Infusion Of Endothelial Progenitor Cells Research Articles

Infusion of Cultured Endothelial Progenitor Cells Boosts Hematopoietic Recovery and Increases the Expression of LepR+ Cells after Irradiation

The hemopoietic system's function of producing blood cells and immune cells during thorough life relies on the Maintainance and differentiation of hematopoietic stem cells (HSCs). HSCs activities were tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow. However, during the routine treatment of hematopoietic diseases, such as chemotherapy and radiotherapy, the niche was inevitably impaired which is one of the essential causes of hematopoietic suppression. Our previous research results showed that infusion of cultured endothelial progenitor cells (EPCs) could boost hematopoietic recovery after HSCT. And the latent mechanisms could be that EPCs can differentiate into endothelial cells (ECs) in vivo. However, the roles of EPCs play in the bone marrow niche and the specific mechanism remain unknown. Here we designed further to investigate the effect and mechanism of EPCs in boosting bone marrow microenvironment recovery after total body irradiation (TBI). This may offer a new cue for therapy of hematopoietic suppression after the treatment of malignant hematological diseases. Bone marrow-derived Primary EPCs isolation and culture were described as before. Briefly, Bone marrow cells from Balb/c or C57BL/6J mice aged 6-8 weeks were cultured with EGM-2 medium which was changed in 36 h, the adherent cells formed colonies identified as elongated sprouting cells radiating from a central core of round cells and identified as VEGFR2+; CD133+; CD144+; SCA1+ by FACS on days 4-6. To assess the effects of the EPCs in boosting hematopoiesis, Balb/c mice underwent TBI with a lethal dose and were injected withEPCs or the same volume of PBS via tail vein in 4-6 hours. The survival of the EPC group was elevated ( survival: 8/9 vs 0/13; p＜0.001). C57BL/6J mice underwent TBI with a sublethal dose and were injected with the cultured EPCs isolated from C57BL/6-Tg(CAG-EGFP) mice or the same volume of PBS via tail vein in 4-6 hours. Results showed that EPCs boost the recovery of hematopoietic cells in the bone marrow and peripheral blood cells; Immunofluence (IF) staining showed that Infused EPCs with GFP fluorescence were home to bone marrow and differentiated into ECs which were marked with endomucin (EMCN); Moreover, assessments of the alteration of bone marrow niche using H&E staining revealed that infusion of EPCs reduced the accumulation of fat and using multi-analyte flow assay showed that mice infused with EPCs express a higher level of M-CSF and TGF-β in plasma while the level of SCF and IL-6 both in plasma and bone marrow supernatant were lower compared to TBI. Furthermore, to assess the change of sinusoidal niche, we used IF staining and found that the sinusoids were dilated and perisinusoidal stromal cells identified as LepR+ were massively lost on the 7th day after TBI while the dilated sinusoidal lumens were narrowed (11.05±1.789 vs 20.86±4.573，p=0.0258) and LepR+ cells were upregulated on the 21st day after the infusion of EPCs which indicated that the sinusoidal niche has a better recovery compared to TBI. In Conclusion, EPCs could differentiate into endothelial cells in vivo boosting the recovery of the hematopoietic system. And the potential mechanism is that EPCs repair the bone marrow sinusoidal niche damaged by irradiation.

Endothelial progenitor cells and coronary artery disease: Current concepts and future research directions.

Vascular injury is a frequent pathology in coronary artery disease. To repair the vasculature, scientists have found that endothelial progenitor cells (EPCs) have excellent properties associated with angiogenesis. Over time, research on EPCs has made encouraging progress regardless of pathology or clinical technology. This review focuses on the origins and cell markers of EPCs, and the connection between EPCs and coronary artery disease. In addition, we summarized various studies of EPC-capturing stents and EPC infusion therapy, and aim to learn from past technology to predict the future.

Combination of Mesenchymal Stem Cell and Endothelial Progenitor Cell Infusion Accelerates Injured Intestinal Repair by Regulating Gut Microbiota after Hematopoietic Cell Transplantation

Mesenchymal stem cells (MSC) have been widely applied for repairing intestinal barrier function and restoring immune homeostasis for pretransplantation conditioning, yet the repair process is often impaired or delayed owing to a lack of vascularity. How combined therapy with MSC and endothelial progenitor cells (EPC) for the intestinal microenvironment and repair remain unclear. In this study, BALB/c mice received syngeneic bone marrow transplantation with or without MSC or EPC infusion. The findings show that the MSC+EPC mice had greater blood capillary distribution and higher expression of tight junction protein (occludin) in the small intestinal tract. Meanwhile, the MSC+EPC cotreatment increased IL-17A levels and decreased IFN-γ levels at the early stage after transplantation. Furthermore, the MSC+EPC treatment motivated p38 mitogen-activated protein kinase (MAPK) and enhanced heat shock protein 27 (HSP27) activation, which subsequently promoted intestinal epithelial cell proliferation and down-regulated apoptosis-related molecule caspase 3 expression. Finally, the high-throughput sequencing of gut microbiota (16S) showed that the MSC+EPC treatment can inhibit the Enterococcus population (<0.5%) and stabilize the Akkermansia population (~15%), with the Akkermansia population showing significant positive correlations with p38 MAPK/phos-p38, HSP27/phos-HSP27, IL-17A, and occludin. Taken together, our results show that MSC+EPC combined therapy is beneficial for the repair of injured intestine and drives gut microbial community stability by regulating the intestinal microenvironment.

Proventive and Therapentic Effects of Endothelial Progenitor Cells on Monocrotaline-Induced Hepatic Vein Occlusion Disease

To investigate the preventive and therapeutic effects of endothelial progenitor cells on monocrotaline-induced hepatic vein occlusion disease in mice. C57BL/6 mice were randomly divided into 3 groups: saline group (n=15), monocrotaline group (n=15), and endothelial progenitor cell infusion group (n=15). Liver function (TBIL, ALT, AST), liver index, and serum levels of TNF-α and IL-6 were measured on the 8th day after intragastric administration. Hepatic sinusoidal endothelial cells, hepatic central venous endothelial cells and hepatocytes were observed by both HE and immunohistochemical staining. Hepatic fibrosis was observed by Masson's trichrome staining. By the light microscopy, the liver of the monocrotaline group showed moderate to the severe injuries of hepatic sinusoidal and central venous endothelial cells, and hepatic venous congestion. Masson staining showed moderate to severe hepatic fibrosis of central vein and hepatic sinus. In the endothelial progenitor cell group, hepatic sinusoidal and central venous endothelial cell injuries, and the fibrosis of central hepatic vein and hepatic sinus were mild to moderate. Hepatic venous congestion was reduced in comparison with that in the mice of the monocrotaline group. Compared with the endothelial progenitor cell group, the liver index was higher, the liver function was more abnormal, and the serum expression levels of TNF-α and IL-6 were higher in the monocrotaline group. The monocrotaline-induced damage of hepatic sinusoidal and central venous endothelial cells is an linitiating factor for hepatic vein occlusive disease. Infusion of endothelial progenitor cells can play a role in preventing and treating hepatic vein occlusion.

Proventive and Therapentic Effects of Endothelial Progenitor Cells on Monocrotaline-Induced Hepatic Vein Occlusion Disease

To investigate the preventive and therapeutic effects of endothelial progenitor cells on monocrotaline-induced hepatic vein occlusion disease in mice. C57BL/6 mice were randomly divided into 3 groups: saline group (n=15), monocrotaline group (n=15), and endothelial progenitor cell infusion group (n=15). Liver function (TBIL, ALT, AST), liver index, and serum levels of TNF-α and IL-6 were measured on the 8th day after intragastric administration. Hepatic sinusoidal endothelial cells, hepatic central venous endothelial cells and hepatocytes were observed by both HE and immunohistochemical staining. Hepatic fibrosis was observed by Masson's trichrome staining. By the light microscopy, the liver of the monocrotaline group showed moderate to the severe injuries of hepatic sinusoidal and central venous endothelial cells, and hepatic venous congestion. Masson staining showed moderate to severe hepatic fibrosis of central vein and hepatic sinus. In the endothelial progenitor cell group, hepatic sinusoidal and central venous endothelial cell injuries, and the fibrosis of central hepatic vein and hepatic sinus were mild to moderate. Hepatic venous congestion was reduced in comparison with that in the mice of the monocrotaline group. Compared with the endothelial progenitor cell group, the liver index was higher, the liver function was more abnormal, and the serum expression levels of TNF-α and IL-6 were higher in the monocrotaline group. The monocrotaline-induced damage of hepatic sinusoidal and central venous endothelial cells is an linitiating factor for hepatic vein occlusive disease. Infusion of endothelial progenitor cells can play a role in preventing and treating hepatic vein occlusion.

Infusion of Mesenchymal Stem Cells Combined with Epithelial Progenitor Cells Promotes Injured Intestinal Repairing after Hematopoietic Cell Transplantation

Pre-transplant chemoradiotherapy can impair intestinal barrier function and disrupt immune homeostasis, and thus increase the incidence of infection and other related complications. Mesenchymal stem cells (MSCs) have the potential to rescue Inflammatory Bowel Disease and intestinal graft versus host disease owing to their immunosuppressive capabilities. However, limited studies regarding MSC administration has been reported to aggravate T cell-mediated tissue injury. Plus, endothelial progenitor cells (EPCs) could improve endothelium repair, facilitate hematopoietic reconstitution and alleviate complications associated with hematopoietic cell transplantation (HCT). The key goals of this study were to i) identify the role of MSC derived soluble and contact dependent factors and how these affect intestinal injury; ii) investigate the effect of MSC combined EPC therapy for repairing the injured intestine. In this study, BALB/c mice were randomly divided into five groups, namely, total body irradiation only, bone marrow transplantation (BMT), MSC(BMT with 1 × 106MSC infusion), EPC(BMT with 5 × 105 EPCs infusion), and MSC+EPC (BMT with 1 × 106 MSC and 5 × 105 EPCs infusion). Results showed that the best performance of intestine was found in the MSC+EPC treated group, which showed more epithelial and goblet cells, and less apoptosis cells and adjacent crypts fusion in intestine morphology. EPC or MSC only group improved the intestinal injuries slightly compared with BMT group. The higher MECA-32 expression level was found in the intestinal tissue of MSC+EPC and MSC groups compared with other treated groups. The tight junction molecules occludin had highest expression level in the intestinal epithelial cells of MSC+EPC group, followed by MSC group and then EPC groups, but the expression level in BMT group was extremely low. Further study demonstrated that in MSC+EPC group the phosphorylated P38 enhanced heat shock protein HSP27 activation, which promoted cytoskeleton reconstruction and intestinal epithelial cells proliferation; and phosphorylated P38 also down-regulated the expression of apoptosis-related molecule caspase3. Moreover, the multiple effects of MSCs on cellular immunity may reflect their diverse influences on the different T-cell subpopulations. MSC+EPC infusion increased the number of IL-17A secreting cells (Th17 and Tc17) in mesenteric lymph nodes early after HCT, and decreased Th1 cells at day 10 and delayed expanding of Tc1 from day 10 to day 15. The soluble IL-17A in intestinal tissue was significantly increased after MSC+EPC infusion in according with IL-17A secreting cells in mesenteric lymph nodes. Furthermore, the gut bacterial information analyzed by high throughput sequencing uncovered that MSC and MSC+EPC groups had higher bacterial diversity and richness compared with other groups, and the bacterial community structures exhibited big changes regarding different treatment. Co-occurrence analysis demonstrated that the genus Akkermansia inducing PD-1 expression of T cells had significant correlation with phosphorylation of P38 and HSP27 in MSC+EPC and EPC treated groups, which indicated Akkermansia may be a key bacteria participanting the intestinal repairing in MSC and MSC+EPC groups. In conclusion, this study confirmed that the MSC+EPC infusion can obviously repair injured intestine, and gut bacteria Akkermansia correlated with phosphorylation of P38 and HSP27 expression and participated in intestinal repairing. Disclosures No relevant conflicts of interest to declare.

Clinical Assessment of Intravenous Endothelial Progenitor Cell Transplantation in Dogs

Endothelial progenitor cells (EPCs) have been applied for cell therapy because of their roles in angiogenesis and neovascularization in ischemic tissue. However, adverse responses caused by EPC therapy have not been fully investigated. In this study, a human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated using Ficoll-Hypaque. There were four experimental groups: 10 ml saline infusion group (injection rate; 3 ml/min), 10 ml saline bolus group (injection rate; 60 ml/min), 10 ml EPCs infusion group (2 x 105 cells/ml, injection rate; 3 ml/min), 10 ml EPCs bolus group (2 × 105 cells/ml, injection rate; 60 ml/min). Clinical assessment included physical examination and laboratory examination for intravenous human EPC transplantation in dogs. The results revealed no remarkable findings in vital signs among the dogs used. In blood analysis, platelet counts in saline infusion groups were significantly higher than in the EPC groups within normal ranges, and no significant differences were observed except K+, Cl– and blood urea nitrogen/urea. In ELISA assay, no significant difference was observed in serum tumor necrosis factor alpha. The serum concentration of vascular endothelial growth factor was significantly higher in EPC groups than in saline groups, and interleukin 10 was significantly up-regulated in the EPC infusion group compared with other groups. In conclusion, we demonstrated that no clinical abnormalities were detected after intravenous transplantation of human EPCs in dogs. The transplanted xenogenic EPCs might be involved in anti-inflammatory and angiogenic functions in dogs.

Endothelial progenitor cells improve functional recovery in focal cerebral ischemia of rat by promoting angiogenesis via VEGF

To investigate the role of venous infusion of endothelial progenitor cells (EPCs) in the reendothelialization of acute focal cerebral ischemia model in rats. And explore the mechanism of VEGF to promote angiogenesis of functional recovery in focal cerebral ischemia of rat. A model of middle cerebral artery occlusion (MCAo) was used to mimic ischemia following EPCs extraction from the same donor rats. EPCs were characterized by CD34, CD45 and CD133 expressions, and confirmed by uptake of fluorescently labeled Dil-ac-LDL and FITC-UEA-1 and flow cytometry analysis. EPCs were expanded in vitro and injected into the jugular vein of the same donor animals daily for 5 days after ischemia surgery. EPC-treated animals received approximately 1 × 106 cells, while control animals received PBS. Animals were evaluated the functional recovery, endothelial cell proliferation, vascular distribution, and VEGF levels. The EPC-treated group showed lower infarct volume and a significant recovery of neurological function. We also observed increased vascular distribution through bromodeoxyuridine (BrdU) staining and high plasma VEGF levels in the EPC-treated group compared to control groups. Our results provided direct evidence that auto-graft EPCs can improve neurological outcome and revascularization after ischemic stroke and indicated an important role of VEGF in this process. Our study suggested that EPCs may have potential therapeutic applications for the ischemic cerebrovascular disease.

Effects of allogeneic hematopoietic stem cell transplantation in combination with infusion of endothelial progenitor cells on bone marrow inflammatory injury

目的探索异基因造血干细胞移植联合内皮祖细胞（EPC）输注对骨髓炎性损伤的影响。方法6~8周龄BALB/c（H-2Kd）小鼠经致死剂量全身照射（TBI）预处理后建立异基因骨髓移植模型（BMT组），并在BMT基础上同时输注体外培养的EPC，建立骨髓联合EPC移植模型（EPC组）。移植后第7、14、21、28天分别获取各组小鼠骨髓细胞，流式细胞术检测EPC培养比率及两组嵌合率变化，通过骨髓细胞和外周血细胞计数比较两组骨髓造血情况，Western blot法检测骨髓巨噬细胞表面特异性标志CD68表达水平；实时定量PCR及Western blot法分析骨髓炎性复合体nlrp1、nlrp6、caspase-1 mRNA及蛋白表达水平。结果移植后第7天BMT组嵌合率为（83.69±1.26）%，明显低于EPC组的（91.65±2.77）%（P<0.05）。在移植后第10、15、20天，EPC组小鼠外周血中的WBC、网织红细胞、PLT均高于BMT组（P值均<0.05），在第7、21天EPC组骨髓中原始红细胞较BMT组高（P值均<0.05）。HE染色显示，移植后第7、14、21天，EPC组骨髓微环境恢复较BMT组快。Western blot检测显示EPC组骨髓CD68蛋白表达水平较BMT组低。EPC组nlrp1、nlrp6、casepase-1 mRNA及蛋白表达水平在移植后各时间点接近正常表达水平，与BMT组相比，差异均有统计学意义（P值均<0.05）。结论异基因造血干细胞移植联合输注EPC可减少炎性细胞的浸润及减少炎性复合体的活化，促进骨髓造血重建。

Endothelial progenitor cells improve the quality of transplanted hematopoietic stem cells and maintain longer term effects in mice.

Engraftment of hematopoietic stem cells (HSCs) is a pre-requisite for the success of hematopoietic stem cell transplantation (HSCT). Fetal blood cell (FBC)-derived endothelial progenitor cells (EPCs) are known to facilitate HSC reconstitution in the early phase. However, longer term effects on HSCs remain unclear. The purpose of this study was to evaluate the effect of EPCs on the quality of transplanted hematopoietic stem cells in mouse HSCT model. BALB/c mice were randomly divided into four groups, namely, control, total body irradiation only, HSCT, and HSCT + EPCs (with infusion of 5 × 105 EPCs). Mice was sacrificed on days 7, 14, 21, and 28 post-HSCT for the analysis of the bone marrow pathology by H&E staining, measurement of c-kit+sca-1+, c-kit+, apoptosis, and necrosis by flow cytometry as well as colony formation assay. Secondary transplantation involved the injection of transplanted BALB/c-derived HSCs into new TBI-treated BALB/c mice. Compared with HSCT, EPCs infusion promoted the differentiation and reduced apoptosis of transplanted HSCs, possibly through promotion of vascular repair of the bone marrow microenvironment via differentiation into the bone marrow endothelial cells. Significantly, EPCs' effect on HSCs was maintained for a long period as demonstrated using a secondary transplantation approach. These data revealed EPCs improved the quality and quantity of transplanted HSCs and maintained their effects over the longer term, suggesting a novel approach to improve HSCT efficiency and outcomes.

Endothelial progenitor cells inhibit platelet function in a P-selectin-dependent manner.

BackgroundThe role of endothelial progenitor cells (EPCs) in vascular repair is related to their recruitment at the sites of injury and their interaction with different components of the circulatory system. We have previously shown that EPCs bind and inhibit platelet function and impair thrombus formation via prostacyclin secretion, but the role of EPC binding to platelet P-selectin in this process has not been fully characterized. In the present study, we assessed the impact of EPCs on thrombus formation and we addressed the implication of P-selectin in this process.MethodsEPCs were generated from human peripheral blood mononuclear cells cultured on fibronectin in conditioned media. The impact of EPCs on platelet aggregation and thrombus formation was investigated in P-selectin deficient (P-sel−/−) mice and their wild-type (WT) counterparts.ResultsEPCs significantly and dose-dependently impaired collagen-induced whole blood platelet aggregation in WT mice, whereas no effects were observed in P-sel−/− mice. Moreover, in a ferric chloride-induced arterial thrombosis model, infusion of EPCs significantly reduced thrombus formation in WT, but not in P-sel−/− mice. Furthermore, the relative mass of thrombi generated in EPC-treated P-sel−/− mice were significantly larger than those in EPC-treated WT mice, and the number of EPCs recruited within the thrombi and along the arterial wall was reduced in P-sel−/− mice as compared to WT mice.ConclusionThis study shows that EPCs impair platelet aggregation and reduce thrombus formation via a cellular mechanism involving binding to platelet P-selectin. These findings add new insights into the role of EPC-platelet interactions in the regulation of thrombotic events during vascular repair.

Infusion of endothelial progenitor cells ameliorates liver injury in mice after haematopoietic stem cell transplantation.

Injury to liver sinusoidal endothelial cells (LSECs) is thought to be the initial factor for Hepatic veno-occlusive disease, a severe complication after haematopoietic stem cell transplantation (HSCT). Endothelial progenitor cells (EPCs) have the capacity to differentiate into endothelial cells and play a critical role in vasculogenesis, tissue regeneration and repair. Whether EPCs infusion ameliorates LSECs injury remains unclear. The aim of this study was to evaluate the effects of EPCs on liver injury in mice after HSCT. Mice received HSCT without or with EPCs infusion (HSCT + EPCs). Untreated mice were used as control. Liver and whole blood were collected post HSCT and used for the analysis of pathology of liver sinusoidal endothelial cells (LSECs) and hepatocytes, liver ultrastructure, function, level of IL-6, TNF-α and platelet activation. Severe LSECs injury, hepatocyte damage, abnormal liver function was observed in HSCT group. In addition, increased P-selectin expression and secretion of IL-6, TNF-α was also found. However, all the above changes were alleviated in HSCT + EPCs at all the time points and normalized at the endpoint. Meanwhile, EPCs-induced repair of LSECs and hepatocytes was totally inhibited by the addition of anti-VE-cadherin antibody. EPCs infusion ameliorated the damage to LSECs and hepatocytes as well as reduced secretion of IL-6, TNF-α and inhibited platelet activation after HSCT, leading to improved liver function, suggesting EPCs might be a new therapeutic strategy in the prophylaxis of liver injury after HSCT.

Transplantation of cryopreserved human umbilical cord blood-derived endothelial progenitor cells induces recovery of carotid artery injury in nude rats

IntroductionTransplantation of endothelial progenitor cells (EPCs) restores endothelial function in patients with endothelial dysfunction and initial denudation. The goal of the present study was to determine the effect of cryopreserved human umbilical cord blood (UCB)-derived EPC infusion on the repair of carotid artery injury in nude rats.MethodsMononuclear cells (MNCs) from human cryopreserved UCB and peripheral blood (PB) of patients with cardiovascular diseases and healthy volunteers were cultured in a conditioned medium. The in vitro migration, proliferation, adhesion, and survival capacities, as well as paracrine cytokine release of EPCs were investigated. EPC homing, induced reendothelialization, and the effect on neointima formation were also assessed in vivo.ResultsPatient-derived PB EPCs (PPB-EPCs) displayed decreased migration, proliferation, adhesion, and survival capabilities as compared to PB-EPCs from healthy volunteers (HPB-EPCs) and cryopreserved UCB-EPCs. However, there was no difference in the release of vascular endothelial growth factor (VEGF) and stromal cell derived factor 1 (SDF-1) between the three groups. Two weeks after transplantation, more labeled UCB-EPCs and HPB-EPCs than PPB-EPCs were found by cell tracking in the injury zone. Administration of PPB-EPCs, HPB-EPCs, and UCB-EPCs enhanced reendothelialization and inhibited neointima formation compared to the saline control. However, UCB-EPC and HPB-EPC infusion showed a greater improvement than PPB-EPCs.ConclusionsCryopreserved UCB-MNCs derived EPCs and HPB-EPCs show better responses to cytokines and vascular injury than PPB-EPCs. Thus, cryopreservation and delivery of cryopreserved autogenous UCB-EPCs or HPB-EPCs may be a promising vasculoprotective approach for patients with multiple cardiovascular risk factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0022-4) contains supplementary material, which is available to authorized users.

EPCs in vascular repair: How can we clear the hurdles between bench and bedside?

Endothelial progenitor cells (EPCs) play a fundamental role in the maintenance and repair of damaged vascular endothelium, as well as in new blood vessel formation. Based on this function of EPCs, it has been hypothesized that transfusion of these cells could be an approach to treat vascular disease. While this concept has subsequently been proven in animal models clinical trials have not been encouraging. These discrepancies have limited translation of EPCs from bench to bedside. In this review, by analyzing the reported data from the animal models and clinical trials, we describe the main factors limiting the clinical effects of EPCs infusion and the unfavorable in vivo reactions of the receipts. To facilitate future clinical application of EPCs, a series of strategy to overcome the obstacles have been suggested.

Kidney and heart: TGFβ1-mediated cross-talk

Rzeźnik et al. [1] in their recent paper published in ‘Kardiologia Polska’ found that revascularisation of unilateral or bilateral renal artery stenosis (RAS) leads to improvement in several echocardiographic parameters reflecting heart structure and function, as well as impacts on plasma transforming growth factor β1 (TGFβ1) and B-type natriuretic peptide. At least some of the effects on the heart (such as LVMI reduction) did not depend on the degree of blood pressure lowering. As can be concluded from the publication, patients did not suffer from symptomatic heart failure (HF; at least in NYHA class III–IV); however, it has been demonstrated recently that RAS revascularisation may also be associated with improvement in NYHA class and reduction in the risk of hospitalisation for HF in patients with concomitant RAS and HF [2]. The message delivered by this paper is very important for both nephrologists and cardiologists. TGFβ1 remains one of the key profibrotic growth factors in renal pathology. It is produced by damaged mesangial cells in the glomerulus (and is crucial for the development of glomerular sclerosis), injured tubular cells and resident interstitial fibroblasts (resulting in interstitial fibrosis). HF in the course of atherosclerotic RAS may develop according to least three different scenarios: (1) Both HF and RAS are consequences of the same, devastating, pathology, namely advanced atherosclerosis; (2) RAS contributes to heart remodelling and dysfunction due to significantly elevated and difficult-to-control hypertension; (3) Humoral mediators released from injured (ischaemic) renal parenchyma may remotely damage the heart (and this may be the case with TGFβ1). The role of TGFβ1 in the development of several heart diseases resulting in myocardial fibrosis has been well recognised for some years [3]. Experimental data suggest that RAS triggers the synthesis and release of several inflammatory and vasoconstrictive agents from the kidney, in addition to the activation of the renin–angiotensin–aldosterone (RAA) axis. These include, among others, endothelin, interleukin-6, TNFα and TGFβ1. It has been demonstrated that disruption of the Smad3 protein gene (signalling pathway essential for TGFβ1 effects) prevents fibrosis of the kidney supplied by stenotic artery even if stenosis persists [4]. Infusion of endothelial progenitor cells into the artery with stenosis significantly decreases plasma levels of several proinflammatory cytokines, prevents myocardial damage and remodelling, even in stenosis remains uncorrected and there is no change in systemic blood pressure [5]. TGFβ1 is one of the key effector molecules involved in epithelial-to-mesenchymal transition, the process of phenotypic change of epithelial tubular cells into fibroblast-like cells — now considered one of the most important mechanisms that lead to kidney fibrosis. A similar process has also been described for cardiac endothelium and was called ‘endothelial-to-mesenchymal transition’ (Endo-MT) [6]. These and other findings should lead to caution when interpreting apparently ‘negative’ trials published recently on RAS which did not demonstrate any substantial be nefit associated with renal artery revascularisation ± stenting vs. medical therapy — effects on blood pressure and/or kidney function were exactly the same in both types of intervention. Now it is tempting to speculate that successful RAS revascularisation might be a much more definite strategy compared to everyday use of RAA axis blockade in lowering TGFβ1 and thus in limiting the impact of this growth factor on the heart and the kidneys. It seems however plausible to expect some lag time between intervention and expected cardiac benefits, not necessarily apparent in the short time following intervention. The findings of Rzeźnik et al. [1] may hold some promise for more delayed, but possible, benefit following RAS revascularisation for the heart, and possibly for the kidneys.

Analysis of cd45- [cd34+/kdr+] Endothelial Progenitor Cells as Juvenile Protective Factors in a Rat Model of Ischemic-Hemorrhagic Stroke

BackgroundIdentification of juvenile protective factors (JPFs) which are altered with age and contribute to adult-onset diseases could identify novel pathways for reversing the effects of age, an accepted non-modifiable risk factor to adult-onset diseases. Since endothelial progenitor cells (EPCs) have been observed to be altered in stroke, hypertension and hypercholesterolemia, said EPCs are candidate JPFs for adult-onset stroke. A priori, if EPC aging plays a ‘master-switch JPF-role’ in stroke pathogenesis, juvenile EPC therapy alone should delay stroke-onset. Using a hypertensive, transgenic-hyperlipidemic rat model of spontaneous ischemic-hemorrhagic stroke, spTg25, we tested the hypothesis that freshly isolated juvenile EPCs are JPFs that can attenuate stroke progression and delay stroke onset.Methodology/Principal FindingsFACS analysis revealed that cd45- [cd34+/kdr+] EPCs decrease with progression to stroke in spTg25 rats, exhibit differential expression of the dual endodthelin-1/VEGFsp receptor (DEspR) and undergo differential DEspR-subtype specific changes in number and in vitro angiogenic tube-incorporation. In vivo EPC infusion of male, juvenile non-expanded cd45-[cd34+/kdr+] EPCs into female stroke-prone rats prior to stroke attenuated progression and delayed stroke onset (P<0.003). Detection of Y-chromosome DNA in brain microvessels of EPC-treated female spTg25 rats indicates integration of male EPCs into female rat brain microvessels. Gradient-echo MRI showed delay of ischemic-hemorrhagic lesions in EPC-treated rats. Real-time RT-PCR pathway-specific array-analysis revealed age-associated gene expression changes in cd45-[cd34+/kdr]EPC subtypes, which were accelerated in stroke-prone rats. Pro-angiogenic genes implicated in intimal hyperplasia were increased in stroke-prone rat EPCs (P<0.0001), suggesting a maladaptive endothelial repair system which acts like a double-edged sword repairing while predisposing to age-associated intimal hyperplasia.Conclusions/SignificanceAltogether, the data demonstrate that cd45-[cd34/kdr+]EPCs are juvenile protective factors for ischemic hemorrhagic stroke as modeled in the spTg25-rat model. The ability to delay stroke onset emphasizes the importance of EPC-mediated roles in vascular health for ischemic-hemorrhagic stroke, a high unmet need.

Transfusion of CXCR4-Primed Endothelial Progenitor Cells Reduces Cerebral Ischemic Damage and Promotes Repair in db/db Diabetic Mice

This study investigated the role of stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) axis in brain and endothelial progenitor cells (EPCs), and explored the efficacy of CXCR4 primed EPCs in treating ischemic stroke in diabetes. The db/db diabetic and db/+ mice were used in this study. Levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were measured. Brain SDF-1α and CXCR4 expression were quantified at basal and after middle cerebral artery occlusion (MCAO). In in vitro study, EPCs were transfected with adenovirus carrying null (Ad-null) or CXCR4 (Ad-CXCR4) followed with high glucose (HG) treatment for 4 days. For pathway block experiments, cells were pre-incubated with PI3K inhibitor or nitric oxide synthase (NOS) inhibitor for two hours. The CXCR4 expression, function and apoptosis of EPCs were determined. The p-Akt/Akt and p-eNOS/eNOS expression in EPCs were also measured. In in vivo study, EPCs transfected with Ad-null or Ad-CXCR4 were infused into mice via tail vein. On day 2 and 7, the cerebral blood flow, neurologic deficit score, infarct volume, cerebral microvascular density, angiogenesis and neurogenesis were determined. We found: 1) The levels of plasma SDF-1α and circulating CD34+CXCR4+ cells were decreased in db/db mice; 2) The basal level of SDF-1α and MCAO-induced up-regulation of SDF-1α/CXCR4 axis were reduced in the brain of db/db mice; 3) Ad-CXCR4 transfection increased CXCR4 expression in EPCs and enhanced EPC colonic forming capacity; 4) Ad-CXCR4 transfection prevented EPCs from HG-induced dysfunction (migration and tube formation) and apoptosis via activation of PI3K/Akt/eNOS signal pathway; 4) Ad-CXCR4 transfection enhanced the efficacy of EPC infusion in attenuating infarct volume and promoting angiogenesis and neurogenesis. Our data suggest that Ad-CXCR4 primed EPCs have better therapeutic effects for ischemia stroke in diabetes than unmodified EPCs do.

Infusion of Endothelial Progenitor Cells Accelerates Hematopoietic and Immune Reconstitution, and Ameliorates the Graft-Versus-Host Disease After Hematopoietic Stem Cell Transplantation

Hematopoietic stem cells transplantation (HSCT) causes endothelial cell damage, disrupting hematopoietic microenviroment and leading to various complications. We hypothesized that infusion of endothelial progenitor cells (EPCs) may improve endothelium repair, facilitate hematopoietic reconstitution, and alleviate complications associated with HSCT. C57Bl6, and BALB/c mice received total body irradiation followed by infusion of C57Bl6-derived bone marrow (BM) cells, with or without concomitant infusion of C57Bl6-derived EPCs. The time course of hematopoietic and immune reconstitution and the severity of the graft-versus-host disease (GVHD) were monitored. Further, to confirm that EPCs promote endothelial cell recovery, HSCT mice were treated with anti-VE-cadherin antibody targeting the endothelium. The EPCs-treated mice exhibited accelerated recovery of BM vasculature, cellularity, hematopoietic stem and progenitor cell recovery, improved counts of lymphocyte subsets in peripheral blood, and facilitated spleen structure reconstruction. EPCs infusion also ameliorated the GVHD in the C57Bl6 → BALB/c allo-HSCT model. Systemic administration of anti-VE-cadherin antibody significantly delayed hematological and immune reconstitution in the EPCs-infused mice. In conclusion, our data demonstrate that infusion of EPCs augments the hematopoietic and immune reconstitution, and alleviates the GVHD. These findings further highlight the relationship between the microvascular recovery, hematopoietic and immune reconstitution, and the GVHD.

Abstract 3962: Hematopoietic Endothelial Progenitor Cells Enhance Motor Function And Motor Map Integrity Following Cerebral Ischemia.

Stroke is the leading cause of disability in the Unites States resulting in upper extremity motor impairments. The present study examined the efficacy of systemically administered hematopoietic endothelial progenitor cells (EPCs) for ameliorating motor impairments and cortical dysfunction following cerebral ischemia. Methods: Baseline motor performance of Forty one male adult rats was established on a single pellet reaching task. Animals were assigned to one of five experimental conditions that counterbalanced baseline motor performance. Animals in the Stroke condition (n=7) received infusion of endothelin-1 onto the middle cerebral artery contralateral to the preferred paw. Animals in the PRE (n=7) and POST (n=7) conditions received the same infusion of endothelin-1 one but also received ten million EPCs injected into the tail vein either thirty minutes before (PRE) or immediately after (POST) infusion. The EPC’s were enriched from the bone marrow of a separate group of donor rats using nanoparticles tagged with LIN negative and CD90 markers. SHAM (n=9) animals only received EPC infusion and CONTROLS received no stroke or EPC infusion. One, three and five weeks following stroke all animals were tested on the skilled reaching task after which intracortical microstimulation was used to derive maps of forelimb movement representations within the motor cortex contralateral to the preferred paw. All assessments were performed in a blinded manner. Results: showed POST animals performed significantly better on single pellet reaching than animals in all other stroke conditions on weeks 3 and 5. Control animals scored a reaching accuracy of 41±8.1% and 46±11% at 3 and 5 weeks, respectively. Stroke decreased this task to 12±2.1% and 18±2.8%, respectively (p&lt;0.05, compared to controls at both time points). Injection of EPC’s following reperfusion led to an increase in reaching accuracy to 27±2.6% and 32±2.4%, at 3 and 5 weeks, respectively (p&lt;0.05, compared to stroked non-EPC cohort at 5 weeks). Further, POST animals had significantly larger forelimb motor maps than animals in all other stroke conditions. Infarct size did not significantly differ between the stroke conditions. The results show that post stroke treatment of EPCs does not reduce infarct size but does enhance the functional integrity of residual cortical tissue that in turn supports enhanced behavioral outcome.

Adriamycin Nephropathy: A Failure of Endothelial Progenitor Cell-Induced Repair

Adriamycin-associated nephropathy (AAN) remains poorly understood. We hypothesized that adriamycin affects endothelial progenitor cells (EPCs), leading to impaired regeneration. We analyzed renal hematopoietic stem cells (HSCs) and EPCs in mice with AAN and examined the potential contribution of adoptive transfer of intact EPCs to the repair processes. FACS analyses revealed that populations of HSCs and EPCs were scarcely represented in control kidneys and did not change numerically in kidneys obtained from mice with AAN. The observed defect in engraftment was attributable to the decreased viability and increased senescence of EPCs. Adoptive transfer of intact EPCs improved proteinuria and renal function, with a threefold decrease in mortality. Infusion of EPCs to adriamycin-treated mice reduced plasma levels of interleukin-1alpha and -beta and granulocyte-colony stimulating factor as well as increased the level of vascular endothelial growth factor with concomitant improvement of vascular density and reduction of apoptosis. An additional mechanism of tissue repair is proposed based on tunneling nanotube formation between EPCs and endothelial cells exposed to adriamycin, leading to the multiple rounds of exchange between EPCs and mature cells. In conclusion, AAN is associated with development of EPC incompetence; adoptive transfer of intact EPCs blunts morphological and functional manifestations of AAN; and the proposed mechanisms of repair by EPCs include direct incorporation into blood vessels, paracrine signaling, and tunneling nanotube renewal of mitochondrial pool in endothelial cells.