Atherogenic Activation of Human Vascular Smooth Muscle Cells by Monosodium Urate Crystals

: Gout is strongly associated with atherosclerosis and other cardiovascular comorbidities. Furthermore, sites of extra-articular monosodium urate (MSU) crystal deposits in gout can include heart valves and atherosclerotic artery plaques, but with unclear effects therein. Hence, we seminally explored cultured vascular smooth muscle cell (VSMC) responsiveness to MSU crystals. To limit confounding effects, we cultured human aortic VSMCs under serum-free conditions to assess MSU crystal effects on VSMC differentiation and function, differentially expressed genes (DEGs) via RNA sequencing, and selected atherogenic changes in cytokines and the lipidome. MSU crystals induced p38 phosphorylation, IL-6, and VSMC vacuolization with dysregulated autophagy. MSU-crystal-induced DEGs included decreased late-stage autophagosome maturation mediator GABARAPL1, decreased physiologic VSMC differentiation regulators (LMOD1 and SYNPO2), increased ATF4, CHOP, and the intrinsic apoptosis signaling pathway in response to ER stress, and neointimal atherogenic nuclear receptors (NR4A1 and NR4A3). MSU crystals alone increased the levels of cholesterol biosynthetic intermediates 14-demethyl-lanosterol (14-DML), desmosterol, and zymosterol. Adding MSU crystals increased oxidized LDL’s capacity to increase intracellular 27-OH cholesterol, and MSU crystals and oxidized LDL synergistically induced a marked release of arachidonate. In conclusion, MSU crystals deposited in arterial media and neointima have the potential to dysregulate VSMC differentiation and proteostasis, and to induce further atherogenic effects, which include enhanced VSMC loading of oxidized cholesterol intermediates and release of IL-6 and arachidonic acid (AA).


Introduction
Gout is strongly associated with atherosclerosis and other cardiovascular comorbidities [1,2]. Furthermore, MSU crystals have been reported to deposit in a broad range of extra-articular sites in gout, which include certain cardiac and arterial tissues [3], and in the renal medulla in patients with relatively severe forms of tophaceous gout [4]. Also, case studies of patients with chronic, tophaceous disease have reported MSU crystal deposits in cardiac valves, the myocardium, and the cardiac conducting system [3,[5][6][7][8][9]. Moreover, small, tophus-like intimal and adventitial coronary artery lesions have also been reported [10]. Notably, inflammatory macrophages express xanthine oxidase (XO) [11], and XO has been reported to be significantly upregulated in the macrophage-rich atherosclerotic plaques of patients with symptomatic central nervous system (CNS) ischemia, compared to plaques of those without CNS symptoms [12,13]. XO and urate are particularly enriched in the shoulder and subendothelial regions of such carotid atherosclerotic plaques, and increased XO and urate aggregate in plaque [13].
Using a highly sensitive micro-optical coherence tomography approach augmented by cross-polarized light microscopy and uricase treatment, MSU crystals have been detected in 2.7. RNA Sequencing Cells were collected and subjected to RNA sequencing analysis, performed by LC Sciences (Houston, TX, USA). STRING application-Protein Query was employed to generate a node network of functionally enriched differentially expressed genes (DEGs) whose defined biological processes, cellular content, and molecular functions were analyzed by gene ontology (GO).

Statistical Analyses
GraphPad PRISM 9 (San Diego, CA, USA) was used for statistical analyses. All data were subjected to the normality test. For normally distributed data, either unpaired Student's t-test (comparing 2 groups), one-way (comparing 2 ≥ groups with 1 variable), or two-way analysis of variance (ANOVA) (comparing 2 ≥ groups with 2 independent variables) with Tukey's multiple comparisons tests were performed. The data are expressed as mean ± SD and p < 0.05 was considered statistically significant.

Statistical Analyses
GraphPad PRISM 9 (San Diego, CA, USA) was used for statistical analyses. All data were subjected to the normality test. For normally distributed data, either unpaired Student's t-test (comparing 2 groups), one-way (comparing 2 ≥ groups with 1 variable), or two-way analysis of variance (ANOVA) (comparing 2 ≥ groups with 2 independent variables) with Tukey's multiple comparisons tests were performed. The data are expressed as mean ± SD and p < 0.05 was considered statistically significant.

VSMC IL-6 Release Induced by Human VSMCs
VSMC proliferation was stimulated by the positive control PDGF (10 ng/mL), but not by MSU crystals ( Figure 1A). The mitogen-activated protein kinases (MAPKs) transduce the proliferative effects of PDGF in VSMCs [28]. Here, PDGF induced rapid p38 phosphorylation and relatively transient c-Jun N-terminal kinase (JNK) phosphorylation, whereas MSU crystals induced p38 but not JNK phosphorylation (Supplementary Figure S1). PDGF also induced a marked release of the atherogenic cytokines monocyte chemoa ractant protein (MCP)-1 and IL-6 ( Figure 1B,C). By comparison, MSU crystals robustly induced IL-6 but did not induce MCP-1 ( Figure 1B  Differential effects of MSU crystals and PDGF on proliferation and cytokine production in human VSMCs. Three different donors of human VSMCs (passage number <3-4) were stimulated with MSU crystals (0.1 and 0.2 mg/mL) and PDGF (10 ng/mL) for 24 h under serum-free conditions. Cell proliferation was evaluated (A) using the CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega) and production of MCP-1 (B) and IL-6 (C) was measured by ELISA analysis of the conditioned media. One-way ANOVA with Tukey's post hoc comparison test was used for data analysis. Cell proliferation was evaluated (A) using the CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega) and production of MCP-1 (B) and IL-6 (C) was measured by ELISA analysis of the conditioned media. One-way ANOVA with Tukey's post hoc comparison test was used for data analysis.

Marked VSMC Vacuolar Changes Induced by MSU Crystals
MSU crystals, but not PDGF, induced the robust formation of vacuolar structures in VSMCs (Figure 2A and Supplementary Figure S2). To test if the vacuoles represented lipid deposits such as in VSMC macrophage-like foam cell development [29][30][31][32], or simply vacuolar changes related to ingested crystals and associated frustrated autophagy [33,34], we treated the cells with bafilomycin A1, a specific vacuolar H + ATPase (V-ATPase) inhibitor. We performed immunostaining with an antibody to LC3B (an autophagosome marker) conjugated with Alexa Fluor 488, and a lysosomal tracker (LysoTracker, Red). As shown in Figure 2B, we thereby documented vacuole formation in VSMCs in response to MSU crystals. In addition, the membranes of most vacuoles stained positively for LC3B (green). Few vacuoles had positive staining for both LC3B and LysoTracker (yellow) in SMCs treated with MSU crystals for 24 h.
MSU crystals, but not PDGF, induced the robust formation of vacuolar structures in VSMCs (Figure 2A and Supplementary Figure S2). To test if the vacuoles represented lipid deposits such as in VSMC macrophage-like foam cell development [29][30][31][32], or simply vacuolar changes related to ingested crystals and associated frustrated autophagy [33,34], we treated the cells with bafilomycin A1, a specific vacuolar H + ATPase (V-ATPase) inhibitor. We performed immunostaining with an antibody to LC3B (an autophagosome marker) conjugated with Alexa Fluor 488, and a lysosomal tracker (LysoTracker, Red). As shown in Figure 2B, we thereby documented vacuole formation in VSMCs in response to MSU crystals. In addition, the membranes of most vacuoles stained positively for LC3B (green). Few vacuoles had positive staining for both LC3B and LysoTracker (yellow) in SMCs treated with MSU crystals for 24 h. Bafilomycin A1 (25 nM) blunted vacuole formation in response to MSU crystals in SMCs. Bafilomycin A1 inhibits autophagy by preventing autophagosome-lysosome fusion [35]. Moreover, not only LC3B-II, the phosphatidylethanolamine (PE)-conjugated form of LC3B, but also p62, which can directly bind to LC3, and is selectively degraded by autophagy, accumulated in VSMCs after 24 h treatment with MSU crystals (Supplementary Figure S2). The results suggested frustrated autophagy flux in a failed autophagic process.

mRNA Changes Induced by MSU Crystals in Human VSMCs
RNA sequencing assayed for differentially expressed genes (DEGs) in human VAMCs in response to MSU crystals. As shown in Figure 3A, MSU crystals upregulated 43 genes (Log2FC ≥ 1) and downregulated 21 genes (Log2FC ≤ −1). STRING enrichment analysis of a network of the 43 upregulated genes indicated one major cluster containing Bafilomycin A1 (25 nM) blunted vacuole formation in response to MSU crystals in SMCs. Bafilomycin A1 inhibits autophagy by preventing autophagosome-lysosome fusion [35]. Moreover, not only LC3B-II, the phosphatidylethanolamine (PE)-conjugated form of LC3B, but also p62, which can directly bind to LC3, and is selectively degraded by autophagy, accumulated in VSMCs after 24 h treatment with MSU crystals (Supplementary Figure S2). The results suggested frustrated autophagy flux in a failed autophagic process.

mRNA Changes Induced by MSU Crystals in Human VSMCs
RNA sequencing assayed for differentially expressed genes (DEGs) in human VAMCs in response to MSU crystals. As shown in Figure 3A, MSU crystals upregulated 43 genes (Log2FC ≥ 1) and downregulated 21 genes (Log2FC ≤ −1). STRING enrichment analysis of a network of the 43 upregulated genes indicated one major cluster containing CHAC1, ATF4, DDIT3, TRIB3, and SLC7A5 genes (indicated in red in Figure 3B). Gene ontology (GO) analysis of the network revealed the CHOP-ATF4 complex, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), and the intrinsic apoptosis signaling pathway in response to endoplasmic reticulum (ER) stress as the major enriched signaling pathways in the GO terms of cellular component, molecular function, and biological process, respectively ( Figure 3C). MSU-crystal-induced changes in mRNA levels of two members of the nuclear receptors of the subfamily 4 group A1 and A3 (NR4A1 and NR4A3), known to be induced by atherogenic stimuli in VSMCs [36], were shown in another cluster (NR4A1, NR4A3, FOSB, and DUSP, indicated in yellow in Figure 3B). sine monophosphate (cAMP) response element-binding protein (CREB), and the intrinsic apoptosis signaling pathway in response to endoplasmic reticulum (ER) stress as the major enriched signaling pathways in the GO terms of cellular component, molecular function, and biological process, respectively ( Figure 3C). MSU-crystal-induced changes in mRNA levels of two members of the nuclear receptors of the subfamily 4 group A1 and A3 (NR4A1 and NR4A3), known to be induced by atherogenic stimuli in VSMCs [36], were shown in another cluster (NR4A1, NR4A3, FOSB, and DUSP, indicated in yellow in Figure 3B). GABARAPL1 (GABAA-receptor-associated protein-like 1), which suppresses autophagic flux, was downregulated (Log2FC= −1.7) in response to MSU crystals. These results potentially reflected a compensatory change to the observed impairment of autophagy flux in MSU-crystal-treated VSMCs (Supplementary Figure S3). Last, we observed that MSU crystals induced downregulation of the VSMC cytoskeletal and differentiation and function regulators LMOD1 (leiomodin 1) and SYNPO2 (synaptopodin 2).

Effects of MSU Crystals on Intracellular Cholesterol Metabolism in Human VSMCs
In the cytosol of VSMCs treated with MSU crystals for 24 h, we observed increased staining by BODIPY495/503, a lipophilic fluorescent probe that labels cellular neutral lipid contents, particularly so for those localized to lipid droplets (Supplementary Figure S4). MSUcrystal-induced formation of vacuoles under these conditions was not associated with detectable staining of BODIPY inside the vacuoles. Targeted lipidomics examined the effects of MSU crystals on cellular lipid metabolism in VSMCs. Since MSU crystals avidly bind GABARAPL1 (GABAA-receptor-associated protein-like 1), which suppresses autophagic flux, was downregulated (Log2FC= −1.7) in response to MSU crystals. These results potentially reflected a compensatory change to the observed impairment of autophagy flux in MSU-crystal-treated VSMCs (Supplementary Figure S3). Last, we observed that MSU crystals induced downregulation of the VSMC cytoskeletal and differentiation and function regulators LMOD1 (leiomodin 1) and SYNPO2 (synaptopodin 2).

Effects of MSU Crystals on Intracellular Cholesterol Metabolism in Human VSMCs
In the cytosol of VSMCs treated with MSU crystals for 24 h, we observed increased staining by BODIPY495/503, a lipophilic fluorescent probe that labels cellular neutral lipid contents, particularly so for those localized to lipid droplets (Supplementary Figure S4). MSU-crystal-induced formation of vacuoles under these conditions was not associated with detectable staining of BODIPY inside the vacuoles. Targeted lipidomics examined the effects of MSU crystals on cellular lipid metabolism in VSMCs. Since MSU crystals avidly bind LDL, with effects on phagocyte activation [37], these studies were performed in the presence of atherogenic oxLDL [38].

Impact of MSU Crystals on VSMC Intracellular Free Fa y Acids (FFAs)
We analyzed a panel of 33 FFAs, including medium-chain fa y acids (MCFAs) and long-chain fa y acids (LCFAs) (Figure 5A

Effects of MSU Crystals on VSMC Eicosanoid Release
We used RP-UPLC-MS to analyze a panel of 80 eicosanoids from VSMCs treated with MSU crystals ( Figure 6A) in conditioned media. Where indicated, oxLDL was present. Of the 15 eicosanoids affected by treatment with MSU crystals or oxLDL, approximately two-thirds were derived from AA via enzymatic (e.g., cyclooxygenases (COXs) and lipoxygenases (LOXs)) and non-enzymatic oxidation ( Figure 6B). Certain AA-derived, eicosapentaenoic acid (EPA)-derived, docosahexaenoic acid (DHA)-derived, and linolenic acid (LA)-derived metabolites were increased only by oxLDL. Notably, levels of AA were not altered by MSU crystals or oxLDL alone. By contrast, the marked induction of AA release by the combination of MSU crystals and oxLDL suggested a synergistic effect. The AA derivative 6-keto-Prostaglandin F1a (6K PGF1a) was increased significantly by MSU crystals alone, and this was enhanced when oxLDL was also present. Last, levels of prostaglandin E2 (PGE2) and 13,14-Dihydro-15-keto-PGF2a (dhk PGF2a) were increased by the combination of MSU crystals and oxLDL. Gout Urate Cryst. Depos. Dis. 2023, 1, FOR PEER REVIEW 8

Discussion
MSU crystals have been detected within coronary atherosclerotic plaques, potentially a consequence of enhanced lesion XO and urate content. This seminal study revealed the potential of MSU crystals to activate VSMCs in vitro, with effects that can enhance atherogenesis. A prime example was the observation that MSU crystals induced IL-6 release by VSMCs. IL-6 atherogenic effects include the promotion of inflammation and foam cell formation, and stimulation of altered VSMC differentiation, including ectopic osteoblastic differentiation in the artery wall and associated arterial calcification [40,41].
In this study, VSMC RNA sequencing and GO analysis demonstrated that MSU crystals upregulated the mRNA of the CHOP-ATF4 complex, CREB, and the intrinsic apoptosis signaling pathway in response to ER stress as the major cellular component, molecular function, and biological process, respectively. In this light, the ER stress response modulates atherogenesis and is particularly upregulated in advanced atherosclerotic plaques [42]. Despite the beneficial effect of a transient unfolded protein response (UPR), prolonged ERstress-induced CHOP expression is involved in VSMC apoptosis both in vitro and in vivo, and it promotes thinning of the protective collagen cap of the plaque, and consequent plaque vulnerability [23,24,[42][43][44]. The ER stress effector ATF4 plays a critical role in the pathogenesis of arterial lesion calcification through increased phosphate uptake in VSMCs [45]. The nuclear transcription factor CREB, which is downregulated in vascular disease, is a central modulator of cell proliferation, differentiation, adaptation, and survival under stress [46].
A particularly striking VSMC response to MSU crystals was the robust development of vacuoles, associated with evidence for impaired autophagic flux. RNA sequencing demonstrated that the VSMC mRNA of GABARAPL1, which suppresses autophagic flux [47], was downregulated in response to MSU crystals. This result suggested an adaptive response to impaired autophagy. In prior studies, MSU crystals induced autophagy of osteoblasts and chondrocyte autophagy [48,49], which, like VSMCs, are not professional phagocytes. In VSMCs, autophagy is not simply induced by metabolic stress but also by diverse stimuli, such as oxidative stress and oxidized lipids, as well as a number of cytokines and growth factors [23,24]. VSMC autophagy is intimately involved in promoting survival as the cells undergo the ER stress response and phenotypic switching [23,24].
Additional RNA sequencing findings in this study buttressed the potential atherogenic effects of MSU crystals. First, we observed that MSU crystals decreased the mRNA of SYNPO2 and of LMOD1, a downstream target of the VSMC master regulator serum response factor (SRF)-Myocardin (MYOCD) complex [50]. Both LMOD1 and SYNPO2 play major roles in the maintenance of smooth muscle actin polymerization and contractile differentiation, and reduced LMOD1 and SYNPO2 is linked with altered VSMC phenotype in arterial disease in vivo [50,51]. Second, NR4A1 and NR4A3 mRNA were upregulated in response to MSU crystals. These NR4A receptors are increased in VSMCs by atherogenic stimuli and upregulated in human atherosclerotic plaque neointima [36].
Lipidomics demonstrated that MSU crystals alone increased the levels of the cholesterol biosynthetic intermediates 14-DML, desmosterol, and zymosterol ( Figure 4B-D). Oxysterols are associated with nearly every atherogenesis pathway, are present in human atherosclerotic plaques, and are held to play an active role in plaque development [38]. Conversely, since desmosterol inhibits NLRP3 inflammasome and macrophage activation and limits atherosclerosis [39], not all the MSU-crystal-induced changes in the VSMC sterol panel were atherogenic.
LDL binds avidly to MSU crystals and markedly inhibits MSU-crystal-induced activation of neutrophils [37]. However, LDL undergoes oxidation in atherosclerotic lesions, is taken up robustly by VSMCs and macrophages, and is atherogenic [38]. Here, the combination of MSU crystals and oxLDL significantly increased the VSMC levels of 27-OHC, the major oxysterol in advanced atherosclerotic lesions [52]. This suggested the possibility, which warrants testing, that OxLDL bound to MSU crystals may undergo increased VSMC uptake and further oxidation. Elevation of 27-OHC promotes murine experimental atherosclerotic lesion formation without altered lipid status [52]. Furthermore, 27-OHC attenuates estrogen-related atheroprotection and elevates vascular inflammation via estrogen receptor alpha [52].
Lipidomics revealed that MSU crystals alone had little effect on intracellular FFAs. However, MSU crystals and oxidized LDL synergistically induced a marked release of arachidonate, while decreasing intracellular linoleic acid (18:2), palmitoleic acid (16:1), oleic acid (18:1), arachidonic acid (20:4), erucic acid (22:1), and docosahexaenoic acid (DHA 22:6). We did not determine if the decrease in the levels of particular intracellular FFAs was due to increased degradation or reduced synthesis of FFAs. The release of 6K PGF1a was increased significantly by MSU crystals alone. Levels of released PGE2, which is a central mediator of the inflammatory response, and dhk PGF2a, a metabolite of PGF2a that increases following tissue injury, were also increased by the combination of MSU crystals and oxLDL.
The ω-6 PUFA arachidonic acid is a substrate for the biosynthesis of PGs and leukotrienes (LTs) that mediate inflammation, including in the vasculature [53]. That said, arachidonic acid can also be used to biosynthesize lipoxin A4 (LXA4), which participates in the resolution of inflammation. Arachidonic acid metabolites modulate vascular tone and contribute to cardiovascular diseases, including hypertension, atherosclerosis, and myocardial infarction [53]. The polyunsaturated ω-6 FA linoleic acid can be converted to longer-chain anti-inflammatory ω-3 FAs, such as EPA and DHA, or to longer-chain ω-6 FAs, such as AA [54].
The limitations of this seminal study are principally related to its exploratory and in vitro nature, the focus on the effects of MSU crystals, and the requisite contraction of the experimental template to render it manageable. In this context, though MSU crystals are reported in atherosclerotic plaques, it was beyond the scope of the work to compare the effects of the other crystal types and cells found in plaques or to assess combinations of MSU with cholesterol or BCP crystals. MSU crystals can prime the subsequent enhancement of innate immune tissue inflammatory reactions through effects on macrophages and fibroblasts [22]. Thus, it would also be of interest to directly assess in vivo if MSU crystals can enhance atherogenesis and plaque instability, and if MSU crystals share or diverge from the effects of crystals of cholesterol and BCP on these processes. Importantly, complex, extensive distinctions exist in atherogenesis-regulating effects between individual eicosanoids, reviewed in great detail elsewhere [55][56][57][58], that could ultimately be relevant to the seminal findings of this study. Last, our experimental template did not test for MSU crystal effects on all potentially atherogenic VSMC cytokine release responses, calcification, and changes in cell differentiation, such as senescence.

Conclusions
We conclude that MSU crystals induced severe VSMC vacuole formation and altered cell homeostasis mechanisms, including frustrated autophagy, that were associated with altered transcriptional pathways, IL-6 release, intracellular accumulation of atherogenic oxysterols, and eicosanoid release.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/gucdd1030016/s1, Figure S1: Rapid induction of phosphorylation of p38 but not JNK by MSU crystals in human SMCs; Figure S2: MSU crystals but not PDGF robustly induced vacuole formation in human VSMCs; Figure S3: Time-dependent accumulation of LC3B and p62 by MSU crystals in human VSMCs; Figure S4