Secondary Logo

Journal Logo

Basic Science Aspects

Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase

Xie, Keliang*; Wang, Weina*†; Chen, Hongguang*; Han, Huanzhi*; Liu, Daquan; Wang, Guolin*; Yu, Yonghao*

Author Information
doi: 10.1097/SHK.0000000000000365

Abstract

INTRODUCTION

Sepsis is a very common clinical complication of systemic inflammatory response caused by a variety of infections; because of its complex pathogenesis, there is no effective treatment (1). During sepsis, vascular endothelial cells are activated as the first liner. Then it arouses a series of pathophysiological changes and a variety of inflammatory cytokines and adhesion molecules are released. Abundant studies show that inflammatory mediators such as tumor necrosis factor or thrombin can lead to cytoskeletal filamentous fibers polymerization into stress fibers, regulate cell contraction, and increase the permeability of endothelial cells (2). The inflammatory cells are mediated from the blood vessels migrating to the site of injury and infection. The inflammatory effect is expanded, and this is also the key to the systemic inflammatory response syndrome and multiple organ failure (3). Vascular endothelial cadherin (VE-cadherin) as the vascular endothelial cell–specific adhesion protein, involved in formation of the connection between the normal cells, regulates the permeability of the vascular endothelium (4).

The Ras homolog gene family member A/Rho-associated coiled-coil protein kinase (RhoA/ROCK) signaling pathway could regulate a range of fundamental cellular functions, such as cellular apoptosis, migration and cell adhesion (5). There is increasing evidence supporting the hypothesis that ROCK is an important component of signaling pathways involved in the regulation of the inflammatory response (6). This pathway regulates the actin cytoskeleton and integrity between cell connections, including the adherence junction like VE-cadherin or tight junction like zonula occludens-1 (ZO-1). Rho/ROCK pathway plays a crucial role in the regulation of endothelial cell permeability (7–9). Rho-associated coiled-coil protein kinase inhibitor, Y-27632 or fasudil, can reduce the expression of adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1), monocyte chemoattractant protein 1, and intercellular adhesion molecule 1 (ICAM-1) (5), which mediate tight junction disassembly or VE-cadherin redistribution to affect cell permeability (10, 11).

Molecular hydrogen (H2) has a very distinct advantage over other antioxidants as its selective antioxidant roles (12). H2 shows a very effective treatment on a variety of diseases, including inflammation, ischemia-reperfusion injury, type 2 diabetes, cancer, atherosclerosis, and so on (13). We have found that H2 inhalation can significantly improve survival rate and multiple organ damage in polymicrobial sepsis and zymosan-induced generalized inflammation via regulation of oxidative stress and inflammatory response (14, 15). In the present study, we explored the role and mechanism of hydrogen-rich medium in the regulation of adhesion of monocytes/polymorphonuclear neutrophils (PMNs) to endothelial cells and vascular endothelial permeability.

MATERIALS AND METHODS

Cell culture and grouping

Human umbilical vein endothelial cells (HUVECs) and human monocytic cell line U937 were purchased from ATCC Company (USA). Cells were routinely maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2 in Dulbecco modified Eagle medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, NY, USA). After detachment of 80% confluent HUVECs (2 × 106/mL) from the flasks with 0.025% trypsin, the cells were grown in 6-well plates and were subjected to the next experiment. The passaging of monocytes cells was done by centrifugation.

Neutrophils were isolated from whole blood of healthy human volunteers by Ficoll-Paque density gradient. Isolated red blood cells and neutrophils were subjected to 3% dextran separation, with purification of neutrophils by hypotonic lysing. Isolated neutrophils were resuspended in RPMI 1640 at a concentration of 5 × 106 cells/mL. Morphologic examination with Wrights-Giemsa staining confirmed that the purity of the neutrophil preparations was greater than 95%.

In the first part, HUVECs were grown to confluence in 6-well plates and treated with or without lipopolysaccharide (LPS) or hydrogen-rich medium for 6, 12, and 24 h, respectively. Then U937 (5 × 106/mL, 2 mL per well) were added into the plates to coculture for 90 min. Human umbilical vein endothelial cells were subjected to 4 conditions: normal cell culture medium as control (N group); saturated hydrogen-rich medium (0.6-mmol/L hydrogen) (H2 group); LPS (1 μg/mL) (16) as the model group (LPS group); LPS + saturated hydrogen-rich medium as the treatment group (LPS + H2 group). In the second part, HUVECs were treated as the first part for 24 h and cocultured with U937cells for 90 min. Cells were subjected to 5 groups: normal cell culture medium as control (N group); LPS (1 μg/mL) as the model group (LPS group); LPS + saturated hydrogen-rich medium (LPS + H2 group); LPS + Y-27632 (10 μmol/L) (Y-27632 is the inhibitor of ROCK); and LPS + Y-27632 (20 μmol/L) (17).

Analysis of adhesion of monocytes to endothelial cells

The cell adhesion assay was performed by Wright-Giemsa. Human umbilical vein endothelial cells were grown to confluence in 6-well plates and treated with LPS or hydrogen-rich medium for 6, 12, and 24 h. Then U937 (5 × 106/mL, 2 mL per well) were added into the plates. After coculture of 90 min, the monolayer was gently washed with phosphate-buffered saline 3 times to remove the unbound monocytes. The Wright-Giemsa AB liquid was dropped, respectively. Then, they were rinsed with tap water and dried. The number of adherent cells was expressed as optical microscope. Three fields were captured per experimental condition. Individual treatment was performed in duplicates, and the entire set of experiments was repeated 3times.

Enzyme-linked immunosorbent assay

Cells were processed as before. After coculture of 90 min, concentrations of VCAM-1 and E-selectin in culture medium were measured with commercially available enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, Calif) according to the manufacturer’s instructions. The sensitivities of the VCAM-1 and E-selectin ELISA were 1 ng/mL and 4 μg/mL, respectively. All experiments were performed according to the manufacturers’ instructions (17).

Transepithelial/endothelial electrical resistance measurement

Human umbilical vein endothelial cells were grown to confluence on porous polyester membrane inserts (6.5 mm diameter, 0.4 mm pore size; Transwell, Corning, Cambridge, Mass). Cells were processed as previously described. For experimental treatment, transepithelial/endothelial electrical resistance (TEER) measurement was performed using an EVOM volt-ohmmeter (World Precision Instruments, Sarasota, Fla). The resistance value of an empty culture insert (no cells) was subtracted. A decrease in TEER indicates an increase in monolayer permeability, whereas an increase in TEER signifies an increase in monolayer integrity. Data were collected from duplicate inserts per treatment in each experiment. All steps were performed according to Warfel et al. (18).

Western blotting

Cells were lysed with ice-cold radioimmunoprecipitation buffer, centrifuged at 10,000 rpm for 5 min at 4°C, and the supernatants were collected. Protein concentrations in the supernatants were measured using the bicinchoninic acid protein assay kit (Pierce Chemical Company, USA). Cell homogenates (40 μg of protein) were separated on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride, then washed with Tris-buffered saline, blocked with 5% skimmed milk powder, and incubated with the appropriate primary antibody at dilutions recommended by the supplier. Then, the membrane was washed, and primary antibodies were detected with secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature. The blots were then developed with SuperSignal-enhanced chemiluminescent substrate solution (Pierce Chemical Company, Rockford, Ill). Anti–β-actin, anti-ROCK1, anti–VE-cadherin antibody used in this study were purchased from Abcam Company (Abcam, USA).

Statistical analysis

All values are presented as mean ± standard deviation (SD). The intergroup differences were tested by one-way analysis of variance. The statistical analysis was performed using SPSS version 16.0 software (SPSS Inc, Chicago, Ill). In all tests, P < 0.05 was considered statistically significant.

RESULTS

Effects of hydrogen-rich medium on LPS-induced adhesion of monocytes/PMNs to endothelial cells

Cytoplasm and nucleus contain different chemical compositions, have different affinity for various dyes, respectively, so different cells can be distinguished after dying. Adhesion of monocytes and/or PMNs to endothelial cells is the initial inflammation phenomena. For determination of LPS concentration in this study, we first investigated the adhesion of monocytes-endothelial cells after different concentrations of LPS stimulation. We found that LPS-induced adhesion of monocytes to endothelial cells in a dose-dependent manner (see Figure, Supplemental Digital Content 1, at https://links.lww.com/SHK/A279, showing effects of different concentrations of LPS on adhesion of monocytes to endothelial cells. Human umbilical vein endothelial cells and human monocytes line U937 were cultured. Cells were challenged with phosphate-buffered saline or LPS (0.01, 0.1, 1 μg/mL) for 24 h. The cell adhesion assay was performed by Wright-Giemsa. Results are presented as mean ± SD (n = 6 each group). *P < 0.05 vs. group N, P < 0.05 vs. group LPS (0.01 μg/mL), P < 0.05 vs. group LPS (0.1 μg/mL). In this study, we used LPS stimulation at a dose of 1 μg/mL. In the LPS group, the monocyte adhesion significantly increased compared with N group (P < 0.05), whereas in the LPS + H2 group, it was significantly reduced (P < 0.05). They showed the same trend in 3 time points (Fig. 1). Moreover, we also found that hydrogen-rich medium significantly attenuated the adhesion of PMNs to endothelial cells after LPS stimulation (Fig. 2). These data suggest that hydrogen-rich medium ameliorates the adhesion of monocytes/PMNs to endothelial cells induced by LPS stimulation.

Fig. 1
Fig. 1:
Effects of hydrogen treatment on monocytes adhesion in LPS-stimulated HUVECs. In group N, cells were cultured in the normal medium; in group H2, cells were cultured in hydrogen-saturated medium; in group LPS, cells were cultured in the normal medium and stimulated by LPS; in group LPS + H2, cells were cultured in hydrogen-saturated medium and stimulated by LPS. Human umbilical vein endothelial cells (HUVECs) and human monocytes line U937 were cultured. Cells were challenged with 1 μg/mL of LPS or PBS with or without 0.6-mmol/L hydrogen-rich medium for 6, 12, and 24 h. Cell adhesion assay was performed by Wright-Giemsa. Results are presented as mean ± SD (n = 6 each group at each time point). *P < 0.05 vs. group N; P < 0.05 vs. group LPS.
Fig. 2
Fig. 2:
Effects of hydrogen treatment on polymorphonuclear leukocytes (PMNs) adhesion in LPS-stimulated HUVECs. In group N, cells were cultured in the normal medium; in group LPS, cells were cultured in the normal medium and stimulated by LPS; in group LPS + H2, cells were cultured in hydrogen-saturated medium and stimulated by LPS. Human umbilical vein endothelial cells and neutrophils were cultured. Cells were challenged with 1 μg/mL of LPS or PBS in the absence or presence of 0.6-mmol/L hydrogen-rich medium for 6, 12, and 24 h. Cell adhesion assay was performed by Wright-Giemsa. Results are presented as mean ± SD (n = 6 each group at each time point). *P < 0.05 vs. group N; P < 0.05 vs. group LPS.

Effects of hydrogen-rich medium on LPS-induced expression of adhesion molecules

Various adhesion molecules and chemokines regulate monocyte migration, adhesion, and accumulation and ultimately destroy the endothelial cells to increase its permeability. Enzyme-linked immunosorbent assay results showed that after LPS stimulation, the concentrations of VCAM-1 and E-selectin in cell culture supernatants significantly increased (P < 0.05),whereas in the LPS + H2 group, the levels of adhesion molecules were reduced (P < 0.05; Fig. 3).

Fig. 3
Fig. 3:
Effects of hydrogen treatment on the concentration of VCAM-1 and E-selectin after LPS stimulation. Grouping method was the same as in Figure 1. Cells were challenged with 1 μg/mL of LPS or phosphate-buffered saline in the absence or presence of 0.6-mmol/L hydrogen-rich medium. At 6, 12, and 24 h after LPS administration, VCAM-1 and E-selectin concentrations in culture medium were measured with commercially available ELISA kits. Results are presented as mean ± SD (n = 6 each group at each time point). *P < 0.05 vs. group N; P < 0.05 vs. group LPS.

Impact of hydrogen-rich medium on LPS-induced endothelial permeability

The TEER of the cultured endothelial cell layer could reflect endothelial permeability. Endothelial cells are a variable resistor in the placed electrodes in upper and lower Transwell chamber, respectively. The measured resistance reflected the functional state of intercellular connections through changes in TEER values to determine the change in the situation as well as the connection between cell barrier function. The TEER of endothelial cells gradually increased and stabilized at a certain level after culture for 3 days. In the LPS group, the TEER values significantly decreased over time (P < 0.05). In the LPS + H2 group, the TEER values increased markedly (P < 0.05; Fig. 4). Hydrogen-rich medium could improve the high permeability of endothelial cells induced by LPS stimulation.

Fig. 4
Fig. 4:
Effects of hydrogen treatment on the endothelial permeability induced by LPS. Grouping method was the same as in Figure 1. Human umbilical vein endothelial cells were grown to confluence on porous polyester membrane (6.5-mm diameter, 0.4-mm pore size, Transwell). Transepithelial/endothelial electrical resistance measurements were performed at 6, 12, and 24 h after LPS administration using an EVOM volt-ohmmeter. Decrease in TEER indicates an increase in monolayer permeability, whereas an increase in TEER signifies an increase in monolayer integrity. Data were collected from duplicate inserts per treatment in each experiment. Results are presented as mean ± SD (n = 6 each group at every time point). *P < 0.05 vs. group N; P < 0.05 vs. group LPS.

Effects of hydrogen-rich medium on the expression and distribution of VE-cadherin induced by LPS

Vascular endothelial cadherin as endothelial cell–specific cadherin is connected to the transmembrane part of the endothelial cell adhesion complex, connected by actin skeleton and muscle cytoplasmic tail, playing an important role in the maintenance of a single-cell layer. In this study, Western blot analysis showed that LPS could decrease the expression of VE-cadherin (P < 0.05), whereas the hydrogen-rich medium significantly inhibited the decrease of VE-cadherin induced by LPS (P < 0.05; Fig. 5). Fluorescence results showed that the red linear fluorescent reflected the VE-cadherin, and the blue fluorescent staining represented nuclei. In the control and hydrogen-rich medium groups, VE-cadherin was around the periphery of the nucleus, forming a continuous monolayer barrier. In the LPS group, VE-cadherin distributed in the cell junctions was not complete, and the fluorescence intensity was weak; whereas compared with the LPS group, VE-cadherin showed more complete, uniform distribution, and the fluorescence intensity was significantly higher in the LPS + H2 group (Fig. 6).

Fig. 5
Fig. 5:
Effects of hydrogen treatment on the expression of VE-cadherin induced by LPS. Grouping method was the same as in Figure 1. Vascular endothelial cadherin as endothelial cell–specific cadherin is connected to the transmembrane part of the endothelial cell adhesion complex. Expression of VE-cadherin was measured at 24 h after LPS administration by Western blotting. Results are presented as mean ± SD (n = 6 each group). *P < 0.05 vs. group N; P < 0.05 vs. group LPS.
Fig. 6
Fig. 6:
Effects of hydrogen treatment on the location of VE-cadherin induced by LPS. Grouping method was the same as in Figure 1. Vascular endothelial cadherin was determined at 24 h after LPS administration by immunofluorescence staining. Red fluorescence represents VE-cadherin positive expression and blue fluorescence represents 4’,6-diamidino-2-phenylindole (DAPI)-positive cells (original magnification ×200).

Effects of ROCK inhibitors and hydrogen-rich medium on LPS-induced adhesion molecules

Cellular adhesion molecules are a member of the immunoglobulin superfamily involved in leukocytes across the blood vessel walls in inflammation. Rho/ROCK signaling pathway is an important pathway regulating the permeability of the endothelial cells. Y-27632, as a selective inhibitor of Rho/ROCK signaling pathway, is now widely used. Compared with the control (N) group, adhesion molecules in the LPS group were significantly elevated, whereas in the LPS + H2 group, the levels of adhesion molecules decreased compared with the LPS group (P < 0.05). Y-27632 also affected the levels of adhesion molecules. There was a decreased level of adhesion molecules in the LPS + Y-27632 group compared with the LPS group. This effect was more significantly attenuated by Y-27632 at a dose of 20 μmol/L than 10 μmol/L (P < 0.05; Fig. 7).

Fig. 7
Fig. 7:
Effects of ROCK inhibitor or hydrogen treatment on VCAM-1 and E-selectin concentration induced by LPS. Y-27632 as a selective synthetic inhibitor of Rho/ROCK signaling pathway could regulate levels of inflammatory molecules. Concentration of Y-27632 is 10 μmol/L and 20 μmol/L in this part, respectively. Cells were challenged as mentioned before in “Materials and Methods”. Levels of VCAM-1 and E-selectin were detected at 24 h after LPS administration. Results are presented as mean ± SD (n = 6 each group). *P < 0.05 vs. group N; P < 0.05 vs. group LPS; P < 0.05 vs. group LPS+ Y-27632 (10 μmol/L).

Effects of ROCK inhibitors and the hydrogen-rich medium on the endothelial permeability

The TEER value could reflect endothelial cell barrier function. In the LPS group, the TEER value was decreased (P < 0.05). In the LPS + H2 group, hydrogen-rich medium showed a protective effect on endothelial barrier function (P < 0.05). Y-27632 could also reduce endothelial permeability and increase TEER value (P < 0.05). Compared to Y-27632 (10 μmol/L), Y-27632 (20 μmol/L) had a more significant increase in TEER value (P < 0.05; Fig. 8). Y-27632 performed a protective effect on endothelial permeability.

Fig. 8
Fig. 8:
Effects of ROCK inhibitor or hydrogen treatment on endothelial permeability induced by LPS. Cells were challenged as mentioned in Figure 7. The TEER value was measured at 24 h after LPS administration. Results are presented as mean ± SD (n = 6 each group). *P < 0.05 vs. group N; P < 0.05 vs. group LPS; P < 0.05 vs. group LPS+ Y-27632 (10 μmol/L).

Effects of hydrogen-rich medium and Y-27632 on the expression of VE-cadherin and ROCK

There are many signaling pathways in regulating endothelial barrier function, including Rho/ROCK pathway. In the LPS group, ROCK was significantly activated (P < 0.05) and had an increased expression, participating in the regulation of endothelial permeability. After administration of the ROCK inhibitor Y-27632 (20 μmol/L), it showed an inhibition of ROCK protein expression (P < 0.05) and a protective effect on endothelial barrier. In the LPS + H2 group, ROCK expression level was also reduced (P < 0.05; Fig. 9). Rho-associated coiled-coil protein kinase also could regulate tight junctions and adhesion junctions adjusting cell permeability. Y-27632 as a ROCK inhibitor could inhibit the increased endothelial permeability and regulate the VE-cadherin level (P < 0.05; Fig. 10). Hydrogen-rich medium could regulate the vascular endothelial barrier perhaps through influencing the ROCK expression.

Fig. 9
Fig. 9:
Effects of ROCK inhibitor or hydrogen treatment on the expression of ROCK. Cells were challenged as mentioned in Figure 7. In this part, the concentration of Y-27632 was 20 μmol/L. Expression of ROCK was measured at 24 h after LPS administration by Western blotting. Results are presented as mean ± SD (n = 6 each group). *P < 0.05 vs. group N; P < 0.05 vs. group LPS.
Fig. 10
Fig. 10:
Effects of ROCK inhibitor or hydrogen treatment on the expression of VE-cadherin. Cells were challenged as mentioned in Figure 9. Expression of VE-cadherin was measured at 24 h after LPS administration by Western blotting. Results are presented as mean ± SD (n = 6 each group). *P < 0.05 vs. group N; P < 0.05 vs. group LPS.

DISCUSSION

The present study demonstrated that hydrogen-rich medium could attenuate adhesion of LPS-induced monocytes/PMNs to endothelial cells and increases in VCAM-1 and E-selectin expression. In addition, hydrogen-rich medium also could increase TEER and increase VE-cadherin expression. Our data suggested that ROCK inhibitor Y-27632 had a similar effect on adhesion molecules, TEER, and VE-cadherin. Hydrogen-rich medium also could lessen the expression of ROCK. Therefore, hydrogen regulated monocyte adhesion and vascular endothelial cell permeability by regulating ROCK expression.

Hydrogen is a colorless, odorless, and inert gas. Recently, basic and clinical researches have shown that hydrogen is an important physiological regulatory factor with antioxidant, anti-inflammatory, and antiapoptotic properties (19). In addition, hydrogen-rich water can reduce the burn-induced lung injury, paraquat-induced lung injury, and the hyperoxic lung injury (20). Hydrogen showed a very effective treatment on a variety of diseases, including type 2 diabetes, cancer, atherosclerosis, and other diseases (13). We would explore the effect and mechanism of hydrogen-rich medium on cell adhesion and endothelial permeability.

During septic shock, LPS induces contraction of endothelial cells and the formation of intercellular gaps that allow soluble and particulate blood components to leak from blood vessel adhesion molecules such as ICAM-1. These CAMs mediate the firm adhesion of leukocytes to the endothelium and subsequent transmigration to the inflammatory sites and contribute to the adhesion of activated lymphocytes and monocytes to cells in acute inflammatory tissues (21). The interaction between monocytes and the molecules expressed on the endothelial cell surface recruits lymphocytes and monocytes into the vascular wall, facilitates the adhesion of monocytes to endothelial cells, and up-regulates chemoattractant and adhesion molecules expression and monocytes migration through endothelial cells (22). We used LPS to stimulate endothelial cells to induce an inflammatory response. After LPS stimulation, the levels of adhesion molecules were elevated, endothelial adhesion of monocytes increased, while hydrogen could reduce the levels of adhesion molecules and inhibit monocyte adhesion. In addition, we also found that hydrogen could decrease the adhesion of PMNs to endothelial cells.

The endothelial barrier serves to separate blood in vessels from surrounding tissues and to control the exchange of cells and tissue fluids between them (23). Barrier hyperpermeability, which is necessary to provide access to inflamed tissues by leukocytes, is also accompanied by excessive tissue swelling because of fluid extravasation from vascular (24–26). The endothelium quickly responds to certain stimuli, changes in paraendothelial barrier function, cell spreading, and cell migration. These processes depend on a balanced regulation between cell adhesion and remodeling and essentially rely on both the cadherin/catenin complex and the associated actin filaments (27, 28). Vascular endothelial cadherin, as the major component of adherence junction, is a single-span transmembrane protein that is unique to endothelial cells, and it promotes homophilic interaction between neighboring cells (4). Vascular endothelial cadherin and claudin-5 are major components of adherens and tight junctions, respectively, and various agents induce decreases in their expressions associated with increases in the paracellular permeability of endothelial cells (29, 30).

Transepithelial/endothelial electrical resistance measurement is a sensitive and relatively simple method to assess the permeability of endothelial cell. The measured resistance reflected the functional state of intercellular connections, through changes in TEER values to determine the change in the situation as well as the connection between the cell barrier function of culture. The TEER of endothelial cells gradually increased after culture of 3 days; it stabilized at a certain level. After LPS stimulation, the TEER significantly decreased as it meant the destruction of cell permeability, and over time, the damage increased gradually. After hydrogen treatment, the TEER values increased, indicating decreased cell permeability.

Rho-associated coiled-coil protein kinase belongs to the AGC family of serine/threonine kinases and is a major downstream effector of the small GTPase RhoA. Recent studies suggest that the RhoA/ROCK pathway may contribute to diabetic vascular complications (31). Previous reports have demonstrated that vascular barrier integrity is controlled by modulating cytoskeletal proteins, actins, and myosin via the activation of the Rho family of small GTPase (32). Inhibition of Rho/ROCK signaling may have therapeutic potential in preventing diabetes associated with vascular inflammation and atherogenesis (5). Rho-associated coiled-coil protein kinase has also been implicated in the regulation of vascular tone and proliferation as well as smooth muscle contraction, cell adhesion, and cell motility (33). There is evidence that inhibition of ROCKs causes activation of endothelial nitric oxide synthase and reduction of vascular inflammation (34). Rho/ROCK pathway activation in endothelial cells and leucocytes participates in the regulation of genes such as intercellular adhesion molecule-1 (35). We examined the impact of ROCK inhibitor Y-27632 on the expression of adhesion molecules and the endothelial permeability. The results showed that Y-27632 can reduce the expression of adhesion molecules and attenuate the damage of endothelial permeability, similar to the regulation of hydrogen-rich medium. Hydrogen-rich medium could also down-regulate ROCK protein, indicating that hydrogen-rich medium influencing endothelial permeability and adhesion of monocytes might be related to its regulation of ROCK expression.

REFERENCES

1. Rubenfeld GD, Herridge MS: Epidemiology and outcomes of acute lung injury. Chest 131: 554–562, 2007.
2. McKenzie JA, Ridley AJ: Roles of Rho/ROCK and MLCK in TNF-alpha-induced changes in endothelial morphology and permeability. J Cell Physiol 213: 221–228, 2007.
3. Matsuda N, Hattori Y: Vascular biology in sepsis: pathophysiological and therapeutic significance of vascular dysfunction. J Smooth Muscle Res 43: 117–137, 2007.
4. Dejana E, Orsenigo F, Lampugnani MG: The role of adherens junctions and VE-cadherin in the control of vascular permeability. J Cell Sci 121: 2115–2122, 2008.
5. Li H, Peng W, Jian W, Li Y, Li Q, Li W, Xu Y: ROCK inhibitor fasudil attenuated high glucose-induced MCP-1 and VCAM-1 expression and monocyte-endothelial cell adhesion. Cardiovasc Diabetol 11: 65, 2012.
6. Okamoto H, Yoshio T, Kaneko H, Yamanaka H: Inhibition of NF-kappaB signaling by fasudil as a potential therapeutic strategy for rheumatoid arthritis. Arthritis Rheum 62: 82–92, 2010.
7. Shen Q, Wu MH, Yuan SY: Endothelial contractile cytoskeleton and microvascular permeability. Cell Health Cytoskelet 2009: 43–50, 2009.
8. van Nieuw Amerongen GP, Beckers CM, Achekar ID, Zeeman S, Musters RJ, van Hinsbergh VW: Involvement of Rho kinase in endothelial barrier maintenance. Arterioscler Thromb Vasc Bio l27: 2332–2339, 2007.
9. Holeiter G, Bischoff A, Braun AC, Huck B, Erlmann P, Schmid S, Herr R, Brummer T, Olayioye MA: The RhoGAP protein deleted in liver cancer 3 (DLC3) is essential for adherens junctions integrity. Oncogenesis 1: e13, 2012.
10. Zheng YJ, Zhou B, Ding G, Wang ZC, Wang XQ, Wang YL, Tang YQ: Effect of serum from patients with severe acute pancreatitis on vascular endothelial permeability. Pancreas 42: 633–639, 2013.
11. Xie H, Xue YX, Liu LB, Liu YH, Wang P: Role of RhoA/ROCK signaling in endothelial-monocyte-activating polypeptide II opening of the blood-tumor barrier: role of RhoA/ROCK signaling in EMAP II opening of the BTB. J Mol Neurosci 46: 666–676, 2012.
12. Hong Y, Chen S, Zhang JM: Hydrogen as a selective antioxidant: a review of clinical and experimental studies. J Int Med Res 38: 1893–1903, 2010.
13. Sun H, Chen L, Zhou W, Hu L, Li L, Tu Q, Chang Y, Liu Q, Sun X, Wu M, et al.: The protective role of hydrogen-rich saline in experimental liver injury in mice. J Hepatol 54: 471–480, 2011.
14. Xie K, Yu Y, Zhang Z, Liu W, Pei Y, Xiong L, Hou L, Wang G: Hydrogen gas improves survival rate and organ damage in zymosan-induced generalized inflammation model. Shock 34: 495–501, 2010.
15. Xie K, Yu Y, Pei Y, Hou L, Chen S, Xiong L, Wang G: Protective effects of hydrogen gas on murine polymicrobial sepsis via reducing oxidative stress and HMGB1 release. Shock 34: 90–97, 2010.
16. Chen HG, Xie KL, Han HZ, Wang WN, Liu DQ, Wang GL, Yu YH: Heme oxygenase-1 mediates the anti-inflammatory effect of molecular hydrogen in LPS-stimulated RAW 264.7 macrophages. Int J Surg 11: 1060–1066, 2013.
17. Venkatesh D, Fredette N, Rostama B, Tang Y, Vary CP, Liaw L, Urs S: RhoA-mediated signaling in Notch-induced senescence-like growth arrest and endothelial barrier dysfunction. Arterioscler Thromb Vasc Biol 31: 876–882, 2011.
18. Warfel JM, Steele AD, D’Agnillo F: Anthrax lethal toxin induces endothelial barrier dysfunction. Am J Pathol 166: 1871–1881, 2005.
19. Huang CS, Kawamura T, Toyoda Y, Nakao A: Recent advances in hydrogen research as a therapeutic medical gas. Free Radic Res 44: 971–982, 2010.
20. Fang Y, Fu XJ, Gu C, Xu P, Wang Y, Yu WR, Sun Q, Sun XJ, Yao M: Hydrogen-rich saline protects against acute lung injury induced by extensive burn in rat model. J Burn Care Res 32: e82–e91, 2011.
21. Peters K, Unger RE, Brunner J, Kirkpatrick CJ: Molecular basis of endothelial dysfunction in sepsis. Cardiovasc Res 60: 49–57, 2003.
22. Piga R, Naito Y, Kokura S, Handa O, Yoshikawa T: Short-term high glucose exposure induces monocyte-endothelial cells adhesion and transmigration by increasing VCAM-1 and MCP-1 expression in human aortic endothelial cells. Atherosclerosis 193: 328–334, 2007.
23. Bogatcheva NV, Verin AD: The role of cytoskeleton in the regulation of vascular endothelial barrier function. Microvasc Res 76: 202–207, 2008.
24. Qiao RL, Wang HS, Yan W, Odekon LE, Del Vecchio PJ, Smith TJ, Malik AB: Extracellular matrix hyaluronan is a determinant of the endothelial barrier. Am J Physiol 269: C103–C109, 1995.
25. Bae JS, Lee W, Rezaie AR: Polyphosphate elicits pro-inflammatory responses that are counteracted by activated protein C in both cellular and animal models. J Thromb Haemost 10: 1145–1151, 2012.
26. Hansson GK, Libby P: The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol 6: 508–519, 2006.
27. Cavey M, Lecuit T: Molecular bases of cell-cell junctions stability and dynamics. Cold Spring Harb Perspect Biol 1: a002998, 2009.
28. Yonemura S: Cadherin-actin interactions at adherens junctions. Curr Opin Cell Biol 23: 515–522, 2011.
29. Aslam M, Ahmad N, Srivastava R, Hemmer B: TNF-alpha induced NFκB signaling and p65 (ReIA) overexpression repress Cldn5 promoter in mouse brain endothelial cells. Cytokine 57: 269–275, 2012.
30. Komarova Y, Malik AB: Regulation of endothelial permeability via paracellular and transcellular transport pathways. Annu Rev Physiol 72: 463–493, 2010.
31. Bu DX, Rai V, Shen X, Rosario R, Lu Y, D’Agati V, Yan SF, Friedman RA, Nuglozeh E, Schmidt AM: Activation of the ROCK1 branch of the transforming growth factor-beta pathway contributes to RAGE-dependent acceleration of atherosclerosis in diabetic ApoE-null mice. Circ Res 106: 1040–1051, 2010.
32. McLaughlin JN, Shen L, Holinstat M, Brooks JD, Dibenedetto E, Hamm HE: Functional selectivity of G protein signaling by agonist peptides and thrombin for the protease-activated receptor-1. J Biol Chem 280: 25048–25059, 2005.
33. Noma K, Oyama N, Liao JK: Physiological role of ROCKs in the cardiovascular system. Am J Physiol 290: C661–C668, 2006.
34. Ocaranza MP, Rivera P, Novoa U, Pinto M, Gonzalez L, Chiong M, Lavandero S, Jalil JE: Rhokinase inhibition activates the homologous angiotensin-converting enzyme-angiotensin-(1–9) axis in experimental hypertension. J Hypertens 29: 706–715, 2011.
35. Noma K, Rikitake Y, Oyama N, Yan G, Alcaide P, Liu PY, Wang H, Ahl D, Sawada N, Okamoto R, et al.: ROCK1 mediates leukocyte recruitment and neointima formation following vascular injury. J Clin Invest 118: 1632–1644, 2008.
Keywords:

Sepsis; hydrogen gas; vascular endothelial permeability; rho kinase; adhesion molecules; ELISA—Enzyme-linked immunosorbent assay; HUVECs—Human umbilical vein endothelial cells; ICAM-1—Intercellular adhesion molecule 1; LPS—Lipopolysaccharide; MCP-1—Monocyte chemoattractant protein 1; PMNs—Polymorphonuclear leukocytes; RhoA—Ras homolog gene family member A; ROCK—Rho-associated coiled-coil protein kinase; SD—Standard deviation; TEER—Transepithelial/endothelial electrical resistance; VCAM-1—Vascular cell adhesion molecule 1

Supplemental Digital Content

© 2015 by the Shock Society