Effect of flavonoids hydroxygenkwanin on vascular smooth muscle cell proliferation, 1 migration, and neointimal formation

23 Background: Restenosis and atherosclerosis are chronic inflammatory disease. Abnormal vascular 24 smooth muscle cell (VSMC) proliferation and migration play crucial roles in neointimal hyperplasia 25 and restenosis progression in response to stimulation with various inflammatory cytokines, such as 26 platelet-derived

library was validated and quantified with a Qsep100 automated DNA analyzer (Bioptic,Taiwan,206 China) and a Qubit 3.0 fluorometer (Invitrogen). Libraries were sequenced with the HiSeq platform. 207

Evans blue staining 208
Seven days after transluminal mechanical injury, 50 µl of Evans blue (Sigma) (10%) were 209 administered intravenously before euthanasia. After fifteen minutes, the mice were perfused, and the 210 injured femoral arteries were gently dissected to evaluate the denuded vessel area. The ratio of the 211 Evans blue-stained area to the total femoral artery area was calculated. 212

Immunoprecipitation 213
VSMCs were pretreated with HGK or MK2206 for 1 h and then treated with PDGF-BB for 214 another 6 min. The treated cell lysates (400 μg) were incubated with 4 μg of an antibody specific for 215 the Rac family small GTPase 1 (Rac1) at 4°C overnight. Then, the cell lysates were incubated with 216 50 μl of Protein A agarose beads (Sino Biological, Chesterbrook, PA, USA) at 4°C for another 2 h. 217 Afterward, the beads were washed three times with lysis buffer, and the bound proteins were then 218 eluted by heating (95°C, 10 min) and analyzed using SDS-PAGE and Western blotting. The 219 membrane was incubated first with the appropriate anti-Rac1 and anti-ATP7A antibodies overnight 220 at 4°C and then with the appropriate HRP-conjugated secondary antibodies for 1 h at room 221 temperature. Immunoreactivity was detected with enhanced chemiluminescence and quantified 222 using Gel-Pro software. 223

Statistical analyses 224
Statistical analyses were conducted only for studies with a group size of at least 5. All values are 225 provided as the means ± standard deviations (SDs). Statistical comparisons were performed using 226 two-tailed Student's t test and one-way analysis of variance (ANOVA) followed by Tukey's post hoc 227 test. Significance was defined as a p value<0.05.  Figure 1C). These data indicated the structure of HGK 238 ( Figure 1D). 239

HGK reduces platelet-derived growth factor-BB (PDGF-BB)-induced VSMC 240 proliferation and migration by inhibiting the AKT pathway 241
Abnormal VSMC proliferation and migration play important roles in neointimal hyperplasia and 242 restenosis. After chemokine stimulation, contractile VSMCs transform into synthetic VSMCs. 243 PDGF, a potent growth factor present in the injured vessel wall, contributes to VSMC phenotypic 244 switching and VSMC proliferation and migration involved in vascular remodeling by activating 245 multiple intracellular signal transduction pathways. Activated synthetic VSMCs exhibit increased 246 proliferation, migration and extracellular matrix production (3). The matrix metallopeptidase 9 247 (MMP-9) expression level is an indicator of restenosis (13). Western blot analysis showed that 248 compared with treatment with PDGF-BB alone, combined treatment with 10 μM HGK significantly 249 reduced PDGF-BB-induced expression of synthetic VSMC markers (MMP-9 and Connexin-43 250 (Cx43)) and increased expression of VSMC contractile marker α-smooth muscle actin (α-SMA) 251  Figure 1C). Accumulating 258 evidence has shown that AKT activation plays an important role in VSMC proliferation and 259 migration (14). In the present study, compared to PDGF-BB treatment alone, combined treatment 260 with HGK markedly reduced PDGF-BB-induced AKT phosphorylation ( Figure 2F). In addition, 261 treatment with HGK reduced PDGF-BB-induced VSMC proliferation, the expression of 262 proliferation-regulating proteins (Cyclin D1, CDK4, Cyclin E and CDK2), the expression of 263 MMP-9, migration, and lamellipodia formation and increased the expression level of P27. These

HGK reduces tumor necrosis factor-α (TNF-α)-induced VSMC inflammation and 285 thrombosis by inhibiting the AKT pathway 286
Endothelial dysfunction and injury trigger the infiltration and adhesion of inflammatory 287 leukocytes and induce excessive fibrin deposition to cause thrombosis and restenosis (17). 288 Numerous studies have shown that TNF-α production increases and aggravates restenosis 289 progression after angioplasty or vessel injury (18). We evaluated the inhibitory effects of HGK on 290 inflammation and thrombosis in TNF-α-treated VSMCs and found that a high concentration of HGK 291 significantly inhibited the TNF-α-induced expression of inflammatory adhesion molecules (such as 292 intercellular adhesion protein 1 (ICAM-1), E-selectin and vascular cell adhesion protein 1 293 (VCAM-1)) and thromboinflammatory factors (TF and plasminogen activator inhibitor 1 (PAI-1)) 294 compared with that in the TNF-α treatment group ( Figure 4A-B). We further investigated the 295 molecular mechanisms underlying the inhibitory effects of HGK on TNF-α induced inflammation 296 and thrombosis. The AKT pathway plays a key role in inflammation and thrombosis (19). Our data 297 showed that HGK markedly inhibited TNF-α-induced AKT phosphorylation ( Figure 4C). The 298 inhibitory effects of HGK on the TNF-α-induced expression of VSMC inflammatory adhesion 299 molecules (ICAM-1 and E-selectin) and thromboinflammatory factors were similar to those of 300 MK2206 ( Figure 4D-E). In addition, the monocyte adhesion assay showed that compared with 301 TNF-α treatment alone, HGK pretreatment significantly reduced the increase in monocyte adhesion 302 to TNF-α-treated VSMCs. The involvement of ICAM-1, E-selectin and VCAM-1 in the adhesion of 303 monocytes to TNF-α-treated VSMCs was examined by pretreating cells with 1 μg/ml anti-VCAM-1, 304 anti-E-selectin and anti-VCAM-1 antibodies for 1 h before an incubation with TNF-α for another 24 305 h. Pretreatment with antibodies specific for adhesion molecules resulted in a significant reduction in 306 the adhesion of monocytes to TNF-α-treated VSMCs compared to that in the groups treated with 307 TNF-α and cotreated with TNF-α and the IgG isotype control. These results indicated that ICAM-1, 308 adhesion of monocytes to TNF-α-treated VSMCs was also inhibited by 1 μM MK2206 ( Figure 4F), 310 and these inhibitory effects were similar to those of pretreatment with antibodies specific for 311 inflammatory adhesion molecules or HGK. Therefore, HGK inhibits both monocyte adhesion to 312 TNF-α-stimulated VSMCs and thrombosis by inhibiting ICAM-1, E-selectin, TF and PAI-1 313 expression and this effect may be partially mediated by regulating the AKT pathway. inhibitory effects on PDGF-BB-or TNF-α-treated VSMCs were similar between the HGK and 321 MK2206 treatment groups. These results indicated that HGK inhibits VSMC proliferation, 322 migration and inflammation by suppressing mTOR-S6K signaling through the AKT pathway. 323 We further investigated the molecular mechanisms underlying the inhibitory effects of HGK on 324 VSMC proliferation, migration and inflammation. PDK1 has been reported to play an important role 325 in regulating proliferation, migration and inflammation by activating AKT via phosphorylation at 326 threonine 308 (Thr308) and serine 473 (Ser473) (21). The docking analysis showed that HGK has a 327 strong binding affinity for PDK1 ( Figure 6A). In addition, HGK treatment decreased PDK1 328 phosphorylation in PDGF-BB-and TNF-α-treated VSMCs ( Figure 6B). Moreover, PDGF-BB-or 329 TNF-α-induced phosphorylation of AKT at Ser473 and Thr308 was also inhibited by the HGK 330 pretreatment. Similar inhibitory effects were observed on the group pretreated with BX795 (a PDK1 331 inhibitor) ( Figure 6C-D). Based on these results, HGK decreases PDGF-BB-or TNF-α-induced 332 AKT phosphorylation in VSMCs through the PDK1 pathway. 333 Cell viability and wound healing assays were performed to further elucidate the role of PDK1 in 334 inhibitory effects of HGK on PDGF-BB-treated VSMC proliferation and migration were similar to 336 those of the BX795 pretreatment and HGK/BX795 cotreatment. In addition, HGK inhibited the 337 proliferation and migration of VSMCs treated with PS48 (a PDK1 activator). ( Figure 7A VSMCs ( Figure 8A-B) to a similar degree. Furthermore, compared to the control treatment, PS48 347 treatment significantly induced the phosphorylation of mTOR and S6K, and compared to PS48 348 treatment, HGK treatment substantially reduced the levels of mTOR and S6K phosphorylation 349 ( Figure 8C). Therefore, HGK inhibits VSMC proliferation, migration and inflammation mainly by 350 inhibiting PDK1 activation and subsequently regulating the AKT/mTOR/S6K pathway. 351

HGK enhances the chemotaxis of circulating endothelial progenitor cells (EPCs) and 352 reendothelialization after mouse femoral artery denudation 353
Homing of circulating EPCs and reendothelization inhibit neointimal hyperplasia formation and 354 restenosis progression (22). Peripheral blood was collected 5 days after endothelial denudation to 355 evaluate the therapeutic effects of HGK on the homing of circulating EPCs and reendothelization. 356 The flow cytometry analysis showed that HGK treatment increased the number of circulating EPCs 357 in the blood ( Figure 9A). Similar results were observed in the Transwell assay ( Figure 9B). These 358 findings showed that HGK treatment increases the chemotaxis of circulating EPCs. In addition, 359 Evans blue staining showed that HGK reduced the degree of vascular injury at 7 days after 360 reendothelization in denuded femoral arteries at 28 days after endothelial denudation ( Figure 9D). 362 The aforementioned data showed that HGK promotes EPC chemotaxis and reendothelization. 363

HGK reduces restenosis in vivo 364
Endothelial denudation was performed to evaluate the therapeutic effect of HGK on neointimal 365 hyperplasia. Compared to the vehicle treatment, the HGK treatment significantly reduced 366 neointimal hyperplasia, collagen deposition, and the proliferation of VSMCs and macrophages and 367 increased the elastin content in denuded femoral arteries at 28 days after endothelial denudation 368 ( Figure 10A-B). MMP-9 is a marker of synthetic VSMCs and a key factor regulating smooth muscle 369 cell proliferation and migration (23). After angioplasty or vessel injury, the activation of 370 inflammation has been shown to play an important role in neointimal growth. Previous studies 371 showed that TNF-α production and the number of infiltrated macrophages increased soon after 372 angioplasty or vessel injury and that these increases were maintained for at least 2 weeks, thereby 373 contributing to arterial restenosis (24). Immunohistochemical staining showed that the HGK 374 treatment reduced MMP-9, macrophage marker and TNF-α expression levels in denuded femoral 375 arteries ( Figure 10C). Inflammation, macrophage activation, monocyte adhesion, platelet activation 376 and thrombus formation are also involved in neointimal hyperplasia, restenosis and thrombosis 377 progression, and HGK also reduced the expression levels of adhesion molecules, TF and PAI-1 in 378 denuded femoral arteries ( Figure 10D). In addition, the phospho-PDK1 level was markedly reduced 379 in denuded arteries from the HGK treatment group compared with those from the vehicle treatment 380 group ( Figure 10E). circulating EPCs on day 5 after femoral artery denudation and accelerated reendothelization in the 413 present study. Furthermore, the results of in vitro assays revealed that HGK increased EPC 414 chemotaxis. Thus, HGK has considerable therapeutic potential for EPC mobilization and endothelial 415 injury repair, mainly through its antioxidant effects. 416 AKT is a serine/threonine protein kinase with multiple phosphorylation sites and is responsible 417 for multiple behaviors of VSMCs, such as proliferation, migration and differentiation (30). Thr308, 418 which is located in the kinase catalytic region of AKT, is phosphorylated by PDK1, and this 419 phosphorylation is required for AKT activation (31). Then, phospho-AKT (Thr308) phosphorylates 420 AKT at Ser473 via mTORC2 by phosphorylating SIN1 at Thr86 and enhancing mTORC2 kinase 421 activity (32). AKT has a broad range of downstream targets, and accumulating evidence has shown 422 that the AKT/mTOR/S6K signaling pathway is involved in the regulation of several biological 423 processes, such as survival, proliferation, migration and inflammation (33). According to the 424 molecular docking analysis, PDK1 is a major molecular target of HGK. Consistent with this 425 observation, HGK markedly reduced the phosphorylation of PDK1 in PDGF-BB-or TNF-α-treated 426 VSMCs. The increase in AKT (Ser473/Thr308) phosphorylation was substantially inhibited after a 427 PDK1 inhibitor pretreatment, and these effects were similar to those of the HGK pretreatment. The BX795 (a PDK1 inhibitor) for 1 h and were then treated with 20 ng/ml PDGF-BB or 10 ng/ml 563 TNF-α for another 15 or 10 min, respectively. Western blot analyses showed that HGK and BX795 564 decreased PDGF-BB-or TNF-α-induced AKT phosphorylation at Ser473 (S473) and Thr308 (T308). 565 The values are presented as the means ± SDs. *p < 0.05 compared with the CTRL group. †p < 0.05 566 compared with the PDGF or TNF-α group. N=6-9. CTRL, control group. Statistical comparisons 567 were performed using one-way ANOVA. were performed using one-way ANOVA. 582