Biology subjects

Pober, J. S.

Publications and source records attributed to Pober, J. S..

7 recordsLinked to original sources

bioRxiv2026

Simvastatin Preserves Human Microvascular Endothelial Barrier Function Through Mevalonate-Dependent Regulation of RhoB Localization and Activation

Endothelial barrier failure contributes to edema, tissue hypoperfusion, and organ dysfunction during inflammatory critical illness. RhoB has emerged as a regulator of tight junction disruption in human microvascular endothelial cells, but whether clinically used drugs can modulate RhoB-dependent barrier failure remains unclear. We tested whether simvastatin preserves human dermal microvascular endothelial cell barrier function during TNF stimulation and examined the role of mevalonate-dependent RhoB localization and activation. Simvastatin pretreatment attenuated TNF-induced loss of transendothelial electrical resistance and preserved junctional ZO-1 and claudin-5 organization. Simvastatin reduced membrane-associated and increased nuclear RhoB, decreased active RhoB, and suppressed myosin light chain phosphorylation. RhoB silencing phenocopied the barrier-protective effect of simvastatin, and combined RhoB silencing and simvastatin did not provide additive protection. Mevalonate supplementation reversed simvastatin-mediated preservation of barrier function, restored peripheral RhoB localization, and restored MLC phosphorylation. These findings identify mevalonate-dependent RhoB spatial regulation as a statin-sensitive mechanism contributing to inflammatory microvascular barrier dysfunction.

cell biology↗

bioRxiv2026

Oncostatin M-driven leak in human endothelial cells with preserved tight junctions

Blood vessels must preserve blood volume while permitting exchange of nutrients. Endothelial cells line the lumen of all blood vessels and form the intercellular junctions that govern vascular permeability. In acute critical illness the barrier fails, fluid escapes into tissues, and the resulting loss of volume and impaired nutrient exchange cause organ failure. Several inflammatory cytokines are known to disrupt junctions in cultured cells, yet therapies targeting these models have failed. How blood vessels respond to critical illness has remained largely elusive because endothelial cells are generally inaccessible. Here we profile endothelial cells recovered directly from critically ill children to show that oncostatin M drives a previously unrecognized mode of vascular leak. Oncostatin M increases permeability by forming narrow, nanoscale channels within the overlapping borders of adjacent cells, around intact tight junctions. These channels permit passage of small and intermediate sized solutes. Their formation depends on non-canonical signaling in which the kinase JAK1 activates two required parallel pathways (via ERK1 and AKT1) that converge on the transcription factor c-Fos. Inhibiting JAK1, either parallel pathway or depleting c-Fos, prevents oncostatin M-induced loss of barrier function. These results identify a permeability mechanism distinct from the junction disassembly driven by the cytokines that have dominated this field and implicate kinases already targeted by approved drugs as potential points of intervention. More broadly, vascular barriers are transiently loosened during immune-cell trafficking and metastasis, raising the possibility that diverse forms of vascular permeability arise through regulated nanoscale remodeling rather than junctional breakdown.

cell biology↗

bioRxiv2025

Endothelial cell MHC molecules are necessary and sufficient to reject 3D-printed human skin grafts in an advanced human immune system mouse

Vascularized skins were 3D-printed using single donor human fibroblasts, pericytes, keratinocytes and endothelial cells (ECs), the latter either unmodified (WT-ECs) or deleted of MHC molecules (KO-ECs). Adult MISTRG6 immunodeficient mice neonatally inoculated with adult human hematopoietic stem cells (HSCs) received printed skin allogeneic to the HSCs and were boosted 3 weeks post-grafting with human peripheral blood mononuclear cells (PBMCs) autologous to the HSCs. HSC inoculation alone produced low levels of circulating human myeloid and lymphoid cells without affecting grafts; PBMC boosting dramatically increased circulating human CD4+ T cells and boosted CD8+ T cells only in mice with WT-EC grafts. These grafts became infiltrated by human macrophages, dendritic cells, CD4+ and CD8+ T cells, and showed evidence of rejection. Shared T cell clones were present in skin and spleen. KO-EC grafts had minimal infiltration of graft or spleen without rejection despite MHC molecule expression on other graft cell types.

immunology↗

bioRxiv2025

A T-cell Intrinsic Role for APOL1 Risk Alleles in Allograft Rejection

African Americans have an increased risk of kidney disease due to exonic variants in Apolipoprotein-L1 (G1 and G2). These prevalent variants have also been linked with kidney rejection, but outside of association with African ancestry, underpinning causal mechanisms are unknown. We investigated T-cell function using transgenic mice with physiologic expression of wild type (G0-), G1-, or G2-APOL1. Mice with variant APOL1 showed greater CD8+T-cell activation with expansion of a central memory (TCM) subset. Stimulated G1-CD8+T-cells showed enhanced proliferation and cytokine production, which reversed with APOL1 inhibition. In MHC-mismatched cardiac transplants, G1-mice demonstrated greater CD8+T-cell infiltration and reduced survival. Bulk transcriptome of G1-CD8+T-cells, and single-cell transcriptome of graft infiltrating TCMs, showed enrichment of canonical T-cell receptor (TCR) pathways including Ca2+-signaling. G1-CD8+T-cells demonstrated baseline ER-Ca2+ depletion followed by sustained increases in cytosolic-Ca2+ upon TCR stimulation. G1-CD8+T-cells were more sensitive to Ca2+ chelation, or store-operated Ca2+ entry inhibition, and relatively resistant to calcineurin antagonism vs. G0-CD8+T-cells. Analogously, in a kidney transplant cohort, APOL1-variant recipients developed rejection when they had elevated peripheral TCMs before transplantation and despite significantly higher tacrolimus levels vs G0/G0-AAs with rejection. In summary, we unravel an excitatory T-cell intrinsic mechanism for APOL1 exonic variants, causally linking them with kidney rejection.

immunology↗

bioRxiv2024

The Multiple Roles of Gamma Interferon in Intraepithelial T Cell-Villous Enterocyte Interactions in Active Celiac Disease

We compared duodenal biopsies showing active celiac disease (CeD) to normal controls using single cell RNA sequencing, cyclic immunofluorescence, RNAScope and proximity ligation assays. There is increased infiltration of villous but not crypt epithelium T cells bearing either {beta} or {gamma}{delta} T cell receptors (TCRs) in CeD. Both T cell subsets are activated cytotoxic T lymphocytes (CTLs) and surprisingly are the predominant mucosal source of IFN{gamma}. In response to this IFN{gamma},villous but not crypt enterocytes show an IFN{gamma} signature, including nuclear phospho-STAT1 protein, class II HLA molecules and IFN{gamma}-inducible chemokines known to recruit CTLs (e.g., CCL3, CCL4, CXCL10, and CXCL11) and receptors for these chemokines are expressed on the infiltrating CTLs. Villous enterocytes also display increased HLA-E and HLA-B mRNAs and proteins. Bioinformatic analyses (NICHES) and proximity ligation assays show frequent binding of both {beta} and {gamma}{delta} TCRs with enterocyte HLA-E or HLA-B, but not HLA-DR. In contrast, NKG2C, proposed as an alternative trigger of CTL activation, is infrequently-expressed and shows few interactions with HLA-E. Our data suggest that activated intraepithelial CTLs produce IFN{gamma} which recruits additional CTLs and increases antigen-dependent killing of villous epithelium using either conventional or HLA-E antigen presentation. SignifanceAnalyses of diagnostic biopsies from celiac disease patients reveals critical roles for IFN{gamma} actions on enterocytes and a surprising role of HLA-E in antigen presntation. Activated cytotoxic T lymphocytes (CTLs) that are the primary source of mucosal IFN{gamma}, leading to IFN{gamma} signaling in villous enterocytes, including expression of phospho-STAT1 protein, class II HLA molecules, and chemokines that attract CTLs. Bioinformatic ligand receoptor analyses and proximity ligation assays showed frequent interactions between intraepithelial CTLs and enterocyte HLA-E or HLA-B, indicating that IFN{gamma} production by the CTLs likely promotes further CTL recruitment and antigen-dependent killing of the villous epithelium.

immunology↗

bioRxiv2022

3D bioprinting of an implantable xeno-free vascularized human skin graft

Bioengineered tissues or organs produced using matrix proteins or components derived from xenogeneic sources pose risks of allergic responses, immune rejection, or even autoimmunity. Here, we report successful xeno-free isolation, expansion, and cryopreservation of human endothelial cells, fibroblasts, pericytes and keratinocytes from a single donor. We further demonstrate the bioprinting of a human skin substitute with a dermal layer containing xeno-free cultured human endothelial cells (EC), fibroblasts, and pericytes in a xeno-free bioink containing human collagen type I and fibronectin layered in a biocompatible polyglycolic acid (PGA) mesh and subsequently seeded with xeno-free human keratinocytes to form an epidermal layer. Following implantation of such bilayered skin grafts on the dorsum of immunodeficient mice, keratinocytes form a mature stratified epidermis with rete ridge-like structures. The ECs and pericytes form human EC-lined perfused microvessels within 2 weeks after implantation, preventing graft necrosis, and eliciting further perfusion of the graft by angiogenic host microvessels. In summary, we describe the fabrication of a bioprinted vascularized bilayered skin substitute under completely xeno-free culture conditions demonstrating feasibility of a xeno-free approach to complex tissue engineering.

bioengineering↗

bioRxiv2019

Differential functional roles of fibroblasts and pericytes in the formation of tissue-engineered microvascular networks in vitro

AimsFormation of a perfusable microvascular network (VN) is critical for tissue engineering of solid organs. Stromal cells can support endothelial cell (EC) self-assembly into a VN, but distinct stromal cell populations may play different roles in this process. Here we investigated the effects that two widely used stromal cells populations, fibroblasts (FBs) and pericytes (PCs), have on VN formation. Methods and resultsWe examined the effects of adding defined stromal cell populations on the self-assembly of ECs derived from human endothelial colony forming cells (ECFCs) into perfusable VNs in fibrin gels cast within a microfluidics chamber. ECs alone fail to fully assemble a perfusable VN. Human lung FBs stimulate the formation of EC lined VNs within microfluidic devices. RNA-seq analysis suggested that FBs produce high levels of hepatocyte growth factor (HGF), and addition of recombinant HGF improved VN formation within devices. Human placental PCs could not substitute for FBs, but in the presence of FBs, PCs closely associated with ECs, formed a common basement membrane, extended microfilaments intercellularly, and reduced microvessel diameters. ConclusionsDifferent stromal cell types provide different functions in microvessel assembly by ECs. FBs support VN formation by providing paracrine growth factors whereas PCs directly interact with ECs to modify microvascular morphology. Statement of ContributionNatalia Kosyakova, Derek Kao, William G. Chang were primarily responsible for the conception, design, interpretation of experiments, and drafting of the manuscript. Francesc Lopez-Giraldez carried out analysis of RNA-seq data. Susann Spindler and Gregory Tietjen assisted with microvessel analysis software. Morven Graham and Xinran Liu assisted with the electron microscopy. Kevin J. James and Jee Won Shin assisted with data collection. Jordan Pober assisted with a critical review of manuscript and experimental design.

cell biology↗