Center for Vascular Biology

Murphy Lab

Project 1:in vivo function of endothelial splice factors identified by informatics approaches and in vitro screens as important regulators of endothelial activation under disturbed flow.

Figure shows top 200 TNF induced genes in murine aortic endothelial cells with or without CRISPR-mediated deletion of Ptbp1 or a rescue with human PTBP1 cDNA.

This set of projects uses mouse models with endothelial-specific deletion of splice factors implicated by expression and motif analysis in the endothelial response to innate immune cell recruitment, and activity in regulation of endothelial inflammation in our in vitro CRISPR-KO screens. We hypothesize that these splice factors alter the endothelial transcriptome post-transcriptionally, through effects on RNA-splicing, stability or translation to modify the endothelial interaction with immune cells in chronic inflammation. Our recent publication, describing a key function for the splice factor Ptbp1 in endothelial NFkB signaling, myeloid recruitment and plaque formation is an example of this class of RNA-binding proteins. Several other proteins are being examined using these assays, and each appears to have unique effects on immune cell behavior.

Project 2:Define the signaling from innate immune cells leading to splice factor activation within the endothelium

Figure shows heat map of splicing changes induced by innate immune cells in the arterial intima of the carotid artery (Murphy et al. eLife 2018). GPIba antibody was used to deplete platelets from mice prior to induction of low and disturbed flow. Note that, even in low flow conditions, platelet depletion blocks splicing changes in the endothelium. Adjacent images show, a DIC image of an aortic endothelial cell monolayer, which is overlaid with bone marrow derived monocytes from a reporter mouse (red).

Splice factor activity in the endothelium is induced by innate immune cell recruitment (platelet & myeloid dependent) in conditions of low and disturbed flow. We now know that activation of these splice factors is important in priming the endothelium for subsequent activation. We do not yet know the mediators of the signaling from innate immune cells to the endothelium which lead to increased expression and activation of these splice factors, but we do know that this process can be recapitulated in vitro, which provides us with an opportunity to identify these mediators. This project will focus on endothelial immune co-culture, screening for the effect of candidate mediator receptors on the endothelium for their effect on the expression and activation of the splice factors of interest.

Project 3: Identification of splicing changes induced by splice factor activation leading to effects on immune responses in vivo

Figure shows the RNA-sequencing analysis from the carotid intima of mice exposed to disturbed flow or not. This splice factor had little effect on total RAN expression (upper panels, FPKM = Fragments Per Kilobase of transcript per Million mapped reads).  However, there was a large effect on RNA splicing, primarily affected the genes and pathways in the inset.

This set of projects is focused on analysis of altered splice isoforms within the endothelium induced by activation of the splice factors above. We have good data showing that, for some of these splice factors, there is little change in RNA expression when we look at all of the transcripts produced from a single gene, but that there are large changes in the specific splice isoforms that are produced. As these splice isoforms are enriched in pathways related to the immune phenotypes we have observed, we hypothesize that differential splicing of these transcripts contributes to the altered immune responses we have observed in vitro and in vivo. This work will use informatics and CRISPR-based screening approaches to define the key splicing events linking splice factor function to phenotype. Extrapolation of this work, to examine splicing QTLs (sQTLs) in human datasets will provide an important link to human disease.

Project 4: Interrogation of splice factor dysfunction in neurodegenerative disease using our novel methods of endothelial nuclei enrichment and single cell analysis

Figure cell-type specific expression of Erg in the brain in endothelial cells (from Zhang et al. 2016), and our use of an Erg specific antibody to isolate a high Erg+ population of endothelial nuclei from archived frozen brain tissues. RNA-sequencing analysis of these nuclei reveals enrichment endothelial specific transcripts (green) and a good concordance with whole brain endothelial cells. Lower panels show RNA expression levels of example markers of brain endothelial cells and microglia in the indicated sorted populations. The last panel shows analysis of the Fn transcript, and splice isoforms, in Erg+ nuclei. Details on these approaches can be found in our pre-print below.

Splicing dysregulation, caused by defects in RNA-binding splice factors (e.g. TDP-43 and MATR3), is considered a hallmark and potential driver of neuronal dysfunction and cognitive decline in Alzheimer’s (AD) and Related Dementia (ADRD). Although substantial evidence suggests that genetic causes of splicing dysfunction are not limited to neuronal cells, splicing defects in the endothelium have not been examined. As the endothelium is a principal component of the blood brain barrier, and defects in the blood brain barrier function increase with age and early in the progression of AD and ADRD, we believe that understanding splice factor function in the endothelium in aging and disease states could provide new approaches to targeting age and dementia related declines in cognitive function. We recently developed a method to specifically extract endothelial nuclei, based on the expression of an endothelial transcription factor in these nuclei (Erg), and are using this to profile the endothelial transcriptome across aging and disease states. This will be used to identify dysfunctional splicing patterns within the endothelium, and candidate regulatory splice factors. These will be functionally validated in vitro using our established screening assays and in vivo, using a novel combination of Br1-AAV for brain endothelial delivery of gRNA with a mouse model of endothelial specific Cas9 expression.

Project 5: Analysis of the impact of common disease-causing mutations on endothelial splicing patterns and function in mouse models

Figure shows Sclo1c1-CreER activity in brain endothelial cells, allowing the specific deletion of splice factors, such as Tardbp (TDP-43) in the brain endothelium for phenotypic characterization. Adjacent figure shows in vivo multiphoton imaging of dextran labeled vasculature through a cranial window. This approach is used to assess endothelial barrier functions, leukocyte recruitment and blood flow patterns in vivo.

Several genetic mutations are known to directly or indirectly affect TDP-43 (Tardbp) function during the progression of neurogenerative disease. These include mutations in TDP-43 itself, leading to increased cytoplasmic aggregation, as well as mutations in other pathways (e.g. Proganulin, GRN) which indirectly lead to TDP-43 dysfunction. Although earlier work in zebrafish morpholino knockdowns suggested a potential direct effect on vascular growth and development, very little is known about TDP-43 function within the brain endothelium. As several studies have indication that TDP-43 dysfunction in disease may not be limited to the endothelium, we propose that an analysis of endothelial cell functions will be important in understanding blood brain barrier dysfunction in neurodegenerative disease. We will use novel mouse models we have generated to examine TDP-43 functions within the endothelium and brain endothelium in vivo, and to examine the effect of commonly linked mutations, like those in GRN, to TDP-43 function in the endothelium. This work will focus on blood brain barrier properties, inflammatory status and responses, and neurovascular coupling. We will use a combination of mouse genetics, cellular and molecular characterization and intravital multi-photon imaging to achieve our goals.