Combined tacrolimus and melatonin effectively protected kidney against acute ischemia-reperfusion injury.

Acute kidney injury (AKI) is commonly encountered and causes high mortality in hospitalized patients; however, effective therapies for AKI have still not been established. Accordingly, we performed a rodent model with acute renal ischemia-reperfusion (IR) and tested the hypothesis that combined tacrolimus and melatonin therapy could be superior to either one for protecting the kidney against IR injury. Adult-male SD rat (n = 30) were equally categorized into group 1 (receiving laparotomy only), group 2 (IR treated by 3.0 cc/normal-saline), group 3 [IR + tacrolimus/0.5 mg/kg by intravenous administration at 30 minutes and at days 1/2/3 after IR], group 4 (IR + melatonin/50 mg/kg by intra-peritoneal administration at 30 minutes and 25 mg/kg at days 1/2/3 after IR] and group 5 (IR + tacrolimus +melatonin). By day 3 after IR, the creatinine/BUN levels and ratio of urine protein to urine creatinine were highest in group 2, lowest in group 1 and significantly lower in group 5 than in groups 3/4 (all P < .0001), but they did not differ between the groups 3/4. The protein expressions of oxidative-stress (p47phox/NOX-1/NOX-2/NOX-4), upstream (TLR4/MAL/MyD88/TRAF6/ASK1/MKK4/MKK7/NF-κB) and downstream (IL-6/INF-γ/MMP-9/IL-1ß) inflammatory signaling, MAPK-family-signaling cascades(ERK1/2, JNK/p38/c-JUN), apoptotic/autophagic (p53/caspase 3/mitochondrial-Bax, ratio of LC3B-II/LC3B-I), and mitochondrial-damaged (cyclophilin D/cytochrome C/DRP1) biomarkers, and the expressions of inflammatory-immune cells (F4/80, CD14/CD3/CD8) as well as the kidney injured score exhibited an identical pattern of creatinine level (all P < .0001). In conclusion, combined tacrolimus and melatonin therapy was better than either single one on protecting the kidney functional and anatomical integrity against IR injury through suppressing inflammation and the generation of oxidative stress.

Losing Regulation of the Extracellular Matrix is Strongly Predictive of Unfavorable Prognostic Outcome after Acute Myocardial Infarction.

This study tested the hypothesis that MMP-9-/-tPA-/- double knock out (i.e., MTDKO) plays a crucial role in the prognostic outcome after acute myocardial infarction (AMI by ligation of left-coronary-artery) in MTDKO mouse. Animals were categorized into sham-operated controls in MTDKO animals (group 1) and in wild type (B6: group 2), AMI-MTDKO (group 3) and AMI-B6 (group 4) animals. They were euthanized, and the ischemic myocardium was harvested, by day 60 post AMI. The mortality rate was significantly higher in group 3 than in other groups and significantly higher in group 4 than in groups 1/2, but it showed no difference in the latter two groups (all p < 0.01). By day 28, the left-ventricular (LV) ejection fraction displayed an opposite pattern, whereas by day 60, the gross anatomic infarct size displayed an identical pattern of mortality among the four groups (all p < 0.001). The ratio of heart weight to tibial length and the lung injury score exhibited an identical pattern of mortality (p < 0.01). The protein expressions of apoptosis (mitochondrial-Bax/cleaved-caspase3/cleaved-PARP), fibrosis (Smad3/T-GF-ß), oxidative stress (NOX-1/NOX-2/oxidized-protein), inflammation (MMPs2,9/TNF-α/p-NF-κB), heart failure/pressure overload (BNP/ß-MHC) and mitochondrial/DNA damage (cytosolic-cytochrome-C/γ-H2AX) biomarkers displayed identical patterns, whereas the angiogenesis markers (small vessel number/CD31+cells in LV myocardium) displayed opposite patterns of mortality among the groups (all p < 0.0001). The microscopic findings of fibrotic/collagen deposition/infarct areas and inflammatory cell infiltration of LV myocardium were similar to the mortality among the four groups (all p < 0.0001). MTDKO strongly predicted unfavorable prognostic outcome after AMI.

Daily melatonin protects the endothelial lineage and functional integrity against the aging process, oxidative stress, and toxic environment and restores blood flow in critical limb ischemia area in mice.

We tested the hypothesis that daily melatonin treatment protects endothelial lineage and functional integrity against the aging process, oxidative stress/endothelial denudation (ED), and toxic environment and restored blood flow in murine critical limb ischemia (CLI). In vitro study using HUVECs, in vivo models (ie, CLI through left femoral artery ligation and ED through carotid artery wire injury), and model of lipopolysaccharide-induced aortic injury in young (3 months old) and aged (8 months old) mice were used to elucidate effects of melatonin treatment on vascular endothelial integrity. In vitro study showed that menadione-induced oxidative stress (NOX-1/NOX-2), inflammation (TNF-α/NF-kB), apoptosis (cleaved caspase-3/PARP), and mitochondrial damage (cytosolic cytochrome c) in HUVECs were suppressed by melatonin but reversed by SIRT3-siRNA (all P < .001). In vivo, reduced numbers of circulating endothelial progenitor cells (EPCs) (C-kit/CD31+/Sca-1/KDR+/CXCR4/CD34+), and angiogenesis (Matrigel assay of bone marrow-derived EPC and ex vivo aortic ring cultures) in older (compared with younger) mice were significantly reversed through daily melatonin administration (20 mg/kg/d, ip) (all P < .001). Aortic vasorelaxation and nitric oxide release were impaired in older mice and reversed in age-match mice receiving melatonin (all P < .01). ED-induced intimal/medial hyperplasia, reduced blood flow to ischemic limb, and angiogenesis (reduced CD31+/vWF+ cells/small vessel number) were improved after daily melatonin treatment (all P < .0001). Lipopolysaccharide-induced aortic endothelial cell detachment, which was more severe in aged mice, was also alleviated after daily melatonin treatment (P < .0001). Daily melatonin treatment protected both structural and functional integrity of vascular endothelium against aging-, oxidative stress-, lipopolysaccharide-, and ischemia-induced damage probably through upregulating the SIRT signaling pathway.

Shock Wave Therapy Enhances Mitochondrial Delivery into Target Cells and Protects against Acute Respiratory Distress Syndrome.

This study tested the hypothesis that shock wave therapy (SW) enhances mitochondrial uptake into the lung epithelial and parenchymal cells to attenuate lung injury from acute respiratory distress syndrome (ARDS). ARDS was induced in rats through continuous inhalation of 100% oxygen for 48 h, while SW entailed application 0.15 mJ/mm2 for 200 impulses at 6 Hz per left/right lung field. In vitro and ex vivo studies showed that SW enhances mitochondrial uptake into lung epithelial and parenchyma cells (all p < 0.001). Flow cytometry demonstrated that albumin levels and numbers of inflammatory cells (Ly6G+/CD14+/CD68+/CD11b/c+) in bronchoalveolar lavage fluid were the highest in untreated ARDS, were progressively reduced across SW, Mito, and SW + Mito (all p < 0.0001), and were the lowest in sham controls. The same profile was also seen for fibrosis/collagen deposition, levels of biomarkers of oxidative stress (NOX-1/NOX-2/oxidized protein), inflammation (MMP-9/TNF-α/NF-κB/IL-1ß/ICAM-1), apoptosis (cleaved caspase 3/PARP), fibrosis (Smad3/TGF-ß), mitochondrial damage (cytosolic cytochrome c) (all p < 0.0001), and DNA damage (γ-H2AX+), and numbers of parenchymal inflammatory cells (CD11+/CD14+/CD40L+/F4/80+) (p < 0.0001). These results suggest that SW-assisted Mito therapy effectively protects the lung parenchyma from ARDS-induced injury.

Combined Therapy with SS31 and Mitochondria Mitigates Myocardial Ischemia-Reperfusion Injury in Rats.

Myocardial ischemia-reperfusion (IR) injury contributes to adverse cardiac outcomes after myocardial ischemia, cardiac surgery, or circulatory arrest. In this study, we evaluated the ability of combined SS31-mitochondria (Mito) therapy to protect heart cells from myocardial IR injury. Adult male SD rats (n = 8/each group) were randomized: group 1 (sham-operated control), group 2 (IR, 30-min ischemia/72 h reperfusion), group 3 (IR-SS31 (2 mg intra-peritoneal injection at 30 min/24 h/48 h after IR)), group 4 (IR-mitochondria (2 mg/derived from donor liver/intra-venous administration/30 min after IR procedure)), and group 5 (IR-SS31-mitochondria). In H9C2 cells, SS31 suppressed menadione-induced oxidative-stress markers (NOX-1, NOX-2, oxidized protein) while it increased SIRT1/SIRT3 expression and ATP levels. In adult male rats 72 h after IR, left ventricular ejection fraction (LVEF) was highest in sham-operated control animals and lowest in the IR group. LVEF was also higher in IR rats treated with SS31-Mito than untreated IR rats or those treated with Mito or SS31 alone. Areas of fibrosis/collagen-deposition showed the opposite pattern. Likewise, levels of oxidative-stress markers (NOX-1, NOX-2, oxidized protein), inflammatory markers (MMP-9, CD11, IL-1ß, TNF-α), apoptotic markers (mitochondrial-Bax, cleaved-caspase-3, PARP), fibrosis markers (p-Smad3, TGF-ß), DNA-damage (γ-H2AX), sarcomere-length, and pressure/volume overload markers (BNP, ß-MHC) all showed a pattern opposite that of LVEF. Conversely, anti-apoptotic (BMP-2, Smad1/5) and energy integrity (PGC-1α/mitochondrial cytochrome-C) markers exhibited a pattern identical to that of LVEF. This study demonstrates that the combined SS31-Mito therapy is superior to either therapy alone for protecting myocardium from IR injury and indicates that the responsible mechanisms involved increased SIRT1/SIRT3 expression, which suppresses inflammation and oxidative stress and protects mitochondrial integrity.

Impact of Double Loading Regimen of Clopidogrel on Final Angiographic Results, Incidence of Upper Gastrointestinal Bleeding and Clinical Outcomes in Patients with STEMI Undergoing Primary Coronary Intervention.

This study tested the therapeutic impact of double-loading dose (i.e., 600 mg) versus standard-loading dose (i.e., 300 mg) of clopidogrel on ST-segment-elevation-myocardial-infarction (STEMI) patients undergoing primary-coronary-intervention (PCI).Between January 2005 and December 2013, a total of 1461 STEMI patients undergoing PCI were consecutively enrolled into the study and categorized into group 1 (600 mg/clopidogrel; n = 508) and group 2 (300 mg/clopidogrel; n = 953). We assessed angiographic thrombolysis-in-myocardial-infarction (TIMI) flow in the infarct-related-artery, 30-day mortality and upper-gastrointestinal-bleeding (UGIB) within 30 days as primary-endpoints and later incidents of UGIB as secondary-endpoints.The results showed that the incidences of advanced Killip score (defined as ≥ score 3) upon presentation (23.8% versus 24.6%) and advanced heart failure (defined as ≥ NYHAFc-3) (10.2% versus 10.4%) did not differ between groups 1 and 2 (all P > 0.4). Primary-endpoints, which were final TIM-3 flow (91.3% versus 91.7%) in the infarct-related-artery, incidences of 30-day mortality (5.8% vs. 7.1%), and UGIB ≤ 30 day (7.8% versus 8.9%) did not differ between group 1 and group 2 (all P > 0.33). The secondary-endpoints which were incidences of ≥ 30-day < one-year (5.2% versus 4.7) and > one-year (8.9% versus 10.1%) UGIB did not differ between groups 1 and 2 (all P > 0.45). One-year mortality did not differ between two groups (10.74% versus 12.9%) (P > 0.25). Multiple-stepwise-logistic-regression analysis showed that age and advanced-Killip score were independently predictive of 30-day mortality (all P < 0.001).Double-loading dose of clopidogrel did not confer an additional benefit to the final angiograph results, 30-day/one-year clinical outcomes; and age and advanced Killip-score were powerful predictors of 30-day mortality.

Early Administration of Carvedilol Protected against Doxorubicin-Induced Cardiomyopathy.

This study tested for the benefits of early administration of carvedilol as protection against doxorubicin (DOX)-induced cardiomyopathy. Thirty male, adult B6 mice were categorized into group 1 (untreated control), group 2 [DOX treatment (15 mg/every other day for 2 weeks, i.p.], and group 3 [carvedilol (15 mg/kg/d, from day 7 after DOX treatment for 28 days)], and euthanized by day 35 after DOX treatment. By day 35, the left ventricular ejection fraction (LVEF) was significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1, whereas the left ventricular (LV) end-diastolic and LV end-systolic dimensions showed an opposite pattern to the LVEF among the three groups. The protein expressions of fibrotic (Smad3, TGF-ß), apoptotic (BAX, cleaved caspase 3, PARP), DNA damage (γ-H2AX), oxidative stress (oxidized protein), mitochondrial damage (cytosolic cytochrome-C), heart failure (brain natriuretic peptide), and hypertrophic (ß-MHC) biomarkers of the LV myocardium showed an opposite pattern to the LVEF among the three groups. The protein expressions of antifibrotic (BMP-2, Smad1/5), α-MHC, and phosphorylated-Akt showed an identical pattern to the LVEF among the three groups. The microscopic findings of fibrotic and collagen-deposition areas and the numbers of γ-H2AX(+) and 53BP1(+) cells in the LV myocardium exhibited an opposite pattern, whereas the numbers of endothelial cell (CD31(+), vWF(+)) markers showed an identical pattern to the LVEF among the three groups. Cardiac stem cell markers (C-kit(+) and Sca-1(+) cells) were significantly and progressively increased from group 1 to group 3. Additionally, the in vitro study showed carvedilol treatment significantly inhibited DOX-induced cardiomyoblast DNA (CD90/XRCC1(+), CD90/53BP1(+), and r-H2AX(+) cells) damage. Early carvedilol therapy protected against DOX-induced DNA damage and cardiomyopathy.

Administered circulating microparticles derived from lung cancer patients markedly improved angiogenesis, blood flow and ischemic recovery in rat critical limb ischemia.

BACKGROUND: We hypothesized that lung cancer patient's circulating microparticles (Lc-MPs) could promote angiogenesis, blood flow in ischemic zone and ischemic recovery in rat critical limb ischemia (CLI). METHODS: To investigate the impact of MP therapy on reversing the setting of CLI, adult-male Sprague-Dawley rats (n=50) equally randomized into sham control (SC) (group 1), SC-Lc-MPs (1.0 x 10(7) particles) (group 2), CLI (group 3), CLI-Hs-MPs (MPs from healthy-subject) (group 4), and CLI-Lc-MPs (group 5) were sacrificed by post-CLI day-14. RESULTS: In vitro study showed that Lc-MPs enhanced VEGFR2 expression, angiogenesis, nitric-oxide production, and endothelial cell proliferation (all p<0.005). By days 7 and 14, Laser Doppler showed significantly higher ischemic/normal blood-flow ratio in groups 1 and 2 compared with group 3, and was significantly higher in group 4 and further elevated in group 5 (p<0.0001). Numbers of small vessels and endothelial markers (CD31(+) and vWF(+) cells) and protein expressions (eNOS, CD31) exhibited a pattern identical to Lasre Doppler among the five groups (all p<0.001). Pro-angiogenic factors (VEGF, CXCR4, SDF-1α, HGF) at cellular and protein levels showed a significant step-wise increase from groups 1 and 2 to groups 3, 4, and 5 (all p<0.001). Protein expressions of fibrotic (Smad3, TGF-ß) and apoptotic (mitochondrial Bax, cleaved caspase 3, and PARP) biomarkers displayed an opposite pattern compared to that of Laser Doppler, whereas the protein expressions of anti-fibrotic (Smad1/5, BMP-2) and anti-apoptotic (Bcl-2) biomarkers showed an identical pattern compared with that of Laser Doppler among groups 1 to 3, and 5 (all p<0.001). CONCLUSION: Administration of Lc-MPs augmented angiogenesis and restored blood flow in a rat of CLI.

Protective effect of melatonin-supported adipose-derived mesenchymal stem cells against small bowel ischemia-reperfusion injury in rat.

We tested the hypothesis that combined melatonin and autologous adipose-derived mesenchymal stem cells (ADMSC) was superior to either alone against small bowel ischemia-reperfusion (SBIR) injury induced by superior mesenteric artery clamping for 30 min followed by reperfusion for 72 hr. Male adult Sprague Dawley rats (n = 50) were equally categorized into sham-operated controls SC, SBIR, SBIR-ADMSC (1.0 × 10(6) intravenous and 1.0 × 10(6) intrajejunal injection), SBIR-melatonin (intraperitoneal 20 mg/kg at 30 min after SI ischemia and 50 mg/kg at 6 and 18 hr after SI reperfusion), and SBIR-ADMSC-melatonin groups. The results demonstrated that the circulating levels of TNF-α, MPO, LyG6+ cells, CD68+ cells, WBC count, and gut permeability were highest in SBIR and lowest in SC, significantly higher in SBIR-ADMSC group and further increased in SBIR-melatonin group than in the combined therapy group (all P < 0.001). The ischemic mucosal damage score, the protein expressions of inflammation (TNF-α, NF-κB, MMP-9, MPO, and iNOS), oxidative stress (NOX-1, NOX-2, and oxidized protein), apoptosis (APAF-1, mitochondrial Bax, cleaved caspase-3 and PARP), mitochondrial damage (cytosolic cytochrome C) and DNA damage (γ-H2AX) markers, as well as cellular expressions of proliferation (PCNA), apoptosis (caspase-3, TUNEL assay), and DNA damage (γ-H2AX) showed an identical pattern, whereas mitochondrial cytochrome C exhibited an opposite pattern compared to that of inflammation among all groups (all P < 0.001). Besides, antioxidant expressions at protein (NQO-1, GR, and GPx) and cellular (HO-1) levels progressively increased from SC to the combined treatment group (all P < 0.001). In conclusion, combined melatonin-ADMSC treatment offered additive beneficial effect against SBIR injury.

Inhibition of dipeptidyl peptidase-IV enzyme activity protects against myocardial ischemia-reperfusion injury in rats.

BACKGROUND: We investigated whether attenuating dipeptidyl peptidase-IV (DPP4) enzyme activity protected rat heart from ischemia-reperfusion (IR) injury (40-min left anterior descending coronary artery ligation followed by 72 h reperfusion). METHODS AND RESULTS: Adult male Fischer 344 rats (n = 24) were equally divided into sham-control (WT-SC), WT-IR, and WT-IR-Sita (oral sitagliptin 400 mg/kg/day for 3 days) groups, whereas adult male DPP4-deficiency (DPP4(D)) rats (n = 16) were equally divided into DPP4(D)-SC and DPP4(D)-IR groups. Animals were sacrificed at 72 h after reperfusion with collection of heart specimens. Infarct area (H&E), collagen deposition (Sirius-red stain), fibrotic area (Masson's trichrome), and fluorescent-ROS intensity (H2DCFDA-labeling myocardium) of left ventricle were significantly higher in WT-IR than those in other groups, significantly higher in WT-IR-Sita and DPP4(D)-IR groups than in WT-SC and DPP4(D)-SC groups (all p < 0.001), but there was no difference between the latter two groups. Protein expressions of oxidative stress (oxidized protein), reactive oxygen species (NOX-1, NOX-2), inflammation (TNF-α, NF-κB, MMP-9, VCAM-1), apoptosis (mitochondrial Bax, cleaved caspase-3 and PARP), myocardial damage markers (cytosolic cytochrome-C, γ-H2AX), and number of inflammatory cells (CD14+, CD68+, CD40+ cells) showed a pattern identical to that of histological changes among all groups (all p < 0.005), whereas markers of anti-apoptosis (Bcl-2) and mitochondrial integrity (mitochondrial cytochrome-C) as well as left ventricular ejection fraction showed an opposite pattern (all p < 0.001). Protein expressions of anti-oxidants (HO-1, NQO-1), angiogenesis factors (SDF-1α, CXCR4), and glycogen-like-peptide-1-receptor were significantly higher inWT-IR-Sita and DPP4(D)-IR than those in other groups (all p <0.001). CONCLUSION: Abrogation of DPP4 activity protects against myocardial IR injury and preserved heart function.

Levels and values of lipoprotein-associated phospholipase A2, galectin-3, RhoA/ROCK, and endothelial progenitor cells in critical limb ischemia: pharmaco-therapeutic role of cilostazol and clopidogrel combination therapy.

OBJECTIVE: We tested the hypothesis that clopidogrel and cilostazol combination therapy could effectively attenuate systemic inflammatory reaction, facilitate proliferation of circulating endothelial progenitor cell (EPC), and improve the clinical outcomes of critical limb ischemia (CLI) in patients unsuitable for surgical revascularization or percutaneous transluminal angioplasty (PTA). METHODS: A total 55 patients (mean age, 72 years; 56% female) were consecutively enrolled. Clopidogrel and cilostazol combination therapy was administered throughout the study period. RESULTS: As compared with the baseline, circulating endothelial progenitor cell level (as shown by flow cytometry) was significantly increased (p<0.003), whereas the CLI-related ulcers and painfulness were significantly improved (all p<0.01) by day 90 after treatment. On the other hand, after clopidogrel and cilostazol combination therapy, galectin-3 level, lipoprotein-associated phospholipase A2 gene expression, and RhoA/ROCK-related protein expression in peripheral blood mononuclear cells were significantly suppressed (all p<0.01). Eventually, by day 90, 5 patients (9.1%) died of other etiologies, 3 (5.5%) withdrew from the study, 6 (10.9%) required amputation, and the remaining 41 had satisfactory clinical improvement with complete wound healing in 9 (16.4%) patients. CONCLUSION: The results of the present study highlight that clopidogrel and cilostazol combination therapy may be considered to be an alternative method for treating patients with CLI unsuitable for surgical revascularization or PTA.

Prompt bone marrow-derived mesenchymal stem cell therapy enables early porcine heart function recovery from acute myocardial infarction.

Impact of early bone marrow-derived mesenchymal stem cell (BMDMSC) implantation on left ventricular (LV) function after AMI was studied.Twelve mini-pigs were equally divided into placebo (AMI through left coronary artery ligation) and cell-treated groups [BMDMSCs (3.0 × 10(7)) implanted into infarct area (IA)] with myocardium harvested by post-AMI day 90. Six healthy animals served as controls.On post-AMI day 90, magnetic resonance imaging showed a lower LV ejection fraction but higher LV dimensions in the placebo group (P < 0.003) that also had increased IAs but reduced wall thickness (P < 0.005). Pro-apoptotic gene expressions (Bax, caspase-3) and apoptotic nucleus number in IAs and peri-IAs were highest in the placebo group (P < 0.001). Inflammatory biomarker expressions (MMP-9, oxidized protein, CD40+ cells) were highest, whereas those of angiogenesis (VEGF, CD31+ cells, SDF-1α, CXCR4) and myocardium-preservation (connexin43, troponin-I, cytochrome-C) were lowest in the placebo group (P < 0.01).BMDMSC implantation preserved LV function and alleviated remodeling at post-AMI day 90.

Combined Phloretin and Human Platelet-rich Plasma Effectively Preserved Integrities of Brain Structure and Neurological Function in Rat after Traumatic Brain Damage.

BACKGROUND: This study tested whether phloretin (a brain-edema inhibitors) would augment therapeutic impact of human-derived platelet-rich plasma (hPRP) on attenuating brain-hemorrhagic volume (BHV) and preserving the neurologic function in rodent following acute traumatic brain damage (TBD). METHODS: Rats (n=40) were separated into group-1 (sham-control), group-2 (TBD), group-3 [TBD + phloretin (80mg/kg/dose by intra-peritoneal administration at 30min and days 2/3 followed-by TBD), group-4 (TBD + PRP/80µL by left intra-carotid-artery injection at 3h after TBD) and group-5 (TBD + phloretin + hPRP) and cerebral tissues were harvested by day 28 after TBD. RESULTS: The brain MRI at day 28 revealed that the BHV was lowest in group 1, highest in group 2 and significantly lower in group 5 than in groups 3/4, but it was similar between groups 3/4, whereas neurological function displayed a opposite pattern of BHV among the groups (all p&amp;amp;lt;0.0001). By 72h, the protein levels of upstream (HGMB1/TLR-2/TLR-4/MyD88/Mal/TRAM/ TRIF/TRAF6/IKK-α/IKK-ß/p-NF-κB) and downstream (IL-1ß/TNF-α/iNOS) inflammation signalings, apoptosis (caspase3/PARP) and fibrosis (Smad3/TGF-ß) biomarkers and flow cytometric assessment of inflammation cells (CD11b/c+//Ly6G+/PMO+) and early (AN-V+/PI-)/late (AN-V+/PI+) mononuclear-cell apoptosis displayed a similar manner of BHV among the groups (all p&amp;amp;lt;0.0001). Cell number of inflammatory (CD68+/MMP9+) and brain-swelling/myelin-damaged (AQP4+/ GFAP+) mediators revealed a similar way, whereas the neuronal-myelin (Doublecortin+/NeuN/nestin) mediators exhibited an inverse manner of BHV among the groups (all p&amp;amp;lt;0.0001). CONCLUSION: Combination of phloretin and hPRP regimen was better than just one treatment to offer synergic benefits for protecting the brain against TBD.

Sildenafil improves long-term effect of endothelial progenitor cell-based treatment for monocrotaline-induced rat pulmonary arterial hypertension.

BACKGROUND AIMS: We hypothesized that the long-term therapeutic effect of combined sildenafil and bone marrow-derived endothelial progenitor cells (BMDEPCs) on monocrotaline (MCT)-induced rat pulmonary arterial hypertension (PAH) is superior to either treatment alone. METHODS: Male Sprague-Dawley rats (n = 40) were equally divided into normal controls, MCT (65 mg/kg, subcutaneously) only, MCT + sildenafil (25 mg/kg/day, orally), MCT + BMDEPCs (2.0 × 10(6) autologous cells, intravenously) and MCT + sildenafil+ BMDEPCs. BMDEPCs and sildenafil were given on day 21 after MCT administration. Animals were sacrificed by day 90 after MCT administration. RESULTS: The apoptotic (caspase 3, Bax) and inflammatory (tumor necrosis factor-α, matrix metalloproteinase-9) biomarkers in right ventricle and lung and pulmonary expressions of fibrotic biomarkers (transforming growth factor-ß, p-Smad3) and connexin 43 protein were lower in monotherapy groups (i.e., MCT + sildenafil and MCT + BMDEPCs) and further decreased in normal controls and combined treatment groups (i.e., MCT + sildenafil + BMDEPCs) compared with untreated animals (i.e., MCT only) (all P < 0.01). Expressions of anti-fibrotic biomarkers (bone morphogenetic protein-2, p-Smad1/5) and numbers of alveolar sacs and arterioles in lung were higher in monotherapy groups and further increased in normal controls and combined treatment groups compared with untreated animals (all P < 0.005). In right ventricle, connexin 43 and α-myosin heavy chain (MHC) expressions were higher in the monotherapy groups and further elevated in normal controls and combined treatment groups compared with untreated animals, whereas ß-MHC exhibited the opposite pattern (all P < 0.01). Right ventricular systolic pressure and weight were lower in the monotherapy animals and further reduced in normal controls and combined treatment groups compared with untreated animals (all P < 0.0001). CONCLUSIONS: Combined therapy with BMDEPCs and sildenafil was superior to either treatment alone in attenuating rodent MCT-induced PAH.

Innate immune response after acute myocardial infarction and pharmacomodulatory action of tacrolimus in reducing infarct size and preserving myocardial integrity.

BACKGROUND: This study investigated the association between innate immune reaction and myocardial damage after acute myocardial infarction (AMI) and anti-inflammatory role of tacrolimus in reducing infarct size. Male mini-pigs (n=18) were equally categorized into sham control (SC), untreated AMI (by ligation of left anterior descending coronary artery), and AMI-Tacrolimus (AMI-Tac) (0.5 mg intra-coronary injection 30 minutes post-AMI). Cardiac magnetic resonance imaging (MRI) was performed at post-AMI days 2, 5 and 21 before sacrificing the animals. RESULTS: By post-AMI day 21, left ventricular ejection fraction (LVEF) was lowest in untreated AMI animals, significantly higher in SC than in AMI-Tac group (all p<0.003). Infarct areas at basal, middle, and apical levels, numbers of CD14+ and iNOS+ cells in infarct area (IA) and peri-IA, and protein expression of CD14, CD68, and Ly6g from circulating inflammatory cells showed an opposite pattern compared with that of LVEF in all groups (all p<0.005). Protein expressions of MCP-1, MIP-1, TNF-α, NF-κB, iNOS, and IL-12 in IA and peri-IA exhibited an identical pattern compared to that of CD14, CD68, and Ly6g from circulating inflammatory cells (all p<0.01). Expressions of myocardial damage biomarkers in IA and peri-IA [γ-H2AX, ß-myosin heavy chain (MHC), Smad3, TGF-ß] were highest in AMI and higher in AMI-Tac than in SC, whereas expressions of myocardial integrity biomarkers (connexin43, mitochondrial cytochrome-C, α-MHC, BMP-2, Smad1/5) were opposite to those of damage biomarkers (all p<0.001). CONCLUSION: Innate immune responses were markedly augmented and LVEF was significantly reduced after AMI but were remarkably improved after tacrolimus treatment.

High Flow-Mediated Vasodilatation Predicts Pulmonary Edema in Liver Transplant Patients.

BACKGROUND: Early pulmonary edema is common after orthotopic liver transplantation. Associated pathogenic mechanisms might involve increased activity of cardiac-inhibitory systems due to increased vasodilator production, mainly nitric oxide (NO). NO is primarily responsible for flow-mediated vasodilatation (FMD). We investigated the incidence of pulmonary edema in liver transplant patients and its correlation with FMD. METHODS: We prospectively evaluated traditional risk factors, Doppler echocardiographic findings, derived hemodynamic data, and brachial artery nitroglycerin-induced vasodilatation (NTD) and FMD within 1 week prior to liver transplantation in 54 consecutive liver transplant patients with cirrhosis. Post-transplantation chest roentgenography was performed daily. In-hospital outcomes, transfusion volume of blood components, and hemodynamic data during surgery and at the intensive care unit were analyzed. RESULTS: Twenty-nine patients (53.7%) developed radiological pulmonary edema within 1 week of transplantation. Diffuse-type interstitial and alveolar pulmonary edema constituted 13 cases (24.1%). Patients with pulmonary edema had higher pretransplantation Child-Turcotte-Pugh scores (p = 0.01), cardiac output (p = 0.03), FMD (p < 0.01), NTD (p = 0.01), and FMD/NTD ratio (p = 0.02). Although the total volume of intravenous fluid transfused was higher in the pulmonary edema group, the net fluid retention during surgery was statistically insignificant. The lengths of intensive care unit stay and hospitalization, as well as mortality rates, were not different in these groups. CONCLUSIONS: The high incidence of pulmonary edema after living donor liver transplantation was associated with a high FMD and FMD/NTD ratio at pretransplantation. FMD is the only significant predictor associated with pulmonary edema. However, we observed no alteration in mortality rates. KEY WORDS: Cirrhotic cardiomyopathy; Flow-mediated vasodilatation; Liver transplantation; Pulmonary edema.

Homocysteine Levels in Severe OSA Patients Before and After TORS-OSA Surgery.

OBJECTIVE: The increased risk of cardiovascular diseases owing to a high level of serum homocysteine has been widely reported. Literature has demonstrated that patients with obstructive sleep apnea/hypopnea syndrome (OSA) had a higher homocysteine level than control group. This study aimed to investigate the alteration of serum homocysteine levels in severe OSA patients receiving transoral robotic surgery (TORS). STUDY DESIGN: Retrospective chart review. SETTING: Tertiary academic medical center. METHODS: Data of polysomnography (PSG) and serum homocysteine levels before and at least 3 months after the surgery were collected and analyzed via paired t tests. A subgroup analysis based on the preoperative homocysteine level (≥15 mcmol/L, as hyperhomocysteinemia group) was conducted to compare the intergroup differences of homocysteine decrease. Pearson's correlation was used to survey the relationships between the changes of major PSG parameters and the levels of homocysteine decrease at baseline and after TORS-OSA surgery. RESULTS: Two hundred sixty-one patients with severe OSA were enrolled. There were significant improvements in major PSG parameters after TORS-OSA surgery. Homocysteine levels significantly decreased from 12.1 ± 3.9 to 11.4 ± 3.7 mcmol/L (difference = -0.7 ± 2.8 mcmol/L, p = .001) postoperatively, which was shown in the hyperhomocysteinemia group (difference = -2.9 ± 4.7 mcmol/L, p = .007) to a greater extent. Pearson's correlation revealed that ΔODI (oxygen desaturation index/h) was the predominant estimate with a positive association with Δhomocysteine (r = 0.525, p = .012). CONCLUSION: TORS-OSA surgery could decrease homocysteine levels in OSA patients. The effects were more relevant in severe OSA patients with abnormal preoperative homocysteine levels.

Inflammatory biomarkers of neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios in 563 severe OSA patients before and after surgery.

BACKGROUND: Evidence has proved that high neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were risk factors for cardiovascular comorbidities. The alterations of NLR and PLR following obstructive sleep apnea (OSA) treatment were under studied and thus should be investigated. This study aimed to evaluate the changes of inflammatory biomarkers including NLR and PLR in severe OSA patients after surgical interventions of the upper airway, and their relationships with improvements in polysomnographic (PSG) parameters. METHODS: This retrospective cohort study included 563 consecutive severe OSA patients at a tertiary academic medical center who received OSA surgery, as well as underwent pre- and post-operative polysomnographic (PSG) examinations and blood tests. The changes of major PSG estimates, NLR, and PLR before and at least 3 months after OSA surgery were analyzed using paired t-tests with subgroup analyses. Pearson's correlations were performed to discover which PSG parameter contributed to the improvement of the values. RESULTS: After OSA surgery, the major PSG estimates, NLR and PLR dropped significantly in the overall population. In those with a higher preoperative NLR (pre-operative NLR≧3) and PLR (pre-operative PLR≧150), the mean (SD) difference of NLR (- 0.8 [1.6], 95% CI - 1.5 to - 0.2) and PLR (- 41.6 [40], 95% CI - 52.8 to - 30.5) were even more substantial. The changes of the "apnea, longest (r = 0.298, P = .037)" and "hypopnea, longest (r = 0.321, P = .026)" were found significantly related to the improvement of PLR. CONCLUSION: NLR and PLR did significantly drop in severe OSA patients following OSA surgery, and this could be related to the alterations of sleep indices. The findings could possess clinical importance for severe OSA patients after OSA surgeries in reducing possible OSA-associated cardiovascular comorbidities.

Obesity suppresses circulating level and function of endothelial progenitor cells and heart function.

BACKGROUND AND AIM: This study tested the hypothesis that obesity suppresses circulating number as well as the function of endothelial progenitor cells (EPCs) and left ventricular ejection fraction (LVEF). METHODS: High fat diet (45 Kcal% fat) was given to 8-week-old C57BL/6 J mice (n = 8) for 20 weeks to induce obesity (group 1). Another age-matched group (n = 8) were fed with control diet for 20 weeks as controls (group 2). The animals were sacrificed at the end of 20 weeks after obesity induction. RESULTS: By the end of study period, the heart weight, body weight, abdominal fat weight, serum levels of total cholesterol and fasting blood sugar were remarkably higher in group 1 than in group 2 (all p<0.01). The circulating level of EPCs (C-kit/CD31, Sca-1/KDR, CXCR4/CD34) was significantly lower in group 1 than in group 2 (p<0.03) at 18 h after critical limb ischemia induction. The angiogenesis and migratory ability of bone marrow-derived EPCs was remarkably impaired in group 1 compared to that in group 2 (all p<0.01). The repair ability of aortic endothelium damage by lipopolysaccharide was notably attenuated in group 1 compared with that in group 2 (p<0.01). Collagen deposition (Sirius red staining) and fibrotic area (Masson's Trichrome staining) in LV myocardium were notably increased in group 1 compared with group 2 (p<0.001). LVEF was notably lower, whereas LV end-diastolic and end-systolic dimensions were remarkably higher in group 1 than in group 2 (all p<0.001). CONCLUSIONS: Obesity diminished circulating EPC level, impaired the recovery of damaged endothelium, suppressed EPC angiogenesis ability and LVEF, and increased LV remodeling.

Impact of apoptotic adipose-derived mesenchymal stem cells on attenuating organ damage and reducing mortality in rat sepsis syndrome induced by cecal puncture and ligation.

BACKGROUND: We tested whether apoptotic adipose-derived mesenchymal stem cells (A-ADMSCs) were superior to healthy (H)-ADMSCs at attenuating organ damage and mortality in sepsis syndrome following cecal ligation and puncture (CLP). METHODS: Adult male rats were categorized into group 1 (sham control), group 2 (CLP), group 3 [CLP + H-ADMSC administered 0.5, 6, and 18 h after CLP], group 4 [CLP + A-ADMSC administered as per group 3]. RESULTS: Circulating peak TNF-α level, at 6 h, was highest in groups 2 and 3, and higher in group 4 than group 1 (p < 0.0001). Immune reactivity (indicated by circulating and splenic helper-, cytoxic-, and regulatory-T cells) at 24 and 72 h exhibited the same pattern as TNF-α amongst the groups (all p < 0.0001). The mononuclear-cell early and late apoptosis level and organ damage parameters of liver (AST, ALT), kidney (creatinine) and lung (arterial oxygen saturation) also displayed a similar pattern to TNF-α levels (all p < 0.001). Protein levels of inflammatory (TNF-α, MMP-9, NF-κB, ICAM-1), oxidative (oxidized protein) and apoptotic (Bax, caspase-3, PARP) biomarkers were higher in groups 2 and 3 than group 1, whereas anti-apoptotic (Bcl-2) biomarker was lower in groups 2 and 3 than in group 1 but anti-oxidant (GR, GPx, HO-1, NQO-1) showed an opposite way of Bcl-2; these patterns were reversed for group 4 (all p < 0.001). Mortality was highest in group 3 and higher in group 2 than group 4 than group 1 (all p < 0.001). CONCLUSIONS: A-ADMSC therapy protected major organs from damage and improved prognosis in rats with sepsis syndrome.