Abstract
Aims
The identification of growth factors and cytokines with angiogenic activity has enabled new therapeutic treatments for a variety of diseases; this concept is called therapeutic angiogenesis . The vascular endothelial growth factor (VEGF) is the most critical regulator of vascular formation. In the present study, we were interested in the therapeutic angiogenesis effect using plasmid transfer of human complementary DNA VEGF 165 (phVEGF 165 ) in experimental skin and cartilage trauma.
Methods
Ten BALB/c mice were used for cartilage injury model. At 6 weeks of age, all mice were ear-punched, resulting in 2-mm–diameter puncture through the center of both pinnae. Each mouse got phVEGF 165 injection into the first ear and vector without insert or saline injection into the second one. The healing process was followed. The hollow diameter was measured on days 0, 14, and 42. Histological sections of experimental and control pinnae were taken from days 1, 3, 5, 7, 9, 11, 13, 15, 20, and 30 after experimental injury for hematoxylin and eosin and periodic acid Schiff staining and for human VEGF immunocytochemistry. The expression of human VEGF was also checked by real-time polymerase chain reaction in formalin-fixed, paraffin-embedded tissue sections.
Key Findings
In BALB/c mouse strain, a significant angiogenesis promotion and cartilage repair were observed after phVEGF 165 injection into the punched ear area.
Significance
We suggest that administering phVEGF 165 leads to faster cartilage regeneration even if not only on the angiogenic basis.
1
Introduction
Angiogenic stimulation may lead to angiogenesis in both nonischemic and ischemic tissues, providing an alternative treatment of severe chronic limb or myocardial ischemia. The identification of growth factors and cytokines with angiogenic activity has enabled new therapeutic treatments for a variety of diseases; this concept is called therapeutic angiogenesis . The vascular endothelial growth factor (VEGF) is the most critical regulator of vascular formation.
Gene transfer of VEGF constructs has been shown to induce functionally significant angiogenesis in numerous preclinical and clinical studies of angiogenic therapy for ischemic heart disease and peripheral arterial disease . The concept that angiogenic processes might be useful in the treatment of vascular diseases has been proposed for variety of ischemic conditions, not only affecting the heart or the lower limb , but also skin flaps , peripheral nerves , tracheal grafts , and the bone .
Angiogenesis development assays are crucial in the preclinical phase of gene therapy tests. Various in vitro and in vivo assays have been developed. The in vitro angiogenesis assays include the rat aortic assay , chorioallantoic membrane assay , corneal assay , and collagen and Matrigel assay .
Animal models for this kind of testing have been introduced including work with rat, pig, mice, etc. A widely used animal model is the rabbit hind limb acute ischemia model . In this model, the superficial femoral artery is removed from its proximal origin as a branch of the external iliac artery to the site where it bifurcates into saphenous and popliteal arteries. Another model of chronic limb ischemia uses a modification of the previous one . The direct assessments of angiogenesis after surgery are usually performed by microangiography, Doppler ultrasonography, or immunohistochemical staging .
Here, we tested the effect of therapeutic angiogenesis using plasmid transfer of complementary DNA (cDNA) encoding the 165–amino acid isoform of VEGF (phVEGF 165 ) in the experimental skin and cartilage trauma and the feasibility of a novel angiogenesis assay based on monitoring the angiogenesis in a mouse auricle. In this context, experiments were designed to determine the potential of phVEGF 165 to augment the angiogenesis in traumatic tissue and to facilitate the cartilage regeneration.
2
Material and methods
2.1
Animals
The angiogenesis response and the cartilage regeneration after hVEGF 165 plasmid administration were investigated using BALB/c mice. All of the used protocols have been approved by the Ethical Committee of the Academy of Sciences of the Czech Republic.
2.2
Plasmid phVEGF preparation
Human promyelocytic leukemia HL-60 cell line was stimulated for higher production of VEGF by the tumor-promoting agent phorbol-12-myristate-13-acetate . A cDNA was generated via reverse transcription by means of oligo(dT) 23 primers (Enhanced Avian RT-PCR kit; Sigma-Aldrich, St Louis, MO, USA).
The specific 611–base pair fragment of VEGF cDNA was obtained using VEGF-specific primers (5′-CCTCCGAAACCATGAACTTT-3′, 5′-GGAGGCTCCTTCCTCCTG-3′) . The fragment was amplified by Expand High Fidelity PCR System (Roche Diagnostics, Mannheim, Germany) and cloned via T-cloning into pTARGET Mammalian Expression Vector (Promega, Madison, WI, USA). phVEGF 165 was propagated through transformation and cultivation of Escherichia coli JM109 competent cells. The plasmid DNA was isolated from grown bacterial cultures with GenElute Endotoxin-free Plasmid Maxiprep Kit (Sigma-Aldrich) according to the directions of the manufacturer. To confirm the identity of the prepared plasmid, the VEGF-coding region from each pooled batch was resequenced.
2.3
Auricle—angiogenesis model
The angiogenesis induction was performed on the anesthetized mice pinnae (auricle—the cartilaginous structure of the external ear). Mice were anesthetized with a ketamine (50 mg/kg) and xylazine (5 mg/kg). Injection of phVEGF 165 in saline solution, vector without insert, or pure saline solution (controls) was applied into the region of the central artery at the base of each auricle. Each mouse had an injection containing phVEGF 165 into the first ear and with vector without insert or saline injection into the second ear (total volume of 200 μL, DNA 20 ng/μL).
2.4
Evaluation of the revascularization in the auricle
The progress of the angiogenesis was documented by Nikon Coolpix 5000 (Nikon, Tokyo, Japan). Quantitative angiographic analysis of vessel development was performed as follows. The total number of ear arteries was counted individually by the software Corel PHOTO-PAINT (Corel Corporation, Fremont, USA) as the number of pixels of a red color and the ratio of all areas of the ear in percentages. The documentation was prepared under specific constant distance of the tested samples apart. An angiographic score was calculated for each ear on the day before the application of phVEGF 165 or saline and on days 7 and 21 after the injection.
2.5
The auricle model of cartilage injury
Ten BALB/c mice were used for cartilage injury model. At 6 weeks of age, all mice were ear-punched, resulting in 2-mm–diameter puncture through the center of both pinnae. Measurements of the hollow diameter were taken on days 0, 14, and 42.
2.6
Statistical analysis
Results were expressed as mean ± SD. Statistical significance of differences was evaluated using the analysis of variance test.
2.7
Histological examination
Histological sections of experimental and control pinnae from days 1, 3, 5, 7, 9, 11, 13, 15, 20, and 30 after experimental injury were taken for hematoxylin and eosin and periodic acid Schiff staining and for VEGF immunocytochemistry.
2.8
Immunocytochemistry
Histological sections (7 μ m thick) of formaldehyde (4%)-fixed and paraffin-embedded pinnae were cut serially and mounted on the poly- l -lysine (Sigma-Aldrich)–coated slides. The slides were dewaxed in xylene and rehydrated in descending concentration of ethanol. The sections were exposed to microwaves (2 × 5 minutes at 750 W) in target retrieval solution, pH 9 (DakoCytomation, Glostrup, Denmark). The sections were then incubated in distilled water containing solution of H 2 O 2 (1%) for 10 minutes and with normal goat serum (5% in phosphate-buffered saline) for 60 minutes to block endogenous peroxidase activity and nonspecific immunoglobulin binding, respectively. Following incubation, monoclonal mouse anti-human VEGF clone VG1 (DakoCytomation, Dako, Carpinteria, USA) was diluted 1:25 in 1.5% normal goat serum in phosphate-buffered saline at room temperature in moist chamber for 30 minutes. The binding of primary antibody was visualized with EnVision System− HRP anti-mouse labeled polymer (DakoCytomation) and DAB+ Substrate Chromogen System (DakoCytomation). The slides were counterstained with hematoxylin and mounted in Faramount (Dako, Carpintenteria, USA).
Sections from the mouse embryo were used as the positive control. In the negative control experiment, the anti-VEGF antibody was omitted.
2.9
Real-time polymerase chain reaction
RNA was converted to cDNA using Taq-Man reverse transcription reagents (Applied Biosystems, Carlsbad, USA). A reaction mix for real-time polymerase chain reaction (PCR) was made with TaqMan Universal PCR master mix, water, and Assays-on-Demand gene expression products for human VEGF and 18S RNA (all Applied Biosystems). Twenty microliters of reaction mix was aliquotted to the wells on a real-time PCR plate. Each sample was analyzed in duplicate. A volume of 5 μ L of cDNA was added to each well. Wells were sealed with optical caps. A no-template control contained water instead of cDNA. The PCR was run on the 7000 real-time PCR system (Applied Biosystems) using standard conditions. Expressions of all genes were normalized to RNA loading for each sample using the 18S RNA as an internal standard. The quantity of messenger RNA was given as 2 −ΔΔct . ΔΔct was calculated as follows: ΔΔct = Δct (target) − Δct (reference).
2
Material and methods
2.1
Animals
The angiogenesis response and the cartilage regeneration after hVEGF 165 plasmid administration were investigated using BALB/c mice. All of the used protocols have been approved by the Ethical Committee of the Academy of Sciences of the Czech Republic.
2.2
Plasmid phVEGF preparation
Human promyelocytic leukemia HL-60 cell line was stimulated for higher production of VEGF by the tumor-promoting agent phorbol-12-myristate-13-acetate . A cDNA was generated via reverse transcription by means of oligo(dT) 23 primers (Enhanced Avian RT-PCR kit; Sigma-Aldrich, St Louis, MO, USA).
The specific 611–base pair fragment of VEGF cDNA was obtained using VEGF-specific primers (5′-CCTCCGAAACCATGAACTTT-3′, 5′-GGAGGCTCCTTCCTCCTG-3′) . The fragment was amplified by Expand High Fidelity PCR System (Roche Diagnostics, Mannheim, Germany) and cloned via T-cloning into pTARGET Mammalian Expression Vector (Promega, Madison, WI, USA). phVEGF 165 was propagated through transformation and cultivation of Escherichia coli JM109 competent cells. The plasmid DNA was isolated from grown bacterial cultures with GenElute Endotoxin-free Plasmid Maxiprep Kit (Sigma-Aldrich) according to the directions of the manufacturer. To confirm the identity of the prepared plasmid, the VEGF-coding region from each pooled batch was resequenced.
2.3
Auricle—angiogenesis model
The angiogenesis induction was performed on the anesthetized mice pinnae (auricle—the cartilaginous structure of the external ear). Mice were anesthetized with a ketamine (50 mg/kg) and xylazine (5 mg/kg). Injection of phVEGF 165 in saline solution, vector without insert, or pure saline solution (controls) was applied into the region of the central artery at the base of each auricle. Each mouse had an injection containing phVEGF 165 into the first ear and with vector without insert or saline injection into the second ear (total volume of 200 μL, DNA 20 ng/μL).
2.4
Evaluation of the revascularization in the auricle
The progress of the angiogenesis was documented by Nikon Coolpix 5000 (Nikon, Tokyo, Japan). Quantitative angiographic analysis of vessel development was performed as follows. The total number of ear arteries was counted individually by the software Corel PHOTO-PAINT (Corel Corporation, Fremont, USA) as the number of pixels of a red color and the ratio of all areas of the ear in percentages. The documentation was prepared under specific constant distance of the tested samples apart. An angiographic score was calculated for each ear on the day before the application of phVEGF 165 or saline and on days 7 and 21 after the injection.
2.5
The auricle model of cartilage injury
Ten BALB/c mice were used for cartilage injury model. At 6 weeks of age, all mice were ear-punched, resulting in 2-mm–diameter puncture through the center of both pinnae. Measurements of the hollow diameter were taken on days 0, 14, and 42.
2.6
Statistical analysis
Results were expressed as mean ± SD. Statistical significance of differences was evaluated using the analysis of variance test.
2.7
Histological examination
Histological sections of experimental and control pinnae from days 1, 3, 5, 7, 9, 11, 13, 15, 20, and 30 after experimental injury were taken for hematoxylin and eosin and periodic acid Schiff staining and for VEGF immunocytochemistry.
2.8
Immunocytochemistry
Histological sections (7 μ m thick) of formaldehyde (4%)-fixed and paraffin-embedded pinnae were cut serially and mounted on the poly- l -lysine (Sigma-Aldrich)–coated slides. The slides were dewaxed in xylene and rehydrated in descending concentration of ethanol. The sections were exposed to microwaves (2 × 5 minutes at 750 W) in target retrieval solution, pH 9 (DakoCytomation, Glostrup, Denmark). The sections were then incubated in distilled water containing solution of H 2 O 2 (1%) for 10 minutes and with normal goat serum (5% in phosphate-buffered saline) for 60 minutes to block endogenous peroxidase activity and nonspecific immunoglobulin binding, respectively. Following incubation, monoclonal mouse anti-human VEGF clone VG1 (DakoCytomation, Dako, Carpinteria, USA) was diluted 1:25 in 1.5% normal goat serum in phosphate-buffered saline at room temperature in moist chamber for 30 minutes. The binding of primary antibody was visualized with EnVision System− HRP anti-mouse labeled polymer (DakoCytomation) and DAB+ Substrate Chromogen System (DakoCytomation). The slides were counterstained with hematoxylin and mounted in Faramount (Dako, Carpintenteria, USA).
Sections from the mouse embryo were used as the positive control. In the negative control experiment, the anti-VEGF antibody was omitted.
2.9
Real-time polymerase chain reaction
RNA was converted to cDNA using Taq-Man reverse transcription reagents (Applied Biosystems, Carlsbad, USA). A reaction mix for real-time polymerase chain reaction (PCR) was made with TaqMan Universal PCR master mix, water, and Assays-on-Demand gene expression products for human VEGF and 18S RNA (all Applied Biosystems). Twenty microliters of reaction mix was aliquotted to the wells on a real-time PCR plate. Each sample was analyzed in duplicate. A volume of 5 μ L of cDNA was added to each well. Wells were sealed with optical caps. A no-template control contained water instead of cDNA. The PCR was run on the 7000 real-time PCR system (Applied Biosystems) using standard conditions. Expressions of all genes were normalized to RNA loading for each sample using the 18S RNA as an internal standard. The quantity of messenger RNA was given as 2 −ΔΔct . ΔΔct was calculated as follows: ΔΔct = Δct (target) − Δct (reference).
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