Transglutaminase 2 moderates the expansion of mouse abdominal aortic aneurysms

Objective Previously published work has indicated that transcripts encoding transglutaminase 2 (TG2) increase markedly in a rat model of abdominal aortic aneurysm. This study determines whether TG2 and the related TG, factor XIII-A (FXIII-A), protect against aortic aneurysm development in mice. Methods C57BL/6J wild-type, Tgm2–/– knockout, F13a1–/– knockout, and Tgm2–/–/F13a1–/– double knockout mice were subjected to laparotomy and periaortic application of CaCl2. Results Tgm2–/– mice showed slightly greater aortic dilatation at 6 weeks after treatment when compared with wild type. However, vessels from Tgm2–/– mice, but not wild-type mice, continued to dilate up to 6 months after injury and by 24 weeks, a greater number of Tgm2–/– mice had developed aneurysms (16/17 vs 10/19; P = .008). Laparotomy resulted in a high death rate in F13a1–/– knockout mice, more frequently from cardiac complications than from hemorrhage, but among F13a1–/– mice that survived for 6 weeks after CaCl2 treatment, abdominal aortic aneurysm diameter was unaltered relative to wild-type mice. Laparotomy resulted in a higher death rate among Tgm2–/–/F13a1–/– double knockout mice, owing to an increased frequency of delayed bleeding. Surprisingly, Tgm2–/–/F13a1–/– double knockout mice showed a trend toward decreased dilatation of the aorta 6 weeks after injury, and this finding was replicated in Tgm2–/–/F13a1–/– mice subjected to carotid artery injury. Levels of transcripts encoding TG2 were not increased in the aortas of injured wild-type or F13a1–/– knockout mice relative to uninjured mice, although changes in the levels of other transcripts accorded with previous descriptions of the CaCl2 aneurysm model in mice. Conclusions Knockout of Tgm2, but not F13a1 exacerbates aortic dilatation, suggesting that TG2 confers protection. However, levels of TG2 messenger RNA are not acutely elevated after injury. FXIII-A plays a role in preventing postoperative damage after laparotomy, confirming previous reports that it prevents distal organ damage after trauma. TG2 promotes wound healing after surgery and, in its absence, the bleeding diathesis associated with FXIII-A deficiency is further exposed.

open and endovascular aneurysm repair carry significant risk 2 and effective medical treatments that mitigate AAA development are not available currently. 3 Targeting the proteases that degrade aortic proteins could potentially moderate aneurysm development. However, several proteases may need to be simultaneously inhibited to halt disease progression, 4 and although absence of matrix metalloproteinase (MMP)9 inhibits aneurysm development in animal models, [5][6][7] administration of the MMP inhibitor, doxycycline, to patients with small aneurysms failed to decrease aneurysm growth. 8 Alternatively, it may be possible to promote the expression of enzymes that mediate arterial protection and repair. TGs comprise a family of eight enzymes that introduce N ε (g-glutamyl) lysine isopeptide cross-links between and within protein chains, conferring mechanical stability and proteolytic resistance. 9 Among these, extracellular TG2 10 cross-links matrix proteins including fibronectin 11,12 and possibly isoforms of collagen. 11 In addition, intracellular TG2 regulates gene expression and hence decisions of cell fate. 13 Although many cells express TG2 basally, expression is induced in response to proinflammatory stimuli 14 and when macrophages polarize to a reparative M2 phenotype. 15 Accordingly, TG2 is present in injured tissues, such as the vulnerable shoulder regions of human atherosclerotic plaques. 16,17 A second TG, blood clotting factor XIII-A (FXIII-A), circulates in plasma as the heterotetramer FXIII-A 2 B 2 and is present within the cytosol of cell types including megakaryocytes, platelets, and macrophages as the homodimer FXIII-A 2 . 18 Plasma FXIII-A 2 B 2 cross-links matrix proteins including fibrin, fibronectin, collagen, and vitronectin 19 and contributes to placental maintenance 20 and dermal repair. 21 Macrophage FXIII-A expression is induced upon differentiation to a reparative M2 phenotype 22 and cooperates with plasma FXIII-A 2 B 2 to stabilize myocardial scars 23 and with macrophage-derived TG2 to facilitate inward arterial remodelling. 24 Munezane et al 25 reported that TG2 expression increases when infrarenal aortic aneurysms are induced in rats. We previously observed that lack of TG2 lessens the resistance of carotid arteries to the mechanical strain of ligation. 26 Given that weakened vessels are prone to aneurysm development, 27 this finding might imply that TG2 would confer protection. We are unaware of evidence that FXIII-A directly influences aneurysm development, although various studies have proposed that FXIII-A overlaps in function with TG2 (eg, 28 ). Therefore, to address the roles of TG2 and FXIII-A in aneurysm development, we have induced aneurysms in the infrarenal aorta ofTgm2 e/e knockout, F13a1 e/e knockout, and Tgm2 e/e /F13a1 e/e double knockout mice.

METHODS
Animal housing, husbandry, and procedures were conducted in accordance with guidelines and regulations of the University of Leeds and of the United Kingdom Home Office. Mice had ad libitum access to water and to a standard chow diet (softened for 24 to 48 hours after surgery).
Generation of TG-deficient mice. The breeding and genotyping of Tgm2 þ/e mice 29 and of F13a1 e/e mice and Tgm2 e/e /F13a1 e/e mice each back-crossed onto a more than 97.5% C57BL/6J background 26 have been described previously. Equal numbers of male and female mice at 8 to 10 weeks of age were used for each procedure.
Harvesting and biochemical analysis of arteries. Mice were anaesthetized with isoflurane and then subjected to perfusion exsanguination via the left ventricle with 5 mL phosphate-buffered saline (Sigma Aldrich, Dorset, UK). The descending aorta to the iliac bifurcation was snap frozen in liquid N 2 , stored at e80 C, and either (i) processed to determine messenger RNA (mRNA) levels 26 or (ii) dehydrated to constant weight under vacuum at 80 C, lysed for 48 hours at 4 C in 750 mL of Na 2 HPO 4 (50 mmol.L À1 ), NaCl (50 mmol.L À1 ), 1% Triton X-100, 0.1% SDS, pH 7.4, and subjected to centrifugation (14,000Âg for 10 minutes at 4 C). The supernatant fraction was assayed to determine protein (bicinchoninic acid assay; Sigma-Aldrich) and lactate dehydrogenase activity ( Tgm2 e/e mice show a small increase in aortic diameter 6 weeks after CaCl 2 injury relative to control mice while vessels from Tgm2 e/e mice, but not wild-type mice, continued to dilate up to 24 weeks after injury. Tgm2 e/e /F13a1 e/e double knockout mice show a trend toward decreased aortic dilatation and this finding was replicated in Tgm2 e/e /F13a1 e/e mice subject to carotid artery injury. d Take Home Message: Transglutaminase 2 (TG2) has been proposed to play a role in the repair of blood vessels. Our results do not confirm that TG2 expression is acutely increased after aneurysm development, but instead suggest that the absence of TG2 exacerbates experimental aneurysm development.
Wire myography. Abdominal aortas (n ¼ 4) were dissected in cold Hanks buffered salt solution 30 and two rings from each (1 mm in length) were mounted in a myograph (610 mol/L; Danish Myograph Technology, Hinnerup, Denmark), equilibrated for 30 minutes, and then placed under normalized tension in KrebseHenseleit buffer gassed with 5% CO 2 /95% O 2 at 37 C. 31 The contractile responses to KCl (60 mmol.L À1 ) and to phenylephrine (1 mmol.L À1 ) were verified, after which the response to increasing concentrations of phenylephrine was determined. The median effective concentration values were estimated using Origin software (OriginLab Corporation, Northampton, Mass). Finally, endothelial integrity was confirmed by showing that carbachol (1 mmol.L À1 ) induced relaxation of vessels that had contracted in response to phenylephrine (1 mmol.L À1 ).
Aneurysm induction. Mice were anaesthetized and then either the abdominal aorta was exposed or the right common carotid artery was exposed and isolated from surrounding tissue using a silicone strip (Eddingtons Ltd, Hungerford, UK). Subsequently CaCl 2 (0.5 mol.L À1 ) or NaCl (0.15 mol.L À1 ) was applied to the artery (2 Â 7 minutes), and the exposed area was rinsed with NaCl (0.15 mol.L À1 ). The mice received an intraperitoneal injection of Buprenorphine 0.1 to 1.0 mg/kg (Vetergesic, Reckitt Benckiser, Slough, UK) before recovery. Arteries were imaged in situ before CaCl 2 treatment and at termination (6 weeks or 24 weeks) using the OPMI-PICO video micrometer (Carl Zeiss AG, Jena, Germany). Vessel measurements were independently determined by two investigators using Image-Pro software (MediaCybernetics, Rockville, Md).
Histology. Arteries for histologic examination were perfusion-fixed in 4% paraformaldehyde in 50 mmol.L À1 Na 2 HPO 4 , 150 mmol.L À1 NaCl, pH 7.4. Serial transverse sections were cut at 5-mm intervals from the left renal artery to iliac bifurcation and stained with Miller's elastic Van Gieson, hematoxylin and eosin, picrosirius red, or alizarin red S and were imaged using an Olympus BX61WI inverted microscope with an XC10-IR camera under the control of CellSens software (Olympus, Tokyo, Japan). Fibrillar collagen density adjacent to damaged and undamaged regions from Van Gieson stained aortic sections was determined by excitation at 800 nm with a Chameleon laser and collecting the second harmonic signal (400 nm) through a 10Â objective lens and an EF SP 485 IRþþ filter onto a Zeiss LSM NDD R2 detector using a Zeiss 710 multiphoton microscope and analyzing 10 Â 10 pixel square regions using ImageJ.
All image analysis was carried out in a blinded manner.
Quantification of messenger RNA. Mouse aortas (n ¼ 8-10) were disrupted in TRIzol (ThermoFisher Scientific) using a TissueLyser II (Qiagen, Hilden, Germany), The nuclei acid fraction was precipitated from the aqueous layer and mRNA processed for reverse transcriptase polymerase chain reaction, as previously described, 26 using the primer pairs shown in the Supplementary Table (online only). Transcript levels were normalized to those encoding ribosomal protein subunit-32, using the 2 eDCt method. 32 Immunohistochemistry. Immunofluorescent detection of CD163 and FXIII-A antigens in sections of mouse aorta was carried out as previously described. 33 Statistical analysis. Unless stated, all are is presented as mean 6 standard deviation. All values were analyzed using Prism 7 (GraphPad software, San Diego, Calif). Comparisons between each group were carried out using the unpaired Student t test (with Welch's correction) or one-way analysis of variance (with Bonferroni correction) for multiple groups. A Kruskal-Wallis test with Dunn's multiple comparison test was used for any data that were not normally distributed; normality was assessed using Shapiro-Wilk testing. Contingency data (proportion of mice developing an aneurysm; defined as increase in diameter of >50%) was analyzed using Fisher's exact test of proportions. Significance was accepted where the P was less than .05.

RESULTS
Baseline properties of mouse aortas are similar between genotypes. The mRNAs encoding TG2, FXIII-A. and TG3 were the most abundant TG transcripts in the wild-type aorta. Other TG mRNAs were present at very low levels or were undetectable (TG6). Knockout of the F13a1 gene, the Tgm2 gene, or both did not result in induction of other Tgm genes to a level expected to compensate for either gene knockout. Immunofluorescence confirmed that FXIII-A was present in CD163 positive macrophages of aorta (Fig 1), as previously described in skin 34 and heart. 33 The aortic structure was similar between genotypes, illustrated by representative images of wild-type and Tgm2 e/e /F13a1 e/e aortas (Fig 2, A). The mean lamellar number (n ¼ 4) did not differ between genotypes (C57Bl/6J, 4.76 6 0.54; Tgm2 e/e , 4.38 6 0.49; F13a1 e/e , 4.60 6 0.49; Tgm2 e/e /F13a1 e/e , 4.64 6 0.54). Measurements of elastin were consistent with published values 35,36 and did not vary between genotypes. Pepsinsoluble collagen was measured by the Biocolor assay, which preferentially detects newly synthesized collagen, and showed no statistically significant difference between groups (Fig 2, B). Elastin and collagen could not be assayed in the same sample, precluding estimation of errors in the ratio. However the ratio (average (soluble collagen/protein)/(average [soluble elastin/protein]) seemed to be higher in Tgm2 e/e /F13a1 e/e mice (0.0633) than in other genotypes (C57Bl/6J, 0.0398; Tgm2 e/e , 0.0458; F13a1 e/e , 0.0455 [n ¼ 10-12]). DNA content and lactate dehydrogenase activity, measured as indices of cellularity, were decreased in F13a1 e/e and Tgm2 e/e / F13a1 e/e aortas (Fig 2, C).
The basal tension exerted by excised aortas from Tgm2 e/ e /F13a1 e/e mice tended to be lower than that exerted by wild-type, Tgm2 e/e or F13a1 e/e mice (P ¼ .072). The median effective concentration for the phenylephrine response and the maximal increase in aortic tension were similar between genotypes (Fig 3). In summary, while the properties of aortas from F13a1 e/e and Tgm2 e/e single knockout mice appeared similar to C57Bl/6J wild-type mice, there were detectable differences in the basal state of Tgm2 e/ e /F13a1 e/e double knockout aortas that could affect their response to aneurysm development.
Laparotomy is followed by failure to thrive and high mortality in mice lacking FXIII-A. Despite recovering from surgery, a high proportion of F13a1 e/e mice failed to regain weight and died, often within 1-5 days (Fig 4, A, B). Necropsy frequently showed blackened, thrombus-filled atria (Fig 4, C) and/or multiple discrete areas of focal necrosis in the bowel, spleen, and liver. Postoperative mortality was further increased in Tgm2 e/ e /F13a1 e/e mice over F13a1 e/e mice (Table) and the increase was largely attributable to delayed bleeding. A comparable death rate among NaCl-treated (shamoperated) Tgm2 e/e /F13a1 e/e mice indicated that deaths were not a consequence of aneurysm development.
Knockout of Tgm2 exacerbates aneurysm progression, but FXIII-A does not exhibit functional redundancy with TG2. Exposure to CaCl 2 caused surface inflammation and an increase in external aortic diameter in all genotypes (Fig 5, A). Transverse sections showed elastic fiber breakage (Fig 5, B) and were used to measure internal vessel circumference. Regression analysis indicated that the ratio D (internal circumference) /D (external diameter) was 4.1 (Fig 5, C), which confirmed that luminal dilatation contributed to the increase in external diameter (expected value ¼ p). Although a regression analysis cannot exclude that wall thickening also contributed to the increase in external diameter, this finding was not apparent by visual inspection of the sections, whereas wall thickening in the absence of dilatation would have generated a line of zero gradient. Tgm2 e/e mice showed slightly greater aortic dilatation 6 weeks after treatment (67.5 6 30.2%) than wild-type mice (60.0 6 36.3%; P ¼ .097) (Fig 5, D). Aneurysms further progressed in Tgm2 e/e mice in the period up to 24 weeks (89.5 6 34.5% dilatation [P ¼ .025] relative to Tgm2 e/e mice at 6 weeks), but showed minimal progression in wild-type mice (64.1 6 30.2% dilatation at 24 weeks [P ¼ .57] relative to wildtype at 6 weeks). Defining aneurysms as a 50% increase in aortic diameter, 37 16 of the 17 Tgm2 e/e mice developed aneurysms at 24 weeks as opposed to 10 of the 19 wildtype mice (P ¼ .008).
In view of the possibility that FXIII-A might act redundantly with TG2 to influence aneurysm development, aortic dilatation was also measured 6 weeks after CaCl 2 treatment in F13a1 e/e mice and Tgm2 e/e /F13a1 -/mice. Dilatation was essentially equal in F13a1 e/e mice (59.4 6 32.9%) and wild-type mice, but tended to be less in Tgm2 e/e /F13a1 e/e mice (43.1 6 27.5%; P ¼ .097). Although Transglutaminase (TG) expression in aortas from C57Bl/6J wild-type mice and transglutaminase knockout mice. Transcripts encoding TG2 and factor XIII-A (FXIII-A) were detected in C67Bl/6J wild-type (WT) mice but were absent from the relevant knockout mice. Transcripts encoding TG3 were detectable in WT mice but did not increase in knockout mice. Transcripts encoding TG1, TG4, TG5, and TG7 were present in WT mouse aortas at very low concentrations relative to TG2 expression (main graph) and did not increase in Tgm2 e/e or F13a1 e/e knockout mice (lower inset). FXIII-A protein was present in CD163 positive cells (macrophages) in WT mice, but was not detected in CD163 macrophages in F13a1 e/e knockout mice (upper inset). mRNA, Messenger RNA. the cause of death after laparotomy in Tgm2 e/e /F13a1 e/e mice seemed to be unrelated to aneurysm development, it seemed possible that Tgm2 e/e /F13a1 e/e mice that would otherwise have developed a large AAA were more likely to die prematurely than those developing a small aneurysm. To address this phenomenon, we also induced aneurysms in the carotid artery and all mice survived 6 weeks after CaCl 2 treatment. Similar to the abdominal aorta, dilatation of the carotid artery was less in Tgm2 e/e /F13a1 e/e mice (20.7 6 15.9%) than wildtype mice (43.1 6 34.8%; P ¼ .028) (Fig 6), suggesting that the decrease in aortic dilatation in Tgm2 e/e /F13a1 e/ e mice was not biased by mortality. Although the basis for this unexpected decrease in dilatation is unclear, the results exclude the possibility that FXIII-A acts redundantly with TG2 to inhibit aneurysm protection.
In view of the high mortality associated with laparotomy in mice lacking FXIII-A, and the lack of evidence that FXIII-A protects against aneurysm development, aneurysm development at 24 weeks was not assessed in either F13a1 e/e or Tgm2 e/e /F13a1 e/e mice.
Collagen density is decreased adjacent to areas of elastic breakage and is decreased in Tgm2 e/e mice relative to wild-type mice. The fractional areas within aneurysms of wavy elastin (normal), straightened elastin (damaged), or broken elastin 6 weeks after CaCl 2 treatment did not differ significantly between genotypes (Fig 7, A). However, elastin breakage tended to be decreased in Tgm2 e/e /F13a1 e/e mice in accord with their reduced dilatation. In each genotype, the density of fibrillar collagen was decreased adjacent to regions of elastic fiber flattening and was further decreased adjacent to regions of elastic fiber breakage (Fig 7, B, C). Fibrillar collagen density at regions of similar damage did not differ between genotypes after 6 weeks, but was decreased adjacent to intact and flattened regions in 24-week aneurysms in Tgm2 e/e mice (relative to wild-type mice), consistent with the increased aneurysm size seen at this time point.
Despite evidence that either FXIII-A or TG2 activity is necessary for ectopic calcification, 38 fragmented elastin underwent calcification 39 in all genotypes including Tgm e/e /F13a1 e/e mice. Calcification persisted for at least 24 weeks in wild-type (Fig 8, A) and Tgm2 e/e (not shown) mice.
Gene expression studies revealed marked alterations in aneurysmal arteries from mice lacking FXIII-A. Because visual inspection could not easily determine the boundary between injured and uninjured tissue, RNA was isolated from the aorta extending from the aortic arch to the iliac bifurcation to ensure comparability between samples. Serial sectioning suggested that at least 25% of the excised tissue was injured (not shown). Apart from the targeted genes in knockout mice, transcript levels did not show consistent differences between genotypes. Normalized levels of transcripts including MMP12, MT1-MMP (MMP14), and heme oxygenase increased after aneurysm development, particularly in the gene knockout mice, consistent with the invasion of inflammatory macrophages (Fig 9). In contrast, transcripts encoding FXIII-A and CD163 (present in resident macrophages) did not show statistically significant changes in expression. Further, TG2 mRNA did not increase after aneurysm induction in either wild-type or F13a1 e/e mice (Fig 9).
Transcripts including vimentin, smooth muscle a-actin and collagen III (possibly also collagen I; P ¼ .056) showed decreases in aneurysmal arteries from both F13a1 knockout and Tgm2/F13a1 double knockout mice relative to uninjured aortas from mice of the same genotypes. MMP2 mRNA was also decreased in injured relative to uninjured aortas in these mice, whereas MMP9 showed no significant difference in the levels between the genotypes. Fibrillin I mRNA levels decreased, whereas fibrillin 2 mRNA increased in FXIII-A-deficient mice, suggesting reversion to a more fetal pattern of gene expression 40 ; there were also reciprocal changes between TIMP1 (expression increased in gene knockout mice) and TIMP2 (expression decreased) (Fig 9).
The mRNAs for TG1 and TG7 increased in each of the three mouse gene knockout lines (P < .01 in each case), but the low initial concentrations of these mRNAs, relative to the mRNAs encoding TG2 and FXIII-A (Fig 1), mean that it is unlikely that they had increased sufficiently to compensate for either knockout. Although TG3 was detected at appreciable levels in uninjured aorta, no statistically significant changes in TG3 mRNA were observed upon aneurysm induction (P > .7).

DISCUSSION
The risk that AAA poses to human health has prompted the development of several small animal models, including induction of AAA by periarterial application of CaCl 2 . 37, 41 Munezane et al 25 reported that aneurysms of   the rat infrarenal aorta induced by combined CaCl 2 and luminal elastase treatment showed a marked rise in TG2 expression and suggested that this might constitute a protective response.
Here we have tested whether TG2 influences CaCl 2induced AAA development by comparing wild-type and Tgm2 e/e mice. Tgm2 e/e mice showed a marginal increase in dilatation relative to wild-type mice 6 weeks after CaCl 2 treatment, but dilatation then continued from 6 to 24 weeks in the Tgm2 e/e but not wild-type mice, suggesting a role for TG2 in arterial protection or repair. Because FXIII-A might act redundantly with TG2 to mediate protection or repair, we also induced aneurysms in Tgm2 e/e /F13a1 e/e and F13a1 e/e mice. Dilatation was unaltered in F13a1 mice, whereas, in contrast with expectations, dilatation was decreased in Tgm2 e/e /F13a1 e/e mice. We cannot account for the decreased dilatation of aortas from Tgm2 e/e /F13a1 e/e mice, although the basal tension and the elastin/collagen ratio of their aortas appeared different from other genotypes. Tgm2 e/e /F13a1 e/e mice also develop red blood cell extravasation and myocardial fibrosis, 26 whereas altered fibronectin metabolism was noted in a separate cohort of Tgm2 e/e /F13a1 e/e mice. 28 It is unclear whether common Fig 5. A, Representative images of aortas from wild-type mice captured: (i) before treatment; (ii) 6 weeks after the application of 0.15 mol.L À1 NaCl, native image; (iii) contrast inversion of image (ii); and (iv) 6 weeks after the application of 0.5 mol.L À1 CaCl 2 . Scale bars represent 1 mm. Treatment with CaCl 2 , but not NaCl, induced surface inflammation and focal dilatation. Contrast inversion afforded better resolution of the vessel margins and facilitated the measurement of the external diameter. B, Transverse section (stained with hematoxylin and eosin and imaged at 10Â original magnification) of a C57Bl/6J wild-type aorta obtained 6 weeks after exposure to 0.5 mol.L À1 CaCl 2 . The ventral face (marked with arrows and shown in the inset) displays elastin breakage and fiber loss. C, The external diameter of CaCl 2 -treated arteries from wild-type mice is linearly related to the vessel circumference, confirming luminal dilatation. D, The percentage change in aortic diameter from baseline (mean 695% confidence intervals) is shown at 6 weeks (crossed symbol) and 24 weeks (24 weeks, filled symbol) after treatment with 0.5 mol.L À1 CaCl 2 . There was no statistically significant difference between genotypes at 6 weeks of age (analysis of variance; P ¼ .097), although there was a tendency for dilatation to be less in Tgm2 e/e /F13a1 e/e double knockout mice. At 24 weeks, the mean aortic diameter had further increased in the Tgm2 e/e mice (6 weeks vs 24 weeks; P ¼ .025), causing the average diameter to be greater than that in the C57Bl/6J wild-type mice (P ¼ .031). KO, Knockout.
factors underlie the fibrosis, alterations in fibronectin metabolism, and the arterial changes seen here. Regardless of this finding, our results show that FXIII-A does not act redundantly with TG2 to protect against aneurysm development in the CaCl 2 model. Human aneurysms frequently exhibit a decreased elastin/collagen ratio, owing to preferential degradation of elastin coupled, in some instances, with increased collagen deposition. 27,42 We observed lower periaortic collagen density adjacent to the injured face of mouse aneurysms, both at 6 and 24 weeks after injury, suggesting that compensatory collagen deposition had not occurred. Periaortic collagen density was also lower at 24 weeks in Tgm2 e/e mice than in wild-type mice, corresponding with the increased dilatation in these animals. A decrease in TG2-mediated cross-linking and consequent increase in susceptibility to proteolysis could explain the decreased density of collagen in the Tgm2 e/e mice, although verifying this finding by quantifying isopeptide cross-links remains difficult. 43,44 Alternatively, the absence of intracellular TG2 may have altered cellular function 13 and hence the deposition of collagen.
Although we observed calcification of mouse AAA, rat AAA did not calcify in the study of Munezane et al 25 and this difference in aneurysm morphology was accompanied by differences in gene expression profiles. In particular, we did not observe increased levels of mRNAs encoding TG2, MMP2, or MMP9, 25 although similar to Longo et al 45 we observed an increase in MMP12 mRNA. Our results indicate that an increase in TG2 mRNA levels is not an invariable response to vessel dilatation, and similarly using a microarray approach, Biros et al 46 found that TG2 mRNA did not increase within human AAA, but that it did increase in aortic obstructive and Tgm2 e/e /F13a1 e/e (not shown) carotid arteries following injury. Scale bar represents 100 mm.
disease. We have stained AAA sections from four patients with a polyclonal antibody to TG2 and have observed intense areas of striated staining in isolated areas of inflammation, but also large areas devoid of staining (not shown). Therefore, localized expression of TG2 could be important in the response to disease, even if an average increase in mRNA expression across the whole lesion is not evident A proportion of mice that lacked FXIII-A died within a few days of laparotomy. Necropsy frequently revealed cardiac pathology (eg, atrial thrombosis) in C57Bl/6J F13a1 e/e mice, sometimes associated with distal ischemia, and with clots that may have embolized from the heart. In addition to this pathology, Tgm2 e/e / F13a1 e/e mice showed an increased frequency of hemorrhage relative to F13a1 e/e mice, that may have resulted from delayed bleeding caused by surgical injury or postoperative stress. It seems that compromised tissue protection and repair in the absence of TG2 exposes the bleeding diathesis associated with FXIII-A deficiency, similar to the situation where TG2 deficiency exacerbates extravasation and myocardial fibrosis associated with FXIII-A deficiency. 26 The cause of the nonhemorrhagic deaths is uncertain, but FXIII-A also maintains endothelial barrier function 26,47 and can protect against systemic organ injury. 48,49 Impaired barrier function may have rendered F13a1 knockout mice susceptible to cardiac damage after laparotomy. We also observed large decreases in the mRNAs encoding certain structural proteins after aneurysm induction, particularly in mice lacking FXIII-A and note that Kothapalli et al 50   Collagen density is decreased adjacent to areas of medial damage. A, There is no significant difference between genotypes in the percentage distribution of regions of apparently healthy media, straightened elastic lamina, or broken elastic lamina in aortas examined 6 weeks after CaCl 2 treatment. B, Representative Miller's elastic Van Gieson stained images of the media (above) and second harmonic images of fibrillar collagen in the adjacent adventitia (below) in areas in which the lamina appeared: (i) healthy, (ii) straightened or (iii) broken. C, The density of fibrillar collagen, measured in arbitrary units (AU) by second harmonic signal at 400 nm decreased adjacent to areas of laminar damage in mice of all genotypes, but there is no difference between genotypes 6 weeks after CaCl 2 treatment (plain bars). However, when measured 24 weeks after CaCl 2 treatment, the collagen density is significantly reduced in Tgm2 knockout (KO) mice relative to wild-type mice, adjacent to areas of healthy and straightened elastin (patterned bars). expression in cells explanted from CaCl 2 -induced rat aortic aneurysms, although presumably these cells expressed FXIII-A. Further work is needed to determine whether common pathways underlie the unexpected changes in gene expression in aneurysmal aortas from F13a1 e/e knockout mice and the changes in endothelial barrier function, and hence mouse survival.
Of the three models commonly used to induce AAA in mice, the relevance of the angiotensin II-hyperlipidemia protocol has been questioned. 51 The infusion of elastase into the aortic lumen causes rapid dilatation, but would risk hemorrhage in mice lacking FXIII-A. Further, ligation necessary for elastase infusion is expected to preferentially damage Tgm2 e/e vessels, 26 complicating the analysis of aneurysm development. We therefore chose the CaCl 2 protocol to minimize manipulation of the aorta. A limitation of the CaCl 2 model is that murine aneurysms do not accumulate luminal thrombus, possibly because of rapid fibrinolysis. 52,53 Second, CaCl 2 -induced aneurysms rarely rupture and cannot model protection against this outcome. Third, mice do not express proelafin/trappin-2, a neutrophil elastase inhibitor, which is cross-linked by TG(s) to the extracellular matrix 54 and which may confer protection in human arterial lesions. 17 A point of difference between the C57BL/6J mice used here and the mixed strain mice used previously is that ligation caused carotid artery rupture in 50% of mixed strain apoE e/e /Tgm2 e/e /F13a1 e/e triple knockout mice and elastic breakage without rupture in apoE e/e / Tgm2 e/e mice, 26 whereas ligation did not cause carotid rupture in either C57BL/6J Tgm2 e/e /F13a1 e/e or C57BL/6J apoE e/e /Tgm2 e/e /F13a1 e/e mice (n ¼ 10). Nevertheless, ligation induced the deposition of additional elastic lamellae in C57BL/6J mice lacking TG2 (not shown), presumably to reinforce the intrinsically weaker vessel against mechanical stress, and showing that the elastase protocol might prove problematic in these mice. We suspect that rupture depended on the additional stress Intense staining for calcium is apparent at the injured ventral face of the aorta in all genotypes 6 weeks after CaCl 2 treatment. Representative images from wild-type and Tgm2 e/e /F13a1 e/e double knockout (KO) mice are shown. Calcium deposition was also apparent 24 weeks after CaCl 2 deposition, when in some mice, medial degeneration and calcification were apparent around the whole circumference of the vessel. B, Aorta from two representative C57Bl/6J wild-type mice examined 24 hours after CaCl 2 treatment. Alizarin red S staining showed occasional small yellow orange Ca 2þ containing crystals in the lamellae, but not widespread Ca 2þ deposition, verifying that CaCl 2 treatment did not immediately flood the aorta with Ca 2þ ions, but initiated tissue damage. Transcript levels in aortas in control and aneurysmal aortas. Transcript levels in aortas collected 6 weeks after CaCl 2 treatment were determined by reverse transcriptase polymerase chain reaction and normalized to the levels of ribosomal protein large subunit (Rpl)32. Mean values are shown with 95% confidence intervals. Data are presented on a logarithmic scale for clarity. Statistically significant increases upon aneurysm induction were observed in messenger RNAs (mRNAs) encoding heme oxygenase 1, matrix metalloproteinase 12 (MMP12) and membrane-type MMP (MT-MMP), comparing aneurysmal and nonoperated aortas mice from all genotypes. For other transcripts including CD163, tissue type plasminogen activator (tPA), transforming growth factor (TGF)-b, no significant changes in levels were observed between aneurysmal and non-operated aortas. Surprisingly marked decreases in mRNAs encoding a-smooth muscle actin (a-SMA) and collagen III were seen in both Tgm2 e/e /F13a1 e/ e mice and F13a1 e/e mice and significant decreases in mRNAs encoding elastin and MMP2 were also apparent in these genotypes. Probability values shown here compare aneurysmal and nonaneurysmal aortas from all mice deficient in factor XIII-A (FXIII-A). HO-1, Heme oxygenase 1; TG, Transglutaminase. caused by neointimal deposition, which was occlusive in the mixed strain mice, but, as reported, 55,56 was minimal or absent in C57BL/6J mice.
Regardless of these caveats, we conclude that the basal level of TG2 expression in aorta appears sufficient to limit mouse AAA development. We did not detect induction of TG2 mRNA expression upon injury, but do not exclude the possibility that localized increases in TG2 expression may have occurred but were not apparent in total tissue measurements. Experiments were not done that would confirm that the replacement or enhancement of TG2 would be protective; however, because TG2 expression is one of a limited number of enzymes implicated in tissue repair (and its expression can be induced pharmacologically 10 ), further studies are merited to determine whether TG2 may prove a future viable target for the management of human AAA.