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Loss of Timp3 Gene Leads to Abdominal Aortic Aneurysm Formation in Response to Angiotensin II*

  • Ratnadeep Basu
    Footnotes
    Affiliations
    Departments of Physiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

    Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, Alberta T6G 2S2, Canada
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  • Dong Fan
    Affiliations
    Departments of Physiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

    Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, Alberta T6G 2S2, Canada
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  • Vijay Kandalam
    Footnotes
    Affiliations
    Departments of Physiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

    Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, Alberta T6G 2S2, Canada
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  • Jiwon Lee
    Affiliations
    Departments of Physiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

    Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, Alberta T6G 2S2, Canada
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  • Subhash K. Das
    Affiliations
    Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, Alberta T6G 2S2, Canada

    Department of Medicine, Division of Cardiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
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  • Xiuhua Wang
    Affiliations
    Departments of Physiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

    Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, Alberta T6G 2S2, Canada
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  • Troy A. Baldwin
    Footnotes
    Affiliations
    Departments of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
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  • Gavin Y. Oudit
    Footnotes
    Affiliations
    Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, Alberta T6G 2S2, Canada

    Department of Medicine, Division of Cardiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
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  • Zamaneh Kassiri
    Correspondence
    A new investigator of the Heart and Stroke Foundation of Canada and an AI-HS scholar. To whom correspondence should be addressed: Dept. of Physiology, University of Alberta, 474 HMRC, Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-9283; Fax: 780-492-975
    Affiliations
    Departments of Physiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

    Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, Alberta T6G 2S2, Canada
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  • Author Footnotes
    * This work was supported by Canadian Institute of Health Research Grant 84279 and a Heart and Stroke Foundation of Canada grant (to Z. K.).
    1 Supported by an Alberta Innovates-Health Solutions (AI-HS) studentship.
    2 An AI-HS scholar.
    3 An AI-HS clinician-scientist.
Open AccessPublished:November 09, 2012DOI:https://doi.org/10.1074/jbc.M112.425652
      Aortic aneurysm is dilation of the aorta primarily due to degradation of the aortic wall extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs), the proteases that degrade the ECM. Timp3 is the only ECM-bound Timp, and its levels are altered in the aorta from patients with abdominal aortic aneurysm (AAA). We investigated the causal role of Timp3 in AAA formation. Infusion of angiotensin II (Ang II) using micro-osmotic (Alzet) pumps in Timp3−/− male mice, but not in wild type control mice, led to adverse remodeling of the abdominal aorta, reduced collagen and elastin proteins but not mRNA, and elevated proteolytic activities, suggesting excess protein degradation within 2 weeks that led to formation of AAA by 4 weeks. Intriguingly, despite early up-regulation of MMP2 in Timp3−/−Ang II aortas, additional deletion of Mmp2 in these mice (Timp3−/−/Mmp2−/−) resulted in exacerbated AAA, compromised survival due to aortic rupture, and inflammation in the abdominal aorta. Reconstitution of WT bone marrow in Timp3−/−/Mmp2−/− mice reduced inflammation and prevented AAA in these animals following Ang II infusion. Treatment with a broad spectrum MMP inhibitor (PD166793) prevented the Ang II-induced AAA in Timp3−/− and Timp3−/−/Mmp2−/− mice. Our study demonstrates that the regulatory function of TIMP3 is critical in preventing adverse vascular remodeling and AAA. Hence, replenishing TIMP3, a physiological inhibitor of a number of metalloproteinases, could serve as a therapeutic approach in limiting AAA development or expansion.

      Introduction

      Abdominal aortic aneurysm (AAA)
      The abbreviations used are:
      AAA
      abdominal aortic aneurysm
      Ang II
      angiotensin II
      ECM
      extracellular matrix
      GT
      Gomori trichrome
      MMP
      matrix metalloproteinase
      TIMP
      tissue inhibitor of metalloproteinase
      VVG
      Verhoff-Van Gieson
      MMPi
      MMP inhibitor
      BM
      bone marrow.
      is a degenerative vascular disorder characterized by dilation of the aorta due to destructive remodeling of the aortic wall and degradation of the fibrillar proteins of the vascular extracellular matrix (ECM). AAA has remained an unresolved clinical problem because β-blockers (
      • Baxter B.T.
      • Terrin M.C.
      • Dalman R.L.
      Medical management of small abdominal aortic aneurysms.
      ,
      • Propanolol Aneurysm Trial Investigators
      Propranolol for small abdominal aortic aneurysms: results of a randomized trial.
      ), statin therapy (
      • Ferguson C.D.
      • Clancy P.
      • Bourke B.
      • Walker P.J.
      • Dear A.
      • Buckenham T.
      • Norman P.
      • Golledge J.
      Association of statin prescription with small abdominal aortic aneurysm progression.
      ), and angiotensin-converting enzyme inhibitors have been ineffective in controlling AAA expansion, and AAA remains as the 13th leading cause of death in Western countries (
      • Golledge J.
      • Norman P.E.
      Current status of medical management for abdominal aortic aneurysm.
      ,
      • Isselbacher E.M.
      Thoracic and abdominal aortic aneurysms.
      ,
      • Danyi P.
      • Elefteriades J.A.
      • Jovin I.S.
      Medical therapy of thoracic aortic aneurysms: are we there yet?.
      ). Surgical and mechanical interventions are the only effective treatments to prevent AAA rupture, but that is only recommended in severe AAA cases where the inherent risk of surgery is less than the risk of aortic rupture (
      • Baxter B.T.
      • Terrin M.C.
      • Dalman R.L.
      Medical management of small abdominal aortic aneurysms.
      ,
      • Danyi P.
      • Elefteriades J.A.
      • Jovin I.S.
      Medical therapy of thoracic aortic aneurysms: are we there yet?.
      ). The 10-year survival for patients deemed unfit for surgical repair is less than 25% (
      • United Kingdom EVAR Trial Investigators
      • Greenhalgh R.M.
      • Brown L.C.
      • Powell J.T.
      • Thompson S.G.
      • Epstein D.
      Endovascular repair of aortic aneurysm in patients physically ineligible for open repair.
      ). Additionally, the recent Tromsø study indicates that the incident of AAA is greater in males compared with female patients (
      • Singh K.
      • Bønaa K.H.
      • Jacobsen B.K.
      • Bjørk L.
      • Solberg S.
      Prevalence of and risk factors for abdominal aortic aneurysms in a population-based study: the Tromsø study.
      ,
      • Forsdahl S.H.
      • Solberg S.
      • Singh K.
      • Jacobsen B.K.
      Abdominal aortic aneurysms, or a relatively large diameter of non-aneurysmal aortas, increase total and cardiovascular mortality: the Tromsø study.
      ), and similar findings have been reported in animal models (
      • Zhang X.
      • Thatcher S.E.
      • Rateri D.L.
      • Bruemmer D.
      • Charnigo R.
      • Daugherty A.
      • Cassis L.A.
      Transient exposure of neonatal female mice to testosterone abrogates the sexual dimorphism of abdominal aortic aneurysms.
      ). As such, understanding the molecular mechanism underlying AAA development, expansion, and rupture is critical in developing effective therapies for this disease.
      Arterial ECM is primarily composed of collagen and elastin fibers that provide significant structural support and recoil properties for the arteries (
      • Cummins P.M.
      • von Offenberg Sweeney N.
      • Killeen M.T.
      • Birney Y.A.
      • Redmond E.M.
      • Cahill P.A.
      Cyclic strain-mediated matrix metalloproteinase regulation within the vascular endothelium: a force to be reckoned with.
      ,
      • Wagenseil J.E.
      • Mecham R.P.
      Vascular extracellular matrix and arterial mechanics.
      ). Matrix metalloproteinases (MMPs) degrade the ECM structural proteins, whereas their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), keep their activity in check. An imbalance in the interaction between MMPs and TIMPs has been reported in the aorta of patients with abdominal aneurysm (
      • Knox J.B.
      • Sukhova G.K.
      • Whittemore A.D.
      • Libby P.
      Evidence for altered balance between matrix metalloproteinases and their inhibitors in human aortic diseases.
      ). Among the four TIMPs identified in mammals, TIMP3 is ECM-bound whereby it can exert tissue-specific effects (
      • Leco K.J.
      • Khokha R.
      • Pavloff N.
      • Hawkes S.P.
      • Edwards D.R.
      Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues.
      ,
      • Yu W.H.
      • Yu S.
      • Meng Q.
      • Brew K.
      • Woessner Jr., J.F.
      TIMP-3 binds to sulfated glycosaminoglycans of the extracellular matrix.
      ). TIMP3 protein levels are reduced in the aorta of patients with Marfan syndrome with increased rate of aortic rupture (
      • Ikonomidis J.S.
      • Jones J.A.
      • Barbour J.R.
      • Stroud R.E.
      • Clark L.L.
      • Kaplan B.S.
      • Zeeshan A.
      • Bavaria J.E.
      • Gorman 3rd, J.H.
      • Spinale F.G.
      • Gorman R.C.
      Expression of matrix metalloproteinases and endogenous inhibitors within ascending aortic aneurysms of patients with Marfan syndrome.
      ). Timp3 mRNA levels are increased in dilated aorta from patients with aortic aneurysm, whereas other TIMPs were not altered (
      • Tsarouhas K.
      • Soufla G.
      • Apostolakis S.
      • Zaravinos A.
      • Panagiotou M.
      • Khoury M.
      • Hassoulas J.A.
      • Tsatsakis A.M.
      • Spandidos D.A.
      Transcriptional regulation of TIMPs in ascending aorta aneurysms.
      ), However, TIMP3 protein levels were not measured in this study. In addition, a significant interaction between polymorphisms of Timp3, but not Timp1 or Timp2, occurs in patients with AAA and with a positive family history of AAA (
      • Ogata T.
      • Shibamura H.
      • Tromp G.
      • Sinha M.
      • Goddard K.A.
      • Sakalihasan N.
      • Limet R.
      • MacKean G.L.
      • Arthur C.
      • Sueda T.
      • Land S.
      • Kuivaniemi H.
      Genetic analysis of polymorphisms in biologically relevant candidate genes in patients with abdominal aortic aneurysms.
      ). Hence, the causal role of TIMP3 in AAA development remains to be determined.
      Our study examined the role of Timp3 in angiotensin II (Ang II)-induced vascular remodeling and formation of AAA. We found that Ang II infusion led to AAA in mice lacking Timp3 but not in WT mice. Although deletion of Mmp2 in Timp3−/− mice resulted in heightened inflammation and more severe AAA, broad spectrum inhibition of MMPs prevented AAA formation.

      DISCUSSION

      Abdominal aortic aneurysm is a common and lethal vascular disorder as about 6–9% of the elderly population have an AAA (
      • Danyi P.
      • Elefteriades J.A.
      • Jovin I.S.
      Medical therapy of thoracic aortic aneurysms: are we there yet?.
      ,
      • Ruddy J.M.
      • Jones J.A.
      • Spinale F.G.
      • Ikonomidis J.S.
      Regional heterogeneity within the aorta: relevance to aneurysm disease.
      ). Although small aneurysms can be managed conservatively with imaging and controlling of risk factors such as smoking and hypertension, patients continue to experience significant morbidity and mortality from ruptured aneurysms (
      • Golledge J.
      • Norman P.E.
      Current status of medical management for abdominal aortic aneurysm.
      ,
      • Danyi P.
      • Elefteriades J.A.
      • Jovin I.S.
      Medical therapy of thoracic aortic aneurysms: are we there yet?.
      ). Development and expansion of AAA result from disruption of the orderly structure of the aortic wall and ECM. Elastin and collagen fibers, which are the main structural proteins of the aortic ECM, underlie the recoil properties and impart strength to the vessel wall, respectively (
      • Cummins P.M.
      • von Offenberg Sweeney N.
      • Killeen M.T.
      • Birney Y.A.
      • Redmond E.M.
      • Cahill P.A.
      Cyclic strain-mediated matrix metalloproteinase regulation within the vascular endothelium: a force to be reckoned with.
      ,
      • Wagenseil J.E.
      • Mecham R.P.
      Vascular extracellular matrix and arterial mechanics.
      ). TIMP3 is the only ECM-bound TIMP, and its altered levels have been linked to aortic rupture (
      • Ikonomidis J.S.
      • Jones J.A.
      • Barbour J.R.
      • Stroud R.E.
      • Clark L.L.
      • Kaplan B.S.
      • Zeeshan A.
      • Bavaria J.E.
      • Gorman 3rd, J.H.
      • Spinale F.G.
      • Gorman R.C.
      Expression of matrix metalloproteinases and endogenous inhibitors within ascending aortic aneurysms of patients with Marfan syndrome.
      ) and aortic aneurysm (
      • Tsarouhas K.
      • Soufla G.
      • Apostolakis S.
      • Zaravinos A.
      • Panagiotou M.
      • Khoury M.
      • Hassoulas J.A.
      • Tsatsakis A.M.
      • Spandidos D.A.
      Transcriptional regulation of TIMPs in ascending aorta aneurysms.
      ), and its polymorphism showed strong association with AAA in patients with a family history of AAA (
      • Ogata T.
      • Shibamura H.
      • Tromp G.
      • Sinha M.
      • Goddard K.A.
      • Sakalihasan N.
      • Limet R.
      • MacKean G.L.
      • Arthur C.
      • Sueda T.
      • Land S.
      • Kuivaniemi H.
      Genetic analysis of polymorphisms in biologically relevant candidate genes in patients with abdominal aortic aneurysms.
      ). In this study, we provide evidence for the causal role of Timp3 deficiency in the development of AAA by demonstrating that mice lacking Timp3 are more susceptible to Ang II-induced AAA. We found a rise in TIMP3 levels in the abdominal aorta of WT mice following Ang II infusion. This is consistent with the finding in patients with aortic aneurysm that the increase in Timp3 mRNA was a compensatory response to the augmented MMP activity (
      • Tsarouhas K.
      • Soufla G.
      • Apostolakis S.
      • Zaravinos A.
      • Panagiotou M.
      • Khoury M.
      • Hassoulas J.A.
      • Tsatsakis A.M.
      • Spandidos D.A.
      Transcriptional regulation of TIMPs in ascending aorta aneurysms.
      ).
      We used the Ang II-infused model of aortic aneurysm formation in the absence of dyslipidemia and/or metabolic syndrome (
      • Ruddy J.M.
      • Jones J.A.
      • Spinale F.G.
      • Ikonomidis J.S.
      Regional heterogeneity within the aorta: relevance to aneurysm disease.
      ). Ang II is a physiological hormone that is elevated in patients with cardiovascular diseases (
      • Tomaschitz A.
      • Pilz S.
      • Ritz E.
      • Morganti A.
      • Grammer T.
      • Amrein K.
      • Boehm B.O.
      • März W.
      Associations of plasma renin with 10-year cardiovascular mortality, sudden cardiac death, and death due to heart failure.
      ,
      • Javed U.
      • Deedwania P.C.
      Angiotensin receptor blockers: novel role in high-risk patients.
      ,
      • Böhm M.
      Angiotensin receptor blockers versus angiotensin-converting enzyme inhibitors: where do we stand now?.
      ) and has been shown to exert direct effects on vascular remodeling and function in numerous studies (
      • Satoh K.
      • Nigro P.
      • Matoba T.
      • O'Dell M.R.
      • Cui Z.
      • Shi X.
      • Mohan A.
      • Yan C.
      • Abe J.
      • Illig K.A.
      • Berk B.C.
      Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms.
      ,
      • Flamant M.
      • Placier S.
      • Dubroca C.
      • Esposito B.
      • Lopes I.
      • Chatziantoniou C.
      • Tedgui A.
      • Dussaule J.C.
      • Lehoux S.
      Role of matrix metalloproteinases in early hypertensive vascular remodeling.
      ,
      • Brands M.W.
      • Banes-Berceli A.K.
      • Inscho E.W.
      • Al-Azawi H.
      • Allen A.J.
      • Labazi H.
      Interleukin 6 knockout prevents angiotensin II hypertension: role of renal vasoconstriction and janus kinase 2/signal transducer and activator of transcription 3 activation.
      ). The Ang II infusion model allowed us to examine the role of TIMP3 in the adverse remodeling of the aortic wall leading to aortic aneurysm formation. In this study, we report the following. First, the regulatory function of TIMP3 is essential in preventing AAA development. Second, despite the greater early rise in MMP2 activation in the Timp3−/− abdominal aorta, specific deletion of MMP2 led to inflammation, elevated MMP9 levels, and exacerbated AAA. Third, WT bone marrow reconstitution prevented inflammation, MMP9 up-regulation, and AAA in the double deficient mice. Fourth, simultaneous inhibition of a number of ECM-degrading proteinases (MMPs) could be a more effective approach in treating AAA.
      The saline-infused Timp3−/− mice showed MMP activities and aortic structure comparable with WT-saline group and did not develop AAA, suggesting that under normal conditions the remaining TIMPs (-1, -2, and -4) are sufficient to keep the activity of MMPs under control in the absence of TIMP3. However, in the presence of a pathological stimulus such as Ang II, TIMP3 is critical in regulating the proteolytic activities for optimal remodeling in the abdominal aortic wall. TIMP3 has been reported to hinder the activation of pro-MMP2 (72 kDa) into its cleaved (64-kDa) form (
      • English J.L.
      • Kassiri Z.
      • Koskivirta I.
      • Atkinson S.J.
      • Di Grappa M.
      • Soloway P.D.
      • Nagase H.
      • Vuorio E.
      • Murphy G.
      • Khokha R.
      Individual Timp deficiencies differentially impact pro-MMP-2 activation.
      ), and consistently, we found that the absence of TIMP3 promoted activation of pro-MMP2 into its 64-kDa form in the Timp3−/−-Ang II aortas. However, Timp3−/−/Mmp2−/− mice revealed that MMP2 activation is not the deriving factor in AAA formation in these mice. The more severe AAA in Timp3−/−/Mmp2−/−-Ang II mice is particularly interesting because MMP2 has been strongly linked to AAA in patients (
      • Goodall S.
      • Crowther M.
      • Hemingway D.M.
      • Bell P.R.
      • Thompson M.M.
      Ubiquitous elevation of matrix metalloproteinase-2 expression in the vasculature of patients with abdominal aneurysms.
      ,
      • Crowther M.
      • Goodall S.
      • Jones J.L.
      • Bell P.R.
      • Thompson M.M.
      Localization of matrix metalloproteinase 2 within the aneurysmal and normal aortic wall.
      ), and Mmp2−/− mice are protected against CaCl2-induced AAA (
      • Longo G.M.
      • Xiong W.
      • Greiner T.C.
      • Zhao Y.
      • Fiotti N.
      • Baxter B.T.
      Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms.
      ). However, in the absence of TIMP3, MMP2 deletion resulted in adverse outcomes such as inflammation and MMP9 up-regulation. MMP2 has been shown to play a role in early stages of vascular remodeling such as in aneurysm formation (
      • Goodall S.
      • Crowther M.
      • Hemingway D.M.
      • Bell P.R.
      • Thompson M.M.
      Ubiquitous elevation of matrix metalloproteinase-2 expression in the vasculature of patients with abdominal aneurysms.
      ,
      • Crowther M.
      • Goodall S.
      • Jones J.L.
      • Bell P.R.
      • Thompson M.M.
      Localization of matrix metalloproteinase 2 within the aneurysmal and normal aortic wall.
      ), whereas MMP9 is suggested to be involved in later stages such as aneurysm expansion (
      • Goodall S.
      • Crowther M.
      • Hemingway D.M.
      • Bell P.R.
      • Thompson M.M.
      Ubiquitous elevation of matrix metalloproteinase-2 expression in the vasculature of patients with abdominal aneurysms.
      ,
      • Freestone T.
      • Turner R.J.
      • Coady A.
      • Higman D.J.
      • Greenhalgh R.M.
      • Powell J.T.
      Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm.
      ). MMP9 has been identified as the key elastase involved in vascular degeneration and aneurysm (
      • Thompson R.W.
      • Holmes D.R.
      • Mertens R.A.
      • Liao S.
      • Botney M.D.
      • Mecham R.P.
      • Welgus H.G.
      • Parks W.C.
      Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages.
      ,
      • Pyo R.
      • Lee J.K.
      • Shipley J.M.
      • Curci J.A.
      • Mao D.
      • Ziporin S.J.
      • Ennis T.L.
      • Shapiro S.D.
      • Senior R.M.
      • Thompson R.W.
      Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.
      ), and it can also degrade other vascular ECM proteins such as basement membrane collagen type IV (
      • Murphy G.
      • Cockett M.I.
      • Ward R.V.
      • Docherty A.J.
      Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan. A quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and -2 and punctuated metalloproteinase (PUMP).
      ). Mice lacking Mmp9 are protected against elastase-induced AAA (
      • Thompson R.W.
      • Holmes D.R.
      • Mertens R.A.
      • Liao S.
      • Botney M.D.
      • Mecham R.P.
      • Welgus H.G.
      • Parks W.C.
      Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages.
      ) as well as CaCl2-induced AAA (
      • Longo G.M.
      • Xiong W.
      • Greiner T.C.
      • Zhao Y.
      • Fiotti N.
      • Baxter B.T.
      Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms.
      ). In this study, we demonstrate that reconstitution of WT bone marrow in Timp3−/−/Mmp2−/− mice suppressed infiltration of inflammatory cells, reduced MMP9 levels, and prevented the Ang II-induced AAA formation, whereas no MMP2 was detected in Timp3−/−/Mmp2−/−-chimera mice. In agreement with our findings, it has been reported that reconstitution of WT macrophages in Mmp9−/− mice, but not in Mmp2−/− mice, led to the development of AAA in the Mmp9−/− mice that are otherwise resistant to CaCl2-induced AAA; therefore, the macrophage-derived MMP9 and the MMP2 produced by mesenchymal cells were proposed to work in concert to mediate AAA (
      • Longo G.M.
      • Xiong W.
      • Greiner T.C.
      • Zhao Y.
      • Fiotti N.
      • Baxter B.T.
      Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms.
      ). Furthermore, production of MMP2 by mesenchymal cells has been shown to be enhanced by the presence of inflammatory cells (
      • Davis V.
      • Persidskaia R.
      • Baca-Regen L.
      • Itoh Y.
      • Nagase H.
      • Persidsky Y.
      • Ghorpade A.
      • Baxter B.T.
      Matrix metalloproteinase-2 production and its binding to the matrix are increased in abdominal aortic aneurysms.
      ). This is consistent with our observation of elevated MMP2 levels in WT-chimera mice (containing Timp3−/−/Mmp2−/− bone marrow) in which the only possible source of MMP2 is in fact the native cells and not the Timp3−/−/Mmp2−/− inflammatory cells.
      Elastin degradation products in AAA samples from patients exhibit chemotactic activity that attracts macrophages to the site of AAA, leading to inflammation (
      • Hance K.A.
      • Tataria M.
      • Ziporin S.J.
      • Lee J.K.
      • Thompson R.W.
      Monocyte chemotactic activity in human abdominal aortic aneurysms: role of elastin degradation peptides and the 67-kD cell surface elastin receptor.
      ). Because the inflammatory cells such as neutrophils and macrophages produce MMP9 (
      • Cuadrado E.
      • Ortega L.
      • Hernández-Guillamon M.
      • Penalba A.
      • Fernández-Cadenas I.
      • Rosell A.
      • Montaner J.
      Tissue plasminogen activator (t-PA) promotes neutrophil degranulation and MMP-9 release.
      ,
      • Lambert J.M.
      • Lopez E.F.
      • Lindsey M.L.
      Macrophage roles following myocardial infarction.
      ) and other ECM-degrading metalloproteinases (
      • Shapiro S.D.
      • Campbell E.J.
      • Senior R.M.
      • Welgus H.G.
      Proteinases secreted by human mononuclear phagocytes.
      ,
      • Shapiro S.D.
      • Campbell E.J.
      • Welgus H.G.
      • Senior R.M.
      Elastin degradation by mononuclear phagocytes.
      ), this initiates a vicious cycle, leading to further destruction of the aortic wall structure, aortic expansion, and more severe AAA. TIMP1 has been shown be protective against aortic aneurysm (
      • Ikonomidis J.S.
      • Gibson W.C.
      • Butler J.E.
      • McClister D.M.
      • Sweterlitsch S.E.
      • Thompson R.P.
      • Mukherjee R.
      • Spinale F.G.
      Effects of deletion of the tissue inhibitor of matrix metalloproteinases-1 gene on the progression of murine thoracic aortic aneurysms.
      ,
      • Eskandari M.K.
      • Vijungco J.D.
      • Flores A.
      • Borensztajn J.
      • Shively V.
      • Pearce W.H.
      Enhanced abdominal aortic aneurysm in TIMP-1-deficient mice.
      ,
      • Allaire E.
      • Forough R.
      • Clowes M.
      • Starcher B.
      • Clowes A.W.
      Local overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat model.
      ), whereas Timp2 deficiency posed beneficial outcomes in a CaCl2 model of AAA (
      • Xiong W.
      • Knispel R.
      • Mactaggart J.
      • Baxter B.T.
      Effects of tissue inhibitor of metalloproteinase 2 deficiency on aneurysm formation.
      ).
      The current study is the first report on the role of TIMP3 and AAA. In this study, we demonstrate that lack of TIMP3 triggers adverse remodeling of aortic wall and AAA formation in response to Ang II. In addition, inhibition of one specific MMP did not provide beneficial outcomes in limiting AAA progression, whereas inhibition of a number of ECM-targeting MMPs was a more effective approach. Consistent with our findings, doxycycline, a broad spectrum MMP inhibitor, has been a promising treatment in preventing AAA expansion and rupture (
      • Baxter B.T.
      • Pearce W.H.
      • Waltke E.A.
      • Littooy F.N.
      • Hallett Jr., J.W.
      • Kent K.C.
      • Upchurch Jr., G.R.
      • Chaikof E.L.
      • Mills J.L.
      • Fleckten B.
      • Longo G.M.
      • Lee J.K.
      • Thompson R.W.
      Prolonged administration of doxycycline in patients with small asymptomatic abdominal aortic aneurysms: report of a prospective (Phase II) multicenter study.
      ,
      • Mosorin M.
      • Juvonen J.
      • Biancari F.
      • Satta J.
      • Surcel H.M.
      • Leinonen M.
      • Saikku P.
      • Juvonen T.
      Use of doxycycline to decrease the growth rate of abdominal aortic aneurysms: a randomized, double-blind, placebo-controlled pilot study.
      ,
      • Hackmann A.E.
      • Rubin B.G.
      • Sanchez L.A.
      • Geraghty P.A.
      • Thompson R.W.
      • Curci J.A.
      A randomized, placebo-controlled trial of doxycycline after endoluminal aneurysm repair.
      ,
      • Hackmann A.E.
      • Rubin B.G.
      • Sanchez L.A.
      • Geraghty P.A.
      • Thompson R.W.
      • Curci J.A.
      A randomized, placebo-controlled trial of doxycycline after endoluminal aneurysm repair.
      ,
      • Lindeman J.H.
      • Abdul-Hussien H.
      • van Bockel J.H.
      • Wolterbeek R.
      • Kleemann R.
      Clinical trial of doxycycline for matrix metalloproteinase-9 inhibition in patients with an abdominal aneurysm: doxycycline selectively depletes aortic wall neutrophils and cytotoxic T cells.
      ), whereas other treatments including β-blockers, angiotensin-converting enzyme inhibitors, and statins have been ineffective (
      • Baxter B.T.
      • Terrin M.C.
      • Dalman R.L.
      Medical management of small abdominal aortic aneurysms.
      ,
      • Propanolol Aneurysm Trial Investigators
      Propranolol for small abdominal aortic aneurysms: results of a randomized trial.
      ,
      • Ferguson C.D.
      • Clancy P.
      • Bourke B.
      • Walker P.J.
      • Dear A.
      • Buckenham T.
      • Norman P.
      • Golledge J.
      Association of statin prescription with small abdominal aortic aneurysm progression.
      ,
      • Golledge J.
      • Norman P.E.
      Current status of medical management for abdominal aortic aneurysm.
      ,
      • Danyi P.
      • Elefteriades J.A.
      • Jovin I.S.
      Medical therapy of thoracic aortic aneurysms: are we there yet?.
      ). Overall, our study provides evidence that TIMP3 is critical in constructive vascular remodeling, and as such, targeted overexpression of TIMP3 could serve as a promising therapeutic approach in preventing aneurysm formation or controlling its expansion.

      Acknowledgments

      We thank Bing Zhang for technical support and the Cardiovascular Research Center core facility at University of Alberta.

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