GDF11 Protects against Endothelial Injury and Reduces Atherosclerotic Lesion Formation in Apolipoprotein E-Null Mice

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1 originl rticle The Americn Society of Gene & Cell Therpy Protects ginst Endothelil Injury nd Reduces Atherosclerotic Lesion Formtion in Apolipoprotein E-Null Mice Wen Mei, Gungd Xing, Yixing Li 2, Hun Li, Lingwei Xing 3, Junyn Lu, Lin Xing, Jing Dong nd Min Liu Deprtment of Endocrinology, Wuhn Generl Hospitl of Gungzhou Commnd, Wuhn, Hubei Province, Chin; 2 Rdition-Dignostic/Oncology School of Medicine, Emory University, Atlnt, Georgi, USA; 3 Mthemtics nd Sttistics Deprtment, Georgi Stte University, Atlnt, Georgi, USA Growth differentition fctor () reduces crdic hypertrophy, improves cerebrl vsculture nd enhnces neurogenesis in geing mice. Higher growth differentition fctor /8 (/8) is ssocited with lower risk of crdiovsculr events in humns. Here, we showed tht deno-ssocited viruses- nd recombinnt protein improve endothelil dysfunction, decrese endothelil poptosis, nd reduce inflmmtion, consequently decrese therosclerotic plques re in polipoprotein E / mice. Moreover, deno-ssocited viruses- nd recombinnt stbilize therosclerotic plques by selectively decresing in mcrophges nd T lymphocytes, while incresing in collgen nd vsculr smooth muscle cells within plques. In ddition, inhibit plmitic cid-induced endothelil poptosis nd meliorte plmitic cid-induced inflmmtory response in RAW264.7 mcrophges in vitro. Mechnisticlly, ctivtes the TGF-β/Smd2/3, AMPK/endothelil nitricoxide synthse (enos) while suppresses JNK nd NF-κB pthwys. In humns, circulting /8 is positively ssocited with flow-medited endothelium-dependent diltion in overweight subjects. We concluded tht denossocited viruses- nd recombinnt protect ginst endothelil injury nd reduce therosclerosis in polipoprotein E / mice, thus my be providing novel pproch to the tretment of therosclerosis. Received My 26; ccepted 28 July 26; dvnce online publiction October 26. doi:.38/mt.26.6 INTRODUCTION Growth differentition fctor () belongs to the trnsforming growth fctor β (TGF-β) superfmily. exerts its function by intercting with ctivin type IIA nd IIB receptors 2 nd type I receptor Alk5 (ref. 3) to induce phosphoryltion of Smd2/3. Recent studies showed tht is circulting fctor tht declines with ge, nd restoring systemic levels reverses ge-relted dysfunction in mouse hert, 4 skeletl muscle, 5 nd brin. 6 However, recent publictions from Egermn et l. nd Smith et l. rgued tht inhibits skeletl muscle regenertion nd hs no effect on crdic hypertrophy. 7,8 Very importntly, new dt demonstrted tht circulting growth differentition fctor /8 (/8) levels decrese with ge in mice s well s other mmmlin species nd tht incresing /8 levels with exogenous regultes crdiomyocyte size. 9 Furthermore, humn study showed tht plsm concentrtions declined with ge in nondibetic control subjects, nd the other two clinicl reports reveled tht higher or /8 levels from lrge humn cohorts re ssocited with lower risk of crdiovsculr events nd deth, suggesting the pthwy s potentil new trget to improve dverse crdiovsculr outcomes ssocited with ging.,2 Thus, future studies re needed to prove the potentil beneficil effects of on ge- ssocited crdiovsculr diseses. Atherosclerosis, chronic inflmmtory disese of rteril wll, is n ging-relted disese, nd ging is one of the min risk fctors for therosclerosis. 3 Atherosclerosis is highly prevlent in older persons hving ctstrophic consequences in their qulity of life nd incresing disbility nd mortlity in this popultion. 4,5 However, the potentil role of on therosclerosis hs not been investigted. Adeno-ssocited viruses (AAV) medite stble gene expression nd re suitble for diseses for which long-term trnsgene expression is needed. 6 AAV cn trnsduce wide vriety of cell types nd trnsgene expression hs been reported t lest in the retin, skeletl muscle, vsculr smooth muscle, nd centrl nervous system. In this study, we evluted the potentil role of the AAV-medited gene trnsfer (AAV-) injected vi til vein nd exogenous recombinnt on therosclerosis in polipoprotein E-null (poe / ) mice. The AAV- ws first expressed in the liver nd subsequent relesed into the circultion. It ws found tht AAV- nd recombinnt were ble to improve endothelil dysfunction, decrese endothelil poptosis nd reduce inflmmtion ctivity, consequently decrese therosclerotic plques re s well s stbilize therosclerotic plques in poe / mice. Correspondence: Gungd Xing, Deprtment of Endocrinology, Wuhn Generl Hospitl of Gungzhou Commnd, Wuluo Rod 627, Wuhn 437, Hubei Province, Chin. E-mil: Gungd64@hotmil.com vol. 24 no., nov. 26

2 The Americn Society of Gene & Cell Therpy nd Atherosclerosis TR CMV polya TR b AAV- AAV- c AAV- e f d Serum /8 (pg/ml) AAV- Serum /8 (pg/ml) Time (weeks) from inocultion 4 2 Time (weeks) from injection Figure Delivery of cdna to poe / mice using AAV vector. ApoE / mice (n = 3) preinoculted with 2 vp of AAV- or through the til vein. Rndomly three mice from nd AAV- groups were killed 4 weeks fter injection nd the expression of in liver nd ort were detected by immunofluorescence. () AAV vector used in this study. TR indictes terminl repet sequences; CMV, cytomeglovirus immedite erly promoter; nd polya, polydenyltion site. (b) Expression of ws evluted by immunofluorescence nd Western blot in MAECs trnsduced with AAV-. Scle br, 5 μm. (c nd d) All dt were repeted three times. Immunofluorescence of liver nd ort sections of mice treted with AAV- t 4 weeks fter injection. Scle br, 5 μm (c) nd Scle br, μm (d). (e nd f) Serum /8 ws nlyzed by enzyme-linked immunosorbent ssy (ELISA) in poe / mice. Dt were shown s men ± SD. Student s t-test ws performed to compre differences between two groups. P <.5 versus ; P <.5 versus vehicle group. AAV, deno-ssocited viruses; AAV-, AAV-medited gene trnsfer; poe /, polipoprotein E null;, growth differentition fctor ; GFP, green fluorescent protein; MAECs, mice ortic endothelil cells; SD, stndrd devition. RESULTS Persistence of expression in AAV- treted poe / mice As n lterntive strtegy of dministrtion, we hve constructed n AAV vector-expressing (Figure ) to nlyze possible dvntges of sustined in vivo expression of compred with injections with recombinnt humn. The AAV- vector ws used to encpsidte virl prticles in serotype 2. Efficiency of trnsduction nd expression of the trnsgene ws initilly demonstrted by immunofluorescence nd Western blot in cultured mice ortic endothelil cells (MAECs) from poe / mice, demonstrting tht the trnsgene is constructed successfully (Figure b). In this study, poe / mice received single injection with high-titer (~ 2 virl genome prticles) AAV- or AAVgreen fluorescent protein (GFP) through the til vein. To ssess the in vivo dissemintion of AAV-, three nimls were killed 4 weeks fter injection nd the expression of in liver nd ort were detected by immunofluorescence (Figure c,d). In both cses, mice injected with AAV- showed lrge number of res positive for expression of. We lso nlyzed the serum /8 levels by enzyme-linked immunosorbent ssy nd found tht protein relese to the bloodstrem remined constnt up to 2 weeks (Figure e) (n = mice in ech group). The influence of AAV- nd recombinnt on metbolic chrcteristics in nimls Some clinicl studies showed tht higher circulting or /8 levels re ssocited with lower risk of crdiovsculr events nd deth.,2 It is estblished tht metbolic chrcteristics re ssocited with crdiovsculr diseses. Thus, we investigted the effects of AAV- nd recombinnt on metbolic profiles in vivo. Firstly, compred with the norml chow control group, the high ft diet (HFD) control group hd higher levels of body weight, serum free ftty cids (FFA) t, 4, 8, nd 2 weeks, nd lower levels of /8 t 4, 8, nd 2 weeks (see Supplementry Figure Sb d). Secondly, to investigte the chnges of circulting /8 levels with ge in mice fed with HFD, the results showed tht circulting /8 levels hve trend of decrese Moleculr Therpy vol. 24 no. nov

3 nd Atherosclerosis The Americn Society of Gene & Cell Therpy t week when compred with 8 weeks (P >.5), which significntly lower t 4, 8, nd 2 weeks s compred with 8 weeks nd week (P <.5) (see Supplementry Figure Sd). Thirdly, to explore the chnges of circulting /8 in mice induced by different interventions, serum /8 levels were mesured t week (n = 3 mice in ech group), 4 weeks (n = 3 mice in ech group), nd 2 week time point (n = mice in ech group). Serum /8 levels declined with ge in the nd vehicle groups (P <.5) nd were significntly elevted in the AAV- nd recombinnt groups t 4 nd 2 weeks time points (P <.5) (Figure e,f). Fourthly, body weight, systolic blood pressure, nd other metbolic prmeters were nlyzed t different experiment time points. The body weight incresed drmticlly from 8 weeks (7.3 ±.7 g, n = 2 mice) to weeks (23.4 ±.9 g, n = 92 mice) (P <.) in intervention poe / mice. The body weight nd systolic blood pressure levels were not sttisticlly different mong, AAV-, vehicle nd -treted groups t nd 2 weeks (see Supplementry Tble S). After different tretments for 2 weeks, s shown in Supplementry Tble S2, AAV- nd hd no significnt effects on fsting glucose, glycosylted hemoglobin (HbAc) nd high density lipoprotein. However, the serum levels of fsting insulin, interleukin-6 (IL-6), tumor necrosis fctor-α (TNF-α), c-rective protein, oxidized low density lipoprotein, totl cholesterol, triglycerides nd FFA were significntly decresed t the end of this study in AAV- nd groups when compred with the or vehicle groups. Fifthly, to test the effects of AAV- nd recombinnt on insulin sensitivity in mice fed with HFD, intrperitonel glucose tolernce test nd insulin tolernce test were performed before nd 2 weeks fter AAV- or others tretments. Before these interventions, there ws no significnt difference on glucose nd insulin tolernce mong the four groups. But fter 2 weeks of interventions, the glucose nd insulin tolernce were improved significntly when compred with the or vehicle groups (see Supplementry Figures S2 nd S3). AAV- nd recombinnt protected ginst endothelil injury in vivo Previous study reveled tht tretment induces migrtion nd in vitro tube formtion of peripherl blood endothelil progenitor cells. 7 Thus, the effects of AAV- nd recombinnt on endothelil injury in vivo were detected in this study. Firstly, the protective roles of AAV- nd on endothelil function in vivo were mesured t 4 weeks. Both AAV- nd tretments improved the vsculr endothelium-dependent relxtion in response to cetylcholine when compred with the or vehicle groups (7.32 ± 3.89% versus 87.7 ± 4.3%; 73.6 ± 3.63% versus ± 3.52% relxtion t 4 mmol/l cetylcholine, respectively; four ortic rings from ech mice, n = 3 mice in ech group, P <.5) (Figure 2). In contrst, the vsculr endothelium-independent relxtion in response to sodium nitroprusside did not differ mong the 4 groups (Figure 2b). Secondly, poptosis of endothelil cells hs been suggested to ply unfvorble role in therosclerosis. 8 Thus, the effects of AAV- nd recombinnt on the poptosis of endothelil cell in vivo were explored t 2 weeks. The seril plque sections from the thorcic ort were costined for terminl deoxynucleotidyl trnsferse biotin-dutp nick end lbeling nd CD3, n endothelil cell-specific mrker. As shown in Figure 2c,d, both AAV- nd tretments hd sprse terminl deoxynucleotidyl trnsferse-medited dutp-biotin nick end lbeling -positive cells tht seldom coloclized with endothelil cells compred with the or vehicle tretments. It mens tht greter more integrity of endothelil cells lid on the plque surfce of AAV- nd -treted mice. In ddition, electron microscopy of rteril endothelium reveled rteries from the nd vehicle groups hd extensive endothelil dmge chrcterized by nucler condenstion nd cytoplsmic vcuoliztion, while most rteril endothelium from the AAV- nd groups ws intct (Figure 2e). These findings suggest tht AAV- nd recombinnt decresed endothelil injury in poe / mice fed HFD. AAV- nd recombinnt ttenuted therosclerotic lesion formtion in poe / mice Endothelil injury is the erly pthophysiologic chnge of therosclerosis, pproches tht protect ginst endothelil injury my be therpeutic for therosclerosis. 9 Therefore, we investigted whether AAV- nd recombinnt llevite therosclerosis besides protecting vsculr endothelium t 2 weeks in this study. Firstly, the lipid-rich therosclerotic lesion re, including both en fce nd cross sections nlyses, ws significntly smller in the AAV- group compred with the group (en fce: 4.54 ± 2.86% versus 38. ± 4.43%, cross sections: 9.6 ±.63% versus 23.2 ± 2.76%, P <.5) (Figure 3,c). Similrly, the recombinnt significntly decresed the plque re compred with the vehicle group (en fce: 7.8 ± 2.7% versus 3.23 ± 3.2%, cross sections:.32 ±.47% versus 9.87 ± 2.%, P <.5) (Figure 3,c). Secondly, we sought to investigte whether, besides decresing the development of therosclerotic lesions, AAV- nd recombinnt tretment lso ffected their cellulr composition. For this purpose, seril plque sections from the ortic rch were nlyzed by immunohistochemistry to quntittively evlute the vsculr smooth muscle cells (VSMCs), collgen content, mcrophges, T lymphocytes, mtrix metlloproteinse (MMP)-2 nd MMP-9. As shown in Figure 3e,f, the reltive contents of VSMCs nd collgen were higher in the AAV- nd groups, possibly contributing to the stbility of therosclerotic plques. In contrst, AAV- nd tretments significntly reduced the re of mcrophges nd T lymphocytes infiltrtion in plques compred with the or vehicle groups (Figure 3e,f). Furthermore, the expression levels of MMP-2 nd MMP-9 were lower in the AVV- nd groups (see Supplementry Figure S4). These findings pper prticulrly relevnt becuse plque infiltrting mcrophges re through production of metlloproteinses to induce plque destbiliztion. 2 It is estblished tht therosclerosis is n inflmmtory disese of rteril wll. 2 Next, we ssessed the inflmmtory cytokines in orts by rel-time polymerse chin rection. As shown in Figure 3g, tretment drmticlly reduced the mrna expression levels of IL-6, IL-β, TNF-α, monocyte chemotctic vol. 24 no. nov. 26

4 The Americn Society of Gene & Cell Therpy nd Atherosclerosis Relxtion (%) AAV- b Relxtion (%) AAV Ach (log mol/l) SNP (log mol/l) c TUNEL CD3-TUNEL DAPI merged d AAV- Apoptotic endothelil cells % totl endothelil cells AAV- e AAV- Figure 2 AAV- nd improved endothelil function nd decresed endothelil cells poptosis in poe / mice. Four thorcic ort rings from ech poe / mice (n = 3 mice from ech group) were equilibrted for hour t prelod tension of.5 g in Krebs buffer nd then precontrcted with norepinephrine (NE, 6 mmol/l). Once stedy stte ws chieved, vsodiltion responses were evluted by cumultive concentrtion-response curves to () cetylcholine (ACh, 9 4 mmol/l) nd (b) sodium nitroprusside (SNP, 9 4 mmol/l). (c) Five continuous sections from ech poe / mice (n = 4 mice from ech group) were costined with terminl deoxynucleotidyl trnsferse-medited dutp-biotin nick end lbeling (TUNEL) (poptotic cells, green), nti-cd3 (endothelil cells, red) nd 4,6-dimidino-2-phenylindole (DAPI) (nuclei; blue). (d) The percentge of poptotic endothelil cells per totl endothelil cells. (e) Electron microscopy ws performed on thorcic orts using ultrthin sections nd exmined with Nikon EclipseE8 light microscope. Arrow shows endothelil cell (EC), IEL: internl elstic lmin. Scle br, 2 μm. Dt were shown s men ± SD. Differences between 2 groups were tested with Student s t-test. P <.5 versus ; P <.5 versus vehicle group. AAV, deno-ssocited viruses; AAV-, AAV-medited gene trnsfer; poe /, polipoprotein E null;, growth differentition fctor ; GFP, green fluorescent protein; SD, stndrd devition. peptide- (MCP-), interferon (IFN)-γ, but incresed the levels of IL- in orts (P <.5). induced the prolifertion nd inhibited plmitic cid-induced poptosis of endothelil cells in vitro As is known, high-ft diets re commonly used in rodents to induce obesity, increse serum FFA nd induce lipotoxicity in vrious orgns. High levels of circulting FFA re ble to induce inflmmtion effect nd impir endothelil function. 22 Our niml results lso showed tht serum FFA incresed significntly in control group fed with HFD compred with norml chow. Therefore, PA ws used to tret cells in the in vitro experiments. As shown in bove, AAV- nd cn protect ginst endothelil injury in mice fed with HFD. Next, we questioned whether endothelil cells cn benefit from tretment in vitro. Firstly, the prolifertion of MAECs mesured by EdU-kit did not differ mong the vehicle, nd SB43542 ( μmol/l, TβR-Smd2/3 inhibitor) groups t -dy nd 3-dy time points (Dt not shown). However, 5-dy tretment of MAECs with incresed their prolifertion by 37.2% s compred with tht of vehicle, but not in the presence of SB43542 (see Supplementry Figure S5,b). Secondly, With regrd to the effect of on the migrtion of MAECs nd mcrophges, no significnt differences were found in the cell numbers mong the vehicle, nd SB43542 groups (see Supplementry Figure S5c e). Moleculr Therpy vol. 24 no. nov

5 nd Atherosclerosis The Americn Society of Gene & Cell Therpy b Lesion re % en fce re AAV- c e CD9 CD68 Collgen α-sma AAV- AAV- AAV- f d α-sma positive stining re CD68 positive stining re Lesion re % cross section AAV- AAV- AAV- Collgen content % of plque re CD9 positive stining re AAV- AAV- g Reltive mrna levels IL-6 IL-β TNF-α MCP- INF-γ IL Figure 3 AAV- nd ttenuted therosclerotic lesion formtion, meliorted plque components nd inflmmtory cytokines in orts of poe / mice. () Oil red O stining of the whole ort (n = 4 mice in ech group) ws performed to nlysis the en fce therosclerotic lesions re. (b) Quntittive nlysis of. (c) Hemtoxylin nd eosin stining (n = 4 mice in ech group) ws performed to quntify luminl crosssectionl re involved by therosclerotic plque. (d) Quntittive nlysis of c. (e) Immunohistochemicl stining of α-sma, nti-cd68, nd nti- CD9 were performed to nlysis the VSMCs, mcrophges nd T lymphocytes expression levels. Scle br, 2 μm. Msson s trichrome stining ws performed to quntifiction of collgen content. Scle br, μm. (f) Quntittive nlysis of e. (g) mrna expression levels of IL-6, IL-β, TNF-α, MCP-, IFN-γ, nd IL- mesured by quntittive rel-time polymerse chin rection (PCR) in the bdominl ort (n = 6 mice in ech group) nd expressed reltive to β-ctin. Dt were shown s men ± SD. Differences between two groups were tested with Student s t-test. P <.5 versus AAV- GFP; P <.5 versus vehicle group. AAV, deno-ssocited viruses; AAV-, AAV-medited gene trnsfer; poe /, polipoprotein E null;, growth differentition fctor ; GFP, green fluorescent protein; TNF-α, tumor necrosis fctor-α; IL, interleukin; IFN-γ, interferon-γ; VSMCs, vsculr smooth muscle cells; SD, stndrd devition. Before the next experiment, we explored the time- nd dose-dependent of PA-induced vibility by MTT. As shown in Supplementry Figure S6, we chose PA.4 mmol/l nd 24 hours s the optimum concentrtion nd time condition in the following study. Compred with the vehicle group, the percentge of poptotic MAECs in PA group ws significntly incresed, nd the effect ws blocked by cotretment with (Figure 4,b). Consistent with these findings, we lso found tht significntly decresed the poptotic proteins (cleved-cspse-3 nd bx) expression nd incresed the ntipoptotic protein (bcl-2) expression in PA treted MAECs (Figure 4c,d). reduced PA-induced inflmmtory cytokines expression in RAW264.7 mcrophges It hs been shown tht mcrophges represent key cellulr component of innte immunity, which hs been shown to promote therosclerosis initition nd progression. 23 Previous study showed tht HFD significntly induced the mrna expression of inflmmtory fctors in peritonel mcrophges in 93 vol. 24 no. nov. 26

6 The Americn Society of Gene & Cell Therpy nd Atherosclerosis 4 4 PA Propidium iodide PA PAL-NAME 4 4 PASIS Apototic cells (%) PA (.4 mmol/l) (5 ng/ml) L-NAME (5 µmol/l) SIS3 (3 µmol/l) Bcl-2/Bx rtio.5.5 Annexin V-FITC b c d 2 Bcl-2 Bx β-ctin PA (.4 mmol/l) (5 ng/ml) L-NAME (5 µmol/l) SIS3 (3 µmol/l) Cleved-cspse 3 β-ctin PA (.4 mmol/l) (5 ng/ml) L-NAME (5 µmol/l) SIS3 (3 µmol/l) Figure 4 reduced the plmitic cid-induced poptosis of MAECs. () Representtive imges of poptotic cells stined with nnexin-v-fitc nd propidium iodide in MAECs pretreted with or without L-NAME (5 μmol/l) or SIS3 (3 μmol/l) for 3 minutes before tretment with (5 ng/ml) for 3 minutes, then treted with PA (.4 mmol/l) or vehicle for 24 hours. (b) The percentge of poptotic cells. (c nd d) Expression of Bcl-2, Bx, nd cleved-cspse 3 in MAECs were determined by western blot with the bove tretments in comprison to β-ctin. Dt were shown s men ± SD. Anlysis of vrince (ANOVA) followed by LSD t-test ws used to compre the differences mong different groups. Ech experiment repeted five times. P <.5., growth differentition fctor ; MAECs, mice ortic endothelil cells; SD, stndrd devition; LSD, lest significnt difference. poe / mice. 24 Therefore, next we questioned whether meliorted PA-induced inflmmtory cytokines expression levels of IL-6, TNF-α, MCP-, nd IL- in RAW264.7 mcrophges. Similrly, we explored the time- nd dose-dependent responses of PA-induced inflmmtion (evluted by IL-6 nd TNF-α production) by enzyme-linked immunosorbent ssy. As shown in Supplementry Figure S6b,c, we chose PA.4 mmol/l nd 6 hours s the optimum concentrtion nd time condition for mcrophges incubtion in the following study. After stimulting 6 hours, significntly ttenuted PA-induced inflmmtion response (IL-6, TNF-α, MCP-, nd IL-). However, Pretretment with SB43542 significntly ttenuted the inhibitory effects of on PA-induced inflmmtory response (see Supplementry Figure S7). The possible signling mechnisms of protective effects of on therosclerosis in vivo nd in vitro In the lst experiments, we explored the possible signling mechnisms of protective effects of on therosclerosis in vivo nd in vitro. Firstly s one member of the TGF-β superfmily, binds to specific receptors with serine threonine kinse ctivity, leding to intrcellulr signling through the ctivtion of the cnonicl pthwy Smd 2/3. To explore whether Smd 2/3 is involved in the protective effects of AAV- nd on rtery in our study, proteins extrcted from orts were nlyzed by Western blot. As shown in Figure 5,b, both AAV- nd tretments significntly incresed Smd2/3 phosphoryltion cscde (P <.5). In the in vitro experiments, treting MAECs nd RAW264.7 cells with (5 ng/ml) ctivted the cnonicl TGF-β/Smd signling pthwy, reveled by n increse in the Smd2/3 phosphoryltion cscde, wheres this effect could be blocked by coincubtion with SB43542 (Figure 5c f). Secondly, we explored the noncnonicl signling pthwy of in our study. In the in vivo experiment, proteins extrcted from thorcic orts fter 2 weeks of or others tretments were nlyzed by Western blot, nd the results showed tht AAV- nd tretments significntly incresed enos phosphoryltion when compred with the AAV- GFP or vehicle groups (Figure 6,b). Nitric oxide (NO), produced by enos, plys pivotl role in regulting endothelil cell function, poptosis, nd exhibits thero-protective effects. 25,26 Accordingly, AAV- nd tretments significntly incresed the NO levels compred with the or vehicle groups (see Supplementry Figure S8), which re consistent Moleculr Therpy vol. 24 no. nov

7 nd Atherosclerosis The Americn Society of Gene & Cell Therpy P-Smd2/3 Smd2/3 AAV- b Reltive P-Smd2/Smd AAV- Reltive P-Smd3/Smd AAV- c TGF-β inhibitor d p-smd2/3 DAPI/ P-Smd2/3 % p-smd2/3 cells SB43542 e f p-smd2/3 DAPI/ P-Smd2/3 % p-smd2/3 cells Figure 5 ctivted TGF-β/Smd pthwys in vivo nd in vitro. () Expression of Smd2/3 nd phosphorylted Smd2/3 in the orts of poe / mice were determined by western blot 2 weeks post or others tretments. (b) Quntittive nlysis of. Dt were shown s men ± SD. Differences between two groups were tested with Student s t-test. n = 3 mice in ech group. P <.5 versus ; P <.5 versus vehicle group. (c nd e) Representtive imges of the percentge of phosphorylted-smd2/3 cells in c MAECs nd e RAW mcrophges treted with either (5 ng/ml) for 3 minutes or pretreted with SB43542 ( μmol/l) for 3 minutes, nd then treted with (5 ng/ml) for 3 minutes. (d nd f) Quntittive nlysis of c nd e. Dt were shown s men ± SD. Anlysis of vrince (ANOVA) followed by LSD t-test ws used to compre the differences mong different groups. Ech experiment repeted five times. P <.5 versus vehicle; P <.5 versus group. AAV, deno-ssocited viruses;, AAV-green fluorescent protein;, growth differentition fctor ; MAECs, mice ortic endothelil cells; SD, stndrd devition; TGF-β, trnsforming growth fctor; LSD, lest significnt difference. with enos phosphoryltion levels in orts of four groups. The phosphoryltion of enos cn be regulted by severl kinses. 27 Thus, we exmined the chnges in the levels of camp-dependent protein kinse (PKA), denosine monophosphte-ctivted protein kinse (AMPK) nd Akt/protein kinse B in orts. AAV- nd tretments for 2 weeks incresed AMPK phosphoryltion when compred with the or vehicle groups, but did not influence the protein expression of PKA nd Akt (Figure 6,b). We next verified this noncnonicl signling pthwy in cultured MAECs. First of ll, the time-course of phosphorylted endothelil nitricoxide synthse (P-eNOS) expression in vitro showed tht ctivted P-eNOS expression fter 5 minutes tretment (5 ng/ml), reched the highest P-eNOS expression fter 3 minutes, reduced P-eNOS expression fter 36 hours tretment (see Supplementry Figure S9). Secondly, we found incubtion of MAECs with 5 ng/ml for 3 minutes incresed AMPK nd enos phosphoryltion. However, the ctivtory effects of on AMPK nd enos phosphoryltion were blocked Compound C (n AMPK inhibitor) or L-NAME (n enos inhibitor) (see Supplementry Figure S9b,c). As expected, the expressions of PKA nd Akt did not differ mong different tretment groups (see Supplementry Figure S9d,e). These findings indicte tht could ctivte AMPK/eNOS signling pthwy. Next, we mesured the production of NO in culture medi nd found significntly incresed NO synthesis. However, the -induced increse in NO production ws ttenuted in the presence of Compound C or L-NAME (see Supplementry Figure S8b). More importntly, pretretment MAECs with L-NAME or SIS3 ( Smd3 inhibitor), significntly ttenuted the inhibitory effects of on PA-induced endothelil poptosis (Figure 4,b). This suggested tht the ntipoptotic effect of on MAECs my through the TGF-β/Smd nd AMPK/eNOS pthwys. Thirdly, pretretment with significntly inhibited PA-induced phosphoryltion of c-jun N-terminl kinse (JNK) nd nucler fctor-kpp B (NF-κB) p65 nucler trnsloction, but hd no vol. 24 no. nov. 26

8 The Americn Society of Gene & Cell Therpy nd Atherosclerosis P-PKA PKA P-Akt Akt AAV b Reltive P-PKA/PKA AAV- Reltive P-Akt/Akt AAV- P-AMPK AMPK P-eNOS e-nos Reltive P-AMPK/AMPK AAV- Reltive P-eNOS/eNOS AAV- c d e P-ERK/2 ERK/2 P-JNK JNK NF-kB P65 nucler trnsloction PA (.4 mmol/l) (5 ng/ml) SB43542 ( µmol/l) PA (.4 mmol/l) (5 ng/ml) SB43542 ( µmol/l) PA (.4 mmol/l) (5 ng/ml) SB43542 ( µmol/l) Figure 6 ctivted AMPK/eNOS signling in vivo nd inhibited inflmmtion signling pthwys in RAW264.7 mcrophges. () Expression of PKA, P-PKA, Akt, P-Akt, AMPK, P-AMPK, enos, nd P-eNOS in the orts of poe / mice were determined by western blot 2 weeks post AAV- or others tretments. (b) Quntittive nlysis of. Dt were shown s men ± SD. Differences between two groups were tested with Student s t-test. n = 3 mice in ech group. P <.5 versus ; P <.5 versus vehicle group. (c-e) Expression of ERK/2, P-ERK/2, JNK, P-JNK, nd NF-κB P65 nucler trnsloction levels in RAW264.7 mcrophges were determined by western blot. Cells were pretreted with SB43542 for 3 minutes, nd then treted with (5 ng/ml) for hour followed by stimultion with PA (.4 mmol/l). Dt were shown s men ± SD. Anlysis of vrince (ANOVA) followed by LSD t-test ws used to compre the differences mong different groups. Ech experiment repeted five times. P <.5. AAV, deno-ssocited viruses;, AAV-green fluorescent protein;, growth differentition fctor ; SD, stndrd devition; AMPK, denosine monophosphte-ctivted protein kinse; P-eNOS, phosphorylted endothelil nitricoxide synthse; ERK/2, extrcellulr signl-regulted kinse/2; JNK. c-jun N-terminl kinse; NF-κB, nucler fctor-kpp B; LSD, lest significnt difference; PA, plmitic cid. effect on PA-induced phosphoryltion of extrcellulr signlregulted kinse/2 (ERK/2) in RAW264.7 mcrophges. The ddition of SB43542 significntly ttenuted the inhibitory effects of on PA-induced phosphoryltion of JNK nd NF-κB p65 ctivtion (Figure 6c e). These results indicte tht could reduce inflmmtory cytokines in mcrophges vi inhibition of intrcellulr inflmmtory signling, including JNK nd NF-κB p65. Humn studies Circulting /8 is positively relted with endothelium-dependent vsodiltion in overweight subjects. The clinicl chrcteristics nd biochemicl dt of the control nd overweight subjects were summrized in online Supplementry Informtion (see Supplementry Tble S3). Compred with the control subjects, circulting /8 levels were significntly lower (P =.2) in overweight subjects. By dividing the distribution of flow-medited endothelium-dependent rtery dilttion (FMD) in overweight subjects, compred with subjects in the lowest qurtile of FMD levels, those in the highest hd significntly higher /8 concentrtions (P for trend <.) (see Supplementry Tble S4). Moreover, the /8 levels in the qurtile 3 nd qurtile 4 were significntly higher s compred with those in the qurtile fter djustment for ge nd body mss index (Figure 7). Multiple stepwise regression nlysis showed tht /8, ge, body mss index, low density lipoprotein cholesterol, FFA, c-rective protein, homeostsis model ssessment of insulin resistnce nd fsting insulin were found to be independently ssocited with FMD levels (ll P <.5) (see Supplementry Tble S5). The correltion nlysis showed tht circulting /8 levels were ssocited with FMD levels (r =.452, P <.) (Figure 7b). In ddition, circulting /8 concentrtions declined with ge (Figure 7c) (P <. for trend). By logistic regression nlysis the odds rtio for lower Moleculr Therpy vol. 24 no. nov

9 nd Atherosclerosis The Americn Society of Gene & Cell Therpy 25 b 3 r =.452 c Serum /8 (pg/ml) Qurtile Qurtile2 Qurtile3 FMD (%) Qurtile4 Serum /8 (pg/ml) FMD (%) Serum /8 (pg/ml) Ages (yer) Figure 7 Circulting /8 is positively relted with endothelium-dependent vsodiltion in overweight subjects. () Circulting /8 concentrtions in different qurtiles of FMD levels in overweight subjects. Dt were shown s men ± SD. Anlysis of vrince (ANOVA) followed by LSD t-test ws used to compre the differences mong different groups. P <. versus Qurtile. (b) Correltion nlysis to evlute correltion of circulting /8 with FMD in overweight subjects. Correltion ws performed using Person s method. (c) The chnges of plsm /8 with ges in this study group. The /8 levels declined with ge (P <. for trend). ANOVA followed by LSD t-test ws used to compre the differences mong different groups., growth differentition fctor ; SD, stndrd devition; FMD, flow-medited endothelium-dependent rtery dilttion; LSD, lest significnt difference. FMD levels ws reduced by 4.4% per pg/ml increse in the /8 concentrtion (OR (95% CI);.856 ( )) (see Supplementry Tble S6). DISCUSSION The mjor finding of this study ws tht (i) AAV- nd exogenous recombinnt improved endothelil injury nd reduced therosclerosis lesion formtion in poe / mice fed HFD; (ii) AAV- nd exogenous recombinnt enhnced mture plque stbility vi incresing VSMCs nd collgen content nd reducing mcrophges, MMPs ctivities nd inflmmtory cytokine expression; (iii) the moleculr mechnisms underlying these beneficil effects my involve the ctivtion of the cnonicl Smd2/3 pthwy nd the noncnonicl signling pthwys including ctivtion of AMPK/eNOS nd inhibition of inflmmtion pthwys; nd (iv) circulting /8 levels re positively ssocited with FMD in mle overweight subjects. To the best of our knowledge, our study hs provided for the first time tht AAV-medited gene trnsfer nd systemic dministrtion of hve protective role ginst therosclerosis progression. It is estblished tht nd GDF8 shred 9% protein sequence homology. Accordingly, recent studies from Egermn et l. nd Smith et l. reported tht key regents tht recognized lso recognize GDF8. 7,8 Egermn et l. reported tht they developed -specific immunossy nd found trend towrd incresed levels in serum of both ged rts nd humns by Western blot. However, other dt showed tht the increse of serum in mice reported by Egermn et l. is not but predominntly immunoglobulin light chin (the ~25 kd bnd by Western blot), immunoglobulins hve long been known to increse with ge in C57BL/6 mice. 9 They lso found tht circulting /8 levels decline with ge in multiple mmmlin species. In this study, we lso found circulting /8 levels decline with ge in mice nd in mle overweight subjects. Recently, clinicl study showed tht mrkedly reduced risk of incident hert filure hospitliztion, stroke, myocrdil infrction, nd ll-cuse deth in those with higher circulting /8 levels in two independent cohorts cross two continents totling,899 subjects with stble CHD. 2 In niml studies, Loffredo et l. demonstrted the ntihypertrophic effect of in mice by showing tht dministrtion of. mg/kg/dy of for 28 dys reduced crdic mss in ged mice. 4 However, Smith et l. rgued tht dily injections of biologiclly ctive r for 28 dys hd no effect on overll crdic structure nd crdic pump function in 24-month-old C57BL/6 mice. 8 To further chrcterize the ntihypertrophic effect of, Loffredo et l performed dose titrtion study in young nd ged mice nd observed vol. 24 no. nov. 26

10 The Americn Society of Gene & Cell Therpy nd Atherosclerosis significnt dose-dependent decrese of hert weight, normlized to tibi length, fter only 9 dys of tretment in mice receiving.5 or. mg/kg/dy. 9 In this study, we found tht improved endothelil dysfunction, decresed endothelil poptosis in vivo nd in vitro, nd reduced therosclerosis plque including the en fce nd cross section res s well s enhnced mture plque stbility in poe / mice. Moreover, our humn study showed tht circulting /8 levels re positively ssocited with FMD in mle overweight subjects. Tken together, the dt indicted tht AAV- nd exogenous recombinnt cn improve endothelil injury nd reduce therosclerosis lesion formtion. No significnt differences on body weight, blood pressure, fsting blood glucose, nd HbAc were found mong the four groups fter 2 weeks of intervention, indicting tht the protection of rtery contributed by AAV- nd exogenous recombinnt cnnot be explined by these clssic crdiovsculr risk fctors lone. The mechnism of AAV- nd protecting ginst endothelil injury nd reducing therosclerotic lesion formtion in poe / mice my underlie the following spects. A previous study reported tht tretment incresed primry brin cpillry endothelil cells prolifertion by 22.9% s compred with controls. 6 Next, we questioned the effects of AAV- nd exogenous recombinnt on endothelil cell, which is crucil during therosclerotic lesion formtion. 22 The present niml studies demonstrted tht AAV- nd exogenous recombinnt tretment llevited the impirment of endothelium-dependent vsodiltion induced by HFD, decresed the poptosis of endothelil cells nd so tht preserved the endothelil cell coverge on the plque surfces. Consistent with the in vivo dt, our in vitro experiments showed tht significntly reduced the poptosis in PA treted MAECs. Recently, nother study showed tht hd no effect on prolifertion nd migrtion of humn umbilicl vein endothelil cell nd rt ortic endothelil cells fter 72 hours of tretment. 28 We lso found hd no effect on the migrtion of MAECs nd mcrophges, however, we found significntly promoted MAECs prolifertion fter 5 dys of tretment, which mkes endothelil cells hve certin bility to self-repir. The discrepncy my be due to different tretment time. Thus, bsed on these dt, we cn suggest tht AAV- nd recombinnt could protect ginst endothelil injury, consequently meliorte therosclerosis. Atherosclerosis is considered s n inflmmtory disese nd inflmmtory responses medited by inflmmtory cytokines re prticipted nd considered importnt in ll stges of therosclerosis. 29 This study showed tht elevted levels significntly reduced the mrna expression levels of inflmmtory meditors nd incresed ntiinflmmtory (IL-) cytokines in orts nd decresed plsm inflmmtory cytokines in HFD fed mice, s well s ttenuted inflmmtory rection induced by PA in RAW264.7 mcrophges. Therefore, the nti-inflmmtion of my lrgely contribute to the decresed number of mcrophges nd T-lymphocytes in the plque tissue nd its protective effect of rtery. Previous studies found tht inflmmtion results in insulin resistnce, nd the improvement of insulin sensitivity llevites endothelil dysfunction nd therosclerosis. 3,3 In this study, glucose nd insulin tolernce tests in poe / mice fed by HFD displyed impirment in both glucose tolernce nd insulin sensitivity, nd AAV- nd exogenous tretment for 2 weeks significntly improved insulin nd glucose tolernce when compred with the nd vehicle (refer to intrperitonel glucose tolernce test nd insulin tolernce test results), suggesting tht AAV- nd recombinnt cn improve insulin resistnce in poe / mice fed by HFD. As result, the fsting insulin concentrtions in AAV- nd recombinnt tretment groups decresed significntly. Therefore, the improvement of glucose tolernce nd insulin sensitivity cn prtilly explin the endothelium-protective s well s ntitherosclerotic effects of AAV- nd recombinnt. Pthologicl studies hve demonstrted tht not only the lesion size but lso the plque components ply key role in the development of cute coronry syndrome nd rteriosclerosis. 32 Vulnerble plque is chrcterized by incresed contents of mcrophges nd T lymphocytes nd reduced contents of collgen nd VSMCs. Our dt suggested tht one of the key mechnisms by which AAV- nd exogenous elicits plque stbiliztion is decresing the number of plque-infiltrting mcrophges, becuse mcrophge lysis in dvnced lesions hs been involved in the genertion of necrotic cores, which promote plque instbility. 2,33 In ddition, the loss of VSMCs in the fibrous cp represents criticl mechnism in trnsforming stble plque into rupture-prone lesions. This study showed tht AAV- nd recombinnt tretment showed tendency to increse the number of α-sma-positive cells in the fibrous cp within the therosclerotic plques in poe / mice, which is relevnt for plque stbility. Tken together, sustined in vivo expression of endogenous or injection not only drmticlly ttenuted the totl extension of the plques but lso contributed to stbilize therosclerotic plques by selectively decresing in mcrophges nd T lymphocytes nd substntil incresing in collgen nd VSMCs in the ortic plques. Moreover, mcrophges in plques my secrete proteolytic enzymes, such s MMPs, which my digest the extrcellulr mtrix nd weken the fibrous cp. 34 Indeed, AAV- nd recombinnt tretment significntly inhibited MMP-2 nd MMP-9 protein expression in plques in this study. Thus, there is possible direct link between the decresed of mcrophges nd MMPs nd incresed of collgen s well s VSMCs in these lesions, indicting tht is powerful cytokine for ltering plque components towrd stble plque phenotype. Next, we questioned the moleculr signl pthwys for the protective effect of on rtery. It is well known tht exerts its function by intercting with ctivin type IIA nd IIB receptors 2 nd type I receptor Alk5 (ref. 3) to induce cnonicl Smd signling pthwy. This study showed tht AAV- nd exogenous ctivted phosphoryltion of Smd 2/3 in orts of poe / mice nd consequently induced less plques compred with the nd vehicle. The in vitro experiments showed tht enhnced the phosphoryltion of Smd 2/3 in MAECs nd RAW mcrophges, nd the ntipoptotic effects of could be ttenuted by the Smd3 inhibitor. In ddition, TGF-β nd BMP receptor ctivtion results in ctivtion Moleculr Therpy vol. 24 no. nov

11 nd Atherosclerosis The Americn Society of Gene & Cell Therpy of severl other non-smd signling pthwys in context-dependent mnner. Non-Smd signling pthwys cn involve the ERK/2, JNK, nd the Akt pthwy, nd these cn crosstlk with the Smd pthwys. 35 This study showed tht improved endothelil dysfunction by incresing the phosphoryltion of AMPK nd enos nd consequently incresed serum NO concentrtions in poe / mice. Consistent with the niml experiments, incresed the phosphoryltion of AMPK nd enos, nd production of NO in cultured MAECs, wheres the specific inhibitor of AMPK or enos, such s Compound C or L-NAME, ttenutes these effects of. Furthermore, pretretment with L-NAME significntly ttenuted the inhibitory effects of on endothelil poptosis. Of note, previous studies suggested tht the control of dysregulted mitogen-ctivted protein kinse cscde nd NF-κB ctivtion could be potentil trget in the tretment of therosclerosis. 36,37 This study further showed tht significntly inhibited PA-induced ctivtion of JNK nd NF-κB nd consequently inhibited the inflmmtory cytokines expression in RAW264.7 mcrophges. Overll, our dt suggest tht protects rtery through the cnonicl (Smd2/3) nd noncnonicl (AMPK/eNOS, JNK, nd NF-κB) signling pthwys. Due to the low toxicity nd efficient nd long-term trnsduction in vivo, AAV vectors re currently evluted in mny nimls nd clinicl studies. 38,39 Intriguingly, AAV vector genome relized liver trnsduction nd subsequent relesed into the circultion, which is the most likely mechnism. This study demonstrtes successful in vivo trnsduction vi AAV2 vector which fcilitted sustined overexpression nd obtined similr results on ntitherosclerotic effect compred with repeted intrperitonel injection of recombinnt, strongly suggesting tht AAV represents potentil lterntive to recombinnt protein injection. Some limittions should be mentioned here. Firstly, myosttin (GDF8) is close structurl homologue of, with 9% mino cid sequence identity shred in their mture ctiveforms. 4 Thus, our ssy for mouse nd humn serum does not distinguish circulting nd GDF8. As result, we did not ccurtely determine the chnges of in mice nd in humn. Secondly, since ltertions of circulting monocytes nd lymphocytes might ffect therosclerotic plque formtion directly. However, in this study, we did not mesure the circulting cells in mice, such we cnnot evlute the chnges of circulting cells before nd fter intervention. Thirdly, we did not explore the Akt/mmmlin trget of rpmycin (mtor) pthwy, becuse some studies showed tht Akt/mTOR pthwy ws involved in crdiovsculr diseses. 4 Fourthly, the number of study subjects in humn is reltively smll. It is difficult to exclude bis in the results, which should be confirmed in lrge studies. In conclusion, AAV- nd recombinnt llevited therosclerotic lesions formtion vi protecting ginst endothelil injury, decresing plque-infiltrting inflmmtory cells nd lleviting inflmmtion rections. The signl mechnisms underlying these effects my involve the cnonicl (TGF-β/Smd) nd noncnonicl (AMPK/eNOS, JNK, nd NF-κB) signling pthwys. Thus, AAV- or recombinnt my open new therpeutic strtegy for treting therosclerosis. MATERIALS AND METHODS AAV- construct. The cdna (GenBnk ccession number NM_272.) ws obtined by reverse trnscriptse-polymerse chin rection mplifiction. The cdna ws inserted into the AAV2 vector plsmid psnav under the control of the constitutive cytomeglovirus promoter to construct psnav/. The psnav/ ws trnsfected into 293 T-cells nd lrge-scle raav production nd purifiction were described previously. 42 Virl preprtions used for niml trnsduction hd titers between 2 nd 3 virl genome prticles per ml. Animls. The experiments conformed to the Ntionl Institutes of Helth Guidelines for the Use of Lbortory Animls. All niml experimentl protocols were pproved by the niml ethics committee of the Wuhn Generl Hospitl of Gungzhou Commnd. One hundred nd two mle 4-week-old poe / mice (The Jckson Lbortory, Br Hrbor, ME) were housed in specific-pthogen-free environment with 2-hour light/drk cycle, nd unrestricted ccess to wter. Of those mice 25 were selected rndomly s norml chow control group, fed with norml chow from 4-week-old to the end of the study, the rest of those mice were fed with HFD (45% kcl ft, 35% kcl crbohydrtes, nd 2% kcl protein) from 4-week-old to the end of the study. Of those, 25 mice fed with HFD were selected rndomly s HFD control group. The HFD control group nd the norml chow control group were just for the evlution of weight, FFA nd serum /8 t different time points (8,, 4, 8, nd 2 weeks) (see Supplementry Figure Sb-d) nd 5 mice were killed t ech time point. After 8 weeks of HFD, the rest 52 mice were rndomly divided into four groups: AAV- nd tretment groups (received single injection of 25 ul purified AAV- or 25 ul through the til vein), vehicle nd recombinnt tretment groups (received dily intrperitonel dministrtion of ph = 3, 25 ul citrte buffer or 25 ul citrte buffer contining (. mg/kg, PeproTech, Rocky Hill, NJ)). 4 After 4 weeks of tretment, three mice from ech group were nesthetized by n intrperitonel injection of pentobrbitl sodium (6 mg/kg body weight) nd killed for the mesurements of endothelil function nd serum /8 in ll groups nd the expression of livers nd orts in AAV- nd groups. After 2 weeks of tretment, the rest of nimls (n = mice in ech group) were nesthetized by the sme wy nd killed for blood tests nd histologicl exmintion. Glucose nd insulin tolernce test. Intrperitonel glucose tolernce test nd insulin tolernce test were performed before dy nd 2 weeks fter different interventions in 4 groups (n = 3 mice before tretment nd n = mice fter tretment) ccording to our previous report. 43 Endothelil function ssessment. The thorcic orts were hrvested under the stereo-microscope (Olympus, Tokyo, Jpn) nd cut into 4 mm rings with specil cre to preserve the endothelium. The endotheliumdependent nd endothelium-independent vsodiltion responses were mesured ccording to our previous report. 43 Atherosclerosis nlysis nd immunohistochemistry. To evlute the therosclerotic lesions, two complementry pproches (en fce whole nd histologicl section nlyses) were performed ccording to our previous report. 43 After tretment for 2 weeks, whole ort extending from the scending ort to the bdominl bifurction ws opened longitudinlly nd stined with Oil-red-O (Sigm-Aldrich, St.Louis, MO) to nlyze the en fce therosclerotic lesions re (n = 4 mice in ech group). To quntify luminl cross-sectionl re involved by therosclerotic plque, sections obtined every 2 μm from the ortic rch (n = 4 mice in ech group) were stined with hemtoxylin nd eosin. For detection of trgeted protein expression levels in ll groups, the immunohistochemicl nlysis ws used in seril plque sections from the ortic rch (n = 4 mice in ech group). The immunohistochemistry ssys for CD68, CD9, α-smooth muscle ctin, MMP-2 nd MMP-9 were shown in online Supplementry Informtion vol. 24 no. nov. 26

12 The Americn Society of Gene & Cell Therpy nd Atherosclerosis Anlysis of poptosis in vivo. According to our previous report, 43 endothelil cell poptosis in thorcic orts ws detected by double stin with terminl deoxynucleotidyl trnsferse-medited dutp-biotin nick end lbeling nd nti-cd3. Electron microscopy ws performed on thorcic segments using ultrthin sections nd exmined with Nikon EclipseE8 light microscope (Nikon, Tokyo, Jpn). Cell cultures. MAECs for in vitro experiments were isolted from orts of poe / mice (6 8 weeks of ge) s described. 44 RAW264.7 cells were purchsed from the Cell Bnk of the Chinese Acdemy of Sciences (Shnghi, Chin). The detils for cell cultures were vilble in online Supplementry Informtion. Cell signling nlysis. For psmad ssys, MAECs nd RAW264.7 cells were divided into three tretment groups: vehicle-treted group, -treted group (5 ng/ml) nd SB43542 ( μmol/l). Cells were pretreted with SB43542 for 3 minutes, nd then treted with for 3 minutes. For P-eNOS ssys, MAECs were divided into five groups nd pretreted with inhibitors ginst AMPK (Compound C, 5 μmol/l, Compound C group) or enos (L-NAME, 5 μmol/l, L-NAME group) or both of them for 3 minutes before (5 ng/ml) ws dded. After cultured for 3 minutes, the protein expression levels of P-AMPK, AMPK, P-eNOS, enos, P- PKA, PKA, P-Akt, nd Akt were ssessed by Western blot. Inflmmtory nlysis. RAW264.7 cells were divided into four groups: vehicle-treted group, PA-treted group, PA -treted group (5 ng/ml), nd PA SB43542 ( μmol/l) group. Cells were pretreted with TβR inhibitor for 3 minutes, nd then treted with for hour followed by stimultion with PA (.4 mmol/l). After tretment for 3 minutes, the protein expression levels of P-ERK/2, ERK/2, P-JNK, JNK, nd NF-κB p65 nucler trnsloction were ssessed by Western blot. After the sme tretment for 6 hours, culture medi levels of IL-6, TNF-α, nd MCP- were determined using enzyme-linked immunosorbent ssy ccording to the mnufcturer s instructions (R&D Systems, Minnepolis, MN). The ssessments of other prmeters. The Body weight, blood pressure nd blood biochemicl nlysis, glucose nd insulin tolernce test for mouse, poptosis nlysis, immunohistochemistry, NO mesurement, rel time polymerse chin rection, Western blot nlysis, prolifertion nd migrtion nlysis in vitro re described in online Supplementry Informtion. Humn studies Subjects. From July 24 to July 25, totl of 4 successive newly dignosed Chinese mle overweight subjects from Wuhn re (ged 3 65 yers, men 48.2 ± 9.5), who referred to our hospitl for helth exmintion, were selected rndomly in this study. Overweight subjects were defined s body mss index 25, nd <3 kg/m 2 (WHO criteri). The detils for inclusion nd exclusion criteri were shown in online Supplementry Informtion. Biochemicl mesurements. Blood smples were obtined from prticipnts fter 2-hour fst. Aliquots of serum nd plsm were stored t 8 C nd were not thwed until nlyzed. Serum /8 concentrtions were mesured in duplicte by using the enzyme-linked immunosorbent ssy kits (Elbscience, Wuhn, Chin), in ccordnce with the mnufcturer s instructions. The sensitivity of the ssy ws pg/ml nd the liner rnge of the stndrd ws 5.625, pg/ml. The intr- nd interssy coefficients of vrition were <%. The detils for other biochemicl prmeters were shown in online Supplementry Informtion. Ultrsound study of the brchil rtery. The vsculr studies of the brchil rtery were performed noninvsively in the AM following controlled diet, s described in our previous publictions. 45,46 Sttisticl nlysis. Dt re expressed s men ± SD. Differences between two groups were tested with unpired Student s t-test. Dt of multiple groups were compred using one-wy nlysis of vrince followed by lest significnt difference t-test. A P-vlue <.5 ws considered sttisticlly significnt. All nlyses were performed with SPSS 2. (IBM, Somers, NY). The sttisticl methods for humn study were shown in online Supplementry Informtion. SUPPLEMENTARY MATERIAL Figure S. influenced the body weight, serum free ftty cids nd /8 levels in mice fed with norml chow or high-ft diet. Figure S2. AAV- nd improved glucose tolernce in poe-/- mice. Figure S3. improved insulin tolernce in poe-/- mice. Figure S4. AAV- nd reduced mtrix metlloproteinse-2 (MMP-2) nd MMP-9 protein expression in plques. Figure S5. induced the prolifertion of MAECs but hd no effect on the migrtion of MAECs nd mcrophges. Figure S6. Identifiction of the optimum incubtion condition in cell experiments. Figure S7. reduced plmitic cid (PA)-induced inflmmtory cytokines expression in RAW264.7 mcrophges. Figure S8. promoted nitric oxide (NO) production in vivo nd in vitro. Figure S9. ctivted AMPK-eNOS signling in cultured MAECs. Tble S. Body weight nd blood pressure levels t week nd 2 week of the experiments in poe/ mice. Tble S2. The metbolic chrcteristics t the end of the experiments in poe/ mice. Tble S3. Clinicl nd biochemicl chrcteristics in control nd in overweight subjects. Tble S4. Clinicl nd biochemicl chrcteristics in overweight subjects ccording to the qurtiles of FMD levels. Tble S5. Multiple stepwise regression nlysis (forwrd) with FMD level s dependent vrible. Tble S6. The odds rtio for the lower FMD levels ccording to /8 levels. Supplementry Informtion ACKNOWLEDGMENTS This work ws supported by Ntionl Nturl Science Foundtion of Chin (number nd number 85773). Gungd Xing performed the reserch design nd the clinicl experiment; Wen Mei conducted the niml experiment; Yixing Li, Hun Li, Min Liu, nd Lin Xing performed the in vitro experiment; Lingwei Xing conducted dt nlysis; Junyn Lu nd Jing Dong conducted the mnuscript writing. The uthors declre no competing finncil interests. References. 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