Proteasome inhibitor bortezomib prevents proliferation and migration of pulmonary arterial smooth muscle cells

Pulmonary vascular remodeling is a key pathological process of pulmonary arterial hypertension (PAH), characterized by uncontrolled proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). Bortezomib (BTZ) is the first Food and Drug Administration (FDA)‐approved proteasome inhibitor for multiple myeloma treatment. Recently, there is emerging evidence showing its effect on reversing PAH, although its mechanisms are not well understood. In this study, anti‐proliferative and anti‐migratory effects of BTZ on PASMCs were first examined by different inducers such as fetal bovine serum (FBS), angiotensin II (Ang II) and platelet‐derived growth factor (PDGF)‐BB, while potential mechanisms including cellular reactive oxygen species (ROS) and mitochondrial ROS were then investigated; finally, signal transduction of ERK and Akt was examined. Our results showed that BTZ attenuated FBS‐, Ang II‐ and PDGF‐BB‐induced proliferation and migration, with associated decreased cellular ROS production and mitochondrial ROS production. In addition, the phosphorylation of ERK and Akt induced by Ang II and PDGF‐BB was also inhibited by BTZ treatment. This study indicates that BTZ can prevent proliferation and migration of PASMCs, which are possibly mediated by decreased ROS production and down‐regulation of ERK and Akt. Thus, proteasome inhibition can be a novel pharmacological target in the management of PAH.


| INTRODUCTION
Pulmonary arterial hypertension (PAH), a severe progressive disorder with high morbidity and mortality, is mainly caused by the increase of pulmonary vascular resistance (PVR). 1,2The main cause of increased PVR in most patients with PAH is due to fixed vascular obstruction with loss of the cross-sectional area, which leads to reduced cardiac output, right heart failure, and premature death. 3Vasodilators are the mainstay of treatment for PAH; however, vasodilators treatment does not directly improve vascular obstruction nor resolve right ventricular hypertrophy caused by fibrosis, ischemia, and microvascular rarefaction. 4,5A new treatment paradigm for PAH is crucially needed to address the severe disease caused by dysfunction of the right ventricle and pulmonary vasculature.
Pulmonary vascular remodeling, a key pathological process of PAH, includes hyperplasia of the media and neomuscularization in the subendothelial layer, driven by uncontrolled proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). 1 Oxidative stress, an imbalance between reactive oxygen species (ROS) production and the antioxidant capacity of cells, has also been reported to contribute to the pathogenesis of PAH in several ways, such as pulmonary vascular remodeling, dysfunction of pulmonary endothelial cells, proliferation of PASMCs, and right ventricular hypertrophy.Antioxidant therapy has also become a major area of research in the treatment of PAH. 6 Several studies have shown that the dysregulation of PASMCs can be triggered by numerous environmental and extracellular factors, such as hypoxia, tumor necrosis factor, angiotensin II (Ang II), platelet-derived growth factor (PDGF), and endothelial (ET)-1. 7In addition, Ang II and PDGF are considered to be the potent mitogen and chemoattractant for PASMCs and contribute essentially to the progression of PAH. 8,9oteasomes are multimeric protease complexes, comprising a 20S core catalytic complex with 19S regulatory subunits at each end.
The 20S core catalytic complex contains three active sites, including chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (CP-L) active sites. 10There is emerging evidence showing that proteasome has a role in the pathogenesis of PAH.For example, a proteomic analysis reveals that proteasome subunit beta 6 is involved in pulmonary vascular remodeling in rats and knockdown of this subunit using siRNA prevented PASMC proliferation. 11Similarly, a recent animal study of PAH also demonstrated upregulation of proteasome in the mitochondrial proteomic analysis. 12The exact mechanisms of proteasome in PAH is not fully understood.However, proteasome inhibition has also been shown to increase anti-oxidative capacity by increasing antioxidant proteins such as the superoxide dismutase type 1 (SOD1) and catalase, and reducing the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression. 13,14In addition, proteasome inhibition may also effectively scavenge ROS by increasing the expression and activity of nitric oxide synthase, and promoting nitric oxide production.BTZ is the first Food and Drug Administration (FDA)-approved proteasome inhibitor for the treatment of multiple myeloma. 15It is known that inhibition of proteasome function ameliorates the development of PAH 16 and proteasome inhibitor inhibits the proliferation and migration of vascular smooth muscle cells. 2,17,18BTZ attenuates hypoxia-induced PASMCs proliferation by restoring mitofusin-2 expression, and inhibits right ventricular hypertrophy and pulmonary vascular remodeling both in hypoxia-and monocrotalineinduced PAH animal models. 2,19Even though anti-proliferative effects of BTZ in PASMCs has been reported, 19 the mechanism is not well understood.Furthermore, it remains unclear whether it can inhibit migration, another major factor mediating vascular remodeling.
In this study, the role of BTZ on proliferation, migration, ROS production, and the phosphorylation of ERK and Akt in Ang II-and PDGF-BB-induced PASMCs models have accordingly been clarified.(Cat #ARG62346, Hsinchu, Taiwan).Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and all other tissue culture reagents were obtained from GIBCO BRL Life Technologies (Grand Island, NY, USA).

| Preparation of PASMCs
PASMCs were isolated from the pulmonary artery of 16 male Wistar rats (6-to 8-week-old; 180-200 g) purchased from BioLASCO Taiwan Co., Ltd.(Taipei, Taiwan).These rats were sacrificed in different experiments.This study was approved by the Institutional Animal Care and Use Committee of the Kaohsiung Medical University (IACUC Approval No: 108118).Animals were cared for in accordance with Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health.All rats were housed in a temperature-controlled environment (22 ± 2 C) with a relative humidity of 55 ± 10% and a 12-h light/12-h dark cycle with free access to food and sterile tap water.PASMCs were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin and incubated in a humidified 37 C incubator with 5% CO 2 .Cells were passaged at 70%-80% confluence by dissociation from plates with 0.25% trypsin-EDTA and passages 2-5 were used in the experiments.The purity of PASMCs was examined by immunofluorescence staining for α-smooth muscle actin (>95% of cells stained positive) and lack of staining for vimentin. 1

| MTT assay
Cells were seeded at a density of 3 Â 10 3 cells per well in 96 well tissue culture plates.After serum starvation for 24 h, cells were treated with different concentrations of BTZ to detect the toxicity of BTZ to PASMCs.In addition, effects of BTZ in FBS-, Ang II-, and PDGF-BB-stimulated proliferation were also detected.Cells were pre-treated with or without BTZ for 1 h, followed by treatment with FBS (10%), Ang II (100 nM), and PDGF-BB (20 ng/mL) for 24 h.MTT solution was added to the medium for 2 h.The culture medium was then removed and the cells were dissolved in isopropanol and shaken for 10 min.The amount of MTT formazan was quantified at absorbance of 540 and 630 nm using an ELISA reader (DYNEX Technologies, Denkendorf, Germany).
Cells were seeded at a density of 5 Â 10 4 cells per well in the upper chamber.After serum starvation for 24 h, FBS (10%), Ang II (100 nM), and PDGF-BB (20 ng/mL) were added to medium for 24 h, with or without pre-treatment of BTZ for 1 h in the lower chamber.Nonmigrated cells on the upper membrane surface were removed and those on the lower surface were fixed in methanol and stained with Giemsa (Cat #GS500, Sigma-Aldrich, Inc., St. Louis, MO, USA).The cell numbers per six high-power fields (200Â HPF) were counted and the mean numbers of cells were used to express migration ability.

| Wound healing assay
Cells were seeded at a density of 2 Â 10 5 cells per well in 6 well tissue culture plates.Vertical lines were scratched using a 200 μL pipette tip, and the floating cells were removed by washing three times in 1Â PBS.After serum starvation for 24 h, cells were pre-treated with or without BTZ for 1 h, followed by treatment with FBS (10%), Ang II (100 nM), and PDGF-BB (20 ng/mL) for 24 h and images were acquired by a light microscopy (Nikon Ti2-U).Quantitative calculations were performed using Image J software.

| DCFH-DA assay
Cells were seeded at a density of 5 Â 10 3 cells per well in 96-well tissue culture plates.After serum starvation for 24 h, cells were pretreated with or without BTZ for 1 h, followed by treatment with FBS (10%), Ang II (100 nM), and PDGF-BB (20 ng/mL) for 24 h.Cells were stained with 5 μM of DCFH-DA for 30 min at 37 C, and then immediately examined under a fluorescence microscope (Nikon Ti2-U) at an excitation/emission of 485/535 nm.Results were presented as relative fluorescence intensity normalized to control.

| MitoSOX assay
Cells were seeded at a density of 5 Â 10 3 cells per well in 96-well tissue culture plates.After serum starvation for 24 h, cells were pretreated with or without BTZ for 1 h, followed by treatment with FBS (10%), Ang II (100 nM), and PDGF-BB (20 ng/mL) for 24 h.Cells were stained with 5 μM of MitoSOX, a fluorescent probe specific for mitochondrial superoxide, for 1 h at 37 C, and fixed by 1% formaldehyde after washed with PBS.Fluorescence was examined under a fluorescent microscope (Nikon Ti2-U) at an excitation/emission of 510/580 nm.Results were presented as relative fluorescence intensity normalized to control.

| Western blot
Cells were washed by cold 1Â PBS, and then lysed by lysis buffer (Cat #78501, M-PER™, Mammalian Protein Extraction Reagent, Pierce, USA).The lysed cells were centrifuged at 13,000Â g for 30 min at 4 C, then, supernatant proteins were collected and stored at À80 C until analysis.Equivalent amount of protein (25 μg/mL) was loaded on SDS-polyacrylamide gel electrophoresis (10%-14%) and transferred to polyvinylidene difluoride membranes.Membranes were incubated in a blocking buffer (5% nonfat dry milk) for 1 h at room temperature, followed by incubation in primary antibody for 2 h at room temperature.After washing off the primary antibody, the blots were incubated with horseradish peroxidase-linked secondary antibody (Chemicon, Inc., Temecula, CA) for 1 h at room temperature, and the immuno-reactive bands were detected by chemiluminescence reagents (Cat #NEL103E001EA, PerkinElmer Life Sciences, Inc., Waltham, MA).β-actin was probed as a control to ensure equal protein loading.All primary antibodies were used at a dilution of 1:1000, and secondary antibodies were used at a dilution of 1:5000 in Western blot.

| Statistical analysis
Data were analyzed by GraphPad Prism5.0 (GraphPad Software, Inc., San Diego, CA, USA).One-way ANOVA was used to compare the means of different groups and the Tukey multiple comparison test was used as a post hoc test following ANOVA.Data are shown as means ± standard error (SEM) from at least three independent experiments.p < 0.05 was considered statistically significant.
PASMCs were treated with different concentrations of BTZ for 24 h.

| BTZ attenuated FBS-, Ang II-, and PDGF-BBinduced migration of PASMCs
1][22] To investigate effects of BTZ on Ang II and PDGF-BB-induced migration, Boyden Chamber assay and wound healing assay were used to detect cell migration ability.PASMCs were pre-treated with BTZ (10 nM) for 1 h, followed by treatment with FBS (10%), Ang II (100 nM) and PDGF-BB (20 ng/mL) for 24 h, and migration ability was examined.Results showed that the number of migrated cells in Boyden Chamber assay was significantly higher in induced groups (FBS, Ang II, and PDGF-BB) than that in the control group, and BTZ reduced FBS-, Ang II-, and PDGF-BB-induced numbers of migrated cells (Figure 2).In wound healing assay, the wound closure rate was also significantly higher in induced groups (FBS, Ang II, and PDGF-BB) than that in the control group, and BTZ decreased FBS-, Ang II-, and PDGF-BB-induced wound closure rate (Figure 3).These results indicated that BTZ significantly attenuated FBS-, Ang II-, and PDGF-BB-induced migration of PASMCs.

| BTZ attenuated FBS-, Ang II-, and PDGF-BBinduced ROS production of PASMCs
Excessive ROS production causes oxidative stress, contributing to the pathogenesis of PAH. 6 To investigate effects of BTZ on ROS production, cellular ROS production and mitochondrial ROS production were detected by DCFH-DA assay and MitoSOX assay, respectively.Furthermore, PASMCs were pre-treated with BTZ (10 nM) for 1 h, followed by treatment with FBS (10%), Ang II (100 nM) and PDGF-BB (20 ng/mL) for 24 h, and then ROS production was detected.Results showed that BTZ attenuated FBS-, Ang II-, and PDGF-BB-induced cellular ROS production (Figure 4).Similarly, BTZ also attenuated FBS-, Ang II-, and PDGF-BB-induced mitochondrial ROS production (Figure 5).Furthermore, we found that NAC, a ROS scavenger, attenuated FBS-, Ang II-, and PDGF-BB-induced cellular ROS production and mitochondrial ROS production.These results indicate that BTZ has similar ROS-reducing effects as NAC.

| BTZ attenuated Ang II-and PDGF-BBinduced the phosphorylation of ERK and Akt in PASMCs
ERK and Akt activation play important roles in vascular remodeling. 23 investigate effects of BTZ on ERK and Akt activation, the phosphorylation of ERK and Akt was detected by Western Blot.PASMCs were pre-treated with BTZ (10 or 50 nM) for 1 h, followed by treatment with Ang II (100 nM) and PDGF-BB (20 ng/mL) for 15 min, and the phosphorylation of ERK and Akt was detected.Results showed that BTZ inhibited Ang II-and PDGF-BB-induced phosphorylation of ERK and Akt in PASMCs (Figure 6).In summary, a proposed mechanism in this study is shown in Figure 7. Abnormal proliferation and migration of vascular smooth muscle cells are characteristic of many proliferative vascular diseases such as PAH, atherosclerosis, and restenosis.In addition, targeting oxidative stress is considered as a preventive and therapeutic approach for vascular remodeling. 241][22] In this study we demonstrated that BTZ attenuated FBS-, Ang II-and PDGF-BB-induced proliferation, migration, and ROS production.In addition, the phosphorylation of ERK and Akt induced by Ang II and PDGF-BB was also inhibited by BTZ in PASMCs.Our study revealed that BTZ attenuated Ang II and PDGF-BB-induced proliferation, migration, and ROS production, which was associated with down-regulation of ERK and Akt in PASMCs.
The imbalance between ROS production and the antioxidant capacity of cells results in oxidative stress, contributing to the pathogenesis of PAH. 6 ROS production appears to be critical for proliferation and migration of smooth muscle cells and is essential in vascular remodeling of PAH. 1,25There are many sources of ROS, including the mitochondrial respiratory chain, the NADPH oxidases, xanthine oxidase, lipoxygenases, and nitric oxide synthases. 25Mitochondria and NADPH oxidases are the major sources of cellular ROS production in vascular smooth muscle cells, 25,26 and approximately 90% of cellular F I G U R E 3 BTZ attenuated FBS-, Ang II-, and PDGF-BB-induced wound healing migration of PASMCs.PASMCs were pretreated with or without BTZ (10 nM) for 1 h, followed by treatment with (A) FBS (10%), (B) Ang II (100 nM), and (C) PDGF-BB (20 ng/mL) for 24 h, and then migration ability was detected by wound healing assay.Values represent mean ± SEM, n = 3. ###p < 0.001, compared with the control group.**p < 0.01, ***p < 0.001, compared with the induced groups (FBS, Ang II, and PDGF-BB).
ROS production is generated in mitochondria 26,27 ; therefore, we focused on effects of BTZ on mitochondrial ROS production in this study.
There was contradictory effect of anti-oxidative in different cells or organs.Some previous studies have shown that in cancer cell lines BTZ can induce ROS 28,29 ; however, in a recent animal study of myocardial ischemia reperfusion injury, BTZ could reduce ROS by augmenting oxidative stress related protein levels of superoxide dismutase, catalase and glutathione.Mechanistically, they found that BTZ promoted nuclear translocation of transcriptional factor nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 expression. 30In our study, BTZ attenuated ROS production induced by three inducers in PASMCs, suggesting cell-specific differences in BTZ's effects on ROS.
BTZ is an anti-cancer agent that has been reported to induce endoplasmic reticulum (ER) stress, to activate unfolded protein response, and trigger apoptosis and autophagy in several cancer cells.Currently, there is no study investigates that BTZ treatment can induce ER stress or unfolded protein responses in the PASMCs, however, ROS mediates several critical aspects of the ER stress response.Of the many cell types, myeloma cells rely heavily on the secretory pathway of ER, which is sensitive to proteasomal inhibition, so appear to be particularly sensitive to BTZ.Recent reports indicate that proteasome inhibitors have diverse effects on nonmalignant cells, and their effects depend on dosage and cell type.Biphasic dosage effects have been reported for proteasome inhibitors in different cells, including astrocytes and endothelial cells, whereby they afford protection at low doses and induce apoptosis at high doses. 31K and Akt activation play important roles in cell proliferation, and has been increasingly recognized as a regulator of vascular remodeling. 23Previous studies have shown that the expression levels of F I G U R E 5 BTZ attenuated FBS-, Ang II-, and PDGF-BB-induced mitochondrial ROS production in PASMCs.PASMCs were pretreated with BTZ for 1 h, followed by treatment with (A) FBS (10%), (B) Ang II (100 nM), and (C) PDGF-BB (20 ng/mL) for 24 h, and then mitochondrial ROS production was detected by MitoSOX assay.Values represent mean ± SEM, n = 6.###p < 0.001, compared with the control group.**p < 0.01, ***p < 0.001, compared with the induced groups (FBS, Ang II, and PDGF-BB).phosphorylated ERK and Akt was upregulated with Ang II and PDGF-BB treatment, which resulted in the promotion of PASMCs proliferation. 1,32Our results showed that BTZ attenuated Ang II-and PDGF-BB-induced phosphorylation of ERK and Akt, suggesting that BTZ inhibited proliferation and migration, at least in part through the ERK and Akt pathways.
Several studies have also investigated the possible mechanisms by which BTZ regulates cell proliferation and migration.A previous study indicates that BTZ attenuates Ang II-induced hypertensive response in rats, with associated decreased aortic wall-to-lumen ratio, collagen deposition, MMP2 activity, proteasomal chymotrypsin-like activity, Ki67 staining, ROS generation, VCAM-1 immunoreactivity, and expression of TIMP1 and TIMP2. 33In PASMCs, our previous study suggests that BTZ inhibits proliferation by overexpression of mitofusin-2. 2Likewise, BTZ inhibits proliferation by inhibiting caveolin-1/calcium signaling axis in human PASMCs. 34In addition, BTZ reduces proliferation and migration of retinal pigment epithelium cells by the NF-κB signaling pathway. 35Compared with previous studies in various cells, our study shows that in PASMCs, BTZ also conveys anti-proliferative and anti-migratory effects.Furthermore, the present study shows novel mechanisms underlying these antiremodeling effects by BTZ, including attenuation of mitochondrial ROS production and deactivation of MAPK and Akt.
In addition, despite the apparent success of BTZ in the treatment of multiple myeloma, some patients still do not respond to BTZ therapy or develop drug resistance. 36BTZ inhibits the CT-L activity, but does not have any significant effect on T-L and CP-L activity. 37hibition of CT-L by BTZ leads to the compensatory activation of T-L and CP-L, resulting in resistance to BTZ.The development of an irreversible pan-proteasome inhibitor is considered an effective approach to overcome drug resistance. 2 For example, carfilzomib is a next-generation FDA-approved proteasome inhibitor and exerts its inhibitory function by binding to the CT-L subunit. 38Unlike BTZ, the binding of carfilzomib to the CT-L subunit is irreversible, and carfilzomib maintains its cytotoxic potential in the BTZ-resistant cell lines. 39In addition, marizomib is another next-generation irreversible proteasome inhibitor. 37As marizomib inhibits all three major proteolytic activities (CT-L activity, T-L activity and CP-L activity), it can target proteasomes more broadly. 6,40Marizomib has been tested in clinical trials in a variety of cancers such as refractory multiple myeloma, leukemia, lymphoma, glioblastoma, and malignant glioma. 6ether the newly developed next-generation proteasome inhibitors have more potential than BTZ in the treatment of PAH deserves further study in the future.
There are two limitations in this study.First, there is no animal experiment in this study.However, previous studies have shown that in animal models of mice and rats, BTZ could attenuate right ventricular systolic pressure, suppress right ventricular hypertrophy and thickening of pulmonary vascular walls. 16,19Thus in our in vitro study, we aimed to investigating cellular and molecular mechanisms underlying its anti-remodeling effects.Second, we did not compare the efficacy of BTZ with NAC in the same experiments, since our study design was not to determine if BTZ is the best ROS-reducing agent.Instead, we assume that in addition to ROS-reduction, there are other potential synergistic mechanisms, such as signal transduction, underlying the anti-remodeling effects of BTZ and may warrant further investigation.
In conclusion, our study demonstrated the therapeutic potential and mechanism of proteasome inhibition in Ang II-and PDGF-BB-induced dysregulation of PASMCs, shedding some lights in novel therapeutic strategies for PAH.Further clinical investigations are required to substantiate these important findings.

F
I G U R E 6 BTZ attenuated Ang II-and PDGF-BB-induced phosphorylation of ERK and Akt in PASMCs.PASMCs were pretreated with BTZ for 1 h, followed by treatment with (A) Ang II (100 nM) and (B) PDGF-BB (20 ng/mL) for 15 min, and then the phosphorylated and total forms of ERK and Akt was detected by Western Blot.Value represents the mean ± SEM, n = 3. #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the control group.*p < 0.05, **p < 0.01 compared with the induced groups (Ang II and PDGF-BB).F I G U R E 7 Proposed diagram of BTZ attenuating Ang II-and PDGF-BB-induced proliferation and migration of PASMCs.BTZ attenuates proliferation, migration, ROS production, and the phosphorylation of ERK and Akt induced by Ang II and PDGF-BB in PASMCs, suggesting a possible therapeutic role of BTZ in pulmonary vascular remodeling and providing novel therapeutic strategies for PAH.mtROS, mitochondrial ROS.