Center for Vascular Biology

Murphy Lab

Project 1: Analysis of alternative splicing in flow-driven arterial inflammation

Figure shows the consistency in gene expression and in splicing response among different biological replicates of endothelium exposed to low flow in vivo

Using an established model of flow-induced arterial injury, we have recently performed large scale RNA-sequencing analysis of an endothelial enriched fraction in normal and disturbed flow conditions. We have identified thousands of changes in alternative splicing, and a handful of potential upstream regulatory splice-factors. A large portion of these splicing changes are responsive to the recruitment of innate immune cells. We have mouse lines for the conditional deletion of three of these splice factors from the endothelium, allowing us to assess their functions in vivo.

This project involves phenotypic analysis of the consequences of perturbing these splice factors in vivo, with an emphasis on understanding the effects on immune response.

Project 2: Alternative splicing in cross-talk with myeloid cell

Macrophages co-cultured with splicing-mutated endothelial cells

Figure shows a DIC image of an aortic endothelial cell monolayer, which is overlaid with bone marrow derived monocytes from a reporter mouse (red). Profiling of the co-cultured monocytes and their differentiated progeny has revealed striking differences in phenotype, depending on the alternative splicing status of the endothelial cells they are cultured on.

We have developed methods for the conditional expansion of aortic endothelial cells in culture. These cells, and versions of these cells with alterations in specific splice factors or alternatively spliced genes are being used in co-culture experiments with myeloid cells. Pilot experiments have shown strong effects on the phenotype of wild-type myeloid cells cultured with endothelial cells altered in either the expression of the splice factors we have identified in vivo, or the alternative splicing events they regulate. This interesting result suggests how splicing responses in the endothelium, which are responsive to innate immune cell recruitment, shape chronic inflammation in arterial injury and likely other inflammatory processes as well.

This project involves in vitro culture of endothelial cells with mutations in the alternative splicing response, and the isolation and characterization of immune cells by FACs and molecular biology.

Project 3: Identification of splice regulators by candidate screen

Figure shows the relative level of alternative exon inclusion (increasing from left to right) by flow cytometry. Red=baseline, yellow=mutant cells, with impaired inclusion, blue=wild-type cells, with normal inclusion.

The list of regulatory splice factors identified in vivo was done by bioinformatics – specifically an enrichment in the regulatory motifs they bind to among the regulated splicing events. While this has revealed a clear role for the splice factors we are testing in Project 1, we have a handful of other factors which have been less characterized are therefore very interesting. We have established a method to screen the function of these factors in the regulation of a few key splicing events by flow cytometry. This screen will allow us to identify factors that result in either the increased or the decreased inclusion of specific exons in the final RNA in primary and cultured aortic endothelial cells.

This project involves pooled screens, using shRNA and CRISPR depletion approaches, to determine which factors affect the splicing response. We will be using FACs as the screen, and NextGen sequencing enrichment as the readout. "Hits," or novel regulators of splicing, will then be further interrogated in vitro and potentially in vivo.

Project 4: Regulation of NF-kappa B signaling by alternative splicing

For the vast majority of alternative splicing events we have identified, the function remains unknown. However, many of them target key processes in endothelial cells. One of the processes we are most interested in is NF-kappaB signaling. This pathway has a pivotal role in the response of the vascular endothelium to low and disturbed flow, and thus alterations of this pathway induced by changes in splicing are likely to impact the response of the vasculature to chronic flow-induced injury. We will examine these splice variants in our in vitro model, to determine which impact NF-kappaB induction, and then define the mechanism for this regulation.

This project involves the expression cDNA encoding the splice variants, and shRNA or CRISPR mediated suppression of specific splice isoforms. Outcomes will be assessed in a pooled fashion, using murine and human aortic endothelial cells engineered to express NF-kappaB reporters.

<--Molecular components of the NF-kappa B signaling pathway.

Figure reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology, 8, 49-62 (doi:10.1038/nrm2083), copyright 2007