Dennis W. Wilson

Mechanisms and Pathology of Monocrotaline Pulmonary Toxicity

Monocrotaline (MCT) is an 11-membered macrocyclic pyrrolizidine alkaloid (PA) that causes a pulmonary vascular syndrome in rats characterized by proliferative pulmonary vasculitis, pulmonary hypertension, and cor pulmonale. Current hypotheses of the pathogenesis of MCT-induced pneumotoxicity suggest that MCT is activated to a reactive metabolite(s) in the liver and is then transported by red blood cells (RBCs) to the lung, where it initiates endothelial injury. While several lines of evidence support the requirement of hepatic metabolism for pneumotoxicity, the mechanism and relative importance of RBC transport remain undetermined. The endothelial injury does not appear to be acute cell death but rather a delayed functional alteration that leads to disease of the pulmonary arterial walls by unknown mechanisms. The selectivity of MCT for the lung, as opposed to that of other primarily hepatotoxic PAs, appears likely to be a consequence of the differences in hepatic metabolism and blood kinetics of MCT. A likely candidate for a reactive metabolite of MCT is the dehydrogenation product monocrotaline pyrrole (MCTP). Secondary or phase II metabolism of MCT through glutathione (GSH) conjugation has been characterized recently and appears to represent a detoxification pathway. The role of inflammation in the progression of MCT-induced pulmonary vascular disease is uncertain. Both perivascular inflammation and platelet activation have been proposed as processes contributing to the response of the vascular media. This review presents the experimental evidence supporting these hypotheses and outlines additional questions that arise from them.

Comparison of Nonciliated Tracheal Epithelial Cells in Six Mammalian Species: Ultrastructure and Population Densities

Three types of nonciliated epithelial cells in mammalian conducting respiratory airways are thought to be secretory: mucous (goblet) cells, serous epithelial cells, and Clara cells. Mucous and serous cells are considered to be the secretory cells of the trachea. Clara cells are considered to be the secretory cells of the most distal conducting airways or bronchioles. To ascertain if mucous and serous epithelial cells are common to the tracheal epithelium of mammalian species, we characterized the ultrastructure and population densities of tracheal epithelial cells in six species: hamster (H), rat (Rt), rabbit (Rb), cat (C), Bonnet monkey (M. radiata) (B), and sheep (S). Following fixation by airway infusion with glutaraldehyde/paraformaldehyde, tracheal tissue was processed for light and electron microscopy (EM) by a selective embedding technique. Tracheal epithelium over cartilage was quantitated by light microscopy and characterized by transmission EM. Mucous cells were defined by abundant large nonhomogeneous granules, numerous Golgi complexes, basally located nuclei and granular endoplasmic reticulum (GER). The percentage of mucous cells in the tracheal epithelium was: H (0%), Rt (0.5%), Rb (1.3%), C (20.2%), B (8%), S (5.1%). Serous cells had homogeneous, electron-dense granules and extensive GER. Serous cells were present only in rats (39.2%). Clara cells had homogeneous electron-dense granules, abundant agranular endoplasmic reticulum (AER) and basal GER. Clara cells were found in hamsters (41.4%) and rabbits (17.6%). In sheep trachea, 35.9% of the epithelial cells had small electron-lucent granules, abundant AER and numerous Golgi complexes. In Bonnet monkey trachea, 16% of the epithelial cells had small electron-lucent granules, numerous polyribosomes, perinuclear Golgi apparatus and moderate GER. In cat trachea, 5.4% of the epithelial cells lacked granules, and had moderate numbers of mitochondria, moderate amounts of polyribosomes, a central nucleus, and long luminal microvilli. The percentage of the tracheal epithelial population occupied by basal, ciliated and nonciliated cells was: H (5.6%, 47.5%, 46.7%), Rt (13.4%, 40.6%, 45.9%), Rb (28.2%, 43.0%, 28.3%), C (37.3%, 36.1%, 26.7%), B (31%, 41%, 28%), S (28.5%, 30.6%, 41%). We conclude: 1) mucous and serous cells are not common to the tracheal epithelial lining of all mammalian species; 2) there is significant interspecies heterogeneity in the abundance, distribution and ultrastructure of tracheal secretory cells; 3) potential differences in the roles of nonciliated cells in tracheal function exists within tracheal epithelial populations and between species.

Progressive inflammatory and structural changes in the pulmonary vasculature of monocrotaline-treated rats

The progression of changes in the bronchus-associated and intraacinar pulmonary arteries of rats treated with a single dose of monocrotaline (60 mg/kg) was evaluated by quantitative light and electron microscopy. The relative volume of vessel wall components was normalized to the surface area of the adventitial sheath. An increased relative volume of media was evident in intraacinar pulmonary arteries by 4 hr post-treatment. This increase may represent vascular smooth muscle contraction. Significant increases in adventitial mononuclear inflammatory cells were evident by 8-16 hr post-treatment in intraacinar pulmonary arteries and veins but not until 14 days post-treatment in major, bronchus-associated pulmonary arteries. Inflammatory cell influxes were associated with increased relative volume of adventitia, largely due to increased extracellular space. By 22 days posttreatment, there was right ventricular hypertrophy and a marked mononuclear vasculitis in major and intraacinar pulmonary arteries as well as intraacinar veins (confirmed as such by vascular perfusion of carbon/gelatin). There was increased relative medial volume in both major and intraacinar pulmonary arteries associated with increased extracellular matrix composed largely of collagen. Intraacinar veins developed intimal plaques of smooth muscle in a collagenous matrix. We conclude that (1) adventitial inflammation precedes morphologic evidence of medial changes in monocrotaline-induced pulmonary hypertension, (2) involvement of intraacinar arteries precedes that of major bronchus associated arteries, and (3) both pulmonary arteries and veins are involved in monocrotaline-induced pulmonary vascular disease in the rat.

17β-Estradiol Attenuates Development of Angiotensin II-Induced Aortic Abdominal Aneurysm in Apolipoprotein E-Deficient Mice

Objectives—Angiotensin II (Ang II) promotes vascular inflammation, accelerates atherosclerosis, and induces abdominal aortic aneurysm (AAA). These changes were associated with activation of nuclear factor (NF)-&kgr;B–mediated induction of proinflammatory genes. The incidence of AAA in this model was higher in male than in female mice, and the vascular effects of estrogen may be associated with anti-inflammatory actions. The present study was undertaken to test the hypothesis that estrogen can attenuate Ang II–induced AAA in apolipoprotein E–deficient mice via its anti-inflammatory mechanism. Methods and Results—Infusion of Ang II (1.44 mg/kg per d for 1 month) induced AAA in 90% of the animals (n=20) with an expansion of the suprarenal aorta (diameter 1.9±0.14 mm versus <1 mm in normal mice). In mice treated with 17&bgr;-estradiol (E2, 0.25-mg subcutaneous pellets), Ang II induced AAA only in 42% of the animals (n=19) with a significant reduction of average diameters of the suprarenal aorta (1.5±0.14 mm). E2 also decreased the expressions of intracellular adhesion molecule-1, vascular cellular adhesion molecule-1, E-selectin, monocyte chemotactic protein-1, and macrophage-colony stimulating factor in the aorta. Conclusions—These data suggest that attenuation of AAA by E2 is associated with inhibition of proinflammatory gene expression.

Role of triglyceride-rich lipoproteins in diabetic nephropathy

Diabetic nephropathy is an increasingly important cause of morbidity and mortality worldwide. A large body of evidence suggests that dyslipidemia has an important role in the progression of kidney disease in patients with diabetes. Lipids may induce renal injury by stimulating TGF-β, thereby inducing the production of reactive oxygen species and causing damage to the glomeruli and glomerular glycocalyx. Findings from basic and clinical studies strongly suggest that excess amounts of a variety of lipoproteins and lipids worsens diabetes-associated microvascular and macrovascular disease, increases glomerular injury, increases tubulointerstitial fibrosis, and accelerates the progression of diabetic nephropathy. The increasing prevalence of obesity, type 2 diabetes mellitus, and diabetic nephropathy means that interventions that can interrupt the pathophysiological cascade of events induced by lipoproteins and lipids could enable major life and cost savings. This Review discusses the structural, cellular, and microscopic findings associated with diabetic nephropathy and the influence of lipoproteins, specifically triglyceride-rich lipoproteins (TGRLs), on the development and perpetuation of diabetic nephropathy. Some of the accepted and hypothesized mechanisms of renal injury relating to TGRLs are also described.

Infection kinetics, prostacyclin release and cytokine-mediated modulation of the mechanism of cell death during bluetongue virus infection of cultured ovine and bovine pulmonary artery and lung microvascular endothelial cells.

Bluetongue virus (BTV) infection causes a haemorrhagic disease in sheep, whereas BTV infection typically is asymptomatic in cattle. Injury to the endothelium of small blood vessels is responsible for the manifestations of disease in BTV-infected sheep. The lungs are central to the pathogenesis of BTV infection of ruminants; thus endothelial cells (ECs) cultured from the pulmonary artery and lung microvasculature of sheep and cattle were used to investigate the basis for the disparate expression of bluetongue disease in the two species. Ovine and bovine microvascular ECs infected at low multiplicity with partially purified BTV were equally susceptible to BTV-induced cell death, yet ovine microvascular ECs had a lower incidence of infection and produced significantly less virus than did bovine microvascular ECs. Importantly, the relative proportions of apoptotic and necrotic cells were significantly different in BTV-infected EC cultures depending on the species of EC origin and the presence of inflammatory mediators in the virus inoculum. Furthermore, BTV-infected ovine lung microvascular ECs released markedly less prostacyclin than the other types of ECs. Results of these in vitro studies are consistent with the marked pulmonary oedema and microvascular thrombosis that characterize bluetongue disease of sheep but which rarely, if ever, occur in BTV-infected cattle.

Endothelial NO Synthase Deficiency Promotes Smooth Muscle Progenitor Cells in Association With Upregulation of Stromal Cell-Derived Factor-1α in a Mouse Model of Carotid Artery Ligation

Background—Endothelial NO deficiency (endothelial NO synthase [eNOS]–knockout [KO]) enhanced smooth muscle cell (SMC)–rich neointimal lesion formation in a mouse model of carotid artery ligation (CAL). Recent evidence indicated that stromal cell-derived factor-1α (SDF-1α)–mediated recruitment of circulating SMC progenitor cells substantially contributed to the SMC-rich neointimal hyperplasia induced by vascular injury. The goal of this study was to investigate the effects of eNOS deficiency on the expression of SDF-1α and mobilization of circulating SMC progenitor cells in CAL model. Methods and Results—Two- to 3-month-old C57BL/6J wild-type (WT) and eNOS-KO mice were evaluated 1, 2, or 4 weeks after CAL. CAL-induced expression of SDF-1α, as detected by immunohistochemical staining and further quantified by ELISA in the ligated carotid arteries, was moderate and transient with a peak at 1 week in WT mice. SDF-1α expression was significantly higher at 1 week and persisted through 2 weeks in eNOS-KO mice. CAL was associated with increased circulating stem cell antigen-1+ (Sca-1+)/c-Kit−/Lin− cells (interpreted as SMC progenitor cells), which peaked at 1 week in WT mice. This effect was also significantly greater and longer-lasting in eNOS-KO than WT mice. The number of circulating Sca-1+/c-Kit−/Lin− cells was positively correlated with the expression of SDF-1α but not vascular endothelial growth factor in the ligated carotid arteries. Furthermore, immunostaining showed abundant Sca-1–positive cells in the adventitia of the 1-week ligated carotid arteries from eNOS-KO mice but not in WT mice. We also determined that eNOS deficiency enhanced CAL-induced intimal cell proliferation in the ligated arteries as detected by proliferating cell nuclear antigen staining but did not induce cell apoptosis as detected by staining for active caspase-3. Conclusion—Our results indicate that eNOS deficiency exacerbates CAL-induced expression of SDF-1α and its receptor CXCR4. This is correlated with an increase in Sca-1+ cells in peripheral blood and adventitia, which may contribute to vascular remodeling and SMC-rich neointimal lesion formation. This suggests that constitutive eNOS inhibits SDF-1α expression and provides an important vasculoprotective mechanism for intact endothelium to limit SMC proliferation and recruitment in response to vascular injury.

Involvement of cytochrome P450 3A in the metabolism and covalent binding of 14C‐monocrotaline in rat liver microsomes

The metabolism and covalent binding of 14C‐monocrotaline in Sprague–Dawley (SD) rat liver microsomes was investigated using the inducers dexamethasone, clotrimazole, pregnenolone‐16α‐carbonitrile, and phenobarbital. Monocrotaline is a pyrrolizidine alkaloid (PA) that causes a syndrome in rats that is a model for human primary pulmonary hypertension. It has been documented that bioactivation of PAs (dehydrogenation to reactive pyrroles) in the liver by cytochromes P450 is required for their toxicity. Covalent binding of these reactive pyrroles to tissue macromolecules has been hypothesized to correspond to PA toxicosis. We correlated metabolism and total microsomal covalent binding of 14C‐monocrotaline with cytochrome P450 3A using the aforementioned inducers, troleandomycin (a cytochrome P450 3A inhibitor), erythromycin N‐demethylase assay of cytochrome P450 3A activity, and Western blots employing anti‐rat cytochrome P450 3A antibodies. In addition, autoradiography of membranes electroblotted from SDS‐PAGE demonstrated the formation of radiolabeled adducts with specific protein(s). The most intensely radiolabeled protein bands have an apparent molecular weight of ∼52 kDa, which was similar to the molecular weight detected by anti‐rat cytochrome P450 3A antibodies in the Western blots. No radiolabeled proteins were detected in microsomes pretreated with troleandomycin.

Endotoxin and polycyclic aromatic hydrocarbons in ambient fine particulate matter from Fresno, California initiate human monocyte inflammatory responses mediated by reactive oxygen species

In urban areas, a correlation between exposure to particulate matter (PM) from air pollution and increased cardiovascular morbidity and mortality has been observed. Components of PM include bacterial contaminants, transition metals, salts, polycyclic aromatic hydrocarbons (PAH), and carbonaceous material, which could interact with various cell types to produce systemic responses when inhaled. We examined the effects of PM collected from Fresno, California on activation of human monocytes and their interaction with vascular endothelium, a key event in atherogenesis. PM exposure increased cytokine expression and secretion from monocytes and enhanced monocyte adhesion to human aortic endothelial cells, both of which were attenuated by neutralizing endotoxin. PM also increased monocyte CYP1a1 expression, and inhibition of the aryl hydrocarbon receptor reduced the CYP1a1 and inflammatory responses. PM-treated monocytes accumulated intracellular reactive oxygen species (ROS), and antioxidants attenuated inflammatory and xenobiotic responses. Finally, supernatants from PM-treated pulmonary microvascular endothelial cells induced monocyte inflammatory responses that were not a consequence of endotoxin transfer. These results suggest that certain components of urban PM, namely endotoxin and PAH, activate circulating monocytes directly or indirectly by first stimulating other cells such as pulmonary endothelial cells, providing several mechanisms by which PM inhalation could induce pulmonary and/or systemic inflammation.

Isolation and identification of a pyrrolic glutathione conjugate metabolite of the pyrrolizidine alkaloid monocrotaline

This report describes the isolation and identification of a monocrotaline-derived, glutathione-conjugated pyrrole obtained from the bile of male Sprague-Dawley rats. Bile obtained from rats given an intravenous bolus of 14C-monocrotaline was fractionated using a series of chromatographic separations. Initial purification with cholestyramine resin removed bile acid and pigment contaminants. Subsequent anion exchange and reversed-phase HPLC separations yielded several fractions that contained the 14C label and tested positive for pyrroles using Ehrlichs reagent. These fractions were analyzed using fast-atom-bombardment tandem mass spectrometry (FAB MS/MS). In addition to glutathione-conjugated dehydroretronecine, at least one other pyrrole present had similar ionic properties. The latter was not present in amounts sufficient for positive identification.