Monocrotaline pyrrole induces pulmonary endothelial damage through binding to and release from erythrocytes in lung during venous blood reoxygenation

Monocrotaline has been widely used to establish an animal model of pulmonary hypertension, most frequently in rats. An important feature of this model resides in the selectivity of monocrotaline injury toward the pulmonary vascular endothelium versus the systemic vasculature when administrated at standard dosage. The toxic metabolite of monocrotaline, monocrotaline pyrrole, is transported by erythrocytes. This study aimed to reveal whether partial pressure of oxygen of blood determined the binding and release of monocrotaline pyrrole from erythrocytes in rats with one subcutaneous injection of monocrotatline at the standard dosage of 60 mg/kg. Our experiments demonstrated that monocrotaline pyrrole bound to and released from erythrocytes at the physiological levels of partial pressure of oxygen in venous and arterial blood, respectively, and then aggregated on pulmonary artery endothelial cells. Monocrotaline pyrrole-induced damage of endothelial cells was also dependent on partial pressure of oxygen. In conclusion, our results demonstrate the importance of oxygen partial pressure on monocrotaline pyrrole binding to erythrocytes and on aggregation and injury of pulmonary endothelial cells. We suggest that these mechanisms contribute to pulmonary selectivity of this toxic injury model of pulmonary hypertension.

Single-Dose Monocrotaline Pyrrole Injection as a Model of Pulmonary Endothelial Injury In Mice

Monocrotaline (MCT) is a plant substance that induces severe pulmonary hypertension in several animals except for mice. The aim of our study was to state whether monocrotaline pyrrole (MCTp), the main monocrotaline metabolite, could induce significant injury in mouse lung when given intravenously. MCTp caused moderate pulmonary inflammation, remodelling of small distal vessels (percentage of muscularized arteries: 33,5 vs 20,6%, p≤0,0006) and a right ventricular dysfunction (RVSP 27,8mmHg vs 16,4mmHg, p≤0,0001; Fulton index 0,35 vs 0,26, p≤0,0007). These vascular effects were associated with a decrease in eNOS protein expression in lung tissues and resolved after 45 days. In conclusion, we developed a model of endothelial dysfunction and transient pulmonary hypertension in mice.

Golgi, trafficking, and mitosis dysfunctions in pulmonary arterial endothelial cells exposed to monocrotaline pyrrole and NO scavenging

Although the administration of monocrotaline (MCT) into experimental animals is in widespread use today in investigations of pulmonary arterial hypertension (PAH), the underlying cellular and subcellular mechanisms that culminate in vascular remodeling are incompletely understood. Bovine pulmonary arterial endothelial cells (PAECs) in culture exposed to monocrotaline pyrrole (MCTP) develop “megalocytosis” 18–24 h later characterized by enlarged hyperploid cells with enlarged Golgi, mislocalization of endothelial nitric oxide synthase away from the plasma membrane, decreased cell-surface/caveolar nitric oxide (NO), and hypo- S-nitrosylation of caveolin-1, clathrin heavy chain, and N-ethylmaleimide-sensitive factor. We investigated whether MCTP did in fact affect functional intracellular trafficking. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) and the NO donor diethylamine NONOate were used for comparison. Both MCTP and c-PTIO produced distinctive four- to fivefold enlarged PAECs within 24–48 h with markedly enlarged/dispersed Golgi, as visualized by immunostaining for the Golgi tethers/matrix proteins giantin, GM130, and p115. Live-cell uptake of the Golgi marker C5 ceramide revealed a compact juxtanuclear Golgi in untreated PAECs, brightly labeled enlarged circumnuclear Golgi after MCTP, but minimally labeled Golgi elements after c-PTIO. These Golgi changes were reduced by NONOate. After an initial inhibition during the first day, both MCTP and c-PTIO markedly enhanced anterograde secretion of soluble cargo (exogenous vector-expressed recombinant horseradish peroxidase) over the next 4 days. Live-cell internalization assays using fluorescently tagged ligands showed that both MCTP and c-PTIO inhibited the retrograde uptake of acetylated low-density lipoprotein, transferrin, and cholera toxin B. Moreover, MCTP, and to a variable extent c-PTIO, reduced the cell-surface density of all receptors assayed (LDLR, TfnR, BMPR, Tie-2, and PECAM-1/CD31). In an important distinction, c-PTIO enhanced mitosis in PAECs but MCTP inhibited mitosis, even that due to c-PTIO, despite markedly exaggerated Golgi dispersal. Taken together, these data define a broad-spectrum Golgi and subcellular trafficking dysfunction syndrome in endothelial cells exposed to MCTP or NO scavenging.

Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole

Investigations of regulated S-nitrosylation and denitrosylation of vasorelevant proteins are a newly emergent area in vascular biology. We previously showed that monocrotaline pyrrole (MCTP)-induced megalocytosis of pulmonary arterial endothelial cells (PAECs), which underlies the development of pulmonary arterial hypertension, was associated with a Golgi blockade characterized by the trapping of diverse vesicle tethers, soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs), and soluble NSF-attachment proteins (SNAPs) in the Golgi; reduced trafficking of caveolin-1 (cav-1) and endotheial nitric oxide (NO) synthase (eNOS) from the Golgi to the plasma membrane; and decreased caveolar NO. We have investigated whether NSF, the ATPase involved in all SNARE disassembly, might be the upstream target of MCTP and whether MCTP might regulate NSF by S-nitrosylation. Immunofluorescence microscopy and Golgi purification techniques revealed the discordant decrease of NSF by ∼50% in Golgi membranes after MCTP despite increases in α-SNAP, cav-1, eNOS, and syntaxin-6. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide failed to affect the initiation or progression of MCTP megalocytosis despite a reduction of 4,5-diaminofluorescein diacetate fluorescence and inhibition of S-nitrosylation of eNOS as assayed using the biotin-switch method. Moreover, the latter assay not only revealed constitutive S-nitrosylation of NSF, eNOS, cav-1, and clathrin heavy chain (CHC) in PAECs but also a dramatic 70–95% decrease in the S-nitrosylation of NSF, eNOS, cav-1, and CHC after MCTP. These data point to depletion of NSF from Golgi membranes as a mechanism for Golgi blockade after MCTP and to denitrosylation of vasorelevant proteins as critical to the development of endothelial cell megalocytosis.