Experiment
Growth and maintenance of blood vessels are mediated by many factors. The current study used a novel method to follow angiogenesis in mice that reduces environmental variance by measuring endothelial cell growth from culture of isolated skin biopsies that varies depending on the genetic source of the tissue.
The study model was designed to uncover differences in the early steps of angiogenesis, including processes, such as endothelial cell sprouting and proliferation. Skin biopsies were isolated from postnatal animals (3-day-old mice in the current study) that were embedded in Matrigel and grown in a media that encouraged endothelial cell growth from tissue biopsies with the vessel-specific growth factor VEGF. After 7 days of culture, endothelial cells were quantified by fixing the ex vivo culture, staining with a DNA-specific dye, and counting the total number of endothelial cells that grew.
A (C57BL/6J x FVB/NJ) x FVB/NJ backcross was used to generate 174 N2 animals that were evaluated for vessel growth. Cultures from C57BL/6J animals exposed to 20 ng/ml VEGF grew an average of 1150 cells from 2 mM punch biopsies, where as FVB biopsies were more proliferative with an average of 1785 cells. Genotype and phenotype data were correlated by use of R/QTL with the use of the sex of individuals as an interactive covariate.
A novel QTL, reaching genome-wide significance of P<0.05 (based on 5000 permutations of the dataset), was detected on proximal mouse Chr. 8. QTL Scvg1 (subcutaneous vessel growth QTL 1) mapped to Chr 8, peaking at marker rs33166255 with a LOD score of 4.01. Scvg1 effects were independent of sex.
Two suggestive QTL were detected on Chrs 2 and 7. The Chr 2 QTL peaked at rs13476359 with a LOD score of 3.19 in the vicinity of Angvq2, a corneal angiogenesis QTL previously detected in the region. The Chr 7 QTL peaked at rs13479186 with a LOD score of 2.42 in the same region as Mmtg3, a QTL reported to modify tumor vessel growth in FVB mice.
A focus on differentially expresses genes that might be responsible for the variance attributed to Scvg1, in the 95% CI, revealed 2 members of the defensin cluster, Defb1 and Defb14, and 170016D06Rik, Cdc16, Efnb2, Ckap2, Vps36 and Ap3m2 [Supp Table]. Only Defb1, Ckap2 and Vps36 reproduced significant expression differences in independent triplicate skin biopsies from B6 and FVB animals.
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