Allen M. Gown

Characterization of the early lesion of 'degenerative' valvular aortic stenosis. Histological and immunohistochemical studies.

BackgroundNonrheumatic stenosis of trileaflet aortic valves, often termed senile or calcific valvular aortic stenosis, is considered a “degenerative” process, but little is known about the cellular or molecular factors that mediate its development. Methods and ResultsTo characterize the developing aortic valvular lesion, we performed histological and immunohisto-chemical studies on Formalin-fixed and methanol-Carnoys-fixed parafflin-embedded aortic valve leaflets or on frozen sections obtained at autopsy from 27 adults (age, 46 to 82 years) with normal leaflets (n=6), mild macroscopic leaflet thickening (n= 15), or clinical aortic stenosis (n=6). Focal areas of thickening (“early lesions”) were characterized by (1) subendothelial thickening on the aortic side of the leaflet, between the basement membrane (PAS-positive) and elastic lamina (Verhoeff–van Gieson), (2) the presence of large amounts of intracellular and extracellular neutral lipids (oil red O) and fine, stippled mineralization (von Kossa), and (3) disruption of the basement membrane overlying the lesion. Regions of the fibrosa adjacent to these lesions were characterized by thickening and by protein, lipid, and calcium accumulation. Control valves showed none of these abnormalities. Immunohistochemical studies were performed using monoclonal antibodies directed against macrophages (anti-CD68 or HAM-56), and contractile proteins of smooth muscle cells or myofibroblasts (anti-α-actin and HHF-35) or rabbit polyclonal antiserum against T lymphocytes (anti-CD3). In normal valves, scattered macrophages were present in the fibrosa and ventricularis, and occasional muscle actin-positive cells were detected in the proximal portion of the ventricularis near the leaflet base, but no T lymphocytes were found. In contrast, early lesions were characterized by the presence of an inflammatory infiltrate composed of non-foam cell and foam cell macrophages, occasional T cells, and rare α-actin-positive cells. In stenotic aortic valves, a similar but more advanced lesion was seen. ConclusionsThe early lesion of “degenerative” aortic stenosis is an active inflammatory process with some similarities (lipid deposition, macrophage and T-cell infiltration, and basement membrane disruption) and some dissimilarities (presence of prominent mineralization and small numbers of smooth muscle cells) to atherosclerosis.

Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation.

Mesangial cell proliferation is common in glomerulonephritis but it is unclear if proliferation is associated with any in vivo alteration in phenotype. We investigated whether mesangial of mesangial proliferative nephritis induced with antibody to the Thy-1 antigen present on mesangial cells. At day 3 glomeruli displayed de novo immunostaining for alpha-smooth muscle actin in a mesangial pattern, correlating with the onset of proliferation, and persisting until day 14. An increase in desmin and vimentin in mesangial regions was also noted. Immunoelectron microscopy confirmed that the actin-positive cells were mesangial cells, and double immunolabeling demonstrated that the smooth muscle actin-positive cells were actively proliferating. Northern analysis of isolated glomerular RNA confirmed an increase in alpha and beta/gamma actin mRNA at days 3 and 5. Complement depletion or platelet depletion prevented or reduced proliferation, respectively; these maneuvers also prevented smooth muscle actin and actin gene expression. Studies of five other experimental models of nephritis confirmed that smooth muscle actin expression is a marker for mesangial cell injury. Thus, mesangial cell proliferation in glomerulonephritis in the rat is associated with a distinct phenotypic change in which mesangial cell assume smooth muscle cell characteristics.

Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.

Haemophilia B, or factor IX deficiency, is an X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5–30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery1. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral2 and adenoviral3 vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression3,4. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells5. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues6‐12. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.

PDGF B-chain in neurons of the central nervous system, posterior pituitary, and in a transgenic model.

Platelet-derived growth factors (PDGFs) are growth-regulatory molecules that stimulate chemotaxis, proliferation, and increased metabolism of primarily connective tissue cells. In a survey of normal tissues, we found specific immunostaining for PDGF B-chain in neurons, principal dendrites, some axons, and probable terminals throughout the brain, in the dorsal horn of the spinal cord, and in the posterior pituitary of a nonhuman primate (Macaca nemestrina). PDGF activity was extracted from brain cortex and posterior pituitary, and ubiquitous expression of transcripts for the two chains of PDGF and both PDGF receptors was detected throughout the brain and posterior pituitary. A transgenic model was also evaluated in which the chloramphenicol acetyltransferase gene was placed under transcriptional control of the PDGF B-chain promoter. The transgene was preferentially expressed within neural cell bodies in the cortex, hippocampus, and cerebellum. PDGF may act as a neuronal regulatory agent. Neuronal release of PDGF could contribute to nerve regeneration and to glial proliferation that leads to gliosis and scarring.

Detection of Chlamydia pneumoniae in aortic lesions of atherosclerosis by immunocytochemical stain.

Recent evidence has shown the presence of Chlamydia pneumoniae antigens and nucleic acid in coronary artery atheromas from autopsy patients in South Africa. In this study, the immunocytochemical technique was used to demonstrate C pneumoniae antigens in atheromas of the aorta in autopsy patients from retrospective aortic atherosclerosis studies at the University of Washington. The patients were 34 to 58 years old. Immunoperoxidase staining using Chlamydia-specific monoclonal antibodies showed one of four fatty streaks and six of 17 fibrous plaques were positive for C pneumoniae antigens; four control aortic tissues were negative. Two of the positive plaques were from the same patient. Double-label immunocytochemical staining using Chlamydia- and tissue type-specific monoclonal antibodies demonstrated the antigens in the cytoplasm of macrophages and smooth muscle cells in the atheromatous lesion. This study suggested a wider involvement of C pneumoniae organisms in atherosclerotic lesions of the arterial system than has previously been documented.

Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray.

The development of cancer is the result of a series of molecular changes occurring in the cell. These events lead to changes in the expression level of numerous genes that result in different phenotypic characteristics of tumors. In this report we describe the assembly and utilization of a 5766 member cDNA microarray to study the differences in gene expression between normal and neoplastic human ovarian tissues. Several genes that may have biological relevance in the process of ovarian carcinogenesis have been identified through this approach. Analyzing the results of microarray hybridizations may provides new leads for tumor diagnosis and intervention.

Fatty streak initiation in Watanabe Heritable Hyperlipemic and comparably hypercholesterolemic fat-fed rabbits.

Morphologic and immunocytochemical studies were conducted to determine the sequence of cellular interactions that occur during the initiation of the fatty streak in the aorta of Watanabe Heritable Hyperlipemic rabbits and comparably hypercholesterolemic fat-fed rabbits. Watanabe rabbits from 3.5 weeks gestation to 2 months of age and fat-fed rabbits from 1 week to 2 months duration of hypercholesterolemia were compared utilizing light microscopic and scanning and transmission microscopic techniques. In both groups of animals, the earliest detectable events were an increase in monocyte adherence and subendothelial migration followed by formation of a single layer of intimal macrophage-derived foam cells. Immunocytochemical studies using macrophage-specific and muscle-actin-specific monoclonal antibodies support the morphologic data which suggests that the early fatty streak in both the Watanabe and fat-fed rabbits is predominantly composed of macrophage-derived foam cells. Thus, the absence of functional low density lipoprotein receptors in the Watanabe rabbit and differences in the distribution of cholesterol among the lipoproteins in the Watanabe and fat-fed rabbits do not appear to alter the initial responses of the cells of the artery wall to chronic hypercholesterolemia.

Immunocytochemical analysis of cellular components in atherosclerotic lesions. Use of monoclonal antibodies with the Watanabe and fat-fed rabbit.

We have performed immunocytochemical investigations into the distribution of smooth muscle cells and macrophages in the atherosclerotic lesions of the Watanabe heritable hyperlipidemic (WHHL) and fat-fed rabbits. We used monoclonal antibodies specific for muscle cells and macrophages, the latter with a new macrophage-specific monoclonal antibody designated RAM11. Scattered macrophages were observed in the subendothelium in areas of grossly normal aorta. Raised lesions were divided into four major groups, based on qualitative aspects of cell localization: fatty streak, composed predominantly of macrophages; intimal thickening, composed predominantly of smooth muscle cells that displayed considerable morphological heterogeneity with an admixture of macrophages; early fibrous plaques, characterized by approximately equal numbers of smooth muscle cells and macrophages; advanced fibrous plaques, composed of a fibrous cap containing flat smooth muscle cells overlying a macrophage-rich zone of atheromatous debris. These studies demonstrate the nature of the lesions in the two rabbit models and the usefulness of monoclonal antibodies in analyzing the cellular composition of atherosclerotic lesions.

Fatty streak expansion and maturation in Watanabe Heritable Hyperlipemic and comparably hypercholesterolemic fat-fed rabbits.

This study focuses on the expansion and maturation of the fatty streak in the aorta of Watanabe Heritable Hyperlipemic rabbits and comparably hypercholesterolemic fat-fed rabbits between 2 and 6 months duration of hypercholesterolemia. In both groups of animals, the fatty streaks expanded due to: 1) the formation of multiple layers of a mixed population of macrophage-derived foam cells and lipid-containing smooth muscle cells, 2) the hypertrophy of the macrophage-derived foam cells, 3) the continued accumulation of extracellular matrix, 4) the insudation of plasma components. Immunocytochemical studies utilizing macrophage-specific and muscle-actin-specific monoclonal antibodies indicated that the expanding and mature fatty streaks in both the Watanabe and fat-fed rabbits were primarily composed of macrophage-derived foam cells. Hypertrophy of those foam cells situated immediately beneath the endothelium was associated with retraction of the endothelium and exposure of the intimal foam cells to the circulation. Endothelial retraction with exposure of intimal foam cells may facilitate entry of blood cells and lipoproteins into the lesions and formation of mural thrombi on the surfaces of the exposed cells. Biochemical analyses of the cholesterol content of the arteries indicated that both unesterified cholesterol and cholesteryl esters were deposited to a comparable degree in both the Watanabe and fat-fed rabbits. Thus, the absence of the low density lipoprotein receptor in Watanabe rabbits does not appear to directly influence the accumulation of cholesterol in the artery wall.

Malignant vascular tumors of the serous membranes mimicking mesothelioma. A report of 14 cases.

Malignant endothelial neoplasms involving the serous membranes are rare, and only a few cases have been documented. We report 14 patients with epithelioid hemangioendothelioma (EHE) or epithelioid angiosarcoma (EA) diffusely involving the pleural, peritoneal, or pericardial cavities, resulting in a picture closely resembling mesothelioma. The mean age at diagnosis was 52 (range, 34-85). The patients included two women and one man with peritoneal tumors, eight men with pleural tumors, and three men with pericardial tumors. A shared histological appearance was a diffuse sheet-like and clustered pattern of tumor growth with variable degrees of vascular differentiation. A tubulopapillary growth pattern, often seen in mesothelioma, was prominent in four cases. Nine cases showed a variable number of spindle cells, some neoplastic, others reactive, focally producing a biphasic growth pattern, further suggesting mesothelioma. Initial interpretations included mesothelioma, adenocarcinoma, and, in one case with prominent spindle-cell components, leiomyosarcoma. Immunohistochemically, strong vimentin staining and negative or weak to moderate cytokeratin staining were observed in all 14 cases. The tumor cells coexpressed at least two of the four endothelial markers used in the study (CD31, CD34, von Willebrand factor, and Ulex europaeus agglutinin-I [UEA-I)]. Detection of abortive vessel formation was facilitated by staining for collagen type IV. Markers of mesothelial, epithelial, muscular, and neuronal differentiation were all negative in the subset of cases studied. As a control group, 39 mesotheliomas and more than 60 adenocarcinomas of various origins were studied using the same antibody panel. This group revealed strong keratin staining, moderate or negative vimentin staining, and no expression of any of the endothelial-lineage markers, with the exception of positive staining for UEA-I in occasional adenocarcinomas. Clinically, these endothelial tumors were highly aggressive; 12 patients presented with disseminated disease, and most died within months of the initial presentation. These findings indicate that, although uncommon, EHE/EA should be included in the differential diagnosis of serous membrane neoplasms with histological and clinical features of malignant mesothelioma. The diagnosis of an endothelial neoplasm can be suspected by the presence of abortive vessel formation and by the strong expression of vimentin, with absent or low-level expression of cytokeratin. The demonstration of immunoreactivity for two or more endothelial-associated markers is essential in confirming the diagnosis.