Background
Patients with sepsis commonly suffer from abnormal activation of the coagulation system, including diffuse intravascular coagulation and microthrombi formation. Anti-coagulant-mediated regulation of thrombosis-dependent endothelial injury and repair following sepsis may represent a therapeutic approach for patients with sepsis [
1]. Sepsis-associated acute lung injury (ALI) is a common and severe consequence of infection, which contributes to significant morbidity and mortality in critically-ill patients [
2]. A large number of studies show that lung endothelial cells are key modulators and orchestrators of ALI [
3]. Lipopolysaccharide (LPS), the main component of the outer membrane of Gram-negative bacteria, can cause sepsis-associated ALI and endothelial barrier dysfunction [
4].
Endothelial permeability is controlled by dynamic cytoskeletal remodeling and inter-cell junction protein complexes, which are also tightly linked to the cytoskeleton [
5]. Numerous studies demonstrated that the actin cytoskeleton is critical for controlling endothelial barrier function and vascular permeability. Rho GTPases are the foremost mediators of regulation of actin remodeling, which is involved in endothelial barrier stability in response to different stimuli [
6‐
8]. The Rho guanine nucleotide exchange factor, GEF-H1, acts as a microtubule-dependent regulator and couples microtubules to the Rho GTPase-dependent actin cytoskeleton [
9]. An increasing body of evidence corroborates the role of microtubule dynamics in regulation of endothelial permeability [
10,
11].
Unfractionated heparin (UFH), a widely used anticoagulant drug, participates in the regulation of multifarious biological functions [
12]. We previously demonstrated that LPS induced remodeling of the F-actin cytoskeleton and formation of stress fibers, which increased human umbilical vascular endothelial cell (HUVEC) permeability; in contrast, UFH protected cells from endothelial hyperpermeability [
13]. Furthermore, we also showed that UFH attenuated pulmonary vascular hyperpermeability in mouse model of sepsis via the RhoA/Rho kinase (ROCK) pathway [
14]; however, the mechanism of action was not clear. Therefore, we investigated whether UFH ameliorates LPS-induced endothelial barrier dysfunction by inhibiting MT disassembly, and regulating GEF-H1 expression and F-actin remodeling.
The p38 MAPK pathway is involved in various biological functions, including endothelial barrier function in response to exogenous and endogenous stimuli [
15,
16]. We observed that UFH regulated anti-inflammatory activity by inhibiting p38 MAPK activation [
17,
18]. Hence, we investigated whether the p38 MAPK pathway was involved in UFH-mediated attenuation of LPS-induced MT disassembly and endothelial barrier dysfunction.
Methods
Reagents and antibodies
LPS from Escherichia coli 055:B5 was obtained from Sigma (St. Louis, MO, USA), and UFH was obtained from Shanghai No.1 Biochemical & Pharmaceutical Co. (China). Rabbit polyclonal α-tubulin, GEF-H1, p-MYPT1, p38 MAPK, and p-p38 MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal acetylated-α-tubulin and the p38/MAPK inhibitor SB203580 were obtained from Abcam (Cambridge, MA, USA). Nocodazole was purchased from Sigma-Aldrich (St. Louis, MO). Mouse monoclonal antibody against GAPDH was from Invitrogen (Carlsbad, CA, USA). Tetraethyl rhodamine isothiocyanate (TRITC)-phalloidin (Solarbio, China), donkey anti-mouse Cy2 (Jackson, USA), and donkey anti-rabbit Alexa Fluor® 488 (Abcam, USA) were used for immunofluorescence.
Animal studies
Male C57BL/6 mice weighing 20–25 g were obtained from the Experimental Animal Center of China Medical University. Mice were randomized into four groups: vehicle, UFH, LPS and LPS + UFH. Briefly, mice were anesthetized and given a 30 mg/kg body weight LPS (in 100 μL of saline) intraperitoneally. Subcutaneous injection of 8 units UFH diluted in sterile saline (LPS + UFH group) or equal volume sterile saline (LPS group) was administered 30 min prior to the LPS injection. The dose of LPS and UFH used in vivo are identical to that used previously [
14,
19]. After 6 h, the animals were killed under anesthesia and lung tissue samples were collected for subsequent experiments. Evans blue extravasation, lung wet/dry (W/D) weight ratio, and histological assessment of lung injury was conducted as previously described [
14]. The animal protocols were formulated in accordance with the guidelines of our University Experimental Animal Administration Committee.
Cells and cell treatment
HPMECs were supplied by ScienCell Research Laboratories (USA). HPMECs were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Invitrogen, USA), and cultured at 37 °C in a laboratory CO2 incubator with a humidified atmosphere of 5% CO2–95% air. HPMECs were utilized at passages 4–8 in all experiments.
Cell viability assay
Methyl thiazoyltetrazolium (MTT) was adopted to detect the toxicity of LPS on HPMECs. The cells (1–2 × 104 cells/well) were plated on 96-well plates and stimulated with different concentrations LPS for 6 h. Then HPMECs were washed with PBS and incubated with 200 μl MTT medium (1 mg/ml) at 37 °C for 4 h. Afterwards, the medium was discarded and dimethyl sulfoxide (DMSO) (150 μl/well) was used to solubilize formazan. Optical densities (OD) were measured at 490 nm using a plate-reading spectrophotometer. The value of each well was presented as a percentage of the control group. The experiments were repeated five times and the data were calculated as means ± SD.
Measurement of transendothelial permeability
HPMECs (1.5 × 10
5 cells/well) were seeded on the 24-well Transwell system (Greiner Bio-One, 0.4-mm pore size, 6.5-mm diameter, transparent, Costar,The Netherlands). The cells were cultured for 4 days and the media was replaced with serum-starved medium for 2 h before exposure to any inhibitor/stimulator. The endothelial cell barrier function was evaluated by measurements of transendothelial electrical resistance (TEER) across confluent cells utilizing Millicell-ERS (MERS00002, Millipore, Bedford,USA). Transendothelial permeability was analyzed using Chemicon’s in vitro vascular permeability assay, which utilizes fluorescein isothiocyanate–dextran (FITC-dextran, 40-kDa, Sigma, USA) [
20]. The Transwell plate was removed from the incubator to quantify the passage of FITC-dextran across the monolayer. Using a micropipetter, media (50 μl) was removed from the bottom chamber of each Transwell and transferred to a 96-well plate. The 96-well fluorescence plate reader was used at an excitation wavelength of 488 nm and emission wavelength of 515 nm. Data was represented graphically as raw data in the arbitrary fluorescence units generated by the plate reader.
Immunofluorescence and image analysis
HPMECs were seeded on glass coverslips and maintained as described above. The cells were then fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 5 min, washed in phosphate buffered saline (PBS), and blocked with 5% bovine serum albumin (BSA) for 1 h. The coverslips were incubated with rabbit anti-α-tubulin (1:100) or mouse anti-acetylated-α-tubulin primary antibody (1:200) at 4 °C overnight for determining the MT and acetylated tubulin structure. The coverslips were washed with PBS, followed by incubation with secondary antibodies for 1 h at 37 °C. The actin cytoskeleton was examined by immunofluorescence staining with TRITC-phalloidin for 30 min at 37 °C. The nuclei of the HPMECs were counterstained by DAPI. HPMECs were imaged by microscopy (Olympus BX61, Tokyo, Japan). Images obtained using the 60× objective were processed using the Image J software (National Institute of Health, Bethesda, MD) to outline the cell borders and compute the unoccupied area [
13].
Extraction and quantification of tubulin fractions
The monomeric and polymerized tubulin fractions were extracted from HPMECs using a method previously described by Putnam [
21]. HPMECs cultured in 6-well culture plates were washed in an MT stabilization buffer (MTSB, 37 °C). MTSB contained 0.1 M piperazine-1, 4-bisethanesulfonic acid (PIPES), 2 mM ethylene glycol-bis (β-aminoethylether)-N,N,N9,N9-tetraacetic acid (EGTA), 0.1 mM ethylene diamine tetraacetic acid (EDTA), 0.5 mM MgCl
2, and 20% glycerol (pH 6.8). HPMECs were incubated with MTSB + 0.1% Triton X-100 + protease inhibitor cocktail (1:100, Biotool) + phenylmethylsulphonyl fluoride (PMSF, 1:100, Beyotime Biotechnology) + phosphatase inhibitor cocktail (1:100, Biotool). The monomeric and polymeric tubulin fractions were quantified by western blot analysis.
Western blot analysis
The monomeric and polymeric tubulin fractions were prepared as described above. The protein extracts were probed using rabbit anti-α-tubulin antibody (1:1000). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked for 1 h in 5% BSA in Tris-buffered saline Tween 20 (TBST) and incubated overnight with primary antibodies against acetylated tubulin (1:2000), GEF-H1 (1:1000), p-MYPT1 (1:1000), p38 (1:1000), and p-P38 (1:1000). After washing with TBST, the membranes were incubated with horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse IgG (Bio-Rad). GAPDH was reprobed as a loading control. The immunocomplexes were developed using the enhanced chemiluminescence (ECL) Plus kit (Amersham). The Image J software (NIH) was used to quantify the intensity values of the bands. Each experiment was repeated thrice.
Statistical analysis
All statistical analyses were performed using Graphpad Prism 6.0 software. The results were expressed as mean ± SD. Statistical analyses were conducted using one-way ANOVA followed by post hoc analysis. P < 0.05 indicated a statistical significance.
Discussion
Our study elucidates a novel mechanism via which UFH exerts protective effects in endothelial barrier dysfunction of ALI. This mechanism suggests that UFH protects the endothelial barrier by stabilizing MTs, inhibiting GEF-H1, and remodeling actin in vitro and in vivo, which may be related to its inhibition of LPS-induced activation of p38 MAPK. This provides a new theoretical basis for the use of UFH in the treatment of sepsis-associated ALI in the clinic.
UFH possesses several biological properties beyond its anticoagulant activity, and has been reported to produce clinical benefit in patients with sepsis [
24,
25]. Our previous study demonstrated that pretreatment of mice with sepsis with UFH strongly ameliorates inflammation and coagulation [
19]. Sepsis is a common disease and is often complicated with ALI. Treatment with UFH can also attenuate inflammatory responses in a rat model of LPS-induced ALI by downregulating nuclear factor-κB signaling pathway [
26]. As for the dose of LPS used to study sub-lethal lung injury, a variety of different dosages of LPS have been used, such as 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 30 mg/kg and 40 mg/kg [
27‐
31]. Low doses of LPS may not be sufficient to induce injury, while high doses may be lethal. In our study, the dose of LPS used was 30 mg/kg, which was referred to previous studies from Kawasaki et al. [
30] and Han et al. [
14]. An in vivo study showed that UFH pretreatment by inhalation inhibits alveolar procoagulant reaction and early inflammatory response, promotes fibrinolysis, and alleviates pulmonary pathology in rats with endotoxemia-induced lung injury, which is especially more effective than administration of UFH after LPS challenge [
32]. It was suggested that UFH pre-treatment was more effective on lung injury. Based on this report and the results of our previous studies, UFH was administered before LPS injection in this study. Additionally, our previous in vitro study showed that the levels of Tie2, an endothelial cell-specific tyrosine kinase receptor affecting stable vasculature, was markedly reduced by 6-h LPS stimulation, which showed no obvious difference when compared with the levels obtained under 12-h LPS stimulation [
13]. An in vivo study demonstrated that levels of pulmonary vascular endothelial growth factor (VEGF) at different time points after LPS injection (1, 3, 6, and 10 h) were down-regulated, especially at 6 h after LPS treatment [
33]. In the present study, we observed that HPMECs stimulated by LPS for more than 6 h showed marked decreased in the TEER of HPMEC monolayer. Based on the above results, we assessed changes in vascular injury at 6 h post-LPS in this study. However, 6 h post-LPS may not be the timepoint of peak injury in mice, and that lack of an injury timecourse is a weakness of the study.
Loss of endothelial barrier integrity is central to the pathogenesis of sepsis-associated ALI [
34]. nocodazole, as a microtubule poison, destroys the vascular endothelial barrier [
35]. Our results showed that nocodazole increased the permeability of HPMECs, expression of GEF-H1 and MYPT1 phosphorylation and F-actin remodeling, suggesting microtubule disassembly is sufficient to cause the increase in GEF-H1 and p-MYPT1.The microtubule stabilizer, taxol, inhibits thrombin or TGF- β1-induced activation of Rho GTPases in endothelial barrier dysfunction during lung injury [
36,
37]. A study showed that GEF-H1 overexpression antagonized the heparin-mediated inhibition of pulmonary artery smooth muscle cell (PASMC) proliferation, indicating that the inhibitory effect of heparin was exerted partially via the GEF-H1/RhoA/ROCK/p27 signaling pathway and GEF-H1 down-regulation [
38]. Additionally, a previous report revealed that siRNA-mediated depletion of GEF-H1 in HeLa cells prevents nocodazole-induced microtubule disassembly and RhoA activation and cell contractility were rescued by reintroduction of siRNA-resistant GEF-H1, suggesting GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA [
35]. In our study, UFH pretreatment blocked the LPS-induced increase in GEF-H1 expression in LPS-induced ALI mice and HPMECs. Our results indicated that UFH ameliorates LPS-induced endothelial barrier dysfunction by inhibiting GEF-H1 expression. We also confirmed that UFH ameliorates nocodazole-induced MTs disassembly and endothelial barrier dysfunction via increasing the acetylation of tubulin and decreasing the expression of GEF-H1 and MYPT1 phosphorylation, implying the protective effects of UFH can be reversed by downstream activation of microtubule disassembly.
Myosin phosphatase regulates the interaction of actin and myosin in response to small GTPase Rho signaling, and Rho activity inhibits myosin phosphatase via ROCK. MYPT1 is one of the subunits of myosin phosphatase, phosphorylation of which results in cytoskeletal reorganization. In this study, we observed that UFH pretreatment remarkably decreased LPS-induced increase in MYPT1 phosphorylation both in vitro and in vivo. This is in agreement with the results of our previous study, which showed that UFH treatment significantly attenuated the increase in p-MYPT1 levels in the lung tissues of LPS-challenged mice [
14].
The Rho-Rho kinase cascade triggers MYPT1 phosphorylation, which might lead to actin reorganization and stress fiber formation, resulting in the formation of paracellular gaps and increase in endothelial permeability. GEF-H1 links changes in microtubule integrity to a Rho-dependent regulation of the actin cytoskeleton. Our previous study showed that pretreatment with UFH decreased the formation of stress fiber and intracellular gaps induced by LPS [
13], which is in agreement with the results of this study.
GEF-H1 is uniquely positioned for transducing signals after MT depolymerization. It is bound to MTs and is released and activated upon MT depolymerization. The LPS-induced decrease in dynamic MT pool occurs as a result of posttranslational modifications of tubulin [
39]. Further, our study showed that UFH administration increased the levels of polymerized tubulin and decreased monomeric tubulin levels in LPS-challenged cells. Additionally, UFH pretreatment increased acetylated tubulin levels both in vitro and in vivo, which could be the mechanism underlying UFH-mediated prevention of LPS-induced MT instability.
The p38 MAPK pathway plays a crucial role in the regulation of cell growth, differentiation, proliferation, apoptosis, invasion, and metastasis. MTs act as docking sites and reservoirs for various proteins, including MAPKs, PI3Ks, and GTPases. p38 MAPK signaling was affected by microtubule state [
40]. SB-203580, a p38 MAPK inhibitor, attenuated nocodazole-induced MT depolymerization, actin remodeling, and endothelial barrier dysfunction [
41]. Previous studies showed SB-203580 decreased p38 phosphorylation [
42,
43], which was in accordance with our result in this study. However, some studies reported that phosphorylation of p38 MAPK was not influenced by SB 203580 [
44,
45]. Therefore, the involvement of p38 still need to be confirmed by depletion experiment using siRNA technique. Zhou et al. showed that the p38/mitogen-activated protein kinase pathway is implicated in LPS-induced microtubule depolymerization via up-regulation of microtubule-associated protein 4(MAP4) phosphorylation in human vascular endothelium; however, SB203580 did not completely block the LPS-induced MAP4 phosphorylation in ECs, suggesting other LPS-related mechanisms are involved in LPS-induced MT disassembly [
46]. Sepsis-related inflammatory factors promoted endothelial cell activation, dysfunction, and apoptosis via activation of the p38 MAPK pathway [
47]. UFH was reported to enhance the barrier function of resting endothelium [
48‐
50]. An early study has revealed that UFH suppress inflammatory-mediated tissue factor expression and increase the anticoagulant properties of macro- and micro-vascular endothelial cells [
51]. Our previous studies showed that UFH exerted its anti-inflammatory effect by inhibiting the p38 MAPK pathway [
17,
18], which were in agreement with the results of this study. p38 MAPK, a downstream effectors of RhoA-GTPase signaling, was activated by RhoA [
52], while increasing of GEF-H1 expression could lead to the activation of RhoA. The underlying mechanisms of UFH inhibition on p38 MAPK may be related to the inhibition of GEF-H1 expression. In addition, a previous report uncovered that microtubule disassembly by nocodazole induced rapid decreases in transendothelial electrical resistance and actin cytoskeletal remodeling [
53], which was consistent with our results. In this study, we observed that UFH prevented MT destabilization caused by LPS-induced actin microfilament cytoskeletal changes and barrier dysfunction in HPMECS, which is similar to the effects of SB 203580 treatment. This indicates that UFH protected cells from LPS-stimulated pulmonary microvascular endothelial dysfunction by stabilizing MTs involved with the p38 MAPK pathway. However, MT dynamics is regulated by other pathways [
6,
46,
47], and whether UFU acts in regulating endothelial barrier activity requires further investigation.
It is generally known that sugar-based endovascular structure is essential for proper microvascular physiology, which gets damaged by SIRS mainly due to the oxidative stress [
54]. Our previous studies had found that UFH or N-desulfated/re-N-acetylated heparin (NAH) can protect glycocalyx from shedding in septic shock model of beagle dogs or mice [
55,
56]. The effect of UFH on endothelial barrier protection in the ALI model may be implicated with building bricks for self-reconstitution of the glycocalyx .
Compared to young mice, aged mice exhibit increased severity of lung injury, as demonstrated by higher diffuse alveolar damage and alveolar wall thickening, and increased alveolar and vascular permeability [
57]. Future studies on UFH’s effect on LPS-induced ALI in aged mice are warranted. Meanwhile, confirmation could also be performed in future studies of female mice. In the present study, UFH was administered before LPS injection, which is clinically irrelevant. Therefore, a clinically relevant study, in which UFH will be administered after LPS induction, is still required in future.