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1 Department of Medicine Endocrinology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA
2 Department of Genetics, School of Medicine, Yale University, New Haven, Connecticut 06510, USA
3 Arthritis and Immunology Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, Oklahoma 73104, USA
(Requests for offprints should be addressed to K Lu; Email: kangmo.lu{at}yale.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Vascular endothelial growth factor (VEGF) is a potent mediator of vascular permeability and angiogenesis in a variety of human pathological situations. The increased VEGF levels are responsible for the retinal vascular leakage or retinal vascular hyperpermeability, and retinal NV (Aiello et al. 1998, Antonetti et al. 1999, Gardner et al. 2000, Ishida et al. 2003). A number of clinical and animal studies have shown that VEGF plays a pivotal role in normal and pathological angiogenesis, for example, the development of DR (Chiarelli et al. 2000, Caldwell et al. 2003). The up-regulated expression of retinal VEGF and its receptors correlates to retinal vascular leakage in oxygen-induced retinopathy (OIR) and STZ-induced diabetic rats (Adamis et al. 1994, Pierce et al. 1995). The inhibition of VEGF and VEGF receptors has been found to prevent retinal NV in diabetic and OIR animal models (Gilbert et al. 1998, Qaum et al. 2001).
Genetic variations in the VEGF mRNA, VEGF promoter, and VEGF receptors are involved in diabetic microvascular complications (Awata et al. 2002, Ikeda 2003, Yang et al. 2003, Ray et al. 2004). The VEGF –460C polymorphism reversed from the transcriptional start site in the promoter region is associated with proliferative DR and a positive independent predictive factor for the development of proliferative DR (Ray et al. 2004). The C(–634)G polymorphism reversed from the translational start site in the 5'UTR of the VEGF gene is strongly associated with an increased risk of retinopathy (Awata et al. 2002). VEGF variants with an intact receptor binding interface inhibit VEGF-stimulated receptor phosphorylation and proliferation of human endothelial cells (Siemeister et al. 1998).
Our recent studies have found that there is a strain difference in susceptibility to retinal vascular leakage between Brown Norway (BN) and Sprague–Dawley (SD) rats in response to ischemia and diabetes (Gao et al. 2001, 2002, Zhang et al. 2005). Under the same stress conditions, BN rats have higher levels of VEGF mRNA and protein with lower protein levels of pigment epithelium-derived factor (PEDF) in the retina and more severe retinal vascular leakage and NV than SD rats. However, the mRNA level of PEDF was not changed in OIR animal models. PEDF may be one of the factors affecting VEGF expression. We hypothesize that VEGF, a potent vascular permeability factor, is a candidate gene responsible for susceptibility to retinal vascular leakage in the OIR-BN model. In this study, we searched the polymorphisms of VEGF gene between BN and SD rats and compared the expression levels of VEGF and vascular permeability in the retina of BN, SD, and F2 progeny of BN and SD crossbreeding.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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BN and SD rats were purchased from Charles River Laboratories (Wilmington, MA, USA). BN males and SD females were crossbred to produce the F1 generation rats (BN/SD) with BN and SD strain rat background. To generate F2 animals, F1 BN/SD males and females were intercross-bred. Care, use, and treatment of all animals in this study were in strict agreement with the guidelines for the Care and Use of Laboratory Animals by the University of Oklahoma.
Oxygen-induced retinopathy model
The induction of OIR models was performed as described by Smith et al. with some modifications as in our previous method (Zhang et al. 2001). Briefly, newborn BN, SD, and BN/SD F2 rats were randomly assigned to experimental and control groups. On postnatal day 7 (P7), rats were exposed to hyperoxia (75% O2) for 5 days (P7–P12) and then returned to room air for 4 days (P16) to induce OIR-rats because our previous studies have shown that all oxygen-treated rats on postnatal day 16 (P16) developed severe retinal vascular leakage (Zhang et al. 2005). Control rats were kept in constant normoxia conditions.
Evaluation of retinal vascular permeability
Retinal vascular permeability was quantified by measuring albumin leakage from blood vessels into the retina following the previous methods (Bhatia et al. 1998, Xu et al. 2001, Ishida et al. 2003) with modifications. The rat was anesthetized by inhalation with isoflurane. The fluorescein isothiocyanate (FITC)-labeled albumin in phosphate buffer solution (PBS; 10 mg/kg body weight, Sigma) was injected into the femoral vein under microscopic inspection. After the injection, the rats were kept in room temperature for 3 h. Then the rat chest cavity was opened and blood was collected through the right atrium. To clear the remaining intravascular FITC-albumin, each rat was perfused via the left ventricle with 50 ml of 1xPBS (pH 7.4), which was pre-warmed to 37 °C to prevent vasoconstriction. Immediately after the perfusion, the eyes were enucleated and the retinas were carefully dissected under an operating microscope. The retina was homogenized in 200 µl PBS containing the proteinase inhibitor cocktail (Roche Diagnosis). The FITC-albumin leaked into the retina was extracted by sonication and centrifugation. The fluorescence intensity (FI) of FITC-albumin from supernatant and serum was measured at excitation wave 485 nm/emission wave 530 nm in Vallac Victor3 multilabel counter (Perkin–Elmer Life and Analytical Science, Turkin, Finland). Retinal protein levels in the same wells were measured in the multilabel counter at A280 and A490. The leaked FITC-albumin levels in the retina were normalized by serum fluorescence intensity and retinal protein level. Retinal vascular permeability was calculated as the following formula: Retinal FITC-albumin (µg)/serum FITC-albumin concentration (µg/µl)/retinal protein level (A280)/circulation time. The results of retinal vascular permeability were expressed as retinal FI/serum FI/retinal A280.
ELISA for retinal VEGF levels
The ELISA kits for rat VEGF were purchased from R&D Systems, Inc (Minneapolis, MN, USA). The retinal samples as mentioned above were used for the measurement of VEGF levels. ELISA for VEGF was performed according to the manufacturer’s instructions. The retinal VEGF levels were normalized by retinal protein levels at A280.
Genomic DNA purification
Rat genomic DNA was extracted using the salt precipitation method (Klett et al. 2004). Rat tail (about 0.5 cm) was digested in 500 µl lysis buffer (50 mM Tris–HCl (pH 8.0), 100 mM EDTA, 125 mM NaCl, 1% SDS, 200 µg Proteinase K) at 55 °C with rocking overnight. The saturated NaCl (200 µl) was added into digested solution with vigorous shaking for at least 60 s. Mixed solution was spun down at maximal speed in top microcentrifuge for 20 min. The supernatant was transferred to a new tube with an equal volume 100% ethanol, mixed by inversion and spun down. The DNA pellet was washed in 70% ethanol and suspended in 1000 µl Tris–EDTA buffer. DNA concentration was quantified in Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc. Wilmington, DE, USA).
DNA sequencing
The VEGF gene DNA fragments from BN, SD, and F2 BN/SD rats were amplified by PCR using rat genomic DNA, Master PCR kits (Roche Diagnosis), and primer sets as shown in Table 1
. The PCR products were purified using PCR product purification kits (Roche Diagnosis). The purified DNAs were amplified using internal primers and sequencing PCR kit and were sequenced on the ABI 3730 capillary DNA sequencer by the Oklahoma Medical Research Foundation Sequencing Core.
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The SNP of the upstream stimulating factor (USF)-binding site in the VEGF promoter region between BN and SD rats was analyzed using Kaufman’s method with some modifications (Kaufman et al. 2006) in the following steps. The polymorphism of USF-binding site and primer design are shown in Fig. 4A and B.
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Step 2: PCRs cleanup
PCR products were treated with shrimp alkaline phosphatase (SAP) to remove excess dNTPs. SAP (0.2 Unit; Roche Diagnosis) in 5 µl of 50 mM Tris–HCl buffer (pH 7.4) was added into the PCR product solution. The reaction solution was incubated at 37 °C for 30 min and denatured at 70 °C for 15 min to inactive the enzyme.
Step 3: primer extension
For primer extension reaction, dATP-missed dNTP was used in the PCR to stop primer extension at adenine base. USF Extension-R primer was synthesized and labeled with fluorescence IR Dye 700 by Li-Cor, Inc (Lincoln, NE, USA). The primer extension was performed in 5 µl PCR buffer containing 2 µl SAP-treated PCR products, 0.1 pmol Dye 700-labeled extension primer, 250 nM dCTP, dGTP, and dTTP, and 1xPCR buffer. Primer extension PCR was 94 °C, 2 min for denaturing, and 10 cycles of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 30 s.
Step 4: fragmentation analysis
The PCR products of primer extension were loaded on 15% polyacrylamide gels and analyzed on a Li-Cor 4200 automated DNA sequencing system (Li-Cor) following the manufacturer’s recommendations. The genotypes of USF SNP in F2 progeny were analyzed by sequencing system software as shown in Fig. 5C.
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Data are shown as mean ± S.D. Student’s t-test was used to determine the statistical significance of differences between the groups of animals. Significance was set at P<0.05. The association of retinal VEGF levels with retinal vascular permeability was tested using ANOVA linear regression analysis program.
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Results |
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Our previous studies have shown that retinal permeability is significantly increased, with a peak at P16 in OIR-BN rats, when compared with the age-matched normal control BN and SD rats. The increase in permeability is greater in OIR-BN rats than in OIR-SD rats (Zhang et al. 2005). To determine whether this susceptibility will be inherited to offspring generations, F2 progeny with BN and SD background was generated. Before crossbreeding of inbred BN rats and outbred SD rats, the retinal vascular permeability was assayed at P16 in control, OIR-BN and SD rats to confirm that the breeding BN rats are susceptible to retinal vascular leakage in response to ischemia. There was no difference in the basal levels of retinal vascular permeability between normal control SD and BN rats. In OIR-SD rats, retinal vascular permeability was 1.74-fold higher than that in the control SD rats (P<0.05, n=4; Fig. 1A). However, retinal vascular permeability in OIR-BN rats was 4.97-fold higher than that in the age-matched control BN rats (P<0.001, n=4) and 2.85-fold higher than that in OIR-SD rats (P<0.001, n=4; Fig. 1A). The results suggest that BN rats develop more severe vascular leakage than SD rats under the same ischemia stress.
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). However, in OIR-BN rats, retinal VEGF levels were approximately tenfold higher than those in normal control BN and OIR-SD rats (P<0.001, n=4; Fig. 1B). Distribution and association of retinal VEGF protein levels with retinal vascular permeability in F2 progeny BN/SD rats in response to ischemia
To determine if retinal VEGF levels are correlated to retinal vascular permeability, the retinal vascular permeability and VEGF contents at P16 were quantified in 97 F2 SD/BN rats exposed to 75% oxygen for 5 days and to room air for 4 days. The retinal VEGF contents in oxygen-treated F2 rats had a linear distribution in the range of 22–666 pg/mg protein (Fig. 2A). The retinal VEGF levels were significantly positively correlated to retinal vascular permeability in F2 SD/BN rats in response to ischemia (n=97, R=0.7575, P<0.001; Fig. 2B). The F2 rats with extreme high retinal vascular permeability had approximately fivefold higher retinal VEGF contents than in those with extreme low retinal vascular permeability (Fig. 3A and B).
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Since retinal VEGF contents were significantly associated with retinal vascular leakage in oxygen-treated F2 BN/SD rats, it is proposed that VEGF be one of the candidate genes responsible for susceptibility to vascular leakage in OIR-BN rats. First, we searched the rat sequence database (http://rgd.mcw.edu/tool-entry.shtml and http://www.ncbi.nlm.nih.gov/) to determine if there is any polymorphism of the VEGF gene between SD and BN rat strains. There are two SNPs (rs8152485 and rs8148303) of the VEGF gene between BN and SD rat strains according to the existing information in the database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=snp). The code of VEGF SNP (rs8148303, A/G) does not change the amino acid residue. Another VEGF SNP (rs8152485) is A/G (BN/SD) at 209 of the VEGF mRNA sequence from transcription initiation site, which causes amino acid substitution, tyrosine (Tyr)/cysteine (Cys) (BN/SD) at position 70 of the peptide sequence. Different from the GenBank information, we did not detect any polymorphism of the entire coding region of the VEGF mRNA between breeding BN and SD rats by amplifying and sequencing the VEGF cDNA from BN and SD rats.
In order to determine whether there is any polymorphism in the rat VEGF promoter region, a 2.3 kb fragment of the VEGF promoter was amplified by PCR from the BN and SD rat genomic DNA and sequenced (Table 1). One SNP (G/T) was found at –1234 in the VEGF promoter region between BN and SD rat strains. When compared with the SD rat sequence, BN rats lack the binding site of USF in the VEGF promoter region (Fig. 4A).
No differences in retinal VEGF contents and retinal vascular permeability between the BN, SD, and BN/SD alleles of the VEGF promoter SNP in F2 progeny
To determine if the genetic variation of VEGF USF-binding site polymorphism is associated with the susceptibility to vascular leakage in the BN rat strain, the retinal VEGF contents and vascular permeability were measured in F2 BN/SD rats in response to ischemia. The VEGF USF-binding site SNP was genotyped by primer extension method using genomic DNA from oxygen-treated F2 rats. The retinal vascular permeability showed no significant differences between the heterozygous BN/SD, homozygous BN, and SD alleles of VEGF USF SNP (Fig. 5A). The distributions of retinal VEGF contents between BN, SD, and BN/SD allele rats were 22–666 (n=28), 28–534 (n=17), and 25–601 (n=39) pg/µg protein respectively (Fig. 5B). There is no significant difference in retinal VEGF levels between the BN, SD, and BN/SD alleles of the VEGF SNP.
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Discussion |
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The molecular mechanisms for the different susceptibility to vascular leakage in response to ischemia and diabetes are not clear. Multiple genes, such as VEGF, PEDF, aldose reductase (AR), nitric oxide synthase (NOS2A and NOS3), protein kinase C-ß (PKC-ß), etc, are implicated in the pathogenesis of DR. Alterations of these growth factors and their receptors in diabetes have been identified in both experimental and clinical DR (Casey & Li 1997, Gerhardinger et al. 2001). VEGF is a potent mediator of vascular permeability and angiogenesis, and a potent mitogen with a unique specificity for endothelial cells in a variety of human pathological conditions. The increased VEGF levels are responsible for the retinal vascular leakage or retinal vascular hyperpermeability, and retinal NV. A number of clinical and animal studies have shown that VEGF plays a pivotal role in the development of DR (Chiarelli et al. 2000, Caldwell et al. 2003). The up-regulated expression of retinal VEGF and its receptors correlate to high retinal vascular permeability (RVP) in OIR and STZ-induced diabetic rats (Adamis et al. 1994, Aiello et al. 1994, Pierce et al. 1995). It has been known that VEGF expression is stimulated by hypoxia (Gao et al. 2002, Zhang et al. 2005), high glucose levels (Iglesias-de la Cruz et al. 2002, Omori et al. 2004), advanced glycosylation end products (Yamagishi et al. 2002, Yokoi et al. 2005), IGF-I (Hellstrom et al. 2001), and angiotensin II (Funatsu et al. 2002). Inhibition of VEGF and VEGF receptors has been shown to prevent retinal NV in diabetic and OIR animal models (Gilbert et al. 1998, Qaum et al. 2001). The present study demonstrated that OIR-BN rats have retinal VEGF levels more than ten times higher than those in OIR-SD rats. OIR F2 BN/SD strain rats have a continuous distribution of retinal VEGF levels, which is positively correlated to retinal vascular permeability. The F2 rats with extreme high retinal vascular permeability have retinal VEGF levels five times higher than those with extreme low retinal vascular permeability.
The susceptibility for vascular leakage may be a polygenic mode of inheritance. VEGF would be a strong candidate gene responsible for susceptibility to vascular leakage. It has been reported that the C(–634)G polymorphism in the 5'UTR of the VEGF gene is strongly associated with an increased risk of retinopathy (Awata et al. 2002). The VEGF –460C polymorphism-based transcriptional start site is associated with proliferative DR and a positive independent predictive factor for the development of proliferative DR (Ray et al. 2004). VEGF mutants at Cys51Ser and Cys60Ser have low potency in inducing proliferation in human umbilical vein endothelial cells (Leenders et al. 2001). We hypothesize that VEGF is a candidate gene responsible for the higher susceptibility to vascular leakage in OIR-BN rats. Two SNPs of the VEGF gene between BN and SD rat strains are found in the rat sequence database. One SNP (rs8148303, A/G) does not change peptide residue. Another VEGF SNP (rs8152485, A/G), located at 209 of the VEGF mRNA sequence from transcript initiation site leads to an amino acid change tyrosine (Tyr)/cysteine (Cys) (BN/SD) at 70 of the peptide sequence. However, our studies did not detect these SNP in our breeding BN and SD rat strains. No polymorphism of the VEGF mRNA was detected in the full-length retinal VEGF cDNAs from BN and SD rats.
In order to determine whether VEGF promoter polymorphisms result in elevated retinal VEGF in OIR-BN rats, we amplified and sequenced 2.3 kb DNA fragments of the VEGF promoter from the genomic DNAs of BN and SD rats. One SNP (G/T) was identified at –1234 in the promoter region of VEGF between BN and SD rat strains. The single nucleotide mutation in the VEGF promoter in BN rats results in a loss of the binding site for the USF in the VEGF promoter region. USF, known as major late transcription factor, consists of two related polypeptides of 43 kDa (USF1) and 44 kDa (USF2), and is expressed in every tissue. USF1 regulates several genes related to glucose and lipid metabolism (Casado et al. 1999). It has been reported that USF1 is associated with the familial combined hyperlipidemia and affects the complex lipid phenotype (Pajukanta et al. 2004, Coon et al. 2005). USF proteins induce human TGF-ß1 gene activation via the glucose-response element (Weigert et al. 2004). In order to determine whether the genetic variation of the USF transcription factor-binding site in the VEGF promoter region affects VEGF expression in OIR-BN rats, we performed genotyping of USF-binding site SNP allele and measured the retinal VEGF levels and vascular permeability in 97 F2 rats in response to ischemia. Our studies have shown that all of the three rats with the USF SNP allele have similar distribution of retinal VEGF levels and vascular permeability. These results suggest that the USF SNP per se does not affect VEGF expression and that VEGF promoter and mRNA are not responsible for the strain difference in susceptibility to vascular leakage in OIR-BN and OIR-SD rats. The other upstream and intronic transcript factors regulating the VEGF mRNA expression or mediators affecting VEGF protein stability may be associated with vascular leakage in BN rat strain.
In summary, OIR-BN rats have higher retinal VEGF levels and more severe retinal vascular leakage than OIR-SD rats. VEGF levels are positively correlated to vascular leakage in OIR F2 BN/SD rats. No SNP was found in the VEGF gene that is associated with different susceptibilities to vascular leakage. These observations suggest that the VEGF promoter and not mRNA is associated with increased retinal VEGF level and vascular permeability; instead, the yet to be identified upstream factors and mediators regulating VEGF expression are responsible for the genetic difference for the retinal vascular leakage. This study lays a solid ground for further elucidation of the genetic basis for the different susceptibilities to DR.
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Acknowledgements |
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References |
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Received in final form 6 January 2007
Accepted 17 January 2007
Made available online as an Accepted Preprint 24 January 2007
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, BN allele;
, SD allele;
, BN/SD allele. Horizontal lines represent the average values. The retinal vascular permeability of F2 rats in homozygous BN, homozygous SD, and heterozygous BN/SD alleles were 69.67±25.93, 56.68±22.71, and 53.25±24.21 respectively. There is no difference of retinal vascular permeability between BN, SD, and BN/SD alleles. (B) The distribution of retinal VEGF levels in F2 rats with homozygous BN, homozygous SD, and heterozygous BN/SD alleles of the VEGF USF SNP respectively. The retinal VEGF levels of F2 rats in the BN, SD, and BN/SD alleles were 242.18±167.88, 208.40±162.48, and 220.54±160.0 respectively. All of the three alleles of F2 rats have the similar distribution in retinal vascular permeability and VEGF contents.
P<0.001.
