Overexpression of miR-19a and miR-20a in iPS-MSCs preserves renal function of chronic kidney disease with acute ischaemia-reperfusion injury in rat.

This study tested the hypothesis that therapy with double overexpression of miR-19a-3p and miR-20a-5p (miRDOE ) to human inducible pluripotent stem cell-derived mesenchymal stem cells (iPS-MSCs) was superior to iPS-MSCs alone for preserving renal function in rat with pre-existing chronic kidney disease (CKD), followed by ischaemia-reperfusion (IR) injury. In vitro study demonstrated that the protein expressions of oxidative stress (NOX-1/NOX-2/NOX4/oxidized protein/p22phox), inflammatory downstream signalling (TLR2&4/MyD88/TRAF6/IKK-ß/p-NFκB/IL-1ß/IL-6/MMP-9) and cell apoptosis/death signalling (cleaved caspase-3/mitochondrial Bax/p-ERKs/p-JNK/p-p38) at time-points of 24-hour/48-hour cell cultures were significantly increased in p-Cresol-treated NRK-52E cells than in the control that was significantly reversed by miR-19a-3p-transfected iPS-MSC (all P < .001). Animals were categorized into group 1 (sham-operated control), group 2 (CKD-IR), group 3 (CKD-IR + oligo-miRDOE of iPS-MSCs/6.0 ×105 /intra-renal artery transfusion/3 hours after IR procedure), group 4 (CKD-IR + iPS-MSCs) and group 5 (CKD-IR + miRDOE of iPS-MSCs/6.0 ×105/ intra-renal artery transfusion/3 hour after IR procedure). By day 35, the creatinine/BUN levels were lowest in group 1, highest in group 2 and significantly lower in group 5 than in groups 3 and 4 (all P < .0001) but they showed no difference between the latter two groups. The protein expressions of oxidative stress, inflammatory downstream signalling and cell apoptosis/death signalling exhibited an identical pattern of creatinine level among the five groups (all P < .00001). Also, the microscopic findings demonstrated that the kidney injury score/fibrotic area/number of inflammatory cells (CD14+/CD68+) exhibited an identical pattern of creatine level (all P < .0001). The miRDOE of iPS-MSCs was superior to iPS-MSCs for preserving the residual kidney function and architecture in CKD-IR rat.

Double overexpression of miR-19a and miR-20a in induced pluripotent stem cell-derived mesenchymal stem cells effectively preserves the left ventricular function in dilated cardiomyopathic rat.

BACKGROUND: This study tested the hypothesis that double overexpression of miR-19a and miR-20a (dOex-mIRs) in human induced pluripotent stem cell (iPS)-derived mesenchymal stem cells (MSCs) effectively preserved left ventricular ejection fraction (LVEF) in dilated cardiomyopathy (DCM) (i.e., induced by doxorubicin) rat. METHODS AND RESULTS: In vitro study was categorized into groups G1 (iPS-MSC), G2 (iPS-MSCdOex-mIRs), G3 (iPS-MSC + H2O2/100uM), and G4 (iPS-MSCdOex-mIRs + H2O2/100uM). The in vitro results showed the cell viability was significantly lower in G3 than in G1 and G2, and that was reversed in G4 but it showed no difference between G1/G2 at time points of 6 h/24 h/48 h, whereas the flow cytometry of intra-cellular/mitochondrial oxidative stress (DCFA/mitoSOX) and protein expressions of mitochondrial-damaged (cytosolic-cytochrome-C/DRP1/Cyclophilin-D), oxidative-stress (NOX-1/NOX2), apoptotic (cleaved-caspase-3/PARP), fibrotic (p-Smad3/TGF-ß), and autophagic (ratio of LC3B-II/LC3BI) biomarkers exhibited an opposite pattern of cell-proliferation rate (all p< 0.001). Adult-male SD rats (n=32) were equally divided into groups 1 (sham-operated control), 2 (DCM), 3 (DCM + iPS-MSCs/1.2 × 106 cells/administered by post-28 day's DCM induction), and 4 (DCM + iPS-MSCdOex-mIRs/1.2 × 106 cells/administered by post-28 day's DCM induction) and euthanized by day 60 after DCM induction. LV myocardium protein expressions of oxidative-stress signaling (p22-phox/NOX-1/NOX-2/ASK1/p-MMK4,7/p-JNK1,2/p-cJUN), upstream (TLR-4/MAL/MyD88/TRIF/TRAM/ TFRA6/IKKα/ß/NF-κB) and downstream (TNF-α/IL-1ß/MMP-9) inflammatory signalings, apoptotic (cleaved-PARP/mitochondrial-Bax), fibrotic (Smad3/TGF-ß), mitochondrial-damaged (cytosolic-cytochrome-C/DRP1/cyclophilin-D), and autophagic (beclin1/Atg5) biomarkers were highest in group 2, lowest in group 1 and significantly lower in group 4 than in group 3, whereas the LVEF exhibited an opposite pattern of oxidative stress (all p< 0.0001). CONCLUSION: iPS-MSCdOex-mIRs therapy was superior to iPS-MSC therapy for preserving LV function in DCM rat.

Quality and quantity culture effectively restores functional and proliferative capacities of endothelial progenitor cell in end-stage renal disease patients.

BACKGROUND: Endothelial cell dysfunction plays the crucial role in initiation and propagation of obstructive arteriosclerosis which ultimately causes arterial obstructive syndrome. Additionally, severe endothelial progenitor cells (EPC) dysfunction is always found in those of end-stage renal disease (ESRD) patients. This study tested the hypothesis that a novel method, named "quality and quantity (QQ) culture", could successfully improve the EPC proliferation and function in ESRD patients. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMNCs) were isolated from age-matched control subjects (i.e., normal renal function) (group 1) and ESRD patients (group 2), followed by culture in either conventional EPC culture for one month or in QQ culture for 7 days, respectively. The result showed that as compared to the conventional EPC culture method, the EPC population and M2-like population/ratio (M2/M1) were significantly enriched in QQ culture both in groups 1 and 2 (all p < 0.001), but these parameters did not differ between the groups. As compared with conventional EPC culture, the angiogenesis capacity and colony formation were significantly increased in QQ culture (all p < 0.001), but they showed no difference between groups 1 and 2. In RAW264.7 macrophages treated by liposaccharide, the gene expressions and ELISA findings of pro-inflammatory cytokines (IL-1ß/IL-6/TGF-ß) and inflammatory mediator (iNOS) were significantly reduced in QQ culture than in conventional EPC culture in groups 1 and 2 (all p < 0.001), but they showed no difference between the groups. CONCLUSIONS: This study demonstrated that QQ culture enhanced number, proliferation, and angiogenesis of EPCs and anti-inflammatory capacity in ESRD patients.

Circulatory Rejuvenated EPCs Derived from PAOD Patients Treated by CD34+ Cells and Hyperbaric Oxygen Therapy Salvaged the Nude Mouse Limb against Critical Ischemia.

This study tested whether circulatory endothelial progenitor cells (EPCs) derived from peripheral arterial occlusive disease (PAOD) patients after receiving combined autologous CD34+ cell and hyperbaric oxygen (HBO) therapy (defined as rejuvenated EPCs) would salvage nude mouse limbs against critical limb ischemia (CLI). Adult-male nude mice (n = 40) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood prior to CD34+ cell and HBO treatment (EPCPr-T) by intramuscular injection at 3 h after CLI induction) and group 4 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood after CD34+ cell and HBO treatment (EPCAf-T) by the identical injection method). By 2, 7 and 14 days after the CLI procedure, the ischemic to normal blood flow (INBF) ratio was highest in group 1, lowest in group 2 and significantly lower in group 4 than in group 3 (p < 0.0001). The protein levels of endothelial functional integrity (CD31/von Willebrand factor (vWF)/endothelial nitric-oxide synthase (eNOS)) expressed a similar pattern to that of INBF. In contrast, apoptotic/mitochondrial-damaged (mitochondrial-Bax/caspase-3/PARP/cytosolic-cytochrome-C) biomarkers and fibrosis (Smad3/TGF-ß) exhibited an opposite pattern, whereas the protein expressions of anti-fibrosis (Smad1/5 and BMP-2) and mitochondrial integrity (mitochondrial-cytochrome-C) showed an identical pattern of INBF (all p < 0.0001). The protein expressions of angiogenesis biomarkers (VEGF/SDF-1α/HIF-1α) were progressively increased from groups 1 to 3 (all p < 0.0010). The number of small vessels and endothelial cell surface markers (CD31+/vWF+) in the CLI area displayed an identical pattern of INBF (all p < 0.0001). CLI automatic amputation was higher in group 2 than in other groups (all p < 0.001). In conclusion, EPCs from HBO-C34+ cell therapy significantly restored the blood flow and salvaged the CLI in nude mice.

P-cresyl sulfate causes mitochondrial hyperfusion in H9C2 cardiomyoblasts.

Increased circulating level of uraemic solute p-cresyl sulphate (PCS) in patients with chronic kidney disease (CKD) is known to increase myocardial burden relevant to mitochondrial abnormalities. This study aimed at investigating mitochondrial response to PCS in H9C2 cardiomyoblasts. H9C2 cardiomyoblasts were treated with four different concentrations of PCS (3.125, 6.25, 12.5 and 25.0 µg/mL) to study the changes in cell proliferation, cell size and mitochondrial parameters including morphology, respiration, biogenesis and membrane potential. The lowest effective dose of PCS (6.25 µg/mL) induced mitochondrial hyperfusion with enhanced mitochondrial connectivity, mitochondrial oxygen consumption rates, mitochondrial mass, mitochondrial DNA copy number and increased volume of cardiomyoblasts. After PCS treatment, phosphorylation of energy-sensing adenosine monophosphate-activated protein kinase (AMPK) was increased without induction of apoptosis. In contrast, mitochondrial mass was recovered after AMPK silencing. Additionally, mitochondrial hyperfusion and cell volume were reversed after cessation of PCS treatment. The results of the present study showed that low-level PCS not only caused AMPK-dependent mitochondrial hyperfusion but also induced cell enlargement in H9C2 cardiomyoblasts in vitro.

MicroRNA-214 modulates the senescence of vascular smooth muscle cells in carotid artery stenosis.

BACKGROUND: MicroRNAs control gene expression by post-transcriptional inhibition. Dysregulation of the expressions of miR-199a/214 cluster has been linked to cardiovascular diseases. This study aimed at identifying potential microRNAs related to vascular senescence. METHODS: Seven candidate microRNAs (miR-19a, -20a, -26b, -106b, - 126, - 214, and - 374) related to cell proliferation were tested for their expressions under CoCl2-induced hypoxia in vascular smooth muscle cells (VSMCs). After identification of miR-214 as the candidate microRNA, telomere integrity impairment and cell cycle arrest were examined in VSMCs by using miR-214 mimic, AntagomiR, and negative controls. To investigate the clinical significance of miR-214 in vascular diseases, its plasma level from patients with carotid artery stenosis (CAS) was assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: CoCl2 treatment for 48 h suppressed cell proliferation and angiogenesis as well as enhanced cell senescence in VSMCs. Besides, miR-214 level was elevated in both intracellular and exosome samples of VSMCs after CoCl2 treatment. Manipulating miR-214 in VSMCs demonstrated that miR-214 not only inhibited angiogenic and proliferative capacities but also promoted senescence through the suppression of quaking. Additionally, circulating miR-214 level was upregulated in CAS patients with high low-density lipoprotein cholesterol (LDL-C) value. CONCLUSION: Our findings suggested that miR-214 plays a role in the modulation of VSMC angiogenesis, proliferation, and senescence with its plasma level being increased in CAS patients with elevated LDL-C value, implying that it may be a vascular senescence marker and a potential therapeutic target for vascular diseases.

Synergistic effect of combined melatonin and adipose-derived mesenchymal stem cell (ADMSC)-derived exosomes on amelioration of dextran sulfate sodium (DSS)-induced acute colitis.

This study tested the hypothesis that melatonin (Mel) and adipose-derived mesenchymal stem cell-derived exosomes effectively suppress dextran sulfate sodium (DSS)-induced acute inflammatory colitis (AIC) in rats. To determine whether Mel-exosome treatment could ameliorate the severity of AIC, we treated Sprague Dawley rats with DSS-induced AIC with Mel, exosomes, or combined Mel-exosome therapy and evaluated the effects on AIC. First, to induce an inflammatory response in vitro, we treated HT-29 cells with lipopolysaccharide (LPS) and evaluated the response to Mel and/or exosome treatment. We found that expression of NOX-1, NOX-2, MMP-9, NF-κB, iNOS, ICAM-1, and COX-2 was significantly higher in HT-29 cells treated with LPS than in control cells, and was significantly reduced by either exosome or Mel treatment (P<0.001 for all). In vivo, flow cytometric analysis showed that, compared to untreated rats with AIC, the number of circulating inflammatory cells was lowest in rats treated with combined Mel-exosome treatment than in rats treated with either Mel or exosomes alone (P<0.0001). Compared with controls, as well as Mel or exosome treatment alone, combined Mel-exosome treatment ameliorated the effects of DSS-induced AIC as evidenced by changes in the expression of markers for inflammation, oxidative stress, apoptosis, and fibrosis (P<0.0001 for all). Additionally, histopathological findings showed that colon injury score, expression of inflammatory and DNA-damage markers, and bloody stool were all improved following combined Mel-exosome treatment (P<0.0001 for all). In conclusion, combined Mel-exosome treatment significantly protected the rat colon against DSS-induced AIC injury.

Long-term Therapeutic Effects of Extracorporeal Shock Wave-Assisted Melatonin Therapy on Mononeuropathic Pain in Rats.

We evaluated the ability of extracorporeal shock wave (ECSW)-assisted melatonin (Mel) therapy to offer an additional benefit for alleviating the neuropathic pain (NP) in rats. Left sciatic nerve was subjected to chronic constriction injury (CCI) to induce NP. Animals (n = 30) were randomized into group 1 (sham-operated control), group 2 (CCI only), group 3 (CCI + ECSW), group 4 (CCI + Mel) and group 5 (CCI + ECSW + Mel). By days 15, 22 and 29 after CCI, the thermal paw withdrawal latency (TPWL) and mechanical paw withdrawal threshold (MPWT) were highest in group 1, lowest in group 2, significantly higher in group 5 than in groups 3 and 4, but they showed no difference between the later two groups (all p < 0.0001). The protein expressions of inflammatory (TNF-α, NF-κB, MMP-9, IL-1ß), oxidative-stress (NOXs-1, -2, -4, oxidized protein), apoptotic (cleaved-caspase3, cleaved-PARP), DNA/mitochondrial-damaged (γ-H2AX/cytosolic-cytochrome C), microglia/astrocyte activation (ox42/GFAP), and MAPKs [phosphorylated (p)-p38, p-JNK, p-ERK] biomarkers in dorsal root ganglia neurons (DRGs) and in spinal dorsal horn were exhibited an opposite pattern of TPWL among the five groups (all p < 0.0001). Additionally, protein expressions of Nav.1.3, Nav.1.8 and Nav.1.9 in sciatic nerve exhibited an identical pattern to inflammation among the five groups (all p < 0.0001). The numbers of cellular expressions of MAPKs (p-ERK1/2+/peripherin + cells, p-ERK1/2+/NF200 + cells and p-JNK+/peripherin + cells, p-JNK+/NF200 + cells) and voltage-gated sodium channels (Nav.1.8+/peripherin + cells, Nav.1.8+/NF200 + cells, Nav.1.9+/peripherin + cells, Nav.1.9+/NF200 + cells) in small and large DRGs displayed an identical pattern to inflammation among the five groups (all p < 0.0001). In conclusion, the synergistic effect of combined ECSW-Mel therapy is superior to either one alone for long-term improvement of mononeuropathic pain-induced by CCI in rats.

Combined Adipose-Derived Mesenchymal Stem Cells and Low-Energy Extracorporeal Shock Wave Therapy Protect the Brain From Brain Death-Induced Injury in Rat.

This study tested the hypothesis that combined adipose-derived mesenchymal stem cell (ADMSC) and low-energy extracorporeal shock wave (ECSW) therapy could protect brain from brain death (BD)-induced injury. Adult male Sprague Dawley rats were categorized into group 1 (sham control), group 2 (BD), group 3 (BD + ECSW [0.15 mJ/mm2/300 impulses] applied to the skull surface 3 hours after BD induction), group 4 (BD + ADMSC [1.2 × 106 cell] by intravenous injection 3 hours after BD induction) and group 5 (BD + ECSW + ADMSC). By 6 hours after BD induction, circulating/spleen levels of immune cells (CD3/CD4+, CD8/CD4+, Treg+) and circulating levels of inflammatory cells (MPO/Ly6G/CD11a/b) and soluble mediators (TNF-α/IL-6) were lowest in group 1 and significantly progressively reduced from groups 2 to 5 (all p < 0.0001). Brain protein expressions of inflammatory (TNF-α/NF-κB/MMP-9/IL-1ß), apoptotic (caspase-3/PARP/mitochondrial-BAX), oxidative stress/DNA-damage (NOX-1/NOX-2/oxidized protein/γ-H2AX) biomarkers exhibited an identical pattern, whereas anti-oxidant (SIRT1/SIRT3) and mitochondrial-integrity (mitochondrial-cytochrome-C) biomarkers exhibited an opposite pattern to inflammatory biomarkers among the 5 groups (all p < 0.0001). The cellular expressions of inflammatory/brain-edema (F4/80/CD14+/GFAP/AQP4) biomarkers exhibited an identical pattern to inflammation among the 5 groups (all p < 0.0001). In conclusion, ECSW-ADMSC therapy is superior to either alone for attenuating brain from BD-induced damage.

Extracorporeal shockwave against inflammation mediated by GPR120 receptor in cyclophosphamide-induced rat cystitis model.

BACKGROUND: We tested the hypothesis that extracorporeal shockwave treatment (ESWT) can abolish inflammation and restore urothelial barrier integrity in acute interstitial cystitis by upregulating the fatty acid receptor GPR120. METHODS: A total of 30 female Sprague-Dawley rats were categorized into five groups: (1) sham-operated rats (SC); (2) rats treated with ESWT (SC + ESWT); (3) rats with bladder irritation using 150 mg/kg cyclophosphamide through intraperitoneal injection; (4) cyclophosphamide rats treated with ESWT (cyclophosphamide+ESWT); (5) cyclophosphamide rats treated with GPR120 agonist (cyclophosphamide+GW9508). RESULTS: On Day 3, urine and bladder specimens were collected for biochemical, histopathological, immunological, and immunoblotting analysis. Following stimulation with cyclophosphamide, the inhibition of the elevated levels of TAK1/NF-κB and phospho-TAK1/NF-κB by ESWT and GPR120 agonists in RT4 cells was associated with a suppression of NF-κB translocation from the cytosol to the nucleus. Accordingly, this anti-inflammatory effect was abolished by GPR120 antagonist and knockdown of GPR120. Histologically, bladder inflammation in cyclophosphamide-treated rats was suppressed by GW9508 or ESWT. Masson's trichrome and Sirius red staining revealed that cyclophosphamide treatment enhanced synthesis of extracellular matrix in rats that was reversed by GW9508 or ESWT. Upregulated pro-inflammatory mediators and cytokines in the cyclophosphamide-treated rats were also suppressed in the GW9508- or ESWT-treated rats. The significantly increased inflammatory cell infiltration as well as the impaired urothelial integrity of the bladder after cyclophosphamide treatment were reversed by treatment with GW9508 or ESWT. CONCLUSIONS: These findings suggest that GPR120, the sensing receptor for ESWT, may be useful in the treatment of interstitial cystitis by inhibiting inflammatory response in bladder cells.

Inducible pluripotent stem cell-derived mesenchymal stem cell therapy effectively protected kidney from acute ischemia-reperfusion injury.

This study tested whether inducible pluripotent stem cell (iPSC)-derived mesenchymal stem cell (MSC) therapy could effectively protect kidney from acute ischemia (1 h) - reperfusion (5 day) injury (IRI). Male-adult SD-rats (n = 24) were equally categorized into groups 1 (sham-control), 2 [sham-control + iPSC-MSC (1.2 × 106 cells/rat)], 3 (IR only) and 4 (IR + iPSC-MSC). Blood urine nitrogen/creatinine levels and ratio of urine protein to creatinine, kidney weight and expressions of inflammation (TNF-α/NF-κB), oxidative-stress (NOX-1/NOX-2/oxidized protein) and apoptosis (mitochondrial-Bax/cleaved caspase-3/PARP) were significantly higher in group 3 than in groups 1, 2 and 4 and significantly higher in group 4 than in groups 1 and 2 (all P<0.0001), but showed no differences between groups 1 and 2, whereas the protein expressions of anti-inflammation (IL-4/IL-10) and endothelial (CD31/vWF) markers exhibited an opposite pattern to inflammation among the four groups (all P<0.0001). Protein expressions of angiogenesis (VEGF/CXCR4/SDF-1α) markers progressively increased from groups 1 to 4 (all P<0.0001). Cellular expressions of kidney injury score/DNA-damage (γ-H2AX)/apoptotic nuclei and glomerulus-tubular-damage (KIM/FSP-1) displayed an identical pattern to inflammation, whereas the cellular expressions of glomerulus-tubular-integrity (dystroglycan/podocin/p-cadherin/synaptopodin/ZO-1/fibronectin) revealed an opposite pattern to inflammation among the four groups (all P<0.0001). In conclusion, iPSC-derived MSC therapy effectively protected kidney against IRI.

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.

Correlation between Therapeutic Efficacy of CD34+ Cell Treatment and Directed In Vivo Angiogenesis in Patients with End-Stage Diffuse Coronary Artery Disease.

BACKGROUND: This study was aimed at testing the association between the therapeutic efficacy of CD34+ cell treatment in patients with end-stage diffuse coronary artery disease as reflected in angiographic grading and results of directed in vivo angiogenesis assay (DIVAA) on their isolated peripheral blood mononuclear cell- (PBMC-) derived endothelial progenitor cells (EPCs). METHODS: Angiographic grades (0: <5%; 1: 5-35%; 2: 35-75%; 3: >75%) which presented the improvement of vessel density pre- and post-CD34+ treatment were given to 30 patients with end-stage diffuse coronary artery disease having received CD34+ cell treatment. The patients were categorized into low-score group (angiographic grade 0 or 1, n = 12) and high-score group (angiographic grade 2 or 3, n = 18). The percentages of circulating EPCs with KDR+/CD34+/CD45-, CD133+/CD34+/CD45-, and CD34+ were determined in each patient using flow cytometry. PBMC-derived EPCs from all patients were subjected to DIVAA through a 14-day implantation in nude mice. The DIVAA ratio (i.e., mean fluorescent units in angioreactors with EPCs/mean fluorescent units in angioreactors without EPCs) was obtained for each animal with implanted EPCs from each patient. RESULTS AND CONCLUSIONS: The number of EPCs showed no significant difference among the two groups. The DIVAA ratio in the high-score group was significantly higher than that in the low-score group (p = 0.0178). Logistic regression revealed a significant association between the DIVAA ratio and angiographic grading (OR 3.12, 95% CI: 1.14-8.55, p = 0.027). The area under the ROC curve (AUC) was 0.8519 (p = 0.0013). We proposed that DIVAA may be a reliable tool for assessing coronary vascularization after CD34+ cell treatment.

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.

Effective protection against acute respiratory distress syndrome/sepsis injury by combined adipose-derived mesenchymal stem cells and preactivated disaggregated platelets.

This study assessed whether combining adipose-derived mesenchymal stem cells (ADMSC) with preactivated, disaggregated shape-changed platelets (PreD-SCP) was superior to either therapy alone for protecting rat lung from acute respiratory distress syndrome (ARDS) complicated by sepsis. ARDS and sepsis were induced through 100% oxygen inhalation and peritoneal administration of 1.5 mg/kg lipopolysaccharide (LPS), respectively. Adult-male Sprague-Dawley rats (n=40) were randomized into sham-control (SC), ARDS-LPS, ARDS-LPS-ADMSC (1.2x106 cells), ARDS-LPS-PreD-SCP (3.0x108, intravenous administration), and ARDS-LPS-ADMS/PreD-SCP groups, and were sacrificed 72 h after 48 h ARDS induction. Lung injury scores (LIS) and collagen deposition were highest in ARDS-LPS, lowest in SC, higher in ARDS-LPS+ADMSC than in ARDS-LPS+PreD-SCP and ARDS-LPS+ADMS/PreD-SCP, and higher in ARDS-LPS+PreD-SCP than in ARDS-LPS+ADMS/PreD-SCP (all p<0.0001). Alveolar-sac numbers, oxygen saturation, endothelial marker levels, and mitochondrial cytochrome-C levels exhibited opposite patterns with respect to LIS (all p<0.001). Levels of inflammatory, oxidative-stress, apoptosis, mitochondrial/DNA damage, and MAPK and Akt signaling markers exhibited patterns identical to that of LIS (all p<0.001). Anti-oxidant and anti-inflammatory protein levels increased progressively from SC to ARDS-LPS+ADMS/PreD-SCP (all p<0.0001). These findings indicate combined ADMSC/PreD-SCP was superior to either therapy alone for protecting rat lung from ARDS-sepsis injury.

Combination therapy with extracorporeal shock wave and melatonin markedly attenuated neuropathic pain in rat.

This study tested the hypothesis that combination therapy using extracorporeal shock wave (ECSW)-melatonin (Mel) was superior to either alone at ameliorating neuropathic pain (NP). NP was induced by chronic constriction injury (CCI) to the left sciatic nerve in rats. Animals were categorized into sham control (group 1), CCI only (group 2), CCI-ECSW (group 3), CCI-Mel (group 4) and CCI-ECSW-Mel (group 5). By days 2 and 8 after CCI, the mechanical paw withdrawal threshold (MPWT)/thermal paw withdrawal latency (TPWL) were highest in group 2, lowest in group 1, significantly lower in group 5 than in groups 3 and 4 (all p<0.0001), and not significantly different between groups 3 and 4. The protein expressions of inflammatory (TNF-α/NF-κB/MMP-9/IL-1ß/GFAP/ox42), oxidative-stress (NOX-1/NOX-2/NOX-4/oxidized protein), DNA/mitochondrial-damaged (γ-H2AX/cytosolic mitochondria), apoptotic (cleaved capase-3/PARP), and MAPK family biomarkers (p-P38/p-JNK/p-ERK1/2) in dorsal root ganglia and spinal dorsal horn expressed a similar pattern of MPWT/TPWL among the five groups, except for significantly higher in group 4 than in group 3 (all p<0.0001). The protein expressions of Nav.1.3, Nav.1.8 and Nav.1.9 in sciatic nerve displayed an identical pattern to inflammation among the five groups (all p<0.001). Pain facilitated cellular expressions (p-P38+/peripherin+ cells, P38+/NF200+ cells) displayed an identical pattern to inflammation among the five groups (all p<0.0001). In conclusion, ECSW-Mel combination therapy markedly ameliorated NP induced by CCI.

Exendin-4-assisted adipose derived mesenchymal stem cell therapy protects renal function against co-existing acute kidney ischemia-reperfusion injury and severe sepsis syndrome in rat.

This study tested the hypothesis that combined therapy with exendin-4 (Ex4) and autologous adipose-derived mesenchymal stem cells (ADMSCs) was superior to either alone for protecting renal function against acute kidney ischemia-reperfusion (IR; 40-min ischemia/27-h reperfusion) injury when complicated by sepsis syndrome (SS; by cecal-ligation-puncture). Adult-male Sprague-Dawley rats (n=40) were equally divided into group 1 (sham-control), group 2 (IR-SS), group 3 (IR-SS + Ex4, 10 µg/kg subcutaneously 30 min after reperfusion and daily for 3 days), group 4 [IR-SS + ADMSC (1.2 × 106)], and group 5 (IR-SS + Ex4 + ADMSC). The circulating levels of BUN and creatinine and the ratio of urine protein to creatinine were highest in group 2, lowest in group 1, significantly higher in groups 3 and 4 than group 5, and significantly higher in group 3 than in group 4 (all P<0.0001). Microscopic findings of kidney injury score, inflammatory cells (CD14+, F4/80+), and expressions of glomerular-damage indicators (FSP-1+/WT-1+) and renal tubular-damage indicators (KIM-1+/snail+) showed an identical pattern, whereas expressions of indices of glomerular-integrity (ZO-1+/p-cadherin+/podocin+/synaptopodin+) and angiogenesis (CD31+/vWF+/number of small vessels) biomarkers demonstrated an opposite pattern, to that of creatinine level (all P<0.001). Protein expressions of inflammatory (MMP-9/IL-1ß/TNF-α/TLR-2/TLR-4), apoptotic (cleaved caspase-3/PARP/mitochondrial Bax), and oxidative-stress (NOX-1/NOX-2/oxidized protein) biomarkers exhibited an identical pattern, whereas anti-inflammatory (IL-10/IL-4) biomarkers displayed an opposite pattern, to that of creatinine level (all P<0.001). In conclusion, combined Ex4 and ADMSC therapy significantly protected kidney from acute IR-SS injury.

Investigated the safety of intra-renal arterial transfusion of autologous CD34+ cells and time courses of creatinine levels, endothelial dysfunction biomarkers and micro-RNAs in chronic kidney disease patients-phase I clinical trial.

This was a phase I clinical trial to investigate the safety of autologous peripheral-blood-derived CD34+ cell therapy for patients with chronic kidney disease (CKD-treatment) (i.e., at Stages III and IV). Between November 2014 and October 2015, a total of 10 study patients were prospectively enrolled into this phase I trial. Patients who failed to enroll into the trial in the initial state of eligibility assessment were served as CKD-control group (n = 9). The health-control group was composed of 10 volunteers for the purposes of comparing (1) circulation level of endothelial progenitor cells (EPCs), (2) angiogenesis ability, and (3) anti-apoptotic miRNAs between healthy subjects and CKD patients. CD34+ cells (5.0 x 107) were transfused into right-renal artery after subcutaneous G-CSF injection (5µg/kg/twice a day for 4 days). Circulating EPC number, angiogenesis capacity (i.e., Matrigel assay) and anti-apoptotic miRNAs (miR-374a-5p/miR-19a-3p/ miR-106b-5p/miR-26b-5p/ miR-20a-5p) were significantly lower in CKD patients than in healthy subjects (all p < 0.001). Flow-cytometric analysis of renal-vein blood samplings (i.e., at 0/5/10/30 mins after cell transfusion) showed the EPC level was significantly progressively increased (p < 0.001). Procedural safety was 100% with all patients uneventfully discharged and one-year survival rate was 100%. The paired-t test showed serum creatinine maintained the same level between the baseline and at the end of one-year follow-up (all p > 0.4), whereas the net increase between initial and final creatinine level was higher in CKD-control than in CKD-treatment. In conclusion, CD34+ cell therapy was safe and maintained the renal function in stationary state at the end of study period.

SS31 therapy effectively protects the heart against transverse aortic constriction-induced hypertrophic cardiomyopathy damage.

This study tested the hypothesis that SS31 therapy could effectively protect the heart against transverse aortic constriction (TAC)-induced hypertrophic cardiomyopathy (HCM) damage. Adult-male B6 mice (n=36) were equally divided into sham-operated control (group 1), TAC only (group 2) and TAC+SS31 (group) (2.0 mg/kg/day by intra-peritoneal administration from day 28 after TAC induction) and euthanized by day 60. In vitro results showed that SS31 markedly suppressed angiotensin-II induced protein expressions of BNP/ß-MHC, ATM, p-P38 and P53 and ATP damage in H9C2 cells, and protein expression of pro-collagen-I/CTGF in fibroblasts (all P<0.001). By day 60, left ventricular ejection fraction (LVEF) and sarcomere length were significantly lower in group 2 than groups 1 and 3, and significantly lower in group 3 than in group 1, whereas the LEVDd/LVESd and ratio of heart weight to tibial length showed an opposite pattern to LVEF (all P<0.0001). Microscopic findings of numbers of apoptotic nuclei, inflammatory (CD14+, F4/80+) and oxidative-stress (H2DCFDA+) biomarkers, disorganized score of endocardium, and fibrotic and collagen-deposition areas showed an opposite pattern to LVEF among the three groups (all P<0.0001). The protein expressions of inflammatory (PDGF/TNF-α/NF-κB/COX-2), oxidative-stress (NOX-1/NOX-2/oxidized protein), fibrotic (TGF-ß/Smad3) apoptotic (cleaved-caspase-3/cleaved-PARP), pressure/volume overload (BNP/ß-MHC), CTGF, mitochondrial-damaged (cytosolic cytochrome-C), p-ERK1/2, p-Akt and PI3K signaling showed an opposite pattern to LVEF among the three groups (all P<0.001). The protein expression of anti-oxidants (HO-1/Nrf2) were significantly progressively increased in groups 1 to 3 (all P<0.001). In conclusion, SS31 therapy effectively protected the heart against TAC-induced damage.

Shock Wave Enhances Angiogenesis through VEGFR2 Activation and Recycling.

Although low-energy shock wave (SW) is adopted to treat ischemic diseases because of its pro-angiogenic properties, the underlying mechanism remains unclear. This study aimed at testing whether SW-induced angiogenesis may be through endothelial vascular endothelial growth factor receptor 2 (VEGFR2) signaling and trafficking. Phosphorylation of VEGFR2-Akt-eNOS axis and production of nitric oxide (NO) were determined in human umbilical vein endothelial cells (HUVECs) treated with SW. Carotid artery in ob/ob mice was treated with SW before evaluation with sprouting assay. Critical limb ischemia was induced in ob/ob mice to evaluate blood flow recovery after SW treatment. Tube formation and migration assays were also performed with/without SW treatment in the presence/absence of SU5416 (VEGFR2 kinase inhibitor) and siRNA-driven silencing of VEGFR2. Chloroquine was used for disrupting endosome, and Rab11a controlling slow endocytic recycling was silenced with siRNA in vitro. Following SW treatment, augmented ligand-independent phosphorylation in VEGFR2-Akt-eNOS axis and endogenous NO production, increased cellular migration and tube formation, elevated sprouting of carotid artery and blood flow in ischemic limb in ob/ob mice were noted. Moreover, SU5416 and VEGFR2 silencing both inhibited SW-induced angiogenesis. SW-induced angiogenesis, which was accompanied by increased VEGFR2 protein expression without transcriptional change, was suppressed by chloroquine and Rab11a silencing. We concluded that SW enhanced angiogenesis via ligand-independent activation of VEGFR2 and further prolonged through endosome-to-plasma membrane recycling in endothelial cells.