Edmond Sabo

RAGE, LRP-1, and amyloid-beta protein in Alzheimer’s disease

The receptor for advanced glycation end products (RAGE) is thought to be a primary transporter of β-amyloid across the blood–brain barrier (BBB) into the brain from the systemic circulation, while the low-density lipoprotein receptor-related protein (LRP)-1 mediates transport of β-amyloid out of the brain. To determine whether there are Alzheimer’s disease (AD)-related changes in these BBB-associated β-amyloid receptors, we studied RAGE, LRP-1, and β-amyloid in human elderly control and AD hippocampi. In control hippocampi, there was robust RAGE immunoreactivity in neurons, whereas microvascular staining was barely detectable. LRP-1 staining, in contrast, was clearly evident within microvessels but only weakly stained neurons. In AD cases, neuronal RAGE immunoreactivity was significantly decreased. An unexpected finding was the strongly positive microvascular RAGE immunoreactivity. No evidence for colocalization of RAGE and β-amyloid was seen within either microvessels or senile plaques. A reversed pattern was evident for LRP-1 in AD. There was very strong staining for LRP-1 in neurons, with minimal microvascular staining. Unlike RAGE, colocalization of LRP-1 and β-amyloid was clearly present within senile plaques but not microvessels. Western blot analysis revealed a much higher concentration of RAGE protein in AD hippocampi as compared with controls. Concentration of LRP-1 was increased in AD hippocampi, likely secondary to its colocalization with senile plaques. These data confirm that AD is associated with changes in the relative distribution of RAGE and LRP-1 receptors in human hippocampus. They also suggest that the proportion of amyloid within the brains of AD patients that is derived from the systemic circulation may be significant.

Association of the membrane estrogen receptor, GPR30, with breast tumor metastasis and transactivation of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases function as a common signaling conduit for membrane receptors that lack intrinsic enzymatic activity, such as G-protein coupled receptors and integrins. GPR30, an orphan member of the seven transmembrane receptor (7TMR) superfamily has been linked to specific estrogen binding, rapid estrogen-mediated activation of adenylyl cyclase and the release of membrane-tethered proHB-EGF. More recently, GPR30 expression in primary breast adenocarcinoma has been associated with pathological parameters commonly used to assess breast cancer progression, including the development of extramammary metastases. This newly appreciated mechanism of cross communication between estrogen and EGF is consistent with the observation that 7TMR-mediated transactivation of the EGFR is a recurrent signaling paradigm and may explain prior data reporting the EGF-like effects of estrogen. The molecular details surrounding GPR30-mediated release of proHB-EGF, the involvement of integrin beta1 as a signaling intermediary in estrogen-dependent EGFR action, and the possible implications of these data for breast cancer progression are discussed herein.

Disruption of Interleukin-1 Signaling Improves the Quality of Wound Healing

In this study, we investigated the role of interleukin (IL)-1 signaling in wound healing. IL-1 receptor type I (IL-1R) knockout (KO) mice showed reduced fibrosis in both cutaneous and deep tissue wounds, which was accompanied by a reduction in inflammatory cellular infiltration in cutaneous but not in deep tissue wounds. There were no differences in either total collagenolytic activity or in the expression of selected matrix metalloproteinases or tissue inhibitors of metalloproteinases between the wound fluids from wild-type or IL-1R KO mice. However, wound fluids from IL-1R KO mice contained lower levels of IL-6 compared with wild-type controls. In addition, the infusion of IL-6 into wounds in IL-1R KO mice did not increase fibrosis. Skin wounds in IL-1R KO animals had lower levels of collagen and improved restoration of normal skin architecture compared with skin wounds in wild-type mice. However, neither the tensile strength of incisional skin wounds nor the rate of closure of excisional wounds differed between IL-1R KO and wild-type animals. The reduced fibrotic response in wounds from IL-1R KO mice could be reproduced by the administration of an IL-1R antagonist. These findings suggest that pharmacological interference with IL-1 signaling could have therapeutic value in the prevention of hypertrophic scarring and in the treatment of fibrotic diseases.

Lack of UCP2 reduces Fas-mediated liver injury in ob/ob mice and reveals importance of cell-specific UCP2 expression

Fatty liver is vulnerable to conditions that challenge hepatocellular energy homeostasis. Lipid‐laden hepatocytes highly express uncoupling protein‐2 (UCP2), a mitochondrial carrier that competes with adenosine triphosphate (ATP) synthesis by mediating proton leak. However, evidence for a link between UCP2 expression and susceptibility of liver to acute injury is lacking. We asked whether absence of UCP2 protects ob/ob mice from Fas‐mediated acute liver damage. UCP2‐deficient ob/ob mice (ob/ob:ucp2−/−) and UCP2‐competent littermates (ob/ob:ucp2+/+) received a single dose of agonistic anti‐Fas antibody (Jo2). Low‐dose Jo2 (0.15 mg/kg intraperitoneally) caused less serum alanine aminotransferase (ALT) elevation and lower apoptosis rates in ob/ob:ucp2−/− mice. High‐dose Jo2 (0.40 mg/kg intraperitoneally) proved uniformly fatal; however, ob/ob:ucp2−/− mice survived longer with less depletion of liver ATP stores, indicating that fatty hepatocytes may benefit from lack of UCP2 during Jo2 challenge. Although UCP2 reportedly controls mitochondrial oxidant production, its absence had no apparent effect on fatty liver tissue malondialdehyde levels augmented by Jo2. This finding prompted us to determine UCP2 expression in Kupffer cells, a major source of intrahepatic oxidative stress. UCP2 expression was found diminished in Kupffer cells of untreated ob/ob:ucp2+/+ mice, conceivably contributing to increased oxidative stress in fatty liver and limiting the impact of UCP2 ablation. In conclusion, whereas UCP2 abundance in fatty hepatocytes exacerbates Fas‐mediated injury by compromising ATP stores, downregulation of UCP2 in Kupffer cells may account for persistent oxidative stress in fatty liver. Our data support a cell‐specific approach when considering the therapeutic effects of mitochondrial uncoupling in fatty liver disease. (HEPATOLOGY 2006;44:592–601.)

DIFFERENTIATING THE UNDIFFERENTIATED: IMMUNOHISTOCHEMICAL PROFILE OF MEDULLARY CARCINOMA OF THE COLON WITH AN EMPHASIS ON INTESTINAL DIFFERENTIATION

Undifferentiated or medullary carcinoma is characterized by its distinct histologic appearance and relatively better prognosis compared to poorly differentiated colonic carcinoma. These 2 entities may be difficult to differentiate by light microscopy alone. Only limited immunohistochemical studies investigating medullary carcinoma have been reported. These studies suggest a loss of intestinal differentiation, exemplified by a high percentage of CDX2 negativity. Our aim was to further characterize the immunohistochemical profile of medullary carcinoma, with particular emphasis on intestinal markers. Paraffin blocks from 16 cases of medullary carcinoma and 33 cases of poorly differentiated colonic carcinoma were retrieved, and tissue microarrays were constructed and stained with an immunohistochemical panel including CDX2, CK7, CK20, p53, intestinal trefoil factor 3, chromogranin, synaptophysin, MLH-1, MUC-1, MUC-2, and calretinin. A significantly higher proportion of medullary carcinomas, as opposed to poorly differentiated colonic carcinomas, showed loss of staining for MLH-1 and for the intestinal transcription factor CDX2, in accordance with previous studies. MLH-1 staining was present in only 21% of medullary carcinoma cases compared with 60% of the poorly differentiated colonic carcinoma cases (P = .02), whereas CDX2 was positive in 19% of medullary carcinomas and 55% of poorly differentiated colonic carcinomas (P = .03). Interestingly, calretinin staining was strongly positive in 73% of medullary carcinomas compared to only 12% of poorly differentiated colonic carcinomas (P < .0001). Evidence of intestinal differentiation by MUC-1, MUC-2, and TFF-3 staining was seen in 67%, 60%, and 53% of the medullary carcinomas, respectively. These 3 markers were frequently positive in many of the CDX2-negative medullary carcinoma cases. Medullary carcinoma of the colon retains a significant degree of intestinal differentiation as evidenced by its high percentage of staining for MUC-1, MUC-2, and TFF-3. Calretinin, MLH-1, and CDX2 may help to differentiate medullary carcinoma from poorly differentiated colonic carcinoma of the colon.

Postnatal Expansion of the Pancreatic β-Cell Mass Is Dependent on Survivin

OBJECTIVE—Diabetes results from a deficiency of functional β-cells due to both an increase in β-cell death and an inhibition of β-cell replication. The molecular mechanisms responsible for these effects in susceptible individuals are mostly unknown. The objective of this study was to determine whether a gene critical for cell division and cell survival in cancer cells, survivin, might also be important for β-cells. RESEARCH DESIGN AND METHODS—We generated mice harboring a conditional deletion of survivin in pancreatic endocrine cells using mice with a Pax-6-Cre transgene promoter construct driving tissue-specific expression of Cre-recombinase in these cells. We performed metabolic studies and immunohistochemical analyses to determine the effects of a mono- and biallelic deletion of survivin. RESULTS—Selective deletion of survivin in pancreatic endocrine cells in the mouse had no discernible effects during embryogenesis but was associated with striking decreases in β-cell number after birth, leading to hyperglycemia and early-onset diabetes by 4 weeks of age. Serum insulin levels were significantly decreased in animals lacking endocrine cell survivin, with relative stability of other hormones. Exogenous expression of survivin in mature β-cells lacking endogenous survivin completely rescued the hyperglycemic phenotype and the decrease in β-cell mass, confirming the specificity of the survivin effect in these cells. CONCLUSIONS—Our findings implicate survivin in the maintenance of β-cell mass through both replication and antiapoptotic mechanisms. Given the widespread involvement of survivin in cancer, a novel role for survivin may well be exploited in β-cell regulation in diseased states, such as diabetes.

Expression Analysis of Barrett's Esophagus–Associated High-Grade Dysplasia in Laser Capture Microdissected Archival Tissue

Purpose: Identifying genes differentially expressed in nondysplastic BE (NDBE) from those expressed in high-grade dysplasia (HGD) should be of value in improving our understanding of this transition and may yield new diagnostic and/or prognostic markers. The aim of this study was to determine the differential transcriptome of HGD compared with NDBE through gene microarray analysis of epithelial cells microdissected from archival tissue specimens. Experimental Design: Laser capture microdissection was used to isolate epithelial cells from adjacent inflammatory and stromal cells. Epithelial mRNA was extracted from areas of NDBE and HGD in matched biopsies from 11 patients. mRNA was reverse transcribed and applied on Affymetrix cDNA microarray chips customized for formalin-exposed tissue. For a subset of these genes, differential gene expression was confirmed by real-time PCR and immunohistochemistry. Results: There were 131 genes overexpressed by at least 2.5-fold in HGD versus NDBE and 16 genes that were underexpressed by at least 2.5-fold. Among the overexpressed genes are several previously shown to be increased in the neoplastic progression of BE, as well as novel genes such as lipocalin-2, S100A9, matrix metallopeptidase 12, secernin 1, and topoisomerase IIα. Genes decreased in dysplastic epithelium include MUC5AC, trefoil factor 1 (TFF1), meprin A, and CD13. Real-time PCR validated the changes in expression in 24 of 28 selected genes. Immunohistochemistry confirmed increased protein expression for topoisomerase IIα, S100A9, and lipocalin-2 and decreased expression of TFF1 across the spectrum of BE-associated dysplasia from NDBE through adenocarcinoma. Conclusions: This is the first study to identify epithelial genes differentially expressed in HGD versus NDBE in matched patient samples. The genes identified include several previously implicated in the pathogenesis of BE-associated dysplasia and new candidates for further investigation.

Computerized morphometry as an aid in determining the grade of dysplasia and progression to adenocarcinoma in Barrett's esophagus

The aims of this study were to use computerized morphometry in order to differentiate between the degree of dysplasia and to predict progression to invasive adenocarcinoma in Barretts esophagus (BE). Biopsies from 97 patients with BE graded by a consensus forum of expert gastrointestinal pathologists were available for morphometrical analysis. The study group included 36 biopsies negative for dysplasia (ND), none of which progressed to carcinoma; 16 indefinite for dysplasia (IND) and 21 low-grade dysplasia (LGD), of which three progressed in each group and 24 high-grade dysplasia (HGD), of which 15 progressed to invasive carcinoma. Computerized morphometry was used for measuring indices of size, shape, texture, symmetry and architectural distribution of the epithelial nuclei. Low-grade dysplasia was best differentiated from the ND group by nuclear pseudostratification (P=0.036), pleomorphism (P<0.01), and chromatin texture (margination, P<0.01) and from the HGD group by nuclear area (P<0.01), pleomorphism (P<0.01), chromatin texture (margination, P<0.01), symmetry (P<0.01), and orientation (P=0.027). These results were validated on a new set of cases (n=55) using a neural network model, resulting in an accuracy of 89% for differentiating between the ND and LGD groups and 86% for differentiating between the LGD and HGD groups. Within the HGD group, univariate significant predictors of the progression interval to carcinoma were: indices of nuclear texture (heterogeneity: P=0.0019, s.d.-OD: P=0.005) and orientation: P=0.022. Nuclear texture (heterogeneity) was the only independent predictor of progression (P=0.004, hazard=11.54) by Coxs multivariate test. This study proposes that computerized morphometry is a valid tool for determining the grade of dysplasia in BE. Moreover, histomorphometric quantification of nuclear texture is a powerful tool for predicting progression to invasive adenocarcinoma in patients with HGD.

Uncoupling protein-2 modulates the lipid metabolic response to fasting in mice

Uncoupling protein-2 (UCP2) regulates insulin secretion by controlling ATP levels in beta-cells. Although UCP2 deficiency improves glycemic control in mice, increased expression of UCP2 interferes with glucose-stimulated insulin secretion. These observations link UCP2 to beta-cell dysfunction in type 2 diabetes with a perplexing evolutionary role. We found higher residual serum insulin levels and blunted lipid metabolic responses in fasted ucp2(-/-) mice, supporting the concept that UCP2 evolved to suppress insulin effects and to accommodate the fuel switch to fatty acids during starvation. In the absence of UCP2, fasting initially promotes peripheral lipolysis and hepatic fat accumulation at less than expected rates but culminates in protracted steatosis, indicating diminished hepatic utilization and clearance of fatty acids. We conclude that UCP2-mediated control of insulin secretion is a physiologically relevant mechanism of the metabolic response to fasting.

Primary mucin-producing tumours of the salivary glands: a clinicopathological and morphometric study.

Yakirevich E, Sabo E, Klorin G, Alos L, Cardesa A, Ellis G L, Shumway B S & Gnepp D R
(2010) Histopathology 57, 395–409
Primary mucin‐producing tumours of the salivary glands: a clinicopathological and morphometric study