Novel Proangiogenic Effect of Factor XIII Associated With Suppression of Thrombospondin 1 Expression

Cells

Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and maintained in EGM-2 medium (Clonetics) by standard procedures as described previously.¹⁰ Cells from the first and second passages were used for the Matrigel tube formation assays, and cells from the fourth to sixth passages were used for all other experiments.

Human microvascular dermal (neonatal) endothelial cells (HMVEC-d) were purchased from Clonetics and cultured in EGM-MV medium (Clonetics) according to the manufacturer’s instructions. Cells from the fourth passage were used for the experiments.

FXIII Activation

The source of FXIII was FXIII concentrate, Fibrogammin-P (Aventis Behring). After reconstitution, 2 mL of 100 U/mL (approximately 1000 μg/mL) FXIII was incubated with thrombin immobilized on Affi-gel 10 beads. One milliliter of packed bead volume contained 200 U of bovine thrombin. Ten millimolar CaCl₂ was added, and the mixture was incubated at 37°C for 2 hours. FXIII activation was monitored by measuring FXIII activity using a chromogenic assay (Berichrome, Dade Behring). FXIIIa was inactivated by treatment with 3 mmol/L iodoacetamide (Sigma) for 30 minutes at 22°C to block transglutaminase activity; free iodoacetamide was then removed by dialysis.

Matrigel Assay

HUVECs (1×10⁵/ well) were plated on Matrigel (Becton Dickinson) previously diluted 1:1 with medium M199 and allowed to adhere for 2 hours at 37°C. Either FXIII or FXIIIa was then added at concentrations of 10, 20, or 50 μg/mL. In some experiments, 50 μg/mL FXIIIa was added together with polyclonal goat anti-FXIII antibody (Affinity Biologicals; 50 μg/mL). Goat anti TSP-1 antibody (Santa Cruz; 50 μg/mL) was added either alone or in combination with 50 μg/mL FXIIIa in other experiments. Endothelial array or tube formation in Matrigel was visualized by light microscopic examination.

HUVEC Migration Assays

Migration of ECs was measured using 2 assays, the Boyden migration assay and the wound migration assay. The migration assay was performed by a modification of the procedure described previously using a Boyden chamber (Neuro Probe).¹¹ HUVECs grown overnight in EBM media (Clonetics) were treated with trypsin, resuspended in EBM media at 100 000 cells/300 μL, and added to the upper chamber. Added to the lower chamber was 50 μg/mL FXIII, FXIIIa, or iodoacetamide-treated FXIIIa. A polycarbonate filter (8-μm pores) was placed between upper and lower chambers. The chamber was incubated for 24 hours at 37°C. After incubation, the filter was removed and the cells migrating through the filter and adhering to the lower chamber were incubated for 1 hour at 37°C with 4 μg/mL Calcein AM (Molecular Probes) in Hanks’ buffered saline and counted in a plate reader (SpectraMax Gemini, Molecular Devices Corp). The wound-migration assay has been previously described and used with slight modification.¹² Briefly, monolayers of HUVECs were grown in Petri dishes. A small incision was made adjacent to the confluent cell-dense region using a small razor blade. Cells were removed from approximately half of the plate by gentle scraping. After wounding, the cells were allowed to migrate for 24 hours. Migration of the cells across the sharp wound edge to the cell-free region was assessed by phase microscopy and counting the cells grown in cell culture medium in the absence or presence of 50 μg/mL FXIII, FXIIIa, or iodoacetamide-treated FXIIIa.

[³H]-Thymidine Incorporation

HUVECs were seeded on 24-well tissue culture plates at a density of 1×10⁴ cells/well. After 24 hours, the cell culture media was replaced with serum-free media, and 48 hours after seeding, 50 μg/mL FXIII, FXIIIa, iodoacetamide-treated FXIIIa, or 10 ng/mL bFGF was added. Seventy-two hours after seeding, 2% FBS was added to the media along with 1 μCi/well [³H]-thymidine, and the cells were incubated for an additional 16 hours. After this time period, the cells were washed twice with PBS and incubated in 10% TCA overnight at 4°C. The cells were then washed twice with 100% ethanol and dried. Precipitated DNA and protein were solubilized with 1 N NaOH, and the radioactivity was counted in a liquid scintillation counter.

MTT Assay

HUVECs were seeded in a 96-well tissue culture plates at a density of 5×10³ cells/well. After 24 hours, the cell culture media was replaced with serum-free media, and 48 hours after seeding, 50 μg/mL FXIII, FXIIIa, iodoacetamide-treated FXIIIa, or 10 ng/mL bFGF was added. Seventy-two hours after seeding, 2% FBS was added to the media and incubated for 16 hours. After this incubation, 1 mg /mL MTT (3-[4,5-dimethylthiazol-2yl]-2-5-diphenyl-2H-tetrazolium bromide; Sigma) was added to the media and the culture incubated for an additional 3 hours. After the incubation, the medium was removed, the cells were solubilized in isopropanol containing 0.04 N HCl and 10% Triton X-100, and the amount of the dye released from the cells was measured at 560 nm by ELISA reader (Molecular Devices). Final quantification of dye release was obtained after subtraction of absorbance at 650 nm.

Apoptosis Assay

Apoptosis in HUVECs was determined by TUNEL using an in situ cell death detection kit (Roche) in which dUTP is labeled with fluorescein to allow the direct detection of the labeled DNA strand breaks. Cells grown overnight in complete EGM-2 medium (Clonetics) were treated with 50 μg/mL FXIII, FXIIIa, or iodoacetamide-treated FXIIIa for 24 hours, after which they were fixed, permeabilized, and labeled according to the manufacturer’s instructions. To highlight the nuclei, the cells were also stained with DAPI in mounting media (Vector) and covered with cover slips. The apoptotic cells were counted by direct visualization using fluorescence microscopy.

Gene Array Analysis

Total RNA from HUVECs treated with 50 μg/mL FXIIIa for 16 hour at 37°C or of control, untreated cells was isolated using the Qiagen RNA isolation kit (Qiagen). RNA was then reverse transcribed into cDNA by MMLV reverse transcriptase (Promega) in the presence of 50 μCi [α-³²]P-dCTP. Each radiolabeled cDNA sample was hybridized to a gene array membrane containing 100 genes associated with angiogenesis using the hybridization buffer supplied by the manufacturer. Hybridization conditions and washings of the membranes were performed according to the manufacturer’s instructions (Superarray Inc). The membranes were exposed to Kodak X-ray film for 16 hours at −70°C, and the autoradiograms were compared.

Western Blot Analysis of TSP-1 in Conditioned Medium

HUVECs or HMVEC-d were incubated with FXIIIa at concentrations of 10, 20, or 50 μg/mL for 16 hours at 37°C. Conditioned medium of FXIIIa-treated and untreated cells was collected, centrifuged for 5 minutes at 700g, and incubated with heparin-Sepharose (5 mL medium/0.5 mL of packed beads). The beads were then washed with 10 mL TBS (0.15 mol/L NaCl and 20 mmol/L Tris-HCl, pH 7.5) followed by washing with 10 mL TBS, 0.25 mol/L NaCl, and 20 mmol/L Tris-HCl, pH 7.5. The beads were boiled in 0.2 mL nonreducing electrophoresis sample buffer, and the eluted protein was subjected to 3% to 8% SDS-PAGE gel (Novex). The SDS gel was electrophoretically transferred to an Immobilon membrane (Millipore). The blot was blocked in a 3% BSA solution in TBS (TBS/BSA) for 16 hour at 4°C and incubated with a monoclonal anti-TSP-1 antibody (Sigma) diluted 1:500 in TBS/BSA for 2 hours at 22°C. The blot was washed with TBS containing 0.1% Tween-20 (TBS/Tween) and incubated with peroxidase-conjugated goat anti-mouse immunoglobulins (Jackson ImmunoResearch Laboratories) at 1:2000 in TBS/Tween for 1 hour at 22°C. After washing in TBS/Tween, the blot was developed using an enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech).

Analysis of VEGF Level in Conditioned Medium

Supernatants of HUVECs grown either in the absence or presence of FXIIIa (50 μg/mL) were analyzed for VEGF levels using the VEGF DuoSet ELISA kit (R&D Systems).

In Vivo Model of Rabbit Cornea Vascularization

New Zealand albino male rabbits (3.0 kg in weight) were used in this study. Surgery and care were performed according to ARVO guidelines for animal research. General anesthesia was achieved by intramuscular injection of xylazine 2% and ketamine HCl. Using a previously described model,¹³ 2 μL containing either 20 μg FXIIIa or PBS was injected subepithelially in the cornea of the rabbits using a Hamilton syringe. The site of the injection was 2 mm away from the limbus, at the site of the attachment of the superior rectus muscle. In each of the 4 rabbits studied, FXIIIa was injected into the right cornea and a similar volume of PBS (negative control) into the left cornea. Clinical examination of the cornea was performed before injection, immediately after injection, and then every 24 hours up to 96 hours after the maximal effect was observed. At this stage, the rabbits were killed by lethal injection of pentobarbital. The corneas were excised, fixed in 4% paraformaldehyde, and stained with H&E for histological evaluation by light microscopy and with GSLI-isolectin B₄ (Vector) for evaluation of blood vessels.¹⁴ Some of the fixed slides were stained with anti-thrombospondin antibody for immunohistological analysis.

Immunohistochemical Analysis of TSP

Sections from FXIIIa- or PBS-treated rabbit corneas were incubated with polyclonal goat anti-human TSP-1 (1:100 in TBS/BSA; Santa Cruz Biotechnology) for 2 hours at 22°C. The slides were then washed with TBS and incubated with biotinylated anti-goat IgG for 1 hour at 22°C, followed by washing in TBS and incubation with avidin-conjugated peroxidase (ABC kit; Vector) for 1 hour at 22°C and stained with 3-amino-9-ethyl carbazole substrate (Zymed). The slides were counterstained with hematoxylin.

Statistical Analysis

The data were analyzed by one-way ANOVA with post hoc comparisons. P<0.05 was considered significant.

Effect of FXIIIa on Endothelial Tube Formation

HUVECs were plated on Matrigel in the presence or absence of FXIII or FXIIIa, and array formation was analyzed 16 hours after plating. Addition of 20 to 100 μg/mL of FXIIIa caused a marked, dose-dependent increase in endothelial cell content, resulting in a significant increase in tube thickness compared with that observed in untreated samples (Figures 1A, 1C, and 1D). No effect was observed on addition of nonactivated (Figure 1B) or acetamide-treated (Figure 1E) FXIIIa at a concentration of 50 μg/mL (≈5 U/mL). This effect of FXIIIa was abolished by a polyclonal anti-FXIII antibody (50 μg/mL; Figure 1F).

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Figure 1. Effect of FXIII on tube formation in Matrigel. 1×10⁵ HUVECs were plated on Matrigel and incubated with control medium (A), medium containing 50 μg/mL of FXIII (B), medium containing 20 μg/mL of FXIIIa (C), medium containing 50 μg/mL of FXIIIa (D), medium containing 50 μg/mL of iodoacetamide-treated FXIIIa (E), or medium containing 50 μg/mL of FXIIIa and 20 μg/mL of polyclonal anti-FXIII (F) for 16 hours at 37°C and processed as described in Methods.

Effect of FXIII on Endothelial Cell Migration

In the wound migration assay, migration of the cells across the sharp wound edge to the cell-free region was assessed. As shown in Figure 2, 50 μg/mL FXIIIa increased the migration of the cells greater than 7-fold (747±328% of control) (P<0.001 by ANOVA). Treatment of the cells with nonactivated FXIII had no effect on migration. Iodoacetamide-treated FXIIIa decreased migration by 30%. Similar results were obtained using the Boyden-migration assay (Figure 2); 50 μg/mL FXIIIa increased migration ≈2.5-fold (258±72% of control) (P<0.001 by ANOVA), whereas no significant change in migration was observed with treatment of the cells with similar concentrations of FXIII or iodoacetamide-treated FXIIIa.

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Figure 2. Effect of FXIII on HUVEC migration using Boyden chamber (black bars) and wound-migration (white bars) assays. In the Boyden assay, each data point is presented as a percent of control from the average±SD of 15 samples in each group. *P<0.001 by ANOVA. In the wound-migration assay, 5 to 10 random fields were examined. Each data point is presented as a percent of control from the average±SD of 12 samples in each group. *P<0.001 by ANOVA.

Effect of FXIII on Endothelial Cell Proliferation

[³H]-thymidine incorporation and MTT uptake were used to determine HUVEC proliferation in the presence or absence of FXIII. As shown in Figure 3A, 50 μg/mL FXIIIa increased the proliferation of HUVECs by approximately 64% (164±5% of control) (P<0.001 by ANOVA). In comparison, addition of 10 ng/mL bFGF (positive control) enhanced cell proliferation by approximately 93% (193±25 of control). Similarly, in MTT uptake experiments, an increase in cell proliferation by 70% on exposure to 50 μg/mL FXIIIa was observed (170±17%; Figure 3B). By contrast, neither FXIII nor iodoacetamide-treated FXIIIa significantly affected proliferation.

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Figure 3. Effect of FXIII on HUVEC proliferation. HUVECs were seeded and processed as described in Methods. A, [³H]-thymidine incorporation; B, MTT assay. Each data point is presented as a percent of control from the average±SD of 6 samples in each treatment group. *P<0.001 by ANOVA.

Effect of FXIII on Endothelial Cell Apoptosis

The effect of FXIII on apoptosis of HUVECs was studied by the TUNEL assay. Figure 4 shows that only in cells exposed to FXIIIa (50 μg/mL) did the number of apoptotic cells decrease significantly compared with untreated cells (0.21±0.09% versus 1.08±0.16%, respectively; P<0.001 by ANOVA). The apoptotic response to the same concentration of FXIII or iodoacetamide-treated FXIIIa-induced apoptosis is similar to that of the untreated cells.

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Figure 4. Effect of FXIII on HUVEC apoptosis (TUNEL assay). The apoptotic cells were counted by direct visualization using fluorescence microscopy. To calculate the percentage of apoptotic cells in each case, 5 to 10 random fields were examined. Each bar represents average±SD of 8 samples in each treatment group. *P<0.001 by ANOVA.

Effect of FXIIIa on VEGF Production and Secretion

To explore the mechanism underlying the proangiogenic effect of FXIIIa, we measured VEGF in the supernatant of cultured HUVECs. In addition, the mRNA levels of either VEGF or VEGF receptor-2 (VEGFR2) from HUVECs were assessed by reverse transcriptase–polymerase chain reaction. Neither VEGF protein level nor steady-state VEGF or VEGFR2 mRNA levels were affected by FXIIIa (data not shown).

Effect of FXIIIa on Endothelial Cell Gene Expression

After treatment of HUVECs with FXIIIa, a modest reduction in the mRNA of the following genes was observed: endoglin, bFGF, integrin α₅ subunit, PLGF, and SPARC (osteonectin); however, the most striking finding was the almost complete disappearance of thrombospondin 1 mRNA (Figure I, please see http://atvb.ahajournals.org).

Effect of FXIIIa on TSP-1 Synthesis

As shown by Figure 5A, TSP-1 protein was markedly reduced in a dose-dependent manner in the conditioned medium obtained from each HUVEC and HMVEC-d cultures grown in the presence of FXIIIa compared with controls. The basal amount of TSP1 secreted from HMVEC-d was much lower than that of HUVEC (Figure 5, lanes 0). Blockade of the transglutaminase activity of FXIIIa by iodoacetamide reversed the attenuating effect of FXIIIa on TSP-1 secretion in both cell types (Figure 5B).

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Figure 5. Effect of FXIIIa on TSP-1 protein level in HUVECs and HMVECs in the absence (A) or presence (B) of the transglutaminase inhibitor iodoacetamide. TSP-1 purified by heparin-affinity chromatography from conditioned media of HUVEC or HMVEC-d grown either in the absence of FXIIIa (line 0) or in the presence of FXIIIa (A) or iodoacetamide-treated FXIIIa (Iodo-FXIII) (B) at the concentrations (μg/mL) indicated above each lane was subjected to SDS-PAGE and Western blot analysis by a monoclonal anti-TSP-1 antibody B7.

To confirm the role of TSP-1 in the effects observed in Matrigel assays, HUVECs were plated on Matrigel in the presence or absence of either anti-TSP-1 antibody or FXIIIa, and tube formation was analyzed 16 hours after plating. Addition of 50 μg/mL of anti-TSP-1 antibody caused enhancement of endothelial cell proliferation similar to that observed with 50 μg/mL FXIIIa (Figures IIA through IIC, please see http://atvb.ahajournals.org).

In Vivo Effect of FXIII on Rabbit Cornea Neovascularization

FXIIIa was injected into the right eye of each of the 4 rabbits as outlined in Methods. The effect of FXIIIa on corneal vascularization was compared with that observed after injection of PBS into the left eye. In contrast to the left eyes treated with PBS, blood vessel formation was obvious in the right eyes of all 4 rabbits 48 hours after injection of FXIIIa (Figure III, please see http://atvb.ahajournals.org).

Histological section of the cornea shown in Figure 6B demonstrates the rich network of capillaries grown in the cornea of eyes treated with FXIII compared with those treated with PBS (Figure 6A). Staining of the FXIIIa–treated sections of the cornea with endothelial cell marker isolectin B₄ showed positive stain in the membranes of endothelial cells lining well-formed but also abortive blood vessels (Figure 6C). Immunohistochemical staining of the PBS-injected (control) corneas (Figure 6D) for TSP-1 disclosed positive staining in the stromal fibers (collagen fibers) and a few stromal cells (young fibroblasts). In contrast, in FXIIIa-treated corneas, the stromal elements were negative for TSP-1 (Figure 6E).

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Figure 6. Representative histological section of rabbit cornea excised 96 hours after injection of PBS (A and D) or F XIIIa (B, C, and E). A and B, H&E stain, magnification ×400. Blood vessels are denoted by arrows. C, GSLI-isolectin B₄ staining. Immunohistochemical staining for TSP-1 and counterstaining with hematoxylin of cornea sections injected with PBS (D) or FXIIIa (E).