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(Investigative Ophthalmology and Visual Science. 2004;45:441-447.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0869
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Mouse Strain–Dependent Heterogeneity of Resting Limbal Vasculature

Candy K. Chan,1,2 Lucia N. Pham,2 Cynthia Chinn,2 Christine Spee,3 Stephen J. Ryan,2,3 Rosemary J. Akhurst,4 and David R. Hinton1,2,3

1From the Departments of Pathology, and 3Ophthalmology and 2The Arnold and Mabel Beckman Macular Research Center at the Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, California; and the 4Mt. Zion Cancer Research Institute, University of California San Francisco, San Francisco, California.


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Heterogeneity of the extent of angiogenesis induced by exogenous growth factors may be determined by genetic influences. Because angiogenesis is the formation of new vessels from preexisting ones, strain-related influences on naïve resting limbal vessel phenotype and gene expression were determined in mice having divergently low and high angiogenic responses.

METHODS. Resting limbal vessel surface area and density and extent of bFGF-induced corneal angiogenesis were determined in C57BL/6J, BALB/cJ, F1 intercross {equiv} C57BL/6J X 129S3/SvIM, and 129S3/SvIM mouse strains by quantitative three-dimensional reconstruction confocal microscopy. Strain-related influences on pro- and antiangiogenic gene expression in naïve cornea were determined by quantitative real-time RT-PCR.

RESULTS. The strain-dependent rank order of resting limbal vessel surface area and resting vessel density paralleled bFGF-induced neovascularization: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (P < 0.0006). Pigment epithelium–derived factor (PEDF) was increased more than 67-fold compared to Ang-2 in resting cornea of both C57BL/6J and 129S3/SvIM strains (P < 0.0001; P < 0.0001), suggesting a strongly antiangiogenic environment. The corneas of the C57BL/6J mice demonstrated 1.8-, 1.5-, and 1.7-fold increased mRNA levels for Flt-1, VEGF, and bFGF, respectively (P < 0.02; P < 0.04; P < 0.02); however, TSP-1 expression was increased 2.4-fold compared with 129S3/SvIM (P < 0.0004).

CONCLUSIONS. Strain-dependent differences in the resting limbal vessel surface area and density correlated with heterogeneity in the extent of bFGF-induced angiogenesis. Differences in pro- and antiangiogenic gene expression levels in resting cornea may influence vascular limbal phenotype during quiescence and may predict susceptibility to angiogenesis-dependent diseases.

Angiogenesis is involved in many physiologic events, such as wound healing and the female reproductive cycle and pathologic processes, including corneal neovascularization, proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), and cancer.1 2 Normally, resting vessels remain in a state of quiescence in which a tenuous balance exists between angiogenic stimulators and inhibitors. However, angiogenesis may occur when expression favors proangiogenic stimulators or reduces levels of antiangiogenic growth factors.3 Often, the extent of neovascularization is associated with severity in angiogenesis-dependent diseases.4 5 6

Genetic factors have been implicated in the progression of many angiogenesis-dependent diseases. In patients with diabetes, Mexican American populations have increased risk for PDR compared with non-Hispanic whites after controlling for duration and management of diabetes.7 Among those with AMD, vision loss due to choroidal neovascularization is more prevalent in white than in black populations, suggesting a genetic component.8 Experiments using inbred mouse strains in a corneal micropocket assay indicate that the heterogeneity in the extent of angiogenesis in response to exogenous growth factor may be determined by genetic factors.9 10

Angiogenesis is defined as the growth of new vessels from preexisting ones. Thus, genetic factors that determine strain-dependent differences in angiogenic vessels may also influence differences in the resting vasculature from which new vessels are derived. The normally avascular cornea provides an ideal environment to study strain-related influences in the resting limbal vessels and to correlate findings with strain-related differences after angiogenic stimuli. Normally confined to the circumferential periphery to maintain optical clarity, the blood supply to the cornea arises from the ciliary arteries that divide and terminate in the pericorneal plexus in the limbal area.1 When corneal neovascularization occurs, new vessels form from capillaries and venules of the limbal plexus.11 12 We evaluated the strain dependence of resting vessel surface area and resting vessel density in the normal limbus of naïve mice previously reported to have divergent responses to VEGF- and bFGF-induced angiogenesis.9 10 Strain-related influences on gene expression were then determined in naïve cornea from mouse strains with low (C57BL/6J) and high (129S3/SvIM) resting vessel surface area and density.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Experimental Animals
Weight- and age-matched (10–12 weeks) male mice (The Jackson Laboratory, Bar Harbor, ME) were used for all experiments. The following strains of mice were used: C57BL/6J (inbred, pigmented: black); BALB/cJ (inbred, albino); 129S3/SvIM inbred, pigmented: brown); F1 {equiv} C57BL/6J X 129S3/SvIM (F1 cross, pigmented: dark brown). All experiments were performed in accordance with the USC Animal Care and Use Committee and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Corneal Angiogenesis Assay
A murine corneal neovascularization assay was used to assess in vivo angiogenesis, as described previously.13 In this model, the normally avascular cornea is vascularized from the adjacent limbal vessels when stimulated by bFGF. Briefly, weight- and age-matched adult male mice were anesthetized with ketamine (160 mg/kg) and xylazine (8 mg/kg). A midline keratotomy was made with a breakable surgical blade (Electron Microscopy Sciences, Fort Washington, PA) and a corneal pocket dissected out toward the limbal vessels using an angled corneal knife (Surgical Specialties Corp., Reading, PA). A slow-release sucralfate-hydron polymer NCC (Hydro Med Sciences, Cranbury, NJ) pellet (0.4 x 0.4 mm) containing 75 ng bFGF or saline was implanted 1.0 mm from the limbus. A polymyxin B-bacitracin ointment (Akorn, Inc., Buffalo Grove, IL) was immediately applied to the eye after the procedure. Periodic corneal examinations and photography was performed using a slit lamp (Carl Zeiss Meditec, Oberkochen, Germany) with an attached 35-mm camera (Contax; Kyocera, Meerbusch, Germany). On postoperative day 7, mice were prepared for quantification of corneal neovascularization.

Confocal Microscopic Quantification of Integrated Intravascular Surface Area
Resting limbal vessels and bFGF-induced corneal neovascularization were quantified in the same manner by confocal microscopy. Mice (both nonsurgical and postprocedure day 7) were anesthetized and the femoral vein dissected and injected intravenously with 200 µg endothelium-specific, FITC-conjugated Griffonia simplicifolia lectin I (GSLI; Vector Laboratories, Burlingame, CA).14 After 30 minutes, the mice were killed and the eyes enucleated and fixed in 4% paraformaldehyde overnight at 4°C. The conjunctiva, iris, and ciliary body were dissected from the cornea. Radial incisions were made to facilitate flatmounting of the specimens. Confocal microscopy scanning (2.5x objective) was performed through the entire thickness of the vascularized cornea at 10-µm scanning plane intervals (Carl Zeiss Meditec). Integrated vascular surface area to determine implicit vascular volume was obtained by three-dimensional (3-D) reconstruction and analysis of planar confocal visual slices (LSM 510) using the microscope’s 3D software (3D for LSM510; Carl Zeiss Meditec). Units for implicit volume were designated "volsurfarea" (in square micrometers) by the microscope’s software and were reported as the mean ± SEM.

Limbal Vessel Density Quantification
To determine limbal vessel density in the resting, unstimulated vasculature, non–surgically altered corneas were evaluated by fluorescence microscopy after they were labeled with endothelium-specific, FITC-conjugated GSLI. For each cornea, the number of primary and secondary vessel branches was summed for three random fields of view (10x objective, 0.8-mm2 area). Values reported represent the mean number of vessel branches ± SEM.

Relative Quantitative Real-Time RT-PCR
Relative quantitative expression of mRNA in the normal naïve adult cornea was evaluated using real-time RT-PCR. Both corneas from each animal were pooled to isolate total RNA. RNA extraction reagent (TRIzol; Invitrogen Life Technologies, Carlsbad, CA) was used to isolate RNA, and the contaminating genomic DNA was removed with a kit (DNA-free; Ambion, Austin, TX). Reverse transcription was performed using 1 µg total RNA, oligo(dT)15 primer (Promega Corp., Madison, WI), and AMV reverse transcriptase (Promega). Real-time PCR reactions were performed in triplicate with a sequence detection system (GeneAmp 5700; Applied Biosystems, Foster City, CA). Each 25 {lambda} PCR reaction contained cDNA template, SYBR Green PCR Master Mix (Applied Biosystems), and 167 nM gene-specific primers. Reaction conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of denaturation at 95°C for 15 seconds with annealing and extension at 60°C for 1 minute. Ribosomal L-32 was used as a reference (housekeeping) gene.

Angiogenic factors studied were vascular endothelial growth factor (VEGF), VEGF receptors Flt-1 and Flk-1, and VEGF coreceptor neuropilin-1 (NPL); angiopoietin-1 (Ang-1) and -2 (Ang-2) and Ang receptor Tie-2; and basic fibroblast growth factor (bFGF). The antiangiogenic growth factors studied were pigment epithelium–derived factor (PEDF) and thrombospondin-1 (TSP-1). The following primer sets were designed on computer (Primer Express software; Applied Biosystems): L-32 5'-TGGTTTTCTTGTTGCTCCCATA-3' and 5'-GGGTGCGGAGAAGGTTCAA-3'; VEGF 5'-CATCTTCAAGCCGTCCTGTGT-3' and 5'-CTCCAG-GGCTTCATCGTTACA-3'; NRP 5'-CAGAGTTCCCGACATACGGTTT-3' and 5'-TCCCAGTGGCAGAATGTCTTG-3'; Flt-1 5'-CGGCTGTCCATGAAAGTGAA-3' and 5'- TTGCAGGCGAGCCATCTT-3'; Flk-1 5'-ACTGCAGTGATTGCCATGTTCT-3' and 5'- CCTTCATTGGCCCGCTTAA-3'; Ang-1 5'-CAGCAGCAAGTGGTTATGTCATG-3' and 5'-TTGACCTCAGAAGGCTCCAAA-3'; Ang-2 5'-GACTTCCAGAGGACGTGGAAAG-3' and 5'-CTCATTGCCCAGCCAGTACTC-3'; Tie2 5'-CATCCCTCACCTG-CATTGC-3' and 5'-GCTTCAAAGTCCCTTCCTATGGT-3'; bFGF 5'-CCCACCAGGCCACTTCAA-3' and 5'-GATGGATGCGCAGGAAGAA-3'; PEDF 5'-CACCCGACTTCAGCAAGATTACT-3' and 5'-TCGAAAGCAGCCCTGTGTT-3'; and TSP-1 5'-ACTACGCTGGCTTTGTATTC-3' and 5'-GGACT-GGGTGACTTGTTTCC-3'. Detection of product formation was set in the center of the linear portion of PCR amplification. The cycle at which each reaction reached the set threshold (CT) was determined. Amplification efficiencies between primer pairs for genes of interest compared with that for the L-32 reference gene was evaluated by amplifying a dilution range of cDNA template. A plot of {Delta}CT on the y-axis and log (cDNA) on the abscissa generated a linear curve with slope < 0.01, verifying comparable primer pair amplification efficiencies between L-32 and genes of interest. To assess relative abundance of genes expressed in the cornea, comparable primer pair efficiencies were also determined for all permutations between genes of interest in comparison with each other. Relative multiples of change in mRNA expression were determined by calculation of {Delta}{Delta}CT.15 Results are reported as mean difference in relative multiples of change in mRNA expression ± SEM.

Statistical Analysis
All statistical analyses were performed using SAS (SAS Institute, Cary, NC). Values reported in figures represent mean ± SEM. Each eye was treated as an independent event for statistical analysis of corneal vessels. The Shapiro-Wilk statistic was used to test for normality of the measurements within each group. If one or more of the groups were found to follow a non-normal distribution, nonparametric statistics were used. For nonparametric distributions, The Kruskal-Wallis test was used to determine overall differences between groups, and Wilcoxon rank sum was used to test for differences between two groups. For normal distributions, parametric tests used were analysis of variance and paired t-tests. For gene expression data, comparisons between groups were made using independent t-tests for equal or unequal variances. Accepted level of significance for all tests was {alpha} = 0.05.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Strain-Dependency of bFGF-Induced Corneal Neovascularization
Mouse strain effects on extent of bFGF-induced angiogenesis was confirmed by 3-D reconstruction confocal microscopy. Corneal angiogenesis was induced using sucralfate-hydron polymer pellets containing 75 ng bFGF. These pellets were implanted into corneal pockets of inbred C57BL/6J (n = 6), BALB/cJ (n = 8), and 129S3/SvIM (n = 8) mice and F1 cross-bred ({equiv}C57BL/6J X 129S3/SvIM; n = 8) mice. Inbred mouse strains were chosen to represent previously reported similarly divergent responders to both bFGF and VEGF.9 10 A dose of 75 ng bFGF was used because data produced in our laboratory (not shown) and those reported by others showed a maximum neovascular response in the C57BL/6J strain (a low angiogenic responder) at this dose.9 On day 7 after bFGF pellet implantation, strain-dependent induction of angiogenesis from the limbus across a previously avascular cornea toward the bFGF pellet was assessed (Fig. 1) . All eyes implanted with bFGF pellets demonstrated a neovascular response. For all eyes implanted with saline pellets, no angiogenic response was observed (data not shown). Among animals evaluated, the most exuberant neovascular response was observed in the 129S3/SvIM strain and the least extensive in C57BL/6J (Fig. 1A) mice. Intermediate responses were seen in BALB/cJ and F1 cross-bred animals. Vessels induced in the 129S3/SvIM strain were longer and more dense and formed a more extensive network across the cornea that those in C57BL/6J mice. Neovascularization in the C57BL/6J and F1 genetic backgrounds was decreased 1.7- and 1.4-fold, respectively, compared with the 129S3/SvIM strain (P < 0.01; P < 0.009); BALB/cJ was increased 1.4-fold compared with C57BL/6J (P < 0.02). Overall, bFGF-induced angiogenesis resulted in the following strain-dependent rank order: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (Kruskal-Wallis, P < 0.0002).



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  		FIGURE 1. Strain-dependent angiogenesis induced by bFGF. (A) Induction of angiogenesis (day 7) using a mouse corneal micropocket assay with 75 ng bFGF hydron polymer pellets in C57BL/6J (n = 6), BALB/cJ (n = 8), 129S3/SvIM (n = 8), and F1 {equiv} C57BL/6J X 129S3/SvIM (n = 8) strains. (B) Single representative planar confocal microscopy images of neovascularization visualized by endothelium-specific FITC-GSLI for each strain. (C) Quantification of angiogenesis by 3-D integration of confocal microscopy images. Neovascularization was increased in the 129S3/SvIM strain by 1.7-, 1.2-, and 1.4-fold, respectively, compared with C57BL/6J, BALB/cJ, and F1 mice. (*P < 0.01, {dagger}P < 0.009 compared with 129S3/SvIM; §P < 0.02 compared with C57BL/6J) Kruskal-Wallis analysis resulted in the following strain-dependent rank order: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (P < 0.0002).

 
Resting Limbal Vascularity
Because limbal vessels are the source of new vessel growth after bFGF pellet implantation in the corneal angiogenesis assay, strain dependence of resting limbal vessels in nonstimulated, non–surgically manipulated mice was evaluated in detail (Fig. 2) . More prominent vessels with increased vessel branches were observed by slit lamp in 129S3/SvIM than in C57BL/6J mice (Fig. 2A) . No significant differences between sizes of eyes were found between these inbred strains. Observations under confocal microscopy revealed an increased limbal vascular network in 129S3/SvIM mice compared with all other animals evaluated (Fig. 2B) . Complete labeling of the limbal vasculature in all animals was confirmed under high magnification (10x objective) before quantification. Resting vascularity was increased in 129S3/SvIM mice 11.7-, 4.1-, and 2.7-fold, respectively, over that in the C57BL/6J, BALB/cJ, and F1 strains (P < 0.002; P < 0.04; P < 0.05; Fig. 2C ). Rank order of resting limbal vascularity determined using Kruskal-Wallis analysis revealed a similar strain-dependent rank order as that found for angiogenic vessels: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (P < 0.0006). Thus, the amount of vessels present before angiogenic stimulation may influence the extent of angiogenesis observed after stimulation with bFGF.



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  		FIGURE 2. Resting limbal vascularity was strain dependent. (A) Slit lamp observation of resting limbal vessels in naïve C57BL/6J and 129S3/SvIM strains. (B) Single representative planar confocal images of the innate, resting limbal vasculature using endothelium-specific FITC-GSLI. Each image is only weakly fluorescent, but multiple images at 10-µm levels can be accurately quantitated to give an implicit volume using 3-D reconstruction software. (C) Quantification of innate limbal vascularity by 3-D reconstruction confocal microscopy for C57BL/6J (n = 10), BALB/cJ (n = 8), 129S3/SvIM (n = 13), and F1 {equiv} C57BL/6J X 129S3/SvIM (n = 10). Resting vascularity was increased in 129S3/SvIM mice 11.7-, 4.1-, and 2.7-fold, respectively, compared with C57BL/6J, BALB/cJ, and F1 animals (*P < 0.002, {dagger}P < 0.04, {ddagger}P < 0.05 compared with 129S3/SvIM; §P < 0.02 compared with C57BL/6J). Resting vessels demonstrated a strain-dependent rank order that paralleled that of bFGF-induced angiogenesis: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (Kruskal-Wallis, P < 0.0006).

 
Normalization of Resting Vessels
To determine whether the strain dependence observed in resting vessel surface area can account entirely for strain-dependent differences in bFGF-induced angiogenesis, each strain was normalized to its corresponding resting vessel surface area (Table 1) . After normalization of resting vessel surface area, angiogenic differences among the four mouse strains remained statistically significant (probabilities calculated by Wilcoxon rank sum analysis). However, C57BL/6J, the strain with the least total surface area of vessels after bFGF stimulation, showed an angiogenic-to-resting vessel surface area ratio of 36.5 compared with 129S3/SvIM with an angiogenic-to-resting vessel surface area ratio of 5.5 (P < 0.01). BALB/cJ and F1 mice demonstrated angiogenic-to-resting vessel surface area ratios of 18.5 and 10.7, respectively, compared with 129S3/SvIM (P < 0.005; P < 0.005). In fact, the rank order for the increase in angiogenic-to-resting vessel surface area becomes: C57BL/6J > BALB/cJ, F1 > 129S3/SvIM (P < 0.0001). These results suggest that differences in the amount of resting vessels before stimulation cannot account entirely for strain-related differences in angiogenic vessels. More important, decreased levels of resting vessels may be more responsive to angiogenic stimuli and produce a greater ratio of new vessels compared with the number of preexisting vessels from which they are derived.


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  		TABLE 1. Resting Vessel Normalization of bFGF-Induced Angiogenesis

 
Resting Limbal Vessel Density
To characterize further the strain-related influences on the phenotype of resting vessels, vessel density was evaluated in C57BL/6J (n = 10), BALB/cJ (n = 8), F1 (n = 10), and 129S3/SvIM (n = 13) mice (Fig. 3) . Increased density of limbal vessel arcades were observed in the 129S3/SvIM strain compared with all other strains evaluated, whereas C57BL/6J mice demonstrated the most sparsely distributed vessel arcades (Fig. 3A) . The density of vessel arcades in BALB/cJ and F1 mice showed intermediate phenotypes between those of 129S3/SvIM and C57BL/6J mice. Quantification of vessel density was determined by summing the number of primary and secondary vessel branch points for three random fields of view (0.8 mm2). The number of primary vessel branches was increased in 129S3/SvIM mice 1.9-, 1.4-, and 1.2-fold, respectively, over the number in C57BL/6J, BALB/cJ, and F1 mice (P < 0.0001, P < 0.0001, P < 0.04; Fig. 3B ). The number of secondary vessel branches was similarly increased (1.8-, 1.5-, and 1.3-fold) in 129S3/SvIM animals compared with C57BL/6J, BALB/cJ, and F1, respectively (P < 0.0001, P < 0.0001, P < 0.003; Fig. 3C ). The number of both primary and secondary vessel branches demonstrated a strain-dependent rank order similar to that found for both resting and angiogenic vessels. These results suggest that vessel branching/density contribute to the strain dependence of resting vascularity.



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  		FIGURE 3. Resting limbic vessel density was strain dependent. (A) Single representative planar images of limbal vessels for C57BL/6J (n = 10), BALB/cJ (n = 8), 129S3/SvIM (n = 8), and F1 {equiv} C57BL/6J X 129S3/SvIM (n = 9). (B) The number of primary vessel branches were increased in 129S3/SvIM mice 1.9-, 1.4-, and 1.2-fold, respectively, compared with C57BL/6J, BALB/cJ, and F1 mice (*P < 0.04, {dagger}P < 0.0001). (C) Secondary vessel branches were increased in 129S3/SvIM animals 1.8-, 1.5-, and 1.3-fold, respectively, compared with C57BL/6J, BALB/cJ, and F1 ({dagger}P < 0.0001, {ddagger}P < 0.003).

 
Expression of Pro- and Antiangiogenic Factors in Resting Vessels
Strain-related influences on the expression of pro- and antiangiogenic genes in the naïve cornea were assessed in C57BL/6J (n = 9) and 129S3/SvIM (n = 10) strains (Fig. 4) . Comparable efficiencies between RT-PCR primer pair sets were determined for all permutations between genes of interest to validate intergene comparisons. Gene expression was determined relative to Ang-2, a gene that is expected to be expressed at low levels in the resting vessels, and upregulated early in the angiogenic response.



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  		FIGURE 4. Expression of pro- and antiangiogenic genes in resting vessels is strain dependent. Gene expression from corneas (both corneas pooled from each animal) was determined in C57BL/6J ({blacksquare}, n = 9 mice) and 129S3/SvIM ({square}, n = 10 mice) inbred strains using quantitative real-time PCR. Proangiogenic genes in both VEGF and angiopoietin systems were expressed at lower levels than were angiogenic inhibitors PEDF and TSP-1. No strain-related differences were observed in expression levels in the angiopoietin system. NRP was increased more than threefold compared with Ang-2 in both inbred strains (*P < 0.0001). Flk-1 expression was similar to that of Ang-2. No strain-related differences were seen in either NRP or Flk-1 expression. VEGF and bFGF expression were increased 1.5- and 1.7-fold, respectively, in C57BL/6J compared with 129S3/SvIM mice ({dagger}P < 0.04; {ddagger}P < 0.02). VEGF was expressed at levels similar to those of Ang-2 in both strains. bFGF decreased more than 10- and 18-fold in C57BL/6J (*P < 0.0001) and 129S3/SvIM, respectively (*P < 0.0001), compared with Ang-2. Flt-1 mRNA levels were decreased 2.4- and 4.6-fold in C57BL/6J (*P < 0.0001) and 129S3/SvIM (*P < 0.0001), respectively, compared with Ang-2. In addition, flt-1 mRNA levels were increased 1.8-fold in C57BL/6J compared with 129S3/SvIM mice ({ddagger}P < 0.02). PEDF expression was increased more than 67-fold compared with Ang-2 in both inbred strains (*P < 0.0001). Also highly expressed in both strains, TSP-1 was increased 15.8- and 6.6-fold in C57BL/6J and 129S3/SvIM strains, respectively, compared with Ang-2 (*P < 0.0001; *P < 0.0001). In addition, TSP-1 mRNA levels were increased 2.4-fold in C57BL/6J compared with 129S3/SvIM animals (§P < 0.0004).

 
A striking result was the relatively high levels of expression of antiangiogenic, compared with proangiogenic, genes in both strains. PEDF expression was increased more than 67-fold compared with Ang-2 in both C57BL/6J and 129S3/SvIM strains (P < 0.0001; P < 0.0001), although no strain-related differences were seen. Similarly, TSP-1 was increased 15.8- and 6.6-fold in C57BL/6J and 129S3/SvIM strains, respectively, compared with Ang-2 (P < 0.0001; P < 0.0001). When strains were compared, TSP-1 mRNA levels were increased 2.4-fold in C57BL/6J (P < 0.0004), the less vascular strain, compared with that in 129S3/SvIM.

In contrast, the proangiogenic genes were expressed at relatively lower levels. Both inbred strains demonstrated low expression of Ang-1 and -2 and their receptor Tie2; however, no strain-related differences were seen. When members of this group were compared, Ang-1 and Tie2 decreased more than 5- and 27-fold, respectively, compared with Ang-2 in both inbred strains (P < 0.0001; P < 0.0001). Similarly, bFGF was expressed at extremely low levels in both C57BL/6J and 129S3/SvIM; however, when compared, expression was increased 1.7-fold in C57BL/6J compared with 129S3/SvIM (P < 0.02). VEGF was expressed in resting limbal vessels at levels similar to those in Ang-2; however, VEGF was increased 1.5-fold in C57BL/6J compared with 129S3/SvIM (P < 0.04). The VEGF receptor Flt-1 was also increased 1.8-fold in C57BL/6J compared with 129S3/SvIM (P < 0.02). No strain-related differences in gene expression were observed in NRP or Flk-1.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In this study we provide evidence that genetic background influences the extent of the resting vasculature in the naïve corneal limbus. The summed vascular surface area of resting limbal vessels in naïve cornea specimens demonstrated the strain-dependent rank order 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (P < 0.0006). Vessel density, determined from both primary and secondary vessel branching demonstrated a strain-dependence parallel to that of the extent of resting vessels. Corneal tissue thickness revealed no significant differences among mouse strains (data not shown), suggesting that an increased need for tissue perfusion alone cannot be implicated. In addition, observations of the primary feeding limbal vessel did not show remarkable differences in vessel caliber among inbred strains. This is not surprising because increases in vessel density increase total vascular surface area more efficiently than increases in either vessel caliber or vessel length. Thus, strain-related differences in vessel density may contribute to strain-related differences in the extent of resting vessels.

We then demonstrated that the strain-related rank order for summed vascular surface area of resting limbal vessels (129S3/SvIM > BALB/cJ, F1 > C57BL/6J; P < 0.0002) was identical with the strain-related rank order found for bFGF- or VEGF-induced corneal neovascularization reported herein for bFGF and previously for both growth factors.9 10 These results suggest that the heterogeneity in the extent of resting vessels is associated with, and may potentially predict, the heterogeneity of the extent of angiogenic vessels.

An interesting finding was that C57BL/6J (36.5) had a higher ratio of angiogenic-to-resting vessel surface area than did 129S3/SvIM (5.5; P < 0.01). However, the total vessel surface area after stimulation with bFGF remained lower than that in the 129S3/SvIM strain before normalization for resting vessel surface area. This suggests that the amount of preexisting resting vessels before angiogenic stimuli may be a factor that limits new vessel formation but remains an important contribution to the extent of angiogenesis produced.

The low variation in phenotype for resting vessels and vessel density observed in the F1 {equiv} C57BL/6J X 129S3/SvIM intercross suggests that genetic influences on the extent of resting vessels (as with other complex physiologic and pathologic processes) likely follow polygenic inheritance patterns.10 Characterization of gene expression in naïve corneas revealed a prominent expression of antiangiogenic genes. Whereas no strain-related differences were observed in PEDF expression in naïve cornea specimens, mRNA levels were increased more than 67-fold compared with Ang-2 in both C57BL/6J and 129S3/SvIM mice (P < 0.0001; P < 0.0001). In addition, TSP-1 expression was increased more than 15.8- and 6.6-fold in C57BL/6J and 129S3/SvIM compared with Ang-2 (P < 0.0001). High expression of angiogenic inhibitors is consistent with the primarily avascular phenotype in the cornea. Moreover, TSP-1 mRNA levels were increased 2.4-fold in C57BL/6J compared with 129S3/SvIM mice (P < 0.0004). Resting vessels in the 129S3/SvIM strain demonstrated decreased levels of TSP-1 expression compared with C57BL/6J, consistent with observations that TSP-1 null mice showed increased vessel densities in both the dermis and pancreatic islets.16 Thus, the decreased expression of TSP-1 in 129S3/SvIM mice may explain their higher resting vascular densities and the greater extent of angiogenesis that follows after exogenous proangiogenic growth factor stimulation. Clinical observations have reported that decreased levels of angiogenic inhibitors in aqueous humor of diabetic patients is predictive of progression of diabetic retinopathy.17

Proangiogenic growth factors were expressed at much lower levels in both strains than were PEDF or TSP-1. Ang-1 and -2 and their receptor Tie-2 were all expressed at low levels and no strain-related differences were seen. The relative level of Ang-1 (5-fold; P < 0.0001) and Tie2 (>27-fold; P < 0.0001) was decreased compared with Ang-2 in both strains, consistent with similar relative expression levels reported in normal prostate.18 The proangiogenic factor bFGF, also expressed at very low levels, increased (1.5-fold, P < 0.04) in resting vessels of C57BL/6J compared with 129S3/SvIM mice. In addition, expression of both VEGF and its receptor Flt-1 were increased in C57BL/6J compared with 129S3/SvIM mice (P < 0.02), whereas no differences were seen in expression of Flk-1 or neuropilin. The relatively increased but low expression of several proangiogenic growth factors in C57BL/6J resting vessels may explain why this less-vascular strain showed a higher change in neovascular area after angiogenic stimulation (although it never achieved the extent of angiogenesis found in 129S3/SvIM mice). During angiogenesis, bFGF-induced upregulation of Flk-1 protein, and mRNA expression has been reported.19 After angiogenic stimuli, already elevated levels of bFGF and VEGF in resting vessels of C57BL/6J mice may potentiate synergistic effects between bFGF and VEGF signaling, resulting in an increased ratio of angiogenic-to-resting vessel surface area formation compared with 129S3/SvIM.

TSP-1 expression is found in corneal endothelium, whereas PEDF is expressed in both normal corneal epithelium and endothelium.20 21 The relative abundance of angiogenic inhibitors compared with proangiogenic factors is consistent with the fact that the cornea is largely avascular. Very high levels of PEDF expression compared with angiogenic stimulators (>67-fold) in the normal cornea may explain the permissive effects on angiogenesis with inhibition of PEDF in the cornea.22 Because TSP-1 levels were also highly increased compared with those of all angiogenic stimulators evaluated in the naïve cornea (although not as elevated as those of PEDF levels), inhibition of TSP-1 expression may also provide a permissive environment for angiogenesis.

Identification of single nucleotide polymorphisms (SNPs) which correlate with functional differences in gene expression and or vessel phenotype may provide valuable insight toward predicting susceptibility to a spectrum of angiogenesis-dependent diseases. Studies have reported SNPs in PEDF and TSP-1 genes that have been implicated in increased risk for ocular and coronary disease.23 24 Among healthy individuals, differences in VEGF mRNA expression and protein levels have been associated with SNPs in the 3' untranslated region of the VEGF gene.25 26

This study presents evidence that genetic influences may determine both phenotype of resting vessels and molecular equilibrium between angiogenic stimulators and inhibitors in their environment that could provide a blueprint for prognostic and preventive therapies toward angiogenesis-dependent diseases.


   Acknowledgements
 
The authors thank Laurie Labree, statistical consultant for our National Eye Institute-supported Biostatistics Core, for computational advice and statistical analyses; Fernando Gallardo and Lawrence Rife for technical assistance with the animal experiments; and Ernesto Barron and Anthony Rodriguez for technical assistance with confocal microscopy.


   Footnotes
 
Supported by the Arnold and Mabel Beckman Foundation (DRH); National Eye Institute Grants EY01545 (DRH, SJR) and EY03040 (DRH, SJR); National Institute of General Medical Science Grant GM60514-01 (RJA); the American Heart Association (RJA); the March of Dimes (RJA), and Research to Prevent Blindness (Doheny Eye Institute).

Submitted for publication August 11, 2003; revised October 7, 2003; accepted October 17, 2003.

Disclosure: C.K. Chan, None; L.N. Pham, None; C. Chinn, None; C. Spee, None; S.J. Ryan, None; R.J. Akhurst, None; D.R. Hinton, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: David R. Hinton, Departments of Pathology and Ophthalmology, University of Southern California, Keck School of Medicine, 2011 Zonal Avenue, HMR 209, Los Angeles, CA 90033; dhinton{at}usc.edu.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Chang JH, Gabison EE, Kato T, Azar DT. Corneal neovascularization. Curr Opin Ophthalmol. 2001;12:242–249.[CrossRef][Medline][Order article via Infotrieve]
  2. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653–660.[CrossRef][ISI][Medline][Order article via Infotrieve]
  3. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27–31.[CrossRef][ISI][Medline][Order article via Infotrieve]
  4. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1–8.[Abstract]
  5. Bressler SB, Maguire MG, Bressler NM, Fine SL. The Macular Photocoagulation Study Group: relationship of drusen and abnormalities of the retinal pigment epithelium to the prognosis of neovascular macular degeneration. Arch Ophthalmol. 1990;108:1442–1447.[Abstract]
  6. Epstein RJ, Stulting RD, Hendricks RL, Harris DM. Corneal neovascularization: pathogenesis and inhibition. Cornea. 1987;6:250–257.[ISI][Medline][Order article via Infotrieve]
  7. Harris MI, Klein R, Cowie CC, Rowland M, Byrd-Holt DD. Is the risk of diabetic retinopathy greater in non-Hispanic blacks and Mexican Americans than in non-Hispanic whites with type 2 diabetes?. A U.S. population study. Diabetes Care. 1998;21:1230–1235.[Abstract]
  8. Sommer A, Tielsch JM, Katz J, et al. Racial differences in the cause-specific prevalence of blindness in east Baltimore. N Engl J Med. 1991;325:1412–1417.[Abstract]
  9. Rohan RM, Fernandez A, Udagawa T, Yuan J, D’Amato RJ. Genetic heterogeneity of angiogenesis in mice. FASEB J. 2000;14:871–876.[Abstract/Free Full Text]
  10. Rogers MS, Rohan RM, Birsner AE, D’Amato RJ. Genetic loci that control vascular endothelial growth factor-induced angiogenesis. FASEB J. .Express article. 10.1096/Fj.03-0246Fje. Published on September 4, 2003.
  11. Burger PC, Chandler DB, Klintworth GK. Experimental corneal neovascularization: biomicroscopic, angiographic, and morphologic correlation. Cornea. 1985;4:35–41.[Medline][Order article via Infotrieve]
  12. Yaylali V, Ohta T, Kaufman SC, Maitchouk DY, Beuerman RW. In vivo confocal imaging of corneal neovascularization. Cornea. 1998;17:646–653.[CrossRef][ISI][Medline][Order article via Infotrieve]
  13. Kenyon BM, Voest EE, Chen CC, Flynn E, Folkman J, D’Amato RJ. A model of angiogenesis in the mouse cornea. Invest Ophthalmol Vis Sci. 1996;37:1625–1632.[Abstract/Free Full Text]
  14. Asahara T, Chen D, Takahashi T, et al. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2 modulate VEGF-induced postnatal neovascularization. Circ Res. 1998;83:233–240.[Abstract/Free Full Text]
  15. Fink L, Seeger W, Ermert L, et al. Real-time quantitative RT-PCR after laser-assisted cell picking. Nat Med. 1998;4:1329–1333.[CrossRef][ISI][Medline][Order article via Infotrieve]
  16. Crawford SE, Stellmach V, Murphy-Ullrich JE, et al. Thrombospondin-1 is a major activator of TGF-beta1 in vivo. Cell. 1998;93:1159–1170.[CrossRef][ISI][Medline][Order article via Infotrieve]
  17. Boehm BO, Lang G, Volpert O, et al. Low content of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor predicts progression of diabetic retinopathy. Diabetologia. 2003;46:394–400.[ISI][Medline][Order article via Infotrieve]
  18. Shih SC, Robinson GS, Perruzzi CA, et al. Molecular profiling of angiogenesis markers. Am J Pathol. 2002;161:35–41.[Abstract/Free Full Text]
  19. Hata Y, Rook SL, Aiello LP. Basic fibroblast growth factor induces expression of VEGF receptor KDR through a protein kinase C and p44/p42 mitogen-activated protein kinase-dependent pathway. Diabetes. 1999;48:1145–1155.[Abstract]
  20. Adams JC. Thrombospondins: multifunctional regulators of cell interactions. Annu Rev Cell Dev Biol. 2001;17:25–51.[CrossRef][ISI][Medline][Order article via Infotrieve]
  21. Ogata N, Wada M, Otsuji T, Jo N, Tombran-Tink J, Matsumura M. Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization. Invest Ophthalmol Vis Sci. 2002;43:1168–1175.[Abstract/Free Full Text]
  22. Dawson DW, Volpert OV, Gillis P, et al. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science. 1999;285:245–248.[Abstract/Free Full Text]
  23. Koenekoop R, Pina AL, Loyer M, et al. Four polymorphic variations in the PEDF gene identified during the mutation screening of patients with Leber congenital amaurosis. Mol Vis. 1999;5:10.[Medline][Order article via Infotrieve]
  24. Topol EJ, McCarthy J, Gabriel S, et al. Single nucleotide polymorphisms in multiple novel thrombospondin genes may be associated with familial premature myocardial infarction. Circulation. 2001;104:2641–2644.[Abstract/Free Full Text]
  25. Renner W, Kotschan S, Hoffmann C, Obermayer-Pietsch B, Pilger E. A common 936 C/T mutation in the gene for vascular endothelial growth factor is associated with vascular endothelial growth factor plasma levels. J Vasc Res. 2000;37:443–448.[CrossRef][ISI][Medline][Order article via Infotrieve]
  26. Watson CJ, Webb NJ, Bottomley MJ, Brenchley PE. Identification of polymorphisms within the vascular endothelial growth factor (VEGF) gene: correlation with variation in VEGF protein production. Cytokine. 2000;12:1232–1235.[CrossRef][ISI][Medline][Order article via Infotrieve]



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