Lipoxin Alleviates Diabetic Vascular Calcication via the YAP Pathway

Background: Vascular calcication is highly prevalent in patients with diabetes and has detrimental consequences. However, no effective prevention and treatment methods are currently available. Extensive evidence has demonstrated the protective effect of lipoxin (LX) against vascular diseases. However, whether LX prevents diabetic vascular calcication remains unknown. Here, we tested the hypothesis that LX alleviated osteogenic differentiation and subsequent calcication of vascular smooth muscle cells (VSMCs). Methods: In vitro, human aortic smooth muscle cells (HASMCs) were incubated in osteogenic medium (OM) with advanced glycation end products (AGEs) and LX to further determine the underlying mechanisms. An in vivo diabetic mouse model was established using a combination of a high-fat diet and multiple formulations of low-dose streptozotocin (STZ). Cell culture, alkaline phosphatase (ALP) staining, ALP activity, Alizarin red staining, von kossa staining, determination of calcium content, western blot analysis, immunohistochemistry, and immunouorescence staining and statistical analysis were used in our study. Results: AGEs dose-dependently induced calcication and expression of osteogenesis-related markers, including Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and type I collagen (COL1), coupled with the activation of yes-associated protein (YAP). Mechanistically, YAP activation enhanced the AGE-induced osteogenic phenotype and calcication, but inhibition of YAP signalling alleviated this trend. Consistent with the in vitro results, diabetes promoted YAP expression as well as the subcellular localisation of the protein in the nucleus in the arterial tunica media. Interestingly, treatment with LX reduced vascular osteogenesis and calcication in diabetic mice, which was correlated with the reduced YAP levels. In addition, LX signicantly inhibited COL1 accumulation and modulated the extracellular matrix. Our results further demonstrated that a pharmacological agonist of YAP reversed LX-mediated protection against osteogenic phenotypic conversion and calcication in VSMCs. Conclusions: These results demonstrate that LX attenuates transdifferentiation and calcication of VSMCs in diabetes mellitus via the YAP signalling axis, suggesting that LX is a potent therapeutic strategy to prevent diabetic vascular calcication. it also suggests LX as a therapeutic strategy for diabetic vascular calcication. Our study used ample evidence to conrm that the diabetic mice and AGE-induced VSMCs exhibited extensive osteogenesis and calcication, coupled with more increased YAP expression. In addition, we provide evidence that LX prevents diabetic vascular calcication via YAP, which potentially explains the mechanism by which LX regulates collagen content. Our study therefore shows that preventing diabetic vascular calcication via the LX and YAP pathways represents a novel therapeutic option for the prevention and treatment of vascular disease.


Background
Vascular calci cation is a common and severe comorbidity and complication of chronic diseases, including diabetes mellitus [1], chronic kidney disease [2], and ageing [3]. Although vascular calci cation has been demonstrated to be an active, cell-mediated, and reversible process [4], no treatments have been developed to prevent, attenuate, or reverse vascular calci cation [5]. Vascular calci cation is common in diabetics and has detrimental consequences [6], which increases the risk of cardiovascular and all-cause mortality [7,8] and is an independent risk factor for the prediction of cardiac events [9]. Cumulative evidence suggests that advanced glycation end products (AGEs), which are metabolites caused by high glucose levels [10], are involved in the initiation and propagation of vascular calci cation [11,12].
Currently, several lines of evidence have suggested that YAP expression increases in response to arterial injury [35], and YAP inhibition may inhibit phenotypic conversion and maintain the contractile phenotype of VSMCs [36]. Furthermore, diabetes may induce the expression of YAP in retinal tissues of a streptozotocin (STZ)-treated mouse model [37]. Therefore, we speculated YAP levels may increase in diabetic mouse vasculature, and we hypothesised that nding e cient drugs to suppress YAP activation may alleviate osteogenic differentiation and subsequent calci cation.
Notably, G-protein-coupled receptor (GPCR) ligands have been shown to activate or inhibit YAP in a manner dependent on G protein activation [38], and the receptor of lipoxin (LX) belongs to the GPCR family [39]. LX may exert robust immunoregulatory and anti-in ammatory activities [40] via formyl peptide receptor 2 and A-type lipoxin receptor (FPR2/ALX) [41]. LX is a specialised pro-resolving lipid mediator (SPM) [42] that has recently been shown to play a role in the functioning of vascular wall cells, such as VSMCs [43,44]. In some studies, LX was reported to modulate the VSMC phenotype [45] and attenuate diabetes-associated atherosclerosis [46]. However, the mechanisms underlying this process remain unclear. Thus, we investigated the protective effect of LX and whether LX may function as a therapeutic target for diabetic vascular calci cation.
Antibodies against RUNX2 and smooth muscle actin (SMA) were acquired from Abcam (Cambridge, UK). Antibodies against osteopontin (OPN) and type collagen (COL1) were purchased from Santa Cruz (Santa Cruz, CA, USA).

Cell culture
HASMCs were cultured in smooth muscle cell medium supplemented with 2% FBS, 1% smooth muscle cell growth supplement, and 1% penicillin/streptomycin solution. All experiments were performed using HASMCs at passage 3-6.

Cell viability assay
VSMCs were cultured in 96-well plates for 24 h and treated with different concentrations of AGEs (0-400 μg/mL) or LX (0-100 nM) in OM for 1, 3, and 7 days. After treatment, 10 μL CCK8 (Dojindo Laboratories, Kumamoto, Japan) was added to 100 μL fresh medium in each well and incubated for 2 h at 37 °C. A microplate reader (Tecan, Mannedorf, Switzerland) was used to measure the absorbance at 450 nm.

Animal and experimental models
Six-week-old male C57BL/6J mice susceptible to STZ were acclimatised for one week to the conditions of the animal room. All animal experiments were performed in compliance with the Animal Ethics Procedures and Guidelines of the People's Republic of China and were approved by the Harbin Medical University Animal Ethics Committee. After one week of adaptation, the mice were randomly divided into four groups: (1) control (CON) group, (2) LX group, (3) diabetes mellitus (DM) group, and (4) DM+LX group. Diabetic mice were induced as previously described [50,51] and fed with a commercial high-fat diet for four weeks, followed by intraperitoneal injection of low-dose STZ (50 mg/kg) for ve days during the last week of the high-fat diet. Nondiabetic mice were fed a normal diet and injected with buffer solution. The diabetic model was successfully established in mice with fasting blood glucose (FBG) levels of ≥ 11.1 mM two weeks after the induction of diabetes [52]. Mice were treated with either ethanol (0.1%) or LXA 4 (5 μg/kg) twice weekly by intraperitoneal injection two weeks after injection of the rst STZ dose or buffer solution. FBG levels and body weight were monitored after STZ injection during the study to validate the diabetic status of the mice. At the experimental end points, the mice were euthanised, and the aortic arch and descending aorta were dissected under a microscope.

2.6Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin sensitivity test (IPIST)
For both IPGTT and IPIST, blood samples were collected from the tail vein after 16 h fasting. To conduct IPGTT, blood glucose levels were assessed as threshold glucose levels (0 min) before intraperitoneal injection of glucose load (2 g/kg) [53]. Blood glucose levels were estimated at 30, 60, 90, and 120 min following injection with a calibrated glucometer (Roche, Basel, Switzerland). Similar to IPGTT, the IPIST was established with intraperitoneal injection of insulin at a dose of 0.75 U/kg [54], after which blood glucose was measured 30, 60, 90, and 120 min after injection.

ALP staining and ALP activity assay
VSMCs were xed with 4% paraformaldehyde and stained with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium mixture (Beyotime, Shanghai, China) for 30 min. After washing, photographs were taken under a digital microscope (Olympus, Tokyo, Japan). The ALP activity assay kit (Beyotime) uses p-nitrophenyl phosphate as a phosphatase substrate, which turns yellow (λ max = 405 nm) when dephosphorylated by ALP. As previously described [55], total proteins of cells were rst extracted by centrifugation in lysis buffer, and then their ALP activity was measured colourimetrically. The results were normalised to total protein levels determined using a bicinchoninic acid (BCA) assay kit (Beyotime).
2.8 Alizarin red staining, von Kossa staining, and determination of calcium content VSMCs were xed with 4% paraformaldehyde and stained with Alizarin red staining solution (Cyagen, Guangzhou, China) for 10 min. Tissues were xed in 4% paraformaldehyde, embedded in para n, and sectioned at a thickness of 5 μm. After conventional dehydration, sections were stained with Alizarin red solution for 10 min and washed with distilled water. After xylene treatment, the slides were sealed with gel resin.
VSMCs and tissue slices were incubated with 1% silver nitrate solution for 30 min under ultraviolet light. They were then washed with 5% sodium thiosulfate solution for 2 min and counterstained with Nuclear Fast Red for 3 min. Images were obtained using a microscope (Olympus).
Calcium content in VSMCs and aortas was quanti ed with a calcium colorimetric assay (Beyotime), and total protein concentration was measured using the BCA method. Calcium content was normalised to the total protein concentration.

Western blot analysis
Proteins were extracted from cells or tissues with RIPA buffer, fractionated by SDS-polyacrylamide gel, and transferred to polyvinylidene di uoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in the blocking agent (5% non-fat dried milk in Tris-buffered saline with Tween 20) for 1 h and incubated overnight at 4 °C with a primary antibody. After three cycles of cleaning with Tris-buffered saline-Tween 20, the membranes were incubated with appropriate secondary antibodies and observed by enhanced chemiluminescence. ImageJ software was used for analysis.

Immunohistochemistry
Brie y, sections were dewaxed, rehydrated, blocked for endogenous peroxidase activity and nonspeci c binding, and incubated overnight with primary antibodies (YAP, 1:200) at 4 °C. After three cycles of washing with phosphate-buffered saline, the sections were incubated with secondary antibody at 37 °C for 30 min. Bound secondary antibodies were detected using diaminobenzidine solution.

Immuno uorescence staining
For immuno uorescence staining analysis, VSMCs and slides were stained with antibodies speci c to RUNX2, SMA, or YAP.

Statistical analysis
All data are expressed as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8. Independent experiments were performed using different cell batches. Differences between groups were determined using the Student's t-test or one-way analysis of variance (ANOVA). Results with two or more variables were analysed using a two-way ANOVA. Where normality could not be con rmed, data were analysed using the Mann-Whitney U test. Statistical signi cance was de ned as P < 0.05.

Results
To determine a suitable concentration of AGEs for osteogenic differentiation and the subsequent VSMC calci cation, CCK-8 assay was used to test cell viability. We cultured VSMCs with AGEs (0-400 μg/mL) for 1, 3, and 7 days in OM. As shown in Supplementary Figure 1A, AGEs did not affect VSMC viability at all concentrations tested. Thus, we may use 25-400 μg/mL of AGEs in the follow-up study.
To assess the capacity of calci cation induced by AGEs in VSMCs, we rst investigated whether AGEs may increase the activity of ALP, an early marker of osteogenic differentiation and calcium nodule formation [56]. Therefore, we examined ALP staining and calcium nodule staining in control media (CM) and in OM with AGEs (0-400 μg/mL). ALP activity of VSMCs in the CM group was markedly lower than that in the OM group. Our results further demonstrated that elevated concentrations of AGEs increased ALP activity ( Figure 1A). Interestingly, VSMCs in CM were devoid of nodular staining, whereas nodular staining gradually increased with AGE concentration ( Figure 1B). In addition, von Kossa staining showed granular calci cation localised in the cell and extracellular matrix regions of the cultures (Supplementary Figure 1B). In contrast with 0 μg/mL AGEs, marked augmentation of ALP activity and calcium content was observed when VSMCs were treated with 100, 200, or 400 μg/mL AGEs, as indicated in Figure 1C and 1D.
As expected, AGEs reduced the expression of the smooth muscle cell-speci c contractile marker SMA and elevated the expression of osteogenic markers RUNX2, OPN, and COL1 ( Figure 1E and 1F). These results indicated that 100, 200, and 400 μg/mL AGEs dramatically induced VSMC calci cation. YAP expression was profoundly induced in parallel with the expression of osteogenesis-related markers. Furthermore, YAP serine 127 phosphorylation signi cantly decreased as the concentrations of AGEs increased ( Figure 1G), suggesting that YAP was activated by AGEs.

AGEs activated YAP under calcifying conditions
To identify the role of AGEs during VSMC calci cation, VSMCs induced with 100 μg/mL AGEs were monitored by speci c staining and western blotting at the following stages of calci cation: the early stage (day 7), the medium stage (day 9), and the late stage (day 14). The staining of ALP was observed at the early stage of calci cation in VSMCs induced by 100 μg/mL AGEs (Figure 2A). Calci cation induced by 100 μg/mL AGEs was time-dependent. Notably, calci cation nodules were not detectable at the early stage of calci cation. However, sparse and spotted precipitates were observed in VSMCs and the surrounding extracellular matrix at the medium stage of calci cation ( Figure 2B and 2C), suggesting the involvement of the extracellular matrix in vascular calci cation.
During calci cation formation, RUNX2 expression gradually increased, and conversely, the levels of SMA reduced ( Figure 2D and 2E). During the early stage of calci cation, YAP levels were low. YAP expression was moderately elevated during the middle stage of calci cation but signi cantly elevated during the late stage. As expected, immunoblot analysis validated the decreased expression of phosphorylated YAP ( Figure 2F). These ndings indicate that YAP activation may play a role in the process of VSMC calci cation.

AGE-induced VSMC calci cation through a YAPdependent pathway
To determine the potential involvement of YAP in AGE-induced VSMC calci cation, we applied the YAP inhibitor ATV [57] and the YAP agonist LPA [58] in OM. We observed ATV attenuated calcium deposition, and LPA increased calci ed deposits in OM. These ndings suggest YAP can modulate VSMC calci cation. As expected, AGEs induced severe calcium deposition in OM, which was signi cantly attenuated by ATV. In addition, LPA further increased calcium deposits in the presence of AGEs, as shown in Figure 3A-C.
To investigate the underlying mechanisms of YAP-mediated VSMC calci cation, we examined the protein expression levels. RUNX2 levels were upregulated by LPA, whereas SMA levels were downregulated. However, ATV induced the opposite trend. Furthermore, ATV reversed the AGE-induced increase in RUNX2 levels, and LPA further aggravated the increased levels of RUNX2 in response to AGEs ( Figure 3D-F).

LX attenuated vascular calci cation in diabetic mice
To evaluate whether LX may attenuate vascular calci cation in DM, a diabetic mouse model was established using a combination of a high-fat diet and multiple low-dose STZ. LX was intraperitoneally injected twice weekly from weeks 6-25 of the experiment, as described in the methods section ( Figure 4A). Notably, all diabetic mice had lower body weight and increased FBG levels compared to nondiabetic mice ( Figure 4B and Supplementary Figure 2A). Glucose homeostasis was evaluated using IPGTT and IPIST at the end of the study. For IPGTT, compared to nondiabetic mice, the diabetic mice showed delayed glucose clearance and high blood glucose levels for 120 min (Supplementary Figure 2B). For IPIST, all mice exhibited reduced blood glucose levels, but the diabetic mice had higher blood glucose levels than the nondiabetic mice (Supplementary Figure 2C). In both IPGTT and IPIST, the area under the curve (AUC) value in diabetic mice was signi cantly increased compared with that in nondiabetic mice. The diabetic mice given multiple low-dose STZ following a high-fat diet had impaired glucose tolerance and insulin resistance (P < 0.0001) (Supplementary Figure 2D and 2E). Interestingly, treatment with LX had no signi cant effect on blood glucose levels in IPGTT and IPIST, indicating that the role of LX in diabetic mice was not induced by glycaemic control (P > 0.05).
Next, we evaluated the effect of LX on medial arterial calci cation in diabetic mice. Calcium precipitation is a major event in VSMC calci cation. Von Kossa staining and alizarin red staining in the aortas indicated multifocal calci cation lesions in diabetic mice ( Figure 4C and 4D). However, LX treatment signi cantly lowered aortic calci cation in the DM+LX group compared to that in the DM group. Elastin Van Gieson staining also demonstrated that vessels of diabetic mice exhibited more attening and degrading elastin bres and collagen accumulation compared to those of the nondiabetic mice ( Figure  4D). Aortic calcium content analysis further con rmed that calcium deposition was higher in diabetic mice than in the nondiabetic mice. However, treatment with LX mitigated these effects ( Figure 4E).
Transdifferentiation of the osteogenic phenotype of VSMCs is another key event in vascular calci cation. LX alleviated the loss of the contractile phenotype markers SMA and the gain of the osteogenic marker RUNX2, as demonstrated by IF and WB ( Figure 4F-H). Interestingly, in healthy control mouse vessels, YAP was expressed slightly. However, the YAP levels in diabetic mice increased signi cantly and were primarily localised in the nuclei but were reduced moderately with LX treatment (Figure 4F and 4I). Consistent with the in vitro data during osteogenic phenotypic drift of AGE-induced VSMCs, YAP expression was profoundly induced in parallel with the expression of osteogenesis-related markers.

LX inhibited AGE-induced calci cation in VSMCs
We rst con rmed the effect of LX on cell viability using CCK-8 assay. The results suggested that LX at the concentrations used had little effect on VSMC viability, as compared to OM alone (Supplementary Figure 3A).
We observed that staining intensity decreased with increasing LX concentration, as depicted by ALP staining, Alizarin red staining, and von Kossa staining ( Figure 5A, 5B, and Supplementary Figure 3B). These quantitative results of ALP activity and calcium content further demonstrated the dose-dependent inhibitory effect of LX on AGE-induced VSMC calci cation. For example, compared to VSMCs treated with 0 nM LX, VSMCs treated with 100 nM LX showed signi cantly decreased ALP activity (1.609 ± 0.4541 U/mg protein) and calcium content (45.870 ± 5.655 μg/mg protein) ( Figure 5C and 5D).
We then explored whether low doses of LX distinctly affect differentiation to an osteogenic phenotype in AGE-induced VSMCs. The expression of the contractile and osteogenic phenotype genes was changed signi cantly after LX treatment in AGE-induced VSMCs ( Figure 5E and 5F). Inhibition of osteogenic signalling by LX was accompanied by YAP inhibition ( Figure 5G).

LX exerted an anti-calci cation role by inhibiting AGEsinduced YAP activation
To con rm that LX may suppress calci cation induced by AGEs via YAP, VSMCs were cultured with AGEs plus LX or LPA. AGEs enhanced ALP staining, Alizarin red staining, and von Kossa staining. However, LX alleviated the enhanced staining induced by AGEs, and this was rescued by LPA ( Figure 6A-C).
LX signi cantly ameliorated AGEs-induced osteogenic transdifferentiation in VSMCs, and the decreasing trend was counteracted by LPA ( Figure 6D-G). As depicted in Figure 6H, YAP was predominantly localised to the nucleus in the cultured VSMCs, in line with previous results [35]. AGEs enhanced YAP and RUNX2 staining; however, LX alleviated this effect. As expected, LPA may rescue the LX-mediated decreased trend of YAP and RUNX2 expression.

Discussion
There are three major striking observations in our results that support the novel protective effect of LX against diabetic vascular calci cation via YAP. First, the expression and subcellular localisation of YAP in the nucleus is enhanced in VSMCs induced by AGEs and in vascular tissues of diabetic mice, accompanied by increased expression of osteogenic markers. Second, this study is the rst to demonstrate that LX attenuates diabetic vascular osteogenesis and calci cation via YAP signalling and subsequent coordinated expressional regulation. Third, the underlying mechanism involves extracellular matrix remodelling. We propose a novel mechanism for the protective effect of LX against vascular calci cation by inhibiting extracellular matrix generation.
To our knowledge, this is the rst study to evaluate YAP expression in AGE-induced VSMCs. We observed AGEs had a dose-dependent effect on YAP expression, which increased with increasing AGE concentrations ( Figure 1E and 1G). In addition, the phosphorylated YAP (pYAP, S127) signal also decreased with increasing AGE concentrations, suggesting that YAP signalling is activated during diabetic conditions. Our results showed AGEs enhanced calcium deposition ( Figure 1B, 1D, and Supplementary Figure 1B) and osteogenic phenotypic markers ( Figure 1E), and these correlated with the upregulation of YAP accumulation. We further excluded the possibility that AGEs induced calcium deposition by in uencing VSMC viability (Supplementary Figure 1A). A diabetic mouse model was established using a combination of a high-fat diet and multiple low-dose STZ, which imitated the natural history of DM (impaired glucose tolerance and insulin resistance) (Supplementary Figure 2B-E). The model also exhibited similar metabolic features, including signi cantly elevated levels of AGEs [59,60], which gradually developed vascular calci cation in the media [61]. YAP was expressed at low levels and localised mostly in the cytoplasm of vascular tissues of nondiabetic mice ( Figure 4I), which is consistent with the results of the only other study related to YAP and vascular calci cation [62]. In contrast, YAP was expressed at high levels and localised in the nucleus in the arterial tunica media of diabetic mice, coupled with the in vitro results indicating that YAP upregulation was induced by local VSMCs ( Figure 1E). These results implied diabetic conditions caused YAP activation, which further induced enhanced vascular calci cation. A previous study con rmed that VSMCs expressed higher levels of YAP in response to injury stimuli to further contribute to neointima formation [35]. Extended periods of increased YAP expression led to vascular diseases, such as vascular calci cation and neointima formation. This novel nding has promising implications for preventing diabetic vascular calci cation by targeting YAP.
Osteogenic phenotype shift in VSMCs is a key event in the mechanism of vascular calci cation [63]. Accumulating evidence has con rmed that YAP plays a crucial role in the phenotypic conversion of VSMCs, which modulates vascular remodelling [3,35]. Nevertheless, the mechanism by which YAP modulates osteogenic reprogramming remains to be clari ed.
Osteogenic phenotypic transition is characterised by the downregulation of contractile markers and upregulation of osteogenic-related markers [3,63]. Our results illustrated that AGEs signi cantly induced osteogenic phenotypic transition in vitro, and this was accompanied by YAP upregulation (Figure 1E). We further identi ed that pharmacological inhibition of YAP mitigated this trend, suggesting that AGEs induced osteogenic phenotypic switching via YAP signalling ( Figure 3D). Consistent with these results, we also found that in vivo YAP upregulation was related to enhanced trans-differentiation to the osteogenic phenotype of VSMCs in diabetic vessels ( Figure 4E-I). Our results support the notion that YAP signalling is a major driver of the osteogenic phenotypic conversion of VSMCs. This nding agrees with previous reports showing that suppression of YAP maintains smooth muscle cell-speci c contractile markers [64][65][66].
However, the only other study associated with YAP and vascular osteogenic differentiation showed that VSMCs lost the contractile phenotype and underwent osteogenic differentiation in SM-YAP/Taz-KO mice, followed by vascular calci cation. These apparent discrepancies could be due to the different states of VSMCs, including pathological or physiological conditions. In VSMCs of normal mature arteries, YAP is retained in the cytoplasm and interacts with DVL3 to avoid nuclear translocation and osteogenic differentiation. However, in many disease states, YAP is recruited to the nucleus [67-69]. These results further con rmed that subcellular localisation of YAP affected its ability to function, which is in line with previous studies [70,71]. The results of this study show that the subcellular localisation of YAP in the nucleus is increased in the vasculature of diabetic mice ( Figure 4I) and in AGE-induced VSMCs ( Figure   6H), which offers a novel mechanism for its nuclear accumulation.
Resolvin, protectin, and maresin, with LX, are referred to as SPMs, which carry out robust immunoregulatory and proresolving activities [72][73][74]. The link between LX and vascular calci cation has not been previously investigated; however, SPMs and their receptors prevent vascular calci cation [75][76][77]. We have demonstrated for the rst time that LX has a protective effect against vascular calci cation in response to diabetes in vivo and in vitro. In a mouse model, arterial medial calci cation induced by chronic dysregulated glucose metabolism due to diabetes was alleviated by LX ( Figure 4C-E). Similar to the in vitro data, LX inhibited AGE-induced VSMC calci cation in a dose-dependent manner ( Figure 5B, 5D, and Supplementary Figure 3B). We also observed that adding the YAP agonist masked the protection of LX from calci cation in VSMCs ( Figure 6B and 6C). Despite in rodent models, studies on the bene ts of eicosapentaenoic acid (EPA) [78,79], the precursor of SPMs, and results describing its clinical effect on vascular calci cation have been con icting [80, 81]. One explanation for this discrepancy is the distinct components and variables of EPA. However, the present results offer another plausible explanation that EPA supplementation enhanced the formation of multiple SPMs, including LX [82], which directly prevented vascular calci cation.
We observed that LX attenuated the increased expression of osteogenesis-related markers and the decreased expression of smooth muscle cell-speci c contractile marker in vivo in diabetic mouse vessels ( Figure 4F and 4G). Similar results were observed in vitro in VSMCs induced by AGEs ( Figure 5E and 5F). These trends were reversed by LPA ( Figure 6D-F), suggesting that suppression of VSMC transdifferentiation to an osteogenic phenotype via YAP is a pivotal mechanism by which LX inhibits VSMC calci cation. Our results are the rst to demonstrate how LX regulates calcium deposition in VSMCs in response to diabetic conditions and con rms YAP as a key driver of this process. Several reports have shown that LX mediates a protective role in vascular disease [46,83,84], and this is consistent with the role of LX in vascular calci cation observed in diabetic mouse vessels.
Cumulative evidence has demonstrated that the extracellular matrix plays a role in vascular calci cation [85][86][87]. We observed more collagen deposition in the calci ed arteries of diabetic mice than in control mice. Moreover, elastic Van Gieson staining results also showed that elastin bres were attened and degraded in the diabetic vasculature ( Figure 4D). Consistent with previous studies, our results further illustrated the link between arterial medial calci cation and altering the extracellular matrix, such as elastin degradation [88] and collagen deposition [89] in the diabetic arterial wall.
Notably, LX prevented collagen deposition in diabetic mice, as depicted by Van Gieson staining. In addition, western blot results showed that the diabetic aorta treated with LX exhibited less COL1, which accounted for 65% of the total collagen in the normal vessel [90] ( Figure 4F).  [92]. This is in line with literature showing that YAP signalling plays a role in extracellular matrix remodelling, especially in regulating collagen production [92].

Conclusion
This study provides several new insights into the role of YAP in vascular medial calci cation; it also suggests LX as a therapeutic strategy for diabetic vascular calci cation. Our study used ample evidence to con rm that the diabetic mice and AGE-induced VSMCs exhibited extensive osteogenesis and calci cation, coupled with more increased YAP expression. In addition, we provide evidence that LX prevents diabetic vascular calci cation via YAP, which potentially explains the mechanism by which LX   The results represent n = 3 per group. Scale bar = 200 μm. (C, D) Quantitative analysis of ALP activity for 7 days and calcium content for 14 days in VSMCs was measured using a colorimetric assay (n = 3). (E-G) Effects of AGEs on the expression of Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), type collagen (COL1), smooth muscle actin (SMA), and yes-associated protein (YAP) for 14 days.

Supplementary Files
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