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Laminar restriction of retinal macrophagic response to optic nerve axotomy in the rat.

Following optic nerve transection, most of the retinal ganglion cells die. Their debris is promptly cleared by phagocytic cells. It is currently not known to what extent peripherally derived macrophages contribute to this activity. Using antibodies OX42 and ED-1, phagocytic cells were labeled in the retinas of optic nerve lesioned adult rats. To distinguish whether the cells were reactive microglial or macrophagic in origin, blood-borne monocytes were labeled with fluorescent microspheres while in the systemic circulation. Macrophages invaded the retina, but only in the nerve fiber layer, sparing the ganglion cell and other layers. These macrophages engulfed only the axonal debris from dying ganglion cells, not their degenerating cell bodies. These results indicate that although peripherally derived monocytic cells are recruited into the retrogradely degenerating retina, their role in clearing debris is limited to the optic fiber layer.

Animals↗

Expression of emmprin by oral squamous cell carcinoma.

A transmembrane glycoprotein recently identified on some tumor cells, extracellular matrix metalloproteinase inducer (EMMPRIN), has been shown to induce metalloproteinase (MMP) production by peritumor fibroblasts (PTF). We examined biopsy specimens of normal human oral mucosa and oral squamous cell carcinoma (SCC) for expression of EMMPRIN. In normal mucosa, EMMPRIN was expressed at the cell membrane throughout the epithelium with a slight enhancement along the basal cell layer. In oral SCC, EMMPRIN was expressed at the cell membrane throughout the entire lesion. Immunofluorescence microscopy localized EMMPRIN to the cell membrane in a highly invasive oral SCC cell line in agreement with our in vivo observations. Function-blocking antibodies to EMMPRIN significantly inhibited oral SCC cell migration on tenascin-C (TN-C) and fibronectin as well as invasion through a reconstituted basement membrane (RBM). We previously showed that soluble factors from SCC cells and PTF are required for deposition of a TN-C matrix. To determine whether EMMPRIN may modulate the release or expression of these soluble factors, we again used function-blocking antibodies. Antibodies to EMMPRIN completely inhibited the organization of TN-C matrices and partially reduced the deposition of FN matrices by oral SCC cell /PTF co-cultures. In addition, antibodies to EMMPRIN perturbed the expression of MMP-2. Moreover, antibodies to MMP-2 perturbed oral SCC cell invasion of an RBM by approx. 75%. Our results demonstrate that EMMPRIN is highly expressed in oral SCC, facilitates tumor cell motility, and mediates TN-C matrix deposition. Taken together, these results suggest that EMMPRIN may help regulate oral squamous cell carcinoma invasion.

Antigens, CD↗

CD147.

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Amino Acid Sequence↗

Regulation of MMP-1 and MMP-2 production through CD147/extracellular matrix metalloproteinase inducer interactions.

Extracellular matrix metalloproteinase inducer (EMMPRIN; CD147) is a heavily glycosylated protein containing two immunoglobulin superfamily domains. It is enriched on the surface of tumor cells and stimulates the production of matrix metalloproteinases (MMPs) by adjacent stromal cells. Here we use CD147 transfectants and immobilized recombinant CD147-Fc fusion protein to show that CD147/FMMPRIN engages in a homophilic interaction, predominantly through the first immunoglobulin domain. Anti-CD147 antibody 8G6 and recombinant CD147-Fc fusion protein markedly inhibited not only homophilic interaction, but also the production of secreted MMP-2 by breast cancer cell line MDA-435 and the MMP-2-dependent invasion of MDA-435 cells through reconstituted basement-membrane Matrigel. Purified native CD147 induced the production of secreted MMP not only by dermal fibroblasts (MMP-1) but also by MDA-435 cells themselves (MMP-2), suggesting homophilic CD147-binding may occur in the context of both heterotypic and homotypic cell-cell interactions. Purified deglycosylated CD147 failed to induce MMP-1 or MMP-2, but instead antagonized the MMP-1-inducing activity of purified native CD147. Our results suggest that homophilic CD147 interactions may play a key role in MMP-2 production and tumor cell invasion, and that perturbation of this molecule may have potential therapeutic uses in the prevention of MMP-2 and MMP-1-dependent cancer metastasis.

Animals↗

Status epilepticus causes necrotic damage in the mediodorsal nucleus of the thalamus in immature rats.

Status epilepticus (StE) in immature rats causes long-term functional impairment. Whether this is associated with structural alterations remains controversial. The present study was designed to test the hypothesis that StE at an early age results in neuronal loss. StE was induced with lithium-pilocarpine in 12-d-old rats, and the presence of neuronal damage was investigated in the brain from 12 hr up to 1 week later using silver and Fluoro-Jade B staining techniques. Analysis of the sections indicated consistent neuronal damage in the central and lateral segments of the mediodorsal nucleus of the thalamus, which was confirmed using adjacent cresyl violet-stained preparations. The mechanism of thalamic damage (necrosis vs apoptosis) was investigated further using TUNEL, immunohistochemistry for caspase-3 and cytochrome c, and electron microscopy. Activated microglia were detected using OX-42 immunohistochemistry. The presence of silver and Fluoro-Jade B-positive degenerating neurons in the mediodorsal thalamic nucleus was associated with the appearance of OX-42-immunopositive activated microglia but not with the expression of markers of programmed cell death, caspase-3, or cytochrome c. Electron microscopy revealed necrosis of the ultrastructure of damaged neurons, providing further evidence that the mechanism of StE-induced damage in the mediodorsal thalamic nucleus at postnatal day 12 is necrosis rather than apoptosis. Finally, these data together with previously described functions of the medial and lateral segments of the mediodorsal thalamic nucleus suggest that some functions, such as adaptation to novelty, might become compromised after StE early in development.

Animals↗

Influence of beta1 integrins on epidermal squamous cell carcinoma formation in a transgenic mouse model: alpha3beta1, but not alpha2beta1, suppresses malignant conversion.

Although aberrant integrin expression has been documented in many epithelial tumors, little is known about how integrins influence neoplastic progression. To examine this issue, transgenic mice in which the alpha2beta1 or alpha3beta1 integrin was expressed in the suprabasal epidermal layers via the involucrin promoter were subjected to skin carcinogenesis. Equal numbers of benign squamous papillomas were observed in transgenic and wild-type animals. However, the frequency of conversion of papillomas to malignant squamous cell carcinomas was much lower in alpha3beta1 transgenic than in alpha2beta1 transgenic and wild-type mice. No differences were observed in apoptosis or in the expression of endogenous integrins in transgenic and wild-type papillomas. However, alpha3beta1 transgenic papillomas displayed a diminished proliferative capacity and were more highly differentiated as judged by BrdUrd incorporation and keratin 10 expression, respectively, than alpha2beta1 transgenic and wild-type papillomas. Two proteins that associate with alpha3beta1 and not alpha2beta1 are extracellular matrix metalloproteinase inducer and CD81. Extracellular matrix metalloproteinase inducer expression correlated inversely with the degree of differentiation in normal epidermis and in transgenic and wild-type papillomas. Up-regulation of CD81 was observed in 100% of wild-type and 88% of alpha2beta1 transgenic papillomas but in only 25% of alpha3beta1 transgenic papillomas. CD81 was undetectable in untreated epidermis and strongly expressed in all transgenic and wild-type squamous cell carcinomas. Our results demonstrate that the alpha3beta1 integrin can suppress malignant conversion, and that the mechanism may involve CD81.

9,10-Dimethyl-1,2-benzanthracene↗

Expression of serine protease inhibitor 3 in ocular tissues in endotoxin-induced uveitis in rat.

PURPOSE: To ascribe the serine protease inhibitor 3 (SPI-3) as an ocular acute inflammatory molecule and to clarify its producing cells in an endotoxin-induced uveitis (EIU) model. METHODS: Male Wistar rats were injected with lipopolysaccharide (LPS), and the expression of SPI-3 mRNA in the ocular tissues was examined by in situ hybridization (ISH) and Northern blot analysis. A combination of ISH and immunohistochemistry (IHC) were performed to prove the colocalization of SPI-3 mRNA and either glial fibrillary acidic protein (GFAP) or OX-42. The expression of phosphorylated STAT3 (pSTAT3) was demonstrated by IHC and Western blot after LPS injection. The colocalization of SPI-3 mRNA and pSTAT3 was finally examined by the double labeling of ISH and IHC. RESULTS: After LPS injection, the expression of SPI-3 mRNA in ocular tissues was quickly upregulated and reached a peak between 12 and 24 hours after injection. An intense mRNA signal was observed in epithelial cells of the iris and ciliary body and the innermost retinal layer. In the retina, SPI-3 mRNA was colocalized with GFAP, demonstrating that the cells expressing SPI-3 mRNA were astrocytes. After LPS treatment, SPI-3 mRNA and pSTAT3 were colocalized in retinal astrocytes, and pSTAT3 expression appeared slightly earlier than that of SPI-3 mRNA. CONCLUSIONS: Ocular inflammation induced the transient expression of SPI-3 mRNA in retinal astrocytes and epithelial cells in the iris and ciliary body, particularly during early phase of the inflammation. Simultaneously, the activation of STAT3 (phosphorylation of STAT3) occurred slightly earlier in astrocytes. This supports the previous in vitro results that SPI-3 expression is induced in a STAT3-mediated manner. SPI-3 may have some crucial roles in preventing some degenerative proteolysis, which is induced by inflammatory stimuli.

Acute-Phase Proteins↗

Nerve injury proximal or distal to the DRG induces similar spinal glial activation and selective cytokine expression but differential behavioral responses to pharmacologic treatment.

The specific mechanisms by which nervous system injury becomes a chronic pain state remain undetermined. Historically, it has been believed that injuries proximal or distal to the dorsal root ganglion (DRG) produce distinct pathologies that manifest in different severity of symptoms. This study investigated the role of injury site relative to the DRG in (1) eliciting behavioral responses, (2) inducing spinal neuroimmune activation, and (3) responding to pharmacologic interventions. Rats received either an L5 spinal nerve transection distal to the DRG or an L5 nerve root injury proximal to the DRG. Comparative studies assessed behavioral nociceptive responses, spinal cytokine mRNA and protein expression, and glial activation after injury. In separate studies, intrathecal pharmacologic interventions by using selective cytokine antagonists (interleukin-1 [IL-1] receptor antagonist and soluble tumor necrosis factor [TNF] receptor) and a global immunosuppressant (leflunomide) were performed to determine their relative effectiveness in these injury paradigms. Behavioral responses assessed by mechanical allodynia and thermal hyperalgesia were almost identical in the two models of persistent pain, suggesting that behavioral testing may not be a sensitive measure of injury. Spinal IL-1beta, IL-6, IL-10, and TNF mRNA and IL-6 protein were significantly elevated in both injuries. The overall magnitude of expression and temporal patterns were similar in both models of injury. The degree of microglial and astrocytic activation in the L5 spinal cord was also similar for both injuries. In contrast, the pharmacologic treatments were more effective in alleviating mechanical allodynia for peripheral nerve injury than nerve root injury, suggesting that nerve root injury elicits a more robust, centrally mediated response than peripheral nerve injury. Overall, these data implicate alternate nociceptive mechanisms in these anatomically different injuries that are not distinguished by behavioral testing or the neuroimmune markers used in this study.

Animals↗

[Monitoring gene expression profile changes in endometrial cancer using cDNA microarray technology].

OBJECTIVE: To study the difference in gene expression between normal endometrial tissue and endometrial cancer tissue so as to investigate the mechanism of pathogenesis of endometrial cancer. METHODS: The tissues from 30 patients with endometrial cancer and normal endometrial tissues from the same patients were analyzed by cDNA microarray technology (including 588 genes). General RNA was extracted from these tissues and probes were made to be hybridized with cDNA array membrane. The CD147 expressions in normal endometrial tissue and in endometrial cancer tissue were identified by immunohistochemistry. RESULTS: The endometrial cancer associated genes - IRP, Wnt-5A, 5T4 oncofetal antigen precursor, aggrecan core protein precursor, CD147, TIMP-2, were expressed highly. CONCLUSION: cDNA microarray technology can simultaneously monitor the different expressions of genes from two different tissues. CD147 might be useful in assessing the progression and infiltration of endometrial cancer.

Antigens, CD↗

Osteopontin in kainic acid-induced microglial reactions in the rat brain.

The present study was performed to investigate the spatial and temporal expression of osteopontin (OPN) mRNA in the rat brain after kainic acid-induced seizures, and to determine whether this phenomenon is associated spatiotemporally with the microglial reaction. The expression of OPN mRNA was detected using an in situ hybridization technique and Northern blot analysis. Following intraperitoneal injection of kainic acid (10 mg/kg), OPN mRNA was expressed in selective vulnerable areas, including the hippocampus, thalamus, hypothalamus, amygdala, and entorhinal cortex. Comparison of the morphology and localization with the established microglial marker OX-42 in the adjacent sections positively identified the OPN-expressed cells as microglia. Furthermore, double labeling experiments revealed that OPN mRNA expression was confined to ameboid-like cells among microglia stained with GSI-B4, an another microglial marker. These findings from a rat model of seizure support the notion that OPN can be synthesized in a subpopulation of reactive microglial cells. It can therefore be assumed that in the response of the brain to excitotoxic injury, synthesis of OPN occurs generally in a subset of activated microglia.

Animals↗

EMMPRIN (CD 147) is expressed in Hodgkin's lymphoma and anaplastic large cell lymphoma. An immunohistochemical study of 60 cases.

BACKGROUND: Matrix metalloproteinases are involved in tumor invasion and metastatic spread. Expression of metalloproteinases is induced by the extracellular matrix metalloproteinase inducer (EMMPRIN). Their activity can be inhibited by several tissue inhibitors of metalloproteinases (TIMP1-to-4). Whereas TIMP-1 expression is described for high-grade malignant lymphomas and seems to be correlated with unfavourable prognosis, there are no data on the expression of EMMPRIN in malignant lymphoma. MATERIALS AND METHODS: We investigated 60 cases of Hodgkin's lymphoma and anaplastic large cell lymphoma for TIMP-1 and EMMPRIN expression using immunohistochemistry. RESULTS: EMMPRIN was expressed in all but four cases. EMMPRIN and TIMP-1 were co-expressed in two-thirds of the Hodgkin's lymphomas and anaplastic large cell lymphomas. CONCLUSION: This is the first report on the expression of EMMPRIN in malignant lymphoma. We speculate that EMMPRIN and TIMP-1 may be important in the pathogenesis of anaplastic large cell lymphoma and Hodgkin's disease.

Antigens, CD↗

[Expression of matrix metalloproteinases in metastatic lesions of cutaneous squamous cell carcinoma].

OBJECTIVE: To investigate the expression of matrix metalloproteinases (MMPs) and its correlation with basigin/CD147 expression in metastatic lesions of cutaneous Sequamous cell carcinoma (SCC). METHODS: Expressions of MMPs and basigin/CD147 were examined by DAB and ALP immunohistochemical staining in 14 metastatic SSCs, and immunoblot analysis was used to observe the basigin/CD147 expression in metastatic samples. RESULTS: Normal skin and lymph nodes were basically negative for MMPs, but expressions of MMP-1, MMP-2, MMP-3 and MTl-MMP were increased in both tumor cells and stromal cells in metastatic SCC. Strongly positive or positive staining was observed in 4 or 8 of metastatic SCCs for basigin/CD147 in tumor cells respectively. Significant correlations were found between basigin/CD147 expression in tumor cells and MMP-1, MMP-2, MMP-3, MTl-MMP expression in surrounding stromal cells (p-values: 0.012, 0.024, 0.047 and 0.026, respectively). Immunoblot analysis revealed that basigin/CD147 expression increased in metastatic SCC. CONCLUSION: These results suggest that MMPs produced by both tumor cells and stromal cells play a role in metastasis of cutaneous SCC, and increased MMPs in stromal cells might be induced by basigin/CD147 expressed in tumor cells.

Antigens, CD↗

Overexpression of extracellular matrix metalloproteinase inducer in multidrug resistant cancer cells.

Multidrug resistant (MDR) cancer cells overexpressing P-glycoprotein (P-gp) display variations in invasive and metastatic behavior. We previously reported that these properties of MDR cancer cell lines overexpressing P-gp could be altered by chemotherapeutic drugs or MDR modulators (R. S. Kerbel et al., Cancer Surv., 7: 597-629, 1988). To attempt to clarify the mechanism(s) underlying these observations, we studied the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein enriched on the surface of tumor cells that can stimulate the production of matrix metalloproteinases (MMPs), in sensitive and MDR cancer cells. Using immunofluorescence staining and fluorescence-activated cell sorting analysis, we found that EMMPRIN expression was increased in MDR carcinoma cell lines, MCF-7/AdrR, KBV-1, and A2780Dx5, as compared to their parental counterparts. The MDR cell lines produced more matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9), as determined by zymography, Western blot, and reverse transcription-PCR. Treatment of MDR cells with an anti-EMMPRIN antibody inhibited the activity of MMP-1, MMP-2, and MMP-9. In MDR cell line MCF-7/AdrR, an increased in vitro invasive ability was observed as compared with the sensitive line MCF-7, and EMMPRIN antibody could inhibit the in vitro invasion in drug-resistant cells. In addition, the expression and activity of MMP-1, MMP-2, and MMP-9 in MDR cells were decreased by treatment with U-0126, an inhibitor of mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/Erk). Our results suggest that during the development of MDR, the expression of EMMPRIN is responsible for the increased activity of MMP in MDR cell lines.

Antigens, CD↗

[Bio-panning of antagonistic peptides against HAb18G/CD147 and their function of anti-hepatoma invasion].

OBJECTIVE: To screen out the HAb18G/CD147 binding peptides and find out an antagonist against hepatoma invasion. METHODS: HAb18G/CD147 was purified by affinity chromatographic method and the antigen binding peptides acquired by bio-panning a phage-displayed 12-peptide library. After obtaining the sequence of the selected phage-displayed peptides, all the 9 peptides were synthesized by solid-phase method and identified by mass spectrograph. The peptides' anti-metastatic function was tested by Boyden Chamber assay. RESULTS: The purified HAb18G/CD147, identified by Western blot (molecular weight about 65 kd) could be used to bio-pan the phage-displayed peptide library. After 3 rounds of bio-panning, 9 positive phage clones were selected and sequenced. The synthesized peptides had uneven inhibitory activities and three of them were able to markedly inhibit the hepatoma cell invasion (P < 0.01). The most effective peptide decreased by 90.1% of hepatoma cells migrating through the Boyden Chamber membrane as compared with the control. CONCLUSION: Bio-panning the phage-displayed peptide library can be used successfully to screen out the antigen binding peptides. Hepatoma metastatic potential can be inhibited by peptide antagonist which could be a good foundation of developing peptide therapeutic agent against hepatoma metastasis.

Animals↗

Matrix-metalloproteinases in Hodgkin lymphoma.

BACKGROUND: Classical Hodgkin lymphomas are characterized by relatively few tumour cells and prominent proliferation of plasma cells, histiocytes, lymphocytes and eosinophils. In addition there is a varying degree of sclerosis, which is especially prominent in nodular sclerosis. These morphological peculiarities led to the idea that the interaction between tumour cells and bystander cells as well as the extracellular matrix may be important in Hodgkin lymphomas. MATERIALS AND METHODS: Thirty-four classical Hodgkin lymphomas (CHL) were analysed regarding the expression of EMMPRIN, MMP-2, -7, -9, -10 and-11 using immunohistochemistry. RESULTS: The tumour cells were positive for EMMPRIN in 100% of the cases. In 82% of CHL the Hodgkin and Reed-Sternberg cells (HRS) were negative for MMP-2. In contrast the surrounding non-neoplastic cells were MMP-2-positive in 71% of the cases. The HRS cells stained positive for MMP-7 in 68% of CHL, whereas only a few surrounding cells were positive for this marker. In all but one case (97%) the HRS cells were negative for MMP-9. However, the surrounding cells stained positive in 32%, thus resembling the staining pattern for MMP-2. Only scattered cells of both populations, HRS cells as well as bystander cells, stained for MMP-10 and -11, and no specific staining pattern was observed. CONCLUSION: Our data indicate a complex interaction between tumour cells and bystander cells with regard to metalloproteinases. The expression of EMMPRIN in the tumour cells may induce the expression of MMP-2 in the surrounding non-neoplastic cells. MMP-2 can be activated by MMP-7, which is expressed in the tumour cells. It is tempting to speculate that an interruption of this cycle could be of therapeutic benefit.

Antigens, CD↗

[Expression of extracellular matrix metalloproteinase inducer in human bladder transitional cell carcinoma].

BACKGROUND & OBJECTIVE: Matrix metalloproteinases(MMPs) play critical roles in the process of tumorigenesis, tumor invasion and metastasis. Extracellular matrix metalloproteinase inducer (EMMPRIN) derived from cancer cell can induce stromal fibroblasts to synthesize MMPs, which advances the invasion and metastasis of cancer. This study was designed to investigate the roles of EMMPRIN in the tumorigenesis, invasion and metastasis of bladder carcinoma by detecting the expression of EMMPRIN in human normal and carcinomatous bladder tissues. METHODS: The expression of EMMPRIN was examined in 45 bladder carcinoma tissues from 45 patients and normal bladder mucosa from 9 persons by immunohistochemistry SP method. The relationship between EMMPRIN expression and clinicopathologic parameters was analyzed. RESULTS: EMMPRIN protein was localized at membrane and cytoplasm of carcinomatous epithelial cell, but was not expressed in normal bladder epithelial or tumor stromal cell. The overall positive rate of EMMPRIN in 45 cases of bladder carcinoma was 73%. The positive rates of EMMPRIN in grade II, grade III and recurrent carcinomas were 84%, 85%, and 100%, respectively, significantly higher than those in grade I and non-recurrent carcinomas (14% and 56%, respectively) (P< 0.05). The intensive positive rate of EMMPRIN in lymph node metastasis group (86%) was significantly higher than that in no lymph node metastasis group (24%) (P< 0.05). CONCLUSION: EMMPRIN was overexpressed in human bladder transitional cell carcinoma, and related to the malignant degree and recurrence. It could be a useful indicator for the assessment of potent invasion and metastasis of bladder carcinoma.

Adult↗

Regulation of extracellular matrix metalloproteinase inducer and matrix metalloproteinase expression by amphiregulin in transformed human breast epithelial cells.

Amphiregulin (AR) and epidermal growth factor effects on expression and activity of the extracellular matrix metalloproteinase inducer (EMMPRIN) were examined in NS2T2A1 breast tumor cells. Both growth factors induced mRNA and protein expression of EMMPRIN, and matrix metalloproteinase (MMP) -2 and -9 enzymatic activity. The induction of EMMPRIN by AR was mediated by epidermal growth factor receptor (EGFR) tyrosine kinase activation and inhibited by ZD1839. AR and EGFR antisense (AS) cDNAs inhibited EMMPRIN expression and MMP activity. Coculture of NS2T2A1V expressing AR- or EGFR-AS with fibroblasts and endothelial cells showed a decreased MMP activity. In parallel, nude mice tumors derived from AR and EGFR-AS cells revealed reduced level of EMMPRIN and MMP activity. AR and epidermal growth factor, therefore, regulate EMMPRIN and its MMP-mediated expression, identifying EGFR signaling as critical to this regulation.

Amphiregulin↗

[Effects of L-nitroarginine on the recovery of traumatic facial paralysis].

OBJECTIVE: To study the effects of constitutive nitric oxide synthase inhibitor L-nitroarginine on the recovery of traumatic facial paralysis in rats and the changes of the expression of cNOS and OX42 in the facial nucleus. METHODS: L-nitroarginine was intraperitoneally injected into rats and the recovery of facial paralysis was observed at different time point. and the changes of cNOS and OX42 positive neurons were studied in facial nucleus. RESULTS: Treatment of L-nitroarginine could remarkably inhibit the recovery of traumatic facial paralysis. The cNOS immunoactivity was obvious inhibited in facial nucleus, while the OX42 immunoactivity was obvious increased. CONCLUSION: Endogenous nitric oxide may play an important mediator role on the recovery of traumatic facial paralysis.

Animals↗