Angiotensin II and αvβ3 Integrin Expression in Rat Neonatal Cardiac Fibroblasts
Abstract—We recently demonstrated that αvβ3 integrins are involved in the mechanisms of angiotensin II (Ang II)–induced DNA synthesis and collagen gel contractions in rat cardiac fibroblasts (CFBs), cellular mechanisms that are relevant for cardiac remodeling. The aim of the present study was to elucidate the effect of Ang II and other growth factors on the regulation of the αvβ3 integrins in fibroblasts from neonatal rat hearts. The αvβ3 integrin receptor expression was significantly increased (P<0.05) at the mRNA level after treatment with Ang II, transforming growth factor-β1 (TGF-β1), and platelet-derived growth factor (PDGF) for 8 and 16 hours. The surface expression of the αv and β3 integrin subunits was elevated after 32 and 48 hours (P<0.05) as determined with flow cytometry. To investigate fibroblast motility, we performed chemotaxis experiments with transwell chambers. Ang II was chemotactic for CFBs, as tested with checkerboard experiments. The chemotactic effect was concentration dependent and was completely blocked by Ang II type 1 receptor blockers but not by Ang II type 2 receptor blocker PD 123319. Ang II– and PDGF-BB–mediated chemotaxis could be significantly inhibited by RGD peptides and the blocking antibodies against αvβ3 integrin (both P<0.01). Adhesion of CFBs to vitronectin was partially inhibited by an antibody to αvβ3 integrin but was mainly mediated by an αvβ5 integrin. Relevant in vivo expression of αvβ3 integrin by CFBs was confirmed with in situ hybridization with probes for αv and β3 mRNA in rat hearts. The present study demonstrates that the expression of αvβ3 integrin is augmented by Ang II, PDGF, and TGF-β1 in neonatal CFBs. Furthermore, this integrin is involved in the chemotaxis, motility, and adhesion of CFBs. The present findings support the current concept that integrins participate in the control of fibroblast behavior during cardiac remodeling mechanisms.
Integrin heterodimers, which belong to a family of transmembrane receptor molecules, provide the direct adhesive link between the cells and the surrounding matrix and are transducing signals that are involved in various cell functions.1 2 In the heart, the interaction of cardiac fibroblasts (CFBs) with their surrounding matrix is critical for repair mechanisms, including synthesis of matrix proteins, proliferation, collagen gel contractions, and cell motility.
Angiotensin II (Ang II) and transforming growth factor-β1 (TGF-β1) have been shown to be critical for cardiac remodeling and tissue repair.3 Ang II induces fibronectin, laminin, and TGF-β1 mRNA via its Ang II type 1 receptor (AT1) in CFBs.4 5 AT1 receptor expression, ACE activity, and accumulation of fibrillar collagens are increased in healing rat myocardium after myocardial infarction.6 7 These processes are accompanied by an augmented expression of TGF-β1 in the myocardium.8 TGF-β1 has been shown to regulate integrins in different cell lines, including rabbit smooth muscle and endothelial cells.9 10 11
Burgess and coworkers12 demonstrated that Ang II increases CFB-mediated collagen gel contractions, which are partially mediated via β1 integrins. We recently demonstrated that via binding to integrin αvβ3 and its binding motif arginine-glycine-aspartic acid (RGD), osteopontin (OP) also contributes to the regulation of Ang II–induced DNA synthesis and collagen gel contractions in cultured CFBs from neonatal and adult rats.13 Furthermore, we could demonstrate that increased OP expression is associated with cardiac hypertrophy in rat and human hearts.14 The present data indicate that integrins might be important players in cardiac tissue repair and remodeling processes. However, the regulation of integrin expression and its further functional relevance in regard to cellular remodeling mechanisms have not been sufficiently investigated in CFBs.
In the present study, we demonstrate that αvβ3 integrin is upregulated by Ang II, platelet-derived growth factor (PDGF), and TGF-β1 in CFBs and that this integrin contributes to fibroblast adhesion to vitronectin and mediates Ang II–directed chemotaxis and motility of these cells.
Culture medium, glutamine, antibiotics, HEPES, PDGF-BB, FBS, rat vitronectin, rat fibronectin, and gelatin were purchased from Sigma Chemical Co. Culture plasticware was purchased from Becton Dickinson. TGF-β1 was purchased from R&D Laboratories. The transwell chambers were obtained from Costar. Collagen I was obtained from Cetrix. Sprague-Dawley rats were obtained from Charles River. Antibodies against human αv (AB1930) and human β3 (AB1932) were obtained from Chemicon. Antibody against rat β3 integrin F11 was purchased from PharMingen. The monoclonal antibody against vimentin was obtained from Immunotech. The monoclonal antibodies against αvβ5 (P1F6) and αv (VNR 139) were obtained from GIBCO BRL. Ang II and peptide examers GRGDSP and GRGESP were obtained from Bachem.
Neonatal CFBs were prepared from Sprague-Dawley rats 1 to 3 days after birth and characterized as previously described.4 Briefly, neonatal hearts were dissected free of atria, minced, and subjected to trypsin and DNase II digestion. The isolated cells were preplated for 30 minutes in DMEM/F12 and 5% FBS. During this period of time, the nonmyocytes attached to the plate while the myocytes remained floating, thus separating the 2 populations. The attached nonmyocytes were grown in DMEM/F12 with 10% FBS until they reached confluency, at which time they were detached through trypsin treatment (0.5%) and split 1:4. All experiments were performed in the second and third passages after starvation in serum-free DMEM/F12 containing insulin (5 μg/mL), transferrin (5 μg/mL), and selenium (5 ng/mL) for 48 hours. Thereafter, cells were incubated with Ang II (0.1 μmol/L), PDGF-BB (10 ng/mL; Sigma), TGF-β1 (20 ng/mL; Sigma), or vehicle control for various time intervals, as indicated in Results.
Cells were incubated with primary antibody, washed, resuspended in the appropriate FITC-conjugated secondary antibody (Sigma), and analyzed for fluorescence on an FACScan flow cytometer (Becton Dickinson). The x and y axes represent log fluorescent intensity and cell number, respectively.
Chemotaxis experiments were performed as described previously.15 CFB migration was examined in transwell cell culture chambers with a gelatin-coated polycarbonate membrane with 8-μm pores. Preconfluent fibroblasts were suspended in DMEM/0.4% FBS to a concentration of 5.0×105 cells/mL. Cells were pretreated with antibodies or nonspecific IgG for 30 minutes at 20°C. DMEM/0.4% FCS (0.6 mL) was added to the lower compartment. Cell suspension (0.1 mL; final 50 000 cells/well, diameter 6.5 mm) was added to the upper compartment, and cells were then incubated at 37°C (95% air/5% CO2). Chemotaxis was induced by the addition of PDGF-BB or Ang II to the lower compartment. After 4 hours, the filters were fixed with methanol (10 minutes at 4°C), followed by counterstaining with hematoxylin. The number of cells per ×320 high-power field that migrated to the lower surface of the filters was determined microscopically. Four randomly chosen high-power fields were counted per filter. Experiments were performed in duplicate or triplicate and were repeated at least 3 times.
Adhesion assays were performed as described previously.16 Adhesive substrates (rat fibronectin, rat vitronectin, and collagen I; Sigma Chemical Co) were added to 96-well plates (Maxisorb; Nunc) and incubated overnight at 4°C. Nonspecific binding was blocked with 1% BSA at 37°C for 1 hour. Cells were detached by minimal trypsinization (0.05% trypsin; GIBCO), placed into medium containing 5% serum, and centrifuged. The cells were washed 3 times in DMEM/0.5% BSA. Cells (30 000) were added to each well in the presence of antibodies or hexamer peptides (GRGDSP and GRGESP). Plates were then incubated for 60 minutes at 37°C. Thereafter, nonadherent cells were washed away with PBS, and the remaining cells were fixated with 4% paraformaldehyde for 5 minutes and then stained with 0.5% toluidine blue in 4% paraformaldehyde for 5 minutes, rinsed with water, and solubilized with 100 μL of 1% SDS. Optical density was read at a wavelength of 595 nm with an ELISA reader.
Isolation and Analysis of RNA
Total RNA was isolated from CFBs using the acid guanidinium hemocyanate–phenol–chloroform method.17 RNA was size-fractionated with electrophoresis through a denaturing 1% agarose gel, transferred to nitrocellulose membranes, and hybridized with cDNA probes labeled with 32P-dCTP (3000 Ci/mmol) through random priming. The cDNAs for rat αv and β3 integrins were kindly provided by Dr Gideon Rodan (Merck). For detection of OP mRNA, the 2B7 plasmid, which contains a 1.0-kb cDNA fragment for rat OP, was used.13 14 The hybridization signals of the specific mRNAs of interest were normalized to those of CHO-B, a constitutively expressed gene, to correct for differences in loading or transfer.18 CHO-B cDNA is originally isolated from Chinese hamster ovary cells and corresponds to an RNA ubiquitously expressed in mammalian tissues that does not exhibit regulation as a function of growth or development. Quantification of Northern blots was performed through densitometric analysis with NIH Image 1.60 software for Macintosh personal computers. Several autoradiographic film exposures (from 12 hours to 4 days) were used to ensure the densities of the signals were linear on each film.
In Situ Hybridization
The plasmids with the cDNA for rat and β3 integrins were linearized through digestion with a restriction enzyme. Riboprobe fragments of 350 (αv) and 430 (β3) bp were generated through transcription of linearized cDNA with T3- and T7-polymerase with digoxigenin-labeled UTP (Boehringer-Mannheim) as substrate. Formalin-fixed, paraffin-embedded 6-μm-thick sections of adult and neonate rat hearts were deparaffinized, and procedures were performed as described previously.14
The labeled avidin-biotin method was used for detection as described previously.14 Biotinylated secondary antibodies were applied (Zymed), followed by an incubation with streptavidin-peroxidase. Peroxidase activity was detected with aminoethyl carbazole as a chromogen (liquid AEC Kit; Zymed). Slides were counterstained with hematoxylin.
ANOVA and paired or unpaired t test were performed for statistical analysis, as appropriate. A value of P<0.05 was considered to be statistically significant. Data were expressed as mean±SEM, if not stated otherwise.
CFBs Express αvβ3 Integrin
Northern blot analysis with cDNA for rat αv and β3 mRNA integrins demonstrated mRNA levels for both integrin subunits in cultured CFBs (Figure 1A⇓). Treatment of quiescent CFBs with Ang II (0.1 μmol/L), PDGF-BB (10 ng/mL), or TGF-β1(20 ng/mL) significantly increased αv and β3 integrin mRNA levels after 8 and 16 hours as determined with densitometric analysis from 3 different experiments (Figure 1B⇓). At 32 hours, no significant differences were observed (Figure 1B⇓). We also studied the expression of a ligand of αvβ3, OP, as an indicator for fibroblast activation by Ang II and the other growth factors. In these experiments (Figure 1⇓), OP mRNA levels showed a significant time-dependent increase between 8 and 32 hours, as we described previously.13 No significant differences were seen in experiments with 4-hour stimulation with these growth factors (data not shown). Vehicle control did not change mRNA levels for both integrins in cultured rat CFBs.
To investigate the effect of growth factors on integrin surface expression, rat CFBs were treated for 12, 24, 32, and 48 hours with Ang II (0.1 μmol/L), Ang II and losartan (DUP, 10 μmol/L) combined, PDGF-BB (20 ng/mL), TGF-β1 (10 ng/mL), or vehicle, and the expression was determined with flow cytometry. After 12- and 24-hour treatments, no significant changes in expression were seen. After 32 hours, PDGF and Ang II induced significant upregulation of β3 integrins, which increased further after 48 hours (P<0.05 versus control, Figure 2⇓, left). A 48-hour treatment with Ang II, PDGF-BB, and TGF-β1 induced a clear shift in fluorescence activity and an increase in mean channel fluorescence due to increased integrin expression for both integrins (Figure 2⇓, right). Coincubation of Ang II and losartan completely prevented changes in surface expression of both integrins after Ang II treatment for 32 and 48 hours.
Ang II–Directed Chemotaxis of Cardiac Fibroblasts Is αvβ3 Dependent
Ang II is chemotactic for CFBs as demonstrated in the checkerboard experiment (Table⇓). Ang II demonstrated a concentration-dependent chemotactic effect, which was significant at the concentration of 10 nmol/L Ang II (P<0.05, Figure 3A⇓). Coincubation with the AT1 receptor blockers losartan and irbesartan completely inhibited the chemotactic effect (10 μmol/L each, P<0.01), whereas incubation with PD123319 (10 μmol/L) did not affect Ang II–directed chemotaxis (Figure 3B⇓). To investigate the role of αvβ3 integrin function in CFBs, we performed migration experiments in a transwell chamber system. Ang II (0.1 μmol/L) and PDGF-BB (10 ng/mL) were used as chemoattractants (Figures 3C⇓ and 3D⇓). To avoid interference with the adhesion process occurring in the first 30 minutes, cells were first added to the upper chamber. After 40 minutes, pretreatment of CFBs was started for an additional 20-minute period with a nonspecific IgG (25 μg/mL); F11 (25 μg/mL); an blocking antibody of β3 integrin, GRGDSP, which blocks the integrin binding pouch of αvβ3 integrin; or GRGESP as a control peptide (both GRGDSP and GRGESP at 100 μmol/L). Nonspecific IgG or GRGESP did not affect CFB chemotaxis (Figures 3C⇓ and 3D⇓). In contrast, the antibody F11 against αvβ3 integrin and the competitive peptide GRGDSP inhibited chemotaxis by >50% (P<0.05).
Adhesion of CFBs to Vitronectin Is Partially Mediated by αvβ3 Integrin
The function of αvβ3 integrin-mediated adhesion to vitronectin, collagen I, and fibronectin was tested in an adhesion assay (Figure 4⇓). CFBs demonstrated reproducible binding to all matrices. Maximal adhesion was observed on plates coated with 5 μg/mL vitronectin, 10 μg/mL collagen I, and 20 μg/mL fibronectin (data not shown). The effect was tested of F11 (25 μg/mL), the blocking antibody of β3 integrin GRGDSP, which blocks the integrin binding pouch of αvβ3 integrin, or GRGESP as control peptide (both at 100 μmol/L). Furthermore, we used a blocking antibody against αvβ5 integrin (P1F6), which is another receptor for vitronectin. Adhesion to collagen I and fibronectin was not significantly attenuated by either the blocking antibodies or the GRGDSP hexamers (Figure 4⇓). In contrast, the adhesion of CFBs to vitronectin-coated layers was significantly reduced by F11 (P<0.05 versus IgG) for ≈20%. Adhesion to vitronectin was mainly inhibited by GRGDSP and an antibody against αvβ5 integrin (P1F6) but not by nonspecific IgG or control peptides with the GRGESP sequence.
In Vivo Expression of αvβ3 in Rat Hearts
We performed in situ hybridization with rat heart sections from neonatal and 8-week-old Sprague-Dawley rats (n=4). Consistent expressions of mRNA transcripts were found for both integrins in all sections. Intense signals of transcripts for αv integrin were seen in cardiomyocytes, in fibroblast areas, and in endocardium (Figure 6). Transcripts for β3 were generally less intense and were observed in perivascular tissue, in vessel walls, and in cardiomyocytes (Figure 5⇓). The major sources for both integrins in the heart were cardiomyocytes and CFBs. Signals for both mRNA transcripts were found in areas typical for fibroblasts. Comparable results were also observed in sections from neonatal rat hearts (n=4, data not shown). Interestingly, strong expression for both integrins was observed in perivalvular tissues of the atrioventricular valves in neonatal and adult rat hearts (Figures 5A⇓ to 5C).
In the present investigation, we demonstrate that Ang II via the AT1 receptor, PDGF, and TGF-β1 increased αvβ3 integrin in cultured neonatal rat CFBs, as shown with flow cytometry and Northern blot analysis. These growth factors induced upregulation of mRNA levels for both integrin subunits after 8 to 16 hours of stimulation and corresponded to the upregulation of OP mRNA levels, a potential ligand for αvβ3 integrin. TGF-β1 has been reported previously to upregulate β3 integrins in rabbit smooth muscle and endothelial cells10 11 and in some cell lines.9 To our knowledge, it is the first evidence that Ang II increases cellular integrin expression in CFBs. The present finding might be of pathophysiological relevance, because Ang II and TGF-β1 are critical for cardiac growth and repair mechanisms.3 19 20
Using in situ hybridization, we could detect in vivo expression of both subunits, αv and β3 integrin mRNAs, in cardiac tissues and in regions that are typical for fibroblasts. In addition, the in situ hybridization revealed strong expression of αv RNA in cardiomyocytes, whereas β3 mRNA expression was modest in cardiomyocytes. This indicates that αvβ3 integrin is not exclusively expressed by fibroblasts, but it might also be expressed by cardiomyocytes. We recently demonstrated that via binding to integrin αvβ3 and its binding motif arginine-glycine-aspartic acid (RGD), OP regulates Ang II–induced DNA synthesis and collagen gel contractions in cultured CFBs from neonatal and adult rats.13 Furthermore, increased OP expression is associated with cardiac hypertrophy in rat and human hearts.14 This study demonstrates that the expression of αvβ3 is upregulated by Ang II and other growth factors in CFBs and that this receptor is potentially important for mediation of fibroblast functions, such as DNA synthesis and collagen gel contractions.13 Therefore, we tested in the second part of this study how αvβ3 is involved in adhesion to extracellular matrix proteins and fibroblast motility and chemotaxis.
Vitronectin is an adhesive plasma protein that is found in tissues after injury and during repair processes.21 The αvβ3 and αvβ5 integrins mediate adhesion of rat neonatal CFBs to vitronectin via its RGD sequence. Our experiments demonstrated that αvβ5 is the major vitronectin receptor on these cells and that αvβ3 integrin mediated only ≈20% of adhesion. In the present study, neither RGD peptides nor F11, the blocking antibody of αvβ3, impaired the adhesion of CFBs to collagen I or fibronectin. Binding to vitronectin was mediated via the RGD sequence. These data correspond to the recently published study by MacKenna and coworkers,22 who also found RGD-dependent adhesion in adult CFBs. They also reported that block of αvβ3 did not affect the adhesion of adult fibroblasts to collagen I and fibronectin. Both extracellular matrix proteins contain binding sites, other than the RGD motif, to bind to other integrin receptors. Furthermore, it has been reported that the ligand affinity varies depending on cell type and divalent cations concentrations.23
The third finding of the present study is that αvβ3 integrin is important for Ang II–directed motility of CFBs. In addition, we could demonstrate that Ang II (like PDGF-BB) is chemotactic for CFBs and affects the motility of these cells. This effect was mediated via the AT1 receptor, whereas the AT2 receptor blocker PD 123319 did not interfere with Ang II–directed fibroblast motility. This is a novel aspect of Ang II–mediated functions on CFBs, which adds to the present understanding of Ang II as an important mediator of cardiac repair and fibrosis.3 The concentration of Ang II that attracts CFBs was in the physiological range. Chemotaxis, or directed cell migration, is a very complex mechanism that combines cellular behaviors of adhesion, spreading, and contraction. Furthermore, the cell uses an intracellular machinery, which is able to sense low gradients of chemoattractant protein.24 The effect of Ang II, as well as PDGF, on migration was significantly reduced in the presence of RGD hexamers or in the presence of an blocking antibody against αvβ3 integrin. In the present study, we started experiments of chemotaxis as early as 1 hour after the placement of cells into the chamber system so as not to interfere with the primary attachment and adhesion process, which is not αvβ3 dependent on gelatin. The experiments demonstrated that αvβ3 integrins are involved in the regulation of Ang II–directed fibroblast migration.
In the present study, we could demonstrate that Ang II induces αvβ3 integrin expression in CFBs and regulates fibroblast migration, which is mediated by αvβ3 integrin. These findings further elucidate the important relationship between Ang II and integrins. The present data underscore the potential pathophysiological relevance of cell/matrix interaction for cardiac remodeling and repair mechanisms.
This work was supported by Deutsche Forschungsgemeinschaft grant GR 1368/2-1.
- Received July 1, 1999.
- Revision received July 21, 1999.
- Accepted November 22, 1999.
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