Intra-Coronary Administration of Tacrolimus Improves Myocardial Perfusion and Left Ventricular Function in Patients with ST-Segment Elevation Myocardial Infarction (COAT-STEMI) Undergoing Primary Percutaneous Coronary Intervention.

BACKGROUND: Ischemia-reperfusion injury following acute ST-segment elevation myocardial infarction (STEMI) is strongly related to inflammation. However, whether intracoronary (IC) tacrolimus, an immunosuppressant, can improve myocardial perfusion is uncertain. METHODS: A multicenter double-blind randomized controlled trial was conducted in Taiwan from 2014 to 2017. Among 316 STEMI patients with Killip class ≤ 3 undergoing primary percutaneous coronary intervention (PCI), 151 were assigned to the study group treated with IC tacrolimus 2.5 mg to the culprit vessel before first balloon inflation, and the remaining 165 were assigned to the placebo group receiving IC saline only. The primary endpoint was percentage of post-PCI TIMI-3 flow. The primary composite endpoints included achievement of TIMI-3 flow, TIMI- myocardial perfusion (TMP) grade, or 90-min ST-segment resolution (STR). The secondary endpoints were left ventricular ejection fraction (LVEF) and 1-month/1-year major adverse cardio-cerebral vascular events (MACCEs) (defined as death, myocardial infarction, stroke, target-vessel revascularization or re-hospitalization for heart failure). RESULTS: Although post-PCI TIMI-3 epicardial flow and MACCE rate at 1 month and 1 year did not differ between the two groups, TMP grade (2.54 vs. 2.23, p < 0.001) and 90-min STR (67% vs. 61%, p < 0.001) were significantly higher in the tacrolimus-treated group than in the placebo group. The STEMI patients treated with tacrolimus also had significantly higher 3D LVEF and less grade 2 or 3 LV diastolic dysfunction at 9 months compared to those without. CONCLUSIONS: IC tacrolimus for STEMI improved coronary microcirculation and 9-month LV systolic and diastolic functions. However, the benefit of tacrolimus on clinical outcomes remains inconclusive due to insufficient patient enrollment.

Changes in Right Ventricle Function After Mitral Valve Repair Surgery.

BACKGROUND: Right ventricular (RV) dysfunction can occur after cardiac surgery and persist for years. We assessed perioperative RV systolic function in patients undergoing mitral valve (MV) repair and further compared minimally invasive robotic-assisted mitral valve repair (MIMVr) vs standard 'open' MV repair (MVr). Speckle tracking (RV free wall strain [RVS]) was used as a sensitive echocardiography method to assess RV function. METHODS: Retrospective analysis, over 3 years, of consecutive patients (n = 158) referred to Mayo Clinic (Rochester, MN, USA). Preoperative, pre-discharge and 1 year transthoracic echocardiograms were reviewed. A prospective pilot study was performed for sample size estimation. Primary outcome was RV free wall strain (RVS). RESULTS: Right ventricular free wall strain declined after MV repair surgery (-22.6 ± 7% vs -15 ± 6%, p < 0.001). There were smaller reductions in RVS in MIMVr vs MVr group (-6.0 ± 9% vs -10.3 ± 8%, p < 0.01), which persisted after adjusting for baseline values (RVS treatment effect 1.5%, p = 0.007). There was greater recovery in MIMVr vs MVr group at 1 year follow-up vs pre-surgery values (-3.4 ± 9% vs -8.1 ± 8% respectively, p < 0.001, RVS treatment effect 1.7%, p = 0.001). Bypass time was higher in the MIMVr group (80min ± 22 vs 40min ± 20, p < 0.0001). The echo findings remained significant correcting for age, pulmonary pressures and change in ejection fraction. CONCLUSIONS: Right ventricular systolic dysfunction is common after MV repair surgery. Deterioration in RV contraction is less pronounced following MIMVr vs MVr and is associated with enhanced RV functional recovery at 1 year, albeit not to preoperative levels. This may potentially be associated with clinical functional improvement but further studies are warranted to investigate this.

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.

Deletion of RasGRF1 Attenuated Interstitial Fibrosis in Streptozotocin-Induced Diabetic Cardiomyopathy in Mice through Affecting Inflammation and Oxidative Stress.

BACKGROUND: Diabetic cardiomyopathy (DCM) is characterized by cardiac fibrosis and stiffness, which often develops into heart failure. This study investigated the role of Ras protein-specific guanine nucleotide releasing factor 1 (RasGRF1) in the development of DCM. METHODS: Forty-eight mice were divided into four groups (n = 12 per group): Group 1: Wild-type (WT) mice, Group 2: RasGRF1 deficiency (RasGRF1-/-) mice. Group 3: Streptozotocin (STZ)-induced diabetic WT mice, Group 4: STZ-induced diabetic RasGRF1-/- mice. Myocardial functions were assessed by cardiac echography. Heart tissues from all of the mice were investigated for cardiac fibrosis, inflammation, and oxidative stress markers. RESULTS: Worse impaired diastolic function with elevation serum interleukin (IL)-6 was found in the diabetic group compared with the non-diabetic groups. Serum IL-6 levels were found to be elevated in the diabetic compared with the non-diabetic groups. However, the diabetic RasGRF1-/- mice exhibited lower serum IL-6 levels and better diastolic function than the diabetic WT mice. The diabetic RasGRF1-/- mice were associated with reduced cardiac inflammation, which was shown by lower invading inflammation cells, lower expression of matrix metalloproteinase 9, and less chemokines compared to the diabetic WT mice. Furthermore, less oxidative stress as well as extracellular matrix deposition leading to a reduction in cardiac fibrosis was also found in the diabetic RasGRF1-/- mice compared with the diabetic WT mice. CONCLUSION: The deletion of RasGRF1 attenuated myocardial fibrosis and improved cardiac function in diabetic mice through inhibiting inflammation and oxidative stress.

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.

Melatonin pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia-reperfusion injury in rats through suppression of mitochondrial permeability transition.

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia-reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e. nontreatment, menadione, and menadione-melatonin treatment, 4.0 × 10(5) each), while in vivo study used adult male Sprague Dawley rats (n = 40) equally divided into sham-control (SC), IR (60-min left-lobe ischemia + 72-hr reperfusion), IR-Mel (melatonin at 30 min/6/8 hr after reperfusion), IR-Mito (mitochondria 15,000 µg/rat 30 min after reperfusion), and IR-Mel-Mito. Following menadione treatment in vitro, oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (cleaved caspase-3/PARP), DNA damage (γ-H2AX/CD90/XRCC1), mitochondria damage (cytosolic cytochrome c) biomarkers, and mitochondrial permeability transition were found to be lower, whereas mitochondrial cytochrome c were found to be higher in hepatocytes with melatonin treatment compared to those without (all P < 0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group, but lowest in SC group and higher in IR-Mito group than that in groups IR-Mel and IR-Mel-Mito, and higher in IR-Mel group than that in IR-Mel-Mito group after 72-hr reperfusion (all P < 0.003). Protein expressions of inflammatory (TNF-α/NF-κB/IL-1ß/MMP-9), oxidative stress (NOX-1/NOX-2/oxidized protein), apoptotic (caspase-3/PARP/Bax), and mitochondria damage (cytosolic cytochrome c) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome c) showed an opposite pattern compared to that of liver injury score (all P < 0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO(+) /CD68(+) /CD14(+) cells), and DNA damage (γ-H2AX(+) cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all P < 0.001) among five groups. Melatonin-supported mitochondria treatment offered an additional benefit of alleviating hepatic IR injury.

Administration of antioxidant peptide SS-31 attenuates transverse aortic constriction-induced pulmonary arterial hypertension in mice.

AIM: Antioxidant peptide SS-31 is a class of cell-permeable small peptides, which selectively resides on the inner mitochondrial membrane and possesses intrinsic mitochondrial protective capacities. In this study we investigated the therapeutic effects of antioxidant peptide SS-31 on transverse aortic constriction (TAC)-induced pulmonary arterial hypertension (PAH) in a murine model. METHODS: Adult male mice were divided into 3 groups: sham-operated mice, TAC mice, and TAC+SS-31 mice that underwent TAC surgery and received SS-31 (2 mg/d, ip) for 60 d. The right ventricular systolic blood pressure (RVSBP) was measured on d 60 prior to sacrificing the mice; then their right heart and lung tissues were collected for histological and biochemical examinations. Lung injury scores were defined by the increased crowded area and decreased number of alveolar sacs. RESULTS: TAC mice showed significantly higher RVSBP compared with sham-operated mice, the elevation was substantially suppressed in TAC+SS-31 mice. The same pattern of changes was found in pulmonary levels of oxidative stress proteins (NOX-1/NOX-2/oxidized proteins), cytosolic cytochrome c, biomarkers related to inflammation (MMP-9/TNF-α/iNOS), calcium overload index (TRPC1, 2, 4, 6), apoptosis (mitochondrial BAX, cleaved caspase 3/PARP), fibrosis (Smad3/TGF-ß), hypoxic (HIF-1α), DNA damage (γ-H2AX) and endothelial function (eNOS/ET-1R), as well as in lung injury score, number of muscularized vessels in lungs, number of TRPC1(+) and HIF-1α(+) cells in pulmonary artery, and number of γ-H2AX(+) and Ki-67(+) cells in lung parenchyma. An opposite pattern of changes was observed in pulmonary anti-fibrotic markers (Smad1/5, BMP-2), number of small vessels, and number of alveolar sacs. In contrast, the levels of antioxidant proteins (HO-1/NQO-1/GR/GPx) in lung parenchyma were progressively and significantly increased from sham-operated mice, TAC mice to TAC+SS-31 mice. CONCLUSION: Antioxidant peptide SS-31 administration effectively attenuates TAC-induced PAH in mice.

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.

Systemic combined melatonin-mitochondria treatment improves acute respiratory distress syndrome in the rat.

Despite high in-hospital mortality associated with acute respiratory distress syndrome (ARDS), there is no effective therapeutic strategy. We tested the hypothesis that combined melatonin-mitochondria treatment ameliorates 100% oxygen-induced ARDS in rats. Adult male Sprague-Dawley rats (n = 40) were equally categorized into normal controls, ARDS, ARDS-melatonin, ARDS with intravenous liver-derived mitochondria (1500 µg per rat 6 hr after ARDS induction), and ARDS receiving combined melatonin-mitochondria. The results showed that 22 hr after ARDS induction, oxygen saturation (saO2 ) was lowest in the ARDS group and highest in normal controls, significantly lower in ARDS-melatonin and ARDS-mitochondria than in combined melatonin-mitochondria group, and significantly lower in ARDS-mitochondria than in ARDS-melatonin group. Conversely, right ventricular systolic blood pressure and lung weight showed an opposite pattern compared with saO2 among all groups (all P < 0.001). Histological integrity of alveolar sacs showed a pattern identical to saO2 , whereas lung crowding score exhibited an opposite pattern (all P < 0.001). Albumin level and inflammatory cells (MPO+, CD40+, CD11b/c+) from bronchoalveolar lavage fluid showed a pattern opposite to saO2 (all P < 0.001). Protein expression of indices of inflammation (MMP-9, TNF-α, NF-κB), oxidative stress (oxidized protein, NO-1, NOX-2, NOX-4), apoptosis (mitochondrial Bax, cleaved caspase-3, and PARP), fibrosis (Smad3, TGF-ß), mitochondrial damage (cytochrome C), and DNA damage (γ-H2AX+) exhibited an opposite pattern compared to saO2 in all groups, whereas protein (HO-1, NQO-1, GR, GPx) and cellular (HO-1+) expressions of antioxidants exhibited a progressively increased pattern from normal controls to ARDS combined melatonin-mitochondria group (all P < 0.001). In conclusion, combined melatonin-mitochondrial was superior to either treatment alone in attenuating ARDS in this rat model.

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.

Minimizing door-to-balloon time is not the most critical factor in improving clinical outcome of ST-elevation myocardial infarction patients undergoing primary percutaneous coronary intervention.

OBJECTIVES: We tested the hypothesis that, as compared with conventional door-to-balloon, shortened door-to-balloon time would further improve 30-day outcome in ST-elevation myocardial infarction patients undergoing primary stenting. DESIGN: Retrospective cohort study SETTING: Academic tertiary care hospital with approximately 2600 beds PATIENTS: Between January 2008 and December 2009, 266 ST-elevation myocardial infarction patients underwent primary stenting with conventional Door-to-baloon were consecutively enrolled as group 1, while 293 ST-elevation myocardial infarction patients underwent primary stenting with shortened door-to-balloon between January 2010 and December 2011 were consecutively enrolled as group 2. INTERVENTION: Shorten door-to-balloon time. MEASUREMENTS AND MAIN RESULTS: The results showed that time from chest pain onset to door did not differ between two groups (p > 0.1), whereas door-to-balloon time was significantly reduced in group 2 compared with that in group 1 (p < 0.0001). The prevalences of successful reperfusion, acute and subacute stent thrombosis, 30-day death or combined endpoint (defined as congestive heart failure ≥ New York Heart Association functional class 3 or 30-d death), and left ventricular function did not differ between two groups (all p > 0.05), whereas the peak creatine phosphokinase level was significantly reduced in group 2 (< 0.05). Further analysis showed that shortening the chest pain-to-reperfusion time to less than 240 minutes was the most important factor in improving left ventricular function (p < 0.001) and 30-day combined endpoint. Multivariate analysis showed that congestive heart failure greater than or equal to New York Heart Association functional class 3, poor left ventricular function, and age (all p < 0.001) along with unsuccessful reperfusion (p = 0.25) were independently predictive of 30-day mortality. CONCLUSION: Shortening the duration between chest pain onset and reperfusion to less than 4.0 hours was critical in reducing myocardial necrosis and improving heart function and 30-day mortality.

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.

Additional benefit of combined therapy with melatonin and apoptotic adipose-derived mesenchymal stem cell against sepsis-induced kidney injury.

This study tested whether combined therapy with melatonin and apoptotic adipose-derived mesenchymal stem cells (A-ADMSCs) offered additional benefit in ameliorating sepsis-induced acute kidney injury. Adult male Sprague-Dawley rats (n = 65) were randomized equally into five groups: Sham controls (SC), sepsis induced by cecal-ligation and puncture (CLP), CLP-melatonin, CLP-A-ADMSC, and CLP-melatonin-A-ADMSC. Circulating TNF-α level at post-CLP 6 hr was highest in CLP and lowest in SC groups, higher in CLP-melatonin than in CLP-A-ADMSC and CLP-melatonin-A-ADMSC groups (all P < 0.001). Immune reactivity as reflected in the number of splenic helper-, cytoxic-, and regulatory-T cells at post-CLP 72 hr exhibited the same pattern as that of circulating TNF-α among all groups (P < 0.001). The histological scoring of kidney injury and the number of F4/80+ and CD14+ cells in kidney were highest in CLP and lowest in SC groups, higher in CLP-melatonin than in CLP-A-ADMSC and CLP-melatonin-A-ADMSC groups, and higher in CLP-A-ADMSC than in CLP-melatonin-A-ADMSC groups (all P < 0.001). Changes in protein expressions of inflammatory (RANTES, TNF-1α, NF-κB, MMP-9, MIP-1, IL-1ß), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), fibrotic (Smad3, TGF-ß) markers, reactive-oxygen-species (NOX-1, NOX-2), and oxidative stress displayed a pattern identical to that of kidney injury score among the five groups (all P < 0.001). Expressions of antioxidants (GR+, GPx+, HO-1, NQO-1+) were lowest in SC group and highest in CLP-melatonin-A-ADMSC group, lower in CLP than in CLP-melatonin and CLP-A-ADMSC groups, and lower in CLP-melatonin- than in CLP-A-ADMSC-tretaed animals (all P < 0.001). In conclusion, combined treatment with melatonin and A-ADMSC was superior to A-ADMSC alone in protecting the kidneys from sepsis-induced injury.

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.

Simvastatin attenuates the additive effects of TNF-α and IL-18 on the connexin 43 up-regulation and over-proliferation of cultured aortic smooth muscle cells.

Statin therapy is known to down-regulate inflammatory activities in atheromatous tissues of animals. The aims of this study were to examine the regulatory role of interleukin-18 (IL-18) in the connexin 43 (Cx43) and the proliferation of cultured aortic smooth muscle cells (SMCs) as well as to elucidate the underlying therapeutic mechanism of simvastatin. Vytorin therapy significantly alleviated high-cholesterol diet-induced hypercholesterolemia, suppressed neointimal hyperplasia, macrophage infiltration, and Cx43 and IL-18 expression in rabbit aortic walls. In vitro study using an aortic SMC line showed that IL-18 up-regulated constitutive Cx43 expression and potentiated tumor necrosis factor-α (TNF-α)-triggered Akt and MAPK signaling pathways. Simvastatin treatment alone reduced constitutive Cx43 levels and prevented the TNF-α-induced IL-18 up-regulation. Mechanistic investigation using kinase-specific inhibitors showed that simvastatin pretreatment attenuated TNF-α-elicited Akt and ERK1/2 phosphorylation, whereas PI3K and all MAPK activities were also implied in the additive effect of TNF-α and IL-18 on Cx43 up-regulation. Proliferation assay indicated that IL-18 stimulated SMC proliferation and synergized the TNF-α-stimulated cell proliferation. Likewise, simvastatin treatment suppressed the SMC over-proliferation induced not only by TNF-α alone, but also by simultaneous treatment with TNF-α and IL-18. The suppression of simvastatin in SMC proliferation was not mediated through mitochondrial related pro-apoptogenesis under both scenarios. In conclusion, simvastatin attenuates the additive effects of TNF-α and IL-18 on Cx43 up-regulation and over-proliferation of aortic SMCs, mainly through the blockade of Akt signaling pathway. These findings may fortify the rationale underlying the atheroprotective mechanism of statin therapy.

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.

Sitagliptin therapy enhances the number of circulating angiogenic cells and angiogenesis-evaluations in vitro and in the rat critical limb ischemia model.

BACKGROUND AIMS: We tested the hypothesis that sitagliptin is capable of increasing blood flow in the rat critical limb ischemia (CLI) model by enhancement of angiogenesis. METHODS: Adipose tissue from adult-male Fischer 344 rats (n = 6) were cultured in endothelial progenitor cell culture medium for 14 d with (25 µmol/L) or without sitagliptin. CLI was induced by ligation of the left femoral artery. Rats (n = 32) were equally separated into four groups: untreated controls (group 1), sitagliptin (4 mg/kg per day; group 2), CLI (group 3) and CLI with sitagliptin (group 4). RESULTS: In vitro, 7 and 14 d after cell culture, endothelial progenitor cell biomarkers assessed by flow cytometry (Sca-1/CD31+, CXCR4+, c-kit+ and CD34+ cells) and Western blot (vascular endothelial growth factor, CXCR4 and stromal-derived factor [SDF]-1α) were remarkably higher in group 4 than in the other groups (all P < 0.01). In vivo, 2 and 14 d after the CLI procedure, circulating angiogenic cell (Sca-1/CD31+, Sca-1+ and CD31+) numbers were significantly higher in group 4 than in the other groups (all P < 0.001). Additionally, the messenger RNA and protein expression of angiogenic biomarkers (CXCR4, SDF-1α and vascular endothelial growth factor), immunofluorescent staining of angiogenic cells (CXCR4+, SDF-1α+, CD31+, von Willebrand factor + cells) and immunohistochemical staining of small vessel numbers in the ischemic area were significantly higher in group 4 than in the other groups (all P < 0.01). Furthermore, laser Doppler showed that the ratio of ischemic/normal blood flow was remarkably higher group 4 than in group 3 by days 14 and 28 after the CLI procedure (all P < 0.01). CONCLUSIONS: Sitagliptin therapy enhances circulating angiogenic cell numbers, angiogenesis and blood flow in the CLI area.

Value and level of circulating endothelial progenitor cells, angiogenesis factors and mononuclear cell apoptosis in patients with chronic kidney disease.

BACKGROUND: Chronic renal failure on dialysis can reduce the number of circulating endothelial progenitor cells (EPCs), but this biomarker has not been fully investigated in patients with chronic kidney disease (CKD). A link between CKD and increased mononuclear cell apoptosis (MCA) in circulation has been reported but the effect of vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1α, two angiogenesis factors, on circulating EPC levels in CKD has not been clarified. This study examined the relationships between the numbers of circulating EPCs and the severity of CKD, degree of MCA and serum levels of VEGF and SDF-1α in CKD patients. METHODS: The numbers of circulating EPCs (CD31/CD34+, CD62E/CD34+, KDR/CD34+, CXCR4/CD34+) were measured in 166 patients with varying degrees of CKD under regular treatment at an outpatient department and in 30 volunteer control subjects. RESULTS: CKD patients had significantly lower numbers of EPCs (p < 0.007), higher MCA in circulation and higher serum levels of VEGF and SDF-1 compared with the control subjects (all p < 0.001). Compared with patients with early CKD (stages I-III), patients with late CKD [stage IV-V or end-stage renal disease (ESRD)] had significantly lower numbers of EPCs (CXCR4/CD34+), higher MCA, and elevated serum levels of VEGF and SDF-1α (all p < 0.01). Serum VEGF level but not MCA or SDF-1α was strongly correlated with increased numbers of circulating EPCs. Multivariate analysis showed that ESRD along with lower serum albumin was independently predictive of lower numbers of circulating EPCs (p < 0.04). CONCLUSION: Circulating EPCs were markedly reduced in CKD patients. ESRD was strongly and independently predictive of decreased numbers of circulating EPCs.

Interleukin-18 and Gelsolin Are Associated with Acute Kidney Disease after Cardiac Catheterization.

Patients undergoing cardiac catheterization are at high risk of post-procedure acute kidney injury (AKI) and may experience persistent renal damage after an initial insult, a state known as acute kidney disease (AKD). However, the association between AKD and urinary renal biomarkers has not yet been evaluated in this population. We enrolled 94 patients who underwent elective cardiac catheterization to investigate patterns of urinary renal biomarkers and their associations with post-procedure AKD. Serial urinary renal biomarker levels were measured during pre-procedure, early post-procedure (12-24 h), and late post-procedure (7-10 days) periods. In our investigation, 42.55% of the enrolled patients developed AKD during the late post-procedure period. While the liver-type free-fatty-acid-binding protein level increased sharply during the early post-procedure period, it returned to baseline during the late post-procedure period. In contrast, interleukin-18 (IL-18) levels increased steadily during the post-procedure period. Early post-procedure ratios of IL-18 and gelsolin (GSN) were independently associated with subsequent AKD (odds ratio (95% confidence interval), 4.742 (1.523-14.759) for IL-18 ratio, p = 0.007; 1.812 (1.027-3.198) for GSN ratio, p = 0.040). In conclusion, post-procedure AKD is common and associated with early changes in urinary IL-18 and GSN in patients undergoing cardiac catheterization.