Monotropein promotes angiogenesis and inhibits oxidative stress-induced autophagy in endothelial progenitor cells to accelerate wound healing.

Attenuating oxidative stress-induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress-induced mitochondrial dysfunction and stimulate bone marrow-derived EPC (BM-EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM-EPCs and prevented tert-butyl hydroperoxide (TBHP)-induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM-EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury-related wounds.

Advances of Stem Cell Therapeutics in Cutaneous Wound Healing and Regeneration.

Cutaneous wound healing is a complex multiple phase process, which overlaps each other, where several growth factors, cytokines, chemokines, and various cells interact in a well-orchestrated manner. However, an imbalance in any of these phases and factors may lead to disruption in harmony of normal wound healing process, resulting in transformation towards chronic nonhealing wounds and abnormal scar formation. Although various therapeutic interventions are available to treat chronic wounds, current wound-care has met with limited success. Progenitor stem cells possess potential therapeutic ability to overcome limitations of the present treatments as it offers accelerated wound repair with tissue regeneration. A substantial number of stem cell therapies for cutaneous wounds are currently under development as a result of encouraging preliminary findings in both preclinical and clinical studies. However, the mechanisms by which these stem cells contribute to the healing process have yet to be elucidated. In this review, we emphasize on the major treatment modalities currently available for the treatment of the wound, role of various interstitial stem cells and exogenous adult stem cells in cutaneous wound healing, and possible mechanisms involved in the healing process.

Endothelial progenitor cell­derived extracellular vesicle­meditated cell­to­cell communication regulates the proliferation and osteoblastic differentiation of bone mesenchymal stromal cells.

Bone tissue engineering is a promising treatment strategy to increase bone regeneration. Endothelial progenitor cells (EPCs) and bone marrow stromal cells (BMSCs) are commonly used to promote vessel formation and osteoblastic differentiation in tissue engineering. Previous studies have demonstrated that EPCs regulate both proliferation and differentiation of BMSCs. However, the underlying mechanism remains unclear. Understanding this mechanism is critical to developing more effective treatments. The role of extracellular vesicles in cell­to­cell communication has attracted substantial attention. These small vesicles deliver proteins, DNA, and RNA and consequently regulate the commitment, function, and differentiation of target cells. In the present study, EPC­derived extracellular vesicles (EPC­EVs were isolated using gradient ultracentrifugation and ultrafiltration and the influence of EPC­EVs on BMSC osteoblastic differentiation and proliferation was examined in vitro. The results indicated that EPC­EVs regulate the osteoblastic differentiation of BMSCs by inhibiting the expression of osteogenic genes and increasing proliferation in vitro. It is suggested that the results regarding the role of EPC­EVs will provide a novel way to explain the crosstalk between EPCs and BMSCs.

Evaluation of velvet antler total protein effect on bone marrow­derived endothelial progenitor cells.

Lu Rong, velvet antler (VA), is a traditional Chinese medicine, which is used as a food supplement and therapeutic drug in China, Japan, Russia, New Zealand and Southeast Asia. The regenerative characteristics of VA have resulted in great research interest, particularly regarding the fields of organ grafting and stem cell differentiation. Various VA proteomic studies verified that proteins act as the primary bioactive components of VA. The present study aimed to investigate if VA proteins (VA­pro) influence endothelial progenitor cell (EPC) viability. Various methods have previously been used to investigate VA­pro, including freeze­drying technology, ultrasonic wave methods, high performance liquid chromatography­mass spectrometry, EPCs extraction and culture. Results demonstrated that VA­pro promoted EPCs proliferation and migration, particularly at a concentration of 1 mg/ml. Furthermore, VA­pro increased the activation level of Notch1 intracellular domain and Hes1, and the level of phosphorylated­Akt and phosphorylated­mechanistic target of rapamycin. VA­pro may therefore affect EPC viability via regulation of the Notch and Akt signaling pathways. The present study revealed the effects and potential molecular mechanism of VA­pro on EPCs, and suggested an association between VA regeneration characteristics and the optimization of EPC viability. These findings may contribute to EPC transplantation research and aid in providing a novel treatment method for vascular diseases in the future.

Effect of vitamin D on endothelial progenitor cells function.

BACKGROUND: Endothelial progenitor cells (EPCs) are a population of bone marrow-derived cells, which have an important role in the process of endothelialization and vascular repair following injury. Impairment of EPCs, which occurs in patients with diabetes, was shown to be related to endothelial dysfunction, coronary artery disease (CAD) and adverse clinical outcomes. Recent evidence has shown that calcitriol, the active hormone of vitamin D, has a favorable impact on the endothelium and cardiovascular system. There is limited data on the effect of vitamin D on EPCs function. AIM: To examine the in vitro effects of Calcitriol on EPCs from healthy subjects and patients with diabetes. METHODS: Fifty-one patients with type 2 diabetes (60±11 years, 40% women, HbA1C: 9.1±0.8%) and 23 healthy volunteers were recruited. EPCs were isolated and cultured with and without calcitriol. The capacity of the cells to form colony-forming units (CFUs), their viability (measured by MTT assay), KLF-10 levels and angiogenic markers were evaluated after 1 week of culture. RESULTS: In diabetic patients, EPC CFUs and cell viability were higher in EPCs exposed to calcitriol vs. EPCs not exposed to calcitriol [EPC CFUs: 1.25 (IQR 1.0-2.0) vs. 0.5 (IQR 0.5-1.9), p < 0.001; MTT:0.62 (IQR 0.44-0.93) vs. 0.52 (IQR 0.31-0.62), p = 0.001]. KLF-10 levels tended to be higher in EPCs exposed to vitamin D, with no differences in angiopoietic markers. In healthy subjects, calcitriol supplementation also resulted in higher cell viability [MTT: 0.23 (IQR 0.11-0.46) vs. 0.19 (0.09-0.39), p = 0.04], but without differences in CFU count or angiopoietic markers. CONCLUSION: In patients with diabetes mellitus, in vitro vitamin D supplementation improved EPCs capacity to form colonies and viability. Further studies regarding the mechanisms by which vitamin D exerts its effect are required.

Human Stem Cell-Derived Endothelial-Hepatic Platform for Efficacy Testing of Vascular-Protective Metabolites from Nutraceuticals.

Atherosclerosis underlies many cardiovascular and cerebrovascular diseases. Nutraceuticals are emerging as a therapeutic moiety for restoring vascular health. Unlike small-molecule drugs, the complexity of ingredients in nutraceuticals often confounds evaluation of their efficacy in preclinical evaluation. It is recognized that the liver is a vital organ in processing complex compounds into bioactive metabolites. In this work, we developed a coculture system of human pluripotent stem cell-derived endothelial cells (hPSC-ECs) and human pluripotent stem cell-derived hepatocytes (hPSC-HEPs) for predicting vascular-protective effects of nutraceuticals. To validate our model, two compounds (quercetin and genistein), known to have anti-inflammatory effects on vasculatures, were selected. We found that both quercetin and genistein were ineffective at suppressing inflammatory activation by interleukin-1ß owing to limited metabolic activity of hPSC-ECs. Conversely, hPSC-HEPs demonstrated metabolic capacity to break down both nutraceuticals into primary and secondary metabolites. When hPSC-HEPs were cocultured with hPSC-ECs to permit paracrine interactions, the continuous turnover of metabolites mitigated interleukin-1ß stimulation on hPSC-ECs. We observed significant reductions in inflammatory gene expressions, nuclear translocation of nuclear factor κB, and interleukin-8 production. Thus, integration of hPSC-HEPs could accurately reproduce systemic effects involved in drug metabolism in vivo to unravel beneficial constituents in nutraceuticals. This physiologically relevant endothelial-hepatic platform would be a great resource in predicting the efficacy of complex nutraceuticals and mechanistic interrogation of vascular-targeting candidate compounds. Stem Cells Translational Medicine 2017;6:851-863.

The proangiogenic potential of a novel calcium releasing composite biomaterial: Orthotopic in vivo evaluation.

Insufficient angiogenesis remains a major hurdle in current bone tissue engineering strategies. An extensive body of work has focused on the use of angiogenic factors or endothelial progenitor cells. However, these approaches are inherently complex, in terms of regulatory and methodologic implementation, and present a high cost. We have recently demonstrate the potential of electrospun poly(lactic acid) (PLA) fiber-based membranes, containing calcium phosphate (CaP) ormoglass particles, to elicit angiogenesis in vivo, in a subcutaneous model in mice. Here we have devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a (Hydroxypropyl)methyl cellulose (HPMC) matrix, with the capacity to release calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. The bone regeneration kinetics was dependent on the Ca2+ release rate, with the faster Ca2+ release composite gel showing improved bone repair at 3weeks, in relation to control. In the same line, improved angiogenesis could be observed for the same gel formulation at 6weeks post implantation. This methodology allows to integrate two fundamental processes for bone tissue regeneration while using a simple, cost effective, and safe approach. STATEMENT OF SIGNIFICANCE: In current bone tissue engineering approaches the achievement of sufficient angiogenesis, during tissue regeneration, is a major limitation in order to attain full tissue functionality. Recently, we have shown that calcium ions, released by the degradation of calcium phosphate ormoglasses (CaP), are effective angiogenic promoters, in both in vitro and in a subcutaneous implantation model. Here, we devised an injectable composite, containing CaP glass-ceramic particles, dispersed within a HPMC matrix, enabling the release of calcium in a more sustained fashion. We show that by tuning the release of calcium in vivo, in a rat bone defect model, we could improve both bone formation and increase angiogenesis. This simple and cost effective approach holds great promise to translate to the clinics.

Effect of High-Dose Atorvastatin Reload on the Release of Endothelial Progenitor Cells in Patients on Long-Term Statin Treatment Who Underwent Percutaneous Coronary Intervention (from the ARMYDA-EPC Study).

Endothelial progenitor cells (EPCs) may concur to endogenous vascular repair. Previous studies have reported that statin treatment increases EPC levels. We investigated whether this occurs in patients on long-term statin treatment who underwent percutaneous coronary interventions (PCIs). In a phase A study, 53 patients (atorvastatin reload [AR] 80 mg 12 hours before + 40 mg 2 hours before PCI, n = 27; placebo [P], n = 26) were evaluated for EPC mobilization as CD45dim/CD34+/CD133+/KDR+ cell number by flow cytometry. Assays were run at randomization (12 hours before PCI, R), immediately before PCI (T0) at 8 (T8) and 24 hours (T24). In phase B study, 50 patients (AR, n = 25; P, n = 25) were evaluated for early colony formation by Hill colony forming unit (CFU) assay, with sampling at randomization and 24 hours later. In phase A, EPCs levels were similar at randomization between 2 arms (0.23% [0.14 to 0.54] of total events in AR vs 0.22% [0.04 to 0.37] in P group; p = 0.33). At PCI, EPC levels were higher in AR arm (0.42% [0.06 to 0.30] vs 0.19% [0.06 to 030]; p = 0.009). Higher EPC levels in AR group were also found at 8 and 24 hours. In phase B, EPC CFUs/well numbers at randomization were similar in the 2 arms (8 [6 to 12] in AR vs 12 [6 to 20] in P group, p = 0.109). EPC CFU/well at 24 hours became significantly higher in AR arm (17 [10 to 23] vs 5 [2 to 13], p = 0.002). In conclusion, high-dose AR before PCI in patients on long-term statin therapy promptly increases EPCs mobilization, which are capable of early colony formation and may contribute to cardioprotection.

[Effect of Naoxintong capsule on endothelial progenitor cell mobilization and homing following bone marrow transplantation in a mouse hind limb ischemia model].

Endothelial progenitor cells (EPCs) are precursor cells of endothelial cells. Signal molecules produced by ischemia and hypoxia can promote mobilization of bone marrow EPCs to peripheral circulation and formation of novel blood vessels in tissues that are damaged during heart attack. Naoxintong capsule (NXT) has the functions of promoting blood circulation, removing blood stasis, promoting the circulation of qi and relieving pain. The various components in NXT have protective effects on blood vessels and can effectively improve the symptoms of ischemia. However, its effect on EPCs is not clear. To study the intervention effect of NXT on mobilization and homing of peripheral blood EPCs, green fluorescent protein (GFP) transgenic mice were used for bone marrow transplantation (BMT) and then unilateral hind limb ischemia model (UHLI) were constructed. For BMT, wild-type ICR mice were irradiated by CS137 and then injected with 4×106 bone marrow cells isolated from GFP mice. The bone marrow reconstitution of recipients was assessed by quantification of GFP bone marrow-derived cells (BMDC) from transplanted mice 4 weeks after BMT. The UHLI model was duplicated by ligating femoral artery and divided into three groups: the model group, the NXT group (model+NXT) and the positive control group (model+simvastatin). Flow cytometry was used to detect the proportion of GFP positive cells and the peripheral blood EPCs levels at 1, 3, 7, 14 days before and after surgery. Ischemic tissue of gastrocnemius muscle was excised at 3 and 7 days after operation for immunofluorescence staining to detect the number of GFP+ cells. The bone marrow chimerism was achieved at day 28 after BMT. There was no significant difference in the percentage of GFP positive cells between BMT mice and GFP transgenic mice. NXT and simvastatin could significantly increase the number of peripheral blood EPCs 1,3 days after surgery. Three and seven days after operation, the number of homing EPCs was significantly higher in NXT group and positive control group than that in model group (P<0.001). In conclusion, NXT can obviously promote the mobilization and homing of EPCs.

[Promotion of Function of Endothelial Progenitor Cells with Shexiang Baoxin Pill Treatment under Shear Stress].

The aim of this study was to investigate whether shear stress could promote function of endothelial progenitor cells (EPCs) with Shexiang Baoxin Pill (SBP) treatment in vitro, and to study whether shear stress contributed to vascular injury repair by EPCs. EPCs were isolated and characterized; EPCs' proliferation, migration, adhesion, tube formation and eNOS protein level in vitro were investigated by culturing confluent EPCs in 4 mg/mL SBP under physiological shear stress (15 dyne/cm2) for up to 24 hours. Afterwards, EPCs were transfused into rats after wire-induced carotid artery injury augmented re-endothelialization. The results showed that, compared to the SBP group, the shear stress+SBP group obviously enhanced EPCs proliferation, migration, adhesion, tube formation and eNOS protein expression in vitro (P<0.01). After one week, immunofluorescence staining showed that endothelial regeneration rate obviously enhanced in shear stress+SBP group (P<0.01). The present study demonstrates that shear stress can promote function of endothelial progenitor cells treated with SBP, which improves the vascular injury repair potentials of EPCs.

Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells following Finnish Sauna: A Pilot Study.

BACKGROUND: Sauna bathing is claimed to provide benefits for patients suffering from cardiovascular diseases. The current study aims at analyzing the induction of potential regenerative processes by quantifying the mobilization of bone marrow-derived stem cells into the peripheral blood of healthy adults following Finnish sauna. MATERIALS AND METHODS: Twenty healthy unbiased male volunteers (20-30 years old) were exposed to a Finnish sauna bath (3 × 10 min, 90°C). Venous blood samples were drawn before (baseline), immediately, and 6 h as well as 24 h after the sauna bath. Blood analysis included isolation of mononuclear cells, cell staining with mononuclear antibodies, and fluorescence-activated cell sorting (FACS). For baseline and 24 h post-sauna samples colony-forming unit-Hill assays were applied to quantify endothelial progenitor cells (EPC). RESULTS: Flow cytometry revealed an upregulation of circulating CD45+/CD309+ progenitor cells immediately after the sauna bath, however without reaching statistical significance. Circulating cell numbers of the CD45+CD34+, CD45+CD34+CD133+, and CD45+CD34+CD117+ populations did not show clear enhancements following sauna. EPC colony formation tended to be enhanced after sauna as compared to baseline values. CONCLUSION: Peripheral EPC numbers exhibited a moderate increase following Finnish sauna in a cohort of healthy young men. Furthermore, sauna bathing tended to increase EPC colony-forming capacity. These rather weak responses to thermotherapy might indicate a ceiling effect. In individuals exhibiting cardiovascular risk factors the effects may be more pronounced.

Tripterine treatment improves endothelial progenitor cell function via integrin-linked kinase.

BACKGROUND/AIMS: Atherosclerosis is associated with dysfunction of endothelial progenitor cells (EPCs). Tripterine, a chemical compound derived from the Chinese medicinal plant Tripterygium wilfordii Hook, displays anti-inflammatory properties in several animal models. We hypothesized that tripterine can improve EPC function and thus the efficiency of EPC transplantation. METHODS AND RESULTS: Tripterine preconditioning (2.5 µM, 4 h) improved EPC proliferation, tube formation, migration, and adhesion, and reduced apoptosis in cells cultured in ox-LDL (200 µg/ml). Tripterine restored integrin-linked kinase (ILK) levels downregulated by ox-LDL in EPCs, suggesting the involvement of the ILK/Akt pathway. Small interfering RNA-mediated depletion of ILK and dominant-negative ILK transduction inhibited the phosphorylation of the ILK downstream signaling targets protein kinase B/Akt and glycogen synthase kinase 3-beta (GSK-3ß), and reduced ß-catenin and cyclin D1 expression. In atherosclerotic mice injected with green fluorescent protein-labeled EPCs to evaluate EPC function, tripterine decreased aortic lesions and plaque deposition, and injection of tripterine-treated EPCs restored ILK levels. CONCLUSION: The present results suggest that tripterine improves vascular function in atherosclerosis by enhancing EPC function through a mechanism involving the ILK signaling pathway.

Protective effects of tanshinone â ¡A on endothelial progenitor cells injured by tumor necrosis factor-α.

Tanshinone ⅡA (Tan ⅡA) is a Traditional Chinese Medicine commonly used in Asian and Western countries for the prevention and treatment of cardiovascular disorders, such as atherosclerosis. Endothelial dysfunction and associated inflammatory processes have a critical role in the development of atherosclerosis. Endothelial progenitor cells (EPCs) have been demonstrated to be involved in certain aspects of the endothelial repair process. The present study aimed to investigate the putative protective effects of Tan ⅡA on EPCs injured by tumor necrosis factor­α (TNF­α). The potential effects of Tan ⅡA on TNF-α-stimulated EPC proliferation, migration, adhesion, in vitro tube formation ability and paracrine activity were investigated in the current study. The results indicated that TNF­α impaired EPC proliferation, migration, adhesion capacity and vasculogenesis ability in vitro as well as promoted EPC secretion of inflammatory cytokines, including monocyte chemoattractant protein­1 (MCP­1), interleukin­6 (IL­6) and soluble CD40 ligand (sCD40L). However, Tan ⅡA was able to reverse these effects. In conclusion, these findings demonstrated that Tan ⅡA may have the potential to protect EPCs against damage induced by TNF­α. Therefore, these results may provide evidence for the pharmacological basis of Tan ⅡA and its potential use in the prevention and treatment of early atherosclerosis associated with EPC and endothelial damage.

Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells.

Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with diabetes and cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. The aim of this study was to investigate whether Icariin, a natural bioactive component known from traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress-induced cell dysfunction in bone marrow-derived EPCs (BM-EPCs), and, if so, through what mechanisms. We observed that treatment of BM-EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2 O2 )-induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species levels and restored mitochondrial membrane potential. Icariin downregulated endothelial nitric oxide synthase 3, as well as nicotinamide-adenine dinucleotide phosphate-oxidase expression upon H2 O2 induction. These antiapoptotic and antiautophagic effects of Icariin are possibly mediated by restoring the loss of mammalian target of rapamycin /p70S6K/4EBP1 phosphorylation as well as attenuation of ATF2 and ERK1/2 protein levels after H2 O2 treatment. In summary, favorable modulation of the angiogenesis and redox states in BM-EPCs make Icariin a promising proangiogenic agent both enhancing vasculogenesis and protecting against endothelial dysfunction.

Roles of Chinese medicine bioactive ingredients in the regulation of cellular function of endothelial progenitor cells.

To improve the function of endothelial progenitor cells (EPCs) is one of the goals in Chinese traditional therapy to treat various cardio-celebrovascular diseases. In the past decades, scholars in the field of traditional Chinese medicine (TCM) have found fifteen active compounds to regulate the function of EPC. These metabolites are extracted from thirteen, plant-based Chinese medicine, with majority of them as potent reductive or oxidative hydrophilic molecules containing phenyl groups. These active compounds either enhance the mobilization of EPC, or inhibit their apoptosis through different signaling pathways. In this review, the molecular structure, biophysical properties, and the plant sources of these active ingredients and their regulatory effects on the function of EPC are summarized, aiming to reveal the modern basis of Chinese medicine for promoting blood circulation and removing blood stasis at the progenitor cell level.

[Effect of tongguan capsule on the number of endothelial progenitor cells in the peripheral blood of patients with coronary artery disease after PCI].

OBJECTIVE: To observe the effect of Tongguan Capsule (TC) on the number of endothelial progenitor cells (EPCs) in the peripheral blood of patients with coronary artery disease (CAD) after percutaneous coronary intervention (PCI). METHODS: Recruited were 60 CAD patients undergoing PCI who were admitted and treated at ICU and the Heart Center of Guangdong Provincial Hospital of Traditional Chinese Medicine from March to October 2010. They were assigned to the treatment group (treated by TC) and the control group (treated by placebos) according to the random digit table, 30 cases in each group. They took TC or placebos from the day of PCI, three pills each time, three times a day, for three consecutive months. The numbers of peripheral blood CD34 and vascular endothelial growth factor receptor-2 (VEGFR2) positive cells were detected before PCI and 3 months after PCI respectively. The echocardiography was performed before PCI and 3 months after PCI respectively to determinate the left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV), stroke volume (SV), cardiac output (CO), and left ventricular ejection fraction (LVEF). The wall motion score index (WMSI) was assessed in the two groups. RESULTS: There was no statistical difference in the number of EPCs, LVEF,WMSI, or SV in the two groups before PCI (P > 0.05). The number of EPCs increased in both the two groups after 1 month of PCI (P < 0.05). It was obviously higher in the treatment group than in the control group (P < 0.05). The LVEF both increased in the two groups 3 months after PCI (P < 0.05, P < 0.01). The WMSI decreased and SV increased in the treatment group (P < 0.05). The improvement of LVEF and WMSI was better in the treatment group than in the control group (P < 0.05). CONCLUSION: TC could up-regulate the number of EPCs in the peripheral blood of CAD patients after PCI, and improve their cardiac functions.