Angiogenesis, the formation of new blood vessels from existing vasculature, is a complex process that is essential for normal embryonic development. Current models for experimental evaluation of angiogenesis often use tissue from large vessels like the aorta and umbilical vein, which are phenotypically distinct from microvasculature. We demonstrate that the utilization of skin to measure microvascular angiogenesis in embryonic and adult tissues is an efficient way to quantify microvasculature angiogenesis. We validate this approach and demonstrate its added value by showing significant differences in angiogenesis in monogenic and polygenic mouse models. We discovered that the pattern of angiogenic response among inbred mouse strains in this ex vivo assay differ from the strain distributions of previous in vivo angiogenesis assays. The difference between the ex vivo and in vivo assays may be related to systemic factors present in whole animals. Expression analysis of cultured skin biopsies from strains of mice with opposing angiogenic response were performed to identify pathways that contribute to differential angiogenic response. Increased expression of negative regulators of angiogenesis in C57Bl/6J mice was associated with lower growth rates.

The Kidd locus phenotype Jk(a-b-) was detected in 0.9 percent of Polynesians living in New Zealand. Over a period of 13 years, nine examples of anti-Jk3 were detected, one of which caused a delayed hemolytic transfusion reaction. Other examples resulted in mild hemolytic disease of the newborn. The anti-Jk3 reacted as an inseparable antibody, confirmed that inheritance of the Jk(a-b-) phenotype was best explained by the presence of a silent Jk allele.

Epilepsy has many causes and comorbidities affecting as many as 4% of people in their lifetime. Both idiopathic and symptomatic epilepsies are highly heritable, but genetic factors are difficult to characterize among humans due to complex disease etiologies. Rodent genetic studies have been critical to the discovery of seizure susceptibility loci, including Kcnj10 mutations identified in both mouse and human cohorts. However, genetic analyses of epilepsy phenotypes in mice to date have been carried out as acute studies in seizure-naive animals or in Mendelian models of epilepsy, while humans with epilepsy have a history of recurrent seizures that also modify brain physiology. We have applied a repeated seizure model to a genetic reference population, following seizure susceptibility over a 36-d period. Initial differences in generalized seizure threshold among the Hybrid Mouse Diversity Panel (HMDP) were associated with a well-characterized seizure susceptibility locus found in mice: Seizure susceptibility 1. Remarkably, Szs1 influence diminished as subsequent induced seizures had diminishing latencies in certain HMDP strains. Administration of eight seizures, followed by an incubation period and an induced retest seizure, revealed novel associations within the calmodulin-binding transcription activator 1, Camta1. Using systems genetics, we have identified four candidate genes that are differentially expressed between seizure-sensitive and -resistant strains close to our novel Epileptogenesis susceptibility factor 1 (Esf1) locus that may act individually or as a coordinated response to the neuronal stress of seizures.

Objective: MRL/MpJ mice are known for enhanced healing, but mechanistic details or how specific aspects of wounding (e.g., angiogenesis) contribute to healing are unknown. While previous studies investigated the systemic effects of immunity in MRL/MpJ healing, few have focused on tissue-intrinsic effects. Approach: Ex vivo skin biopsies from MRL/MpJ and C57BL/6J mice were cultured in ex vivo conditions that favor endothelial cell growth to compare their angiogenic potential. We localized enhanced angiogenesis quantitative trait loci (QTL) in an F2 intercross. We then performed an expression analysis in cultured skin biopsies from MRL/MpJ and C57BL/6J mice to determine the pathways that are associated with the capacity for differential growth.

Efforts to understand the basic mechanisms of angiogenesis, that is, the formation of new blood vessels from existing vasculature, have been limited by the methods that are currently used to measure vessel growth. Although in vivo assays provide the best environment in which to track angiogenesis, inherent difficulties in obtaining reproducible data limit the power of this approach. Limitations include: environmental variations between experimental animals, induction of inflammatory responses by surgical methods, and labor-intensive blood vessel quantification procedures. A better assay would measure vessel growth in one animal at multiple time points and would focus on minimization of artifacts induced by experimental manipulation.

Identification of genetic factors that modify complex traits is often complicated by gene-environment interactions that contribute to the observed phenotype. In model systems, the phenotypic outcomes quantified are typically traits that maximize observed variance, which in turn, should maximize the detection of quantitative trait loci (QTL) in subsequent mapping studies. However, when the observed trait is dependent on multiple interacting factors, it can complicate genetic analysis, reducing the likelihood that the modifying mutation will ultimately be found. Alternatively, by focusing on intermediate phenotypes of a larger condition, we can reduce a model's complexity, which will, in turn, limit the number of QTL that contribute to variance. We 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. This method, in combination with a backcross breeding strategy, is intended to reduce genetic complexity and limit the phenotypic effects to fewer modifier loci. We determined that our approach was an efficient means to generate recombinant progeny and used this cohort to map a novel s.c. angiogenesis QTL to proximal mouse chromosome (Chr.) 8 with suggestive QTL on Chr. 2 and 7. Global mRNA expression analysis of samples from parental reference strains revealed β-defensins as potential candidate genes for future study.

Progress towards understanding the basic mechanisms of angiogenesis, the formation of new blood vessels from existing vasculature, have been limited by the current methods that are used to measure vessel growth. While in vivo assays provide the best environment to follow angiogenesis, inherent difficulties in obtaining reproducible data limit the power of this approach. Limitations can be attributed to several factors including variations between animals, labor intensive vessel quantification procedures and surgical methods that induce inflammatory responses that exacerbate angiogenic response. A better assay would focus on approaches that enable the measurement of vessel growth in one animal at multiple time points, while minimizing artifacts induced by experimental manipulation. MethodsAnimals All animals were handled in accordance with approved IACUC (institutional animal care and use committee) procedures. FVB/N-Tg (TIE2-lacZ)182Sato/J mice were anesthetized with isoflurane. The hind paws were then injected with 15µl of high Concentration MATRIGEL TM (BD Biosciences) containing 100 ng/ml VEGF165 and 300 ng/ml basic FGF (R&D Systems Inc.). We chose to evaluate the anterior glaborous skin of the hind leg proximal to the subtalar joint as this region is easily isolated on the microscope stage and does not require hair removal prior to intravital imaging. Vital signs were monitored with the MouseOx system, Starr Life Science Corp. (Oakmount PA) throughout all procedures. Animals were given a tail vein injection of 70kD FITC conjugated dextran (Sigma Aldrich, St, Louis MO) before each imaging session.Imaging Mice were restrained and imaged on a custom-machined microscope stage as shown in Fig. 1. The temperature of the mice was maintained by flowing 37ºC water through tubing embedded on the stage. Mouse paws were imaged with a Leica SP5 confocal equipped for multi-photon imaging (Mai Tai laser, Spectra Physics, Tucson, AZ). The MP laser was tuned to 920nm for imaging the FITC labeled dextran and 810 nm for imaging expressed GFP. A 20x (0.70NA) water objective lens was used to collect all Z stacks (1.2µm/slice).Image processing and analysis Image stacks were processed with a 3D Gaussian filter (ImagePro, Media Cybernics) to reduce noise and smooth motion introduced into individual sections by the animal's breathing and heart beat. Vessel segments were traced on composite images between branch points with ImageJ version 1.04g using the neuron J plugin. Measurements were quantified by counting the total number of branch points (as a surrogate of total individual segments), total vessel length and average vessel length.