Loose Extracellular Matrix Research Articles

Evidence for Rapid Accumulation and Persistently Disordered Architecture of Fibrillar Collagen in Human Coronary Restenosis Lesions

The pattern of collagen deposition after coronary angioplasty could significantly influence recurrent lesion formation. Traditional histologic assessments of coronary restenosis lesions have not identified abundant collagen fibers in restenotic tissue; however, these methods can suffer from lack of sensitivity and are not quantitative. We analyzed collagen architecture in 40 coronary lesions retrieved from patients by directional atherectomy, by exploiting the birefringent properties of fibrillar collagen. Picrosirius red-stained sections were illuminated with circularly polarized light, and fiber content and thickness were quantified by digital image analysis. Fifteen of 19 restenosis lesions (79%) and 1 of 21 native atherosclerosis lesions (5%) displayed a pattern of reactive intimal modeling, characterized by stellate-shaped smooth muscle cells variably oriented in a loose extracellular matrix. There was an apparent paucity of collagen fibers in these regions based on staining with Movat's pentachrome, a traditional connective tissue stain. However, circular polarization light microscopy revealed an extensive distribution of collagen fibers in restenosis tissue, occupying 79.9% ± 11.8% of the section area. Despite this high collagen content, the restenosis lesions were distinct from de novo atherosclerosis lesions in having a disordered collagen alignment, reduced fiber packing (p <0.05), and thinner fibers (4.3 ± 1.7 vs 9.2 ± 4.3 μm, p <0.001). Fiber diameter was greater in lesions retrieved between 3 and 17 months after angioplasty than in lesions retrieved between 1 week and 3 months (p <0.05). However, fiber disorientation was evident in all lesions retrieved after 1 week, with little similarity to that of native plaque. Lesions retrieved within 1 week of angioplasty represented a distinct group with identical collagen features as in de novo atherosclerosis lesions, implying a different mechanism of restenosis in that population. We conclude that human coronary restenosis involves rapid accumulation of collagen fibers, which are persistently disordered. This may be critical in the development of restenosis and could significantly influence therapeutic attempts to control the process. (Am J Cardiol 1996;78:633–637)

Ultrastructural characteristics of human atherectomy tissue from coronary and lower extremity arterial stenoses

In animal studies, smooth muscle cell phenotype conversion has been suggested to be an essential prerequisite for subsequent migratory and proliferative events leading to (neo)intima formation. To determine ultrastructural characteristics of individual smooth muscle cells and to relate them to specific lesion types and intimai cell density, we compared atherectomy samples from 17 restenotic and 32 primary coronary and peripheral lesions using transmission electron microscopy and histology. Ultrastructural analysis of cell-rich tissue, predominantly of restenotic origin, revealed smooth muscle cells full of synthetic organelles. Moreover, these cells were frequently found to be surrounded by loose extracellular matrix and partially fragmented basement membrane components. In contrast, plaques exhibiting low cell density, as exclusively seen with primary lesions, displayed an extensive buildup of extracellular matrix containing sparse numbers of microfilament-rich smooth muscle cells. The central finding of our study is a morphometrically quantitated, twofold greater (p < 0.001) volume fraction of synthetic organelles (V s) within smooth muscle cells in restenotic versus primary plaques, indicating a more dedifferentiated cellular phenotype as a typical feature of restenotic lesions. Equally enhanced V s values were seen for restenotic coronary and peripheral plaques. No V s decrease was observed during time after angioplasty (2.2 to 30 months) regardless of previous revascularization procedures (balloon angioplasty or atherectomy). Despite intra- and interlesional variability, V s and intimal cell density were strongly correlated (r = 0.74; p < 0.001). This correlation was observed more often with clinical restenoses and, importantly, in a portion (10% to 15%) of primary lesions. Data from restenotic lesions indicate that a dedifferentiated smooth muscle cell phenotype, pericellular matrix disintegration, and intimal hypercellularity are long-lasting biologic responses to previous smooth muscle cell injury. Similar tissue characteristics expressed in several primary lesions suggest that comparable pathogenic mechanisms are related to the progression and/or acuity of chronic lesions.

Use of human tissue specimens obtained by directional atherectomy to study restenosis

Directional atherectomy has provided the opportunity to study the pathology of restenosis in human tissue specimens from live patients. The restenosis lesion is characterized by two distinctive features: a focus of hypercellularity, comprised of cells with phenotypic features of proliferative vascular smooth muscle cells (SMCs), and a rich, loose extracellular matrix (ECM). Analysis of restenosis lesions by in situ hybridization, immunohistochemistry, and cell culture has disclosed evidence of activated SMCs, and in many cases—particularly lesions from the peripheral vasculature—ongoing cellular proliferation. The ECM of restenosis lesions is biglycan rich and decorin poor, a finding that is associated with increased expression of transforming growth factor beta (TGF-β). While certain restenosis lesions contain foci of microangiogenesis, the pathogenetic significance of this feature remains uncertain.

Osseointegration of titanium implants in the tibia. Electron microscopy of biopsies from 4 patients.

We studied the ultrastructure of bone tissue around implants of pure titanium inserted into the tibia in 4 patients with arthrosis or rheumatoid arthritis. Three main appearances of the interface were noted. First, a close contact between titanium and calcified bone with living osteocytes inside the newly-formed bone was observed in all samples. Secondly, a close contact was also seen between the implant and osteoid, the newly formed collagenous matrix being either uncalcified or calcifying. Thirdly, a loose extracellular matrix with fibrillar and nonfibrillar materials was sometimes observed between bone mineral and implant. There was no inflammatory reaction at the interface. We concluded that the titanium implants were osseointegrated, but the calcification of the bone tissue was not complete even after 20 months. However, mineralization of osteoid and living bone cells revealed the presence of an active tissue.

Extracellular matrix antigen of human muscle defined by a monoclonal antibody

A monoclonal antibody (7.1B3) has been produced in mice previously immunised with a glycoprotein fraction prepared from human myotube cells grown in tissue culture. The antibody precipitates an antigen complex of four glycoprotein components from detergent extracted human muscle myotube cultures. These are two chains of about 205K MW and two chains of about 130–140K MW. The antigen complex is trypsin-sensitive collagenase insensitive and bands as a complex of 10S on sucrose gradients in NP40. Indirect immunofluorescence studies on cell cultures of myoblasts and myotubes showed that the antigen existed in a number of locations: as a granular stain on the cell surface of myotubes, and as short streaks of immunoreactivity in the extracellular matrix. Clonal populations of myoblasts and myotubes gave a similar result showing that 7.1B3 is specifically synthesized by cells of the myogenic lineage. Double indirect immunofluorescence staining with 7.1B3 antibody and fibronectin antibody showed no overlap of immunoreactivity indicating that these two antigens are different. 7.1B3 antigen was found during human fetal muscle development in vivo at all time points studied from 15 weeks onwards. Immunoreactivity was localised to the loose extracellular matrix and in the endomysium. Perymysium staining was very prominent at 25 weeks and in adult muscle. Double staining of 7.1B3 antibody and α-bungarotoxin was carried out to determine whether 7.1B3 was present in synaptic junctions. Although 7.1B3 was found in the endomysium it is excluded from synaptic areas. We conclude that 7.1B3 antibody