Table Of ContentJBC Papers in Press. Published on November 9, 2012 as Manuscript M112.425652
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.425652
MS ID#: JBC/2012/425652R1
Loss of Timp3 leads to abdominal aortic aneurysm formation in response to Angiotensin II
Ratnadeep Basu1,4, Dong Fan1,4, Vijay Kandalam1,4, Jiwon Lee1,4, Subhash Das2,4,
Xiuhua Wang1,4, Troy A Baldwin3, Gavin Y. Oudit2,4, Zamaneh Kassiri1,4
From 1Department of Physiology, 2Department of Medicine/Division of Cardiology,
3Dept. of Medical Microbiology & Immunology, University of Alberta, Edmonton, AB, Canada.
4Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, AB, Canada.
* Running Title: TIMP3 and abdominal aortic aneurysm.
To whom correspondence should be addressed: Zamaneh Kassiri, Associate Professor, Department of
Physiology, University of Alberta, 474 HMRC, Edmonton, Alberta, Canada, T6G 2S2; Tel: (780) 492-
9283, Fax: (780) 492-975; E-mail: [email protected].
Key words: Tissue inhibitor of metalloproteinase, Matrix metalloproteinase, extracellular matrix, aortic
aneurysm, elastin.
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ow
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Background: TIMP3 is ECM-bound and is Intriguingly, despite early upregulation of lo
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implicated in patients with abdominal aortic Mmp2 in Timp3-/-AngII aortas, additional de
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aneurysm (AAA). deletion of Mmp2 in these mice (Timp3-/-/Mmp2- fro
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Results: Timp3-deficiency triggers AAA in /-) resulted in exacerbated AAA, compromised h
response to Ang II. Additional deletion of Mmp2 survival due to aortic rupture, and ttp://w
exacerbated AAA with enhanced inflammation. inflammation in the abdominal aorta. w
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Broad-spectrum Mmp-inhibition prevented AAA Reconstitution of WT bone marrow in Timp3-/- .jb
in both genotypes. /Mmp2-/- mice reduced inflammation and c.o
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Conclusion: Timp3 is protective against Ang II- prevented AAA in these animals following Ang b/
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mediated adverse remodeling. II infusion. Treatment with a broad-spectrum g
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Significance: Replenishment of Timp3 in Mmp inhibitor (PD166793) prevented the Ang s
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aneurysmal aorta could prevent aneurysm II-induced AAA in Timp3-/- and in Timp3-/- n N
expansion and rupture. /Mmp2-/- mice. Our study demonstrates that the ov
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regulatory function of Timp3 is critical in
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SUMMARY preventing adverse vascular remodeling and r 2
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Aortic aneurysm is dilation of the aorta AAA. Hence, replenishing Timp3, a , 2
0
primarily due to degradation of the aortic wall physiological inhibitor of a number of 18
extracellular matrix (ECM). Tissue inhibitor of metalloproteinases, could serve as a therapeutic
metalloproteinases (TIMPs) inhibit Mmps, the approach in limiting AAA development or
proteases that degrade the ECM. Timp3 is the expansion.
only ECM-bound Timp and its levels are
altered in the aorta from patients with Abdominal aortic aneurysm (AAA) is a
abdominal aortic aneurysm (AAA). We degenerative vascular disorder characterized by
investigated the causal role of Timp3 in AAA dilation of the aorta due to destructive remodeling
formation. Infusion of angiotensin II (Ang II), of the aortic wall and degradation of the fibrillar
using micro-osmotic (Alzet) pumps, in Timp3-/- proteins of the vascular extracellular matrix
male mice, but not in wild type control mice, (ECM). AAA has remained an unresolved clinical
led to adverse remodeling of the abdominal problem since β-blockers (1,2), statin therapy (3),
aorta, reduced collagen and elastin proteins but and angiotensin converting enzyme (ACE)
not mRNA, and elevated proteolytic activities inhibitors have been ineffective in controlling
suggesting excess protein degradation within 2 AAA expansion, and AAA remains as the 13th
weeks that led to formation of AAA by 4 weeks. leading cause of death in Western countries (4-6).
1
Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
MS ID#: JBC/2012/425652R1
Surgical and mechanical interventions are the only EXPERIMENTAL PROCEDURES
effective treatments to prevent AAA rupture, but Experimental Animals and Procedures- Wildtype
that is only recommended in severe AAA cases (WT) mice were purchased from Jackson
where the inherent risk of surgery is less than the Laboratories. We cross-bred Timp3-/- (19) and
risk of aortic rupture (1,6). The ten-year survival Mmp2-/- (20) mice to generate Timp3-/-/Mmp2-/-
for patients deemed unfit for surgical repair is less mice. Timp3-/-/Mmp2-/- mice are healthy and
than 25% (7). Additionally, the recent Tromsø fertile. All mice are in C57BL/6 background.
study indicates that the incident of AAA is greater Alzet micro-osmotic pumps (Model 1002, Durect
in males compared to female patients (8,9), while Co.) were implanted subcutaneously in male mice
similar findings have been reported in animal of indicated gentoypes to deliver 1.5 mg/kg/day of
models (10). As such understanding the molecular Ang II (21-23) or saline (control). Broad spectrum
mechanism underlying AAA development, Mmp inhibitor (PD166793, MMPi) was
expansion and rupture is critical in developing administered orally by daily gavage as before (30
effective therapies for this disease. mg/kg/d) (24,25). All experiments were conducted
Arterial ECM is primarily comprised of in accordance with the guidelines of the
collagen and elastin fibres which provide University of Alberta Animal Care and Use
significant structural support and recoil properties Committee (ACUC) and the Canadian Council of
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for the arteries (11,12). Matrix metalloproteinases Animal Care. o
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(Mmps) degrade the ECM structural proteins Bone Marrow Reconstitution - Bone marrow lo
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while their inhibitors, tissue inhibitor of (BM) reconstitution was performed to generate de
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metalloproteinases (Timps) keep their activity in chimera mice as described previously (26). Donor fro
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check. An imbalance in the interaction between WT and Timp3-/-/Mmp2-/- mice received 100 μg of h
Mmps and TIMPs has been reported in the aorta of purified anti-CD8 Ab (clone 2.43) intraperitonealy ttp://w
patients with abdominal aneurysm (13). Among on days −2 and −1 before BM harvest to deplete w
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the four Timps identified in mammals, TIMP3 is CD8 T-cells. BM was harvested from the femur, .jb
ECM-bound whereby it can exert tissue-specific tibia, and humerus in EasySep media (PBS, 2% c.o
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effects (14,15). Timp3 protein levels are reduced FCS, 2 mM EDTA) and was passed through 70 b/
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in the aorta of patients with Marfan syndrome μm nylon cell strainers (Fisherbrand). Recipient g
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with increased rate of aortic rupture (16). Timp3 WT and Timp3-/-/ Mmp2-/- mice were sublethally es
mRNA levels are increased in dilated aorta from irradiated (1,000Gy), and received 10×106 BM t on
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patients with aortic aneurysm whereas other cells of the opposite genotype via tail vein o
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TIMPs were not altered (17), however, Timp3 injection. WT BM was reconstituted in Timp3-/-/ m
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protein levels were not measured in this study. In Mmp2-/- mice (Timp3-/-/ Mmp2-/--chimera mice), er 2
addition, a significant interaction between and Timp3-/-/ Mmp2-/- BM in WT (WT-chimera 4, 2
0
polymorphism of Timp3, but not Timp1 or Timp2, mice). Mice were provided with antibiotic water 18
occurs in patients with AAA and with a positive (40 mg neomycin, 15 mg polymyxin per 1 L) for 4
family history of AAA (18). Hence, the causal weeks post-injection, and 7 weeks were allowed
role of Timp3 in AAA development remains to be for reconstitution before Alzet pumps were
determined. implanted for Ang II infusion.
Our study examined the role of Timp3 in Ultrasonic Imaging of Abdominal Aorta-
angiotensin II (Ang II)-induced vascular Ultrasonic images of the abdominal aorta were
remodeling and formation of AAA. We found that obtained in mice anesthetized with isoflurane,
Ang II infusion led to AAA in mice lacking Timp3 using the Vevo 770 high resolution imaging
but not in WT mice. While deletion of Mmp2 in system equipped with the real-time
Timp3-/- mice resulted in heightened inflammation microvisualization scanhead (RMV 704; Visual
and more severe AAA, broad spectrum inhibition Sonics, Toronto, Canada) (27). The aortic
of Mmps prevented AAA formation. diameters were measured by M-mode, at the
suprarenal region where aneurysm was detected
(in Timp3-/- and Timp3-/-/ Mmp2-/- mice) (28). The
maximum aortic lumen diameter (aortic systolic
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diameter corresponding to cardiac systole) and the Statistical Analyses- The statistical analyses
minimum aortic lumen diameter (aortic diastolic were performed using SPSS software (Chicago,
diameter corresponding to cardiac diastole) Illinois, Version 10.1). We performed normality
monitored by simultaneous ECG recordings, were test (Shapiro-Wilk) and homogeneity of variance
measured and used to calculate aortic expansion test (Levene) to confirm normal distribution of all
index [(Systolic aortic diameter- Diastolic aortic data. To compare data sets with two factors
diameter)/systolic diameter X 100] (29). Three (genotype and treatment) we performed two-way
independent measurements were made for each analysis of variance (ANOVA) (Fig. 1-6). In
mouse. Figures 5-8, to compare 3 levels of the genotype,
Histological Analyses and Scanning Electron two-way ANOVA was followed by Bonferroni
Microscopy - Abdominal aortas were fixed in 10% post hoc testing if the results attained statistical
formalin, paraffin-embedded and used for Gomori significance (p<0.05). In Figure 7B, the effects of
Trichrome (GT), Verhoeff-Van Geison (VVG). MMPi in each genotype was tested using unpaired
Tissue processing and scanning electron t-test. Survival data (Fig. 4B) were compared
microscopy was done as before (25). Neutrophil using Kaplan-Meier survival curves followed by
and macrophage staining was performed as the log rank test adjusted with the Bonferroni
described previously (30) using anti-mouse method. Averaged values represent mean ± SEM.
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neutrophil antibody with Cy3-labeled anti-rat Statistical significance was recognized at p<0.05. o
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secondary antibody, and F4/80 macrophage lo
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antibody with Cy3-labeled anti-rat secondary RESULTS de
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antibody which was pseudo-coloured to appear Timp3-deficient mice exhibit adverse aortic fro
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green for visual differentiation from neutrophil remodelling after 2 weeks of angiotensin II h
staining. We used nuclear staining by DAPI to infusion - In WT mice, 2 weeks of Ang II infusion ttp://w
confirm the presence of infiltrating cells in significantly elevated Timp3 mRNA and protein w
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positively stained regions. Superimposed staining levels in the abdominal aorta (Fig. 1Ai and 1Aii). .jb
for neutrophil or macrophage with DAPI are To examine the role of Timp3 in Ang II-induced c.o
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presented. aortic remodeling, we subjected mice lacking b/
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Protein Extraction, Western Blot Analysis, Timp3 to Ang II infusion, we assessed the g
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Gelatin Zymography and mRNA analysis - Flash- predominant arterial structural proteins, elastin es
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frozen abdominal aorta was used for protein or and fibrillar collagen type I (12). Verhoeff-Van n
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RNA extraction. Western blot was performed to Gieson staining showed disorganized and o
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detect collagen type I (COL1A1) (Santa Cruz disrupted elastin fibres with a greater frequency of m
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Biotechnology) and α-elastin (Abcam Inc.), and in elastin fibre disruption in the abdominal aorta of r 2
vitro gelatin zymography as before (30). Timp3-/--Ang II compared to WT-Ang II mice 4, 2
0
Coomassie blue-stained SDS/PAGE gel (after (Fig. 1B, 1C). The Ang II-induced reduction in 18
proteins were transferred to PVDF membrane) elastin protein content was greater in Timp3-/-
was used as loading control. In situ gelatin abdominal aorta (Figure 1Di-ii) despite a larger
zymography was performed on 14µm cryosections increase in mRNA synthesis of elastin in these
as described (31). Total collagenase and gelatinase mice (Fig. 1Diii). Gomori Trichrome staining
activities were measured using Enzchek similarly revealed disorganized collagen structures
collagenase (Cat. No. D-12060) and elastase (Cat. (greenish-blue, Fig. 1E), and a drastic decrease in
No. E-12056) activity assay kits (from Millipore), collagen type I protein levels (Fig. 1Fi-ii) despite a
as before (30). Briefly, 50 µg of protein extract marked rise in its mRNA synthesis (Fig. 1Fiii) in
was incubated with fluorescent-labeled collagen Timp3-/--Ang II compared to WT-Ang II
elastin substrate. The generated fluorescence abdominal aortas. These data suggest that the
signal was recorded for 4 hours (Ex.=485nm, decrease in elastin and collagen protein levels in
Em.=515nm), and the slope of the curves were Timp3-deficient aorta is due to post-translational
used to calculate the collagenase or elastase degradation of these proteins and not reduced
activities. mRNA expression was measured using synthesis.
TaqMan real-time PCR (Table 1) (30,32).
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MS ID#: JBC/2012/425652R1
Enhanced Mmp2 activation in Timp3-/- mice Deletion of Mmp2 in Timp3-/- mice led to
following 2 weeks of Angiotensin II infusion - exacerbated AAA – We observed that after 2
Timp3 is a potent inhibitor of a number of active weeks of Ang II-infusion, Mmp9 levels increased
Mmps, and can inhibit cell-surface activation of similarly in both genotypes, whereas the increase
Mmp2 (33), hence its absence can lead to in Mmp2 levels was greater in the Timp3-/-
uncontrolled proteolytic activities (34). In situ compared to WT abdominal aorta (Fig. 2B). In
gelatin zymography showed that Ang II-infusion order to determine the contribution of the elevated
resulted in a stronger gelatinase activity in Timp3-/- Mmp2 activation in development of AAA in
compared to WT aorta (Fig. 2A). To determine the Timp3-/--Ang II mice, we generated Timp3-/-
contribution of individual gelatinases, namely /Mmp2-/- double-deficient mice. Saline-infused
Mmp2 and Mmp9, we performed in vitro gelatin Timp3-/- and Timp3-/-/Mmp2-/- mice showed similar
zymography which revealed a markedly greater aortic structure and expansion index compared to
Mmp2 activation in Timp3-/--Ang II abdominal WT mice (data not shown). Intriguingly, Timp3-/-
aorta, as indicated by a stronger 64kDa band, /Mmp2-/- mice exhibited more severe AAA after 4
while Mmp9 levels rose similarly in both weeks of Ang II-infusion compared to Timp3-/-
genotypes (Fig. 2Bi-ii). The increase in Mmp2 mice (Fig. 4A) as dilation of the abdominal aorta
activation was also associated with increased exceeded 50% of the original aortic diameter in
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mRNA expression (Fig. 2Ci), while Mmp9 mRNA 75% of these mice. In addition, while Timp3-/- o
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did not increase (Fig. 2Cii). Expression analysis of mice exhibited compromised survival post-Ang II lo
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other Mmps showed a greater increase in Mmp13 infusion, mortality was increased further in Timp3- de
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levels in Timp3-/--Ang II group (Fig. 2Ciii) while /-/Mmp2-/- mice (Fig. 4Bi). Routine autopsy fro
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membrane type Mmp (MT1-MMP) was elevated examination revealed that aortic rupture at the h
similarly in both genotypes (Fig. 2Civ). supra-renal region of the aorta where aneurysmal ttp://w
Timp3-deficient mice developed abdominal dilation was detected (Fig. 4Bii), was the cause of w
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aortic aneurysm (AAA) after 4 weeks of Ang II the increased mortality in these mice. .jb
infusion - Next, we examined the long term Morphometric analysis of the full cross-section of c.o
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outcome of these structural remodelling in the abdominal aorta in saline-infused WT compared b/
Timp3-/- aorta. After 4 weeks of Ang II infusion, to Ang II-infused WT, Timp3-/- and Timp3-/- y g
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Timp3-/- mice, but not WT mice, developed an /Mmp2-/- mice clearly shows the dilation in es
aneurysm in the abdominal aorta and not in the abdominal aorta in Timp3-/- mice, and a strikingly t on
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ascending or descending thoracic aorta (Fig. 3A). greater dilation in the Timp3-/-/Mmp2-/- mice (Fig. o
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Aneurysm is defined as a dilation of 50% or more 4C). Histological analyses at higher m
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in the abdominal aortic diameter (1,35), measured magnifications further revealed deterioration of r 2
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by ultrasound imaging. In WT mice, Ang II- the aortic wall at the site of aneurysm as indicated , 2
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induced dilation of the abdominal aorta did not by disrupted elastin fibres (VVG staining) and 18
exceed 20% of the original aortic diameter, collagen structure (GT staining) which were more
whereas 60% of Timp3-/- mice exhibited greater severe in Timp3-/-/Mmp2-/- mice (Fig. 4C). Aortic
than 50% aortic dilation in the supra-renal region. wall structures in saline-infused mice of the three
Representative ultrasonic images (Fig. 3B) and genotypes were comparable (data not shown).
averaged diameter of the abdominal aorta (Fig. Scanning electron microscopy allowed us to
3Ci-ii) demonstrate marked aortic dilation at the perform a more detailed evaluation of the
supra-renal region in Ang II-infused Timp3-/- structural remodelling of the abdominal aortic
compared to the parallel WT group. Aortic walls. Ang II infusion led to remodelling of the
systolic expansion index, a measure of aortic aortic wall in WT mice as indicated by compactly
elasticity and recoil property during systole and folded elastin lamellae (Fig. 4Di) which was more
diastole (29), was significantly suppressed in the evident at a higher magnification (Fig. 4Dii).
aneurysmal aorta in Timp3-/--Ang II mice (Fig. Timp3-deficient mice, on the other hand, exhibited
3Ciii) confirming the compromised structural and disrupted medial elastic lamellae and disrupted
functional integrity of the Timp3-deficient aorta. fibrillar structures (open arrows in Fig. 4Di) and
this structural deterioration was worsened in
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MS ID#: JBC/2012/425652R1
Timp3-/-/Mmp2-/--Ang II mice (Fig. 4Di-ii). (IL-1β), interleukin-6 (IL-6), and monocyte
Structure and diameter of the abdominal aorta in chemotactic protein-1 (Mcp-1) were significantly
saline-infused mice were comparable among the elevated in the aneurysmal aorta of Timp3-/-
genotypes (data not shown). These findings /Mmp2-/- mice compared to parallel WT and
demonstrate that despite the marked increase in Timp3-/- groups (Fig. 5Di-iii).
Mmp2 activation in Timp3-/- aortas early post-Ang Inflammation is a key factor in exacerbated
II infusion, its deletion indeed aggravated the AAA in Timp3-/-/Mmp2-/--Ang II mice - In order to
structural deterioration and the adverse determine the mechanism underlying the
remodeling in the abdominal aorta which resulted worsening of AAA in Timp3-/-/Mmp2-/--Ang II
in worsened AAA. mice, and the contribution of the observed
Timp3-/-/Mmp2-/--Ang II mice exhibited heightened inflammation in these aortas, we
heightened inflammation in abdominal aorta - reconstituted WT bone marrow in Timp3-/-/Mmp2-
Further examination of the Timp3-/-/Mmp2-/- mice /- mice, and vice versa. We generated Timp3-/-
revealed that Ang II-induced total collagenase /Mmp2-/--chimera mice by reconstituting WT bone
activity was the largest in the abdominal aorta of marrow in these mice, and WT-chimera mice by
Timp3-/- mice and was reduced significantly in reconstituting Timp3-/-/Mmp2-/- bone marrow in
Timp3-/-/Mmp2-/- group (Fig. 5Ai). This is WT mice. After 7 weeks of reconstitution
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consistent with Mmp2 also being a potent followed by 4 weeks of Ang II infusion, we found ow
collagenase (36). However, the Ang II-induced that Timp3-/-/Mmp2-/--chimera mice did not nlo
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rise in gelatinase activity was markedly greater in develop aortic aneurysm (Fig. 6A) as the diameter de
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Timp3-/-/Mmp2-/- compared to Timp3-/- abdominal of abdominal aorta (Fig. 6Bi-ii) and aortic fro
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aorta despite the absence of MMP2 (Fig. 5Aii). expansion index (Fig. 6Biii) in these mice were h
Mmp2 and Mmp9 are the classically known comparable to saline-infused animals. ttp://w
gelatinases, and as such the rise in total gelatinase Consistently, minimal infiltration of neutrophils w
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activity in the absence of Mmp2 implied an and macrophages were detected (Fig. 6Cii), while .jb
increase in Mmp9 activity. In vitro gelatin Mmp9 levels were significantly reduced (Fig. 6D) c.o
zymography confirmed a striking upregulation in in the Timp3-/-/Mmp2-/--chimera-Ang II mice. WT- brg/
Mmp9 levels in Ang II-infused Timp3-/-/Mmp2-/- chimera mice exhibited neutrophil and y g
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abdominal aortas compared to all other groups macrophage infiltration which was not detected in es
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(Fig. 5Bi-ii). This was also accompanied by Ang II-induced intact WT mice (Figure 6Ci). In n
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elevated mRNA expression of Mmp9 (Fig. 5Biii). vitro gelatin zymography showed increased ov
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The increase in MMP9 levels in WT and Timp3-/- MMP9 and a striking increase in MMP2 levels in m
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aortas after 2 weeks of Ang II infusion (Fig. 2B), the WT-chimera (Figure 6D). These data indicates r 2
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was dissipated by 4 weeks. This could perhaps be that inflammation was the underlying cause of , 2
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due to an early inflammatory response which exacerbated AAA in Timp3-/-/Mmp2-/--Ang II 18
occurred irrespective of the genotype. Mmp9 is a mice.
well known elastase that can degrade vascular Multiple Mmp inhibition prevented AAA in
elastin lamella (37) leading to invasion of Timp3-/- and Timp3-/-/Mmp2-/- mice - To further
inflammatory cells and inflammation (38) and investigate the contribution of the elevated
exacerbation of AAA (22). Immunofluorescent protease activity as the underlying mechanism for
staining for inflammatory cells, neutrophils and AAA in Timp3-/- and Timp3-/-/Mmp2-/- mice, we
macrophages (superimposed with DAPI-nucleus treated these mice with a broad-spectrum MMP
staining), showed enhanced infiltration of inhibitor, PD166793 (MMPi) (24,39) during the
neutrophils and macrophages in the abdominal course of Ang II infusion. Interestingly,
aorta of Ang II-infused Timp3-/-/Mmp2-/- mice simultaneous inhibition of a number of Mmps with
(Fig. 5C). Timp3-/--Ang II group showed a less this inhibitor blocked AAA development in
severe macrophage infiltration, while no Timp3-/- and significantly suppressed AAA
infiltrating cells were detected in WT-Ang II formation in Timp3-/-/Mmp2-/- mice (Fig. 7A).
abdominal aorta (Figure 5C). Consistently, mRNA Ultrasonic measurement of abdominal aortic
levels of inflammatory markers, interleukin-1β diameter showed that MMPi-treatment was
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MS ID#: JBC/2012/425652R1
strongly effective in preventing the Ang II- We used the Ang II-infused model of aortic
induced dilation of the abdominal aorta in Timp3-/- aneurysm formation in the absence of
and Timp3-/-/Mmp2-/- mice (Fig. 7Bi-ii), while dyslipidemia and/or metabolic syndrome (40).
aortic recoil property, as measured by systolic Ang II is a physiological hormone that is elevated
expansion index, was significantly restored in in patients with cardiovascular diseases (41-43)
MMPi-treated Timp3-/--Ang II mice and to a lesser and has been shown to exert direct effects on
extent in Timp3-/-/Mmp2-/--Ang II mice (Fig. vascular remodelling and function in numerous
7Biii). These improvements were well reflected in studies (22,44,45). The Ang II infusion model
the abdominal aortic wall structures as shown by allowed us to examine the role of Timp3 in the
intact elastin (VVG) and collagen (GT) structures adverse remodeling of the aortic wall leading to
(Fig. 8A), and preserved elastin lamella aortic aneurysm formation. In this study we report
organization as seen in the scanning electron that, first, the regulatory function of Timp3 is
microscopy images (Fig. 8B). Consistently, MMPi essential in preventing AAA development;
treatment prevented the elevation of collagenase second, despite the greater early rise in Mmp2
and gelatinase activities in the abdominal aorta of activation in the Timp3-/- abdominal aorta, specific
Ang II-infused Timp3-/- and Timp3-/-/Mmp2-/- mice deletion of MMP2 led to inflammation, elevated
(Fig. 8C). These data demonstrate the protective Mmp9 levels and exacerbated AAA; third, WT
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function of MMPi against Ang II-induced aortic bone marrow reconstitution prevented o
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wall deterioration which otherwise leads to AAA inflammation, Mmp9 upregulation and AAA in the lo
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in Timp3-/- and Timp3-/-/Mmp2-/- mice. double-deficient mice; and fourth, simultaneous de
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inhibition of a number of ECM-degrading fro
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DISCUSSION proteinases (Mmps) could be a more effective h
and leAthbadlo vmaisncaull aar odrtiisco rdaenre uarsy sambo uits 6a- 9%co mofm tohne approTachhe ins atrleinaeti-ningf uAsAedA . T imp3-/- mice showed ttp://ww
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elderly population have an AAA (6,40). While comparable Mmp activities and aortic structure to .jb
small aneurysms can be managed conservatively WT-saline group and did not develop AAA, c.o
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with imaging and controlling of risk factors such suggesting that under normal conditions, the b/
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as smoking and hypertension, patients continue to remaining Timps (-1, -2 and -4) are sufficient to g
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experience significant morbidity and mortality keep the activity of Mmps under control in the es
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from ruptured aneurysms (4,6). Development and absence of Timp3. However, in the presence of a n
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expansion of AAA result from disruption of the pathological stimulus such as Ang II, Timp3 is o
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orderly structure of the aortic wall and ECM. critical in regulating the proteolytic activities for m
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Elastin and collagen fibres are the main structural optimal remodeling in the abdominal aortic wall. r 2
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proteins of the aortic ECM, which underlie the Timp3 has been reported to hinder the activation , 2
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recoil properties and impart strength to the vessel of pro- Mmp2 (72kDa) into its cleaved (64 kDa) 18
wall, respectively (11,12). Timp3 is the only form (33), and consistently, we found that absence
ECM-bound Timp, and its altered levels have been of Timp3 promoted activation of pro- Mmp2 into
linked to aortic rupture (16) and aortic aneurysm its 64kDa form in the Timp3-/--AngII aortas.
(17) while its polymorphism showed strong However, Timp3-/-/Mmp2-/- mice revealed that
association with AAA in patients with a family Mmp2 activation is not the deriving factor in AAA
history of AAA (18). In this study we provide formation in these mice. The more severe AAA in
evidence for the causal role of Timp3-deficiency Timp3-/-/Mmp2-/--Ang II mice is particularly
in development of AAA by demonstrating that interesting since Mmp2 has been strongly linked to
mice lacking Timp3 are more susceptible to Ang AAA in patients (46,47), and Mmp2-/- mice are
II-induced AAA. We found a rise in Timp3 levels protected against CaCl -induced AAA (48).
2
in the abdominal aorta of WT mice following Ang However in the absence of Timp3, Mmp2 deletion
II infusion. This is consistent with the finding in resulted in adverse outcomes such as
patients with aortic aneurysm which reported that inflammation and Mmp9 upregulation. Mmp2 has
the increase in Timp3 mRNA was a compensatory been shown to play a role in early stages of
response to the augmented Mmp activity (17). vascular remodeling such as in aneurysm
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MS ID#: JBC/2012/425652R1
formation (46,47), while Mmp9 is suggested to be leading to inflammation (53). Since the
involved in later stages such as aneurysm inflammatory cells such as neutrophils and
expansion (46,49). Mmp9 has been identified as macrophages produce Mmp9 (54,55) and other
the key elastase involved in vascular degeneration ECM-degrading metalloproteinases (56,57), this
and aneurysm (37,50) which can also degrade initiates a vicious cycle leading to further
other vascular ECM proteins such as the basement destruction of the aortic wall structure, aortic
membrane collagen type IV (51). Mice lacking expansion and more severe AAA. Timp1 has been
Mmp9 are protected against elastase-induced AAA shown be protective against aortic aneurysm (58-
(37), as well as CaCl -induced AAA (48). In this 60), whereas Timp2-deficiency posed beneficial
2
study, we demonstrate that reconstitution of WT outcomes in a CaCl model of AAA (61).
2
bone marrow in Timp3-/-/Mmp2-/- mice suppressed The current study is the first report on the
infiltration of inflammatory cells, reduced MMP9 role of Timp3 and AAA. In this study we
levels and prevented the Ang II-induced AAA demonstrate that lack of Timp3 triggers adverse
formation, while no MMP2 was detected in remodeling of aortic wall and AAA formation in
Timp3-/-/Mmp2-/--chimera mice. In agreement with response to Ang II. In addition, inhibition of one
our findings, it has been reported that specific MMP did not provide beneficial outcomes
reconstitution of WT macrophages in Mmp9-/- in limiting AAA progression, whereas inhibition
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mice, but not in Mmp2-/- mice, led to development of a number of ECM-targeting MMPs was a more o
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of AAA in the Mmp9-/- mice which are otherwise effective approach. Consistent with our findings, nlo
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resistant to CaCl -induced AAA, therefore, the doxycycline, a broad spectrum MMP inhibitor, de
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macrophage-derived Mmp9 and the Mmp2 has been a promising treatment in preventing fro
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produced by mesenchymal cells were proposed to AAA expansion and rupture (62-65), whereas h
work in concert to mediate AAA (48). Further, other treatments including β-blockers, ACE ttp://w
production of Mmp2 by mysenchymal cells has inhibitors and statins have been ineffective (1- w
w
been shown to be enhanced by the presence of 4,6). Overall, our study provides evidence that .jb
inflammatory cells (52). This is consistent with Timp3 is critical in constructive vascular c.o
rg
our observation of elevated Mmp2 levels in WT- remodeling and as such, targeted overexpression b/
chimera mice (containing Timp3-/-/Mmp2-/- bone of Timp3 could serve as a promising therapeutic y g
u
marrow) as the only possible source of Mmp2 in approach in preventing aneurysm formation or es
t o
these animals is in fact the native cells and not the controlling its expansion. n
N
Timp3-/-/Mmp2-/- inflammatory cells. ov
e
Elastin degradation products in AAA m
b
e
samples from patients exhibit chemotactic activity r 2
4
that attracts macrophages to the site of AAA , 2
0
18
7
MS ID#: JBC/2012/425652R1
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