Cytopathology of malignant mesothelioma. Reappraisal of the diagnostic value of collagen cores

The identification of malignant mesothelial cells in cytological smears prepared from serous effusions is still hampered by the lack of features specific for mesothelial differentiation. We examined the diagnostic value of collagen cores within clusters of tumour cells in cytological smears prepared from effusions from 43 patients with malignant mesothelioma and of 62 cases of metastatic adenocarcinoma. In Giemsa-stained smears collagen cores were detected in 51% of the cases of malignant mesothelioma and in none of the smears with metastatic adenocarcinoma. Using the Azan stain, collagen cores were detected in 64% of the malignant mesotheliomas and 4% of the adenocarcinomas. Immunoelectron microscopy demonstrated that the collagen cores are largely composed of collagen type III fibrils and some elastin embedded in a homogenous extracellular matrix. It can be concluded that the presence of collagen cores within clusters of tumour cells is highly suggestive of mesothelial differentiation and a common finding in malignant mesothelioma.     

Methods in elastic tissue biology: elastin isolation and purification

Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60-70 kDa monomer called tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin 'contaminates' are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and tropoelastin. It also addresses key aspects of studying tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.     

The content of elastin in the uterine cervix

Mature, crosslinked elastin has been isolated from 4 human and 12 monkey uterine cervices. A modification of previous methods for determination of elastin content was devised to quantitate the low amounts of elastin in the crude connective tissue of uterine cervices. The percentage of elastin was found to range between 0.9 and 2.4% and did not appear to change at various stages of gestation.     

Role of plasma and serum proteases in the degradation of elastin

Accelerated proteolysis of tropoelastin and elastin occurs in the major arteries of chicks fed copper-deficient diets. Signs of elastin degradation are not obvious in normal arteries of copper-supplemented chicks. It is proposed that the sources of proteases that effect elastin degradation are from plasma and serum. Both calcium-dependent proteases and kallikrein were effective in degrading tropoelastin and partially crosslinked insoluble elastin into peptides similar to those detected in aortic extracts from copper-deficient chicks. As dietary copper deficiency progresses it is also possible to detect elastin peptides in plasma.     

Biosynthesis of lysine-derived elastin crosslinks in aortic cell culture.

Culture of an established line of aortic medial cells in the presence of L-[¹⁴C] lysine for 72 hours, beginning on the twenty-first day after transfer, has resulted in the incorporation of label into a residue, insoluble after autoclaving. Acid hydrolysates of this residue with or without reduction by NaBH₄ were subjected to ion exchange chromatography. Several radioactive lysine-derived residues were identified, by comparison to standards, as the distinctive crosslinks of elastin, isodesmosine, desmosine, merodesmosine and lysinonorleucine. This confirms the synthesis of elastin in aortic cell culture and establishes the formation of insoluble crosslinked elastin. Differences in the heights of the peaks in the reduced and nonreduced elastin indicate the probable occurrence of dehydromerodesmosine and dehydrolysino-norleucine as well and suggest that these may be intermediates in crosslink formation.     

Accumulation and regulation of elastin in the rat uterus

The relative levels of elastin-specific mRNA were used as a measure of tropoelastin expression in uteri from pregnant Sprague-Dawley rats. The levels of elastin-specific mRNA were also correlated with values for net tropoelastin production and net deposition of mature, crosslinked elastin. The total content of uterine elastin increased throughout gestation, reaching maximal levels at Day 19 of gestation, which were three times those of nongravid tissue. Following involution, the elastin content decreased rapidly to near baseline values by 5 days postpartum. The content of soluble elastin, estimated using an enzyme-linked immunosorbent assay, paralleled in part the increase in elastin deposition and elastin mRNA levels. Uterine elastin metabolism appears to be unlike that in other elastic tissues, e.g., lung and large blood vessels. In most elastin containing tissues, the protein is synthesized during discrete developmental periods and is not readily degraded. However, uterine elastin is continuously expressed, and appears to be in a continual cycle of degradation and replacement.     

Adherence,proliferation and collagen turnover by human fibroblasts seeded into different types of collagen sponges

We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted non-crosslinked collagen, (2) reconstituted collagen that was chemically crosslinked with either glutaraldehyde, aluminium alginate or acetate, and (3) native collagen fibres, with or without other extracellular matrix molecules (elastin hydrolysate, hyaluronic acid or fibronectin). The non-crosslinked reconstituted collagen was degraded rapidly by human fibroblasts. Teh chemically crosslinked materials proved to be cytotoxic. Native collagen fibres were stable. In the absence of ascorbic acid, the addition of elastin hydrolysate to this type of matrix reduced the rate of collagen degradation. Both elastin hydrolysate and fibronectin partially prevented fibroblast-mediated contraction. Hyaluronic acid was only slightly effective in reducing the collagen degradation rate and more fibroblast-mediated contraction of the material was found than for the native collagen fibres with elastin hydrolysate and fibronectin. In the presence of ascorbate, collagen synthesis was enhanced in the native collagen matrix without additions and in the material containing elastin hydrolysate, but not in the material with hyaluronic acid. These results are indicative of the suitability of tissue substitutes for in vivo application.     

Elastin metabolism during recovery from impaired crosslink formation

Accelerated proteolysis of tropoelastin and elastin occurs in the arteries of chicks rendered nutritionally copper-deficient. The process results in part from decreased elastin crosslinking. Repletion of copper-deficient chicks with copper causes a deposition of elastin that is proteinase resistant. Resistance to proteolysis is conferred within 48 h of dietary copper repletion. Deposition of aorta elastin to near normal values occurs after 3-4 days in copper-repleted chicks. Moreover, elastolysis was enhanced when the content of dehydrolysinonorleucine in elastin was abnormally low. The chemical modification of lysyl residue in elastin by citroconylation, however, did not influence the rate of elastolysis. We have shown previously that tropoelastin messenger RNA activity and synthesis are not influenced by dietary copper deprivation (1986, Biochem. J. 236, 17-23). Rather, as demonstrated herein, the decrease in elastin content in arteries of copper-deficient birds appears to be more the result of enhanced degradation. Restoration of normal crosslinking restores deposition and imparts resistance to elastolysis. Moreover, serum appears to be a good source of elastolytic proteinases when the elastin substrate is partially or abnormally crosslinked.     

Elastic fiber production in cardiovascular tissue-equivalents.

Elastic fiber incorporation is critical to the success of tissue-engineered arteries and heart valves. Elastic fibers have not yet been observed in tissue-engineered replacements fabricated in vitro with smooth muscle cells. Here, rat smooth muscle cells (SMC) or human dermal fibroblasts (HDF) remodeled collagen or fibrin gels for 4 weeks as the basis for a completely biological cardiovascular tissue replacement. Immunolabeling, alkaline extraction and amino acid analysis identified and quantified elastin. Organized elastic fibers formed when neonatal SMC were cultured in fibrin gel. Fibrillin-1 deposition occurred but elastin was detected in regions without fibrillin-1, indicating that a microfibril template is not required for elastic fiber formation within fibrin. Collagen did not support substantial elastogenesis by SMC. The quantity of crosslinked elastic fibers was enhanced by treatment with TGF-beta1 and insulin, concomitant with increased collagen production. These additives overcame ascorbate's inhibition of elastogenesis in fibrin. The elastic fibers that formed in fibrin treated with TGF-beta1 and insulin contained crosslinks, as evidenced by the presence of desmosine and an altered elastin labeling pattern when beta-aminopropionitrile (BAPN) was added. These findings indicate that in vitro elastogenesis can be achieved in tissue engineering applications, and they suggest a physiologically relevant model system for the study of three-dimensional elastic structures.     

Circular dichroism studies of an elastin crosslinked peptide.

Circular dichroic studies of a desmosine crosslinked peptide reveal a hitherto undescribed elastin spectrum possessing a weak negative band at 230–235 nm, a weak positive band at 215 nm, and a maximum negative band at 190 nm. The spectrum is sensitive to both pH and temperature displaying increased ellipticity of the 215-nm band at acidic pH and low temperature. The general shape of the spectrum and its behaviour toward temperature changes suggest the presence of an extended helical conformation. Susceptibility of insoluble elastin to digestion with chymotrypsin is increased tenfold at low temperature (4°C), supporting the contention that the conformational change of the type described above occurs in insoluble elastin. Such changes in conformation would result in increased availability of aromatic amino-acid resiudues to peptide bond cleavage.     

Characterization of a Crosslinked Elastomeric‐Protein Inspired Polypeptide

Materials inspired by natural proteins have a great appeal in tissue engineering for their biocompatibility and similarity to extracellular matrix (ECM). Chimeric polypeptides inspired by elastomeric proteins such as silk, elastin, and collagen are of outstanding interest in the field. A recombinant polypeptide constituted of three different blocks, each of them having sequences derived from elastin, resilin, and collagen proteins, was demonstrated to be a good candidate as biomaterial for its self‐assembling characteristics and biocompatibility. Herein, taking advantage of the primary amine functionalities present in the linear polypeptide, we crosslinked it with 1,6‐hexamethylene‐diisocyanate (HMDI). The characterization of the obtained polypeptide was realized by CD spectroscopy, AFM, and SEM microscopies. The obtained results, although not conclusive, demonstrate that the crosslinked polypeptide gave rise to porous networks, thin nanowires, and films not observable for the linear polypeptide. Chirality 28:606–611, 2016. © 2016 Wiley Periodicals, Inc.     

Structural insights into the elastin mimetic (LGGVG)6 using solid-state 13C NMR experiments and statistical analysis of the PDB

Elastin is a crosslinked hydrophobic protein found in abundance in vertebrate tissue and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this study, the central residues of the (LGGVG)(6) elastin mimetic are targeted. Using a combination of a statistical analysis based on structures in the Brookhaven Protein Data Bank (PDB), 1D cross-polarization magic-angle-spinning (CPMAS) NMR spectroscopy, and 2D off-magic-angle-spinning (OMAS) spin-diffusion experiments, it is determined that none of the residues are found in a singular regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, there are multiple conformations and significant disorder. Furthermore, the conformational ensembles are not reflective of proteins generally, as in the PDB, suggesting that the structure distributions in elastin mimetics are unique to these peptides and are a salient feature of the functional model of the native protein.     

Crosslinked elastic fibers are necessary for low energy loss in the ascending aorta

In the large arteries, it is believed that elastin provides the resistance to stretch at low pressure, while collagen provides the resistance to stretch at high pressure. It is also thought that elastin is responsible for the low energy loss observed with cyclic loading. These tenets are supported through experiments that alter component amounts through protease digestion, vessel remodeling, normal growth, or in different artery types. Genetic engineering provides the opportunity to revisit these tenets through the loss of expression of specific wall components. We used newborn mice lacking elastin (Eln^−/−) or two key proteins (lysyl oxidase, Lox^−/−, or fibulin-4, Fbln4^−/−) that are necessary for the assembly of mechanically-functional elastic fibers to investigate the contributions of elastic fibers to large artery mechanics. We determined component content and organization and quantified the nonlinear and viscoelastic mechanical behavior of Eln^−/−, Lox^−/−, and Fbln4^−/− ascending aorta and their respective controls. We confirmed that the lack of elastin, fibulin-4, or lysyl oxidase leads to absent or highly fragmented elastic fibers in the aortic wall and a 56–97% decrease in crosslinked elastin amounts. We found that the resistance to stretch at low pressure is decreased only in Eln^−/− aorta, confirming the role of elastin in the nonlinear mechanical behavior of the aortic wall. Dissipated energy with cyclic loading and unloading is increased 53–387% in Eln^−/−, Lox^−/−, and Fbln4^−/− aorta, indicating that not only elastin, but properly assembled and crosslinked elastic fibers, are necessary for low energy loss in the aorta.     

The visceral pericardium: macromolecular structure and contribution to passive mechanical properties of the left ventricle

Much attention has been focused on the passive mechanical properties of the myocardium, which determines left ventricular (LV) diastolic mechanics, but the significance of the visceral pericardium (VP) has not been extensively studied. A unique en face three-dimensional volumetric view of the porcine VP was obtained using two-photon excitation fluorescence to detect elastin and backscattered second harmonic generation to detect collagen, in addition to standard light microscopy with histological staining. Below a layer of mesothelial cells, collagen and elastin fibers, extending several millimeters, form several distinct layers. The configuration of the collagen and elastin layers as well as the location of the VP at the epicardium providing a geometric advantage led to the hypothesis that VP mechanical properties play a role in the residual stress and passive stiffness of the heart. The removal of the VP by blunt dissection from porcine LV slices changed the opening angle from 53.3 +/- 10.3 to 27.3 +/- 5.7 degrees (means +/- SD, P < 0.05, n = 4). In four porcine hearts where the VP was surgically disrupted, a significant decrease in opening angle was found (35.5 +/- 4.0 degrees ) as well as a rightward shift in the ex vivo pressure-volume relationship before and after disruption and a decrease in LV passive stiffness at lower LV volumes (P < 0.05). These data demonstrate the significant and previously unreported role that the VP plays in the residual stress and passive stiffness of the heart. Alterations in this layer may occur in various disease states that effect diastolic function.     

Ultrastructural studies of cells in body cavity effusions

Transmission electron microscopic examination of benign (16 cases) and malignant (2 cases) mesothelial cells and metastatic carcinoma (10 cases) was performed. These studies showed long, slender, branching and bushy microvilli with high length-to-diameter ratios to be the most important distinguishing features of the mesothelial cells. Cytoplasmic intermediate filaments were present in all mesothelial cells as well as in carcinoma cells. The mesothelial cells showed an absence of mucin vacuoles, intracellular lumens and luminal tight junctions, which are seen in adenocarcinoma cells. The pinocytotic vesicles were found to be more numerous in the mesothelial cells. Lipid vacuoles, lysosomes, Golgi apparatus and intercellular lumens appeared to be variably present in all mesothelial and carcinoma cells. The methodology is discussed and pertinent literature reviewed.     

Insights into a putative hinge region in elastin using molecular dynamics simulations

The resiliency and elasticity of vertebrate tissues are traced to elastin, a crosslinked protein with extensive hydrophobic regions. There is little discussion in the literature on the structure and dynamics of the alanine-rich crosslinking regions of elastin that comprise a significant part of the native protein. In particular, the region encoded by exons 21 and 23, a contiguous splice form found in all types of human elastin, is believed to be strategically positioned for proper function of the protein, namely, in the reversible elongation and contraction of tissue. Hence, molecular dynamics (MD) calculations on the EX21/23 domain are reported here. This crosslinking domain has been assumed to adopt an architecture in which the putative hinge region links two α-helices. In this paper, we use a homology-based approach to obtain starting structures in the hinge region. The subsequent MD brings new insights into the possibility of fluctuations between “open” and “closed” states, as well as distinguishing structural features of the latter. The significance of these findings towards an enhanced understanding of structure–function relationships in elastin and the elastic fiber is discussed.     

Studies on coexistence and intercellular communication

Mouse ascites tumor cells (MAT-cells) were co-cultured with mesothelial cells of the mouse. During early stages of coexistence (30–60 min) the mesothelial cells show finger-like protrusions formed mostly at their flanks pointing towards MAT-cells in the neighbourhood. Obviously there is a directional response of the mesothelial cells to the existence of MAT-cells. The mesothelial cells are the more active partner at the beginning of coexistence. Later on MAT-cells also develop fingerlike protrusions which grow towards the mesothelial cells. A network of fingerlike protrusions is formed in the region between MAT-cells and mesothelial cells when they come near to each other. The advantages of the system serving as a model for investigations on intercellular communication are discussed.     

Isolation and characterization of rabbit peritoneal mesothelial cells

Four enriched mesothelial cell populations distinguished by different size and density were obtained when a unit gravity sedimentation procedure was applied to pronase-dispersed rabbit peritoneal mesothelial cells. Cell integrity was confirmed by trypan blue, ultrastructural and biosynthetic analyses, as well as by explantation in tissue culture. The enriched mesothelial cells displayed the immunocytochemical, ultrastructural and growth characteristics of native mesothelial cells. This isolation and separation procedure should provide a valuable experimental tool to study the pathobiology of mesothelial cells.     

Modification of arterial elastin in vivo. Effects of age and diet on changes in the N-terminal amino acid content of aorta elastin

We have previously demonstrated that aorta elastin, a highly crosslinked protein, does not undergo turnover that is easily measured in vivo. Therefore, it was hypothesized that when proteolysis of elastin occurs, a positive increase in N-terminal amino acids should result. Such an increase would represent elastin-derived fragments held covalently in situ. A cyanate carbamylation procedure was used to estimate the changes in N-terminal amino acids in aorta elastin. To provide tissue for the studies, Japanese quail (3 weeks old) were fed diets with or without the addition of 1% cholesterol. It was found that, in normal birds, the number of N-terminal amino acid residues increased from two to approximately three residues per 800 total residues (or mole of tropoelastin) throughout sexual development (3 to 8 weeks, post-hatching), with little increase thereafter. In hypercholesterolemic birds, the rate of appearance of new N-terminal residues, particularly glutamine or glutamic acid, appeared enhanced throughout early development, but by sexual maturity the number of N-terminal amino acid residues in aorta elastin from cholesterol-fed birds was similar to that for the control birds. For each of the elastin samples analyzed, approximately one residue of glycine was recovered per 800 total residues. Other amino acids that predominated as N-terminal residues were serine, aspartic and glutamic acids.     

Surface configuration of mesothelial cells in effusions. A comparative light microscopic and scanning electron microscopic study.

Surface configuration of mesothelial cells identified by light microscopy (LM) has been studied by scanning electron microscopy (SEM). It has been shown that mesothelial cells may have a variable SEM appearance. The surfaces of a small proportion of mesothelial cells are covered by regular microvilli (MV) and show openings of the pinocytotic vesicles. The surfaces of the majority of these cells are covered by vesicles or blebs. An intermediate population of mesothelial cells, i.e., cells displaying side-by-side blebs and MV, has also been observed. The latter cells no longer display pinocytotic vesicles. Occasional mesothelial cells have smooth surfaces. It has been shown by LM and transmission electron microscopy that cells with blebs are viable and capable of mitotic activity. It is concluded that mesothelial cells, detached from their epithelial setting, lose microvilli and pinocytotic vesicles and acquire surface blebs. The possible relationship between mesothelial cells and macrophages based on surface features has been discussed.