The bone marrow endothelial progenitor cell response to septic infection.

Early increase in the level of endothelial progenitor cells (EPCs) in the systemic circulation occurs in patients with septic infection/sepsis. The significance and underlying mechanisms of this response remain unclear. This study investigated the bone marrow EPC response in adult mice with septic infection induced by intravenous injection (i.v.) of Escherichia coli. For in vitro experiments, sorted marrow stem/progenitor cells (SPCs) including lineage(lin)-stem cell factor receptor (c-kit)+stem cell antigen-1 (Sca-1)-, lin-c-kit+, and lin- cells were cultured with or without lipopolysaccharides (LPSs) and recombinant murine vascular endothelial growth factor (VEGF) in the absence and presence of anti-Sca-1 crosslinking antibodies. In a separate set of experiments, marrow lin-c-kit+ cells from green fluorescence protein (GFP)+ mice, i.v. challenged with heat-inactivated E. coli or saline for 24 h, were subcutaneously implanted in Matrigel plugs for 5 weeks. Marrow lin-c-kit+ cells from Sca-1 knockout (KO) mice challenged with heat-inactivated E. coli for 24 h were cultured in the Matrigel medium for 8 weeks. The marrow pool of EPCs bearing the lin-c-kit+Sca-1+VEGF receptor 2 (VEGFR2)+ (LKS VEGFR2+) and LKS CD133+VEGFR2+ surface markers expanded rapidly following septic infection, which was supported by both proliferative activation and phenotypic conversion of marrow stem/progenitor cells. Increase in marrow EPCs and their reprogramming for enhancing angiogenic activity correlated with cell-marked upregulation of Sca-1 expression. Sca-1 was coupled with Ras-related C3 botulinum toxin substrate 2 (Rac2) in signaling the marrow EPC response. Septic infection caused a substantial increase in plasma levels of IFN-γ, VEGF, G-CSF, and SDF-1. The early increase in circulating EPCs was accompanied by their active homing and incorporation into pulmonary microvasculature. These results demonstrate that the marrow EPC response is a critical component of the host defense system. Sca-1 signaling plays a pivotal role in the regulation of EPC response in mice with septic infection.

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.

Diversity of Vascular Niches in Bones and Joints During Homeostasis, Ageing, and Diseases.

The bones and joints in the skeletal system are composed of diverse cell types, including vascular niches, bone cells, connective tissue cells and mineral deposits and regulate whole-body homeostasis. The capacity of maintaining strength and generation of blood lineages lies within the skeletal system. Bone harbours blood and immune cells and their progenitors, and vascular cells provide several immune cell type niches. Blood vessels in bone are phenotypically and functionally diverse, with distinct capillary subtypes exhibiting striking changes with age. The bone vasculature has a special impact on osteogenesis and haematopoiesis, and dysregulation of the vasculature is associated with diverse blood and bone diseases. Ageing is associated with perturbed haematopoiesis, loss of osteogenesis, increased adipogenesis and diminished immune response and immune cell production. Endothelial and perivascular cells impact immune cell production and play a crucial role during inflammation. Here, we discuss normal and maladapted vascular niches in bone during development, homeostasis, ageing and bone diseases such as rheumatoid arthritis and osteoarthritis. Further, we discuss the role of vascular niches during bone malignancy.

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.

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).

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.

Insufficient production of IL-10 from M2 macrophages impairs in vitro endothelial progenitor cell differentiation in patients with Moyamoya disease.

Moyamoya disease (MMD) is well known to be caused by insufficient cerebral vascular formation. However, the essential pathogenesis has not yet been identified. Using our recently developed technique of generating vasculogenic and anti-inflammatory cultures, we investigated endothelial progenitor cell (EPC) expansion and differentiation under the cytokine milieu generated by the peripheral blood mononuclear cells (PBMNCs) of the operated and non-operated MMD patients. EPC colony forming assay of the cultured PBMNCs disclosed the decline of the definitive EPC colony numbers in the both MMD patients. The level of interleukin-10 (IL-10) was lower in secretory cytokines from the cultured PBMNCs of MMD patients than that in that of controls using a cytometric bead array. The addition of human recombinant IL-10 to PBMNCs cultured from MMD patients restored the EPC colony forming potential of MMD PBMNCs. Following phorbol myristate acetate stimulation of the cultured PBMNCs, flow cytometry revealed a decrease in intracellular IL-10 storage in the main cell populations of the PBMNCs cultured from MMD patients relative to those cultured from controls. The present data provide the expected mechanism of vascular malformation in MMD pathogenesis originated from the insufficient production of IL-10 secreting cells from PBMNCs fostering EPC expansion and differentiation.

Keloid patients have higher peripheral blood endothelial progenitor cell counts and CD34+ cells with normal vasculogenic and angiogenic function that overexpress vascular endothelial growth factor and interleukin-8.

BACKGROUND: One suggested reason for aberrant wound healing in keloid scars is chronic inflammation of the dermis. We hypothesized that excessive blood vessel formation and high capillary density in keloid tissue is caused by dysfunction of endothelial progenitor cells. METHODS: We compared the number of circulating endothelial progenitor cells and vasculogenic and angiogenic capacity, as well as secretory function, of circulating CD34+ cells in keloid patients and healthy individuals. RESULTS: Compared to mononuclear cell cultures from healthy donors, cultures of peripheral blood mononuclear cells obtained from keloid patients showed a more than twofold increase in the number of peripheral blood EPCs (fibronectin-adhering cells that phagocytized acetylated low-density lipoprotein and bound Ulex europaeus agglutinin-I lectin). However, there was no difference in colony-forming ability and participation in in vitro angiogenesis between circulating CD34+ cells isolated from keloid patients and healthy individuals. This means that circulating CD34+ /endothelial progenitor cells in keloid patients have normal vasculogenic and angiogenic function. However, CD34+ cells derived from keloid patients demonstrated a more than sevenfold expression of the interleukin-8 gene and a more than fivefold expression of the vascular endothelial growth factor gene than CD34+ cells derived from healthy individuals. CONCLUSIONS: These results support the role of vascular endothelial growth factor and interleukin-8 in increased recruitment of endothelial progenitor cells in keloid patients.

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.

Transplantation of Endothelial Progenitor Cells Attenuates Lipopolysaccharide-Induced Lung Injury via Inhibiting the Inflammatory Secretion of Neutrophils in Rats.

BACKGROUND: Endothelial progenitor cells (EPCs) are special types of stem cells and are a potential novel therapeutic approach in acute lung injury (ALI). Transplantation of EPCs can ameliorate the inflammatory state by reducing adhesion and exudation of inflammatory cells. However, the mechanism underlying the effect of EPCs on inflammatory response modulation remains unclear. The aim of the present study was to investigate the effect of EPCs on the modulation of neutrophils in vitro and in vivo. MATERIALS AND METHODS: EPCs were cocultured with neutrophils after lipopolysaccharide stimulation in vitro or transplanted into ALI rats, and neutrophil inflammatory mediators including tumor necrosis factor-α, interleukin-1ß, neutrophil elastase, myeloperoxidase and matrix metalloproteinases-9 were detected by enzyme-linked immunosorbent assay, an myeloperoxidase detection kits, reverse transcription-polymerase chain reaction and western blotting. RESULTS: The results showed that EPCs significantly downregulated the expression of inflammatory mediators when cocultured with neutrophils in vitro or in vivo. CONCLUSIONS: These findings demonstrated that EPCs contributed to lung injury in ALI rats by downregulating neutrophil inflammatory mediators.

Reduced mRNA and Protein Expression Levels of Tet Methylcytosine Dioxygenase 3 in Endothelial Progenitor Cells of Patients of Type 2 Diabetes With Peripheral Artery Disease.

Endothelial progenitor cells (EPCs) with immunological properties repair microvasculature to prevent the complications in patients with diabetes. Epigenetic changes such as DNA methylation alter the functions of cells. Tet methylcytosine dioxygenases (TETs) are enzymes responsible for the demethylation of cytosine on genomic DNA in cells. We hypothesized that EPCs of diabetic patients with peripheral artery disease (D-PAD) might have altered expression levels of TETs. Subjects who were non-diabetic (ND, n = 22), with diabetes only (D, n = 29) and with D-PAD (n = 22) were recruited for the collection of EPCs, which were isolated and subjected to analysis. The mRNA and protein expression levels of TET1, TET2, and TET3 were determined using real-time PCR and immunoblot, respectively. The TET1 mRNA expression level in ND group was lower than that in the D and D-PAD groups. The TET3 mRNA level in the ND group was higher than that in the D group, which was higher than that in the D-PAD group. The TET1 protein level in the D-PAD group, but not the D group, was higher than that in the ND group. The TET2 protein level in the D-PAD group, but not the D group, was lower than that in the ND group. The TET3 protein level in the ND group was higher than that in the D group, which was higher than that in the D-PAD group, which is the lowest among the three groups. The changes of TETs protein levels were due to the alterations of their transcripts. These probably lead to epigenetic changes, which may be responsible for the reductions of EPCs numbers and functions in patients with the D-PAD. The expression pattern of TET3 mRNA and TET3 protein in EPCs may be a biomarker of angiopathy in diabetic patients.

Low Levels of Endothelial Progenitor Cells and Their Association with Systemic Inflammation and Monocyte Activation in Older HIV-Infected Men.

Endothelial progenitor cells (EPCs) repair damaged vascular endothelium, and low circulating EPC levels have been associated with cardiovascular disease (CVD). CD34+/KDR+ EPCs are commonly reported in the literature and CD34+/CD133+/KDR+ EPCs are rare in circulation but highly specific for endothelial lineage. HIV-infected (HIV+) adults have chronic inflammation and increased CVD risk, but the relationship between CVD, vascular inflammation, and EPCs in HIV remains unclear. In a pilot study, EPCs were measured in 57 HIV+ men [≥50 years old, HIV-1 RNA <50 copies/ml on antiretroviral therapy (ART)] by real-time flow cytometry using cellular immaturity (CD34 and/or CD133) and endothelial commitment (KDR) markers. Fasting inflammatory biomarker levels were measured by ELISA. Median age was 57 years; CD4+ T lymphocyte count was 570 cells/mm3. Prevalent CVD risk factors included 16% diabetes, 28% hypertension, 53% dyslipidemia, and 33% smoking. Median (interquartile range) EPC values were CD34+/KDR+ 0.1 (0.0-0.9) cells/105 peripheral blood mononuclear cells (PBMCs) and CD34+/CD133+/KDR+ 0.1 (0.0-0.9) cells/105 PBMCs. We observed a high prevalence of undetectable CD34+/KDR+ (40%) and CD34+/CD133+/KDR+ EPCs (44%). Men with undetectable EPCs were more likely to have ≥2 CVD risk factors, lower interleukin-6 (IL-6), and higher sCD163 levels. In these older HIV+ men on suppressive ART, CD34+/KDR+ and CD34+/CD133+/KDR+ EPC levels were low and often undetectable. Undetectable EPC levels were associated with greater CVD risk factor burden, lower IL-6 (consistent with decreased EPC production stimulus), and higher sCD163 (consistent with monocyte activation and prior CVD associations) levels, suggesting a potential relationship between EPCs and atherosclerotic burden in this population.

Hypoxia induced mitogenic factor (HIMF) triggers angiogenesis by increasing interleukin-18 production in myoblasts.

Inflammatory myopathy is a rare autoimmune muscle disorder. Treatment typically focuses on skeletal muscle weakness or inflammation within muscle, as well as complications of respiratory failure secondary to respiratory muscle weakness. Impaired respiratory muscle function contributes to increased dyspnea and reduced exercise capacity in pulmonary hypertension (PH), a debilitating condition that has few treatment options. The initiation and progression of PH is associated with inflammation and inflammatory cell recruitment and it is established that hypoxia-induced mitogenic factor (HIMF, also known as resistin-like molecule α), activates macrophages in PH. However, the relationship between HIMF and inflammatory myoblasts remains unclear. This study investigated the signaling pathway involved in interleukin-18 (IL-18) expression and its relationship with HIMF in cultured myoblasts. We found that HIMF increased IL-18 production in myoblasts and that secreted IL-18 promoted tube formation of the endothelial progenitor cells. We used the mouse xenograft model and the chick chorioallantoic membrane assay to further explore the role of HIMF in inflammatory myoblasts and angiogenesis in vivo. Thus, our study focused on the mechanism by which HIMF mediates IL-18 expression in myoblasts through angiogenesis in vitro and in vivo. Our findings provide an insight into HIMF functioning in inflammatory myoblasts.

Pinocembrin ex vivo preconditioning improves the therapeutic efficacy of endothelial progenitor cells in monocrotaline-induced pulmonary hypertension in rats.

Pulmonary hypertension is still not curable and the available current therapies can only alleviate symptoms without hindering the progression of disease. The present study was directed to investigate the possible modulatory effect of pinocembrin on endothelial progenitor cells transplanted in monocrotaline-induced pulmonary hypertension in rats. Pulmonary hypertension was induced by a single subcutaneous injection of monocrotaline (60mg/kg). Endothelial progenitor cells were in vitro preconditioned with pinocembrin (25mg/L) for 30min before being i.v. injected into rats 2weeks after monocrotaline administration. Four weeks after monocrotaline administration, blood pressure, electrocardiography and right ventricular systolic pressure were recorded. Rats were sacrificed and serum was separated for determination of endothelin-1 and asymmetric dimethylarginine levels. Right ventricles and lungs were isolated for estimation of tumor necrosis factor-alpha and transforming growth factor-beta contents as well as caspase-3 activity. Moreover, protein expression of matrix metalloproteinase-9 and endothelial nitric oxide synthase in addition to myocardial connexin-43 was assessed. Finally, histological analysis of pulmonary arteries, cardiomyocyte cross-sectional area and right ventricular hypertrophy was performed and cryosections were done for estimation of cell homing. Preconditioning with pinocembrin provided a significant improvement in endothelial progenitor cells' effect towards reducing monocrotaline-induced elevation of inflammatory, fibrogenic and apoptotic markers. Furthermore, preconditioned cells induced a significant amelioration of endothelial markers and cell homing and prevented monocrotaline-induced changes in right ventricular function and histological analysis compared with native cells alone. In conclusion, pinocembrin significantly improves the therapeutic efficacy of endothelial progenitor cells in monocrotaline-induced pulmonary hypertension in rats.

Kinetics of circulating progenitor cell mobilization during submaximal exercise.

Circulating progenitor cells (CPCs) are a heterogeneous population of stem/progenitor cells in peripheral blood that includes hematopoietic stem and progenitor cells (HSPCs and HSCs), endothelial progenitor cells (EPCs), and mesenchymal stem cells (MSCs) that are involved in tissue repair and adaptation. CPC mobilization during exercise remains uncharacterized in young adults. The purpose of this study was to investigate the kinetics of CPC mobilization during and after submaximal treadmill running and their relationship to mobilization factors. Seven men [age = 25.3 ± 2.4 yr, body mass index = 23.5 ± 1.0 kg/m2, peak O2 uptake (V̇o2peak) = 60.9 ± 2.74 ml·kg-1·min-1] ran on a treadmill for 60 min at 70% V̇o2peak Blood sampling occurred before (Pre), during [20 min (20e), 40 min (40e), 60 min (60e)], and after exercise [15 min (15p), 60 min (60p), 120 min (120p)] for quantification of CPCs (CD34+), HSPCs (CD34+/CD45low), HSCs (CD34+/CD45low/CD38-), CD34+ MSCs (CD45-/CD34+/CD31-/CD105+), CD34- MSCs (CD45-/CD34-/CD31-/CD105+), and EPCs (CD45-/CD34+/CD31+) via flow cytometry. CPC concentration increased compared with Pre at 20e and 40e (2.7- and 2.4-fold, respectively, P < 0.05). HSPCs and HSCs increased at 20e compared with 60p (2.7- and 2.8-fold, respectively, P < 0.05), whereas EPCs and both MSC populations did not change. CXC chemokine ligand (CXCL) 12 (1.5-fold; P < 0.05) and stem cell factor (1.3-fold; P < 0.05) were increased at 40e and remained elevated postexercise. The peak increase in CPCs was positively correlated to concentration of endothelial cells during exercise with no relationship to CXCL12 and SCF. Our data show the kinetics of progenitor cell mobilization during exercise that could provide insight into cellular mediators of exercise-induced adaptations, and have implication for the use of exercise as an adjuvant therapy for CPC collection in hematopoietic stem cell transplant.NEW & NOTEWORTHY Using a comprehensive evaluation of circulating progenitor cells (CPCs), we show that CPC mobilization during exercise is related to tissue damage, and not plasma concentrations of CXC chemokine ligand 12 and stem cell factor. These data have implications for the use of exercise interventions as adjuvant therapy for CPC mobilization in the context of hematopoietic stem cell transplant and also support the role of mobilized progenitor cells as cellular mediators of systemic adaptations to exercise.

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.

CD31+ Cells From Peripheral Blood Facilitate Bone Regeneration in Biologically Impaired Conditions Through Combined Effects on Immunomodulation and Angiogenesis.

Controlled revascularization and inflammation are key elements regulating endogenous regeneration after (bone) tissue trauma. Peripheral blood-derived cell subsets, such as regulatory T-helper cells and circulating (endothelial) progenitor cells, respectively, can support endogenous tissue healing, whereas effector T cells that are associated with an aged immune system can hinder bone regeneration. CD31 is expressed by diverse leukocytes and is well recognized as a marker of circulating endothelial (precursor) cells; however, CD31 is absent from the surface of differentiated effector T cells. Thus, we hypothesized that by separating the inhibitory fractions from the supportive fractions of circulating cells within the peripheral blood (PB) using the CD31 marker, bone regeneration in biologically compromised conditions, such as those observed in aged patients, could be improved. In support of our hypothesis, we detected an inverse correlation between CD31+ cells and effector T cells in the hematomas of human fracture patients, dependent on the age of the patient. Furthermore, we demonstrated the regenerative capacity of human PB-CD31+ cells in vitro. These findings were translated to a clinically relevant rat model of impaired bone healing. The transplantation of rat PB-CD31+ cells advanced bone tissue restoration in vivo and was associated with an early anti-inflammatory response, the stimulation of (re)vascularization, and reduced fibrosis. Interestingly, the depletion or enrichment of the highly abundant CD31+/14+ monocytes from the mixed CD31+ cell population diminished tissue regeneration at different levels, suggesting combined effects within the PB-CD31+ subsets. In summary, an intraoperative enrichment of PB-CD31+ cells might be a novel option to facilitate endogenous regeneration under biologically impaired situations by supporting immunomodulation and vascularization. © 2016 American Society for Bone and Mineral Research.