Abstract 7: In vivo Transcriptional Profiling of Endothelial Cells Identifies Vascular Bed-Specific Changes Upon Lps-Induced Endotoxemia

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Audrey Cleuren ◽  
Martijn van der Ent ◽  
Kristina Hunker ◽  
Hui Jiang ◽  
Andrew Yee ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cellular Response ◽  
Inflammatory Responses ◽  
Differential Expression Analysis ◽  
Response To Therapy ◽  
Microbial Pathogens ◽  
P Value ◽  
Transcriptional Changes ◽  
Vascular Beds

Endothelial cells (ECs) form a critical barrier between blood and parenchymal cells and play an important role in many pathologic conditions, including sepsis. ECs are highly adaptive to their microenvironment and also act as a critical responder to microbial pathogens. Though ECs are thought to display extensive heterogeneity, detailed profiling of the in vivo EC gene expression program has been limited by the challenges of isolating ECs from complex tissues and the phenotypic drift associated with manipulation and expansion of ECs in vitro . We applied an in vivo system in which a conditional hemagglutinin-epitope tag is targeted into the mouse ribosomal protein Rpl22 locus and specifically activated in ECs, allowing immunoisolation of endothelial ribosome-associated mRNA. Both EC-selected and total mRNA from tissue lysates (brain, heart, kidney, liver and lung) were subjected to RNA sequencing followed by differential expression analysis to determine EC-enriched transcripts. These analyses were performed under physiologic conditions as well as in LPS injected mice to study transcriptional changes induced in ECs following endotoxin exposure. LPS-induced endotoxemia resulted in striking changes in the EC transcriptome (~800 per tissue), and included transcripts associated with known sepsis related pathophysiology, including impaired hemostasis, leukocyte recruitment and increased vascular permeability. Gene ontology analysis of transcriptional changes shared between ECs of different tissues identified cellular response to LPS among the highest enriched biologic processes (adjusted p-value 5.2E-5), together with immune (2.0E-14) and inflammatory responses (4.4E-12). Novel transcripts not previously associated with ECs or endotoxemia were also identified, as well as a subset of genes uniquely expressed in distinct vascular beds. In conclusion, our findings demonstrate remarkable heterogeneity of the EC transcriptome across multiple vascular beds in vivo . The EC response to endotoxin challenge is also highly heterogeneous across vascular beds and provides new insight into the endothelial response to infectious challenges, as well as identifying potentially useful biomarkers for the onset of sepsis and response to therapy.

Abstract 116: Slit-Robo Signaling Regulates Lipopolysaccharide-induced Endothelial Inflammation and Expression of Slit/Robo is Dysregulated During Endotoxemia

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Helong Zhao ◽  
Appakkudal Anand ◽  
Ramesh Ganju
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Model Studies ◽  
Endothelial Inflammation ◽  
Anti Inflammatory ◽  
Whole Heart

Abstract Introduction: Lipopolysaccharide (LPS) is one of the critical factors which induce endothelial inflammation during the pathogenesis of atherosclerosis, endocarditis and sepsis shock induced heart injury. The secretory Slit2 protein and its endothelial receptors Robo1 and Robo4 have been shown to regulate mobility and permeability of endothelial cells, which could be functional in regulating LPS induced endothelial inflammation. Hypothesis: We hypothesized that in addition to regulating permeability and migration of endothelial cells, Slit2-Robo1/4 signaling might regulate other LPS-induced endothelial inflammatory responses. Methods and Results: Using Human Umbilical Vein Endothelial Cells (HUVEC) culture, we observed that Slit2 treatment suppressed LPS-induced secretion of pro-inflammatory cytokines (including GM-CSF), cell adhesion molecule upregulation and monocyte (THP-1 cell) adhesion. With siRNA knock down techniques, we further confirmed that this anti-inflammatory effect is mediated by the interaction of Slit2 with its dominant receptor in endothelial cells, Robo4, though the much lesser expressed minor receptor Robo1 is pro-inflammatory. Our signaling studies showed that downstream of Robo4, Slit2 suppressed inflammatory gene expression by inhibiting the Pyk2 - NF-kB pathway following LPS-TLR4 interaction. In addition, Slit2 can induce a positive feedback to its expression and downregulate the pro-inflammatory Robo1 receptor via mediation of miR-218. Moreover, both in in vitro studies using HUVEC and in vivo mouse model studies indicated that LPS also causes endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 and upregulating the pro-inflammatory Robo1 during endotoxemia, especially in mouse arterial endothelial cells and whole heart. Conclusions: Slit2-Robo1/4 signaling is important in regulation of LPS induced endothelial inflammation, and LPS in turn causes inflammation by interfering with the expression of Slit2, Robo1 and Robo4. This implies that Slit2-Robo1/4 is a key regulator of endothelial inflammation and its dysregulation during endotoxemia is a novel mechanism for LPS induced cardiovascular pathogenesis.

Disruption of endothelial Pfkfb3 ameliorates diet-induced murine insulin resistance

2021 ◽  
Author(s):  
Qiuhua Yang ◽  
Jiean Xu ◽  
Qian Ma ◽  
Zhiping Liu ◽  
Yaqi Zhou ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Endothelial Cells ◽  
High Fat Diet ◽  
Inflammatory Responses ◽  
Critical Role ◽  
Endothelial Inflammation ◽  
Glucose Clearance ◽  
Deficient Mice

Overnutrition-induced endothelial inflammation plays a crucial role in high fat diet (HFD)-induced insulin resistance in animals. Endothelial glycolysis plays a critical role in endothelial inflammation and proliferation, but its role in diet-induced endothelial inflammation and subsequent insulin resistance has not been elucidated. PFKFB3 is a critical glycolytic regulator, and its increased expression has been observed in adipose vascular endothelium of C57BL/6J mice fed with HFD in vivo, and in palmitate (PA)-treated primary human adipose microvascular endothelial cells (HAMECs) in vitro. We generated mice with Pfkfb3 deficiency selective for endothelial cells to examine the effect of endothelial Pfkfb3 in endothelial inflammation in metabolic organs and in the development of HFD-induced insulin resistance. EC Pfkfb3-deficient mice exhibited mitigated HFD-induced insulin resistance, including decreased body weight and fat mass, improved glucose clearance and insulin sensitivity, and alleviated adiposity and hepatic steatosis. Mechanistically, cultured PFKFB3 knockdown HAMECs showed decreased NF-κB activation induced by PA, and consequent suppressed adhesion molecule expression and monocyte adhesion. Taken together, these results demonstrate that increased endothelial PFKFB3 expression promotes diet-induced inflammatory responses and subsequent insulin resistance, suggesting that endothelial metabolic alteration plays an important role in the development of insulin resistance.

scholarly journals Neutrophils roll on adherent neutrophils bound to cytokine-induced endothelial cells via L-selectin on the rolling cells.

1994 ◽  
Vol 180 (5) ◽  
pp. 1785-1792 ◽  
Author(s):  
R F Bargatze ◽  
S Kurk ◽  
E C Butcher ◽  
M A Jutila
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Leukocyte Recruitment ◽  
Neutrophil Accumulation ◽  
Adhesion Proteins ◽  
Rapid Accumulation ◽  
Vascular Adhesion

Specific arrest of neutrophils in venules is central to their rapid accumulation during local inflammatory responses. Initial neutrophil rolling on endothelium is mediated by leukocyte L-selectin and the inducible vascular adhesion proteins P- and E-selectin. This rolling is a prerequisite for endothelial-dependent neutrophil arrest. Here we describe rolling of neutrophils on the surface of previously arrested neutrophils and demonstrate that this interaction involves L-selectin exclusively on rolling cells. The adherent neutrophil support of L-selectin-dependent neutrophil rolling in vivo can promote continuous and augmented leukocyte recruitment at sites of previous neutrophil accumulation.

scholarly journals RIPK1 Expression Associates with Inflammation in Early Atherosclerosis in Humans and Can be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice

Circulation ◽  
2020 ◽  
Author(s):  
Denuja Karunakaran ◽  
My-Anh Nguyen ◽  
Michele Geoffrion ◽  
Dianne Vreeken ◽  
Zachary Lister ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Activation ◽  
Inflammatory Responses ◽  
Early Stage ◽  
Oxidized Ldl ◽  
Loss Of Function ◽  
Aortic Sinus ◽  
Atherosclerotic Lesions

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. Previously, we showed that macrophages in the atherogenic plaque undergo RIPK3-MLKL-dependent programmed necroptosis in response to sterile ligands such as oxidized LDL and damage-associated patterns (DAMPs) and necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1, which acts as a master switch that controls whether the cell undergoes NFκB-dependent inflammation, caspase-dependent apoptosis or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is largely driven by NFκB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NFκB-dependent inflammation in early atherogenic lesions and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and using loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 anti-sense oligonucleotides (ASO) to Apoe -/- mice fed a cholesterol-rich (Western) diet for 8 weeks. Results: We find RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 ASOs led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, p<0.01) and plasma inflammatory cytokines (IL-1α, IL-17A, p<0.05) compared to controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NFκB, TNFα, IL-1α) and in vivo LPS- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin and monocyte attachment. Conclusions: We have identified RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

scholarly journals The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

2019 ◽  
Author(s):  
Audrey C.A. Cleuren ◽  
Martijn A. van der Ent ◽  
Hui Jiang ◽  
Kristina L. Hunker ◽  
Andrew Yee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Single Cell ◽  
Blood Vessels ◽  
Rna Sequencing ◽  
Specific Gene ◽  
Sequencing Analysis ◽  
Single Cell Rna Sequencing ◽  
Vascular Beds

AbstractEndothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression following EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single cell suspensions for fluorescence-activated cell sorting (FACS) or single cell RNA sequencing analysis. Comparison of our EC-TRAP to published single cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.SignificanceEndothelial cells (ECs), which line all vertebrate blood vessels, are highly heterogeneous across different tissues. The present study uses a genetic approach to specifically tag mRNAs within ECs of the mouse, thereby allowing recovery and sequence analysis to evaluate the EC-specific gene expression program directly from intact organs. Our findings demonstrate marked heterogeneity in EC gene expression across different vascular beds under both normal and disease conditions, with a more accurate picture than can be achieved using other methods. The data generated in these studies advance our understanding of EC function in different blood vessels and provide a valuable resource for future studies.

scholarly journals Endothelium Response to Iron Sucrose Nanoparticles in Static Versus Dynamic Culture Model

2021 ◽  
Author(s):  
Niusha Nikravesh ◽  
Liliane Diener ◽  
Savvina Chortarea ◽  
Alexandra Rippl ◽  
Peter Wick
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Dynamic Condition ◽  
Biological Fluids ◽  
Static Condition ◽  
Factors Affecting ◽  
Noticeable Increase ◽  
In Cells

Abstract Nanomedicines are promising therapeutic compounds allowing the development of new treatment approaches. However, important factors affecting the behavior of nanoparticles in vivo cannot be simulated in conventional static models. Dynamic cell cultures, where cells are cultivated in the presence of shear stress, have the potential to bridge this gap by mimicking critical features of physiological conditions. Iron-carbohydrate nanoparticles are a dominant treatment for iron deficiency unresponsive to oral iron supplements. Compared to the data available from clinical studies, little is known about the interaction of these particles with endothelial cells. Our approach implements and compares a microchannel-based dynamic human endothelium model to the static culture to explore potential differences in cells response after exposure to iron nanoparticles. Differences in cellular uptake are observable using Prussian blue assay. There is a noticeable increase on VCAM-1 and ICAM-1 gene expression on endothelial cells activated by inflammatory responses, in cells exposed to nanoparticles under static condition. Results show that cytotoxicity caused by iron particles is significantly lower under dynamic condition compared to static cultures. We demonstrate that inclusion of dynamic flow and biological fluids are positive steps towards nanoparticle evaluation in a physiologically relevant in vitro model.

Cellular response to hypoxia involves signaling via Smad proteins

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2253-2260 ◽  
Author(s):  
Hong Zhang ◽  
Hasan O. Akman ◽  
Eric L. P. Smith ◽  
Jin Zhao ◽  
Joanne E. Murphy-Ullrich ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Cellular Response ◽  
Transforming Growth Factor ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Hypoxia Inducible Factor ◽  
Transforming Growth Factor Β ◽  
Smad Proteins ◽  
Umbilical Vein Endothelial Cells

The transforming growth factor-β (TGF-β) family of cytokines regulates vascular development and inflammatory responses. We have recently shown that exposure of human umbilical vein endothelial cells (HUVECs) to hypoxia (1% O2) increases gene expression and bioactivation of TGF-β2 and induces its downstream effectors, Smad proteins (Smads), to associate with DNA. In the present study, we show that hypoxia-induced TGF-β2 gene expression is dependent on thrombospondin-1–mediated bioactivation of latent TGF-β. Blocking TGF-β2 but not TGF-β1 in hypoxic endothelial cell cultures inhibited induction of the TGF-β2 gene, indicating that an autocrine mechanism driven by bioactivation of TGF-β2 leads to its gene expression in hypoxic HUVECs. Exposure of HUVECs to hypoxia resulted in phosphorylation and nuclear transportation of Smad2 and Smad3 proteins as well as stimulation of transcriptional activities of Smad3 and the transcription factor hypoxia-inducible factor-1α and culminated in up-regulation of TGF-β2 gene expression. Autocrine regulation of TGF-β2 production in hypoxia may involve cross-talk between Smad3 and HIF-1α signaling pathways, and could be an important mechanism by which endothelial cells respond to hypoxic stress.

scholarly journals Role of P-Selectin Cytoplasmic Domain in Granular Targeting In Vivo and in Early Inflammatory Responses

1998 ◽  
Vol 143 (4) ◽  
pp. 1129-1141 ◽  
Author(s):  
Daqing W. Hartwell ◽  
Tanya N. Mayadas ◽  
Gaëtan Berger ◽  
Paul S. Frenette ◽  
Helen Rayburn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Inflammatory Responses ◽  
Cytoplasmic Domain ◽  
Leukocyte Rolling ◽  
Storage Pool ◽  
Storage Granules

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (ΔCT mice). The deletion did not affect the sorting of P-selectin into α-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The ΔCT–P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the ΔCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

scholarly journals miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses

2009 ◽  
Vol 106 (37) ◽  
pp. 15819-15824 ◽  
Author(s):  
Gang Liu ◽  
Arnaud Friggeri ◽  
Yanping Yang ◽  
Young-Jun Park ◽  
Yuko Tsuruta ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Inflammatory Responses ◽  
Inflammatory Cells ◽  
Negative Regulator ◽  
Microbial Pathogens ◽  
Cellular Activation ◽  
Mouse Macrophages ◽  
Non Coding Rnas ◽  
Maximal Induction

Toll-like receptors (TLRs) are major receptors that enable inflammatory cells to recognize invading microbial pathogens. MicroRNAs are small non-coding RNAs that play important regulatory roles in a variety of biological processes. In this study, we found that a microRNA, miR-147, was induced upon stimulation of multiple TLRs and functioned as a negative regulator of TLR-associated signaling events in murine macrophages. We first demonstrated that the NMES1 transcript was a functional primary miR-147. miR-147 was induced in LPS-stimulated mouse macrophages and under in vivo conditions in the lungs of LPS-treated mice. Expression of miR-147 was greater after cellular activation by TLR4 than after engagement of either TLR2 or TLR3, suggesting that maximal induction of miR-147 required activation of both NF-κB and IRF3. TLR4-induced miR-147 expression was both MyD88- and TRIF-dependent. The miR-147 promoter was responsive to TLR4 stimulation and both NF-κB and STAT1α bound to the miR-147 promoter. miR-147 mimics or induced expression of miR-147 decreased, whereas miR-147 knockdown increased inflammatory cytokine expression in macrophages stimulated with ligands to TLR2, TLR3, and TLR4. These data demonstrate a negative-feedback loop in which TLR stimulation induces miR-147 to prevent excessive inflammatory responses.

Sulforaphane Reduces HMGB1-Mediated Septic Responses and Improves Survival Rate in Septic Mice

2017 ◽  
Vol 45 (06) ◽  
pp. 1253-1271 ◽  
Author(s):  
In-Chul Lee ◽  
Dae Yong Kim ◽  
Jong-Sup Bae
Keyword(s):  
Endothelial Cells ◽  
Survival Rate ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Cecal Ligation ◽  
High Mobility ◽  
Umbilical Vein Endothelial Cells

Sulforaphane (SFN), a natural isothiocyanate present in cruciferous vegetables such as broccoli and cabbage, is effective in preventing carcinogenesis, diabetes, and inflammatory responses. Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity is emerging as an attractive therapeutic strategy in the management of severe sepsis or septic shock. In this study, we examined the effects of SFN on HMGB1-mediated septic responses and survival rate in a mouse sepsis model. The anti-inflammatory activities of SFN were monitored based on its effects on lipopolysaccharide (LPS)- or cecal ligation and puncture (CLP)-mediated release of HMGB1. The antiseptic activities of SFN were determined by measuring permeability, leukocyte adhesion and migration, and the activation of pro-inflammatory proteins in HMGB1-activated human umbilical vein endothelial cells (HUVECs) and mice. SFN inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. SFN also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with SFN reduced CLP-induced release of HMGB1 and sepsis-related mortality and pulmonary injury in vivo. Our results indicate that SFN is a possible therapeutic agent that can be used to treat various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway.

Sign in / Sign up

Export Citation Format

Share Document