Viable Circulating Endothelial Cells Research Articles

Endothelial Progenitor Cells as Biomarkers of Cardiovascular Pathologies: A Narrative Review.

Endothelial progenitor cells (EPC) may influence the integrity and stability of the vascular endothelium. The association of an altered total EPC number and function with cardiovascular diseases (CVD) and risk factors (CVF) was discussed; however, their role and applicability as biomarkers for clinical purposes have not yet been defined. Endothelial dysfunction is one of the key mechanisms in CVD. The assessment of endothelial dysfunction in vivo remains a major challenge, especially for a clinical evaluation of the need for therapeutic interventions or for primary prevention of CVD. One of the main challenges is the heterogeneity of this particular cell population. Endothelial cells (EC) can become senescent, and the majority of circulating endothelial cells (CEC) show evidence of apoptosis or necrosis. There are a few viable CECs that have properties similar to those of an endothelial progenitor cell. To use EPC levels as a biomarker for vascular function and cumulative cardiovascular risk, a correct definition of their phenotype, as well as an update on the clinical application and practicability of current isolation methods, are an urgent priority.

Angio-Phenotype of Pediatric Acute Lymphoblastic Leukemia and Pharmacodynamic Evaluation of the Antiangiogenic Activity of Corticosteroids.

Corticosteroids are essential in the treatment of pediatric acute lymphoblastic leukaemia (ALL). Corticosteroid anti-angiogenic activity is postulated but unproven.Objectives: To assess the kinetics of cellular and plasma angiogenic surrogate markers during 7-day prednisone pre-phase/induction therapy in the AIEOP ALL 2000 protocol.Methods: 17 diagnosed common ALL and 12 normal controls were enrolled. Total and viable circulating endothelial cells (CECs) and progenitors (CEPs), vascular endothelial growth factor receptors (VEGFR2, R3), endoglin (CD105), VECadherin (CD144) expression were analyzed by four color flow-cytometry on peripheral blood. Plasma circulating VEGF, VEGFRs and endoglin were analyzed by ELISA. Gene expression of VE-Cadherin, Nanog, RGS5, VEGFR2 and R3 were quantified by Real Time RT-PCR.Results: Total CECs (31 ±8cells/uL), viable CECs (12 ± 5 cells/uL) and CEPs (0.91 ±0.91 cells/uL) were found to be significantly higher in pediatric controls than in adult. At baseline patients demonstrated a statistically higher number of CECs (CD45− CD31+ CD146+ cells) and CEPs (CD133+ cells) p=0.01 and 0.03, compared to controls. CD105, VEGFR-3 and VEGFR-2 expression on leukemic blasts was 91%, 27% and 0% of patients, respectively.After 7 days of prednisone, a statistically significant reduction of total and viable CECs (p= 0.01) and CEPs ( p<0.0001) as well as endothelial cells expressing VEGFR2, R3, and CD105 (p=0.01, p=0.03, p=0.02) was observed. Plasma VEGFR2, VEGFR3, and endoglin showed a trend to decrease at day 8 and 33 but did not reach significance. A statistically significant reduction of blast was also observed.The pro-apototic effect of prednisone was greater on blast compared to lymphocytes.VEGFR-3 gene expression was significantly increased in patients at diagnosis compared to pediatric controls, and significantly reduced on day 8.Conclusions:Increase in CECs, CEPS confirm a pro-angiogenic activity at diagnosis in common ALL. The anti-leukemic effects of prednisone may be linked to its pro-apoptotic and anti-angiogenic activities. Total and viable CECs, CEPs, expression of endoglin and VEGFR3 on blast appear to be suitable angiogenic surrogate biomarkers.

Dynamics of circulating endothelial cells and endothelial progenitor cells in breast cancer patients receiving cytotoxic chemotherapy

BackgroundThe abundance of circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CEPs), which serve as surrogate markers for angiogenesis, may be affected by chemotherapy. We studied their dynamic change during consecutive cycles of chemotherapy.MethodsWe collected blood samples from 15 breast cancer patients, who received a total of 56 courses of systemic chemotherapy, and measured the CECs, viable CECs (V-CECs), and CEPs by six-color flow cytometry within the seven days prior to chemotherapy, twice a week during the first and second cycles of chemotherapy, and then once a week during the subsequent cycles.ResultsThe CEC, V-CEC, and CEP levels all significantly decreased from day 1 of treatment to the first week of chemotherapy. After one week of chemotherapy, the CEC and V-CEC levels returned to a level similar to day 1. The CEP level remained significantly reduced after the first week of chemotherapy, but gradually rebounded until the next course of chemotherapy. After six cycles of chemotherapy, the total number of CEC and V-CEC cells trended toward a decrease and the CEP cells toward an increase. Clinical factors, including the existence of a tumor, chemotherapy regimens, and the use of granulocyte colony stimulating factor, did not significantly affect these results.ConclusionsThe CEC and CEP counts change dynamically during each course of chemotherapy and after the chemotherapy cycles, providing background data for any future study planning to use CECs and CEPs as surrogate markers of angiogenesis in antiangiogenesis treatments combined with chemotherapy.

Enhanced anti-tumor and anti-angiogenic effects of metronomic cyclophosphamide combined with Endostar in a xenograft model of human lung cancer

Standard chemotherapy for advanced NSCLC has reached a therapeutic plateau. More effective strategies must be explored. The purpose of this study was to evaluate the role of metronomic chemotherapy combined with an angiogenesis inhibitor in non-small cell lung cancer (NSCLC). A total of 114 BALB/c nude mice were inoculated subcutaneously with human NSCLC cells (A549), and when xenograft tumors were palpable, mice were randomly injected with saline as controls (Ctrl), or treated with metronomic cyclophosphamide (MET CPA), recombinant human endostatin, Endostar (Endo), MET CPA combined with Endostar (MET CPA + Endo) or maximum tolerance dose of CPA (MTD CPA), respectively. The growth of xenograft tumors and mouse survival were monitored. The frequency of peripheral blood circulating endothelial cells (CECs), microvessel density (MVD) and pericyte coverage was determined using flow cytometry and immunofluorescence staining. In comparison with the controls, treatment with either drug significantly inhibited the growth of xenograft tumors in mice. Treatment with MET CPA or Endostar, but not with MTD CPA, significantly reduced the frequency of peripheral blood total and viable CECs and the value of MVD. Endostar also considerably reduced pericyte coverage in xenograft tumors. Moreover, MET CPA combined with Endostar further reduced the frequency of peripheral blood CECs, the value of MVD, and pericyte coverage, with concomitant delay in tumor growth and extension of mouse survival. Our results indicate that MET CPA combined with Endostar results in enhanced anti-tumor and anti-angiogenic effects in a xenograft model of human lung cancer. Combined therapy with metronomic chemotherapy and an angiogenesis inhibitor may serve as a promising treatment strategy for patients with advanced NSCLC.

Temsirolimus (TEM) maintenance therapy after response of castration-resistant prostate cancer (CRPC) to docetaxel (TAX).

e15177^ Background: There are no standard treatments for men who have demonstrated a good response to TAX without signs of progression. The mTOR pathway is involved in many aspects of CRPC, and mTOR inhibitors have shown significant anti-CRPC activity in preclinical testing. Therefore, we designed a phase II trial to explore whether maintenance therapy with TEM has the potential to prolong response to TAX without compromising quality of life. Methods: For this single-arm, multicenter phase II trial we recruited CRPC pts with documented treatment response by PSA (&gt; 50% decline from baseline) or RECIST (1.0) criteria following ≥ 6 to 10 cycles of TAX (75 mg/m2 q3wks). Subjects received weekly TEM (25 mg iv x 4/cycle). The primary endpoint was time to treatment failure (TTF; RECIST or symptomatic progression). Secondary endpoints included safety (NCI-CTCAE v3.0), quality of life (FACT-P, PPI), changes in PSA, objective tumor response rate, and overall survival. We were also studying surrogate markers of TEM activity, including the enumeration of circulating endothelial cells (CEC) as a potential indicator of the antiangiogenic effects of TEM. Results: 19 pts have been enrolled to data after a median of 7 cycles of TAX. The mean age was 68 years (range 51-82), and the median baseline PSA 17.3 (0.02-380.7). 15 pts had received prior local radiation therapy, 6 surgery. 17 pts had bone metastases, 9 visceral and 8 nodal disease. Pts received a median of 4 cycles of TEM (maximum 23 and continuing), resulting in a median TTF of 5.8 (95%CI 3.8-9.2) months. No PSA responses were seen (time to PSA progression 1.8 months), but 1 PR. 8 pts achieved SD. 12 pts have been discontinued due to treatment failure (RECIST [4], symptomatic [7], combined [1]), and 3 for other reasons, including TEM-associated toxicity (lymphedema [1]). TEM has been generally well tolerated without unexpected adverse events or negative impact on quality of life. The median overall survival has not yet been reached. There is a trend for TEM to reduce viable CEC amid large inter/intrapatient CEC variability. Conclusions: TEM maintenance therapy in CRPC pts that have responded to TAX appears to be well tolerated and results in meaningful prolonged TTF.

A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood

Blood circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells (CPCs) represent two cell populations that are thought to play important roles in tissue vascularization. CECs and CPCs are currently studied as surrogate markers in patients for more than a dozen pathologies, including heart disease and cancer. However, data interpretation has often been difficult because of multiple definitions, methods and protocols used to evaluate and count these cells by different laboratories. Here, we propose a cytometry protocol for phenotypic identification and enumeration of CECs and CPCs in human blood using four surface markers: CD31, CD34, CD133 and CD45. This method allows further phenotypic analyses to explore the biology of these cells. In addition, it offers a platform for longitudinal studies of these cells in patients with different pathologies. The protocol is relatively simple, inexpensive and can be adapted for multiple flow cytometer types or software. The procedure should take 2-2.5 h, and is expected to detect 0.1-6.0% viable CECs and 0.01-0.20% CPCs within blood mononuclear cell population.

Differential circulation kinetics during antiangiogenic therapy of four distinct blood cell populations expressing endothelial markers

3038 Background: Circulating endothelial cells (CECs) are evaluated as potential biomarker of antiangiogenic therapy. CD146 is an endothelial marker, but its use as a single marker to detect CECs is questionable. In addition, several sub-populations of CECs are thought to exist. Methods: We analyzed the expression of CD31, CD34, CD45, CD133 and CD146 on mononuclear blood cells and primary tissue endothelial cells by flow cytometric and immunohistochemical analyses. Furthermore, we investigated by flow cytometry the phenotype of blood cells expressing endothelial markers and evaluated their changes VEGF blockade by bevacizumab in ten rectal cancer patients enrolled in a Phase I trial. Percent values obtained at days 3 and 12 after bevacizumab infusion were compared with pre-treatment values in individual patients using the Wilcoxon signed-rank test. Results: In the blood, CD146 primarily marked cells with lymphoid markers (CD3 and CD4). CD146 was largely undetectable on blood CD31+CD45- CECs or CD34+CD133+ progenitor cells. In contradistinction, CD146 was detectable in tissues on both cellular components of the vessel wall: CD31brightCD45- endothelial cells and CD31-CD45- pericytes. The CD31+CD45- CEC population contained two distinct populations: CD31brightCD34+CD45- (viable CECs) and CD31dimCD45- (mostly non-viable CECs). Unlike CD31brightCD45- CECs and CD34+CD133+ progenitor cells, CD146+ and CD31dimCD45- cell concentration in the peripheral blood of cancer patients did not decrease during VEGF blockade. Conclusions: In the blood of cancer patients, we identified 4 distinct populations using endothelial markers. CD146 identified T lymphocytes. Within the CD31+CD45- CEC population, there were 2 distinct subpopulations, while the rare progenitor populations localized primarily within the CD45+ hematopoietic cell pool. The changes in blood concentration of these 4 cell types during anti-VEGF therapy were distinct in individual patients. These results underscore the need for further characterization and identification of new markers for CEC subpopulations for their development as biomarkers of antiangiogenic therapy. [Table: see text]

Differential CD146 Expression on Circulating Versus Tissue Endothelial Cells in Rectal Cancer Patients: Implications for Circulating Endothelial and Progenitor Cells As Biomarkers for Antiangiogenic Therapy

Circulating endothelial cells (CECs) and progenitor cells are currently evaluated as potential biomarkers of antiangiogenic therapy. CD146 is considered a panendothelial-specific marker, but its utility as a CEC marker in cancer patients remains unclear. We analyzed the expression of CD146 on mononuclear blood cells, primary tissue endothelial cells, and malignant and normal tissues by flow cytometric and immunohistochemical analyses. Furthermore, we measured the circulation kinetics of CD146+ cells before, and then 3 and 12 days after vascular endothelial growth factor (VEGF) antibody blockade by bevacizumab infusion in rectal cancer patients enrolled in a phase I trial. In the peripheral blood of these cancer patients, over 90% of the CD146+ cells were CD45+ hematopoietic cells. CD146 expression was primarily detected on a subset of CD3+CD4+ lymphocytes, and was undetectable on CD34+CD133+CD45(dim) progenitor cells or CD31(bright)CD45- viable CECs. In contradistinction, CD146 was detectable in tissues on both cellular components of tumor vessel wall: CD31(bright)CD45- endothelial cells and alpha-SMA+ pericytes. Unlike viable CECs and progenitor cells, CD146+ cell concentration in the peripheral blood of cancer patients did not decrease during VEGF blockade. CD146 is fairly homogeneously expressed on vascular endothelium but not on viable CECs or progenitor cells. The vast majority of CD146+ blood cells are lymphocytes, and VEGF blockade by bevacizumab did not significantly change their number in rectal cancer patients. These results underscore the need for further characterization and identification of new markers for CEC subpopulations for their development as biomarkers of antiangiogenic therapy.