Fibroblast Extracellular Matrix Research Articles

Tamoxifen ameliorates renal tubulointerstitial fibrosis by modulation of estrogen receptor α-mediated transforming growth factor-β1/Smad signaling pathway.

After insult to the kidney, a renal fibrotic process is initiated with sustained inflammation, fibroblast activation and accumulation of extracellular matrix (ECM). Tamoxifen has been used as an anti-estrogen for the prevention and treatment of breast cancer. In this study, we investigated the protective effects of tamoxifen on unilateral ureteral obstruction (UUO)-induced renal tubulointerstitial fibrosis and its molecular mechanism. Renal fibrosis was induced by UUO in 7-week-old C57BL/6 mice. Tamoxifen (50 mg/kg) was given by oral gavage for 5 days before induction of renal fibrosis. Tamoxifen treatment was continued for 14 days after UUO operation. Histologic changes were examined by periodic acid-Schiff stain and Masson's trichrome stain. Expression of α-smooth muscle actin, vimentin, type I collagen, fibronectin and cell adhesion molecules were evaluated by immunohistochemistry and western blot analysis. We also evaluated the effect of tamoxifen on estrogen receptor (ER)-α-mediated transforming growth factor (TGF)-β1/Smad signaling pathway in vitro. Renal tubular injury and fibrosis were increased after UUO. Tamoxifen treatment significantly decreased UUO-induced renal tubular injury and fibrosis. Renal fibroblast activation, ECM deposition and inflammation were significantly increased after ureteral ligation. However, tamoxifen treatment significantly decreased UUO-induced renal fibroblast activation, ECM deposition and inflammation by suppression of TGF-β1/Smad signaling pathway in vivo. Tamoxifen decreased TGF-β1-induced fibroblast proliferation and cell migration by modulating ERα-mediated TGF-β1/Smad signaling pathway in vitro. These findings indicate that tamoxifen has a beneficial effect on UUO-induced tubulointerstitial fibrosis by suppression of renal fibroblast activation via modulation of ERα-mediated renal TGF-β1/Smad signaling pathway.

Human cardiac fibroblast extracellular matrix remodeling: dual effects of tissue inhibitor of metalloproteinase-2.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) is an endogenous inhibitor of matrix metalloproteinases (MMPs) that attenuates maladaptive cardiac remodeling in ischemic heart failure. We examined the effects of TIMP-2 on human cardiac fibroblast activation and extracellular matrix (ECM) remodeling. Human cardiac fibroblasts within a three-dimensional collagen matrix were assessed for phenotype conversion, ECM architecture and key molecular regulators of ECM remodeling after differential exposure to TIMP-2 and Ala+TIMP-2 (a modified TIMP-2 analogue devoid of MMP inhibitory activity). TIMP-2 induced opposite effects on human cardiac fibroblast activation and ECM remodeling depending on concentration. TIMP-2 activated fibroblasts into contractile myofibroblasts that remodeled ECM. At higher concentrations (>10 nM), TIMP-2 inhibited fibroblast activation and prevented ECM remodeling. As compared to profibrotic cytokine transforming growth factor (TGF)-beta1, TIMP-2 activated fibroblasts and remodeled ECM without a net accumulation of matrix elements. TIMP-2 increased total protease activity as compared to TGF-beta1. Ala+TIMP-2 exposure revealed that the actions of TIMP-2 on cardiac fibroblast activation are independent of its effects on MMP inhibition. In the presence of GM6001, a broad-spectrum MMP inhibitor, TIMP-2-mediated ECM contraction was completely abolished, indicating that TIMP-2-mediated fibroblast activation is MMP dependent. TIMP-2 functions in a contextual fashion such that the effect on cardiac fibroblasts depends on the tissue microenvironment. These observations highlight potential clinical challenges in using TIMP-2 as a therapeutic strategy to attenuate postinjury cardiac remodeling.

Insidious Changes in Stromal Matrix Fuel Cancer Progression

Reciprocal interactions between tumor and stromal cells propel cancer progression and metastasis. A complete understanding of the complex contributions of the tumor stroma to cancer progression necessitates a careful examination of the extracellular matrix (ECM), which is largely synthesized and modulated by cancer-associated fibroblasts. This structurally supportive meshwork serves as a signaling scaffold for a myriad of biologic processes and responses favoring tumor progression. The ECM is a repository for growth factors and cytokines that promote tumor growth, proliferation, and metastasis through diverse interactions with soluble and insoluble ECM components. Growth factors activated by proteases are involved in the initiation of cell signaling pathways essential to invasion and survival. Various transmembrane proteins produced by the cancer stroma bind the collagen and fibronectin-rich matrix to induce proliferation, adhesion, and migration of cancer cells, as well as protease activation. Integrins are critical liaisons between tumor cells and the surrounding stroma, and with their mechano-sensing ability, induce cell signaling pathways associated with contractility and migration. Proteoglycans also bind and interact with various matrix proteins in the tumor microenvironment to promote cancer progression. Together, these components function to mediate cross-talk between tumor cells and fibroblasts ultimately to promote tumor survival and metastasis. These stromal factors, which may be expressed differentially according to cancer stage, have prognostic utility and potential. This review examines changes in the ECM of cancer-associated fibroblasts induced through carcinogenesis, and the impact of these changes on cancer progression. The implication is that cancer progression, even in epithelial cancers, may be based in large part on changes in signaling from cancer-associated stromal cells. These changes may provide early prognostic indicators to further stratify patients during treatment or alter the timing of their follow-up visits and observations.

Protease activated receptor-1 deficiency diminishes bleomycin-induced skin fibrosis.

Accumulating evidence shows that protease-activated receptor-1 (PAR-1) plays an important role in the development of fibrosis, including lung fibrosis. However, whether PAR-1 also plays a role in the development of skin fibrosis remains elusive. The aim of this study was to determine the role of PAR-1 in the development of skin fibrosis. To explore possible mechanisms by which PAR-1 could play a role, human dermal fibroblasts and keratinocytes were stimulated with specific PAR-1 agonists or antagonists. To investigate the role of PAR-1 in skin fibrosis, we subjected wild-type and PAR-1-deficient mice to a model of bleomycin-induced skin fibrosis. PAR-1 activation leads to increased proliferation and extra cellular matrix (ECM) production, but not migration of human dermal fibroblasts (HDF) in vitro. Moreover, transforming growth factor (TGF)-β production was increased in keratinocytes upon PAR-1 activation, but not in HDF. The loss of PAR-1 in vivo significantly attenuated bleomycin-induced skin fibrosis. The bleomycin-induced increase in dermal thickness and ECM production was reduced significantly in PAR-1-deficient mice compared with wild-type mice. Moreover, TGF-β expression and the number of proliferating fibroblasts were reduced in PAR-1-deficient mice although the difference did not reach statistical significance. This study demonstrates that PAR-1 contributes to the development of skin fibrosis and we suggest that PAR-1 potentiates the fibrotic response mainly by inducing fibroblast proliferation and ECM production.

Extracellular matrix dependence of organization of the cardiomyocyte contractile apparatus

In the process of cardiomyocyte cultivation, there occurs rearrangement of their contractile apparatus with transformation of typical myofibrils into structures of nonmuscle type and with subsequent restoration of the initial organization. The causes and mechanisms of the rearrangements described are unknown. In this study, it was shown that, at cultivation of cardiomyocytes on the individual proteins of the extracellular matrix (ECM), as well as on the matrix produced either by cardiac fibroblasts or by similar cardiomyocytes, the period in which the contractile apparatus is in a converted state is shortened, whereas soluble factors of the conditioned cultural medium do not produce any obvious effect on the above-indicated rearrangements. Using the method of extracellular matrix isolation adapted for cardiomyocytes, we have confirmed that, in the process of cultivation, cardiomyocytes themselves produce ECM that differs from ECM of cardiac fibroblasts by composition and varies with culturing time.

The Expression of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Idiopathic Interstitisal Pneumonia

Background: Idiopathic interstitial pneumonia is characterized by fibroblast proliferation and extracellular matrix (ECM) accumulation. Matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs) have been shown to regulate remodeling of the ECM, which indicates that they are important factors in the process of lung fibrosis. Therefore, we evaluated the expression of MMPs and TIMPs in tissues obtained from patients with idiopathic interstitial pneumonia and control tissues. Methods: Thirty-seven patients who were diagnosed with IIP (22: IPF, 13: NSIP, 2: COP) and 5 controls were enrolled in this study. The MMP-2 and -9 activity in lung tissue obtained from these patients was analyzed using gelatin zymography and the levels of TIMP-1 and -2 were measured by western blotting. We also evaluated the expression of MMP-2 and -9, as well as that of TIMP-1 and -2 in lung tissue using immunohistochemistry. Results: The levels of MMP-2 and MMP-9 were significantly increased in patients with IPF compared to those with NSIP and COP. The activities of TIMP-1 and -2 were also higher in patients with IPF than NSIP/COP patients and control subjects. There were no significant differences observed in the activities of MMPs and TIMPs obtained from patients with NSIP/COP and control subjects. The immunohistochemical analysis showed that TIMP-2 and MMP-2 were strongly stained at the fibroblasts of the fibroblastic foci in patients with IPF. Conclusions: These results suggest that over-expression of gelatinases and TIMPs in patients with IPF are important factors in the irreversible fibrosis that is associated with lung parenchyma.

Curcumin Ameliorates Renal Fibrosis by Inhibiting Local Fibroblast Proliferation and Extracellular Matrix Deposition

Renal fibrosis is mainly characterized by activation and proliferation of interstitial fibroblasts and by excessive synthesis and accumulation of extracellular matrix (ECM) components, including fibronectin (FN) and collagen. This study investigated the effects of curcumin on proliferation of renal interstitial fibroblasts and their underlying mechanisms in vivo and in vitro. ECM components were visualized by Sirius red and immunohistochemistry staining and quantified by western blot analysis in mice with unilateral ureteral obstruction (UUO). Duplex staining for proliferating cell nuclear antigen and α-smooth muscle actin (α-SMA), as well as MTT and flow cytometry assays, were performed to measure fibroblast proliferation. Protein expression of phosphorylated Smad2/3 (p-Smad2/3) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were assessed by western blotting. Curcumin treatment decreased the accumulation of type I collagen and FN in the kidney of animals with UUO. Activation of rat renal interstitial fibroblasts (NRK-49F) was induced by TGF-β1. Curcumin treatment inhibited fibroblast proliferation and the cell cycle was arrested in the G1 phase. Curcumin treatment upregulated the expression of PPAR-γ and downregulated the expression of p-Smad2/3. These results suggest that curcumin treatment ameliorates renal fibrosis by reducing fibroblast proliferation and ECM accumulation mediated by PPAR-γ and Smad-dependent TGF-β1 signaling.

Regulation of the muscle fiber micro environment by activated satellite cells during hypertrophy

Our aim in the current study was to determine the necessity of satellite cells for long-term muscle growth and maintenance. We utilized a transgenic Pax7-DTA mouse model, allowing for the conditional depletion of > 90% of satellite cells with tamoxifen treatment. Synergist ablation surgery, where removal of synergist muscles places functional overload on the plantaris, was used to stimulate robust hypertrophy. Following 8 wk of overload, satellite cell-depleted muscle demonstrated an accumulation of extracellular matrix (ECM) and fibroblast expansion that resulted in reduced specific force of the plantaris. Although the early growth response was normal, an attenuation of hypertrophy measured by both muscle wet weight and fiber cross-sectional area occurred in satellite cell-depleted muscle. Isolated primary myogenic progenitor cells (MPCs) negatively regulated fibroblast ECM mRNA expression in vitro, suggesting a novel role for activated satellite cells/MPCs in muscle adaptation. These results provide evidence that satellite cells regulate the muscle environment during growth.

Proliferative effect and osteoinductive potential of extracellular matrix coated on cell culture plates

Different cell/tissue derived extracellular matrix (ECM) display subtle differences that might provide important cues for proliferation and differentiation of cells in vitro or in vivo. However, the bioactivities of different ECMs in vitro were not fully understood. In this study, osteoblasts-derived and fibroblast-derived ECM-coated cell culture dishes were prepared respectively by culturing osteoblastic MC3T3-E1 cells and rat fibroblast then decellularizing the cultures. We investigated the bioactivities of the two different ECMs coated on cell culture plates using cellular, biochemical and molecular method. The proliferative activity of the bone marrow-derived mesenchymal stem cells (BMSCs) cultured on osteoblast-ECM was lower than for BMSCs grown on fibroblast-ECM. Compared with the BMSCs cultured on fibroblast-derived ECM, the cells grown on osteoblastic ECM showed enhanced alkaline phosphatase (ALP) activity, higher BMP-2 and osteopontin protein levels, increased secreted calcium content, and higher levels of runt-related transcriptional factor 2 (Runx 2) and osteocalcin (OCN) mRNA. Knockdown of BMP-2 or FGF-2 with shRNA transfection hardly effected osteoblastic differentiation or proliferation of MC3T3-E1 seeded on osteoblast-ECM or fibroblast-ECM. Therefore, the osteoblastic ECM had better osteoinductive potential and lower proliferative effect than fibroblastic ECM, and the two ECM presented enough bioactivity, knockdown of growth factors had no significant effect on differentiation and proliferation of re-seeded cells.

Cellular re- and de-programming by microenvironmental memory: why short TGF-β1 pulses can have long effects

BackgroundFibrosis poses a substantial setback in regenerative medicine. Histopathologically, fibrosis is an excessive accumulation of collagen affected by myofibroblasts and this can occur in any tissue that is exposed to chronic injury or insult. Transforming growth factor (TGF)-β1, a crucial mediator of fibrosis, drives differentiation of fibroblasts into myofibroblasts. These cells exhibit α-smooth muscle actin (α-SMA) and synthesize high amounts of collagen I, the major extracellular matrix (ECM) component of fibrosis. While hormones stimulate cells in a pulsatile manner, little is known about cellular response kinetics upon growth factor impact. We therefore studied the effects of short TGF-β1 pulses in terms of the induction and maintenance of the myofibroblast phenotype.ResultsTwenty-four hours after a single 30 min TGF-β1 pulse, transcription of fibrogenic genes was upregulated, but subsided 7 days later. In parallel, collagen I secretion rate and α-SMA presence were elevated for 7 days. A second pulse 24 h later extended the duration of effects to 14 days. We could not establish epigenetic changes on fibrogenic target genes to explain the long-lasting effects. However, ECM deposited under singly pulsed TGF-β1 was able to induce myofibroblast features in previously untreated fibroblasts. Dependent on the age of the ECM (1 day versus 7 days’ formation time), this property was diminished. Vice versa, myofibroblasts were cultured on fibroblast ECM and cells observed to express reduced (in comparison with myofibroblasts) levels of collagen I.ConclusionsWe demonstrated that short TGF-β1 pulses can exert long-lasting effects on fibroblasts by changing their microenvironment, thus leaving an imprint and creating a reciprocal feed-back loop. Therefore, the ECM might act as mid-term memory for pathobiochemical events. We would expect this microenvironmental memory to be dependent on matrix turnover and, as such, to be erasable. Our findings contribute to the current understanding of fibroblast induction and maintenance, and have bearing on the development of antifibrotic drugs.

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts

BackgroundThe extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines.ResultsOur results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments.ConclusionsOverall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

Satellite cells regulate the skeletal muscle environment by inhibiting fibroblast function

Interaction between satellite cells and the skeletal muscle extracellular matrix (ECM) has been demonstrated, but a role for satellite cells in regulating the ECM during hypertrophy has not been defined. We utilized mouse primary myoblasts and fibroblasts in co‐culture in addition to a genetically modified mouse model (Pax7‐DTA) to investigate the role of satellite cells in the regulation of muscle ECM. The Pax7‐DTA mouse allows for the conditional depletion of satellite cells in adult muscle following tamoxifen administration. Vehicle/tamoxifen treated animals were randomized to sham or synergist ablation surgery, leading to overload of the plantaris muscle for 1 and 8 weeks. Microarray analysis after 1 week of surgery showed the loss of satellite cells was associated with increased expression of numerous collagens and ECM structural genes. Depletion of satellite cells also led to attenuated hypertrophy and fibrosis of the plantaris with increases in both ECM deposition and fibroblast number following 8 weeks of overload. Co‐culturing mouse myoblasts with fibroblasts led to decreased mRNA expression of Col Ia2, IIIa1, VIa2 and Fibronectin compared to fibroblasts cocultured with fibroblasts, suggesting a paracrine role of myoblasts that inhibits fibroblast ECM production. We conclude that a novel function of satellite cells both at rest and during hypertrophy is to regulate muscle fibroblasts and the ECM.Funding: NIAMS R01AR060701, R21AG34453 and the Jeane B. Kempner Postdoctoral Award.

Translational control of the fibroblast-extracellular matrix association: An application to pulmonary fibrosis.

Pulmonary fibrosis is a severe lung disease characterized by sustained propagation of lung fibroblasts and relentless accumulation of extracellular matrix (ECM). Idiopathic pulmonary fibrosis (IPF) is the most severe chronic form of pulmonary fibrosis and results both in the gradual exchange of normal lung parenchyma with fibrotic tissue and in the irreversible impairment of gas exchange in the lung. Despite the urgency for novel therapies in IPF treatment, there is no effective and proven medical therapy available. Molecular mechanisms underlying IPF pathogenesis include aberrant ECM signaling through the canonical integrin/PI3K/Akt/mTORC1 signal transduction pathway. One important and well-characterized downstream effector of this pathway is the cellular protein synthesis machinery. Here we will review the recent advances in our understanding of the function of ECM and integrin receptor signaling in development of IPF and will present evidence indicating that the dysregulation of the eIF4F-mediated translational apparatus is an important factor in the development and progression of IPF and other fibrotic disorders. We further discuss the perspectives and challenges to curbing this deadly disease by targeting aberrant translation.

PEGylated human plasma fibronectin is proteolytically stable, supports cell adhesion, cell migration, focal adhesion assembly, and fibronectin fibrillogenesis

Delayed wound healing in many chronic wounds has been linked to the degradation of fibronectin (FN) by abnormally high protease levels. We sought to develop a proteolytically stable and functionally active form of FN. For this purpose, we conjugated 3.35 kDa polyethylene glycol diacrylate (PEGDA) to human plasma fibronectin (HPFN). Conjugation of PEGDA to HPFN or HPFN PEGylation was characterized by an increase of approximately 16 kDa in the average molecular weight of PEGylated HPFN compared to native HPFN in SDS-PAGE gels. PEGylated HPFN was more resistant to α chymotrypsin or neutrophil elastase digestion than native HPFN: after 30 min incubation with α chymotrypsin, 56 and 90% of native and PEGylated HPFN respectively remained intact. PEGylated HPFN and native HPFN supported NIH 3T3 mouse fibroblast adhesion and spreading, migration and focal adhesion formation in a similar manner. Fluorescence microscopy showed that both native and PEGylated HPFN in the culture media were assembled into extracellular matrix (ECM) fibrils. Interestingly, when coated on surfaces, native but not PEGylated HPFN was assembled into the ECM of fibroblasts. The proteolytically stable PEGylated HPFN developed herein could be used to replenish FN levels in the chronic wound bed and promote tissue repair.

Abstract B34: Angiotensin-(1-7) attenuates the proliferation of cancer-associated fibroblasts in triple negative breast cancer

Abstract The interaction between cancer cells and the surrounding microenvironment transforms the stroma into an abnormal phenotype, altering normal function, tissue architecture, cellular morphology, and extracellular matrix-cell interactions that directly contribute to formation of neoplasia. About 80% of reactive stroma associated with breast carcinoma is comprised of activated myofibroblasts or cancer-associated fibroblasts which secrete extracellular matrix proteins resulting in the desmoplasia that is associated with advanced breast tumor progression. Cancer-associated fibroblasts, transformed by cytokines such as transforming growth factor-β (TGF-β), secrete tumor promoting growth factors and extracellular matrix components, to promote tumor initiation, growth, and metastases. Inhibition of cancer-associated fibroblast proliferation and function may be an effective therapeutic strategy, since these cells play a vital role in carcinogenesis. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, seven amino acid peptide hormone of the renin-angiotensin system with anti-proliferative and anti-fibrotic properties. We showed that Ang-(1-7) inhibited the growth of MDA-MB-231 human triple negative breast tumors growing in the mammary fat pads of athymic mice with a concomitant 3-fold decrease in interstitial tumor fibrosis (n=9-11, p<0.0001). Cancer-associated fibroblasts were isolated from orthotopic breast tumors, to assess the effect of Ang-(1-7) on fibroblast proliferation. The isolated tumoral fibroblasts stained positive for fibronectin, vimentin, collagen I, and β-smooth muscle actin, confirming that the isolated cells were activated myofibroblasts. Ang-(1-7) significantly reduced the growth of cancer-associated fibroblasts isolated from orthotopic triple negative breast tumors (n=4-5, p<0.05). The decrease in cancer-associated fibroblast proliferation was accompanied by attenuation of the activation of the growth-promoting mitogen-activated protein kinases (MAPK) ERK1 and ERK2 (n=3, p<0.001) as quantified by Western blot hybridization. In contrast, incubation of the heptapeptide hormone resulted in a 2.5-fold increase in the MAPK phosphatase DUSP1 (dual specificity phosphatase 1) in cancer-associated fibroblasts (n=3, p<0.05). These studies suggest that Ang-(1-7) promotes the dephosphorylation of MAPK by up-regulating DUSP1 to reduce this proliferative signaling pathway in cancer-associated fibroblasts and prevent tumor growth. TGF-β is a potent stimulator of fibroblast activation and plays an important role in promoting fibrosis, including activation of the MAP kinase pathway to stimulate fibroblast proliferation. TGF-β activates tumor-promoting cancer-associated fibroblasts by phosphorylation of Smad2/3 which translocates to the nucleus to induce pro-fibrotic gene expression. Stimulation of the Smad signaling pathway by TGF-β results in extracellular matrix protein synthesis and deposition as well as its own production, creating an autocrine cycle of fibroblast activation and extracellular matrix deposition. TGF-β was quantified by Western blot hybridization in protein homogenates from myofibroblasts isolated from orthotopic breast tumors. TGF-β stimulation of cancer-associated fibroblasts significantly increased phospho-Smad2/3 which was reduced approximately 60% by treatment with Ang-(1-7) (n=3, p<0.001), indicating that the heptapeptide inhibits both the growth of stromal fibroblasts as well as the signaling pathways that participate in extracellular matrix protein production. These findings suggest that Ang-(1-7) may serve as a first-in-class chemotherapeutic agent for triple negative breast cancer in part through a reduction in the proliferation and function of cancer-associated fibroblasts in the tumor microenvironment. Citation Format: Alison L. Arter, Patricia E. Gallagher, E. Ann Tallant. Angiotensin-(1-7) attenuates the proliferation of cancer-associated fibroblasts in triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B34.

Defective alterations in the collagen network to prostacyclin in COPD lung fibroblasts

BackgroundProstacyclin analogs are potent vasodilators and possess anti-inflammatory properties. However, the effect of prostacyclin on extracellular matrix (ECM) in COPD is not well known. Collagen fibrils and proteoglycans are essential ECM components in the lung and fibroblasts are key players in regulating the homeostasis of ECM proteins. The aim was to study the synthesis of prostacyclin and its effect on fibroblast activity and ECM production, and in particular collagen I and the collagen-associated proteoglycans biglycan and decorin.MethodsParenchymal lung fibroblasts were isolated from lungs from COPD patients (GOLD stage IV) and from lungs and transbronchial biopsies from control subjects. The prostacyclin analog iloprost was used to study the effect of prostacyclin on ECM protein synthesis, migration, proliferation and contractile capacity of fibroblasts.ResultsTGF-β1 stimulation significantly increased prostacyclin synthesis in fibroblasts from COPD patients (p < 0.01), but showed no effect on fibroblasts from control subjects. Collagen I synthesis was decreased by iloprost in both control and COPD fibroblasts (p < 0.05). Conversely, iloprost significantly altered biglycan and decorin synthesis in control fibroblasts, but iloprost displayed no effect on these proteoglycans in COPD fibroblasts. Proliferation rate was reduced (p < 0.05) and contractile capacity was increased in COPD fibroblasts (p < 0.05) compared to control fibroblasts. Iloprost decreased proliferative rate in control fibroblasts (p < 0.05), whereas iloprost attenuated contraction capacity in both COPD (p < 0.01) and control fibroblasts (p < 0.05).ConclusionsIloprost reduced collagen I synthesis and fibroblast contractility but did not affect the collagen-associated proteoglycans or proliferation rate in fibroblasts from COPD patients. Enhanced prostacyclin production could lead to improper collagen network fibrillogenesis and a more emphysematous lung structure in severe COPD patients.

Extracellular Matrix of Mechanically Stretched Cardiac Fibroblasts Improves Viability and Metabolic Activity of Ventricular Cells

Background: In heart, the extracellular matrix (ECM), produced by cardiac fibroblasts, is a potent regulator of heart,s function and growth, and provides a supportive scaffold for heart cells in vitro and in vivo. Cardiac fibroblasts are subjected to mechanical loading all the time in vivo. Therefore, the influences of mechanical loading on formation and bioactivity of cardiac fibroblasts, ECM should be investigated.Methods: Rat cardiac fibroblasts were cultured on silicone elastic membranes and stimulated with mechanical cyclic stretch. After removing the cells, the ECMs coated on the membranes were prepared, some ECMs were treated with heparinase II (GAG-lyase), then the collagen, glycosaminoglycan (GAG) and ECM proteins were assayed. Isolated neonatal rat ventricular cells were seeded on ECM-coated membranes, the viability and lactate dehydrogenase (LDH) activity of the cells after 1-7 days of culture was assayed. In addition, the ATPase activity and related protein level, glucose consumption ratio and lactic acid production ratio of the ventricular cells were analyzed by spectrophotometric methods and Western blot.Results: The cyclic stretch increased collagen and GAG levels of the ECMs, and elevated protein levels of collagen I and fibronectin. Compared with the ECMs produced by unstretched cardiac fibroblasts, the ECMs of mechanically stretched fibroblasts improved viability and LDH activity, elevated the Na+/K+-ATPase activity, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and SERCA 2a protein level, glucose consumption ratio and lactic acid production ratio of ventricular cells seeded on them. The treatment with heparinase II reduced GAG levels of these ECMs, and lowered these metabolism-related indices of ventricular cells cultured on the ECMs.Conclusions: Mechanical stretch promotes ECM formation of cardiac fibroblasts in vitro, the ECM of mechanically stretched cardiac fibroblasts improves metabolic activity of ventricular cells cultured in vitro, and the GAG of the ECMs is involved in regulating metabolic activity of ventricular cells.

Macrophage-fibroblast interplay: a target for neuropeptide-based treatment of fibrotic disease?

Background Fibrosis is defined as an excessive accumulation of extracellular matrix (ECM) components with an associated loss of normal tissue architecture and function. Fibrosis occurs in many different auto-inflammatory (auto-immune) diseases. Current therapies targeting fibrosis are limited and we are in great need for novel therapeutic regimens. In fibrosis, abnormal fibroblast proliferation and ECM production by fibroblasts is stimulated by factors released by different cell types, amongst which macrophages. Macrophages produce many pro-fibrotic factors, such as IL-6, CCL2, PDGF and TGF-beta. In rodent models of rheumatoid arthritis and inflammatory bowel disease, somatostatin analogues exerted strong anti-inflammatory effects, by inhibition of proinflammatory cytokine secretion. Somatostatin analogues bind to somatostatin receptors (sst) and we demonstrated sst subtype 2 expression on human macrophages. In the present study we investigated the effects of somatostatin, octreotide, a clinically used somatostatin analogue, and cortistatin, an endogenously expressed somatostatin-like peptide, on macrophage-induced fibroblast proliferation.

Transgenic overexpression of matrix metalloproteinase-9 in macrophages attenuates the inflammatory response and improves left ventricular function post-myocardial infarction

Following myocardial infarction (MI), activated macrophages infiltrate into the necrotic myocardium as part of a robust pro-inflammatory response and secrete matrix metalloproteinase-9 (MMP-9). Macrophage activation, in turn, modulates the fibrotic response, in part by stimulating fibroblast extracellular matrix (ECM) synthesis. We hypothesized that overexpression of human MMP-9 in mouse macrophages would amplify the inflammatory and fibrotic responses to exacerbate left ventricular dysfunction. Unexpectedly, at day 5 post-MI, ejection fraction was improved in transgenic (TG) mice (25±2%) compared to the wild type (WT) mice (18±2%; p<0.05). By gene expression profiling, 23 of 84 inflammatory genes were decreased in the left ventricle infarct (LVI) region from the TG compared to WT mice (all p<0.05). Concomitantly, TG macrophages isolated from the LVI, as well as TG peritoneal macrophages stimulated with LPS, showed decreased inflammatory marker expression compared to WT macrophages. In agreement with attenuated inflammation, only 7 of 84 cell adhesion and ECM genes were increased in the TG LVI compared to WT LVI, while 43 genes were decreased (all p<0.05). These results reveal a novel role for macrophage-derived MMP-9 in blunting the inflammatory response and limiting ECM synthesis to improve left ventricular function post-MI.

A crimp-like microarchitecture improves tissue production in fibrous ligament scaffolds in response to mechanical stimuli

The aim of this study was to determine the influence of a crimp-like microarchitecture within electrospun polymer scaffolds on fibroblast extracellular matrix (ECM) production when cultured under dynamic conditions. Electrospun poly(l-lactide-co-d,l-lactide) scaffolds possessing a wave pattern similar to collagen crimp (amplitude: 5μm and wavelength: 46μm) were seeded with bovine fibroblasts and mechanically stimulated under dynamic uniaxial tension. The effect of strain amplitude (5%, 10% and 20%) was investigated in a short-term stimulation study. The 10% strain amplitude in the stimulated crimp-like fibre scaffold increased only collagen synthesis, while the 20% strain amplitude increased both collagen and sulphated proteoglycan synthesis compared to stimulated uncrimped (straight) fibre scaffolds and unloaded controls (crimp-like static fibre scaffolds). Alternatively, mechanical stimulation of fibroblasts seeded on uncrimped fibre scaffolds induced significant fibroblast proliferation compared to the stimulated crimp-like fibre scaffolds and no-load controls. Long-term, dynamic mechanical stimulation of fibroblasts seeded on crimp-like fibre scaffolds at 10% strain amplitude resulted in significantly up-regulated collagen accumulation and down-regulated sulphated proteoglycan accumulation. Additionally, the fibroblasts seeded on dynamically stimulated crimp-like fibre scaffolds appeared to form bundles that resembled fascicles, a characteristic hierarchical feature of the native ligament. Our findings demonstrate that fibroblasts seeded on crimp-like fibrous scaffolds respond more favourably (increased ECM synthesis and fascicle formation) to dynamic mechanical loading compared to those grown on scaffolds containing uncrimped (straight) fibres.