Angiotensin II and α_vβ₃ Integrin Expression in Rat Neonatal Cardiac Fibroblasts

Materials

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 α_v (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 α_vβ₅ (P1F6) and α_v (VNR 139) were obtained from GIBCO BRL. Ang II and peptide examers GRGDSP and GRGESP were obtained from Bachem.

Cell Culture

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.

Flow Cytometry

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

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

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 α_v 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 (α_v) 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

Immunohistochemistry

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.

Statistical Analysis

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 α_vβ₃ Integrin

Northern blot analysis with cDNA for rat α_v 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 α_v 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 α_vβ₃, 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.

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Figure 1.

A, Representative Northern analysis demonstrates presence of mRNA for β₃ at 4.5 kb, for α_v at 4.2 kb, and for OP at 1.1 kb in cultured CFBs after treatment with Ang II (AII; 0.1 μmol/L), PDGF-BB (10 ng/mL), TGF-β1 (20 ng/mL), and vehicle control (Co) for 8, 16, and 32 hours. Hybridization signal CHO-b (1.1 kb), a ubiquitously expressed nonregulated housekeeping gene (see Methods), was used as an internal standard for RNA loading. Hybridization with a probe against rat osteopontin was used as internal control and showed a clear increase after stimulation with growth factors. B, Densitometric analysis from 3 different experiments with CFBs. *P<0.05 vs control.

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.

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Figure 2.

Mean channel fluorescence for the binding of an anti-α_v rabbit antiserum (top) and an anti-β₃ rabbit antiserum (bottom) was determined from CFBs by flow cytometry. CFBs were treated with diluent (Co), Ang II (A II; 0.1 μmol/L), Ang II (0.1 μmol/L) and the AT₁ receptor blocker losartan (DUP; 10 μmol/L), TGF-β1 (20 ng/mL), and PDGF-BB (10 ng/mL) for 32 (left) at 48 hours (right). The experiment was performed with 4 sets of cells from different preparations. Treatment with growth factors induced a significant increase in binding sites for both antisera. The AT₁ receptor blocker losartan (DUP) completely inhibited the effect of Ang II. *P<0.05 vs control.

Ang II–Directed Chemotaxis of Cardiac Fibroblasts Is α_vβ₃ 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 α_vβ₃ 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 α_vβ₃ 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 α_vβ₃ integrin and the competitive peptide GRGDSP inhibited chemotaxis by >50% (P<0.05).

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Figure 3.

A, Using a transwell system chemotactic effect of Ang II (AII) was tested with different Ang II concentrations between 0.1 nmol/L and 1 μmol/L on CFBs. A concentration-dependent chemotactic effect of Ang II was observed which was significant at 10 nmol/L (n=8, mean±SEM, *P<0.05 vs control). B, Ang II (A II; 1 μmol/L)–mediated chemotaxis was tested in presence of AT₁ receptor blockers losartan (DUP) and irbesartan (Irb) and the AT₂ receptor blocker PD 123319 (each at 10 μmol/L). Both AT₁ receptor blockers completely inhibited the Ang II–mediated effect, whereas PD 123319 did not affect Ang II–directed chemotaxis (n=5, mean±SEM, *P<0.05 vs control, #P<0.05 vs Ang II alone). C, Effect of α_vβ₃ blockade on PDGF- and Ang II (D)–directed chemotaxis. Chemotaxis toward both gradients was inhibited by blocking antibodies against α_vβ₃ (F11, 50 μg/mL) and by GRGDSP (100 μmol/L). IgG (50 μg/mL) or control peptide hexamer GRGESP did not affect the gradient directed motility of CFBs (n=6, mean±SE, *P<0.01 vs Ang II+IgG).

Adhesion of CFBs to Vitronectin Is Partially Mediated by α_vβ₃ Integrin

The function of α_vβ₃ 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 α_vβ₃ integrin, or GRGESP as control peptide (both at 100 μmol/L). Furthermore, we used a blocking antibody against α_vβ₅ 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 α_vβ₅ integrin (P1F6) but not by nonspecific IgG or control peptides with the GRGESP sequence.

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Figure 4.

Adhesion of CFBs (30 000/well) to plates coated with collagen I (10 μg/mL), fibronectin (20 μg/mL), and vitronectin (5 μg/mL) in presence of nonspecific IgG (25 μg/mL), a non–function-blocking antibody against α_v (aVNR, 25 μg/mL), the β₃ integrin–blocking antibody F11 (25 μg/mL), P1F6 (2 μg/mL), and the peptidhexamers GRGDSP and GRGESP (both 100 μmol/L). GRGDSP, F11, and P1F6 significantly inhibited adhesion to gelatin. IgG, aVN, and the control hexamer GRGESP did not affect adhesion. Adhesion to vitronectin was predominantly inhibited by P1F6 and the GRGDSP hexamer. F11 showed a smaller but significant effect on vitronectin adhesion (data expressed in percent adhesion of untreated fibroblasts, mean±SEM, n=4 different experiments in quadruplicate, *P<0.05 vs control, **P<0.01 vs control).

In Vivo Expression of α_vβ₃ 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 α_v 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).

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Figure 5.

A, Immunohistochemistry performed with a vimentin antibody, which demonstrates a high number of positive cells in the mitral leaflet area (×200). B, In situ hybridization of an adjacent section with antisense riboprobe against α_v integrin demonstrates presence of transcripts in these cells and in cardiomyocytes (×300). C, Control hybridization with sense probe did not show significant nonspecific signals (×100, counterstained with hematoxylin). D, In situ hybridization with antisense riboprobe against β₃ integrin mRNA demonstrates presence of transcripts in perivascular cells (arrows) and in vascular endothelial and smooth muscle cells. Some transcripts were also found in cardiomyocytes. E, Control hybridization with sense probe did not show significant nonspecific signals (×300, counterstained with hematoxylin).