Bishop, T. F. | bioRxiv Science

bioRxiv2026

A Large Animal Model of Heritable Pulmonary Arterial Hypertension UsingGene-edited BMPR2 Sheep

Pulmonary Arterial Hypertension (PAH) is a rare vascular disorder characterized by elevated pressure in pulmonary arteries, eventually leading to right ventricular failure. Approximately 50% of pediatric disease and 20% of adult disease can be linked to a genetic mutation, with nearly 70% of these cases involving mutations in the bone morphogenetic protein receptor type 2 (BMPR2) locus. Investigations using rodent models have made significant advances in our understanding of BMPR2 signaling; however, limited data exist regarding the onset and course of PAH, and etiologies for phenotypic expression in these patients remain unknown. In this work, we describe the development of a novel ovine model of heritable PAH. Because homozygous disruption of BMPR2 is embryonic lethal, we developed heterozygous BMPR2 sheep by using a PAM-disrupting synonymous single stranded oligodeoxyribonucleotide alongside a single guide RNA and Cas9 mediated gene editing strategy. The resulting BMPR2(+/-) lambs demonstrated cardiac and pulmonary vascular pathology that are consistent with BMPR2 mutation-driven PAH observed in humans. Given the genetic and physiological similarities of BMPR2(+/-)sheep to humans with heritable PAH, this large animal model will serve as a vital platform for mechanistic molecular studies and will provide a much-needed pre-clinical model for extensive treatment evaluations.

molecular biology↗

bioRxiv2018

The Germ Theory of Regeneration

Amphibians have remarkable regenerative capabilities, but the mechanisms they use to regenerate are largely unknown. Identifying these mechanism would be of great interest for applications in medicine. Reactive oxygen species (ROS) are continually produced and required during tadpole tail regeneration (1). Production of ROS and tadpole tail regeneration are prevented by NADPH oxidase (Nox) inhibitors, suggesting Nox complexes as the source of ROS. However, the role of ROS and the mechanism of their sustained production throughout regeneration, were not known. NF-{kappa}B is a rapid-acting transcription factor with the potential to dramatically alter the activity and function of a cell (2). NF-{kappa}B is necessary for maintaining the undifferentiated state of human embryonic stem cells (3), human induced pluripotent stem cells (4) and mesenchymal stem cells (5), so may similarly be involved in maintaining the de-differentiated state of regeneration blastema cells. In the absence of an activating signal, NF-{kappa}B is sequestered in the cytoplasm by IkB (inhibitor of NF-{kappa}B), preventing its nuclear localisation and activity. The IkB kinase (IKK) complex inhibits IkB in response to multiple extracellular stimuli, but ROS can also inhibit IkB (6). Nuclear NF-{kappa}B directly activates transcription of several genes encoding Nox proteins (7, 8), so could thereby facilitate ROS production. A positive-feedback loop was hypothesised where ROS inhibit IkB to help maintain continual NF-{kappa}B activity and, in turn, facilitate the continual production of ROS by activating the transcription of Nox-encoding genes. Here we demonstrate the involvement of microorganisms in the initiation of tadpole tail regeneration. Microorganisms offer sources of ligands for toll-like receptor (TLR) pathway activation and consequently, IKK complex activity. It was also suggested that sustained NF-{kappa}B activity allows the continual expression of the genes encoding Nox4 in blastema cells and Nox2 in professional phagocytes. These findings provide potential targets for the activation of regeneration in non-regenerative animals.

developmental biology↗