Surface Of Basal Lamina Research Articles

Abstract B23: Further defining reactive stroma in prostate cancer

Abstract Quiescent fibroblasts and smooth muscle make up the mesenchymally-derived component of normal human stroma and function to maintain tissue homeostasis by forming a reactive stroma phenotype in the presence of injury. Reactive stroma initiates early during the evolution of human prostate cancer, with foci being observed during premalignant prostatic intraepithelial neoplasia (PIN). This microenvironment response is characterized by an accumulation of vimentin positive cells that co-express several reactive stroma markers. Reactive stroma co-evolves with development of cancer foci and is typified by accumulation of myofibroblasts that express tenascin-C and pro-collagen I. Our previous studies have shown that the degree of reactive stroma is a significant predictor of biochemical recurrent disease (elevation of PSA) using either archival prostatectomy specimens or pre-operative needle biopsy material. Patients with grade 3 exhibit a significantly reduced time to biochemical recurrent disease. Moreover, we have shown significant differences in gene expression profiles in human reactive stroma grade 3 relative to normal human prostate stroma. Our previous studies have shown that reactive stroma is tumor-promoting in prostate cancer models. TGF-β has emerged as a key factor in mediating the microenvironment perturbations in a variety of cancers, however its corresponding role in reactive stroma initiation has not been examined. It is our hypothesis that prostate cancer reactive stroma is composed of myofibroblasts / CAFs and is induced from tissue-fixed and/or bone marrow derived cells (BMDCs) in a TGF-β dependent manner. Our data shows that foci of reactive stroma in human prostate cancer consist of CD34+/vimentin+ progenitor cells that are juxtaposed to the sub basal lamina surface at the stromal-epithelial junction. Bone marrow transplants with CFP reporter donors into athymic mice containing LNCaP xenografts shows a time dependent increase in recruited fibroblast-like cells of marrow origin. Mechanistic studies using a transgenic mouse overexpressing TGF-β in prostatic epithelium demonstrate foci of inflammation in nerve ganglia and vessel structures. These mice also display distinct stromal proliferative lesions reminiscent of changes seen in a subtype of human prostate cancer. These histopathological changes occur in an age-dependent manner and seem to become more prevalent after a year of age in transgenic mice. Further mechanistic studies examining TGF-β signaling and the role of BMDCs are underway to examine reactive stroma induction and progression in prostate cancer. We propose a model of reactive stroma potential, affected by activation of local progenitors and subsequent recruitment of circulating progenitors to initiate and sustain a reactive microenvironment adjacent to cancer foci. We also propose that this microenvironment is selective for more aggressive cancer phenotypes and therefore is a potential target of opportunity for more advanced prognostics and novel therapeutics. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B23

Scanning electron microscopy examination of endothelium morphology in human carotid plaques

In recent years, experimental and clinical evidence has been provided regarding endothelial dysfunction and its contribution to the inflammatory process leading to atherosclerotic plaque formation. All the techniques employed direct to the study of endothelial dysfunction, however usually require isolation of endothelial cells and therefore tissue manipulation and destruction, with subsequent loss of information regarding morphology and topographical distribution of the lesions. By means of scanning electron microscopy (SEM), we have investigated the characteristics of the endothelial layer in carotid specimens obtained from subjects undergoing carotid endarterectomy. Carotid specimens obtained from 6 patients undergoing carotid endarterectomy for stenosis ≥ 70% were fixed, prepared and examined by use of SEM in a direct mode. All the plaque specimens showed increased thickness of subendothelial connective tissue with respect to post-mortem tissue obtained from an healthy subject. Endothelial layers were typically detached from the basal lamina surface and infiltrating cells (mainly erythrocytes and, possibly, monocytes) could be identified. Endothelial cells in proximity of the plaques had irregular shape, with prominent nuclei. In several areas, the endothelial layer was completely absent and basal lamina completely uncovered. In the present study, by using SEM analysis, the morphological features of dysfunctional endothelium in human carotid plaques were extensively documented at the ultrastructural level. SEM is a powerful investigational technique which allows tridimensional examination of specimens without disruption of the originary morphology.

A Re-appraisal of the Morphophenotype and Basal Lamina Coverage of Cytotrophoblasts in Human Term Placenta

A recent study of human placental villi [Mori et al., The cytotrophoblast layer of human chorionic villi becomes thinner but maintains its structural integrity during gestation, Biol Reprod 76 (2007) 164–172] concluded that cytotrophoblast (CT) cells occupy 80% of the basal lamina (BL) surface at term and that syncytiotrophoblast (ST) does not make direct contact with the BL. Based on SPINT-1 localisation using immunofluorescence on cryosections, these conclusions run counter to previous light and electron microscopic data suggesting that term CT cells cover no more than about 24% of the BL surface. To resolve these discrepancies, we have undertaken a stereological study of term placenta using transmission electron microscopy (TEM) and a novel immunofluorescence approach. Test line lattices were randomly superimposed on TEM images of villous trophoblast from 13 normal term placentae. Intersections with the test lines were counted to assess the fractional surface of BL occupied by CT cells. After trypsin-mediated removal of syncytium, cells in whole-mounted term and first trimester villi were stained with cytokeratin 7 to identify CT and then visualised by confocal microscopy. CT formed an almost continuous layer in the first trimester. In contrast, term CT cells and their processes were found to cover only 44% (SD 14%) of the BL surface with intervening regions occupied by ST. TEM and confocal images were consistent with the concept of a network of ‘octopoid’ CT cells with fine processes extending from a central cell body. Our estimates of CT coverage are lower than the recent immunofluorescence estimate but greater than earlier TEM estimates. The former may have been biased by overprojection (section thickness) effects whilst the latter may be underestimates due to failure to include the fine CT cell processes. We conclude that CT cells transform from a cuboidal phenotype early in gestation to flattened cells with multiple interconnecting processes. The CT layer thins but maintains a functional network within which cells intercommunicate without compromising substance transfer via the syncytium.

P0-Cre Transgenic Mice for Inactivation of Adhesion Molecules in Schwann Cells.

Normal peripheral nerve myelination depends on Schwann cell-basal lamina interactions. An important component of Schwann cell basal lamina is laminin-predominantly laminins 2 and 4. Mutations in the alpha 2 chain common to these two isoforms are associated with dysmyelination in mouse (dy) and man (congenital muscular dystrophy). Thus, laminin 2 and 4 receptors are also likely to be important for myelin formation. Several laminin 2/4 receptors are detected at the basal lamina surface of myelin-forming Schwann cells, namely, α6β4 and α6β1 integrins and dystroglycan. The evidence linking these receptors to myelination is suggestive, but not conclusive. Genetic studies have not yet confirmed a role for these molecules in myelin formation. Natural or targeted inactivation of α6, β4, and β1 integrins and of dystroglycan have profound effects on other tissues causing embryonic or perinatal death before myelination. Therefore, to conditionally inactivate these receptors specifically in myelin-forming Schwann cells, we have constructed and initially characterized a P0-Cre transgene that activates Cre-mediated recombination of loxP-containing genes in peripheral nerve.

Basement membrane associated changes in the rat ventral prostate following castration.

This study focuses on the basement membrane associated modifications that take place after androgen blockade, by studying some of its main components, through histochemical, immuno-histochemical and Western blotting tests, and its ultrastructural aspects. It was demonstrated that laminin and collagen type IV remain associated with a thickened basement membrane and that there is an apparent increase in heparan sulfate content 21 days after castration. Ultrastructurally, basal lamina appeared extensively folded and pleated. It was also observed that detachment of epithelial cells is not dependent of basal lamina degradation and that the free basal lamina surfaces are folded by the action of adjacent cells. We have also observed some aspects of smooth muscle cell degeneration and death, that lead to modifications of the associated basal lamina. In this case, residual basal lamina also shows extensive folding. The results suggested that degradation of excess basement membrane does not occur or is a very slow process within the period examined, and that basement membrane is left re-organized but ultrastructurally and compositionally unaffected.

Quantitative Pathology of Nasal Passages in Rats Exposed to Urban Levels of Air Pollution

In order to assess the adverse effects of urban levels of air pollution, rats were used as biological indicators in a chronic exposure experiment. Animals were housed for 6 months in the center of São Paulo (the largest South American town) and compared to controls kept for the same period in a clean area. Pollution levels were obtained from a state air pollution monitoring station, 200 m distant from exposure location, which provided the levels of CO, SO 2, particulates, and ozone. The nasal septum was submitted to quantitative analysis of morphological and histochemical parameters, comprising the measurement of volume of epithelium and lamina propria per unity of surface of basal lamina, the amount of mucus stored in the surface epithelium, the volume densities of neutral and acidic mucus in the lamina propria glands, and the densities of secretory and ciliated cells in the epithelium. The results obtained in the present investigation suggest that chronic exposure to urban levels of air pollution induces secretory hypertrophy, combined with a shift toward acidic mucus secretion and ciliary damage. The results are consistent with the idea that prolonged exposure to low levels of air pollution deteriorates respiratory defenses against infectious agents and may cause an increase in respiratory morbidity and perhaps mortality.

Migration of perivascular cells into the neuropil and their involvement in beta-amyloid plaque formation.

Our recent ultrastructural studies of amyloid angiopathy in biopsy specimens from Alzheimer's disease patients showed that perivascular cells and perivascular microglia are involved in the production of amyloid fibrils. Further examination of the walls of the vessels with and without amyloid deposits presented in this report reveals numerous mononuclear cells with a broad spectrum of morphological appearances. Some of these cells produce amyloid in the vascular wall and migrate into the neuropil. Others do not produce amyloid in this location but also migrate through the vascular basal lamina and position themselves on the external surface of basal lamina or in the neuropil outside the vascular astrocytic end-feet processes. The presence of clusters or rows of six or more of these cells in the position of perivascular microglial cells suggests their proliferation in the perivascular region. After leaving the perimeter of the vessel wall, perivascular cells become the perivascular, neuropil, and satellite microglia cells. Migrating perivascular cells become the microglia, which are engaged in amyloid fibril formation and development of classical and primitive plaques.

Ultrastructure and permeability of the Schwann cell layer surrounding the giant axon of the squid.

The ultrastructure of the Schwann cell layer surrounding the giant axon of the squid Alloteuthis subulata is described, and the permeability of extracellular compartments assessed by exposure to electron-dense tracers. Morphometric analysis is used to deduce the number, size and shape of the Schwann cells, and the routes for ion flux across the Schwann cell layer. Axons (mean diameter 233 microns) were surrounded by a 1-2 microns thick layer of Schwann cells which were approximately 1 micron thick, approximately 70 microns long and approximately 23 microns wide. There were around 62,000 Schwann cells per cm2 axon surface. The outer (abaxonal) surface of the Schwann cells was invaginated, with evidence for a covering of fine Schwann cells processes; the inner (adaxonal) surface of the Schwann cells was less folded. The percentage area occupied by mesaxonal cleft openings to the axon and to the basal lamina was 0.02% and 1.09% respectively. A system of tubules, the glial tubular system, occupied 3.9% of the Schwann cell volume, and opened to both axonal and basal lamina surfaces, with more elaborate lattice-like clusters towards the basal side of the cell. Tubule openings accounted for 0.26% of the surface area facing the axon and 0.37% of the area facing the basal lamina (where there was greater clustering of openings). The electron dense tracers horseradish peroxidase, ionic lanthanum and tannic acid filled mesaxon clefts, glial tubular system and periaxonal space. If ion flux occurred via the mesaxonal clefts, a theoretical series resistance (Rsth) of > 20 omega cm2, would be predicted, whereas if it occurred via the tubular system, the figure would be < 2 omega cm2, closer to physiological estimates. The results presented show that the glial tubular system is likely to be the major route for ion flux into and across the Schwann cell layer, and for clearance of K+ from the periaxonal space during periods of axonal stimulation. The implications for K+ homeostasis in the axonal microenvironment are discussed.

Comparison of osmium/sonication and edta/sonication microdissection techniques in exposing the adepithelial basal lamina surface of developing rat colon

We have used two epithelial-stripping techniques in our studies of the basal lamina in the developing rat colon. The first involves prolonged osmication followed by sonication; the second uses chelation of calcium by EDTA followed by sonication. Both techniques remove the epithelium from the basal lamina; however, the EDTA/sonication technique appears to produce a cleaner adepithelial surface of the basal lamina. In addition, the fine structure of the basal lamina appears to be better preserved in specimens prepared by the EDTA/sonication technique. In contrast, the basal lamina of specimens prepared by the osmium/sonication technique had a shattered appearance that we believe is due to an increase in the fragility of the delicate fetal basal lamina.

Histochemical aspects of the Schwann cell basal lamina.

Schwann cell basal laminae of mouse sciatic nerve were studied histochemically using tannic acid (TA) and ruthenium red (RR) in tissue sections. In addition, basal laminae isolated by sonication were examined using ferritin-conjugated lectin for carbohydrate residues and by immunohistochemistry for laminin. Isolated basal laminae were also examined by RR. The outer (interstitial) surface of the basal laminae were observed by scanning electron microscopy (SEM).By TA staining, the basic lattice-like structure of lamina densa was revealed, and fine and thick filamentous fuzzy structures were seen to project from both sides of this lattice.By RR staining, there were dark-stained spots of proteoglycans, arranged at 30-100 nm intervals, on the outer surface of the basal lamina. These spots are considered to be primarily responsible for the attachment of basal lamina to the surrounding connective tissue matrix.Out of 12 lectins examined, WGA, RCA-I and ConA bound to the isolated basal laminae: WGA tended to bind to the inner surface, while the other two lectins bound almost equally to both sides.Laminin appeared to be more abundant on the inner surface than on the outersurface of the basal lamina.SEM revealed a somewhat fuzzy but unexpectedly smooth outer surface of basal lamina. There were small pits scattered randomly on the basal lamina.These findings are discussed from the point of view of the polarity of the basal lamina, and its role in cell adhesion to the surrounding extracellular matrices.

Ozone-induced adaptive and reactive cellular changes in respiratory bronchioles of bonnet monkeys.

To characterize the response of respiratory bronchioles (RBs) to chronic high ambient levels of ozone, bonnet monkeys were exposed for 90 days to 0, 0.4, or 0.64 ppm ozone (UV photometric standard; 3 monkeys/exposure). Morphologic changes in respiratory bronchiolar epithelium and interstitium were evaluated quantitatively at both the light and transmission electron microscopic levels. Significant changes in respiratory bronchioles following exposure included: a thicker wall and a narrower lumen, a thicker epithelial compartment and a much thicker interstitial compartment, shifts in epithelial cell populations with many more nonciliated bronchiolar epithelial cells and fewer squamous type I epithelial cells, larger nonciliated bronchiolar epithelial cells with a larger complement of cellular organelles associated with protein synthesis, greater amounts of both interstitial fibers and amorphous ground substance, greater numbers of interstitial smooth muscle cells per epithelial basal lamina surface area, and greater volumes of interstitial smooth muscle, macrophages, mast cells, and neutrophils per epithelial basal lamina surface area. These observations imply that chronic ozone exposure causes a concentration-dependent reactive peribronchiolar inflammatory response and an adaptive response consisting of hypertrophy and hyperplasia of the nonciliated bronchiolar cell.

Significance of basal lamina for regeneration of injured lung.

To study the relationship between basal lamina and regenerating cells, lungs of dogs were injured with intravenous injection of oleic acid. The lesion produced is characterized by desintigration of cells and by preservation of basal lamina. Within three days, regeneration with epithelial and endothelial cells, and probably with septal cells begins from the adjacent viable portions of the lung. Guided by the epithelial and endothelial basal lamina, these cells proliferate along the basal lamina surfaces, which have been denuded of cells, and in three weeks almost completely re-establish the structure and function of the lung. The new epithelial and endothelial cells grow directly upon the original basal lamina and do not deposit a new layer. Observations are presented which indicate that specific recognition systems may exist between denuded basal lamina and regenerating cells.