Regulation of vascular smooth muscle cell calcification by syndecan-4/FGF-2/PKCα signalling and cross-talk with TGFβ

Abstract Aims Vascular calcification is a major cause of morbidity and mortality. Fibroblast growth factor-2 (FGF-2) plays an instructive role in osteogenesis and bone development, but its role in vascular calcification was unknown. Therefore, we investigated the involvement of FGF-2 in vascular calcification and determined the mechanism by which it regulates this process. Methods and results We demonstrate that FGF-2 expression is increased in vascular smooth muscle cells (VSMCs) induced to deposit a mineralized matrix by incubation with β-glycerophosphate. FGF-2 is also localized to sites of calcification within human atherosclerotic plaques. The expression of syndecan-4, a heparan sulfate proteoglycan which regulates FGF-2 signalling, is also increased in mineralizing VSMCs and co-localizes with FGF-2 in human calcified atherosclerotic plaques. Exogenous FGF-2 inhibits VSMC mineralization, and this inhibition is reduced when syndecan-4 expression is knocked-down using siRNA. Biochemical inhibition of FGFR signalling using a pan FGFR inhibitor (BGJ398) or knocking-down syndecan-4 expression in VSMCs using siRNA increases VSMC mineralization. These increases are prevented by inhibiting transforming growth factor-β (TGFβ) signalling with SB431542, suggesting cross-talk between FGF-2 and TGFβ signalling is crucial for the regulation of VSMC mineralization. Syndecan-4 can also regulate FGF-2 signalling directly via protein kinase Cα (PKCα) activation. Biochemical inhibition of PKCα activity using Gö6976, or siRNA-mediated suppression of PKCα expression increases VSMC mineralization; this increase is also prevented with SB431542. Finally, the ability of FGF-2 to inhibit VSMC mineralization is reduced when PKCα expression is knocked-down. Conclusion This is the first demonstration that syndecan-4 promotes FGF-2 signalling, and in turn, suppresses VSMC mineralization by down-regulating TGFβ signalling. Our discoveries that FGF-2 and syndecan-4 expression is increased in mineralizing VSMCs and that PKCα regulates FGF-2 and TGFβ signalling in VSMCs suggests that the syndecan-4/FGF-2/TGFβ signalling axis could represent a new therapeutic target for vascular calcification.


Introduction
Vascular calcification is the formation of mineralized tissue, bone and/or cartilage within the vessel wall. Most patients with cardiovascular disease have some calcification, although it is most prevalent in patients with chronic kidney disease, type 2 diabetes mellitus and atherosclerosis. 1,2 Calcification is not only highly prevalent in these diseases, but there is now substantial evidence that it contributes to the morbidity and mortality associated with these common conditions. 3,4 Vascular calcification is an active cell-regulated process, involving the osteogenic differentiation of vascular smooth muscle cells (VSMCs), VSMC apoptosis, calcifying matrix vesicle/exosome release, and matrix mineralization. 5,6 Existing approaches for the prevention of vascular calcification are limited; therefore, there is an urgent need to identify new therapeutic targets to treat this devastating pathology.
The fibroblast growth factors (FGFs) are a large family of secreted glycoproteins that can be classified as either paracrine-or endocrine-acting. Paracrine FGFs, such as FGF-2, are readily sequestered to the extracellular matrix by heparan sulfate proteoglycans (HSPGs) which limits their diffusion within the extracellular space. For signal propagation, the paracrine FGFs bind to a cell surface FGF-receptor (FGFR1-5) in a ternary complex consisting of FGF, FGFR and HPSGs leading to the activation of downstream signalling events via phospholipase Cc and protein kinase C (PKC), Ras-Erk1/2 or PI3K-Akt. 7 FGF-2 is a critical regulator of osteogenesis and bone development, 8,9 although its role in this process is complex. Bone formation and mineralization are reduced in FGF-2-null mice. 10,11 However, whilst short-term FGF-2 treatment stimulates matrix mineralization in calvarial osteoblasts 12,13 and mesenchymal stem cells, 14 continuous FGF-2 treatment inhibits mineralization by these cells. [12][13][14][15][16] These studies suggest that FGF-2 is required to promote bone mineralization, but then must be down-regulated so mineralization can proceed.
Previous studies have shown that short-term FGF-2 treatment stimulates the expression of osteogenic markers in rat VSMCs. 17 However, the potential role of FGF-2 in VSMC mineralization is currently unknown. Therefore, this study investigated whether FGF-2 regulates VSMC mineralization. We demonstrate that FGF-2/FGFR signalling plays an inhibitory role in this process by interacting with syndecan-4 and down-regulating transforming growth factor-b (TGFb) signalling in VSMCs.

Methods
Detailed experimental protocols are in the Supplementary material online.

Immunohistochemistry
Human atherosclerotic coronary arteries were used for the detection of FGF-2 (n = 7) and syndecan-4 (n = 5) by immunohistochemistry. 18 Calcification was detected using von Kossa staining. Images were acquired using a 20x/0.80 Plan Apo objective using the 3 D Histech Pannoramic 250 Flash II slide scanner. Human tissue was obtained with informed consent and with approval from the Local and National Research Ethics Committees (STH 16346, 12/NW/0036). This study conforms to the Declaration of Helsinki.

Cell culture
Bovine VSMCs were isolated from aortic explants obtained from a local abattoir, and routinely cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2 mM L-glutamine, 100 U/ mL penicillin, 1.4 lM streptomycin, 1 mM sodium pyruvate, 1x nonessential amino acids and 10% (v/v) fetal calf serum (FCS), referred to as 10% FCS-DMEM. For mineralization assays, cells were cultured in 10% FCS-DMEM until confluent (day 0), and then in 10% FCS-DMEM and 3 or 5 mM b-glycerophosphate (b-GP) for up to 18 days. 19 Controls were cultured without b-GP. Four preparations of uncloned VSMCs isolated from different animals were used for these studies; different batches of cells were used in independent experiments. Unless otherwise stated, in vitro studies used bovine VSMCs. Cells were used between passage 10-13.
Human coronary artery VSMCs were routinely cultured in medium 231 supplemented with smooth muscle growth supplement (Gibco, Life Technologies, UK). For mineralization assays, cells were cultured in medium 231 supplemented with smooth muscle growth supplement until confluent (day 0), and then with 5 mM b-GP and 0.9 mM calcium chloride for up to 40 days. The final concentration of calcium chloride in the human VSMC calcifying media was 2.5 mM. Controls were cultured without b-GP and additional calcium chloride. Two preparations of human VSMCs (passage 6-7) were used for these studies; different batches of cells were used in independent experiments.

Small interfering RNAs (siRNAs)
VSMCs were transfected with siRNAs against syndecan-4 (S459980, Ambion V R , Life Technologies, UK) or PKCa (SI01965138, Qiagen, UK) using RNAiMAX (Invitrogen TM , Life Technologies, UK). A random control siRNA (#1027281; Qiagen, UK) was the control. All siRNAs were used at a final concentration of 20 nM. For signalling assays, VSMCs were cultured for up to 7 days, with repeated siRNA transfections every 48-72 h. For mineralization assays, VSMCs were transfected twice with siRNA (with 48-72 h between transfections) prior to b-GP treatment. During b-GP treatment, siRNAs were removed after 4 h and fresh medium containing b-GP was added to the cells between transfections.

RNA isolation and quantitative polymerase chain reaction (qPCR)
RNA was isolated using the RNeasy Mini Kit (Qiagen) and cDNA synthesized using Taqman V R Reverse Transcription Reagents (Invitrogen TM , Life Technologies). qPCR was performed using SYBR Green PCR master mix (Applied Biosystems, Life Technologies) and the CFX96 or CFX384 Real-Time PCR system (Bio-Rad, UK). Primer sequences are provided in the Supplementary material online. All samples were amplified in duplicate and averaged to produce one data-point. The expression of each gene was normalized to the reference genes [ribosomal protein L12 (RPL12) and peptidylprolyl isomerase A (PPIA)] using the comparative C t method.

Statistical analysis
Data are presented as the mean ± standard error of the mean (SEM). Data were normalized where required using log 10 and statistical comparisons were made using t-tests or one-way ANOVA. Data with two or more variables were analysed with a 2-way ANOVA. Where normality could not be confirmed, data were analysed using a Mann-Whitney ttest. A value of P < 0.05 was considered statistically significant.

FGF-2 inhibits mineral deposition by VSMCs
FGF-2 plays an instructive role in osteogenesis, [12][13][14] but the potential involvement of FGF-2 in vascular calcification was unknown. Therefore, to investigate FGF-2 expression during VSMC mineralization, a wellestablished in vitro model of vascular calcification was used. 19 VSMCs deposited a mineralized matrix when cultured from confluence (day 0) in the presence of b-GP, and the extent of mineralization increased with time ( Figure 1A). No mineralization was detected in controls cultured without b-GP ( Figure 1A).
RNA and protein were isolated from VSMCs at specific time-points: early mineralization (days 9-10), mid mineralization (days 10-12), and late mineralization (days [12][13][14][15][16][17][18]. RNA and protein were also isolated from VSMCs cultured without b-GP at these same time-points. FGF-2 mRNA (40-fold increase; Figure 1B) and protein (2.5-fold increase; Figure 1C) expression were significantly increased in b-GP-treated VSMCs at late mineralization when compared to controls at the same time-point. In contrast, FGF-2 expression was not increased in VSMC preparations that do not deposit a mineralized matrix in the presence of b-GP ( Figure 1D) confirming that the changes observed in FGF-2 are either necessary for, or are a consequence of, VSMC mineralization and are not due to extended culture in the presence of b-GP.
To determine whether FGF-2 regulates VSMC mineralization, VSMCs were cultured with b-GP plus vehicle or FGF-2 (25 or 50 ng/mL). Exogenous FGF-2 significantly reduced b-GP-induced mineralization when compared to the vehicle and b-GP control ( Figure 2A). The addition of FGF-2 at different time-points during the mineralization protocol (i.e. 0, 2, 4 or 6 days after addition of b-GP) also significantly reduced b-GP-induced mineralization in VSMCs compared to controls (see Supplementary mate rial online, Figure S1), suggesting FGF-2 can also suppress matrix mineralization when added to cells which are primed to mineralize.
We next investigated the role of FGF-2-dependent fibroblast growth factor receptor (FGFR) signalling during VSMC mineralization using the pan-FGFR inhibitor BGJ398. BGJ398 markedly inhibited FGF-2-induced Akt and Erk1/2 phosphorylation in VSMCs ( Figure 2B); BGJ398 also increased b-GP-induced VSMC mineralization compared to the vehicle and b-GP control (4.5-fold increase with 1 lM BGJ398; Figure 2C). This result was verified in mineralizing human VSMCs ( Figure 2D). BGJ398 did not induce VSMC mineralization in the absence of raised phosphate levels (see Supplementary material online, Figure S2A), nor did it induce mineralization in preparations of VSMCs that do not mineralize in the presence of b-GP (see Supplementary material online, Figure S2B), suggesting that inhibition of FGFR signalling does not drive VSMC mineralization on its own, but it accelerates mineralization in VSMCs that are already primed to mineralize.

FGF/TGFb cross-talk regulates mineral deposition by VSMCs
The above results demonstrate FGF-2 and FGFR signalling reduce matrix mineralization in VSMCs, but how FGF-2 mediates this effect was unknown. Recent studies have shown that inhibiting FGF signalling increases TGFb signalling in VSMCs. 21,22 As TGFb1 accelerates mineral deposition by calcifying vascular cells, 23 we next investigated the relationship between FGF and TGFb signalling in VSMC mineralization.
To confirm TGFb signalling regulates VSMC mineralization, VSMCs were cultured with b-GP plus vehicle or TGFb1 (0.1 or 1 ng/mL). Exogenous TGFb1 significantly increased b-GP-induced VSMC mineralization when compared to the vehicle and b-GP control ( Figure 3A). In contrast, inhibiting endogenous TGFb signalling using the type 1 TGFb receptor (TGFbR1) kinase inhibitor, SB431542 (0.1 or 1 mM), significantly reduced VSMC mineralization when compared to the vehicle and b-GP control ( Figure 3B).
TGFbR activation leads to Smad2 phosphorylation. 21,22 Previous studies have shown that decreased Smad2 phosphorylation co-localizes with increased FGFR1 phosphorylation in the medial layer of atherosclerotic human coronary arteries. 22 In the late stages of matrix mineralization, Smad2 phosphorylation was significantly reduced in b-GP-treated VSMCs when compared to controls at the same time-point (four-fold decrease; Figure 3C). This decrease in Smad2 phosphorylation coincided with increased FGF-2 expression in b-GP-treated VSMCs (compare Figures 1B,  1C and 3C).
To confirm FGF regulates TGFb signalling in VSMCs, FGFR signalling was inhibited using BGJ398 and cells were incubated with TGFb1 for up to 60 min. TGFb1-induced Smad2 phosphorylation was significantly increased in VSMCs treated with BGJ398 after 30 and 60 min ( Figure 3D); this increase was prevented by co-incubation with SB431542 ( Figure 3D). The relationship between FGF and TGFb signalling in matrix mineralization was also studied by incubating VSMCs with both inhibitors. As before, BGJ398 significantly increased b-GP-induced VSMC mineralization compared to the vehicle and b-GP control ( Figure 3E); this increase was prevented by co-incubation with SB431542 ( Figure 3E). Together these results suggest FGFR inhibition increases matrix mineralization by up-regulating TGFb signalling in VSMCs.

Syndecan-4 expression co-localizes with FGF-2 in calcified vessels
Syndecan-4 is a transmembrane HSPG that functions as an adhesion receptor and growth factor co-receptor, eliciting signals in response to the extracellular microenvironment. 24,25 Atherosclerotic plaque susceptibility is increased in syndecan-4/low-density lipoprotein receptor double knock-out mice fed a high-fat diet 26 ; but its role in vascular calcification was unknown. As syndecan-4 is a critical regulator of FGF-2 signalling, 27,28 we next investigated the potential involvement of syndecan-4 in VSMC mineralization, and examined whether syndecan-4 regulates FGF-2/TGFb signalling in this process.
Syndecan-4 mRNA expression was markedly increased in b-GPtreated VSMCs, with a five-fold increase at late mineralization compared to the same time-point controls ( Figure 4A). This increase in syndecan-4 mRNA expression coincided with increased FGF-2 expression and decreased Smad2 phosphorylation in b-GP-treated VSMCs (compare Figures 1B, 1C, 3C and 4A). In contrast, syndecan-1 expression was significantly decreased in b-GP-treated VSMCs compared to controls, with a two-fold decrease at late mineralization ( Figure 4A). No significant changes were detected in syndecan-2 or syndecan-3 mRNA expression ( Figure 4A). Furthermore, syndecan-1 and syndecan-4 mRNA expression did not change when VSMC preparations which do not deposit a mineralized matrix in the presence of b-GP were analysed ( Figure 4B).
Syndecan-4 and FGF-2 expression in human atherosclerotic arteries was also examined. FGF-2 and syndecan-4 staining was localized to areas directly adjacent to, and within, calcified regions of atherosclerotic arteries (representative images of three atherosclerotic lesions from two different donors are shown in Figure 4Ci-iii). No staining was observed in the rabbit IgG controls (Figure 4Ci-iii). stained with alizarin red (bar = 500 mm) and mineral deposition quantified by dye elution (n = 6 independent experiments). FGF-2 expression was measured using (B) qPCR (data expressed relative to day 0; n = 9 independent experiments) and (C) immunoblotting of cell lysates (FGF-2 is expressed relative to bactin; n = 6 independent experiments). Molecular weight markers are shown. (D) Two different preparations of non-mineralizing VSMCs were cultured ± 5 mM b-GP from confluence (day 0) for up to 14 days. RNA was collected from cells at day 0, 9, 11, and 14. These time-points were chosen as they correspond to the time for mineralizing VSMCs (1 A) to reach early, mid or late mineralization. FGF-2 mRNA expression was measured using qPCR (data expressed relative to day 0; n = 6 independent experiments).

FGF-2 inhibits mineral deposition via syndecan-4
To determine the role of syndecan-4 in VSMC mineralization, siRNA was used to knock-down syndecan-4 expression ( Figure 5A). Syndecan-4 knock-down significantly increased b-GP-induced VSMC mineralization compared to control siRNA-treated cells cultured with b-GP ( Figure  5B). Knocking-down syndecan-4 expression in VSMCs cultured in control media did not induce matrix mineralization on its own (see Supplementary material online, Figure S3A). Furthermore, knockingdown syndecan-4 expression in a preparation of VSMCs that do not mineralize in the presence of b-GP did not induce matrix mineralization (see Supplementary material online, Figure S3B).
Inhibiting FGFR signalling increases TGFb signalling in VSMCs ( Figure  3D) and inhibiting TGFb signalling prevents FGFR inhibition from increasing VSMC mineralization ( Figure 3E). Therefore, to determine if TGFb signalling is also responsible for the increased matrix mineralization in syndecan-4 knock-down VSMCs, syndecan-4 siRNA-transfected VSMCs were cultured with b-GP and vehicle or SB431542 (1 lM). Control siRNA-transfected VSMCs cultured with b-GP were used as controls. Knocking-down syndecan-4 expression in VSMCs significantly increased b-GP-induced matrix mineralization; this increase was prevented by coincubation with SB431542 ( Figure 5D). These results suggest syndecan-4 and FGF-2 both suppress matrix mineralization by down-regulating TGFb signalling in VSMCs.
Knocking-down PKCa with siRNA ( Figure 7B) or inhibiting PKCa activity with Gö6976 (1 lM) ( Figure 7C) significantly increased b-GPinduced VSMC mineralization compared to the relevant controls. Gö6976 also increased mineralization in human VSMCs ( Figure 7D). Knocking-down PKCa expression in VSMCs cultured in control media (see Supplementary material online, Figure S3A), or culturing VSMCs in control media with Gö6976 (see Supplementary material online, Figure  S3C) did not induce matrix mineralization. Also Gö6976 did not induce mineralization in a preparation of VSMCs that do not mineralize in the presence of b-GP (see Supplementary material online, Figure S3D). These results suggest that loss or inhibition of PKCa is not a driver of VSMC mineralization per se, but it accelerates mineralization in VSMCs which are already primed to mineralize.
To further define the link between FGF-2/syndecan-4 and PKCa in regulating VSMC mineralization, control siRNA-and PKCa siRNAtransfected VSMC were cultured with vehicle or FGF-2 in the presence of b-GP. As before, FGF-2 reduced mineralization whereas PKCa knock-down markedly increased mineralization ( Figure 7E). PKCa knock-down reduced the inhibitory effect of FGF-2 on mineralization ( Figure 7E), suggesting the FGF-2/syndecan-4 signalling axis may, at least in part, regulate VSMC mineralization via PKCa. FGF-2 was still able to inhibit mineralization in PKCa knock-down VSMCs ( Figure 7E), suggesting that FGF-2 can also signal via other downstream signalling pathways.
To determine if increased TGFb signalling mediates the increased mineralization in PKCa knock-down VSMCs, PKCa siRNA-transfected VSMCs were cultured with b-GP and vehicle or SB431542 (1 lM). Control siRNA-transfected VSMCs cultured with b-GP were controls. SB431542 prevented PKCa knock-down from increasing b-GP-induced matrix mineralization in VSMCs ( Figure 7F), suggesting loss of PKCa increases matrix mineralization by up-regulating TGFb signalling in VSMCs.

Discussion
We demonstrate the expression of FGF-2 and its co-receptor, syndecan-4, are increased in mineralizing VSMCs and at sites of calcification in vehicle ± 3 mM b-GP, or SB431542 (0.1 or 1 lM) and 3 mM b-GP, stained with alizarin red (bar = 500 mm) and mineral deposition quantified (n = 3 independent experiments). (C) VSMCs were incubated ± 3 mM b-GP for up to 18 days. Cell lysates were isolated at late VSMC mineralization and immunoblotted for phosphorylated Smad2 (pSmad2), total Smad2, and b-actin on the same membrane. Molecular weight markers are shown. The pSmad2/Smad2 ratio is expressed relative to b-actin (n = 6 independent experiments). human atherosclerotic plaques, and that biochemical inhibition of FGFR signalling or knocking-down syndecan-4 expression increases VSMC mineralization. Importantly, syndecan-4 is, at least in part, responsible for the inhibition of VSMC mineralization by FGF-2, suggesting syndecan-4 expression is increased in mineralizing VSMCs to maintain FGF-2 signalling. We also show syndecan-4 and FGF-2 signalling suppress the deposition of a mineralized matrix by down-regulating TGFb signalling. Finally, we demonstrate that PKCa, which is activated in a cytoplasmic domaindependent manner by syndecan-4, 29,30 regulates FGF-2/TGFb signalling and mineralization in VSMCs. Together, these results demonstrate a novel feedback mechanism whereby mineralizing VSMCs increase FGF-2 and syndecan-4 expression and down-regulate TGFb signalling to prevent more extensive calcification (Figure 8).
This is the first demonstration that FGF-2 expression is increased in mineralizing VSMCs in vitro, and that FGF-2 is localized to calcified regions of human atherosclerotic plaques. Consistent with these findings, FGF-2 is expressed adjacent to calcified regions in valve leaflets. 31  and mineralization of osteoprogenitors, 32 and is expressed at sites of bone formation in vivo. 33 FGF-2 plays a complex role in osteoblast mineralization, with its effects dependent on the timing and duration of signalling. [12][13][14] Consistent with these studies, we show that continuous FGF-2 treatment inhibits b-GP-induced VSMC mineralization. The addition of FGF-2 after the commencement of b-GP treatment also reduces VSMC mineralization, supporting the suggestion that increases in FGF-2 expression during late mineralization may put a 'brake' on this process. Indeed, FGF-2 stimulates the osteogenic potential of calvarial osteoblasts and mesenchymal stem cells during the early stages of differentiation, but must then be down-regulated for mineralization to proceed. [12][13][14] It is well established that syndecan-4 is a critical regulator of FGF-2 signalling, 27,28 but it is currently unknown how the transcription of syndecan-4 and FGF-2 is regulated in mineralizing VSMCs. Previous studies have reported that FGF-2 synergizes with Runx2 to enhance syndecan-4 mRNA expression in calvarial osteoblasts, 34 but we found that 24-h FGF-2 treatment has no effect on syndecan-4 mRNA expression in VSMCs (see Supplementary material online, Figure S4). It is possible, therefore, that raised levels of Runx2 are required for FGF-2 to induce syndecan-4 expression in these cells e.g. as observed during VSMC osteogenic differentiation and mineralization.
A role for TGFb1 in vascular calcification was first suggested by Watson et al. who reported TGFb1 increased mineralized nodule formation in bovine calcifying vascular cells. 23 More recent studies have shown that inhibition of TGFbR1 using SB431542 inhibits VSMC mineralization. 35,36 We also show that TGFb1 accelerates mineral deposition by bovine VSMCs, whereas SB431542 inhibits it. Furthermore, we show FGF-2 expression is increased in mineralizing VSMCs and TGFb signalling is concomitantly reduced to minimize further calcification. Previous studies in VSMCs have shown that suppressing FGF signalling results in reduced let-7 microRNA, leading to increased TGFbR1 receptor expression and TGFb signalling activation. 22 It is therefore possible that FGF-2/TGFb cross-talk may also be mediated via let-7 microRNA in mineralizing VSMCs.
Several studies have suggested PKCa normally acts to suppress bone formation. 37,38 Consistent with a previous study in mouse VSMCs, 39 we show that inhibiting PKCa activity with Gö6976 or knocking-down PKCa expression increases VSMC mineralization. Moreover, we demonstrate that this increase in mineralization is prevented by inhibiting TGFbR1 signalling. The crucial role of PKCa in regulating mineralization is further highlighted by our demonstration that knocking-down PKCa reduces the ability of FGF-2 to inhibit VSMC mineralization. Overexpressing PKCa in an osteoblastic cell line reduces alkaline phosphatase activity and the expression of osteogenic marker genes in these cells 37 ; however, the effects of over-expressing PKCa on osteoblast or VSMC mineralization are unknown. As PKCa is downstream of FGF-2/ syndecan-4, a possible focus for therapeutic targeting in vascular calcification may be the modulation of PKCa activation/signalling in VSMCs.
A potential limitation of our study is that the signalling data were obtained following short-term incubation of the VSMCs with growth factors and/or inhibitors; whereas the mineralization data were obtained following incubation of the cells with these same reagents for up to 14 days. However, although caution should be taken when extrapolating between these two sets of data, our study clearly demonstrates that signalling and mineralization are both affected by these treatments.
Whilst our results indicate an important role for syndecan-4 in regulating FGF-2/TGFb signalling during VSMC mineralization, other PGs could also regulate these signalling pathways in this process. Indeed, our data show the expression levels of several other PGs are modulated during VSMC mineralization (see Supplementary material online, Figure S5). For example, glypican-4 expression is also up-regulated in b-GP-treated VSMCs (see Supplementary material online, Figure S5). Glypican-4 binds FGF-2 40 and may therefore also affect FGF-2/FGFR signalling in VSMCs, although glypican-4 wouldn't directly activate PKCa. Decorin has also been shown to promote mineralization by increasing TGFb signalling in VSMCs. 35 Future studies could, therefore, determine whether these PGs also regulate FGF-2 and TGFb cross-talk during VSMC mineralization.
In conclusion, our study has identified a novel potential therapeutic target pathway in the control of vascular disease. We highlight syndecan-4/FGF-2/TGFb signalling as a critical regulator of VSMC mineralization. Intriguingly, both syndecan-4 and FGFR signalling appear to be important in this process. It remains to be determined whether syndecan-4 and FGFR regulate mineralization in convergent or parallel pathways. It is possible that syndecan-4 may act to prevent excessive mineralization via two mechanisms: (a) interacting as a co-receptor for FGF-2 and inducing down-stream signalling via FGFR and (b) via interaction with PKCa. These pathways may then coalesce to suppress mineralization induced by TGFb. Although this dual activity of syndecan-4 is well established in other systems (e.g. during neural induction 41 ) its role here is of particular importance given the current need for novel drugs to treat vascular disease.

Supplementary material
Supplementary material is available at Cardiovascular Research online.