Cathelicidin Modulates Vascular Smooth Muscle Cell Phenotypic Switching through ROS/IL-6 Pathway

Vascular smooth muscle cells (VSMC) are stromal cells of the blood vessels and their differentiation is thought to be essential during atherosclerosis. Cathelicidin-related antimicrobial peptides (CRAMP) are suggested to play a role in the development of atherosclerosis. Even so, the relationship of CRAMP and VSMC remains unclear. The present study was to determine whether CRAMP regulates VSMC phenotypic transformation and underlying mechanisms. We demonstrated that CRAMP could reverse platelet-derived growth factor-BB (PDGF-BB)-induced VSMC phenotypic transformation, evidencing by increasing α-smooth muscle actin (α-SMA), smooth muscle 22α (SM22α) and decreasing of proliferation and migration. Further studies showed that CRAMP inhibited nuclear factor κB (NF-κB)-induced autocrine of interleukin-6 (IL-6), which further activated of janus kinase 2 (JAK2)/signal transducer and activator 3 (STAT3). Meanwhile, our data showed that CRAMP can significantly inhibit PDGF-BB enhanced intracellular reactive oxygen species (ROS) level which further affected the NF-κB signaling pathway, indicating that CRAMP can regulate the phenotypic transformation of VSMC by regulating oxidative stress. These results indicated that CRAMP regulated the differentiation of VSMC by inhibiting ROS-mediated IL-6 autocrine, suggesting that targeting CRAMP is a potential avenue for regulating the differentiation of VSMC and treatment of atherosclerosis.


Primary VSMC Culture
VSMC were harvested from normal rat aortas using the explant technique. The VSMC were cultured routinely in Dulbecco's modified Eagle's medium (DMEM; Hyclone Laboratories Inc., Logan, UT, USA) containing 10% fetal bovine serum (FBS), supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 • C with a humidified atmosphere of 5% CO 2 . The primary VSMC were identified using smooth muscle α-actin antibody. For all experiments, VSMC (2-5 passages) were used following by quiescence for 12 h.

Cell Viability Assay
Cells were seeded into 96-well plates and allowed to adhere for 24 h. After being treated with different doses of CRAMP for 48 h, cells were subjected to viability detection by using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's specifications. In brief, cells in each well were incubated with 10 µL MTT working solution at 37 • C for 4 h. The absorbance of each well at 490 nm was measured using an Epoch Microplate Reader (BIO-TEK, Winooski, VT, USA).

Western Blot
Tissues and cells were lysed by using lysis buffer and centrifuged at 13,000× g, 4 • C. Samples incubated with a sodium dodecyl sulfate (SDS) sample loading buffer were heated on the boiling water bath for 5 min, then subjected to 12% SDS-polyacrylamidegel (PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes. After being blocked in 5% fat-free milk at room temperature (RT) for 1 h, membranes were incubated overnight at 4 • C with primary antibodies, followed by HRP-conjugated secondary antibodies for 1 h at RT. Finally, membrane-bound antibodies were detected using a chemiluminescence reagent. The total protein content of loading was monitored by reprobing the same blots with loading control.

Animal Experiments
C57BL/6 mice were purchased from JOINN Laboratories (Suzhou). CRAMP knockout C57BL/6 mice were preserved in our laboratory. All animal experimental procedures were approved by the Jiangnan University Experimental Animal Management and Animal Welfare Ethics Committee (IACUC Issue Number: JN. No20191015c0401218). Mice were raised under conventional controlled conditions (22 • C, 55% humidity and day-night rhythm) and had free access to a standard diet and tap water. All mice were allowed to acclimate to these conditions for at least 2 days before inclusion in experiments.

Enzyme-Linked Immunosorbent Assay (ELISA) Assay
IL-6 ELISA detection kit (SBJ-M0657) were purchased from SenBeiJia Biological Technology Co., Ltd. (Nanjing, Jiangsu, China). Labeled antibodies and the biotin were co-incubated with the test samples. The optical density (OD) values of the samples were detected using a BioTek microplate reader. Standard curves were plotted according to standard OD values, and test sample concentrations were calculated from the standard curve.

ROS Detection
ROS was detected by using the commercial assay kit. Briefly, cell extracts were incubated with ROS specific dye, 2, 7-dichlorofuorescin diacetate (DCFH-DA), at 37 • C for 30 min, and then were centrifuged, washed and suspended in PBS. ROS were detected by using Epoch Microplate Reader (BIO-TEK, VT, USA) at 525 nm and Fluorescence microscope. Dihydroethidine hydrochloride (5 µM, Molecular Probes) was topically applied to the freshly cut frozen aortic sections (10 µm) for 30 min at 37 • C to reveal the presence of ROS as red fluorescence (585 nm) by Fluorescence microscopy.

Statistical Analysis
All statistical analysis was carried out by using GraphPad Prism software. Error bars for in vitro and in vivo analysis represent the standard deviation among intra-class data collected from more than 3 independent experiments. Data were analyzed by using analysis of variance (ANOVA). Statistical significance was determined using unpaired Student's two-tailed t-test for two data sets and using a one-way ANOVA and followed by least significance difference multiple comparison tests. Statistical significance was defined as * p < 0.05; ** p < 0.01; *** p < 0.001.

CRAMP Inhibits PDGF-BB-Induced VSMC Phenotypic Transformation, Proliferation and Migration
VSMC phenotypic change to dedifferentiated state was a key step in arterial neointimal hyperplasia during the formation of restenosis [29]. To investigate the function of CRAMP on VSMC phenotypic transformation, we first detected the cytotoxity of CRAMP on VSMC. The MTT assay showed that CRAMP have almost no effects on VSMC at the maximum dose at 1000 ng/mL ( Figure 1A). Furthermore, the western blot results showed that CRAMP concentration-dependently reversed PDGF-BB-mediated the decrease of α-SMA and SM22α expression ( Figure 1B). These results suggested that CRAMP could inhibit PDGF-BB-induced VSMC phenotypic transformation.
We then detected the effects of CRAMP on VSMC proliferation and migration. As showed in Figure 2A,B CRAMP significantly inhibited PDGF-BB-enhanced cell viability of VSMC. The EdU assay also showed that CRAMP could decrease PDGF-BB-mediated VSMC proliferation. Followingly, we detected the wound healing assay and transwell assay, and the results showed that CRAMP could significantly inhibit both PDGF-BB-induced VSMC migration and invasion. Above data suggested that CRAMP could inhibit PDGF-BB-elevated VSMC proliferation and migration.

CRAMP Inhibited PDGF-Mediated IL-6/STAT3 Activation
Activation of ERK1/2 and STAT3 plays an effective role in VSMC phenotypic switching [30][31][32][33][34][35]. To find out the mechanisms of CRAMP in regulating VSMC phenotypic modulation, we first examined the effects of CRAMP on ERK1/2 and STAT3 activation. As showed in Figure 3A, the phosphorylation of ERK1/2 and STAT3 were significantly enhanced when treated with PDGF-BB, while the level of p-STAT3 but not p-ERK1/2 was inhibited when treated with both PDGF-BB and CRAMP.

CRAMP Prevented PDGF-BB-Enhanced ROS by Targeting NOX1
Intracellular ROS accumulation is critical for NF-κB activation [37]. Our results showed that pretreated with CRAMP inhibited PDGF-BB could significantly increase the ROS level, and CRAMP alone could also enhance the ROS level after 4 h, but at the same time, CRAMP could inhibit the effect of PDGF-BB ( Figure 6A,B). Since CRAMP can affect ROS levels in VSMC, we wonder whether CRAMP plays a role in major ROS producing enzyme NADPH oxidase (NOX). The results showed that the protein abundance of NOX1, NOX2 and NOX4 increased in PDGF-BB treated cells. CRAMP can significantly inhibit the increase of NOX1 induced by PDGF-BB, but has no significant effect on NOX2 and NOX4 ( Figure 6C). Next, we used ROS scavenger N-acetyl-L-cysteine (NAC) to confirm the role of NOX1-ROS in PDGF-BB-mediated phenotypic transformation, as showed in Figure 6D, NAC could significantly reverse the reduction of α-SMA and SM22α which induced by PDGF-BB. These results indicate that CRAMP can inhibit PDGF-BB function by regulating ROS levels in VSMC and targeting NOX1. Figure 6. CRAMP prevented PDGF-BB-enhanced ROS by targeting NOX1. (A) VSMC were pretreated with CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) at different times (0.5, 1, 2, 4, 6 h). The level of ROS was detected using fluorescence microplate reader. Data of 3 independent experiments is presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control, n = 5. (B) VSMC were pretreated with CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) for 4 h. The level of ROS was detected by immunofluorescence detection. (C) VSMC were pretreated with CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) for 24 h following by immunoblotting with anti-NOX1, anti-NOX2 and anti-NOX4 antibodies. (D) VSMC were pretreated with CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) for 24 h followed by immunoblotting with anti-α-SMA and anti-SM22α antibody. Data of 3 independent experiments is presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control, n = 3.

CRAMP Repressed Intimal Hyperplasia and Suppressed ROS/IL-6 Generation In Vivo
Compared with the Sham group, significant intimal hyperplasia was observed both in the wild type (WT) and CRAMP knockout (CRAMP −/− ) mice, and the proportion of intimal hyperplasia in the CRAMP −/− mice was higher than that in the WT mice ( Figure 7A). Administration of CRAMP obviously reduced the ratio of neointimal area to media area in injured carotid arteries of both WT and CRAMP −/− mice ( Figure 7A). The phenotypic transformation marker α-SMA are significantly reduced in both WT and CRAMP −/− model groups, and CRAMP can significantly inhibit this phenomenon ( Figure 7B). Meanwhile, the ROS level and IL-6 level were also significantly enhanced in both WT and CRAMP −/− model groups, and CRAMP could significantly block it ( Figure 7C,D). All these results demonstrated that CRAMP repressed intimal hyperplasia and suppressed ROS/IL-6 generation.

Discussion
Atherosclerosis is a chronic progressive inflammatory disease and a leading cause of death worldwide [41][42][43]. In the samples of atherosclerosis patients, the content of LL-37 (CRAMP in mice) was much higher than that of normal volunteers [44], suggesting that LL-37 may play a role in the development of atherosclerosis. In present study, we explored the role of CRAMP in regulating the phenotype transformation and the underlying mechanism of VSMC exposed to PDGF-BB. Our results demonstrated that CRAMP treatment blocked NF-κB nuclear translocation, IL-6 autocrine and thus the expression of phenotypic transformation marker, α-SMA and SM22α in VSMC. Collectively these findings demonstrated a role in attenuated phenotype transformation, proliferation and migration of VSMC in response to PDGF-BB. Results from the present study also suggested that CRAMP participate oxidative stress, inhibiting ROS and following regulating NF-κB/IL-6 cascade. Additional studies will be required to test the direct target of CRAMP and to address the role of CRAMP for the regulation of ROS production in response to other stimuli.
IL-6 is a pleiotropic cytokine. Several studies indicated that IL-6 has critical pathophysiological roles in cardiovascular diseases, such as atherosclerosis [45,46]. Nevertheless, it has been suggested that locally secreted IL-6 is involved in the VSMC proliferation in response to PDGF [47]. Similarly, our results showed that under the stimulation of PDGF-BB, VSMC can secrete IL-6, which can regulate the phenotypic transformation of VSMC, and this process is realized by CRAMP by regulating oxidative stress, that is, inhibiting the production of ROS.
Previous studies have demonstrated that incubation of VSMC with PDGF-BB triggers the rat sarcoma (Ras)/ rapidly accelerated fibrosarcoma (Raf)/ mitogen-activated protein kinase kinase (MEK)/ extracellular regulated protein kinases (ERK) kinase cascade, which leads to Elk-1 phosphorylation and the inhibition of SMC, specific marker genes, via the displacement of myocardin from smooth muscle cell-specific prompters [48]. Our present results showed that PDGF-BB enhanced the phosphorylation of ERK1/2 but CRAMP failed to block the activation although it has the similar effects with PDGF-BB all alone. Additional studies will be needed to unravel whether CRAMP has functions on other mitogen-activated protein kinases including p38 and JNK, and how CRAMP exerts functions on VSMC alone. Furthermore, accumulating evidence supports an important role for the activation of STAT3 in PDGF-BB-induced VSMC proliferation and migration [49]. Similarly, our results demonstrated that PDGF-BB regulates VSMC phenotypic transformation through the IL-6-STAT3 pathway and CRAMP could block the activation.
The NOX catalytic subunits-NOX1, NOX2, and NOX4-share a conserved structure and associate with p22phox on the cell membranes and are expressed in the vascular wall cells in rodents and humans [50]. NOX1 is predominantly expressed in VSMC and NOX1 plays a critical role in VSMC function in response to pathophysiological stimuli. The NOX1 expression is induced in neointimal SMC after vascular injury where it mediates cell migration, proliferation, and extracellular matrix production, while NOX1 deletion significantly reduces neointima hyperplasia [51][52][53]. The NF-κB pathway is the main pathway through which NOX1 plays its role, while ROS are often considered as the second messenger to mediate the activation of NF-κB [54,55]. The activation of NF-κB by ROS, specifically ROS generated by NOX, has been shown in VSMCs and other cells, and induced IL-6 release [56,57]. There have been many reports about the relationship between CRAMP/LL-37 and ROS. CRAMP/LL-37 can regulate the immune response by increasing the production of NOS and ROS during the treatment of infection [58]. LL-37 also increased platelet-neutrophil aggregates formation and activated neutrophil through promoting the production of ROS [59]. Recent studies showed that CWA was able to reduce LPS-induced ROS accumulation both in macrophages and in mice [27] and effectively decreased the level of ROS in E. coli K88-induced macrophages [26]. The results above indicated that the regulatory effect of CRAMP on ROS varies according to different states. Our current results also indicated that CRAMP can effectively inhibit ROS production induced by PDGF-BB. The relationship between CRAMP and ROS under other conditions remains to be further studied.
Herein, we have demonstrated that CRAMP modulates phenotypic transformation in VSMC. One potential mechanism by which this occurs is via CRAMP/autocrine IL-6/JAK2/STAT3 cascade; However, we also provided evidence that CRAMP regulates ROS and ROS-mediated NF-κB activation. These data suggest critical mechanism by which CRAMP regulates oxidation stress and cell differentiation. Given the reported relevance of CRAMP in atherosclerosis, the present findings which demonstrate a strong impact of CRAMP on the activation of ROS/NF-κB/IL-6 studies and provide new insights into the mechanisms by which CRAMP can regulate oxidation signaling in cardiovascular diseases like atherosclerosis.