Integrin binding angiopoietin-1 monomers reduce cardiac hypertrophy

The FASEB Journal • Research Communication

Integrin binding angiopoietin-1 monomers reducecardiac hypertrophy

Susan M. Dallabrida,*,† Nesreen S. Ismail,* Elke A. Pravda,* Emily M. Parodi,*Renee Dickie,‡ Ellen M. Durand,* Jean Lai,‡ Flavia Cassiola,* Rick A. Rogers,*,‡

and Maria A. Rupnick1,*,†,§,¶

*Division of Vascular Biology, Children’s Hospital, Boston, Massachusetts, USA; †Harvard MedicalSchool Affiliates, Boston, Massachusetts, USA; ‡Harvard School of Public Health, Harvard University,Boston, Massachusetts, USA; §Chemical Engineering Department, Massachusetts Institute ofTechnology, Cambridge, Massachusetts, USA; and ¶Division of Cardiovascular Medicine, Brighamand Women’s Hospital, Boston, Massachusetts, USA

ABSTRACT Angiopoietins were thought to be endothe-lial cell-specific via the tie2 receptor. We showed thatangiopoietin-1 (ang1) also interacts with integrins oncardiac myocytes (CMs) to increase survival. Because ang1monomers bind and activate integrins (not tie2), wedetermined their function in vivo. We examined mono-mer and multimer expressions during physiological andpathological cardiac remodeling and overexpressed ang1monomers in phenylephrine-induced cardiac hypertro-phy. Cardiac ang1 levels (mRNA, protein) increasedduring postnatal development and decreased with phen-ylephrine-induced cardiac hypertrophy, whereas tie2phosphorylations were unchanged. We found that most orall of the changes during cardiac remodeling were inmonomers, offering an explanation for unchanged tie2activity. Heart tissue contains abundant ang1 monomersand few multimers (Western blotting). We generatedplasmids that produce ang1 monomers (ang1–256), in-jected them into mice, and confirmed cardiac expression(immunohistochemistry, RT-PCR). Ang1 monomers local-ize to CMs, smooth muscle cells, and endothelial cells. Inphenylephrine-induced cardiac hypertrophy, ang1–256 re-duced left ventricle (LV)/tibia ratios, fetal gene expres-sions (atrial and brain natriuretic peptides, skeletal actin,�-myosin heavy chain), and fibrosis (collagen III), andincreased LV prosurvival signaling (akt, MAPKp42/44), andAMPKT172. However, tie2 phosphorylations were un-changed. Ang1–256 increased integrin-linked kinase, a keyregulator of integrin signaling and cardiac health. Collec-tively, these results suggest a role for ang1 monomers incardiac remodeling.—Dallabrida, S. M., Ismail, N. S.,Pravda, E. A., Parodi, E. M., Dickie, R., Durand, E. M.,Lai, J., Cassiola, F., Rogers, R. A., Rupnick, M. A. Integrinbinding angiopoietin-1 monomers reduce cardiac hyper-trophy. FASEB J. 22, 3010–3023 (2008)

Key Words: cardiac myocytes � endothelial cells � integrin-linked kinase � AMP-activated protein kinase � Tie2

Aging societies, increasingly prevalent risk factorssuch as obesity and diabetes, and improved survivalfrom acute coronary syndromes have produced a grow-

ing population with reduced cardiac function. Compro-mised cardiac function triggers a process of cardiacremodeling whereby progressive changes in heart com-position and structure occur to counterbalance stressand salvage function. Although initially stabilizing, thechanges cannot offset the impairment. The evolvingcardiac phenotype becomes maladaptive and decom-pensates, manifesting as heart failure. Once cardiacreserve is exhausted, mortality rates exceed 50%. Withheart failure the fastest growing form of cardiovasculardisease in developed countries, the need for new ad-vances is pressing. As a final common pathway, cardiacremodeling has emerged as a primary therapeutictarget for heart failure of all causes.

Ang1 is vital to cardiovascular development, with aburgeoning role in heart disease. It is widely reportedas an endothelial cell (EC) -specific regulator of vesselmaturation because its receptor, tie2, is almost exclu-sive to EC (1, 2). However, our studies (3) and others(4) are revising this view. We showed that ang1 acts oncardiac and skeletal myocytes as a potent survival factorvia select integrins, resulting in enhanced adhesion andactivation of prosurvival signaling. Thus, ang1 is well-positioned at the vessel/tissue interface to mediatecardiac remodeling and preserve function.

Establishing that ang1/cardiac myocyte (CM) inter-actions promote myocyte survival invites reinterpreta-tion of in vivo studies showing that ang1 is cardiopro-tective to include broader mechanisms of action. Ang1knockout mice have embryonic lethal cardiac defects(impaired endocardial development, trabeculae forma-tion, vessel maturation) (5). In myocardial infarction(MI) models, ang1 overexpression reduced infarctzones and preserved ejection fractions (6). These ben-efits were attributed solely to the role of ang1 inangiogenesis, causing increased capillary density (6–8).However, our data suggest that direct actions of ang1

1 Correspondence: Children’s Hospital, Vascular BiologyDivision, 1 Blackfan Cir., Karp Bldg. RB 11–211, Boston, MA02115, USA. E-mail: [email protected]

doi: 10.1096/fj.07-100966

3010 0892-6638/08/0022-3010 © FASEB

on CMs and smooth muscle cells (SMCs) contribute tothe protective effects.

Ang1 protein structure is complex, with quaternarymultimeric forms unique among growth factors. Fromamino to carboxyl terminus, the domains are superclus-tering (9), coiled-coil (oligomerization) (10), linker(binds ang1 to matrix) (11), and fibrinogen-like (bindstie2) (9, 12, 13). Coiled-coil domains join monomersinto disulfide-linked ang1 homo-oligomers that mul-timerize about the superclustering domains (9, 10).

Ang1’s quaternary protein structure is a determinantof its receptor targeting. Ang1 tetramers and pentamersbind and activate tie2 (9). Ang1 trimers bind solubletie2-Fc without phosphorylating tie2 (10, 12). Ang1dimers weakly bind soluble tie2-Fc (63-fold�tetramer)(9) and do not phosphorylate EC tie2 (12). Ang1monomers show little (290-fold�tetramer) (9) to no(12) binding to soluble tie2-Fc and do not activate ECtie2 but bind integrin �5�1 (13).

The findings that ang1 monomers bind integrins(13), whereas tie2 activation requires at least tetramers(9, 12), suggested to us that ang1 forms may bedifferentially regulated in vivo during remodeling andamong organs. Here, we show that ang1 monomers arein adult tissues, shift during cardiac remodeling, andlocalize to CMs, SMCs, and ECs in vivo. Recombinantang1 monomers reduced murine cardiac hypertrophy,activated prosurvival signaling and AMPKT172 (masterregulator of cardiac energetics and metabolism), andintegrin-linked kinase (ILK) (central mediator of CMcontractility and cell survival) (14). Thus, we proposethat differential expression of ang1 quaternary forms isa unique regulatory mechanism for directing receptor/cell interactions in vivo and that ang1 monomer/integrin interactions have a cardioprotective role inremodeling.

MATERIALS AND METHODS

Animals

The investigation conforms with the U.S. National Institutesof Health Guide for the Care and Use of Laboratory Animals.C57BL/6J (C57) mice (Jackson Laboratory, Bar Harbor, ME,USA) were given free access to water and standard chow.Where indicated, 7-wk-old male mice received phenylephrinehydrochloride (PE) (Sigma, St. Louis, MO, USA) in saline (75mg/kg/day) or saline (control) via subcutaneous minios-motic pumps (Alzet, Cupertino, CA, USA). CMs were isolatedfrom Sprague-Dawley rats (Charles River Laboratory, Wil-mington, MA, USA).

Recombinant Ang1 constructs

Ang1 was cloned from C57 mouse heart as we described (15).Using pcDNA3.1/V5-His TOPO/TA (Invitrogen, Carlsbad,CA, USA) vector, we cloned in N-terminal hemagluttininsecretion signal (MKTIIALSYIFCLVFA) and FLAG tag (DYK-DDDDK) fused to mouse ang1 from amino acids 256–498(ang1–256/pcDNA). The vector contains C-terminal V5 and6X polyhistidine tags. Plasmids were double-purified with

Endotoxin-free plasmid DNA purification kits (Qiagen, Va-lencia, CA, USA) and sequenced. Mice were retroorbitallyinjected with ang1–256/pcDNA or pcDNA (40 �g) usingInVivo Jet-polyethylenimine (PEI) transfection agent plusglucose (ISC Bioexpress, Kaysvilla, UT, USA).

Cell culture and isolations

C2C12 myoblast cell lines were maintained as we described(3). Myoblasts were differentiated into myocytes by conflu-ency or by 3% horse serum in Dulbecco modified Eaglemedium (DMEM) (16). Rat neonatal CMs (RNCMs) and ratneonatal cardiac fibroblasts (RNCFs) were isolated fromventricles of 2-day-old pups. RNCMs were cultured as de-scribed (3) except for preplatings (2�, 1 h), cytosine-�-D-arabinofuranoside (c�Da) (10 �g/ml) added to media, and1% gelatin coating on plates. RNCFs were isolated usingpublished methods (17). Media was changed to low-glucose(LG) -DMEM with 10% fetal bovine serum (FBS) and 1%penicillin–streptomycin–glutamine (PSG). Cells were grownto confluency and passaged to eliminate residual nonfibro-blasts. Rat adult cardiac myocytes (RACMs) were isolatedfrom 8-wk-old rat ventricles using published procedures (18).Hearts were sterilely removed, hung by the aorta (23-gaugeblunt needle), and secured (4.0-gauge suture). Hearts wereretrograde perfused with Ca2�-free Krebs-Henseleit buffer(KHB), 11 mM glucose, and 25 mM NaHCO3 at pH 7.4 untilclear and perfusion digested (20 min, 37°C) with Enzyme Isolution [0.33 mg/ml type II collagenase (Worthington,Lakewood, NJ, USA), 0.3 mg/ml type II hyaluronidase, Ca2�-free KHB, 11 mM glucose, and 25 mM NaHCO3]. Ventricleswere minced and further digested with Enzyme I, 0.02 mg/mltrypsin type IX, and 0.02 mg/ml DNase (Worthington) (20min, 37°C with shaking). Tissues were filtered (80-�m nylonmesh) into 1:1 Ca2�-free KHB/LG-DMEM and centrifuged,supernatant was removed, and the cell pellet was resuspendedin 1:1 Ca2�-free KHB/LG-DMEM. Cells were placed in Ca2�-free KHB with 6.5% bovine serum albumin (BSA) (FisherScientific, Morris Plains, NJ, USA) until myocytes settled.Supernatant was removed and myocytes were plated on 10�g/ml laminin-coated plates in Medium199 with 5 mMcreatine, 2 mM carnitine, 5 mM taurine, 10 �M c�Da, 4%FBS, and 1% PSG (37°C, 5% CO2, 1 day).

Polymerase chain reaction (PCR)

Real-time primers for mouse [ang1, ang2, tie1, tie2, atrialnatriuretic peptide (ANP), brain natriuretic peptide (BNP),�-myosin heavy chain (�MHC), skeletal actin, smooth muscleactin (SMA), collagen III, and GAPDH] (19) and reverse-transcription PCR (RT-PCR) mouse [ang1, ang2, tie2, plate-let/endothelial cell adhesion molecule (PECAM), vascularendothelial growth factor (VEGF) (20), and VEGF receptors1 and 2 (VEGFR1, VEGFR2)] and rat [ang1 (forward mouseang1, ref. 15; reverse 5�, ref. 21)] and GAPDH (IntegratedDNA Technology, Coralville, IA, USA) were used for PCR asdescribed (15). Ang1–256 mRNA was amplified with forward5�-TACAAAGACGATGACGACAAGGACACA-3� and reverse 5�-AGGGTTAGGGATAGGCTTACCTTCGAA-3� primers. GAPDHmRNA levels were stable among groups and used for normal-ization.

Western blotting and cell signaling

Protein lysates were made as we described (3), except 50 mMNaF was added. Some lysates were PNGaseF-treated (Sigma)following manufacturer’s instructions to assess for N-linkedglycosylation. SDS-PAGE analyses were conducted under non-

3011ANGIOPOIETIN-1 MONOMERS REDUCE CARDIAC HYPERTROPHY

reduced and reduced conditions. Tissue (100 �g) and recom-binant human ang1 (rhAng1) lysates (R&D Systems, Minne-apolis, MN, USA) received sample loading buffer (Bio-Rad,Hercules, CA, USA). Some lysates received reducing agent(Bio-Rad reducing agent or 0.435 M �Me) and were heated (5min, 95°C). Samples were loaded on 3–8% Tris-acetatecriterion gels (Bio-Rad), separated by SDS-PAGE, and trans-ferred to nitrocellulose (Perkin Elmer, Boston, MA, USA).Membranes were probed for ang1 with the following antibod-ies: 100–401-403 (Rockland, Gilbertsville, PA, USA), MAB050or AF923 (R&D Systems), A0604 (Sigma), sc6319 (SantaCruz, Santa Cruz, CA, USA), ab8451 (Abcam, Cambridge,MA, USA), and AB3120 (Chemicon/Millipore, Temecula,CA, USA). Blots were blocked (30 min), incubated in primaryantibody in block (1 h), rinsed 3� in 10 mM Tris-base with150 mM NaCl and 0.1% Tween20 (TBST), incubated inappropriate horseradish peroxidase (HRP) -conjugated sec-ondary antibody [anti-mouse, anti-rabbit immunoglobulin G(IgG); 1:1500] in block (1 h), rinsed (3�, TBST), andexposed to chemiluminescence using ECL kits (Amersham,Piscataway, NJ, USA, or Perkin Elmer). Blots were reprobedfor GAPDH (Santa Cruz) as we described (3). Blots wereprobed for phosphorylation of aktS473, MAPKp42(T202)/44(T204), AMP-activated protein kinase (AMPK)T172,AMPKS485/S491, ILK (Cell Signaling, Danvers, MA, USA), andFAKT397 (BD Pharmingen, Billerica, MA, USA); stripped withRestore Stripping Buffer (Pierce Biotechnology, Rockford,IL, USA); and reprobed for total akt, AMPK �1/�2 (CellSignaling), MAPKp42/44 (Chemicon/Millipore), FAK (BDPharmingen), or GAPDH (Santa Cruz) as we described (3).

Tie2 immunoprecipitation

Protein lysates (1000 �g) from mouse left ventricle (LV) wereincubated (overnight, 4°C) with 7 �g mouse anti-tie2 mono-clonal clone AB33 (Upstate, Waltham, MA, USA), protein GPLUS agarose beads (Santa Cruz) were added (4 h, 4°C),pellets were rinsed (modified RIPA buffer, 3�), and sampleloading buffer with Bio-Rad reducing agent was added. Mem-branes were probed with mouse anti-phosphotyrosine mono-clonal recombinant 4G10 (1:200; Upstate) and HRP-linkedanti-mouse IgG (1:2000). Blots were stripped with RestoreStripping Buffer (45 min), rinsed in PBS with 0.05%Tween20, and reprobed with rabbit anti-tie2 polyclonal sc324(1:400; Santa Cruz) and HRP-linked anti-rabbit IgG (1:1500).

Ang1 immunoprecipitation

C2C12 myocytes, RNCMs, and RNCFs were cultured in anti-biotic-free media (3 days). Media (90 ml) was collected andcentrifuged, and protease and phosphatase inhibitors wereadded as we described (3). Media was precleared with agarosebeads, and anti-ang1 antibody (2 �g; R&D monoclonal) wasadded (2 h, 4°C) followed by protein A/G PLUS agarosebeads (overnight, 4°C). Beads were centrifuged and washedin modified RIPA buffer (3 min, 3�), and sample loadingbuffer was added. Samples were divided in half, prepared asnonreduced and reduced (0.435 M �Me), and analyzed bySDS-PAGE. Membranes were probed with anti-ang1 antibody.Negative controls included substituting mouse IgG for pri-mary antibody. When these control samples were probed withang1 antibodies on a Western blot, there were no bands(unpublished observations).

Hanging heart perfusions

Mice were given intraperitoneal injections of heparin (4 U/g)with 2.5% avertin. For some studies, anesthetized mice were

retroorbitally injected (30-gauge needle) with 10% fluores-cein isothiocyanate (FITC) -tomato lectin (Vector, Burlin-game, CA, USA) in PBS and given 5 min to allow the lectin tocirculate. Hearts were removed, dipped in KHB with 25 mMNaHCO3 and 1.4 mM CaCl2, and hung by the aorta (23-gaugeblunt needle). Aortas were tied (4.0-gauge suture), retro-grade perfused (KHB, 25 mM NaHCO3, 1.4 mM CaCl2, 40mM KCl, and 4 mM sodium nitroprusside dihydrate) untilexiting perfusates were cleared, and perfusion fixed (2%formaldehyde in PBS, 10 min). Where indicated, hearts werethen perfused with tissue-marking dye (Triangle Biomedical,Durham, NC, USA). Hearts were trimmed to the ventricles.The left and right ventricles (LVs and RVs) were filled withand then immersed into warmed (37°C) TissueTek optimalcutting temperature (OCT) compound (Fisher Scientific),and frozen (–80°C).

Immunohistochemistry

Endogenous LV Ang1

Ventricles embedded in OCT compound were cryosectioned(5 �m, –20°C) and mounted on Superfrost Plus slides.Sections were air-dried (warming plate), postfixed (ice-coldacetone, 2 min), washed in TBS, and blocked in appropriatenormal serum buffer. Tissues were incubated (overnight,4°C) in primary antibodies ang1 (Rockland and Santa Cruz)or goat polyclonal troponin I (Santa Cruz) (1:100) in anti-body diluent (Dako, Carpinteria, CA, USA). Negative controlsincubated in the appropriate IgG or serum (Vector) showedno signal (unpublished observations). Slides were washed inTBS, blocked (serum buffer), and incubated (2 h) in rabbitanti-goat Texas Red (Jackson Immunoresearch, West Grove,PA, USA), rabbit anti-goat Alexa Fluor 647 (Invitrogen), orgoat anti-rabbit Alexa Fluor 568-conjugated secondary anti-bodies (Invitrogen) (1:200, serum block buffer). Slides werewashed (TBS) and coverslips were placed (Fluoromount Gantifade mounting media; Southern Biotech, Birmingham,AL, USA).

Alternatively, 100-�m-thick sections were cut using anoscillating tissue slicer, washed (buffer with 0.1% TritonX-100) and incubated in secondary antibody (4 h). Slideswere scanned in fluorescence mode with a Leica PL APO �40NA 1.25 oil-immersion objective lens using a Leica TCS-NTlaser scanning confocal microscope (Leica Microsystems,Wetzlar, Germany) fitted with argon and krypton lasers atroom temperature. Serial optical sections of ventricle wererecorded beginning at the ventricular apex, using confocalmicroscopy with a photomultiplier tube detector and Leicaconfocal software to capture images digitally. Where indi-cated, the resultant stacks were rendered in three dimensionsusing VoxelView 2.5.1 software (Vital Images, Minnetonka,MN, USA).

Recombinant Ang1–256

Mouse heart tissues were fixed in 4% paraformaldehyde andPBS, dehydrated through a graded ethanol series, embeddedin paraffin, and cut into 8-�m sections. Sections were rehy-drated, and epitopes were retrieved with 10 mM sodiumcitrate buffer and 0.05% Tween-20 (pH 6.0) (20 min, 95°C),rinsed in PBS, and blocked with normal goat serum in PBS(0.5 h); then primary antibody or relevant control (IgG ornormal serum) was added in block. FLAG rabbit polyclonalantibody (Sigma) was added (1:50; overnight, 4°C). SMAmouse monoclonal antibody (Sigma; 1:100, 45 min), vonWillebrand factor (vWF) rabbit polyclonal antibody (Dako;1:150, 1 h), or phalloidin-conjugated to Alexa Fluor 488

3012 Vol. 22 August 2008 DALLABRIDA ET AL.The FASEB Journal

(Invitrogen; 1:100, 0.5 h) was added. Slides were rinsed(PBS), and the appropriate secondary antibodies were added:for FLAG, goat anti-rabbit Alexa Fluor 647 (1:200, 1 h); forSMA, goat anti-mouse Alexa Fluor 568 (1:200, 45 min); forVWF, biotinylated anti-rabbit IgG (Vector; 1:100, 1 h) andstrepavidin-conjugated Alexa Fluor 568 (1:200, 1 h). Slideswere rinsed and Vectashield Mounting Medium with DAPIwas added (Vector). Optical sections were taken with a LeicaTCS SP2-AOBS attached to a DMIRE-2 inverted microscopewith an �63 objective (Leica Microsystems).

Statistical analysis

Results are reported as mean � sd; 2-tailed Student’s t testswith 2-sample equal variance and P � 0.05 were consideredsignificant.

RESULTS

Ang1 mRNA levels shift during cardiac remodeling

We examined angiopoietin ligands and receptors indifferent-aged C57 mouse hearts during physiologicalremodeling (RT-PCR analysis using GAPDH as controland PECAM as EC marker). Ang1 mRNA levels in-

creased with age in the LV, RV, and atria (AT), whereasang2, tie2, and PECAM levels were stable (Fig. 1A).Western blotting showed parallel increases in ang1 LVprotein levels (Fig. 1B) with stable tie2 protein levels(unpublished observations). Rat neonatal CMs hadtrace ang1 mRNA, whereas adult CMs expressed ang1strongly (Fig. 1C).

To quantify ang1 mRNA increases, we conductedreal-time PCR with LV, RV, and AT from different-agedmice [(–1 dayembryonic day 20), 1, 2, 4, 7, 14, 21, 56,133, and 238 days]. Relative fold increases in ang1 weregraphed alongside increasing chamber weights. Ang1mRNA levels increased over 1000-fold in LV (Fig. 1D)and RV (Fig. 1E), with smaller atrial increases (Fig. 1F).A small spike in LV and RV ang1 occurred between days1 and 4, which returned to near baseline, then climbed.During the fetal to adult transition, similar ang1 in-creases (LV, RV, AT) were found in female and malemice with stable ang2 and tie2 mRNA levels (unpub-lished observations).

To assess ang1 in pathological cardiac remodeling,mice were given PE to induce hypertrophy, or saline ascontrol. PE increased LV/tibia ratios (Fig. 1G) and LVmRNA levels of ANP, BNP, �MHC, collagen III, skeletal

Ang1

GAPDH

Neonatal Adult

Cardiac myocytesAtriaLV7d 14d 56d 7d 14d 56d 7d 14d 56d

RV

Ang1

GAPDH

Ang2

Tie2

VEGF188VEGF164

PECAM

VEGF120

VEGFR1

VEGFR2 Den

sito

met

ric U

nits

Age of Mice (Days)

1600

800

0

*

*

2 14 91

LV

2 d 14 d 91 d

GAPDH

Ang1

B C

Age of Mice (Days)

10 20

60

30

0

40

20

00

Ang

1 m

RN

A F

old

Cha

nge

1600

1200

800

400

0

LV W

eight (mg)

120

80

40

0

LV

-1 50 100 150 200 250

Ang

1 m

RN

A F

old

Cha

nge

1200

800

400

0

RV

Weight (m

g)

40

30

20

10

0

RV

-1 50 100 150 200 250

10 20

20

10

0

80

40

00

Age of Mice (Days)

Ang

1 m

RN

A F

old

Cha

nge 20

15

10

5

0

AT

Weight (m

g)

9

6

3

0

Age of Mice (Days)

-1 50 100 150 200 250

AT

Age of Mice (Days)

10 20

9

6

0

4

2

00

3

Treatment (Days)

3 7 14 28 42

20

15

10

5

0Ang

1 m

RN

A F

old

Cha

nge

**

* * *

Control PE

A

HD

E

F

GTreatment Control Phenylephrine

(Days) LV/tibia LV/tibia Value

3 5.00 + 0.13 5.74 + 0.26 0.002

7 5.58 + 0.13 0.008

14 4.97 + 0.23 5.48 + 0.19 0.028

28 6.00 + 0.46 0.003

42 5.05 + 0.36 5.63 + 0.14 0.005

Hypertrophic Response to Phenylephrine

p

Ang1 mRNA Chamber Weight

Figure 1. Ang1 mRNA levels change duringcardiac remodeling. A) RT-PCR analysis ofmRNA transcripts in mouse LV, RV, and AT.From neonate to adult, ang1 and VEGF188mRNA expressions increase, whereas VEGF120mRNA decreases. B) Western blotting showscorresponding increases in ang1 protein levelsin mouse LV with age. *P � 0.05 vs. 2 days. C)Blots show ang1 mRNA levels in rat CMs: tracelevels in neonatal rats; abundant in adults.Studies done in duplicate; n 4/group (A–C). D–F) Real-time PCR analysis of ang1mRNA in mice: levels in LV (D), RV (E), andAT (F) increase with age. Insets (dashedboxes) magnify days –1 to 21. Studies done intriplicate; n 5/group. G, H) Analysis of micetreated with PE or saline (control): PE in-

creases LV/tibia ratios (G); real-time PCR shows LV ang1 mRNA levels decrease with PE-induced hypertrophy.


actin, and SMA (Supplemental Fig. 1A–F), and de-creased ang1 mRNA levels in LV (Fig. 1H) and RV,whereas ang2 and tie2 were stable (real-time PCR)(unpublished observations).

Integrin-binding ang1 monomers are abundant inheart tissue

Using immunoprecipitation and Western blotting, wefound that tie2 phosphorylation was stable in different-aged (Fig. 2A) and PE-treated (Fig. 2B) mice, despitesubstantial shifts in ang1 (Fig. 1). Thus, we proposedthat integrins are the target for ang1 shifts.

We first established whether integrin-binding ang1monomers and various multimers are present in hearttissue. We defined the ang1 forms recognized by avail-able ang1 antibodies (monoclonal: R&D Systems andSigma; polyclonal: Abcam, Chemicon/Millipore, R&DSystems, Rockland, and Santa Cruz) using rhAng1(R&D Systems). Using Western blotting under nonre-ducing conditions, monoclonal (R&D and Sigma) andpolyclonal (R&D and Santa Cruz) ang1 antibodiesdetected several multimers (Fig. 2C). Using a 6�polyhistidine antibody to the polyhistidine tag onrhAng1, we detected a similar pattern of multimers asobserved with ang1 antibodies (Fig. 2C). In reducedconditions, rhAng1 monomer was found at 64 kDa(Rockland, Abcam, and Chemicon polyclonals) and 70kDa (R&D and Sigma monoclonals; Santa Cruz, R&D,and Rockland polyclonals), similar to a prior report (12).

Ang1 has potential glycosylation sites. We treatedrhAng1 with PNGase-F and conducted Western blottingwith ang1 antibodies. In reduced conditions, PNGase-F-treated rhAng1 monomer shifted from 70 to 55 kDa(R&D and Sigma monoclonal; Santa Cruz polyclonal;Fig. 2D) as was previously shown (22). However, theRockland anti-ang1 antibody detects ang1 monomer at64 kDa. PNGase-F treatment did not alter the mobilityof the 64 kDa monomer, indicating that this form is notglycosylated (Fig. 2D).

To establish whether ang1 monomers and multimersexist in vivo, we conducted Western blotting with ang1and GAPDH (control) antibodies on adult mouse (Fig.2E–G) and human tissues (Fig. 2H; USBiological,Swampscott, MA, USA, and Imgenex, San Diego, CA,USA; unpublished observations). Also, rhAng1 (nonre-duced and reduced) was run on these blots to confirmthe location of ang1 bands (Fig. 2H). In nonreducedconditions, mouse tissues probed with R&D (Fig. 2E)and Sigma (Fig. 2F) monoclonal antibodies revealedang1 monomers (70 and 55 kDa), dimmers, and trim-ers, but few larger forms. Human tissues had only traceang1 multimers (R&D and Sigma monoclonals, unpub-lished observations), perhaps due to degradation orinstability of higher complexes (9, 12, 13) or poorepitope recognition.

With �Me, all ang1 forms reduced to 55 kDa withR&D (Fig. 2E) and Sigma (Fig. 2F) monoclonal anti-ang1, and PNGase-F treatment shifted all ang1 forms to55 kDa (Supplemental Fig. 2A, B), similarly to rhAng1.

Mouse LV and skeletal muscle had trace ang1 multi-mers (Fig. 2E, F). Multimers were abundant in kidney,lung, and adipose tissue, but without appreciable ex-pression in brain.

Using Rockland anti-ang1, mouse LV highly ex-pressed 64 kDa monomer vs. other tissues (Fig. 2G),and ang1 monomers were in all human tissues tested(Fig. 2H). PNGase-F did not alter mobility of the 64 kDaform in tissue or with rhAng1 (Supplemental Fig. 2C).Thus, integrin-binding ang1 monomers are broadlyproduced in vivo with tissue-specific differential expres-sions of ang1 forms.

Nonendothelial cells produce ang1, so we assessedwhether CMs, cardiac fibroblasts, and skeletal myocytesproduce ang1 monomers and multimers. We collectedrat neonatal CM, cardiac fibroblast, and differentiatedC2C12 skeletal myocyte media and immunoprecipi-tated ang1. In nonreduced conditions, Western blot-ting showed that neonatal CMs, cardiac fibroblasts, andC2C12 myocytes produced ang1 monomer (70 kDa),dimer, trimer, tetramer, and trace larger forms as wereseen with rhAng1 (Sigma anti-ang1, Supplemental Fig.2D; R&D anti-ang1, unpublished observations). In re-duced conditions, different ang1 forms converted to 55kDa monomer.

Ang1 monomers shift during cardiac remodeling andinteract with CMs and ECs in vivo

We conducted Western blotting with LV protein lysatesfrom different-aged mice or mice treated with PE orsaline (control). Under nonreduced conditions, 64 kDa(Rockland polyclonal, Fig. 3A), 55 kDa, and 70 kDa(R&D monoclonal, Fig. 3C) ang1 monomers increasedduring the neonatal to adult transition. At 10 days, 110kDa ang1 dimers increased, but ang1 dimers (140 kDa)and trimers were stable (Fig. 3C). With hypertrophy, innonreduced conditions (Fig. 3B), 64 kDa (Rocklandanti-ang1) monomer declined, but 55 and 70 kDamonomer, dimer, and trimer (R&D monoclonal anti-ang1) levels were stable (Fig. 3D). Reduced conditionsconverted various ang1 forms to monomers (unpub-lished observations). Thus, shifts in ang1 during phys-iological and pathological cardiac remodeling are pri-marily in integrin-binding monomers.

To identify in vivo interactions of ang1 forms withcardiac cells, we conducted fluorescent immunohisto-chemistry on perfusion-fixed mouse LV (Fig. 3E). Con-focal images of CMs labeled with troponin I conjugatedto Alexa Fluor 647 (red) show a typical ladder pattern(Fig. 3E, F). ECs perfusion stained with FITC-conju-gated ulex lectin (green) border CMs (Fig. 3E, F).Anti-ang1 (Santa Cruz; detects multimers and mono-mers; 70 and 55 kDa) conjugated to Alexa Fluor 647(red) appeared to label CMs (autofluorescent green)and the vasculature (Fig. 3E, G). Anti-ang1 (Rockland;detects monomers; 70 and 64 kDa) conjugated to AlexaFluor 568 (red) also appeared to label CMs (ladder andpunctuate pattern) and ECs (Fig. 3E, H). Rabbit IgGsubstituted for primary antibody was used as negative


Figure 2. Tie2 activation is stable during cardiac remodeling and heart has abundant ang1 monomers compared to other tissues.A, B) Tie2 phosphorylation [immunoprecipitation and Western blotting for phosphotyrosine (pTyr) and tie2] shows no changeduring physiological (A) and pathological (PE-induced) (B) LV remodeling, remaining stable in neonatal through adult (A)and control vs. hypertrophic LVs (B). Studies done in triplicate; n 3/group. C, D) Results of Western blotting conducted withrhAng1 (0.09 �g) in nonreduced (NR) and reduced (R) (�Me) conditions. Blots were probed with anti-ang1 monoclonal(mono) or polyclonal (poly) antibodies in duplicate studies. The ang1 monomers and multimers detected vary (C). PNGase-Ftreatment of rhAng1 reveals that some ang1 forms were glycosylated (D). E–H) Western blot analysis of protein lysates frommouse (E–G) and human (H) LV, skeletal muscle (sk. mus), kidney, lung, epididymal fat (ep. fat), and brain tissue, evaluatedin nonreduced and reduced (�Me) conditions. Blots were probed with anti-ang1 [monoclonal: R&D (E) and Sigma (F);polyclonal: Rockland (G, H)] and anti-GAPDH antibodies in duplicate studies. Ang1 monomers and multimers are seen inmouse (E–G) and human (H) adult tissues. Heart and skeletal muscle show low multimer levels (E, F) and high monomer levels(G) vs. other tissues.


Figure 3. Ang1 monomers shift during cardiac remodeling. Western blotting was conducted under nonreduced conditions withLVs from neonate to adult mice (A, C) or mice with PE-induced hypertrophy vs. saline-treated controls (con) (B, D). Blots wereprobed with anti-ang1 Rockland (Rk) polyclonal (A, B) or R&D monoclonal antibodies (C, D). Studies done in duplicate; n 3/group. A) Ang1 monomers increase with age. *Left to right: P 0.015, 0.036, 0.013, 0.00002, 0.0007. B) Ang1 monomersdecline during PE-induced remodeling. *P � 0.03. C) Western blotting with anti-ang1 (R&D monoclonal) shows that 55 kDamonomers (*left to right: P0.023, 0.017, 0.011), 70 kDa monomers (**left to right: P0.038, 0.029), and 110 kDa dimers (#left


control (Fig. 3E, I). We propose that ang1 monomersinteract with adult CMc and cardiac ECs in vivo, likelyvia integrins.

Ang1–256 monomer expression in heart tissue

To determine the function of integrin-binding ang1monomers in cardiac remodeling, we generated atruncated recombinant ang1 form (ang1–256) knownto generate only monomers (12). Ang1–256 consists ofang1’s amino acids 256–498, N-terminal hemagluttininsecretion signal and FLAG tag, and C-terminal V5 andpolyhistidine tags (Fig. 4A). Using pcDNA plasmid-based expression, we transfected human embryonickidney (HEK) 293 cells with ang1–256/pcDNA andpurified ang1–256 protein. Western blotting with ananti-V5 antibody showed a single ang1 monomer band(34 kDa) under nonreduced and reduced conditions(Fig. 4D), verifying that ang1–256/pcDNA generatesonly ang1 monomers. We retroorbitally injected ang1–256/pcDNA or pcDNA vector alone using In Vivo-Jet-PEI into C57 mice and examined the expression ofang1–256 mRNA (RT-PCR) and protein (fluorescentimmunohistochemistry and confocal imaging). Thismethod of delivering plasmids to heart tissue is veryeffective, reliable, efficient, and nontoxic, and is usedin clinical trials to deliver cancer gene therapy (ISCBioexpress) (23–25). Ang1–256 mRNA was detected inmouse LV at all time points tested (Fig. 4B). Controlmice injected with pcDNA alone were negative forang1–256 mRNA, as were LVs from mice injected withang1–256 where cDNA synthesis reactions were con-ducted without reverse transcriptase (Fig. 4B). Ang1–256 plasmid was used as a positive control. Ang1–256protein expression was detected using FLAG antibodylinked to Alexa Fluor 647 (green), and nuclei werestained with DAPI (blue). Ang1–256 protein was ob-served at all time points, with the highest expression onday 1 (Fig. 4C). Ang1–256 appeared to be present inCMs and cardiac ECs. Negative controls includedpcDNA alone-injected mice (control) and rabbit IgGsubstituted for primary antibody in LV sections fromang1–256-injected mice.

Ang1–256 protein localizes to CMs, SMCs, and ECs

We conducted fluorescent immunohistochemistry andconfocal imaging to discern the location of ang1–256 onday 1 in mouse hearts. Phalloidin-linked Alexa Fluor 488(red) -stained F-actin, vWF antibody linked to biotinylated

anti-rabbit IgG and strepavidin-linked Alexa Fluor 488-labeled ECs, �SMA antibody linked to anti-mouse AlexaFluor 568-labeled SMCs, FLAG antibody linked to anti-rabbit Alexa Fluor 647-tagged ang1–256, and nuclei werelabeled with DAPI. Negative controls included LV ang1–256 (day 1) where the appropriate sera or IgGs weresubstituted for primary antibodies. These sections had noappreciable signal (unpublished observations). Inmerged images, we found that ang1–256 localizes withphalloidin on CMs, vWF on ECs, and SMA on SMCs,appearing yellow in each case (Fig. 5).

Ang1–256 reduces PE-induced cardiac hypertrophy

Ang1 monomer levels decrease in PE-induced cardiachypertrophy (Fig. 3B), so we determined whether replace-ment of ang1 monomer attenuates cardiac hypertrophy.We retroorbitally injected C57 mice with ang1–256/pcDNA or pcDNA alone, and the next day implantedosmotic pumps containing PE or saline. Mice in controland PE groups received pcDNA. Mice in control andang1–256 groups received saline-filled pumps. On day 6,we found that PE increased LV/tibia ratios, whereas micegiven PE � ang1–256 had significantly lower LV/tibiaratios (Fig. 6A). We examined mRNA levels (real-timePCR) of fetal genes that increase during cardiac hypertro-phy. PE increased mRNA levels of ANP (Fig. 6B), BNP(Fig. 6C), collagen III (Fig. 6D), skeletal actin (Fig. 6E),and �MHC (Fig. 6F). In contrast, ang1–256 decreasedmRNA levels for each of those fetal genes (Fig. 6B–F) aswell as for SMA (Fig. 6G) mRNA levels � PE treatment.Ang1–256 protein completely blocked the PE-inducedincreases in collagen III, skeletal actin, and �MHC, main-taining normal transcript levels.

Ang1–256 does not change tie2 phosphorylation, butup-regulates ILK

Ang1 monomers do not bind or activate tie2. However,Ang1 binding to EC �5�1 integrin in vitro induces com-plexation and activation of tie2 (26). Thus, we deter-mined whether ang1–256 plasmid-based expression inheart tissue changes tie2 phosphorylation by conductingtie2 immunoprecipitation and Western blotting on heartsfrom C57 mice injected with ang1–256/pcDNA orpcDNA (control). At 1 and 2 days after plasmid injection,levels of tie2 phosphorylation were similar in hearts fromcontrol and ang1–256/pcDNA-injected mice (Fig. 7A).We also measured tie2 phosphorylation in hearts of micewith PE-induced cardiac hypertrophy. On day 6, levels of

to right: P0.029, 0.014) increase with age, whereas other dimmers and trimers are stable (arrows indicate ang1 forms). D) InPE-induced remodeling, blots probed with anti-ang1 (R&D monoclonal) show no change in monomer (55 and 70 kDa), dimer,and trimer levels. E) Fluorescent immunohistochemistry and confocal imaging of ang1 forms in perfusion-fixed mouse LV;studies done in triplicate. F) CMs are labeled with troponin-I linked to Texas Red (red) and ECs are tagged with FITC-lectin(green). G) LV labeled with anti-ang1 (Santa Cruz) conjugated to Alexa Fluor 647 (red), and CMs autofluoresce (green).Labeling of CMs and ECs indicates that ang1 multimers interact with both cell types. H) Anti-ang1 (Rockland) conjugated toAlexa Fluor 568 (red), which recognizes only monomers, immunostains CMs and ECs in vivo. I) Rabbit sera (negative control)were substituted for anti-ang1 (Rockland). Scale bars 10 �m.


tie2 phosphorylation were similar among the groups (Fig.7B). We examined (Western blotting) the effect of ang1–256 on mouse LV ILK protein levels. Ang1–256 � PEincreased ILK (Fig. 7C), a major regulator of integrinsubunit �1- and �3-based signaling, survival, and CMcontractility (14), which suggests that ang1–256 actionsare integrin mediated.

Ang1–256 monomer activates Akt, MAPKp42/44,and AMPK

To assess the effect of ang1–256 monomer on cardiacfunction, we examined major cardioprotective regula-tors of survival and energetics (akt, MAPKp42/44, AMPK,and FAK). We tracked LV levels of phosphorylated vs.total receptor over time (day 1–6) (Fig. 8A, C, E) and�PE-induced cardiac hypertrophy (Fig. 8B, D, F).Ang1–256 monomer phosphorylated aktS473 and

AMPKT172 with the largest increases on day 1, and lesserincreases on days 2–6 (Fig. 8A, C) and �PE treatment(Fig. 8B, D). PE-induced decreased AMPKT172 phosphor-ylation (Fig. 8D) is consistent with increased proteinsynthesis during pathological cardiac hypertrophy andreduces cardiac energetics. Ang1–256-mediated increases inAMPKT172 phosphorylation suggest reduced protein syn-thesis. Ang1–256 monomer increased phosphorylation ofMAPKp42(T202)/44(T204) on day 1 only (Fig. 8E–F). Ang1–256 monomer did not alter FAKY397 or AMPKS485/S491phosphorylation on days 1–6 or �PE-induced cardiachypertrophy (unpublished observations).

DISCUSSION

Ang1 mRNA/protein (Fig. 1) levels shift during physi-ological and pathological cardiac remodeling without

Figure 4. Ang1–256 mono-mers are expressed in hearttissue. A) Recombinant trun-cated ang1–256 has a modi-fied influenza hemagluttininsecretion signal (HS) andFLAG (F) tag fused to ang1 atamino acid 256. Ang1–256has 6 amino acids of thecoiled coil domain (C) andthe linker (L) and fibrino-gen-like domains of ang1 fol-lowed by C-terminal V5 and6� polyhistidine (H) tags. B,C) Ang1–256/pcDNA (ang1–256) or pcDNA control (con)were retroorbitally injectedinto C57 mice in triplicatestudies. cDNA synthesis reac-tions conducted �RT withang1–256/pcDNA plasmid(positive control) show LV

ang1–256 mRNA at all time points (B). Fluorescent immunohistochemistry and confocal imaging of mouse heart tissueshows ang1–256 protein at all time points (C). Ang1–256 was labeled using FLAG antibody linked to Alexa Fluor 647, andnuclei were labeled using DAPI. Negative controls included substituting rabbit IgG for primary antibody and injectingmice with pcDNA vector (control) (1 day). Scale bars 20 �m. D) Western blot analysis of HEK293 cells transfected withang1–256/pcDNA. Ang1–256 proteins were purified by anti-FLAG M1 affinity column chromatography (Sigma),dialyzed, and concentrated. Western blots probed with V5 antibody show ang1–256 monomer protein at the predictedmolecular mass (34 kDa) in nonreduced (NR) and reduced (R) conditions.


changes in tie2 phosphorylation (Figs. 2 and 7). Thisdisparity was also seen in an MI model (27). Thesefindings support a novel mechanism of action for ang1in heart tissue, which our prior work suggested isintegrin-mediated (3). Herein we show it is associatedwith changes in ILK, a molecule central to integrin-based signaling.

We show that integrin-binding ang1 monomers areabundant in mouse and human heart tissue (Fig. 2)and that they shift during cardiac remodeling (Fig. 3)and bind CMs and ECs (Fig. 3). These findings suggesta major role for ang1 monomer/integrin interactionsin cardiac health and disease. Ang1–256 increased ILKin mouse LV (Fig. 7C). ILK binds the cytoplasmic tail of�1 and �3 integrins, and we identified CM �1 and �3subunits as those that interact with ang1 (3). ILK isabundant in human and mouse heart tissue and regu-lates physiological cardiac hypertrophy (28). ILK abla-tion in murine heart tissue causes cardiomyopathy andheart failure (28). In humans, loss of ILK causes adilated cardiomyopathy. ILK promotes cell survival viaaktS473. We showed that full-length ang1 (3) and ang1–256 monomers phosphorylate aktS473 (Fig. 8A, B) inmouse heart tissue and isolated CMs. ILK is importantto CMs, which rely on ILK- and integrin-based interac-tions to support the force of contraction. Increased ILK

blocks cardiac fibrosis, and we show that ang1–256blocked PE-induced increases in collagen III (Fig. 6D).

Here, we introduce a new regulatory mechanism forang1 receptor targeting that is based on changes indifferent ang1 quaternary forms. EC tie2 activationrequires ang1 tetramers or larger (9, 29). Cartilageoligomeric matrix protein fused to ang1 linker/fibrin-ogen-like domains (COMP-ang1) forms mainly tetra-mers and pentamers and increases EC tie2 phosphory-lation more than native ang1 (29, 30). COMP-ang1, butnot native ang1, tracks with tie2 in adult heart andother tissues. Our data may explain this finding, in thatwe show native ang1 in mouse and human heart andother tissues is predominately composed of monomers,dimers, and trimers (Fig. 2), and these forms may actchiefly via integrins.

Ang1 monomer and multimer expressions were tis-sue specific. Multimers were abundant in adipose tis-sue, lung, and kidney (Fig. 2E, F). Monomers wereprevalent in heart tissue (Fig. 2G, H). In support of thisfinding, COMP-ang1 weakly stains heart tissue butprominently labels lung tissue (30). Further, braintissue has little tie2 (31), and we did not detect ang1multimers in brain tissue, only monomers (Fig. 2).Ang1 has an antipermeability effect in brain tissue, andintegrin-binding recombinant ang1 monomers blocked

Figure 5. Ang1–256 protein in heart tissue localizes to CMs, SMCs, and ECs. Ang1–256/pcDNA (ang1–256) was retroorbitallyinjected into C57 mice (1 day), and fluorescent immunohistochemistry with confocal imaging was conducted on heart tissue intriplicate studies. CMs are labeled with Alexa Fluor 488-linked phalloidin (red), ECs are tagged with vWF antibody andbiotinylated anti-rabbit IgG linked to strepavidin-linked Alexa Fluor 568 (red), SMCs are tagged with �-SMA antibody linked toAlexa Fluor 568 (red), ang1–256 is immunostained with FLAG antibody conjugated to Alexa Fluor 647 (green), and nuclei arestained with DAPI (blue). Merged images show that ang1–256 localizes to CMs, ECs, and SMCs. Scale bars 50 �m.


trans-EC permeability (13). Thus, our data suggest anew role for ang1 monomer/integrin interactions inbrain tissue.

We found ang1 monomers and dimers in all tissuestested (Fig. 2), however, other studies using solubletie2-Fc identified ang1 trimers, tetramers, and pentam-ers (10, 12). Because monomers and dimers show weakor no tie2 binding, they may not be detected withtie2-Fc. Use of soluble tie2-Fc to block the actions ofang1 may not disrupt monomer and dimer functionsand may prevent ang1 from binding integrins and tie1.

Also, our studies demonstrated the importance of ang1antibody selection, as antibodies differ in the ang1forms they detect (Fig. 2).

Akt and MAPKp42/44 (32) activities are decreased infailing human hearts (33). Akt phosphorylation pre-vents CM apoptosis and positively affects CM inotro-pism (34). Our studies show that integrin-bindingang1–256 monomer retains at least some of thefunctions of full-length ang1 (3), including akt andMAPKp42/44 activation (Fig. 8A, B, E, F). Anotherrecombinant truncated ang1 that produces monomers

Figure 6. Ang1–256 monomer reduces cardiac hypertrophy in vivo. Ang1–256/pcDNA (ang1–256) or pcDNA (control) was retroorbitally injected into C57mice. On day 2, mice were given PE or saline via pumps (n4/group; studiesdone in duplicate). A) At day 6, PE increased LV/tibia ratios, and ang1–256monomer blunted PE-induced increases in LV/tibia ratios. *P 0.009;**P0.02. B–G) RT-PCR analysis shows PE up-regulated mRNA levels of ANP(B), BNP (C), collagen III (D), skeletal actin (E), and (F) �MHC, but not SMA(G). Ang1–256 monomer reduced ANP, BNP, collagen III, skeletal actin,�MHC, and SMA mRNA levels in the absence or presence of PE. *P � 0.03;**P � 0.02; ***P 0.006.

Figure 7. Ang1–256 monomer does not change LV tie2 phosphorylation, but increases ILK. A–C) Ang1–256/pcDNA (ang1–256)or pcDNA (control) was retroorbitally injected into C57 mice. A, B) Results of tie2 immunoprecipitation and Western blottingfor pTyr and tie2 (studies done in duplicate) show that tie2 phosphorylations were unchanged in adult heart tissue on days 1and 2 after plasmid injection (A) and on day 6 in PE-induced cardiac hypertrophy (B). C) Six days after plasmid injection,Western blotting of mouse LV shows that ILK protein levels were increased by ang1–256, either alone or with PE-induced cardiachypertrophy. Studies done in duplicate; n 4/group. *P 0.01; **P 0.03. ILK was normalized to GAPDH.


Figure 8. Ang1–256 monomer activates Akt, AMPK, and MAPKp42/44. Ang1–256/pcDNA (ang1–256) or pcDNA (control) wasretroorbitally injected into C57 mice. On day 2 (B, D, F), mice were given PE or saline (control) via pumps. Western blottingon mouse hearts measured phosphorylated vs. total protein (n4/group; studies done in duplicate). Ang1–256 activated aktS473(A) and AMPKT172 (C) at 1 (*P�0.01), 2 (**P�0.03), 3 (#P�0.05), and 6 (##P�0.04) days, and MAPKp42(T202)/44(T204) (E) onday 1 (*P0.05) only. On day 6 (B, D, F), ang1–256 monomers phosphorylated LV akt S473 (B) and AMPKT172 (D) without (*P�0.02) or with PE treatment (**P�0.01), but did not alter MAPKp42/44 phosphorylation (F).


and dimers and binds integrins, but does not phosphor-ylate tie2, also has some capacities of full-length ang1(promotes EC adhesion, activates MAPKp42/44, andreduces EC permeability) (13). Besides CMs, ang1–256localized to cardiac ECs and SMCs, indicating thatthese monomers act on vasculature, likely via integrins.Ang1 binds human umbilical vein endothelial cell(HUVEC) integrins (4), and ang1-driven angiogenesisin chorioallantoic membrane assays was inhibited com-pletely by anti-�5�1 and partly by anti-�v�3 function-blocking antibodies (26). Thus, ang1 monomer/integrininteractions may mediate some EC functions in vivo.

Here, we introduced new functions for ang1–256monomers, including AMPK phosphorylation and de-creased cardiac hypertrophy and fetal gene expressions.These new cardioprotective actions have several implica-tions, all of which are consistent with improved cardiacfunction and energetics in injured myocardium.

AMPK phosphorylation is largely determined by �subunit phosphorylation at threonine 172 (35) andincreases energy production and inhibits apoptosis,protecting the heart during stress (36). AMPK is a keyregulator of energetics and is known as the “guardianof energy status” (37) in heart tissue. AMPK responds tostress by activating ATP-generating pathways (glycolysis,glucose uptake, fatty acid oxidation, and glycogenoly-sis) and down-regulating ATP-consuming pathways(protein synthesis and hypertrophy).

MI, hypertension, cardiac hypertrophy, and heartfailure are associated with increases in collagen III.Increased collagen III causes fibrosis and myocardialstiffening (38). Ang1–256 monomers blunt PE-inducedincrease in collagen III, which may decrease patholog-ical cardiac remodeling, preserving LV function.

In summary, we show that integrin-binding ang1monomers have a regulatory role in cardiac remodel-ing. Ang1–256 monomers localize to CMs, ECs, andSMCs and block PE-induced cardiac hypertrophy, acti-vating cardioprotective signaling pathways. These dataintroduce a novel mechanism of ang1 regulation,whereby quaternary forms are differentially expressedin vivo as a function of tissue type and remodeling stateto direct receptor/cell type interactions. Such a mech-anism may serve to coordinate ang1’s actions as a CMsurvival factor and vascular regulator during cardiacremodeling to preserve function and impede the devel-opment of heart failure.

This work was supported by U.S. National Institutes ofHealth (NIH) grant K02-HL071840-01, American Heart Asso-ciation grant-in-aid 0455824T, grant R21-CA107976-01, and aThomas Smith award to M.A.R.; grant K01-DK063970-01 anda David M. Bray Scholars in Medicine award to S.M.D; andNIH grant P01-CA45548 and philanthropic funds provided toDr. Judah Folkman (Children’s Hospital, Boston, MA, USA).

REFERENCES

1. Dumont, D. J., Yamaguchi, T. P., Conlon, R. A., Rossant, J., andBreitman, M. L. (1992) tek, a novel tyrosine kinase gene located

on mouse chromosome 4, is expressed in endothelial cells andtheir presumptive precursors. Oncogene 7, 1471–1480

2. Korhonen, J., Partanen, J., Armstrong, E., Vaahtokari, A., Ele-nius, K., Jalkanen, M., and Alitalo, K. (1992) Enhanced expres-sion of the tie receptor tyrosine kinase in endothelial cellsduring neovascularization. Blood 80, 2548–2555

3. Dallabrida, S. M., Ismail, N., Oberle, J. R., Himes, B. E., andRupnick, M. A. (2005) Angiopoietin-1 promotes cardiac andskeletal myocyte survival through integrins. Circ. Res. 96, e8–24

4. Carlson, T. R., Feng, Y., Maisonpierre, P. C., Mrksich, M., andMorla, A. O. (2001) Direct cell adhesion to the angiopoietinsmediated by integrins. J. Biol. Chem. 276, 26516–26525

5. Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre,P. C., Davis, S., Sato, T. N., and Yancopoulos, G. D. (1996)Requisite role of angiopoietin-1, a ligand for the TIE2 receptor,during embryonic angiogenesis. Cell 87, 1171–1180

6. Takahashi, K., Ito, Y., Morikawa, M., Kobune, M., Huang, J.,Tsukamoto, M., Sasaki, K., Nakamura, K., Dehari, H., Ikeda, K.,Uchida, H., Hirai, S., Abe, T., and Hamada, H. (2003) Adeno-viral-delivered angiopoietin-1 reduces the infarction and atten-uates the progression of cardiac dysfunction in the rat model ofacute myocardial infarction. Mol. Ther. 8, 584–592

7. Siddiqui, A. J., Blomberg, P., Wardell, E., Hellgren, I., Eskan-darpour, M., Islam, K. B., and Sylven, C. (2003) Combination ofangiopoietin-1 and vascular endothelial growth factor genetherapy enhances arteriogenesis in the ischemic myocardium.Biochem. Biophys. Res. Commun. 310, 1002–1009

8. Chen, S. L., Zhang, B. R., Mei, J., Xu, Z. Y., Zhu, J. L., Cai, K. H.,Huang, S. D., and Liu, Y. L. (2003) Induction of angiogenesis inischemic myocardium by adenovirus mediated angiopoietin-1 genetransfer, an experimental study. Zhonghua Yi Xue Za Zhi 83, 637–640

9. Davis, S., Papadopoulos, N., Aldrich, T. H., Maisonpierre, P. C.,Huang, T., Kovac, L., Xu, A., Leidich, R., Radziejewska, E.,Rafique, A., Goldberg, J., Jain, V., Bailey, K., Karow, M., Fandl, J.,Samuelsson, S. J., Ioffe, E., Rudge, J. S., Daly, T. J., Radziejewski,C., and Yancopoulos, G. D. (2003) Angiopoietins have distinctmodular domains essential for receptor binding, dimerizationand superclustering. Nat. Struct. Biol. 10, 38–44

10. Procopio, W. N., Pelavin, P. I., Lee, W. M., and Yeilding, N. M.(1999) Angiopoietin-1 and -2 coiled coil domains mediatedistinct homo-oligomerization patterns, but fibrinogen-like do-mains mediate ligand activity. J. Biol. Chem. 274, 30196–30201

11. Xu, Y., and Yu, Q. (2001) Angiopoietin-1, unlike angiopoietin-2,is incorporated into the extracellular matrix via its linkerpeptide region. J. Biol. Chem. 276, 34990–34998

12. Kim, K.-T., Choi, H.-H., Steinmetz, M. O., Maco, B., Kammerer,R. A., Ahn, S. Y., Kim, H.-Z., Lee, G. M., and Koh, G. Y. (2005)Oligomerization and multimerization are critical for angiopoi-etin-1 to bind and phosphorylate tie2. J. Biol. Chem. 280,20126–20131

13. Weber, C. C., Cai, H., Ehrbar, M., Kubota, H., Martiny-Baron,G., Weber, W., Djonov, V., Weber, E., Mallik, A. S., Fussenegger,M., Frei, K., Hubbell, J. A., and Zisch, A. H. (2005) Effects ofprotein and gene transfer of the angiopoietin-1 fibrinogen-likereceptor-binding domain on endothelial and vessel organiza-tion. J. Biol. Chem. 280, 22445–22453

14. Hannigan, G. E., Coles, J. G., and Dedhar, S. (2007) Integrin-linked kinase at the heart of cardiac contractility, repair, anddisease. Circ. Res. 100, 1408–1414

15. Dallabrida, S. M., Zurakowski, D., Shih, S. C., Smith, L. E.,Folkman, J., Moulton, K. S., and Rupnick, M. A. (2003) Adiposetissue growth and regression are regulated by angiopoietin-1.Biochem. Biophys. Res. Commun. 311, 563–571

16. McMahon, D. K., Anderson, P. A., Nassar, R., Bunting, J. B.,Saba, Z., Oakeley, A. E., and Malouf, N. N. (1994) C2C12 cells:biophysical, biochemical, and immunocytochemical properties.Am. J. Physiol. 266, C1795–1802

17. Kim, N. N., Villarreal, F. J., Printz, M. P., Lee, A. A., andDillmann, W. H. (1995) Trophic effects of angiotensin II onneonatal rat cardiac myocytes are mediated by cardiac fibro-blasts. Am. J. Physiol. Endocrinol. Metab. 269, E426–E437

18. Mitcheson, J. S., Hancox, J. C., and Levi, A. J. (1998) Culturedadult cardiac myocytes: future applications, culture methods,morphological and electrophysiological properties. Cardiovasc.Res. 39, 280–300

19. Schoenfeld, J. R., Vasser, M., Jhurani, P., Ng, P., Hunter, J. J.,Ross, J., Jr., Chien, K. R., and Lowe, D. G. (1998) Distinct


molecular phenotypes in murine cardiac muscle development,growth, and hypertrophy. J. Mol. Cell. Cardiol. 30, 2269–2280

20. Kim, I., Ryan, A. M., Rohan, R., Amano, S., Agular, S., Miller,J. W., and Adamis, A. P. (1999) Constitutive expression of VEGF,VEGFR-1, and VEGFR-2 in normal eyes. Invest. Ophthalmol. Vis.Sci. 40, 2115–2121

21. Goede, V., Schmidt, T., Kimmina, S., Kozian, D., and Augustin,H. G. (1998) Analysis of blood vessel maturation processesduring cyclic ovarian angiogenesis. Lab. Invest. 78, 1385–1394

22. Davis, S., Aldrich, T. H., Jones, P. F., Acheson, A., Compton,D. L., Jain, V., Ryan, T. E., Bruno, J., Radziejewski, C., Maison-pierre, P. C., and Yancopoulos, G. D. (1996) Isolation ofangiopoietin-1, a ligand for the TIE2 receptor, by secretion-trapexpression cloning. Cell 87, 1161–1169

23. Bragonzi, A., Boletta, A., Biffi, A., Muggia, A., Sersale, G.,Cheng, S. H., Bordignon, C., Assael, B. M., and Conese, M.(1999) Comparison between cationic polymers and lipids inmediating systemic gene delivery to the lungs. Gene Ther. 6,1995–2004

24. Chemin, I., Moradpour, D., Wieland, S., Offensperger, W. B.,Walter, E., Behr, J. P., and Blum, H. E. (1998) Liver-directedgene transfer: a linear polyethlenimine derivative mediateshighly efficient DNA delivery to primary hepatocytes in vitroand in vivo. J. Viral. Hepat. 5, 369–375

25. Ferrari, S., Pettenazzo, A., Garbati, N., Zacchello, F., Behr, J. P.,and Scarpa, M. (1999) Polyethylenimine shows properties ofinterest for cystic fibrosis gene therapy. Biochim. Biophys. Acta1447, 219–225

26. Cascone, I., Napione, L., Maniero, F., Serini, G., and Bussolino,F. (2005) Stable interaction between alpha5beta1 integrin andTie2 tyrosine kinase receptor regulates endothelial cell responseto Ang-1. J. Cell Biol. 170, 993–1004

27. Sandhu, R., Teichert-Kuliszewska, K., Nag, S., Proteau, G., Robb,M. J., Campbell, A. I., Kuliszewski, M. A., Kutryk, M. J., andStewart, D. J. (2004) Reciprocal regulation of angiopoietin-1and angiopoietin-2 following myocardial infarction in the rat.Cardiovasc. Res. 64, 115–124

28. White, D. E., Coutu, P., Shi, Y. F., Tardif, J. C., Nattel, S., St.Arnaud, R., Dedhar, S., and Muller, W. J. (2006) Targetedablation of ILK from the murine heart results in dilatedcardiomyopathy and spontaneous heart failure. Genes Dev. 20,2355–2360

29. Cho, C. H., Kammerer, R. A., Lee, H. J., Steinmetz, M. O., Ryu,Y. S., Lee, S. H., Yasunaga, K., Kim, K. T., Kim, I., Choi, H. H.,

Kim, W., Kim, S. H., Park, S. K., Lee, G. M., and Koh, G. Y.(2004) COMP-Ang1: a designed angiopoietin-1 variant withnonleaky angiogenic activity. Proc. Natl. Acad. Sci. U. S. A. 101,5547–5552

30. Cho, C. H., Kammerer, R. A., Lee, H. J., Yasunaga, K., Kim, K. T.,Choi, H. H., Kim, W., Kim, S. H., Park, S. K., Lee, G. M., andKoh, G. Y. (2004) Designed angiopoietin-1 variant, COMP-Ang1, protects against radiation-induced endothelial cell apo-ptosis. Proc. Natl. Acad. Sci. U. S. A. 101, 5553–5558

31. Wong, A. L., Haroon, Z. A., Werner, S., Dewhirst, M. W.,Greenberg, C. S., and Peters, K. G. (1997) Tie2 expression andphosphorylation in angiogenic and quiescent adult tissues. Circ.Res. 81, 567–574

32. Ravingerova, T., Barancik, M., and Strniskova, M. (2003) Mito-gen-activated protein kinases: a new therapeutic target in car-diac pathology. Mol. Cell. Biochem. 247, 127–138

33. Baba, H. A., Stypmann, J., Grabellus, F., Kirchhof, P., Sokoll, A.,Schafers, M., Takeda, A., Wilhelm, M. J., Scheld, H. H., Takeda,N., Breithardt, G., and Levkau, B. (2003) Dynamic regulation ofMEK/Erks and Akt/GSK-3beta in human end-stage heart failureafter left ventricular mechanical support: myocardial mechano-transduction-sensitivity as a possible molecular mechanism. Car-diovasc. Res. 59, 390–399

34. Latronico, M. V. G., Costinean, S., Lavitrano, M. L., Peschle, C.,and Condorelli, G. (2004) Regulation of cell size and contractilefunction by AKT in cardiomyocytes. Ann. N. Y. Acad. Sci. 1015,250–260

35. Kemp, B. E., Mitchelhill, K. I., Stapleton, D., Michell, B. J.,Chen, Z. P., and Witters, L. A. (1999) Dealing with energydemand: the AMP-activated protein kinase. Trends Biochem. Sci.24, 22–25

36. Russell, R. R., 3rd, Li, J., Coven, D. L., Pypaert, M., Zechner, C.,Palmeri, M., Giordano, F. J., Mu, J., Birnbaum, M. J., and Young,L. H. (2004) AMP-activated protein kinase mediates ischemicglucose uptake and prevents postischemic cardiac dysfunction,apoptosis, and injury. J. Clin. Invest. 114, 495–503

37. Hardie, D. G. (2004) AMP-activated protein kinase: the guard-ian of cardiac energy status. J. Clin. Invest. 114, 465–468

38. Fedak, P. W., Verma, S., Weisel, R. D., and Li, R. K. (2005)Cardiac remodeling and failure From molecules to man (PartII). Cardiovasc. Pathol. 14, 49–60

Received for publication December 19, 2007.Accepted for publication April 10, 2008.


Integrin binding angiopoietin-1 monomers reduce cardiac hypertrophy

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