mechanism in ameliorating lung fibrosis.—Cipolla, E., Fisher, A. J., Gu, H., Mickler, E. A., Agarwal, M., Wilke, C. A., Kim, K. K., Moore, B. B., Vittal, R. IL‐17A deficiency mitigates bleomycin‐induced complement activation during lung fibrosis. FASEB J. 31, 5543–5556 (2017). www.fasebj.org

Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis

While our previous studies suggest that limiting bleomycin-induced complement activation suppresses TGF-β signaling, the specific hierarchical interactions between TGF-β and complement in lung fibrosis are unclear. Herein, we investigated the mechanisms underlying TGF-β-induced complement activation in the pathogenesis of lung fibrosis. C57-BL6 mice were given intratracheal instillations of adenoviral vectors overexpressing TGF-β (Ad-TGFβ) or the firefly gene-luciferase (Ad-Luc; control). Two weeks later, mice with fibrotic lungs were instilled RNAi specific to receptors for C3a or C5a-C3ar or C5ar, and sacrificed at day 28. Histopathological analyses revealed that genetic silencing of C3ar or C5ar arrested the progression of TGF-β-induced lung fibrosis, collagen deposition and content (hydroxyproline, col1a1/2); and significantly suppressed local complement activation. With genetic silencing of either C3ar or C5ar, in Ad-TGFβ-injured lungs: we detected the recovery of Smad7 (TGF-β inhibitor) and diminished local release of DAF (membrane-bound complement inhibitor); in vitro: TGF-β-mediated loss of DAF was prevented. Conversely, blockade of the TGF-β receptor prevented C3a-mediated loss of DAF in both normal primary human alveolar and small airway epithelial cells. Of the 52 miRNAs analyzed as part of the Affymetrix array, normal primary human SAECs exposed to C3a, C5a or TGF-β caused discrete and overlapping miRNA regulation related to epithelial proliferation or apoptosis (miR-891A, miR-4442, miR-548, miR-4633), cellular contractility (miR-1197) and lung fibrosis (miR-21, miR-200C, miR-31HG, miR-503). Our studies present potential mechanisms by which TGF-β activates complement and promotes lung fibrosis.