Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice

Abstract

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF(-/-) and heterozygous CTGF(+/-) mice to establish involvement of CTGF in neovascularization. CTGF(-/-) mice die around birth. Therefore, embryonic CTGF(-/-), CTGF(+/-), and CTGF(+/+) bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF(+/-) and CTGF(+/+) mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.

Figure 1

Retinal flat mount of mouse eye with oxygen-induced retinopathy showing blood vessels after perfusion with fluorescein isothiocyanate (FITC) coupled to high-molecular-weight dextran. The retina was divided into three concentric zones: the inner zone around the optic disc (A), the middle zone (B), and the outer zone (C) for scoring of avascular areas on a central—peripheral axis. Vascular proliferations were quantified by counting blood vessel tufts and cluster formation (presumed extra-retinal neovascularization) in each of 12 equally sized sections of the retina (numbers). Bar = 1 μm.

Figure 2

Induction of choroidal neovascularization in mice by laser photocoagulation.

Figure 3

Estimation of choroidal neovascularization after induction by laser photocoagulation in hematoxylin-stained cryostat sections of the area containing the entire extent of the burn in mouse eyes. Neovascularization is determined by the ratio of the thickness of the lesion (B) to the adjacent intact pigmented choroidal layer (C).

Figure 4

Vascular outgrowth of metatarsals as stained immunohistochemically with anti-CD31 antibodies after being cultured in the absence (A, C, E) or presence (B, D, F) of vascular endothelial growth factor (VEGF). (A, B) connective tissue growth factor (CTGF)^+/+ metatarsals; (C, D) CTGF^+/− metatarsals; (E, F) CTGF^−/− metatarsals. Vascular outgrowth is enhanced by VEGF independently of the CTGF genotype. Bar = 1 μm.

Figure 5

Graphic representation of the differences in median area of vascular outgrowth (expressed as numbers of pixels × 1000) between the metatarsals cultured with and without VEGF for CTGF^+/+, CTGF^+/−, and CTGF^−/− mice. Data are expressed as median with range. The box indicates values from the 25th to the 75th quartile.

Figure 6

Median scores with range of vascular pathology in retinal whole mounts of CTGF^+/+ and CTGF^+/− mice in the oxygen-induced retinopathy model. There is no significant difference in neovascular response between CTGF^+/+ and CTGF^+/− mice.

Figure 7

Confocal images of FITC—dextran-perfused retinas showing the vasculature in a CTGF^+/+ (A) and a CTGF^+/− mouse (B). Bar = 80 μm.

Figure 8

Induction of choroidal neovascularization by laser in retina of CTGF^+/+ and CTGF^+/− mice. There is no difference between the two groups as determined by B/C ratio (A) and surface area of choroidal neovascularization, expressed as μm² × 1000 (B).