Platelet–Vessel Wall Interactions in the Microcirculation

Continued population growth and clear evidence of climate change are placing unprecedented strain on food production and health care systems worldwide. Agricultural production goals that highlight environmental sustainability are at the forefront of social dialog, with animal agriculture routinely targeted for inefficiencies, welfare concerns and impact on climate. Genomic selection has transformed animal production and the rate of genetic gain in each generation continues to increase. Nonetheless, it is apparent that the rate of production gains will fall short of the necessary goals of increased efficiency and adaptation to the changing environment. Genetic engineering may have an important role in enhancing livestock production as well as providing new models for research in human health. The CRISPR/Cas and related systems for targeted gene editing has revolutionized livestock genetic engineering by increasing the rate of targeted mutation, thus drastically reducing the cost of producing founder animals with the desired trait. Intentional insertion of well-known mutations into genes such as myostatin have been shown to impart the expected increase in meat production, but have also resulted in undesirable associated traits affecting fertility, marbling and the skeleton. Thus, the approach to increasing meat production may require nuances such as promotor targeting and multiple gene targeting to obtain the desired production characteristics. Efforts to increase disease resistance could have dramatic impacts on animal welfare and production rates, potentially saving millions of animals from morbidity and mortality. However, confronting a single disease with each genetic alteration will be costly and inefficient. Considerations on how broad scale disease resistance could be achieved through genetic engineering will be instrumental in developing livestock with advantages to avoid infection and/or transmission of multiple pathogens. Gene editing also allows correction of mutations that arise in livestock through natural mechanisms while under intense genomic selection for increased production. Removing disease causing mutations in otherwise high value livestock will allow continued production from these lines without concern for inheriting the causative mutation. The same process can be utilized to insert genetic mutations known to phenocopy human conditions providing valuable large animal models of human genetic disease. Increased milk, meat, or eggs per animal unit will sustainably improve high quality, nutrient dense food production while large animal models of human disease will provide a more translational biomedical model for accelerated development of drugs and treatments. These goals are achievable, but require continued investment in innovation, clear regulatory framework for commercialization, education, and public engagement to ensure success.

The International Society for Biological and Environmental Repositories Presents Abstracts from Its Annual Meeting

The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune diseases and cancer: contribution of germ-free and gnotobiotic animal models of human diseases.

A set of unique sequences in bacterial genomes, responsible for protecting bacteria against bacteriophages, has recently been used for the genetic manipulation of specific points in the genome. These systems consist of one RNA component and one enzyme component, known as CRISPR ("clustered regularly interspaced short palindromic repeats") and Cas9, respectively. The present review focuses on the applications of CRISPR/Cas9 technology in the development of cellular and animal models of human disease. Making a desired genetic alteration depends on the design of RNA molecules that guide endonucleases to a favorable genomic location. With the discovery of CRISPR/Cas9 technology, researchers are able to achieve higher levels of accuracy because of its advantages over alternative methods for editing genome, including a simple design, a high targeting efficiency and the ability to create simultaneous alterations in multiple sequences. These factors allow the researchers to apply this technology to creating cellular and animal models of human diseases by knock-in, knock-out and Indel mutation strategies, such as for Huntington's disease, cardiovascular disorders and cancers. Optimized CRISPR/Cas9 technology will facilitate access to valuable novel cellular and animal genetic models with respect to the development of innovative drug discovery and gene therapy.

Flow-based assays for global assessment of hemostasis. Part 1: Biorheologic considerations.

The burden of cardiovascular and metabolic diseases worldwide is staggering. The emergence of systems approaches in biology promises new therapies, faster and cheaper diagnostics, and personalized medicine. However, a profound understanding of pathogenic mechanisms at the cellular and molecular levels remains a fundamental requirement for discovery and therapeutics. Animal models of human disease are cornerstones of drug discovery as they allow identification of novel pharmacological targets by linking gene function with pathogenesis. The zebrafish model has been used for decades to study development and pathophysiology. More than ever, the specific strengths of the zebrafish model make it a prime partner in an age of discovery transformed by big-data approaches to genomics and disease. Zebrafish share a largely conserved physiology and anatomy with mammals. They allow a wide range of genetic manipulations, including the latest genome engineering approaches. They can be bred and studied with remarkable speed, enabling a range of large-scale phenotypic screens. Finally, zebrafish demonstrate an impressive regenerative capacity scientists hope to unlock in humans. Here, we provide a comprehensive guide on applications of zebrafish to investigate cardiovascular and metabolic diseases. We delineate advantages and limitations of zebrafish models of human disease and summarize their most significant contributions to understanding disease progression to date.

Asparaginyl endopeptidase, or legumain, is a lysosomal cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes. Legumain is also up-regulated in a number of human cancers, and recent studies suggest that it may play important functional roles in the process of tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging reagents. Here we present the design, optimization, and in vivo application of fluorescently labeled activity-based probes (ABPs) for legumain. We demonstrate that optimized aza-peptidyl Asn epoxides are highly selective and potent inhibitors that can be readily converted into near-infrared fluorophore-labeled ABPs for whole body, noninvasive imaging applications. We show that these probes specifically label legumain in various normal tissues as well as in solid tumors when applied in vivo. Interestingly, addition of cell-penetrating peptides to the probes enhanced cellular uptake but resulted in increased cross-reactivity toward other lysosomal proteases as the result of their accumulation in lysosomes. Overall, we find that aza-peptidyl Asn ABPs are valuable new tools for the future study of legumain function in more complex models of human disease.

Lactosylceramide (LacCer), also known as CD17/CDw17, is a member of a large family of small molecular weight compounds known as glycosphingolipids. It plays a pivotal role in the biosynthesis of glycosphingolipids, primarily by way of serving as a precursor to the majority of its higher homolog sub-families such as gangliosides, sulfatides, fucosylated-glycosphingolipids and complex neutral glycosphingolipids—some of which confer “second-messenger” and receptor functions. LacCer is an integral component of the “lipid rafts,” serving as a conduit to transduce external stimuli into multiple phenotypes, which may contribute to mortality and morbidity in man and in mouse models of human disease. LacCer is synthesized by the action of LacCer synthase (β-1,4 galactosyltransferase), which transfers galactose from uridine diphosphate galactose (UDP-galactose) to glucosylceramide (GlcCer). The convergence of multiple physiologically relevant external stimuli/agonists—platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), stress, cigarette smoke/nicotine, tumor necrosis factor-α (TNF-α), and in particular, oxidized low-density lipoprotein (ox-LDL)—on β-1,4 galactosyltransferase results in its phosphorylation or activation, via a “turn-key” reaction, generating LacCer. This newly synthesized LacCer activates NADPH (nicotinamide adenine dihydrogen phosphate) oxidase to generate reactive oxygen species (ROS) and a highly “oxidative stress” environment, which trigger a cascade of signaling molecules and pathways and initiate diverse phenotypes like inflammation and atherosclerosis. For instance, LacCer activates an enzyme, cytosolic phospholipase A2 (cPLA2), which cleaves arachidonic acid from phosphatidylcholine. In turn, arachidonic acid serves as a precursor to eicosanoids and prostaglandin, which transduce a cascade of reactions leading to inflammation—a major phenotype underscoring the initiation and progression of several debilitating diseases such as atherosclerosis and cancer. Our aim here is to present an updated account of studies made in the field of LacCer metabolism and signaling using multiple animal models of human disease, human tissue, and cell-based studies. These advancements have led us to propose that previously unrelated phenotypes converge in a LacCer-centric manner. This LacCer synthase/LacCer-induced “oxidative stress” environment contributes to inflammation, atherosclerosis, skin conditions, hair greying, cardiovascular disease, and diabetes due to mitochondrial dysfunction. Thus, targeting LacCer synthase may well be the answer to remedy these pathologies.

Regulatory acceptance of animal models of disease to support clinical trials of medicines and advanced therapy medicinal products

This review so far has considered the role of vascular cells in the generation of reactive oxygen species (ROS) and novel approaches to their detection in integrated systems such as animal models of vascular disease and humans. We now turn attention to evidence consistent with a role for ROS in models of human disease and in discrete patient populations. We also briefly comment on the outcomes of clinical trials of antioxidants. Mainly, these have been disappointing. However, it is premature to conclude that the oxidation hypothesis of disease causality has been adequately tested. Animal studies, for the most part, support a fundamental role for ROS in cardiovascular disease. Any controversy could in part reflect the use of ineffective antioxidants and the selection of models in which ROS generation is of marginal relevance to the measured outcome. Both of these issues require a quantitatively accurate measurement of drug effect (ie, antioxidant capacity) before hypotheses relating to the role of oxidant stress can be addressed rationally. These issues pertain to clinical trials also, as we will discuss. However, animal studies permit administration of much more powerful antioxidants (eg, rate constant for interaction of superoxide dismutase (SOD) with O2·− ≈1.6×109 · mol/L−1 · s−1) than is possible in humans (eg, rate constant for vitamin E ≈0.59 mol/L−1 · s−1) or, like in the case of vitamin E, doses of the antioxidant in excess of those usually applied in clinical research. ### Atherosclerosis Animal models of atherosclerosis have documented that all the constituents of the plaque produce and use ROS. Lesion formation is associated with the accumulation of lipid peroxidation products1,2 and induction of inflammatory genes,3 inactivation of NO·resulting in endothelial dysfunction, 4,5 activation of matrix metalloproteinases,6 and increased smooth muscle cell growth.7 …

Companion animals, particularly dogs, are being increasingly appreciated as useful animal models of human diseases, as many naturally occurring canine diseases resemble diseases in people. Traditionally, laboratory rodents have been...

8 - Animal Models of Human Disease: Opportunities and Limitations

Spontaneous Animal Models of Human Disease

Nitric oxide (NO) is released by endothelial cells that line the inner walls of healthy blood vessels at fluxes ranging from 0.5 x 10(-10) to 4.0 x 10(-10) mol cm(-2) min(-1), and this continuous NO release contributes to the extraordinary thromboresistance of the intact endothelium. To improve the biocompatibility of blood-contacting devices, a biomimetic approach to release/generate NO at polymer/blood interfaces has been pursued recently (with NO donors or NO generating catalysts doped within polymeric coatings) and this concept has been shown to be effective in preventing platelet adhesion/activation via several in vivo animal studies. However, there are no reports to date describing any quantitative in vitro assay to evaluate the blood compatibilities of such NO release/generating polymers with controlled NO fluxes. Such a methodology is desired to provide a preliminary assessment of any new NO-releasing material, in terms of the effectiveness of given NO fluxes and NO donor amounts on platelet activity before the more complex and costly in vivo testing is carried out. In this article, we report the use of a lactate dehydrogenase assay to study in vitro platelet adhesion on such NO-releasing polymer surfaces with varying NO fluxes. Reduced platelet adhesion was found to correlate with increasing NO fluxes. The highest NO flux tested, 7.05 (+/-0.25) x 10(-10) mol cm(-2) min(-1), effectively reduced platelet adhesion to nearly 20% of its original level (from 14.0 (+/-2.1) x 10(5) cells cm(-2) to 2.96 (+/-0.18) x 10(5) cells cm(-2)) compared to the control polymer coating without NO release capability.

Over the past decade remarkable progress has been made in our knowledge of the pathobiology of human disease through the use of genetically engineered mice. The ability to manipulate the murine genome by use of gain of function or loss of function strategies is providing fundamental insights into the mechanisms which underlie atherosclerosis (), hypertension (), cardiac hypertrophy and failure (). The power of this approach derives from the ability to precisely overexpress or ablate a gene of interest and examine the phenotypic consequences during embryogenesis, development, maturation and senescence. In contrast to conventional experimental animal models of human disease which employ some form of environmental stress and elicit a complex array of molecular and biochemical events, genetic engineering involves a single known molecular perturbation which produces a phenotype that may or may not recapitulate human disease. Gene targeting and transgenic analysis involves a candidate gene approach. The gene of interest is believed to potentially be involved in the pathogenesis of a disease state. These approaches are now being complemented by viral or chemical mutagenesis studies which produce random disruptions in the mouse genome. This strategy holds the promise of discovering new genes responsible for a pathologic state or a previously unrecognized role in the pathogenesis of disease for known genes.