Monoclonal antibody PAL-E specific for endothelium

A monoclonal antibody, PAL-E, is described that is specific for endothelial cells. The monoclonal antibody, an IgG2a, markedly stains endothelium of capillaries, medium-sized and small veins, and venules in frozen sections of human and some animal tissues tested. It reacts not at all or only weakly with endothelium of large, medium-sized, and small arteries, arterioles, and large veins and does not stain the endothelial lining of lymphatic vessels and sinus histiocytes. The cellular staining pattern and tissue staining were different from those obtained with antifactor VIII R:AG antiserum and Ulex europaeus I lectin. Blocking experiments indicated that these three reagents recognize different endothelial binding sites. Therefore, PAL-E is a new staining reagent for endothelium in frozen sections. Based on immunoelectronmicroscopic observations, the antigenic determinant recognized by PAL-E is associated with endothelial vesicles.     

An immunohistochemical study of mesothelial cell seeding for knitted Dacron.

Six greyhounds underwent bilateral femoral artery replacement with knitted Dacron, one side seeded with omental digest at graft preclotting, the other acting as an unseeded control. Grafts were removed at 24 hours and two months. Tissue was examined using a monoclonal antibody (MNF116) directed against a broad range of human cytokeratins to differentiate mesothelial cells (MC) from microvascular endothelial cells (MEC), which stained only with a polyclonal antibody directed against von Willebrand Factor (anti-vWF). Cells released from omentum by collagenase stained with MNF116 and reacted poorly with anti-vWF. Identical cells were observed to be within the interstices of seeded but not control knitted Dacron. Few remained in seeded grafts (n = 2) removed at 24 hours and none at two months (n = 4).     

Adenomatoid tumours: an immunohistochemical and ultrastructural appraisal of their histogenesis

The histogenesis of adenomatoid tumour has continued to provoke debate since Golden and Ash suggested the term in 1945 for a characteristic benign neoplasm typically found in the uterus, fallopian tube or epididymis. Endothelial, epithelial, mesonephric, müllerian and mesothelial histogenesis have been suggested. The balance of evidence suggests mesothelial derivation, but two recent studies point to endothelial origin for at least some of these tumours. Twenty-two histologically typical adenomatoid tumours were studied by electron microscopy, mucin histochemistry and immunohistochemistry. Ultrastructurally, all cases showed vacuolated cells bearing long bushy microvilli and the features were not those of endothelial cells. Glandular spaces contained acid mucopolysaccharide consistent with hyaluronic acid. Immunohistochemical double labelling techniques showed the cells lining such spaces to contain cytokeratin in the absence of factor VIII related antigen and receptors for Ulex europaeus I lectin which were expressed in the endothelium of tumour blood vessels. The evidence points to mesothelial histogenesis in all cases examined.     

Gastrointestinal stromal tumors and KIT-positive mesenchymal cells in the omentum

Gastrointestinal stromal tumor (GIST) is currently considered to be derived from the interstitial cells of Cajal (ICC). To test the hypothesis that omental mesenchymal tumor is also a type of GIST, we evaluated the expression of specific molecules in GIST, and c-kit gene mutation in omental mesenchymal tumors, and we identified a possible counterpart of ICC in the omentum. Immunohistochemically, all of the omental mesenchymal tumors (n = 5) were positive for both KIT and CD34, and three of the five tumors were also positive for an embryonic form of smooth-muscle myosin heavy chain (SMemb). Polymerase chain reaction-single-strand conformational polymorphism analysis (PCR-SSCP) and direct sequencing revealed mutations in c-kit gene exon 11 in all five tumors. As for the ICC counterparts in the omentum, there were some KIT-positive mesenchymal cells resembling ICC at the surface of the omentum. Double fluorescence immunostaining, using anti-KIT polyclonal antibodies and monoclonal antibodies against other molecules, demonstrated that KIT-, CD34- and SMemb-positive cells were present just beneath the mesothelial cells of the omentum. These results show that omental mesenchymal tumor corresponds to GIST of the omentum, and that KIT-positive bipolar mesenchymal cells may be a counterpart of ICC in the gastrointestinal tract. Identification of a new type of KIT-positive mesenchymal cell in the omentum may lead to the discovery of a new physiological role for this organ.     

Lobular capillary hemangioma. An immunohistochemical study including steroid hormone receptor status.

Twenty-one lobular capillary hemangiomas (LCH), including lesions from six pregnant patients, were examined by immunohistochemical analysis. Antibodies to estrogen and progesterone receptor proteins were used to determine whether these steroid hormones play a direct role in LCH development and growth. All 21 LCHs were negative for both receptor proteins. Contrary to the association of LCH with pregnancy and oral contraceptive use, the absence of these steroid receptors strongly suggests that estrogen or progesterone are not directly involved in the formation of these lesions. All 21 LCHs were stained with Ulex europaeus lectin and with a panel of antibodies directed against cytokeratin, vimentin, Factor VIII, collagen Type IV, and muscle-specific actin. Endothelial cells in LCH, both in cellular proliferations with poorly formed lumens and in well-formed capillaries, were labeled by Factor VIII, Ulex europaeus lectin, and vimentin. A population of concentrically arranged, perivascular spindle-shaped cells was highlighted by positive staining for muscle-specific actin, collagen Type IV, and vimentin. The spindled cells were associated with both well-developed and immature vessels in all lesions. The appearance, immunophenotype, and location of these cells is consistent with a pericytic origin. Although the intimate association of both endothelial cells and pericytes suggests a reactive, as opposed to neoplastic, origin, other benign vascular tumors traditionally considered neoplastic have a similar bicellular composition. Accordingly, the findings neither support nor refute a neoplastic origin for LCH.     

Adrenomedullin is an autocrine regulator of endothelial growth in human endometrium

Human endometrium is a mucosa served by a microvascular blood supply that involves benign angiogenesis under the control of ovarian steroids throughout reproductive life. Adrenomedullin is a multifunctional 52-amino acid peptide involved in numerous physiological and pathological processes, including angiogenesis, growth regulation, differentiation, vasodilation and smooth muscle relaxation. We have previously shown that adrenomedullin is present in the human uterus. To investigate further the role of adrenomedullin in human endometrial angiogenesis, a method for the isolation and culture of non-pregnant endometrial endothelium was developed. Enzymatic dispersion and 'Percoll' gradient centrifugation, followed by positive selection using Ulex europaeus agglutinin-coated immunomagnetic beads, yielded pure isolates of endothelium. The cells formed a typical 'cobblestone' monolayer within 5-7 days and expressed the classic endothelial markers, CD31 and von Willebrand factor. The presence of adrenomedullin immunoreactivity in endometrial endothelial cells was shown by immunohistochemistry both in vitro and in vivo. Adrenomedullin promotes growth of endothelial cells as measured by [methyl-(3)H] thymidine uptake. Adrenomedullin also induced cyclic AMP in endometrial endothelial cells. These results demonstrate, for the first time, that adrenomedullin is an autocrine growth factor for human endometrial endothelial cells and is thus involved in endometrial angiogenesis.     

Endothelial markers in malignant vascular tumours of the liver: superiority of QB-END/10 over von Willebrand factor and Ulex europaeus agglutinin 1.

A new monoclonal antibody, QB-END/10, raised against the CD34 antigen in human endothelial cell membranes and haemopoietic progenitor cells, was studied for its usefulness as a marker of neoplastic vascular cells in 21 angiosarcomas and seven malignant haemangioendotheliomas of the liver. QB-END/10 was both more sensitive and more specific than Von Willebrand factor (VWF) and Ulex europaeus 1 agglutinin (UEA-1) in labelling endothelial cells and it did not cross react with epithelia as UEA-1 often does. Staining was uniformly strong and clear in all histological variants of these two tumours. QB-END/10 should prove particularly useful in the differential diagnosis of malignant vascular tumours of the liver.     

Immunocytochemical typification of mesothelial cells in effusions: In vivo and in vitro models

We have performed immunocytochemical, immunoelectron microscopy. Western blot, and culture techniques using monoclonal antibodies against cytokeratin, vimentin, and desmin on 17 benign and 20 malignant effusions of pleural and ascitic origin. Triple coexpression of these three antigens was observed in benign reactive mesothelial cells as well as in one case of mesothelioma. All metastatic adenocarcinoma cells were consistently negative to desmin and positive to cytokeratin and vimentin. Present results were helpful to distinguish reactive and malignant mesothelioma from metastatic carcinoma cells in effusions. © 1994 Wiley-Liss, Inc.     

Angiosarcoma of the larynx

The morphological and immunohistochemical features of a laryngeal angiosarcoma are described. Initially, the tumour was interpreted as a poorly differentiated squamous cell carcinoma. The laryngectomy specimen contained an extensive and haemorrhagic tumour consisting of irregular and dissecting vascular spaces delineated by pleomorphic endothelial cells. In addition to these obvious angiosarcomatous areas, islands of more compact growing tumour cells were present, reminiscent of a poorly differentiated squamous cell carcinoma. On immunohistochemistry, the tumour cells expressed factor VIII, Ulex europaeus I lectin, CD31 and vimentin. There was no expression of cytokeratin or epithelial membrane antigen. Angiosarcoma of the larynx is very rare and should be differentiated from a pseudo-angiosarcomatous carcinoma.     

Stromal cells in cerebellar haemangioblastomas: an immunocytochemical study

The nature of the stromal cells in formalin-fixed paraffin-embedded material from 23 cerebellar haemangioblastomas was investigated using antisera to intermediate filaments (glial fibrillary acidic protein, vimentin and desmin), histiocytic markers (alpha 1-antitrypsin, alpha 1-antichymotrypsin and lysozyme), glycolytic enzymes (alpha and gamma enolase and aldolase C4) and the endothelial markers, factor VIII related antigen and Ulex europaeus I lectin. Most stromal cells stained positively for vimentin and the glycolytic enzymes. Occasional process-bearing cells within the stroma stained strongly for glial fibrillary acidic protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. No stromal cell staining for desmin, lysozyme or the endothelial markers was observed, although the latter stained the vascular endothelium within all neoplasms. The findings do not support previous suggestions of an endothelial or histiocytic origin for the stromal cells. They appear to be a heterogeneous population including entrapped reactive astrocytes and locally-derived non-angiogenic cells of neuroectodermal (pial) origin.     

Heterogenous expression of endothelial cell markers in infantile hemangioendothelioma. Immunohistochemical study of two solitary cases and one multiple one

Three cases of infantile hemangioendothelioma were immunohistochemically studied with the use of antibodies against von Willebrand factor (vWF), Ulex europaeus I lectin (UEA I), vimentin, thrombomodulin (TM), and actin, as endothelial cell (EC) markers. Because of a broad variety of histologic features, the growth pattern of the tumor cells was subclassified into the following four subtypes: capillary, sinusoidal, cavernous, and myxomatous parts. The solitary tumor from patients 1 and 2 was composed of these four components, but the multiple tumor in the patient 3 consisted of capillary and sinusoidal parts. Immunohistochemically, vWF and vimentin were dominantly expressed in the ECs of the cavernous and myxomatous parts. UEA I had strongly positive results in all histologic types, except the myxomatous part. Expression of vWF and vimentin in neoplastic EC suggests that functional differentiation of the tumor tissue occurs in the myxomatous and cavernous parts and may be related to the spontaneous regression of the infantile hemangioendothelioma.     

The histogenesis of angiosarcoma of the face and scalp: an immunohistochemical and ultrastructural study

Thirteen cases of angiosarcoma of the face and scalp have been examined using immunohistochemistry and electron microscopy. Endothelial cell markers have been employed in an immunoperoxidase technique on tissue that has either been routinely processed, periodate-lysine paraformaldehyde fixed (PLP) and cold processed, or fixed in methacarn. A consistent pattern of endothelial cell labeling was only achieved in the PLP fixed tissue. In this fixative the angiosarcomas were factor VIII related antigen negative, Ulex europaeus lectin positive, laminin positive, unlabelled by the monoclonal antibody PAL-E, and positively labelled by the monoclonal antibody EN4. Ultrastructural examination of four cases showed evidence of vascular lumina in all tumours. Weibel-Palade bodies were seen in only one case but three tumours showed some evidence of tight junction formation and marginal folding. Thus, our cell marker studies can be interpreted as consistent with a lymphatic derivation for this type of angiosarcoma but in contra-distinction the ultrastructural studies showed tumour channels with features suggestive of blood vessel differentiation.     

Sclerosing hemangioma of the lung. An immunohistochemical study of intermediate filaments and endothelial markers

Three cases of so-called pulmonary sclerosing hemangioma have been studied for endothelial markers (alkaline phosphatase, adenosine triphosphatase, factor VIII-related antigen, and Ulex europaeus I lectin), for intermediate filaments (keratin, vimentin), and for carcinoembryonic and epithelial membrane antigen. Not one of the neoplasms expressed endothelial markers, carcinoembryonic antigen, or keratin reactivity. The tumor cells showed a positive reaction for epithelial membrane antigen and vimentin. The findings exclude an endothelial origin for this group of tumors and favored an epithelial origin as the probable genesis of the neoplastic proliferation.     

Role of mesothelial and submesothelial stromal cells in matrix remodeling following pleural injury.

The pleural response to injury is a complex and poorly understood multifactorial process that can result in the development of fibrosis or obliteration of the pleural space. Pleural fibroblasts are considered the main source of extracellular matrix but cell culture studies have demonstrated synthesis of matrix components by mesothelial cells. We assessed the mesothelial cell contribution to extracellular matrix during pleural healing using immunohistochemical technique. Paraffin-embedded tissue of 3 normal adult lungs and 7 adults with active pleuritis were studied using monoclonal antibodies to cytokeratin, type IV collagen, vimentin, and type I procollagen (PCI). Normal pleural had a single layer of cytokeratin-positive and PCI-negative mesothelium over a thin, continuous type IV collagen-positive basement membrane and PCI-negative submesothelial stroma. Areas of active pleuritis showed loss of the continuous linear staining with anti-type IV collagen antibody. Coexpression of cytokeratin, vimentin and PCI was identified in spindle and/or cuboidal cells located in the fibrin layer, submesothelial connective tissue layer, or on the pleural surface. These findings suggest that reactive mesothelial cells play an active role in the production of extracellular matrix during pleural injury, and that disruption of the submesothelial basement membrane is a key event in determining subsequent fibrous organization of pleural exudate.     

Culture and characterization of microvascular endothelial cells derived from human brain

Primary cultures of human cerebral endothelial cells were established from microvessels isolated from cortical fragments removed at surgery for seizure disorder and from brains at autopsy. A uniform population of cells growing in close association to each other formed confluent monolayers by 7 to 10 days in culture. They contained factor VIII/Von Willebrand antigen, the most specific marker for cells of endothelial origin, and showed lectin-binding sites for Ulex europaeus agglutinin characteristic of human endothelium. Cultured cells formed thin, continuous monolayers, contained few pinocytotic vesicles, and were joined together by tight junctional complexes. More than 99% of the intercellular junctions restricted the transendothelial passage of horseradish peroxidase. Monolayers of human brain microvessel endothelial cells thus resemble cerebral endothelium in vivo and should provide a useful in vitro model for studies of the biology of these cells and their role in the pathogenesis of certain human central nervous system diseases associated with abnormal blood-brain barrier function.     

Nephroblastoma. A comparative immunocytochemical and lectin-histochemical study

Nephroblastomas (Wilms' tumors) from a dog, a bird, a pig, and a child were subjected to comparative immunocytochemical and lectin-histochemical analysis along with normal renal tissues from the same animals. Primary rabbit and mouse anti-human antibodies directed at intermediate-filament proteins, neuron-specific enolase, S100 protein, and epithelial membrane antigen were employed, as were biotinylated peanut agglutinin, soybean agglutinin, wheat germ agglutinin, and lectins from Dolichos biflorus and Ulex europaeus. The human neoplasm showed positivity for cytokeratin and epithelial membrane antigen and bound peanut, soybean, and wheat germ agglutinins in epithelial areas. Among the animal tumors, the porcine and canine nephroblastomas showed immunoreactivity for cytokeratin and vimentin in epithelial and blastematous areas, respectively. In addition, they were positive for S100 protein in epithelial foci. All of these results were substantiated by staining patterns in nonhuman kidneys. None of the neoplasms or renal tissues showed reactivity to the other antigens that were assessed. In the porcine tumor, endothelial cells bound D biflorus lectin, and epithelial areas were stained by U europaeus lectin. The avian nephroblastoma bound peanut, soybean, and wheat germ agglutinins, while the canine neoplasm showed no lectin-histochemical reactivity. These data appear to reflect limited immunohistological and histochemical similarities between nephroblastomas of different vertebrates.     

QBEND/10, a new monoclonal antibody to endothelium: assessment of its diagnostic utility in paraffin sections

The immunoreactivity of a new monoclonal antibody to endothelium. QBEND/10, in formalin-fixed, paraffin-embedded sections from a variety of vascular and lymphatic tumours is described and compared to that of two other endothelial markers, von Willebrand factor and Ulex europaeus agglutinin, type 1. All the benign tumours of blood vascular origin showed immunoreactivity whereas only five out of eight lymphangiomas demonstrated a weak focal reaction with QBEND/10. Primitive lumina in epithelioid and spindle cell haemangioendotheliomas were highlighted in all the cases. Tumour cells in angiosarcoma forming vasoformative areas and solid areas showed immunopositivity to QBEND/10 in 17/23 and 13/24 cases respectively, and complementary immunoreactivity for von Willebrand factor was observed. Proliferating vessels and the majority of spindle cells in Kaposi's sarcoma were positive in all 40 cases. Only one of 54 cases of carcinoma showed luminal reaction to QBEND/10. However, 17 of 45 spindle cell tumours displayed a positive reaction. QBEND/10 is an additional marker for demonstrating endothelial differentiation and has some advantages over currently available antibodies.     

Malignant peritoneal mesothelioma in childhood

We present a case of diffuse malignant peritoneal mesothelioma, initially misdiagnosed as benign. Electron microscopy and immunocytochemistry proved helpful diagnostically. Using monoclonal antibodies against cytokeratin and vimentin, we compared neoplastic with normal and reactive mesothelia and we found coexpression of these two intermediate filaments in the reactive and neoplastic mesothelial but not in the normal mesothelia, supporting the suggestion that surface mesothelial cells are derived from multipotential submesothelial cells.     

Endothelial cell markers in vascular neoplasms: an immunohistochemical study comparing factor VIII-related antigen, blood group specific antigens, 6-keto-PGF1 alpha, and Ulex europaeus 1 lectin

Markers for endothelial cells including Ulex europaeus 1 lectin, blood group A, B, and H, and the prostaglandin metabolite 6-keto-PGF1 alpha were evaluated in paraffin secretions from formalin-fixed benign and malignant vascular neoplasms using a variety of immunohistochemical techniques, and results compared with staining for factor VIII-related antigen. Staining for Ulex appeared more sensitive than factor VIII-related antigen in identifying poorly differentiated neoplasms including haemangiosarcomas and spindle cell proliferations in Kaposi's sarcoma. Staining for blood group related antigens correlated with blood group in all cases. Ulex europaeus 1 lectin was the only marker for endothelial cells in lymphangiomas.     

Coexpression of intermediate filament polypeptides in human fetal and adult tissues

Tissues from human fetuses (12 to 14 weeks) were studied by using immunohistochemical methods, with special emphasis on coexpression of intermediate filaments. Well-characterized antibodies, monoclonal as well as polyclonal were used. Indirect immunoperoxidase staining disclosed simultaneous expression of cytokeratin and vimentin, or vimentin and desmin in several tissues, whereas some other tissues coexpressed three classes of intermediate filaments, i.e., cytokeratin, vimentin, and desmin. Coexpression of cytokeratin and vimentin was seen in immature tubules of the kidney localized in the blastema and in one case in a small area of the epithelium of the tongue. Coexpression of vimentin and desmin was found in stromal cells of the medulla of the kidney, in stromal cells of the decidua (maternal tissue) and in muscle cells in blood vessels of small intestine, kidney and decidua. Coexpression of cytokeratin, vimentin, and desmin was present in mesothelial cells of serosa, pleura and pericardium, in stroma of umbilical cord and placental villi, in muscle cells of small intestine, tongue, and heart, and in muscle cells of blood vessels of lung, heart, umbilical cord, and placental villi. Mesothelium and reactive submesothelial stroma cells also coexpressed three classes of intermediate filament polypeptides. In some cases, immunoperoxidase results were confirmed by double labeling immunofluorescence microscopy or by immunoblotting experiments. The results of this study indicate that coexpression of different types of intermediate filaments is a more general phenomenon in fetal tissues than previously realized and it also occurs in some reactive proliferative lesions in the adult.