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Pam3Cys-treated HUVEC before and after TLR2 knockdown

ABSTRACT: The vascular endothelium is integrally involved in the host response to infection and in organ failure during acute inflammatory disorders such as sepsis. Gram-negative and gram-positive bacterial lipoproteins circulate in sepsis and can directly activate the endothelium by binding to endothelial cell (EC) Toll-like receptor 2 (TLR2). In this report, we perform the most comprehensive analysis to date of the immune-related genes regulated after activation of endothelial TLR2 by bacterial di- and triacylated lipopeptides. We found that TLR2 activation specifically induces the expression of IL6, IL8, CSF2, CSF3, ICAM1 and SELE by human umbilical vein ECs and human lung microvascular ECs. These proteins participate in neutrophil recruitment, adherence and activation at sites of inflammation. Significantly, our studies demonstrate that bacterial lipopeptides activate human EC exclusively through TLR2, that TLR2-mediated EC responses are specifically geared towards recruitment, activation, and survival of neutrophils and not mononuclear leukocytes, and that ECs do not require priming by other inflammatory stimuli to respond to bacterial lipopeptides. This study suggests that endothelial TLR2 may be an important regulator of neutrophil trafficking to sites of infection in general, and that direct activation of lung endothelial TLR2 may contribute to acute lung injury during sepsis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE30117 | GEO | 2011/06/23

SECONDARY ACCESSION(S): PRJNA144041

REPOSITORIES: GEO

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Project description:The vascular endothelium constitutes the inner lining of the blood vessel and is directly involved in the control of blood fluidity, vascular tone, platelet aggregation, regulation of immunity, inflammation, and angiogenesis. Malfunction and injuries of the endothelium can cause cardiovascular diseases, which are the leading causes of death globally. The impairment of the endothelium is a hallmark in other diseases as well such as sepsis, stroke, tumor growth, insulin resistance, venous thrombosis, and chronic kidney failure. Generation of effective sources to replace injured endothelial cells (ECs) could have significant clinical impact and somatic cell sources like peripheral or cord blood cannot credibly supply enough endothelial cell progenitors for multitude of treatments. Pluripotent stem cells are a promising source for a reliable EC supply that have the potential to repair, reconstruct, replace damaged cells, and revascularize ischemic areas in order to restore tissue function and treat vascular diseases. We have developed methods to differentiate induced pluripotent stem cells (iPSCs) efficiently and robustly across multiple iPSC lines into non-tissue-specific pan vascular ECs (iECs) with high purity. These iECs present with canonical endothelial cell markers and exhibit measures of endothelial cell functionality with uptake of acetylated low-density lipoprotein (Dil-Ac-LDL) and tube formation. Using proteomic analysis, we revealed the iECs are more proteomically similar to established umbilical vein ECs (HUVECs) than to iPSCs. Post-translational modifications (PTMs) were most shared between HUVECs and iECs, and potential targets for increasing the proteomic similarity of iECs to HUVECs were identified. PTM analysis can be used in the future to glean additional insights and generate novel hypotheses. Here we demonstrate an efficient robust method to differentiate iPSCs into functional ECs, and for the first time provide a comprehensive protein expression profile of iECs, which indicates their similarities with a widely used immortalized HUVECs, allowing for further mechanistic studies of EC development, signaling and metabolism for future regenerative applications.

Project description:An early aspect of sepsis is dysregulated activation of endothelial cells (EC), initiating a cascade of inflammatory signaling leading to leukocyte adhesion/migration and organ damage. Therapeutic targeting of ECs has been hampered by concerns regarding ECs heterogeneity from different organs. Using a combination of in vitro and in silico analysis, we present a comprehensive analysis of proteomic changes in mouse lung, kidney and liver ECs following exposure to a clinically relevant cocktail of proinflammatory cytokines. Mouse lung, liver and kidney ECs were incubated with TNFα/IL-1β/IFNγ for 4 or 24 hrs to model inflammatory responses during sepsis. Quantitative label-free global proteomics was performed on the ECs to identify differentially expressed proteins (DEP). Proteins with an abundance ratio >2.0 or <0.5 and an adjusted p<0.05 were characterized as upregulated or downregulated respectively. Gene Ontology (GO) classification was used to determine biological processes that regulate EC function during sepsis and PANTHER was used to classify the molecular function of the identified proteins. Finally, an interactive pathway was developed to investigate signaling within ECs and across organs. Proteomic analysis identified both unique and shared DEPs between the ECs specific to lung, liver and kidney. Using GO, 5 Biological Processes (BP) for each of the organ specific ECs were identified. Interestingly, 4 of the top 5 GO BPs were observed in all 3 ECs at 4 and 24 hrs: defense response to other organism, innate immune response, response to bacterium and response to cytokine. However, these BPs were found to be differentially regulated. For example, at 4 and 24 hrs, lung ECs which have the highest number and highest fold expression of upregulated proteins (189 and 316 respectively), also have higher numbers of unique upregulated proteins (124 vs 194) compared to liver (98 vs 121) and kidney ECs (109 vs 136). The liver showed the greatest number of conserved proteins between 4 and 24 hrs (56) compared to lung (50) and kidney (51). The number of common proteins between all three ECs increases from 38 at 4 hrs to 79 at 24 hrs, indicating more uniformity in ECs proteomic expression during the progression of sepsis. The PANTHER database was used to classify proteins in different functional groups (e.g. defense/immunity) at 4 and 24 hrs. Thus, BPs and PANTHER hits can provide insight into why some organs are more susceptible to sepsis early on and show that as sepsis progresses, some protein expression patterns become more uniform while additional organ specific proteins are expressed. Proteomic analysis of organ-specific ECs provides further understanding of how sepsis affects multiple organs across time and supports future proteomic, temporal studies of EC dysfunction.

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Pam3Cys-treated HUVEC before and after TLR2 knockdown

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