Among aortic diseases, aortic aneurysm is a great health

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1 Divergent Roles of Matrix Metalloproteinase 2 in Pathogenesis of Thoracic Aortic Aneurysm Mengcheng Shen, Jiwon Lee, Ratnadeep Basu, Siva S.V.P. Sakamuri, Xiuhua Wang, Dong Fan, Zamaneh Kassiri Downloaded from by guest on October 16, 2017 Objective Aortic aneurysm, focal dilation of the aorta, results from impaired integrity of aortic extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are traditionally known as ECM-degrading enzymes. has been associated with aneurysm in patients and in animal models. We investigated the role of in thoracic aortic aneurysm using 2 models of aortic remodeling and aneurysm. Approach and Results Male 10-week-old -deficient ( / ) and wild-type mice received angiotensin II (Ang II, 1.5 mg/kg/day) or saline (Alzet pump) for 4 weeks. Although both genotypes exhibited dilation of the ascending aorta after infusion, / mice showed more severe dilation of the thoracic aorta and thoracic aortic aneurysm. The induced increase in elastin and collagen (mrna and protein) was markedly suppressed in / thoracic aorta and smooth muscle cells, whereas only mrna levels were reduced in / - abdominal aorta. Consistent with the absence of, proteolytic activities were lower in / - compared with wild-type- thoracic and abdominal aorta. -deficiency suppressed the activation of latent transforming growth factor-β and the Smad2/3 pathway in vivo and in vitro. Intriguingly, / mice were protected against CaCl 2 -induced thoracic aortic aneurysm, which triggered ECM degradation but not synthesis. Conclusions This study reveals the dual role of in ECM degradation, as well as ECM synthesis. Moreover, the greater susceptibility of the thoracic aorta to impaired ECM synthesis, compared with vulnerability of the abdominal aorta to aberrant ECM degradation, provides an insight into the regional susceptibility of the aorta to aneurysm development. (Arterioscler Thromb Vasc Biol. 2015;35: DOI: /ATVBAHA ) Key Words: aortic aneurysm, thoracic matrix metalloproteinase 2 Among aortic diseases, aortic aneurysm is a great health concern with fatal or devastating outcomes. It is often asymptomatic until it ruptures causing significant morbidities and >80% mortality. 1 3 It is the 15th most common cause of death in the Western world. 4 Pharmacological treatments, such as β-blockers, 5,6 angiotensin II () receptor blockers, 6,7 converting enzyme inhibitors, 6,8,9 and statins, 10,11 have not shown consistent and significant improvements in clinical outcomes. Surgical repair can stabilize large aneurysms but are often limited to cases where the risk of rupture exceeds that of the surgical intervention, 12 and even then subsequent mortality and morbidity remain high Therefore, better understanding of the molecular mechanism responsible for development and progression of aortic aneurysm may be necessary in developing effective treatments for this devastating disease. See accompanying editorial on page 752 Weakened aortic wall and compromised integrity of the extracellular matrix (ECM) are among the common features in different types of aortic aneurysm. The predominant ECM structural proteins in the aorta are elastin that provides the recoil property of the aortic wall and collagen that provides the tensile strength to withstand the high blood pressure during cardiac systole. Physiological turnover of the aortic ECM includes degradation of its existing proteins by matrix metalloproteinases (MMPs) 16 to be replaced by newly synthesized proteins. Activity of MMPs is tightly regulated by their physiological inhibitors, tissue inhibitor of metalloproteinases. Among MMPs, is capable of degrading multiple ECM components, including collagen and elastin, 16,17 and has been strongly linked to excess ECM degradation in aneurysmal aorta as its levels are elevated in thoracic and abdominal aortic aneurysms (AAAs) deficient mice are protected against AAA in a model of adventitial CaCl 2 exposure 25 ; however the role of in thoracic aortic aneurysm (TAA) has not yet been explored. Thoracic aorta consists of greater elastin lamellae layers and a greater elastin:collagen Received on: June 17, 2014; final version accepted on: January 22, From the Department of Physiology, University of Alberta, Edmonton, Alberta, Canada; and Cardiovascular Research Center, Mazankowski Alberta Heart Institute, Edmonton, Alberta, Canada. These authors contributed equally to this article. The online-only Data Supplement is available with this article at Correspondence to Zamaneh Kassiri, MSc, PhD, Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. z.kassiri@ualberta.ca 2015 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at DOI: /ATVBAHA

2 Shen et al and Thoracic Aortic Aneurysm 889 Downloaded from by guest on October 16, 2017 Nonstandard Abbreviations and Acronyms AAA ECM FBN1 LTBP MMP SMC TAA TGF-β abdominal aortic aneurysm angiotensin II extracellular matrix fibrillin-1 latent TGF-β binding protein matrix metalloproteinase smooth muscle cell thoracic aortic aneurysm transforming growth factor-β wild-type ratio that reverses in the abdominal aorta. 26,27 Given the differential ECM composition and histological characteristics of the thoracic versus abdominal aorta, 15 could play regionally distinct roles in remodeling of the aorta. In this study, we used 2 experimental models of aortic remodeling and aneurysm, infusion that triggers ECM synthesis, as well as degradation, and adventitial CaCl 2 exposure that only triggers ECM degradation. is a physiological hormone elevated in patients with cardiovascular diseases, which leads to vascular remodeling, 31 whereas adventitial CaCl 2 exposure is an established experimental model of aortic aneurysm in rodents 32,33 and in large mammals. 34 The findings in this study reveal a dual function of in ECM synthesis, as well as degradation, and differential susceptibility of the thoracic and abdominal aorta to reduced synthesis versus excess proteolysis of ECM proteins. Materials and Methods Materials and Methods are available in the online-only Data Supplement. Experimental Animals and Procedures Wild-type (; The Jackson Laboratory) and -deficient ( / ) 35 mice in C57BL/6 background received (Sigma- Aldrich) or saline (control) for 4 weeks. Systolic blood pressure was measured using the tail-cuff method (iitc Life Science, Woodland Hills, CA) as before. 39 TAA by CaCl 2 exposure was induced in and / mice as described. 32,40 All animal experiments were performed in accordance with Canadian Council on Animal Care Guidelines and regulations of Animal Policy and Welfare committee at the University of Alberta. Statistical Analyses Two-way ANOVA was performed to compare data sets with 2 main factors (genotype and treatment). Statistical analyses were performed using SPSS software (Version 10.1; Chicago, IL). Normality test (Shapiro Wilk) was performed to confirm normal distribution of all data sets. Averaged values are presented as mean±sem. Statistical significance was recognized at P<5. Results Infusion Led to Formation of TAA in / Mice but Not in Mice After 4 weeks of infusion, uniform remodeling was detected throughout the aorta in mice, whereas a striking aneurysmal dilation was detected in the thoracic aorta in / mice (Figure 1A). AAA was not detected in either genotype (Figure I in the online-only Data Supplement). Representative ultrasonic images (Figure 1B), averaged internal aortic diameter of ascending aorta (Figure 1C), the arch (Figure 1D), and the internal (i) and maximal external diameter ( of descending thoracic aorta (Figure 1E) show that although aortic dilation was detected in -Ang-II, it was markedly greater in / - group. We used these 2 measurements to document aortic dilation because accurate measurement of the inner aortic diameter using trichromestained sections can be challenging and influenced by the steps involved in tissue processing; therefore, inclusion of the maximal outer diameter is required to confirm aortic dilation. Baseline aortic diameters in saline-infused groups were comparable between the genotypes. Interestingly, the aortic arch diameter during cardiac systole (maximum diameter) was increased similarly between - and / - mice (Figure 1Di), whereas aortic diameter during cardiac diastole (minimum diameter) was significantly greater in / - compared with - mice (Figure 1D, suggesting impaired recoil properties of the aorta in the -deficient mice. Consistent with the suppressed aortic wall motion observed in the M-mode images from / - (Figure 1B, aortic recoil index was markedly reduced in / - compared with -Ang II mice (Figure 1Diii; Figure IIAii in the online-only Data Supplement). The TAA in / mice was despite similar induced hypertension (Figure IIB in the online-only Data Supplement), and similar changes in receptor levels in the thoracic aorta after 2 or 4 weeks of infusion (Figure IIC in the online-only Data Supplement). Aberrant Structural Remodeling in Thoracic Aorta of Infused -Deficient Mice We investigated the structural remodeling in the thoracic aorta (Figure 2A 2D) by assessing the integrity of the aortic wall (Gomori trichrome; Figure 2Ai and 2A, elastin lamella (Verhoeff Van Gieson; Figure 2Ai, smooth muscle cell (SMC) density (calponin; Figure 2Aiv), and fibrillar collagen arrangement (picrosirious red; Figure 2Av). Compared with the saline-infused mice (Figure 2A), infusion resulted in uniform thickening of the medial layer in mice (Figure 2Bi and 2B with intact elastin lamella (Figure 2Bi, a proportionate increase in SMC density (Figure 2Biv) and adventitial fibrosis (Figure 2Bv). In / mice, however, compared with the saline-infused group (Figure 2Ci 2Civ), infusion resulted in excess dilation of the aortic lumen (Figure 2Di and 2D, loss of elastin lamella (Figure 2Di, loss of SMCs (Figure 2Div), and disrupted fibrillar collagen structure (Figure 2Dv). The abdominal aorta in both genotypes showed uniform (constructive) remodeling with intact elastin and collagen fibers, after 4 weeks of infusion (Figure IA and IB in the online-only Data Supplement). Therefore, -deficiency led to a striking structural disruption and aneurysmal dilation in the thoracic but not in the abdominal aorta after infusion.

3 890 Arterioscler Thromb Vasc Biol April 2015 Downloaded from by guest on October 16, 2017 Figure 1. Four weeks of angiotensin II () infusion led to thoracic aortic aneurysm in / but not in mice. A, Whole aorta pictures from wild-type () and / mice after saline or infusion. Arrows point to the aneurysmal areas. B, Representative ultrasound images showing the thoracic aorta in B-mode (i) and M-mode ( in indicated groups. C, Ascending aortic diameter measured by ultrasound. D, Averaged aortic diameter (arch) during cardiac systole (i) and cardiac diastole (, and aortic recoil index: ([systolic diameter diastolic diameter]/systolic diameter) 100; n=9 per group per genotype. E, Inner diameter measured from trichrome-stained crosssections (i) and maximal outer diameter measured from whole aorta ex vivo ( in descending thoracic aorta. C E, n=7 to 8 per group per genotype. P<5 compared with the corresponding saline group. P<5 compared with the - group. Reduced Elastin and Collagen Levels in / - Thoracic Aorta Are Because of Reduced Synthesis To investigate the induced molecular events underlying the marked structural disruption in the / aortic wall, we measured the protein levels for elastin and collagen, the 2 main ECM structural proteins. Western blot analysis showed that infusion increased elastin and collagen protein levels in but not in / thoracic aorta (Figure 3Ai and 3A. To determine whether the lower levels of elastin and collagen type I in / - aorta are because of reduced synthesis or increased degradation, we first measured the mrna levels for these molecules. In mice, infusion increased the elastin and collagen type

4 Shen et al and Thoracic Aortic Aneurysm 891 Downloaded from by guest on October 16, 2017 Figure 2. Adverse remodeling in the thoracic aortic wall in / mice after angiotensin II () infusion. Full cross-section (i), Gomori trichrome (, Verhoeff Van Gieson (i, calponin/dapi staining (iv), and picrosirious red (PSR) staining (v) on saline- (A) and infused wild-type () mice (B) and saline- (C) and infused / (D) mice. Elastin autofluorescence appears green in (iv and v). I mrna levels by 4-fold and 5-fold, respectively, whereas in the / group, the induced increase in these mrnas was markedly suppressed (Figure 3Bi and 3B. Next, we investigated how the lack of altered the proteolytic activities in the thoracic aorta. Gelatin zymography showed increased pro- (72 kda) and active levels (64 kda) in - compared with -saline aortas and confirmed the lack of in the aortas from - deficient mice (Figure 4Ai 4Ai. Consistently, mrna levels of were elevated only in - aortas (Figure 4Bi), whereas MMP9 mrna remained comparable among all groups (Figure 4B. infusion did not alter

5 892 Arterioscler Thromb Vasc Biol April 2015 Downloaded from by guest on October 16, 2017 A i) Elastin (70 kda) Loading control Collagen type I (130 kda) Loading control B i) Elastin / HPRT (R.E.) Elastin (A.U.) ProteinLevels mrna Levels i the levels of collagenases, MMP7, MMP13, and increased collagenase membrane type 1 MMP (Figure 4Biii 4Bv) and elastase MMP12 (Figure 4Bvi) similarly in and / mice. Collagenase MMP8 was negligible in both genotypes and remained unaltered after infusion (data not shown). Total elastase activity was increased significantly in -Ang II but to a lesser extent in / - aortas (Figure 4C) consistent with the absence of -mediated proteolysis. In the abdominal aorta, the protein levels for elastin and collagen were comparable in infused mice of either genotype despite the markedly reduced mrna levels in / -Ang II compared with - mice (Figure IC and ID in the online-only Data Supplement). Similar to the thoracic aorta, total elastase activity was significantly lower in the abdominal Collagen type I (A.U.) Collagen 1α1 / HPRT (R.E.) ProteinLevels mrna Levels Figure 3. Reduced elastin and collagen levels in angiotensin II () induced / aortas. A, Representative western blots (i) and averaged protein quantification for elastin ( and collagen type 1 (i in the thoracic aorta from the indicated groups. Coomassie blue stained gel served as loading control; n=6 per group per genotype. B, Averaged mrna levels for elastin (i) and collagen ( in the thoracic aorta from indicated groups; n=8 per group per genotype. P<5 compared with corresponding saline group. P<5 compared with wild-type ()- group. A.U. indicates arbitrary units; HPRT, hypoxanthine-guanine phosphoribosyltransferase; and R.E., relative expression aorta from / - compared with - mice (Figure IE in the online-only Data Supplement). Therefore, despite lower mrna synthesis, protein levels of these ECM proteins were sustained in / - abdominal aorta because of reduced degradation. In vitro assessment of SMCs isolated from thoracic and abdominal aorta showed that, consistent with our in vivo findings, the induced elastin and collagen mrna synthesis were suppressed in / thoracic SMCs. However, the abdominal SMCs of neither genotype showed a rise in mrna in response to (Figure IIIA in the online-only Data Supplement). Collectively, these data indicate that the lack of results in reduced production of elastin and collagen in the thoracic and abdominal aorta. However, in the abdominal aorta, the suppressed proteolytic activity is sufficient to sustain the protein content for elastin and collagen, whereas in the thoracic aorta, these ECM proteins are more predominantly dependent on their synthesis. Impaired Activation of Latent Transforming Growth Factor-β and the Smad2/3 Pathway in -Deficient Aortas To determine the underlying molecular mechanism for the impaired elastin and collagen synthesis in / -Ang II mice, we examined the transforming growth factor-β1 (TGFβ1) signaling pathway, a well-known growth factor and cytokine that mediates the synthesis of ECM structural proteins. Activation of TGFβ requires its proteolytic release from its latent complex (latent-tgfβ binding protein [LTBP] TGFβ). We found that the induced rise in active (25 kda) TGFβ1 was significantly reduced in / compared with aortas (Figure 5Ai and 5A, and consistently, activation of the downstream Smad2/3 pathway was suppressed in / - aortas as evident by reduced phospho- Smad2/3 levels and the phospho:total Smad2/3 ratio in this group (Figure 5Bi and 5B. Assessment of LTBP-1 showed a greater full length (180 kda):cleaved (130 and 80 kda) ratio of this protein in / - aortas (Figure 5Ci and 5C, indicating hindered LTBP-1 cleavage in the absence of. In vitro, thoracic SMCs from / mice demonstrated a similar pattern, indicating the suppressed levels of active TGFβ1 (Figure IIIB in the online-only Data Supplement) and phospho:total Smad2/3 ratio (Figure IIIC in the online-only Data Supplement) compared with -SMCs in response to. These data demonstrate impaired TGFβ1 processing, bioavailability, and suppressed activation of the Smad2/3 pathway in infused -deficient aortas and thoracic SMCs. -Deficiency Is Protective Against CaCl 2 -Induced TAA To further establish that plays a divergent role in ECM homeostasis by contributing to both production and degradation of the ECM proteins, we used the well-established CaCl 2 exposure model that induces TAA primarily through promoting ECM degradation First, we confirmed that CaCl 2 -treatment in vivo results in activation of pro- to its cleaved form (Figure IVA in the online-only Data

6 Shen et al and Thoracic Aortic Aneurysm 893 A Pro- i Active iv) MMP9 i) MMP9 (90 kda) Pro- (72 kda) Active (64 kda) Loading control Con Band intensity (A.U.) N.D. N.D. Band intensity (A.U.) N.D. N.D. Band intensity (A.U.) 1.8 Downloaded from by guest on October 16, 2017 B i) /HPRT (R.E) N.D. N.D. MMP9/HPRT (R.E) v) vi) MT1-MMP/HPRT (R.E) MMP12/HPRT (R.E) i MMP7/HPRT (R.E) C Total Elastase Activity (A.U.) iv) MMP13/HPRT (R.E) Figure 4. Reduced proteolytic activities in angiotensin II () infused / thoracic aorta. A, Representative gelatin zymography (i) and averaged band intensity for promatrix metalloproteinase 2 (;, active (i, and MMP9 (iv) in thoracic aorta of indicated groups. Coomassie blue stained gel was served as loading control; n=6 per group per genotype. B, mrna expression levels of (i), MMP9 (, MMP7 (i, MMP13 (iv), membrane type 1 MMP (v), and MMP12 (vi); n=6 to 8 per group per genotype. C, Fluorescent-based elastase activity in the thoracic aorta of indicated groups; n=5 per group per genotype. P<5 compared with the corresponding saline group. P<5 compared with the wild-type ()- group. A.U. indicates arbitrary units; con=positive control; HPRT, hypoxanthineguanine phosphoribosyltransferase; N.D., not detected; and R.E., relative expression. Supplement) and elevated mrna synthesis (Figure IVB in the online-only Data Supplement). After 6 weeks of adventitial CaCl 2 exposure, all mice (n=16) developed TAA, whereas none of the / mice (n=14) exhibited dilation of the thoracic aorta (Figure 6A), while neither genotype exhibited aortic rupture. Histological analysis of the thoracic aortas confirmed lumenal dilation, structural degradation, interrupted elastin fibers in the medial layer, and adventitial fibrosis in but not in / mice (Figure 6Bi and 6B. Measurement of the internal diameter (i) and maximal external diameter ( of the descending thoracic aorta further confirmed the presence of aneurysmal dilation only in but not in / mice (Figure 6C). Consistent with the degradation of the medial ECM in the -CaCl 2 group, elastase activity was significantly elevated in but not in / aortas (Figure 6D). CaCl 2 treatment did not alter the mrna expression levels of elastin nor collagen in either genotype (Figure 6Ei and 6B. Therefore, the CaCl 2 -induced aortic aneurysm is primarily driven by excess degradation of the ECM (without enhanced synthesis), and the absence of

7 894 Arterioscler Thromb Vasc Biol April 2015 the proteolytic function of offers a protective effect against aneurysm formation in this model. Discussion Aortic aneurysm is the outcome of maladaptive remodeling of vascular ECM. MMPs, particularly, are best known as ECM-degrading enzymes, and as such, they are often associated with adverse outcomes in vascular pathologies. 20,21,41 In this study, we demonstrate 2 opposing roles for in aortic wall remodeling whereby it contributes to production in addition to degradation of the ECM structural proteins. is a peptide hormone upregulated in several cardiovascular diseases and can trigger a range of cellular responses, including synthesis of ECM proteins, as well as enhanced proteolytic activities through activation of MMPs, such as. 15,42,43 Adventitial CaCl 2 exposure is an established model of thoracic 32,33 and AAAs, 44 and as we and others have shown, is associated with early activation of and aberrant ECM degradation. 44,45 By using these 2 distinct models of aortic remodeling and aneurysm, we studied the comparative role of in constructive versus destructive remodeling of the aortic ECM. Here, we report that in addition to serving as a potent ECM protease, is a key factor for optimal ECM synthesis in the aortic wall through activation of latent TGFβ1 and the downstream Smad2/3 pathway. promotes constructive ECM turnover by triggering proteolysis of the existing ECM proteins and de novo synthesis of ECM proteins to replace the degraded proteins. In the absence of, although the mediated proteolysis is reduced, the suppressed production of elastin and collagen Downloaded from by guest on October 16, 2017 A B C i) i) i) TGFβ1 (25 kda) Coomassie Blue (Loading control) P-Smad2 (58 kda) P-Smad3 (52 kda) Smad2 (58 kda) Smad3 (52 kda) Coomassie Blue (Loading control) LTBP-1 (180 kda) LTBP-1 (130 kda) LTBP-1 (80 kda) Coomassie Blue (Loading control) TGFβ1 (A.U.) Protein Levels Full length LTBP / cleaved LTBP ratio A.U. P-Smad2/3 / total Smad2/3 ratio Figure 5. Impaired angiotensin II () induced transforming growth factor-β (TGFβ) activation in matrix metalloproteinase 2 () deficient aortas. A, Representative western blot (i) and averaged protein levels ( for active TGFβ in the thoracic aorta of indicated groups. B, Representative western blots for phospho-smad2 and phoshpho-smad3 (i) and averaged phospho-to-total Smad 2/3 in the indicated groups. C, Representative western blot for full-length and cleaved latent-tgfβ binding protein-1 (LTBP-1; i) and averaged full length:cleaved ratio for LTBP1 in the indicated groups. Coomassie blue stained gels were used as the loading control. P<5 compared with the corresponding saline group. P<5 compared with the wild-type ()- group; n=6 per group per genotype. A.U. indicates arbitrary units. A.U.

8 Shen et al and Thoracic Aortic Aneurysm 895 Downloaded from by guest on October 16, 2017 leaves the need for replenishment of degraded ECM components unmet, leading to adverse ECM remodeling and aortic aneurysm. The presence of TAA but not AAA in / - mice, despite similar reductions in mrna synthesis of elastin and collagen in both regions, could be because the suppressed induced proteolysis in the absence of is sufficient to preserve the elastin and collagen protein contents in the abdominal aorta, whereas in the thoracic aorta, the ECM integrity is more adversely affected by the reduced production of these proteins. This could reflect a differential ECM turnover rate in thoracic versus abdominal aorta. Thoracic and abdominal aortas have different characteristics that alter their susceptibility to different pathological stimuli. 15,46,47 For instance, although the wall thickness-to-lumen size is consistent throughout the aorta, the thoracic aortic wall consists of a greater number of elastin lamellae, whereas in the abdominal aorta, the elastin lamellae are comprised of a thicker layer of SMCs. Based on the higher content of elastin in the thoracic aorta, this region could be more susceptible to defects in ECM synthesis, whereas the abdominal aorta would be more vulnerable to excess ECM degradation. We have reported that Ang II infusion resulted in AAA (but not TAA) in mice lacking tissue inhibitor of metalloproteinase-3, a potent MMP inhibitor, because of increased proteolytic degradation and despite uninterrupted synthesis of ECM proteins. 48 Similarly, the lack of tissue inhibitor of metalloproteinase-1 49 or tissue inhibitor of metalloproteinase-2 44 exacerbated aortic dilation in different models of AAA. It is also plausible that the turnover rate for the ECM proteins in abdominal aorta is lower than that in the thoracic region, and therefore, as long as the existing ECM structure is preserved (by reduced proteolysis), the abdominal aorta would not be affected by reduced ECM synthesis. Notably, genetic disorders that interfere with collagen or elastin production or assembly are often associated with TAA and less frequently with AAA. 24 This differential susceptibility of the abdominal aorta to excess proteolysis could also suggest potential therapeutic advantages for reducing MMP activities in patients with AAA by MMP inhibitors, such as doxycycline, although recent reports have been contradictory. 50,51 TGFβ is a multifunctional cytokine and growth factor contributing to several diseases, 52 and therefore, its activation is tightly regulated and involves many steps. TGFβ isoforms are transcribed with the latency-associated proprotein forming the small latent complex that is secreted as the large latent complex covalently bound to LTBP via a disulphide bond. After secretion, latent TGFβ (LTBP TGFβ) is sequestered in the ECM through covalent binding of LTBPs to ECM proteins, such as fibrillin-1 (FBN1). TGFβ activation requires the release of the 25-kDa homodimer from the latent complex. Proteolytic cleavage of LTBP1 from the ECM and the subsequent release of TGFβ1 from the latency-associated proprotein complex yield bioavailability of TGFβ, activation of its receptors, the downstream Smad2/3 signaling pathway, and subsequent transcriptional regulation of matrix-associated proteins, such as elastin and collagen Among MMPs,, MMP9, and membrane type 1 MMP have been reported to be able to cleave and activate the latent TGFβ In our study, infusion did not alter MMP9 levels in the aorta, whereas the increase in membrane type 1-MMP was comparable in and / aortas, hence indicating a key role of that was elevated in infused aortas but absent in the / mice. TGFβ can also trigger noncanonical, Smad-independent, signaling pathways, such as the mitogen-activated protein kinase pathways. 60,61 We assessed the activation of the extracellular signal regulated protein kinase and c-jun N-terminal kinase pathways, determined by phosphorylation of extracellular signal regulated protein kinase 1/2 and c-jun N-terminal kinase 1/2, and did not find a significant induced activation in either genotype (data not shown). It is critical to note that the TAA observed in / -Ang II mice is mediated by a distinct molecular mechanism from that identified in Marfan syndrome associated TAA The primary cause of TAA in Marfan syndrome is a mutation or loss of FBN1, the building unit of microfibrils that form the scaffold for elastin assembly. FBN1 is also one of the main ECM proteins to which LTBP binds to sequester latent TGFβ in the ECM. With loss of FBN1, as it occurs in the Fbn +/ mouse model of Marfan syndrome, sequestration of latent TGFβ in the ECM is compromised leading to increased bioavailability of TGFβ and therefore enhanced activation of the TGFβ mediated pathways. 66 This is clearly the opposite scenario to our findings in / mice where the lack of hinders the release of ECM-bound latent TGFβ and therefore reduces its bioavailability and the subsequent ECM production. This could also explain the protective effects of -deficiency in FBN1 +/ mice that exhibited excess active TGFβ (and reduced latent TGFβ) levels. 67 The heterozygous loss of FBN1 ( 50%) in these mice would result in a reduced fraction of TGFβ that would exist in the latent form (FBN1-bound). Therefore, loss of in these mice would prevent the proteolytic release of the remaining FBN1-bound latent TGFβ molecules, thereby controlling the otherwise overactivated TGFβ pathway resulting in improved outcomes. Our findings are consistent with an earlier study that reported interruption of the TGFβ pathway, through a loss-of-function mutation of TGFβ2, led to TAA formation in patients and in animal models. 68 Interestingly, mutations in TGFβ receptors also led to TAA but through increased TGFβ mediated Smad pathway and increased collagen production, 47,69 indicating that TGFβ can interact with multiple receptors to mediate its effects. Therefore, excess bioavailability or suppressed activation of TGFβ can lead to impaired ECM turnover and thereby enhanced susceptibility to TAA. In conclusion, this study demonstrates an intriguing dual role of in vascular ECM turnover where it is a key factor in mediating ECM synthesis in addition to ECM degradation. This further highlights the complexity of the function of MMPs, which extends well beyond their classically known ECM-degrading role. Furthermore, the regional susceptibility of the aorta to aneurysm could be partly explained by the greater susceptibility of the thoracic aorta to interruptions of ECM synthesis, whereas pathologies associated with excess ECM degradation would render the abdominal aorta susceptible to aneurysm formation.

9 896 Arterioscler Thromb Vasc Biol April 2015 Downloaded from by guest on October 16, 2017 Figure 6. Adventitial CaCl2 exposure did not result in thoracic aortic aneurysm in matrix metalloproteinase 2 () deficient mice. A, Whole aorta pictures from wild-type (; i) and / mice ( after 6 weeks of CaCl2 exposure. Arrows point to areas of CaCl2 exposure. B, Trichrome, Verhoeff van Gieson (VVG), and picrosirius red (PSR) staining of the thoracic aorta of (i) and -deficient ( mice are shown in (A). C, Inner diameter measured from trichrome-stained cross-sections (i) and maximal outer diameter measured from whole aorta ex vivo ( in descending thoracic aorta in the indicated groups measured ex vivo in perfusefixed aortas (n=7 per group per genotype). D, Total elastase activity in the descending thoracic aorta of indicated groups (n=5 per group per genotype). E, mrna levels of collagen type I (i) and elastin ( in the descending aorta of the indicated groups (n=5 7 per group per genotype). P<5 vs corresponding saline group. P<5 vs -CaCl2. HPRT indicates hypoxanthine-guanine phosphoribosyltransferase.

10 Shen et al and Thoracic Aortic Aneurysm 897 Downloaded from by guest on October 16, 2017 Sources of Funding This study was supported by Operating Grant from Heart and Stroke Foundation to Z. Kassiri. Z. Kassiri is an Alberta Innovates-Health Solutions Scholar. M. Shen is supported by China Scholarship Council. None. Disclosures References 1. Best VA, Price JF, Fowkes FG. Persistent increase in the incidence of abdominal aortic aneurysm in Scotland, Br J Surg. 2003:90: Filipovic M, Goldacre MJ, Roberts SE, Yeates D, Duncan ME, Cook- Mozaffari P. Trends in mortality and hospital admission rates for abdominal aortic aneurysm in England and Wales, Br J Surg. 2005:92: Lindsay ME, Dietz HC. Lessons on the pathogenesis of aneurysm from heritable conditions. Nature. 2011;473: doi: / nature National Center for Injury Prevention and Control. WISQRS Leading Causes of Death Reports, ncipc/leadcaus10.html. 5. Propanolol Aneurysm Trial Investigators. 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Cook JR, Carta L, Galatioto J, Ramirez F. Cardiovascular manifestations in Marfan syndrome and related diseases; multiple genes causing similar phenotypes. Clinical Genet. 2014;87: Xiong W, Meisinger T, Knispel R, Worth JM, Baxter BT. MMP-2 regulates Erk1/2 phosphorylation and aortic dilatation in Marfan syndrome. Circ Res. 2012;110:e92 e101. doi: /CIRCRESAHA Lindsay ME, Schepers D, Bolar NA, et al. Loss-of-function mutations in TGFB2 cause a syndromic presentation of thoracic aortic aneurysm. Nat Genet. 2012;44: doi: /ng Gallo EM, Loch DC, Habashi JP, et al. Angiotensin II-dependent TGF-β signaling contributes to Loeys Dietz syndrome vascular pathogenesis. J Clin Invest. 2014;124: doi: /JCI Significance Aortic aneurysm, focal dilation of the aorta, is one of the major vascular diseases and a great health concern. If not detected early or not managed, it can lead to fatal or devastating outcomes. Aortic aneurysm results from deterioration of the aortic wall and the aortic extracellular matrix (ECM). Matrix metalloproteinases are classically known to degrade ECM proteins, and a rise in matrix metalloproteinases is often linked to adverse events in vascular diseases. In this study, we report that matrix metalloproteinase 2, which is elevated in aneurysmal aorta, plays a dual role by promoting synthesis, in addition to degradation of vascular ECM. Moreover, this study provides evidence that the thoracic aorta is susceptible to impaired ECM synthesis, whereas the abdominal aorta can be more adversely influenced by a rise in ECM degradation. This finding provides novel insight into the factors that determine the site of aortic aneurysm formation.

12 Downloaded from by guest on October 16, 2017 Divergent Roles of Matrix Metalloproteinase 2 in Pathogenesis of Thoracic Aortic Aneurysm Mengcheng Shen, Jiwon Lee, Ratnadeep Basu, Siva S.V.P. Sakamuri, Xiuhua Wang, Dong Fan and Zamaneh Kassiri Arterioscler Thromb Vasc Biol. 2015;35: ; originally published online February 5, 2015; doi: /ATVBAHA Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2015 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online at:

13 (A) i) - - Trichrome Trichrome 200 μm 50 μm 200 μm 50 μm VVG PSR VVG PSR 50 μm 50 μm 50 μm 50 μm (B) i) - Trichrome - Trichrome 200 μm 50 μm 200 μm 50 μm VVG PSR VVG PSR 50 μm 50 μm 50 μm 50 μm (C) i) Elastin (70 kda) Col type I (130 kda) Loading control Loading control (D) 2.6 i) 2.4 Elastin/HPRT (R.E.) Elastin mrna Collagen 1α1/HPRT (R.E.) Collagen I mrna Supplemental Figure I - Similar -induced remodeling in and -deficient abdominal aorta. Cross-sectional images of abdominal aorta from (A) and -deficient mice (B) following saline (i) or -infusion (, stained with trichrome, Verhoeff van Gieson (VVG), and picrosirius red (PSR). C) Western blot for elastin (i) and collagen type I ( in the abdominal aorta of the indicated groups. D) mrna expression levels of elastin (i) and Collagen type I ( in the indicated group (n=6-8/group/genotype). E) Total elastase activity measured by fluorescent-based activity assay in the indicated groups (n=5-6/group/genotype). p<5 vs corresponding saline group, p<5 vs -AngII group. (E) Total Elastase Activity (A.U.) Total Elastase Activity 1.8

14 (A) Ascending Aorta i) Systolic Aortic Diameter (mm) Aortic Recoil Index (%) (B) 180 Systolic Blood Pressue (mmhg) Time (days) (C) i) AT1/HPRT AT2/HPRT wks 4wks 2wks 4wks 0 2wks 4wks 2wks 4wks Supplemental Figure II - A) Aortic diameter of ascending aorta (measured by ultrasound) during cardiac systole (i) and diastole ( after 4 weeks of saline or infusion in both genotypes (n=7-9/group/genotype). B) Systolic blood pressure in and -deficient mice before and after infusion (n=9/genotype). C) mrna expression of Angiotensin receptors (AT1 and AT2) in the thoracic aorta of and -deficient mice treated with saline or for 2 weeks and 4 weeks (n=7-8/group/genotype). p<5 compared to corresponding saline. p<5 compared to corresponding group.

15 (A) i) Thoracic SMC Abdominal SMC Elastin/HPRT (R.E.) Collagen 1α1/HPRT (R.E.) Elastin/HPRT (R.E.) Collagen 1α1/HPRT (R.E.) (B) Thoracic SMC i) TGFβ1 (25 kda) Coomassie blue (Loading control) TGFβ1 (A.U.) AngII AngII (C) Thoracic SMC i) p-smad2 (58 kda) p-smad3 (52 kda) Smad2 (58 kda) Smad3 (52 kda) Coomassie blue (Loading control) psmad2/3-to-smad2/3 ratio (A.U.) AngII AngII Supplemental Figure III - Production of elastin and collagen, and activation of the TGFβ-Smad pathway in aortic smooth muscle cells (SMCs) from and -deficient mice. A) mrna levels of elastin and collagen type I in thoracic (i) and abdominal ( SMCs from each genotype. B) Representative Western blot (i) and averaged quantification ( for cleaved (active) TGFβ in and -deficient thoracic SMCs. C) Representative phospho- and total-smad2/3 (i), and averaged phospho-to-total ratio ( in the indicated groups. n= 6 culture plates/group/genotype (from 3 individual mice per genotype). p<5 compared to corresponding saline. p<5 compared to - group.

16 (A) i) mrna 0.9 MMP9 mrna 0.7 /HPRT (R.E.) MMP9/HPRT (R.E.) CaCl 2 CaCl 2 CaCl 2 CaCl 2 (B) 2wks CaCl2 4wks 6wks +ve Control MMP9 Pro- Active Loading control (coomassie blue) Supplemental Figure IV - Expression and activation of are elevated in CaCl2-induced aortic aneurysm. A) mrna expression of (i) and MMP9 ( in thoracic aortas treated with saline or CaCl2 (6 wks) in and mice (n=4-6/group/genotype). B) Gelatin Zymography in aortas from mice following saline or different durations of CaCl2 treatment as indicated. p<5 compared to corresponding saline group. p<5 compared to -CaCl2 group.