Generational analysis reveals that TGF-beta 1 inhibits the rate of angiogenesis in vivo by selective decrease in the number of new vessels

Quantitative analysis of vascular generational branching demonstrated that transforming growth factor-beta 1 (TGF-beta 1), a multifunctional cytokine and angiogenic regulator, strongly inhibited angiogenesis in the arterial t ree of the developing quail chorioallantoic membrane (CAM) by inhibition of the normal increase in the number of new, small vessels. The cytokine was applied uniformly in solution at embryonic day 7 (E7) to the CAMs of quail embryos cultured in petri dishes. After 24 h the rate of arterial growth wa s inhibited by as much as 105% as a function of increasing TGF-beta 1 conce ntration. Inhibition of the rate of angiogenesis in the arterial tree by TG F-beta 1 relative to controls was measured in digital images by three well- correlated, computerized methods. The first computerized method, direct mea surement by the computer code VESGEN of vascular morphological parameters a ccording to branching generations G(1) through G(greater than or equal to 5 ), revealed that TGF-beta 1 selectively inhibited the increase in the numbe r density of small vessels, N-v greater than or equal to 5 (382 +/- 85 cm(- 2) for specimens treated with 1 mu g TGF-beta 1/CAM for 24 h, compared to 5 83 +/- 99 cm(-2) for controls), but did not significantly affect other para meters such as average vessel length or vessel diameter. The second and thi rd methods, the fractal dimension (D-f) and grid intersection (rho(v)), are statistical descriptors of spatial pattern and density. According to D-f a nd rho(v), arterial density increased in control specimens from 1.382 +/- 0 .007 and 662 +/- 52 cm(-2) at E7 (0 h) to 1.439 +/- 0.013 and 884 +/- 55 cm (-2) at E8 (24 h), compared to 1.379 +/- 0.039 and 650 +/- 111 cm(-2) for s pecimens treated with 1 mu g TGF-beta 1/CAM for 24 h. TGF-beta 1 therefore regulates vascular pattern and the rate of angiogenesis in a unique "finger print" manner, as do other major angiogenic regulators that include VEGF, F GF-2 (bFGF), and angiostatin. TGF-beta 1 did not stimulate angiogenesis sig nificantly at low cytokine concentrations, which suggests that this quail C AM model of angiogenesis is not associated with an inflammatory response. ( C) 2000 Academic Press.