Construction of a Bicistronic Proangiogenic Expression Vector and Its Application in Experimental Angiogenesis in Vivo

Manipulation of angiogenesis in vivo is an example of successful gene therapy strategies. Overexpression of angiogenic genes like VEGF, FGF or PDGF causes new vessel formation and improves the clinical state of patients. Gene therapy is a very promising procedure but requires large amounts of pharmaceutical-grade plasmid DNA. In this regard we have constructed a bicistronic plasmid DNA vector encoding two proangiogenic factors, VEGF165 and FGF-2. The construct (pVIF) contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) which permits both genes to be translated from a single bicistronic mRNA. The IRES sequence allows for a high efficiency of gene expression in vivo. The pVIF vector was characterized in vitro and in vivo. In vivo angiogenesis studies showed that the bicistronic vector encoding two proangiogenic factors induces the formation of new vessels significantly more than pVEGF165 or pFGF-2 alone. In our opinion the combined pro-angiogenic approach with VEGF165 and FGF-2 is more powerful and efficient than single gene therapy. We also postulate that IRES sequence can serve as a useful device improving efficiency of gene therapy. Angiogenesis is a multistep process involved in many physiological and pathological phenomena (Battegay, 1995; Szala & Radzi-kowski, 1997). The formation of new vessels is postulated to be a crucial point for tumo-rigenesis and metastasis, whereas deterioration of the arterial system is one of the rea-In the last decade significant development of angiogenic gene therapy methods has been observed (Isner & Asahara, 1998; Harjai et al., 2002). Many hopes are linked with application

Angiogenesis is a multistep process involved in many physiological and pathological phenomena (Battegay, 1995;Szala & Radzikowski, 1997).The formation of new vessels is postulated to be a crucial point for tumorigenesis and metastasis, whereas deterioration of the arterial system is one of the reasons of ischemic diseases (Battegay, 1995;Szala & Radzikowski, 1997).Therefore, manipulation of angiogenesis in vivo may be a successful gene therapy strategy (Zhang & Harris, 1998;Hayes, 1999;Harjai et al., 2002).In the last decade significant development of angiogenic gene therapy methods has been observed (Isner & Asahara, 1998;Harjai et al., 2002).Many hopes are linked with application of various expression vectors encoding angiogenic factors for the treatment of heart or hind limb ischemia (Isner & Asahara, 1998;Dulak et al., 1999;Harjai et al., 2002).Overexpression of angiogenic genes, for example VEGF, PDGF or FGF can cause the formation of new vessels and improve the clinical state of patients (Isner et al., 1996a;1996b;Isner & Asahara, 1998).In our previous study we described promising results of in vitro and in vivo experiments using pHVEGF165 vector (Ma³ecki et al., 2001).The clinical state of patients suffering from serious heart ischemia improved after intracardiac injection of our plasmid encoding VEGF165 (Ko³sut et al., 2003).Since VEGF as well as FGF belong to the family of proangiogenic growth factors a vector encoding two proangiogenic proteins would have an advantage over two or three single gene plasmids.In the present work we have prepared a new construct -a bicistronic DNA vector encoding two angiogenic factors, namely VEGF165 and FGF-2.It could be expected that combination of VEGF165 and FGF-2 would have higher proangiogenic efficiency than monocistronic plasmids.The new construct (pVIF) contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV).This sequence permits both genes of interest (VEGF165 and FGF-2) to be translated separately from a single bicistronic mRNA.Additional advantages of bicistronic vectors are lower cost of preparation and lower level of endotoxins in the single bicistronic vector preparation than in two monocistronic plasmids.We also expected that combined gene therapy with two angiogenic factors would induce more new vessels in vivo than could be obtained with monocistronic plasmid.

Construction and preparation of bicistronic pVIF vector.
The structure of the pSEC expression vector is shown in Fig. 1.
The cloning procedure begun from the cloning of two expression vectors, pFGF-2 and pVEGF/IRES, encoding FGF-2 and VEGF165/IRES, respectively.Amplified fragments were inserted into pSEC expression vector (Invitrogen, Holland).The pSEC expression vector (Fig. 1) was amplified in Escherichia coli DH5 strain growing on LB medium (Sigma, U.S.A.) containing ampicillin (100 mg/ml).The plasmid was isolated with the EndoFree Plasmid Mega Kit (QIAGEN, Germany).Purity of the empty plasmid was confirmed spectrophotometrically and by agarose gel electrophoresis.The isolated plasmid was then digested with appropriate restriction enzymes.
A) Construction of pSEC/FGF-2 expression vector.A fragment of DNA encoding FGF-2 was amplified using cDNA from HUVEC (total cDNA of HUVEC was a gift of Dr. Józkowicz from Collegium Medicum, Kraków, Poland).PCR amplification of cDNA was performed using specific primers for FGF-2 carrying EcoRI and XhoI restriction The functional elements of the vector are as follows: CMV, human cytomegalovirus immediate-early promoter/enhancer; MCS, multiple cloning site; BGH, bovine growth hormone polyadenylation signal; pSV40, early promoter and origin; Zeo, Zeocin resistance gene; Amp, ampicillin resistance gene.The bicistronic expression cassette was cloned into the MCS site.
sites (Table 1).The product was digested with EcoRI and XhoI, separated by electrophoresis on 1.2% agarose gel with ethidium bromide (0.5 mg/ml) and isolated using Qiagen Gel Extraction Kit (QIAGEN, Germany).The obtained product was then ligated with EcoRIand XhoI-digested pSEC vector for 16 h at 16°C in the presence of T4 ligase (Amershem, U.K.).The ligation mixture was transferred into a competent E. coli.The bacterial culture was incubated on LB agar followed by LB medium supplemented with ampicillin.After incubation individual clones were isolated with QIAprep Spin Miniprep Kit (QIAGEN, Germany) and analysed by digestion with restrictases.Finally, the pSEC/FGF-2 vector was sequenced.

B) Construction of pSEC/VEGF/IRES expression vector.
Construction of the pSEC/VEGF165 expression vector was described previously (Ma³ecki et al., 2001).The plasmid pSEC/VEGF165/IRES was obtained after amplification by PCR (Table 1) and subcloning of the EcoRI/IRES/NotI fragment from the pMIG retroviral vector containing the sequence of the internal entry site (IRES) into the plasmid pSEC/VEGF cleaved by EcoRI and NotI (pMIG vector was a gift of Dr. Skórski from Temple University, Philadelphia, U.S.A.).The IRES sequence is derived from encephalomyocarditis virus (ECMV) and permits the translation of two open reading frames from one messenger RNA.The obtained vector was isolated and analysed as described above.

C) Construction of bicistronic pSEC/ VEGF165/IRES/FGF-2 expression vector.
The NotI-ATG/Igk/FGF-2/XhoI sequence from pSEC/FGF-2 vector was amplified by PCR (F 1 +R; Table 1) and cloned into pSEC/VEGF165/IRES vector digested with NotI and XhoI.The obtained vector was analysed as described previously.The structure of the cloned bicistronic expression cassette containing the VEGF165 gene followed by IRES sequence and FGF-2 gene is shown in Fig. 2.

PCR analysis of the pVIF bicistronic vector.
The pVIF bicistronic vector as well as pSEC, pVEGF and pFGF were analysed by standard PCR.Amplification of cDNA cloned into pSEC vector was performed using specific primers for VEGF165, IRES and FGF-2.The sequences of the primers and the reaction conditions are shown in Table 1.The products were separated by electrophoresis on 1.2% agarose gel with ethidium bromide (0.5 mg/ml).
Cell culture and transfection procedure.The mouse sarcoma cell line L1 was used in the experiments.Cells were cultured in Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 0.1 mg/ml penicillin + streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .The pVIF vector was transfected into L1 cells by lipotransfection according to the manufacturer's protocol (Przybyszewska et al., 1998).Zeocin resistant clones of the L1 transfected cells were analysed directly for VEGF and FGF overexpression.
Western blot assay.VEGF165 and FGF-2 proteins were detected in the conditioned medium of pVIF-transfected L1 cells by Western blotting with the use of anti-VEGF (Santa Cruz Biotechnology, Inc., sc-152) and anti-FGF (Santa Cruz Biotechnology, Inc., sc-1390) antibodies, respectively.The conditioned medium was 20 times concentrated using 10 kDa cut off centrifugal filter devices (Amicon, U.S.A.).
In vivo angiogenesis assay.Adult inbred female BALB/c mice (9-11 weeks old) received six intradermal injections of 10 mg of naked pVIF, pVEGF and pFGF-2 vectors.Three and thirteen days later, the mice were sacrificed, and new blood vessel formation on the inner surface of the skin was counted as described by Sidky and Auerbach (1975).

Cloning and validation of VEGF and FGF cDNAs
The structure of the bicistronic expression cassette cloned into the pSEC vector is shown in Fig. 2. The obtained pVIF vector was amplified in E. coli DH5.The plasmid was isolated with EndoFree Plasmid Mega Kit and its quality was confirmed spectrophotometrically and electrophoretically.Restriction digestion mapping and DNA sequencing of this vector confirmed the presence of VEGF165 and FGF-2 cDNAs.Restriction digestion results of the pVIF vector are shown in Fig. 3.The presence of VEGF165, IRES and FGF-2 inserts in the pVIF vector was also confirmed by PCR analysis (Fig. 4).

Western blotting study
To determine the expression/secretion of VEGF165 and FGF-2 in vitro, mouse sarcoma cells (L1) were transfected with pVIF.Western blot analysis showed a high level of VEGF and FGF proteins in the conditioned medium The elements of the cassette are as follows: CMV, human cytomegalovirus immediate-early promoter/enhancer; ATG, initiation codon; Ig, leader sequence; VEGF, vascular endothelial growth factor 165 gene; TGA, termination codon; IRES, internal ribosome entry site; FGF, basic fibroblast growth factor (FGF-2).
of the pVIF L1 transfected cells.As shown by quantitative analysis (1D Image Analysis, Kodak) of the VEGF and FGF bands, the pVIF transfectants revealed an about 25% higher level of VEGF than FGF.The results are shown in Fig. 5A-C.

In vivo angiogenesis assay
To determine the effect of the bicistronic proangiogenic pVIF vector on the formation of new vessels in vivo, mice were intradermally injected with pVIF.As seen in Table 2 the formation of new vessels after intradermal injection of the naked proangiogenic vectors increased significantly compared with the injection of a plasmid without an angiogenic gene (empty plasmid).The bicistronic pVIF vector induced generation of new vessels twice that induced by monocistronic vectors, pVEGF or pFGF.These results confirm the ability of the pVIF vector to produce two angiogenic proteins in vivo.The IRES sequence enables a high efficiency of expression of both genes.The pVIF vector was analysed by standard PCR method.Presence of the VEGF165, IRES and FGF-2 inserts was confirmed by amplification of the cDNA according to data shown in Table 1.The size of the cDNAs is shown in Fig. 1, VEGF165 (549 bp); 2, IRES (601 bp); 3, FGF-2 (460 bp); m, size marker (pK03/HinfI).states.It is a multistep process that plays a crucial role in the development of vascular supply in normal tissue, e.g. in reproduction and wound healing.Angiogenesis is also involved in many ischemic diseases like heart disease, peripheral vascular disease, rheumatoid arthritis, and tumor growth and metastasis (Battegay, 1995;Szala & Radzikowski, 1997).Manipulation of angiogenesis is seen as a promising strategy for treatment of many diseases such as ischemic heart and peripheral vascular disease (Isner & Asahara, 1998;Harjai et al., 2002;Ma³ecki & Janik, 2002;Ko³sut et al., 2003).A multitude of angiogenic molecules have been well characterized (Battegay, 1995;Ma³ecki & Janik, 1999).Many researches have found that transfer of angiogenic genes, e.g.VEGF or FGF, into ischemic tissues caused the formation of new vessels and improved the clinical state of patients (Isner et al., 1996a;1996b;Garcia-Martinez et al., 1999;Harjai et al., 2002).Most of the current gene therapy approaches are compromised by the inability to deliver 1, L1 non transfected cells; 2, L1 cells transfected with empty pSEC vector; 3, L1 cells transfected with bicistronic pVIF vector.

Angiogenesis -formation of new blood vessels -occurs in physiological and pathological
Table 2.The number of blood vessels found in animals injected intradermally with plasmid DNA encoding angiogenic factors.
Empty pSEC vector and 0.9% NaCl served as control.Results represent the mean ±S.D.

A B C
genes efficiently to obtain sustained expression.One of the methods used is direct injection of naked plasmid DNA into muscles.It is a very efficient procedure but requires large amounts of pharmaceutical-grade plasmid DNA.We have constructed a bicistronic plasmid DNA vector to induce angiogenesis in vivo via high expression of two proangiogenic factors, namely VEGF165 and FGF-2.The pVIF vector was tested in vitro and in vivo.As seen in Figs.3-4 digestion mapping and PCR analysis confirmed the presence of VEGF165 and FGF-2 cDNAs.Western blotting analysis (Fig. 5) showed that secretion of VEGF165 and FGF-2 proteins to the conditioned medium of the pVIF transfected L1cells was increased significantly comparing with non transfected cells.All cells positive for VEGF also expressed FGF.The IRES sequence permits both genes of interest -VEGF165 and FGF-2 -to be translated efficiently from a single bicistronic mRNA (Wiznerowicz et al., 1998;Martinez-Salas, 1999;Vagner et al., 2001).It is worth noticing that expression of FGF-2 was lower than VEGF165.These observations are consistent with those obtained in other studies.Mizuguchi et al. (2000) described that IRES-dependent second gene expression can be lower than of the first gene in a bicistronic vector.These findings were also confirmed by Kapturczak et al. (2002) who showed similar differences in the degree of ex-pression levels for studied genes.To determine the effect of the bicistronic pVIF vector on angiogenesis in vivo mice were implanted intradermally with naked plasmid DNA and the formation of new vessels was monitored.
Our studies demonstrate convincingly the increased number of newly formed vessels in animals injected with pVIF.The proposed system is based upon studies where combined expression of angiogenic cytokines has been used to achieve efficient angiogenesis in vivo.
Ibukiyama showed that both exogenous VEGF and FGF can significantly promote collateral vessel development (Ibukiyama, 1996).It seems clear that combined angiogenic therapy can be an effective strategy for the treatment of ischemic disease.Similar to other researchers we postulate that IRES sequence can serve as a useful biotechnological tool improving the efficiency of gene therapy (Gurtu et al., 1996;Attal et al., 1999;Mizuguchi et al., 2000;de Felipe, 2002;Singh et al., 2002).In our opinion this sequence may be also helpful in angiogenic gene therapy.As we show here, the combined use of VEGF165 and FGF-2 is more powerful and efficient than single gene therapy.Additionally, the pVIF vector permits a lower cost of preparation and high level of purity.The content of endotoxins in the final pVIF preparations is almost always two times lower than in the mixture of two vectors (pVEGF and pFGF) isolated separately.
Figure 1.A schematic map of pSEC vector.

Figure 2 .
Figure 2. The bicistronic expression cassette cloned into the pSEC vector.
Figure 5.Western blot analyses of the VEGF165 (A) and FGF-2 (B) proteins secreted to the medium of pVIF transfected L1 cells.The bands seen in Figs.A and B were analysed by densitometry (C).