Human Endothelial Progenitor Cells Protect the Kidney against Ischemia-Reperfusion Injury via the NLRP3 Inflammasome in Mice.

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and progression to chronic kidney disease (CKD). However, no effective therapeutic intervention has been established for ischemic AKI. Endothelial progenitor cells (EPCs) have major roles in the maintenance of vascular integrity and the repair of endothelial damage; they also serve as therapeutic agents in various kidney diseases. Thus, we examined whether EPCs have a renoprotective effect in an IRI mouse model. Mice were assigned to sham, EPC, IRI-only, and EPC-treated IRI groups. EPCs originating from human peripheral blood were cultured. The EPCs were administered 5 min before reperfusion, and all mice were killed 72 h after IRI. Blood urea nitrogen, serum creatinine, and tissue injury were significantly increased in IRI mice; EPCs significantly improved the manifestations of IRI. Apoptotic cell death and oxidative stress were significantly reduced in EPC-treated IRI mice. Administration of EPCs decreased the expression levels of NLRP3, cleaved caspase-1, p-NF-κB, and p-p38. Furthermore, the expression levels of F4/80, ICAM-1, RORγt, and IL-17RA were significantly reduced in EPC-treated IRI mice. Finally, the levels of EMT-associated factors (TGF-ß, α-SMA, Snail, and Twist) were significantly reduced in EPC-treated IRI mice. This study shows that inflammasome-mediated inflammation accompanied by immune modulation and fibrosis is a potential target of EPCs as a treatment for IRI-induced AKI and the prevention of progression to CKD.

The heterogeneity of cancer endothelium: The relevance of angiogenesis and endothelial progenitor cells in cancer microenvironment.

Tumor-associated vessels constitution is the result of angiogenesis, the hallmark of cancer essential for tumor to develop in dimension and to spread throughout the organism. Tumor endothelium is configured as an active functioning organ capable of determine interaction with the immune response and all the other components of the variegate cancer microenvironment, determining reciprocal influence. Angiogenesis is here analyzed in its molecular and cellular mechanisms, multiple mediators and principal players, represented by Endothelial Cells. It is discussed the striking heterogeneity of cancer endothelium, due to morphological and molecular aberrations that it often presents and its multiple origin. Among the cells that participate to the composition of tumor vasculature, Endothelial Progenitor Cells represent an important source for physical sustain and paracrine signaling in the process of angiogenesis. Treatment options are reviewed, with particular focus on novel therapeutic strategies for overcoming tumor resistance to anti-angiogenic agents.

TNFα priming through its interaction with TNFR2 enhances endothelial progenitor cell immunosuppressive effect: new hope for their widespread clinical application.

BACKGROUND: Bone marrow derived endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) involved in neo-angiogenesis and endothelial homeostasis and are considered as a circulating reservoir for endothelial repair. Many studies showed that EPCs from patients with cardiovascular pathologies are impaired and insufficient; hence, allogenic sources of EPCs from adult or cord blood are considered as good choices for cell therapy applications. However, allogenic condition increases the chance of immune rejection, especially by T cells, before exerting the desired regenerative functions. TNFα is one of the main mediators of EPC activation that recognizes two distinct receptors, TNFR1 and TNFR2. We have recently reported that human EPCs are immunosuppressive and this effect was TNFα-TNFR2 dependent. Here, we aimed to investigate if an adequate TNFα pre-conditioning could increase TNFR2 expression and prime EPCs towards more immunoregulatory functions. METHODS: EPCs were pre-treated with several doses of TNFα to find the proper dose to up-regulate TNFR2 while keeping the TNFR1 expression stable. Then, co-cultures of human EPCs and human T cells were performed to assess whether TNFα priming would increase EPC immunosuppressive and immunomodulatory effect. RESULTS: Treating EPCs with 1 ng/ml TNFα significantly up-regulated TNFR2 expression without unrestrained increase of TNFR1 and other endothelial injury markers. Moreover, TNFα priming through its interaction with TNFR2 remarkably enhanced EPC immunosuppressive and anti-inflammatory effects. Conversely, blocking TNFR2 using anti-TNFR2 mAb followed by 1 ng/ml of TNFα treatment led to the TNFα-TNFR1 interaction and polarized EPCs towards pro-inflammatory and immunogenic functions. CONCLUSIONS: We report for the first time the crucial impact of inflammation notably the TNFα-TNFR signaling pathway on EPC immunological function. Our work unveils the pro-inflammatory role of the TNFα-TNFR1 axis and, inversely the anti-inflammatory implication of the TNFα-TNFR2 axis in EPC immunoregulatory functions. Priming EPCs with 1 ng/ml of TNFα prior to their administration could boost them toward a more immunosuppressive phenotype. This could potentially lead to EPCs' longer presence in vivo after their allogenic administration resulting in their better contribution to angiogenesis and vascular regeneration. Video Abstract.

Increased circulating endothelial progenitor cells (EPCs) in prepubertal children born prematurely: a possible link between prematurity and cardiovascular risk.

BACKGROUND: Endothelial progenitor cells (EPCs) ensure vascular integrity and neovascularization. No studies have investigated EPCs in preterm-born children beyond infancy. METHODS: One hundred and thirty-six prepubertal children were enrolled: 63 preterm and 73 born at term (controls). Circulating CD34(+)/VEGFR-2(+)/CD45(-) and CD34(+)/VEGFR-2(+)/CD45dim EPCs were measured in preterm-born children compared to controls. Body mass index (BMI), waist-to-hip ratio (WHR), neck circumference, systolic and diastolic blood pressure (SBP and DBP, respectively), fasting glucose, insulin, lipid profile, common carotid and abdominal aortic intima-media thickness (cIMT and aIMT, respectively), endothelium-dependent brachial artery flow-mediated dilation (FMD), and echocardiographic parameters were also assessed. RESULTS: Circulating CD34(+)/VEGFR-2(+)/CD45(-) and CD34(+)/VEGFR-2(+)/CD45dim EPCs were significantly higher in preterm-born children compared to controls (p < 0.001 and p < 0.001, respectively). In total study population and in the preterm-born group, EPCs were significantly lower in children born to mothers with gestational diabetes compared to non-diabetic mothers. Prematurity was associated with higher WHR, neck circumference, SBP, DBP, cIMT, aIMT, mean pressure, and velocity of pulmonary artery; the peak velocity of the brachial artery was significantly lower in children born prematurely. In multiple regression analysis, preterm birth and maternal gestational diabetes were recognized as independent predictors of EPCs. CONCLUSIONS: Circulating EPCs were increased in prepubertal preterm-born children in comparison with peers born full-term. Maternal gestational diabetes was associated with a decrease in EPCs. IMPACT: Mounting evidence supports the adverse effect of prematurity on cardiovascular health. However, the underlying mechanisms that could lead to endothelial dysfunction in preterm-born individuals are not fully understood. Endothelial progenitor cells (EPCs) ensure vascular integrity, normal endothelial function and neovascularization. No studies have investigated the EPCs counts in peripheral blood beyond infancy in children born prematurely. Circulating EPCs were significantly higher in preterm-born prepubertal children compared to controls, thus indicating that prematurity is possibly associated with endothelial damage. In total study population and in the preterm-born group, maternal gestational diabetes was associated with decreased EPCs concentrations.

Endothelial Progenitor Cells and Rheumatoid Arthritis: Response to Endothelial Dysfunction and Clinical Evidences.

Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disease characterized by the swelling of multiple joints, pain and stiffness, and accelerated atherosclerosis. Sustained immune response and chronic inflammation, which characterize RA, may induce endothelial activation, damage and dysfunction. An equilibrium between endothelial damage and repair, together with the preservation of endothelial integrity, is of crucial importance for the homeostasis of endothelium. Endothelial Progenitor Cells (EPCs) represent a heterogenous cell population, characterized by the ability to differentiate into mature endothelial cells (ECs), which contribute to vascular homeostasis, neovascularization and endothelial repair. A modification of the number and function of EPCs has been described in numerous chronic inflammatory and auto-immune conditions; however, reports that focus on the number and functions of EPCs in RA are characterized by conflicting results, and discrepancies exist among different studies. In the present review, the authors describe EPCs' role and response to RA-related endothelial modification, with the aim of illustrating current evidence regarding the level of EPCs and their function in this disease, to summarize EPCs' role as a biomarker in cardiovascular comorbidities related to RA, and finally, to discuss the modulation of EPCs secondary to RA therapy.

Murine sca1/flk1-positive cells are not endothelial progenitor cells, but B2 lymphocytes.

Circulating sca1+/flk1+ cells are hypothesized to be endothelial progenitor cells (EPCs) in mice that contribute to atheroprotection by replacing dysfunctional endothelial cells. Decreased numbers of circulating sca1+/flk1+ cells correlate with increased atherosclerotic lesions and impaired reendothelialization upon electric injury of the common carotid artery. However, legitimate doubts remain about the identity of the putative EPCs and their contribution to endothelial restoration. Hence, our study aimed to establish a phenotype for sca1+/flk1+ cells to gain a better understanding of their role in atherosclerotic disease. In wild-type mice, sca1+/flk1+ cells were mobilized into the peripheral circulation by granulocyte-colony stimulating factor (G-CSF) treatment and this movement correlated with improved endothelial regeneration upon carotid artery injury. Multicolor flow cytometry analysis revealed that sca1+/flk1+ cells predominantly co-expressed surface markers of conventional B cells (B2 cells). In RAG2-deficient mice and upon B2 cell depletion, sca1+/flk1+ cells were fully depleted. In the absence of monocytes, sca1+/flk1+ cell levels were unchanged. A PCR array focused on cell surface markers and next-generation sequencing (NGS) of purified sca1+/flk1+ cells confirmed their phenotype to be predominantly that of B cells. Finally, the depletion of B2 cells, including sca1+/flk1+ cells, in G-CSF-treated wild-type mice partly abolished the endothelial regenerating effect of G-CSF, indicating an atheroprotective role for sca1+/flk1+ B2 cells. In summary, we characterized sca1+/flk1+ cells as a subset of predominantly B2 cells, which are apparently involved in endothelial regeneration.

Systemic lupus erythematosus, endothelial progenitor cells and intracellular Ca2+ signaling: A novel approach for an old disease.

Systemic lupus erythematosus (SLE) is an autoimmune multisystem disease featured by an increased cardiovascular risk that may lead to premature patient's death. It has been demonstrated that SLE patients suffer from early onset endothelial dysfunction which is due to the impairment of endogenous vascular repair mechanisms. Vascular integrity and homeostasis are maintained by endothelial progenitor cells (EPCs), which are mobilized in response to endothelial injury to replace damaged endothelial cells. Two main EPCs subpopulations exist in peripheral blood: endothelial colony forming cells (ECFCs), which represent truly endothelial precursors and can physically engraft within neovessels, and myeloid angiogenic cells (MACs), which sustain angiogenesis in a paracrine manner. Emerging evidence indicates that ECFCs/MACs are down-regulated and display compromised angiogenic activity in SLE, thereby contributing to the pathogenesis of this disease. Intracellular calcium (Ca2+) signaling plays a crucial role in maintaining vascular integrity by stimulating migration, proliferation and tube formation in both ECFCs and MACs. Herein, we illustrate the evidences that support the role played by EPCs dysfunction in SLE. Subsequently, we discuss about the hypothesis that the Ca2+ handling machinery is compromised in SLE-derived ECFCs and MACs, thereby resulting in their reduced pro-angiogenic activity. Finally, we speculate about the proposal to exploit intracellular Ca2+ signaling to improve ECFCs' reparative phenotype and suggest this strategy as a new approach to treat SLE patients.

The TNF/TNFR2 signaling pathway is a key regulatory factor in endothelial progenitor cell immunosuppressive effect.

BACKGROUND: Endothelial progenitor cells (EPCs) are non-differentiated endothelial cells (ECs) present in blood circulation that are involved in neo-vascularization and correction of damaged endothelial sites. Since EPCs from patients with vascular disorders are impaired and inefficient, allogenic sources from adult or cord blood are considered as good alternatives. However, due to the reaction of immune system against allogenic cells which usually lead to their elimination, we focused on the exact role of EPCs on immune cells, particularly, T cells which are the most important cells applied in immune rejection. TNFα is one of the main activators of EPCs that recognizes two distinct receptors. TNFR1 is expressed ubiquitously and its interaction with TNFα leads to differentiation and apoptosis, whereas, TNFR2 is expressed predominantly on ECs, immune cells and neural cells and is involved in cell survival and proliferation. Interestingly, it has been shown that different immunosuppressive cells express TNFR2 and this is directly related to their immunosuppressive efficiency. However, little is known about immunological profile and function of TNFR2 in EPCs. METHODS: Using different in-vitro combinations, we performed co-cultures of ECs and T cells to investigate the immunological effect of EPCs on T cells. We interrupted in the TNFα/TNFR2 axis either by blocking the receptor using TNFR2 antagonist or blocking the ligand using T cells derived from TNFα KO mice. RESULTS: We demonstrated that EPCs are able to suppress T cell proliferation and modulate them towards less pro-inflammatory and active phenotypes. Moreover, we showed that TNFα/TNFR2 immune-checkpoint pathway is critical in EPC immunomodulatory effect. CONCLUSIONS: Our results reveal for the first time a mechanism that EPCs use to suppress immune cells, therefore, enabling them to form new immunosuppressive vessels. Furthermore, we have shown the importance of TNFα/TNFR2 axis in EPCs as an immune checkpoint pathway. We believe that targeting TNFR2 is especially crucial in cancer immune therapy since it controls two crucial aspects of tumor microenvironment: 1) Immunosuppression and 2) Angiogenesis. Video Abstract. (MP4 46355 kb).

Role of fibrocytes and endothelial progenitor cells among low-differentiated CD34+ cells in the progression of lung sarcoidosis.

BACKGROUND: Sarcoidosis is a multisystemic granulomatous disease with still unknown etiology. Our previous studies showed a significantly higher percentage of CD34 + cells in the peripheral blood in patients with sarcoidosis (SA) compared to the control group. The objective of the present study was to characterized of the CD34 + cell population in peripheral blood in patients with SA with reference to the control group. Moreover in patients with SA, fibrocytes and endothelial cells were analysed and their relationship to the fibrosis process based on assessment of diffusing capacity for carbon monoxide (DLCO). METHODS: Data from patients diagnosed with SA at Military Institute of Medicine (Warsaw, Poland) between January 2018 and December 2019 were collected and analysed ongoing basis. Peripheral blood was collected from 26 patients with newly diagnosed pulmonary SA and 16 healthy subjects. The immunomagnetic method and flow cytometry were used. Among the CD34+ progenitor cells were assessed: low-differentiated cells, hematopoietic progenitor cells and endothelial progenitor cells. The Statistica 12.0 software was used for a statistical analysis. RESULTS: We observed a significantly higher percentage of low-differentiated cells (13.8 vs. 2.3, P = 0.001) and endothelial cells (0.3 vs. 0.0, P = 0.001) in patients with SA compared to the control group. In the study group the median proportion of fibrocytes was 1.877% (0.983-2.340) in patients with DLCO< 80%, while in patients with DLCO> 80% was 0.795% (0.139-1.951) (P = 0.72). The median proportion of endothelial progenitor cells was higher in patients with DLCO< 80%: 0.889% (0.391-1.741), than in patients with DLCO> 80%: 0.451% (0.177-0.857) (P = 0.44). CONCLUSIONS: In conclusion we demonstrated for the first time the immunophenotype of peripheral CD34 + cells with the degree of their differentiation. The study confirmed the involvement of low differentiated cells and endothelial cells in patients with SA.

Autologous CD34+ Cell Therapy for Ischemic Tissue Repair.

In 1997, the seminal manuscript by Asahara, Murohara, Isner et al outlined the evidence for the existence of circulating, bone marrow-derived cells capable of stimulating and contributing to the formation of new blood vessels. Consistent with the paradigm shift that this work represented, it triggered much scientific debate and controversy, some of which persists 2 decades later. In contrast, the clinical application of autologous CD34 cell therapy has been marked by a track record of consistent safety and clinical benefit in multiple ischemic conditions. In this review, we summarize the preclinical and clinical evidence from over 700 patients in clinical trials of CD34 cell therapy.

Lower resting and exercise-induced circulating angiogenic progenitors and angiogenic T cells in older men.

Aging is associated with a dysfunctional endothelial phenotype as well as reduced angiogenic capabilities. Exercise exerts beneficial effects on the cardiovascular system, possibly by increasing/maintaining the number and/or function of circulating angiogenic cells (CACs), which are known to decline with age. However, the relationship between cardiorespiratory fitness (CRF) and age-related changes in the frequency of CACs, as well as the exercise-induced responsiveness of CACs in older individuals, has not yet been determined. One-hundred seven healthy male volunteers, aged 18-75 yr, participated in study 1. CRF was estimated using a submaximal cycling ergometer test. Circulating endothelial progenitor cells (EPCs), angiogenic T cells (TANG), and their chemokine (C-X-C motif) receptor 4 (CXCR4) cell surface receptor expression were enumerated by flow cytometry using peripheral blood samples obtained under resting conditions before the exercise test. In study 2, 17 healthy men (8 young men, 18-25 yr; 9 older men, 60-75 yr) were recruited, and these participants undertook a 30-min cycling exercise bout at 70% maximal O2 consumption, with CACs enumerated before and immediately after exercise. Age was inversely associated with both CD34+ progenitor cells ( r2 = -0.140, P = 0.000) and TANG ( r2 = -0.176, P = 0.000) cells as well as CXCR4-expressing CACs (CD34+: r2 = -0.167, P = 0.000; EPCs: r2 = -0.098, P = 0.001; TANG: r2 = -0.053, P = 0.015). However, after correcting for age, CRF had no relationship with either CAC subset. In addition, older individuals displayed attenuated exercise-induced increases in CD34+ progenitor cells, TANG, CD4+, TANG, and CD8+CXCR4+ TANG cells. Older men display lower CAC levels, which may contribute to increased risk of cardiovascular disease, and older adults display an impaired exercise-induced responsiveness of these cells. NEW & NOTEWORTHY Older adults display lower circulating progenitor cell and angiogenic T cell counts compared with younger individuals independently of cardiometabolic risk factors and cardiorespiratory fitness. Older adults also display impaired exercise-induced mobilization of these vasculogenic cells.

VEGF gene therapy cooperatively recruits molecules from the immune system and stimulates cell homing and angiogenesis in refractory angina.

BACKGROUND: New vessels are formed in response to stimuli from angiogenic factors, a process in which paracrine signaling is fundamental. OBJECTIVE: To investigate the cooperative paracrine signaling profile in response to Vascular Endothelial Growth Factor (VEGF) gene therapy in patients with coronary artery disease (CAD) and refractory angina. METHOD: A cohort study was conducted in which plasma was collected from patients who underwent gene therapy with a plasmid expressing VEGF 165 (10) and from surgical procedure controls (4). Blood samples were collected from both groups prior to baseline and on days 3, 9 and 27 after the interventions and subjected to systemic analysis of protein expression (Interleukin-6, IL-6; Tumor Necrosis Factor-α, TNF-α; Interleukin-10, IL-10; Stromal Derived Factor-1 α, SDF-1α; VEGF; Angiopoietin-1, ANGPT-1; and Endothelin-1, ET-1) using the enzyme-linked immunosorbent assay (ELISA). RESULTS: Analysis showed an increase in proinflammatory IL-6 (p=0.02) and ET-1 (p=0.05) on day 3 after gene therapy and in VEGF (p=0.02) on day 9. A strong positive correlation was found between mobilization of endothelial progenitor cells and TNF-α on day 9 (r=0.71; p=0.03). Furthermore, a strong correlation between ß-blockers, antiplatelets, and vasodilators with SDF-1α baseline in the group undergoing gene therapy was verified (r=0.74; p=0.004). CONCLUSION: Analysis of cooperative paracrine signaling after VEGF gene therapy suggests that the immune system cell and angiogenic molecule expression as well as the endothelial progenitor cell mobilization are time-dependent, influenced by chronic inflammatory process and continuous pharmacological treatment.

Endothelial precursor cell cross-match using Tie-2-enriched spleen cells.

BACKGROUND: Non-HLA antibodies against human endothelial progenitor cells (EPC) in pre-transplant recipient serum can have a deleterious influence on the graft. EPC enriched from peripheral blood have been commonly used for EPC cross-matching. In the present study, we describe cross-matches using EPC enriched from fresh or frozen-thawed spleen cell preparations, thereby widening the sample source for deceased-donor cross-matching and retrospective studies. METHODS: EPC cross-matches were performed retrospectively using spleen cells and the flow cytometric XM-ONE cross-match test kit. RESULTS: Healthy controls (n = 28) showed no IgG antibodies against EPC. When sera of 11 random dialysis patients were studied, 2 patients (18%) exhibited IgG EPC antibodies. When pre-transplant sera of 20 kidney graft recipients with good long-term graft outcome (serum creatinine 1.0 ± 0.2 mg/dL measured 2463 ± 324 days post-transplant) were investigated using frozen-thawed and then separated Tie-2-enriched spleen cells of the original transplant donor, 3 patients (15%) had pre-transplant IgG EPC antibodies. When pre-transplant sera of 5 patients with intra-operative graft loss were studied employing the original donor spleen cells, 4 (80%) patients showed IgG EPC antibodies. CONCLUSIONS: Cross-matches with spleen cell-derived EPC using the XM-ONE assay are technically possible. Our very preliminary experience suggests clinical relevance.

Endothelial-to-mesenchymal transition in lipopolysaccharide-induced acute lung injury drives a progenitor cell-like phenotype.

Pulmonary vascular endothelial function may be impaired by oxidative stress in endotoxemia-derived acute lung injury. Growing evidence suggests that endothelial-to-mesenchymal transition (EndMT) could play a pivotal role in various respiratory diseases; however, it remains unclear whether EndMT participates in the injury/repair process of septic acute lung injury. Here, we analyzed lipopolysaccharide (LPS)-treated mice whose total number of pulmonary vascular endothelial cells (PVECs) transiently decreased after production of reactive oxygen species (ROS), while the population of EndMT-PVECs significantly increased. NAD(P)H oxidase inhibition suppressed EndMT of PVECs. Most EndMT-PVECs derived from tissue-resident cells, not from bone marrow, as assessed by mice with chimeric bone marrow. Bromodeoxyuridine-incorporation assays revealed higher proliferation of capillary EndMT-PVECs. In addition, EndMT-PVECs strongly expressed c-kit and CD133. LPS loading to human lung microvascular endothelial cells (HMVEC-Ls) induced reversible EndMT, as evidenced by phenotypic recovery observed after removal of LPS. LPS-induced EndMT-HMVEC-Ls had increased vasculogenic ability, aldehyde dehydrogenase activity, and expression of drug resistance genes, which are also fundamental properties of progenitor cells. Taken together, our results demonstrate that LPS induces EndMT of tissue-resident PVECs during the early phase of acute lung injury, partly mediated by ROS, contributing to increased proliferation of PVECs.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood.

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual-platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re-evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Accessory cells for ß-cell transplantation.

Despite recent advances, insulin therapy remains a treatment, not a cure, for diabetes mellitus with persistent risk of glycaemic alterations and life-threatening complications. Restoration of the endogenous ß-cell mass through regeneration or transplantation offers an attractive alternative. Unfortunately, signals that drive ß-cell regeneration remain enigmatic and ß-cell replacement therapy still faces major hurdles that prevent its widespread application. Co-transplantation of accessory non-islet cells with islet cells has been shown to improve the outcome of experimental islet transplantation. This review will highlight current travails in ß-cell therapy and focuses on the potential benefits of accessory cells for islet transplantation in diabetes.

Clinical relevance of preformed IgG and IgM antibodies against donor endothelial progenitor cells in recipients of living donor kidney grafts.

BACKGROUND: Literature reports suggest that non-HLA-antibodies against human endothelial progenitor cells (EPC) can be detected in pre-transplant recipient serum and that EPC antibodies can have a deleterious influence on the graft. METHODS: We investigated 71 renal transplant recipients from living donors for a possible influence of pre-transplant donor-specific IgG and/or IgM recipient antibodies against EPC of the donor using the flow cytometric XM-ONE cross-match. RESULTS: Eight of the 71 patients developed acute biopsy-proven rejection. Two of these patients showed IgM antibodies against EPC prior to transplantation while the other six patients had neither IgG nor IgM EPC antibodies. Conversely, pre-transplant IgG or IgM antibodies against EPC were detected in 19 patients without acute rejection (3 × both IgG and IgM, 1 × IgG and 15 × IgM). The remaining 44 patients had neither EPC antibodies nor experienced rejection. Comparing serum creatinine levels at one month and one yr post-transplant within and among the three patient groups revealed that serum creatinine levels were similar in patients with or without EPC antibodies (p > 0.05). CONCLUSION: In this series of 71 recipients with living donor kidneys, pre-transplant EPC antibodies detected with the XM-ONE test kit were neither associated with acute rejection nor with graft function at one month or one yr.

Residential Proximity to Major Roadways Is Associated With Increased Levels of AC133+ Circulating Angiogenic Cells.

OBJECTIVES: Previous studies have shown that residential proximity to a roadway is associated with increased cardiovascular disease risk. Yet, the nature of this association remains unclear, and its effect on individual cardiovascular disease risk factors has not been assessed. The objective of this study was to determine whether residential proximity to roadways influences systemic inflammation and the levels of circulating angiogenic cells. APPROACH AND RESULTS: In a cross-sectional study, cardiovascular disease risk factors, blood levels of C-reactive protein, and 15 antigenically defined circulating angiogenic cell populations were measured in participants (n=316) with moderate-to-high cardiovascular disease risk. Attributes of roadways surrounding residential locations were assessed using geographic information systems. Associations between road proximity and cardiovascular indices were analyzed using generalized linear models. Close proximity (<50 m) to a major roadway was associated with lower income and higher rates of smoking but not C-reactive protein levels. After adjustment for potential confounders, the levels of circulating angiogenic cells in peripheral blood were significantly elevated in people living in close proximity to a major roadway (CD31(+)/AC133(+), AC133(+), CD34(+)/AC133(+), and CD34(+)/45(dim)/AC133(+) cells) and positively associated with road segment distance (CD31(+)/AC133(+), AC133(+), and CD34(+)/AC133(+) cells), traffic intensity (CD31(+)/AC133(+) and AC133(+) cells), and distance-weighted traffic intensity (CD31(+)/34(+)/45(+)/AC133(+) cells). CONCLUSIONS: Living close to a major roadway is associated with elevated levels of circulating cells positive for the early stem marker AC133(+). This may reflect an increased need for vascular repair. Levels of these cells in peripheral blood may be a sensitive index of cardiovascular injury because of residential proximity to roadways.

Endothelial Progenitor Cells (EPC) Count by Multicolor Flow Cytometry in Healthy Individuals and Diabetes Mellitus (DM) Patients.

BACKGROUND: The present study aimed to determine circulating Endothelial Progenitor Cell (EPC) counts by multicolor flow cytometry in healthy individuals and diabetic subjects by means of forming an analysis procedure using a combination of monoclonal antibodies (moAbs), which would correctly detect the circulating EPC count. METHODS: The circulating EPC count was detected in 40 healthy individuals (20 Female, 20 Male; age range: 26 - 50 years) and 30 Diabetes Mellitus (DM) patients (15 Female, 15 Male; age range: 42 - 55) by multicolor flow cytometry (FCM) in a single-tube panel consisting of 5 CD45/CD31/CD34/CD309/ SYTO® and 16 monoclonal antibodies. RESULTS: Circulating EPC count was 11.33 (7.89 - 15.25) cells/µL in the healthy control group and 4.80 (0.70 - 10.85) cells/µL in the DM group. EPC counts were significantly lower in DM cases that developed coronary artery disease (53.3%) as compared to those that did not (p < 0.001). CONCLUSIONS: In the present study, we describe a method that identifies circulating EPC counts by multicolor flow cytometry in a single tube and determines the circulating EPC count in healthy individuals. This is the first study conducted on EPC count in Turkish population. We think that the EPC count found in the present study will be a guide for future studies.

Interruption of CD40 Pathway Improves Efficacy of Transplanted Endothelial Progenitor Cells in Monocrotaline Induced Pulmonary Arterial Hypertension.

BACKGROUND/AIMS: Transplantation of endothelial progenitor cells (EPCs) plays a therapeutic role in pulmonary arterial hypertension (PAH). Meanwhile, recruitment of progenitors has potential inflammatory effects and exaggerates vascular injury. CD40 pathway is identified as a major player in vascular inflammatory events. In this study, we investigated the role of CD40 pathway in regulating early outgrowth EPC functions, and searched for improvements in PAH cell therapy. METHODS: EPCs were isolated from rat bone marrow and cultured for 7 days. After treatment with soluble CD40 ligand (sCD40L) for 24 hours, EPC migration, adhesion, proliferation, paracrine and vasculogenesis functions were tested. Rat PAH model was founded by subcutaneous injection of monocrotaline (MCT). Control EPCs or lentivirus vectors (Lv)-shRNA-CD40 EPCs were infused via tail vein at day 7, 14, and 21 after MCT injection. Therapeutic effects were evaluated at day 28. RESULTS: sCD40L dose-dependently impaired EPC migration, adhesion, proliferation, and vasculogenesis functions. However, paracrine effects of soluble intercellular adhesion molecule-1, vascular endothelial growth factor and interleukin-6 were dose-dependently improved by sCD40L. Control EPC-derived conditioned medium protected endothelial cell in vitro vasculogenesis, while sCD40L-pretreated ones showed detrimental effects. After MCT injection, sCD40L levels in rat serum increased gradually. Other than in vitro results, benefits of both two EPC treatments were obvious, even taken at day 21. Benefits of control EPCs wore off over time, but those of Lv-shRNA-CD40 EPCs were more effective and enduring, as characterized by both ameliorated rat hemodynamic and reversed vascular remodeling. Furthermore, Lv-shRNA-CD40 EPCs integrated into endothelium better, rather than into adventitia and media. CONCLUSION: sCD40L impaired protective effects of EPCs. Traditional EPC treatments were limited in PAH, while interruption of CD40 pathway of transplanted cells could apparently improve the therapeutic efficacy.