Brent W. Beaumont

Changes in elastic fibres in the small airways and alveoli in COPD

Small airways are the major site of airflow obstruction in chronic obstructive pulmonary disease (COPD). This is attributed to loss of elastin in alveoli and fibrosis in small airways. In the present study, it was hypothesised that changes to elastic fibres in alveoli might be paralleled by a similar reduction in elastic fibres in small airways. Tissue blocks from patients who had lobectomy for bronchial carcinoma were studied. Patients were classified as COPD (forced expiratory volume in one second (FEV1) <80% predicted, FEV1/forced vital capacity (FVC) <0.7) or controls (FEV1 ≥80% pred, FEV1/FVC ≥0.7). Elastic fibres were visualised using Elastic van Gieson staining and the volume fraction (v/f) of elastic fibres was determined as a percentage of tissue volume using point counting. Elastic fibre networks were also visualised by confocal microscopy. The v/f for elastic fibres in alveoli was 18.6% for COPD and 32.8% in controls. In the airways the v/f was 14.6% for COPD and 25.5% in controls. FEV1% predicted was correlated with v/f in both alveoli and small airways. The volume fraction of elastic fibres was reduced to a similar extent in small airways and alveoli in chronic obstructive pulmonary disease and both were correlated with the extent of airflow obstruction. Loss of elastic fibres in small airways may contribute to the development of airflow obstruction in chronic obstructive pulmonary disease.

Inhibition of Versican Synthesis by Antisense Alters Smooth Muscle Cell Phenotype and Induces Elastic Fiber Formation In Vitro and in Neointima After Vessel Injury

The proteoglycan versican is implicated in several atherogenic events, including stimulation of vascular smooth muscle cell (VSMC) growth and migration, retention of lipoproteins, and promotion of thrombogenesis. A high content of intimal versican also correlates with a low content of elastin, suggesting an inhibitory role for versican in elastogenesis. To determine whether reduced production of versican can be used to enhance elastogenesis, we transduced Fischer rat VSMC (FRSMC) with a versican antisense sequence using the retroviral vector LXSN. Stable expression of versican antisense (LVaSN) significantly reduced versican production, induced a flattened morphology, reduced cell proliferation and migration, increased tropoelastin synthesis, increased elastin binding protein (S-Gal/EBP), and increased deposition of elastic fibers in long-term cultures. Add-back of chondroitin sulfate chains, or versican, decreased S-Gal/EBP and elastic fiber formation. LVaSN cells seeded into balloon catheter-injured rat carotid arteries formed neointimae containing low levels versican, increased amounts of S-Gal/EBP, and increased elastin deposits 7 days postinjury. At 4 weeks, neointimae formed from LVaSN cells were highly structured and contained multiple layers of elastic fibers and lamellae. These results indicate a central role for versican and its constituent chondroitin sulfate chains in controlling cell phenotype, elastogenesis, and intimal structure.

Changes in elastin, elastin binding protein and versican in alveoli in chronic obstructive pulmonary disease

BackgroundCOPD is characterised by loss of alveolar elastic fibers and by lack of effective repair. Elastic fibers are assembled at cell surfaces by elastin binding protein (EBP), a molecular chaperone whose function can be reversibility inhibited by chondroitin sulphate of matrix proteoglycans such as versican. This study aimed to determine if alveoli of patients with mild to moderate COPD contained increased amounts of versican and a corresponding decrease in EBP, and if these changes were correlated with decreases in elastin and FEV1.MethodsLung samples were obtained from 26 control (FEV1 ≥ 80% predicted, FEV1/VC >0.7) and 17 COPD patients (FEV1 ≥ 40% – <80% predicted, FEV1/VC ≤ 0.7) who had undergone a lobectomy for bronchial carcinoma. Samples were processed for histological and immuno-staining. Volume fractions (Vv) of elastin in alveolar walls and alveolar rims were determined by point counting, and versican and EBP assessed by grading of staining intensities.ResultsElastin Vv was positively correlated with FEV1 for both the alveolar walls (r = 0.66, p < 0.001) and rims (r = 0.41, p < 0.01). Versican was negatively correlated with FEV1 in both regions (r = 0.30 and 0.32 respectively, p < 0.05), with the highest staining intensities found in patients with the lowest values for FEV1. Conversely, staining intensities for EBP in alveolar walls and rims and were positively correlated with FEV1 (r = 0.43 and 0.46, p < 0.01).ConclusionPatients with mild to moderate COPD show progressively increased immuno-staining for versican and correspondingly decreased immuno-staining for EBP, with decreasing values of FEV1. These findings may explain the lack of repair of elastic fibers in the lungs of patients with moderate COPD. Removal of versican may offer a strategy for effective repair.

Comparison of deposits of versican, biglycan and decorin in saphenous vein and internal thoracic, radial and coronary arteries: correlation to patency.

BackgroundMatrix proteoglycans versican, biglycan and decorin are important determinants of vessel‐wall structure and pathology. Thickened myxoid intimas typical of restenosis and early atherosclerosis are enriched in versican and biglycan, proteoglycans that promote proliferation and migration of smooth muscle cells and bind lipoproteins. In contrast, compact fibrous intimas are characterized by decorin. ObjectiveTo compare the distribution patterns of these matrix proteoglycans, and changes induced by organ culture in coronary artery, saphenous vein, internal thoracic artery (ITA), and radial artery, and correlate differences to patency. MethodsVessels were collected at the time of bypass surgery and heart transplantation and either fixed for immunohistochemistry or prepared for organ culture. Vessels in culture were labelled with [3H]‐glucosamine and processed for autoradiography and immunohistochemistry. Distribution patterns for proteoglycans and radio‐labelling were determined morphometrically. ResultsDistribution profiles in coronary artery and saphenous vein were similar, with relatively high levels of subendothelial versican and biglycan and low levels of decorin. In culture subendothelial incorporation of [3H]‐glucosamine and immunostaining for versican and biglycan, but not decorin, were significantly increased. In contrast, the thin intima of the ITA was relatively enriched in decorin compared with the medial layers and in culture intimal staining for decorin increased markedly compared with a modest increase for biglycan and no change for versican. There was an even distribution in radial artery of all three proteoglycans across the intima without subendothelial accumulations. In culture there was an increase in staining intensity for proteoglycans of the radial artery. Neither the ITA nor radial artery exhibited an increase in subendothelial incorporation of [3H]‐glucosamine in culture. ConclusionsThe distributions of proteoglycans, and responses to culture correlate to the known differences in patency between grafted saphenous vein and ITA and predict that the radial artery will outperform the saphenous vein but might not be as good as the ITA for long‐term patency.

Matrix proteoglycans and remodelling of interstitial lung tissue in lymphangioleiomyomatosis

The interstitial lung disease lymphangioleiomyomatosis (LAM) is characterized by diffuse proliferation of smooth muscle cells (SMCs), which in many patients show TSC2 (tuberin) gene mutations, in addition to thickening of interstitial tissues, loss of alveoli, and the development of cystic spaces. While SMC proliferation is the defining feature of LAM, a significant proportion of LAM lung tissue consists of expanded interstitial connective tissue that is negative for smooth muscle actin and TSC2 mutations. The importance of this actin‐negative interstitial tissue to the pathophysiology of LAM is not clear. The present study has determined the contribution of this interstitial tissue to LAM lung volume by morphometric analysis and has examined its cell and matrix proteoglycan composition by immunohistochemistry. Lung tissue from nine LAM patients and four control subjects was examined. LAM lung contained twice as much interstitial tissue as control lung (27% versus 13% of total lung volume), with SMCs accounting for less than 25% of the interstitial volume. Areas of interstitial tissue stained strongly for the matrix proteoglycans versican and biglycan. Decorin was prominent in association with collagen bundles. SMCs did not stain, or stained lightly, for proteoglycans. Versican and biglycan deposits were closely associated with actin‐negative interstitial fibroblasts identified by prolyl 4‐hydroxylase immuno‐staining. Comparatively normal alveolar walls in LAM lung also stained strongly for versican and had a reduced elastin content. Thickened interstitial regions contained significant amounts of elastin (∼13% of interstitial volume) but with fibres in disorganized patterns. Elastic fibres were absent from areas that stained strongly for versican and biglycan. These areas also showed weak staining for elastin binding protein (EBP), consistent with proteoglycan‐induced shedding of EBP and inhibition of elastic fibre formation. These findings point to a significant contribution from matrix proteoglycans to the expanded and remodelled interstitial lung tissue of LAM patients. Copyright

The Macrophage Is the Predominant Inflammatory Cell in Renal Allograft Intimal Arteritis

Background. Intimal arteritis defines acute vascular rejection in the Banff 97 schema. The arteritis is generally considered to be lymphocytic, although the cellular infiltrate in tubulitis is composed of both lymphocytes and macrophages. This study aimed to determine the extent of macrophage involvement in renal allograft intimal arteritis. Methods. We obtained archival biopsy material from 57 biopsies of 34 renal allografts transplanted between March 1999 and February 2002. All biopsies were diagnostic. We examined clinical and histological parameters. Biopsies were graded using the Banff 97 criteria. We identified macrophages and memory T cells using immunohistochemistry for CD68 and CD45RO, respectively. Results. In all, 24 biopsies showed borderline rejection, and 12 biopsies showed grade IA, 13 showed grade IB, and 8 showed grade II or III acute rejection. Both lymphocytes and macrophages were present in the tubulointerstitium in all grades of acute rejection. We identified intimal arteritis in 10 vessels in eight biopsies. The infiltrating cells invariably included CD68-positive cells; however, we saw intimal CD45RO-positive cells in only seven vessels. There were significantly more CD68-positive cells than CD45RO-positive cells (mean, 9.5 vs. 4.4 positive cells per vessel, P< 0.01). CD45RO cells were never the predominant component of the intimal inflammatory infiltrate. Conclusions. In the intimal arteritis of biopsies graded as Banff II or III acute rejection, the infiltrating cells were predominantly macrophages. T cells were in the minority. This finding challenges the assumption that the mononuclear cells in intimal arteritis are predominantly lymphocytic.

Effect of TGF-β1 Antisense S-Oligonucleotide on Synthesis and Accumulation of Matrix Proteoglycans in Balloon Catheter-Injured Neointima of Rabbit Carotid Arteries

Arterial matrix proteoglycans (PG) are necessary for the maintenance of viscoelastic properties of the vessel wall, but excess levels, particularly of versican and biglycan in primary and restenotic intimal thickenings, are correlated with increased tissue volume and with atherogenicity. There is good evidence that the primary stimulus to increased PG synthesis, including versican and biglycan, is transforming growth factor-β1 (TGF-β1). The aim of this study was to determine the effects of reducing endogenous TGF-β1 on rates and patterns of PG synthesis and on versican, biglycan and decorin accumulation in vivo. Rabbit common carotid arteries subjected to balloon catheter injury were treated with a TGF-β1 antisense phosphorothioate oligonucleotide applied in a pluronic gel to the adventitia. Control animals received a nonsense oligonucleotide or gel alone. TGF-β1 antisense (1) significantly (p < 0.005) inhibited, at day 2, the balloon catheter-induced increase in TGF-β1 mRNA relative to β-actin mRNA; (2) inhibited intimal thickening at 23 days by ∼40% (p < 0.05); (3) inhibited (p < 0.05) PG synthesis, measured by autoradiographic detection of [3H]glucosamine, in the media of day 2 ballooned carotids and in the subendothelial zone of day 23 neointima, and (4) decreased immunostaining intensity for versican (p < 0.03) and TGF-β1 (p < 0.001) in the neointima. Biglycan was reduced to a lesser extent but not significantly and decorin was not affected. Proliferating cell nuclear antigen indices were variable and not significantly changed. These findings confirm a role for TGF-β1 in developing neointima and demonstrate a specific effect on the synthesis, distribution, and accumulation of matrix PG, particularly versican.

Neointima Formed by Arterial Smooth Muscle Cells Expressing Versican Variant V3 Is Resistant to Lipid and Macrophage Accumulation

Objective—Extracellular matrix (ECM) of neointima formed following angioplasty contains elevated levels of versican, loosely arranged collagen, and fragmented deposits of elastin, features associated with lipid and macrophage accumulation. ECM with a low versican content, compact structure, and increased elastic fiber content can be achieved by expression of versican variant V3, which lacks chondroitin sulfate glycosaminoglycans. We hypothesized that V3-expressing arterial smooth muscle cells (ASMC) can be used to form a neointima resistant to lipid and macrophage accumulation associated with hypercholesterolemia. Methods and Results—ASMC transduced with V3 cDNA were seeded into ballooned rabbit carotid arteries, and animals were fed a chow diet for 4 weeks, followed by a cholesterol-enriched diet for 4 weeks, achieving plasma cholesterol levels of 20 to 25 mmol/L. V3 neointimae at 8 weeks were compact, multilayered, and elastin enriched. They were significantly thinner (57%) than control neointimae; contained significantly more elastin (118%), less collagen (22%), and less lipid (76%); and showed significantly reduced macrophage infiltration (85%). Mechanistic studies demonstrated that oxidized low-density lipoprotein stimulated the formation of a monocyte-binding ECM, which was inhibited in the presence of V3 expressing ASMC. Conclusion—These results demonstrate that expression of V3 in vessel wall creates an elastin-rich neointimal matrix that in the presence of hyperlipidemia is resistant to lipid deposition and macrophage accumulation.

Structural Heterogeneity of the Diffuse Intimal Thickening and Correlation with Distribution of TGF-β1

This study examines the ultrastructural features of diffuse intimal thickenings of human coronary arteries and correlates structural heterogeneity along the radial axis with the distribution and known actions of transforming growth factor beta 1 (TGF-beta 1). Morphometric and immunohistochemical data were collected from thickenings of varying widths, sampled at autopsy from 19 persons ranging in age from 3 months to 81 years. Thickenings were characterised by an inner proteoglycan layer (PGL), up to approximately 70 mm wide, and an underlying variable-width musculofibrous layer (MFL). The PGL was characterised by low volume fractions (v/fs) for collagen, elastin, basement membranes and cells and a high v/f for matrix space; v/fs for the MFL components were more evenly distributed. Proteoglycans were visualised by ruthenium red staining, quantified and sized. Densities of large (versican; > 20 nm) and small (< 20 nm) granules changed little across intimal thickenings. Mean diameters of matrix space granules increased with increasing intimal thickness and notably were significantly (p < 0.001) larger in the PGL than the MFL. In contrast, diameters of collagen-associated small granules (decorin) did not differ between the PGL and MFL. TGF-beta 1 staining was detected in 70% of vessels examined and occurred almost exclusively in the PGL, although showed a patchy distribution. Both the distribution of TGF-beta 1 and its known differential effects on versican and decorin synthesis suggest that it may play a significant role in the formation and maintenance of the PGL.

Changes in the morphology and synthetic activity of cultured rat tail tendon

SummaryIsolated single fascicles from tail tendons of young rats were freed of epitenon cells and cultured in vitro for up to 7 days. The tissue remained viable, as judged by the structural integrity of cell organelles and the ability to synthesize DNA and glycosaminoglycans (GAG). The rate of DNA synthesis peaked after 2 days in culture and decreased slowly thereafter. Concomitantly, an increase in cell number was noted at the periphery of the fascicle. GAG production also increased during culture, sulphated GAG being increased proportionately more than hyaluronic acid. Dermatan sulphate was the predominant sulphated GAG in freshly isolated fascicles, but in cultured tissue, the newly synthesized sulphated GAG was more sensitive to degradation by chondroitinase AC and had an increased electrophoretic mobility. Fine structural changes were observed in cultured tissues such as the retraction of cell processes. rounding up of cell bodies and the appearance of gaps between collagen fibrils. Cultured tenocytes also frequently contained apparently phagocytized collagen fibrils which were not seen in freshly isolated fascicles, and this appearance was suggestive of collagen degradation occurring in vitro, although no change in the total hydroxyproline content was noted. The data show that when individual fascicles are cultured in vitro they undergo a process of matrix remodelling which has features in common with events occurring in vivo when tendons have been surgically manipulated.