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Pathogenesis of abdominal aortic aneurysms: Possible role of differential production of proteoglycans by smooth muscle cells  James Melrose, PhD, John.

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Presentation on theme: "Pathogenesis of abdominal aortic aneurysms: Possible role of differential production of proteoglycans by smooth muscle cells  James Melrose, PhD, John."— Presentation transcript:

1 Pathogenesis of abdominal aortic aneurysms: Possible role of differential production of proteoglycans by smooth muscle cells  James Melrose, PhD, John Whitelock, PhD, Qian Xu, BSc, Peter Ghosh, DSc  Journal of Vascular Surgery  Volume 28, Issue 4, Pages (October 1998) DOI: /S (98) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

2 Fig. 1 Composite agarose polyacrylamide gel electrophoresis (CAPAGE) of abdominal aortic aneurysm (AAA), arterial occlusive disease (AOD), and normal smooth muscle cell media proteoglycans from monolayer culture. An ovine articular cartilage (AC) proteoglycan sample consisting of the chondroitin sulfate (CS)–rich and keratan sulfate–rich aggrecan populations (Agg 1, Agg 2) and the small dermatan sulfate–substituted proteoglycans decorin and biglycan (DS PG) was run as an internal standard. The gel was stained with toluidine blue. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

3 Fig. 2 Demonstration of perlecan (A), heparan sulfate–substituted proteoglycans (B), biglycan (C), and keratan sulfate–substituted (D) smooth muscle cell proteoglycans separated by means of CAPAGE and identified by means of immunoblotting with monoclonal antibodies (MAb) specific to each proteoglycan type (see Table I). Differences in proteoglycan expression levels were quantitated by means of densitometric scanning of particular proteoglycan bands on the blots with Scan Analysis software (Biosoft, Cambridge, United Kingdom, Ferguson, Mo). These results are shown in the lower half of the figure below the lane of interest. Each lane was scanned six times; the histogram represents averaged values per lane ± SD expressed in relative terms as arbitrary absorbance units. AAA, abdominal aortic aneurysm; AOD, arterial occlusive disease; N, normal arterial tissue; SMCs, smooth muscle cells; CS Std, chondroitin sulfate standard; HUAEC, human umbilical artery endothelial cell; AF, annulus fibrosus; AC, articular cartilage. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

4 Fig. 3 Affinity and immunoblots of abdominal aortic aneurysm (AAA), arterial occlusive disease (AOD), and normal (N) smooth muscle cell media proteoglycans separated by means of CAPAGE. The same articular cartilage (AC) proteoglycan sample as in Fig. 1 was used as was a human fibroblast media proteoglycan sample (Hum Fib PG). Arrows at left (V-1, V-2) depict the elution positions of the two AAA smooth muscle cell versican isoforms. Arrows at right show the elution positions of the two articular cartilage aggrecan populations. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

5 Fig. 4 Demonstration of native (A) and deglycosylated (B) free core protein of a keratan sulfate proteoglycan synthesized by smooth muscle cells in culture. Anion exchange–purified media proteoglycans (30 μg/lane) were subjected to electrophoresis on 4% to 12% PAGE gels. A, Gel was electroblotted to nitrocellulose, and the native keratan sulfate-proteoglycan was identified by means of immunoblotting with monoclonal antibody 5-D-4 (anti-keratan sulfate). B, Gel was stained directly for protein with Coomassie R250 to identify the free core protein of the keratan sulfate proteoglycan (approximately 55 kd). +, Sample predigested with keratanase, which degrades the glycosaminoglycan component of the proteoglycan. –, Sample not predigested. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

6 Fig. 5 Enzyme-linked immunosorbent assay of perlecan levels in media samples from monolayer culture of smooth muscle cells. The number of smooth muscle cells in culture at each time point was determined by means of detaching cells from monolayers with trypsin/EDTA and counting the cells with a hemocytometer. Perlecan levels (inset) were quantified relative to a binding curve constructed with affinity-purified HUAEC perlecan and A76 monoclonal mouse anti-human perlecan core protein. Each time point represents a mean value calculated from six replicate samples of the smooth muscle cell cultures. Bars indicate standard deviation. The ELISA standard curve was constructed from triplicate replicates of each dilution of perlecan standard. Assay Zap software (Biosoft) was used for calculation of results from this standard curve. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

7 Fig. 6 Demonstration of heparan sulfate–substituted proteoglycan species in smooth muscle cell media samples from day 7 of passage 5. Anion exchange–purified smooth muscle cell media proteoglycans were subjected to electrophoresis on 4% to 12% SDS PAGE gels and electroblotted to nitrocellulose. Selected samples were predigested with heparitinase before this step. Heparan sulfate proteoglycan species were identified on immunoblots with the following monoclonal antibodies: mouse anti-human perlecan core protein (A76); anti–native heparan sulfate (10-E-4), and anti–D-unsaturated heparan sulfate stub epitopes generated by means of the action of heparitinase on the heparan sulfate side chains of heparan sulfate proteoglycan species. Journal of Vascular Surgery  , DOI: ( /S (98) ) Copyright © 1998 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

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