Mobilization Of Progenitor Cells Research Articles

Oxygen Dose: A new perspective

IntroductionThis study followed up on our findings, published in 2019, of stem progenitor cell mobilization following intermittent low level hyperoxia (42%) in an animal model. This most recent study in human participants used a never before tested small dose of intermittent hyperbaric air that has been used for many years as a placebo control in hyperbaric oxygen (HBOT) research. Because of the reports of a placebo response using this small dose in HBOT research performed by the Department of Defense and conflicting reports disputing the placebo response, coupled with the lack of evidence in the literature that this dose had ever been tested, we questioned if this a placebo or a dose response.In this research human participants breathed room air in a portable hyperbaric chamber (mountain sickness chamber) (Vitaeris 320 Oxyhealth LLC) at 1.3 atmospheres absolute (ATA), daily for 10 days. This hyperbaric hyperoxia level is similar to 28% oxygen concentration in normobaria.We hypothesized that the low dose of intermittent hyperoxia would mobilize stem cells in humans similar to our animal study.MethodsEight adult subjects were exposed to 1.3 atmospheres absolute (ATA) in a mountain sickness bag daily for 60‐minutes, 10‐times (M‐F) over a 12‐day period. Venous blood draws were taken at four time points. (1) Immediately preceding the first treatment (control), (2) Immediately following the first treatment, (3) Prior to the 10th treatment, and (4) 72‐hours after final treatment. We analyzed blood samples using flow cytometry for changes in the expression of surface markers CD45, CD34 and CD133 and performed a statistical analysis using a Friedman test followed by a paired Wilcoxon signed‐rank test.ResultsWe found a nearly 2 fold increase in the expression of CD45dimCD34+CD133‐prior to the 10th treatment (9.8% to 18.9% = 194% increase) and a 3 fold increase 72 hours after the 10th treatment (9.8% to 29.6% = 303% increase). We also found CD45dimCD34‐133+ decreased by 85% between the control time point and prior to the 10th treatment (15% to 2.3% = 85% decrease) and further decreased to a decrease of 84%, 72 hours after the final treatment (15% to 2.0% = 87% decrease).ConclusionsWe conclude that breathing room air at 1.3 ATA daily for 60 minutes over a 2 week period has significant effects on the mobilization of stem progenitor cells and therefore is physiologically active. These findings suggest that 1.3 ATA hyperbaric air breathed daily is a dose treatment and not a placebo nor a sham. These data suggest a re‐evaluation of its use as a placebo control in scientific research is warranted.

Superior physical and mental health of healthy volunteers before and five\xa0years after mobilized stem cell donation

BackgroundSafety, tolerability and efficacy of granulocyte colony-stimulating factor (G-CSF) for mobilization of hematopoietic stem and progenitor cells (HSPCs) from healthy donors have been conclusively demonstrated. This explicitly includes, albeit for smaller cohorts and shorter observation periods, biosimilar G-CSFs. HSPC donation is non-remunerated, its sole reward being “warm glow”, hence harm to donors must be avoided with maximal certitude. To ascertain, therefore, long-term physical and mental health effects of HSPC donation, a cohort of G-CSF mobilized donors was followed longitudinally.MethodsWe enrolled 245 healthy volunteers in this bi-centric long-term surveillance study. 244 healthy volunteers began mobilization with twice-daily Sandoz biosimilar filgrastim and 242 underwent apheresis after G-CSF mobilization. Physical and mental health were followed up over a period of 5-years using the validated SF-12 health questionnaire.ResultsBaseline physical and mental health of HSPC donors was markedly better than in a healthy reference population matched for ethnicity, sex and age. Physical, but not mental health was sharply diminished at the time of apheresis, likely due to side effects of biosimilar G-CSF, however had returned to pre-apheresis values by the next follow-up appointment after 6 months. Physical and mental health slightly deteriorated over time with kinetics reflecting the known effects of aging. Hence, superior physical and mental health compared to the general healthy non-donor population was maintained over time.ConclusionsHSPC donors are of better overall physical and mental health than the average healthy non-donor. Superior well-being is maintained over time, supporting the favorable risk–benefit assessment of volunteer HSPC donation.Trial registration National Clinical Trial NCT01766934

SDF-1α loaded bioengineered human amniotic membrane-derived scaffold transplantation in combination with hyperbaric oxygen improved diabetic wound healing

Based on its multifactorial nature, successful treatment of diabetic wounds requires combinatorial approach. In this regard, we hypothesized that engraftment of a bioengineered micro-porous three-dimensional human amniotic membrane-scaffold (HAMS) loaded by SDF-1α (SHAMS) in combination with hyperbaric oxygen (HBO), throughout mobilization and recruitment of endothelial progenitor cells (EPCs), could accelerate wound healing in rats with type 1 diabetes mellitus. To test this hypothesis, 30 days after inducting diabetes, an ischemic wound was created in rat skin and treatments were performed for 21 days. In addition to wounded non-diabetic (ND) group, diabetic animals were randomly divided into non-treated (NT-D), HBO-treated (HBO-D), HBO-treated plus HAMS transplantation (HBO+HAMS-D) or HBO-treated in combination with SHAMS transplantation (HBO+SHAMS-D) groups. Our results on post-wounding days 7, 14 and 21 showed that the wound closure, volume of new dermis and epidermis, numerical density of basal cells of epidermis, fibroblasts and blood vessels, number of proliferating cells, deposition of collagen and biomechanical properties of healed wound were considerably higher in both HBO+HAMS-D and HBO+SHAMS-D groups in comparison to those of the NT-D and HBO-D groups, and were the highest in HBO+SHAMS-D ones. The transcripts for Vegf, bFgf, and Tgf-β genes were significantly upregulated in all treatment regimens compared to NT-D group and were the highest for HBO+SHAMS-D group. This is while expression of Tnf-α and Il-1β as well as cell density of neutrophil and macrophage decreased more significantly in HBO+SHAMS-D group as compared with NT-D or HBO-D groups. Overall, it was found that using both HAMS transplantation and HBO treatment has more impact on diabetic wound healing. Moreover, SDF-1α loading on HAMS could transiently improve the wound healing process, as compared with the HBO+HAMS-D group on day 7 only.

An ApoA-I Mimic Peptide of 4F Promotes SDF-1α Expression in Endothelial Cells Through PI3K/Akt/ERK/HIF-1α Signaling Pathway.

Atherosclerosis (AS) seriously impairs the health of human beings and is manifested initially as endothelial cells (ECs) impairment and dysfunction in vascular intima, which can be alleviated through mobilization of endothelial progenitor cells (EPCs) induced by stromal-cell-derived factor-1α (SDF-1α). A strong inverse correlation between HDL and AS has been proposed. The aim of the present work is to investigate whether 4F, an apolipoprotein A-I (apoA-I, major component protein of HDL) mimic peptide, can upregulate SDF-1α in mice and human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. The protein levels of SDF-1α were measured by ELISA assay. Protein levels of HIF-1α, phosphorylated Akt (p-Akt), and phosphorylated ERK (p-ERK) were evaluated by Western blotting analysis. The results show that L-4F significantly upregulates protein levels of HIF-1α, Akt, and ERK, which can be inhibited by the PI3K inhibitor, LY294002, or ERK inhibitor, PD98059, respectively. Particularly, LY294002 can downregulate the levels of p-ERK, while PD98059 cannot suppress that of p-Akt. D-4F can upregulate the levels of HIF, p-Akt, and p-ERK in the abdominal aorta and inferior vena cava from mice. These results suggest that 4F promotes SDF-1α expression in ECs through PI3K/Akt/ERK/HIF-1α signaling pathway.

Tumor-Associated Macrophages in Hepatocellular Carcinoma Pathogenesis, Prognosis and Therapy.

Simple SummaryHepatocellular carcinoma (HCC) constitutes a major health burden, accounting for >80% of primary liver cancers globally. Inflammation has come into the spotlight as a hallmark of cancer, and it is evident that tumor-associated inflammation drives the involvement of monocytes in tumor growth and metastasis. Tumor-associated macrophages (TAMs) actively participate in tumor-related inflammation, representing the main type of inflammatory cells in the tumor microenvironment, setting the crosstalk between tumor and stromal cells. Infiltrating TAMs exert either anti-tumorigenic (M1) or pro-tumorigenic (M2) functions. In most solid human tumors, increased TAM infiltration has been associated with enhanced tumor growth and metastasis, while other studies showcase that under certain conditions, TAMs exhibit cytotoxic and tumoricidal activity, inhibiting the progression of cancer. In this review, we summarize the current evidence on the role of macrophages in the pathogenesis and progression of HCC and we highlight their potential utilization in HCC prognosis and therapy.Hepatocellular carcinoma (HCC) constitutes a major health burden globally, and it is caused by intrinsic genetic mutations acting in concert with a multitude of epigenetic and extrinsic risk factors. Cancer induces myelopoiesis in the bone marrow, as well as the mobilization of hematopoietic stem and progenitor cells, which reside in the spleen. Monocytes produced in the bone marrow and the spleen further infiltrate tumors, where they differentiate into tumor-associated macrophages (TAMs). The relationship between chronic inflammation and hepatocarcinogenesis has been thoroughly investigated over the past decade; however, several aspects of the role of TAMs in HCC development are yet to be determined. In response to certain stimuli and signaling, monocytes differentiate into macrophages with antitumor properties, which are classified as M1-like. On the other hand, under different stimuli and signaling, the polarization of macrophages shifts towards an M2-like phenotype with a tumor promoting capacity. M2-like macrophages drive tumor growth both directly and indirectly, via the suppression of cytotoxic cell populations, including CD8+ T cells and NK cells. The tumor microenvironment affects the response to immunotherapies. Therefore, an enhanced understanding of its immunobiology is essential for the development of next-generation immunotherapies. The utilization of various monocyte-centered anticancer treatment modalities has been under clinical investigation, selectively targeting and modulating the processes of monocyte recruitment, activation and migration. This review summarizes the current evidence on the role of TAMs in HCC pathogenesis and progression, as well as in their potential involvement in tumor therapy, shedding light on emerging anticancer treatment methods targeting monocytes.

DNA methylation is involved in the microRNA-886-3p gene expression and plays a potential role in hematopoietic stem and progenitor cell mobilization through affecting SDF-1

The role of the sympathetic nervous system (SNS) in hematopoietic stem and progenitor cell (HSPC) mobilization has been largely investigated. However, there is a critical need for the identification of the underlying contributing factors to improve HSPC yield for transplantation. It has been demonstrated that miR-886-3p targets stromal-derived factor-1 (SDF-1), the central mediator of mobilization, and therefore may play a part in this process. Besides, miR-886-3p expression can be epigenetically regulated through DNA methylation modifications inits gene promoter. Here, to assess the contribution of miR-886-3p and other epigenetic factors in HSPC mobilization, human bone marrow-derived mesenchymal stem cells (MSCs) were treated with the β-adrenergic agonist of isoprenaline. The expression of miR-886-3p and SDF-1and the gene promoter methylation status of this miRNA were then respectively evaluated through the appropriate PCR techniques. As expected, despite a transient initial increase in SDF-1mRNA level, its expression reduced, and miR-886-3plevel remarkably increased 48 h following treatment. The gene promoter methylation pattern of miR-886-3p also changed from a full methylated state to a partially methylated one. Together, our findings suggest that miR-886-3p can be epigenetically regulated and through suppressing the expression of SDF-1 play an active role in the SNS-mediated HSPC mobilization.

Stem cells mobilization by cardiopulmonary bypass after coronary artery bypass grafting.

Expression of haematopoietic stem and progenitor cells occurs as a result of an inflammatory reaction caused by a damaged organ. Patients undergoing coronary artery bypass surgery (CABG) using cardiopulmonary bypass (CPB) are exposed to an inflammatory reaction, which may be a trigger in the mobilization of regenerative cells. To assess the impact of CPB on stem and progenitor cells mobilization in patients scheduled for CABG. Twenty patients with stable coronary disease, who were scheduled for CABG, were included in the study. Peripheral blood samples were collected perioperatively: 2 h before surgery, before CPB, at CPB weaning, 24 h postoperatively, and on the 6th postoperative day. Analyses of immune-labelled cells were carried out on an ImageStream (IS) system. The following cell populations were identified during cardiac surgery: haematopoietic stem cells (HSC), mesenchymal cells (MSC), endothelial progenitors (EPC), and very small embryonic-like cells (VSEL). The profile of cell mobilization coincides with the perioperative inflammatory response. Mobilization of stem and progenitor cells induced by CPB did not occur in any of the isolated cell lines (p > 0.05). The expression profile of stem and progenitor cells was related with the inflammatory reaction associated with traumatic stress in all cell lines. Type 2 diabetes is a disease that is hampering the trend of MSC mobilization. Mobilization of haematopoietic stem and progenitor cells was not related with CPB. The inflammatory reaction was associated with perioperative trauma. Cell release was inhibited in patients with diabetes.

3057 – HIGH SALT DIETS PROMOTE A TH17 CELL DRIVEN GUT-BONE AXIS TO REGULATE HAEMATOPOIESIS AND ATHEROSCLEROSIS

Salt intake is often associated with increased risk of cardiovascular disease through its relationship with hypertension. However, a high salt diet (HSD) can directly modulate immune cells. We found that a HSD increases IL-17A-producing T helper (TH17) cells in the bone marrow (BM), which promotes hematopoietic stem and progenitor cells (HSPCs) mobilization into the spleen, expanding the population of atherogenic monocytes to impact atherosclerosis. Next, we wanted to explore how a HSD increases TH17 cells in the BM. As salt is first sensed in the gut, we questioned if TH¬17 cells arose from the gut to influence the BM. We placed WT mice on a normal salt diet (NSD) or a HSD for 6 weeks and discovered that a HSD promotes gut permeability, increases gut TH17 cells and causes microbial dysbiosis. To explore how the HSD-induced effects in the gut influences HSPC mobilization, we first blocked TH17 cell migration from the gut with anti-CCL20. This resulted in a reduction in TH17 cells in the BM and HSPC mobilization. We then blocked intestinal permeability using AT-1001, demonstrating that dampening permeability prevented HSD-induced HSPC mobilization. Moreover, restoring the microbiome with prebiotics increased microbial diversity, decreased gut permeability and TH17 cell abundance, which was associated with a reduction in BM TH17 cells and HSPC mobilization. Finally, to determine how dampening gut inflammation influences HSD-induced atherosclerosis, we placed Apoe-/- mice on a NSD or HSD with or without prebiotics for 12 weeks. We found that supplementing HSD-fed mice with prebiotics not only decreased TH17 cells in the gut, blood, and BM, but also prevented HSPC mobilization from the BM to the blood and spleen, thereby reducing myelopoiesis and atherosclerosis. This suggests that targeting the gut using prebiotics can reduce TH17-driven myelopoiesis and atherosclerosis. Salt intake is often associated with increased risk of cardiovascular disease through its relationship with hypertension. However, a high salt diet (HSD) can directly modulate immune cells. We found that a HSD increases IL-17A-producing T helper (TH17) cells in the bone marrow (BM), which promotes hematopoietic stem and progenitor cells (HSPCs) mobilization into the spleen, expanding the population of atherogenic monocytes to impact atherosclerosis. Next, we wanted to explore how a HSD increases TH17 cells in the BM. As salt is first sensed in the gut, we questioned if TH¬17 cells arose from the gut to influence the BM. We placed WT mice on a normal salt diet (NSD) or a HSD for 6 weeks and discovered that a HSD promotes gut permeability, increases gut TH17 cells and causes microbial dysbiosis. To explore how the HSD-induced effects in the gut influences HSPC mobilization, we first blocked TH17 cell migration from the gut with anti-CCL20. This resulted in a reduction in TH17 cells in the BM and HSPC mobilization. We then blocked intestinal permeability using AT-1001, demonstrating that dampening permeability prevented HSD-induced HSPC mobilization. Moreover, restoring the microbiome with prebiotics increased microbial diversity, decreased gut permeability and TH17 cell abundance, which was associated with a reduction in BM TH17 cells and HSPC mobilization. Finally, to determine how dampening gut inflammation influences HSD-induced atherosclerosis, we placed Apoe-/- mice on a NSD or HSD with or without prebiotics for 12 weeks. We found that supplementing HSD-fed mice with prebiotics not only decreased TH17 cells in the gut, blood, and BM, but also prevented HSPC mobilization from the BM to the blood and spleen, thereby reducing myelopoiesis and atherosclerosis. This suggests that targeting the gut using prebiotics can reduce TH17-driven myelopoiesis and atherosclerosis.

Mobilization of Hematopoietic Stem and Progenitor Cells During Dengue Virus Infection

Mobilization of Hematopoietic Stem and Progenitor Cells During Dengue Virus Infection

Spiperone Stimulates Regeneration in Pulmonary Endothelium Damaged by Cigarette Smoke and Lipopolysaccharide.

BackgroundEndothelial dysfunction and destruction of the pulmonary microcirculation are important pathogenic factors in chronic obstructive pulmonary disease (COPD). In COPD, bronchial obstruction is associated with endothelial dysfunction. Thus, new pharmacological treatment options aimed at restoring the pulmonary endothelium represent a clinical need in COPD therapy. Notch1 has been shown to protect cells against apoptosis, inflammation, and oxidative stress caused by cigarette smoke extract (CSE). Therefore, drug which effect on Notch1 may be a potential therapeutic target for COPD in the future.MethodsIn this study, we assessed the potential of spiperone to mediate regeneration of pulmonary endothelium in model of pulmonary emphysema induced by a CSE and lipopolysaccharide (LPS) in female C57BL/6 mice.ResultsSpiperone increased the number of capillaries as well as the expression of the CD31 in the alveolar tissue compared to the controls. Moreover, application of spiperone prevented alveolar wall destruction (DI), and reduced the area of emphysema. Lastly, we demonstrated that spiperone positively influenced mobilization and migration of endothelial progenitor cells (EPC, CD45−CD34+CD31+), CD309+-endothelial cells, and angiogenesis precursors (CD45−CD117+CD309+) into the lung. Spiperone administration significantly reduced the number Notch1 positive CD309+-endothelial cells and Notch1+ EPCs.ConclusionOverall, our results suggest that spiperone mediates endothelial regeneration in an animal model of COPD. Thus, it could represent a novel therapeutic approach for treatment of emphysema associated with COPD.

Endothelial Progenitor Cells: An Appraisal of Relevant Data from Bench to Bedside.

The mobilization of endothelial progenitor cells (EPCs) into circulation from bone marrow is well known to be present in several clinical settings, including acute coronary syndrome, heart failure, diabetes and peripheral vascular disease. The aim of this review was to explore the current literature focusing on the great opportunity that EPCs can have in terms of regenerative medicine.

Vascular repair and regeneration in cardiometabolic diseases.

Chronic cardiometabolic assaults during type 2 diabetes (T2D) and obesity induce a progenitor cell imbalance in the circulation characterized by overproduction and release of pro-inflammatory monocytes and granulocytes from the bone marrow alongside aberrant differentiation and mobilization of pro-vascular progenitor cells that generate downstream progeny for the coordination of blood vessel repair. This imbalance can be detected in the peripheral blood of individuals with established T2D and severe obesity using multiparametric flow cytometry analyses to discern pro-inflammatory vs. pro-angiogenic progenitor cell subsets identified by high aldehyde dehydrogenase activity, a conserved progenitor cell protective function, combined with lineage-restricted cell surface marker analyses. Recent evidence suggests that progenitor cell imbalance can be reversed by treatment with pharmacological agents or surgical interventions that reduce hyperglycaemia or excess adiposity. In this state-of-the-art review, we present current strategies to assess the progression of pro-vascular regenerative cell depletion in peripheral blood samples of individuals with T2D and obesity and we summarize novel clinical data that intervention using sodium-glucose co-transporter 2 inhibition or gastric bypass surgery can efficiently restore cell-mediated vascular repair mechanisms associated with profound cardiovascular benefits in recent outcome trials. Collectively, this thesis generates a compelling argument for early intervention using current pharmacological agents to prevent or restore imbalanced circulating progenitor content and maintain vascular regenerative cell trafficking to sites of ischaemic damage. This conceptual advancement may lead to the design of novel therapeutic approaches to prevent or reverse the devastating cardiovascular comorbidities currently associated with T2D and obesity.

Novel Evidence That Alternative Activation Pathway of Complement Cascade (ComC) Regulates Optimal Homing and Engraftment of Hematopoietic Stem/Progenitor Cells (HSPCs) in Reactive Oxygen Species (ROS) - Nlrp3 Inflammasome-Dependent Manner

Background. We reported in the past that activation of the third (C3) and fifth element (C5) of complement cascade (ComC) is required not only for mobilization of hematopoietic stem progenitor cells (HSPCs) but also for their proper homing and engraftment after transplantation to bone marrow (BM) (Leukemia 2012; 26:106-16). The ComC consists of zymogen proteins that become activated in a cascade-mediated manner by the i) classical, ii) mannan-binding lectin (MBL), or iii) alternative pathway. However, it is not clear which of these pathways plays a crucial role in inducing state of sterile inflammation in recipient BM conditioned for transplantation. Interestingly, the alternative pathway of ComC activation, in contrast to the other two pathways, is not triggered by antibodies or specific structures expressed on the surface of invading microorganisms, but is continuously activated and “ticking” by the spontaneous hydrolysis of the third component of the ComC (C3), which is the most abundant complement protein present in blood plasma. This process of C3 hydrolysis is hyperactivated in response to tissue/organ damage and changes the structure of C3 in order to promote binding of factor B (FB) that initiates the amplification process by which more C3b molecules and C3b-Bb convertases are created responsible for activation of the ComC. We also recently demonstrated that homing and engraftment of HSPCs to BM is mediated by activation of innate immunity pattern recognition receptor Nlrp3 inflammasome (Leukemia 2020; 34:1512-1523). On the other hand reactive oxygen species (ROS) are known activators of Nlrp3 inflammasome. Hypothesis. We hypothesized that sensitive and “continuously ticking” in peripheral blood (PB) alternative pathway of ComC activation facilitates homing and engraftment of HSPCs in response to myeloablation triggered sterile inflammation of hematopoietic microenvironment in donor BM. We also hypothesized that this depends on the activation of the Nlrp3 inflammasome in ROS-dependent manner. Materials and Methods. Activation of ComC and Nlrp3 inflammasome in BM of conditioned for hematopoietic transplantation by lethal irradiation mice was evaluated by C5a ELISA assay and immunofluorescence glow assay measuring activation of Nlrp3 inflammasome product that is activated caspase-1, respectively. We also run shotgun proteomic analysis with BM conditioned media and BMMNCs extracts. The release of ROS was measured by a colorimetric assay. To assess the involvement of alternative pathway of ComC activation, we performed homing and engraftment experiments in wild-type (WT) and FB-deficient mice, that have defect in alternative pathway of ComC activation. FB-KO and WT animals were transplanted with WT BMMNC. We also analyzed changes in the BM microenvironment in response to lethal irradiation in WT and FB-KO mice at mRNA and protein level. Results. We demonstrate for a first time that conditioning for transplantation by myeloablative irradiation induces in BM state of sterile inflammation reflected by activation of ComC, the release of reactive oxygen species (ROS), and activation of Nlrp3 inflammasome in ROS-dependent manner. Moreover, as compared to WT animals, all these pathways were significantly inhibited in FB-KO mice. As a result of this FB-KO animals displayed defective homing and engraftment after transplantation of WT BMMNC. This correlated at molecular level by the decreased expression of cell adhesion molecules and group of structural proteins involved in so called “docking structures” necessary for cell migration and homing as well as for Nlrp3 inflammasome complex and caspase 1, 2, 4 and 6. Moreover, FB-KO mice demonstrated lower level of SDF-1 and KL in BM after myeloablative conditioning for transplantation. Conclusions. We provide for a first time an evidence that myeloablative conditioning for transplantation by lethal irradiation activates in ComC-ROS-Nlrp3 inflammasome - dependent manner a state of sterile inflammation in the BM microenvironment, required for optimal homing and engraftment. This data also explains this phenomenon at molecular level and provides an evidence for a crucial involvement of alternative pathway of ComC activation. DisclosuresNo relevant conflicts of interest to declare.

Mobilized Mouse and Human Peripheral Blood Containing Elevated Numbers of Donor Treg Cells Ameliorates Pre-Clinical GvHD and GVL Is Maintained in an MHC-Matched Allogeneic Murine Model

Mobilized hematopoietic stem / progenitor cells have become the primary option used for adult allogeneic hematopoietic stem cell transplants (aHSCTs) although GVHD remains a major immunologic complication (CIBMTR, 2017). The present studies are directed to developing a translational model for the application of donor regulatory T cells (Tregs) together with the use of mobilized peripheral blood (mPB) for more widespread application of these aHSCTs. We have previously reported that two-pathway Treg expansion strategy markedly elevates Treg numbers and function (Wolf, 2017; Copsel 2018). Additionally, this approach could be effectively applied in a concomitant strategy to amplify Treg suppressor activity in mPB to diminish pre-clinical GVHD (Barreras H, ASTCT Meeting S292 ,2019). Here, we present new findings using mPB transplants examining GVL reactivity in an MHC-matched minor antigen mismatched pre-clinical model and test the notion that human donor Tregs can be employed with mobilized healthy donor PB to effectively regulate xenogeneic GVHD.Donor C3H.SW (H2b) mice were treated with filgrastim for 4 days prior to use for transplants and PB analysis demonstrated significant elevations in c-kit + cells. Some PB donors were concurrently administered the two-pathway Treg expansion strategy (D-6 through D-1) consisting of TL1A-Ig, targeting TNFRSF25, and low dose huIL-2. A significant increase (up to 40%) in the frequency of CD4 +FoxP3 + Tregs occurred during the mobilization process. Pooled C3H.SW PB was collected from both mobilized and Treg unexpanded (“TrUM”) or mobilized and Treg expanded (“TrEM”) donors and transplanted into lethally irradiated, MHC-matched B6 (H2b) recipients. Recipients of TrEM donors exhibited significantly reduced weight loss and clinical GVHD scores compared to recipients of TrUM (Fig.1A). GVL responses were tested in animals administered B6-MLL-AF9 GFP leukemia cells administered at the time of transplant. Notably, recipients of TrEM exhibited comparable GVL activity to TrUM recipients evidenced by B6-MLL-AF9 GFP levels in bone marrow and spleen (Fig.1B).To begin to translate these findings, we tested the use of ex-vivo expanded human donor Tregs (huTregs) to ameliorate xenogeneic GVHD (xGVHD). First, sorted huTregs (CD4 +CD25 +CD127 lo) from a healthy donor were expanded ex-vivo using anti-CD3/anti-CD28 beads 1 week prior to transplant (Fig 2A). next, these expanded huTregs were combined with human mPB from the same healthy donor (6x10 6 PBMC) and transplanted into NSG/ NOD-scid IL2Rgamma null mice. We found that treatment with ex-vivo expanded huTregs resulted in significant reduction of mortality rate and clinical xGVHD (Fig 2B,C). Notably, 1 week post-transplant PB huTregs levels were still elevated and frequency of huCD4 +Tconv cells was diminished supporting xGVHD outcomes (Fig 2D).In total, these findings demonstrated that the use of mPB containing elevated Treg levels significantly reduced pre-clinical GVHD without loss of GVL activity in an MHC-matched allogeneic model. Moreover, utilizing ex-vivo expanded huTregs from a mPB donor and added back to the same donor at the time of transplant ameliorated xGVHD. The observations indicate that during the donor stem / progenitor cell mobilization process, manipulation of donor Tregs using our two-pathway strategy can be successfully accomplished in PB resulting in an effective and translational approach to ameliorate GVHD following aHSCT. In total, the present studies support the notion that in vivo or ex-vivo manipulation of donor tregs together with mobilized peripheral blood could provide therapeutic approaches to improve aHSCT outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Single Intraosseous Simvastatin Application Induces Endothelial Progenitor Cell Mobilization and Therapeutic Angiogenesis in a Diabetic Hindlimb Ischemia Rat Model.

Endothelial progenitor cells have shown the ability to enhance neovascularization. In this study, the authors tested whether intraosseous delivery of simvastatin could mobilize endothelial progenitor cells and enhance recovery in a hindlimb ischemia model. There are eight groups of rats in this study: normal control; type 1 diabetes mellitus control group control without drug intervention; and type 1 diabetes mellitus rats that randomly received intraosseous simvastatin (0, 0.5, or 1 mg) or oral simvastatin administration (0, 20, or 400 mg). All type 1 diabetes mellitus rats had induced hindlimb ischemia. The number of endothelial progenitor cells in peripheral blood, and serum markers, were detected. The recovery of blood flow at 21 days after treatment was used as the main outcome. The authors demonstrated that endothelial progenitor cell mobilization was increased in the simvastatin 0.5- and 1-mg groups compared with the type 1 diabetes mellitus control and simvastatin 0-mg groups at 1, 2, and 3 weeks. Serum vascular endothelial growth factor levels were significantly increased at 2 weeks in the simvastatin 0.5- and 1-mg groups, in addition to the increase of the blood flow and the gastrocnemius weight at 3 weeks. Similar increase can also been seen in simvastatin 400 mg orally but not in simvastatin 20 mg orally. These findings demonstrate that a single intraosseous administration of simvastatin mobilized endothelial progenitor cells at a dose one-hundredth of the required daily oral dose in rats, and this potent mobilization of endothelial progenitor cells markedly improved diabetic limb ischemia by means of neovascularization.

Type I interferon response and vascular alteration in chilblain‐like lesions during the COVID‐19 outbreak*

SummaryBackgroundThe outbreak of chilblain‐like lesions (CLL) during the COVID‐19 pandemic has been reported extensively, potentially related to SARS‐CoV‐2 infection, yet its underlying pathophysiology is unclear.ObjectivesTo study skin and blood endothelial and immune system activation in CLL in comparison with healthy controls and seasonal chilblains (SC), defined as cold‐induced sporadic chilblains occurring during 2015 and 2019 with exclusion of chilblain lupus.MethodsThis observational study was conducted during 9–16 April 2020 at Saint‐Louis Hospital, Paris, France. All patients referred with CLL seen during this period of the COVID‐19 pandemic were included in this study. We excluded patients with a history of chilblains or chilblain lupus. Fifty patients were included.ResultsHistological patterns were similar and transcriptomic signatures overlapped in both the CLL and SC groups, with type I interferon polarization and a cytotoxic–natural killer gene signature. CLL were characterized by higher IgA tissue deposition and more significant transcriptomic activation of complement and angiogenesis factors compared with SC. We observed in CLL a systemic immune response associated with IgA antineutrophil cytoplasmic antibodies in 73% of patients, and elevated type I interferon blood signature in comparison with healthy controls. Finally, using blood biomarkers related to endothelial dysfunction and activation, and to angiogenesis or endothelial progenitor cell mobilization, we confirmed endothelial dysfunction in CLL.ConclusionsOur findings support an activation loop in the skin in CLL associated with endothelial alteration and immune infiltration of cytotoxic and type I IFN‐polarized cells leading to clinical manifestations.

Low-dose metronomic doxorubicin inhibits mobilization and differentiation of endothelial progenitor cells through REDD1-mediated VEGFR-2 downregulation

Low-dose metronomic chemotherapy has been introduced as a less toxic and effective strategy to inhibit tumor angiogenesis, but its anti-angiogenic mechanism on endothelial progenitor cells (EPCs) has not been fully elucidated. Here, we investigated the functional role of regulated in development and DNA damage response 1 (REDD1), an endogenous inhibitor of mTORC1, in low-dose doxorubicin (DOX)-mediated dysregulation of EPC functions. DOX treatment induced REDD1 expression in bone marrow mononuclear cells (BMMNCs) and subsequently reduced mTORC1-dependent translation of endothelial growth factor (VEGF) receptor (Vegfr)-2 mRNA, but not that of the mRNA transcripts for Vegfr-1, epidermal growth factor receptor, and insulin-like growth factor-1 receptor. This selective event was a risk factor for the inhibition of BMMNC differentiation into EPCs and their angiogenic responses to VEGF-A, but was not observed in Redd1-deficient BMMNCs. Low-dose metronomic DOX treatment reduced the mobilization of circulating EPCs in B16 melanoma-bearing wild-type but not Redd1-deficient mice. However, REDD1 overexpression inhibited the differentiation and mobilization of EPCs in both wild-type and Redd1-deficient mice. These data suggest that REDD1 is crucial for metronomic DOX-mediated EPC dysfunction through the translational repression of Vegfr-2 transcript, providing REDD1 as a potential therapeutic target for the inhibition of tumor angiogenesis and tumor progression.

Tetraspanin CD82 regulates S1PR1-mediated hematopoietic stem and progenitor cell mobilization.

SummaryHematopoietic stem and progenitor cell (HSPC) mobilization into the blood occurs under normal physiological conditions and is stimulated in the clinic to enable the isolation of HSPCs for transplantation therapies. In the present study, we identify the tetraspanin CD82 as a novel regulator of HSPC mobilization. Using a global CD82 knockout (CD82KO) mouse, we measure enhanced HSPC mobilization after granulocyte-colony stimulating factor (G-CSF) or AMD3100 treatment, which we find is promoted by increased surface expression of the sphingosine 1-phosphate receptor 1 (S1PR1) on CD82KO HSPCs. Additionally, we identify a disruption in S1PR1 internalization in CD82-deficient HSPCs, suggesting that CD82 plays a critical role in S1PR1 surface regulation. Finally, combining AMD3100 and anti-CD82 treatments, we detect enhanced mobilization of mouse HSPCs and human CD34+ cells in animal models. Together, these data provide evidence that CD82 is an important regulator of HSPC mobilization and suggests exploiting the CD82 scaffold as a therapeutic target to enhance stem cell isolation.

Mobilization of progenitor cells and vessel healing after implantation of SYNERGY in acute coronary syndrome

This study was aimed to compare the vascular healing process of a SYNERGY stent with that of a PROMUS PREMIER stent in patients with acute coronary syndrome (ACS). In 71 patients with ACS, undergoing coronary stent implantation using the SYNERGY stent (n = 52) or PROMUS PREMIER stent (n = 19), we measured circulating CD34+/CD133+/CD45null cells and CD34+/KDR+ cells and observed vascular healing at the stented sites using optical coherence tomography (OCT) and coronary angioscopy. On the day 7, circulating CD34+/CD133+/CD45null cells increased in SYNERGY group (P < 0.0001), while it did not change in PROMUS group. The CD34+/KDR+ cells also increased in SYNERGY group (P < 0.0001) but less significantly in the PROMUS group (P < 0.05). The OCT-based neointimal thickness (P < 0.0005) and neointimal coverage rate (P < 0.05) at 12 months were greater in SYNERGY group, compared with PROMUS group. The coronary angioscopy-based neointimal coverage grade at 12 months was also greater in SYNERGY group (P < 0.001). In overall patients, the change in CD34+/KDR+ cells on the day 7 correlated with the OCT-based neointimal thickness at 12 months (R = 0.288, P < 0.05). SYNERGY stent seems to have potential advantages over PROMUS PREMIER stent for ACS patients in terms of vascular healing process at the stented sites.

Endothelial GABBR2 Regulates Post-ischemic Angiogenesis by Inhibiting the Glycolysis Pathway.

Purpose: Angiogenesis post-ischemia plays an essential role in preventing ischemic damage to tissue by improving the blood recovery. Determining the regulatory mechanism of ischemic angiogenesis, therefore, could provide effective therapeutics for ischemic injury.Materials and Methods: The RNA sequencing (RNA-seq) database was used to predict the association of gamma-aminobutyric acid type B receptor subunit 2 (GABBR2) with endothelial-specific expression. The role of GABBR2 in angiogenesis was verified in vitro by downregulating GABBR2 in human umbilical vein endothelial cells (HUVECs) with lentiviral vectors. Besides, the in vivo effect of GABBR2 on the blood recovery of an ischemic hindlimb was demonstrated by establishing a hindlimb ischemia model in normal and GABBR2 adenoviral vector-infected mice. Then, the mobilization of endothelial progenitor cells (EPCs) in peripheral blood post-ischemia was determined by flow cytometry. Finally, the XF analyzer and Western blot were used to determine the effect of GABBR2 on endothelial metabolism.Results: The RNA-seq results indicated a strong association between GABBR2 and endothelial revascularization, and the upregulation of GABBR2 was detected in both hypoxia-treated HUVECs and ischemic mouse hindlimb. Hypoxia treatment for 6 h increased the proliferation, migration, and tube formation of HUVECs, which were inhibited by GABBR2 knockdown. Additionally, GABBR2 downregulation significantly decreased the blood flow recovery of mouse ischemic hindlimb. The expressions of the EPC markers CD34+ and CD133+ significantly decreased in the peripheral blood in hindlimb post-ischemia. Mechanically, glycolysis-dominated metabolism of HUVECs was compromised by GABBR2 knockdown. Evidences of the decreased expressions of HKII, PFKFB3, and PKM1 also supported the compromised glycolysis induced by GABBR2 downregulation.Conclusion: Our study demonstrated that GABBR2 regulated angiogenesis post-ischemia by inhibiting the glycolysis pathway.