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(The FASEB Journal. 2006;20:1443-1451.)
© 2006 FASEB

Calpain-2 regulation of VEGF-mediated angiogenesis

Yunchao Su*, Zhaoqiang Cui*, Zhaozhong Li* and Edward R. Block*,{dagger}

* Department of Medicine, University of Florida College of Medicine and

{dagger} Research Service, Malcom Randall Veterans Affairs Medical Center, Gainesville, Florida, USA

1Correspondence: Department of Medicine, MSB M452, Box 100225, University of Florida, 1600 S.W. Archer Rd., Gainesville, FL 32610, USA. E-mail: ysu{at}ufl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis is a complex process involving endothelial cell migration, proliferation, and differentiation as well as tube formation. These processes are stimulated by a variety of growth factors such as vascular endothelial growth factor (VEGF). VEGF-induced cytoskeletal reorganization plays a crucial role in the angiogenic processes. In the present study, we evaluated the role of calpain in VEGF-induced angiogenesis in vitro and in vivo. Human pulmonary microvascular endothelial cells (PMEC) were incubated with VEGF (10–60 ng/ml) for 2–24 h, after which we measured calpain activities, protein contents of the calpain subunits and of calpastatin, endothelial monolayer wound repair, tube formation, and actin cytoskeleton changes. Incubation of PMEC with VEGF resulted in dose- and time-dependent increases in calpain activity and protein content of calpain-2. VEGF did not change the protein contents of calpain-1 and the small subunit or of calpastatin. Incubation of PMEC with a VEGF receptor blocker prevented the VEGF-induced increase in calpain activity. Inhibition of calpain activity by siRNA directed against calpain-2 and by overexpression of calpastatin prevented VEGF-induced increases in actin stress fibers in endothelial cells and angiogenesis. Overexpression of calpastatin also inhibits vessel formation in subcutaneous (s.c.) matrigel plugs in mice. These results indicate that calpain mediates VEGF-induced angiogenic effects by modulating actin cytoskeletal organization.—Su, Y., Cui, Z., Li, Z., Block E. R. Calpain-2 regulation of VEGF-mediated angiogenesis.


Key Words: calpains • actin • calpastatin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANGIOGENESIS IS A complex process involving endothelial cell migration, proliferation, and differentiation as well as tube formation (1 2 3) . These processes are stimulated by a variety of growth factors such as vascular endothelial growth factor (VEGF) (2 , 4 , 5) . VEGF is produced by endothelial cells and epithelial cells in a variety of tissues (5 , 6) . In vitro, VEGF stimulates extracellular matrix (ECM) degradation as well as proliferation, migration, and tube formation of endothelial cells (4 , 5 , 7) . In vivo, VEGF has been shown to regulate vascular permeability, which is important for the initiation of angiogenesis (8) . Two high-affinity binding sites for VEGF have been identified on vascular endothelium: VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1) (4 , 5 , 7) . VEGF receptors are associated with receptor tyrosine kinase, which is responsible for the downstream effects of VEGF (7 , 9) . The signal transduction pathway downstream from receptor tyrosine kinase is not clear. Several reports demonstrated that VEGF-induced increase in actin stress fibers plays a crucial role in the angiogenic processes of migration, differentiation, and proliferation of endothelial cells (10 11 12) . The cytoskeletal organization in endothelial cells is regulated by calpains (13 , 14) . Calpains are a family of calcium-activated nonlysosomal neutral cysteine endopeptidases, which act via limited proteolysis of substrate proteins (15 16 17 18 19) . There are at least 15 isozymes in the family (15 , 20) . Calpain-1 (µ-calpain) and calpain-2 (m-calpain) are two major typical calpain isoforms and are responsible for calpain activity in endothelial cells (15 , 17 , 21) . Calpain-1 and calpain-2 isoforms consist of a distinct larger catalytic subunit (~80 kDa) and a common smaller subunit (~30 kDa) that helps regulate catalytic activity (17 , 22) . Calpastatin functions as the major specific endogenous inhibitor for calpain-1 and calpain-2 (19 , 23 , 24) . In the present study we examined the role of calpains in VEGF-induced angiogenic effects. We found that VEGF increases activity and protein contents of calpain-2 in human microvascular endothelial cells and that inhibition of calpain activity by siRNA directed against calpain-2 or by overexpression of calpastatin prevents VEGF-induced increases in actin stress fibers in endothelial cells and angiogenesis, suggesting that calpain plays a critical role in mediating VEGF’s angiogenic effect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Human pulmonary microvascular endothelial cells (PMEC) were purchased from Cambrex Bio Science Rockland, Inc. (East Rutherford, NJ, USA) and cultured according to the instructions provided by the supplier. Third-to-sixth passage cells equilibrated in growth factor-free medium for 24 h were used for all experiments. Cells were identified by and the purity of the preparations were confirmed by phase contrast microscopy and the presence of factor VIII antigen. Cell viability was assessed using trypan blue staining and cell counting.

Calpain activity assay
Calpain activities were assayed by detecting calcium-dependent cleavage of resorufin-labeled casein in vitro and the calpain-mediated degradation of natural substrate spectrin in intact cells as previously reported (25) . To detect calpain-mediated cleavage of resorufin-labeled casein in vitro, cells were scraped and sonicated in buffer A (0.2 M Tris-HCl, pH 7.5, 1 mM dithiothreitol). Then 100 µl of lysates were mixed with 50 µl resorufin-labeled casein substrate solutions (0.4%) and 50 µl buffer B (buffer A plus 10 mM CaCl2). CaCl2 was substituted by 5 mM EDTA in blanks. After incubation for 30 min at 37°C, the reactions were terminated by adding 480 µl of 5% trichloroacetic acid. The mixtures were incubated again at 37°C for 10 min. After centrifugation, 400 µl supernatants were mixed with 600 µl of 0.5 M Tris-HCl (pH 8.8). The fluorescence was measured by spectrophotometry (excitation 574 nm, emission 584 nm). Calpain activity was calculated by subtracting the fluorescence units of samples with EDTA from those with CaCl2. In some experiments, specific calpain inhibitors leupeptin and calpaspatin were used to inhibit calcium-dependent cleavage of resorufin-casein. Our results indicate that leupeptin and calpaspatin caused a 90% inhibition of calcium-dependent cleavage of resorufin-casein, suggesting that this method is reliable to detect the alteration of calpain activity. Calpain activity in intact cells was measured by detecting calpain-specific spectrin breakdown products as reported by Newcomb (26) . Briefly, after treatments the cells were lysed by Western blot sample buffer and subjected to immuno-blot using antibody (Ab) against {alpha}II-spectrin (catalog # FG 6090; clone AA6, AFFINITI Research Products Limited, Mamhead Castle, UK). This Ab recognizes 280 kDa alpha-II-spectrin and a 150 kDa calpain-specific fragment. The calpain activity is expressed as the spectrin proteolytic ratio (150 kDa/280 kDa).

Western blot analysis
After treatments, PAEC were washed with PBS and lysed in boiled sample buffer (0.06 M Tris-HCl, 2% SDS and 5% glycerol, pH 6.8) for 5 min. The lysate proteins (15 to 20 µg) were separated on a 7.5% or 12% SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes. The membranes were incubated in blocking solution overnight at 4°C, then hybridized with primary Ab against spectrin, calpain-1, calpain-2, the small subunit of calpains, and calpastatin at room temperature for 1–2 h. The bands were detected using an immunochemiluminescence method. The density of the blots was quantitated by Bio-Rad Fluor-STM MultiImager.

Construction and transient expression of an adenoviral vector containing calpastatin in PMEC
The recombinant adenovirus encoding porcine calpastatin-CFP (cyan fluorescence protein) fusion protein was constructed using the BD Adeno-XTM Tet-off expression system (Clontech, Palo Alto, CA, USA). The full-length porcine calpastatin gene (Genbank Data Library under accession number AY372988) was inserted in the SacI/EcoR I sites of pEGFP-C1. The resulting plasmid was named calpastatin-CFP. The inserts excised from NheI/SalI sites of calpastatin-CFP were used to construct recombinant adenoviruses containing tetracycline (Tet) -regulated gene inserts according to the manufacturer’s instructions. After package, propagation, purification, and titration, the resulting adenoviruses and Adeno-X Tet-Off regulatory virus were used to coinfect PMEC as previously reported by us (27) .

Gene silencing of calpain-1 and calpain-2
The expression of calpain-1 and calpain-2 was silenced using the small interfering RNA (siRNA) technology. The target sequences for the mRNA of calpain-1 and calpain-2 were 5'-AAGCTAGTGTTCGTGCACTCT-3' and 5'-AAACCAGAGCTTCCAGGAAAA-3', respectively. The siRNA against luciferase mRNA was used as a control. The target sequence for luciferase mRNA was 5'-AACGTACGCGGAATACTTCGA-3'. The siRNAs were custom synthesized by Qiagen (Chatsworth, CA, USA) and transfected into PMEC using Quiagen RNAiFest transfection reagent according to the manufacturer’s protocol. Four days after transfection, protein contents of calpains, tube formation, and actin cytoskeleton of PMEC were evaluated.

Determination of angiogenesis in vitro
Angiogenesis was evaluated in vitro by measuring migration and tube formation as previously reported (25) . Endothelial migration was evaluated by measuring endothelial monolayer wound repair. Briefly, a cell-free wound zone was created by scraping the monolayer with a sterile pipette tip. The wound width of monolayers in millimeters was measured under the microscope. Then monolayers were washed and incubated in 5% CO2 at 37°C. Because the endothelial monolayer required at least 16 h to repair the wound a measurable distance, wound width was measured again after 16 h. Endothelial monolayer wound repair distance was expressed as the width of the wound before treatment subtracted by that after treatment. To do the tube formation assay, 96-well culture plates were coated with 100 µl of matrigel (BD Biosciences Discovery Labware, Bedford, MA, USA) per well, then allowed to polymerize for 30 min at 37°C. PMEC were seeded on coated plates at a density of 2 x 104 cells per well in RPMI 1640 medium containing 1% FBS at 37°C. Endothelial cells start to form tubes at 4 h. Tube formation is optimal after 8 h and begins to fade after 12 h. Therefore, the images of tubes were taken at 8 h at x 100 magnification with a digital output camera (Olympus) attached to an inverted phase-contrast microscope. Total tube length in mm/mm2 in each well was measured.

Determination of angiogenesis in vivo
Angiogenesis was determined in vivo by using a matrigel plug assay as described previously (28 , 29) . C57BL/6 mice (Charles River Laboratory, Wilmington, MA, USA) were used in this study. All mice were male, 2–3 months of age. Animal protocols were approval by the IACUC of the University of Florida and the Malcom Randall Veterans Affairs Medical Center. After inhalation of anesthesia with 15% isoflurane, the abdomen of each mouse was shaved and the skin was prepared with 70% alcohol. 0.6 ml of matrigel containing 100 ng VEGF and 10 U heparin plus sham adenovirus or adenovirus with the calpastatin gene were injected s.c. near the abdominal midline of the mice. After 10 days, the mice were euthanatized by cervical dislocation after inhalation of 15% isoflurane. The skin of the mouse was pulled back to expose the matrigel plug, which remained intact. Hemoglobin (Hb) content in the matrigel plugs was measured using the Drabkin reagent (Sigma, St. Louis, MO, USA) for quantification of blood vessel formation. The concentration of Hb was calculated from a known amount of Hb assayed in parallel. Some plugs were fixed using formalin to observe the cyan fluorescence for confirmation of calpastatin expression.

Staining for actin cytoskeleton
After treatment, PMEC were fixed in 4% paraformaldehyde, then incubated with 0.1% Triton X-100 for 10 min, with 5% goat serum for 30 min, then with Texas red-labeled phalloidin for 1 h. The unbound molecules were washed off and cells were assessed using a Zeiss LSM 510 laser scanning confocal microscope. The cells with actin fiber formation were counted in random observation fields (x400). The ratios of the number of cells with actin stress fiber formation to the total cell numbers in the observation field were calculated. At least 50 cells per treatment were counted.

Statistical analysis
In each experiment, experimental and control endothelial cells were matched for cell line, age, seeding density, number of passages, and number of days postconfluence to avoid variation in tissue culture factors that can influence measurements of angiogenesis and calpain activity. Results are shown as means ± SE for n experiments. Student’s paired t test is used to determine the significance of differences between the means of experimental and control cells. A value of P < 0.05 was taken as significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VEGF increases calpain activity in microvascular endothelial cells
To investigate the role of calpains in VEGF-induced angiogenesis, PMEC were incubated with VEGF (10 and 60 ng/ml) for 24 h, then calpain activities were measured in vitro and in intact cells. As shown in Fig. 1 A, incubation of PMEC with VEGF induces dose-dependent increases in calpain activity. The calpain activities were also measured by detecting its specific spectrin breakdown products in control PMEC and PMEC exposed to VEGF. As shown in Fig. 1B, C , exposure of PMEC to VEGF caused an increase in the calpain-specific breakdown of spectrin, indicating that VEGF increases calpain activity in intact PMEC.


Figure 1
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  		Figure 1. Effects of VEGF on calpain activities. PMEC were incubated with VEGF (10 and 60 ng/ml) for 24 h, after which in vitro calpain activity (A) and calpain-specific spectrin degradation in vivo (B, C) were measured as described in Materials and Methods. B) A representative immunoblot of 150/280 kDa fragments of spectrin. C) Bar graph depicting the changes in the spectrin 150/280 proteolytic ratio. Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. control.

VEGF increases protein content of calpain-2
To study whether VEGF affects protein contents of calpains, we measured the protein contents of calpain-1, calpain-2, the common regulatory small subunit, and calpastatin. We found that incubation of PMEC with VEGF (60 ng/ml) caused time-dependent increases in the protein content of calpain-2 (Fig. 2 ). However, the protein contents of calpain-1, of the small subunit, and of calpastatin were not altered in VEGF-treated endothelial cells (Fig. 2) .


Figure 2
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  		Figure 2. Effects of VEGF on the protein contents of calpains and calpastatin. PMEC were incubated with VEGF (60 ng/ml) for 2–24 h, after which protein contents of calpain-1, calpain-2, the small subunit, and calpastatin were measured. A) A representative immunoblot of proteins. B) A bar graph depicting the changes in protein contents. Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. control (0 incubation time).

VEGFR blocker inhibits VEGF-induced increase in calpain activity
To determine whether the VEGF-induced increase in calpain activity is mediated via the VEGF receptor, we evaluated the effects of 4-[(4'-chloro-2'-fluoro) phenylamino]-6,7-dimethoxyquinazoline (Calbiochem, San Diego, CA, USA), a specific VEGF receptor inhibitor, on VEGF-induced increases in calpain activity. As shown in Fig. 3 , incubation of PMEC with the VEGF receptor inhibitor (20 µM, for 4 h) prevented VEGF-induced increases in calpain activity, suggesting that VEGF increases calpain activity through its receptor.


Figure 3
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  		Figure 3. Effects of a VEGFR inhibitor, 4-[(4'-Chloro-2'-fluoro) phenylamino]-6,7-dimethoxyquinazoline, on VEGF-induced increase in calpain activity. PMEC were incubated with VEGF (60 ng/ml) in the presence and absence of a VEGFR inhibitor (V-inhi, 20 µM) for 4 h, after which in vitro calpain activity (A) and calpain-specific spectrin degradation in vivo (B, C) were measured as described in Materials and Methods. B) A representative immunoblot of 150/280 kDa fragments of spectrin. C) A bar graph depicting the changes in the spectrin 150/280 proteolytic ratio. Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. control.

Effect of calpastatin overexpression on calpain activity, endothelial monolayer wound repair, and endothelial tube formation
As shown in Fig. 4 B, C, infection of PMEC with sham adenovirus did not affect calpain activity. However, infection of PAEC with adenovirus containing a full-length calpastatin cDNA resulted in a dramatic decrease in calpain activity as measured by detecting calcium-dependent cleavage of resorufin-labeled casein in vitro and the calpain-mediated degradation of natural substrate spectrin in intact cells. However, the protein contents of calpain-1 and calpain-2 did not change in calpastatin-overexpressed cells, suggesting that the decrease in calpain activity is caused by the inhibitory effect of calpastatin on calpain (27) . Corresponding to the changes in calpain activity, infection of PMEC with adenovirus containing full-length porcine calpastatin cDNA caused decreases in endothelial monolayer wound repair and endothelial tube formation as well as in VEGF-induced increases in monolayer wound repair and tube formation (Fig. 5 ). These results suggest that calpains play a mediating role in endothelial angiogenesis.


Figure 4
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  		Figure 4. Calpastatin overexpression and its effects on calpain activity. PMEC were infected with or without a sham adenovirus (Sham-adeno) or adenovirus containing a full-length porcine calpastatin gene (Calpastatin-adeno). After 48 h, calpain activities were measured as described in Material and Methods. A) A representative immunoblot of calpastatin from 4 experiments. B) A bar graph depicting the changes in in vitro calpain activity. C) Calpain-specific spectrin degradation in vivo. Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. control.


Figure 5
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  		Figure 5. Effects of calpastatin overexpression on endothelial monolayer wound repair (A) and tube formation (B, C). A) Confluent PMEC were infected with or without (WO) a sham adenovirus (Sham-adeno) or an adenovirus containing a full-length porcine calpastatin gene (Calpastatin-adeno). After 48 h, a cell-free wound zone was created on the monolayers, then the cells were incubated with or without VEGF (60 ng/ml) for 24 h, after which the changes in repair distance were measured. Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. control (with VEGF). #P < 0.05 vs. control in Sham-adeno group. B) PMEC were infected with or without a sham adenovirus (sham) or an adenovirus containing a full-length porcine calpastatin gene (Calpas). After 48 h, PMEC in suspension were seeded on matrigel in 96-well culture plates in the absence and presence of VEGF (60 ng/ml). After 8 h, tube formation was measured. Data shown are representative images from 4 experiments (amplification: x100). C) A bar graph depicting the changes in tube length. Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. control. #P < 0.05 vs. sham group.

Effect of calpain siRNA on endothelial tube formation
To clarify whether calpain-1 or calpain-2 is responsible for the mediating role of calpain in angiogenesis, the protein expression of calpain-1 and calpain-2 was knocked down by using their siRNA. As shown in Fig. 6 , transfection of PMEC with siRNAs targeting the mRNA of calpain-1 and calpain-2 significantly knocked down the protein expression of calpain-1 and calpain-2. Knocking down the protein expression of calpain-2 caused inhibition of endothelial tube formation and a VEGF-induced increase in tube formation. In contrast, knocking down the protein expression of calpain-1 did not affect tube formation or VEGF-induced increase in tube formation in PMEC. These results suggest that calpain-2 rather than calpain-1 mediates VEGF-induced angiogenesis of PMEC.


Figure 6
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  		Figure 6. Effects of knocking down calpains on tube formation of PMEC. PMEC were transfected with a siRNA against the mRNA of calpain-1, calpain-2 or luciferase (Luci). After 96 h, endothelial tube formation was assayed as described in Material and Methods. A) A representative immunoblot of calpain proteins from 4 experiments. B) A bar graph showing the changes in tube length in control PMEC and PMEC transfected with a siRNA directed against the mRNA of calpains or luciferase with or without VEGF (60 ng/ml). Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. vehicle (without VEGF). #P < 0.05 vs. vehicle in group of Luciferase siRNA.

The effects of overexpression of calpastatin and calpain siRNA on VEGF-induced stress fiber formation
To investigate the mechanism for calpain-mediated angiogenesis, alterations of the actin cytoskeleton were studied in PMEC, with calpain inhibition caused by overexpression of calpastatin or by calpains siRNA. As shown in Fig. 7 , incubation of PMEC with VEGF caused formation of stress fibers in the presence and absence of sham adenovirus. Overexpression of calpastatin using an adenovirus containing the calpastatin gene prevented the VEGF-induced increase in stress fiber formation in PMEC. To identify whether calpain-1 or calpain-2 is responsible for VEGF-induced stress fiber formation, we studied alterations of the actin cytoskeleton in PMEC in which calpain-1 or calpain-2 has been knocked down by siRNA directed against the mRNA of calpain-1 or calpain-2. We found that knocking down calpain-1 did not influence VEGF-induced stress fiber formation while knocking down calpain-2 prevented VEGF-induced stress fiber formation (Fig. 8 ). These results suggest that calpain-2 rather than calpain-1 mediates VEGF-induced stress fiber formation.


Figure 7
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  		Figure 7. Effects of calpastatin overexpression on the endothelial actin cytoskeleton. Preconfluent PMEC were infected with or without a sham adenovirus (sham) or adenovirus containing full-length porcine calpastatin gene (Calpas). After 48 h, PMEC were exposed to VEGF (60 ng/ml) for 24 h, then actin cytoskeleton were stained as described in Materials and Methods. A) Representative images from 4 experiments (amplification: x400) showing that cells with calpastatin expression exhibit limited stress fiber formation after VEGF treatment. The red color indicates actin cytoskeleton and the cyan color indicates CFP. B) The ratio of the number of cells with stress fiber formation to total cell number in 6 random observation fields in each group (only CFP-positive cells were counted in sham and Calpas group). Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. vehicle group (without VEGF).


Figure 8
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  		Figure 8. Effects of knocking down calpains on the endothelial actin cytoskeleton. Preconfluent PMEC were transfected with a siRNA directed against the mRNA of calpain-1 (Cal-1), calpain-2 (Cal-2), or luciferase (Luci). After 96 h, PMEC were exposed to VEGF (60 ng/ml) for 24 h, then the actin cytoskeleton was stained as described in Materials and Methods. A) Representative images from 4 experiments (amplification: x400) showing that cells transfected with a siRNA directed against the mRNA of calpain-2 (Cal-2) exhibit limited stress fiber formation after VEGF treatment. B) The ratio of the number of cells with stress fiber formation to total cell number in 6 random observation fields in each group. Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. vehicle group (without VEGF).

The effects of overexpression of calpastatin on VEGF-induced angiogenesis in vivo
We have observed that inhibition of calpain activity by overexpression of calpastatin and by calpain siRNA prevented VEGF-induced increases in endothelial angiogenesis in microvascular endothelial cells in culture. To confirm whether inhibition of calpain blocks angiogenesis in vivo, matrigel plus assay was performed in vivo. Matrigel containing VEGF (100 ng/0.6 ml) with or without sham adenovirus or adenovirus containing a full-length calpastatin cDNA was injected s.c. into C57BL/6 mice; 10 days later the formed matrigel plugs were excised, sectioned, and photographed. Cyan fluorescence was observed in the sections from plugs with VEGF plus sham adenovirus and from plugs with VEGF plus calpastatin adenovirus (data not shown), suggesting expression of CFP and calpastatin. Plugs with VEGF or with VEGF plus sham adenovirus exhibited a dark-red color consistent with new blood vessel formation whereas plugs without VEGF or with VEGF plus calpastatin adenovirus were pale in color, indicating no or limited blood vessel formation (Fig. 9 A). Angiogenesis was quantified by measuring the Hb contents inside the matrigel plugs. As shown in Fig. 9B , overexpression of calpastatin prevents VEGF-induced increases in Hb contents in matrigel plugs corresponding to the color of the matrigel. These results indicate that calpastatin inhibits VEGF-induced angiogenesis in vivo.


Figure 9
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  		Figure 9. The effects of calpastatin overexpression on VEGF-induced angiogenesis in vivo. Matrigel containing VEGF (100 ng/0.6 ml) with or without (WO) sham adenovirus (Sham-adeno), or adenovirus containing full-length calpastatin cDNA (Calpa-adeno) was injected s.c. into C57BL/6 mice, and 10 days later, the formed matrigel plugs were excised and photographed (A). B) Hb contents in matrigel plugs. Results are expressed as mean ± SE; n = 4 experiments. *P < 0.05 vs. control, #P < 0.05 vs. Sham-adeno group.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we have demonstrated that VEGF increases calpain activity in human PMEC primarily due to increased expression of calpain-2. We have also shown that inhibition of calpain activity by overexpression of calpastatin or by siRNA directed against calpain-2 attenuates VEGF-induced increases in angiogenesis and actin stress fiber formation. These results indicate that calpain-2 plays a mediating role in VEGF-induced signaling in endothelial angiogenesis.

Shiraha et al. (30) and Glading (31) have reported that incubation of fibroblasts with epidermal growth factor for 10 min increases calpain-2 activity via a kinase/mitogen-activated protein (ERK/MAP) kinase pathway. Our results indicate that incubation of microvascular endothelial cells with VEGF for 2–24 h induces increases in calpain activity as measured by calcium-dependent cleavage of resorufin-labeled casein in vitro and calpain-specific breakdown of spectrin in intact endothelial cells. The increases in calpain activity correspond to increases in calpain-2 protein content, indicating that increased calpain activity is caused by an increase in calpain-2 protein content. VEGF receptors are associated with receptor tyrosine kinase, which is responsible for the downstream effects of VEGF (7 , 9) . Incubation of PMEC with VEGF receptor tyrosine kinase inhibitor blocks VEGF-induced increases in the activity of calpain, suggesting that calpain acts downstream from the VEGF receptor tyrosine kinase.

Tamada et al. (32) reported that calpain inhibitors leupeptin and SJA6017 reduced basic fibroblast growth factor-induced angiogenesis in guinea pig cornea, suggesting that calpains may be involved in angiogenesis. However, calpains clearly play an important role in the signal transduction of cell migration, differentiation, and proliferation in a variety of cells, including endothelial cells (33 34 35 36) . The mechanism is not clarified but may be related to calpain-induced alteration in the architecture of cell adhesion and cytoskeletal components (37 38 39) . In the present study, we have shown that inhibition of calpain activity by overexpression of calpastatin prevented VEGF-induced increases in endothelial monolayer wound repair, endothelial tube formation, and the formation of actin stress fibers. Moreover, by using a different method, siRNA technology, we have demonstrated that knocking down calpain-2 inhibited VEGF-induced increases in endothelial tube formation and formation of actin stress fibers, whereas knocking down calpain-1 had no effect. These results indicate that calpain-2 plays a fundamental role in VEGF-induced formation of actin stress fibers in lung endothelial cells. The formation of actin stress fibers plays a critical role in endothelial tube formation and angiogenesis (11 , 12) . Several reports indicate that inhibition of calpain activity by overexpression of calpastatin (40) , by the calpain inhibitor calpeptin (40) , or in small subunit knockout fibroblasts (41) inhibits actin stress fiber formation. These studies did not identify which isoform of calpain is important in actin stress fiber formation, because the methods used to create calpain inhibition affect both calpain-1 and calpain-2. Kulkarni et al. (13) reported that calpain-1 cleaves RhoA and generates a fragment that inhibits integrin-induced stress fiber assembly, suggesting that calpain-1 is not responsible for the increase in stress fiber formation. Consistent with these data, we found that knocking down calpain-1 did not prevent VEGF-induced increase in stress fiber formation, but knocking down calpain-2 did prevent VEGF-induced increase in stress fiber formation and tube formation. Hoang et al. (10) and Amerongen et al. (12) reported that RhoA signaling is involved in VEGF-induced endothelial stress fiber formation and angiogenesis. There are reports claiming that calpains play an important role in the activation of the Rho signaling pathway (42 , 43) . Therefore, the role of calpain-2 in VEGF-induced cytoskeleton reorganization and angiogenesis might be related to RhoA signaling pathway in endothelial cells.

We also demonstrated that overexpression of calpastatin inhibits vessel formation in s.c. matrigel plugs, suggesting that inhibition of calpain activity blocks in vivo angiogenesis as well. Therefore, manipulating calpain activity could provide promising therapies for management of pathological angiogenesis, such as those occurring in proliferative retinopathy and cancer, and for tissue repair in which enhanced angiogenesis is preferred.


   ACKNOWLEDGMENTS
 
This work was supported by the Medical Research Service of the Department of Veterans Affairs, NIH grants HL52136 and HL67951, Flight Attendant Medical Research Institute grant 032040, FL DOH grant 04TSP-01, ALAF grant 00053850, and AHA grant 0555322B. We thank Weihong Han for assistance with tissue culture.

Received for publication November 1, 2005. Accepted for publication March 14, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Frelin, C., Ladoux, A., D’angelo, G. (2000) Vascular endothelial growth factors and angiogenesis. Ann. Endocrinol. (Paris) 61,70-74[Medline]
  2. Ferrara, N., Gerber, H. P. (2001) The role of vascular endothelial growth factor in angiogenesis. Acta Haematol. 106,148-156[CrossRef][Medline]
  3. Liekens, S., De Clercq, E., Neyts, J. (2001) Angiogenesis: regulators and clinical applications. Biochem. Pharmacol. 61,253-270[CrossRef][Medline]
  4. Neufeld, G., Cohen, T., Gengrinovitch, S., Poltorak, Z. (1999) Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 13,9-22[Abstract/Free Full Text]
  5. Kliche, S., Waltenberger, J. (2001) VEGF receptor signaling and endothelial function. IUBMB Life 52,61-66[Medline]
  6. Boussat, S., Eddahibi, S., Coste, A., Fataccioli, V., Gouge, M., Housset, B., Adnot, S., Maitre, B. (2000) Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells. Am. J. Physiol. 279,L371-L378
  7. Zachary, I., Gliki, G. (2001) Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovasc. Res. 49,568-581[Abstract/Free Full Text]
  8. Dudek, S. M., Garcia, J. G. (2001) Cytoskeletal regulation of pulmonary vascular permeability. J. Appl. Physiol. 91,1487-1500[Abstract/Free Full Text]
  9. Thuringer, D., Maulon, L., Frelin, C. (2002) Rapid transactivation of the vascular endothelial growth factor receptor KDR/Flk-1 by the bradykinin B2 receptor contributes to endothelial nitric-oxide synthase activation in cardiac capillary endothelial cells. J. Biol. Chem. 277,2028-2032[Abstract/Free Full Text]
  10. Hoang, M. V., Whelan, M. C., Senger, D. R. (2004) Rho activity critically and selectively regulates endothelial cell organization during angiogenesis. Proc. Natl. Acad. Sci. U.S.A. 101,1874-1879[Abstract/Free Full Text]
  11. Linz-McGillem, L. A., Moitra, J., Garcia, J. G. (2004) Cytoskeletal rearrangement and caspase activation in sphingosine 1-phosphate-induced lung capillary tube formation. Stem Cells Dev. 13,496-508[CrossRef][Medline]
  12. Nieuw Amerongen, G. P., Koolwijk, P., Versteilen, A., van and Hinsbergh, V. W. (2003) Involvement of RhoA/Rho kinase signaling in VEGF-induced endothelial cell migration and angiogenesis in vitro. Arterioscler. Thromb. Vasc. Biol. 23,211-217[Abstract/Free Full Text]
  13. Kulkarni, S., Goll, D. E., Fox, J. E. (2002) Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading. J. Biol. Chem. 277,24435-24441[Abstract/Free Full Text]
  14. Deroanne, C. F., Lapiere, C. M., Nusgens, B. V. (2001) In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton. Cardiovasc. Res. 49,647-658[Abstract/Free Full Text]
  15. Perrin, B. J., Huttenlocher, A. (2002) Calpain. Int. J. Biochem. Cell Biol. 34,722-725[CrossRef][Medline]
  16. Huang, Y., Wang, K. K. (2001) The calpain family and human disease. Trends Mol. Med. 7,355-362[CrossRef][Medline]
  17. Suzuki, K., Sorimachi, H., Yoshizawa, T., Kinbara, K., Ishiura, S. (1995) Calpain: novel family members, activation, and physiologic function. Biol. Chem. Hoppe Seyler 376,523-529[Medline]
  18. Forsberg, N. E., Huang, J. (2000) Strategies for regulating calpain activities in living cells. Methods Mol. Biol. 144,319-326[Medline]
  19. Goll, D. E., Thompson, V. F., Li, H., Wei, W., Cong, J. (2003) The calpain system. Physiol. Rev. 83,731-801[Abstract/Free Full Text]
  20. Suzuki, K., Hata, S., Kawabata, Y., Sorimachi, H. (2004) Structure, activation, and biology of calpain. Diabetes 53 Suppl. 1,S12-S18
  21. Fujitani, K., Kambayashi, J., Sakon, M., Ohmi, S. I., Kawashima, S., Yukawa, M., Yano, Y., Miyoshi, H., Ikeda, M., Shinoki, N., Monden, M. (1997) Identification of mu-, m-calpains and calpastatin and capture of mu-calpain activation in endothelial cells. J. Cell. Biochem. 66,197-209[CrossRef][Medline]
  22. Sorimachi, H., Suzuki, K. (2001) The structure of calpain. J. Biochem. (Tokyo) 129,653-664[Abstract/Free Full Text]
  23. Xu, Y., Mellgren, R. L. (2002) Calpain inhibition decreases the growth rate of mammalian cell colonies. J. Biol. Chem. 277,21474-21479[Abstract/Free Full Text]
  24. Wendt, A., Thompson, V. F., Goll, D. E. (2004) Interaction of calpastatin with calpain: a review. Biol. Chem. 385,465-472[CrossRef][Medline]
  25. Su, Y., Cao, W., Han, Z., Block, E. R. (2004) Cigarette smoke extract inhibits angiogenesis of pulmonary artery endothelial cells: the role of calpain. Am. J. Physiol. 287,L794-L800
  26. Newcomb, J. K., Pike, B. R., Zhao, X., Hayes, R. L. (2000) Concurrent assessment of calpain and caspase-3 activity by means of Western blots of protease-specific spectrin breakdown products. Methods Mol. Biol. 144,219-223[Medline]
  27. Cui, Z., Han, Z., Li, Z., Hu, H., Patel, J. M., Antony, V., Block, E. R., Su, Y. (2005) Involvement of calpain-calpastatin in cigarette smoke-induced inhibition of lung endothelial NOS. Am. J. Respir. Cell Mol. Biol. 33,513-520[Abstract/Free Full Text]
  28. Min, J. K., Han, K. Y., Kim, E. C., Kim, Y. M., Lee, S. W., Kim, O. H., Kim, K. W., Gho, Y. S., Kwon, Y. G. (2004) Capsaicin inhibits in vitro and in vivo angiogenesis. Cancer Res. 64,644-651[Abstract/Free Full Text]
  29. Lee, P. C., Kibbe, M. R., Schuchert, M. J., Stolz, D. B., Watkins, S. C., Griffith, B. P., Billiar, T. R., Shears, L. L. (2000) Nitric oxide induces angiogenesis and upregulates alpha (v) beta(3) integrin expression on endothelial cells. Microvasc. Res. 60,269-280[CrossRef][Medline]
  30. Shiraha, H., Glading, A., Chou, J., Jia, Z., Wells, A. (2002) Activation of m-calpain (calpain II) by epidermal growth factor is limited by protein kinase A phosphorylation of m-calpain. Mol. Cell. Biol. 22,2716-2727[Abstract/Free Full Text]
  31. Glading, A., Chang, P., Lauffenburger, D. A., Wells, A. (2000) Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway. J. Biol. Chem. 275,2390-2398[Abstract/Free Full Text]
  32. Tamada, Y., Fukiage, C., Boyle, D. L., Azuma, M., Shearer, T. R. (2000) Involvement of cysteine proteases in bFGF-induced angiogenesis in guinea pig and rat cornea. J. Ocul. Pharmacol. Ther. 16,271-283[Medline]
  33. Han, Y., Weinman, S., Boldogh, I., Walker, R. K., Brasier, A. R. (1999) Tumor necrosis factor-alpha-inducible IkappaBalpha proteolysis mediated by cytosolic m-calpain. A mechanism parallel to the ubiquitin-proteasome pathway for nuclear factor-kappab activation. J. Biol. Chem. 274,787-794[Abstract/Free Full Text]
  34. Carragher, N. O., Frame, M. C. (2002) Calpain: a role in cell transformation and migration. Int. J. Biochem. Cell Biol. 34,1539-1543[CrossRef][Medline]
  35. Carragher, N. O., Westhoff, M. A., Riley, D., Potter, D. A., Dutt, P., Elce, J. S., Greer, P. A., Frame, M. C. (2002) v-Src-induced modulation of the calpain-calpastatin proteolytic system regulates transformation. Mol. Cell. Biol. 22,257-269[Abstract/Free Full Text]
  36. Khorchid, A., Ikura, M. (2002) How calpain is activated by calcium. Nat. Struct. Biol. 9,239-241[CrossRef][Medline]
  37. Franco, S., Perrin, B., Huttenlocher, A. (2004) Isoform specific function of calpain 2 in regulating membrane protrusion. Exp. Cell Res. 299,179-187[CrossRef][Medline]
  38. Franco, S. J., Rodgers, M. A., Perrin, B. J., Han, J., Bennin, D. A., Critchley, D. R., Huttenlocher, A. (2004) Calpain-mediated proteolysis of talin regulates adhesion dynamics. Nat. Cell Biol. 6,977-983[CrossRef][Medline]
  39. Potter, D. A., Tirnauer, J. S., Janssen, R., Croall, D. E., Hughes, C. N., Fiacco, K. A., Mier, J. W., Maki, M., Herman, I. M. (1998) Calpain regulates actin remodeling during cell spreading. J. Cell Biol. 141,647-662[Abstract/Free Full Text]
  40. Dedieu, S., Poussard, S., Mazeres, G., Grise, F., Dargelos, E., Cottin, P., Brustis, J. J. (2004) Myoblast migration is regulated by calpain through its involvement in cell attachment and cytoskeletal organization. Exp. Cell Res. 292,187-200[CrossRef][Medline]
  41. Dourdin, N., Bhatt, A. K., Dutt, P., Greer, P. A., Arthur, J. S., Elce, J. S., Huttenlocher, A. (2001) Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts. J. Biol. Chem. 276,48382-48388[Abstract/Free Full Text]
  42. Fox, J. E. (1999) On the role of calpain and Rho proteins in regulating integrin-induced signaling. Thromb. Haemost. 82,385-391[Medline]
  43. Kulkarni, S., Saido, T. C., Suzuki, K., Fox, J. E. (1999) Calpain mediates integrin-induced signaling at a point upstream of Rho family members. J. Biol. Chem. 274,21265-21275[Abstract/Free Full Text]



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