Absence Of Basement Membrane Research Articles

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs' communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Dermal Pericytes Exhibit Declined Ability to Promote Human Skin Regeneration with Ageing in 3D Organotypic Culture Models.

The well documented decline in the regenerative ability of ageing human skin has been attributed to many factors including genomic instability, telomere shortening, poor nutrient sensing, cellular senescence, and stem cell exhaustion. However, a role for the dermal cellular and molecular microenvironment in skin ageing is just emerging. We previously showed that dermal pericytes co-operate with fibroblasts to improve human skin regeneration in an organotypic skin culture model, and even do so in the absence of fibroblasts. Here, we report that the number of dermal cells, particularly pericytes, declines significantly in human skin of donors aged > 50 years. Notably, aged pericytes promoted epidermal regeneration of neonatal keratinocytes in organotypic cultures and the resulting epithelium exhibited a Ki67+/ΔNp63+ basal layer and terminal differentiation. However, the epithelium lacked several features of homeostasis displaying lower levels of ΔNp63 expression, decreased LAMA5 deposition at the dermo-epidermal junction, and the absence of basement membrane and hemi-desmosome assembly. We conclude that a decline in pericyte incidence and function contribute to an impaired epidermal microenvironment and poor skin regeneration with ageing in the human skin.

Criterios diagnósticos en 400 biopsias de adenocarcinoma de próstata

ObjectiveTo analyze a number of prostate biopsies evaluated by the same pathologist, to find the frequency of minimal histological criteria for prostate cancer and the new changes in the consensus of urologists in the Gleason classification. Materials and MethodsA total of 400 ultrasound-guided biopsies performed using a number 18 needle, and with the hematoxylin-eosin stained sections being evaluated by the same pathologist. ResultsThe histological characteristics in neoplastic glands were stromal infiltration (100%), no basal layer (100%), prominent nucleoli (98%), multiple nucleoli (60%), intraluminal crystalloids (40%), intraluminal amorphous eosinophilic material (70%), collagen micro nodules (1%), glomeruloid structures (10%), perineural invasion (20%), stromal retraction (32%) and invasion of fat (1%). Other associated histological findings were observed, such as atrophy, basal cell hyperplasia, atypical small acinar proliferation. Finally, was the assessment of the Gleason grading was assessed according to the new consensus of urologists seeing an increase in the number of cases with Gleason 7. ConclusionsThe histological diagnosis of prostate cancer based on this study show that the most common criteria are stromal infiltration, absence of basement membrane and prominent nucleoli. Pathologists should be aware, and become familiar with this classification, as well as the changes that have been made over time.© 2012 Sociedad Colombiana de Urología. Published by Elsevier España, S.L. All rights reserved.

Pleomorphic granular cell astrocytoma in the pineal gland: Case report

Pleomorphic granular cell astrocytoma in the pineal region is exceedingly rare, and its clinicopathological features are distinctive. A 67-year-old woman was admitted with a staggering gait. Magnetic resonance imaging revealed a mass lesion at the pineal gland accompanied by obstructive hydrocephalus. Following surgery, pathological examinations demonstrated a pleomorphic granular cell astrocytoma. The patient has been free from recurrence for 24 months after surgery without adjuvant therapy. The specimen exhibited nuclear and cytoplasmic pleomorphism. The nuclei varied in size, shape and coarseness. Variability was also observed in the eosinophilic granular bodies, Rosenthal fibers and spindle-shaped tumor cells. GFAP, S-100 and vimentin were immunohistochemically positive. Reticulin network was absent between the tumor cells, and granular cells with ballooned cytoplasm showing positive staining for PAS. Pleomorphic granular cell astrocytoma is believed to be a form of astrocytoma originating from the pineal gland. Its clinicopathological features resemble those of pleomorphic xanthoastrocytoma. However, it can be differentiated from the latter by the absence of reticulin fibers, absence of basement membrane between adjacent cells, and presence of large numbers of mitochondria.

Prominent expression of metalloproteinases in early stages of ovarian tumorigenesis

The role for matrix metalloproteinases (MMPs) in tumor cells invasion and metastasis is well established, and expression of MMPs is recognized as an indication of tumor cell malignancy. Previous studies suggest that the degradation of the basement membrane is a crucial early step in epithelial transformation and ovarian tumorigenesis. Thus, MMPs may also express and exert a role in preneoplastic lesions of ovarian tissues. We investigated the expression of the major metalloproteinases, gelatinase A, 72 kDa type IV collagenase (MMP-2), and gelatinase B, 92 kDa type IV collagenase (MMP-9), and the presence of basement membrane in ovarian tumors and tissues from prophylactic oophorectomies using immunostaining. MMP expression was also characterized in a panel of ovarian cancer cell lines and several nontumorigenic ovarian surface epithelial primary cells by zymography, Northern, and Western blots. We found, surprisingly, that MMP-2 and MMP-9 are expressed more frequently in early lesions than in established carcinomas. No correlation was found between the expression of MMPs and tumor grades or stages. In preneoplastic lesions, MMP-2 or MMP-9 expression often associates with the absence of basement membrane and morphological alterations. MMP-2 is often expressed in nontumorigenic ovarian surface epithelial cells but reduced or absent in cancer cells. Thus, we conclude that MMPs expression does not correlate with the malignancy of ovarian epithelial cells as generally thought. Rather, increased metalloproteinase expression is an early event in ovarian tumorigenesis and associates with the loss of epithelial basement membrane and morphological transformation. We propose that the increased MMP activity is an etiological factor for ovarian cancer risk. We found that MMPs expression does not correlate with the malignancy of ovarian epithelial cells as generally thought. Rather, increased metalloproteinase expression is an early event in ovarian tumorigenesis. The finding suggests roles of MMP in tumor initiation in addition to invasion, and may impact on the strategy for use of MMP inhibitors in cancer prevention.

Evaluation of basement membrane status in aggressive skin carcinomas with skull base invasion: a case-control study

Some skin carcinomas may be very aggressive. Breached of basement membrane (BM) has been in some situations associated with tumor aggressiveness. In this study, the status of BM in invasion was evaluated in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) with skull base invasion, and it was compared with tumor's good outcome. Integrity or breached of BM was visualized using immunohistochemistry technique with anti-type IV collagen antibody. The pattern of BM was classified as intact, breached, or absent in 24 BCCs and 11 SCCs with skull base invasion. Control group (good outcome) included 23 BCCs and 10 SCCs. Breached BM and absence of BM were respectively noted in 33.33% and 45.83% of BCCs with skull base invasion, compared with 8.33% and 17.395% in the control group ( P < .001). Regarding SCCs, ruptured and absent BMs were, respectively, noted in 36.36% and 63.64% of BCCs with skull base invasion, compared with 30% and 30% in the control group ( P = .075). In this study, destruction of BM was significantly more common in BCCs with skull base invasion, in comparison with those with good outcome. In SCC, this difference was not statistically significant.

Fibroblasts play a regulatory role in the control of pigmentation in reconstructed human skin from skin types I and II.

Human melanocytes in monolayer culture are extremely dependent on a wide range of soluble signals for their proliferation and melanogenesis. The advent of three-dimensional models of reconstructed skin allows one to ask questions of how these cells are regulated within a setting which more closely approximates normal skin. The purpose of this study was to investigate to what extent melanocytes within a reconstructed skin model are sensitive to regulation by dermal fibroblasts, basement membrane (BM) proteins and the addition of alpha-melanocyte-stimulating hormone (alpha-MSH). Sterilized acellular de-epidermized dermis (prepared to retain BM proteins or deliberately denuded of BM by enzymatic treatment) from skin type I or II was reconstituted with fibroblasts, melanocytes and keratinocytes. In all but one case (9/10), cell donors were skin type I or II. The presence of BM antigens was found to be necessary for positional orientation of the melanocytes; in the absence of BM, melanocytes moved into the upper keratinocyte layer pigmenting spontaneously. Addition of fibroblasts suppressed the extent of spontaneous pigmentation of melanocytes within this model. Neither alpha-MSH nor cholera toxin induced pigmentation in this model despite the fact that melanocytes clearly had the ability to synthesize pigment.

Basement membrane pathology: a feature of acute equine laminitis.

Thirty-two dorsal, mid-hoof wall, lamellar sections from 8 Standardbred horses, humanely killed 48 h after the administration of an alimentary carbohydrate overload, were sectioned and examined by light microscopy. Sections were stained with the connective tissue and basement membrane stains periodic acid-Schiff (PAS), Azan and periodic acid silver methanamine (PASM) and with routine haematoxylin and eosin (H&E). Lesions of the epidermal lamellae, attributable to laminitis, were graded in order of increasing severity from Grade N (normal), Grade 1 (mild), Grade 2 (moderate) to Grade 3 (severe and extensive). The grading system was based principally on changes to lamellar basement membrane (BM) which were clearly visible when the connective tissue stains PAS and PASM were used. Earliest changes were rounding of the basal cell nuclei and elongation of secondary epidermal lamellae (SELs). Secondary epidermal lamellae tips were pointed instead of round and the basement membrane had separated from the lamellae. In early Grade 1 lesions, this was obvious at the tips of the SELs where the BM had lifted to form teat-shaped bubbles. The absence of BM at the tips of secondary dermal lamellae, along with varying amounts of connective tissue, was considered a progression in severity and classified as Grade 2. Eventually, even the primary epidermal and primary dermal lamellae separated from each other and the empty shells of isolated BM, in what was once the tip of the primary epidermal lamella, signified that a global separation of the epidermal and dermal lamellae had occurred (Grade 3 lesion). The histopathological grading system correlated well with the degree of lameness at the time of euthanasia, (r2 = 0.94) and apparently described the severity of laminitis accurately. Disintegration of the BM and failure of its attachment to the basal cells of the epidermis appears to be one of the earliest pathological events to occur in acute laminitis and could be the change that initiates the collapse of the lamellar architecture. Histopathological diagnoses of laminitis are strengthened when based on sections stained with at least PAS, in addition to routine H&E and should exhibit evidence of the BM pathology described here.

Evidence that basement membrane prevents apoptosis of Sertoli cells in vitro in the absence of known regulators of Sertoli cell function.

Although the role of basement membrane in the morphological and functional differentiation of Sertoli cells has been well characterized, very little is known about its involvement in Sertoli cell survival and maintenance throughout life. When cultured on laminin or Matrigel, 80-90% of Sertoli cells retained their viability. Sertoli cells prevented from attachment and basement membrane deposition by plating on plastic surfaces coated with polyhydroxyethylmethacrylate (poly-HEMA) exhibited a loss of viability by approximately 50% within 24 h. Addition of soluble laminin did not prevent the loss of viability of Sertoli cells, whereas soluble Matrigel enhanced the survival significantly when added at a concentration of 100 micrograms/ml or more. The addition of FSH, epidermal growth factor, testosterone, retinoic acid, or a mixture of insulin, transferrin, and selenium had no significant effect on the viability of Sertoli cells cultured on polyHEMA for up to 72 h. When all of these hormones and factors were added together, a significantly higher percentage of cell survival was observed at 24, 48, and 72 h, but the percent survival was significantly lower than that seen on either laminin or Matrigel. The nature of cell death occurring in the Sertoli cells plated on polyHEMA was determined by agarose gel analysis that revealed a ladder of approximately 200-base pair DNA multiple fragments. Flow cytometric analysis of propidium iodide-stained cells indicated that most of the cells were apoptotic. Freshly isolated Sertoli cells and adherent cells on basement membrane did not show internucleosomal DNA breakdown or an apoptotic peak in the flow cytometric analysis. These results suggest that basement membrane plays a crucial role in Sertoli cell survival in vitro when it is used as a solid substratum for culture, and in the absence of basement membrane, FSH and other regulators of Sertoli cell function cannot prevent Sertoli cell apoptosis.

Role of basal lamina in Schwann cell glycolipid biosynthesis.

Schwann cells that are deprived of axonal contact switch their glycolipid metabolic pathway from primarily galactocerebroside (GalCe) synthesis to the formation of glucocerebroside (GlcCe) and its homologs. The removal of axonal influence has a dual effect on Schwann cell phenotype; they lose the ability to assemble both myelin and basement membrane. To determine whether a loss of basement membrane directly affects glycolipid expression, we have examined lipid biosynthesis in Schwann cells which were allowed to interact with axons of dorsal root ganglion neurons but which were deprived of the ability to assemble basal lamina. These Schwann cells resemble those from myelinating nerve in that they synthesize a large amount of galactohydroxycerebroside. This suggests that axon contact, even in the absence of basement membrane, is sufficient to induce the GalCe metabolic pathway.

Extracellular matrix regulates expression of the TGF-beta 1 gene.

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell proliferation and modulates the interactions of cells with their extracellular matrix (ECM), in part by inducing the synthesis of various ECM proteins. Three different isoforms of TGF-beta are synthesized in a defined pattern in specific cell populations in vivo. In the specific case of TGF-beta 1, this well-defined and limited expression stands in sharp contrast to its synthesis by virtually all cells in culture. Using mammary epithelial cells as a model system, we evaluated the substratum dependence of the expression of TGF-beta 1. The level of TGF-beta 1 expression is high in cells on plastic, but is strongly downregulated when cells are cultured on a reconstituted basement membrane matrix. In contrast, TGF-beta 2 mRNA levels in cells on either substratum remain unchanged. Using the chloramphenicol acetyl transferase gene as reporter gene under the control of the TGF-beta 1 promoter, we show that transcription from this promoter is suppressed when the cells are in contact with either endogenously synthesized or exogenously administered basement membrane. TGF-beta 1 promoter activity is strongly induced by the absence of basement membrane, i.e., by direct contact of the cells with plastic. This modulation of transcription from the TGF-beta 1 promoter occurs in the absence of lactogenic hormones which allow full differentiation. Our results thus indicate that basement membrane is an important regulator of TGF-beta 1 synthesis, and explain why most cells in culture on plastic express TGF-beta 1 in contrast with the more restricted TGF-beta 1 synthesis in vivo. We propose that there is a feedback loop whereby TGF-beta 1-induced synthesis of basement membrane components is repressed once a functional basement membrane is present. Finally, these results together with our current knowledge of regulation of TGF-beta 1 and TGF-beta 2 synthesis, suggest that, in vivo, TGF-beta 1 may play a major role in regulating the ECM synthesis and the cell-ECM interactions, whereas TGF-beta 2 may be more important in morphogenetic processes.

Proliferating Oral Epithelial Cells in Culture are Capable of Both Extracellular and Intracellular Degradation of Interstitial Collagen

The potential of epithelial cells to degrade interstitial collagen was studied by culturing human masticatory mucosa on decalcified dentin matrix. Morphological changes were observed in the underlying collagen substratum and in the connective tissue of the explant. Degradation of the substratum was initiated two days after the first contact with epithelial cells exhibiting basal cell markers. Electron microscopic studies confirmed extensive collagen degradation in the vicinity of these cells. No collagen degradation was observed underneath the connective tissue portion of the explant. Experiments in which the explant was partially separated from the underlying substratum by a filter further showed that connective tissue was apparently not involved in the collagen degradation by the epithelial cells. Lysis of connective tissue of the explant was observed in association with epithelial cells that showed a disrupted basal lamina and release of vesicular material from the exposed cell membrane. Collagen fibers were visible inside some epithelial cells suggesting intracellular collagenolysis. Primary cultures of human gingival epithelial cells and porcine periodontal ligament epithelial cells (epithelial cell rests of Malassez) that expressed similar basal cell cytokeratins as the active cells of the mucosal explants secreted collagenase, gelatinase and TIMP to the culture medium. They also contained acid collagenolytic proteinases. When cultured on a porous polycarbonate membrane the epithelial cells secreted collagenolytic enzymes from the pores at cell membrane sites lacking basal lamina. These results provide evidence that proliferating basal epithelial cells have a strong capacity for collagen degradation. It seems that the absence of basement membrane is the signal for these cells to secrete matrix degrading enzymes.

Distribution of basement membrane in squamous cell carcinoma of the head and neck

The immunohistologic distribution of the basement membrane components, type IV collagen and laminin, was evaluated in 57 alcohol-fixed, paraffin-embedded tissue sections which demonstrated immunostaining patterns with antigen preservation similar to that of frozen tissue sections. Normal and hyperplastic squamous mucosa, a spectrum of intraepithelial neoplasia, and invasive squamous cell carcinomas of the upper aerodigestive tract were evaluated by this technique. Prominent and continuous basement membrane staining characterized normal and reactive hyperplastic squamous mucosa. The basement membrane varied greatly with epithelial dysplasia and was usually prominent and continuous in mild to moderate dysplasias. In severe dysplasia/carcinoma in situ, the basement membrane was often thinned and occasionally discontinuous. The distribution of basement membrane in invasive carcinomas was also varied. Basement membrane was usually present in invasive tumors with well-defined tumor host borders and cohesive patterns of stromal invasion which were interpreted as foci of histologic differentiation. In contrast, invasive carcinomas with irregular cords or single tumor cells distributed through the host stroma invariably lacked basement membrane at the tumor-stromal interface; this was interpreted as a decreased expression of histologic differentiation. We conclude that 1) severe intraepithelial neoplasia is often associated with irregularities of basement membrane and that the absence of basement membrane does not necessarily define invasive cancer; 2) immunolocalization of basement membrane in invasive carcinomas is common in areas displaying histologic differentiation; and 3) the association of basement membrane distribution and histologic pattern of tumor invasion suggests that squamous cell carcinomas are capable of undergoing focal histologic differentiation after invasion has occurred.

BEHAVIOUR OF BASEMENT MEMBRANE ANTIGENS IN GASTRIC AND COLORECTAL CANCER: Immunohistochemical Study

Eighty gastric and colorectal lesions including cases of dysplasia and carcinoma were studied by immunohistochemical techniques to investigate the behaviour of laminin, type IV collagen and fibronectin. Their distribution was examined on frozen and formalin-fixed, paraffin-embedded samples. In the same lesions, interruption, fragmentation and absence of basement membrane (BM) antigen-staining were observed. Carcinomas with well differentiated glandular structures were always surrounded by a well defined BM as in normal, non-pathological tissues. On the contrary, undifferentiated areas arranged in nests or sheets were usually negative for laminin and type IV collagen. Anomalous BM staining was strictly related to the degree of differentiation of tumor tissue, while no correlation existed between carcinoma staging and BM antigen presence, either in gastric or colorectal neoplasms. Immunostaining with antibody against type IV collagenase showed a massive positivity in the early gastric carcinomas examined, while in colorectal cancer only granulocytes showed it.

Basement membrane Laminin and Type IV Collagen in Various Benign and Malignant Adnexal Tumors of the Skin: An Immunohistochemical Study

Thirty benign and seven malignant adnexal tumors of the skin and one lymph node metastasis were stained for laminin and type IV collagen with rabbit antibodies against the human basement membrane (BM) proteins using the immunoperoxidase technique. Fifteen of the benign sweat gland, sebaceous gland, and hair follicle tumors showed a continuous and distinct BM around the tumor aggregates. The cylindromas and eccrine spiradenomas seemed to produce excessive amounts of BM material, part of which was seen as amorphic patches within the tumor cell clusters, whereas the trichofolliculomas, trichoepitheliomas, and pilomatrixomas showed an absence of BM from many areas. In syringomas, in addition to the tubular structures surrounded by a continuous BM, undifferentiated cell nests containing granular BM material were present. They probably represent primitive structures obtaining during early development into tubules. The seven malignant tumors and the only metastasis studied here all contained small, narrow strips of BM material extracellularly between the infiltrating tumor clusters. Only in two cases was faint staining for laminin found within the cells. The pepsin pretreatment of the formalin-fixed, paraffin-embedded samples had most probably degraded the intracytoplasmic BM material in most cases. The BM defects were found to be associated with malignancy and low differentiation of the adnexal skin tumors, as reported previously for other tumor types, but a partial loss of BM was also associated with high differentiation in some benign adnexal tumors.

Studies on cell migration and axon guidance in the developing distal auditory system of the mouse.

The events that take place along the potential route of distal auditory axons (future vestibular component) prior to and during their outgrowth were examined morphologically using timed mouse embryos. During embryonic (E) day 9.5 a discrete zone of cell death appears in the rostrolateral wall of the otic cup. Necrosis is accompanied by outward migration of epitheloid cells from the same region of the otic wall. Temporally and spatially correlated with these two events is the widening of extracellular spaces between otic neuroepithelial cells and the breakdown of basement membrane. During E 10.5 migrating epitheloid cells condense to form a funnel-shaped configuration. This cellular "funnel" begins narrowly at the dorsorostrolateral wall of the otocyst and broadens as it reaches the auditory ganglion. During E 11.5 through E 12.5, "pioneer" distal auditory axons take a circuitous route and ascend from the auditory ganglion to enter the otocyst. Axons extend toward the otocyst moving along cells of the "funnel," maintaining an orientation similar to that of the cells that compose it. Axon growth cones enter the otocyst at sites devoid of basement membrane and invade the wall of the otocyst moving tangentially along radially arranged cells that bridge the otocyst and the "funnel." These observations demonstrate that a preformed, funnel-shaped tissue exists along the future route of the auditory fibers. We suggest that the "funnel" may influence the growth and directionality of distal auditory axons as they extend from the auditory ganglion to the wall of the otocyst. At the otic wall, the transition provided by "bridge" epitheloid cells, together with the absence of basement membrane at specific sites of the otic wall, provide the auditory axons with a route into the otocyst.