Biology subjects

Bossardi Ramos, R.

Publications and source records attributed to Bossardi Ramos, R..

5 recordsLinked to original sources

bioRxiv2025

Endothelial Cells retain inflammatory memory through chromatin remodeling

Sepsis survivors face a heightened risk of secondary infections and chronic vascular dysfunction despite clinical recovery, yet the underlying mechanisms remain poorly defined. Our study identifies a novel mechanism of endothelial inflammatory memory, wherein transient inflammatory exposure induces durable chromatin remodeling, priming endothelial cells for exaggerated responses to subsequent inflammatory insults. Utilizing a clinically relevant two-hit mouse model, cecal ligation and puncture (CLP) followed by secondary Streptococcus pneumoniae (SP) infection, we reveal persistent transcriptional activation in endothelial cells (ECs), characterized by amplified expression of inflammatory cytokines, adhesion molecules (e.g., ICAM-1), complement factors, and interferon-stimulated genes. Genome-wide ATAC-seq analyses demonstrated stable chromatin accessibility at key inflammatory loci, indicative of epigenetic priming. Mechanistically, we uncovered a critical role for the AP-1 transcription factor JunB in mediating this epigenetic remodeling. JunB knockdown attenuated chromatin accessibility and subsequent transcriptional amplification upon secondary challenge, pinpointing JunB-driven chromatin modifications as central to endothelial reprogramming. Our findings offer mechanistic insights into how transient inflammation creates lasting epigenetic states within the endothelium, highlighting JunB as a potential therapeutic target to mitigate chronic endothelial dysfunction and increased susceptibility to secondary infections post-sepsis.

cell biology↗

bioRxiv2024

Identification of Myeloid Protein Kinase C - Epsilon as a Novel Atheroprotective Gene

BACKGROUNDAtherosclerosis is a chronic inflammatory disease driven by macrophages (MO). Protein Kinase C - epsilon (PKC{varepsilon}) is a serine/threonine kinase involved in diverse cellular processes including migration, growth, differentiation, and survival. PKC{varepsilon} acts in a context dependent manner within the heart, however, its role in atherosclerosis is unknown. METHODSBone marrow derived MO from global PKC{varepsilon} knockout mice were tested for lipid retention and cytokine secretion. Public geneset analysis assessed raw counts of PRKCE in human atheromas to determine translational relevance. A LysM Cre PKC{varepsilon}fl/fl (m{varepsilon}KO) mouse was developed to study the impact of myeloid PKC{varepsilon} on atherosclerosis. After confirming myeloid selective PKC{varepsilon} deletion, human-like hypercholesterolemia was induced and multiple metrics of atherosclerosis were compared in WT and m{varepsilon}KO plaques. RNA sequencing was used to provide unbiased insight into possible mechanisms by which PKC{varepsilon} regulates atherosclerosis. RESULTSPublic geneset analysis of human atherosclerotic plaque tissue revealed that PKC{varepsilon} expression is inversely correlated with plaque vulnerability. Similarly, peritoneal MO from WT hypercholesterolemic mice have significantly lower PKC{varepsilon} expression, providing a translational rational for generation of the m{varepsilon}KO mouse. qPCR revealed no differences between genotypes in the expression of genes related to atherosclerosis, at either steady state or upon lipid loading, suggesting that loss of PKC{varepsilon} does not fundamentally change the basal state and that differences seen are a result of a more complex pathway. Comparing descending aorta and aortic root plaques from WT and m{varepsilon}KO hypercholesterolemic mice revealed that m{varepsilon}KO plaques are larger, have larger foam cells and regions of necrosis, and thinner collagen caps. Upon lipid loading in vitro and in vivo, m{varepsilon}KO MO retained significantly more cholesterol and lipid droplets than WT; Gene Ontology suggests higher expression of genes related to endocytosis in m{varepsilon}KO MO compared to WT. CONCLUSIONSPKC{varepsilon} expression is decreased in vulnerable human plaques and decreases in mouse MO upon lipid loading. m{varepsilon}KO plaques are larger and exhibit markers of vulnerability. With no differences in scavenger receptor (SR) expression, the impact of PKC{varepsilon} deletion is more subtle than simple SR dysregulation. RNA sequencing implicates higher expression of genes involved in endocytosis and m{varepsilon}KO MO have significantly more lipid-containing endosomes. The data define the atherophenotype of m{varepsilon}KO mice and demonstrate that PKC{varepsilon} restricts lipid uptake into MO by a mechanism independent of SR expression. Taken together, these studies identify PKC{varepsilon} as a novel atheroprotective gene, laying the foundation for mechanistic studies on the endocytic signaling networks responsible for the phenotype. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/627650v5_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@1873e8org.highwire.dtl.DTLVardef@161a63eorg.highwire.dtl.DTLVardef@1e95b36org.highwire.dtl.DTLVardef@1634ecd_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA novel murine model in which PKC{varepsilon} is floxed (PKC{varepsilon}fl/fl) on both alleles haas been generated, backcrossed, and deposited into Jackson Laboratories (B6(Cg)-Prkcetm1.1Akv/LennMmjax). Then, PKC{varepsilon}fl/fl LysM Cre (m{varepsilon}KO) mice have been generated. C_LIO_LIDeletion of PKC{varepsilon} has no basal affects on other PKC isoforms, lipid handling markers, or inflammatory markers. C_LIO_LIUpon (in vitro) lipid loading of BMDM or isolating lipid loaded pMACs from hypercholesterolemic mice (in vivo), m{varepsilon}KO BMDMs retain more cholesterol. C_LIO_LILong term (16 wk) hypercholesterolemis generates larger lesions, more necrosis, and thinner plaque caps (i.e., a more vulnerable plaque phenotype) in m{varepsilon}KO mice. C_LIO_LIRNA sequencing suggests PKC{varepsilon} regulates endocytosis mechanisms post initial uptake of LDL and has been confirmed in a model of synchronized endocytosis. C_LIO_LIThese findings identify myeloid PKC{varepsilon} as a novel atheroprotective molecule whose expression impacts multiple metrics of atherosclerosis. This opens new avenues of investigation to uncover the PKC{varepsilon} signaling network responsible for the phenotype. Understanding how myeloid PKC{varepsilon} slows atherosclerosis development will aid in the development of novel preventative and therapeutic treatments. C_LI

immunology↗

bioRxiv2024

Deletion of the endothelial glycocalyx component endomucin leads to impaired glomerular structure and function

BackgroundEndomucin (EMCN), an endothelial-specific glycocalyx component, was found to be highly expressed by the endothelium of the renal glomerulus. We reported an anti-inflammatory role of EMCN and its involvement in the regulation of vascular endothelial growth factor (VEGF) activity through modulating VEGF receptor 2 (VEGFR2) endocytosis. The goal of this study is to investigate the phenotypic and functional effects of EMCN deficiency using the first global EMCN knockout mouse model. MethodsGlobal EMCN knockout mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Flow cytometry was employed to analyze infiltrating myeloid cells in the kidneys. The ultrastructure of the glomerular filtration barrier was examined by transmission electron microscopy, while urinary albumin, creatinine, and total protein levels were analyzed from freshly collected urine samples. Expression and localization of EMCN, EGFP, CD45, CD31, CD34, podocin, albumin, and -smooth muscle actin were examined by immunohistochemistry. Mice were weighed regularly, and their systemic blood pressure was measured using a non-invasive tail-cuff system. Glomerular endothelial cells and podocytes were isolated by fluorescence-activated cell sorting for RNA-seq. Transcriptional profiles were analyzed to identify differentially expressed genes in both endothelium and podocytes, followed by gene ontology analysis of up- and down-regulated genes. Protein levels of EMCN, albumin, and podocin were quantified by Western blot. ResultsEMCN-/- mice were viable with no gross anatomical defects in kidneys. The EMCN-/- mice exhibited increased infiltration of CD45+ cells, with an increased proportion of Ly6GhighLy6Chigh myeloid cells and higher VCAM-1 expression. EMCN-/- mice displayed albuminuria with increased albumin in the Bowmans space compared to the EMCN+/+ littermates. Glomeruli in EMCN-/- mice revealed fused and effaced podocyte foot processes and disorganized endothelial fenestrations. We found no significant difference in blood pressure between EMCN knockout mice and their wild-type littermates. RNA-seq of glomerular endothelial cells revealed downregulation of cell-cell adhesion and MAPK/ERK pathways, along with glycocalyx and extracellular matrix remodeling. In podocytes, we observed reduced VEGF signaling and alterations in cytoskeletal organization. Notably, there was a significant decrease in both mRNA and protein levels of podocin, a key component of the slit diaphragm. ConclusionOur study demonstrates a critical role of the endothelial marker EMCN in supporting normal glomerular filtration barrier structure and function by maintaining glomerular endothelial tight junction and homeostasis and podocyte function through endothelial-podocyte crosstalk.

pathology↗

bioRxiv2022

SOCS3 limits endotoxin-induced endothelial dysfunction by blocking a required autocrine interleukin 6 signal in human umbilical vein endothelial cells

Increased circulating levels of soluble interleukin (IL)-6 receptor (sIL-6R) are commonly observed during inflammatory responses, allowing for IL-6 signaling to occur in cells that express the ubiquitous receptor subunit gp130 but not IL-6R, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-{kappa}B-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6R-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6R in LPS-treated human umbilical vein endothelial cells. We show that cotreatment with sIL-6R dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This co-treatment led to a strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-{kappa}B signaling. Similar results were obtained when sIL-6R was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, while SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-kB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.

cell biology↗

bioRxiv2020

Endothelial SOCS3 maintains homeostasis and promotes survival in endotoxemic mice

SOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKo) with a non-lethal dose of lipopolysaccharide (LPS). SOCS3iEKo mice died 16-24 hours post-injection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I interferon-like program, and high expression of pro-thrombotic and pro-adhesive genes. Consistently, we observed intraluminal leukocyte adhesion and NETosis, as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In HUVEC, pulse-chase experiments showed that SOCS3 protein has a half-life below 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation leads to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrates that the regulation of SOCS3 protein levels is critical to inhibit IL-6-mediated endotheliopathy during shock and provides a promising new therapeutic avenue to prevent MODS though stabilization of endothelial SOCS3. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/424586v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@88eaf4org.highwire.dtl.DTLVardef@f974f1org.highwire.dtl.DTLVardef@16c9fa4org.highwire.dtl.DTLVardef@650229_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗