Angiotensin II–Induced AAA

Mice aged three to four months received either 28 days of Ang II (Sigma Aldrich, St. Louis, MO) infusion at 1000 ng/min/kg or saline infusion from a subcutaneous 1004 model ALZET® mini osmotic pump (DURECT Corporation, Cupertino, CA) as previously described.31 Briefly, an osmotic pump was filled with the saline solution, primed at 37°C for 24 hours in saline, and surgically implanted subcutaneously posterior to the scapula of the mouse. During the implantation procedure, mice were anesthetized with gaseous anesthetic at a flow rate of 1.5 liters per minute of oxygen with 2.5% of isoflurane delivered via a Baines system using a calibrated tabletop anesthetic machine, administered from a rodent nose cone. Depth of anesthesia was monitored by toe pinch response and breathing. Eyes were protected using ocular lubricant. Postsurgical pain control consisted of a subcutaneous injection of buprenorphine. In total eight APOE-KO mice received saline and 15 received Ang II; 11 GZMB/APOE-DKO mice received saline and 14 received Ang II; five PRF1/APOE-DKO mice received saline and 16 received Ang II.

Tissue Collection and Gross Pathological Characterization

At day 28, tissues from the surviving mice were collected. Blood was collected by cardiac puncture after CO₂ euthanasia in EDTA (Sarstedt Monovette, Germany) and red blood cells removed by centrifugation. The mouse was placed on ice, the chest was opened, the right atrium was cut, and a needle was placed in the left ventricle. Sterile saline, and then 4% formalin (Fisher Scientific, Fairlawn, NJ), were perfused at a constant pressure of 100 mm Hg using a pressurized tubing system until no blood was observed exiting the incision in the right atria. The heart, aorta to the iliac bifurcation, and kidneys were dissected from the mouse and photographed. At this point, a gross description of the aorta was made and grouped under the following categories: No pathology; Small localized saccular AAA below diaphragm with or without visible hematoma (28 day survival); Large dissecting AAA beginning in the suprarenal aorta and extending beyond the diaphragm, into the mid-thoracic aorta (28 day survival); Large ruptured AAA with death by exsanguination into abdominal cavity (<28 day survival). Necropsy was performed for all mice that died before 28 days to determine cause of death. Tissues were stored in fresh 10% formalin overnight before being embedded and sectioned.

Murine Tissue Embedding and Sectioning

Aortic segments were isolated from the descending aorta immediately above the diaphragm, and the thoracoabdominal aorta immediately above the renal arteries. APOE-KO, GDKO, and PDKO tissue sections were embedded in paraffin and serial sectioned into 5-μm sections.

Murine Blood and Tissue Histological Analysis

Murine abdominal aortic sections were stained with H&E, Movat pentachrome, or immunohistochemistry performed using rabbit anti-GZMB (Abcam, Cambridge, MA) and rabbit anti–fibrillin-1 (Abcam) as previously described.23 Negative control staining was performed in the same manner, with absence of primary antibody. Fibrillin-1 fragments in mouse blood serum were isolated by immunoprecipitation then analyzed by Western blotting using goat anti-human fibrillin-1 Abs (N-19 and C-19, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). To corroborate gross morphological assessments, histological evaluation for severity of disease was performed by two independent pathologists who were blinded to the experimental conditions.

RNA Extraction and RT-PCR

Human coronary artery smooth muscle cells (HCASMCs; Cambrex, Walkersville, MD) were grown in smooth muscle cell basal medium containing supplements and 5% FBS, according to the supplier’s instructions (LONZA, Walkersville, MD), and were used after reaching 80% confluence. Before treatment, cells were starved for 24 hours in serum-starved media (supplemented smooth muscle cell basal medium [LONZA] + 0.2% FBS). After starvation, cells were treated with 10 nmol/L or 25 nmol/L of GZMB (Alexis Biochemicals, Farmingdale, NY) for 24 hours. Controls were treated with PBS. At 24 hours, cells were lysed with TRIzol (Invitrogen, Carlsbad, CA), RNA was extracted according to manufacturer’s instructions, and total RNA quantified by absorbance at 260 nm. To eliminate genomic DNA contamination, total RNA (1.5 μg) was DNase I-treated (Invitrogen) according to manufacturer’s instructions. cDNA was prepared from 1 μg of total RNA using 50 μmol/L of oligo (dT)₂₀ primer and Superscript III reverse transcriptase (Invitrogen), according to manufacturer’s instructions. All PCR reactions were performed in 25 μl volumes using Platinum TaqDNA polymerase (Invitrogen) and previously published primers for human fibrillin-1 (Forward, 5′-GTGAGATCAACATCAATGGAGC-3′; Reverse, 5′-TTACACACTCCTGGGAACACTTC-3′) and the universal GAPDH primers (Forward, 5′-CATGTTCGTCATGGGTGTGA-3′; Reverse, 5′-GACTGTGGTCATGAGTCCTT-3′).32 Equal aliquots of the PCR products were electrophoresed on a 2% agarose gel, stained with SafeView and photographed.

In Vitro GZMB ECM Cleavage

To determine whether GZMB can cleave SMC ECM, we performed an in vitro experiment as previously described.19 HCASMCs were used, as they are known to secrete fibrillin-1. Cells were cultured to confluence and serum starved for 48 hours to allow for adequate ECM synthesis and secretion, at which time cells were lysed with NH₄OH so that the intact ECM remained on the plate.33 GZMB (80 nmol/L; Axxora, LLC, Farmingdale, NY) was incubated on the ECM for 24 hours at room temperature. Supernatants (containing cleaved ECM) were taken off the plate and ECM still attached to the plate was scraped off, collected, and assessed for fibrillin-1 cleavage by Western blot (Monoclonal antibody from Lab Vision/Neomarkers, Fremont, CA).

Human AAA Tissue Collection and Histological Analysis

Human AAA samples were obtained in accordance with the ethical protocols at the Karolinska Institute (Sweden). Immunohistochemistry for GZMB was performed on formalin-fixed paraffin-embedded sections as described previously.23 Briefly, sections were deparaffinized and rehydrated in xylene and ethanol. Antigen retrieval was performed by boiling slides in citrate buffer (pH 6.0) to expose antigens masked in the fixation process as per recommended protocol from antibody supplier. Background staining was blocked by incubation of sections in 10% horse serum. Sections were incubated in a 1:100 dilution of mouse anti-GZMB (a generous gift from Dr. Joseph Trapani, Melbourne, Australia, previously shown to be monospecific to GZMB34) overnight, followed by incubation with biotinylated horse anti-mouse secondary Ab (Vector laboratories, Burlingame, CA) and ABC reagent (Vector Laboratories). Staining was visualized with the DAB peroxidase substrate (Vector Laboratories), and nuclei were counterstained with hematoxylin. Negative control staining was performed in the same manner, with absence of primary antibody.

Human TAA Tissue Collection and GZMB Colocalization

Formalin-fixed paraffin-embedded human TAA samples were obtained in accordance with the ethical protocols at the Biobank Cardiovascular Registry, St. Paul’s Hospital, Vancouver, BC. Sections were de-paraffinized and rehydrated in xylene and ethanol. Antigen retrieval was performed by boiling slides in citrate buffer (pH 6.0) to expose antigens masked in the fixation process as per recommended protocol from antibody supplier. Background staining was blocked by incubation of sections in 10% goat serum. Sections were incubated in a 1:100 dilution of rabbit anti-GZMB (Abcam) overnight, followed by incubation in biotinylated goat anti-rabbit secondary Ab (Vector laboratories) and ABC reagent (Vector laboratories). GZMB staining was visualized with TSA™ Plus Fluorescence Systems cyanine 3 tyramide (PerkinElmer Life Sciences, Inc., Boston, MA). To assess for macrophages, slides were incubated with avidin and biotin to prevent interaction of labeling reagents and blocked with 10% horse serum. Sections were incubated in a 1:100 mouse anti-MAC387 (AbD Serotec, UK) overnight followed by incubation in biotinylated horse anti-mouse secondary antibody (Vector laboratories) and ABC reagent (Vector Laboratories). MAC387 staining was visualized with TSA™ Plus Fluorescence Systems fluorescein tyramide (PerkinElmer Life Sciences, Inc.). Confocal images of fluorescently labeled tissue sections were acquired with a Leica AOBS SP2 laser scanning inverted confocal microscope (Leica, Heidelberg, Germany). Excitation beams were produced by Ar (488 nm for fluorescein) and HeNe (543 nm for cyanine 3) lasers (Leica AOBS SP2 module), respectively. Images from these dual stained samples were acquired sequentially to eliminate cross talk between the emission signals and acquired images were overlaid using Volocity software (Improvisions, UK). Similarly, for colocalization of GZMB in lymphocytes, slides were first stained for GZMB with mouse anti-GZMB (Dr. Joseph Trapani) then incubated with avidin and biotin to prevent interaction of labeling reagents and blocked with 10% goat serum. Sections were incubated in a 1:100 mouse anti-CD3 (Abcam) overnight followed by incubation in biotinylated goat anti-rabbit secondary antibody (Vector laboratories) and ABC reagent (Vector laboratories). CD3 staining was visualized with TSA™ Plus Fluorescence Systems fluorescein tyramide (PerkinElmer Life Sciences, Inc.). Confocal images were acquired in a similar manner as described above. To assess GZMB colocalization in mast cells, slides were first stained for GZMB as described above, but visualized with Vector Red substrate (Vector Laboratories). Slides were then counterstained with Alcian blue to differentiate mast cells.

GZMB Deficiency Reduces Medial Disruption

As previously observed,13 medial disruption characterized by the loss of elastin is a predominant pathological feature of AAA; both in human aneurysms and in Ang II–induced murine AAA. In APOE-KO and PDKO mice, severe elastin breakage was frequently observed in conjunction with aneurysm development (Figure 3, C–H). Incidence of AAA and dissection was much reduced in GZMB-deficient mice. Severe medial disruption and subsequent remodelling, as seen in APOE-KO and PDKO mice, was not observed in GDKO mice (Figure 3, A and B).

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Figure 3

GZMB deficiency reduces medial disruption. Formalin-fixed tissues collected from mice that survived to 28 days were assessed using Movat Pentachrome as described. A and B: Representative aorta from GDKO mouse with no medial disruption and minimal adventitial thickening. C and D: Aorta from APOE-KO with elastin breakage, pronounced adventitial thickening, but no visible hematoma. E and F: Aorta from PDKO mouse with small AAA, elastin breakage, remodeled medial hematoma. G and H: Aorta from PDKO mouse with dissection, elastin breakage, and pronounced dilation. Scale bars: ×4 = 1 mm, ×40 = 50 μm.

GZMB Is Elevated in Ang II–Induced AAA

GZMB was elevated in the aortas of APOE-KO and PDKO mice that developed AAA after Ang II infusion (Figure 4A–F). No GZMB immunopositivity was observed in healthy age-matched controls that received saline infusion. GZMB was particularly abundant in the thrombus, both in trapped lymphocytes and in the extracellular space, as well as in the adventitia of large dissecting AAA.

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Figure 4

GZMB immunostaining in APOE-KO and PDKO AAA. GZMB-immunopositivity is observed in the medial thrombus of an APOE-KO mouse that suffered a small saccular AAA (A and B) and the adventitia of a PDKO mouse that suffered a large dissecting AAA (C and D). Trapped lymphocytes in the thrombus are indicated by arrows. GZMB staining is not observed in APOE-KO mice that received saline infusion (E and F). Scale bars: ×4 = 1 mm, ×40 = 50 μm.

Fibrillin-1 Staining Is Reduced in APOE-KO Mice as Compared with GDKO Mice

Immunostaining with anti–fibrillin-1 antibody revealed greater levels of fibrillin-1 in the tunica media of GDKO mice versus APOE-KO in Ang II treatment groups. Interestingly, in areas around the thrombus of AAA, minimal fibrillin-1 staining was observed suggesting that these areas could be weakened because of a lack of fibrillin-1 (Figure 5, A and B).

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Figure 5

A: Fibrillin-1 immunostaining in APOE-KO AAA is reduced compared with GDKO. A and B: Decreased fibrillin-1 staining, as indicated by red color, was observed in Ang II–infusion APOE-KO mice versus Ang II–infusion GDKO mice. Scale bar = 50 μm. C: GZMB cleaves fibrillin-1 from HCASMC-generated ECM in vitro. HCASMCs were cultured to confluency, lysed, and ECM was biotinylated as described. Human GZMB, isolated from IL-2 stimulated lymphocytes was then added to biotinylated ECM, and supernatants were collected to assess ECM fragments that were released from the plate. GZMB-mediated fibrillin-1 cleavage was attenuated by the GZMB inhibitor DCI but not by the inhibitor solvent control DMSO. Fibrillin-1 fragments from the supernatant are indicated by an arrow. Gel is representative of six independent experiments. D: Fibrillin-1 fragmentation is prevented in GDKO mouse serum compared with APOE-KO after Ang II infusion. Fibrillin-1 in mouse serum was isolated by immunoprecipitation and analyzed by Western blotting. APOE-KO mice with aneurysm show a distinct band at approximately 45 kDa that is not observed in GDKO on Ang II and APOE-KO on saline. E: In vitro fibrillin-1 expression is not directly affected by GZMB treatment. HCASMCs were treated with GZMB and assessed for fibrillin-1 transcription by RT-PCR. No difference was observed in cells that received treatment compared with controls.

Granzyme B Cleaves Fibrillin-1 in Vitro

HCASMCs were cultured to confluency and serum starved for 48 hours at which time cells were lysed and GZMB activity was determined as described. When treated with GZMB, a 210-kDa fibrillin-1 fragment was detected in the supernatant indicating that fibrillin-1 is susceptible to GZMB-mediated proteolytic cleavage (Figure 5C). As the antibody is monoclonal, the other fragment was not recognizable as it did not contain the epitope. We also observed an increase in full-length fibrillin-1 in the supernatant suggesting that GZMB was also weakening the adhesion of this protein to other ECM proteins or cleaving other proteins. The full-length protein was observed to dissociate from the plate in all six repetitions of the assay (data not shown). Fibrillin-1 cleavage was not observed in the plates that received PBS alone, or in the presence of the serine protease inhibitor, 3,4-Dichloroisocoumarin (DCI), confirming fibrillin-1 is indeed a substrate for GZMB. The latter two treatments also were associated with reduced full-length fibrillin-1 in the supernatant.

Fibrillin-1 Fragmentation Is Prevented in GDKO Mice Blood Serum

Mouse blood serum levels of fibrillin-1 were assessed by immunoprecipitation and Western blotting. All samples tested have a band at approximately 50 kD representing immunoglobulin heavy chain; however, it was observed that samples from APOE-KO mice with aneurysms after infusion with Ang II have a distinct second band at approximately 45 kDa that is not seen in samples from GDKO mice that received similar treatment or in APOE-KO mice that received the saline control, suggesting that significant fibrillin-1 fragmentation is prevented in GDKO compared with APOE-KO after Ang II infusion (Figure 5D).

GZMB does not Alter in Vitro Fibrillin-1 Expression

Fibrillin-1 transcription levels in HCASMCs after treatment with GZMB were assessed by RT-PCR (Figure 5E). It was found that there was no difference in fibrillin-1 expression in HCASMCs treated with GZMB compared with controls, suggesting that GZMB does not have any direct effect on fibrillin-1 transcription levels.

GZMB Is Elevated in Human AAA

Human AAA tissue and healthy nonatherosclerotic aorta tissue were assessed for the presence of GZMB. No GZMB immunopositivity was observed in control healthy aorta (Figure 6A), whereas GZMB was abundantly present in AAA tissue (Figure 6, B–G). GZMB positivity was noted in phagocytic cells trapped in the intima and fibrin platelet-red cell thrombus, and also in thrombic material attached to the deep atheroma. GZMB was also noted in cells in proximity to the medial neovasculature, in adventitial collagen layers, in remote adventitia containing nerves, and particularly in large collections of lymphocytes in the adventitia. Moderate staining was observed extracellularly and in the occasional SMC. GZMB positivity was also found in thrombic material with red cells, suggestive of GZMB in the plasma. Intense granular cytoplasmic GZMB was also identified in neuroganglion cells of the remote adventitia.

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Figure 6

GZMB expression in nonatherosclerotic human aorta versus human AAA. No GZMB immunopositivity was observed in control healthy aorta (A), whereas GZMB was abundantly present in AAA tissue (B–F). GZMB positivity was observed in the media and adventitia but particularly in the adventitial lymphocytes and extracellular connective tissue layers of AAA tissue (B). Staining in the medial thrombus (C) was largely extracellular, however, within the intraluminal thrombus (D–E), GZMB positivity was noted in cellular elements trapped and scattered throughout the fibrin platelet red cell thrombus. GZMB was also noted in remote adventitia containing nerves, and in large collections of lymphocytes in the adventitia. Intense granular cytoplasmic GZMB was identified in nerve ganglion cells of the remote adventitia (F). Negative control, no GZMB primary antibody, shows lack of non-specific staining (G). Scale bar ×40 = 50 μm.

We thank Amrit Samra and Crystal Leung for technical assistance with histology, Thomas Abraham for assistance with confocal imaging, and Dr. Joseph Trapani for the anti-human GZMB Ab. We also thank Dr. Alan Daugherty for technical assistance and for providing us with the murine AAA protocol.