[Pulmonary Vascular Remodeling Characteristics of Pulmonary Arterial Hypertension Mouse Model Induced by Left Pneumonectomy and Jugular Vein Injection of Monocrotaline Pyrrole].

Objective: To compare and investigate the differences and characteristics of pulmonary vascular remodeling in three mouse models of pulmonary arterial hypertension (PAH) constructed by left pneumonectomy, jugular vein injection of monocrotaline pyrrole, and left pneumonectomy combined with jugular vein injection of monocrotaline pyrrole, to explore for a PAH animal model that approximates the clinical pathogenesis of PAH, and to create a model that will provide sound basis for thorough investigation into the pathogenesis of severe PAH. Methods: 59 male C57/BL mice (10-12 weeks, 24-30 g) were randomized into four groups, a control group ( n=9), a group that had left pneumonectomy (PE, n=15), a group that had jugular vein injection of monocrotaline pyrrole (MCTP, n=15), and the last group that had left pneumonectomy combined with jugular injection of monocrotaline pyrrole (P+M, n=20). To evaluate the effect of modeling and the characteristics of pulmonary vascular remodeling, hemodynamic and morphological parameters, including right ventricular systolic pressure (RVSP), right ventricle/(left ventricle plus septum) (RV/LV+S), percent of wall thickness in the pulmonary artery (WT%), muscularization of non-muscular arteries, neointima formation, and vascular obstruction score (VOS), were measured in each group. Results: 1) Compared with those of the control group, the RVSP, RV/LV+S, WT%, and the degree of small pulmonary arteries muscularization in the P+M group were significantly increased ( P<0.01). The MCTP group had just slightly higher findings for these indicators ( P<0.05), while no significant change in these indicators was observed in the PE group ( P>0.05). 2) Neointima formation in the acinus pulmonary arteries, which caused obvious stenosis of the lumen, was observed in the P+M group, the VOS being 1.25±0.80 points ( P<0.001). In contrast, neointima formation was not observed in the MCTP group or the PE groups, the VOS being 0 point ( P>0.05). Conclusion: Left pneumonectomy combined with jugular intravenous injection of MCTP could induce severe PAH formation in mouse. The model provides a good simulation of neointima formation, the characteristic pathological change of clinical severe PAH.

Sympathetic innervation of canine pulmonary artery and morphometric and functional analysis in dehydromonocrotaline-induced models after pulmonary artery denervation.

OBJECTIVES: We aimed to describe the anatomic distribution of periarterial pulmonary sympathetic nerves and to observe the long-term morphometric and functional changes after pulmonary artery denervation (PADN), a novel therapy for pulmonary arterial hypertension (PAH). METHODS: A total of 45 beagles were divided into a sympathetic innervation group (n = 3, 33.3% were females), a PAH group (n = 35, 34.3% were females) and a control group (n = 7, 28.5% were females). The PAH group was randomly divided into no-PADN (n = 7), instant-PADN (n = 7), 1M-PADN (n = 7), 2M-PADN (n = 7) and 3M-PADN (n = 7) subgroups. The sympathetic innervation group was sacrificed to reveal the sympathetic innervation of pulmonary arteries. PAH was induced by injecting dehydromonocrotaline (DHMCT) through the right atrium. The pulmonary capillary wedge pressure, right ventricular systolic pressure, right ventricular mean pressure, pulmonary artery systolic pressure and pulmonary artery mean pressure of each group were continuously measured. The cardiac output was detected to calculate the pulmonary vascular resistance. PAH and control groups were subjected to immunofluorescence assay, sympathetic nerve conduction velocity measurement and transmission electron microscopy. RESULTS: The no-PADN group had significantly higher PVSP, PVMP, pulmonary artery systolic pressure, pulmonary artery mean pressure and pulmonary vascular resistance but lower cardiac output than those of the control group (P < 0.05). Instant-PADN, 1M-PADN, 2M-PADN and 3M-PADN groups had significantly lower PVSP, PVMP, pulmonary artery systolic pressure, pulmonary artery mean pressure and pulmonary vascular resistance but higher cardiac output than those of the no-PADN group (P < 0.05). Most sympathetic nerves were located within 2.5 mm of the intimae of the bifurcation and proximal trunk, mainly in the left trunk. The diameter and cross-sectional area of myelinated fibres in the PAH group were significantly larger than those of the control group. Sympathetic nerve conduction velocity of the PAH group gradually decreased, and nerve fibres were almost demyelinated 3 months after PADN. CONCLUSIONS: PADN effectively relieved dehydromonocrotaline-induced canine PAH and decreased sympathetic nerve conduction velocity.

[Monocrotaline pyrrole induces A549 cells and activates TGF-ß1/SMAD2/SMAD3 pathway to promote proliferation and migration of human pulmonary artery smooth muscle cells].

Objective To explore the effects of A549 cells on the proliferation and migration of human pulmonary arterial smooth muscle cells (HPASMCs) and its mechanism. Methods A549 cells and HPASMCs were cultured in vitro. The A549 cells were randomly divided into four groups: control group, dimethylformamide (DMF) solvent group, monocrotaline pyrrole (MCTP) group, MCTP combined with SB431542 group. The cells were assigned into four groups: HPASMC group, A549 and HPASMC co-culture group, MCTP-stimulated A549 and HPASMC co-culture group, MCTP and SB431542-stimulated A549 and HPASMC co-culture group, and IL-6-stimulated HPASMC group. A549 cell viability was detected by CCK-8 assay. The level of IL-6 in the A549 cell culture supernatant was tested by ELISA. The mRNA levels of SMAD2 and SMAD3 in the A549 cells were detected by real-time PCR. The protein levels of TGF-ß1, SMAD2, SMAD3 and p-SMAD2, p-SMAD3 in the A549 cells were detected by Western blot analysis. The protein levels of TGF-ß1, SMAD2, SMAD3 and p-SMAD2, p-SMAD3 in the A549 cells were examined by Western blot analysis. The protein levels of osteopontin (OPN) and proliferating nuclear antigen (PCNA) in the HPASMCs were determined by Western blot analysis. The migration ability of HPASMCs was measured by wound healing and TranswellTM assay. Results In the A549 cells, compared with the control group, the cell proliferation ability decreased, the production of IL-6 increased, the mRNA levels of SMAD2 and SMAD3, and the expression of TGF-ß1, SMAD2, SMAD3, p-SMAD2, p-SMAD3 proteins significantly increased in the MCTP group. Compared with the MCTP group, the cell proliferation ability increased, the production of IL-6 decreased, the mRNA levels of SMAD 2 and SMAD3, and the expression of TGF-ß1, SMAD2, SMAD3, p-SMAD2, p-SMAD3 proteins significantly decreased in the MCTP and SB431542-stimulated group. In the co-culture system, compared with the HPASMC group, the expression of PCNA and OPN proteins and migration ability did not change significantly in the A549 and HPASMC co-cultured group. The expression of PCNA and OPN proteins significantly increased in the MCTP-stimulated A549 and HPASMC co-culture group, and the cell migration ability increased. Compared with the MCTP-stimulated A549 and HPASMC co-culture group, the expression of PCNA and OPN proteins significantly decreased in MCTP and SB431542-stimulated A549 and HPASMC co-culture group, and the cell migration ability decreased. Compared with the HPASMC group, the migration ability of HPASMCs increased and the expression of PCNA and OPN proteins increased in the IL-6 control group. Conclusion Activation of the TGF-ß1/SMAD2/SMAD3 signaling pathway in A549 cells induced by MCTP increases IL-6 secretion, thus promoting the proliferation and migration of HPASMCs.

Endogenous hydrogen sulfide sulfhydrates IKKß at cysteine 179 to control pulmonary artery endothelial cell inflammation.

BACKGROUND: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. METHODS: Purified recombinant human inhibitor of κB kinase subunit ß (IKKß) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. RESULTS: We showed that hydrogen sulfide (H2S) inhibited IKKß activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKß activity directly via sulfhydrating IKKß at cysteinyl residue 179 (C179) in purified recombinant IKKß protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKß inactivation. Furthermore, to demonstrate the significance of IKKß sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKß. In purified IKKß protein, C179S mutation of IKKß abolished H2S-induced IKKß sulfhydration and the subsequent IKKß inactivation. In human PAECs, C179S mutation of IKKß blocked H2S-inhibited IKKß activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKß abolished the inhibitory effect of H2S on IKKß activation and pulmonary vascular inflammation and remodeling. CONCLUSION: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKß via sulfhydrating IKKß at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

Monocrotaline pyrrole enhanced bone morphogenetic protein 7 signaling transduced by alternative activin A receptor type 2A in pulmonary arterial smooth muscle cells.

BACKGROUND: Increased expression levels of bone morphogenetic protein 7 (BMP7) are associated with poor prognosis in pulmonary hypertension patients. However, whether BMP7 signaling conspire to involve in the proliferation of pulmonary artery smooth muscle cells (PASMC) underlying monocrotaline (MCT) induced pulmonary arterial hypertension (PAH) remain unclear. METHODS AND RESULTS: Western blot experiments found BMP7 was increased in pulmonary arteries isolated from MCT-PAH rat. In addition, monocrotaline pyrrole (MCTP), the putative toxic metabolite of the MCT, increases the expression of BMP7, proliferating cell nuclear antigen (PCNA) and activin A receptor type 2A, but decreases bone morphogenetic protein receptor type 2 in cultured pulmonary artery smooth muscle cells (PASMC). In PASMCs, exogenous BMP7 leads to the decreasing expression of activin A receptor type 2, increasing phosphorylation of p38MAPK and elevation of P21. However, BMP7 treatment results in the increasing expression of activin A receptor type 2A, p38MAPK, and PCNA in bone morphogenetic protein receptor type 2 knockdown PASMCs. Knockdown of activin A receptor type 2A abrogated the MCTP-induced PCNA and cell cycle progression. CONCLUSIONS: MCTP treatment lead to the expression of BMP7, suppression of bone morphogenetic protein receptor type 2 but increasing expression of activin A receptor type 2A, the BMP7 mediated PASMC proliferation via preferential activation of an activin A receptor type 2A signaling axis.

Adipose-derived mesenchymal stromal cells improve hemodynamic function in pulmonary arterial hypertension: identification of microRNAs implicated in modulating endothelial function.

Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial endothelial hyperproliferation and dysfunction. Restoration of endothelial function is a common goal of available treatments. In the present study, human adipose-derived mesenchymal stromal cells (ASCs) were co-cultured with monocrotaline pyrrole-treated human pulmonary arterial endothelial cells (HPAECs); increased proliferation of HPAECs and expression of vascular endothelial growth factor (VEGF) were observed. High throughput sequencing results showed that six microRNAs (miMNAs) of ASCs were significantly dysregulated. In monocrotaline-induced PAH rat models, ASC transplantation improved the right ventricle systolic pressure, right ventricle hypertrophy and pulmonary endothelium hyperproliferation, and four of the six miRNAs were validated in the lung tissue samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these dysregulated miRNAs were involved in the regulation of transcription, signal transduction, negative regulation of cell proliferation through mitogen-activated protein kinase (MAPK) signaling pathway, Wnt signaling pathway, VEGF signaling pathway, cytokine-cytokine receptor interaction, regulation of actin cytoskeleton, transforming growth factor (TGF)-beta signaling pathway and P53 signaling pathway. Our data indicates that the unique six miRNA expression signature could be involved in the PAH endothelial repair by ASCs.

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.

Pyrrolizidine Alkaloid Secondary Pyrrolic Metabolites Construct Multiple Activation Pathways Leading to DNA Adduct Formation and Potential Liver Tumor Initiation.

Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives are hepatotoxic, genotoxic, and carcinogenic phytochemicals. PAs induce liver tumors through a general genotoxic mechanism mediated by a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP)-derived DNA adducts. To date, the primary pyrrolic metabolites dehydro-PAs, their hydrolyzed metabolite DHP, and two secondary pyrrolic metabolites 7-glutathione-DHP (7-GS-DHP) and 7-cysteine-DHP are the known metabolites that can generate these DHP-DNA adducts in vivo and/or in PA-treated cells. Secondary pyrrolic metabolites are formed from the reaction of dehydro-PAs with glutathione, amino acids, and proteins. In this investigation, we determined whether or not more secondary pyrrolic metabolites can bind to calf thymus DNA and to cellular DNA in HepG2 cells resulting in the formation of DHP-DNA adducts using a series of secondary pyrrolic metabolites (including 7-methoxy-DHP, 9-ethoxy-DHP, 9-valine-DHP, 7-GS-DHP, 7-cysteine-DHP, and 7,9-diglutathione-DHP) and synthetic pyrroles for study. We found that (i) many secondary pyrrolic metabolites are DNA reactive and can form DHP-DNA adducts and (ii) multiple activation pathways are involved in producing DHP-DNA adducts associated with PA-induced liver tumor initiation. These results suggest that secondary pyrrolic metabolites play a vital role in the initiation of PA-induced liver tumors.

Inhibition of the RhoA/Rho-associated, coiled-coil-containing protein kinase-1 pathway is involved in the therapeutic effects of simvastatin on pulmonary arterial hypertension.

BACKGROUND: Recent research has shown that statins improve pulmonary arterial hypertension (PAH), but their mechanisms of action are not fully understood. This study aimed to investigate the role of RhoA/ROCK1 regulation in the therapeutic effects of simvastatin on PAH. METHODS: For in vivo experiments, rats (N = 40) were randomly assigned to four groups: control, simvastatin, monocrotaline (MCT), and MCT + simvastatin. The MCT group and MCT + simvastatin groups received proline dithiocarbamate (50 mg/kg, i.p.) on the first day of the study. The MCT + simvastatin group received simvastatin (2 mg/kg) daily for 4 weeks, after which pulmonary arterial pressure was measured by right heart catheterization. The protein and mRNA levels of Rho and ROCK1 were measured by immunohistochemistry, Western blot, and PCR. For in vitro experiments, human pulmonary endothelial cells were divided into seven groups: control, simvastatin, monocrotaline pyrrole (MCTP), MCTP + simvastatin, MCTP + simvastatin + mevalonate, MCTP + simvastatin + farnesyl pyrophosphate (FPP), and MCTP + simvastatin + FPP + geranylgeranyl pyrophosphate (GGPP). After 72 h exposed to the drugs, the protein and mRNA levels of RhoA and ROCK1 were measured by Western blot and PCR. RESULTS: The MCT group showed increased mean pulmonary arterial pressure, marked vascular remodeling, and increased protein and mRNA levels of RhoA and ROCK1 compared to the other groups (P < 0.05). In vitro, the MCTP group showed a marked proliferation of vascular endothelial cells, as well as increased protein and mRNA levels of RhoA and ROCK1 compared to the MCTP + simvastatin group. The MCTP + simvastatin + mevalonate group, MCTP + simvastatin+ FPP group, and MCTP + simvastatin + FPP + GGPP group showed increased mRNA levels of RhoA and ROCK1, as well as increased protein levels of RhoA, compared to the MCTP + simvastatin group. CONCLUSIONS: Simvastatin improved vascular remodeling and inhibited the development of PAH. The effects of simvastatin were mediated by inhibition of RhoA/ROCK1. Simvastatin decreased RhoA/ROCK1 overexpression by inhibition of mevalonate, FPP, and GGPP synthesis.

Involvement of astrocytic CYP1A1 isoform in the metabolism and toxicity of the alkaloid pyrrolizidine monocrotaline.

Monocrotaline (MCT) and its pyrrole derivative, dehydromonocrotaline (DHMC), interact with molecular targets in cells of the central nervous system. DHMC presents higher toxicity than MCT indicating that its metabolism of MCT is a critical step of this alkaloid toxicity. This study sought to elucidate the metabolism and the toxicity of MCT in C6 astrocyte cell line and primary cultures of rat astrocytes by investigating metabolic enzymatic mechanisms of the cytochrome P450 (CYP) system and conjugation with glutathione. Treatment with omeprazole (OMP) (20 µM), a non-specific inducer of CYP450 induced approximately 10-fold increase in CYP1A1 activity after 2 h of treatment. Similarly, the 7-Ethoxyresorufin-O-deethylase (EROD) activity was induced by treatment with MCT (100-500 µM), indicating that the P450 CYP1A1 isoform was active and involved in the metabolism of MCT. Analysis of conjugation with glutathione showed a significant depletion of GSH after MCT (500 µM) treatment, and this was partially reversed by pretreatment with a P450 inhibitor (cimetidine 100 µM). These results suggest that not only the alkaloid MCT but, also its metabolite may deplete GSH. Rosenfeld staining showed intense vacuolization after MCT treatment, which was partially inhibited in the presence of a P450 activator. MTT test showed that association of MCT with OMP induced a reduction in cell viability in C6 and primary astrocytic cells. These results demonstrate that MCT is metabolized by astrocytic CYP1A1 to generate metabolites that can deplete GSH. Moreover, changes in the activity of the P450 enzymes interfere with the cytotoxic effects induced by the alkaloid.

7-cysteine-pyrrole conjugate: A new potential DNA reactive metabolite of pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) require metabolic activation to exert cytotoxicity, genotoxicity, and tumorigenicity. We previously reported that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts are responsible for PA-induced liver tumor formation in rats. In this study, we determined that metabolism of riddelliine and monocrotaline by human or rat liver microsomes produced 7-cysteine-DHP and DHP. The metabolism of 7-glutathionyl-DHP by human and rat liver microsomes also generated 7-cysteine-DHP. Further, reaction of 7-cysteine-DHP with calf thymus DNA in aqueous solution yielded the described DHP-derived DNA adducts. This study represents the first report that 7-cysteine-DHP is a new PA metabolite that can lead to DNA adduct formation.

[Beneficial effects of renal denervation on pulmonary vascular remodeling in experimental pulmonary artery hypertension].

OBJECTIVE: To explore the effects of renal sympathetic denervation (RSD) on pulmonary vascular remodeling in a model of pulmonary arterial hypertension (PAH). METHODS: According to the random number table, 24 beagles were randomized into control, PAH and PAH+RSD groups (n=8 each). The levels of neurohormone, echocardiogram and dynamics parameters were measured. Then 0.1 ml/kg dimethylformamide (control group) or 2 mg/kg dehydromonocrotaline (PAH and PAH+RSD groups) were injected. The PAH+RSD group underwent RSD after injection. At week 8 post-injection, the neurohormone levels, echocardiogram, dynamics parameters and pulmonary tissue morphology were observed. RESULTS: The values of right ventricular systolic pressure (RVSP) and pulmonary arterial systolic pressure (PASP) in PAH and PAH+RSD groups were both significantly higher than those in control group ((42.8±8.7), (30.8±6.8) vs (23.2±5.7) mmHg (1 mmHg=0.133 kPa) and (45.1±11.2), (32.6±7.9) vs (24.7±7.1) mmHg). Meanwhile, the values of RVSP and PASP in PAH group were higher than those in PAH+RSD group (all P<0.01). The levels of serum angiotensin II (Ang II) and endothelin-1 significantly increased after 8 weeks in PAH dogs ((228±41) vs (113±34) pg/ml and (135±15) vs (77±7) pg/ml, all P<0.01). And Ang II and endothelin-1 were higher in lung tissues of PAH group ((65±10) and (96±10) pg/ml) than in those of control group ((38±7) and (54±6) pg/ml) and PAH+RSD group ((46±8) and (67±9) pg/ml) (all P<0.01). Pulmonary tissues had marked collagen hyperplasia and lamellar corpuscles of type 2 alveolar cells were damaged more severely in PAH dogs than in PAH+RSD dogs. CONCLUSIONS: RSD suppresses pulmonary vascular remodeling and decreases pulmonary arterial pressure in experimental PAH. And the effect of RSD on PAH may contribute to decreased neurohormone levels.

Beneficial Effects of Renal Denervation on Pulmonary Vascular Remodeling in Experimental Pulmonary Artery Hypertension.

INTRODUCTION AND OBJECTIVES: Activation of both the sympathetic nervous system and the renin-angiotensin-aldosterone system is closely associated with pulmonary arterial hypertension. We hypothesized that renal denervation decreases renin-angiotensin-aldosterone activity and inhibits the progression of pulmonary arterial hypertension. METHODS: Twenty-two beagles were randomized into 3 groups. The dogs' pulmonary dynamics were measured before and 8 weeks after injection of 0.1mL/kg dimethylformamide (control dogs) or 2mg/kg dehydromonocrotaline (pulmonary arterial hypertension and pulmonary arterial hypertension + renal denervation dogs). Eight weeks after injection, neurohormone levels and pulmonary tissue morphology were measured. RESULTS: Levels of plasma angiotensin II and endothelin-1 were significantly increased after 8 weeks in the pulmonary arterial hypertension dogs and were higher in the lung tissues of these dogs than in those of the control and renal denervation dogs (mean [standard deviation] angiotensin II: 65 [9.8] vs 38 [6.7], 46 [8.1]; endothelin-1: 96 [10.3] vs 54 [6.2], 67 [9.4]; P < .01). Dehydromonocrotaline increased the mean pulmonary arterial pressure (16 [3.4] mmHg vs 33 [7.3] mmHg; P < .01), and renal denervation prevented this increase. Pulmonary smooth muscle cell proliferation was higher in the pulmonary arterial hypertension dogs than in the control and pulmonary arterial hypertension + renal denervation dogs. CONCLUSIONS: Renal denervation attenuates pulmonary vascular remodeling and decreases pulmonary arterial pressure in experimental pulmonary arterial hypertension. The effect of renal denervation may contribute to decreased neurohormone levels.

Reaction of dehydropyrrolizidine alkaloids with valine and hemoglobin.

Pyrrolizidine alkaloid-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids exert toxicity through metabolism to dehydropyrrolizidine alkaloids that bind to cellular protein and DNA, leading to hepatotoxicity, genotoxicity, and tumorigenicity. To date, it is not clear how dehydropyrrolizidine alkaloids bind to cellular constituents, including amino acids and proteins, resulting in toxicity. Metabolism of carcinogenic monocrotaline, riddelliine, and heliotrine produces dehydromonocrotaline, dehyroriddelliine, and dehydroheliotrine, respectively, as primary reactive metabolites. In this study, we report that reaction of dehydromonocrotaline with valine generated four highly unstable 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived valine (DHP-valine) adducts. For structural elucidation, DHP-valine adducts were derivatized with phenyl isothiocyanate (PITC) to DHP-valine-PITC products. After HPLC separation, their structures were characterized by mass spectrometry, UV-visible spectrophotometry, (1)H NMR, and (1)H-(1)H COSY NMR spectral analysis. Two DHP-valine-PITC adducts, designated as DHP-valine-PITC-1 and DHP-valine-PITC-3, had the amino group of valine linked to the C7 position of the necine base, and the other two DHP-valine-PITC products, DHP-valine-PITC-2 and DHP-valine-PITC-4, linked to the C9 position of the necine base. DHP-valine-PITC-1 was interconvertible with DHP-valine-PITC-3, and DHP-valine-PITC-2 was interconvertible with DHP-valine-PITC-4. Reaction of dehydroriddelliine and dehydroheliotrine with valine provided similar results. However, reaction of valine and dehydroretronecine (DHR) under similar experimental conditions did not produce DHP-valine adducts. Reaction of dehydromonocrotaline with rat hemoglobin followed by derivatization with PITC also generated the same four DHP-valine-PITC adducts. This represents the first full structural elucidation of protein conjugated pyrrolic adducts formed from reaction of dehydropyrrolizidine alkaloids with an amino acid (valine). In addition, it was found that DHP-valine-2 and DHP-valine-4, with the valine amino group linked at the C7 position of the necine base, can lose the valine moiety to form DHP.

Endothelial fate mapping in mice with pulmonary hypertension.

BACKGROUND: Pulmonary endothelial injury triggers a reparative program, which in susceptible individuals is characterized by neointima formation, vascular narrowing, and the development of pulmonary arterial hypertension. The neointimal cells in human pathological plexiform lesions frequently coexpress smooth muscle α-actin and the endothelial von Willebrand antigen, creating a question about their cellular lineage of origin. METHODS AND RESULTS: Experimental pulmonary hypertension with neointima formation develops in C57Bl/6 mice subjected to left pneumonectomy followed 1 week later by jugular vein injection of monocrotaline pyrrole (20 µg/µL and 1 µL/g; group P/MCTP). Compared with the group vehicle, by day 35, group P/MCTP developed higher right ventricular systolic pressure (54±5 versus 25±2 mm Hg; P<0.01) and right ventricular hypertrophy (0.58±0.16 versus 0.26±0.05; P<0.01). Transgenic vascular endothelial-cadherin Cre recombinase or Tie-2 Cre mice were intercrossed with mTomato/mGreen fluorescent protein double-fluorescent Cre reporter mice to achieve endothelial genetic lineage marking with membrane-targeted green fluorescent protein. In control mice, few endothelial lineage-marked cells lining the lumen of small pulmonary arteries demonstrate expression of smooth muscle α-actin. Concurrent with the development of pulmonary hypertension, endothelial lineage-marked cells are prominent in the neointima and exhibit expression of smooth muscle α-actin and smooth muscle myosin heavy chain. Human pulmonary arterial hypertension neointimal lesions contain cells that coexpress endothelial CD31 or von Willebrand antigen and smooth muscle α-actin. CONCLUSION: Neointimal cells in pulmonary hypertension include contributions from the endothelial genetic lineage with induced expression of smooth muscle α-actin and smooth muscle myosin heavy chain.

[Research on effects of bone marrow mononuclear cells implantation on model of experimental pulmonary artery hypertension].

In the present study, we carried out intratracheal administration of bone marrow-derived mononuclear cells (BM-MNCs) to dehydromonocrotaline (DMCT)-induced canine pulmonary artery hypertension (PH) of rat model to examine the security and feasibility, and the aim was to discuss the mechanism. All animals (n=30) were randomly divided into 3 groups (n=10 in each group), i. e. control group, PH group and BM-MNCs group. Six weeks after the transplantation, the hemodynamic data and right ventricle weight ratio were significantly improved for those in BM-MNCs group compared with those in PH group. The lung mRNA levels of vascular endothelial growth factor (VEGF) were higher, while preproendothelin-1 (ppET-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were lower compared with those in the PH group (P<0. 05). Immunofluorescence and histochemical results confirmed that 6 weeks after the administration, transplanted BM-MNCs were still alive and could differentiate into pulmonary vascular endothelial cells. These results showed that intratracheal administration of BM-MNCs could obviously reduce or even reverse the DMCT induction of PAH process. The mechanism could be explained as that the function was mainly through the paracrine effect to promote renewable and reduce inflammation.

Pulmonary and hepatic lesions caused by the dehydropyrrolizidine alkaloid-producing plants Crotalaria juncea and Crotalaria retusa in donkeys.

The effects and susceptibility of donkeys to Crotalaria juncea and Crotalaria retusa poisoning were determined at high and low doses. Seeds of C. juncea containing 0.074% of dehydropyrrolizidine alkaloids (DHPAs) (isohemijunceines 0.05%, trichodesmine 0.016%, and junceine 0.008%) were administered to three donkeys at 0.3, 0.6 and 1 g/kg body weight (g/kg) daily for 365 days. No clinical signs were observed and, on liver and lung biopsies, the only lesion was a mild liver megalocytosis in the donkeys ingesting 0.6 and 1 g/kg/day. Two other donkeys that received daily doses of 3 and 5 g seed/kg showed initial respiratory signs 70 and 40 days after the start of the administration, respectively. The donkeys were euthanized following severe respiratory signs and the main lung lesions were proliferation of Clara cells and interstitial fibrosis. Three donkeys ingested seeds of C. retusa containing 5.99% of monocrotaline at daily doses of 0.025, 0.05 and 0.1 g/kg for 365 days. No clinical signs were observed and, on liver and lung biopsies, the only lesion was moderate liver megalocytosis in each of the three donkeys. One donkey that received a single dose of 5 g/kg of C. retusa seeds and another that received 1 g/kg daily for 7 days both showed severe clinical signs and died with diffuse centrilobular liver necrosis. No lung lesions were observed. Another donkey that received a single dose of 2.5 g/kg of C. retusa seeds showed no clinical signs. The hepatic and pneumotoxic effects observed are consistent with an etiology involving DHPAs. Furthermore, the occurrence of lung or liver lesions correlates with the type of DHPAs contained in the seeds. Similarly as has been reported for horses, the data herein suggest that in donkeys some DHPAs are metabolized in the liver causing liver disease, whereas others are metabolized in the lung by Clara cells causing lung disease.

Atorvastatin prevents dehydromonocrotaline-induced pulmonary hypertension in beagles.

BACKGROUND: Pulmonary arterial hypertension is a life-threatening disease characterized by marked and sustained elevation of blood pressure in the lungs. Statins, 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors, have been shown to attenuate the effects of pulmonary hypertension resulting from hypoxia, Monocrotaline exposure, or Monocrotaline exposure in the setting of pneumonectomy. In particular, the effects of Simvastatin have been well studied. Whether other statins, such as Atorvastatin, are capable of preventing dehydromonocrotaline-induced pulmonary hypertension in beagles has not been explored. METHODS: We used eighteen 3-month-old beagles of both genders, weighing 10.3 ± 3.2 kg. The experimental animals were randomized into one of 3 groups: the control group (n = 6), the dehydromonocrotaline (DHMC) + vehicle group (n = 5), and the DHMC + Atorvastatin group (n = 7). The beagles were injected with DHMC (n = 12) on day 1, and from day 5 to day 65 they received Atorvastatin (2 mg/kg, daily by gavage) or vehicle (0.9% saline, daily by gavage) treatment. We used the thermodilution method of hemodynamic measurements at baseline and at day 65 of treatment. At day 65, pulmonary tissue was sampled for morphometry and real-time quantitative PCR. RESULTS: After 65 days, DHMC increased mean pulmonary arterial pressure (mPAP), and this increase was prevented with Atorvastatin treatment (32 ± 11 mmHg vs. 15 ± 3 mmHg, P < .05). Hematoxylin and eosin staining demonstrated less pulmonary endothelium destruction and smooth muscle cell proliferation in the Atorvastatin-treated beagles, compared with the DHMC group. The eNOS mRNA expression was increased in the DHMC group, and this increase was prevented in the Atorvastatin-treated group. In addition, IL-1ß, prepro-ET-1, TNF-α, and VEGF (vascular endothelial growth factor) mRNA expression levels were increased in the lungs of the DHMC group, and these increases were reduced toward normal levels in the Atorvastatin-treated group. CONCLUSION: Atorvastatin prevents the effects of monocrotaline-induced pulmonary hypertension in beagles.

Differential calcium handling in two canine models of right ventricular pressure overload.

BACKGROUND: The purpose of this investigation was to characterize differential right atrial (RA) and ventricular (RV) molecular changes in Ca(2+)-handling proteins consequent to RV pressure overload and hypertrophy in two common, yet distinct models of pulmonary hypertension: dehydromonocrotaline (DMCT) toxicity and pulmonary artery (PA) banding. METHODS: A total of 18 dogs underwent sternotomy in four groups: (1) DMCT toxicity (n = 5), (2) mild PA banding over 10 wk to match the RV pressure rise with DMCT (n = 5); (3) progressive PA banding to generate severe RV overload (n = 4); and (4) sternotomy only (n = 4). RESULTS: In the right ventricle, with DMCT, there was no change in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) or phospholamban (PLB), but we saw a trend toward down-regulation of phosphorylated PLB at serine-16 (p[Ser-16]PLB) (P = 0.07). Similarly, with mild PA banding, there was no change in SERCA or PLB, but p(Ser-16)PLB was down-regulated by 74% (P < 0.001). With severe PA banding, there was no change in PLB, but SERCA fell by 57% and p(Ser-16)PLB fell by 67% (P < 0.001). In the right atrium, with DMCT, there were no significant changes. With both mild and severe PA banding, p(Ser-16)PLB fell (P < 0.001), but SERCA and PLB did not change. CONCLUSIONS: Perturbations in Ca(2+)-handling proteins depend on the degree of RV pressure overload and the model used to mimic the RV effects of pulmonary hypertension. They are similar, but blunted, in the atrium compared with the ventricle.

Dehydropyrrolizidine alkaloids, including monoesters with an unusual esterifying acid, from cultivated Crotalaria juncea (Sunn Hemp cv.'Tropic Sun').

Cultivation of Crotalaria juncea L. (Sunn Hemp cv. 'Tropic Sun') is recommended as a green manure crop in a rotation cycle to improve soil condition, help control erosion, suppress weeds, and reduce soil nematodes. Because C. juncea belongs to a genus that is known for the production of toxic dehydropyrrolizidine alkaloids, extracts of the roots, stems, leaves, and seeds of 'Tropic Sun' were analyzed for their presence using HPLC-ESI/MS. Qualitative analysis identified previously unknown alkaloids as major components along with the expected macrocyclic dehydropyrrolizidine alkaloid diesters, junceine and trichodesmine. The dehydropyrrolizidine alkaloids occurred mainly as the N-oxides in the roots, stems, and, to a lesser extent, leaves, but mainly as the free bases in the seeds. Comprehensive spectrometric and spectroscopic analysis enabled elucidation of the unknown alkaloids as diastereoisomers of isohemijunceine, a monoester of retronecine with an unusual necic acid. The dehydropyrrolizidine alkaloid contents of the roots, stems, and leaves of immature plants were estimated to be 0.05, 0.12, and 0.01% w/w, respectively, whereas seeds were estimated to contain 0.15% w/w.