Role of Uric Acid in Vascular Remodeling: Cytoskeleton Changes and Migration in VSMCs

The mechanisms by which hyperuricemia induces vascular dysfunction and contributes to cardiovascular disease are still debated. Phenotypic transition is a property of vascular smooth muscle cells (VSMCs) involved in organ damage. The aim of this study was to investigate the effects of uric acid (UA) on changes in the VSMC cytoskeleton, cell migration and the signals involved in these processes. MOVAS, a mouse VSMC line, was incubated with 6, 9 and 12 mg/dL of UA, angiotensin receptor blockers (ARBs), proteasome and MEK-inhibitors. Migration property was assessed in a micro-chemotaxis chamber and by phalloidin staining. Changes in cytoskeleton proteins (Smoothelin B (SMTB), alpha-Smooth Muscle Actin (αSMA), Smooth Muscle 22 Alpha (SM22α)), Atrogin-1 and MAPK activation were determined by Western blot, immunostaining and quantitative reverse transcription PCR. UA exposition modified SMT, αSMA and SM22α levels (p < 0.05) and significantly upregulated Atrogin-1 and MAPK activation. UA-treated VSMCs showed an increased migratory rate as compared to control cells (p < 0.001) and a re-arrangement of F-actin. Probenecid, proteasome inhibition and ARBs prevented the development of dysfunctional VSMC. This study shows, for the first time, that UA-induced cytoskeleton changes determine an increase in VSMC migratory rate, suggesting UA as a key player in vascular remodeling.


Introduction
Patients with hyperuricemia (HU) are at high risk for developing hypertension [1], as well as cardiovascular (CV) and kidney diseases [2][3][4]. The strength of data from epidemiologic studies proponing UA as a risk factor for CV and total mortality [5,6] encourages the search for the possible pathogenetic mechanisms of this relationship [7,8]. Although the benefits of urate-lowering therapy (ULT) in reducing CV and renal risk in asymptomatic HU are still a matter of debate [9][10][11][12][13], the amount of evidence demonstrating the involvement of UA in kidney and vascular damage [14] supports the hope for valid therapeutic strategies. Recently, HU has been considered as an independent risk factor of vascular inflammation and remodeling [15], and it has been proposed as a promoter of arterial stiffness in humans [16][17][18]. These findings inspired us to investigate the mechanisms triggered by UA in vessel cells. Several experimental studies identified potential pathways underlying UAinduced vascular changes [19]. These mechanisms include inflammation [20,21], oxidative stress [22] and activation of the renin-angiotensin aldosterone system (RAAS) [14], which together contribute to endothelial dysfunction and the proliferation/phenotypic transition of VSMCs, finally resulting in vascular remodeling, arterial stiffness and cardiovascular disease (CVD) [14,18,22].
VSMCs form the medial layer of major arteries and their contractile ability controls the vessel tone. In physiological condition, VSMCs are quiescent but retain remarkable plasticity [23]. On the contrary, in response to injury, VSMCs can become migratory and downregulate components of the smooth muscle-contractile machinery. It has been proposed that these processes, namely the phenotypic switch, lead to vascular remodeling and atherosclerotic lesions [24].
Although several studies have highlighted the importance of UA as a promoter of cell changes [25][26][27], and new mechanisms for UA-mediated vascular injury have recently been provided [28], a better knowledge of the role played by UA, and of the different pathways involved, may be helpful for devising new "cardiovascular protective therapies" aimed to prevent tissue damage.
In this study, we explore the effects of UA on the VSMCs phenotype. With this purpose, we evaluated UA-induced changes in the expression and arrangement of cytoskeleton components and in the migratory capacity in MOVAS cells, an immortalized VSMC line from murine aorta. We also investigated the pathways involved in these processes by pretreatment of cultured VSMCs with different inhibitors. First, we tested urate transporter blocker (Probenecid) and angiotensin receptor blockers (ARBs), valsartan and losartan. Secondly, bearing in mind that Atrogin-1, a ubiquitin ligase 3 which controls the VSMC phenotype and survival signaling [29], has been previously demonstrated to be upregulated in uremic milieu [30] and to be involved in the proteasome degradation of ubiquitinated proteins in VSMCs [31], we used MG132 (a proteasome inhibitor) to test the involvement of proteasome in UA-induced VSMC alterations. Thirdly, we tested the contribution of MAP kinase in VSMCs migration by U0196, a MEK inhibitor.

Cell Viability and Area
To examine the effects of UA on cell viability, we treated MOVAS with different UA concentrations (6 to 12 mg/dL) for 24-48 h. Cells showed an increase in proliferation regardless of UA concentration. As graphed in Figure 1A, at 24 h, the percentage of cells rose up to + 11-16% in respect to no treated cells (CTR) (p < 0.05) and a similar increment was observed after 48-h of treatment (+8-11%, p < 0.05).
We found the α-SMA protein was upregulated after 24 h UA 9 mg/dL treatment (1.4 fold vs. CTR, p < 0.05), but dropped out after 48 h (−40%, p < 0.01, Figure 3A). These data were confirmed by immunocytochemistry and immunofluorescence. At 48 h, in UA 9 mg/dL treated cells, immunostaining for α-SMA was fainter than in CTR ( Figure 3B) and fluorescence intensity was decreased (−50% vs. CTR, p < 0.01) as graphed in Figure 3C. In addition, a deranged architecture and a disorganized orientation of α-SMA were observed ( Figure 3D).

UA Promoted MOVAS Cells' Migration
To explore the role of UA in the phenotypic modification of MOVAS cells, we studied its effect on migration in the micro-chemotaxis chamber. UA stimulated migration independently by time of exposure ( Figure 4A). Indeed, incubation with 9 mg/dL UA increased the chemotactic index of cells toward a complete medium (24 h: 1.7-1.9 fold vs. CTR, p < 0.0001 48 h: 1.7-2.2 fold vs. CTR, p < 0.05). Accordingly, when cells were stained with Alexa-Fluor 594-conjugated phalloidin, UA induced the F-actin re-arrangement in thinner and poorly oriented fibers localized at cortical level, while in CTR, its compact polymerization in stress fibers along the major cell axis was observed ( Figure 4B). These effects were blunted by 10-20 µm Probenecid ( Figure S1 (Supplementary Materials)). Figure 5A,B show the effects of 9 mg/dL UA on p44/42 MAPK phosphorylation and its involvement in migratory capacity. UA treatment raised the levels of phosphorylation until~2-2.5 folds over a 6-h incubation period (p < 0.05 vs. T0). As a next step, we observed that UA-induced migration was stopped by the MEK-1 and -2 inhibitor U0196 ( Figure 5C,D).

UA Induced Atrogin-1 Expression
In an attempt to identify a possible mechanism by which UA modifies VSMC phenotype, we evaluated the involvement of Atrogin-1, an E3 ubiquitin-ligase that directs the poly-ubiquitination of proteins, on the expression of α-SMA. First of all, we found out that at 48 h, 9 mg/dL, UA increased Atrogin-1 gene expression -2 fold vs. CTR (p < 0.05) ( Figure 6A). In addition, when we measured fluorescence intensity in cells exposed to 9 mg/dL UA and immuno-stained with antibody to Atrogin-1, we found it increased 2.75-fold vs. no treated cells (p < 0.05) ( Figure 6B,C). Then, we performed co-localization studies of Atrogin 1 and α-SMA and we observed that they were oppositely expressed: in CTR, α-SMA was highly expressed (green) and Atrogin-1 (red) was faintly expressed, whereas in UA-treated cells, the red fluorescence was predominant ( Figure 6C). Probenecid treatment blunted these effects ( Figure S2).
Finally, to confirm the role of the ubiquitin-proteasome system (UPS) in VSMC phenotypic modification, we pretreated MOVAS cells with 5 µm MG132 which, at 24 h, inhibited the overexpression of α-SMA, SMT B and SM 22 α (p < 0.01-0.001 vs. UA) ( Figure 7A) and rescued the migratory capacity induced by UA ( Figure 7B). However, L and V had an inhibitory effect on UA-induced migration ( Figure 9A) and this was confirmed by phalloidin staining that was similarly distributed in CTR in L-and V-treated cells, ( Figure 9B). Furthermore, L and V blunted p44/42 MAPK phosphorylation induced by UA ( Figure 10A,B).

Discussion
Several novel findings were made in this study: (1) UA treatment induced structural and functional transition into a proliferative/migratory phenotype of VSMCs; (2) UA determined an increase in the cellular migration rate of VSMCs; (3) Blocking the internalization of UA and the inhibition of proteasome, RAAS or MEK activity abolished the UA-induced cellular changes in VSMCs and their increase in migration rate. The mechanisms by which UA induces VSMCs changes are summarized in Figure 11. Taken together, these results provide compelling evidence that UA is a key player in vascular remodeling by proteasome and RAAS activation.
In human cells, UA has emerged as a major cause of damage in different pathophysiological conditions [21,[25][26][27]. We found that increasing UA promoted the proliferation of VSMCs ( Figure 1A) and determined an increase in cell area ( Figure 1B) in murine cells. Our results confirm [32] and expand previous findings corroborating the hypothesis that UA-induced VSMC hypertrophy is implicated in the development and progression of vascular damage observed in patients with increased UA levels [18,33].
VSMCs mainly has a contractility phenotype, which is responsible for the modulation of vascular tone. They also possess extensive plasticity such that they can be stimulated to transform from a contractile state into proliferative/migratory state [34]. The mature contractile phenotype of VSMCs is morphologically characterized by low numbers of protein synthesis structures, and a high expression of proteins involved in muscle contraction and anchorage including αSMA [35], smooth SM22α [36] and SMT B [37]. Synthetic phenotypes are associated with abnormal mechanical forces, which lead to tissue repair and remodeling.
With this in mind, we analyzed the changes in cytoskeleton protein expression induced by UA. The protein levels of α-SMA, SM22 α and SMT B, after a transitory growth in the earlier hours, were significantly decreased after 48 h of exposure to UA (Figure 2), sustaining the phenotypic switch to a synthetic pathway. The implication of UA in cytoskeleton changes of VSMC is further substantiated by our findings of a derangement of α-SMA architecture and a disorganized orientation of actin fibers in UA treated cells, compared to controls (Figure 3). The emerging role of cell migration as a necessary process in vessel wall remodeling has stimulated strong interest in cellular events and molecular mechanisms of VSMC migration. The migratory-proliferative phenotype is characterized by certain VSMC cytoskeletal structural changes that enable it to migrate to the source of initiating stimuli [38]. For the first time, our study demonstrated UA as a potential trigger for these changes ( Figure 4A). Cell migration begins with the stimulation of cell surface receptors that transduce the external signal to a series of coordinated remodeling events finally altering the structure of the cytoskeleton. Early signaling events provoke actin polymerization, which is critical for generating membrane protrusions at the leading-edge during cell migration [39]. In the present study, UA-treated cells showed cytoskeleton remodeling and rearrangement of F-actin fibers (marked with phalloidin staining), probably causing a detachment of focal contacts at the trailing edge, propelling the cell toward the stimulus ( Figure 4B). These results are of most interest because of the known mechanism by which VSMCs undergoing confined migration may promote the secretory phenotype and further accelerate vascular ageing [40]. The inhibition by Probenecid of this cascade of events, highlighted as the internalization of UA, is essential for the definition of VSMCs' structure ( Figure S1).
Much research exists about the role of biochemical signaling in VSMC migration, which remains challenging for our limited knowledge of biology complexity. Major goals of these studies include the definition of the proximal signals (i.e., UA in our study), as well as the signal transduction pathways that affect cell movement. Intracellular signal-transduction pathways such as mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK p44/42), leading to VSMC proliferation and migration have been demonstrated to be activated by several factors [41,42]. Our study identified UA as one of the potential triggers for these processes ( Figure 5).
The UPS is central for protein turnover as the main pathway for the degradation of proteins related to cell-cycle regulation, signaling and differentiation in eukaryotic cells, including VSMC [43]. Ubiquitination of the target protein is a reversible post-translational modification, which can occur under the action of ubiquitin-ligase enzymes [44]. Several studies suggested that UPS promotes protein degradation, underlying transition from contractile to proliferative VSMC phenotype, with the proteasome inhibitor shifting VSMC away from a synthetic morphology [45]. Our study reveals that Atrogin-1, a cardiac E3 ligase, was two-fold up regulated in VSMCs treated with 9 mg/dL UA compared to no treated cells ( Figure 6). Moreover, a pre-treatment with MG 132, a proteasome inhibitor, prevented all cytoskeleton and migration property changes induced by UA (Figure 7). These findings suggest that in cells exposed to increased UA, the UPS is a strong effector of VSMC phenotypic changes, provoking transition towards a synthetic VSMC phenotype as happened during the development of atherosclerosis and vascular diseases.
Angiotensin II (Ang II) plays a vital role in arterial remodeling through multiple pathways [46]. It accentuates the inflammatory response to the stimuli and promotes the migratory-proliferative activity of VSMC [47]. The present study contributes to the comprehension of the protective mechanisms exerted by RAAS inhibition. We described how ARB treatment (valsartan and losartan 10 µM) inhibited changes in cytoskeleton components (Figure 8), protein overexpression of p44/42 MAPK ( Figure 10) and migration in UA-stimulated VSMCs (Figure 9), indicating a possible mechanism by which ARBs were shown to be able to reduce neointimal hyperplasia.
This study has some limitations. A shift in cell phenotype is observed when VSMCs are removed from their native environment and placed in a culture, presumably due to the absence of the physiological signals that maintain and regulate the VSMC phenotype. Nevertheless, the inhibition both of UPS and RAAS (Figures 7 and 9), significantly blunts the changes in cytoskeleton proteins and cell phenotype we observed, suggesting UA as the through effector of this set of pathologic modifications. Moreover, the experiments were not performed using primary cells. Therefore, the results of the present study need to be confirmed by experiments on primary cell culture and on animal models.

Materials and Methods
The procedures were in accordance with the Declaration of Helsinki. The study was approved by the Ethical Committee of Regione Liguria (Comitato Etico Regionale, CER) on 28 June 2021 (Protocol UA-MSTN-VASC; N. Registro CER Liguria: 129/2021-DB id 11314).

Cell Treatments
MOVAS were grown to sub-confluence and then cultured in starving medium (with 1% FBS) in the presence of Uric Acid (UA) (6-12 mg/dL) (Merck group, Milan, Italy) for 24-48 h. Control cells (CTR) were not treated with UA. Firstly, we evaluated the effects of different concentrations of UA on cell viability and then, on the cell dimensions, the migratory capability and the expression of cytoskeleton proteins (α-SMA, SMT B, SM22α). Furthermore, the role of ubiquitin-proteasome (UPS) on UA effects was studied, evaluating atrogin-1 (a F-box protein or Ubiquitin-protein ligase E3) expression and exposing cells to MG132 (5 µM) (Medchemexpress, DBA Italia s.r.l Seregno, Italy), a potent, reversible and cell-permeable proteasome inhibitor. Probenecid (5-10 µM) (Merck group) was added to the cell culture to test its capability to blunt migration and atrogin-1 expression.
To understand if UA effects were direct or mediated by RAS activation, Losartan (L) or Valsartan (V) (both 10 µM) (Medchemexpress, DBA Italia s.r.l.) were added 1 h earlier than 9 mg/dL AU. Lastly, the UA-induced activation of p44/42 MAPK was studied and its effects on cell migration through pretreatment with U0126 (10 µM) (Merck group).

MTT Assay
This assay for cell viability is based on the reduction of 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) (Merck Group) by mitochondrial dehydrogenase in viable cells to produce a purple formazan product. Each experiment was performed according to the protocol as previously described [27].

mRNA Analysis
MOVAS cells were incubated for 24-48 h with or without 9 mg/dL UA. Total RNA was isolated using the Tri-Reagent (Zymo Research, Aurogene, Rome, Italy). A total of 1 µg RNA was used for cDNA synthesis with SCRIPT RT Supermix (BioRad Laboratories, Segrate, Italy). PCR amplification was carried out in a total volume of 10 µL, containing 1 µL cDNA solution, 5 µL SensiFAST SYBR no ROX mix (Meridian Bioscience, Aurogene, Rome, Italy), 0.08 µL of each primer (Tib Molbiol, Genoa, Italy) and 3.84 µL of nuclease-free water. β-actin was quantified, and used for the normalization of expression values of the other genes.
Assays were run in triplicate on MasterCycler realplex PCR system (Eppendorf, Hamburg, Germany). The primer sequences are reported in Table 1.

Immunocytochemistry and Immunofluorescence
MOVAS grown on chamber slides were incubated for 24-48 h with or without UA (9 mg/dL). After a five minute incubation in cold methanol, cells were exposed to anti α-SMA (Merck Group) and/or anti-atrogin-1 (rabbit polyclonal antibody) (ECM Bioscences, DBA Italia s.r.l.). For immunocytochemistry, cells were probed with an ultra-polymer goat anti-mouse IgG conjugated to HRP (ImmunoReagents Inc, Microtech, Pozzuoli, Italy) and developed with DAB detection kit (Roche, Microtech). Slides were counterstained with hematoxylin and examined by light microscopy.
For immunofluorescence, secondary antibodies conjugated to Alexa Fluor tm 488 and/or 594 were used (Invitrogen, Thermo Fisher Scientific, Monza, Italy). Nuclei were stained with DAPI.
The levels of cell fluorescence were determined by ImageJ and they were calculated as corrected total cell fluorescence (CTCF) using this formula: CTCF = Integrated Density-(Area of selected cell × Mean fluorescence of background readings) (https:// theolb.readthedocs.io/en/latest/index.html, accessed on 20 August 2021).

MOVAS Cell Dimension Analysis
The area of MOVAS cells was evaluated after 24 and 48 h exposure to UA (6-12 mg/dL) and compared with that of CTR. In brief, cells grown on chamber slides (EZ chamber slides; Merck Group) were fixed with 2% paraformaldehyde, stained with Hematoxylin-Eosin and photographed with a Leica Microsystems microscope (GmbH Wetzlar, Germany) (40× magnification). Images were then analyzed using MetaMorph ® NX software (Molecular Devices): the areas of randomly selected single cells were defined using the cursor and automatically estimated. The median values were calculated upon measurement of 100 cells/condition.

F-Actin Detection by Fluorescence Microscopy
Cells grown on EZ-chamber slides (Merck group) were exposed to UA (9 mg/dL) with or without inhibitors for 24-48 h. Cells were fixed with 2% paraformaldehyde, permeabilized with 0.05%Triton × 100 and stained with 5 U/mL Alexa-Fluor 594-conjugated phalloidin (ThermoFisher Scientific, Monza, MB, Italy). Nuclei were counterstained with DAPI. Cells were washed three times with PBS and analyzed by fluorescent microscopy.

Migration Assays
MOVAS were incubated for 24-48 h in the presence or absence of UA 9 mg/dL and/or Probenecid, Losartan, Valsartan and MG-132. Then, cells were tested for migration in a micro-chemotaxis Boyden chamber (Neuro Probe Inc., Gaithersburg, MD, USA) using a 8 µm-pore size, polycarbonate polyvinylpyrrolidone-free filters (Millipore). The lower wells of the chemotaxis chambers were filled with medium alone. After incubation (3 h, 37 • C), the filters were removed from the chambers, fixed and stained with May Grunwald-Giemsa stain (Carlo Erba, Cornaredo, Italy). Each condition was performed in duplicate. The cells of five random oil-immersion fields were counted and the chemotaxis index was calculated from the number of cells migrated to the test samples divided by the number of cells migrated to the control [12].

Statistical Analysis
Data are given as mean ± SEM. Statistical analysis was performed by Student's t-test or one-way Anova with Bonferroni's post-test for multiple group comparison. Statistical significance was set at p < 0.05. All statistical analyses were performed using Graph Pad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA.

Conclusions
We found that even a slight increase in UA levels promotes VSMCs' proliferation and their shift towards synthetic phenotype through proteasome and RAAS activation.
These results provide the evidence of the cellular mechanisms that induce those vascular remodelings and damage [17] which, when they become clinically significant, underlie the increased CV and renal risk of patients with asymptomatic HU. Furthermore, our results suggest a novel role of UPS which can modulate cytoskeleton proteins in UA-stimulated VSMCs. UPS-signaling inhibition might serve as a therapeutic target in the management of vascular damage, and RAAS inhibition might be thought to prevent UA-induced atherosclerosis even in the absence of hypertension. Funding: This work was, in part, supported by grants from CARIGE (Bando Carige-D.R. 3707). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Regione Liguria (protocol code UA-MSTN-VASC, date of approval: 28 June 2021).

Informed Consent Statement: Not applicable.
Data Availability Statement: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.