Jagged/Notch proteins promote endothelial-mesenchymal transition-mediated pulmonary arterial hypertension via upregulation of the expression of GATAs.

This study tested the hypothesis that Jagged2/Notches promoted the endothelial-mesenchymal transition (endMT)-mediated pulmonary arterial hypertension (PAH) (i.e. induction by monocrotaline [MCT]/63 mg/kg/subcutaneous injection) through increasing the expression of GATA-binding factors which were inhibited by propylthiouracil (PTU) (i.e. 0.1% in water for daily drinking since Day 5 after PAH induction) in rodent. As compared with the control (i.e. HUVECs), the protein expressions of GATAs (3/4/6) and endMT markers (Snail/Zeb1/N-cadherin/vimentin/fibronectin/α-SMA/p-Smad2) were significantly reduced, whereas the endothelial-phenotype markers (CD31/E-cadherin) were significantly increased in silenced JAG2 gene or in silenced GATA3 gene of HUVECs (all p < 0.001). As compared with the control, the protein expressions of intercellular signallings (GATAs [3/4/6], Jagged1/2, notch1/2 and Snail/Zeb1/N-cadherin/vimentin/fibronectin/α-SMA/p-Smad2) were significantly upregulated in TGF-ß/monocrotaline-treated HUVECs that were significantly reversed by PTU treatment (all p < 0.001). By Day 42, the results of animal study demonstrated that the right-ventricular systolic-blood-pressure (RVSBP), RV weight (RVW) and lung injury/fibrotic scores were significantly increased in MCT group than sham-control (SC) that were reversed in MCT + PTU groups, whereas arterial oxygen saturation (%) and vasorelaxation/nitric oxide production of PA exhibited an opposite pattern of RVW among the groups (all p < 0.0001). The protein expressions of hypertrophic (ß-MHC)/pressure-overload (BNP)/oxidative-stress (NOX-1/NOX-2) biomarkers in RV and the protein expressions of intercellular signalling (GATAs3/4/6, Jagged1/2, notch1/2) and endMT markers (Snail/Zeb1/N-cadherin/vimentin/fibronectin/TGF-ß/α-SMA/p-Smad2) in lung parenchyma displayed an identical pattern of RVW among the groups (all p < 0.0001). Jagged-Notch-GATAs signalling, endMT markers and RVSBP that were increased in PAH were suppressed by PTU.

Combination of melatonin-delivered endothelial progenitor cells with S-nitroso-N-acetyl-DL-penicillamine for improving critical limb ischemia in the rat.

BACKGROUND: This study tested whether combined shock wave (SW)-facilitated melatonin (Mel) delivered into endothelial progenitor cells (EPCs) (EPCSW-Mel) plus S-nitroso-N-acetyl-DL-penicillamine (SNAP) was superior to merely one modality alone for improving critical limb ischemia (CLI) in rats. METHODS: SD rats (n = 50) were equally categorized into group 1 (sham-control), group 2 (CLI), group 3 (CLI + SNAP), group 4 (CLI + EPCSW-Mel), and group 5 (CLI + EPCSW-Mel + SNAP), and ischemia-involved quadriceps were harvested by day 14. RESULTS: An in vitro study showed that at time points of 24/48/72 h, the cell viability/protein expression of endothelial nitric oxide synthase (eNOS)/and cellular expression of nitric oxide (NO) were highest in EPCs, lowest in EPCs + menadione, and much higher in EPCSW-Mel + Mena than in EPCs + Mena + Mel. Protein levels of oxidative-stress (NOX-1/NOX-2/oxidized protein)/early (AN-V+/PI-)/late (AN-V+/PI+) apoptosis and total intracellular/mitochondrial reactive oxygen species ROS exhibited an antithetical trend of cell viability among the groups (all P<0.0001). Matrigel assay of angiogenesis/positively-stained NO cells showed that they were much higher in EPCs + SNAP than in EPCs only (all P<0.0001). Ex vivo angiogenesis/arterial relaxation of carotid-artery rings were highest in left-common-carotid-artery (LCCA) + SNAP, lowest in LCCA + Mena, and notably higher in LCCA than in LCCA + Mena + SNAP (all P<0.0001). Laser Doppler showed ischemic to normal-blood-flow (INBF) ratio was highest in group 1, lowest in group 2, and it progressively increased from groups 3 to 5 (all P<0.0001). The protein levels of oxidative-stress (NOX-1/NOX-4/oxidized protein)/apoptotic [cleaved-caspase-3/cleaved apoptosis/mitochondrial-damage (cytosolic-cytochrome-C/p-DRP-1)]/fibrotic (Smad3/TGF-ß)/inflammatory (MMP-9/IL-1ß/TNF-α/NF-κB) biomarkers, exhibited an opposite trend, whereas the protein level of endothelial-cell surface markers (CD31/vWF/eNOS) and number of small vessels exhibited an identical pattern of INBF ratio among the groups (all P<0.0001). CONCLUSIONS: Combined EPCSW-Mel and SNAP therapy offered a synergic effect toward rescuing from CLI.

ITRI Biofilm Prevented Thoracic Adhesion in Pigs That Received Myocardial Ischemic Induction Treated by Myocardial Implantation of EPCs and ECSW Treatment.

This study tested the hypothesis that ITRI Biofilm prevents adhesion of the chest cavity. Combined extracorporeal shock wave (ECSW) + bone marrow-derived autologous endothelial progenitor cell (EPC) therapy was superior to monotherapy for improving heart function (left ventricular ejection fraction [LVEF]) in minipigs with ischemic cardiomyopathy (IC) induced by an ameroid constrictor applied to the mid-left anterior descending artery. The minipigs (n = 30) were equally designed into group 1 (sham-operated control), group 2 (IC), group 3 (IC + EPCs/by directly implanted into the left ventricular [LV] myocardium; 3 [+]/3[-] ITRI Biofilm), group 4 (IC + ECSW; 3 [+]/[3] - ITRI Biofilm), and group 5 (IC + EPCs-ECSW; 3 [+]/[3] - ITRI Biofilm). EPC/ECSW therapy was administered by day 90, and the animals were euthanized, followed by heart harvesting by day 180. In vitro studies demonstrated that cell viability/angiogenesis/cell migratory abilities/mitochondrial concentrations were upregulated in EPCs treated with ECSW compared with those in EPCs only (all Ps < 0.001). The LVEF was highest in group 1/lowest in group 2/significantly higher in group 5 than in groups 3/4 (all Ps < 0.0001) by day 180, but there was no difference in groups 3/4. The adhesion score was remarkably lower in patients who received ITRI Biofilm treatment than in those who did not (all Ps <0.01). The protein expressions of oxidative stress (NOX-1/NOX-2/oxidized protein)/apoptotic (mitochondrial-Bax/caspase3/PARP)/fibrotic (TGF-ß/Smad3)/DNA/mitochondria-damaged (γ-H2AX/cytosolic-cytochrome-C/p-DRP1), and heart failure/pressure-overload (BNP [brain natriuretic peptide]/ß-MHC [beta myosin heavy chain]) biomarkers displayed a contradictory manner of LVEF among the groups (all Ps < 0.0001). The protein expression of endothelial biomarkers (CD31/vWF)/small-vessel density revealed a similar LVEF within the groups (all Ps < 0.0001). ITRI Biofilm treatment prevented chest cavity adhesion and was superior in restoring IC-related LV dysfunction when combined with EPC/ECSW therapy compared with EPC/ECSW therapy alone.

Outcomes of combined mitochondria and mesenchymal stem cells-derived exosome therapy in rat acute respiratory distress syndrome and sepsis.

BACKGROUND: The treatment of acute respiratory distress syndrome (ARDS) complicated by sepsis syndrome (SS) remains challenging. AIM: To investigate whether combined adipose-derived mesenchymal-stem-cells (ADMSCs)-derived exosome (EXAD) and exogenous mitochondria (mitoEx) protect the lung from ARDS complicated by SS. METHODS: In vitro study, including L2 cells treated with lipopolysaccharide (LPS) and in vivo study including male-adult-SD rats categorized into groups 1 (sham-operated-control), 2 (ARDS-SS), 3 (ARDS-SS + EXAD), 4 (ARDS-SS + mitoEx), and 5 (ARDS-SS + EXAD + mitoEx), were included in the present study. RESULTS: In vitro study showed an abundance of mitoEx found in recipient-L2 cells, resulting in significantly higher mitochondrial-cytochrome-C, adenosine triphosphate and relative mitochondrial DNA levels (P < 0.001). The protein levels of inflammation [interleukin (IL)-1ß/tumor necrosis factor (TNF)-α/nuclear factor-κB/toll-like receptor (TLR)-4/matrix-metalloproteinase (MMP)-9/oxidative-stress (NOX-1/NOX-2)/apoptosis (cleaved-caspase3/cleaved-poly (ADP-ribose) polymerase)] were significantly attenuated in lipopolysaccharide (LPS)-treated L2 cells with EXAD treatment than without EXAD treatment, whereas the protein expressions of cellular junctions [occluding/ß-catenin/zonula occludens (ZO)-1/E-cadherin] exhibited an opposite pattern of inflammation (all P < 0.001). Animals were euthanized by 72 h post-48 h-ARDS induction, and lung tissues were harvested. By 72 h, flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that the levels of inflammatory cells (Ly6G+/CD14+/CD68+/CD11b/c+/myeloperoxidase+) and albumin were lowest in group 1, highest in group 2, and significantly higher in groups 3 and 4 than in group 5 (all P < 0.0001), whereas arterial oxygen-saturation (SaO2%) displayed an opposite pattern of albumin among the groups. Histopathological findings of lung injury/fibrosis area and inflammatory/DNA-damaged markers (CD68+/γ-H2AX) displayed an identical pattern of SaO2% among the groups (all P < 0.0001). The protein expressions of inflammatory (TLR-4/MMP-9/IL-1ß/TNF-α)/oxidative stress (NOX-1/NOX-2/p22phox/oxidized protein)/mitochondrial-damaged (cytosolic-cytochrome-C/dynamin-related protein 1)/autophagic (beclin-1/Atg-5/ratio of LC3B-II/LC3B-I) biomarkers exhibited a similar manner, whereas antioxidants [nuclear respiratory factor (Nrf)-1/Nrf-2]/cellular junctions (ZO-1/E-cadherin)/mitochondrial electron transport chain (complex I-V) exhibited an opposite manner of albumin among the groups (all P < 0.0001). CONCLUSION: Combined EXAD-mitoEx therapy was better than merely one for protecting the lung against ARDS-SS induced injury.

Intrarenal arterial administration of human umbilical cord-derived mesenchymal stem cells effectively preserved the residual renal function of diabetic kidney disease in rat.

BACKGROUND: This experimental study was designed as a preclinical study for testing the hypothesis that intrarenal arterial (IRA) transfusion of human umbilical cord-derived mesenchymal stem cells (HUCDMSCs) therapy preserved the residual renal function of diabetic kidney disease (DKD) in rat [induction by 5/6 nephrectomy of left kidney and right nephrectomy, followed by intraperitoneal administration of aminoguanidine (180 mg/kg) and streptozotocin (30 mg/kg)]. METHODS: Animals (n = 24) were categorized into group 1 (sham-operated control), group 2 (DKD), group 3 [DKD + HUCDMSCs (2.1 × 105/IRA injection at day 28 after CKD induction)] and group 4 [(DKD + HUCDMSCs (6.3 × 105/IRA injection)]. RESULTS: By day 60 after DKD induction, the kidneys were harvested and the result showed that the creatinine level, ratio of urine protein/urine creatinine and kidney injury score were lowest in group 1, highest in group 2 and significantly lower in group 4 than in group 3 (all p < 0.0001). The protein expressions of apoptotic (cleaved caspase-3/cleaved PARP/mitochondrial Bax), fibrotic (TGF-ß/p-Smad3), autophagic (ratio of LC3B-II/LC3B-I, Atg5/Beclin-1), oxidative stress (NOX-1/NOX-2/oxidized protein/p22phox), mitochondrial/DNA-damaged (cytosolic-cytochrome-C/DRP1/γ-H2AX) and inflammatory (MMP-9/TNF-α/p-NF-κB) biomarkers exhibited an identical pattern, whereas the protein expressions of angiogenesis factors (CD31/vWF/vascularity) exhibited an opposite pattern of creatinine level among the groups (all p < 0.0001). Histopathological findings demonstrated the renal tubular-damaged (KIM-1)/kidney fibrosis area/oxidative stress (8-OHdG + cells) expressed an identical pattern, whereas the podocyte components (ZO-1/synaptopodin/podocin) exhibited an opposite pattern of creatinine level among the groups (all p < 0.0001). No tumorigenesis or immune rejection event was identified. CONCLUSION: IRA injection of xenogeneic MSCs was safe and effectively protected the residual renal function and architectural integrity in DKD rat.

Impact of One Versus Two Consecutive Doses of Endothelial Cells (EPCs) and EPCs-Derived Condition Medium on Protecting Myocardium from Acute Ischemia-Reperfusion Injury in Rat.

This study tested the impact of single dose and two doses of endothelial progenitor cells (EPCs) and EPCs-derived condition medium (CM) on protecting the left-ventricular myocardium (LVM) from acute ischemia-reperfusion (IR) injury. In vitro study showed EPCs and CM had comparably higher capacity for enhancement of angiogenesis as compared with the controls (all P < .001). Adult-male SD rats (n = 36) were equally categorized into groups 1 (sham-operated control), 2 (IR+vehicle), 3 [IR+EPCs/1.2 × 106/intravenous administration at 3 h after IR procedure), 4 (IR+EPCs/1.2 × 106/at 3 h/24 h after IR), 5 (IR+CM/3.0cc/intravenous administration at 3 h after IR), 6 (IR+EPCs/3.0cc/at 3h/24 h after IR), and euthanized by day 3 after IR. The left-ventricular-ejection-fraction, protein and cellular expressions of endothelial-cell markers (CD31/vWF), small vessel number and protein expression of mitochondrial (mitochondrial-cytochrome-C) integrity were highest in group 1, lowest in group 2, significantly higher in group 4 than in groups 3/5/6 and significantly higher in groups 3/6 than in group 5 but they showed no differences in groups3/6, whereas the protein expressions of apoptotic (cleaved-caspase 3/cleaved-PARP), fibrotic (Smad3/TGF-ß), mitochondrial-damaged (cytosolic-cytochrome-C), heart-failed/pressure-overload (BNP), oxidative-stress (p47phox/NOX-1/NOX-2/oxidized protein), and autophagic (LCB3-II/LCB3-I) biomarkers and fibrotic/collagen-deposition areas exhibited an opposite pattern to endothelial-cell markers (all P < .0001). The protein expressions of angiogenesis (VEGF/SDF-1α/CXCR4/HIF-1α) were lowest in group 1, highest in group 4, significantly higher in groups 3/6 than in groups 2/5, significantly higher in group 5 than in group 2, but they showed no difference between groups 3/6 (all P < .0001). These results demonstrate that two consecutive doses of EPC/CM were superior to just one at protecting LVM against IR injury.

Entresto protected the cardiomyocytes and preserved heart function in cardiorenal syndrome rat fed with high-protein diet through regulating the oxidative stress and Mfn2-mediated mitochondrial functional integrity.

This study tested the hypothesis that Entresto (En) therapy protected the cardiomyocytes and heart function in cardiorenal syndrome (CRS) rats fed with high-protein diet (HPD) through regulating the oxidative-stress and Mfn2-mediated mitochondrial functional integrity. En (12.5 µM for the in-vitro study) protected the H9C2-cells against H2O2-induced cell apoptosis, whereas stepwise-increased H2O2 concentrations induced a significant increase in protein expressions of Mfn2/phosphorylated (p)-DRP1/mitochondrial-Bax in H9C2-cells. En downregulated H2O2-induced mitochondrial fission/upregulated mitochondrial fusion and deletion of Mfn2 gene (i.e., shMfn2) to significantly reduce H2O2-induced ROS production. En significantly suppressed and shMfn2 further significantly suppressed both H2O2-reduced mitochondrial-membrane potential and H2O2-induced ROS production/cell apoptosis/mitochondrial damage/mitochondrial-Bax released from mitochondria in H9C2 cells. En significantly reduced protein expressions of Mfn2 and p-DRP1. Additionally, En significantly suppressed and shMfn2 further significantly suppressed the protein expressions of mitochondrial-damaged (DRP1)/oxidative-stress (NOX-1/NOX-2)/apoptosis (mitochondrial-Bax/caspase-3/PARP)/autophagic (LC3B-II/LC3B-I) biomarkers (all p < 0.01). Rats were categorized into group 1 [sham-control + high-protein-diet (HPD)], group 2 (CRS + HPD) and group 3 (CRS+ HPD + En/100 mg/kg/day). By day 63 after CRS induction, the LVEF was significantly lower in group 3 and more significantly lower in group 2 than in group 1, whereas the protein expressions of oxidative-stress (NOX-1/NOX-2/p22phox/oxidized protein)/apoptotic (mitochondrial-Bax/caspase-3/PARP), fibrotic (Smad-3/TGF-ß), autophagic (Beclin-1/Atg5/ratio of LC3B-II/LC3B-I) and mitochondrial-damaged (DRP1/cyclophilin-D/cytosolic-cytochrome-C) biomarkers exhibited an opposite pattern of LVEF among the groups. Downregulation of Mfn2 by En or shMfn2 in cardiomyocytes avoided H2O2 damage and En improved the cardiac function in HPD-feeding CRS rat via adjusting Mfn2-mediated mitochondrial functional integrity.

Early treatment with combination of SS31 and entresto effectively preserved the heart function in doxorubicin-induced dilated cardiomyopathic rat.

BACKGROUND: This study tested the hypothesis that early administration of SS31 and entresto (En) was superior to either one alone on preserving the heart function in setting of dilated cardiomyopathy (DCM) induced by doxorubicin (Dox) [accumulated dosage of 12.5 mg/kg/administered by intraperitoneal (IP) at 4 separated time points within 20 days] in rat. METHODS AND RESULTS: Adult-male SD rats (n = 40) were equally categorized into groups 1 (sham-control), 2 (DCM), 3 (DCM + SS31/0.7 mg/kg/day/IP, since day-14 after DCM induction to day-60), 4 [DCM + En (30 mg/kg/day/orally since day-14 after DCM induction to day-60)] and 5 (DCM + combined SS31-En), and animals were euthanized by day 60. By day 60, left-ventricular ejection-fraction (LVEF) was highest in group 1, lowest in group 2 and significantly higher in group 5 than in groups 3 and 4 (all p < 0.0001), but it showed no difference between groups 3/4. The microscopic study showed that the fibrosis area/cardiomyocyte size and DNA-damaged (γ-H2AX+)/inflammatory (CD14+//CD68+) markers, and flow analysis of inflammatory (Ly6G+/MPO+/CD11b/c+) and early/late apoptosis (AN-V+/PI-//AN-V+/PI+) cells exhibited an opposite pattern of LVEF among the five groups (all p < 0.0001). The protein expressions of inflammatory upstream (TLR2/TLR4/MyD88/Mal/ TRAF6/IKK-α/IKK-ß) and downstream (p-NF-κb/TNF-α/IL-1ß/MMP-9), oxidative-stress/mitochondrial-damaged (NOX-1/NOX-2/cytosolic cytochrome-C/cyclophilin-D/DRP1) and autophagic/apoptotic (ratio of LC3B-II/LC3B-I and mitochondrial-Bax/caspase3/9) signaling pathways also exhibited an opposite pattern of LVEF among the five groups (all p < 0.0001). CONCLUSION: Combined SS31-En therapy was superior to either one alone on protecting the heart structural and functional integrities against Dox-induced DCM damage.