Splice factor polypyrimidine tract-binding protein 1 (Ptbp1) primes endothelial inflammation in atherogenic disturbed flow conditions.

NF-κB-mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through adhesion molecules Icam1 and Vcam1. The endothelium is primed for cytokine activation of NF-κB by exposure to low and disturbed blood flow (LDF)but the molecular underpinnings are not fully understood. In an experimental in vivo model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NF-κB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam1 induction by tumor necrosis factor (TNF)-α stimulation as a readout, we performed a CRISPR Cas9 knockout screen and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no effect on cell growth or response to apoptotic stimuli, but reversed LDF splicing patterns and inhibited NF-κB nuclear translocation and transcriptional activation of downstream targets, including Icam1 and Vcam1. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo, endothelial-specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice, limiting atherosclerosis. This may be mediated, in part, by allowing inclusion of a conserved alternative exon in Ripk1 leading to a reduction in Ripk1 protein. Our data show that Ptbp1, which is induced in a subset of the endothelium by platelet recruitment at regions of LDF, is required for priming of the endothelium for subsequent NF-κB activation, myeloid cell recruitment and atherosclerosis.

A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue.

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow.

OBJECTIVE: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. CONCLUSIONS: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.

Direct CD137 costimulation of CD8 T cells promotes retention and innate-like function within nascent atherogenic foci.

Effector CD8 T cells infiltrate atherosclerotic lesions and are correlated with cardiovascular events, but the mechanisms regulating their recruitment and retention are not well understood. CD137 (4-1BB) is a costimulatory receptor induced on immune cells and expressed at sites of human atherosclerotic plaque. Genetic variants associated with decreased CD137 expression correlate with carotid-intimal thickness and its deficiency in animal models attenuates atherosclerosis. These effects have been attributed in part to endothelial responses to low and disturbed flow (LDF), but CD137 also generates robust effector CD8 T cells as a costimulatory signal. Thus, we asked whether CD8 T cell-specific CD137 stimulation contributes to their infiltration, retention, and IFNγ production in early atherogenesis. We tested this through adoptive transfer of CD8 T cells into recipient C57BL/6J mice that were then antigen primed and CD137 costimulated. We analyzed atherogenic LDF vessels in normolipidemic and PCSK9-mediated hyperlipidemic models and utilized a digestion protocol that allowed for lesional T-cell characterization via flow cytometry and in vitro stimulation. We found that CD137 activation, specifically of effector CD8 T cells, triggers their intimal infiltration into LDF vessels and promotes a persistent innate-like proinflammatory program. Residence of CD137+ effector CD8 T cells further promoted infiltration of endogenous CD8 T cells with IFNγ-producing potential, whereas CD137-deficient CD8 T cells exhibited impaired vessel infiltration, minimal IFNγ production, and reduced infiltration of endogenous CD8 T cells. Our studies thus provide novel insight into how CD137 costimulation of effector T cells, independent of plaque-antigen recognition, instigates their retention and promotes innate-like responses from immune infiltrates within atherogenic foci. NEW & NOTEWORTHY Our studies identify CD137 costimulation as a stimulus for effector CD8 T-cell infiltration and persistence within atherogenic foci, regardless of atherosclerotic-antigen recognition. These costimulated effector cells, which are generated in pathological states such as viral infection and autoimmunity, have innate-like proinflammatory programs in circulation and within the atherosclerotic microenvironment, providing mechanistic context for clinical correlations of cardiovascular morbidity with increased CD8 T-cell infiltration and markers of activation in the absence of established antigen specificity.

Palovarotene reduces heterotopic ossification in juvenile FOP mice but exhibits pronounced skeletal toxicity.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by debilitating heterotopic ossification (HO). The retinoic acid receptor gamma agonist, palovarotene, and antibody-mediated activin A blockade have entered human clinical trials, but how these therapeutic modalities affect the behavior of pathogenic fibro/adipogenic progenitors (FAPs) is unclear. Using live-animal luminescence imaging, we show that transplanted pathogenic FAPs undergo rapid initial expansion, with peak number strongly correlating with HO severity. Palovarotene significantly reduced expansion of pathogenic FAPs, but was less effective than activin A inhibition, which restored wild-type population growth dynamics to FAPs. Palovarotene pretreatment did not reduce FAPs' skeletogenic potential, indicating that efficacy requires chronic administration. Although palovarotene inhibited chondrogenic differentiation in vitro and reduced HO in juvenile FOP mice, daily dosing resulted in aggressive synovial joint overgrowth and long bone growth plate ablation. These results highlight the challenge of inhibiting pathological bone formation prior to skeletal maturation.

Activin-dependent signaling in fibro/adipogenic progenitors causes fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal-dominant disorder characterized by progressive and profoundly disabling heterotopic ossification (HO). Here we show that fibro/adipogenic progenitors (FAPs) are a major cell-of-origin of HO in an accurate genetic mouse model of FOP (Acvr1 tnR206H ). Targeted expression of the disease-causing type I bone morphogenetic protein (BMP) receptor, ACVR1(R206H), to FAPs recapitulates the full spectrum of HO observed in FOP patients. ACVR1(R206H)-expressing FAPs, but not wild-type FAPs, activate osteogenic signaling in response to activin ligands. Conditional loss of the wild-type Acvr1 allele dramatically exacerbates FAP-directed HO, suggesting that mutant and wild-type ACVR1 receptor complexes compete for activin ligands or type II BMP receptor binding partners. Finally, systemic inhibition of activin A completely blocks HO and restores wild-type-like behavior to transplanted Acvr1 R206H/+ FAPs. Understanding the cells that drive HO may facilitate the development of cell-specific therapeutic approaches to inhibit catastrophic bone formation in FOP.