In situ localization and quantification of seventy-two – kilodalton type IV collagenase in aneurysmal, occlusive, and normal aorta William D. McMillan,

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Presentation transcript:

In situ localization and quantification of seventy-two – kilodalton type IV collagenase in aneurysmal, occlusive, and normal aorta William D. McMillan, MD, Bruce K. Patterson, MD, Richard R. Keen, MD, William H. Pearce, MD Journal of Vascular Surgery Volume 22, Issue 3, Pages 295-305 (September 1995) DOI: 10.1016/S0741-5214(95)70144-3 Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Northern blot analysis of mRNA from infrarenal aortic tissue of patients with aortoiliac occlusive disease (AOD), patients with aneurysms (AAA), and control subjects (NA). Radiolabeled cDNA probes for 72 kD type IV collagenase (MMP-2) hybridized to messages of lengths 3.1 kb. Probes for human α-tubulin hybridized to 1.6 kb mRNA in all lanes, indicating adequate amounts of total RNA were loaded in each well. Human foreskin fibroblasts (H.S.-68, ATCC) served as a positive control during the hybridization (+). Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 MMP-2 messenger RNA in abdominal aortic aneurysms (AAA), occlusive aortoiliac tissue (AOD), and normal infrarenal aorta (NA). All signals were normalized to α-tubulin to control for loading differences in total RNA and were expressed as mean ± SD. MMP-2 mRNA levels were increased significantly in aneurysmal tissue when compared with occlusive (p < 0.02) and normal aorta (p < 0.002). No significant differences between MMP-2 occlusive and normal tissue mRNA levels were identified. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Northern blot analysis of mRNA from infrarenal aortic tissue of patients with aneurysms (AAA) or aortoiliac occlusive disease (AOD) and control subjects (NA). Radio-labeled cDNA probes for TIMP-2 hybridized to mRNA of two isoforms with lengths of 3.5 kb and 1.0 kb. Probes for human α-tubulin hybridized to 1.6 kb mRNA in all lanes, indicating that adequate amounts of total RNA were loaded in each well. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 TIMP-2 messenger RNA in abdominal aortic aneurysms (AAA), occlusive aortoiliac tissue (AOD), and normal infrarenal aorta (NA). All signals were normalized to α-tubulin to control for loading differences in total RNA and were expressed as mean ± standard deviation. No significant differences in TIMP-2 tissue mRNA levels between tissue types were identified. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 5 Northern blot analysis of mRNA from cultured normal and aneurysm vascular smooth muscle cells. Radio-labeled cDNA probes for MMP-2 bound to mRNA at 3.1 kb on blots of both aneurysm and normal VSMCs. Likewise, probes bound to two expected isoforms (3.5 kb and 1.0 kb) of TIMP-2 mRNA on Northern blots of aneurysm and normal VSMCs. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Digoxigenin-labeled RNA probes binding to MMP-2 mRNA within aneurysmal tissue sections. Low-power view (A, original magnification x16) demonstrates antisense probes binding to macrophages within adventitial inflammatory infiltrate. No binding of sense probes (negative control) is seen on adjacent section (B, original magnification x16). Higher power views show cytoplasmic staining within macrophages (C, original magnification x60) and adjacent vascular smooth muscle cells (D, original magnification x40). Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Digoxigenin-labeled RNA probes binding to MMP-2 mRNA within aneurysmal tissue sections. Low-power view (A, original magnification x16) demonstrates antisense probes binding to macrophages within adventitial inflammatory infiltrate. No binding of sense probes (negative control) is seen on adjacent section (B, original magnification x16). Higher power views show cytoplasmic staining within macrophages (C, original magnification x60) and adjacent vascular smooth muscle cells (D, original magnification x40). Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Digoxigenin-labeled RNA probes binding to MMP-2 mRNA within aneurysmal tissue sections. Low-power view (A, original magnification x16) demonstrates antisense probes binding to macrophages within adventitial inflammatory infiltrate. No binding of sense probes (negative control) is seen on adjacent section (B, original magnification x16). Higher power views show cytoplasmic staining within macrophages (C, original magnification x60) and adjacent vascular smooth muscle cells (D, original magnification x40). Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 Digoxigenin-labeled RNA probes binding to MMP-2 mRNA within aneurysmal tissue sections. Low-power view (A, original magnification x16) demonstrates antisense probes binding to macrophages within adventitial inflammatory infiltrate. No binding of sense probes (negative control) is seen on adjacent section (B, original magnification x16). Higher power views show cytoplasmic staining within macrophages (C, original magnification x60) and adjacent vascular smooth muscle cells (D, original magnification x40). Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 7 Digoxigenin-labeled RNA probes binding to MMP-2 mRNA within occlusive tissue sections. Low-power view (A, original magnification x16) demonstrates antisense probes binding to macrophages within luminal atherosclerotic plaque and absence of binding within aortic wall. Higher power views (B, original magnification x40 and C, original magnification x80) demonstrate cytoplasmic staining within macrophages adjacent to areas of neovascularization within atherosclerotic plaque. MAC 387 immunohistochemical staining of adjacent tissue section (D, original magnification x40) confirmed macrophages cell type. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 7 Digoxigenin-labeled RNA probes binding to MMP-2 mRNA within occlusive tissue sections. Low-power view (A, original magnification x16) demonstrates antisense probes binding to macrophages within luminal atherosclerotic plaque and absence of binding within aortic wall. Higher power views (B, original magnification x40 and C, original magnification x80) demonstrate cytoplasmic staining within macrophages adjacent to areas of neovascularization within atherosclerotic plaque. MAC 387 immunohistochemical staining of adjacent tissue section (D, original magnification x40) confirmed macrophages cell type. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 7 Digoxigenin-labeled RNA probes binding to MMP-2 mRNA within occlusive tissue sections. Low-power view (A, original magnification x16) demonstrates antisense probes binding to macrophages within luminal atherosclerotic plaque and absence of binding within aortic wall. Higher power views (B, original magnification x40 and C, original magnification x80) demonstrate cytoplasmic staining within macrophages adjacent to areas of neovascularization within atherosclerotic plaque. MAC 387 immunohistochemical staining of adjacent tissue section (D, original magnification x40) confirmed macrophages cell type. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 7 Digoxigenin-labeled RNA probes binding to MMP-2 mRNA within occlusive tissue sections. Low-power view (A, original magnification x16) demonstrates antisense probes binding to macrophages within luminal atherosclerotic plaque and absence of binding within aortic wall. Higher power views (B, original magnification x40 and C, original magnification x80) demonstrate cytoplasmic staining within macrophages adjacent to areas of neovascularization within atherosclerotic plaque. MAC 387 immunohistochemical staining of adjacent tissue section (D, original magnification x40) confirmed macrophages cell type. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 8 Digoxigenin-labeled RNA probes binding to TIMP-2 mRNA within aneurysmal tissue sections. Low-power view (A, original magnification x16) demonstrates diffuse staining within adventitial vascular smooth muscle cells adjacent to inflammatory infiltrates. Higher power view of same area (B, original magnification x80) shows vascular smooth muscle cells staining for TIMP-2 mRNA. High-power view (C, original magnification x80) shows macrophages within an aneurysm adventitial inflammatory infiltrate also staining positively for TIMP-2 mRNA. Alpha-actin immunohistochemical staining of adjacent section (D, original magnification x16) confirmed smooth muscle cell type (note similar pattern of staining outlined by arrows). Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 8 Digoxigenin-labeled RNA probes binding to TIMP-2 mRNA within aneurysmal tissue sections. Low-power view (A, original magnification x16) demonstrates diffuse staining within adventitial vascular smooth muscle cells adjacent to inflammatory infiltrates. Higher power view of same area (B, original magnification x80) shows vascular smooth muscle cells staining for TIMP-2 mRNA. High-power view (C, original magnification x80) shows macrophages within an aneurysm adventitial inflammatory infiltrate also staining positively for TIMP-2 mRNA. Alpha-actin immunohistochemical staining of adjacent section (D, original magnification x16) confirmed smooth muscle cell type (note similar pattern of staining outlined by arrows). Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 8 Digoxigenin-labeled RNA probes binding to TIMP-2 mRNA within aneurysmal tissue sections. Low-power view (A, original magnification x16) demonstrates diffuse staining within adventitial vascular smooth muscle cells adjacent to inflammatory infiltrates. Higher power view of same area (B, original magnification x80) shows vascular smooth muscle cells staining for TIMP-2 mRNA. High-power view (C, original magnification x80) shows macrophages within an aneurysm adventitial inflammatory infiltrate also staining positively for TIMP-2 mRNA. Alpha-actin immunohistochemical staining of adjacent section (D, original magnification x16) confirmed smooth muscle cell type (note similar pattern of staining outlined by arrows). Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 8 Digoxigenin-labeled RNA probes binding to TIMP-2 mRNA within aneurysmal tissue sections. Low-power view (A, original magnification x16) demonstrates diffuse staining within adventitial vascular smooth muscle cells adjacent to inflammatory infiltrates. Higher power view of same area (B, original magnification x80) shows vascular smooth muscle cells staining for TIMP-2 mRNA. High-power view (C, original magnification x80) shows macrophages within an aneurysm adventitial inflammatory infiltrate also staining positively for TIMP-2 mRNA. Alpha-actin immunohistochemical staining of adjacent section (D, original magnification x16) confirmed smooth muscle cell type (note similar pattern of staining outlined by arrows). Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 9 Digoxigenin-labeled antisense RNA probes binding to TIMP-2 mRNA within occlusive aorta. Low-power view (A, original magnification x40) demonstrates cytoplasmic staining with a localized cluster of adventitial smooth muscle cells. Cell type was confirmed by alpha-actin immunohistochemical staining of adjacent section (B, original magnification x80). Sense probes (negative control) did not bind to adjacent sections (C, original magnification x40). Higher power view (D, original magnification x80) demonstrates cytoplasmic staining of vascular smooth muscle cells. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 9 Digoxigenin-labeled antisense RNA probes binding to TIMP-2 mRNA within occlusive aorta. Low-power view (A, original magnification x40) demonstrates cytoplasmic staining with a localized cluster of adventitial smooth muscle cells. Cell type was confirmed by alpha-actin immunohistochemical staining of adjacent section (B, original magnification x80). Sense probes (negative control) did not bind to adjacent sections (C, original magnification x40). Higher power view (D, original magnification x80) demonstrates cytoplasmic staining of vascular smooth muscle cells. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 9 Digoxigenin-labeled antisense RNA probes binding to TIMP-2 mRNA within occlusive aorta. Low-power view (A, original magnification x40) demonstrates cytoplasmic staining with a localized cluster of adventitial smooth muscle cells. Cell type was confirmed by alpha-actin immunohistochemical staining of adjacent section (B, original magnification x80). Sense probes (negative control) did not bind to adjacent sections (C, original magnification x40). Higher power view (D, original magnification x80) demonstrates cytoplasmic staining of vascular smooth muscle cells. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 9 Digoxigenin-labeled antisense RNA probes binding to TIMP-2 mRNA within occlusive aorta. Low-power view (A, original magnification x40) demonstrates cytoplasmic staining with a localized cluster of adventitial smooth muscle cells. Cell type was confirmed by alpha-actin immunohistochemical staining of adjacent section (B, original magnification x80). Sense probes (negative control) did not bind to adjacent sections (C, original magnification x40). Higher power view (D, original magnification x80) demonstrates cytoplasmic staining of vascular smooth muscle cells. Journal of Vascular Surgery 1995 22, 295-305DOI: (10.1016/S0741-5214(95)70144-3) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions