Serpina3n attenuates granzyme B-mediated decorin cleavage and rupture in a murine model of aortic aneurysm

Abstract

Granzyme B (GZMB) is a proapoptotic serine protease that is released by cytotoxic lymphocytes. However, GZMB can also be produced by other cell types and is capable of cleaving extracellular matrix (ECM) proteins. GZMB contributes to abdominal aortic aneurysm (AAA) through an extracellular, perforin-independent mechanism involving ECM cleavage. The murine serine protease inhibitor, Serpina3n (SA3N), is an extracellular inhibitor of GZMB. In the present study, administration of SA3N was assessed using a mouse Angiotensin II-induced AAA model. Mice were injected with SA3N (0-120 μg/kg) before pump implantation. A significant dose-dependent reduction in the frequency of aortic rupture and death was observed in mice that received SA3N treatment compared with controls. Reduced degradation of the proteoglycan decorin was observed while collagen density was increased in the aortas of mice receiving SA3N treatment compared with controls. In vitro studies confirmed that decorin, which regulates collagen spacing and fibrillogenesis, is cleaved by GZMB and that its cleavage can be prevented by SA3N. In conclusion, SA3N inhibits GZMB-mediated decorin degradation leading to enhanced collagen remodelling and reinforcement of the adventitia, thereby reducing the overall rate of rupture and death in a mouse model of AAA.

Figure 1

SA3N inhibits mGZMB enzymatic activity. Mouse GZMB (20 nM)-mediated cleavage of the colorimetric substrate Ac-IEPD-pNA (1 mM) was inhibited by increasing concentrations of SA3N. Percent activity was determined as the change in initial rate relative to the initial rate in the absence of SA3N. IC₅₀=11.83 nM (range 7.925–17.66 nM). Data represent the mean±S.E.M of three experiments

Figure 2

GZMB inhibition increases 28-day survival and reduces rate of aneurysm rupture. (a) Survival. SA3N 120 μg/kg (81.8%, n=11); SA3N 40 μg/kg (83.3%, n=18); SA3N 20 μg/kg (70.0%, n=10); SA3N 4 μg/kg (54.5%, n=11); and saline sham (50.0%, n=12). Log-rank test for trend shows a significant increase in survival with dose of SA3N received (P=0.0207). (b) Total aneurysm incidence and rupture. SA3N 120 μg/kg (incidence 63.3% rupture 18.2%); SA3N 40 μg/kg (77.8% 16.7%); SA3N 20 μg/kg (90.0% 30.0%); SA3N 4 μg/kg (81.8% 45.5%); saline sham (91.7% 50.0%)

Figure 3

GZMB is abundant in vessels exhibiting medial disruption. Serial sections of abdominal aorta were taken from a sham-treated mouse following aortic rupture and stained for Movat's Pen/tachrome (4 × : a, 40 × : b and c), GZMB (4 × : d, 40 × : e and f) and decorin (4 × : g, 40 × : h and i). GZMB staining by immunohistochemistry (d and f) corresponds to regions of medial disruption and elastin fragmentation (a and c) and loss of decorin in the adventitia (i). The non-dilated side of the aorta has reduced GZMB staining in the media and adventitia (d and e) and corresponds to intact elastic lamellae (a and b) and decorin (g and h), scale bars: 4 × , 500 μm; 40 × , 50 μm

Figure 4

GZMB immunopositivity is reduced in SA3N-treated mice. GZMB staining is minimal in healthy aorta (a) in both the media (represented by ‘M') and the adventitia (represented by ‘Ad'). GZMB expression is increased particularly in the adventitia of sham-treated mice that experienced rupture (b). Reduced GZMB was observed in SA3N-treated mice with no AAA (c) and small, remodelled AAA (d), scale bars: 4 × , 500 μm; 40 × , 50 μm

Figure 5

GZMB cleavage of decorin is prevented by pre-incubation with SA3N in vitro. Purified GZMB was pre-incubated with or without SA3N for 25 min at RT before incubation with recombinant human decorin for 24 h at RT. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and visualized by western blotting for decorin (a) and Ponceau stain (b). In a, full-length decorin is indicated by an asterisk (^*) and is visible at ∼65 kDa in lane 1. Decorin is severely reduced following incubation with GZMB in lane 2. In lane 3, decorin loss is not observed following pre-incubation of GZMB with SA3N. In b, SA3N and SA3N–GZMB complex at approximately 47 and 70 kDa, respectively (indicated by ^) are visible in lanes 2, 3 and 6. Full-length decorin is indicated by the asterisk (^*) at approximately 65 kDa in lanes 4, 5 and 6. Decorin cleavage fragments in lane 5 are marked by arrows on the right side of the membrane

Figure 6

SA3N and GZMB deficiency promotes adventitial thickening and increased decorin content in AAA. Sections stained with picrosirius red are shown in the first column (a, d, g, j, m and p). Tissues immunostained for decorin are shown in the second column (b, e, h, k, n and q) and enlarged for emphasis at 40 × in the third column (c, f, i, l, o and r). When stained with picrosirius red, thick collagen fibers evidenced by strong birefringence (red) under polarized light (a) are visible in the adventitia of healthy normal aorta, as well as robust decorin staining (b and c). Non-ruptured aneurysms from sham-treated mice exhibit bulbous thrombus, reduced decorin staining (h and i) and thinner collagen fibers of lower density (g) as evidenced by predominance of yellow color when stained with picrosirius red and seen under polarized light. Ruptured aneurysms from sham-treated mice demonstrate even greater loss of collagen content (d) and a similarly reduced amount of decorin in the adventitia (e and f) compared with healthy aortas. In comparison, aortas from mice that received SA3N (120 μg/kg) before AII pump implantation demonstrate a significant increase in collagen and a thickened adventitial layer, with a greater proportion of red and orange fibers under polarized light (j). Decorin content in the adventitia is also greatly increased (k and l). Morphology and levels of collagen and decorin in normal, non-diseased aorta from GDKO mice resemble healthy controls (m, n and o) while GDKO that develop small, localized aneurysm display thickened adventitia (p) and increased decorin content (q and r) similar to SA3N-treated mice. Scale bars: 4 × , 500 μm; 40 × , 50 μm

Figure 7

SA3N treatment reduces the loss of collagen fiber density in AII-treated ApoE-KO mice. Adventitial collagen from healthy mouse (a), sham-treated mouse with ruptured aorta (b), SA3N-treated mouse (c) and sham-treated mouse, non-ruptured (NR) aorta (d) were assessed by SHG. Scale bar: 25 μm. SHG signal densities are summarized in e (n=3 per group, ^*P<0.05)