Levels Of Progenitor Cells Research Articles

Different impact of calreticulin mutations on human hematopoiesis in myeloproliferative neoplasms.

Mutations of calreticulin (CALRm) define a subtype of myeloproliferative neoplasms (MPN). We studied the biological and genetic features of CALR-mutated essential thrombocythemia and myelofibrosis patients. In most cases, CALRm were found in granulocytes, monocytes, B and NK cells, but also in T cells. However, the type 1 CALRm spreads more easily than the type 2 CALRm in lymphoid cells. The CALRm were also associated with an early clonal dominance at the level of hematopoietic stem and progenitor cells (HSPC) with no significant increase during granulo/monocytic differentiation in most cases. Moreover, we found that half of type 2 CALRm patients harbors some homozygous progenitors. Those patients were associated with a higher clonal dominance during granulo/monocytic differentiation than patients with only heterozygous type 2 CALRm progenitors. When associated mutations were present, CALRm were the first genetic event suggesting that they are both the initiating and phenotypic event. In blood, type 1 CALRm led to a greater increased number of all types of progenitors compared with the type 2 CALRm. However, both types of CALRm induced an increase in megakaryocytic progenitors associated with a ruxolitinib-sensitive independent growth and with a mild constitutive signaling in megakaryocytes. At the transcriptional level, type 1 CALRm seems to deregulate more pathways than the type 2 CALRm in megakaryocytes. Altogether, our results show that CALRm modify both the HSPC and megakaryocyte biology with a stronger effect for type 1 than for type 2 CALRm.

Angiogenic T cells are decreased in people with type 2 diabetes mellitus and recruited by the dipeptidyl peptidase‐4 inhibitor Linagliptin: A subanalysis from a randomized, placebo‐controlled trial (RELEASE study)

Angiogenic T (Tang) cells are mediators of vascular repair, and are characterized by surface expression of CXCR4. This receptor for stromal cell‐derived factor‐1α (SDF‐1α) is cleaved by dipeptidyl peptidase‐4 (DPP‐4). Tang cell levels were investigated in people with type 2 diabetes mellitus (T2DM) compared with matched healthy controls and after treatment with the DPP‐4 inhibitor Linagliptin. People with T2DM were randomized to 5 mg/day Linagliptin (n = 20) or placebo (n = 21) for 26 weeks. Tang cell frequency was identified in peripheral blood mononuclear cells (CD3+CD31+CXCR4+) and levels of endothelial progenitor cells (EPCs) (CD34+CD133+KDR+) were also assessed in whole blood. Circulating Tang cell levels were significantly lower in people with T2DM compared with the healthy control group. SDF‐1α levels increased significantly in Linagliptin‐treated people with T2DM compared to placebo, and a trend was observed in change of Tang cell levels, while EPC count did not change. In conclusion, circulating Tang cell levels were considerably lower in people with T2DM, while a trend was observed in recruitment of Tang cells after 26 weeks of treatment with Linagliptin. These data suggest that DPP‐4 inhibitors may potentially exert beneficial effects on bone marrow‐driven vascular repair.

Alterations in the Levels of Circulating and Endothelial Progenitor Cells Levels in Young Adults with Type 1 Diabetes: A 2-Year Follow-Up from the Observational METRO Study.

PurposeType 1 diabetes is associated with high risk of cardiovascular disease (CVD). Reduced levels of circulating progenitor cells (CPCs) and endothelial progenitor cells (EPCs) have been indicated as a risk factor for adverse cardiovascular outcomes and death in people at high cardiovascular risk. The aim of the present study was to evaluate the change in CPCs and EPCs levels in a population of young type 1 diabetic patients treated with intensive insulin regimen over a period of 2 years.Patients and MethodsA total of 204 type 1 diabetic patients, of whom 84 treated with insulin pump (CSII) and 120 with multiple daily insulin injections (MDI), completed a 2-year follow-up. Clinical measurements, including the indices of glycemic control and glucose variability, were collected at baseline and after 2 years. Both CPC and EPC cell count were assessed by flow cytometry.ResultsMean age of participants was 24.5 years and mean diabetes duration was 13.6 years. After 2 years, we found a significant reduction of HbA1c (−0.3% versus baseline, P <0.001), associated with decrease in mean amplitude of glucose excursion (MAGE) (−0.5 mmol/L versus baseline, P<0.001), continuous overall net glycemic action (CONGA) (−0.2 mmol/L versus baseline, P=0.006), and blood glucose standard deviation (BGSD) (−0.2 mmol/L versus baseline, P<0.001). The number of all EPCs phenotypes, but not CPC cell count, significantly raised up in the entire population, with higher increase in CSII group. MAGE resulted as an independent predictor for increased levels of both CD34+ (P = 0.020) and CD34+KDR+ (P = 0.004) cell count in the whole population.ConclusionOver a 2-year follow-up, young type 1 diabetic patients showed an increase in circulating EPCs levels, which was higher in patients with CSII. Glucose variability resulted as an independent predictor of the raised levels of EPCs in this selected population.

Aberrant Epithelial Cell Proliferation in Peripheral Airways in Bronchiectasis.

Dilation of bronchi and bronchioles caused by destruction and excessive epithelial remodeling is a characteristic feature of bronchiectasis. It is not known how epithelial progenitor cells contribute to these pathologic conditions in peripheral airways (bronchioles) in bronchiectasis. We aimed to explore the expression levels of signature airway progenitor cells in the dilated bronchioles in patients with bronchiectasis. We obtained the surgically resected peripheral lung tissues from 43 patients with bronchiectasis and 33 control subjects. Immunostaining was performed to determine the expression patterns of thyroid transcription factor-1 (TTF-1, for labeling progenitor cells in distal airways), P63 (basal cells), club cell 10 kDa protein (CC10, club cells), and surfactant protein C (SPC, alveolar type II epithelial cells) in epithelium or sub-epithelium. Here, we reported significantly lower percentage of TTF-1+ cells and CC10+ cells, and higher percentage of P63+ cells within the epithelium of dilated bronchioles compared with control bronchioles. In airway sub-epithelium of the dilated bronchioles, epithelial hyperplasia with disarrangement of TTF-1+ cells yielded cuboidal (100%) and columnar (93.0%) type among bronchiectasis patients. Most progenitor cell markers co-localized with TTF-1. The median (the 1st, 3rd quartile) percentage of P63+TTF-1+, CC10+TTF-1+, and SPC+TTF-1+ cells was 16.0% (8.9, 24.0%), 14.5% (7.1, 20.8%), and 52% (40.3, 64.4%), respectively. For cuboidal epithelial hyperplasia, 91.0% (86.5, 94.0%) of areas co-stained with SPC and TTF-1. Columnar epithelial hyperplasia was characterized by TTF-1 co-staining with P63+TTF-1+ and CC10+TTF-1+ cells. Taken together, aberrant proliferation of airway progenitor cells in both epithelium and sub-epithelium are implicated in bronchiectasis.

The effect of polyphenol-rich chardonnay seed supplements on peripheral endothelial function.

Peripheral endothelial dysfunction (PED) is associated with major adverse cardiovascular events. Similar to cardio-protective Mediterranean diet, Chardonnay seeds are rich in polyphenols, fibers, and grape seed oil. In this randomized double-blinded trial, we investigated safety and incremental benefit of Chardonnay seed polyphenols-rich (PR), compared to polyphenols-free (PF), supplements on PED. 89 patients with PED [reactive hyperemia-peripheral arterial tonometry (RH-PAT) index ≤ 2) were randomized 1:1 to 4.8g (6 capsules)/day PR or PF for 4months. PR and PF capsules had equal amounts of seed oil (~ 7% capsule weight), fibers, and carbohydrates, but only PR capsules contained polyphenols (~ 11% capsule weight). Baseline and follow-up RH-PAT indices (primary endpoint) and endothelial progenitor cells (EPC) levels were measured. Patients were asked to maintain their regular diet and exercise habits during study period. Baseline RH-PAT indices, demographics, lab values, and EPC levels were similar between groups. No significant adverse reactions to PR or PF were reported. Follow-up RH-PAT indices significantly increased (p < 0.05 vs. baseline) in both groups to similar extent (31 ± 39% PR vs. 49 ± 53% PF, p = 0.09). Circulating CD34 + EPCs equally increased (median + 55% vs. + 54%, p = 0.94) in PR and PF groups, respectively. PR and PF Chardonnay seed supplements were safe and improved peripheral endothelial function to similar extent in PED patients. However, there were no incremental benefits of PR over PF on RH-PAT indices or CD34 + EPC levels. Chardonnay seed supplements role in primary prevention of atherosclerosis, as add-on to healthy lifestyle and guidelines directed medical therapy, should be further explored. CLINICALTRIALS. NCT02093455.

Low-density granulocytes and monocytes as biomarkers of cardiovascular risk in systemic lupus erythematosus.

The aim was to evaluate the most relevant cell populations involved in vascular homeostasis as potential biomarkers of SLE-related cardiovascular disease (CVD). Low-density granulocytes (LDGs), monocyte subsets, endothelial progenitor cells, angiogenic T(Tang) cells, CD4+CD28null and Th1/Th17 lymphocytes and serum cytokine levels were quantified in 109 SLE patients and 33 controls in relationship to the presence of subclinical carotid atheromatosis or cardiovascular disease. A second cohort including 31 recent-onset SLE patients was also included. Raised monocyte and LDG counts, particularly those LDGs negative for CD16/CD14 expression (nLDGs), in addition to the ratios of monocytes and nLDGs to high-density lipoprotein-cholesterol (HDLc) molecules (MHR and nLHR, respectively), were present in SLE patients with traditional risk factors or subclinical atheromatosis but not in those who were CV-free, thus revealing their value in the identification of patients at risk of CVD, even at the onset of disease. Accordingly, nLDGs were correlated positively with carotid intima-media thickness (cIMT) and with inflammatory markers (CRP and IL-6). A bias towards more differentiated monocyte subsets, related to increased IFN-α and IL-17 serum levels, was also observed in patients. Intermediate monocytes were especially expanded, but independently of their involvement in CVD. Finally, CD4+CD28null, Th17 and Th1 lymphocytes were increased, with CD4+CD28null and Th17 cells being associated with cIMT, whereas endothelial progenitor and Tang cell levels were reduced in all SLE patients. The present study highlights the potential use of MHR and nLHR as valuable biomarkers of CVD risk in SLE patients, even at diagnosis. The increased amounts of nLDGs, monocytes, Th17 and senescent-CD28null subsets, coupled with reduced pro-angiogenic endothelial progenitor cells and Tang cells, could underlie the development of atheromatosis in SLE.

Clinical significance of endothelial progenitor cells in patients with liver cirrhosis with or without hepatocellular carcinoma.

The role of endothelial progenitor cells in patients with cirrhosis has seldom been investigated. This study was conducted to assess the clinical significance of circulating endothelial progenitor cells in patients with liver cirrhosis with or without hepatocellular carcinoma. A blood sample was collected once from patients with cirrhosis alone (n = 34) or cirrhosis and hepatocellular carcinoma (n = 46) and healthy controls (n = 27) for assessing levels of endothelial progenitor cells and vascular endothelial growth factor. Blood cells staining positive for CD34/CD133/KDR using flow cytometry were characterized as endothelial progenitor cells. Plasma vascular endothelial growth factor was quantified by ELISA. The levels of CD34/KDR-positive endothelial progenitor cells, CD133/KDR-positive endothelial progenitor cells, and vascular endothelial growth factor were higher in patients with cirrhosis ± hepatocellular carcinoma than in healthy controls (P = 0.017, P < 0.001 and P < 0.001, respectively). The levels of endothelial progenitor cells and vascular endothelial growth factor did not show statistical difference according to Child-Turcotte-Pugh class. There was a moderately significant correlation between vascular endothelial growth factor levels and hepatocellular carcinoma stage (ρ = 0.464, P = 0.001). Smoking, ascites, and portal vein thrombosis were independently related to lower levels of circulating CD34/KDR-positive endothelial progenitor cells, higher levels of CD133/KDR-positive endothelial progenitor cells, and higher levels of vascular endothelial growth factor, respectively (P = 0.041, P = 0.023, and P < 0.001, respectively). Circulating endothelial progenitor cells and plasma vascular endothelial growth factor levels were higher in patients with liver cirrhosis ± hepatocellular carcinoma compared to healthy controls. The increase in endothelial progenitor cells and vascular endothelial growth factor may have a possible role in the development of complications, especially ascites and portal vein thrombosis, or in progression of hepatocellular carcinoma.

Cardiovascular risk in patients with plaque psoriasis and psoriatic arthritis without a clinically overt cardiovascular disease: the role of endothelial progenitor cells.

Psoriasis is an autoimmune, chronic disease determined by environmental and genetic factors. The occurrence of psoriasis is accompanied by metabolic diseases, cardiovascular diseases (CVD) and depression, disturbances on interpersonal interactions and a tendency towards social isolation. Regardless of the form of psoriasis and the severity of the disease, early arterial lesions are recorded in arterial vessels of patients. Nevertheless, the chance of CVD is higher in the population of patients with severe psoriasis than in patients with mild to moderate psoriasis. The correlation between the presence of atherosclerotic plaque and psoriatic plaque is partially explained by: (1) a similar inflammatory pathway – via the T helper cells, (2) impaired angiogenesis, and (3) endothelial dysfunction. In the considered tests, the diagnostic tools used showed a reduced level of endothelial progenitor cells in the circulation of patients with psoriasis. Endogenous angiopoietin stimulation in patients with psoriasis leads to deterioration of endothelial regeneration, atherosclerosis which secondarily contributes to the progression of heart failure. Clinical and experimental data confirm the potential of immunomodulatory methods to combat both autoimmune and cardiovascular diseases through the use of immunosuppressive drugs. Full understanding of the way in which CVD develops in patients with autoimmune diseases would enable the implementation of targeted cell therapy allowing the quality and life expectancy of patients to be improved. Modern cellular diagnostic tools allow the use of highly specific biomarkers, which in the near future will enable a reduction in morbidity and mortality due to CVD.

The effect of neoadjuvant chemotherapy on the level of bone marrow progenitor cells in the blood of patients with invasive breast carcinoma

The breast cancer occupies the first place in the structure of women cancer morbidity and mortality for many years. The treatment of this pathology includes two types of chemotherapy: neoadjuvant and adjuvant. Neoadjuvant chemotherapy (NACT) follows surgical treatment and makes it possible to assess the sensitivity of the tumor to the medication. The obtained data can be used to correct the subsequent adjuvant chemotherapy. However, there is a lot of evidence of the ability of NACT to increase the risk of progression of malignant tumors. The bone marrow progenitor cells are components of premetastatic niches. Objective: to assess the effect of neoadjuvant chemotherapy on the level of bone marrow progenitor cells in the blood of patients with breast cancer. In a prospective study were included 31 patients newly diagnosed with invasive breast cancer, of which 17 patients was performed neoadjuvant chemotherapy, 14 patients - without neoadjuvant chemotherapy. The method of multicolor flow cytometry was used to assess the dynamics of bone marrow progenitor cells (hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), endothelial progenitor cells (EPC), mesenchymal stem cells (MSC) in blood of patients with invasive carcinoma of a non-specific type during NACT. It has been shown that neoadjuvant chemotherapy leads to a statistically significant increase the number of endothelial cell precursor cells (EPC) in the blood of patients with invasive breast carcinoma (p = 0.036). The level of mesenchymal stem cells in the blood of patients with invasive breast carcinoma increases at the level of a statistical trend (p = 0.06) during neoadjuvant chemotherapy. Based on these data we can conclude that neoadjuvant chemotherapy enhances the recruitment of bone marrow cells involved in the formation of premetastatic niches.

Fluctuations in circulating endothelial progenitor cell levels and acute cardiac graft rejection.

Endothelial progenitor cells (EPCs) in nontransplant settings have reparative properties. However, their role in heart transplantation (HT) is not well defined. The aim of this study was to prospectively evaluate changes in EPC levels in relation to post‑HT rejection. EPC levels were measured in 27 HT recipients for 6 months after HT. Acute cellular rejection (ACR) or antibody‑mediated rejection (AMR) were assessed by right ventricular endomyocardial biopsy. ACR and AMR were observed in 7 (25.9%) and 6 (22.2%) patients, respectively. The ACR status at 1 month post‑HT did not differ with respect to EPC immediately post‑HT. At 1 month post‑HT in patients without ACR or AMR, EPC levels were significantly reduced compared with the measurements immediately post‑HT (P <0.001). On further follow‑up, EPC levels were similar regardless of the rejection events. Nonetheless, greater changes (coefficient of variation) in EPClog (logarithmic transformation) were associated with the risk of AMR or ACR compared with those without any rejection event (median [lower-upper quartile], 15 [13-18] vs 8 [5-13]; P = 0.02 and 22 [14-26] vs 8 [5-13]; P = 0.01, respectively). The receiver operating characteristic curve showed that the coefficient of variation of EPClog of 12 was the optimal cutoff value for the prediction of rejection (area under the curve = 0.85). Higher levels were associated with greater risk of ACR or AMR (P <0.005). Early reduction of EPC levels was related to a lower risk of ACR or AMR. Greater changes of EPC‑levels during follow‑up were associated with a significantly higher risk of rejection.

Long-Term Hydroxyurea Use Is Associated with Lower Levels of Hematopoietic Stem and Progenitor Cells in Patients with Sickle Cell Disease

Background Sickle cell disease (SCD) is curable by transplantation and potentially by gene therapy, and is generally treated by a combination of blood transfusion and Hydroxyurea (HU). Characterizing the hematopoietic system in SCD patients is important because the long-term effect of HU treatment are not known, and because of lower than expected efficacy of transduction and transplantation in Hematopoietic Stem and Progenitor Cells (HSPCs) in recent gene therapy trials. Previous studies have shown that the number of bone marrow (BM) CD34+ cells is elevated in SCD patients and that HU treatment is associated with decreased level of CD34+ cells in the peripheral blood (PB) and BM relative to steady state patients. However, hematopoiesis in SCD patients naive or treated with HU or transfusion remains poorly understood. Here, we report on the characterization of the HSPC compartment in patients with SCD by prospective isolation of 49f+ long-term Hematopoietic Stem Cells (49f+LT-HSCs), Multipotent Progenitors (MPPs), Common Myeloid Progenitors (CMPs), Megakaryocyte-Erythroid Progenitors (MEPs), and Granulocyte-Monocyte Progenitors (GMPs). Methods After obtaining consent, PB and/or BM were collected from 69 patients with HBSS/SB0, aged 12 to 45years, and 25 healthy adult African American controls. Patients were divided into chronic transfusion therapy (n=19), HU (n=31) and naïve (n=19) groups. Frozen mono-nuclear cells were analyzed by flow cytometry on a BD LSRII using CD 49f, 90 45Ra, 123, 235a, 38, 34, 33 and lineage antibodies. Results FACS analysis revealed that the number PB CD34+ cells was 2.5 CD34+/uL of blood in the HU group as compared to 19 CD34+/uL in the exchange and naive groups, and 7.3 CD34+/uL in the control group (q-value &amp;lt;0.05 in all cases). Analysis with additional markers revealed that the decrease in circulating HSPCs in the HU group affected the entire hematopoietic system since the number of 49f+LT-HSCs, MPPs, CMPs, MEPs were all significantly lower in the HU group. The decrease in cell number in the HU group, however, was not homogeneous. The proportion of LT-HSCs was higher in the HU and transfusion groups when compared to the naive and control groups. The HU group also had the lowest proportion of MPPs and GMPs, as well as the highest proportion of MEPs. We then investigated hematopoiesis as a function of the length of HU treatment to elucidate the long-term treatment effect of this cytotoxic agent. Patients &amp;gt; 18 years of age that had been treated on HU for at least three years exhibited a strong statistically significant negative correlation between years on HU and CD34+/uL (R2 = 0.41), LT-HSC/uL (R2 = 0.35), MPP/uL (R2 =0.43), CMP/uL (R2 = 0.37), MEP/uL (R2 = 0.25) and GMP/uL (R2 0.39, p&amp;lt;0.01 in all cases). Importantly there was no correlation between WBC counts, age, HU dose, or serum erythropoietin level versus the numbers of any HSPC/uL. Lastly, we compared the number of HSPCs in paired PB and BM samples 10 controls and 4 SCD patients. This revealed that the numbers of CD34+, HSCs, MPPs, CMPs, GMPs and MEPs in the PB and BM were well correlated (r2 in 0.6-0.8 range) suggesting that in first approximation, results obtained in the PB reflect changes in the BM rather than changes in egress of HSPCs from the BM. Discussion We have observed lower numbers of circulating CD34+,49f+LT- HSCs, MPPs, CMP, GMP and MEPs in individuals with HBSS on HU therapy when compared to naive, chronic transfusion and, to a lesser extent, controls. Furthermore we observed subtle differences in the proportion of various circulating stem and progenitor cells (HSPCs) suggesting that the various treatments affects hematopoiesis in complex ways. The strong negative correlations between the length of HU treatment and the numbers of HSPCs can be explained either by decreased cell mobilization to the periphery, or by a depletion of the HSPC numbers in the BM overtime. Most patients undergoing gene therapy trials are currently taken off HU and placed on transfusion therapy for several months to increase CD34+ cell collection and LT-HSC transduction efficiency. We observed a greater number of circulating 49f+LT-HSC/uL of blood in the transfusion group than in the HU group, but the proportion of 49f+LT-HSC relative to the number of CD34+ were similar in both groups. Functional studies may help determine whether 49f+LT-HSCs from the transfusion group are qualitatively different from of the HU group and more amenable to gene therapy. Figure Disclosures Manwani: Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Minniti:Doris Duke Foundation: Research Funding.

P4712Hyperbaric oxygen therapy enhanced circulating levels of endothelial progenitor cells and angiogenesis biomarkers, blood flow in ischemic area in patients with peripheral arterial occlusive disease

Abstract Background Arterial atherosclerotic occlusive syndrome remains the leading cause of mortality and morbidity worldwide. Peripheral arterial occlusive disease (PAOD) is a manifestation of atherosclerosis in the lower extremities. In our previous preclinical study, hyperbaric oxygen (HBO) therapy improved ischemic PAOD mainly through an increase of vascular wall permeability and recruiting endothelial progenitor cells (EPCs) to enhance the angiogenesis and blood flow in the ischemic area. Purpose This study tested the hypothesis that hyperbaric oxygen (HBO) therapy enhanced the circulating levels of endothelial progenitor cells (EPCs), soluble angiogenesis factors and blood flow in ischemic area in patients with peripheral arterial occlusive disease (PAOD). Methods 57 consecutive patients with PAOD undergoing the HBO therapy (3 atm for 2h/each time) were prospectively enrolled into the present study. The venous blood sampling was drawn for assessing the circulating levels of EPCs and soluble angiogenesis factors prior to and during five times of HBO therapy. Additionally, skin perfusion pressure (SPP), an indicator of blood flow at ischemic area, was measured by moorVMS-PRES. Results The results demonstrated that the circulating levels of EPCs (CD34+/CD133+/CD45dim, CD31+/CD133+/CD45dim, CD34+) and soluble angiogenesis factors (VEGF/SDF-1α/HGF/FGF) were significantly increased in post-HBO therapy than in pre-HBO therapy (all p&lt;0.01). Additionally, Matrigel assay exhibited that the angiogenesis was significantly increased in post-HBO therapy than prior to the therapy (p&lt;0.001). Furthermore, the SPP was significantly increased in ischemic area (i.e., plantar foot and mean SPP of the ischemic foot) in post-HBO therapy than in pre-HBO therapy (all p&lt;0.01). Importantly, the HBO therapy did not show any complication and all the patients were uneventfully discharged without amputation. Conclusions HBO therapy augmented circulating levels of EPCs and angiogenesis factors as well as improved the blood flow in the ischemic area. Acknowledgement/Funding Kaohsiung Chang Gung Memorial Hospital, Taiwan Society of Stem Cell Research

FUNCTIONAL DISSECTION OF TRANSCRIPTION FACTOR NETWORKS GOVERNING HAEMATOPOIETIC PROGENITOR STATES

Maintenance of the adult haematopoietic system requires carefully balanced cell fate decisions at the level of multipotent progenitor cells. These decisions are controlled by complex transcription factor (TF) networks. While some TF-target connections have been experimentally established, they are mostly inferred from correlations of either gene expression (RNA-Seq) or genome-wide binding events (ChIP-Seq). This limits our understanding and prevents informed manipulation of cell states. To address these issues, we used CRISPR/Cas9 system and small scale RNA-Seq to experimentally assay gene regulatory functions of 38 TFs in the Hoxb8-FL cellular system, which captures in vitro a lymphoid-primed multipotent progenitor (LMPP) like state. Our RNA-Seq analysis following each TF knockout revealed >15,000 functional activating/inhibitory relationships across >7000 genes. Network analysis augmented with genome-wide TF binding information predicted direct interactions, regulatory elements and putative new circuits maintaining the multipotent state. Among many others, this included a novel interplay between co-existing Ebf1 and Gata3 regulating expression of lymphoid and B-cell specific genes. To gain further insights into how TFs regulate cell state transitions, we developed a new computational method that visualises perturbation effects in the context of single cell expression landscapes. Importantly, this avoids using externally curated ontology databases and instead directly employs human or mouse expression data. In the current study, the analysis revealed how each TF is linked with diverse differentiation programs. In addition to revealing known functions, we also identified previously unrecognized associations, such as Meis1 suppression of the monocytic programme. Studies are underway to validate new circuits and their influence on differentiation in primary cells.

Circulating endothelial and progenitor cells in age-related macular degeneration.

To evaluate circulating endothelial and circulating progenitor cells as biomarkers in age-related macular degeneration patients (both exudative and atrophic forms) in order to establish the possible clinical implication of their assessment. We have enrolled 44 age-related macular degeneration patients: 22 patients with a recently diagnosed exudative (neovascular) form (Group A) and 22 patients with an atrophic (dry) form (Group B). The control group consisted of 22 age and sex-matched healthy subjects (Group C). The number of circulating endothelial progenitor cells (CD34+/KDR+, CD133+/KDR+, and CD34+/KDR+/CD133+), circulating progenitor cells (CD34+, CD133+, and CD34+/CD133+), and circulating endothelial cells were determined in the peripheral venous blood samples by flow cytometry. Neovascular age-related macular degeneration patients were evaluated at baseline and 4 weeks after a loading phase of three consequent intravitreal injections of ranibizumab. Comparing age-related macular degeneration patients with the control group, endothelial progenitor cell and circulating progenitor cell levels were not significantly different, while age-related macular degeneration patients showed significantly higher levels of circulating endothelial cells (p = 0.001). Anti-vascular endothelial growth factor treatment with intravitreal ranibizumab was associated with a significant reduction of endothelial progenitor cell levels, with no significant influence on circulating progenitor cells and circulating endothelial cells. We reported higher levels of circulating endothelial cells in age-related macular degeneration patients in comparison with the control group, thereby supporting the hypothesis of an involvement of endothelial dysregulation in the age-related macular degeneration and a reduction of the endothelial progenitor cell level in neovascular age-related macular degeneration patients after three intravitreal injections of ranibizumab.

The effect of post-stroke hyperglycaemia on the levels of brain damage and repair-related circulating biomarkers: the Glycaemia in Acute Stroke Study II.

The aim was to identify whether post-stroke hyperglycaemia (PSH) influences the levels of circulating biomarkers of brain damage and repair, and to explore whether these biomarkers mediate the effect of PSH on the ischaemic stroke (IS) outcome. This was a secondary analysis of the Glycaemia in Acute Stroke II study. Biomarkers of inflammation, prothrombotic activity, endothelial dysfunction, blood-brain barrier rupture, cell death and brain repair processes were analysed at 24-48h (baseline) and 72-96h (follow-up) after IS. The associations of the biomarkers and stroke outcome (modified Rankin Scale score at 3months) based on the presence of PSH were compared. A total of 174 patients participated in this sub-study. Brain-derived neurotrophic factor (BDNF) at admission was negatively correlated with glucose levels. PSH was associated with a trend toward higher levels of endothelial progenitor cells (EPCs) at baseline. The EPCs in the PSH group then decreased in the follow-up samples (-8.5±10.3) compared with the non-PSH group (4.7±7.33; P=0.024). However, neither BDNF nor EPC values had correlation with the 3-month outcome. Higher interleukin-6 at follow-up was associated with poor outcomes (modified Rankin Scale >2) independently of PSH. Post-stroke hyperglycaemia appears to be associated with a negative regulation of BDNF and a different reaction in EPC levels. However, neither BDNF nor EPCs showed significant mediation of the PSH association with IS outcome, and only higher interleukin-6 in the follow-up samples (72-96h) was related to poor outcomes, independently of PSH status. Further studies are needed to achieve definite conclusions.

Amyotrophic lateral sclerosis: characteristics of the immunophenotype of hematopoietic precursor cells as a potential biomarker for early diagnostics of fatal disease

Amyotrophic lateral sclerosis also known as motor neuron disease is a fatal neurodegenerative disease that manifests by degeneration of motor neurons, hypotrophy and atrophy of the muscles. The causes and pathogenesis of amyotrophic lateral sclerosis are not clear so far, the effective therapy is absent. Amyotrophic lateral sclerosis is diagnosed by clinical and neurophysiologic examination and only when over 80% of motor neurons are dead. The multiparameter flow cytometry was used to evaluate the expression of HLA-DR, CD38, CD117, CD13, CD33, CD56, CD90, CD45, CD10, CD71 in 86 samples of the mobilized hematopoietic stem cells from 54 amyotrophic lateral sclerosis cases and in 61 samples of mobilized hematopoietic stem cells from 54 healthy donors. The analysis showed differences in the hematopoietic stem cells subpopulations of amyotrophic lateral sclerosis donors as compared to those of healthy donors and allowed for the introduction of the notion of the amyotrophic lateral sclerosis-specific immu-nophenotypic profile of hematopoietic stem cells membrane antigens. The profile allows for verification of neurospecific immune insufficiency at the level of progenitor cells of the bone marrow and diagnostics of the family and sporadic amyotrophic lateral sclerosis in a molecular-biological way at the earliest stage before clinical manifestation of the disease. We suppose that the amyotrophic lateral sclerosis makes its debut as the disease of hematopoietic stem cells and manifests as pathologic changes at the level of hematopoietic stem cells genome and proteome that are represented in the subpopulation composition of hematopoietic stem cells and their immunophenotypic characteristics, becoming the cause of genetically determined genuine autoimmune origin of the disease so that the motor neuron disease manifests only in the end. However, further research with larger samples and experimental check of the evidence is required.

CD-34+ and VE-cadherin+ endothelial progenitor cells in preeclampsia and normotensive pregnancies.

The objective of our study was to determine levels of endothelial progenitor cells (EPCs) in preeclampsia and normotensive pregnant women. Prospective cohort study of women with preeclampsia and normotensive pregnancies. EPCs were estimated by flow cytometry. Multiple linear regression was used to assess the association of EPCs with preeclampsia adjusting for maternal age, body mass index (BMI), gestation and ethnicity. Levels of EPCs in preeclampsia and normotensive pregnancies, with CD-34 and vascular endothelial (VE)-cadherin as markers of EPCs. VE-cadherin is an endothelial cell adhesion molecule used to delineate endothelial lineage of EPCs. There were thirty women in the preeclampsia group and thirty-three in the normotensive group. The two groups were similar except for the BMI and blood pressures, which were higher in preeclampsia. On multiple linear regression, EPCs numbers were significantly higher by 29 (95% confidence interval 11.7-46.6, p = 0.001) in preeclampsia compared to the normotensive group. There was significant positive correlation between EPCs and systolic blood pressure in preeclampsia (Spearman correlation coefficient 0.39, p = 0.03). Although widely used in cardiovascular disease other than preeclampsia, this is the first study using VE-cadherin as a marker of endothelial lineage to define EPCs in preeclampsia. Our results suggest the higher number of EPCs in preeclampsia may be a response of the bone marrow to endothelial injury.

Red Blood Cell Distribution Width, Vascular Aging Biomarkers, and Endothelial Progenitor Cells for Predicting Vascular Aging and Diagnosing/Prognosing Age-Related Degenerative Arterial Diseases.

The emerging evidence emphasizes red blood cell distribution width (RDW) as optimal prognostic biomarker for cardiovascular diseases. However, several clinical biases impede its clinical application. Recent recommendations suggest combining RDW with other biomarkers. Accordingly, we propose evaluating the well-recognized biomarkers of vascular aging (i.e., the leukocyte telomere length and telomerase activity, and reduced levels of endothelial progenitor cells [EPCs]) with RDW, for predicting the risk for vascular aging and onset and prognosis of age-related degenerative arterial diseases, such as sporadic ascending aorta aneurysm (AAA), characterized to have an increased incidence in old people. Consequently, in this study (and for the first time), we simultaneously investigated the relationship between RDW values, systemic inflammatory molecules, mean values of leukocyte telomere length, telomerase activity and EPCs, and the risk for vascular aging and AAA onset and prognosis. To achieve this aim, we selected 80 old and 80 young healthy subjects and 80 AAA cases. Appropriate methodologies were used for assessing blood parameters, aorta alterations, genotyping, impairment of the leukocyte telomere length, and telomerase activity. The main findings obtained demonstrated that increased RDW values along with the augmented blood levels of high-sensitive C-reactive protein and the reduced mean values of both leukocyte telomere length, telomerase activity, and EPCs are independently associated with the high risk for both vascular aging and AAA onset and prognosis. They might be used as the best predictor biomarker profile for vascular aging, and for both diagnosis and outcome of sporadic AAA.

High levels of endothelial progenitor cells and circulating endothelial cells in patients with Behcet’s disease and their relationship to disease activity

AbstractBackground: Behcet’s disease is a multisystemic vasculitis, associated with vascular endothelial dysfunction. Currently, the prognosis is unpredictable, because there is still no valid laboratory marker indicating the disease activity in Behcet’s disease. Endothelial progenitor cells and circulating endothelial cells are newly introduced hematological markers which are presumed to take part in the pathogenesis of vasculitis.Objectives: To evaluate the levels of endothelial progenitor cells and subtypes and circulating endothelial cells in patients with Behcet’s disease and to describe their relationship with the disease activity.Methods: A total of 45 patients with Behcet’s disease and 28 healthy controls were included in the study. Endothelial progenitor cells (CD34+CD133+KDR+ as early endothelial progenitor cells and CD34+KDR+ as late endothelial progenitor cells), and circulating endothelial cells (CD34+CD133+) were measured by flow cytometry.Results: The mean plasma level of endothelial progenitor cells and circulating endothelial cells, vascular endothelial growth factor, matrix metalloproteinase-9, C-reactive protein, and erythrocyte sedimentation rate were significantly higher in patients with Behcet’s disease. All of these parameters except circulating endothelial cells were also found to be higher in patients with active disease than in patients with inactive disease. Early endothelial progenitor cells showed significant correlations with C-reactive protein and circulating endothelial cells.Study limitations: The cross-sectional nature of the study and patient characteristics such as being under treatment, which can affect endothelial progenitor cells numbers.Conclusion: The increase in endothelial progenitor cells may play an essential role in the repair of endothelial injury in Behcet’s disease, especially in the active period of the disease. Thus, endothelial progenitor cells can indicate the disease activity. In addition, endothelial progenitor cells and circulating endothelial cells can be used as endothelial repair and injury markers for Behcet’s disease, respectively.

Circulating endothelial cells as biomarker for cardiovascular diseases

BackgroundEndothelial dysfunction is involved in several cardiovascular diseases. Elevated levels of circulating endothelial cells (CECs) and low levels of endothelial progenitor cells (EPCs) have been described in different cardiovascular conditions, suggesting their potential use as diagnostic biomarkers for endothelial dysfunction. Compared to typical peripheral blood leukocyte subsets, CECs and EPCs occur at very low frequency. The reliable identification and characterization of CECs and EPCs is a prerequisite for their clinical use, however, a validated method to this purpose is still missing but a key for rare cell events. ObjectivesTo establish a validated flow cytometric procedure in order to quantify CECs and EPCs in human whole blood. MethodsIn the establishment phase, the assay sensitivity, robustness, and the sample storage conditions were optimized as prerequisite for clinical use. In a second phase, CECs and EPCs were analyzed in heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction, in arterial hypertension (aHT), and in diabetic nephropathy (DN) in comparison to age‐matched healthy controls. ResultsThe quantification procedure for CECs and EPCs showed high sensitivity and reproducibility. CEC values resulted significantly increased in patients with DN and HFpEF in comparison to healthy controls. CEC quantification showed a diagnostic sensitivity of 90% and a sensitivity of 68.0%, 70.4%, and 66.7% for DN, HFpEF, and aHT, respectively. ConclusionA robust and precise assay to quantify CECs and EPCs in pre‐clinical and clinical studies has been established. CEC counts resulted to be a good diagnostic biomarker for DN and HFpEF.