Articles in PresS. Am J Physiol Heart Circ Physiol (January 30, 2015). doi:10.1152/ajpheart.00470.2014 1

Heat Shock Protein 90 Inhibition by 17-DMAG Attenuates Abdominal Aortic Aneurysm

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Formation in Mice

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Jia Qi1,2*, Ping Yang1*, Bing Yi2, Yan Huo2, Ming Chen2, Jian Zhang1, Jianxin Sun2

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Department of Pharmacy, Xinhua Hospital, Shanghai Jiaotong University, Shanghai, China; 2Center

for Translational Medicine, Thomas Jefferson University, Philadelphia, PA;

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Running Title: Inhibition of Hsp90 Attenuates AAA formation

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*These authors contributed equally to this work.

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Correspondence:

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Jianxin Sun, PhD

Jian Zhang, M.D.

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Center for Translational Medicine

Department of Pharmacy

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Thomas Jefferson University

Xinhua Hospital

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1025 Walnut Street, Room 408

Second Jiaotong University School of Medicine

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Philadelphia, PA 19107, USA

1665 Kongjian Road, Shanghai 200092, China

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Tel: 215-503-9425

Tel: 086-2125077150

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FAX: 215-5035731

FAX: 086-2125077182

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Email Address: [email protected]

Email Address: [email protected]

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Copyright © 2015 by the American Physiological Society.

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ABSTRCT

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Abdominal aortic aneurysm (AAA) is a common degenerative vascular disease whose pathogenesis is

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associated with activation of multiple signaling pathways including Jun N-terminal kinases (JNK) and

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NF-κB. It is now well-recognized that these pathways are chaperoned by the heat shock protein 90

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(Hsp90), suggesting that inhibition of Hsp90 may be a novel strategy for inhibiting AAAs. The aim of

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this study is to Investigate whether inhibition of Hsp90 by 17-DMAG (17-dimethyl

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-aminothylamino-17-demethoxy- geldanamycin) attenuates AngII-induced AAA formation in mice, and

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if so, to elucidate the mechanisms involved. Apolipoprotein E-null mice were infused with AngII to

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induce AAA formation and simultaneously treated by intraperitoneal injection with either vehicle or

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17-DMAG for 4 weeks. AngII infusion induced AAA formation in 80% of mice, which was

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accompanied by increased matrix metalloproteinase (MMP) activity, enhanced tissue inflammation,

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oxidative stress, and neovascularization. Importantly, these effects were inhibited by 17-DMAG

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treatment. Mechanistically, we showed that 17-DMAG prevented the formation and progression of

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AAA through its inhibitory effects on diverse biological pathways including 1) by blocking AngII

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induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and JNK that are

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critically involved in the regulation of MMPs in vascular smooth muscle cells (VSMCs) 2) by

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inhibiting IκB kinase (IKK) expression and expression of MCP-1 and 3) by attenuating

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AngII-stimulated angiogenic processes critical to AAA formation. Our results demonstrate that

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inhibition of Hsp90 by 17-DMAG effectively attenuates AngII–induced AAA formation by

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simultaneously inhibiting vascular inflammation, extracellular matrix degradation, and angiogenesis,

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which are critical in the formation and progression of AAAs.

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Keywords: Aneurysms; angiotensin II; apolipoprotein E-null mouse; heat shock protein 90

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INTRODUCTION

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Abdominal aortic aneurysms (AAAs) occur in approximately 9% of older men and account for

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more than 15000 deaths annually in the United States (9, 51). Pathological processes of AAA are

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complex, but mainly characterized by significant degradation of extracellular matrix including elastin

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and collagen, increased activity of matrix metalloproteinases (MMPs), excessive local inflammation,

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and neovascularization of the media and adventitia(7) (16) (32) ,Currently there is no effective

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pharmacological therapy available to prevent the development and progression of AAA, and surgical

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repair of late-stage disease remains the only effective method of reducing aneurysm-related mortality..

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Therefore, identification of novel molecular targets is of considerable scientific and therapeutic

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interest(32).

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Infusion of Angiotensin II (AngII) with subcutaneous osmotic minipumps in apolipoprotein E-null

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(apoE-/-) mice is a widely used mouse model of AAA, which exhibits multiple characteristics of human

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AAAs, including disruption of media, secretion of MMPs, rupture of elastic layer, macrophage

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infiltration, and neovascularization(2, 10). Moreover, AngII has been shown to activate multiple

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signaling pathways, such as c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB) pathways in

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VSMCs, which are critically involved in AAA formation(11, 38). Accordingly, targeted inhibition of

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c-Jun N-terminal kinase (JNK) pathway, by either genetic or pharmacological approaches, has been

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shown to prevent experimental AAA formation(53). Similarly, inhibition of NF-κB signaling pathway

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has been reported to inhibit macrophage infiltration and inflammation in the adventitia and media of

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AAA model.(33) Nevertheless, these studies highlight the critical importance of both JNK and NF-κB

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pathways in the development of AAA.

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Hsp90 is an evolutionarily conserved and abundant molecular chaperone that participates in

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stabilizing and activating more than 200 proteinsincluding kinases, signaling molecules,and

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transcriptional factors, such as v-Src, Raf-1, ErbB2, HIF-1, HSF1, and some growth factor receptors(41)

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(45).. Because many Hsp90 client proteins are classified as oncogenic proteins, which promote cancer

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cell growth or survival or both(37), a number of highly potent and pharmaceutically improved Hsp90

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inhibitors have been developed, and entered into clinical trials for cancer treatment(45). 17-dimethyl

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-aminothylamino-17-demethoxy- geldanamycin (17-DMAG) is a selective Hsp90 inhibitor, which

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blocks the ATP binding site of Hsp90 to induce the degradation of some clientproteins (54). Recently, 3

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inhibition of Hsp90 by 17-DMAG has been shown to attenuate inflammatory responses and oxidative

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stress in experimental atherosclerosis, indicating that Hsp90 inhibitors may have potentials for

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treatment of certain cardiovascular diseases (27, 28). Since many important signaling proteins,

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including extracellular signal-regulated kinase (ERK), JNK, and NF-κB, which are critically involved

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in formation and progression of AAA, are the Hsp90 client proteins(8) (18), we speculated that

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inhibition of Hsp90 could be an effective therapeutic strategy for the treatment of AAA. Thus, in the

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present study, we investigated whether inhibition of Hsp90 by 17-DMAG attenuates AAA formation in

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mice, and if so, to determine the mechanism(s) involved.

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Materials and Methods

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Mice

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Male, apoE-/- mice at 10 weeks of age on a C57BL/6 background were obtained from the Model Animal

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Research Center of Nanjing University, China and bred in pathogen-free environment. The research

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conforms to the Guide for the care and Use of Laboratory Animals published by the US National

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Institutes of Health (NIH Publication NO.85-23, revised 1996) and the protocol was approved by the

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Institutional Animal care Committee at Shanghai Jiaotong University School of Medicine. Mice were

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sacrificed by using a gradually filling the chamber with CO2.

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Drug treatment

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Osmotic pumps (model 2004, Alzet) containing either AngII (1000 ng/min/kg, Sigma-Aldrich; n=40) or

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saline (n=10) were subcutaneously implanted into 10-week-old male apoE-/- mice as described

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previously(15) (40). AngII-treated mice were intraperitoneal (ip) injected with 5 mg/kg of 17-DMAG

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(Lclab), or vehicle every other day (3 times per week) during 4 weeks. This treatment regime is based

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on the previous notion indicating that low-dose 17-DMAG therapy (5 mg/kg, thrice per week i.p.)

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could efficiently inhibit Hsp90 activity without obvious toxic effects in mice (23) (43). The inhibitory

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effect of 17-DMAG on Hsp90 was determined by the induction of Hsp70 expression(42).

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Blood pressure measurements

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Blood pressure was measured in conscious mice by a tail-cuff system (Bp98A, softron, China).

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Analysis and quantification of AAA

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After perfusion with 4% paraformaldehyde, the abdominal aortas were harvested and immediately 4

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placed in PBS and cleaned of adventitial fat. The outer diameter of the suprarenal aorta was measured

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with a caliper under a dissecting microscope while the aortas were in PBS without physical stretching.

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To quantify aneurysm incidence, an aneurysm was defined as >50% increase in external diameter of

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suprarenal aorta compared to aortas from saline-infused mice, which is consistent with the clinical

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standard to diagnose abdominal aortic aneurysm(47). AAA severity was determined with a

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classification scheme described previously(14), where Type 1 represents a simple dilation of the

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abdominal aorta with an external diameter of 1.5-2 mm, Type 2 represents a AAA with the external

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diameter of 2-3 mm and Type 3 represents a pronounced bulbous containing a thrombus and an external

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aortic diameter of >3 mm. Mice in the Type 4 AAA category were those that died of aneurysmal

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rupture and resultant bleeding in the peritoneal cavity. AAA severity was also evaluated by measuring

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the wet weights of the abdominal aortas.

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Histology and immunohistochemistry

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Anaesthetized mice were perfused with normal saline and fixed with 10% PBS and formalin for 5 min.

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Whole aortas were harvested, fixed for 24 hr and embedded in paraffin, and cross-sections (5 μm) were

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prepared. Paraffin sections were stained with hematoxylin and eosin (H&E) and Vehoeff-van Geisen

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(VVG) for elastin, MAC3 for macrophages, and CD31 for endothelial cells. Antibody binding was

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detected using the Vectastain Elite ABC kit and di-amino benzidine (DAB) staining using

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manufacturer’s instructions (Vector labs, Burlingame, CA). Quantitation of immuno-positive cells was

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performed by determining the ratio of the number of positive cells to the total number of hematoxylin

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positive cells in a defined field on more than 10 slides per mouse.

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Cell culture

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Mouse vascular smooth muscle cells (VSMCs) were cultured in DMEM supplemented with 10% FBS,

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100U/L pencillin and 100ug/ml streptomycin. Human umbilical cord vein endothelial cells (HUVECs)

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were isolated and cultured as previously described(13). In all experiments, the cells were used between

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passages 3 and 8.

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Measurement of MMPs activity and MCP-1 secretion

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The evaluation of MMP-2 and MMP-9 activities in conditioned media form cells cultures or

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homogenates of aortic tissue was performed by zymography as described previously(50). MCP-1

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secretion was measured by ELISA (Pierce, Rockford, IL). 5

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Quantitation of mRNA expression

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Total RNA was extracted from cells or aortic tissue using TRIZOL (Invitrogen). 1μg total RNA was

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used to perform the reverse transcription with High Capacity cDNA Archive Kit (Applied Biosystem).

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Real-time quantitative polymerase chain reaction (PCR) analysis for MCP-1, MMP-2, and MMP-9 was

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performed using TaqMan gene expression assays and the ΔΔCt method with housekeeping gene 18S as

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the endogenous control. The primers used for the qRT-PCR are listed in the Supplemental Table 1.

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Table 1. Primers for PCR Mouse MMP-2 promoter

Mouse MMP-9 promoter

Mouse MMP-2 Mouse MMP-9 18S rRNA Mouse MCP-1 Human VEGF

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5’-AGCTAGTGGCTGCCATATGGAAACTGG-3’ 163 5’-GGTACCTGGTGGGAGCAGAACACACAT-3’

164 165 5’-GTA GTG TAA ACA CAC ACA CACA-3’ and 166 5’-AGT AAA ACG GAA TCA GTG ACC C-3’ for the 167 distal AP-1 site; 168 5’-CCC CAC ACT GTA GGT TCT ATC C-3 and 169 5-ATC CTG CCT CAA AGA GCCT-3’ for the 170 proximal AP-1 site 171 5’- ACCAAGAACTTCCGATTATCCC-3’ 172 5’- CAGTACCAGTGTCAGTATCAGC-3’ 173 5’- GATCCCCAGAGCGTCATTC-3’ 174 5’- CCACCTTGTTCACCTCATTTTG-3’ 175 5’-TCAAGAACGAAAGTCGGAGG-3’ 176 5’-GGACATCTAAGGGCATCAC-3’ 177 5’-GTCCCTGTCATGCTTCTGG-3’ 178 5’-GCTCTCCAGCCTACTCATTG-3’ 179 5’-ATCATGCGGATCAAACCTCACC-3’ 180 5’-GGTCTGCATTCACATCTGCTATGC-3’ 181 182

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Measurement of Malondialdehyde (MDA)

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The protein concentration was measured by using the BCA protein assay kit (Pierce, Rockford, IL).

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MDA levels were measured by an ELISA kit (Mybiosource, San Diego, CA), which applies the

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competitive enzyme immunoassay technique utilizing a monoclonal anti-MDA antibody and an

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MDA-HRP conjugate. Briefly, the assay sample and buffer were incubated together with MDA-HRP

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conjugate in pre-coated plate for 1 hr. After the incubation period, the wells were washed 5 times and

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then incubated with a substrate for HRP enzyme. A stop solution was added and the intensity of color 6

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was measured spectrophotometrically at 450nm in a microplate reader. The intensity of the color is

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inversely proportional to the MDA concentration since MDA from samples and MDA-HRP conjugate

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compete for the anti-MDA antibody binding site.

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Western blot

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Total proteins were extracted from corresponding area of aneurysm tissue or cells. Equal amounts of

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protein lysates were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF)

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membrane and then immunoblotted with antibodies against ERK1/2 (1:1000 dilution; Cell Signaling

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Technology), phosphor-ERK1/2 (1:1000 dilution; Cell Signaling Technology), JNK (1:1000 dilution;

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Cell Signaling Technology), and phosphor-JNK (1:500 dilution; Cell Signaling Technology). Protein

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bands were visualized by Odyssey imagining system (LICOR, Lincoln, NE).

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Electrophoretic mobility shift assay (EMSA)

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EMSA were performed with Odyssey® IRDye® 700 infrared dye labelled double-stranded

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oligonucleotides coupled with the EMSA buffer kit (LI-COR Bioscience) according to manufacturer's

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instructions.

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Chromatin immunoprecipitation (CHIP) assay for AP-1 and MMPs promoters binding

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ChIP assays of VSMCS were performed with a chip kit (Millipore, Billerica, MA) according to

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manufacturer's instructions. Briefly, Chromatin was cross-linked by adding formaldehyde to cell culture

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medium for 10 min at room temperature. Crosslinking reaction was ceased by glycine solution.

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Cross-linked chromatin was sheared by supersonic at 4 °C. Debris was removed by centrifugation and

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supernatants were collected. The purified chromatin was immunoprecipitated with c-Jun and c-Fos

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antibodies from Santa Cruz Biotechnology and normal rabbit IgG from Millipore. The DNA/Protein

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complexes were collected by the use of Protein G-argrose beads. Beads were washed and bound DNA

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was eluted. After reverse cross-linking reaction and proteinase K digestion, the eluted DNA was used in

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35 cycles of PCR amplification with the AP-1 binding site specific primers, which are listed in Table1.

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Statistical analysis

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Quantitative data are presented as mean ± SEM. Parameters between two groups were compared by

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2-tailed Student’s t test. Comparisons of parameters among multiple groups were made by one-way 7

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analysis of variance, and comparisons of different parameters between each group were made by

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Bonferroni’s post-hoc test. Exact Chi-square test was used to assess the statistical significance of

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aneurysm subtype variables. A χ2 test was used to compare AAA incidence and survival rate. A P value

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< 0.05 was considered to be statistically significant.

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RESULTS

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Hsp90 inhibitor 17-DMAG inhibits the incidence and severity of AngII-induced AAAs

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To elucidate whether Hsp90 participates in the development of AAA, we induced the AAA formation

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by the infusion of Ang II to apoE-/- mice for 4 weeks and the expression of Hsp90 was determined by

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western blot. As shown in Figure 1A, Hsp90 expression was significantly increased in the mouse AAA

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lesions. This observation prompted us to investigate whether inhibition of Hsp90 could attenuate the

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AAA formation in mice. To this end,we induced the AAA by the infusion of Ang II to apoE-/- mice and

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treated the mice simultaneously with intraperitoneal injection of either vehicle or 17-DMAG (5 mg/kg,

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every two days) for 4 weeks. The dose of the drug was chosen based on

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treatment and minimal toxicity at dosages under 15 mg/kg administered 3 days per week for 6 weeks

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(23) (43). Hsp90 inhibitors have been shown to disassociate the interaction of Hsp90 with HSF1, which

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in turn induces the expression of Hsp70 (6) . Therefore, assessment of HSP70 induction has been used

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as a useful pharmacodynamic marker of HSP90 inhibition. Indeed, treatment of mice with 5 mg/kg

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17-DMAG markedly increased Hsp70 expression in abdominal aorta by approximately 2-fold (Figure

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1B), but barely affected the body weight of the mice (Figure 1C), suggesting that the current treatment

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regimen is indeed effective for inhibiting Hsp90 in abdominal aorta. Furthermore, AngII infusion for 4

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weeks induced an 80% incidence (16 of 20 mice) of AAA in apoE-/- mice, whereas the incidence was

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significantly reduced to 10% (2 of 20 mice) in 17-DMAG-treated group (Figure 2A and 2B).

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17-DMAG also significantly decreased the severity of AngII-induced AAAs, as determined by a

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classification scheme based on the external diameter of the abdominal aortas (Figure 2C) as well as by

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measuring the wet weights of the abdominal aortas (Figure 2D).

studies showing effective

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17-DMAG attenuates remodeling of the aortic wall, inflammatory responses, and neovascularization

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in AngII-induced AAA

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HE staining of cross sections of the abdominal aortas showed that AngII treatment induced severe

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thickening and considerable destruction of the abdominal aortic walls (Figure 3A). In addition, elastin 8

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staining demonstrated that AngII treatment disrupted the elastin fibers in the suprarenal region of the

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aorta (Figure 3B and 3E) and increased infiltration of Mac-3 positive macrophages (Figure 3C and 3F)

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and formation of capillary vessels, as determined by CD31 immunochemical studies (Figure 3D and

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3G). Remarkably, all these parameters associated with AAA formation observed in AngII-infused mice

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were substantially inhibited by treatment of mice with Hsp90 inhibitor 17-DMAG (Figure 3A-3G).

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17-DMAG attenuates MMP-2 and MMP-9 expression, MDA levels, and MCP-1 secretion in

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AngII-induced AAA

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Given important roles of MMPs, ROS, and MCP-1 in AngII-induced AAA formation (32),we sought to

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investigate the effect of Hsp90 inhibition on expression of MMP-2 and MMP-9, ROS production, and

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MCP-1 secretion in homogenates of AAAs. As expected, MMP-2 and MMP-9 activity in homogenates

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of abdominal aortas were both increased significantly as compared to the abdominal aortas from the

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control group (Figure 4A). Both MMP-2 and MMP-9 activities, however, were significantly lower in

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AngII plus 17-DMAG-treated group as compared with AngII-treated alone (p