RhoA/ROCK1 regulates the mitochondrial dysfunction through Drp1 induced by Porphyromonas gingivalis in endothelial cells

Abstract Porphyromonas gingivalis (P. gingivalis) is a pivotal pathogen of periodontitis. Our previous studies have confirmed that mitochondrial dysfunction in the endothelial cells caused by P. gingivalis was dependent on Drp1, which may be the mechanism of P. gingivalis causing endothelial dysfunction. Nevertheless, the signalling pathway induced the mitochondrial dysfunction remains unclear. The purpose of this study was to investigate the role of the RhoA/ROCK1 pathway in regulating mitochondrial dysfunction caused by P. gingivalis. P. gingivalis was used to infect EA.hy926 cells (endothelial cells). The expression and activation of RhoA and ROCK1 were assessed by western blotting and pull‐down assay. The morphology of mitochondria was observed by mitochondrial staining and transmission electron microscopy. Mitochondrial function was measured by ATP content, mitochondrial DNA and mitochondrial permeability transition pore openness. The phosphorylation and translocation of Drp1 were evaluated using western blotting and immunofluorescence. The role of the RhoA/ROCK1 pathway in mitochondrial dysfunction was investigated using RhoA and ROCK1 inhibitors. The activation of RhoA/ROCK1 pathway and mitochondrial dysfunction were observed in P. gingivalis‐infected endothelial cells. Furthermore, RhoA or ROCK1 inhibitors partly prevented mitochondrial dysfunction caused by P. gingivalis. The increased phosphorylation and mitochondrial translocation of Drp1 induced by P. gingivalis were both blocked by RhoA and ROCK1 inhibitors. In conclusion, we demonstrate that the RhoA/ROCK1 pathway was involved in mitochondrial dysfunction caused by P. gingivalis by regulating the phosphorylation and mitochondrial translocation of Drp1. Our research illuminated a possible new mechanism by which P. gingivalis promotes endothelial dysfunction.


| INTRODUC TI ON
The primary pathogen of periodontitis, 1 Porphyromonas gingivalis (P. gingivalis), is also intimately linked to the progress of other chronic inflammatory diseases in the body, including atherosclerosis, 2 rheumatoid arthritis 3 and Alzheimer's disease 4 . In human clinical trials and mouse models, P. gingivalis is often detected in atherosclerotic plaques. 5,6 Generally, P. gingivalis has been identified as an independent risk factor for atherosclerosis in some studies. [7][8][9] Mitochondria are highly dynamic organelles that continuously perform coordinated fusion and fission movements. Alterations in mitochondrial structure and functions will result from the imbalance between fission and fusion. The previous study of our group found that P. gingivalis infection leads to an increase in endothelial mitochondrial fission. In addition, it is determined that Drp1 mediates P.
gingivalis-induced mitochondrial dysfunction, but the specific mechanism is unclear. 10 Mitochondrial dysfunction is currently recognized as an essential factor of atherosclerosis. 11,12 Lu et al. found that plateletderived growth factor type BB can induce vascular smooth muscle cells (VSMCs) phenotypic switching, proliferation, migration and neointima formation by activating the ROS/NFκB/mTOR/P70S6K signalling pathway, which is one of the pathological processes of atherosclerosis. 13 Yu et al. found mitochondrial DNA damage accelerates the progression of atherosclerosis using human aortic specimens and mouse models of atherosclerosis. 14 Rho family proteins are small G proteins with GTPases, which are widely presented in eukaryotic tissues. RhoA (Ras homologous gene family member A) is one of the most critical Rho family members.
RhoA serves as a molecular switch that cyclically regulates intracellular signalling between an inactive GDP binding conformation and an active GTP binding conformation. 15 Rho-kinase 1 (Rho-related coiled-coil containing protein kinase, ROCK1) is the direct downstream and primary effector substrate of RhoA. 16 Phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1), one of the important physiological substrates of ROCK1, facilitates interaction and phosphorylation of the catalytic domain of ROCK1. 17 The RhoA/ROCK signal mediates the process of cardiovascular diseases by regulating biological processes such as inflammation, differentiation and apoptosis. In addition, some research suggested the RhoA/ ROCK1 pathway regulates mitochondrial fragmentation through Drp1, a large GTPase, 18 which is activated and transported to the surface of mitochondria to regulate mitochondrial fission. Shen et al. found that RhoA/ROCK1 pathway was engaged in phosphorylated Drp1 at the 616th Serine in cardiomyocytes pretreated with TNFα, which promoted mitochondrial fragmentation. 19 Another report showed that in LPS-pretreated mice, ROCK1 inhibitor could improve mitochondrial function by restricting excessive mitochondrial fission through inhibiting Drp1(Ser616) phosphorylation. 20 Although we have learned that mitochondrial dysfunction caused by P. gingivalis infection depended on Drp1. However, the signalling pathway that regulates mitochondrial fragmentation and dysfunction in P. gingivalis-induced endothelial cells remains elusive.
Here, the role of RhoA/ROCK1 pathway and the further involvement of P. gingivalis in mitochondrial dysfunction were explored. Our findings would provide new clues to understand how P. gingivalis facilitated the formation of atherosclerotic lesions. Our studies were carried out on cells in passages 4 to 6.
CCG-1423 (APExBIO) selectively inhibits SRF-mediated transcription of Rho signalling pathway activation. 21 Y-27632 (AbMole Bioscience) is a pharmacologically specific inhibitor of ROCK. 22 They were used to determine the regulatory role of RhoA/ROCK1 pathway in P. gingivalis infection cells. Control cells were those exposed to DMSO only.

| Bacterial culture
Porphyromonas gingivalis ATCC 33277 was inoculated in brain heart infusion broth containing 5% defibrillated sheep's blood, 0.1% vitamin K1 and 0.5% hemin. The bacteria were grown in anaerobic environments with 80% N 2 , 10% O 2 and 10% H 2 . The cells were treated with P. gingivalis at different time points with a multiplicity of infection (MOI) of 100 in the following experiments. Cells that grew under the same conditions without infection were considered as a control.

| Determination and quantification of the opening of mPTP by fluorescence staining and flow cytometry
The openness of mPTP in EA.hy926 cells was tested using the Mitochondrial Permeability Transition Pore Detection Kit Flow cytometry (FACS, Becton-Dickinson) was used to gather fluorescence intensity, which was then analysed using FlowJo 10 analytic software.

| Determination of ATP contents
ATP Assay Kits (Beyotime) were utilized to ascertain the ATP production in the whole lysate of EA.hy926 cells. Cellular ATP levels of every group were computed according to the standard curves and then normalized to the control.

| Western blotting
The protein concentration of cells was measured by a BCA assay.
SDS-polyacrylamide gel electrophoresis was used to isolate the same amount of protein, which was then transferred to a polyvinylidene fluoride membrane with GAPDH (1:3000; Affinity Biosciences) as an internal control. After 5% skim milk blocking, specific primary antibodies were used to detect the target proteins, including rab-

| RhoA activity assay
The RhoA activation kit (STA-403A; Cell Biolabs) was employed to value whether RhoA was activated. The GTP-bound form of RhoA was pulled down by incubating the equivalent amount of protein and a predetermined amount of GST-rhotekin-RBD on a rotator at 4°C for 1 h. The beads were then centrifuged, washed, resuspended in 20 μL loading buffer and boiled. Western blotting was carried out to determine the pulled-down GTP-bound RhoA amount using an anti-RhoA antibody.

| Observation of mitochondrial morphology by transmission electron microscopy (TEM)
Cells were fixed using 2.5% glutaraldehyde for 24 h, followed by 1% osmium tetroxide for 2 h at room temperature. Subsequently, the sample was dehydrated, immersed, embedded, ultrathin sectioned and stained with lead citrate and uranyl acetate. Afterwards, the TEM (H7650; Hitachi) was used to observe the mitochondrial morphology.

| Quantitative analyses of mitochondrial networking
The mitochondrial network refers to forming a highly interconnected network of tubular mitochondria. Cells were plated on the confocal petri dish for 24 h before being stained by MitoTracker Red (Solarbio) in a 37°C condition. Then, confocal laser scanning microscopes (CLSM; GeneTimes) were used to observe the mitochondrial network. Image-Pro Plus 6.0 software was exploited to spatially process the obtained image using the 'top hat' filter to obtain a binary image that removed artefacts. Quantitative analyses of mitochondria were performed to obtain aspect ratio (AR: major axis/minor axis), shape factor value (FF: perimeter 2 /4π•area) and mitochondrial length. 23 A smaller value obtained indicated an increase in mitochondrial fragmentation, while a higher value represented that the shape of the mitochondria had become longer and more complex.

| Statistical analysis
The mean ± SD was used to summarize the results of three separate tests. In SPSS 17.0 software, one-way anova and SNK test of multiple group comparisons were employed for statistical analysis. P-value < 0.05 indicated a statistically significant difference.

| Mitochondrial dysfunction induced by P. gingivalis
According to our earlier research, P. gingivalis caused an accumulation in mtROS, a depolarization in mitochondrial membrane potential (MMP), and a drop in ATP levels. 10 In the current exploration, we continued to investigate how P. gingivalis affects mPTP opening and mtDNA copy number by Calcein AM staining and RT-PCR, respectively. The images showed that the fluorescence intensity of mPTP declined over time in infected cells ( Figure 1A). Flow cytometry analysis corroborated these findings even more. As shown in Figure 1B, when mPTP fluorescence intensity was compared with controls, it was substantially reduced by 57.19%, 75.11% and 80.63% (p < 0.05) after 2, 12 and 24 h following P. gingivalis attack, respectively. Therefore, the conclusion in which P. gingivalis attack conspicuous induced mPTP opening was confirmed. RT-PCR results showed that a significantly reduced mtDNA copy number was present in cells exposed to P. gingivalis ( Figure 1C). Since cells were infected for 2 h, P.
gingivalis had reduced mtDNA copy number, and the reduction was most significant at the 6-h time point (30.27% reduction). After 12 and 24 h of infection, the mtDNA copy number rebounded slightly, and it was still lower than the control group, decreasing by 28.42% and 24.94% (p < 0.05), respectively.

| RhoA activity and RhoA/ROCK1 pathway were activated by P. gingivalis
EA.hy926 cells that express RhoA and ROCK1 were employed, which was indicated by western blotting, to examine the impact of P. gingivalis on the RhoA/ROCK1 pathway. The pull-down assay was utilized to evaluate the activation of RhoA following P. gingivalis exposure.
It was revealed that the levels of RhoA and RhoA-GTP increased significantly and reached a peak 6 h following P. gingivalis challenge

| The RhoA/ROCK pathway was engaged in P. gingivalis-induced mitochondrial fragmentation
To survey and evaluate the regulatory function of the RhoA/ROCK1 signalling in mitochondrial morphology, cells were observed by TEM. We observed that pretreatment with CCG-1423 or Y27632 could inhibit the endothelial mitochondrial swelling and vacuole-like changes caused by P. gingivalis, and most of the mitochondria of the cells returned to normal and rod-shaped ( Figure 3A). Confocal imaging also indicated the inhibition of the fragmentation and punctate changes of mitochondria by pretreatment of CCG-1423 or Y27632

| Mitochondrial dysfunction induced by P. gingivalis was dependent on RhoA/ROCK pathway
The CLSM images in Figure 4A Figure 4B, C, p < 0.05) compared with that in the infected group. Additionally, it was found that CCG-1423 and Y27632 had similar effects on ATP contents.

F I G U R E 3 Effect of RhoA/ROCK pathway inhibition on
Porphyromonas gingivalis (P. gingivalis)-induced mitochondrial fragments. The cells were pretreated with DMSO, 10 μm CCG-1423 and 10 μm Y27632 for 30 min, respectively, and then exposed to P. gingivalis for 6 h. The cells were pretreated with DMSO only and then cultured in the medium were set as a control. (A) Transmission electron microscopy was used to observe the mitochondria morphology. Magnification 30,000; Scale bars: 1 μm. Arrowhead: mitochondria. (B) Before observing by a confocal laser scanning microscope, MitoTracker Red CMXR was operated to label the mitochondrial network. Magnification 2400; Scale bars: 20 μm. (C-E) Summary data of B. Mitochondrial length, aspect ratio and form factor were calculated to estimate the mitochondrial size. Data were presented as the mean ± SD of three independent determinations. *p < 0.05.
Compared with the control group, ATP contents were decreased by 46.62% (p < 0.05) 2 h after infection. However, in comparison with the infected group, pretreatment with CCG-1423 and Y27632 increased the ATP contents by 55.17% and 61.22%, respectively, according to Figure 4C, D (p < 0.05). These results illustrated that RhoA and ROCK1 inhibitors effectively prevented the enhanced F I G U R E 4 Effect of RhoA/ROCK pathway inhibition on mitochondrial dysfunction induced by Porphyromonas gingivalis (P. gingivalis). The cells were pretreated with DMSO, 10 μm CCG-1423 and 10 μm Y27632, respectively, for 30 min before being infected by P. gingivalis (2 h for ATP, 24 h for mPTP and 6 h for mtDNA). The cells pretreated with DMSO only were considered as a control. (A) A confocal laser microscope was used to observe the openness of mPTP. Magnification 400; Scale bars: 50 μm. (B) The openness of mPTP was analysed quantitatively using flow cytometry. (C) mtDNA copy number was determined using real-time PCR. (D) ATP contents were quantified. The data were represented as a change relative to the control group, which had been designed as 100%. Results were presented as the mean ± SD of three independent experiments. *p < 0.05. mitochondrial permeability, bioenergy deficiency and mitochondrial loss caused by P. gingivalis.

| DISCUSS ION
Atherosclerosis is widely known to be the pathological basis of plenty of cardiovascular diseases. Multiple research has demonstrated an association between the activation of the RhoA/ROCK1 pathway and the onset and progression of atherosclerosis. It has been reported that ROCKs mRNA is enhanced in arteriosclerotic arterial lesions of animals and humans. 24,25 The elevated level of ROCK1 inhibits eNOS activity, an effector substrate of ROCK, and NO levels, resulting in impaired endothelial function and vasodilation, thereby accelerating the progression of atherosclerosis. 26,27 In addition, the RhoA/ROCK pathway plays a vital role in the formation of plaques and accelerates the process of atherosclerosis by mediating the differentiation of monocytes into macrophages and then secreting a series of inflammatory mediators. 28 Generally, RhoA/ ROCK1 pathway activation leads to the progression of cardiovascular diseases through inflammation, 29 endothelial dysfunctions, 30 VSMCs contraction, 31 proliferation and migration. 28 Nonetheless, the exact role of RhoA/ROCK in endothelial dysfunction induced by P. gingivalis is still unknown and needs further investigation.
Mitochondria serve as the cell's energy factory and are essential organelles for cell survival. They have received extensive attention from many scholars. Recent research has found that the imbalance of mitochondrial division and fusion leads to changes in mitochondrial dynamics, which is closely associated with atherosclerosis onset and progression. 32 In the early stage of atherosclerosis, there will be changes in cellular inflammation, oxidative stress, endothelial dysfunction and VSMCs proliferation. Interestingly, mitochondrial dysfunction is thought to be related to these changes. 33 The excessive production of ROS caused by mitochondrial dysfunction will oxidize cellular proteins, lipids and DNA. 34 In the mouse model, the mitochondrial DNA damaged by the excessive accumulation of ROS will cause endothelial cell dysfunction and the proliferation of VSMCs, thereby accelerating the progression of atherosclerosis. 35 It is also reported that the mtDNA damage of leukocytes is related to vulnerable plaques in coronary arteries. By assessing mtDNA in leukocytes and plaques in coronary patients, high-risk plaques were associated with leukocyte mtDNA damage, and mtDNA damage was increased in atherosclerotic plaques than in normal arteries. 36 As the RhoA/ROCK1 pathway is pivotal in atherosclerosis progression, and mitochondrial dysfunction is a recognized pathogenic mechanism of atherosclerosis, the relationship between the RhoA/ROCK1 pathway and mitochondrial dynamics has attracted our attention. can recover mitochondrial function, which is followed by the alleviation of the Hutchinson-Gilford progeria syndrome phenotype. 37 Furthermore, it has been found that profilin-1 (a small actin-binding protein induced by advanced glycosylation end products) extensively dispersed in various cells leads to the accumulation of ROS, thereby activating the RhoA/ROCK1 pathway. 38 These findings have shown that the activation RhoA/ROCK1 pathway is closely associated with mitochondrial dysfunction.
Recently, some academics have explored the specific mechanism of RhoA/ROCK1 pathway regulating mitochondrial dysfunction. Drp1, a GTPase, mediates mitochondrial fission by being transferred to mitochondria, which has been widely concerned. 39 Based on all the above viewpoints, it is supposed that the RhoA/ ROCK1 pathway possibly regulates Drp1-mediates mitochondrial dysfunction. However, whether the RhoA/ROCK1 pathway comes into play a role in endothelial mitochondrial dysfunction is unclear regarding periodontal infection. Growing evidence supports the opinion that P. gingivalis is defined as an independent risk factor for atherosclerosis. 48 Its pili and LPS are involved in atherosclerosis formation by supporting the differentiation of monocytes into pro-inflammatory macrophages and migration. 49 Effective manipulation of adaptive immunosuppression through virulence factors is an essential mechanism of atherosclerosis associated with P. gingivalis infection. 50 Here, P. gingivalis was used to observe the activation of RhoA/ROCK1 pathway and to explore the regulatory effect of RhoA/ROCK1 on Drp1, which has not been reported before.
The elevated expression and activation of RhoA by P. gingivalis were shown in this study. We observed ROCK1 activation through quantifying the expression of p-MYPT1 (Thr696), although ROCK1 expression remained unchanged. As the downstream target of ROCK1, the phosphorylation level of MYPT1 increases, indicating ROCK1 activation. 51 Our findings supported the opinion that the activation of RhoA/ROCK1 signalling was induced by P. gingivalis infection. Drp1 has previously been demonstrated to be a crucial protein for maintaining the balance of mitochondrial fission and fusion, essential for sustaining mitochondrial morphological characteristics and functions. 10 Some other scholars have found that Drp1 is the direct substrate of ROCK1 and regulates mitochondrial fission. We hypothesized that RhoA/ROCK1 was the critical signalling pathway connecting P. gingivalis and mitochondrial fission. As predicted, inhibiting the RhoA/ROCK1 pathway downregulated the Drp1 phosphorylation and mitochondrial translocation, significantly alleviating mitochondrial fragmentation and dysfunction. This is in line with the RhoA/ROCK pathway's influence on fibroblast and glomerular endothelial cell mitochondrial fragmentation. 47,52 According to the report, the infection of bovine mammary epithelial cells with Escherichia coli increases mitochondrial fission mediated by Drp1, resulting in decreased MMP, the continuous opening of mPTP, and calcium ion disturbance. 53 56 mtDNA is more vulnerable to ROS attack when exposed to oxidative damage than nuclear DNA. Interestingly, a decrease of mtDNA copy number is proven to lead to endothelial cell dysfunction, which is a characteristic of early events occurring in the pathogenesis of atherosclerosis. 57 Although we established an infection model with viable P. gingivalis and found that P. gingivalis induced mitochondrial dysfunction, we did not further confirm which virulence factor of P. gingivalis was responsible. It is well-accepted that gingipains, LPS, peptidoglycan and flagellin are fundamental virulence factors for P. gingivalis.
Among them, gingipains provide about 85% of the proteolytic activity and have been considered an essential virulence factor for P. gingivalis. 58 Cao et al. treated myocardial cells with gingipains and observed disruption of mitochondrial integrity, inducing mitochondrial pathway apoptosis. 59 P. gingivalis can degrade platelet endothelial cell adhesion molecule 1 and vascular endothelial cadherin through gingipains, leading to vascular injury, increased endothelial permeability and endothelial dysfunction. 60 Here, we conjectured that P. gingivalis expanded the gap and enhanced the permeability of endothelial cells through gingipains, opening up a channel for the invasion of P. gingivalis and its toxic products. Subsequently, RhoA/ ROCK1 signal pathway was activated, leading to mitochondrial dysfunction and endothelial damage. However, further research was required to confirm our assumption.
We have determined here that in P. gingivalis-infected endothelial cells, RhoA/ROCK1 had a new role in the mitochondrial morphology and function depending on Drp1. The findings indicated a potential new mechanism that P. gingivalis promoted the occurrence and progression of atherosclerosis. Drugs that could inhibit the RhoA/ ROCK1 pathway were expected to become new targets for treating atherosclerosis with P. gingivalis infection. To verify the viewpoints, however, further research is required.

| CON CLUS ION
Our findings revealed that RhoA/ROCK1 pathway was activated by P. gingivalis, and it was involved in the mitochondrial dysfunction depending on phosphorylation and mitochondrial translocation of

ACK N O WLE D G E M ENTS
This research was supported by the National Natural Science Foundation of China (No. 81970943).

CO N FLI C T O F I NTER E S T S TATEM ENT
The author declares no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
On reasonable request, the corresponding author could provide the data that support the finding of this article.