Dysregulation of HSG triggers vascular proliferative disorders

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872 NATURE CELL BIOLOGY VOLUME 6 | NUMBER 9 | SEPTEMBER 2004

Dysregulation of HSG triggers vascular proliferativedisorders Kuang-Hueih Chen1,2, Xiaomei Guo2, Dalong Ma3, Yanhong Guo1, Qian Li1, Dongmei Yang2, Pengfei Li1, XiaoyanQiu3, Shaojun Wen1, Rui-Ping Xiao1,2,4 and Jian Tang1

Vascular proliferative disorders, such as atherosclerosis and restenosis, are the most common causes of severe cardiovasculardiseases, but a common molecular mechanism remains elusive. Here, we identify and characterize a novel hyperplasiasuppressor gene, named HSG (later re-named rat mitofusin-2). HSG expression was markedly reduced in hyper-proliferativevascular smooth muscle cells (VSMCs) from spontaneously hypertensive rat arteries, balloon-injured Wistar Kyoto rat arteries, orApoE-knockout mouse atherosclerotic arteries. Overexpression of HSG overtly suppressed serum-evoked VSMC proliferation inculture, and blocked balloon injury induced neointimal VSMC proliferation and restenosis in rat carotid arteries. The HSG anti-proliferative effect was mediated by inhibition of ERK/MAPK signalling and subsequent cell-cycle arrest. Deletion of the p21ras

signature motif, but not the mitochondrial targeting domain, abolished HSG-induced growth arrest, indicating that rHSG-inducedanti-proliferation was independent of mitochondrial fusion. Thus, rHSG functions as a cell proliferation suppressor, whereasdysregulation of rHSG results in proliferative disorders.

Cell hyper-proliferation has long been considered as an important etio-logical factor of cardiovascular diseases and cancer. In particular, vascu-lar proliferative disorders, such as primary atherosclerosis, restenosisafter balloon angioplasty, vein-graft disease and coronary arteriosclero-sis are the most common causes of severe cardiovascular diseases, thecurrent leading cause of death in the United States and the predictednumber one killer worldwide by 2020 (ref. 1). Already, coronary arterydisease is the single largest cause of mortality in the Western world2.Although vascular smooth muscle cells are normally located primarilyin the arterial tunica media and maintained in a non-proliferative statein vivo, injury or mechanical stress of arteries causes migration ofVSMCs into the intima layer of the arterial wall, where the VSMCs pro-liferate and synthesize extracellular matrix proteins, resulting in expan-sion of the arterial intima3–5. This neointimal VSMC proliferationconstitutes a primary cause factor in vascular proliferative disorders3–5.

Mammalian cell proliferation is controlled by extracellular mito-genic stimuli that activate a variety of signalling events involved in theregulation of cell growth and division. Numerous growth factors,cytokines and bioactive peptides — for example, platelet-derivedgrowth factor (PDGF), basic fibroblast growth factor (bFGF),endothelin-1 (ET-1) and angiotensin II — have been implicated inVSMC proliferation6–9. These mitogenic stimuli share a final commonpathway of a proliferative signalling cascade: the cell cycle. The Rasproto-oncogene is a central component of mitogenic signalling path-ways, and is essential for cells to exit a quiescent state (G0) and to passthrough the G1–S transition of the cell cycle10,11. As one of the best

characterized signalling events downstream of Ras, the extracellular-signal-regulated kinase (ERK1/2) cascade — sequentially involvingRas, Raf, MEK1/2 and ERK1/2 mitogen-activated protein kinase(MAPK) — is activated in response to a multitude of mitogenic stim-uli, and drives cell-cycle progression through activation of cyclin-dependent kinases (CDKs). CDKs form holoenzymes with theirregulatory subunits, the cyclins, resulting in phosphorylation of thetumour suppressor protein Rb, and S-phase entry11,12. ERK1/2 sig-nalling is also necessary for the degradation or downregulation ofCDK inhibitors, such as p21waf1 (p21) and p27kip1 (p27), particularlyp27, thereby eliminating their growth-suppressive activities13–15.

Despite increasing knowledge about the cell-cycle regulation ofVSMCs4,16, the molecular mechanism governing VSMC proliferationremains largely elusive. Thus, identifying genetic modifiers of VSMCproliferation is a major focus in cardiovascular biology and medicine.Here, we report the identification and mechanistic characterization ofa gene exhibiting anti-proliferative effects, which was named hyperpla-sia suppressor gene (HSG). Rat HSG (rHSG) was recently renamed ratmitofusin-2 (rMfn-2) by GenBank in 2003, after the identification ofits human homologue, mitofusin-2 (Mfn-2)17. Several studies havedemonstrated that human Mfn-2 and its homologues localize to themitochondrial outer membrane and have an essential role in mito-chondrial fusion, thus regulating mitochondrial morphology andfunction in mammalian cells, yeast and flies17–21. In the present study,however, we demonstrate that rHSG also exhibits a profound anti-pro-liferative effect in vivo and in vitro. The anti-proliferative effect of

1The Institute of Cardiovascular Science & The Institute of Molecular Medicine, Peking University, Beijing 100083, China. 2Laboratory of Cardiovascular Science,National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA. 3Center for Human Disease Genomics and Department of Immunology, HealthCenter of Peking University, Beijing 100083, China.4Correspondence should be addressed to R.-P.X (e-mail: [email protected])

Published online: 22 August 2004, DOI: 10.1038/ncb/1161

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© 2004 Nature Publishing Group

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NATURE CELL BIOLOGY VOLUME 6 | NUMBER 9 | SEPTEMBER 2004 873

rHSG was mediated by cell-cycle arrest in the G0/G1 phases owing toinhibition of the ERK1/2 signalling pathway, whereas disruption ofrHSG mitochondrial targeting by deletion of its transmembranedomain increased, rather than decreased, the inhibitory effects of rHSG.These findings not only reveal a fundamental biological function of theubiquitously expressed and phylogenetically conserved gene, but alsodefine a novel genetic pathway for cell proliferative diseases.

RESULTSCloning and characterization of rHSG cDNAOne strategy for delineating molecular determinants of VSMC prolif-eration is to analyse the gene expression profile in an established exper-imental model system of VSMC hyperplasia. A strain of spontaneouslyhypersensitive rat (SHR) was derived by recurrent selective breeding ofWistar Kyoto rats (WKYs)22. Although SHR and WKY share a similargenetic background, the proliferation rate of VSMCs from SHR is

markedly elevated compared with that of normotensive WKY counter-parts23, resulting in spontaneous hypertension at 6–8 weeks of age, andeventually cardiac hypertrophy and heart failure after 18–24 months inhalf of these rats24. Thus, SHR and WKY provide an established modelsystem for studying VSMC proliferative disorders.

We confirmed the notion that the proliferation rate of SHR VSMCsis greater than that of WKY VSMCs (Fig. 1a). To identify genesinvolved in VSMC proliferation, the gene expression profile of SHRVSMCs was compared with that of WKY VSMCs using a differentialdisplay technique, and a novel gene was identified. We referred to thecDNA fragment highly expressed in WKY but weakly expressed in SHRas hyperplasia suppressor gene (HSG).

The partial (~0.35 kilobase (kb)) cDNA identified from the differen-tial display was cloned into the pGEM-T plasmid vector and sequenced.Using cDNA library screening and 5′ rapid ampilification of cDNAends (RACE), the full-length cDNA was cloned, which consisted of

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Figure 1 rHSG is downregulated in highly proliferative vascular smoothmuscle cells (VSMCs). (a) Growth course of primary cultured VSMCs fromSHR or WKY (n = 4, performed in triplicate; ∗P < 0.001 compared withSHR). (b) rHSG domain structure. (c) Downregulation of rHSG expression atmRNA (top) and protein (Mr of 68K; bottom) levels in cultured SHR VSMCs

compared with that in WKY VSMCs. (d) Expression of rHSG at mRNA andprotein levels in eight different rat tissues, as indicated. (e) The time courseof rHSG mRNA expression, assayed by quantitative real time PCR, inresponse to bFGF, PDGF-BB or ET-1 (n = 3 performed in triplicate;∗P < 0.05, compared with the control value).

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4,151 base pairs (bp) of sequence before a poly(A) tail (seeSupplementary Information, Fig. S1). Analysis of the sequence revealedan open reading frame that encodes a protein of 757 amino acids, with atypical ‘AATAAA’ polyadenylation consensus signal present 5′ of thepoly(A) tail (by 18-bp) and a strong Kozak consensus sequence preced-ing the ATG initiation codon (see Supplementary Information, Fig. S1).

A hydropathy analysis of the protein sequence indicated ahydrophobic transmembrane domain at the position of amino acids599–644. A motif search revealed a p21ras signature motif (aminoacids 77–92), a GTP-binding site motif (P-loop; amino acids98–117), and a possible PKA/PKG phosphorylation site at Ser 442(Fig. 1b). Using the sequence of rHSG, human and mouse homo-logues were cloned, which share 95.2% and 98.4% similarity with therHSG amino-acid sequence, respectively (see SupplementaryInformation, Fig. S2). In addition, rHSG is 50% similar to theDrosophila Fzo gene product21. The highly conserved sequencehomology of rHSG during evolution implies a fundamental biologi-cal function of this ubiquitously expressed gene.

Downregulation of rHSG in hyperplasic VSMCsNorthern blot analysis showed that rHSG expression (the 4.2-kbband) was downregulated in cultured SHR VSMCs compared withthat in VSMCs from age- and gender-matched WKY (Fig. 1c),substantiating the finding from the differential display. Western blot

analysis with an affinity purified antibody that reacts with rHSG,detected two distinct bands with relative molecular masses (Mrs) of86,000 and 68,000 (Fig. 1c). Interestingly, only the protein with an Mrof 68K showed reduced abundance in SHR VSMCs, whereas the 86Kband showed no difference between SHR and WKY groups (Fig. 1c).In addition, northern hybridization of the rHSG fragment to mRNAsfrom eight different rat tissues identified the 4.2-kb single band in alltissues tested, with the highest expression in tissues in which most cellsare terminally differentiated (for example, heart and testis; Fig. 1d).Concomitantly, western blot analysis revealed similar tissue distribu-tion profiles of the 68K rHSG product (Fig. 1d), but no tissue-differ-ence for the 86K product (data not shown).

To determine whether the expression of rHSG can be regulated byproliferative stimuli, rHSG gene expression was examined in WKYVSMCs in response to PDGF-BB, bFGF and the mitogenic peptide ET-1using quantitative real-time polymerase chain reaction (PCR). BothPDGF-BB and bFGF treatments induced a small transient increase(~20%) followed by a robust, time-dependent decrease in the amount ofrHSG mRNA, reaching a steady state inhibition of 70% and 55%, respec-tively, at the 24 h time point (Fig. 1e). In addition, ET-1 stimulationreduced rHSG expression in a time-dependent fashion without a tran-sient increase, reaching about 50% inhibition after 24 h of treatment(Fig. 1e). These results indicate that downregulation of rHSG expressionis a generalized VSMC response to proliferative stimuli.

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< 0.01, **P < 0.001, compared with uninfected or Adv–GFP). (c) Infectionof VSMCs with Adv–rHSG–GFP, but not Adv–GFP, inhibits serum-inducedDNA synthesis assayed by 3H-thymidine incorporation (n = 3, with eightrepeats in each; ∗P < 0.01, ∗∗P < 0.001, compared with Adv–GFP).

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Overexpression of rHSG results in growth arrest in serum-stimulated VSMCsAs downregulation of rHSG expression is associated with anincreased proliferation rate in mitogenic-factor-stimulated WKYVSMCs and in unstimulated SHR VSMCs, we proposed that rHSGinhibits VSMC proliferation. To test this possibility, we constructed areplication-defective adenovirus, Adv–rHSG–GFP, with a c-myc-epitope tag and a green fluorescent protein (GFP) cDNA added tothe C-terminus of the rHSG cDNA. Infection of primary culturedWKY aortic VSMCs with Adv–rHSG–GFP at 50 plaque-formingunits (pfu) cell−1 for 24 h, produced almost 100% GFP-positive cells(data not shown) and increased expression of rHSG at both themRNA and protein levels, as detected by RT–PCR (Fig. 2a, top) andwestern blot analysis (Fig. 2a, bottom), respectively.

To determine whether rHSG affects growth-factor-induced VSMCproliferation, primary cultured WKY VSMCs were made quiescent andinfected with either Adv–rHSG–GFP or Adv–GFP at 50 pfu cell−1 for24h, then stimulated by serum (10% FBS). Serum caused a time-dependent increase of cell number in uninfected VSMCs or thoseinfected with the control virus (Adv–GFP), whereas the increase in cell

number was blunted by ~50% in VSMCs expressing rHSG–GFP(Fig. 2b). Concomitantly, infection of VSMCs with Adv–rHSG–GFP,but not Adv–GFP, attenuated serum-induced DNA synthesis, assayedby 3H-thymidine incorporation, in a titre-dependent manner (Fig. 2c).A similar growth suppressive effect of rHSG was observed in unstimu-lated SHR VSMCs (data not shown).

The role of rHSG in regulating the VSMC cell cycleThe growth suppression effect of rHSG could be the result ofincreased apoptosis or inhibition of cell proliferation. To discriminatethese two possibilities, we examined the possible alteration of the cellcycle in response to increased expression of rHSG using flow cytome-try. Cultured WKY VSMCs were first synchronized and then infectedwith either Adv–rHSG–GFP or Adv–GFP at 50 pfu cell−1 for 24 h,before stimulation with serum to initiate cell-cycle progression.Fluorescence activated cell sorting (FACS) analysis was used to exam-ine cell-cycle distribution. Serum-deprived cells were mainly distrib-uted in the G0/G1 phase (93.4 ± 1.26%; n = 4; Fig. 3a, panel I). Afterserum stimulation for 24 h, approximately 40% of VSMCs infected byAdv–GFP (37.6 ± 4.6%; n = 4; Fig.3 a, panel II) or uninfected VMSCs

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Adv–rHRG–GFP (IV). (b) The average data of cell cycle distributions (n = 4;∗P < 0.001, compared with uninfected or Adv–GFP). (c) rHSG-inducedincreases in p21 and p27 abundance and a decrease in Rb phosphorylation.Similar results were obtained in four experiments.

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(40.3 ± 1.6%; n = 4; Fig. 3a, panel III) progressed into S phase.However, in the presence of serum stimulation, VSMCs infected withAdv–rHSG–GFP remained mostly in the G0/G1 phases with only5.78 ± 2.81% of cells entering S phase (n = 4; Fig. 3a, panel IV). Onaverage, the G0/G1 fraction of cells infected by Adv–rHSG–GFP was90.6 ± 2.0% (n = 4; P < 0.001), compared with 47.8 ± 2.56% (n = 4;P < 0.001) for Adv–GFP-infected cells and 48.8 ± 3.2% (n = 4;P < 0.001) for uninfected cells (Fig. 3b).

The effect of rHSG on cell-cycle arrest was also manifested by rHSG-mediated alterations in key components of the cell-cycle regulatorymachinery. Overexpression of rHSG caused an increase in expressionof the CDK inhibitors p21 and p27, and a significant reduction inphosphorylation of Rb (Fig. 3c), thus permitting accumulation of thehypo-phosphorylated, growth-suppressive form of Rb.

Next, we examined the effect of rHSG on apoptosis, but found noincrease in apoptosis in cells infected with Adv–rHSG–GFP at50 pfu cell−1 for 24 h, compared with those infected with Adv–GFPor uninfected cells (data not shown). Altogether, these findingsindicate that the growth suppressive effect of rHSG is mediated byG1/G0 cell-cycle arrest, rather than increased apoptosis under thepresent experimental conditions.

Binding of rHSG to Ras inhibits the Ras–Raf–ERK1/2 signallingpathwayTo dissect the possible mechanistic pathway that links rHSG with cell-cycle arrest, we sought to determine proteins that bind to rHSG. Usinga yeast two-hybrid assay, we found that HSG could interact with a Rasanalogue (data not shown). This was validated by co-immunoprecipi-tation of rHSG protein with Ras in cultured WKY VSMCs in theabsence or presence of Adv–rHSG–GFP (50 pfu cell−1 for 36 h) orserum stimulation (Fig. 4a).

ERK1/2, a major downstream signalling protein of Ras, is prima-rily stimulated by mitogenic and differentiation factors, and has apivotal role in G1 progression and the G1–S transition11–13. To fur-ther delineate cellular and molecular mechanisms underlyingrHSG-mediated VSMC growth arrest, we evaluated the possible roleof rHSG in regulating the Ras–Raf–MEK–ERK1/2 signalling cas-cade. Overexpression of rHSG markedly inhibited ERK1/2 activa-tion in WKY VSMCs in response to serum stimulation (Fig. 4b).Furthermore, overexpression of rHSG resulted in a robust reduc-tion in the activity of ERK1/2 and its upstream kinase Raf-1 (Fig.4c), indicating a profound inhibitory effect of rHSG on the ERK1/2signalling cascade. This result suggests that rHSG-induced cell-cycle

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Figure 4 Binding of rHSG to Ras inhibits the Ras–Raf–ERK1/2 signallingpathway in WKY VSMCs. (a) Co-immunoprecipitation of rHSG with Ras incultured synchronized or non-synchronized cells in the presence orabsence of Adv–rHSG–GFP. Three similar results were observed. (b) Typical western blots of total (ERK1/2) or phosphorylated (P-ERK1/2)endogenous ERK1/2 in quiescent WKY VSMCs infected withAdv–rHSG–GFP or Adv–GFP and stimulated by serum for 5 min. In the

first and second lanes, 60 and 120 µg of protein were loaded,respectively. (c) The average inhibitory effects of rHSG on ERK1/2 (left) orRaf (right) activity, respectively, (n = 6; ∗P < 0.001, compared withAdv–GFP). (d, e) Enforced expression of rHSG–Ras(∆) cannot inhibitserum-induced ERK1/2 phosphorylation (d) or cell proliferation (e),assayed by the cleavage of the tetrazolium salt MTT in WKY VSMCs (n = 6;∗P < 0.01, compared with Adv–GFP or Adv–rHSG–Ras(∆).

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arrest in the G0/G1 phases is attributable, at least in part, to inhibi-tion of ERK1/2 signalling.

To understand the molecular-structural mechanism underlying theinhibitory effect of rHSG on the Ras–Raf–MEK–ERK1/2 cascade, weconducted a motif search of rHSG and identified a p21ras signaturemotif located at amino acids 77–92 (N-DVKGYLSKVRGISEVL-C),which is highly conserved in the Ras protein family. Remarkably, dele-tion of the p21ras signature motif, rHSG-Ras(∆), abolished rHSG-induced inhibition of ERK1/2 phosphorylation (Fig. 4d) and growthsuppression (Fig. 4e), indicating a crucial role in rHSG-mediated anti-proliferation and inhibition of ERK1/2 signalling.

rHSG-mediated anti-proliferation does not requiremitochondrial targetingIn addition to the p21ras signature motif, rHSG protein has a trans-membrane domain (Fig. 1b), similar to its human homologue, Mfn-2(ref. 17). The transmembrane domain of human Mfn-2 is necessary formitochondrial targeting17,18. Indeed, confocal imaging of the full-length rHSG–GFP fusion protein and a fluorescent mitochondrialmarker, MitoTracker, highlighted the co-localization of rHSG withmitochondria and, more importantly, increased mitochondrial fusion,as manifested by perinuclear mitochondrial clustering in cells infectedwith Adv–rHSG–GFP (Fig. 5a, panels I–III). This observation is consis-tent with the previous notion that Mfn-2 is located at the mitochondr-ial outer membrane and causes perinuclear mitochondrial clusteringwhen overexpressed in mammalian cells17,18. Deletion of the trans-membrane domain (rHSG-TMD(∆)) to disrupt rHSG mitochondrial

targeting resulted in a fairly uniform cytosolic distribution of rHSG-TMD(∆)–GFP fusion protein (Fig. 5a, panels I′-III′). Notably, the dele-tion of the transmembrane domain enhanced rHSG-mediatedinhibition of ERK1/2 signalling and suppression of VSMC prolifera-tion (Fig. 5b, c). In contrast, deletion of the p21ras signature motif didnot alter the intracellular distribution pattern of rHSG (data notshown), but resulted in loss of function of rHSG in terms of its anti-proliferative effect. Altogether, these results not only underscore theimportance of the p21ras signature motif in rHSG-induced inhibitionof the Ras–Raf–MEK–ERK1/2 signalling cascade and growth suppres-sion, but also indicate that the anti-proliferative effect of rHSG is inde-pendent of its function in mitochondrial fusion.

Downregulation of rHSG in restenosis and atherosclerosis Next, we attempted to determine the possible role of rHSG in prevent-ing in vivo VSMC proliferation in response to arterial injury, using a ratcarotid artery injury model in which balloon injury promotes VSMCproliferation25,26. WKY carotid arteries were subjected to ballooninjury and simultaneous infection with either Adv–rHSG–GFP orAdv–GFP from the periarterial sheathes of vessels. First, the efficiencyof the in vivo gene transfer of the c-myc-tagged Adv–rHSG–GFP in theinjured WKY carotid arteries was assessed. Four days after infection,rHSG mRNA expression and c-myc-tagged rHSG protein abundancewere assayed by PCR with reverse transcription (RT–PCR), and west-ern blot analysis with the c-myc monoclonal antibody, respectively. Theconcentration of rHSG mRNA was markedly increased and c-myc-tagged rHSG protein was detected only in arteries infected with

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or rHSG-TMD(∆) (II′); merged imaging in cells expressing rHSG (III) orrHSG-TMD(∆) (III′). (b, c) Deletion of the transmembrane domain (rHSG-TMD(∆)) enhances the inhibitory effects of rHSG on serum-induced ERK1/2activation (b) and cell proliferation (c), (n = 6; ∗P < 0.01, compared withAdv–GFP; ∗∗P < 0.001 compared with Adv–GFP).

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Adv–rHSG–GFP, but not in uninfected or Adv–GFP-infected samples(Fig. 6a). These data indicate that adenoviral infection immediatelyafter balloon injury through periarterial injection, provides an efficientmeans to deliver the rHSG gene into blood vessels in vivo.

To investigate the role of rHSG in regulating VSMC proliferation invivo, WKY carotid arteries were harvested on day 8 after injury forimmunostaining using an antibody reacting with proliferating cellnuclear antigen (PCNA). In the presence of Adv–GFP, the number ofPCNA-positive VSMCs in the media and the intima was increased by 7.6-and 42.1-fold, respectively, compared with uninjured samples (Fig. 6b, c).Remarkably, the injury induced increase in PCNA-positive VSMCs wasalmost fully abolished by overexpression of rHSG (Fig. 6b, c).

Similar to the situation in cultured hyperplasic VSMCs from SHR ormitogenic-factor-stimulated WKY VSMCs, balloon injury inducedhyper-proliferation was accompanied by a marked downregulation ofrHSG expression at protein and mRNA levels, assayed by immunohis-tochemical staining with the anti-rHSG antibody (Fig. 6d, e) andRT–PCR (Fig. 6f), respectively. Interestingly, there was a dynamic reg-ulation of rHSG expression after balloon injury. The reduction inrHSG gene expression emerged at one week, reached its most profoundextent at 2 weeks, and was then partially restored at 3 weeks after injury(Fig. 6f). This is in good agreement with the previously reported tem-poral profile of the VSMC proliferative response in rat carotid arteriesafter balloon injury27.

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Figure 6 Adenoviral gene transfer of rHSG inhibits VSMC proliferation inballoon-injured rat carotid arteries. (a) Expression of c-myc-tagged rHSG atmRNA and protein levels in WKY carotid arteries on day 4 after shamoperation or balloon injury plus Adv–GFP or Adv–rHSG–GFP infection. (b) Representative PCNA staining of arterial cross sections on day 8 aftersurgery. (c) Average data of PCNA staining of VSMCs in media and intima (n

= 6; ∗P < 0.01, compared with Adv–rHSG–GFP or sham). (d, e)Representative rHSG immunostaining of cross-sections from sham-operated(d) or balloon-injured vessels (e) at 3 weeks after surgery, (M, media; I,intima). Brown colour shows rHSG positive staining. (f) Downregulation ofrHSG expression in balloon-injured WKY carotid arteries compared with thatin uninjured vessels (Control).

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Similar results were observed in atherosclerotic carotid arteries froman ApoE-knockout mouse atherosclerosis model. The expression ofrHSG mRNA was progressively reduced in ApoE-knockout mousearteries during the development of atherosclerosis (Fig. 7). Thus, rHSGgene expression is noticeably downregulated in hyperplasic arteriallesions, indicating that the anti-proliferative effect of rHSG may haveimportant pathological as well as physiological relevance.

Overexpression of rHSG prevents balloon angioplasty associatedrestenosisApproximately 30–50% of atherosclerotic coronary arteries treated byconventional balloon angioplasty, coronary stenting, or bypass surgeryocclude owing to restenosis16. As restenosis results mainly from neointi-mal VSMC proliferation, a feature shared by advanced lesions of athero-sclerosis, we explored the possible application of rHSG in the cytostaticgene therapy of balloon angioplasty associated restenosis, as a VSMChyperplasia relevant disorder. WKY arteries were harvested on day 21after balloon angioplasty, and the degree of restenosis was determinedby intima-to-media (I/M) area ratio. Representative examples ofhaematoxylin–eosin staining of cross sections of the infected and unin-fected arteries can be seen in Figure 8a–d. Injured rat carotid arteriesexhibited pronounced neointima formation (Fig. 8b, e), whereas no sig-nificant neointimal expansion was observed in sham-operated arteries(Fig. 8a, e). More importantly, a robust reduction in neointimal thick-ness of arteries infected with Adv–rHSG–GFP was observed (Fig. 8c),but there was no reduction in those infected with Adv–GFP (Fig. 8d).Overexpression of rHSG reduced the I/M area ratio by 90% (0.23 ± 0.21of Adv–rHSG–GFP group compared with 1.89 ± 0.42 of uninfected or2.05 ± 0.73 of Adv–GFP group; n = 8 for each group, P < 0.001; Fig. 8e).Thus, adenoviral gene transfer of rHSG effectively prevents the resteno-sis induced by balloon angioplasty in the rat experimental model.

DISCUSSIONAlthough recent studies have shown that Drosophila and humanhomologues of rHSG have a critical role in mitochondrial fusion inmany cells17–20, we have demonstrated for the first time that rHSG isa powerful regulator of cell proliferation in vivo and in vitro. Thisconclusion is based on several independent lines of evidence. First,rHSG expression at mRNA and protein levels is markedly downregu-lated in highly proliferative VSMCs from the genetic hypertensiveanimal model, SHR, compared with VSMCs from age- and gender-matched WKY (Fig. 1c). Second, an overt downregulation of rHSGexpression in vivo was associated with balloon injury induced VSMChyper-proliferation in rat carotid arteries (Fig. 6d–f) and atheroscle-rosis in ApoE-knockout mouse carotid arteries (Fig. 7b). Third, pro-liferative stimuli, such as PDGF, bFGF and ET-1, reduce rHSGexpression, whilst promoting WKY VSMC proliferation. Finally,overexpression of rHSG inhibits mitogenic-stimuli-mediated prolif-eration of cultured WKY VSMCs, and blocks balloon injury inducedneointimal VSMC proliferation and restenosis in vivo. These obser-vations suggest that normal expression and function of rHSG maycontribute to the maintenance of the non-proliferative state ofVSMCs under physiological conditions, whereas dysregulation ofrHSG expression results in proliferative vascular disorders.

Moreover, our preliminary studies have shown that adenoviralgene transfer of rHSG or its human homologue, Mfn2, has a potentanti-proliferative effect in a variety of cancer cell lines, particularlyin a breast cancer cell line BM-1 (see Supplementary Information,Fig. S3). In fact, the anti-proliferative effect of human HSG is morepotent than that induced by overexpression of p53, a well establishedtumour suppressor28, (K.-H.C., X.-M.G., X.-Y.Q., R.-P.X. and J.T.,unpublished observations). Thus, we conclude that rHSG functionsas a powerful cell proliferation suppressor, and that downregulation

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Figure 7 Downregulation of rHSG in atherosclerotic carotid arteriesfrom ApoE-knockout mice. Histological sections with haematoxylin-eosin (HE) staining (a) and rHSG gene expression (b) in ApoE-knockout

mouse carotid arteries before (control) or after developingatherosclerosis at early and late stages. Similar results were observedin three independent experiments.

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or inactivation of rHSG is probably involved in the pathogenesis ofvascular proliferative disorders and possibly in cancer as well.

To understand the mechanisms underlying rHSG-induced cellgrowth suppression, we have determined the effect of rHSG on thecell cycle, and found that overexpression of rHSG induces cell cyclearrest in the G0/G1 phases mainly by inhibiting theRas–Raf–MEK–ERK1/2 signalling pathway. Specifically, using yeasttwo-hybrid and co-immunoprecipitation assays, we have demon-strated that rHSG binds to Ras in cultured VSMCs in the presence orabsence of serum stimulation. We have also shown that rHSGmarkedly decreases serum-evoked activation of Raf and ERK1/2, andthat the p21ras signature motif has an essential role in rHSG-mediatedinhibition of ERK1/2 signalling and growth arrest. These datastrongly suggest that binding of rHSG to Ras causes a negative regula-tion of the Ras–Raf–MEK–ERK1/2 MAPK signalling pathway.Notably, the profound anti-proliferative effect of rHSG does notrequire mitochondrial membrane targeting, because the deletion ofthe mitochondrial targeting domain enhances rHSG-mediated anti-proliferation in serum-stimulated WKY VSMCs. These findings indi-cate that rHSG or its homologues have multiple vital biologicalactions, as well as their involvement in mitochondrial fusion17–21.

In addition, we have demonstrated that rHSG-induced suppres-sion of VSMC proliferation is associated with an increase in theexpression of CDK inhibitors p21 and p27. The increase in p21 andp27 abundance is most probably the result of rHSG-induced inhibi-tion of ERK1/2 signalling, because sustained activation of ERK1/2 isrequired for the degradation or downregulation of these CDKinhibitors, particularly p27 (refs 13–15). There is much evidence tosuggest that altered p27 levels function as a cause, but not a conse-quence, of a change in cell cycle status29–32. Thus, an increase in p27abundance may constitute an essential signalling step that linksERK1/2 inhibition to rHSG-mediated cell cycle arrest.

Overexpression of rHSG also attenuates phosphorylation of Rb,resulting in the accumulation of hypo-phosphorylated Rb in culturedVSMCs; Rb is one of the major modulators of the G1/S transition inmammalian cells. When hypo-phosphorylated in the G0/G1 phases ofthe cell cycle, Rb blocks cell cycle progression by binding to and sup-pressing the activities of transcription factors such as E2F family mem-bers33,34. Consistent with this, the increase in growth-suppressive,hypo-phosphorylated Rb contributes, at least in part, to rHSG-inducedcell cycle arrest in the G0/G1 phases.

There is much evidence demonstrating that enhanced VSMC prolif-eration is essentially involved in the pathogenesis of atherosclerosis andother vascular proliferative disorders, including restenosis after coro-nary balloon angioplasty3–5,35. Recent studies indicate that coatedstents with anti-proliferative agents, including rapamycin36 andtaxol37, significantly reduce the risk of restenosis after coronary angio-plasty in patients with coronary artery disease. However, smooth-mus-cle-cell proliferation also occurs outside of the coronary arterial tree inthe context of hypertension, resulting in target organ damage through-out the body. Thus, prevention of VSMC pathological proliferation stillremains as a major clinical challenge, which underscores the need fornew therapeutic strategies. Recent progress in molecular medicine hasled to the development of gene therapy as a new therapeutic option forcardiovascular proliferative disorders38.

In the present study, we have used adenoviral gene transfer into thevessel wall, without interruption of blood flow or disruption of theendothelium that occurs in catheter-mediated delivery39–44. Injectionof the recombinant adenovirus into periarterial sheath of rat carotidarteries resulted in a high level of rHSG expression at both mRNA andprotein levels in the injured arteries. Such targeted local rHSG genedelivery immediately after carotid artery balloon angioplasty preventsneointimal and medial wall remodelling to a great extent (Fig. 8a–e).Analyses of the carotid arteries harvested on day 21 post-injury

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Figure 8 Overexpression of rHSG prevents balloon-injury inducedrestenosis in rat carotid arteries. (a–d) Representative histologicalsections of WKY carotid arteries with sham operation (a), or ballooninjury in the absence (b) or presence of either Adv–rHSG–GFP (c)

or Adv–GFP infection (d) at 3 weeks after surgery (original magnification×100). (e) Average data on the ratio of intima/media of the above four groups (n = 8 for each group; ∗P < 0.001, compared withuninfected and Adv–GFP).

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revealed a roughly 90% reduction in the I/M area ratio in response toadenovirus-mediated rHSG gene transfer, without affecting the I/Mratio in the normal surrounding region. The inhibitory effect is medi-ated by inhibiting VSMC proliferation, as evidenced by markedlyreduced PCNA-positive cells (Fig. 6b, c). It should also be noted that theeffect of rHSG to inhibit neointimal formation is much greater than anypreviously reported similar approaches, such as adenoviral gene transferof heme oxygenase-1, gax, Rb or the cell cycle inhibitor p21, in rat, rabbitor pig39–44. In these previous studies, the reduction in I/M ratio isapproximately 40–50%39–44. Thus, rHSG adenoviral gene transfer mayprovide a highly efficient therapeutic intervention against vascular pro-liferative disorders. In addition, recent progress has been made in thedevelopment of tissue-specific adenoviral vectors for arterial gene ther-apy45. Using inducible and tissue-specific expression vectors, local genedelivery of HSG might offer a temporally and spatially well controlledmeans to inhibit or prevent hypertension- and injury induced hyper-proliferation of VSMCs in peripheral organs as well as coronary arteries.

In summary, we have demonstrated for the first time, that rHSG pro-foundly inhibits VSMC proliferation, and that this anti-proliferativeeffect of rHSG is independent of its known function in mitochondrialfusion. rHSG was downregulated in hyper-proliferative VSMCs fromSHR or mitogenic-factor-stimulated WKY VSMCs and in injury- oratherosclerosis-associated hyperplasic arterial lesions. Overexpressionof rHSG markedly inhibited the Ras–Raf–MEK–ERK1/2 signalling cas-cade and resulted in cell cycle arrest in the G0/G1 phases, thus blockingmitogenic stimuli- or injury mediated VSMC proliferation and pre-venting balloon injury induced restenosis. These in vivo and in vitroobservations indicate that rHSG has a previously unappreciated func-tional role in regulating cell proliferation, thereby revealing an impor-tant therapeutic target for the treatment of vascular proliferativedisorders and possibly other hyper-proliferative diseases as well.

METHODSMaterials. bFGF, PDGF-BB and ET-1 were purchased from Calbiochem (La Jolla,CA). Antibodies reacting with p21, p27, phospho-Rb (pRb), c-myc and α-actinwere purchased from Sigma (St. Louis, MO). Complete Proteinase InhibitorCocktail was from Roche Applied Science (Indianapolis, IN). A monoclonal anti-body specifically reacting with the proliferating cell nuclear antigen (PCNA) waspurchased from Zymed Laboratories (San Francisco, CA) and an affinity purifiedanti-chicken rHSG primary antibody was made at GenWay Biotech (San Diego,CA). The specificity of the rHSG antibody was verified by the ability of theimmuno-peptide at a concentration 5-fold over the rHSG antibody to fully abol-ish the antibody induced immunoblots (data not shown). Anti-Pan-Ras mono-clonal antibody (F132) used in the co-immunoprecipitation assay andperoxidase-labelled anti-mouse secondary antibodies were from Santa CruzBiotechnology (Santa Cruz, CA), and goat anti-chicken IgY conjugated gel andanti-chicken secondary antibodies were from GenWay Biotech. Histostain-PlusKit was from Zymed and the PhosphoPlus p44/42 MAP kinase (Thr 202/Tyr204) Antibody Kit was from Cell Signaling Technology (Beverly, MA) and theCell Proliferation Kit I (MTT) was purchased from Roche Applied Science.

Adenoviral constructs. Replication-defective adenoviruses encoding the com-plete rHSG open reading frame (Adv–rHSG–GFP), the p21ras signature motifdeletion mutant (Adv–rHSG–RAS(∆)), or the transmembrane domain dele-tion (Adv–rHSG–TMD(∆)) with 3′ c-myc and GFP tags was constructed byhomologous recombination, as described in our previous studies46. An aden-oviral vector expressing GFP (Adv–GFP) was made as a control virus. Standardviral amplification and caesium chloride purification methods were used toamplify and purify these adenoviruses. The titre for each adenovirus was deter-mined by dilution assay in HEK293 cells.

Animals. Adult male SHR and WKY rats (250–350g) were supplied by the Centerfor Experimental Animals at Peking University, China, or from the animal facilityat the National Institute on Aging (NIA) at the National Institutes for Health

(NIH), USA. All procedures involving experimental animals were performed inaccordance with protocols approved by the committee for animal research ofPeking University or at the NIA and conformed to the Guide for the Care and Useof Laboratory Animals (NIH publication number 86–23, revised 1985).

Primary VSMC culture and adenoviral infection. VSMCs from SHR and WKYthoracic aorta specimens were isolated using a standard enzymatic digestiontechnique, as described 47. VSMCs were cultured in Dulbecco’s modified Eagle’smedium (DMEM) containing 10% fetal bovine serum (FBS, Gibco LifeTechnologies, Rockville, MD). VSMC synchronization was achieved by cultur-ing cells in DMEM with 0.2% FBS for 48 h. Adenoviral infection was thenimplemented by adding an indicated titre of Adv–rHSG–GFP,Adv–rHSG–Ras(∆), Adv–rHSG–TMD(∆), or Adv–GFP. Infection of culturedVSMCs was highly efficient, as evidenced by almost 100% GFP-positive cells foreach adenoviral vector at 50 pfu cell−1 for only 24 h.

Cell counting. The growth curves of VSMCs from SHR and WKY were examinedusing cell counting. Passage-matched WKY and SHR VSMCs were seeded ingrowth medium and counted every day for five days (Fig. 1a). Cells first under-went mitogenic quiescence by serum starvation and viral infection for 24 h at50 pfu cell−1 for experiments shown in Fig. 2. The cell number under these exper-imental conditions was used as the baseline. To examine the status of VSMC pro-liferation, the cells were subsequently stimulated with serum (10% FBS), and thecell number counted every day for four days (Fig. 2b). Each count was an averageof three repeats, whereas each data point was the average of four experiments.

3H-thymidine incorporation. Synchronized VSMCs were infected with eitherAdv–rHSG–GFP or Adv–GFP at 10, 50, 100, 500 or 1000 pfu cell−1. After infec-tion, cells were kept in low serum medium (0.2% FBS) for 24 h and then stimu-lated by the normal serum medium (10% FBS) for 24 h. During the last fourhours of stimulation,VSMCs were pulse-labelled with 3H-thymidine (1 µCi ml−1)to determine 3H-thymidine incorporation as described39.

RNA analysis. Standard northern hybridization was performed to detect rHSGmRNA in eight different normal rat tissues (the Clontech pre-made rat MultipleTissue Northern Blot membrane containing 2 µg of mRNA for each tissue) usinga 1.2-kb rHSG cDNA fragment as the probe. After stripping, the same filter washybridized to the β-actin probe provided with the filter for loading normalization.Total RNA was also isolated from cultured VSMCs or rat carotid arteries, using theRNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instruc-tions. Northern blot analysis with the rHSG cDNA probe and RT–PCR were per-formed using standard protocols (see Supplement Information for details).

Western blot analysis. Proteins were prepared from cultured VSMCs or arteries.For samples infected with Adv–rHSG–GFP, a monoclonal antibody reacting withthe c-myc tag (clone 9E10; Sigma) was used at a 1:1,000 dilution to detect c-myc-tagged rHSG protein. The abundance of rHSG protein in uninfected VSMCsfrom SHR and WKY or in multiple rat tissues was determined by western blotanalysis using an affinity purified chicken antibody that specifically reacts withrHSG (Fig. 1c, d). Phosphorylated Rb (pRb), p21, p27 or α-actin expression wasalso assayed by western blot analysis using a rabbit polyclonal anti-pRb antibody(1:1,000 dilution), a monoclonal anti-p27 antibody (1:200 dilution), an anti-p21antibody (5 µg ml−1) or a rabbit anti-α-actin antibody (1:5,000 dilution).ERK1/2 phosphorylation assays were performed using the PhosphoPlus p44/42MAP kinase (Thr 202/Tyr 204) Antibody Kit (Cell Signaling Technology).

Co-immunoprecipitation. Cells infected with Adv–rHSG–GFP (50 pfu cell−1)for 36 h or uninfected in the presence or absence of serum stimulation werelysed in 0.5 ml of lysis buffer (Cell Signaling Technology) containing CompleteProtease Inhibitor Cocktail (Roche Applied Science). After pre-clearing withProtein A/G-PLUS agarose (Santa Cruz) or goat anti-chicken IgY conjugated gelat 4 οC for 1 h, immunoprecipitations were performed by incubating cell lysateswith anti-Pan-Ras or anti-rHSG antibody at 4 οC overnight. The immune com-plexes were recovered by incubation with Protein A/G-PLUS agarose (SantaCruz) for Ras immunoprecipitation or with goat anti-chicken IgY conjugatedgel (GenWay Biotech) for rHSG immunoprecipitation. After washing withbuffer, beads were boiled in NuPAGE sample buffer (Invitrogen) and immuno-precipitated proteins were analysed by western blotting.

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Propidium iodide staining and FACS analysis. VSMCs were synchronized andinfected with adenoviral vectors for 48 h, and subsequently stimulated to prolif-erate by serum, as described above. After addition of serum for 24 h, the cellswere harvested and fixed overnight with 70% ethanol at −20 οC. Cells were thenpelleted, resuspended in 0.5 ml of PBS containing RNase (100 µg ml−1), incu-bated at 37 οC for 30 min, and stained for 10 min at room temperature withpropidium iodide (50 µg ml−1), and analysed with a FACScan (Becton-Dickinson, San Jose, CA). A total of 1 × 104 cells were counted for each sample,whereas a Double Discriminator Module was used only to detect single cells.Each experiment was repeated four times.

Balloon injury and morphometric analysis of intimal thickening. Balloondenudation of the left common carotid artery of male WKY was performed, asdescribed25,26. Briefly, a 2F embolectomy balloon catheter (Baxter Healthcare,McGaw Park, IL) was inserted into the left common carotid artery via the exter-nal carotid artery. The balloon was inflated with saline to distend the commoncarotid artery and then pulled back to the external carotid artery. After repeatingthis procedure three times, with a 120 ο rotation each time, the catheter was thenremoved. Immediately following injury, 2 × 109 pfu of Adv–rHSG–GFP orAdv–GFP in a total volume of 20 µl were infused into the adventitia of theblood-vessel wall. In our preliminary experiments, we collected rat vessels at 4,7, 14, 21 and 28 days after injury, and found that 3 weeks after balloon injury theneointimal formation was already overt. Thus, we choose three weeks afterinjury as the time point to determine the possible anti-proliferative effect ofrHSG gene transfer. Arteries were collected on day 21 after injury, and embed-ded in paraffin to prepare cross sections. Neointima thickening was assessedusing the intima-to-media area ratio (I/M) measured from haematoxylin- andeosin-stained arterial cross sections with a computer-based Image-ProMorphometric System in a double-blind manner. Six discontinuous sectionsfrom each vessel were measured in a WKY rat, whereas eight WKY rats wereused in each experimental group.

PCNA assay in injured carotid artery. To assess the possible effect of rHSG genetransfer on VSMC proliferation in vivo, a PCNA immunohistochemical assaywas performed on day 8 after injury in sections from injured rat carotid arteriesinfected with Adv–rHSG–GFP or Adv–GFP or uninfected, using mouse anti-PCNA monoclonal antibody (1:200 dilution). Sections were also counterstainedwith haematoxylin. The PCNA-positive cells were determined by cell countswith light microscopy using a computer-based Image-Pro MorphometricSystem by two independent observers in a double-blind manner.

Immunohistochemistry of rHSG. Immunostaining of sections from sham-operated or injured rat carotid arteries in the presence of Adv–rHSG–GFPinfection was performed with the chicken anti-rHSG antibody (1:250 dilu-tion) and Histostain Plus kit (Zymed), following the manufacture’s instruc-tions, on day 21 after balloon injury. The same sections were alsocounterstained with haematoxylin.

Accession numbers. The GenBank accession number of rat HSG is U41803; theaccession numbers of the human and mouse homologues of HSG are AF036536and AF384100, respectively; and the accession number of the Drosophila Fzogene21 is NM_170060.

Note: Supplementary Information is available on the Nature Cell Biology website.

ACKNOWLEDGEMENTSThis work was supported by the National Basic Research Priorities Program 973(G1998051015 and G2000056906), China’s 863 High-Tech National ResearchProgramme, Chinese Young Investigator Award (30225036), Peking University 985Project. The authors would like to thank D. Longo, H. Cheng, E.G. Lakatta, P.Morin, X. Fu and J. Chen for critical reading and discussions. We would also like tothank M. Wang and J. Zhang for excellent technical support.

COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests.

Received 13 January 2004; accepted 8 July 2004Published online at http://www.nature.com/naturecellbiology.

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METHODSRNA Preparation & RT-PCR. Total RNAs were isolated from cultured SHR or WKY VSMCs or vessels using RNeasy Mini kit (QIAGEN). One µg RNA was then reverse transcribed to first-strand cDNA using oligo dT primer and SuperScript II reverse transcriptase (RT) provided in SuperScript First-Strand synthesis kit (Invitrogen, Carlsbad, California) following the manufacturer’s protocol.

The rHSG primers were 5’-GGAGCTGGACAGCTGGATTGAT-3’ (bp 786-807) and 5’-AGCTCCAGCTGCTTGTCCATGA-3’ (bp 1394-1373), generating a 609-bp PCR product. The rat GAPDH primers were 5’-AATGCATCCTGCACCACCAACTGC-3’ and 5’-GGAGGCCATGTAGGCCATGAGGTC-3’, spanning a 555-bp fragment. The PCR condition for both primer pairs was: 1 cycle of 95 oC for 2 min, 35 cycles of 95 oC for 30 sec, 68 oC for 1.5 min and 1 cycle of 68 oC for 7 min.

Real Time Quantitative PCR. To quantitate the expression of rHSG in response to proliferative factors, 3 x 106 synchronized WKY VSMCs/plate were seeded and subjected to serum starvation for 48 h, and then stimulated by bFGF (10 ng/ml), PDGF (10 ng/ml), or ET-1 (5 x 10-8 M) in 0.2% FBS-containing DMEM. Cells were collected at the indicated time points, and RNA isolation and reverse transcription were performed as described above. Real time quantitative PCR was then performed using the Roche LightCycler in combination with SYBR Green dye (Roche Molecular Biochemicals, Mannheim, Germany). PCR reagents excluding the primers were from the LightCycler DNA Master SYBR Green kit (Roche Molecular Biochemicals). The final MgCl2 concentration was 3 mM and the primer concentration was 200 nM. The primers for rHSG were: 5’-CTCAGGAGCAGCGGGTTTATTGTCT-3’ (forward) and 5’-TGTCGAGGGACCAGCATGTCTATCT-3’ (reverse) producing a 412-bp fragment; for GAPDH were: 5’-GGGTGGTGCCAAAAGGGTC-3’ (forward) and 5’-GGAGTTGCTGTTGAAGTCACA-3’ (reverse) amplifying a 532-bp fragment. The PCR profile used for rHSG-1 amplification was: 95 oC 30 sec and 40 cycles of 95 oC 0 sec, 60 oC 5 sec, and 72 oC 17 sec. The profile for GAPDH amplification was: 95 oC 30 sec and 40 cycles of 95 oC 0 sec, 68 oC 5 sec, and 72 oC 17 sec. The amount of SYBR-Green was measured at the end of each cycle. The cycle number at which the emission intensity of the sample rises above the baseline was referred to as Ct (threshold cycle) and was proportional to target concentration. Real time PCR data was the average of three independent experiments, while each experiment was in triplicate.

Differential Display Analysis, Cloning and Sequencing of rHSG cDNA. Differential display of mRNA was performed with RNAmap mRNA Differential Display kit (GenHunter, Brookline, Massachusetts) following manufacturer’s instruction. Twenty PCR amplifications were conducted for VSMCs from SHR or WKY. The amplified cDNAs were separated on a 6% acrylamide sequencing gel. Bands exhibiting differential expression were excised and eluted by boiling the gel for 15 min. The eluted cDNA fragments were re-amplified using the same primer set and then cloned into the plasmid vector pGEM-T (Promega, Madison, Wisconsin). Recombinant plasmid DNA used as template for DNA sequencing was purified by alkali lysis method48. Both strands of the cDNA fragments were sequenced with the fmol DNA Sequencing System (Promega) using T7 and SP6 promoter primers. Comparison of DNA sequence homology with the GenBank and the EMBL data-bases was performed using BLAST49. The insert DNAs were cut out and used for preparation of probes on library screening.

cDNA Library Screening & 5’RACE Reaction. An oligo(dT)-primed rat aorta cDNA library constructed in �gt 11 was purchased from Clontech (Palo Alto, California). Approximately 6 x 105 phages were plated at a density of about 2 x 104 plaque-forming units (pfu)/100-mm plate with Escherichia coli 1090r- strain as host and screened following the manufacturer’s protocols. To obtain the upstream nucleotide sequence of the novel cDNA, 5’-RACE reaction was performed with Marathon-Ready cDNA from rat kidney (Clontech) according to the manufacturer’s instruction. The RACE PCR reaction was primed with an internal gene-specific primer (GSP) and the Marathon Adaptor Primer (AP1). Sequences of these primers were as follows: AP1, 5’-CCATCCTAATACGACTCACTATAGGGC-3’;GSP1, 5’-CCGGGTGATGTCAACTTGCTGGCACAG-3’; and GSP2, 5’-GGTGGCTGCAGTTAGAGCCGGTATAACT-3’. An ~2.2 kb fragment was amplified by PCR. The RACE products were cloned into the vector pGEM-T Easy (Promega) and sequenced with ABI 3700 sequencer using T7, SP6, and synthesized specific primers.


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Figure S1 rHSG is downregulated in highly proliferative vascular smooth muscle cells (VSMCs). Nucleotide sequences (top) and the deduced amino-acid sequence of rHSG.


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Figure S2 HSG Sequence homology among rat (rHSG), mouse (mHSG) and human (hHSG). Alignment of the predicted amino acid sequences of rHSG, hHSG, and mHSG. All cDNAs encode 757 amino acids residues. Amino acid residues are numbered on the right margin. Stars highlight non-identical residues among the three species.


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Figure S3 Anti-proliferative effect of human HSG (hHSG) on breast cancer cell line (BM1). BM1 cell proliferation was assayed by the cleavage of the tetrazolium salt MTT.

Dysregulation of HSG triggers vascular proliferative disorders

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