Regulation of Angiogenesis and Renin Expression in Rats

Medical College Of Wisconsin, Milwaukee WI

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Abstract

A myriad of physiological data confirms that renin is a critical component of the normal angiogenesis response[unreadable] seen in skeletal muscle with electrical stimulation. Using SS-13BN/Mcwi (SS-13BN) consomic rats and a series[unreadable] of congenic rats, we have demonstrated that restoration of a small region surrounding the renin gene confers[unreadable] normal renin levels and restores the normal angiogenic phenotype in the Dahl S (SS) rat. Despite this data, no[unreadable] difference has been observed in the coding region or the classically defined promoter region of the renin gene[unreadable] that would explain the differences in expression. Therefore it is the goal of this application to determine the[unreadable] sequence variants in the region, demonstrate which of these variants impact renin gene regulation in vitro, and[unreadable] using a transgenic approach, demonstrate that this candidate sequence variant eliminates normal renin[unreadable] regulation and the angiogenic phenotype in a subcongenic rat. This will be accomplished through a series of[unreadable] experiments comprising three specific aims. In specific Aim 1, we will begin with a congenic rat line SS.BN-[unreadable] (D13rat123-D13rat101)/Mcwi (referred to as line 9) in which a region of less than 4.5 Mbp surrounding the[unreadable] renin gene region has been introgressed from the BN (Brown Norway) genome onto the SS/JrHsdMcwi (SS)[unreadable] background. Using well-established techniques of marker-assisted selection to identify recombinants, we will[unreadable] reduce this candidate region to 1-2 Mbp. In specific Aim 2, we will identify candidate sequence variants within[unreadable] this reduced region by sequencing the SS and other closely related strains that do not share the antiangiogenic[unreadable] phenotype, including the Dahl salt resistant (SR), the Lyon Normotensive (LN), the Fawn Hooded[unreadable] Hypertensive (FHH) and the BN rats. Using the five strains, we have shown that we can reduce the number of[unreadable] potential causative strain-specific sequence variants within the candidate region to a manageable number,[unreadable] theoretically as low as 10, with a probability of having a false positive candidate mutation remaining below 1%.[unreadable] The candidate variants identified by sequencing and validated by a bioinformatics approach will be tested in vitro.[unreadable] This will be achieved by the use of a cell based system in which endothelial cells derived from the[unreadable] subcongenic line carrying the reduced region (Aim 1) will be transfected with SS Bacterial Artificial[unreadable] Chromosomes (BACs) and renin regulation will be assessed. We will use the surrogate phenotype of serum[unreadable] starvation-induced renin expression to assess the efficacy of the targeted mutation in modulating the renin[unreadable] phenotype. Specific Aim 3 will focus on the final identification and validation of the causative strain-specific[unreadable] sequence variant in this region. Our final experiment will be to use a transgenic rescue approach with a[unreadable] bacterial artificial chromosome transgene harboring the SS allele. Substitution of the allele identified in[unreadable] previous experiments will be performed on the background of the reduced subcongenic to test for confirmation[unreadable] of the loss of the angiogenic phenotype in vivo.