Biology subjects

Reese, B.

Publications and source records attributed to Reese, B..

6 recordsLinked to original sources

bioRxiv2026

Endothelial PTBP1 Deletion in Transplanted Cardiac Tissue Limits Cardiac Allograft Vasculopathy

BackgroundCardiac allograft vasculopathy (CAV) is a leading cause of late graft failure and mortality following heart transplantation, with limited therapeutic options. Endothelial cells (ECs), at the interface between the donor graft and host immune system, play a central role in CAV development. However, the molecular mechanisms driving endothelial dysfunction and vascular remodeling in chronic heart transplant rejection remain poorly understood. MethodsTo characterize endothelial alterations associated with CAV, we isolated nuclei from cardiac tissues of four human donor groups: (1) early post-transplant CAV-negative surveillance biopsies, (2) CAV-negative explanted grafts with acute cellular rejection (ACR), (3) late-stage CAV-positive explanted grafts, and (4) naive non-transplanted control hearts. We applied intranuclear cellular indexing of transcriptomes and epitopes (inCITE-seq) to profile endothelial gene expression together with nuclear protein levels of splice factor polypyrimidine tract-binding protein 1 (PTBP1), a key post-transcriptional regulator of endothelial inflammatory responses. Functional relevance of PTBP1 was assessed using endothelial-specific deletion of Ptbp1 in an F1 hybrid murine model of CAV. ResultsIn human CAV, endothelial cells exhibited increased transforming growth factor-{beta} (TGF-{beta}) signaling and reduced oxidative phosphorylation (OxPhos) transcripts. Nuclear PTBP1 protein levels were markedly elevated in CAV endothelium and were associated with TGF-{beta}-responsive transcriptional programs and correlated with clinical indices of cardiac dysfunction. In murine heart transplants, endothelial-specific deletion of Ptbp1 markedly reduced hallmarks of CAV, including neointimal hyperplasia, fibrosis, and lymphocyte activation. At the molecular level, endothelial Ptbp1 deletion prevented suppression of mitochondrial transcripts and preserved mitochondrial content and integrity under hypoxic stress, attenuating interferon signaling in endothelial cells. ConclusionThese findings identify PTBP1 as a central endothelial regulator linking pro-fibrotic stress to mitochondrial dysfunction and immune activation in chronic cardiac allograft rejection. Targeting endothelial PTBP1 may represent a strategy to limit chronic graft injury while minimizing systemic immunosuppression.

immunology↗

bioRxiv2024

Endothelial Immunosuppression in Atherosclerosis : Translational Control by Elavl1/HuR

Atherosclerotic plaques are defined by the accumulation of lipids and immune cells beneath the endothelium of the arterial intima. CD8 T cells are among the most abundant immune cell types in plaque, and conditions linked to their activation correlate with increased levels of cardiovascular disease. As lethal effectors of the immune response, CD8 T cell activation is suppressed at multiple levels. These checkpoints are critical in dampening autoimmune responses, and limiting damage in cardiovascular disease. Endothelial cells are well known for their role in recruiting CD8 T and other hematopoietic cells to low and disturbed flow (LDF) arterial regions that develop plaque, but whether they locally influence CD8 effector functions is unclear. Here, we show that endothelial cells can actively suppress CD8 T cell responses in settings of chronic plaque inflammation, but that this behavior is governed by expression of the RNA-binding protein Embryonic Lethal, Abnormal Vision-Like 1 (Elavl1). In response to immune cell recruitment in plaque, the endothelium dynamically shifts splicing of pre-mRNA and their translation to enhance expression of immune-regulatory proteins including C1q and CD27. This program is immuno-suppressive, and limited by Elavl1. We show this by Cdh5(PAC)-CreERT2-mediated deletion of Elavl1 (ECKO), and analysis of changes in translation by Translating Ribosome Affinity Purification (TRAP). In ECKO mice, the translational shift in chronic inflammation is enhanced, leading to increased ribosomal association of C1q components and other critical regulators of immune response and resulting in a [~]70% reduction in plaque CD8 T cells. CITE-seq analysis of the remaining plaque T cells shows that they exhibit lower levels of markers associated with T cell receptor (TCR) signaling, survival, and activation. To understand whether the immunosuppressive mechanism occurred through failed CD8 recruitment or local modulation of T cell responses, we used a novel in vitro co-culture system to show that ECKO endothelial cells suppress CD8 T cell expansion--even in the presence of wild-type myeloid antigen-presenting cells, antigen-specific CD8 T cells, and antigen. Despite the induction of C1q mRNA by T cell co-culture in both wild-type and ECKO endothelial cells, we find C1q protein abundantly expressed only in co-culture with ECKO cells. Together, our data define a novel immune-suppressive transition in the endothelium, reminiscent of the transition of T cells to T-regs, and demonstrate the regulation of this process by Elavl1.

cell biology↗

bioRxiv2023

Targeted inCITE-Seq Analysis Identifies the Loss of Nuclear TDP-43 in Endothelium as a Mediator of Blood Brain Barrier Signaling Pathway Dysfunction in Neurodegeneration

AbstractDespite the importance of the endothelium in the regulation of the blood brain barrier (BBB) in aging and neurodegenerative disease, difficulties in extracting endothelial cell (EC) nuclei have limited analysis of these cells. In addition, nearly all Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Degeneration (FTD), and a large portion of Alzheimers Disease (AD) exhibit neuronal TDP-43 aggregation, leading to loss of nuclear function, but whether TDP-43 is similarly altered in human BBB ECs is unknown. Here we utilize a novel technique for the enrichment of endothelial and microglial nuclei from human cortical brain tissues, combined with inCITE-seq, to analyze nuclear proteins and RNA transcripts in a large cohort of healthy and diseased donors. Our findings reveal a unique transcriptional signature in nearly half of the capillary endothelial cells across neurodegenerative states, characterized by reduced levels of nuclear {beta}-Catenin and canonical downstream genes, and an increase in TNF/NF-kB target genes. We demonstrate that this does not correlate with increased nuclear p65/NF-kB, but rather a specific loss of nuclear TDP-43 in these disease associated ECs. Comparative analysis in animal models with targeted disruption of TDP-43 shows that this is sufficient to drive these transcriptional alterations. This work reveals that TDP-43 is a critical governor of the transcriptional output from nuclear p65/NF-kB, which has paradoxical roles in barrier maintenance and also barrier compromising inflammatory responses, and suggests that disease specific loss in ECs contributes to BBB defects observed in the progression of AD, ALS and FTD.

neuroscience↗

bioRxiv2023

Loss of Endothelial TDP-43 Leads to Blood Brain Barrier Defects in Mouse Models of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Loss of nuclear TDP-43 occurs in a wide range of neurodegenerative diseases, and specific mutations in the TARDBP gene that encodes the protein are linked to familial Frontal Temporal Lobar Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Although the focus has been on neuronal cell dysfunction caused by TDP-43 variants, TARDBP mRNA transcripts are expressed at similar levels in brain endothelial cells (ECs). Since increased permeability across the blood brain barrier (BBB) precedes cognitive decline, we postulated that altered functions of TDP-43 in ECs contributes to BBB dysfunction in neurodegenerative disease. To test this hypothesis, we examined EC function and BBB properties in mice with either knock-in mutations found in ALS/FTLD patients (TARDBPG348C and GRNR493X) or EC-specific deletion of TDP-43 throughout the endothelium (Cdh5(PAC)CreERT2; Tardbpff) or restricted to brain endothelium (Slco1c1(BAC)CreERT2; Tardbpff). We found that TARDBPG348Cmice exhibited increased permeability to 3kDa Texas Red dextran and NHS-biotin, relative to their littermate controls, which could be recapitulated in cultured brain ECs from these mice. Nuclear levels of TDP-43 were reducedin vitro and in vivo in ECs from TARDBPG348Cmice. This coincided with a reduction in junctional proteins VE-cadherin, claudin-5 and ZO-1 in isolated ECs, supporting a cell autonomous effect on barrier function through a loss of nuclear TDP-43. We further examined two models of Tardbp deletion in ECs, and found that the loss of TDP-43 throughout the endothelium led to systemic endothelial activation and permeability. Deletion specifically within the brain endothelium acutely increased BBB permeability, and eventually led to hallmarks of FTD, including fibrin deposition, microglial and astrocyte activation, and behavioral defects. Together, these data show that TDP-43 dysfunction specifically within brain ECs would contribute to the BBB defects observed early in the progression of ALS/FTLD.

neuroscience↗

bioRxiv2021

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.

cell biology↗

bioRxiv2021

Identification of Splice Regulators of Fibronectin-EIIIA and EIIIB by Direct Measurement of Exon Usage in a Flow-Cytometry Based CRISPR Screen

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

cell biology↗