Fibroblast Activation Markers Research Articles

Cancer-associated fibroblasts from invasive breast cancer have an attenuated capacity to secrete collagens.

Normal fibroblasts produce extracellular matrix(ECM) components that form the structural framework of tissues. Cancer-associated fibroblasts(CAFs) with an activated phenotype mainly contribute to ECM deposition and construction of cancer masses. However, the stroma of breast cancer tissues has been shown to be more complicated, and the mechanisms through which CAFs influence ECM deposition remain elusive. In this study, we found that the activated fibroblast marker α-smooth muscle actin(α-SMA) was only present in the stroma of breast cancer tissue, and the CAFs isolated from invasive breast cancer sample remained to be activated and proliferative in passages. To further assess the difference between CAFs and normal breast fibroblasts(NFs), MALDITOF/TOF‑MS was used to analyze the secretory proteins of primary CAFs and NFs. In total, 2,903 and 3,023 proteins were identified. Mass spectrum quantitative assay and data analysis for extracellular proteins indicated that the CAFs produce less collagens and matrix-degrading enzymes compared with NFs. This finding was confirmed by western blot analysis. Furthermore, we discovered that reduced collagen deposition was present in the stroma of invasive breast cancer. These studies showed that although CAFs from invasive breast cancer possess an activated phenotype, they secreted less collagen and induced less ECM deposition in cancer stroma. In cancer tissue, the remodeling of stromal structure and tumor microenvironment might, therefore, be attributed to the biological changes in CAFs including their protein expression profile.

SP0179 Biological Changes in Early Systemic Sclerosis

Early systemic sclerosis (SSc) is a newly identified condition characterized by the association of Raynaud's phenomenon (RP) with SSc marker autoantibodies and/or with a scleroderma pattern at nailfold videocapillaroscopy (NVC),...

Blocking sirtuin 1 and 2 inhibits renal interstitial fibroblast activation and attenuates renal interstitial fibrosis in obstructive nephropathy.

Our recent studies revealed that blocking class I/II histone deacetylases (HDACs) inhibits renal interstitial fibroblast activation and proliferation and alleviates development of renal fibrosis. However, the effect of class III HDAC, particularly sirtuin 1 and 2 (SIRT1 and SIRT2), inhibition on renal fibrogenesis remains elusive. Here, we demonstrate that both SIRT1 and SIRT2 were expressed in cultured renal interstitial fibroblasts (NRK-49F). Exposure of NRK-49F to sirtinol, a selective inhibitor of SIRT1/2, or EX527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide), an inhibitor for SIRT1, resulted in reduced expression of fibroblast activation markers (α-smooth muscle actin, fibronectin, and collagen I) as well as proliferation markers (proliferating cell nuclear antigen, cyclin D1, cyclin E) in dose- and time-dependent manners. Treatment with a SIRT2 inhibitor, AGK2 (2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide), also dose- and time-dependently inhibited renal fibroblast activation and, to a lesser extent, cell proliferation. Furthermore, silencing of either SIRT1 or SIRT2 by small interfering RNA exhibited similar inhibitory effects. In a mouse model of obstructive nephropathy, administration of sirtinol attenuated deposition of collagen fibrils as well as reduced expression of α-smooth muscle actin, collagen I, and fibronectin in the injured kidney. SIRT1/2 inhibition-mediated antifibrotic effects are associated with dephosphorylation of epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor-β (PDGFRβ), and signal transducer and activator of transcription 3. Thus, SIRT1/2 activity may contribute to renal fibroblast activation and proliferation as well as renal fibrogenesis through activation of at least EGFR and PDGFRβ signaling. Blocking SIRT1/2 activation may have therapeutic potential for the treatment of chronic kidney disease.

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.

Functional Heterogeneity of Cancer-Associated Fibroblasts from Human Colon Tumors Shows Specific Prognostic Gene Expression Signature

Cancer-associated fibroblasts (CAF) actively participate in reciprocal communication with tumor cells and with other cell types in the microenvironment, contributing to a tumor-permissive neighborhood and promoting tumor progression. The aim of this study is the characterization of how CAFs from primary human colon tumors promote migration of colon cancer cells. Primary CAF cultures from 15 primary human colon tumors were established. Their enrichment in CAFs was evaluated by the expression of various epithelial and myofibroblast specific markers. Coculture assays of primary CAFs with different colon tumor cells were performed to evaluate promigratory CAF-derived effects on cancer cells. Gene expression profiles were developed to further investigate CAF characteristics. Coculture assays showed significant differences in fibroblast-derived paracrine promigratory effects on cancer cells. Moreover, the association between CAFs' promigratory effects on cancer cells and classic fibroblast activation or stemness markers was observed. CAF gene expression profiles were analyzed by microarray to identify deregulated genes in different promigratory CAFs. The gene expression signature, derived from the most protumorogenic CAFs, was identified. Interestingly, this "CAF signature" showed a remarkable prognostic value for the clinical outcome of patients with colon cancer. Moreover, this prognostic value was validated in an independent series of 142 patients with colon cancer, by quantitative real-time PCR (qRT-PCR), with a set of four genes included in the "CAF signature." In summary, these studies show for the first time the heterogeneity of primary CAFs' effect on colon cancer cell migration. A CAF gene expression signature able to classify patients with colon cancer into high- and low-risk groups was identified.

KCa3.1 mediates activation of fibroblasts in diabetic renal interstitial fibrosis

BackgroundFibroblast activation plays a critical role in diabetic nephropathy (DN). The Ca2+-activated K+ channel KCa3.1 mediates cellular proliferation of many cell types including fibroblasts. KCa3.1 has been reported to be a potential molecular target for pharmacological intervention in a diverse array of clinical conditions. However, the role of KCa3.1 in the activation of myofibroblasts in DN is unknown. These studies assessed the effect of KCa3.1 blockade on renal injury in experimental diabetes.MethodsAs TGF-β1 plays a central role in the activation of fibroblasts to myofibroblasts in renal interstitial fibrosis, human primary renal interstitial fibroblasts were incubated with TGF-β1 +/− the selective inhibitor of KCa3.1, TRAM34, for 48 h. Two streptozotocin-induced diabetic mouse models were used in this study: wild-type KCa3.1+/+ and KCa3.1−/− mice, and secondly eNOS−/− mice treated with or without a selective inhibitor of KCa3.1 (TRAM34). Then, markers of fibroblast activation and fibrosis were determined.ResultsBlockade of KCa3.1 inhibited the upregulation of type I collagen, fibronectin, α-smooth muscle actin, vimentin and fibroblast-specific protein-1 in renal fibroblasts exposed to TGF-β1 and in kidneys from diabetic mice. TRAM34 reduced TGF-β1-induced phosphorylation of Smad2/3 and ERK1/2 but not P38 and JNK MAPK in interstitial fibroblasts.ConclusionsThese results suggest that blockade of KCa3.1 attenuates diabetic renal interstitial fibrogenesis through inhibiting activation of fibroblasts and phosphorylation of Smad2/3 and ERK1/2. Therefore, therapeutic interventions to prevent or ameliorate DN through targeted inhibition of KCa3.1 deserve further consideration.

Abstract B47: Characterization of carcinoma-associated fibroblasts from head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with over 350,000 deaths annually. Metastasis remains a major cause of mortality for HNSCC patients. Elucidating the mechanisms leading to invasion and spread of tumor cells is required to find new therapeutic approaches for controlling this disease. The stroma of HNSCCs consists of several non-tumor cell types, of which carcinoma-associated fibroblasts (CAFs) are prominent. We hypothesize that CAFs facilitate the invasion and migration of tumor cells leading to metastasis. Primary cultures of CAFs and their normal adjacent fibroblast (NAF) counterparts were established from 12 matched patient tissue samples using selective conditions. Each resulting cell line was confirmed to be vimentin positive and pan-cytokeratin negative by IHC staining. Characterization of the 12 pairs was performed by microarray analysis, high throughput flow cytometry cell surface marker profiling and intracellular flow cytometry for α-smooth muscle actin (αSMA) expression, a marker of fibroblast activation. The level of α-SMA expression was consistently low in the NAFs but varied widely in the CAFs where lines displayed 5-40% positive cells. Microarray gene expression analysis indentified over 500 differentially expressed genes between CAFs and NAFs. Extracellular matrix proteins (e.g. FN1 & COL11A1) and cell-cell or cell-matrix adhesion proteins (e.g. ITGA11 & ITGB5) were the most highly up-regulated genes in the CAFs, while immuno-modulatory receptors (e.g. IL6R, PTGER4, TGFβR, IL1R) were down-regulated. Co-injection of CAFs with SCC4 tongue carcinoma cells into immuno-compromised mice, resulted in decreased latency of tumor development and a reduced number of cells required to initiate a tumor – 1000 cells with CAF, compared to 10,000 SCC4 cells alone. In vitro co-culture of SCC4 and SCC9 tumor cells in several conditions including CAF and NAF-derived extracellular matrix (ECM), CAF and NAF-conditioned media (CM), and live CAF and NAF feeder layers, resulted in different growth phenotypes. Cells grown on ECM gave rise to tightly packed colonies with defined edges. Cells grown in CM were more scattered with some loose colonies. Cells grown on NAFs formed normal colonies, however, cells grown on matched CAFs took on a migratory phenotype, forming lines of cells along the length of the fibroblasts. Transwell migration assays were also performed where matched CAF and NAF or CAF-CM and NAF-CM were placed in the bottom chamber and SCC4 were seeded in the upper chamber. CAFs and NAFs induced more migration over the control conditions of no cells and un-conditioned media. CAFs tended to induce more cells to migrate compared to NAFs; though in one pair, the NAFs promoted more migration. Interestingly, gene expression data from this pair indicate that the expression of HGF, a potent migration inducer, is almost twice as high in the NAFs versus the CAFs. These results show that CAFs are able to enhance tumor formation and facilitate tumor cell migration. Further experiments using primary patient tumor cells are necessary to confirm this effect. Laser-capture microdissection of tumor and stromal cells, as well as cell sorting of these cell compartments from patient tissue is underway. Microarrays will be performed to identify a mechanism of interaction between these cell populations and will be integrated with our current data. Citation Format: Keira Pereira, Christina Karamboulas, Elzbieta Hyatt, Joshua Paterson, Laurie Ailles. Characterization of carcinoma-associated fibroblasts from head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B47.

Abstract 169: Angiotensin-II Induced Left Ventricular Remodeling Differs Between C57bl/6 Substrains

Background: Although C57Bl/6 mice are widely used in cardiovascular research, little is known about possible substrain differences. We compared the left ventricular (LV) remodeling induced by angiotensin II (angII) in C57Bl/6J and C57Bl/6N mice, these 2 substrains corresponding to two main branches having diverged since 1951. Methods and Results: Male C57Bl/6J and C57Bl/6N mice were treated with angII (350ng/kg/min) or vehicle via mini-osmotic pumps for either 48h (for microarray profiling of gene expression and macrophage cytofluorometry counting) or 15 days (for both gene expression and histology). AngII (15d) induced fibrosis in LV from C57BL/6N (as shown by histology and col1a expression), but not in C57BL/6J. After 48h of treatment, about 100 genes responded in a strain-specific fashion, most responses being specific for C57Bl/6N mice. Among genes showing greater than 2-fold induction by angII (48h) in C57Bl/6N mice, there was enrichment for markers of macrophages activation and M2 polarization (including osteopontin, arginase1 and galectin3). These strain-specific differences were confirmed (both in LV tissues and macrophages isolated from LVs) by Q-RT-PCR, and occurred despite that fact that AngII increased the abundance of Cd11b+ macrophages to the same extent in both strains. Moreover, AngII (48h) increased expression of several markers of fibroblast activation (including Timp1, Lox and tenascin) in C57Bl/6N (both in LV tissue and fibroblasts isolated from LVs), but not in C57Bl/6J. Although one of the best known genetic differences in both substrains is the inactivation of the Nnt gene in C57Bl6/J mice, experiments performed in F2 mice do not indicate that the fibrotic response co-segregates with the Nnt mutation. Conclusions: Although angII-treatment induces macrophage recruitment in the LVs of both C57Bl/6J and C57Bl/6N mice, activation of cardiac macrophages and their M2 pro-reparative polarization (with subsequent activation of fibroblasts and synthesis of collagen) occurs only in the genetic C57Bl/6N background. The absence of macrophage activation and cardiac fibrosis in C57BL/6J may possibly explain some differences in experimental results obtained by various investigators using different C57BL/6 substrains.

SAT0032 The synthetic cannabinoid ajulemic acid exerts potent anti-fibrotic effects in experimental models of systemic sclerosis

BackgroundCannabinoids are able to modulate fibrogenesis in scleroderma. Ajulemic acid (AjA) is a non-psychoactive synthetic analogue of tetrahydrocannabinol (THC), able to bind the peroxisome proliferator-activated receptor-γ (PPAR-γ) (1). Recent evidence...

Epstein-Barr virus infection induces aberrant TLR/MyD88 activation pathway and fibroblast-myofibroblast conversion in systemic sclerosis. (P6322)

Abstract Activated fibroblasts, mainly myofibroblasts are considered the principal mediators of fibrogenesis in systemic sclerosis (SSc), although the origin of persistent activation of fibroblasts and myofibroblasts is unclear. Activation of Interferon by Toll-like receptors in immune cells may play a role in the pathogenesis of inflammation in many autoimmune diseases, including SSc. We investigated whether fibroblasts persistently activated by innate immune and infectious stimuli might induce IFNs and TGFβ, two major markers of inflammation and fibrosis implicated in SSc pathogenesis. Since Epstein-Barr virus has been a leading candidate in triggering several autoimmune diseases, we developed methodology for infecting SSc fibroblasts with this virus. We found that EBV signals through the TLR/MyD88-pathway in infected-fibroblasts, activating a distinct innate immune response characterized by the expression of selected IFN- and TGFβ-inducible genes. Intriguingly, activation of the TLR/MyD88-pathway by CpGODN2006 or R837 ligand, significantly induced IFN-regulated genes, but no expression of TGFβ-regulated genes was detected in TLR/MyD88-stimulated fibroblasts. EBV-TLR/MyD88 aberrant activation induces the expression of selected IRFs, ISGs, 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 pro-fibrotic phenotype in SSc-fibroblasts.

Clinical Year in Review II

Clinical Year in Review II

Synthetic cannabinoid ajulemic acid exerts potent antifibrotic effects in experimental models of systemic sclerosis

BackgroundCannabinoids modulate fibrogenesis in scleroderma. Ajulemic acid (AjA) is a non-psychoactive synthetic analogue of tetrahydrocannabinol that can bind the peroxisome proliferator-activated receptor-γ (PPAR-γ). Recent evidence suggests a key role for...

Elevated expression of angiomodulin (AGM/IGFBP‐rP1) in tumor stroma and its roles in fibroblast activation

Angiomodulin (AGM/IGFBP-rP1), a glycoprotein of about 30 kDa, is overexpressed in tumor vasculature as well as some human cancer cell lines, but it has been suggested to be a tumor suppressor. To elucidate roles of angiomodulin (AGM) in tumor progression, we here examined distribution of AGM in three types of human cancer tissues by immunohistochemistry. The results showed that AGM was overexpressed in the stroma as well as the vasculature surrounding tumor cells in the human cancer tissues. AGM and α-smooth muscle actin (α-SMA) as an activated fibroblast marker were often colocalized in cancer-associated fibroblasts (CAFs). In vitro analysis indicated that transforming growth factor (TGF)-β1 might be an important inducer of AGM in normal human fibroblasts. AGM strongly stimulated the expression of fibronectin and weakly that of α-SMA in normal fibroblasts. AGM significantly stimulated the proliferation and migration of fibroblasts. The AGM-induced expression of fibronectin and α-SMA was blocked by a TGF-β signal inhibitor but neither the stimulation of cell growth nor migration. These results imply that AGM activates normal fibroblasts by TGF-β-dependent and independent mechanisms. These findings also suggest that AGM and TGF-β1 cooperatively or complementarily contribute to the stromal activation and connective tissue formation in human cancer tissues, contributing to tumor progression.

Early systemic sclerosis: short-term disease evolution and factors predicting the development of new manifestations of organ involvement.

IntroductionWe investigated early systemic sclerosis (SSc) (that is, Raynaud's phenomenon with SSc marker autoantibodies and/or typical capillaroscopic findings and no manifestations other than puffy fingers or arthritis) versus undifferentiated connective tissue disease (UCTD) to identify predictors of short-term disease evolution.MethodsThirty-nine early SSc and 37 UCTD patients were investigated. At baseline, all patients underwent clinical evaluation, B-mode echocardiography, lung function tests and esophageal manometry to detect preclinical alterations of internal organs, and were re-assessed every year. Twenty-one early SSc and 24 UCTD patients, and 25 controls were also investigated for serum endothelial, T-cell and fibroblast activation markers.ResultsAt baseline, 48.7% of early SSc and 37.8% of UCTD patients had at least one preclinical functional alteration (P > 0.05). Ninety-two percent of early SSc patients developed manifestations consistent with definite SSc (that is, skin sclerosis, digital ulcers/scars, two or more teleangectasias, clinically visible nailfold capillaries, cutaneous calcinosis, X-ray bibasilar lung fibrosis, X-ray esophageal dysmotility, ECG signs of myocardial fibrosis and laboratory signs of renal crisis) within five years versus 17.1% of UCTD patients (X2 = 12.26; P = 0.0005). Avascular areas (HR = 4.39 95% CI 1.18 to 16.3; P = 0.02), increased levels of soluble IL-2 receptor alpha (HR = 4.39; 95% CI 1.03 to 18.6; P = 0.03), and of procollagen III aminopropeptide predicted disease evolution (HR = 4.55; 95% CI 1.18 to 17; P = 0.04).ConclusionMost early SSc but only a few UCTD patients progress to definite SSc within a short-term follow-up. Measurement of circulating markers of T-cell and fibroblast activation might serve to identify early SSc patients who are more likely to develop features of definite SSc.

Nuclear β-Catenin Is Increased in Systemic Sclerosis Pulmonary Fibrosis and Promotes Lung Fibroblast Migration and Proliferation

Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/β-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of β-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear β-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis-associated advanced pulmonary fibrosis. Forced activation of β-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-β, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of β-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.

Colorectal Cancer Cells Activate Adjacent Fibroblasts Resulting in FGF1/FGFR3 Signaling and Increased Invasion

Cancer-associated fibroblasts expressing fibroblast activation protein (FAP) have been implicated in the invasive behavior of colorectal cancer. In this study, we use FAP expression as a marker of fibroblast activation and analyze the effect of activated fibroblasts on colorectal cancer migration and invasion in experimental cell studies. We also investigated the expression pattern of FAP in cancer-associated fibroblasts during transformation from benign to malignant colorectal tumors. In immunohistochemical analyses, FAP was expressed in fibroblasts in all colorectal cancer samples examined, whereas all normal colon, hyperplastic polyps, or adenoma samples were negative. In in vitro studies, conditioned medium from colon cancer cells, but not adenoma cells, activated fibroblasts by inducing FAP expression. These activated fibroblasts increased the migration and invasion of colon cancer cells in Boyden chamber experiments and in a three-dimensional cell culture model. We identify fibroblast growth factor 1/fibroblast growth factor receptor 3 (FGF1/FGFR-3) signaling as mediators leading to the increased migration and invasion. Activated fibroblasts increase their expression of FGF1, and by adding a fibroblast growth factor receptor inhibitor, as well as an FGF1-neutralizing antibody, we reduced the migration of colon cancer cells. Our findings provide evidence of a possible molecular mechanism involved in the cross talk between cancer cells and fibroblasts leading to cancer cell invasion.

The cannabinoid WIN55, 212-2 abrogates dermal fibrosis in scleroderma bleomycin model

ObjectivesThere is increasing evidence that the endocannabinoid system may be involved in pathological fibrosis, and that its modulation might limit fibrotic responses. The aim of this study was to examine...

Cyclosporine Inhibits Profibrotic Effects of Interleukin-4 and Transforming Growth Factor β on Human Intrahepatic Fibroblasts Cultured In Vitro

Background Hepatic fibrosis, an outcome of chronic liver diseases, is characterized by an accumulation of collagen, which is produced by activated human intrahepatic fibroblasts (HIF). Transforming growth factor (TGF) β is an important inducer of fibrogenesis, in collaboration with other cytokines, such as interleukin (IL) 4. IL-4 is overexpressed in severe recurrent hepatitis C after liver transplantation, exerting profibrotic effects. In contrast, cyclosporine (CsA) had been shown to decrease fibroblast activation and collagen production. We therefore investigated the effects of CsA on TGF-β and IL-4 profibrotic activities on HIF in vitro. Methods Isolated HIF were cultured without or with human TGF-β, human IL-4, CsA, or combined TGF- β+CsA or IL-4+CsA. We performed real-time polymerase chain reaction for collagen types I, III, and IV and alpha-SMA, a marker of fibroblast activation we also measured total collagen in supernates. TGF-β and IL-4 increased the expressions of alpha smooth muscle action (SMA) collagen I, III, and IV mRNAs ( P < .05 vs untreated cells) as well as the overall collagen level in the supernates ( P < .01). CsA decreased the expression of mRNAs encoding alpha-SMA and collagens ( P < .01). Expressions of alpha-SMA and collagens I, III, and IV mRNAs were significantly lower under combined treatments (TGF-β vs TGF-β+CsA [ P < .01] and IL-4 vs IL-4+CsA [ P < .01]). Collagen level was decreased by combined treatments (TGF-β vs TGF-β+CsA [ P < .05] and IL-4 vs IL-4+CsA [ P = .05]). Conclusion CsA inhibited the profibrotic effects of TGF-β and IL-4 by decreasing the activation and production of collagen by HIF. CsA may decrease fibroblast activation and collagen accumulation, exerting beneficial effects on fibrosis progression, particularly among patients with recurrent hepatitis C.

Immunohistochemistry Analysis for Proliferation Marker in IPF Lung Tissue

PURPOSE: Over-abundant fibroblast proliferation is one of the hallmarks of IPF. These fibroblasts are heterogeneous in their distribution and result in the ongoing deposition of excessive extra-cellular matrix leading to progressive pulmonary dysfunction. We sought to analyze the expression and distribution of proliferation marker, Proliferating Cell Nuclear Antigen (PCNA), and activated fibroblast marker, alpha-smooth muscle actin (alpha-SMA), by immunohistochemistry (IHC) within IPF lung tissue.

Lentiviral-Mediated RNA Interference against TGF-Beta Receptor Type II in Renal Epithelial and Fibroblast Cell Populations In Vitro Demonstrates Regulated Renal Fibrogenesis That Is More Efficient than a Nonlentiviral Vector

Background. Lentiviral constructs reportedly can integrate into the genome of non-dividing, terminally differentiated cells and dividing cells, for long-term gene expression. This investigation tested whether a third generation lentiviral-mediated small interfering RNA (siRNA) delivered into renal epithelial and fibroblast cells against type II transforming growth factor-beta receptor (siRNA-TBRII) could better attenuate renal fibrogenesis in comparison with a non-lentiviral construct. Methods. HIV-derived lentiviral and non-lentiviral constructs were used to transfect cells with siRNA-TBRII or siRNA-EGFP control. Human embryonic kidney (HEK-293T), renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) were transfected and gene silencing quantified (fluorescence microscopy, Western blotting, fluorescence-activated cell sorting). Renal fibrogenesis was assessed using extracellular matrix protein synthesis (fibronectin and collagen-III; Western immunoblot), and α-smooth muscle actin (α-SMA) was analysed as a marker of fibroblast activation and epithelial-to-mesenchymal transdifferentiation (EMT). Results. Lentiviral-mediated siRNA-TBRII significantly suppressed TBRII expression in all cell lines, and also significantly suppressed renal fibrogenesis. In comparison with the non-lentiviral construct, lentiviral-mediated siRNA-TBRII produced stronger and more persistent inhibition of collagen-III in NRK-49F cells, fibronectin in all renal cell lines, and α-SMA in renal epithelial cells. Conclusions. Lentiviral vector systems against TBRII can be delivered into renal cells to efficiently limit renal fibrogenesis by sequence-specific gene silencing.