Lipopolysaccharide induces a fibrotic-like phenotype in endothelial cells

Endothelial dysfunction is crucial in endotoxaemia-derived sepsis syndrome pathogenesis. It is well accepted that lipopolysaccharide (LPS) induces endothelial dysfunction through immune system activation. However, LPS can also directly generate actions in endothelial cells (ECs) in the absence of participation by immune cells. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge. However, the mechanism that confers endothelial resistance to LPS is not known. LPS-resistant ECs exhibit a fibroblast-like morphology, suggesting that these ECs enter a fibrotic programme in response to LPS. Thus, our aim was to investigate whether LPS is able to induce endothelial fibrosis in the absence of immune cells and explore the underlying mechanism. Using primary cultures of ECs and culturing intact blood vessels, we demonstrated that LPS is a crucial factor to induce endothelial fibrosis. We demonstrated that LPS was able and sufficient to promote endothelial fibrosis, in the absence of immune cells through an activin receptor–like kinase 5 (ALK5) activity–dependent mechanism. LPS-challenged ECs showed an up-regulation of both fibroblast-specific protein expression and extracellular matrix proteins secretion, as well as a down-regulation of endothelial markers. These results demonstrate that LPS is a crucial factor in inducing endothelial fibrosis in the absence of immune cells through an ALK5-dependent mechanism. It is noteworthy that LPS-induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxaemia-derived sepsis syndrome and other inflammatory diseases.


RNA isolation and quantitative real-time PCR
QPCR experiments were performed to measure CD31, VE-cadherin, α-sma, FSP-1, fibrinogen, and type III collagen mRNA levels in HUVEC cells. Total RNA was extracted with Trizol according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). DNAse I-treated RNA was used for reverse transcription using the Super Script II Kit (Invitrogen, Carlsbad, CA).
Equal amounts of RNA were used as templates in each reaction. QPCR was performed using the SYBR Green PCR Master Mix (AB Applied Biosystems, Foster City, CA). Assays were run using a Rotor-gene system (Corbet Research) instrument. Data are presented as relative mRNA levels of the gene of interest normalized to relative levels of 28S mRNA using the ΔCt method. 2 The following primers pairs were used: CD31, forward 5'-GAGAGTATTACTGCACAGCC-3´ and reverse 5´-GAGCAATGATCACTCCGATG-3´ (expected fragment size 140 bp); VE-
Resolved proteins were transferred to a nitrocellulose or PVDF membrane and non-specific binding was blocked using 5% BSA in PBS for 1 h at pH 7.4. The blocked membrane was incubated with the appropriate primary antibody, washed twice, and incubated with a secondary antibody. Bands were revealed using a peroxidase-conjugated IgG antibody. Tubulin was used as a loading control. For detection of the phosphorylated form of smad2 (p-smad2), we used an antibody against p-smad2 (p-ser 465/467) and total smad2 was used as a loading control.
Peroxidase activity was detected through enhanced chemiluminescence (Bio-Rad, CA) and images were acquired using Fotodyne FOTO/Analyst Luminary Workstations Systems (Fotodyne, Inc., Hartland, WI). Protein content was determined by densitometric scanning of immunoreactive bands and intensity values were obtained by densitometry of individual bands normalized against tubulin or total smad. For a detailed list of antibodies used, see Table S1.

Fluorescent immunocytochemistry and immunohistochemistry
Immunocytochemistry: cells were washed twice with PBS and fixed with 3.7% PFA for 30 min at RT, treated with 50 mmol/L NH 4 Cl for 15 min at RT, permeabilized with 0.1% Triton X-100 in PBS for 30 min at RT, and blocked for 2 h at RT with 3% BSA in PBS. The cells were subsequently washed again and incubated with the first primary antibodies. Then, cells were washed twice and incubated with the first secondary antibodies. For immunofluorescent double staining, the cells were washed with PBS twice and the above staining procedure was repeated for the second set of primary and second secondary antibodies. Samples were mounted with ProLong Gold antifade mounting medium with DAPI (Invitrogen).
Immunohistochemistry: samples obtained from human umbilical cord vein were fixed using Bouin's fixative for 24 h at RT. Samples were permeabilized with 0.1% Triton X-100 in PBS for 40 min at RT and blocked for 3 h at RT with 3% bovine serum albumin in PBS.
Samples were subsequently washed again and incubated with the first primary antibodies. Then, cells were washed twice and incubated with the first secondary antibodies. For immunofluorescent double staining, samples were washed with PBS twice and the above staining procedure was repeated for the second set of primary and second secondary antibodies.
Nuclei were stained using Hoestch 33342 (Sigma). Images were acquired and analyzed using a Floid Cell Imaging Station (Life Technologies™). For a detailed list of antibodies used see Table   S2.

Immunohistochemistry by hematoxylin-eosin stain
Human umbilical cord vein were fixed in 10% buffer formalin solution. The veins were sectioned using a cryostat (Leica), and slices (15 µm) were prepared with hematoxylin-eosin stain and examined with light microscopy.

Cell viability determination
MTT assay: cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay (Invitrogen, Eugene, Oregon, USA), in which cell viability was quantified by the amount of MTT reduction. 1 After different treatments were performed, cells were co-incubated with anhydrous MTT (0.5 mg/mL) for 4 h and then solubilized with an isopropanol/DMSO solution. The optical density value was measured at 540 nm. At least three separate experiments were performed in triplicate. The data are expressed as cell viability normalized against untreated cells.
Propidium iodide incorporation assay: After treatments, total HUVEC cells were harvested by centrifugation at 800 x g for 5 min, and the pellet was suspended in 200 μl PBS.
Then, cells were washed once with PBS and stained with propidium iodide (PI, 10 g/mL) for 20 min at room temperature in the dark. DNA content was analyzed with a flow cytometry system (FACSCanto, BD Biosciences, CA, USA). A minimum of 10,000 cells/sample was analyzed. PI intensity analysis was performed using FACSDiva software (BD Biosciences, CA, USA).

Small interfering RNA against ALK5 and transfection
SiGENOME SMARTpool siRNA (four separated siRNAs per human ALK5 transcript) were purchased from Dharmacon (Dharmacon, Lafayette, CO). The following siRNA were used: human ALK5 (siRNA-ALK5) and non-targeting siRNA (siRNA-CTRL) used as a control. In brief, HUVEC were plated overnight in 24-well plate and then transfected with 5 nmol/L siRNA using DharmaFECT 4 transfection reagent (Dharmacon) used according to the manufacturer's protocols in serum-free medium for 6 hours. After 24 to 48 transfection, experiments were performed.
TGF1 and TGF2 were purchased from R&D Systems. Buffers and salts were purchased from Merck Biosciences (Darmstadt).

Data analysis
All results are presented as the mean±SD. An ANOVA followed by the Bonferroni or Dunn's post hoc tests were used and considered significant at p<0.05.