Differential availability/processing of decorin precursor in arterial and venous smooth muscle cells

Abstract

The existence of specific differentiation markers for arterial smooth muscle (SM) cells is still a matter of debate. A clone named MM1 was isolated from a library of monoclonal antibodies to adult porcine aorta, which in vivo binds to arterial but not venous SM cells, except for the pulmonary vein. MM1 immunoreactivity in Western blotting involved bands in the range of M(r) 33-226 kDa, in both arterial and venous SM tissues. However, immunoprecipitation experiments revealed that MM1 bound to a 100-kDa polypeptide that was present only in the arterial SM extract. By mass spectrometry analysis of tryptic digests from MM1-positive 130- and 120-kDa polypeptides of aorta SM extract, the antigen recognized by the antibody was identified as a decorin precursor. Using a crude decorin preparation from this tissue MM1 reacted strongly with the 33-kDa polypeptide and this pattern did not change after chondroitinase ABC treatment. In vitro, decorin immunoreactivity was found in secreted grainy material produced by confluent arterial SM cells, although lesser amounts were also seen in venous SM cells. Western blotting of extracts from these cultures showed the presence of the 33-kDa band but not of the high-molecular-weight components, except for the 100-kDa monomer. The 100/33-kDa combination was more abundant in arterial SM cells than in the venous counterpart. In the early phase of neointima formation, induced by endothelial injury of the carotid artery or vein-to-artery transposition, the decorin precursor was not expressed, but it was up-regulated in the SM cells of the media underlying the neointima in both models. Collectively, these data suggest a different processing/utilization of the 100-kDa monomer of proteoglycan decorin in arterial and venous SM cells, which is abolished after vein injury.

Fig 1

Screening of MM1 hybridoma. (A) Dot-blot analysis of non-denatured arterial and vein extract with MM1 hybridoma supernatant or 1B8 anti-SM22 antibody (used as control of SM cells). Lanes 1, 1′: aorta extract; lanes 2, 2′: inferior vena cava extract. (B) Immunoperoxidase staining of cryosections from adult porcine thoracic aorta (a,c) and inferior vena cava (b,d) with MM1 hybridoma or 1B8 antibody. Note that AgMM1 is heterogeneously distributed in the medial SM cells whereas SM22 and smooth muscle tissue (asterisk) in the vein is negative. Scale bar, 90 µm.

Fig 2

Confocal microscopy analysis of AgMM1 distribution (in red) in comparison with SM α-actin (in green). Small (a,c,e) and large (b,d,f) branches of the left descending coronary artery are shown. AgMM1 appeared to be present in the extracellular matrix, particularly abundant in the subendothelial space in the small branches of coronary arteries (e) and in the space among SM bundles in the large branches of this vessel (f). The thin white line in (e) identifies the endothelium (ec). sm, Smooth muscle. Scale bar, 50 µm.

Fig 3

Western blotting analysis of MM1 binding in artery and vein extracts (A), MM1-immunoprecipitated arterial and venous antigens (B) and crude decorin from thoracic aorta (C) examined on 7.5% SDS gels. (A) Extracts from artery in lanes 1,1′ and from veins in lanes 2,2′. The electrophoretic pattern is shown in lanes 1,2 and the Western blotting profile in lanes 1′,2′. (B) Immunoprecipitate of antigens reacted with MM1 from arterial (lane 1) and venous (lane 2) extracts. H and L, heavy and light chains of IgGs. Arrows indicate standard molecular weights. Equal amounts of proteins (5 µg) were loaded in the gels. Note the 100-kDa band reacting with MM1 in arterial but not venous extract. (C) Electrophoretic profile of crude decorin (lane 1) and the corresponding Western blotting pattern (lane 2). Note the marked reaction of MM1 with the 33-kDa band.

Fig 4

Confocal microscopy images of double immunofluorescence assay on cultured SM cells from coronary arteries (A) and Western blotting analysis of extracts from arterial and venous SM cells grown in vitro (B). (A) MM1 in red and anti-SM α-actin in green. Arrowheads indicate clusters of different size, apparently not related to the microfilament system. Asterisks indicate MM1-positive secreted material. Scale bar, 25 µm. (B) Western blotting analysis of MM1 binding to arterial (lanes 1,1′) and venous (lanes 2,2′) SM cell antigens examined in 5% (upper panel) and 7.5% (lower panel) SDS gels. Lanes 1,1′ are representative of the electrophoretic pattern and lanes 2,2′ show the corresponding Western blotting profile. Note that the 33- and 100-kDa bands are reactive with MM1 in both extracts. However, in venous SM cells the two bands stain less intensely even if equal amounts of proteins (3 µg) were loaded in the gels. High-molecular-weight MM1-reactive components are lacking in culture.

Fig 5

Distribution of AgMM1 in developing coronary artery (ca) and vein (cv) and aorta (ao). Note that differences in decorin expression in the coronary vessels are evident at embryonic day 30 (E30, panel a) whereas at the same developmental time only very rare foci of MM1-positive cells are seen in the aortic media (panel b, arrow). At E60, AgMM1 is particularly expressed in the region surrounding the aortic endothelium (arrowhead in d) while in the coronary artery, besides SM cells, traces of decorin labelling can also be observed in the interstitial tissue at the interface with the myocardial cells (double arrowheads). Scale bar, 100 µm.

Fig 6

AgMM1 distribution in the intimal cushions of abdominal aorta (a–d) and in intact (e,f) and endothelial-injured (g,h) carotid artery in comparison with SM cell markers. Note that the decorin is almost absent in the intimal cushions (ic; panel a) whereas it is heterogeneously expressed in the underlying media (m). SM-E7, anti-SM myosin (b); NM-F6, anti-MyHC-A_pla1 (c); IST-9, anti-EIIIA fibronectin (d) Reactivities for platelet MyHC-A_pla1, EIIIA fibronectin and SM myosin indicate that intimal cushion tissues contain SM cells of ‘immature’ type (Sartore et al. 1999). In (e), decorin is scattered in the media whereas anti-SM myosin homogeneously stained this wall layer (f). After 14 days from balloon injury, an intimal thickening (it) is formed in the carotid artery, which is positive for SM myosin but almost negative for MM1 (panels g,h). Some immunoreactivity in the SM cells near the internal elastic lamina (iel; large arrowhead in the inset, panel g) can, however, be seen. Almost all the cells of the innermost region of the media now express AgMM1 (asterisk) whereas the outermost layers of this vessel are now AgMM1-negative: av, arterial vasa vasorum. Scale bars: (a)–(d), 70 µm; (e)–(h), 100 µm; insets, 40 µm.

Fig 7

AgMM1 distribution in intact (a,b) and arterially grafted (c,d) internal jugular vein in comparison with SM-E7 anti-SM myosin antibody (b,d). AgMM1 (a,c) is absent from the SM cell layer (sm) in intact vessel (a) but it is expressed in the media of venous SM segment transplanted in the arterial position (c). In the intimal thickening (it), which is formed after vein-to-artery graft and contains SM myosin-positive SM cells, AgMM1 immunostaining is barely detectable (d). a, adventitia; vv, vasa vasorum; t, thrombus. Scale bar, 110 µm.