SDF-1-edited human amniotic mesenchymal stem cells stimulate angiogenesis in treating hindlimb ischaemia.

Although stem cells have extensively been studied as a novel vehicle for tissue repair, their sustained efficacy remains controversial. In this study, we aimed to investigate the angiogenic potency over time of stromal cell-derived factor-1 (SDF-1) gene-edited amniotic mesenchymal stem cells (AMM/S) in a hindlimb ischaemia model. An SDF-1 transgene was inserted into the AMM cell genome via transcription activator-like effector nuclease (TALEN) mediated knock-in, and cell migration, Matrigel tube formation, and in vivo Matrigel plug assays were performed. AMM/S were also transplanted into hindlimb ischaemia model mice. Blood perfusion, therapeutic potential, histology, capillary density and in vivo angiogenic assays were performed. AMM/S exhibited high expression of the SDF-1 gene, and robustly promoted migration, proliferation and microvascular formation. AMM/S transplantation significantly increased blood perfusion and limb loss prevention compared with AMM. AMM/S also significantly inhibited increased capillary density and expression of angiogenic factors in the ischaemic hindlimb. Our study demonstrated that AMM/S provides a significant therapeutic effect in ischaemic hindlimbs by enhancing angiogenesis.

Expression of Matrix Metalloproteinases and Tissue Inhibitor of Matrix Metalloproteinases during Apical Periodontitis Development.

INTRODUCTION: Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) are considered important mediators of the periapical immune response to infection. This study aimed to clarify the putative relationship between MMPs and TIMPs by elucidating the activity of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 in the temporal development of apical periodontitis (AP) in mice. METHODS: AP was induced in the lower first molars of 30 male Kunming mice. The animals were randomly killed at 0, 7, 14, 28, 60, and 90 days after pulp exposure. The jaws were removed and subjected to quantitative real-time reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis. RESULTS: The MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 messenger RNA and protein expression levels increased with periapical inflammation progression (P < .05). The MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 messenger RNA and protein expression levels increased during the acute and chronic stages of periapical lesions, with less MMP-2 and MMP-9 expression levels at the chronic stage (P < .05). The MMP-8 expression increased at the chronic stage of inflammation (P < .05) but not at the acute stage. Immunostained MMP-2 and TIMP-1 were observed in all experimental periods. CONCLUSIONS: MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 were expressed in all periapical samples with varying levels between them. MMP expression could be related to TIMP expression in the temporal development of AP.

ASC and SVF Cells Synergistically Induce Neovascularization in Ischemic Hindlimb Following Cotransplantation.

Previously, we reported the angio-vasculogenic properties of human stromal vascular fraction (SVF) and adipose tissue-derived mesenchymal stem cells (ASCs). In this study, we investigated whether the combination of ASCs and SVF cells exhibited synergistic angiogenic properties. We conducted quantitative (q)RT-PCR, Matrigel plug, tube formation assays, and in vivo therapeutic assays using an ischemic hind limb mouse model. Immunohistochemical analysis was also conducted. qRT-PCR results revealed that FGF-2 was highly upregulated in ASCs compared with SVF, while PDGF-b and VEGF-A were highly upregulated in SVF. Conditioned medium from mixed cultures of ASCs and SVF (A+S) cells showed higher Matrigel tube formation and endothelial cell proliferation in vitro. A+S cell transplantation into ischemic mouse hind limbs strongly prevented limb loss and augmented blood perfusion compared with SVF cell transplantation. Transplanted A+S cells also showed high capillary density, cell proliferation, angiogenic cytokines, and anti-apoptotic potential in vivo compared with transplanted SVF. Our data indicate that A+S cell transplantation results in synergistic angiogenic therapeutic effects. Accordingly, A+S cell injection could be an alternative therapeutic strategy for treating ischemic diseases.

[Effect of 2 bleaching therapies on decoloring of stained dental fluorosis].

PURPOSE: To evaluate the clinical performance of at-home bleaching and its application in combination with cold-light bleaching for esthetic management of fluorosed teeth，providing guidance for clinicians to choose the right treatment regimen and communicate with patients successfully. METHODS: We divided 43 cases with stained dental fluorosis into 2 groups, with 22 cases in the at-home bleaching group and 21 in the combination therapy group. Stained spots were chosen and colorimetric assay was performed using CMYK colorimetric table before treatment. According to the color of the splash, the light and shade were divided into light grade, medium grade and heavy grade. The CMYK data of the selected point and complete decolorization ratio (stain decolorization rate) were recorded after cold-light bleaching, every 2 weeks and half a year later. Meanwhile, tooth sensitivity was recorded using visual analogue scale (VAS). Statistical analysis was performed using SPSS 17.0 software package. RESULTS: In the combination therapy group, the decolorization rates of light, medium and heavy stains were 73.44%, 8.70% and 0% immediately after cold-light bleaching respectively. At 2 weeks, in the at-home group, the postoperative decoloring rate was 64.17% in the light grade group and 4.44% in the medium grade group, respectively. In the combination therapy group, the postoperative decoloring rate was 100% in the light grade group and 63.04% in the medium grade group, respectively (P＜0.01). The postoperative decoloring rate in the heavy grade group was 0%. At 4 weeks, in the at-home group, the postoperative decoloring rate was 100% in the light grade group, 73.33% in the medium grade group and 11.40% in the heavy grade group, respectively. In the combination therapy group, the postoperative decoloring rate was 100% in the medium grade group and 53.00% in the heavy grade group, respectively (P＜0.01). At 6 weeks, in the at-home group, the postoperative decoloring rate was 100% in the medium grade group and 76.32% in the heavy grade group. In the combination therapy group, the postoperative decoloring rate was 94.00% in the heavy grade group (P＜0.01). At 8 weeks, the postoperative decoloring rate was 95.61% in the heavy grade group of at-home group. Half a year after treatment, bleaching decoloration rate had no significant difference compared with that at the end of treatment (P＞0.05). Tooth sensitivity was 45.45% in the at-home group and 80.95% in the combination therapy group (P＜0.05). CONCLUSIONS: Fluorosed teeth show significantly better esthetic appearance after both the at-home bleaching and the combination therapy at 6-8 weeks, and can maintain stable for a long time. Cold-bleaching has faster decoloration speed, with the final decolorization rate of the stains unchanged. In addition, it increases the incidence of postoperative sensitivity of teeth.

CYP2C19 but not CYP2B6, CYP3A4, CYP3A5, ABCB1, PON1 or P2Y12 genetic polymorphism impacts antiplatelet response after clopidogrel in Koreans.

Clopidogrel response variability (CRV) is well documented, and may affect clinical outcomes. Impact of genetic polymorphisms is important for assessing and predicting CRV. The extensive evidence indicates the importance of CYP2C19 variants in reducing efficacy of clopidogrel. This study defined the impact of numerous genetic polymorphisms on CRV before and after percutaneous coronary interventions (PCI) exclusively in a Korean cohort assuming less genetic variability noise. One hundred and thirty-six patients of Korean origin undergoing PCI were included. Platelet reactivity was measured by VerifyNow assay before and after PCI. Genetic polymorphism of seven single nucleotides of CYP2B6, CYP2C19, CYP3A4, CYP3A5, ABCB1, PON1, and P2Y12 were evaluated and matched with platelet reactivity. Carriers of at least one CYP2C19*2 or *3 allele uniformly exhibited higher platelet reactivity compared to 0-carrier pre-PCI (odds ratio 3.1, 95% confidence interval 1.4-6.9, P < 0.01) and post-PCI (odds ratio 3.4, 95% confidence interval 1.7-6.8, P < 0.001). The carriers of other gene allele variants lack uniformed impact on CRV. The Korean carriers of CYP2C19*2 or *3 allele are linked to CRV, whereas CYP2B6, CYP3A4, CYP3A5, ABCB1, PON1, and P2Y12 failed to predict CRV. The exact clinical utility of these findings is uncertain, and requires a large randomized national trial for proof of concept.

Defining predictive values using three different platelet function tests for CYP2C19 phenotype status on maintenance dual antiplatelet therapy after PCI.

Published data suggests that the presence of CYP2C19*2 or *3 loss of function (LOF) alleles is indicative of increased platelet aggregation and a higher risk of adverse cardiovascular events after clopidogrel administration. We sought to determine cut-off values using three different assays for prediction of the CYP2C19 phenotype in Korean percutaneous coronary intervention (PCI) patients. We enrolled 244 patients with drug-eluting stent implantation who were receiving clopidogrel and aspirin maintenance therapy for one month or more. Platelet reactivity was assessed with light transmittance aggregometry (LTA), multiple electrode aggregometry (MEA) and the VerifyNow P2Y12 assay (VN). The CYP2C19 genotype was analyzed by polymerase chain reaction (PCR) and snapshot method. The frequency of CYP2C19 LOF allele carriers was 58.6%. The cut-off values from LTA, MEA and VerifyNow for the identification of LOF allele carriers were as follows: 10 µM ADP-induced LTA ≥ 48 %, VN>242 PRU and MEA ≥ 37 U. Between the three tests, correlation was higher between LTA vs. VN assays (r=0.69) and LTA vs. MEA (r=0.56), with moderate agreement (κ=0.46 and κ=0.46), but between VN assay and MEA, both devices using whole blood showed a lower correlation (r=0.42) and agreement (κ=0.3). Our results provide guidance regarding cut-off levels for LTA, VerifyNow and MEA assays to detect the CYP2C19 LOF allele in patients during dual antiplatelet maintenance therapy.

Predictive values of post-clopidogrel platelet reactivity assessed by different platelet function tests on ischemic events in East Asian patients treated with PCI.

Abstract An accumulating number of studies are revealing that platelet reactivity above specific cut-off scores leads to exponentially increased rates of post-percutaneous coronary intervention (PCI) ischemic events. To evaluate the optimal predictive values for three different platelet function measurement assays of platelet reactivity on early clinical outcomes in Korean patients undergoing PCI, we enrolled 228 patients receiving clopidogrel prior to PCI. Platelet reactivity was measured by light transmittance aggregometry (LTA), VerifyNow P2Y12 assay, and multiple electrode platelet aggregometry (MEA). The primary endpoint was the 30-day occurrence of ischemic events after PCI. MACE occurred in 36 patients (15.8%), including 35 patients (15.4%) with periprocedural MI and the death of one patient (0.4%). ADP-induced LTA and VerifyNow values (pre- and post-PCI) were significantly higher in patients with the subsequent occurrence of periprocedural MI, but the MEA assay data (PCI and post-PCI) displayed no significant differences (pre-PCI p=0.25 and post-PCI p=0.33). ROC curve analysis demonstrated HPR values for LTA (pre-PCI, >66% and post-PCI, >53 %, all p<0.001), VerifyNow (pre-PCI, >347 PRU and post-PCI >272 PRU, all p<0.001) and MEA (pre-PCI, >50 U and post-PCI >39 U, all p>0.05). The platelet reactivity measurements by LTA and the VerifyNow assay can discriminate the risk of 30-day ischemic events after PCI. The predictive cut-off values for adverse events are dependent on sampling time.

Comparison of prasugrel and clopidogrel reloading on high platelet reactivity in clopidogrel-loaded patients undergoing percutaneous coronary intervention (PRAISE-HPR): a study protocol for a prospective randomized controlled clinical trial.

BACKGROUND: Patients with reduced responsiveness to clopidogrel often have diminished platelet inhibition, a factor associated with increased rates of major adverse cardiovascular events. Clinical trials that have focused on reducing high on-treatment platelet reactivity (HPR) with an additional loading dose of clopidogrel have reported varying effects. Prasugrel, a newer thienopyridine, exhibits a more consistent antiplatelet effect and more rapid onset time when compared to clopidogrel. We hypothesize that prasugrel reloading would be more effective than clopidogrel reloading in patients with HPR after an initial loading dose of clopidogrel. METHOD/DESIGN: Comparison of Prasugrel and Clopidogrel Reloading on High Platelet Reactivity in Clopidogrel-loaded Patients Undergoing Percutaneous Coronary Intervention (PRAISE-HPR) is a prospective, randomized, open-label, active controlled study. A total of 76 patients undergoing percutaneous coronary intervention (PCI), with HPR after administration of a loading dose of clopidogrel will be randomly assigned to either prasugrel or clopidogrel groups, and patients in each group will be reloaded with 20 mg of prasugrel or 300 mg of clopidogrel. The primary endpoint will be HPR at 24 hours after PCI, as determined by the VerifyNow assay during the study period. The rate of sustained high platelet reactivity and 30-day clinical outcomes will also be measured. DISCUSSION: PRAISE-HPR is a randomized controlled clinical trial to investigate the efficacy and safety of reloading prasugrel and clopidogrel in suppressing residual high platelet reactivity. The results will be made publicly available in the year 2013. TRIAL REGISTRATION: NCT01609647.

Time-dependent expression patterns of cardiac aquaporins following myocardial infarction.

Aquaporins (AQPs) are expressed in myocardium and the implication of AQPs in myocardial water balance has been suggested. We investigated the expression patterns of AQP subtypes in normal myocardium and their changes in the process of edema formation and cardiac dysfunction following myocardial infarction (MI). Immunostaining demonstrated abundant expression of AQP1, AQP4, and AQP6 in normal mouse heart; AQP1 in blood vessels and cardiac myocytes, AQP4 exclusively on the intercalated discs between cardiac myocytes and AQP6 inside the myocytes. However, neither AQP7 nor AQP9 proteins were expressed in CD1 mouse myocardium. Echocardiography revealed that cardiac function was reduced at 1 week and recovered at 4 weeks after MI, whereas myocardial water content determined by wet-to-dry weight ratio increased at 1 week and rather reduced below the normal at 4 weeks. The expression of cardiac AQPs was up-regulated in MI-induced groups compared with sham-operated control group, but their time-dependent patterns were different. The time course of AQP4 expression coincided with that of myocardial edema and cardiac dysfunction following MI. However, expression of both AQP1 and AQP6 increased persistently up to 4 weeks. Our findings suggest a different role for cardiac AQPs in the formation and reabsorption of myocardial edema after MI.

Pharmacodynamic comparisons for single loading doses of prasugrel (30 mg) and clopidogrel (600 mg) in healthy Korean volunteers.

BACKGROUND: Previous studies involving a loading dose (LD) of 60 mg prasugrel have suggested that active metabolite exposure and pharmacodynamic responses may be higher in persons of Asian ethnicity than in Caucasian subjects. The aim of this study was to determine the pharmacodynamic effect of an LD of 30 mg prasugrel and 600 mg clopidogrel in healthy Korean volunteers. METHODS AND RESULTS: Twelve volunteers were randomly assigned to a prasugrel or a clopidogrel group. Following a 2-week washout period, group designations and treatments were switched (6 per group). Platelet function was serially measured via light transmission aggregometry (LTA), VerifyNow and multiple electrode platelet aggregometry (MEA) assays at baseline and 0.5, 2, 6, and 24h after LD. Inhibition of platelet aggregation (IPA) at 0.5-24 h after prasugrel was significantly higher (P<0.001) than that achieved by clopidogrel. The prasugrel peak IPA at 2 h after LD was 93.7% (±6.2%) compared to the clopidogrel peak IPA at 6h after LD at 65.8% (±17.2%). The VerifyNow and MEA assay yielded results similar to those obtained by LTA. CONCLUSIONS: In healthy Korean subjects, a 30-mg LD of prasugrel yields a more rapid, potent and consistent inhibition of platelet function than a 600-mg LD of clopidogrel.

Robust angiogenic properties of cultured human peripheral blood-derived CD31⁺ cells.

BACKGROUND: Recently, we showed the angio-vasculogenic potential of uncultured human peripheral blood (hPB)-derived CD31(+) cells. However, thus far, the angiogenic property of the cultured hPB-derived CD31(+) (C-31(+)) cells is unknown. Thus, this study aimed to assess the angiogenic potency of C-31(+) cells on experimental ischemia. METHODS: CD31(+) and CD31(-) cells were isolated by magnetic bead separation technique, and cultured in EBM-2 complete medium for 6days. The expression of multiple angiogenic genes in these cells was measured using qRT-PCR. In addition, endothelial progenitor cell culture and matrigel network formation assays were performed. A mouse model of hindlimb ischemia induced by surgical resection of the right femoral artery was used, and the C-31(+) cells were intramuscularly transplanted into the ischemic area. Immunohistochemical analysis was also performed. RESULTS: C-31(+) cells exclusively showed higher colony-forming activity, and gave rise to EPCs. C-31(+) cells also induced higher endothelial network formation, and exhibited higher pro-angiogenic and lower inflammatory gene expression. In our ischemic hindlimb model, transplantation of C-31(+) cells induced increased blood perfusion (0.652±0.03 vs. 0.47±0.04; P<0.01) and increased capillary density (85±5.5 vs. 57±4.1; P<0.01) as compared to C-31(-) cells. In addition, angiogenic factors were markedly upregulated after the transplantation of C-31(+) cells, indicating that C-31(+) cells contributed to the neovascularization. CONCLUSIONS: The high angiogenic and therapeutic potential of C-31(+) cells observed in our ischemic animal model suggests a novel role of hPB-derived cultured CD31(+) cells in the treatment of ischemic cardiovascular diseases.

Effect of different anticoagulants on multiple electrode platelet aggregometry after clopidogrel and aspirin administration in patients undergoing coronary stent implantation: a comparison between citrate and hirudin.

Outcomes of platelet function tests are highly dependent on the type of blood anticoagulant used. The primary objective of this study was to clinically evaluate the platelet function after dual antiplatelet therapy using two different types of anticoagulant (citrate and hirudin). We compared data obtained from multiple electrode platelet aggregometry (MEA) with reference to light transmission aggregometry (LTA) and VerifyNow (VN) assays. Blood samples were obtained from 119 patients on dual antiplatelet therapy at the time of PCI (PCI) and the following morning (post-PCI). The platelet function tests were performed using two anticoagulated (citrate or hirudin) blood types for MEA as well as citrated blood for LTA and VerifyNow assays. ADP-induced MEA values at PCI for citrated and hirudinated anticoagulants were 36.5 ± 14.3 AUC and 41.4 ± 18.2 AUC (p = 0.021) and post-PCI values were 28.2 ± 11.9 AUC and 28.3 ± 12.8 AUC (p = 0.95). Additionally, AA-induced MEA values at PCI by citrated and hirudinated blood was 13.4 ± 7.3 AUC and 17.6 ± 13.4 AUC (p < 0.01). Post-PCI AA-induced MEA values were 12.0 ± 6.7 AUC and 13.5 ± 8.5 AUC (p = 0.12), respectively. Significant correlations were observed between the two anticoagulants used for MEA and LTA or VN values under ADP-induced platelet stimulation. Citrate tubes are clinically adequate for MEA assays and provide a more economical alternative to hirudin for early and/or delayed phases after clopidogrel-loading doses.

Amniotic mesenchymal stem cells with robust chemotactic properties are effective in the treatment of a myocardial infarction model.

BACKGROUND: We previously reported that amniotic mesenchymal stem cells (AMMs) possess high angio-vasulogenic properties. In this study, we investigated the chemotactic abilities of AMMs for improved cardiac function and regenerative angiogenesis. METHODS: The expressions of chemotactic and angiogenic genes were determined by qRT-PCR. Myocardial infarction (MI) was induced in NOD/SCID mice and cells were directly transplanted into the border regions of ischemic heart tissue. Immunohistochemical analysis was also conducted. RESULTS: AMMs significantly expressed the representative chemotactic factor GCP-2, NAP-2 as well as angiogenic factor Hif-1a. AMMs also highly expressed the chemokine receptors CCR2, CCR3 and CCR5. AMM transplantation improved left ventricular function, capillary density, angiogenic cytokine levels, angiopoetin (Ang)-1 and vascular endothelial growth factor (VEGF-A) levels in affected tissue. Immunohistochemical assaying also revealed increased engraftment and endothelial phenotypes. CONCLUSION: Our findings suggest that due to elevated survival and related chemotactic potential, AMMs are a promising stem cell source for the treatment of ischemic cardiovascular disease.

Mesenchymal stem cells overexpressing GCP-2 improve heart function through enhanced angiogenic properties in a myocardial infarction model.

AIMS: In this study, our aim was to evaluate the angio-vasculogenic properties of human adipose tissue-derived mesenchymal stem cells overexpressing the granulocyte chemotactic protein (GCP)-2 (hASCs/GCP-2) and to determine possible therapeutic effects in an experimental ischaemic heart model. METHODS AND RESULTS: Quantitative real-time (qRT)-PCR results revealed that hASCs/GCP-2 expressed significantly higher levels of pro-angiogenic genes, including vascular endothelial growth factor (VEGF)-A, hepatocyte growth factor (HGF), and interleukin (IL)-8, when compared with control-vector transduced hASCs or human umbilical vascular endothelial cells (HUVECs). In addition, the anti-apoptotic insulin-like growth factor (IGF)-1 and Akt-1 were also highly up-regulated in the hASCs/GCP-2 cells. In vitro cell migration and proliferation assays showed that hASCs/GCP-2-derived conditioned media (CM) significantly accelerated the migration and proliferation of fibroblast cells. Examination of in vitro endothelial differentiation showed that hASCs/GCP-2 cells spontaneously formed vascular-like structures and highly expressed endothelial-specific genes and proteins. In vivo study results of our mouse myocardial infarction (MI) model revealed that hASCs/GCP-2 implantation improved the cardiac function and reduced the infarct size. Finally, transplanted hASCs/GCP-2 cells unexpectedly differentiated into endothelial cells and the engraftment rate was significantly higher than control groups. CONCLUSION: We suggest that overexpression of GCP-2 in stem cells has the potential to enhance their angiogenic and survival properties.

Amniotic mesenchymal stem cells enhance wound healing in diabetic NOD/SCID mice through high angiogenic and engraftment capabilities.

Although human amniotic mesenchymal stem cells (AMMs) have been recognised as a promising stem cell resource, their therapeutic potential for wound healing has not been widely investigated. In this study, we evaluated the therapeutic potential of AMMs using a diabetic mouse wound model. Quantitative real-time PCR and ELISA results revealed that the angiogenic factors, IGF-1, EGF and IL-8 were markedly upregulated in AMMs when compared with adipose-derived mesenchymal stem cells (ADMs) and dermal fibroblasts. In vitro scratch wound assays also showed that AMM-derived conditioned media (CM) significantly accelerated wound closure. Diabetic mice were generated using streptozotocin and wounds were created by skin excision, followed by AMM transplantation. AMM transplantation significantly promoted wound healing and increased re-epithelialization and cellularity. Notably, transplanted AMMs exhibited high engraftment rates and expressed keratinocyte-specific proteins and cytokeratin in the wound area, indicating a direct contribution to cutaneous closure. Taken together, these data suggest that AMMs possess considerable therapeutic potential for chronic wounds through the secretion of angiogenic factors and enhanced engraftment/differentiation capabilities.

Impact of platelet function test on platelet responsiveness and clinical outcome after coronary stent implantation: platelet responsiveness and clinical outcome.

BACKGROUND AND OBJECTIVES: The aim of this study was to confirm the predictive cut-off values for P2Y12 reaction units (PRU) and aspirin reaction units (ARU) and to evaluate the clinical impact of VerifyNow® assays. SUBJECTS AND METHODS: From November 2007 to October 2009, 186 eligible patients were prospectively recruited. Post-treatment platelet reactivity was measured by VerifyNow® assays within 12 to 24 hours after intervention, followed by standard dual maintenance dose therapy for 1 year. All patients had scheduled clinical follow-ups at 1, 3, 6, and 12 months. RESULTS: The rate of low responders to clopidogrel, aspirin, and both drugs were 41.4%, 10.2%, and 3.8%, respectively. The predictive factors for low responsiveness to clopidogrel (PRU ≥240) were female sex, age, and non-use of cilostazol medication in our univariate analysis and age ≥65 years and non-use cilostazol in the multivariate analysis. The predictors of low responsiveness to aspirin (ARU ≥550) were male sex and age in both univariate and multivariate analyses. There was no significant difference in the clinical event rate with a cut-off value of PRU ≥240 or ARU ≥550 for 30 days and 1-year (p>0.05). CONCLUSION: Hyporesponsiveness to antiplatelet agents (namely aspirin and clopidogrel) was identified in about half of the patients. The cut-off point of PRU ≥240 or ARU ≥550 did not confer predictive value for 30-day or 1-year clinical event rates in patients who had undergone coronary intervention with drug-eluting stents.

Amniotic mesenchymal stem cells have robust angiogenic properties and are effective in treating hindlimb ischaemia.

AIMS: In this study, we aimed to evaluate whether human amniotic mesenchymal stem cells (AMMs) have angio-vasculogenic properties and to determine their therapeutic effects on experimental ischaemia. Although AMMs are a promising source of stem cells, their angio-vasculogenic properties are not fully understood. METHODS AND RESULTS: We have characterized AMMs by quantitative real-time polymerase chain reaction, Matrigel tube formation assays, and various in vitro endothelial differentiation assays. AMMs expressed significantly higher levels of representative proangiogenic genes, vascular endothelial growth factor-A, angiopoietin-1, hepatocyte growth factor, and fibroblast growth factor-2 (FGF-2) than adipose-derived mesenchymal stem cells. In addition, the anti-apoptotic factor Akt-1 was highly expressed in the AMMs. Cells were directly transplanted into the ischaemic hindlimbs of mice to evaluate their angio-vasculogenic and therapeutic effects. They spontaneously differentiate into vascular-like structures and exhibit endothelial-specific genes and proteins. In an in vivo study on hindlimb ischaemia, implantation of AMMS augmented blood perfusion and capillary density, indicating AMM-augmented neovascularization. The engraftment rate of AMMs was high, and the transplanted AMMs showed vasulogenic potential. CONCLUSION: AMMs are not only markedly angiogenic but also vasculogenic, thus ameliorating hindlimb ischaemia. Our data suggest that AMMs have considerable therapeutic effects on ischaemic hindlimb through high angiogenic and engraftment abilities.

Combined growth factors enhanced angiogenic potential of cord blood-derived mononuclear cells transplanted to ischemic limbs.

Stem cell therapy has recently been limited by poor engraftment and the marginal influence of the administered cells. This study aimed to enhance the survival and angiogenic capacity of human umbilical cord blood (UCB)-derived mononuclear cells (MNCs) and to demonstrate their therapeutic effects on experimental ischemia. A specific culture medium containing five growth factors (Flt-3L, EGF, TPO, FGF and IGF-1) augmented cell proliferation, adhesion potential as well as stimulated MNCs to become progenitor-like cells. In addition, qRT-PCR demonstrated that MNCs cultured with these five growth factors (5f-MNCs) markedly up-regulated multiple angiogenic, arteriogenic and anti-apoptotic factors compared with uncultured MNCs. In an ischemic hindlimb model, the injection of 5f-MNCs prevented limb loss and augmented blood perfusion, capillary density, vascular maturation and angiogenic cytokines in the affected tissues. In addition, the 5f-MNCs exhibited an increased engraftment rate and an endothelial phenotype and stimulated angiogenic factors in ischemic hindlimbs as demonstrated by flow cytometric, immunohistochemical and qRT-PCR analyses. Taken together, these data suggest that 5f-MNCs could be used as a novel therapy for the treatment of ischemic cardiovascular disease due to their increased level of engraftment and angiogenic potential.