The Classical Pink-Eyed Dilution Mutation Affects Angiogenic Responsiveness

Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that affect angiogenic responsiveness in several mouse models. In this study, we used F2 intercrosses between C57BL/6J and the 129 substrains 129P1/ReJ and 129P3/J, as well as the SJL/J strain, where we have identified new QTLs that affect angiogenic responsiveness. In the case of AngFq5, on chromosome 7, congenic animals were used to confirm the existence of this locus and subcongenic animals, combined with a haplotype-based mapping approach that identified the pink-eyed dilution mutation as a candidate polymorphism to explain AngFq5. The ability of mutations in the pink-eyed dilution gene to affect angiogenic response was demonstrated using the p-J allele at the same locus. Using this allele, we demonstrate that pink-eyed dilution mutations in Oca2 can affect both bFGF and VEGF-induced corneal angiogenesis

A Method for Developing Novel 3D Cornea-on-a-Chip Using Primary Murine Corneal Epithelial and Endothelial Cells

Microfluidic-based organ-on-a-chip assays with simultaneous coculture of multi-cell types have been widely utilized for basic research and drug development. Here we describe a novel method for a primary cell-based corneal microphysiological system which aims to recapitulate the basic functions of the in vivo cornea and to study topically applied ocular drug permeation. In this study, the protocols for isolating and cultivating primary corneal epithelial cells and endothelial cells from mouse inbred strain C57BL/6J were optimized, to allow for the development of a primary-cell based microfluidic 3D micro-engineered cornea. This tissue unit, by overcoming the limitations of 2D conventional cell culture, supports new investigations on cornea function and facilitates drug delivery testing

Angiogenic responses in a 3D micro-engineered environment of primary endothelial cells and pericytes

Abstract Angiogenesis plays a key role in the pathology of diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. Understanding the driving forces of endothelial cell migration and organization, as well as the time frame of these processes, can elucidate mechanisms of action of important pathological pathways. Herein, we have developed an organ-specific microfluidic platform recapitulating the in vivo angiogenic microenvironment by co-culturing mouse primary brain endothelial cells with brain pericytes in a three-dimensional (3D) collagen scaffold. As a proof of concept, we show that this model can be used for studying the angiogenic process and further comparing the angiogenic properties between two different common inbred mouse strains, C57BL/6J and 129S1/SvlmJ. We further show that the newly discovered angiogenesis-regulating gene Padi2 promotes angiogenesis through Dll4/Notch1 signaling by an on-chip mechanistic study. Analysis of the interplay between primary endothelial cells and pericytes in a 3D microfluidic environment assists in the elucidation of the angiogenic response

C57BL/6J×129P3/J F2 cross simple interval mapping.

<p>Whole genome simple interval map. Top: likelihood ratio statistic or likelihood of a region being linked to bFGF-induced corneal neovascularization. Dashed line indicates P = 0.05 as determined by bootstrapping. Bottom: predicted additive effect of a region on bFGF-induced corneal neovascularization.</p

Angiogenic response of Congenic Strains.

<p>Differences between parental (leftmost) and congenic strains are statistically significant (P<0.05) except when the bar for the congenic strain is colored white.</p

Mapping the region responsible for <i>AngFq5</i>.

<p>Top: Location of the congenic regions for the congenic strains used is plotted, along with boundary-defining markers for SJL congenics (brown). Blue indicates 129 congenics, with dark blue indicating those congenics that demonstrate a statistically significant difference in angiogenic response. Genes are indicated next by which DNA strand they are coded by. Finally regions of shared haplotype are indicated colored by whether the parental strains have disparate (black) or shared (grey) alleles at <i>AngFq5</i>. Below: Magnification of the region of interest defined by the R82 and R06 congenics. The number of genes, and genes bearing amino-acid altering polymorphisms (cSNPs), is indicated as are the locations of the pink-eyed dilution gene (<i>p</i>) and the tyrosinase gene (<i>c</i>). Scale is in Mbp on build 37.1 of mouse Chromosome 7.</p

Markers showing near-significant and significant association with bFGF-induced corneal neovascularization in a 77-animal C57BL/6J×129P1/ReJ F2 cross.

<p>Marker = marker tested; Chr. = chromosome the marker is on; % = fraction of the experimental variance attributable to genotype at the marker indicated under an additive model; P = likelihood that there is no relationship between the marker genotype and choroidal neovascularization area (by F test), when the data are fit to the simple linear regression model y = b0+b1×+e. The results give the estimates for b0, b1 and the F statistic for each marker. b0 is approximately the average area of C57BL/6J-allele-containing strains. b1 is an indication of the effect of substitution of the 129P1/ReJ allele at that marker. Asterisks indicate uncorrected P<0.05 (*), P<0.01 (**), or P<0.001 (***).</p