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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Shock. 2021 May 1;55(5):660–665. doi: 10.1097/SHK.0000000000001564

Fibrinogen activates PAK1/cofilin signaling pathway to protect endothelial barrier integrity

Feng Wu 1, Amanda Chipman 1, Jing-Fei Dong 2, Rosemary Ann Kozar 1
PMCID: PMC8211399  NIHMSID: NIHMS1712604  PMID: 32433215

Abstract

Introduction:

We recently demonstrated that fibrinogen stabilizes syndecan-1 on the endothelial cell (EC) surface and contributes to EC barrier protection, though the intra-cellular signaling pathway remains unclear. P21 (Rac1) activated kinase 1 (PAK1) is a protein kinase involved in intracellular signaling leading to actin cytoskeleton rearrangement and plays an important role in maintaining endothelial barrier integrity. We therefore hypothesized that fibrinogen binding to syndecan-1 activated the PAK1 pathway.

Methods:

Primary human lung microvascular endothelial cells (HLMEC) were incubated in 10% lactated Ringers (LR) solution or 10% fibrinogen saline solution (5 mg/ml). Protein phosphorylation was determined by Western blot analysis and endothelial permeability measured by FITC-dextran. Cells were silenced by siRNA transfection. Protein concentration was measured in the lung lavages of mice.

Results:

Fibrinogen treatment resulted in increased syndecan-1, PAK1 activation (phosphorylation), cofilin activation (dephosphorylation), as well as decreased stress fibers and permeability when compared with LR treatment. Cofilin is an actin binding protein that depolymerizes F-actin to decrease stress fiber formation. Notably, fibrinogen did not influence myosin light chain (MLC) activation (phosphorylation), a mediator of EC tension. Silencing of PAK1 prevented fibrinogen-induced dephosphorylation of cofilin and barrier integrity. Moreover, to confirm the in-vitro findings, mice underwent hemorrhagic shock and were resuscitated with either LR or fibrinogen. Hemorrhage shock decreased lung p-PAK1 levels and caused significant lung vascular leakage. However, fibrinogen administration increased p-PAK1 expression to near sham levels and remarkably prevented the lung leakage.

Conclusion:

We have identified a novel pathway by which fibrinogen activates PAK1 signaling to stimulate/dephosphorylate cofilin, leading to disassembly of stress fibers and reduction of endothelial permeability.

Introduction

The endothelial glycocalyx provides a protective layer to endothelial cells (1, 2). Hemorrhagic shock causes shedding of the glycocalyx, includes its backbone proteoglycan, syndecan-1. Syndecan-1 shedding contributes to endothelial injury with subsequent hyperpermeability and tissue edema, both of which contribute to organ injury and dysfunction (1, 3–5).

As a cell surface receptor, syndecan-1 engages extra-cellular ligands on the cell surface to regulate various intracellular signaling pathways through its highly conserved cytoplasmic domain. Our previous study demonstrated that fibrinogen functioned as such a ligand (6). Fibrinogen is a glycoprotein found in plasma and plays a key role in hemostasis. We have been interested in its role as an endothelial protector and have shown that it binds to cell surface syndecan-1 to stabilize the glycocalyx (6). Further, this binding decreased endothelial stress fiber formation and resulted in endothelial barrier enhancement. However, much less is known about the signaling mechanism underlying fibrinogen/syndecan-1-induced endothelial barrier protection.

The endothelial barrier is largely influenced by the monolayer tension which is controlled by two types of Rho GTPase molecules, RhoA/C and Rac1/Cdc42 (7–9). RhoA/C activates Rho kinase (RhoK) to phosphorylate myosin light chain phosphatase (MLCP) and thus inhibit its phosphatase function towards myosin light chain (MLC). RhoK therefore increases MLC phosphorylation which leads to the generation of stress fibers and endothelial tension (7, 8). On the other hand, Rac1/Cdc42 can activate P21 (Rac1)-activated kinase 1 (PAK1) signaling to stimulate/dephosphorylate cofilin, leading to disassembly of stress fibers and reduction of endothelial tension (7, 8). We thus hypothesized that fibrinogen binding to syndecan-1 would inhibit MLC phosphorylation and/or activate PAK1/cofilin pathway to enhance endothelial barrier integrity.

Materials and Methods

In-vitro

Primary endothelial cell culture:

Human lung microvascular endothelial cells (HLMEC; Lonza) were cultured as we described previously (6) and were grown to confluence in endothelial basic medium-2 (EBM-2; Lonza) supplemented with 10% fetal bovine serum (FBS), human recombinant epidermal growth factor, human recombinant insulin-like growth factor-1, human basic fibroblast growth factor, vascular endothelial growth factor, hydrocortisone, ascorbic acid, heparin, gentamicin, and amphotericin B. Endothelial cells (passages 6 −9) were serum-starved then treated with 10% lactated Ringers (LR) solution or 10% fibrinogen saline solution (final concentration in medium: 5 mg/ml). The fibrinogen used here was a highly purified preparation of fibrinogen derived from pooled human plasma and was produced by CSL Behring GmbH (RiaSTAP, Germany). For treatment of bleeding patients, the target blood concentrations of fibrinogen therapy are 1.5 to 2.0 mg/ml (10). In the present study, we used 5 mg/ml fibrinogen which is higher than the expected doses in vivo, but our previous results have shown that fibrinogen at 2.5, 5.0 and 10 mg/ml afforded similar protection to endothelial barrier (6).

Transient transfection:

HLMECs were seeded in 6-well plates and grown for 24 h in antibiotic-free EBM-2 containing 5% FBS and supplements. Cells were then transfected by incubation with 100 nM syndecan-1 siRNA, PAK1 siRNA, or scrambled RNA (scRNA) (MilliporeSigma) and 2.5 ul/ml Lipofectamine 2000 (Thermo Fisher Scientific) in antibiotic-free Opti-MEM for 24 h. The medium was then changed to the growth medium, and the cells were cultured for another 48 h prior to assays. The knock-down of respective proteins was validated by Western blot analysis.

Western blotting:

The antibodies included anti-syndecan-1 (sc-12765, Santa Cruz Biotechnolgies), anti-GAPDH (PA1-987, Thermo Scientific), anti-p-PAK1 (#2605, Cell Signaling Technologies), anti-PAK1 (#2604), anti-p-Cofilin (#3311), anti-p-MLC (#3674), and anti-MLC (#8505). Blots were probed with anti-GAPDH antibody for the reference of sample loading.

Hypoxia/reoxygenation (H/R):

H/R was conducted as described previously (11). For normoxia, cells were serum-starved overnight in EBM-2 then cultured in normoxia and 5% CO2. For H/R, cells were serum-starved overnight in EBM-2 then cultured in hypoxia (94% N2, 1% oxygen, and 5% CO2) for 6 h, then oxygenation (i.e., normoxia) for another 3 h.

Endothelial barrier integrity:

HLMECs were plated on gelatin-coated culture inserts (0.4 μm pore size, Falcon) in 24-well companion plates and grown to confluence in EBM-2 containing 5% FBS and supplements. In some experiments, monolayers were transfected with 100 nM PAK1 siRNA or scRNA when seeding the inserts. Subsequently, FITC-labeled dextran (40 kDa, Sigma) was added to the upper chamber at a concentration of 100 ug/mL, and phosphate buffered saline (PBS) to the lower chamber (to prevent the formation of an oncotic pressure gradient) for 1 h. Medium was collected from the lower chamber, and the fluorescence was measured using a fluorimeter (485 nm excitation, 530 nm emission). The fold change in FITC-dextran fluorescence intensity over controls was used as a measure of monolayer permeability. Actin stress fibers were also stained in these cells. Cells were grown on gelatin-coated 8-well chamber slides (Lab-Tek) and fixed in 4% paraformaldehyde then permeabilized with 0.2% Triton X-100. After washing and blocking with 1% bovine serum albumin (BSA), cells were incubated with 5 U/mL of Texas Red-X phalloidin (Molecular Probes) which is specific for F-actin. The immunofluorescence intensity was quantified using Quantity One software (Bio-Rad).

In-vivo

All experimental procedures were approved by the Animal Care and Use Committee of Institutes of the University of Maryland School of Medicine and conducted in compliance with the National Institutes of Health guidelines on the use of laboratory animals. Adult male C57BL/6J mice (9–12 weeks old) were subjected to our validated coagulopathic model of trauma-hemorrhagic shock (1). Under isoflurane anesthesia, a middle line laparotomy incision was made, the intestines were inspected and then the incision was closed. The right femoral artery was cannulated for continuous hemodynamic monitoring and blood withdrawal or resuscitation. Mean arterial blood pressure was recorded via the femoral arterial line at baseline then every 5 min during the shock period. After a 10-min period of equilibration, mice were bled to a mean arterial pressure (MAP) of 35 ± 5 mmHg which was maintained for 90 min. Shams underwent anesthesia and placement of catheters but were not subjected to laparotomy or hemorrhagic shock. Hemorrhage shock animals were resuscitated with 3X shed blood volume of lactated Ringer’s (LR) solution (12) or resuscitated with 1X shed blood volume of fibrinogen saline solution (about 800 mg/Kg body weight) as we have described previously (12). Three hours after the end of shock, animals were sacrificed by exsanguination under isoflurane anesthesia and right lungs were harvested for protein Western blot analysis and left lungs were lavaged with PBS for measuring bronchoalveolar protein leakage. Lavage protein was measured with the BCA Protein Assay (Thermo Fisher Scientific).

Statistical analysis.

Data were expressed as mean ± SE. Values from different groups were analyzed by T test or one-way analysis of variance (ANOVA) with Bonferroni multiple comparison tests and setting significance level at p< 0.05.

RESULTS

In-vitro

Fibrinogen activates PAK1/cofilin but not MLC

Our results first demonstrate that pulmonary endothelial cells treated with fibrinogen had significantly increased syndecan-1 protein compared to LR treatment (Fig.1A–B). Importantly, the increase in syndecan-1 was accompanied by enhanced activation of PAK1 (increased phosphorylation) and cofilin (decreased phosphorylation) in fibrinogen-treated cells (Fig.1A–B). Myosin light chain (MLC) regulates the interaction of actin and myosin to modulate the assembly of the cytoskeleton. PAK1 is reportedly an upstream regulator of MLC, and inhibition of PAK1 has been shown to induce phosphorylation of MLC to increase stress fiber formation (13). We therefore sought to determine if MLC activity was also regulated by fibrinogen. The results indicate that there was no change in expression of MLC or p-MLC after treating cells with fibrinogen or LR (Fig.1A).

Fig. 1.

Fig. 1.

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Fibrinogen (FIB) increases syndecan-1 protein expression and induces PAK1 activation (increased phosphorylation) and cofilin activation (decreased phosphorylation) compared with vehicle control (i.e., Lactated Ringers, LR). The effects of LR and FIB on Sdc1, PAK1 phosphorylation (p-PAK1), cofilin phosphorylation (p-Cofilin), MLC phosphorylation (p-MLC), MLC and GAPDH were determined in human lung microvascular endothelial cells (HLMECs) at 6 h after treatments. (A) Representative Western blots. GAPDH was detected as a loading control. (B) Summaries of band intensities (expressed in relative units). Mean ± SE for four experiments.

Syndecan-1 silencing attenuates activation of PAK1/cofilin pathway

Results demonstrated that syndecan-1 protein expression was largely inhibited after syndecan-1 siRNA transfection (Fig. 2A–B). Syndecan-1 silencing led to both PAK1 inactivation (decreased phosphorylation) and cofilin inactivation (increased phosphorylation) (Fig. 2A–B). The results support a syndecan-1-mediated PAK1/cofilin pathway in fibrinogen-treated cells.

Fig. 2.

Fig. 2.

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Sdc1 silence attenuates FIB-induced PAK1 activation (increased phosphorylation) and cofilin activation (decreased phosphorylation). HLMECs were transfected wither Sdc1 siRNA (Sdc1-si) or scrambled RNA (scRNA) then subjected to the treatments of LR or FIB for 6 h. (A) Western blot analysis for Sdc1, p-PAK1, p-Cofilin, and GAPDH. GAPDH was detected as a loading control. (B) Summaries of band intensities (expressed in relative units). Mean ± SE for four experiments.

PAK1 silencing causes cofilin inactivation (increased phosphorylation)

To verify the importance of upstream regulation of cofilin by PAK1 (14), silencing of PAK1 by siRNA was performed. The increase in PAK1 activation (increased phosphorylation) following fibrinogen treatment was lost, with a marked decrease in p-PAK1 in both LR- and fibrinogen-treated cells. Additionally, PAK1 silencing resulted in cofilin inactivation (increased phosphorylation) in both LR- and fibrinogen-treated cells and confirmed that PAK1 is upstream of cofilin (Fig. 3A–B).

Fig. 3.

Fig. 3.

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PAK1 silence results in cofilin inactivation (increased phosphorylation). HLMECs were transfected wither PAK1 siRNA (PAK-si) or scrambled RNA (scRNA) then subjected to the treatments of LR or FIB for 6 h. (A) Western blot analysis for p-PAK1, PAK1, p-Cofilin, and GAPDH. GAPDH was detected as a loading control. (B) Summaries of band intensities (expressed in relative units). Mean ± SE from four experiments.

Endothelial barrier was disrupted by inhibition of PAK1.

Treatment of endothelial cells with fibrinogen significantly decreased actin stress fiber formation (Fig. 4A–B) and endothelial permeability to FITC-labelled dextran (Fig. 4C) compared to LR-treated cells. However, PAK1 silencing enhanced stress fiber formation and permeability in both fibrinogen- and LR-treated cells compared to their respective scrambled controls (Fig. 4A–C), demonstrating that PAK1 is involved in endothelial barrier protection.

Fig. 4.

Fig. 4.

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PAK1 silence attenuates the beneficial effect of FIB on endothelial barrier integrity. HLMECs were transfected with PAK1 siRNA (PAK-si) or scrambled RNA (scRNA) then subjected to the treatments of LR or FIB for 6 h. Cells were stained for stress fibers or assayed for monolayer permeability. (A) Stress fiber images were captured using a fluorescence microscope with an original magnification of 400. (B) Relative fluorescent intensity of stress fibers. (C) Monolayer permeability was assessed by FITC-labeled dextran. Mean ± SE from four experiments.

H/R inhibits PAK1/cofilin and disrupts endothelial barrier

To further mimic hemorrhagic shock in-vitro, cells were exposed to H/R. H/R significantly decreased syndecan-1 protein compared to normoxia (Fig. 5A–B). The decrease in syndecan-1 was accompanied by decreased activation of PAK1 (reduced phosphorylation) and cofilin (increased phosphorylation) after H/R treatment (Fig. 5A–B). The results further indicate that there was no change in expression of MLC or p-MLC in cells exposed to either normoxia or H/R (Fig. 5A–B). Compared to normoxia, H/R exposure significantly increased actin stress fiber formation (Fig. 6A–B) and endothelial permeability to FITC-labelled dextran (Fig. 6C).

Fig. 5.

Fig. 5.

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H/R decreases Sdc1 expression and PAK1 activation (increased phosphorylation) and cofilin activation (decreased phosphorylation). Sdc1, p-PAK1, p-Cofilin, p-MLC, MLC and GAPDH were determined in HLMECs exposed to normoxia (Norm) or H/R. (A) Representative Western blots. GAPDH was detected as a loading control. (B) Summaries of band intensities (expressed in relative units). Mean ± SE for four experiments.

Fig. 6.

Fig. 6.

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H/R disrupts endothelial barrier integrity. HLMECs were exposed to normoxia (Norm) or H/R then were stained for stress fibers or assayed for monolayer permeability. (A) Stress fiber images were captured using a fluorescence microscope with an original magnification of 400. (B) Relative fluorescent intensity of stress fibers. (C) Monolayer permeability was assessed by FITC-labeled dextran. Mean ± SE from four experiments.

In vivo

PAK1 expression is maintained by fibrinogen and associated with restored barrier function following hemorrhage shock

To confirm and expand our in-vitro findings, mice underwent hemorrhagic shock then were resuscitated with either LR or fibrinogen. In the lung tissues of shock mice resuscitated with LR, levels of p-PAK1 were significantly lower than that observed in sham lungs (Fig. 7A–B). However, when mice were resuscitated with fibrinogen, p-PAK1 expression was markedly increased compared to LR animals, returning to near sham levels (Fig. 7A–B). Moreover, hemorrhagic shock caused significant lung vascular leakage in shock mice resuscitated with LR while fibrinogen administration remarkably prevented the lung leakage (Fig. 7C).

Fig. 7.

Fig. 7.

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Fibrinogen administration induces PAK1 activation (increased phosphorylation) and decreased vascular leakage in mouse shock lungs compared with sham lungs. Mice were subjected to trauma-hemorrhagic shock then resuscitated with LR or FIB. Some mice were subjected to sham surgery. Lungs were harvested 3 h after the end of shock. (A) Representative Western blot examples. GAPDH was detected as a loading control. (B) Summaries of band intensities (expressed in relative units). (C) Summaries of BAL protein concentrations. Mean ± SE from five mice/group.

Discussion

The present study demonstrates that fibrinogen activates PAK1/cofilin signaling pathway rather than inhibits MLC to decrease stress fiber formation and monolayer hyperpermeability. These in vitro findings were confirmed in a mouse model of hemorrhagic shock with an increase in lung PAK1 activation and a decrease in lung leakage following treatment with fibrinogen. Overview of the proposed pathway was shown in Fig. 8.

Fig. 8.

Fig. 8.

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Overview of proposed pathway.

Although there is extensive data on syndecan-1 shedding after hemorrhagic shock, sepsis and other inflammatory insults to the endothelium (1, 5, 15–18), much less is known about intracellular mechanism regulating syndecan-1 expression. Syndecan-1 is a member of the type I transmembrane heparan sulfate proteoglycan superfamily, composed of syndecan-1–4. All members of the syndecan family have three domains, the extracellular domain which is shed following pathologic stimuli, a highly conserved transmembrane and a highly conserved cytoplasmic domain. The cytoplasmic domain interacts with a number of signaling proteins including protein kinases (19, 20). Hayashida et al. reported that syndecan-1 ectodomain shedding is regulated by the small GTPase Rab5. They found that the syndecan-1 cytoplasmic domain interacted with the small GTPase Rab5 to serve as an intra-cellular signal to regulate ectodomain shedding (21). On the other hand, we were interested in determining the intra-cellular signaling pathway that was activated by fibrinogen binding to syndecan-1 and responsible for maintaining or restoring endothelial cell barrier integrity. There have been several reports of syndecan-4 being involved in Rac1 or Rho activity (22–24), but there is a gap in knowledge related to syndecan-1.

The PAK family consists of six members, including PAK1–6. As the major downstream effector of GTP bound Rho-GTPases such as Rac1 and RhoA (25), PAK1 plays a fundamental role in actin cytoskeleton organization, cell shape, and adhesion dynamics, with evidence that it acts through a downstream effector, cofilin (14, 25). Delforme et al. demonstrated that PAK1 mediates cytoskeleton remodeling through activation/dephosphorylation of cofilin (14, 26). Cofilin, also referred to as actin depolymerizing factor, is a member of the family of actin binding proteins and along with other regulatory proteins mediates the response of the actin cytoskeleton to extracellular signals (14, 26). Activated cofilin severs or depolymerizes actin filaments to disassemble stress fibers (27). Using ATP depletion in an in-vitro model of ischemia in endothelial cells, it was observed that cells had higher levels of phosphorylated cofilin and increased F-actin stress fibers (28). Hemorrhage results in fibrinogen loss and tissue hypoxia. Our in vitro data implied that either fibrinogen loss or tissue hypoxia could contribute to endothelial hyperpermeability through interfering with PAK1/cofilin signaling. In cultured human lung microvascular endothelial cells, we observed that fibrinogen supplementation increased syndecan-1 expression and enhanced PAK1/cofilin activation compared to LR-treated cells, findings that correlate with improved barrier integrity. On the other hand, H/R, an in vitro model of tissue hypoxia after hemorrhage, significantly decreased syndecan-1 protein and attenuated PAK1/cofilin activation compared with normoxia. Moreover, H/R significantly increased actin stress fiber formation and endothelial permeability.

Our mouse studies of hemorrhagic shock confirmed in-vitro studies and demonstrated a marked increase in the status of PAK1 phosphorylation in the lungs after hemorrhagic shock in fibrinogen-treated mice. Similar to our in-vitro data, p-PAK1 expression was correlated with reduced vascular leakage. Our data thus implied that fibrinogen activates PAK1 to maintain endothelial integrity in-vitro and in-vivo, a novel finding.

It is known that MLC pathway plays an important role in regulating F-actin stress fiber formation and endothelial tension in the mechanism of thrombin-induced endothelial monolayer hyperpermeability (7–9, 29). Although we observed baseline levels of p-MLC in endothelial cells, treatment of cells with fibrinogen did not alter the p-MLC levels, suggesting that fibrinogen had no effect on the signaling pathway involved in MLC phosphorylation.

In summary, the present study demonstrated that fibrinogen-induced PAK1/cofilin signaling pathway activation, but not MLC inhibition, to decrease stress fiber formation and enhance endothelial barrier integrity, supporting the endothelial protective role of fibrinogen following hemorrhage shock.

Grants:

This work was supported by National Institute of Health RO1GM129533 (R.A.K), NS087296 (J.F.D) and HL119391 (J.F.D).

Footnotes

Disclosures: None of the authors have any conflicts of interest, financial or otherwise, to disclose.

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