Stefania Lenna

The role of endoplasmic reticulum stress and the unfolded protein response in fibrosis

Purpose of reviewTo review the present knowledge of the role of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in the pathogenesis of fibrotic diseases. Recent findingsER stress and UPR occur in a number of diseases associated with organ fibrosis; however, the contribution of these pathways to the fibrotic process has not been systematically investigated. Current studies suggest that prolonged ER stress may lead to fibrosis through activation of CCAAT/enhancer-binding homologous protein-mediated apoptosis, followed by an inflammatory response and release of profibrotic cytokines. A direct profibrotic role of UPR mediators in activation of TGF-&bgr; signaling has been shown in lung fibroblasts. In addition, activation of ER stress and UPR pathways in immune cells contributes to increased production of proinflammatory cytokines. SummaryAlthough limited in scope, current studies strongly suggest that ER stress and UPR may play an important role during development of fibrosis. Further studies are warranted to gain additional insights into the relationship between these processes.

Epstein–Barr Virus Infection Induces Aberrant TLR Activation Pathway and Fibroblast–Myofibroblast Conversion in Scleroderma

Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein–Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV–Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-β1 (TGFβ1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.

Endoplasmic reticulum stress and endothelial dysfunction

Prolonged perturbation of the endoplasmic reticulum (ER) leads to ER stress and unfolded protein response (UPR) and contributes to the pathogenesis of various chronic disorders. This review focuses on the role of ER stress and UPR in endothelial cells and the relevance of these processes to vascular diseases. Chronic activation of ER stress and UPR pathways in endothelial cells leads to increased oxidative stress and inflammation and often results in cell death. Because endothelial cells play a pivotal role in maintaining vascular homeostasis, various pathological conditions interfering with this homeostasis including homocysteinemia, hyperlipidemia, high glucose, insulin resistance, disturbed blood flow, and oxidative stress can lead to endothelial dysfunction in part through the activation of ER stress. We discuss recently discovered aspects of the role of ER stress/UPR in those pathological conditions. We also summarize recent findings implicating ER stress and UPR in systemic hypertension as well as pulmonary arterial hypertension. Finally, this review will highlight a novel role of UPR mediators in the process of angiogenesis.

Interferon-γ promotes vascular remodeling in human microvascular endothelial cells by upregulating endothelin (ET)-1 and transforming growth factor (TGF) β2.

Systemic sclerosis (SSc) is a complex disease characterized by vascular alterations, activation of the immune system and tissue fibrosis. Previous studies have implicated activation of the interferon pathways in the pathogenesis of SSc. The goal of this study was to determine whether interferon type I and/or type II could play a pathogenic role in SSc vasculopathy. Human dermal microvascular endothelial cells (HDMVECs) and fibroblasts were obtained from foreskins of healthy newborns. The RT Profiler PCR Array System was utilized to screen for EndoMT genes. Treatment with IFN‐α or IFN‐γ downregulated Fli1 and VE‐cadherin. In contrast, IFN‐α and IFN‐γ exerted opposite effects on the expression of α‐SMA, CTGF, ET‐1, and TGFβ2, with IFN‐α downregulating and IFN‐γ upregulating this set of genes. Blockade of TGFβ signaling normalized IFN‐γ‐mediated changes in Fli1, VE‐cadherin, CTGF, and ET‐1 levels, whereas upregulation of α‐SMA and TGFβ2 was not affected. Bosentan treatment was more effective than TGFβ blockade in reversing the actions of IFN‐γ, including downregulation of α‐SMA and TGFβ2, suggesting that activation of the ET‐1 pathway plays a main role in the IFN‐γ responses in HDMECs. IFN‐γ induced expression of selected genes related to endothelial‐to‐mesenchymal transition (EndoMT), including Snail1, FN1, PAI1, TWIST1, STAT3, RGS2, and components of the WNT pathway. The effect of IFN‐γ on EndoMT was mediated via TGFβ2 and ET‐1 signaling pathways. This study demonstrates distinct effects of IFN‐α and IFN‐γ on the biology of vascular endothelial cells. IFN‐γ may contribute to abnormal vascular remodeling and fibrogenesis in SSc, partially via induction of EndoMT. J. Cell. Physiol. 228: 1774–1783, 2013.

Increased Expression of Endoplasmic Reticulum Stress and Unfolded Protein Response Genes in Peripheral Blood Mononuclear Cells From Patients With Limited Cutaneous Systemic Sclerosis and Pulmonary Arterial Hypertension

OBJECTIVE Pulmonary arterial hypertension (PAH), a common complication of limited cutaneous systemic sclerosis (lcSSc), is associated with alterations of markers of inflammation and vascular damage in peripheral blood mononuclear cells (PBMCs). Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been implicated in autoimmune and inflammatory diseases. The goal of this study was to assess whether markers of ER stress and the UPR are present in PBMCs from lcSSc patients with PAH. METHODS PBMCs were purified from 36 healthy controls, 32 lcSSc patients with PAH, and 34 lcSSc patients without PAH. Gene expression in healthy control PBMCs stimulated with thapsigargin was analyzed by DNA microarray. Genes were validated by quantitative real-time reverse transcription-polymerase chain reaction in PBMCs from healthy controls and lcSSc patients. RESULTS Several ER stress/UPR genes, including BiP, activating transcription factor 4 (ATF-4), ATF-6, and a spliced form of X-box binding protein 1, were up-regulated in PBMCs from lcSSc patients, with the highest levels in patients with PAH. Thapsigargin up-regulated heat-shock proteins (HSPs) and interferon (IFN)-regulated genes in PBMCs from healthy controls. Selected HSP genes (particularly DnaJB1) and IFN-related genes were also found at significantly elevated levels in PBMCs from lcSSc patients, while IFN regulatory factor 4 expression was significantly decreased. There was a positive correlation between DnaJB1 and severity of PAH (measured by pulmonary artery pressure) (r = 0.56, P < 0.05) and between ER stress markers and interleukin-6 levels (r = 0.53, P < 0.0001) in PBMCs from lcSSc patients. CONCLUSION This study demonstrates an association between select ER stress/UPR markers and lcSSc with PAH, suggesting that ER stress and the UPR may contribute to the altered function of circulating immune cells in lcSSc.

HLA-B35 Upregulates Endothelin-1 and Downregulates Endothelial Nitric Oxide Synthase via Endoplasmic Reticulum Stress Response in Endothelial Cells

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1 mRNA and protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension.

HLA-B35 and dsRNA Induce Endothelin-1 via Activation of ATF4 in Human Microvascular Endothelial Cells

Endothelin 1 (ET-1) is a key regulator of vascular homeostasis. We have recently reported that the presence of Human antigen class I, HLA-B35, contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by upregulating ET-1 and proinflammatory genes. Likewise, a Toll-like receptor 3 (TLR3) ligand, Poly(I:C), was shown to induce ET-1 expression in HDMECs. The goal of this study was to determine the molecular mechanism of ET-1 induction by these two agonists. Because HLA-B35 expression correlated with induction of Binding Immunoglobulin Protein (BiP/GRP78) and several heat shock proteins, we first focused on ER stress and unfolded protein response (UPR) as possible mediators of this response. ER stress inducer, Thapsigargin (TG), HLA-B35, and Poly(I:C) induced ET-1 expression with similar potency in HDMECs. TG and HLA-B35 activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of XBP1, but did not affect the ATF6 pathway. Poly(I:C) also activated eIF2α/ATF4 in a protein kinase R (PKR)-dependent manner. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by all three agonists. Additional experiments have demonstrated that the JNK and NF-κB pathways are also required for ET-1 upregulation by these agonists. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-κB complex was increased in the agonist treated cells. The functional role of c-JUN in responses to HLA-B35 and Poly(I:C) was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. The ER stress/UPR and TLR3 pathways converge on eIF2α/ATF4 during activation of endothelial cells.

Epstein-Barr virus lytic infection promotes activation of Toll-like receptor 8 innate immune response in systemic sclerosis monocytes.

BackgroundMonocytes/macrophages are activated in several autoimmune diseases, including systemic sclerosis (scleroderma; SSc), with increased expression of interferon (IFN)-regulatory genes and inflammatory cytokines, suggesting dysregulation of the innate immune response in autoimmunity. In this study, we investigated whether the lytic form of Epstein-Barr virus (EBV) infection (infectious EBV) is present in scleroderma monocytes and contributes to their activation in SSc.MethodsMonocytes were isolated from peripheral blood mononuclear cells (PBMCs) depleted of the CD19+ cell fraction, using CD14/CD16 negative-depletion. Circulating monocytes from SSc and healthy donors (HDs) were infected with EBV. Gene expression of innate immune mediators were evaluated in EBV-infected monocytes from SSc and HDs. Involvement of Toll-like receptor (TLR)8 in viral-mediated TLR8 response was investigated by comparing the TLR8 expression induced by infectious EBV to the expression stimulated by CL075/TLR8/agonist-ligand in the presence of TLR8 inhibitor in THP-1 cells.ResultsInfectious EBV strongly induced TLR8 expression in infected SSc and HD monocytes in vitro. Markers of activated monocytes, such as IFN-regulated genes and chemokines, were upregulated in SSc- and HD-EBV-infected monocytes. Inhibiting TLR8 expression reduced virally induced TLR8 in THP-1 infected cells, demonstrating that innate immune activation by infectious EBV is partially dependent on TLR8. Viral mRNA and proteins were detected in freshly isolated SSc monocytes. Microarray analysis substantiated the evidence of an increased IFN signature and altered level of TLR8 expression in SSc monocytes carrying infectious EBV compared to HD monocytes.ConclusionThis study provides the first evidence of infectious EBV in monocytes from patients with SSc and links EBV to the activation of TLR8 and IFN innate immune response in freshly isolated SSc monocytes. This study provides the first evidence of EBV replication activating the TLR8 molecular pathway in primary monocytes. Immunogenicity of infectious EBV suggests a novel mechanism mediating monocyte inflammation in SSc, by which EBV triggers the innate immune response in infected cells.

Acid sphingomyelinase deficiency contributes to resistance of scleroderma fibroblasts to Fas-mediated apoptosis

BACKGROUND Scleroderma (SSc) is characterized by excess production and deposition of extracellular matrix (ECM) proteins. Activated fibroblasts play a key role in fibrosis in SSc and are resistant to Fas-mediated apoptosis. Acid sphingomyelinase (ASMase), a major sphingolipid enzyme, plays an important role in the Fas-mediated apoptosis. OBJECTIVE We investigated whether dysregulation of ASMase contributes to Fas-mediated apoptosis resistance in SSc fibroblasts. METHODS Fibroblasts were isolated from SSc patients and healthy controls. Western blot was performed to analyze protein levels and quantitative real time RT-PCR was used to determine mRNA expression. Cells were transiently transfected with siRNA oligos against ASMase or transduced with adenoviruses overexpressing ASMase. Apoptosis was induced using anti-Fas antibody (1 μg/mL) and analyzed using caspase-3 antibody or Cell Death Detection ELISA. RESULTS SSc fibroblasts showed increased resistance to Fas-mediated apoptosis. ASMase expression was decreased in SSc fibroblasts and Transforming Growth Factor beta (TGFβ), the major fibrogenic cytokine involved in the pathogenesis of SSc, downregulated ASMase in normal fibroblasts. Forced expression of ASMase in SSc fibroblasts restored sensitivity of these cells to Fas-mediated apoptosis while blockade of ASMase was sufficient to induce partial resistance to Fas-induced apoptosis in normal fibroblasts. In addition, ASMase blockade decreased activity of protein phosphatase 2A (PP2A) through phosphorylation on Tyr(307) and resulted in activation of extracellular regulated kinase 1/2 (Erk1/2) and protein kinase B (Akt/PKB). CONCLUSION In conclusion, this study suggests that ASMase deficiency promotes apoptosis resistance and contributes to activation of profibrotic signaling in SSc fibroblasts.

The HLA-B*35 allele modulates ER stress, inflammation and proliferation in PBMCs from Limited Cutaneous Systemic Sclerosis patients

IntroductionHLA-B*35 is associated with increased risk of developing pulmonary hypertension in SSc patients. We previously reported that HLA-B*35 induces endothelial cell dysfunction via activation of ER stress/UPR and upregulation of the inflammatory response. Because PBMCs from lcSSc-PAH patients are also characterized by activation of ER stress/UPR and inflammation, the goal of this study was to assess whether the presence of HLA-B*35 contributes to those characteristics.MethodsPBMCs were purified from healthy controls (n = 49 HC) and lcSSc patients, (n = 44 with PAH, n = 53 without PAH). PBMCs from each group were stratified for the presence of HLA-B*35. Global changes in gene expression in response to HLA-B*35, HLA-B*8 or empty lentivirus were investigated by microarray analysis in HC PBMCs. Total RNA was extracted and qPCR was performed to measure gene expression.ResultsER stress markers, in particular the chaperones BiP and DNAJB1 were significantly elevated in PBMC samples carrying the HLA-B*35 allele. IL-6 expression was also significantly increased in HLA-B*35 lcSSc PBMCs and positively correlated with ER stress markers. Likewise, HMGB1 was increased in HLA-B*35-positive lcSSc PBMCs. Global gene expression analysis was used to further probe the role of HLA-B*35. Among genes downregulated by HLA-B*35 lentivirus were genes related to complement (C1QB, C1QC), cell cycle (CDNK1A) and apoptosis (Bax, Gadd45). Interestingly, complement genes (C1QC and C1QB) showed elevated expression in lcSSc without PAH, but were expressed at the low levels in lcSSc-PAH. The presence of HLA-B*35 correlated with the decreased expression of the complement genes. Furthermore, HLA-B*35 correlated with decreased expression of cyclin inhibitors (p21, p57) and pro-apoptotic genes (Bax, Gadd45) in lcSSc B35 subjects. FYN, a tyrosine kinase involved in proliferation of immune cells, was among the genes that were positively regulated by HLA-B*35. HLA-B*35 correlated with increased levels of FYN in lcSSc PBMCs.ConclusionsOur study demonstrates that HLA-B*35 contributes to the dysregulated expression of selected ER stress, inflammation and proliferation related genes in lcSSc patient PBMCs, as well as healthy individuals, thus supporting a pathogenic role of HLA-B*35 in the development of PAH in SSc patients.