Extracellular matrix remodeling in oral submucous fibrosis: its stage-specific modes revealed by immunohistochemistry and in situ hybridization

BACKGROUND: Oral submucous fibrosis (OSF) is a chewing habit-related pre-cancerous condition of the oral mucosa affecting predominantly south Asians. It is histopathologically characterized by epithelial atrophy and fibrosis of the subepithelial connective tissue. Fibrosis extends all the way into the muscle layer, leading to difficulty in mouth opening. However, the dynamics of extracellular matrix (ECM) remodeling with OSF progression is largely unknown. METHODS: Forty biopsy specimens of OSF and 10 of normal buccal mucosa were examined for expression/deposition modes of eight ECM molecules by histochemistry, immunohistochemistry, and in situ hybridization. RESULTS: In the early stage of OSF, tenascin, perlecan, fibronectin, collagen type III were characteristically enhanced in the lamina propria and the submucosal layer. In the intermediate stage, the ECM molecules mentioned above and elastin were extensively and irregularly deposited around muscle fibers. In the advanced stage, such ECM depositions decreased and were entirely replaced with collagen type I only. Their gene expression levels varied with progression of fibrosis, but the mRNA signals were confirmed in fibroblasts in the submucosal fibrotic areas. CONCLUSIONS: The results indicate that the ECM remodeling steps in OSF are similar to each phase of usual granulation tissue formation. Restricted mouth opening may be a result of loss of variety of ECM molecules including elastin into the homogeneity of collagen type I replacing muscle fibers.     

Perlecan Domain V Therapy for Stroke: A Beacon of Hope?

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Differential expression of perlecan receptors, α‐dystroglycan and integrin β1, before and after invasion of oral squamous cell carcinoma

J Oral Pathol Med (2011) 40 : 552–559 Objectives: The deposition of perlecan, a heparan sulfate proteoglycan, is enhanced within oral carcinoma in situ (CIS) foci, while it dynamically switches from CIS foci to the stromal space in squamous cell carcinoma (SCC). Because α‐dystroglycan and integrin β1 have been identified as two of the perlecan receptors, we wanted to determine their differential distributions before and after invasion of oral SCC. Methods: Eighty‐two surgical tissue specimens of oral SCC containing different precancerous stages were examined by immunohistochemistry for perlecan, α‐dystroglycan, integrin β1, and Ki‐67. In addition, α‐dystroglycan mRNA signals were localized by in situ hybridization. Results: In normal epithelia, α‐dystroglycan and integrin β1 were localized on the cell membrane of basal cells, while perlecan was faintly present in the intercellular spaces of parabasal cells. In epithelial dysplasia and CIS, α‐dystroglycan and perlecan were well co‐localized in the epithelial layer, especially in its lower half, and this co‐localization was mostly overlapped with Ki‐67‐positive (+) cell zones. However, in SCC, α‐dystroglycan was localized neither within carcinoma cell nests nor in the stroma, while perlecan disappeared from SCC foci but emerged in the stromal space, leaving integrin β1+ and Ki‐67+ cells only to the periphery of SCC foci. α‐Dystroglycan mRNA signals were basically identical to the α‐dystroglycan protein localizations. Conclusion: The findings suggest that α‐dystroglycan and integrin β1 act as perlecan receptors in oral precancerous lesions prior to invasion, and that the perlecan signals via the two different receptors function in cellular differentiation and proliferation of CIS cells, respectively.     

Perlecan-rich epithelial linings as a background of proliferative potentials of keratocystic odontogenic tumor

Background:  The intraepithelial deposit of perlecan, a basement membrane-type heparan sulfate (HS) proteoglycan, has been demonstrated in neoplastic conditions such as salivary gland tumors, odontogenic tumors, and oral carcinoma in situ . Our aim was to determine whether perlecan turnover was enhanced in the lining cells of keratocystic odontogenic tumor (KCOT), which had been recently renamed from odontogenic keratocyst because of its accumulated evidence of neoplasm, as a possible background for neoplastic proliferation.
Methods:  Ten surgical specimens from each of KCOT, dentigerous cyst, and radicular cyst were examined for the expressions of perlecan core protein, HS chains, heparanase, and Ki-67 by immunohistochemistry and in situ hybridization.
Results:  In KCOT, perlecan core protein and HS chains were localized on the cell border from the parabasal to subkeratinized layers of the lining epithelium. Heparanase was localized in a similar fashion to those for perlecan and HS chains but was within the cytoplasm. mRNA signals for perlecan core protein and heparanase were mostly compatible with their protein signals. Ki-67-positive cells were localized mainly in the second basal cell layers with definitely higher labeling indices (approximately 31.3%, second layer). In contrast to KCOT, dentigerous cysts and radicular cysts had no perlecan, HS chains, and heparanase deposition in their linings with extremely lower Ki-67 indices (0.4–0.8%).
Conclusion:  The result suggests that the characteristic intra-lining-epithelial deposit of perlecan in KCOT, which has never been seen in other cystic jaw lesions, is a new evidence supporting the neoplastic nature of KCOT.     

Maturational changes in laminin, fibronectin, collagen IV, and perlecan in germinal matrix, cortex, and white matter and effect of betamethasone

Germinal matrix is selectively vulnerable to hemorrhage in premature infants, and use of prenatal betamethasone is associated with a lower occurrence of germinal matrix hemorrhage. Because the major components of extracellular matrix of the cerebral vasculature-laminin, fibronectin, collagen IV, and perlecan-provide structural stability to blood vessels, we examined whether the expression of these molecules was decreased in the germinal matrix and affected by betamethasone. In both human fetuses and premature infants, fibronectin was significantly lower in the germinal matrix than in the cortical mantle or white matter anlagen. Conversely, laminin alpha1 gene expression was greater in the human germinal matrix compared with the cortical mantle or white matter. Expression of alpha1- and alpha2(IV) collagen chains increased with advancing gestational age. Low-dose prenatal betamethasone treatment enhanced fibronectin level by 1.5-2-fold whereas a high dose reduced fibronectin expression by 2-fold in rabbit pups. Because fibronectin provides structural stability to the blood vessels, its reduced expression in the germinal matrix may contribute to the fragility of germinal matrix vasculature and the propensity to hemorrhage in premature neonates.     

Electrophysiological studies in a mouse model of Schwartz–Jampel syndrome demonstrate muscle fiber hyperactivity of peripheral nerve origin

Schwartz–Jampel syndrome (SJS) is an autosomal‐recessive condition characterized by muscle stiffness and chondrodysplasia. It is due to loss‐of‐function hypomorphic mutations in the HSPG2 gene that encodes for perlecan, a proteoglycan secreted into the basement membrane. The origin of muscle stiffness in SJS is debated. To resolve this issue, we performed an electrophysiological investigation of an SJS mouse model with a missense mutation in the HSPG2 gene. Compound muscle action potential amplitudes, distal motor latencies, repetitive nerve stimulation tests, and sensory nerve conduction velocities of SJS mice were normal. On electromyography (EMG), neuromyotonic discharges, that is, bursts of motor unit action potentials firing at high rates (120–300 HZ ), were constantly observed in SJS mice in all muscles, except in the diaphragm. Neuromyotonic discharges were not influenced by general anesthesia and disappeared with curare administration. They persisted after complete motor nerve section, terminating only with Wallerian degeneration. These results demonstrate that perlecan deficiency in SJS provokes a neuromyotonic syndrome. The findings further suggest a distal axonal localization of the generator of neuromyotonic discharges. SJS should now be considered as an inherited disorder with peripheral nerve hyperexcitability. Muscle Nerve, 2009     

Extracellular matrix protein expression in cerebrospinal fluid from patients with tropical spastic paraparesis associated with HTLV‐I and Creutzfeldt‐Jakob disease

The cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the CNS, thus biochemical processes in the CNS could potentially be reflected in the CSF. Changes in extracellular matrix (ECM) proteins can be studied through their analysis in the CSF. ECM plays an essential role in CNS homeostasis and several proteins such as laminin (LN), fibronectin (FN), thrombospondin (TS) and heparan sulphate proteoglycan (HS, perlecan) form part of its structure. Possible changes in the levels of these proteins were investigated in two different pathologies —tropical spastic paraparesis/HTLV‐I‐associated myelopathy (TSP/HAM) (n=25) and Creutzfeldt‐Jakob disease (CJD) (n=19)—and compared with those in a control group with or without neurological disease (n=25). CSF analyses were carried out using monoclonal or monospecific polyclonal antibodies. In comparison with the control group, it was found that TSP/HAM patients presented significantly higher levels of LN, TS and HS, while in CJD patients the levels of FN, TS and HS were increased. In CJD patients the HS level was almost double that of the TSP/HAM patients. These results suggest a distinct pattern of ECM proteins in CSF in relation to the type of neurological disease. TSP/HAM is a chronic motor disease that affects the white matter of the spinal cord, while CJD is a subacute dementia that affects cerebral neurons and their synapsis.     

Heparan sulphate proteoglycan expression in human primary liver tumours

Heparan sulphate proteoglycans (HSPGs) play important biological roles in cell–matrix adhesion processes and are essential regulators of growth factor actions (e.g., as co-receptor for hepatocyte growth factor). Since in liver carcinogenesis, interactions between cells, the matrix, and growth factors play a major role, the aim of this study was to investigate whether the distribution pattern of HSPGs is altered in human primary liver tumours. Twenty-two primary liver tumours and five normal liver biopsies were studied, using specific monoclonal antibodies against syndecans-1, -2, -3, and -4; glypican; perlecan; and heparan sulphate chains. Cholangiocarcinomas as well as hepatocellular carcinomas showed an altered immunoreactivity pattern of the different HSPGs in comparison with normal liver parenchyma, probably reflecting the growth regulatory roles of HSPGs. Intracellular positivity for integral membrane HSPGs syndecan-1 and especially syndecan-4 was a constant finding in most tumours, suggesting increased synthesis or internalization of these HSPGs. Syndecan-3 and perlecan expression in tumours was found in an expected distribution pattern. The strong reactivity for syndecan-3 and perlecan in tumoral stromal vessels might suggest a role for these HSPGs in tumoral angiogenesis. In addition, perlecan probably exerts its known growth factor reservoir function also in the stroma of primary liver tumours. © 1998 John Wiley & Sons, Ltd.