Adamts5-/- Mice Exhibit Altered Aggrecan Proteolytic Profiles That Correlate With Ascending Aortic Anomalies

Abstract

Objective: Investigate the requirement of Aggrecan (Acan) cleavage during aortic wall development in a murine model with ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin-type motifs) 5 deficiency and bicuspid aortic valves.

Approach: Mice with altered extracellular matrix remodeling of proteoglycans will be examined for anomalies in ascending aortic wall development. Neo-epitope antibodies that recognize ADAMTS cleaved Acan fragments will be used to investigate the mechanistic requirement of Acan turnover, in aortic wall development.

Results: Adamts5^-/-;Smad2^+/- mice exhibited a high penetrance of aortic anomalies (n=17/17); Adamts5^-/-;Smad2^+/- mice with bicuspid aortic valves (7/17) showed a higher number of anomalies than Adamts5^-/-;Smad2^+/- mice with tricuspid aortic valves. Single mutant Adamts5^-/- mice also displayed a high penetrance of aortic anomalies (n=19/19) compared with wild type (n=1/11). Aortic anomalies correlated with Acan accumulation that was apparent at the onset of elastogenesis in Adamts5^-/- mice. Neo-epitope antibodies that recognize the initial amino acids in the Acan cleaved fragments neo-FREEE, neo-GLGS, and neo-SSELE were increased in the Adamts5^-/- aortas compared with WT. Conversely, neo-TEGE, which recognizes highly digested Acan core fragments, was reduced in Adamts5^-/- mice. However, mice containing a mutation in the TEGE₃₇₃↓₃₇₄ALGSV site, rendering it noncleavable, had low penetrance of aortic anomalies (n=2/4). Acan neo-DIPEN and neo-FFGVG fragments were observed in the aortic adventitia; Acan neo-FFGVG was increased abnormally in the medial layer and overlapped with smooth muscle cell loss in Adamts5^-/- aortas.

Conclusions: Disruption of ADAMTS5 Acan cleavage during development correlates with ascending aortic anomalies. These data indicate that the mechanism of ADAMTS5 Acan cleavage may be critical for normal aortic wall development.

Keywords: aggrecans; animals; aorta; bicuspid; extracellular matrix; mice; protease.

Figure 1:. Mice deficient in the ADAMTS5 protease exhibit ascending aortic anomalies.

Histological sections of adult wild type (TS5^+/+; Smad2^+/+, A, D), ADAMTS5 deficient (TS5^−/−; Smad2^+/+, B, E, G, H, J, K) and ADAMTS5 deficient, heterozygous in the Smad2 allele, (TS5^−/−; Smad2^+/−, C, F, I, L) are depicted. Geometry of the transverse, ascending aorta in wild type mice (A) with altered geometry in ADAMTS5 deficiency (B, double-headed arrows) and a loss of appropriate rotation in the TS5^−/−; Smad2^+/−, that further contributes to defects in the ascending aorta that correlate with ADAMTS5 deficiency. Examples of altered cellularity (E, H, K, closed arrows). Blood cells in the aortic medial layer, breaks in the aortic wall and (G, I, J, K, L, open arrows) consistent with aortic dissection. Bar in A=250μm applies to B, C, F, G; bar in D= 50μm, applies to E, H, K, L; I = 500μm; J=350μm.`

Figure 2:. Mice deficient in the extracellular matrix protease, ADAMTS5, display a constellation of outflow tract related anomalies related to the ascending aorta.

Ascending aortic anomalies are scored for the following genotypes: wild type, Adamts5^+/+ (TS5^+/+;Smad2^+/+, n=11), Adamts5^−/− (TS5^−/−;Smad2^+/+, n=19) and Adamts5^−/−;Smad2^+/− (TS5^−/−;Smad2^+/+, n=17) with a high penetrance of bicuspid aortic valves, as well as control mice (TS5^+/+;Smad2^+/−). Stenosis at the opening of the left ventricle and aortic root, outflow tract (OFT) rotation, membranous interventricular septum (IVS), smooth muscle cell (SMC) loss, or infiltration into the aortic (Ao) wall, red blood cells (RBC) in or around the aortic wall consistent with aortic dissection, *thick aortic wall, quantified in graphs below, and altered aortic geometry. Penetrance is indicated in the ‘Total’ for each anomaly. Penetrance of female (red) and male (blue) is noted to the left of the total of each anomaly examined. The red/blue sex representations of the total examined are noted on the right side of the bold values in the far-right column. Horizontal lines in the graph represent the combined mean, with the error bars representing the standard deviation of the mean. Graphs denote the quantification of the aortic wall thickness with female and male mice combined, then followed by the separation of the sexes (red-female, blue-male) in the right graph. Statistically significant experimental parameters are indicated: *P<0.05; **P<0.01; ***P<0.001, **** P<0.0001.

Figure 3:. Ascending aortic anomalies correlate with aggrecan accumulation in the Adamts5^−/− deficient mice.

Histological sections of adult wild type (TS5^+/+; Smad2^+/+, A, D, G), ADAMTS5 deficient (TS5^−/−; Smad2^+/+, B, E, H) and ADAMTS5 deficient, heterozygous in the Smad2 allele, (TS5^−/−; Smad2^+/−, C, F, I) are shown. Geometry of the transverse, ascending aorta in wild type mice (A) with altered geometry in ADAMTS5 deficiency (B, C, white arrows). Acan localization (green) correlates with aortic anomalies (B, C, white arrows). Boxes in A-C magnified in D-F. Open arrows, endothelial Acan, closed arrows-medial layer Acan. Blue- α smooth muscle actin; red-propidium iodide (A-F). Tropoelastin staining (G, H, I, red) to highlight elastic lamellae fragmentation (H, I, yellow arrows). Quantification of % Acan accumulation from confocal Immunohistological images (J). Western blot of ‘intact’ Acan and reduction of neo-TEGE, N-terminal Acan fragment in adult Adamts5^−/− aortas (K). Quantification of Western Acan and neo-TEGE of Adult Adamts5^+/+ (+/+) and Adamts5^−/− (−/−) in L. Bar in A=200μm applies to B, C; bar in D= 50μm, applies to E-I. **P<0.01; **** P<0.0001.

Figure 4:. Developmental aggrecan accumulation correlates with elastic lamellae formation at embryonic day 14.5.

Histological sections of Adamts5^+/+ (A, C, E), and Adamts5^−/− (B, D, F) are shown. Immunolocalization of Acan (green) blue- α smooth muscle actin (αSMA); red-propidium iodide (PI), (A-F). Boxes in A, B, increased in magnification right panel to show overlap of Acan, and αSMA. Open arrow, increased Acan staining in the valvulosinus. Solid arrow (D) increased Acan on the left side of the Ao in Adamts5^−/− hearts. Quantification of confocal images of postnatal day 7 (P7) Acan immunohistochemistry (G). Western blot of Acan accumulation at P7 (H). Quantification of Western (I). Bar in A=100μm applies to B; bar in C= 100μm, applies to D-F. ***P<0.001, **** P<0.0001.

Figure 5:. Aggrecan neo-epitope antibodies, that detect cleaved Acan fragments, exhibit differential immunolocalization in the Adamts5^−/− ascending aorta at postnatal day 7.

Histological sections of Adamts5^+/+ (A, C, E, G, I, K, M, O), and Adamts5^−/− (B, D, F, H, J, L, N, P). Boxes in A, B, E, F, I, J, M, N, magnified in C, D, G, H, K, L, O, P respectively. Immunolocalization of Acan neo-FREEE (A-D, green), Acan neo-GLGS (E-H, green), Acan neo-SSELE (I-L, green), Acan neo-TEGE Acan (M-P, green); blue- α smooth muscle actin (αSMA); red-propidium iodide (PI). White asterisk-increased localization of initial C-terminal neo-Acan fragments in Adamts5^−/− (B, D, F, H, J, L). Yellow asterisk (O) increased localization of the Acan neo-TEGE fragment in the IGD domain in the N-terminal Acan region that is more prevalent in Adamts5^+/+ (O). Ao-aorta, PA-pulmonary artery. Western blot of neo-GLGS from lysates of 1 mo Adamts5^−/− aortas (Q). Quantification of Western neo-GLGS Adamts5^+/+ (+/+) and Adamts5^−/− (−/−) in R. Western blot of neo-SSELE from lysates of 1 mo Adamts5^−/− aortas (S). Quantification of Western neo-SSELE Adamts5^+/+ (+/+) and Adamts5^−/− (−/−) in T. Bar in A=200μm applies to B, E, F, I, J, M, N; bar in C= 50μm, applies to D, G, H, K, L, O, P. *P<0.05; **** P<0.0001.

Figure 6:. Mice containing a mutation in the aggrecan ADAMTS cleavage site NVTEGE₃₇₃↓₃₇₄ALGSV, termed ‘Jaffa’, exhibit a low penetrance of aortic wall defects.

Histological sections of adult wild type (WT, A, F, K) and Jaffa (B-E, G-J, L-O), mice. Frontal sections of the pulmonary valve (PV) and pulmonary artery (A-E), the ascending aorta (Ao) and aortic valves (denoted by asterisks in F-J). Arrows in C-E indicate stenotic RV, not found in WT. Open arrows (O) show abnormal adventitia in aortic and pulmonary artery. Boxes in F-J, increased in magnification in K-O respectively to highlight the ascending aortic wall. RV-right ventricle; LV-left ventricle. Bar in A=250μm applies to B, C; bar in D= 100μm, applies to E-I.

Figure 7:. Aggrecan neo-epitope antibody neo-DIPEN exhibits a differential pattern of expression in the ascending aorta of postnatal day 7 and adult Adamts5^−/− mice.

Histological sections of Adamts5^+/+ hearts (A, C, E, G, I, K), and Adamts5^−/− hearts (B, D, F, H, J, L). Boxes in A, magnified in C, E; boxes in B, magnified in D, F; boxes in G, magnified in I, K; boxes in H, magnified in J, L. Immunolocalization of Acan neo-DIPEN (A-L, green), at postnatal day 7 (P7, A-F) and adult (G-L). Blue- muscle light chain kinase (MLCK); red-propidium iodide (PI). White asterisk- localization of neo-DIPEN in the adventitia (C, D, I, J). White arrow (F, I, J) aberrant neo-DIPEN staining in the medial layer of the aorta in Adamts5^−/− mice. Ao-aorta; AV-aortic valves; PA-pulmonary artery. Bar in A=200μm applies to B, G, H; bar in C= 50μm, applies to D-F, I-L.

Figure 8:. Aggrecan neo-epitope antibody neo-FFGVG correlates with smooth muscle cell loss and exhibits a differential pattern of expression in the ascending aorta of Adamts5^−/− mice.

Histological sections of Adamts5^+/+ (A, C, E, G, I, K), and Adamts5^−/− (B, D, F, H, J, L). Boxes in A, magnified in C, E; boxes in B, magnified in D, F; boxes in G, magnified in I, K; boxes in H, magnified in J, L. Immunolocalization of Acan neo-FFGVG (A-L, green), at postnatal day 7 (P7, A-F) and adult (G-L). Blue- muscle light chain kinase (MLCK); red-propidium iodide (PI). White asterisk- neo-FFGVG in the adventitia (C, D, I, J). White arrow (E, F, J, L) immunolocalization in the medial layer of the aorta. Ao-aorta; AV-aortic valves; PA-pulmonary artery. Bar in A=200μm applies to B, G, H; bar in C= 50μm, applies to D-F, I-L.

Figure 9:. Summary of differential staining of aggrecan intact and neo-epitope antibodies in the developing and adult ascending aorta of Adamts5^−/− and Adamts5^+/+ mice.

Schematic of Acan core protein (A) with neo-epitope antibodies utilized (green font). Circles denote globular Acan domains, pink bars depict the glycosaminoglycan binding domains, chondroitin sulfate 1 (CS1) and chondroitin sulfate 2 (CS2). Acan antibodies utilized (B) depicted with a schematic showing the expected fragments. ‘Intact Acan’ antibody refers to the region of the epitope utilized for the antibody to Acan presented in Fig. 2. E14.5 (embryonic day 14.5), P7-postnatal day 7, Ad-adult (2mo and older). Medial-aortic medial layer expression; Int-aortic intimal layer expression; SMC-smooth muscle cell. Red font in Adamts5^−/− expression column denotes increased expression in Adamts5^−/− compared to Adamts5^+/+; blue font denotes decreased expression in Adamts5^-/−. In far right Adamts5^−/− localization column, bold black font denotes increased expression within the same region as wild type, Adamts5^+/+. Bold red font in the far-right Adamts5^−/− column, indicates change in localization and increased immunoreactivity of antibodies in Adamts5^−/− hearts.