Adipocyte in vsculr wll cn induce the rupture of dominl ortic neurysm Hiron Kugo 1 *, Nouhiro Zim 1 *, Hiroki Tnk 2 *, Youhei Mouri 1, Kenichi Yngimoto 3, Kohsuke Hymizu 3,4, Keisuke Hshimoto 1, Tkeshi Sski 5, Mski Sno 6, Ttsuro Yt 6, Tetsumei Urno 2, Mitsutoshi Setou 7,8,9, Noki Unno 6, Ttsuy Moriym 1 1 Deprtment of Applied Biologicl Chemistry, Grdute School of Agriculture, Kindi University, 24-3327 Nkmchi, Nr City, Nr 631-855, Jpn 2Deprtment of Medicl Physiology, Hmmtsu University School of Medicine, Shizuok, Jpn 3Humn Life Science R&D Center, Nippon Suisn Kish, Ltd., Tokyo, Jpn 4 Generl Helth Medicl Center, Yokohm University of Phrmcy, Jpn 5 Deprtment of Antomy nd Neuroscience, Hmmtsu University School of Medicine, Jpn 6 Division of Vsculr Surgery, Second Deprtment of Surgery, Hmmtsu University School of Medicine, Jpn 7 Interntionl Mss Imging Center Deprtment of Cellulr nd Moleculr Antomy, nd Preeminent Medicl Photonics Eduction & Reserch Center Deprtment of Systems Moleculr Antomy, Hmmtsu University School of Medicine, 1-2-1 Hndym, Higshi-ku, Hmmtsu, Shizuok 431-3192, Jpn 8 Deprtment of Antomy, The university of Hong Kong, 6/F, Willim MW Mong Block 21 Sssoon Rod, Pokfulm, Hong Kong SAR, Chin 9 Division of Neurl Systemtics, Ntionl Institute for Physiologicl Sciences, 38 Nishigonk Myodiji, Okzki, Aichi, 444-8585, Jpn *These uthors contriuted eqully to this work. *Corresponding uthor Animl Study Nouhiro Zim zim@nr.kindi.c.jp Deprtment of Applied Biologicl Chemistry, Grdute School of Agriculture, Kindi University, 24-3327 Nkmchi, Nr City, Nr 631-855, Jpn Clinicl Study Noki Unno unno@hm-med.c.jp
Division of Vsculr Surgery, Second Deprtment of Surgery, Hmmtsu University School of Medicine, Jpn Keywords: dominl ortic neurysm, rupture, dipocyte, triglyceride, fish oil
Supplementry Informtion Supplementry Tle S1 Diet composition () Diet composition nd () ftty cid composition of coconut oil nd fish oil. Supplementry Fig. S1 Ruptured AAA, pictured efore wshing wy lood. Supplementry Fig. S2 Weight chnge nd serum prmeters () Initil ody weight of rts in the non-ruptured nd ruptured groups. () Body weight gin of rts in the non-ruptured nd ruptured groups. Serum triglyceride (TG) levels (c) nd totl cholesterol levels (d) of rts in the non-ruptured nd ruptured group. Dt re the men ± s.e.m. Non-ruptured (n = 5), ruptured (n = 5). Supplementry Fig. S3 Vsculr wll thickness nd elstin degrdtion score (-d) Representtive imges of HE stining (scle r = 2 µm). (e-h) Representtive imges of EVG stining (scle r = 1 µm). (i) Quntifiction of vsculr wll thickness in the non-ruptured nd ruptured groups. (j) Elstin degrdtion scores in the nonruptured nd ruptured groups. Dt re the men ± s.e.m. Non-ruptured (n = 5), ruptured (n = 5). Vlues with different letters re significntly different (P <.5). Supplementry Fig. S4 Thickness of medil wll (-d) Representtive imges of immunostining for α-smooth muscle ctin (scle r = 1 µm). (e) Quntifiction of α-smooth muscle ctin-positive res of vsculr wll. (f) Quntittive nlysis of medil wll thickness in the non-ruptured nd ruptured groups. Vlues with different letters re significntly different (P <.5). Supplementry Fig. S5 Oservtion of dipocyte nd PPARγ stining Representtive electron microscopic imge of the vsculr wll of the AAA neck () nd sc () from model rt. Representtive opticl imges of the neck (c) nd sc (d)(scle rs = 2 µm). Representtive imges of PPARγ stining of neck (e) nd sc (f)(scle rs = 1 µm). Red: PPARγ. Blue: nucleus. Supplementry Fig. S6 Immunohistochemicl stining for CD163 (-e) Representtive imges of immunostining for CD163 (scle r = 5 µm). N.D.: not detected. (f) Quntifiction of CD163-positive res of the vsculr wll. Vlues with different letters re significntly different (P <.5).
Supplementry Fig. S7 Weight chnge nd serum prmeters () Body weight chnge of rts in the triolein nd fish oil groups. Serum triglyceride (TG) levels () nd totl cholesterol levels (c) in the control, triolein nd fish oil groups. Dt re the men ± s.e.m. Control group (n=1), triolein group (n = 13), fish oil group (n = 12). * P <.5 versus triolein group. Supplementry Fig. S8 Thickness of vsculr wll AAA sc res from the three experimentl groups were divided into two groups: those with res without dipocytes (-)(, e nd h) nd those with dipocytes (+) (c, f nd i). (-i) Representtive imges of HE stining of the control group (-c), triolein group (d-f) nd fish oil group (g-i).(,, d, e, g, h: scle r = 1 µm; c, f, i: scle r = 4 µm). (j) Quntittive nlysis of vsculr wll thickness of the control, triolein nd fish oil groups. Dt re the men ± s.e.m. Control group (n=9), triolein group (n = 1), fish oil group (n = 8). Vlues with different letters re significntly different (P <.5). Supplementry Fig. S9 Elstin degrdtion score nd collgen-positive re AAA sc res from the three experimentl groups were divided into two groups: those with res without dipocytes (-)(, e, h, l, o nd r) nd those with dipocytes (+)(c, f, j, i, m, p nd s). (-i) Representtive imges of EVG stining of the control group (-c), triolein group (d-f) nd fish oil group (g-i)(,, d, e, g, h: scle r = 1 µm; c, f, i: scle r = 2µm). (j) Elstin degrdtion scores in the control, triolein nd fish oil groups. (k-s) Representtive imges of PSR stining of the control group (k-m), triolein group (np) nd fish oil group (q-s)(k, n, q: Scle r = 1 µm, l, o, r: scle r = 4 µm, m, p, s: scle r = 5 µm). (t) Quntifiction of the collgen-positive res of the vsculr wll. Dt re the men ± s.e.m. Control group (n=9), triolein group (n = 1), fish oil group (n = 8). Vlues with different letters re significntly different (P <.5). Supplementry Fig. S1 Thickness of medil wll (-f) Representtive imges of immunostining for α-smooth muscle ctin (scle r = 1 µm). (g) Quntifiction of α-smooth muscle ctin-positive res of the vsculr wll. (h) Quntittive nlysis of medil wll thickness in the control, triolein nd fish oil groups. Vlues with different letters re significntly different (P <.5). Supplementry Fig. S11 Immunohistochemicl stining for MCP-1 nd MAC387 + monocytes/mcrophges
AAA sc res from the three experimentl groups were divided into two groups: those with res without dipocytes (-)(, e, h, l, o nd r) nd those with dipocytes (+)(c, f, j, i, m, p nd s). (-i) Representtive imges of immunostining for MCP-1 (scle r = 5 µm). (j) Quntifiction of MCP-1-positive res of the vsculr wll. (k-s) Representtive imges of immunostining for MAC387 + monocytes/mcrophges (scle r = 5 µm). (t) Quntifiction of MAC387 + monocytes/mcrophge-positive res of the vsculr wll. Dt re the men ± s.e.m. Control group (n=9), triolein group (n = 1), fish oil group (n = 8). Vlues with different letters re significntly different (P <.5). Supplementry Fig S12 Reltionships etween serum lipids, tissue lipids, BMI nd ortic dimeter in humn AAA smples. The mount of triglyceride (TG) in orts versus serum TG levels (), serum totl cholesterol levels () nd ody mss index (BMI) (c). The totl cholesterol mount in orts versus serum TG levels (d), serum totl cholesterol levels (e) nd BMI (f). Aortic dimeter versus serum TG levels (g), serum totl cholesterol levels (h) nd BMI (i). Aorts were divided into two groups: 1) intim nd medi, nd 2) dventiti.
Supplementry Tle S1 Diet composition () Diet composition (%) () Ftty cid coconut oil (%) fish oil (%) choline chloride.25 8 : 7.1-1 : 7.6 - cystine.3 12 : 27.6 - AIN-93 vitmin mix 1 14 : 19.8 5.2 AIN-93G minerl mix 3.5 16 : 14.9 6.6 cellulose 5 16 : 1-9.3 sucrose 1 16 : 2-1.7 16 : 3-2.8 csein 2 16 : 4-4.8 cornstrch 55.75 18 : 6.6.5 coconut oil 4.2 18 : 1 13.3 9.6 1 18:2 n-6 3.1 1.4 18:3 n-3 -.9 18:4 n-3-5.1 2:4 n-6-1.3 2:4 n-3-1.1 2:5 n-3-3.8 22:5 n-6 -.3 22:5 n-3-2.9 22:6 n-3-15.7
Supplementry Fig. S1 Ruptured AAA efore wshing lood.
Serum Triglyceride (mg/dl) Serum Totl Cholesterol (mg/dl) Initil ody weight (g) Body weight gin/dy (g) Supplementry Fig. S2 Weight chnge nd serum prmeters () 3 () 6 25 5 2 4 15 3 1 2 5 1 Rupture - + Rupture - + (c) 2 (d) 8 15 6 1 4 5 2 Rupture - + Rupture - +
Thickness of vsculr wll (µm) Elstin degrdtion score Supplementry Fig. S3 Vsculr wll thickness nd elstin degrdtion score Rupture - + - + HE () () (c) (d) EVG (e) (f) (g) (h) (i) 25 2 c (j) 5 4 15 1 3 2 5 Rupture - + - + 1 Rupture - + - +
α-smooth muscle ctin positive re (%) Thickness of medil wll (µm) Supplementry Fig. S4 Thickness of medil wll Rupture - + - + () () (c) (d) (e) 4 35 3 25 2 15 1 5 Rupture - + - + (f) 12 1 8 6 4 2 Rupture - + - +
Supplementry Fig. S5 Oservtion of Adipocyte nd PPARγ stining () Rt () Rt (c) Opticl imge (e) PPARγ (d) (f)
CD163 positive re (%) Supplementry Fig. S6 Immunohistochemicl stining for CD163 Rupture Adipocyte () - () + - + N.D. (c) (d) (e) (f) 3 25 2 15 1 5 Rupture - + - + + - - - - + Adipocyte c
Serum Triglyceride (mg/dl) Serum Totl Cholesterol (mg/dl) Body weight (g) Supplementry Fig. S7 Weight chnge nd serum prmeters () 4 35 3 25 2 15 1 Control Triolein Fish oil 5 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 (dy) () Control Triolein Fish oil (c) 16 12 14 1 12 1 8 8 6 6 4 4 2 2 Control Triolein Fish oil
Thickness of vsculr wll (µm) Supplementry Fig. S8 Thickness of vsculr wll () Control (d) Triolein (g) Fish oil () (e) (h) (c) (f) (i) (j) 25 c c 2 15 1 c c c 5 Adipocyte - - - - + - + - +
Elstin degrdtion score Collgen positive re (%) Supplementry Fig. S9 Elstin degrdtion score nd collgen-positive re EVG PSR Control Triolein Fish oil Control Triolein () (d) (g) (k) (n) (q) Fish oil () (e) (h) (l) (o) (r) (c) (f) (i) (m) (p) (s) (j) 4 3 2 1 Adipocyte - - - - + - + - + (t) 9 8 7 6 5 4 c 3 cd c cd 2 1 d Adipocyte - - - - + - + - +
α-smooth muscle ctin positive re (%) Thickness of medil wll (µm) Supplementry Fig. S1 Thickness of medil wll () Control (c) Triolein (e) Fish oil () (d) (f) (g) 4 3 2 1 Control Triolein Fish oil (h) 12 1 8 6 4 2 Control Triolein Fish oil
MCP-1 positive re (%) Mc387 positive re (%) Supplementry Fig. S11 Immunohistochemicl stining for MCP-1 nd MAC387 + monocytes/mcrophges MCP-1 Monocyte/Mcrophge Control Triolein Fish oil Control Triolein Fish oil () (d) (g) (k) (n) (q) () (e) (h) (l) (o) (r) (c) (f) (i) (m) (p) (s) (j) 25 2 15 1 5 d c d c d d (t) 35 3 25 2 15 1 5 c c Adipocyte - - - - + - + - + Adipocyte - - - - + - + - +
Aortic dimeter (mm) Aortic dimeter (mm) Aortic dimeter (mm) Totl Cholesterol (mg/g tissue) Totl Cholesterol (mg/g tissue) Totl Cholesterol (mg/g tissue) Triglyceride (mg/g tissue) Triglyceride (mg/g tissue) Triglyceride (mg/g tissue) Supplementry Fig. S12 Reltion etween serum lipids, tissue lipids, BMI nd ortic dimeter () 12 Intim & Medi Adventiti 1 8 R=.1329 6 4 2 R=-.1694 2 4 6 Serum Triglyceride (mg/dl) () 12 Intim & Medi Adventiti (c) Intim & Medi Adventiti 12 1 1 8 R=.146 8 6 6 R=.1742 4 4 2 R=.1617 2 R=.1223 1 2 3 1 BMI 2 3 Serum Totl Cholesterol (mg/dl) (d) (e) (f) 8 Intim & Medi Adventiti 8 Intim & Medi Adventiti 8 Intim & Medi Adventiti 6 4 R=-.128 6 4 R=.1642 6 4 R=-.826 2 R=.943 1 2 3 4 5 Serum Triglyceride (mg/dl) 2 R=.222 1 2 3 Serum Totl Cholesterol (mg/dl) 2 R=.1543 1 2 3 BMI (g) (h) (i) 12 1 8 6 4 2 R=-.1697 1 2 3 4 5 Serum Triglyceride (mg/dl) 12 1 8 6 4 2 R=-.2539 1 2 3 Serum Totl Cholesterol (mg/dl) 12 1 8 6 4 2 R=-.466 1 2 3 BMI