Orally active endothelin receptor antagonist BMS-182874 suppresses neointimal development in balloon-injured rat carotid arteries.

Vascular smooth muscle cell (SMC) proliferation is an important component in the development of restenosis. Because endothelin (ET) has been reported to act as an SMC mitogen, we postulated that the orally active ETA receptor antagonist BMS-182874 would suppress the development of the intimal lesion that develops in rat carotid arteries after balloon injury. Using cultured rat aortic SMC, we noted that ET-1-stimulated increases in [3H]thymidine incorporation were blocked by BMS-182874. To determine the effect of the drug on intimal lesion formation, we treated rats with BMS-182874 (100 mg/kg orally, p.o.) or vehicle once daily for 3 weeks, beginning 1 week before balloon injury. Two weeks after injury, drug-treated rats had a 35% decrease in lesion area and a 34% decrease in the lesion/media ratio as compared with the vehicle-treated rats. In situ hybridization (ISH) analysis of balloon-injured rat carotid arteries showed an increase in ETA receptor mRNA. These data support the concept that ETA receptor activation contributes to intimal lesion formation by promotion of SMC proliferation and suggest a potential use for ETA receptor antagonists in the amelioration of hyperproliferative vascular diseases, including restenosis.

Hepatic nitric oxide production following acute endotoxemia in rats is mediated by increased inducible nitric oxide synthase gene expression.

In the present studies, we analyzed the effects of acute endotoxemia on hepatocyte nitric oxide production and functional activity. Treatment of rats with 5 mg/kg of lipopolysaccharide (LPS), which induces acute endotoxemia, caused an increase in nitric oxide production in the liver, as measured by electron paramagnetic spin trapping, which was evident within 6 hours. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger (m) RNA in hepatocytes and in sinusoidal cells throughout the liver lobule. Acute endotoxemia also caused alterations in hepatic structure, including hypertrophy, vacuolization, and chromosomal emargination, however these changes were not apparent for 24 to 48 hours. Hepatocytes isolated from endotoxemic rats released increased amounts of nitric oxide, measured by nitrite production, in response to interferon gamma (gamma-IFN) alone or in combination with LPS, tumor necrosis factor alpha, macrophage-colony stimulating factor, granulocyte/macrophage-colony stimulating factor, or hepatocyte growth factor. These results show that hepatocytes are sensitized by acute endotoxemia to respond to inflammatory mediators and growth factors. Increased nitrite production by hepatocytes was due to increased expression of iNOS mRNA and protein and was correlated with the time following induction of acute endotoxemia. Thus, cells isolated 48 hours after induction of acute endotoxemia released significantly more nitrite than cells recovered after 6 hours, a response that was not due to alterations in hepatocyte viability. Hepatocytes isolated from endotoxemic rats also exhibited a marked increase in proliferative capacity when compared with cells from control rats. Nitric oxide production by hepatocytes in vitro was associated with inhibition of cell growth and protein synthesis, which was reversed by the nitric oxide synthase inhibitor, NG-monomethyl-l-arginine (L-NMMA). Agarose gel electrophoresis showed extensive cytoplasmic DNA fragmentation in hepatocytes treated with LPS and gamma-IFN, a characteristic of apoptosis, which was also reversed by L-NMMA. These results, together with our findings that treatment of rats with an inhibitor of nitric oxide synthase partially reversed the structural alterations in the liver associated with acute endotoxemia suggest that nitric oxide may contribute to the pathophysiologic response to this bacterially derived toxin.

Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia.

Nitric oxide is a short-lived cytotoxic mediator that has been implicated in the pathogenesis of endotoxin-induced tissue injury and septic shock. In the present studies we determined whether this mediator is produced in the lung during acute endotoxemia. We found that intravenous injection of rats with bacterially derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time-dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6-12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon-gamma (IFN-gamma) and LPS measured by nitrite accumulation in the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN-gamma and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric oxide produced by IMs and AMs can react with superoxide anion to form peroxynitrite. Taken together, the present studies demonstrate that AMs and IMs are activated following acute endotoxemia to produce reactive nitrogen intermediates and that both cell types contribute to inflammatory responses in the lung.

Production of nitric oxide by rat type II pneumocytes: increased expression of inducible nitric oxide synthase following inhalation of a pulmonary irritant.

Nitric oxide is a highly reactive molecule that has been implicated in host defense and tissue injury. In the present studies, we determined whether rat type II alveolar epithelial cells have the capacity to produce this mediator. We found that type II cells synthesize significant quantities of nitric oxide after treatment with the inflammatory cytokines, interferon-gamma (IFN-gamma) and/or interleukin-1 beta (IL-1 beta), or with the combination of IFN-gamma and tumor necrosis factor-alpha. In contrast to rat alveolar macrophages, type II cells were unresponsive to lipopolysaccharide. Production of nitric oxide by type II cells in response to IFN-gamma was dose dependent, reaching a maximum at 100 U/ml, and blocked by NG-monomethyl-L-arginine (L-NMA), a nitric oxide synthase inhibitor. Northern blot analysis demonstrated that nitric oxide production by type II cells was due to expression of mRNA for an inducible form of nitric oxide synthase (iNOS). Following brief exposure of rats to irritant-inducing doses of ozone (2 ppm, 3 h), type II cells were found to produce significantly more nitric oxide than were cells from control animals. This was due to increased expression of iNOS mRNA. Cells from ozone-treated rats were also sensitized to produce more nitric oxide in response to IFN-gamma and IL-1 beta. This was associated with a marked increase in expression of iNOS mRNA and enzyme protein in the cells. We also found that ozone inhalation caused enhanced production of hydrogen peroxide, as well as spontaneous and IFN-gamma-induced cytostasis of type II cells toward P815 mouse mastocytoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin receptor B expression in the rat and rabbit lung as determined by in situ hybridization using nonisotopic probes.

Endothelins are a family of potent vasoactive peptides. Full-length cDNA clones to human endothelin receptor B (ETB) mRNA were random prime-labeled with nucleotides conjugated to digoxigenin for in situ hybridization. The labeled cDNA was used to probe frozen sections of rat and rabbit lung. Detection of the digoxigenin-labeled probe was accomplished by an antibody-enzyme conjugate, anti-digoxigenin alkaline phosphatase. The location of the antibody-antigen complex was visualized as an enzyme-linked color reaction. The hybridization, washings, and detection steps were performed under stringent conditions. The following cell types of the rat and rabbit lung had abundant positive reaction product to the ETB probe: bronchiolar and bronchial epithelium, endothelium of smooth-muscle--walled vessels, and bronchial and bronchiolar-associated lymphoid tissue. Abundant positive reaction product was also observed in cell populations in the lung parenchyma. Additional studies are being performed to identify those populations. The results of this study suggest that in addition to vasoactivity, endothelins play other important roles in the lung.

Immune electron microscopic characterization of monoclonal antibodies to Alzheimer neurofibrillary tangles.

Characterization of eleven monoclonal antibodies (MAbs), raised to isolated sodium dodecyl sulfate (SDS)-treated Alzheimer's neurofibrillary tangles (ANT), has revealed the presence of at least two different epitopes. MAbs were tested for reactivity to ubiquitin and paired helical filaments (PHF) isolated by three different procedures. The effect of protease and/or alkaline phosphatase pretreatment on the reactivity of the MAbs with isolated PHF was also examined. All MAbs that had reacted strongly in the ELISA with sonicated SDS-treated ANT also immune decorated isolated PHF to varying degrees. Two MAbs exhibited a high reactivity to PHF: 3-39 and 5-25. MAb 3-39 was found to recognize a protease sensitive epitope. In contrast MAb 5-25 was found to consistently decorate isolated PHF in all preparations and exhibited a strong reactivity to ubiquitin, and the epitope in isolated PHF was not protease sensitive. Thus structural PHF after protease treatment and detergent treatment contain an antigenic site that is present in ubiquitin.

Delayed acute measles inclusion body encephalitis in a 9-year-old girl: ultrastructural, immunohistochemical, and in situ hybridization studies.

A 9-yr-old girl developed delayed acute measles inclusion body encephalitis, which was different from subacute sclerosing panencephalitis (SSPE) in clinical course. Measles virus was demonstrated by electron microscopy, immunohistochemistry, and in situ hybridization. Contrary to the most previous reports, matrix (M) protein was present in the brain, cerebrospinal fluid, and serum and was demonstrated by Western blot analysis and in situ hybridization. The hybridization was performed by a nonradioactive digoxigenin method.

Nearly ubiquitous tissue distribution of the scrapie agent precursor protein.

The "modified host protein" model of scrapie proposes that the transmissible agent is composed of the degradation-resistant protein, Sp33-37, and that clinical and pathologic signs result from neurotoxic accumulations of this protein. Sp33-37 is an abnormal, amyloidogenic isoform of the normally occurring cellular protein Cp33-37. This study investigated the tissue distribution of Cp33-37 in hamster. In brain, Cp33-37 was most concentrated in the hippocampal formation. Immunohistochemical studies localized Cp33-37 to neurons and surrounding neuropil in hippocampus; septal, caudate, and thalamic nuclei; dorsal root ganglia cells; and large-diameter dorsal root axons. In non-neuronal hamster tissues, Cp33-37 was detected in circulating leukocytes, heart, skeletal muscle, lung, intestinal tract, spleen, testis, ovary, and some other organs. The presence of Cp33-37 in extracerebral tissues indicates that its function is not unique to brain. These results indicate that the molecular substrate for the production of Sp33-37, the scrapie agent, and scrapie amyloid is present in a variety of cerebral and extracerebral sites.

The mRNA encoding the scrapie agent protein is present in a variety of non-neuronal cells.

PrP 27-30, a unique protease-resistant protein associated with scrapie infectivity, derives from the proteolytic cleavage of a larger precursor encoded by a host gene. To identify sites of PrP biosynthesis, in situ hybridization was done using cloned PrP cDNA as a probe. In rodent brain, PrP mRNA was expressed in neurons, ependymal cells, choroid plexus epithelium, astrocytes, pericytes, endothelial cells and meninges of both scrapie-infected and uninfected animals. PrP mRNA was also detected in vitro in isolated brain microglia cells. Pulmonary cells and heart muscle cells contained high levels of this mRNA. Hybridization was not detected in spleen, confirming earlier RNA blot experiments indicating extremely low levels of PrP mRNA in this tissue. Results indicate that PrP mRNA is a normal component in a variety of non-neuronal tissues and may explain the origin of the amyloid plaques present in the subependymal region of scrapie-infected brain.

Postmortem detection of measles virus in non-neural tissues in subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis, a rare, progressive, fatal central nervous system disease of children, is caused by measles virus. Clinical signs occur months to several years after recovery from acute measles infection. It is not known where the virus persists while the disease is inapparent. Involvement of organs outside the central nervous system has rarely been documented. To search for possible peripheral reservoirs of measles virus we used in situ hybridization to probe for measles virus RNA and immunocytochemical studies to localize measles virus antigens ina variety of organs taken at autopsy from confirmed cases of subacute sclerosing panencephalitis. Seven of 9 cadavers were found to contain measles virus RNA or antigens, or both, in at least one location outside the central nervous system. These sites included lymphoid organs such as thymus, spleen, lymph nodes, and tonsil, suggesting a role for lymphocytes in disease pathogenesis. Virus was also detected in kidney, lung, and glandular tissues such as pancreas, adrenal, and pituitary. These reservoirs may provide the antigenic stimulus leading to the elevated response characteristic for subacute sclerosing panencephalitis.