http://informahealthcare.com/cts ISSN: 0300-8207 (print), 1607-8438 (electronic) Connect Tissue Res, 2014; 55(3): 248–256 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03008207.2014.904856
RESEARCH ARTICLE
Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta Chris J. Watson1,2*, Dermot Phelan1,2*, Patrick Collier1,2, Stephen Horgan1,2, Nadia Glezeva1,2, Gordon Cooke1,2, Maojia Xu1,2, Mark Ledwidge1,2, Kenneth McDonald1,2, and John A. Baugh1,2 School of Medicine & Medical Science, UCD Conway Institute, University College Dublin, Ireland and 2Chronic Cardiovascular Disease Management Unit, St Vincent’s Healthcare Group/St Michael’s Hospital, Dublin, Ireland Abstract
Keywords
Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFb1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFb1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.
Cardiac fibroblast, collagen, extracellular matrix, mechanical stretch, transforming growth factor beta
Introduction While constituting only 25% of the volume of the heart, the cardiac interstitium contains approximately two thirds of the total number of cardiac cells in addition to the supporting extracellular matrix (ECM). The predominant cell type within the interstitium is the fibroblast. Historically, the ECM was considered an inert, structural component of the myocardium; however, it is increasingly recognised as a rapidly changing, dynamic substrate which plays a vital role in both cardiac protection and pathology (1). Excessive ECM deposition resulting in cardiac fibrosis drastically impacts the structure and function of the heart in numerous pathological conditions, including, heart failure, ischemic heart disease, aortic stenosis and hypertensive heart disease. Therefore, it is not surprising that understanding the physiology of the cardiac fibroblast as the regulator of ECM synthesis and degradation has generated much interest. To date, published research has mainly utilised animal fibroblasts as in vitro models with a surprising paucity of data using human cardiac fibroblasts (HCF). Within an in vivo setting, the fibroblast exists in an environment surrounded and supported by matrix proteins that change depending on the location within the myocardium
History Received 5 November 2013 Revised 9 March 2014 Accepted 11 March 2014 Published online 8 April 2014
and on different pathological states. Its activity is altered by circulating neurohormones and cytokines, in particular, transforming growth factor beta 1 (TGFb1), a highly potent pro-fibrotic cytokine implicated in the development and progression of cardiac fibrosis. Further, the interplay between surrounding matrix and fibroblast responses to various extracellular stimuli is becoming ever apparent (2,3). Within the heart, the pathological deposition of extracellular matrix components is evident in numerous disease states and directly impacts cardiac structure and function. For example, tissue levels of the common matrix components collagen sub-types I, III, IV, VI, as well as laminin have all been reported to be altered in disease states such as heart failure, post-myocardial infarction and various cardiomyopathies (4). The aim of this research was to describe what effects common matrix proteins including collagen type I, III, IV, VI and laminin, have on HCF biology. In an attempt to replicate as close as possible in vivo conditions, we exposed cells in vitro to strain to assess haemodynamic/stress effects on HCF activity. In addition, we examined the impact of these various matrix substrates on cellular responses to TGFb1.
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Materials and methods Cell culture
*The first two authors contributed equally to the manuscript. Correspondence: Dr. Chris Watson, UCD Conway Institute, University College Dublin, Dublin 4, Ireland. E-mail: [email protected]
Primary human adult cardiac ventricular fibroblast cells (HCF) were purchased from ScienCell Research Laboratories
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(Carlsbad, CA). Cells were derived from one single female donor aged 20. Cells were cultured in Dulbecco’s modified eagles medium (DMEM; Gibco, Paisley, UK), supplemented with 10% Fetal Bovine Serum (Gibco, Paisley, UK) and penicillin–streptomycin antibiotics (Gibco, Paisley, UK) in a 5% CO2 humidified incubator kept at 37 C. Cells between passage 6 and 12 were used for experiments.
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Confirming the biological activity of commercially available TGFb by Western blotting Transforming growth factor beta 1 (TGFb1; R&D systems, Abington, UK) was purchased to investigate the pro-fibrotic effects on human cardiac fibroblast activity under different conditions. TGFb1 was reconstituted in 4 mM HCl as recommended by the manufacturer and the biological activity was assessed by measuring SMAD2 phosphorylation. HCF cells were stimulated with 10 ng/ml TGFb for 0, 15 and 45 min followed by Western blotting analysis. Whole cell protein lysates were generated using RIPA Lysis Buffer (Millipore, Dundee, UK), containing a protease inhibitor cocktail (Roche, Welwyn, UK). Protein concentrations were determined using the BCA Protein Assay Kit (Pierce, Northumberland, UK). About 10 mg of whole cell lysates were denatured, reduced and resolved on SDS-polyacrylamide gels by SDS-PAGE before transfer onto 0.45 mm pore size Immobilon-P polyvinylidene fluoride (PVDF) membranes (Millipore, Dundee, UK). Membranes were incubated with blocking buffer (TBS, 0.25% Tween-20, 0.1% serum from species that secondary antibody was raised in, and 10% fat free skimmed milk) for 1 h at room temperature. Membranes were subsequently probed overnight with either anti-phospho Smad2 (Cell Signaling Technologies, Leiden, Netherlands), or anti-total Smad2 (Cell Signaling Technologies, Leiden, Netherlands). Detection of the specific binding of the primary antibody was achieved using HRP-conjugated secondary antibodies, followed by signal detection with Immobilon Western chemiluminescent HRP substrate (Millipore, Dundee, UK) according to the manufacturer’s instructions. Confirming the pro-fibrotic effect of TGFb on human cardiac fibroblasts by immunocytochemistry Human cardiac fibroblast (HCF) cells (5 104) were seeded onto sterile 10 mm coverslips placed in 12 well plates and grown to 70% confluency. Cells were then placed in serum free media for 48 h prior to treatment with 10 ng/ml TGFb for 72 h. Cells were then fixed and permeabilised with 70% methanol for 1 h. Following fixation, cells were washed with phosphate buffered saline with 0.05% Tween-20 (PBST) and incubated with a primary mouse anti-human alpha Smooth Muscle Actin (aSMA) monoclonal antibody (Sigma, Dorset, UK) for 1 h at room temperature. This was followed by repeated washing with PBST and incubation with a secondary goat anti-mouse polyclonal antibody (AlexaFlur 546) for 40 min at room temperature. Cells were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) at a dilution of 1:10 000 in PBS and left for 5 min in the dark followed by a washing step. Coverslips were then mounted on microscope slides using fluorescence mounting medium and visualised using a fluorescent microscopy (Zeiss Axio
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Imager M1, Cambridge, UK). Images were acquired at the same exposure time for comparison. Confirming the pro-fibrotic effect of TGFb on human cardiac fibroblasts by quantitative real-time PCR Human cardiac fibroblasts (HCF’s) were seeded to a confluency of 70% on 6 well plates. Cells were then placed in serum free media for 48 h prior to treatment with 10 ng/ml TGFb for 72 h. RNA was subsequently extracted using NucleoSpin RNA II Kit (Macherey-Nagel, Duren, Germany). Single strand DNA synthesis was carried out using SuperScript II RT (Invitrogen, Carlsbad, CA). Quantitative real-time PCR (QPCR) was performed using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA) and the Mx3000P System according to the manufacturer’s instructions (Stratagene). One of each pair of primers was designed to bind the exon–exon boundary with the complimentary sequence to that of the boundary on the mRNA. The sequences of the gene-specific primers used are as follows; aSMA, 50 -CGTTACTACTGCTGAGCGTGA-30 (forward), 50 -AACGTTCATTTCCGATGGTG-30 (reverse); collagen 1 a1 (COL1A1), 50 -GAACGCGTGTCATCCCTTG T-30 (forward), 50 -GAACGAGGTAGTCTTTCAGCAACA-30 (reverse); collagen 3 a1 (COL3A1), 50 -AACACGCAAGGCT GTGAGACT-30 (forward), 50 -GAACGAGGTAGTCTTTCA GCAACA-30 (reverse) QPCR reactions were normalized by amplifying the same cDNA with GAPDH primers, 50 -ACAGT CAGCCGCATCTTCTT-30 (forward), 50 -ACGACCAAATCC GTTGACTC-30 (reverse). The PCR cycling program consisted of 40 three-step cycles of 15 s/95 C, 30 s/58 C and 30 s/72 C. Each sample was amplified in duplicate. In order to confirm signal specificity, a melting program was carried out after the PCR cycles were completed. The samples were quantified by comparison with a standard calibration curve created at the same time and the data was normalized by an internal control (GAPDH). Assessing the effect of matrix and mechanical stretch on human cardiac fibroblasts Human cardiac fibroblast (HCF) cells were seeded on BioFlex 6 well culture plates coated with various matrices including collagen I, collagen IV and laminin (Dunn Labortechnik, Asbach, Germany). Untreated Bioflex culture plates were coated with Collagen VI at a concentration of 2 mg/cm2 for complete surface coverage (BD Biosciences, Oxford, UK). Plates were placed on the loading station of a FX-4000T (Flexcell International Corporation, Hillsborough, NC) mechanical stretch machine and exposed to cyclic equibiaxial Heart Simulation strain (1 Hz, maximum elongation 10%) for 72 h in the presence or absence of 10 ng/ml TGFb. Control plates were exposed to the same conditions without being stretched. Statistical analysis All data generated were from three independent experiments. Comparisons between the control and stretched groups were made using independent t-test or ANOVA (Tukey post-hoc analysis), where appropriate, with p values 0.05 considered
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statistically significant. All statistical calculations were performed using Graph Pad prism Software Version 4 (San Diego, CA).
Results Effect of TGFb on human cardiac fibroblast activity
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Under routine cell culture conditions, HCFs do not express detectable levels of phosphorylated SMAD2. Treatment with TGFb significantly induced SMAD2 phosphorylation after 15 and 45 min as confirmed by Western Blot (Figure 1A). TGFb treatment of HCF cells resulted in increased protein Figure 1. TGFb1 induction of a myofibroblast-like phenotype in human cardiac fibroblast cells (HCF) cultured on polystyrene. TGFb treatment (10 ng/ml) induced SMAD2 phosphorylation as indicated by western blotting (A). TGFb treatment of HCF cells resulted in increased protein expression of the myofibroblast differentiation marker aSMA as detected using immunocytochemistry (B). Cells were counterstained with the nuclear stain 40 ,6-diamidino-2-phenylindole (DAPI). Images captured at 20 original magnification. Quantitative real-time PCR was used to assess the impact of TGFb on induction of the fibrosis related genes COL1A1, COL3A1 and aSMA at 6-, 24- and 72-h time points post-treatment (C). Bars represent fold expression changes to serum free control. Data represent mean ± SEM. **p50.01, ***p50.001.
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expression of the myofibroblast differentiation marker aSMA as detected using immunocytochemistry (Figure 1B). When investigating the impact of TGFb on the gene expression profiles at 6, 24 and 72 h post-treatment, aSMA was significantly upregulated 2- to 4-fold at all three time points studied (p50.01). Similarly, COL1A1 gene expression levels were significantly upregulated by approximately 2-fold at 6 and 72 h post-treatment (p50.01). However, COL3A1mRNA expression was not significantly different from serum free control at any time point investigated (Figure 1C). For the subsequent experiments using various matrix proteins as substrates for HCF cells a 72-h analytical time
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point was selected. Although analysis of aSMA gene expression levels post-TGFb treatment was robust at all time points, COL1A1 gene expression was maximal at 72 h. Fluctuation in mRNA levels over the study time course may coincide with increase in protein production having a negative feedback on mRNA levels.
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Effect of extracellular matrix sub-types on basal expression levels and response to TGFb in human cardiac fibroblast cells Human cardiac fibroblast (HCF) seeded for 72 h on collagen type I matrix significantly upregulated expression of COL1A1mRNA by 2-fold (p50.05; Figure 2A). Collagen type I matrix did not influence COL3A1 or aSMA gene expression levels. Collagen type VI matrix also significantly induced COL1A1mRNA expression by 2-fold (p50.05). None of the matrix coatings induced myofibroblast
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transformation as indicated by a lack of significant induction of aSMA or COL3A1 expression levels (Figure 2B and C). When comparing TGFb responses to those observed on polystyrene, there were significant increases in COL1A1mRNA expression (7- to 18-fold) in cells seeded on collagen type I matrix, collagen type IV matrix, collagen type VI matrix and laminin matrix (all p50.05; Figure 3A). No significant upregulation of COL3A1 was demonstrated on any matrix at 72 h post-TGFb-treatment (Figure 3B). The presence of collagen 1 matrix and laminin matrix significantly enhanced the ability of TGFb to induce aSMA gene expression 5- and 7-fold, respectively (p50.05; Figure 3C). To appreciate the relative levels of expression between genes, data are also represented as percentage of housekeeping gene GAPDH expression using the formula 2DCT 100, and is highlighted in Supplemental Figure S1.
Figure 2. Extracellular matrix sub-types can influence basal gene expression levels in human cardiac fibroblast cells. HCF cells were cultured for 72 h on either polystyrene, collagen type 1 matrix, collagen type IV matrix, collagen type VI matrix or laminin and assessed for the impact of substrate on basal gene expression levels of COL1A1 (A), COL3A1 (B) and aSMA (C) gene expression. Bars represent fold basal mRNA expression changes of matrix against polystyrene. Data represent mean ± SEM. *p50.05.
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Effect of stretch on human cardiac fibroblasts seeded on different matrices and the impact of TGFb treatment The impact of mechanical stretch on collagen 1 matrix had no effect on the basal expression levels of either COL1A1, COL3A1 or aSMA gene expression (Figure 4A–C). When comparing TGFb responses on stretched and non-stretched cells on collagen 1 matrix, COL3A1 and aSMA gene expression was significantly less (Figure 5B and C). HCF cells seeded on a collagen IV matrix and exposed to stretch for 72 h significantly upregulated the myofibroblast differentiation marker aSMA by approximately 3-fold, p50.01 (Figure 4C), but had no effect of COL1A1 or COL3A1 gene expression profiles. TGFb responses on collagen IV matrix were not altered in the presence of mechanical stretch (Figure 5A–C). The application of mechanical strain on HCFs seeded on a collagen type VI matrix upregulated basal expression levels of COL1A1, COL3A1 and aSMA gene expression by
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approximately 2-fold (Figure 4A–C). Under these conditions, HCF cells became responsive to TGFb mediated upregulation of COL3A1 gene expression where cells exhibited a 2-fold increase in expression (Figure 5B). Conversely, TGFb-induced aSMA expression was significantly attenuated when cells were exposed to mechanical stretch (Figure 5C). Similar to what was observed in the presence of collagen VI matrix, HCF sells seeded and stretched on a laminin substrate significantly increased basal expression levels of COL1A1, COL3A1 and aSMA gene expression (Figure 4A–C). Mechanical stretch had no impact on TGFb responses compared to non-stretched cells on the same laminin matrix (Figure 5A–C).
Discussion The aetiology of cardiac fibrosis in hypertensive heart disease is attributable to a combination of haemodynamic and hormonal factors (5). As the primary regulator of the
Figure 3. TGFb responses vary depending on type of extracellular matrix sub-types human cardiac fibroblast cells are adhered to. HCF cells were cultured for 72 h on either polystyrene, collagen type 1 matrix, collagen type IV matrix, collagen type VI matrix or laminin and assessed for the impact of substrate on fibroblast response to TGFb. Quantitative real-time PCR was used to assess gene expression changes of COL1A1 (A), COL3A1 (B) and aSMA (C). Bars represent TGFb-induced fold expression changes compared with its untreated control. Magnitude difference in the presence of matrix against polystyrene was assess and data were represent mean ± SEM and *p50.05.
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Figure 4. Mechanical stretch can impact the basal expression levels of human cardiac fibroblast cells seeded on different extracellular matrix sub-types. HCF cells were seeded on either collagen type 1 matrix, collagen type IV matrix, collagen type VI matrix or laminin. Cells underwent either biaxial mechanical stretch for 72 h or were left un-stretched (control). Alterations in COL1A1 (A), COL3A1 (B) and aSMA (C) gene expression were examined using quantitative real-time PCR analysis. Bars represent fold stretch-induced mRNA expression changes compared with non-stretched cells. Data represent mean ± SEM. *p50.05, **p50.01.
ECM, the cardiac fibroblast has engendered a vast amount of research aimed at assessing factors which alter its bioactivity which, in turn, may be utilised as potential therapeutic targets. To the best of our knowledge, concerning factors that regulate the growth and function of cardiac fibroblasts has been acquired from cells in culture. More often than not these cells are fibroblasts from different species most commonly murine cardiac fibroblasts. While fibroblasts were previously thought of as a uniform cell type regardless of the tissue of origin, phenotypic diversity has been demonstrated in fibroblast responses to external stimuli in cells sourced from multiple different tissue types (6,7). It is not surprising therefore that there would be great variability in fibroblast physiology from species to species (8) which questions the reliability of extrapolating from data obtained in animal-derived cells. The aim of this study was to advance our understanding of the biology of the human cardiac fibroblast, examining the complex interaction of multiple factors influencing cellular activity including
mechanical stretch, the underlying matrix and response to the pro-fibrotic cytokine TGFb. The role of the pro-fibrotic agonist TGFb in the process of cardiac fibrosis is well documented. In particular, the role of TGFb in myofibroblast differentiation and activation is becoming ever more apparent where the cells exhibit increased secretary, migratory and proliferative properties and express the contractile protein aSMA (9–12). This is also supported by work carried out by Teekakirikul et al. (13) who, using a mouse model of cardiac fibrosis, highlighted the significance of TGFb in this pathological remodelling by abrogating myocardial fibrosis through the use of TGFb neutralising antibodies. In this study, we found that TGFb upregulated COL1A1 and aSMA mRNA levels in HCF cells cultured on polystyrene which is consistent with published data although it was surprising to observe that COL3A1 was not inducible under these conditions (9,14). Previous studies have highlighted that collagen 3 gene expression can be induced by TGF, however
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Figure 5. Differential TGFb1 responses in human cardiac fibroblast cells exposed to mechanical stretch and various extracellular matrix sub-types. HCF cells were seeded on either collagen type 1 matrix, collagen type IV matrix, collagen type VI matrix or laminin. Cells underwent either biaxial mechanical stretch for 72 h or were left un-stretched (control), in the presence or absence of 10 ng/ml TGFb1. Alterations in COL1A1 (A), COL3A1 (B) and aSMA (C) gene expression were examined using quantitative real-time PCR analysis. Bars represent TGFb-induced fold expression changes in stretched cells compared with TGFb treated non-stretched cells. Data represent mean ± SEM. *p50.05, **p50.01.
these studies have utilised cardiac fibroblasts from diseased patients including those with failing hearts and those undergoing cardiothoracic bypass surgery, unlike reported herein where the primary adult ventricular fibroblast cells were derived from a young female donor (15,16). It is possible that TGF regulation of COL3A1 is influenced by pathological status or environment where the fibroblasts were procured from, where as this may not be the case for COL1A1 regulation. An important regulator of fibroblast activity is the surrounding ECM. Matrix influences cellular activity in a number of ways. By the early 1980s Bissell et al. (17) first described ‘‘dynamic reciprocity’’ between the ECM and the cells in proximity. The fibroblast, as stated previously, maintains ECM homeostasis. In turn, the type of ECM influences the degree of force transmitted to the cell by the beating myocardium. For example, less deformation will
occur in cells surrounded by collagen type I than those surrounded by the more flexible type III collagen. The degree of deformation is interpreted by the cell via a network of actin filaments and microtubules. This mechano-sensory mechanism influences gene activity (3,18–21). Integrins, a family of heterodimeric transmembrane proteins, allow the fibroblast to adhere to the ECM. These consist of a and b chains which combine to form dimers which are specific to different components of the matrix, for example, a1b1 binds to collagen and laminin while a3b1 binds to fibronectin. The clustering of integrins at focal adhesion complexes can stimulate second messenger signalling and influence cell activity. Integrin expression levels can also be influenced by mechanical stretch (22). Different integrin clustering results in different functional activities and may impact cellular responses to the pro-fibrotic cytokine TGFb (23,24). Therefore, when interpreting responses of cells to various
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DOI: 10.3109/03008207.2014.904856
ECM sub-types and mechanical stretch impact HCF responses to TGF
stimuli in vitro, we must consider the effect of the underlying matrix. Our study reveals that all of the matrices studied not only affected the expression of pro-fibrotic genes in human cardiac fibroblast cells at rest, but also affected their response to TGFb. Collagen type I matrix, the most abundant component of the ECM in the heart induced a 2-fold induction of COL1A1 mRNA expression. It also significantly augmented the effect of TGFb on COL1A1 mRNA expression. Collagen type IV matrix, a major component of the basement membrane which surrounds myocyte fibrils also augmented TGFb induction of COL1A1. Although collagen type VI matrix is a minor component of the ECM it appears to play an important role in structural changes associated with hypertension (25) and has been shown to induce myofibroblast differentiation in rat ventricular fibroblasts (26). In our study, it was demonstrated to significantly induce COL1A1 at rest and to augment the effect of TGFb on COL1A1 induction. It did not, however, induce myofibroblast differentiation in human fibroblasts or alter COL3A1 gene expression. Laminin, the major component of the basal lamina providing an integral part of the structural scaffolding of the heart, is more abundant in heart failure (27). Here, we show that HCF cells seeded on laminin have enhanced TGFb induction of COL1A1 and aSMA gene expression. Mechanical load is an important factor in the growth and development of many tissues, for example, bone and skeletal muscle. Responding to mechanical cues is necessary for tissue homeostasis, and mechanical stress is linked to the development and progression of several disease processes (28,29). It is also of primary importance in the regulation of cardiac muscle development. Increasing mechanical load on cardiac tissue, as is the case in hypertension or outflow tract obstruction, results in hypertrophy and reactive fibrosis in vivo. The process by which stretch mediates an increased deposition of collagen is complex and poorly understood. Stretch appears not only to effect fibroblast activity directly, but also induces the autocrine production of pro-fibrotic growth factors. Further complicating matters, the surrounding ECM modulates fibroblast activity and influences a cells response to stretch as described above. To study mechanotransduction and signalling pathways triggered by mechanical stress, stretch devices have been designed (30). These were initially in the form of uniaxial devices which exerted a force in one direction, however, more recent devices exert biaxial strain. Cells are cultured on silicon membranes which can undergo either static stretch or, with the aid of a vacuum, cyclical stretch. In addition, the membranes may be coated with different matrix proteins. Unfortunately, it is difficult to draw definitive conclusions from the literature on the effect of stretch and matrices on human cardiac fibroblast activity for the same reasons given above; to date, most studies use murine fibroblasts are on a small scale and the experimental conditions differ greatly. There are similar findings when one reviews the literature on the effect of mechanical load on collagen synthesis. For example, neither Butt et al. nor Carver et al. found any change in collagen synthesis from fibroblasts exposed to cyclical load while Lee et al. showed an
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inhibition of ECM mRNA’s under such conditions (31–33). On the other hand, more recently, Husse et al. (34) demonstrated a load induced increase in COL1A1 mRNA. Again, this disparity in results may be explained by different experimental conditions. For example, Carver et al. plated their cells on laminin coated plates in the presence of FCS while Butt et al. showed no effect on collagen synthesis in the absence of FCS but an increase in the presence of serum. Not only do different cell types respond differently to mechanical stretch, but the same cells from different tissues also respond differently. Aortic smooth muscle cells increase collagen synthesis in response mechanical load while pulmonary artery smooth muscle cells do not (35,36). Dalla Costa et al. (37) recently reported an increase in aSMA expression in rat cardiac fibroblasts exposed to cyclical stretch. Indeed, mechanotransduction via integrindependent signalling is considered an intrinsic step towards generation of aSMA positive differentiated fibroblasts (38,39). However, again we face a paucity of data regarding the effect of these stimuli on human cardiac fibroblasts. Our data, presented here, attempts to clarify some of these issues. Interestingly, differential cellular responses to stretch were observed depending on the type of matrix the cells were adhered to. Stretching cells on collagen VI or laminin resulted in a significant increase in expression of all three genes studied. Collagen IV matrix also increase aSMA when stretched. Cells were only primed for a TGF-induced COL3A1 expression if they were cultured on collagen VI matrix and stretched. This observation is very curious and physiologically may be a mechanism by which selective deposition of type 3 collagen can occur in disease. Mechanical stretch did not impact TGFb induction of COL1A1 gene expression on any matrix studied. Interestingly, depending on environmental conditions, stretch had a protective impact on cellular responses to TGFb. Stretching cells on a collagen type 1 matrix repressed TGF b induction of aSMA and COL3A1 expression. TGFb induction of aSMA was also reduced in cells stretched on collagen VI matrix. This anti-fibrotic observation may be due to the activation of the protective natriuretic peptide system under certain conditions (40). These data attempt to examine comprehensively the effect of various stimuli (both in isolation and in conjunction with each other) previously documented to alter fibroblast activity. Taken together these data highlight the varied responses of human cardiac fibroblasts to the pro-fibrotic cytokine TGFb which can be impacted by the substrate the cells are adhered to, whether polystyrene or various ECM types, as well as the influence of cells to mechanical stretch. Of note, however, when interpreting these data it must be highlighted that protein levels were not assessed, and additional analytical time points and degrees of stretch would also add value to this work. In addition, increasing experimental replicates as well as reproducing this study in additional primary cell isolates from various donors would lead to a more definitive and comprehensive understanding of human cardiac fibroblast pathological responses as it is possible that cells may behave differently depending on age and cardiovascular health of donors.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The study was supported by the Health Research Board of Ireland [grant number RP/2007/313 to JB and KM].
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20. Petroll WM, Vishwanath M, Ma L. Corneal fibroblasts respond rapidly to changes in local mechanical stress. Invest Ophthalmol Vis Sci 2004;45:3466–74. 21. Mizutani T, Haga H, Kawabata K. Cellular stiffness response to external deformation: tensional homeostasis in a single fibroblast. Cell Motil Cytoskeleton 2004;59:242–8. 22. Kaneko D, Sasazaki Y, Kikuchi T, Ono T, Nemoto K, Matsumoto H, Toyama Y. Temporal effects of cyclic stretching on distribution and gene expression of integrin and cytoskeleton by ligament fibroblasts in vitro. Connect Tissue Res 2009;50:263–9. 23. MacKenna DA, Dolfi F, Vuori K, Ruoslahti E. Extracellular signalregulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts. J Clin Invest 1998;101:301–10. 24. Wilson E, Sudhir K, Ives HE. Mechanical strain of rat vascular smooth muscle cells is sensed by specific extracellular matrix/ integrin interactions. J Clin Invest 1995;96:2364–72. 25. Spiro MJ, Crowley TJ. Increased rat myocardial type VI collagen in diabetes mellitus and hypertension. Diabetologia 1993;36:93–8. 26. Naugle JE, Olson ER, Zhang X, Mase SE, Pilati CF, Maron MB, Folkesson HG, Horne WI, Doane KJ, Meszaros JG. Type VI collagen induces cardiac myofibroblast differentiation: implications for postinfarction remodeling. Am J Physiol Heart Circ Physiol 2006;290:H323–30. 27. Schaper J, Kostin S, Hein S, Elsasser A, Arnon E, Zimmermann R. Structural remodelling in heart failure. Exp Clin Cardiol 2002;7: 64–8. 28. Butcher DT, Alliston T, Weaver VM. A tense situation: forcing tumour progression. Nat Rev Cancer 2009;9:108–22. 29. Wipff PJ, Rifkin DB, Meister JJ, Hinz B. Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix. J Cell Biol 2007;179:1311–23. 30. Lutz R, Sakai T, Chiquet M. Pericellular fibronectin is required for RhoA-dependent responses to cyclic strain in fibroblasts. J Cell Sci 2010;123:1511–21. 31. Lee AA, Delhaas T, McCulloch AD, Villarreal FJ. Differential responses of adult cardiac fibroblasts to in vitro biaxial strain patterns. J Mol Cell Cardiol 1999;31:1833–43. 32. Butt RP, Bishop JE. Mechanical load enhances the stimulatory effect of serum growth factors on cardiac fibroblast procollagen synthesis. J Mol Cell Cardiol 1997;29:1141–51. 33. Carver W, Nagpal ML, Nachtigal M, Borg TK, Terracio L. Collagen expression in mechanically stimulated cardiac fibroblasts. Circ Res 1991;69:116–22. 34. Husse B, Briest W, Homagk L, Isenberg G, Gekle M. Cyclical mechanical stretch modulates expression of collagen I and collagen III by PKC and tyrosine kinase in cardiac fibroblasts. Am J Physiol Regul Integr Comp Physiol 2007;293:R1898–907. 35. Kolpakov V, Rekhter MD, Gordon D, Wang WH, Kulik TJ. Effect of mechanical forces on growth and matrix protein synthesis in the in vitro pulmonary artery. Analysis of the role of individual cell types. Circ Res 1995;77:823–31. 36. Kollros PR, Bates SR, Mathews MB, Horwitz AL, Glagov S. Cyclic AMP inhibits increased collagen production by cyclically stretched smooth muscle cells. Lab Invest 1987;56:410–17. 37. Dalla Costa AP, Clemente CF, Carvalho HF, Carvalheira JB, Nadruz Jr W, Franchini KG. FAK mediates the activation of cardiac fibroblasts induced by mechanical stress through regulation of the mTOR complex. Cardiovasc Res 2010;86:421–31. 38. Geiger B, Spatz JP, Bershadsky AD. Environmental sensing through focal adhesions. Nat Rev Mol Cell Biol 2009;10:21–33. 39. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 2002;3:349–63. 40. Watson CJ, Phelan D, Xu M, Collier P, Neary R, Smolenski A, Ledwidge M, McDonald K, Baugh J. Mechanical stretch up-regulates the B-type natriuretic peptide system in human cardiac fibroblasts: a possible defense against transforming growth factorbeta mediated fibrosis. Fibrogenesis Tissue Repair 2012;5:9–16.
RESEARCH ARTICLE
Extracellular matrix sub-types and mechanical stretch impact human cardiac fibroblast responses to transforming growth factor beta Chris J. Watson1,2*, Dermot Phelan1,2*, Patrick Collier1,2, Stephen Horgan1,2, Nadia Glezeva1,2, Gordon Cooke1,2, Maojia Xu1,2, Mark Ledwidge1,2, Kenneth McDonald1,2, and John A. Baugh1,2 School of Medicine & Medical Science, UCD Conway Institute, University College Dublin, Ireland and 2Chronic Cardiovascular Disease Management Unit, St Vincent’s Healthcare Group/St Michael’s Hospital, Dublin, Ireland Abstract
Keywords
Understanding the impact of extracellular matrix sub-types and mechanical stretch on cardiac fibroblast activity is required to help unravel the pathophysiology of myocardial fibrotic diseases. Therefore, the purpose of this study was to investigate pro-fibrotic responses of primary human cardiac fibroblast cells exposed to different extracellular matrix components, including collagen sub-types I, III, IV, VI and laminin. The impact of mechanical cyclical stretch and treatment with transforming growth factor beta 1 (TGFb1) on collagen 1, collagen 3 and alpha smooth muscle actin mRNA expression on different matrices was assessed using quantitative real-time PCR. Our results revealed that all of the matrices studied not only affected the expression of pro-fibrotic genes in primary human cardiac fibroblast cells at rest but also affected their response to TGFb1. In addition, differential cellular responses to mechanical cyclical stretch were observed depending on the type of matrix the cells were adhered to. These findings may give insight into the impact of selective pathological deposition of extracellular matrix proteins within different disease states and how these could impact the fibrotic environment.
Cardiac fibroblast, collagen, extracellular matrix, mechanical stretch, transforming growth factor beta
Introduction While constituting only 25% of the volume of the heart, the cardiac interstitium contains approximately two thirds of the total number of cardiac cells in addition to the supporting extracellular matrix (ECM). The predominant cell type within the interstitium is the fibroblast. Historically, the ECM was considered an inert, structural component of the myocardium; however, it is increasingly recognised as a rapidly changing, dynamic substrate which plays a vital role in both cardiac protection and pathology (1). Excessive ECM deposition resulting in cardiac fibrosis drastically impacts the structure and function of the heart in numerous pathological conditions, including, heart failure, ischemic heart disease, aortic stenosis and hypertensive heart disease. Therefore, it is not surprising that understanding the physiology of the cardiac fibroblast as the regulator of ECM synthesis and degradation has generated much interest. To date, published research has mainly utilised animal fibroblasts as in vitro models with a surprising paucity of data using human cardiac fibroblasts (HCF). Within an in vivo setting, the fibroblast exists in an environment surrounded and supported by matrix proteins that change depending on the location within the myocardium
History Received 5 November 2013 Revised 9 March 2014 Accepted 11 March 2014 Published online 8 April 2014
and on different pathological states. Its activity is altered by circulating neurohormones and cytokines, in particular, transforming growth factor beta 1 (TGFb1), a highly potent pro-fibrotic cytokine implicated in the development and progression of cardiac fibrosis. Further, the interplay between surrounding matrix and fibroblast responses to various extracellular stimuli is becoming ever apparent (2,3). Within the heart, the pathological deposition of extracellular matrix components is evident in numerous disease states and directly impacts cardiac structure and function. For example, tissue levels of the common matrix components collagen sub-types I, III, IV, VI, as well as laminin have all been reported to be altered in disease states such as heart failure, post-myocardial infarction and various cardiomyopathies (4). The aim of this research was to describe what effects common matrix proteins including collagen type I, III, IV, VI and laminin, have on HCF biology. In an attempt to replicate as close as possible in vivo conditions, we exposed cells in vitro to strain to assess haemodynamic/stress effects on HCF activity. In addition, we examined the impact of these various matrix substrates on cellular responses to TGFb1.
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Materials and methods Cell culture
*The first two authors contributed equally to the manuscript. Correspondence: Dr. Chris Watson, UCD Conway Institute, University College Dublin, Dublin 4, Ireland. E-mail: [email protected]
Primary human adult cardiac ventricular fibroblast cells (HCF) were purchased from ScienCell Research Laboratories
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(Carlsbad, CA). Cells were derived from one single female donor aged 20. Cells were cultured in Dulbecco’s modified eagles medium (DMEM; Gibco, Paisley, UK), supplemented with 10% Fetal Bovine Serum (Gibco, Paisley, UK) and penicillin–streptomycin antibiotics (Gibco, Paisley, UK) in a 5% CO2 humidified incubator kept at 37 C. Cells between passage 6 and 12 were used for experiments.
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Confirming the biological activity of commercially available TGFb by Western blotting Transforming growth factor beta 1 (TGFb1; R&D systems, Abington, UK) was purchased to investigate the pro-fibrotic effects on human cardiac fibroblast activity under different conditions. TGFb1 was reconstituted in 4 mM HCl as recommended by the manufacturer and the biological activity was assessed by measuring SMAD2 phosphorylation. HCF cells were stimulated with 10 ng/ml TGFb for 0, 15 and 45 min followed by Western blotting analysis. Whole cell protein lysates were generated using RIPA Lysis Buffer (Millipore, Dundee, UK), containing a protease inhibitor cocktail (Roche, Welwyn, UK). Protein concentrations were determined using the BCA Protein Assay Kit (Pierce, Northumberland, UK). About 10 mg of whole cell lysates were denatured, reduced and resolved on SDS-polyacrylamide gels by SDS-PAGE before transfer onto 0.45 mm pore size Immobilon-P polyvinylidene fluoride (PVDF) membranes (Millipore, Dundee, UK). Membranes were incubated with blocking buffer (TBS, 0.25% Tween-20, 0.1% serum from species that secondary antibody was raised in, and 10% fat free skimmed milk) for 1 h at room temperature. Membranes were subsequently probed overnight with either anti-phospho Smad2 (Cell Signaling Technologies, Leiden, Netherlands), or anti-total Smad2 (Cell Signaling Technologies, Leiden, Netherlands). Detection of the specific binding of the primary antibody was achieved using HRP-conjugated secondary antibodies, followed by signal detection with Immobilon Western chemiluminescent HRP substrate (Millipore, Dundee, UK) according to the manufacturer’s instructions. Confirming the pro-fibrotic effect of TGFb on human cardiac fibroblasts by immunocytochemistry Human cardiac fibroblast (HCF) cells (5 104) were seeded onto sterile 10 mm coverslips placed in 12 well plates and grown to 70% confluency. Cells were then placed in serum free media for 48 h prior to treatment with 10 ng/ml TGFb for 72 h. Cells were then fixed and permeabilised with 70% methanol for 1 h. Following fixation, cells were washed with phosphate buffered saline with 0.05% Tween-20 (PBST) and incubated with a primary mouse anti-human alpha Smooth Muscle Actin (aSMA) monoclonal antibody (Sigma, Dorset, UK) for 1 h at room temperature. This was followed by repeated washing with PBST and incubation with a secondary goat anti-mouse polyclonal antibody (AlexaFlur 546) for 40 min at room temperature. Cells were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) at a dilution of 1:10 000 in PBS and left for 5 min in the dark followed by a washing step. Coverslips were then mounted on microscope slides using fluorescence mounting medium and visualised using a fluorescent microscopy (Zeiss Axio
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Imager M1, Cambridge, UK). Images were acquired at the same exposure time for comparison. Confirming the pro-fibrotic effect of TGFb on human cardiac fibroblasts by quantitative real-time PCR Human cardiac fibroblasts (HCF’s) were seeded to a confluency of 70% on 6 well plates. Cells were then placed in serum free media for 48 h prior to treatment with 10 ng/ml TGFb for 72 h. RNA was subsequently extracted using NucleoSpin RNA II Kit (Macherey-Nagel, Duren, Germany). Single strand DNA synthesis was carried out using SuperScript II RT (Invitrogen, Carlsbad, CA). Quantitative real-time PCR (QPCR) was performed using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA) and the Mx3000P System according to the manufacturer’s instructions (Stratagene). One of each pair of primers was designed to bind the exon–exon boundary with the complimentary sequence to that of the boundary on the mRNA. The sequences of the gene-specific primers used are as follows; aSMA, 50 -CGTTACTACTGCTGAGCGTGA-30 (forward), 50 -AACGTTCATTTCCGATGGTG-30 (reverse); collagen 1 a1 (COL1A1), 50 -GAACGCGTGTCATCCCTTG T-30 (forward), 50 -GAACGAGGTAGTCTTTCAGCAACA-30 (reverse); collagen 3 a1 (COL3A1), 50 -AACACGCAAGGCT GTGAGACT-30 (forward), 50 -GAACGAGGTAGTCTTTCA GCAACA-30 (reverse) QPCR reactions were normalized by amplifying the same cDNA with GAPDH primers, 50 -ACAGT CAGCCGCATCTTCTT-30 (forward), 50 -ACGACCAAATCC GTTGACTC-30 (reverse). The PCR cycling program consisted of 40 three-step cycles of 15 s/95 C, 30 s/58 C and 30 s/72 C. Each sample was amplified in duplicate. In order to confirm signal specificity, a melting program was carried out after the PCR cycles were completed. The samples were quantified by comparison with a standard calibration curve created at the same time and the data was normalized by an internal control (GAPDH). Assessing the effect of matrix and mechanical stretch on human cardiac fibroblasts Human cardiac fibroblast (HCF) cells were seeded on BioFlex 6 well culture plates coated with various matrices including collagen I, collagen IV and laminin (Dunn Labortechnik, Asbach, Germany). Untreated Bioflex culture plates were coated with Collagen VI at a concentration of 2 mg/cm2 for complete surface coverage (BD Biosciences, Oxford, UK). Plates were placed on the loading station of a FX-4000T (Flexcell International Corporation, Hillsborough, NC) mechanical stretch machine and exposed to cyclic equibiaxial Heart Simulation strain (1 Hz, maximum elongation 10%) for 72 h in the presence or absence of 10 ng/ml TGFb. Control plates were exposed to the same conditions without being stretched. Statistical analysis All data generated were from three independent experiments. Comparisons between the control and stretched groups were made using independent t-test or ANOVA (Tukey post-hoc analysis), where appropriate, with p values 0.05 considered
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statistically significant. All statistical calculations were performed using Graph Pad prism Software Version 4 (San Diego, CA).
Results Effect of TGFb on human cardiac fibroblast activity
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Under routine cell culture conditions, HCFs do not express detectable levels of phosphorylated SMAD2. Treatment with TGFb significantly induced SMAD2 phosphorylation after 15 and 45 min as confirmed by Western Blot (Figure 1A). TGFb treatment of HCF cells resulted in increased protein Figure 1. TGFb1 induction of a myofibroblast-like phenotype in human cardiac fibroblast cells (HCF) cultured on polystyrene. TGFb treatment (10 ng/ml) induced SMAD2 phosphorylation as indicated by western blotting (A). TGFb treatment of HCF cells resulted in increased protein expression of the myofibroblast differentiation marker aSMA as detected using immunocytochemistry (B). Cells were counterstained with the nuclear stain 40 ,6-diamidino-2-phenylindole (DAPI). Images captured at 20 original magnification. Quantitative real-time PCR was used to assess the impact of TGFb on induction of the fibrosis related genes COL1A1, COL3A1 and aSMA at 6-, 24- and 72-h time points post-treatment (C). Bars represent fold expression changes to serum free control. Data represent mean ± SEM. **p50.01, ***p50.001.
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expression of the myofibroblast differentiation marker aSMA as detected using immunocytochemistry (Figure 1B). When investigating the impact of TGFb on the gene expression profiles at 6, 24 and 72 h post-treatment, aSMA was significantly upregulated 2- to 4-fold at all three time points studied (p50.01). Similarly, COL1A1 gene expression levels were significantly upregulated by approximately 2-fold at 6 and 72 h post-treatment (p50.01). However, COL3A1mRNA expression was not significantly different from serum free control at any time point investigated (Figure 1C). For the subsequent experiments using various matrix proteins as substrates for HCF cells a 72-h analytical time
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point was selected. Although analysis of aSMA gene expression levels post-TGFb treatment was robust at all time points, COL1A1 gene expression was maximal at 72 h. Fluctuation in mRNA levels over the study time course may coincide with increase in protein production having a negative feedback on mRNA levels.
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Effect of extracellular matrix sub-types on basal expression levels and response to TGFb in human cardiac fibroblast cells Human cardiac fibroblast (HCF) seeded for 72 h on collagen type I matrix significantly upregulated expression of COL1A1mRNA by 2-fold (p50.05; Figure 2A). Collagen type I matrix did not influence COL3A1 or aSMA gene expression levels. Collagen type VI matrix also significantly induced COL1A1mRNA expression by 2-fold (p50.05). None of the matrix coatings induced myofibroblast
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transformation as indicated by a lack of significant induction of aSMA or COL3A1 expression levels (Figure 2B and C). When comparing TGFb responses to those observed on polystyrene, there were significant increases in COL1A1mRNA expression (7- to 18-fold) in cells seeded on collagen type I matrix, collagen type IV matrix, collagen type VI matrix and laminin matrix (all p50.05; Figure 3A). No significant upregulation of COL3A1 was demonstrated on any matrix at 72 h post-TGFb-treatment (Figure 3B). The presence of collagen 1 matrix and laminin matrix significantly enhanced the ability of TGFb to induce aSMA gene expression 5- and 7-fold, respectively (p50.05; Figure 3C). To appreciate the relative levels of expression between genes, data are also represented as percentage of housekeeping gene GAPDH expression using the formula 2DCT 100, and is highlighted in Supplemental Figure S1.
Figure 2. Extracellular matrix sub-types can influence basal gene expression levels in human cardiac fibroblast cells. HCF cells were cultured for 72 h on either polystyrene, collagen type 1 matrix, collagen type IV matrix, collagen type VI matrix or laminin and assessed for the impact of substrate on basal gene expression levels of COL1A1 (A), COL3A1 (B) and aSMA (C) gene expression. Bars represent fold basal mRNA expression changes of matrix against polystyrene. Data represent mean ± SEM. *p50.05.
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Effect of stretch on human cardiac fibroblasts seeded on different matrices and the impact of TGFb treatment The impact of mechanical stretch on collagen 1 matrix had no effect on the basal expression levels of either COL1A1, COL3A1 or aSMA gene expression (Figure 4A–C). When comparing TGFb responses on stretched and non-stretched cells on collagen 1 matrix, COL3A1 and aSMA gene expression was significantly less (Figure 5B and C). HCF cells seeded on a collagen IV matrix and exposed to stretch for 72 h significantly upregulated the myofibroblast differentiation marker aSMA by approximately 3-fold, p50.01 (Figure 4C), but had no effect of COL1A1 or COL3A1 gene expression profiles. TGFb responses on collagen IV matrix were not altered in the presence of mechanical stretch (Figure 5A–C). The application of mechanical strain on HCFs seeded on a collagen type VI matrix upregulated basal expression levels of COL1A1, COL3A1 and aSMA gene expression by
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approximately 2-fold (Figure 4A–C). Under these conditions, HCF cells became responsive to TGFb mediated upregulation of COL3A1 gene expression where cells exhibited a 2-fold increase in expression (Figure 5B). Conversely, TGFb-induced aSMA expression was significantly attenuated when cells were exposed to mechanical stretch (Figure 5C). Similar to what was observed in the presence of collagen VI matrix, HCF sells seeded and stretched on a laminin substrate significantly increased basal expression levels of COL1A1, COL3A1 and aSMA gene expression (Figure 4A–C). Mechanical stretch had no impact on TGFb responses compared to non-stretched cells on the same laminin matrix (Figure 5A–C).
Discussion The aetiology of cardiac fibrosis in hypertensive heart disease is attributable to a combination of haemodynamic and hormonal factors (5). As the primary regulator of the
Figure 3. TGFb responses vary depending on type of extracellular matrix sub-types human cardiac fibroblast cells are adhered to. HCF cells were cultured for 72 h on either polystyrene, collagen type 1 matrix, collagen type IV matrix, collagen type VI matrix or laminin and assessed for the impact of substrate on fibroblast response to TGFb. Quantitative real-time PCR was used to assess gene expression changes of COL1A1 (A), COL3A1 (B) and aSMA (C). Bars represent TGFb-induced fold expression changes compared with its untreated control. Magnitude difference in the presence of matrix against polystyrene was assess and data were represent mean ± SEM and *p50.05.
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Figure 4. Mechanical stretch can impact the basal expression levels of human cardiac fibroblast cells seeded on different extracellular matrix sub-types. HCF cells were seeded on either collagen type 1 matrix, collagen type IV matrix, collagen type VI matrix or laminin. Cells underwent either biaxial mechanical stretch for 72 h or were left un-stretched (control). Alterations in COL1A1 (A), COL3A1 (B) and aSMA (C) gene expression were examined using quantitative real-time PCR analysis. Bars represent fold stretch-induced mRNA expression changes compared with non-stretched cells. Data represent mean ± SEM. *p50.05, **p50.01.
ECM, the cardiac fibroblast has engendered a vast amount of research aimed at assessing factors which alter its bioactivity which, in turn, may be utilised as potential therapeutic targets. To the best of our knowledge, concerning factors that regulate the growth and function of cardiac fibroblasts has been acquired from cells in culture. More often than not these cells are fibroblasts from different species most commonly murine cardiac fibroblasts. While fibroblasts were previously thought of as a uniform cell type regardless of the tissue of origin, phenotypic diversity has been demonstrated in fibroblast responses to external stimuli in cells sourced from multiple different tissue types (6,7). It is not surprising therefore that there would be great variability in fibroblast physiology from species to species (8) which questions the reliability of extrapolating from data obtained in animal-derived cells. The aim of this study was to advance our understanding of the biology of the human cardiac fibroblast, examining the complex interaction of multiple factors influencing cellular activity including
mechanical stretch, the underlying matrix and response to the pro-fibrotic cytokine TGFb. The role of the pro-fibrotic agonist TGFb in the process of cardiac fibrosis is well documented. In particular, the role of TGFb in myofibroblast differentiation and activation is becoming ever more apparent where the cells exhibit increased secretary, migratory and proliferative properties and express the contractile protein aSMA (9–12). This is also supported by work carried out by Teekakirikul et al. (13) who, using a mouse model of cardiac fibrosis, highlighted the significance of TGFb in this pathological remodelling by abrogating myocardial fibrosis through the use of TGFb neutralising antibodies. In this study, we found that TGFb upregulated COL1A1 and aSMA mRNA levels in HCF cells cultured on polystyrene which is consistent with published data although it was surprising to observe that COL3A1 was not inducible under these conditions (9,14). Previous studies have highlighted that collagen 3 gene expression can be induced by TGF, however
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Figure 5. Differential TGFb1 responses in human cardiac fibroblast cells exposed to mechanical stretch and various extracellular matrix sub-types. HCF cells were seeded on either collagen type 1 matrix, collagen type IV matrix, collagen type VI matrix or laminin. Cells underwent either biaxial mechanical stretch for 72 h or were left un-stretched (control), in the presence or absence of 10 ng/ml TGFb1. Alterations in COL1A1 (A), COL3A1 (B) and aSMA (C) gene expression were examined using quantitative real-time PCR analysis. Bars represent TGFb-induced fold expression changes in stretched cells compared with TGFb treated non-stretched cells. Data represent mean ± SEM. *p50.05, **p50.01.
these studies have utilised cardiac fibroblasts from diseased patients including those with failing hearts and those undergoing cardiothoracic bypass surgery, unlike reported herein where the primary adult ventricular fibroblast cells were derived from a young female donor (15,16). It is possible that TGF regulation of COL3A1 is influenced by pathological status or environment where the fibroblasts were procured from, where as this may not be the case for COL1A1 regulation. An important regulator of fibroblast activity is the surrounding ECM. Matrix influences cellular activity in a number of ways. By the early 1980s Bissell et al. (17) first described ‘‘dynamic reciprocity’’ between the ECM and the cells in proximity. The fibroblast, as stated previously, maintains ECM homeostasis. In turn, the type of ECM influences the degree of force transmitted to the cell by the beating myocardium. For example, less deformation will
occur in cells surrounded by collagen type I than those surrounded by the more flexible type III collagen. The degree of deformation is interpreted by the cell via a network of actin filaments and microtubules. This mechano-sensory mechanism influences gene activity (3,18–21). Integrins, a family of heterodimeric transmembrane proteins, allow the fibroblast to adhere to the ECM. These consist of a and b chains which combine to form dimers which are specific to different components of the matrix, for example, a1b1 binds to collagen and laminin while a3b1 binds to fibronectin. The clustering of integrins at focal adhesion complexes can stimulate second messenger signalling and influence cell activity. Integrin expression levels can also be influenced by mechanical stretch (22). Different integrin clustering results in different functional activities and may impact cellular responses to the pro-fibrotic cytokine TGFb (23,24). Therefore, when interpreting responses of cells to various
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stimuli in vitro, we must consider the effect of the underlying matrix. Our study reveals that all of the matrices studied not only affected the expression of pro-fibrotic genes in human cardiac fibroblast cells at rest, but also affected their response to TGFb. Collagen type I matrix, the most abundant component of the ECM in the heart induced a 2-fold induction of COL1A1 mRNA expression. It also significantly augmented the effect of TGFb on COL1A1 mRNA expression. Collagen type IV matrix, a major component of the basement membrane which surrounds myocyte fibrils also augmented TGFb induction of COL1A1. Although collagen type VI matrix is a minor component of the ECM it appears to play an important role in structural changes associated with hypertension (25) and has been shown to induce myofibroblast differentiation in rat ventricular fibroblasts (26). In our study, it was demonstrated to significantly induce COL1A1 at rest and to augment the effect of TGFb on COL1A1 induction. It did not, however, induce myofibroblast differentiation in human fibroblasts or alter COL3A1 gene expression. Laminin, the major component of the basal lamina providing an integral part of the structural scaffolding of the heart, is more abundant in heart failure (27). Here, we show that HCF cells seeded on laminin have enhanced TGFb induction of COL1A1 and aSMA gene expression. Mechanical load is an important factor in the growth and development of many tissues, for example, bone and skeletal muscle. Responding to mechanical cues is necessary for tissue homeostasis, and mechanical stress is linked to the development and progression of several disease processes (28,29). It is also of primary importance in the regulation of cardiac muscle development. Increasing mechanical load on cardiac tissue, as is the case in hypertension or outflow tract obstruction, results in hypertrophy and reactive fibrosis in vivo. The process by which stretch mediates an increased deposition of collagen is complex and poorly understood. Stretch appears not only to effect fibroblast activity directly, but also induces the autocrine production of pro-fibrotic growth factors. Further complicating matters, the surrounding ECM modulates fibroblast activity and influences a cells response to stretch as described above. To study mechanotransduction and signalling pathways triggered by mechanical stress, stretch devices have been designed (30). These were initially in the form of uniaxial devices which exerted a force in one direction, however, more recent devices exert biaxial strain. Cells are cultured on silicon membranes which can undergo either static stretch or, with the aid of a vacuum, cyclical stretch. In addition, the membranes may be coated with different matrix proteins. Unfortunately, it is difficult to draw definitive conclusions from the literature on the effect of stretch and matrices on human cardiac fibroblast activity for the same reasons given above; to date, most studies use murine fibroblasts are on a small scale and the experimental conditions differ greatly. There are similar findings when one reviews the literature on the effect of mechanical load on collagen synthesis. For example, neither Butt et al. nor Carver et al. found any change in collagen synthesis from fibroblasts exposed to cyclical load while Lee et al. showed an
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inhibition of ECM mRNA’s under such conditions (31–33). On the other hand, more recently, Husse et al. (34) demonstrated a load induced increase in COL1A1 mRNA. Again, this disparity in results may be explained by different experimental conditions. For example, Carver et al. plated their cells on laminin coated plates in the presence of FCS while Butt et al. showed no effect on collagen synthesis in the absence of FCS but an increase in the presence of serum. Not only do different cell types respond differently to mechanical stretch, but the same cells from different tissues also respond differently. Aortic smooth muscle cells increase collagen synthesis in response mechanical load while pulmonary artery smooth muscle cells do not (35,36). Dalla Costa et al. (37) recently reported an increase in aSMA expression in rat cardiac fibroblasts exposed to cyclical stretch. Indeed, mechanotransduction via integrindependent signalling is considered an intrinsic step towards generation of aSMA positive differentiated fibroblasts (38,39). However, again we face a paucity of data regarding the effect of these stimuli on human cardiac fibroblasts. Our data, presented here, attempts to clarify some of these issues. Interestingly, differential cellular responses to stretch were observed depending on the type of matrix the cells were adhered to. Stretching cells on collagen VI or laminin resulted in a significant increase in expression of all three genes studied. Collagen IV matrix also increase aSMA when stretched. Cells were only primed for a TGF-induced COL3A1 expression if they were cultured on collagen VI matrix and stretched. This observation is very curious and physiologically may be a mechanism by which selective deposition of type 3 collagen can occur in disease. Mechanical stretch did not impact TGFb induction of COL1A1 gene expression on any matrix studied. Interestingly, depending on environmental conditions, stretch had a protective impact on cellular responses to TGFb. Stretching cells on a collagen type 1 matrix repressed TGF b induction of aSMA and COL3A1 expression. TGFb induction of aSMA was also reduced in cells stretched on collagen VI matrix. This anti-fibrotic observation may be due to the activation of the protective natriuretic peptide system under certain conditions (40). These data attempt to examine comprehensively the effect of various stimuli (both in isolation and in conjunction with each other) previously documented to alter fibroblast activity. Taken together these data highlight the varied responses of human cardiac fibroblasts to the pro-fibrotic cytokine TGFb which can be impacted by the substrate the cells are adhered to, whether polystyrene or various ECM types, as well as the influence of cells to mechanical stretch. Of note, however, when interpreting these data it must be highlighted that protein levels were not assessed, and additional analytical time points and degrees of stretch would also add value to this work. In addition, increasing experimental replicates as well as reproducing this study in additional primary cell isolates from various donors would lead to a more definitive and comprehensive understanding of human cardiac fibroblast pathological responses as it is possible that cells may behave differently depending on age and cardiovascular health of donors.
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Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The study was supported by the Health Research Board of Ireland [grant number RP/2007/313 to JB and KM].
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