Raffaella Muraro

The Receptor RAGE as a Progression Factor Amplifying Arachidonate-Dependent Inflammatory and Proteolytic Response in Human Atherosclerotic Plaques Role of Glycemic Control

Background—RAGE (receptor for advanced glycation end products [AGEs]) plays a role in diabetic atherosclerosis. Recently, we have demonstrated enhanced expression of cyclooxygenase-2 and PGE synthase-1 (COX-2/mPGES-1) in human symptomatic plaques, and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. However, the specific transmembrane signaling pathway(s) influencing plaque COX-2/mPGES-1 expression is unknown. The aim of this study was to characterize RAGE expression in human plaques and to correlate it with the inflammatory infiltration, COX-2/mPGES-1 and MMP expression, and with clinical evidence of diabetes. Methods and Results—Plaques obtained from 60 patients undergoing carotid endarterectomy were divided into diabetic and nondiabetic according to clinical evidence of type 2 diabetes. Plaques were subjected to analysis of RAGE, NF-&kgr;B, COX-2/mPGES-1, MMP-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, and collagen content by immunohistochemistry and Western blot, whereas zymography was used to detect MMP activity. Immunohistochemistry was used to identify CD68+ macrophages, CD3+ T-lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Diabetic plaques had more (P <0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells, more (P <0.0001) immunoreactivity for RAGE, activated NF-&kgr;B, COX-2/mPGES-1, and MMPs, increased (P <0.0001) gelatinolytic activity, reduced (P <0.0001) collagen content, and increased (P <0.0001) lipid and oxLDL content. Interestingly, RAGE, COX-2/mPGES-1, and MMP expression was linearly correlated with plasma level of HbA1c. Conclusions—In conclusion, this study demonstrates in humans that RAGE overexpression is associated with enhanced inflammatory reaction and COX-2/mPGES-1 expression in diabetic plaque macrophages, and this effect may contribute to plaque destabilization by inducing culprit metalloproteinase expression.

Suppression of the functionally coupled cyclooxygenase-2/prostaglandin E synthase as a basis of simvastatin-dependent plaque stabilization in humans.

Background—The clinical benefits of statins are attributed to changes in plaque composition that lead to reduced metalloproteinase (MMP) activity and plaque stabilization. However, the molecular mechanism of this effect is unclear. Recently, we demonstrated enhanced expression of isoforms of inducible cyclooxygenase (COX) and PGE synthase (COX-2/mPGES) in human symptomatic plaque and provided evidence that this is associated with MMP-induced plaque rupture. The aim of this study was to characterize the effect of simvastatin on inflammatory infiltration and the expression of COX-2/mPGES and MMPs in human carotid plaques. Methods and Results—Seventy patients with symptomatic carotid artery stenosis were randomized to the American Heart Association Step 1 diet plus simvastatin (40 mg/d) or the American Heart Association Step 1 diet alone for 4 months before endarterectomy. Plaques were subjected to analysis of COX-1, COX-2, mPGES, MMP-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, and collagen content by immunocytochemistry, Western blot, and reverse transcription–polymerase chain reaction, whereas zymography was used to detect MMP activity. Immunocytochemistry was also used to identify CD68+ macrophages, CD3+ T-lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Plaques from the simvastatin group had fewer (P <0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells; less (P <0.0001) immunoreactivity for COX-2/mPGES and MMPs; reduced (P <0.0001) gelatinolytic activity; increased (P <0.0001) collagen content; and reduced (P <0.0001) lipid and oxLDL content. Interestingly, COX-2/mPGES inhibition by simvastatin was completely reversed by mevalonate in vitro. Conclusions—This study demonstrates that simvastatin decreases inflammation and inhibits COX-2/mPGES expression in plaque macrophages, and this effect in turn may contribute to plaque stabilization by inhibition of MMP-induced plaque rupture.

Suppression of Rage as a Basis of Simvastatin-Dependent Plaque Stabilization in Type 2 Diabetes

Objective—Receptor for advanced glycation end products (AGEs) (RAGE) plays a central role in the process of plaque rupture in diabetic patients. Recently, it has been reported that RAGE may be downregulated by improving glycemic control. In contrast, despite being well known that RAGE may be induced in human vessels in a glucose-independent fashion, also by myeloperoxidase (MPO)-dependent AGE generation, no data exist regarding the possibility of a pharmacological modulation of glucose-independent RAGE generation. Thus, the aim of this study was to characterize the effect of simvastatin on the expression of RAGE and RAGE-dependent plaque-destabilizing genes in human atherosclerotic plaques. Methods and Results—Seventy type 2 diabetic patients with asymptomatic carotid artery stenosis (>70%) were randomized to American Heart Association (AHA) step 1 diet plus simvastatin (40 mg/d) or AHA step 1 diet alone for 4 months before endarterectomy. Plaque expression of MPO, AGEs, RAGE, NF-&kgr;B, COX-2, mPGES-1, matrix metalloproteinase (MMP)-2 and MMP-9, lipid and oxidized LDL (oxLDL) content, procollagen 1, and interstitial collagen was analyzed by immunohistochemistry and Western blot; zymography was used to detect MMP activity. Plaques from the simvastatin group had less (P<0.0001) immunoreactivity for MPO, AGEs, RAGE, p65, COX-2, mPGES-1, MMP-2, and MMP-9, lipids and oxLDL; reduced (P<0.0001) gelatinolytic activity; increased (P<0.0001) procollagen 1 and collagen content; and fewer (P<0.0001) macrophages, T-lymphocytes, and HLA-DR+ cells. Of interest, RAGE inhibition by simvastatin, observed not only in plaque sections but also in plaque-derived macrophages, was reverted by addition of AGEs in vitro. Conclusions—This study supports the hypothesis that simvastatin inhibits plaque RAGE expression by decreasing MPO-dependent AGE generation. This effect in turn might contribute to plaque stabilization by inhibiting the biosynthesis of PGE2-dependent MMPs, responsible for plaque rupture.

Low-Density Lipoprotein Level Reduction by the 3-Hydroxy-3-Methylglutaryl Coenzyme-A Inhibitor Simvastatin Is Accompanied by a Related Reduction of F2-Isoprostane Formation in Hypercholesterolemic Subjects No Further Effect of Vitamin E

Background—Both statins and vitamin E, by reducing the rate of lipid peroxidation, may interfere with oxidative stress, but the impact of their combination is unknown. Methods and Results—We randomized 43 hypercholesterolemic patients (21 men, 22 women, age 63±11 years) to either simvastatin, to achieve >20% reduction of total cholesterol, or simvastatin plus 600 mg/d vitamin E for 2 months. Patients were then crossed over to the alternative treatment. Lipid parameters documented patients’ compliance to simvastatin, whereas plasma levels of vitamin E documented compliance and absorption of vitamin E. We assessed urinary excretion of the isoprostane 8-iso-prostaglandin F2&agr; (8-iso-PGF2&agr;) as an in vivo index of oxidative stress at baseline and after each month of therapy. 8-Iso-PGF2&agr; was significantly reduced by simvastatin, from 361±148 pg/mg creatinine (mean±SD) at baseline to 239±124 pg/mg creatinine after 1 month. The addition of vitamin E did not reduce such levels any further (256±125 after 1 month). Linear regression analysis showed a weak inverse relationship of 8-iso-PGF2&agr; with vitamin E levels but a much stronger relationship with LDL cholesterol (R2=0.162;P <0.001). Conclusions—In hypercholesterolemic patients, LDL cholesterol is a major correlate of oxidative stress. Concomitant with LDL cholesterol reduction, simvastatin causes a drastic reduction of oxidative stress to a level that is not further reduced by the addition of vitamin E. Results of clinical trials with vitamin E may have been hampered by inadequate knowledge of the background level of lipid peroxidation, which is a major determinant of vitamin E bioactivity.

Frequent overexpression of multiple ErbB receptors by head and neck squamous cell carcinoma contrasts with rare antibody immunity in patients

In an effort to elucidate the role of ErbB receptors in human head and neck squamous cell carcinoma (HNSCC), expression abnormalities and subcellular localization of epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4 were investigated along with EGF and tenascin by immunohistochemistry in 38 carcinomas as compared to adjacent normal mucosa of 24 cases. Although tumour‐specific overexpression affected each ErbB receptor (EGFR 47%, ErbB2 29%, ErbB3 21%, ErbB4 26%), EGFR abnormalities were most prevalent. The latter, and overexpression of more than two ErbB receptors in the same tumour, which always included EGFR, correlated with metastatic disease. ErbB products were specifically detected on the cell membrane and in the cytoplasm. In contrast, ErbB4 was uniquely localized to the nucleus in 7 carcinomas and a tumour‐derived cell line, indicating a role for regulated intramembrane proteolysis resulting in nuclear ErbB4 translocation in HNSCC. Expression of prototype ligand EGF or low‐affinity stromal activator tenascin correlated significantly with EGFR overexpression, implying chronic EGFR activation. Simultaneous overexpression of additional ErbB receptors in most of these cases suggested recurrent involvement of receptor heterodimers. In spite of frequent ErbB receptor alterations, autologous ErbB serum antibodies were rare, with only 1 of 38 tumour patients exhibiting an ErbB2‐specific immune response. Based on upregulation of several known immunosuppressive molecules, scarcity of ErbB‐specific antibodies is consistent with attenuation of natural tumour‐specific immune responses in HNSCC. Copyright

Co-localization of multiple ErbB receptors in stratified epithelium of oral squamous cell carcinoma.

The expression of all four ErbB receptors was compared by immunohistochemistry, using receptor‐specific polyclonal antisera, in 32 invasive, 11 in situ carcinomas, six benign lesions, and 22 samples of histologically normal mucosa adjacent to specimens of carcinoma originating from oral cavity epithelium. Among invasive and in situ carcinoma, EGFR expression was the most prevalent (in 29/32 and 8/11 cases, respectively) followed by ErbB2 (17/32 and 2/11) and ErbB4 (9/32 and 1/10), while ErbB3 was only detected in invasive tumours (12/32). Specific patterns included invasive tumours with expression of EGFR (8/32) or ErbB4 (1/32) alone, as well as different receptor combinations (EGFR+ErbB2, EGFR+ErbB4, EGFR+ErbB2+ErbB3, EGFR+ErbB2+ErbB4, and all four receptors). Simultaneous expression of three or four ErbB receptors correlated with tumour invasion (p=2.2×10−4) and localized in the intermediate epithelial cell layer of well and moderately differentiated tumours. No other significant correlation with clinico‐pathological features was noticed. Some benign lesions and histologically normal mucosa adjacent to carcinomas showed weak immunostaining of EGFR (10/28), ErbB2 (4/28) or ErbB4 (3/28). By comparison, overexpression, as indicated by increased staining intensity, was observed in invasive tumours for EGFR (18/32), ErbB2 (8/32), ErbB4 (3/32), and ErbB3 (3/32). Statistical evaluation demonstrated a significant association of EGFR or ErbB2 overexpression with invasive carcinoma when compared with benign lesions and apparently normal epithelium (p=5.2×10−7 and p=5×10−3, respectively). Tumour‐specific overexpression of ErbB receptors and their co‐expression, most frequently involving EGFR and ErbB2, in the same cell layer of neoplastic epithelium, implicate receptor heterodimers in the pathogenesis of oral squamous carcinoma. Copyright

Immune responses to all ErbB family receptors detectable in serum of cancer patients

Employing NIH3T3 transfectants with individual human ErbB receptor coding sequences as recombinant antigen sources, we detected by immunoblot analysis specific immunoreactivity against all four ErbB receptors among 13 of 41 sera obtained from patients with different types of epithelial malignancies. Overall, serum positivity was most frequently directed against ErbB2 followed by EGFR, ErbB3 and ErbB4. Specificity patterns comprised tumor patients with unique serum reactivity against ErbB2 or ErbB4. Moreover, approximately half of the positive sera exhibited concomitant reactivity with multiple ErbB receptors including EGFR and ErbB2, EGFR and ErbB4, ErbB2 and ErbB3 or EGFR, ErbB2 and ErbB3. Serum reactivity was confirmed for the respective ErbB receptors expressed by human tumor cells and corroborated on receptor-specific immunoprecipitates. Positive sera contained ErbB-specific antibodies of the IgG isotype. Representative immuno-histochemical analysis of tumor tissues suggested overexpression of ErbB receptors for which serum antibodies were detectable in five of six patients. These findings implicate multiple ErbB receptors including ErbB3 and ErbB4 in addition to EGFR and ErbB2 in primary human cancer. Heterogeneity of natural ErbB-specific responses in cancer patients warrants their evaluation in light of immunotherapeutic approaches targeting these receptors.

The BFRF1 Gene of Epstein-Barr Virus Encodes a Novel Protein

ABSTRACT Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are ∼100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in theBamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitts lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription ofBFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

The use of a cationic liposome formulation (DOTAP) mixed with a recombinant tumor-associated antigen to induce immune responses and protective immunity in mice

The cationic liposome DOTAP is a well-known transfection reagent. It has been manufactured and approved for clinical use, is readily available, and can be easily used as an adjuvant. These characteristics prompted us to investigate the effectiveness of DOTAP as an adjuvant to induce immune responses and protective immunity in mice using baculovirus-derived carcinoembryonic antigen (bV-CEA) as a model antigen. Two routes of administration and a dose-response study of bV-CEA were used in BALB/c mice to define the magnitude of the immune response as well as the most effective route of immunization. The results demonstrate differences in antibody titers, immunoglobulin (Ig)G isotype, and T-cell responses between the intravenous (i.v.) or subcutaneous (s.c.) route of immunization. The titer of the anti-CEA antibodies induced by the s.c. immunization was greater than the response by i.v. immunization. The s.c. route enhanced the IgG2a/2b isotype, whereas i.v. immunization elicited primarily IgGl. T-cell proliferation responses and cytokine production paralleled the humoral response (i.e., production was higher in the s.c. immunized animals). No differences in immunological responses were seen using either 25 or 10 μg of bV-CEA three times. An amount of 25 μg of bV-CEA/DOTAP given by s.c. immunization was sufficient in protecting mice from the transplant of syngeneic tumor cells transduced with the human CEA gene. We conclude that the cationic liposome DOTAP may be a useful immunoadjuvant for active anti-tumor immunotherapy in future clinical trials. This study will help to define the most effective way to use such an adjuvant.

An ErbB-3 antibody, MP-RM-1, inhibits tumor growth by blocking ligand-dependent and independent activation of ErbB-3/Akt signaling.

The ErbB receptors, such as ErbB-1 and ErbB-2, have been intensely pursued as targets for cancer therapeutics. Although initially efficacious in a subset of patients, drugs targeting these receptors led invariably to resistance, which is often associated with reactivation of the ErbB-3-PI3K-Akt signaling. This may be overcome by an ErbB-3 ligand that abrogates receptor-mediated signaling. Toward this end, we have generated a mouse monoclonal antibody, MP-RM-1, against the extracellular domain (ECD) of ErbB-3 receptor. Assessment of human tumor cell lines, as well as early passage tumor cells revealed that MP-RM-1 effectively inhibited both NRG-1β-dependent and -independent ErbB-3 activation. The antagonizing effect of MP-RM-1 was of non-competitive type, as binding of [125I]-labeled NRG-1β to ErbB-3 was not influenced by the antibody. MP-RM-1 treatment led, in most instances, to decreased ErbB-3 expression. In addition, MP-RM-1 was able to inhibit the colony formation ability of tumor cells and tumor growth in two human tumor xenograft nude mouse models. Treatment with the antibody was associated with a decreased ErbB-3 and Akt phosphorylation and ErbB-3 expression in the excised tumor tissue. Collectively, these results indicate that MP-RM-1 has the potential to interfere with signaling by ErbB-3 and reinforce the notion that ErbB-3 could be a key target in cancer-drug design.