Vascular Endothelial Growth Factor Induces Activation and Subcellular Translocation of Focal Adhesion Kinase (p125^FAK) in Cultured Rat Cardiac Myocytes

Cell Culture

Primary culture of ventricular cardiac myocytes were prepared from neonatal rats as previously described.²³ Briefly, heart ventricles were aseptically removed from neonatal Wistar rats, minced in calcium-free PBS, and digested with 0.025% trypsin-EDTA in PBS. The isolated cells were washed in DMEM (GIBCO Laboratories) containing 10% FBS and preplated onto plastic dishes for 1 hour to selectively remove fibroblasts. Nonadherent cells (enriched in cardiac myocytes) were collected and replated onto gelatin-coated culture dishes. They were cultured for 2 days in DMEM supplemented with 10% FBS, 50 U/mL penicillin, and 34 μmol/L streptomycin until they were confluent. They were starved for 24 hours before stimulation with VEGF.

The percentage of myocytes was estimated to be ≈90%, as judged by immunoperoxidase staining with a mouse anti-cardiac myosin heavy chain monoclonal antibody (mAb) derived from the CMA-19 clone,²⁴ followed by counterstaining with hematoxylin. Preparation of mouse anti-cardiac myosin heavy chain mAb (CMA19) was previously described.²⁴ Contamination by endothelial cells was estimated to be <1%, as judged by immunoperoxidase staining with mouse monoclonal anti–factor VIII–related antigen antibody (Z002; Zymed Laboratories) (data not shown). Therefore, we concluded that the rest of the contaminating nonmuscle cells (≈10% of total cells) mostly consisted of fibroblasts.

Analyses of Phosphotyrosine Content of KDR/Flk-1, p125^FAK, and Paxillin

The cells were treated with 22.8 pmol/L (1.0 ng/mL) recombinant human VEGF (rhVEGF; Upstate Biotechnology, Inc) for various times as indicated, and then they were frozen in liquid nitrogen and lysed on ice with NP-40 buffer containing (in mmol/L) Tris/HCl 25 (pH 7.6), NaCl 25, Na₃VO₄ 1, sodium pyrophosphate 10, EGTA 0.5, and phenylmethylsulfonyl fluoride 1; 10 nmol/L okadaic acid; and 1% NP-40. The cell lysates were centrifuged, and the supernatants containing detergent-soluble proteins were collected. Proteins were immunoprecipitated at 4°C overnight with mouse anti-phosphotyrosine mAb (PY20; Transduction Laboratories) and protein G–Sepharose (Pharmacia LKB). The immunoprecipitates were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride transfer membranes (NEN Research Products). Polyvinylidene difluoride membranes were blocked with 1% BSA in PBS and then incubated overnight at 4°C with rabbit polyclonal anti-KDR/Flk-1 antibody (C20; Santa Cruz Biotechnology), rabbit polyclonal anti-p125^FAK antibody,²⁵ or mouse anti-paxillin mAb (349; Transduction Laboratories). The preparation of rabbit polyclonal anti-p125^FAK antibody was previously described.²⁵ After incubation with alkaline phosphatase–conjugated anti-rabbit or anti-mouse IgG antibody (both antibodies from Vector Laboratories), the blots were developed with a chemiluminescence detection kit (New England Biolabs). Tyrosine phosphorylation of p125^FAK was confirmed by immunoprecipitation using anti-p125^FAK polyclonal antibody followed by Western blotting with anti-phosphotyrosine mAb (4G10; Upstate Biotechnology, Inc) or anti-p125^FAK polyclonal antibody. The increase in tyrosine phosphorylation of KDR/Flk-1 was quantified by scanning densitometry. For analysis of expression of VEGF receptors, total cell lysates in Laemmli sample buffer were electrophoresed and immunoblotted using rabbit polyclonal anti-KDR/Flk-1 antibody or rabbit polyclonal anti-Flt-1 antibody (C-17; Santa Cruz Biotechnology).

Kinase Assay of p125^FAK

Cardiac myocytes were treated with rhVEGF (22.8 pmol/L) for various times as indicated, and then they were frozen in liquid nitrogen. Immune complex tyrosine kinase assays of p125^FAK were performed with a nonradioactive isotope solid-phase ELISA kit using the exogenous substrate poly(Glu-Tyr)^{15 26 27} (Universal Tyrosine Kinase Assay Kit, Takara Shuzou Co Ltd) according to the manufacturer’s instructions. Briefly, the cell lysates were centrifuged and the supernatants were immunoprecipitated with rabbit polyclonal anti-p125^FAK antibody. The immunoprecipitates were incubated with ATP in the microtiter plate onto which poly(Glu-Tyr) had been precoated. The amount of phosphorylated poly(Glu-Tyr) was measured by ELISA using a horseradish peroxidase–linked anti-phosphotyrosine (PY20) antibody.

Analyses of the Effects of Tyrosine Kinase Inhibitors on Phosphorylation of p125^FAK

Cells were preincubated for 30 minutes with or without either of the tyrosine kinase inhibitors genistein²⁸ (37 μmol/L) and tyrphostin²⁹ (50 μmol/L) (both from GIBCO-BRL), and then they were stimulated with 22.8 pmol/L rhVEGF. The cell lysates were immunoprecipitated with mouse anti-phosphotyrosine mAb (PY20) and subjected to Western analysis using rabbit polyclonal anti-p125^FAK antibody. The increase in tyrosine phosphorylation at the 125-kDa band was quantified by scanning densitometry.

Analyses of Association of p125^FAK With p60^c-src, GRB2, or Shc

After stimulation with 22.8 pmol/L rhVEGF for various times as indicated, the cell lysates were immunoprecipitated with mouse anti-v-Src mAb 2-17 (LA074; Quality Biotech), rabbit polyclonal anti-GRB2 antibody (Santa Cruz Biotechnology), or mouse monoclonal anti-Shc antibody (PG-797; Santa Cruz Biotechnology) along with protein G–Sepharose and subjected to Western analysis using rabbit polyclonal anti-p125^FAK antibody.

Subcellular Fractionation

After stimulation with 22.8 pmol/L rhVEGF for various times as indicated, cytosolic and membrane fractions were prepared from cardiac myocytes according to modified methods previously described.^{30 31} Briefly, cells were washed twice with PBS and then harvested in a buffer containing 20 mmol/L HEPES and 250 mmol/L sucrose (pH 7.4) followed by centrifuging at 1000g for 3 minutes. The supernatant was discarded, and the pellet was suspended in hypotonic Tris buffer containing (in mmol/L) Tris (pH 7.5) 10, MgCl₂ 1, phenylmethylsulfonyl fluoride 1, and Na₃VO₄ 1, and 100 μmol/L leupeptin. The pellet was homogenized by 80 strokes of Dounce homogenizer. The homogenate was centrifuged at 1000g for 15 minutes to remove nuclei and debris. The supernatant was centrifuged at 48 000g for 30 minutes, resulting in a pellet, which was resuspended in hypotonic Tris buffer containing 1% NP-40 and stored as a membrane fraction, and the supernatant, which was recentrifuged at 246 000g for 90 minutes to remove the microsome-rich fraction. The resulting supernatant was stored as a cytosolic fraction. All of the above steps were carried out at 4°C unless otherwise indicated. Protein estimation was carried out using the BCA protein assay reagent (Pierce). The fractions were suspended in Laemmli sample buffer and incubated at 37°C for 30 minutes, and then their aliquots were subjected to SDS-PAGE followed by immunoblotting with polyclonal anti-p125^FAK antibody or rabbit polyclonal anti–rat Na,K-ATPase α₁ fusion protein (Upstate Biotechnology, Inc).

Immunocytochemistry

To investigate whether subcellular distribution of p125^FAK in cardiac myocytes and fibroblasts is altered in response to VEGF, we performed double staining for p125^FAK and cardiac myosin heavy chain. rhVEGF-treated (60 minutes) or untreated cardiac myocytes were fixed in acetone for 5 minutes at room temperature. The cells were incubated first with rabbit polyclonal anti-p125^FAK antibody for 1 hour at 37°C and then incubated sequentially with biotinylated anti-rabbit IgG (Vector Laboratories) for 1 hour at 37°C and FITC-conjugated avidin D (Vector Laboratories) for 30 minutes at 37°C. The cells were then incubated sequentially with mouse anti-cardiac myosin heavy chain mAb (CMA-19) for 1 hour at 37°C and TRITC-conjugated anti-mouse IgG1 antibody (Chemicon International, Inc) for 30 minutes at 37°C. To investigate the relationship between the immunofluorescent staining of p125^FAK and focal adhesions in rhVEGF-treated (60 minutes) cells, we performed double staining for p125^FAK and vinculin. rhVEGF-treated (60 minutes) cells were first stained with rabbit polyclonal anti-p125^FAK antibody and then were stained sequentially with biotinylated anti-rabbit IgG and FITC-conjugated avidin D as described above. The cells were then incubated with mouse anti-vinculin mAb (V-4505; Sigma) for 1 hour at 37°C and TRITC-conjugated anti-mouse IgG1 antibody for 30 minutes at 37°C. The sections were examined and photographed under a fluorescence microscope (Microphoto-FX, Nikon).

Cell-Substrate Adhesion Assays

Cell-substrate adhesion was measured using the electric cell-substrate impedance sensing system (Applied BioPhysics, Inc) previously reported by Giaever and Keese^{32 33} and Ghosh et al.³⁴ In this system, the cells were cultured on a small gold electrode (area, ≈10^–4 cm²) deposited on the bottom of tissue culture vessels. A small alternating current signal (1 μA) at a frequency of 4000 Hz was passed between the small electrode and a larger counter electrode (area, ≈10^–1 cm²) placed at a distance. The voltage between the small and large electrodes was monitored. As cell membranes have very high impedance, the attachment of cells to the small electrode blocks the current, forcing it to flow under the cells, causing an increase in the impedance. Primary cultures of ventricular cardiac myocytes were prepared as above. Cardiac myocytes were seeded on gelatin-precoated electrodes and cultured for 2 days in DMEM supplemented with 10% FBS, 50 U/mL penicillin, and 34 μmol/L streptomycin. They were starved for 24 hours and then stimulated with rhVEGF (34.2 pmol/L). The resistance, which reflects the extent of cell-substrate adhesion, was monitored as described previously.^{35 36}

Cardiac Myocytes Express KDR/Flk-1 and Flt-1

It has been shown that VEGF binds to 2 structurally related tyrosine kinase receptors, Flt-1^{37 38 39} and KDR/Flk-1.^{40 41 42 43} We examined whether cardiac myocytes expressed these receptors by Western analysis. As shown in Figure 1⇓, KDR/Flk-1 and Flt-1 were expressed in the cultured cardiac myocytes. To examine tyrosine phosphorylation of VEGF receptors, the cell lysates from rhVEGF-treated and untreated myocytes were immunoprecipitated with anti-phosphotyrosine mAb (PY20) followed by Western blotting using anti-KDR/Flk-1 or anti-Flt-1 antibodies. KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation, with phosphorylation reaching a maximum level at 5 to 10 minutes with rhVEGF (Figure 2A⇓), whereas Flt-1 was not (data not shown). The increase in tyrosine phosphorylation of KDR/Flk-1 was quantified by scanning densitometry of the autoradiogram and expressed as a ratio of the increase to the initial phosphorylation before addition of rhVEGF. As shown in Figure 2B⇓, tyrosine phosphorylation of KDR/Flk-1 was significantly increased at 5 to 10 minutes after rhVEGF treatment as compared with the initial value.

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Figure 1.

KDR/Flk-1 and Flt-1 are expressed in cardiac myocytes. To investigate whether cardiac myocytes express VEGF receptors KDR/Flk-1 and Flt-1, total cell lysates in Laemmli sample buffer were electrophoresed and immunoblotted using polyclonal anti-KDR/Flk-1 antibody or polyclonal anti-Flt-1 antibody. Mr indicates molecular weight.

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Figure 2.

KDR/Flk-1 is tyrosine phosphorylated in response to VEGF. Cultured rat cardiac myocytes were exposed to rhVEGF (22.8 pmol/L) for the indicated periods, and the cell lysates were immunoprecipitated with an anti-phosphotyrosine mAb (PY20) and then immunoblotted with anti-KDR/Flk-1 antibody (A). In addition, the increase in tyrosine phosphorylation of KDR/Flk-1 was quantified by scanning densitometry of the autoradiograms (B). The initial phosphorylation of KDR/Flk-1 before addition of rhVEGF at 0 minutes is defined as 1.0. Fold induction values represent average of 5 independent experiments. Ab indicates antibody; anti-pTyr I.P., anti-phosphotyrosine immunoprecipitation. *P<0.05, **P<0.01 vs initial value. Results shown are mean±SD.

VEGF Phosphorylates p125^FAK and Paxillin

To examine whether VEGF can stimulate tyrosine phosphorylation of a focal adhesion-associated protein tyrosine kinase p125^FAK, quiescent rat cardiac myocytes were treated with rhVEGF for various time periods and lysed. The cell lysates from the treated myocytes were immunoprecipitated with anti-p125^FAK polyclonal antibody followed by Western blotting with anti-phosphotyrosine mAb (4G10) or anti-p125^FAK polyclonal antibody. Conversely, immunoprecipitates with anti-phosphotyrosine mAb (PY20) were also analyzed by Western blotting with anti-p125^FAK antibody. As shown in Figure 3A⇓ and 3B⇓ (top panel), VEGF significantly increased tyrosine phosphorylation of p125^FAK, which peaked at 5 to 10 minutes after addition of rhVEGF. We confirmed that almost equal amounts of p125^FAK protein were electrophoresed in each reaction (Figure 3A⇓, bottom panel). We also investigated whether VEGF caused tyrosine phosphorylation of paxillin, another focal adhesion-associated protein, which interacts with several proteins, including p125^FAK, members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal protein vinculin. Immunoprecipitates with anti-phosphotyrosine mAb (PY20) from the stimulated cardiac myocytes were analyzed by Western blotting with anti-paxillin mAb. As shown in Figure 3B⇓ (bottom panel), VEGF also caused a significant increase in the tyrosine phosphorylation of paxillin. The tyrosine phosphorylation of paxillin reached a maximum level at 5 to 10 minutes after addition of VEGF and decreased subsequently.

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Figure 3.

VEGF causes tyrosine phosphorylation of p125^FAK and paxillin. Serum-starved cardiac myocytes were treated for the indicated periods with 22.8 pmol/L rhVEGF and lysed with NP-40 buffer. Tyrosine phosphorylation of p125^FAK was analyzed either by immunoprecipitation (I.P.) using anti-p125^FAK polyclonal antibody (Ab) followed by Western blotting with anti–phosphotyrosine (pTyr) mAb (4G10) (A, top panel) or by immunoprecipitation using anti-phosphotyrosine mAb (PY20) followed by Western blotting with anti-p125^FAK antibody (B, top panel). To ascertain equal loading of p125^FAK protein, immunoprecipitates with anti-p125^FAK antibody were analyzed by anti-p125^FAK Western blotting (A, bottom panel). Tyrosine phosphorylation of paxillin was analyzed by immunoprecipitation with anti-phosphotyrosine mAb (PY20) followed by Western blotting using anti-paxillin mAb (B, bottom panel). Arrows indicate positions of p125^FAK and paxillin. Experiments were performed in triplicate. Results shown are representative of 3 independent experiments.

VEGF Stimulates Protein Tyrosine Kinase Activity of p125^FAK

To examine whether the catalytic activity of p125^FAK is stimulated by VEGF, kinase activity of p125^FAK immunoprecipitates from untreated or rhVEGF-treated cardiac myocytes was assayed using poly(Glu-Tyr) as substrate. As shown in Figure 4⇓, VEGF induced activation of p125^FAK as early as 2 minutes, peaking at 5 minutes, which paralleled its tyrosine phosphorylation.

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Figure 4.

VEGF stimulates protein tyrosine kinase activity of p125^FAK. Protein tyrosine kinase activity of p125^FAK immunoprecipitates from untreated or 22.8 pmol/L rhVEGF-treated cardiac myocytes were assayed with a nonradioactive isotope solid-phase ELISA kit using poly(Glu-Tyr) as substrate. The immunoprecipitates were incubated with ATP in the microtiter plate onto which poly(Glu-Tyr) had been precoated. The amount of phosphorylated poly(Glu-Tyr) was measured by ELISA using a horseradish peroxidase–linked anti-phosphotyrosine (PY20) antibody. *P<0.001, **P<0.01 vs control. Results shown are mean±SD.

Tyrosine Kinase Inhibitors Partially Inhibit VEGF-Induced Activation of p125^FAK

Next, to examine whether VEGF-induced tyrosine phosphorylation of p125^FAK was dependent on tyrosine kinase activity, the cardiac myocytes were pretreated with or without either of the tyrosine kinase inhibitors genistein (37 μmol/L) and tyrphostin (50 μmol/L) before addition of VEGF. Representative results of 1 of 3 independent experiments are shown in Figure 5⇓. Both genistein and tyrphostin at least partially inhibited VEGF-induced increase in tyrosine phosphorylation of p125^FAK at 5 minutes.

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Figure 5.

Effects of tyrosine kinase inhibitors genistein and tyrphostin on VEGF-induced tyrosine phosphorylation of p125^FAK. Serum-starved cardiac myocytes were pretreated for 30 minutes with or without 37 μmol/L genistein or 50 μmol/L tyrphostin and were then challenged with 22.8 pmol/L rhVEGF for the indicated periods. Cells were subsequently lysed with NP-40 buffer, and lysates were immunoprecipitated with anti-phosphotyrosine antibody (PY20) and further analyzed by anti-p125^FAK Western blotting. Arrow indicates position of p125^FAK. The increase in tyrosine phosphorylation of p125^FAK was quantified by scanning densitometry of the autoradiogram (the corresponding figures underneath the panel). Initial phosphorylation of p125^FAK before addition of rhVEGF at 0 minutes is defined as 1.0. Fold induction values represent average of 3 independent experiments.

VEGF Induces Association of p125^FAK With Shc, GRB2, and p60^c-src

To determine whether c-Src plays a role in VEGF-induced tyrosine phosphorylation of p125^FAK, immunoprecipitates with anti-v-Src mAb were subjected to SDS-PAGE followed by immunoblotting with anti-p125^FAK antibody. As shown in Figure 6a⇓, VEGF caused a significant increase in the association of p60^c-src with p125^FAK, which peaked at 5 to 10 minutes after addition of VEGF. To determine whether the VEGF-activated p125^FAK signaling complexes contained other known Src homology (SH) 2–containing proteins, such as the SH2/SH3 adapter proteins GRB2 and Shc, we examined the VEGF-induced association of p125^FAK with these adapter proteins. Immunoprecipitates with either anti-GRB2 polyclonal antibody or anti-Shc mAb from VEGF-stimulated cardiac myocytes were subjected to SDS-PAGE followed by immunoblotting with anti-p125^FAK polyclonal antibody. As shown in Figure 6b⇓ and 6c⇓, VEGF caused a significant increase in the association of Shc and GRB2 with p125^FAK. The association peaked at 5 to 10 minutes after addition of VEGF.

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Figure 6.

VEGF induces association of p125^FAK with p60^c-src, GRB2, or Shc. Quiescent cardiac myocytes were treated with 22.8 pmol/L rhVEGF for the indicated periods. Cells were subsequently lysed with NP-40 buffer and immunoprecipitated with anti-v-Src mAb 2-17 (LA074) (a), polyclonal anti-GRB2 antibody (b), or anti-Shc mAb (PG-797) (c). Immunoprecipitates were analyzed by Western blotting with anti-p125^FAK polyclonal antibody. Arrows indicate positions of p125^FAK. Results shown are representative of 3 independent experiments.

VEGF Induces Subcellular Translocation of p125^FAK in Cardiac Myocytes and Fibroblasts

It has been shown that cell adhesion to ECM, such as fibronectin, through integrins causes increased tyrosine phosphorylation of p125^FAK as well as accumulation of p125^FAK in focal adhesions.^{13 44 45 46} We examined the effects of VEGF on subcellular localization of p125^FAK in cardiac myocytes and nonmuscle cells (which mostly consisted of fibroblasts). To distinguish cardiac myocytes from nonmuscle cells, we performed double staining for cardiac myosin heavy chain and p125^FAK by immunofluorescence. Figures 7B⇓ and 7D⇓ show that myofibrils of cardiac myocytes were strongly stained, making it easy to distinguish cardiac myocytes from fibroblasts. As shown in Figure 7A⇓, p125^FAK predominantly localized in the perinuclear region in nonstimulated cardiac myocytes. Only weak fluorescent dots of p125^FAK were seen in some nonstimulated fibroblasts (Figure 7A⇓, arrows). Fluorescent dots of p125^FAK, which mostly localized in the central regions at a higher density, appeared to scatter to the peripheral cytoplasm in cardiac myocytes stimulated with rhVEGF for 60 minutes (Figure 7C⇓, arrowheads). In fibroblasts stimulated with VEGF for 60 minutes, fluorescent dots of p125^FAK were observed to be concentrated in the patchy arrowhead-like structures at the peripheral cytoplasm reminiscent of focal adhesions (Figure 7C⇓, arrow). To compare the localization of p125^FAK staining to focal adhesions in VEGF-treated cells, we performed double staining for p125^FAK (Figure 7E⇓) and vinculin (Figure 7F⇓) by immunofluorescence. Fluorescent dots of p125^FAK showed localization similar to that of vinculin in cardiac myocytes (Figure 7E⇓ and 7F⇓, arrowheads), which suggests that p125^FAK was localized to focal adhesions in response to VEGF, although the rest of the p125^FAK remained in cytoplasm. In fibroblasts, the patchy arrowhead-like fluorescent dots of p125^FAK were clearly observed with higher resolution (Figure 7E⇓, arrow). Incubation of cardiac myocytes with control nonimmune rabbit serum yielded no significant staining (data not shown).

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Figure 7.

VEGF alters the subcellular localization of p125^FAK in cardiac myocytes and fibroblasts. Quiescent cardiac myocytes were given no treatment (A and B) or were stimulated with 22.8 pmol/L rhVEGF for 60 minutes (C and D) and fixed in acetone. To distinguish cardiac myocytes from contaminating fibroblasts, we performed double staining for cardiac myosin heavy chain and p125^FAK by immunofluorescence. Cells were incubated with rabbit polyclonal anti-p125^FAK antibody and then sequentially incubated with biotinylated anti-rabbit IgG and FITC-conjugated avidin D (A and C). Cells were then incubated sequentially with mouse monoclonal anti-cardiac myosin heavy chain antibody (CMA-19) and TRITC-conjugated anti-mouse IgG1 antibody (B and D). For the rhVEGF-treated (60 minutes) cells, we also performed double staining for p125^FAK (E) and vinculin (F) by immunofluorescence. Cells were first incubated sequentially with rabbit polyclonal anti-p125^FAK antibody, biotinylated anti-rabbit IgG, and FITC-conjugated avidin D as above and then incubated with mouse anti-vinculin mAb followed by TRITC-conjugated anti-mouse IgG1 antibody. Arrowheads indicate localization of p125^FAK (C and E) and that of vinculin (F) in cardiac myocytes treated with rhVEGF for 60 minutes. Arrows indicate localization of p125^FAK in fibroblasts, either not treated (A) or treated (C and E) with rhVEGF for 60 minutes. An arrow in panel F indicates corresponding localization of vinculin in a fibroblast treated with rhVEGF for 60 minutes. Bars=10 μm.

Next, to confirm that VEGF induces subcellular translocation of p125^FAK, we analyzed p125^FAK content in subcellular fractions by Western analysis with and without VEGF stimulation. As shown in Figure 8⇓, the amount of p125^FAK in the membrane fraction was increased in response to rhVEGF (Figure 8b⇓, top panel), whereas that in the cytosolic fraction remained almost unchanged (Figure 8a⇓). We confirmed that almost equal amounts of the membrane fraction were electrophoresed in each reaction by Western analysis using anti–Na,K ATPase α₁ subunit antibody (Figure 8b⇓, lower panel).

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Figure 8.

Effect of VEGF on subcellular distribution of p125^FAK. After stimulation with 22.8 pmol/L rhVEGF for the indicated periods, cytosolic and membrane fractions were prepared from cardiac myocytes as described in Materials and Methods and subjected to Western analysis using anti-p125^FAK antibody (a and b, top panels). To confirm that almost equal amounts of membrane fraction were electrophoresed in each reaction, the same amounts of membrane fractions were subjected to Western analysis using anti–Na,K ATPase α₁ subunit (isoform-specific) antibody (b, bottom panel).

VEGF Strengthens Adhesion of Cardiac Myocytes to the ECM

To assess the effects of VEGF on the extent of cell-substrate adhesion, we monitored changes in cell-substrate resistance after addition of rhVEGF, using an electric cell-substrate impedance sensor. Typical results are shown in Figure 9A⇓. The extent of cell-substrate adhesion is expressed as “normalized” resistance, which was defined as a ratio of the resistance to the initial value before addition of rhVEGF. VEGF induced a significant increase in normalized resistance, indicating strengthening of the cell-substrate adhesion at 3 hours after addition of rhVEGF (Figure 9B⇓).

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Figure 9.

Effects of VEGF on adhesion of cardiac myocytes to the ECM. Cardiac myocytes were cultured on the surface of gelatin-coated wells manufactured especially for measurement of resistance. After starvation for 24 hours, they were stimulated with rhVEGF (34.2 pmol/L). A, Typical tracing representative of 4 replicate experiments. In all of these experiments, initial resistance of cardiac myocytes was within the range of 5000 to 7000 Ω. To simplify the comparison, ordinate represents normalized resistance defined as a fraction of the initial resistance before addition of rhVEGF. B, Comparison of percentage increase in normalized resistance at 3 hours after addition of rhVEGF, VEGF(+), with control, VEGF(–). Results shown are mean±SD from 4 independent experiments.