Peter S. Amenta

The mammary gland iodide transporter is expressed during lactation and in breast cancer

The sodium/iodide symporter mediates active iodide transport in both healthy and cancerous thyroid tissue. By exploiting this activity, radioiodide has been used for decades with considerable success in the detection and treatment of thyroid cancer. Here we show that a specialized form of the sodium/iodide symporter in the mammary gland mediates active iodide transport in healthy lactating (but not in nonlactating) mammary gland and in mammary tumors. In addition to characterizing the hormonal regulation of the mammary gland sodium/iodide symporter, we demonstrate by scintigraphy that mammary adenocarcinomas in transgenic mice bearing Ras or Neu oncogenes actively accumulate iodide by this symporter in vivo. Moreover, more than 80% of the human breast cancer samples we analyzed by immunohistochemistry expressed the symporter, compared with none of the normal (nonlactating) samples from reductive mammoplasties. These results indicate that the mammary gland sodium/iodide symporter may be an essential breast cancer marker and that radioiodide should be studied as a possible option in the diagnosis and treatment of breast cancer.

The extracellular matrix in hepatic regeneration.

After partial hepatectomy, as a consequence of hepatocyte proliferation, cell clusters containing 10–14 hepatocytes are formed. These clusters are devoid of sinusoids and extracellular matrix; therefore, many hepatocytes are two to three cells removed from the vascular spaces. Four days after hepatectomy, Ito cells send delicate cell processes between the hepatocytes in the clusters. This “invasion” of the clusters coincides with the activation in Ito cells of genes encoding for several laminin chains. The penetration of Ito cells into the clusters is followed by fenestrated endothelial cells, and in this manner the normal hepatocyte vascular relationship is restored. As soon as the normal vascular structure is reestablished, the laminin genes are turned off. This chain of events is similar to the one taking place during hepatogenesis when continuous capillaries are converted into sinusoids. This similarity in hepatogensis and regeneration suggests that the secreted laminin chains may be signals for the vascu‐larization of the clusters by fenestrated sinusoids. During this process neither entactin nor laminin α chains are secreted. The vascularization of the regenerating clusters contrasts sharply to the vasculari‐zation of cirrhotic nodules. In the latter ease, entactin and perhaps laminin αtl chains are secreted, and the final result is the formation of basement membranes and continuous capillaries rather than fenestrated sinusoids. We suggest that entactin and specific laminin chains play a crucial role in determining the outcome of hepatic injury. Definition of the roles of entactin am! laminin chains in vascularization and modulation of the endothelial phenotype will not only elucidate important aspects of regeneration, but may provide a better understanding of cirrhosis and even suggest therapeutic approaches.—Martinez‐Hernandez, A., Amenta, P. S. The extracellular matrix in hepatic regeneration. FASEBJ. 9, 1401‐1410 (1995)

The hepatic extracellular matrix. I: Components and distribution in normal liver

The unique nature of the hepatic extracellular matrix (ECM) is predicated by the special configuration of the space of Disse. Whereas other epithelial organs have two basement membranes (BM) and a substantial ECM interposed between endothelial and epithelial cells, the liver lobule has no BM and only an attenuated ECM, consisting mostly of fibronectin, some collagen type I, and minor quantities of types III, IV, V, and VI. This configuration, together with the abundant fenestrations and gaps of the sinusoidal endothelial cells, seems ideally suited to facilitate the rapid bidirectional exchange of macromolecules normally taking place between plasma and hepatocytes. During organogenesis, the liver anlage is vascularized by continuous capillaries with BM, but by day 13.5 of development (in the rat) the vessels in the immediate proximity of hepatocytes become fenestrated, lacking specialized junctions and BM, suggesting that the hepatocytes produce signals capable of modulating the endothelial phenotype. In regeneration, hepatocyte proliferation precedes vascular proliferation resulting in the formation of hepatocyte clusters that, temporarily, lack sinusoids. Eventually, vascular proliferation follows and the normal hepatocyte-vascular relationships are restored. During this period laminin synthesis by Ito cells is prominent. As soon as hepatocytes become stable, secretion of the sinusoid phenotype-maintaining factors resumes and laminin synthesis and secretion terminates. The interplay between extracellular matrix and liver cells is essential for normal homeostasis and its modification results in derranged hepatic function.

The Extracellular Matrix is an Integrated Unit: Ultrastructural Localization of Collagen Types I, III, IV, V, VI, Fibronectin, and Laminin in Human Term Placenta

The human term placenta is used extensively as a source of extracellular matrix components. To elucidate the tissue distribution and interrelationships of seven of these components, monospecific antibodies directed against collagen types I, III, IV, V, VI, fibronectin, and laminin were reacted with human term placenta and studied by light and electron immunohistochemistry. Type I collagen was the basic structural unit of human term placenta, present as 30-35 nm, cross-banded fibers, often in the form of large fiber bundles. Type III collagen was present as thin 10-15 nm, beaded fibers often forming a meshwork which encased type I collagen fibers. Types V and VI collagen were present as 6-10 nm filaments, often closely associated with types I and III collagen. Type VI collagen also coated collagen fibers of all diameters, enhancing their periodicity, providing a staining pattern often similar to that observed with anti-fibronectin antibodies. Fibronectin was present in both maternal and fetal plasma and throughout the stroma of the chorionic villus, as both free filaments and coating collagen fibers. Basement membranes contained laminin and type IV collagen, but no fibronectin. In summary, the non-basement membrane proteins studied often codistributed with type I collagen, between and apparently attached to fibers, suggesting that they may act as binding proteins, linking type I fibers and bundles, to themselves and to other structures.

Type XV collagen exhibits a widespread distribution in human tissues but a distinct localization in basement membrane zones.

Abstract.The collagen family of proteins consists of 19 types encoded by 33 genes. One of the more recently discovered collagens is the α1 chain of type XV. Type XV collagen is comprised of a 577-amino-acid, highly interrupted, triple-helical region that is flanked by amino and carboxy noncollagenous domains of 555 and 256 residues, respectively. To address questions of where this collagen is localized and what its function may entail, we produced a bacteria-expressed recombinant protein representing the first half of the type XV collagen carboxy-terminal domain in order to generate highly specific polyclonal antisera. Immunoscreening of an expression library with the affinity-purified antibody revealed three clones coding for part of the type XV triple-helical region and the entire noncollagenous carboxy-terminus. Western blot analysis of human tissue homogenates identified a 116-kDa collagenase-sensitive protein and a 27-kDa collagenase-resistant fragment, whose electrophoretic mobilities were unchanged in the presence and absence of reductant. Northern blot hybridization to human tissue RNAs indicated that type XV has a prevalent and widespread distribution. To determine the precise localization of type XV collagen, immunohistochemical analyses at the light- and electron-microscopic levels were performed. Type XV exhibited a surprisingly restricted and uniform presence in many human tissues as evidenced by a strong association with vascular, neuronal, mesenchymal, and some epithelial basement membrane zones. These data suggest that type XV collagen may function in some manner to adhere basement membrane to the underlying connective tissue stroma.

The hepatic extracellular matrix II. Ontogenesis, regeneration and cirrhosis

The unique nature of the hepatic extracellular matrix (ECM) is predicted by the special configuration of the space of Disse. Whereas other epithelial organs have two basement membranes (BM) and a substantial ECM interposed between endothelial and epithelial cells, the liver lobule has no BM and only an attenuated ECM, consisting mostly of fibronectin (FN), some collagen type I, and minor quantities of types III, IV, V, and VI. This configuration, together with the abundant fenestrations and gaps of the sinusoidal endothelial cells, seems ideally suited to facilitate the rapid bidirectional exchange of macromolecules normally taking place between plasma and hepatocytes. During organogenesis, the liver anlage is vascularized by continuous capillaries with BM, but by day 13.5 of development (in the rat) the vessels in the immediate proximity of hepatocytes become fenestrated, lacking specialized junctions and BM, suggesting that the hepatocytes produce signals capable of modulating the endothelial phenotype. In regeneration, hepatocyte proliferation precedes vascular proliferation resulting in the formation of hepatocyte clusters that, temporarily, lack sinusoids. Eventually, vascular proliferation follows and the normal hepatocyte-vascular relationships are restored. During this period laminin synthesis by Ito cells is prominent. As soon as hepatocytes become stable, secretion of the sinusoid phenotype-maintaining factors resumes and laminin synthesis and secretion terminates. The interplay between extracellular matrix and liver cells is essential for normal homeostasis and its modification results in derranged hepatic function.

Detection of a Shared Colon Epithelial Epitope on Barrett Epithelium by a Novel Monoclonal Antibody

In Barrett epithelium, the typical esophageal stratified squamous epithelium is replaced by metaplastic columnar epithelial cells, usually in the distal esophagus as a complication of severe reflux esophagitis [1, 2]. Adenocarcinoma develops in 8% to 15% of cases [3-5]. The origin and precise nature of this epithelium that contains various cell types [3-7] are unknown. We previously described a unique murine monoclonal antibody, 7E12H12 (IgM isotype), that reacts specifically with normal colonic epithelial cells but not with 13 other epithelial organs, including the small intestinal enterocytes and the gastric and esophageal mucosa [8]. Using the immunoperoxidase assay [8], we examined the immunoreactivity of the 7E12H12 monoclonal antibody against normal and abnormal esophageal mucosa, especially Barrett epithelium and associated adenocarcinoma of the esophagus. Methods Tissue Samples Retrospective Tissue Materials from the Esophagus and Gastroesophageal Junction Fifty-three biopsy specimens taken during endoscopy at different levels of esophagus from 44 persons, and 12 surgical resection specimens for carcinoma of the esophagus arising in Barrett epithelium (7 patients) and squamous cell carcinoma of the esophagus (5 patients) were obtained from the archival material in the Department of Pathology. Prospective Tissue Specimens from the Esophagus, Stomach, and Upper Small Intestine Fifty-one biopsy specimens were obtained from 12 consecutive persons who were evaluated by upper gastrointestinal endoscopy for persistent symptoms of esophageal reflux disorders and acid peptic syndromes. All patients were evaluated by a single gastroenterologist, and biopsy specimens were taken systematically from various sites of the esophagus, stomach, duodenum, and jejunum (Table 1). Among 12 specimens with variable degrees of esophagitis, 6 specimens were taken from the gastroesophageal junction, and the remaining 6 were taken from distal esophagus just proximal to the squamocolumnar junction. Tissue specimens were fixed in formalin, and paraffin blocks were prepared for routine histologic study. Serial sections from each block were processed to study the immunoreactivity against the 7E12H12 monoclonal antibody by the immunoperoxidase method. The pathologist and the investigators who did the immunocytochemical studies were not informed about the history and clinical diagnosis of the patients, and they evaluated their findings independently. Table 1. Histologic Analysis of the Tissue and the Results of Immunoperoxidase Experiments with the 7E12H12 Monoclonal Antibody (IgM Isotype) Immunoperoxidase Method The method of production and characterization of the 7E12H12 monoclonal antibody has been previously reported [8]. An unrelated mouse monoclonal antibody of IgM isotype (MOPC-104E) was used as a control. Normal colonic mucosal biopsy specimens were included in each experiment as a positive control against the 7E12H12 monoclonal antibody. The immunohistochemical analysis was done as previously described [8] with some modifications as described below [9]. The tissues were sectioned (5 microns), mounted on poly-L-lysine-coated slides, deparaffinized by heating at 56 C for 1 hour, immersed in xylene, rehydrated in 100%, 95%, and 70% alcohol, and finally in phosphate-buffered saline (pH, 7.2). Free aldehydes were reduced with 0.05% sodium borohydride in phosphate-buffered saline (pH, 7.2) for 30 minutes at 4 C. Sections were then sequentially incubated with normal swine serum, 7E12H12 monoclonal antibody, or control murine IgM monoclonal antibody, biotinylated swine antimouse IgM (Dakopatts; Carpinteria, California), hydrogen peroxide solution (3%), and streptavidin-peroxidase (Dakopatts), respectively. Tissue sections were washed in phosphate-buffered saline, treated with 3-3 diaminobenzidene hydrochloride (50 g/150 mL of 0.5 mol/L TRIS-buffer; pH, 7.2) for 30 minutes. The sections were washed, counter stained in hematoxylin or toluidine blue for 1 minute, dehydrated in graded ethanol solutions and then in xylene, and mounted for microscopic examination. The presence of clear brown staining of the tissue was graded as positive and its absence as negative. Results The location and histologic diagnosis of the 116 specimens collected retrospectively and prospectively from 53 patients are shown in Table 1. The mean age of these patients was 57 years for the retrospective group and 51 years for the prospective group (range, 14 to 85 years). Of 22 biopsy specimens, 21 (95%) with established diagnoses of specialized Barrett epithelium reacted with the 7E12H12 monoclonal antibody (Figure 1 C, Table 1). These included 19 of 20 retrospective specimens and 2 of 2 prospective specimens. Among these 21 positive patients, three biopsy specimens were taken from the esophagus at 20 to 25 cm, four from 25 to 30 cm, and 14 from 30 cm or below from the incisor teeth, as defined by the endoscopist. The one biopsy specimen that did not react with 7E12H12 monoclonal antibody was from the distal esophagus lower than 30 cm. Figure 1. The immunoreactivity of 7E12H12 monoclonal antibody against the specialized type of Barrett epithelium and colonic mucosal epithelium as shown by the immunoperoxidase assay. A and B. arrowheads C. D and E. F. G and H. I. arrows A and B C D to I The specialized columnar epithelial cells of Barrett epithelium reacted strongly with 7E12H12 monoclonal antibody (Figure 1 C). The reactivity was more intense in the periphery of the cells (probably the membrane area compared with the cytoplasm). The 7E12H12 monoclonal antibody also reacted with some of the goblet cells, including their contents and basolateral regions of the cells. Each of the 11 specimens from normal gastroesophageal junction (squamocolumnar junction) was negative when reacted with the 7E12H12 monoclonal antibody (Figure 1 D and E). The availability of six operative specimens (from patients with cancer) from this area allowed us to obtain multiple tissue samples to evaluate both structure and immunoperoxidase staining. All of the 16 esophageal tissue specimens with normal squamous epithelium were negative when reacted with 7E12H12 monoclonal antibody (Figure 1 C, D, and E). Twelve specimens (six from distal esophagus and six from squamocolumnar junction) obtained from patients with endoscopic and histologic diagnosis of active esophagitis did not react with 7E12H12 monoclonal antibody. Each of the 12 cases of adenocarcinoma arising in Barrett epithelium reacted with 7E12H12 monoclonal antibody and the staining was intense, mostly cytoplasmic (Figure 1 G and H). However, 12 of the 13 esophageal squamous cell carcinomas did not react with 7E12H12 monoclonal antibody (Figure 1)I. Only one specimen showed some focal and patchy staining in the tumor. Normal colonic biopsy specimens that were examined in parallel during each experiment as positive controls consistently reacted with the monoclonal antibody (Figure 1 F). None of the 21 specimens from sites of the stomach (cardia, fundus, body, and antrum), duodenum, and jejunum reacted with the 7E12H12 monoclonal antibody. Discussion Our study is the first report of a unique epitope shared between normal colonic mucosa and distinct specialized type of Barrett epithelium. Most cases of Barrett epithelium have a histologic similarity with incomplete intestinal metaplasia or small-bowel-like histologic findings and occasional gastric or fundic type of epithelium. However, 7E12H12 failed to react with small-bowel or gastric mucosa, confirming previous reports by us [8] and others [10], using both immunoperoxidase and immunofluorescence assays. These data suggest a histogenetic relation between specialized Barrett epithelium and colonic-type epithelium. The origin of specialized Barrett epithelial cells in distal esophagus has been debated because no specific marker distinguishes Barrett epithelium. Metaplastic columnar epithelial cells of Barrett epithelium develop at higher frequency in the distal esophagus as a complication of reflux esophagitis [1, 2]. The cases that show the intestinal type histologic pattern tend to progress to adenocarcinoma at relatively higher frequency than other types of Barrett epithelia [11]. This type of Barrett epithelium has a colon epithelial phenotype. Several independent groups of investigators have reported a higher frequency of colonic neoplasia in patients with Barrett epithelium than in controls [12-14]. The reason for this clinical association is unknown. However, it is intriguing that a common epitope detected by the 7E12H12 monoclonal antibody is shared in these two tissues. Data from both the retrospective and prospective specimens show that the monoclonal antibody 7E (12H)12 reacts frequently (21 of 22) with the specialized type of benign Barrett epithelium but not with any other tissue systematically sampled from different sites of normal esophagus (including gastroesophageal junction); cardia, fundus, body, and antrum of the stomach; duodenum and jejunum. Further, the 7E12H12 monoclonal antibody reacted with all 12 cases of adenocarcinoma derived from Barrett epithelium; however, it did not react with the esophageal squamous cell carcinomas. In addition to its potential as a new tool to study the histogenesis of Barrett epithelium, the 7E12H12 monoclonal antibody may be valuable in the diagnosis of Barrett epithelium. Endoscopic identification of the transition zone can be difficult, especially when the gastric mucosa extends into the distal esophagus and the structure of the gastric mucosa blends with the esophageal mucosa, which occurs in active esophagitis. Although some aids for endoscopic identification of gastroesophageal junction have been described [15, 16], the endoscopist may find that the area of diaphragmatic compression, gastroesophageal junction, and the gastroesophageal muscular junction are not identical. Because the immunoreactive epitope recognized by the 7E12H12

Stent-assisted coiling of wide-necked aneurysms in the setting of acute subarachnoid hemorrhage: experience in 65 patients.

BACKGROUND Stent-assisted coiling in the setting of subarachnoid hemorrhage remains controversial. Currently, there is a paucity of data regarding the utility of this procedure and the risks of hemorrhagic and ischemic complications. OBJECTIVE To assess the utility of stent-assisted coil embolization and pretreatment with antiplatelet agents in the management of ruptured wide-necked aneurysms. METHODS A retrospective study of 65 patients with ruptured wide-necked aneurysms treated with stent-assisted coiling. Patients with hydrocephalus or a Hunt and Hess grade ≥ III received a ventriculostomy before endovascular intervention. Patients were treated intraoperatively with 600 mg of clopidogrel and maintained on daily doses of 75 mg of clopidogrel and 81 mg of aspirin. The Glasgow outcome scale (GOS) score was recorded at the time of discharge. We identified major bleeding complications secondary to antiplatelet therapy and cases of in-stent thrombosis that required periprocedural thrombolysis. RESULTS Of the aneurysms, 66.2% arose within the anterior circulation; 69.2% of patients presented with hydrocephalus or a Hunt and Hess grade ≥ III and required a ventriculostomy. A good outcome (GOS of 4 or 5) was achieved in 63.1% of patients, and the overall mortality rate was 16.9%. There were 10 (15.38%) major complications associated with bleeding secondary to antiplatelet therapy (5 patients, 7.7%) or intraoperative in-stent thrombosis (5 patients, 7.7%). Three (4.6%) patients had a fatal hemorrhage. CONCLUSION Our findings suggest that stent-assisted coiling and routine treatment with antiplatelet agents is a viable option in the management of ruptured wide-necked aneurysms.

Expression and potential role of the extracellular matrix in hepatic ontogenesis: A review

Studies from a number of laboratories have provided information on the temporal and spatial expression of a variety of extracellular matrix (ECM) components in the developing liver and insight into their potential roles in hepatogenesis. Collagen type IV and laminin are present in the basement membranes of the capsular mesothelium, vascular structures of the portal and hepatic vein branches, and the ductular elements of the developing liver. The mesothelial, vascular, and ductular epithelial cells synthesize laminin and type IV collagen. In contrast, fibronectin and type I collagen are restricted to the adjacent or surrounding interstitium of those ductal and vascular elements, but are not within the basement membrane proper.

GROα Is Highly Expressed in Adenocarcinoma of the Colon and Down-Regulates Fibulin-1

Purpose: The growth-related oncogene α (GROα) is a secreted interleukin-like molecule that interacts with the CXCR2 G-protein–coupled receptor. We found that the mRNA and protein products of GROα are more highly expressed in neoplastic than normal colon epithelium, and we studied potential mechanisms by which GROα may contribute to tumor initiation or growth. Experimental Design: Cell lines that constitutively overexpress GROα were tested for growth rate, focus formation, and tumor formation in a xenograft model. GROα expression was determined by Affymetrix GeneChip (241 microdissected colon samples), real-time PCR (n = 32), and immunohistochemistry. Primary colon cancer samples were also employed to determine copy number changes and loss of heterozygosity related to the GROα and fibulin-1 genes. Results: In cell cultures, GROα transfection transformed NIH 3T3 cells, whereas inhibition of GROα by inhibitory RNA was associated with apoptosis, decreased growth rate, and marked up-regulation of the matrix protein fibulin-1. Forced expression of GROα was associated with decreased expression of fibulin-1. Expression of GROα mRNA was higher in primary adenocarcinomas (n = 132), adenomas (n = 32), and metastases (n = 52) than in normal colon epithelium (P < 0.001). These results were confirmed by real-time PCR and by immunohistochemistry. Samples of primary and metastatic colon cancer showed underexpression of fibulin-1 when compared with normal samples. There were no consistent changes in gene copy number of GROα or fibulin-1, implying a transcriptional basis for these findings. Conclusion: Elevated expression of GROα is frequent in adenocarcinoma of the colon and is associated with down-regulation of the matrix protein fibulin-1 in experimental models and in clinical samples. GROα overexpression abrogates contact inhibition in cell culture models, whereas inhibition of GROα expression is associated with apoptosis. Importantly, coexpression of fibulin-1 with GROα abrogates key aspects of the transformed phenotype, including tumor formation in a murine xenograft model. Targeting GRO proteins may provide new opportunities for treatment of colon cancer.