Analysis of necroptosis and its association with pyroptosis in organ damage in experimental pulmonary arterial hypertension.

In this study, a role of cell loss due to necroptosis and its linkage with pyroptosis in organ damage under the conditions of pulmonary arterial hypertension (PAH) was examined. Monocrotaline (MCT) was used to induce PAH in Wistar rats, and depending on the severity of the disease progression, they were further divided into two subgroups: MCT group-sacrificed 4 weeks after MCT administration and ptMCT group-prematurely sacrificed due to rapid deterioration in vital functions (on Day 24,11 ± 0,7). The elevation of respiratory rate and right ventricular (RV) hypertrophy were more evident in ptMCT group, while the heart rate and cardiac haemodynamic stress markers were comparably higher in both diseased groups. Detailed immunoblotting analysis revealed that the upregulation of pThr231 /Ser232 -RIP3 proceeded into necroptosis execution in the RVs, unlike in the lungs of both PAH stages. The elevated pulmonary pThr231 /Ser232 -RIP3 levels in both PAH subgroups were associated rather with GSDMD-mediated pyroptosis. On the contrary, other inflammasome forms, such as AIM2 and NLRC4, were higher in the RV, unlike in the lungs, of diseased groups. The PAH-induced increase in the plasma RIP3 levels was more pronounced in ptMCT group, and positively correlated with RV hypertrophy, but not with haemodynamic stress. Taken together, we indicated for the first time that pThr231 /Ser232 -RIP3 upregulation resulting in two different necrosis-like cell death modes might underlie the pathomechanisms of PAH and that the plasma RIP3 might serve as an additional diagnostic and prognostic marker of cardiac injury under these conditions.

Disease severity-related alterations of cardiac microRNAs in experimental pulmonary hypertension.

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH). We hypothesized that heart-relevant microRNAs, that is myomiRs (miR-1, miR-133a, miR-208, miR-499) and miR-214, can have a role in the right ventricle in the development of PAH. To mimic PAH, male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.); control group received vehicle. MCT rats were divided into two groups, based on the clinical presentation: MCT group terminated 4 weeks after MCT administration and prematurely terminated group (ptMCT) displaying signs of terminal disease. Myocardial damage genes and candidate microRNAs expressions were determined by RT-qPCR. Reduced blood oxygen saturation, breathing disturbances, RV enlargement as well as elevated levels of markers of myocardial damage confirmed PH in MCT animals and were more pronounced in ptMCT. MyomiRs (miR-1/miR-133a/miR-208a/miR-499) were decreased and the expression of miR-214 was increased only in ptMCT group (P < 0.05). The myomiRs negatively correlated with Fulton index as a measure of RV hypertrophy in MCT group (P < 0.05), whereas miR-214 showed a positive correlation (P < 0.05). We conclude that the expression of determined microRNAs mirrored the disease severity and targeting their pathways might represent potential future therapeutic approach in PAH.

Hepatocyte growth factor plays a particular role in progression of overall cardiac damage in experimental pulmonary hypertension.

Background: HGF/MET pathway may have a role in pulmonary hypertension (PH). However, the link between the pathway and development of target organ damage in PH remains elusive. We aimed to demonstrate the relation between plasma HGF and HGF/MET tissue expressions in affected organs during PH progression. Methods: 12 weeks old male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.) to induce PH and sacrificed after 1, 2 and 4 weeks. Controls received saline. mRNA levels of HGF regulatory complex (Hgf, Met, Hgfa, Hai-1, Hai-2) were determined in right and left ventricles (RV, LV), lungs, pulmonary artery and liver by RT-qPCR. HGF protein levels in plasma were analysed by ELISA. Results: PH development was associated with a progressive elevation of HGF plasma levels that correlated with relative RV mass. Furthermore, Hgf mRNA expressions at week 4 were upregulated solely in the cardiac ventricles while being downregulated in a. pulmonalis, lungs and liver. Met and Hai-1/Hai-2 followed a similar pattern and were upregulated in cardiac ventricles, where Hgfa remained unchanged, but downregulated in lungs. Conclusion: We suggest that cardiac overexpression of Hgf might contribute to increased plasma HGF in MCT-induced PH. HGF could be exploited as a cardiospecific biomarker and HGF/MET pathway as a target in drug discovery for PH.

Pegfilgrastim and linagliptin potentiate chemoattraction of Ccr2 and Cd44 stem cells accompanied by alterations of cardiac Hgf, Igf-1 and Mcp-1 in daunorubicin cardiomyopathy.

OBJECTIVE: Daunorubicin (DAU) downregulates cytokines promoting stem cell migration and homing into the heart, reducing cardiac regeneration after anticancer chemotherapy. Pegfilgrastim (PFIL) protects from DAU-induced neutropenia but its cardioprotective potential remains unclear. We tested whether pegfilgrastim and a dipeptidyl peptidase-4 inhibitor linagliptin, potential enhancers of stem cells migration and homing, would improve DAU-cardiomyopathy. METHODS: DAU (7.5 mg/kg, i.v.) was administered to male Wistar rats to induce cardiotoxicity. Pegfilgrastim (100 µg/kg, s.c.) was administered 24h after DAU, and linagliptin was administered orally for 8 weeks (5 mg/kg/day, LINA). Cardiac damage markers (Nppa, Myh6, Myh7, Gp91phox), cytokines (Sdf-1alpha, Mcp-1, Vegf, Hgf, Igf-1), stem cell markers (Cxcr4, Ccr2, Cd34, Cd133, Cd44, Cd105) were determined by qRT-PCR. KEY FINDINGS: Decreased Myh6, elevated Myh7 Nppa, and Gp91phox were not ameliorated by PFIL + LINA. Downregulated expressions of cytokines (Vegf, Sdf-1alpha) and stem cells markers (Cxcr4, Cd34, Cd133, and Cd105) remained decreased after PFIL + LINA. DAU-induced upregulation of Mcp-1, Ccr2 and Cd44 was further potentiated by PFIL + LINA. PFIL + LINA normalised expression of Hgf and Igf-1. CONCLUSIONS: Although PFIL + LINA failed in universal potentiation of stem cells migration and homing, the expression of stem cell markers Ccr2 and Cd44 in the heart potentially increased through the preservation of Hgf, Igf-1 and upregulation of Mcp-1.

Opposite alterations of endothelin-1 in lung and pulmonary artery mirror gene expression of bone morphogenetic protein receptor 2 in experimental pulmonary hypertension.

Aim of the Study: Endothelin-1 (ET-1) overexpression was suggested to play a role in pulmonary hypertension (PH). However, the roles of ET-1 in early stages of PH remain unexplored. We examined the expression of ET-1 and relevant disease progression markers in the pulmonary artery and the lungs during the development of PH induced by monocrotaline (MCT). Material and Methods: Male 12-weeks-old Wistar rats were administered with MCT (60 mg/kg, s.c.) or saline (CON). We measured right ventricular pressure (RVP) by catheterization under tribromoethanol anesthesia; hemoglobin oxygen saturation, breathing rate were measured by pulse oximetry in conscious animals. Rats were sacrificed 1, 2 or 4 weeks after MCT. mRNA levels of ET-1, its receptors, inflammatory markers IL-1beta, TNFalpha, IL-6 and genes related to VSMC proliferation or lung damage (Bmpr2, nestin, Pim1, PAI-1, TGFbeta-1) were analyzed by RT-qPCR. Results: RVP and breathing rate increased and hemoglobin oxygen saturation decreased after MCT only at week 4. Lung weight was increased at all time points. ET-1 was upregulated in the pulmonary artery at weeks 1 and 4, while being clearly suppressed in the lungs at all times. Bone morphogenetic protein receptor 2 followed a similar pattern to ET-1. PAI-1 markedly increased in the MCT lungs (but not pulmonary artery) from week 1 to 4. Nestin peaked at week 2 in both tissues. TGFbeta-1 increased in both tissues at week 4. ET-1 expression did not correlate with other genes, however, Bmpr2 tightly negatively correlated with PAI-1 in the lungs, but not pulmonary artery of MCT groups. Conclusions: ET-1 overexpression in the pulmonary artery preceded development of PH, but it was clearly and unexpectedly downregulated in the lungs of monocrotaline-treated rats and showed no correlation to disease progression markers. We speculate that endothelin-1 may play opposing roles in the lungs vs pulmonary artery in monocrotaline-induced PH.