Angiogenesis and Ovarian Cancer: What Potential Do Different Subtypes of Circulating Endothelial Cells Have for Clinical Application?

Ovarian cancer (OC) remains the most fatal disease of gynaecologic malignant tumours. The neovasculature in the tumour microenvironment principally comprises endothelial cells. Haematogenous cancer metastases are significantly impacted by tumour neovascularisation, which predominantly depends on the tumour-derived endothelial vasculogenesis. There is an urgent need for biomarkers for the diagnosis, prognosis and prediction of drug response. Endothelial cells play a key role in angiogenesis and other forms of tumour vascularisation. Subtypes of circulating endothelial cells may provide interesting non-invasive biomarkers of advanced OC that might have the potential to be included in clinical analysis for patients' stratification and therapeutic management. In this review, we summarise the reported studies on circulating endothelial subtypes in OC, detailing their isolation methods as well as their potential diagnostic, prognostic, predictive and therapeutic utility for clinical application. We highlight key biomarkers for the identification of circulating endothelial cell subtypes and their targets for therapies and critically point out future challenges.

The Effect of Proprotein Convertase Subtilisin Kexin Type 9 Inhibitors on Circulating Endothelial Progenitor Cells in Patients with Cardiovascular Disease.

PURPOSE: Circulating endothelial progenitor cells (cEPCs) are vital to vascular repair by re-endothelialization. We aimed to explore the effect of proprotein convertase subtilisin kexin type 9 inhibitors (PCSK9i) on cEPCs hypothesizing a possible pleiotropic effect. METHODS: Patients with cardiovascular disease (CVD) were sampled for cEPCs at baseline and following the initiation of PCSK9i. cEPCs were assessed using flow cytometry by the expression of CD34(+)/CD133(+) and vascular endothelial growth factor receptor (VEGFR)-2(+), and by the formation of colony-forming units (CFUs) and production of VEGF. RESULTS: Our cohort included 26 patients (median age 68 (IQR 63, 73) years; 69% male). Following 3 months of treatment with PCSK9i and a decline in low-density lipoprotein cholesterol levels (153 (IQR 116, 176) to 56 (IQR 28, 72) mg/dl), p < 0.001), there was an increase in CD34(+)/CD133(+) and VEGFR-2(+) cell levels (0.98% (IQR 0.37, 1.55) to 1.43% (IQR 0.90, 4.51), p = 0.002 and 0.66% (IQR 0.22, 0.99) to 1.53% (IQR 0.73, 2.70), p = 0.05, respectively). Functionally, increase in EPCs-CFUs was microscopically evident following treatment with PCSK9i (1 CFUs (IQR 0.0, 1.0) to 2.5 (IQR 1.5, 3), p < 0.001) with a concomitant increase in EPC's viability as demonstrated by an MTT assay (0.15 (IQR 0.11, 0.19) to 0.21 (IQR 0.18, 0.23), p < 0.001). VEGF levels increased following PCSK9i treatment (57 (IQR 18, 24) to 105 (IQR 43, 245), p = 0.006). CONCLUSIONS: Patients with CVD treated with PCSK9i demonstrate higher levels of active cEPCs, reflecting the promotion of endothelial repair. These findings may represent a novel mechanism of action of PCSK9i.

Deciphering the Role of miR-200c-3p in Type 1 Diabetes (Subclinical Cardiovascular Disease) and Its Correlation with Inflammation and Vascular Health.

Uncomplicated type 1 diabetes (T1DM) displays all features of subclinical cardiovascular disease (CVD) as is associated with inflammation, endothelial dysfunction and low endothelial progenitor cells. MiR-200c-3p has been shown in animal tissues to be pro-atherogenic. We aimed to explore the role of miR-200c-3p in T1DM, a model of subclinical CVD. 19 samples from T1DM patients and 20 from matched controls (HC) were analyzed. MiR-200c in plasma and peripheral blood mononuclear cells (PBMCs) was measured by real-time quantitative polymerase chain reaction. The results were compared with the following indices of vascular health: circulating endothelial progenitor cells, (CD45dimCD34+VEGFR-2+ or CD45dimCD34+CD133+) and proangiogenic cells (PACs). MiR-200c-3p was significantly downregulated in PBMCs but not in plasma in T1DM. There was a significant negative correlation between the expression of miR-200c-3p and HbA1c, interleukin-7 (IL-7), vascular endothelial growth factor-C (VEGF-C), and soluble vascular cell adhesion molecule-1, and a positive correlation with CD45dimCD34+VEGFR-2+, CD45dimCD34+CD133+ and PACs. Receiver operating curve analyses showed miR-200c-3p as a biomarker for T1DM with significant downregulation of miR-200c-3p, possibly defining subclinical CVD at HbA1c > 44.8 mmol/mol (6.2%). In conclusion, downregulated miR-200c-3p in T1DM correlated with diabetic control, VEGF signaling, inflammation, vascular health and targeting VEGF signaling, and may define subclinical CVD. Further prospective studies are necessary to validate our findings in a larger group of patients.

Circulating endothelial progenitor cells during pregnancy in multiple sclerosis.

BACKGROUND: Endothelial progenitor cells (EPCs) have been shown to increase during physiological pregnancy and are believed to play a fundamental role in the process of placentation. Reduced levels of EPCs during pregnancy have been associated with preeclampsia and miscarriage. Women with multiple sclerosis (MS) are not at increased risk of preeclampsia nor of general adverse obstetric outcome, in contrast with some other autoimmune diseases. OBJECTIVE: The aim of this study was to evaluate circulating EPCs levels in pregnant patients with MS. METHODS: CD34+ and CD133+ were longitudinally detected by flow cytometry in the maternal plasma of 29 healthy controls and 9 MS patients and in the cord blood of their newborns. RESULTS: EPCs were affected by pregnancy with the same trend in both groups (CD34+ p = 0.0342; CD133+ p = 0.0347). EPCs during pregnancy were increased in MS (mean ± SD: CD34+ cells 0.038 ± 0.010; CD133+ 0.024 ± 0.009) with respect to healthy controls (mean ± SD: CD34+ cells 0.022 ± 0.006; CD133+ 0.016 ± 0.004), CD34+ p = 0.0004; CD133+ p = 0.0109. EPCs levels of the cord blood of MS patients' newborns mild correlated with maternal EPC levels at delivery (CD34+: spearman's Rho 0.658, p = 0.054; CD133+: spearman's Rho 0.758, p = 0.018). CONCLUSIONS: This work identified increased circulating EPC levels during pregnancy, following the same trend both in MS patients and healthy controls. Despite the similar trend, the levels of circulating EPCs were significantly higher in MS patients with respect to the control population. A correlation was also found in MS patients between cord blood EPCs and circulating EPCs at delivery.

Positive Effects of Heme Oxygenase Upregulation on Adiposity and Vascular Dysfunction: Gene Targeting vs. Pharmacologic Therapy.

OBJECTIVE: Heme oxygenase (HO-1) plays a critical role in adipogenesis and it is important to understand its function in obesity. Many studies have shown that upregulation of HO-1 can affect the biologic parameters in obesity-mediated diabetes, hypertension and vascular endothelial cell function. Thus, we aimed to explore the hypothesis that upregulation of HO-1, using a pharmacologic approach as well as gene targeting, would improve both adiposity and endothelial cell dysfunction by direct targeting of endothelial cells. Our second aim was to compare the short-term effect of a HO-1 inducer, cobalt-protoporphrin IX (CoPP), with the long-term effects of gene targeted therapy on vascular and adipocyte stem cells in obese mice. METHOD: We examined the effect of CoPP on fat pre-adipocytes and mesenchymal stem cells (MSC) in mice fed a high-fat diet (HFD). We also used a lentiviral construct that expressed heme oxygenase (HO-1) that was under the control of an endothelium specific promoter, vascular endothelium cadherin (VECAD) heme oxygenase (VECAD-HO-1). We targeted endothelial cells using vascular endothelium cadherin/green fluorescent protein fusion construct (VECAD-GFP) as the control. Conditioned media (CM) from endothelial cells (EC) was added to fat derived adipocytes. Additionally, we treated renal interlobar arteries with phenylephrine and dosed cumulative increments of acetylcholine both with and without exposure to CoPP. We did the same vascular reactivity experiments with VECAD-HO-1 lentiviral construct compared to the control. RESULTS: CoPP improved vascular reactivity and decreased adipogenesis compared to the control. MSCs exposed to CM from EC transfected with VECAD-HO-1 showed decreased adipogenesis, smaller lipid droplet size and decreased PPAR-γ, C/EBP and increased Wnt 10b compared to the control. HO-1 upregulation had a direct effect on reducing adipogenesis. This effect was blocked by tin mesoporphrin (SnMP). EC treated with VECAD-HO-1 expressed lower levels of ICAM and VCAM compared to the control, suggesting improved EC function. This also improved ACH induced vascular reactivity. These effects were also reversed by SnMP. The effect of viral transfection was much more specific and sustained than the effects of pharmacologic therapy, CoPP. CONCLUSION: This study demonstrates that a pharmacological inducer of HO-1 such as CoPP improves endothelial cell function while dampening adipogenesis, but long-term HO-1 expression by direct targeting of endothelial cells by gene transfer therapy may offer a more specific and ideal solution. This was evidenced by smaller healthier adipocytes that had improved insulin sensitivity, suggesting increased adiponectin levels. HO-1 upregulation reestablished the "crosstalk" between perivascular adipose tissue and the vascular system that was lost in the chronic inflammatory state of obesity. This study demonstrates that gene targeting of EC may well be the future direction in treating obesity induced EC dysfunction, with the finding that targeting the vasculature had a direct and sustained effect on adipogenesis.

Combination of granulocyte colony-stimulating factor and CXCR4 antagonist AMD3100 for effective harvest of endothelial progenitor cells from peripheral blood and in vitro formation of primitive endothelial networks.

Endothelial progenitor cells (EPC) derived from the circulation may be used to enhance neovascularization. Since the combination of granulocyte colony-stimulating factor (GCSF) and CXCR4 antagonist AMD3100 efficiently mobilizes hematopoietic stem cells into peripheral circulation, it may increase the pool of endogenously circulating EPC. We tested this hypothesis by administering GCSF and AMD3100 to adult rabbits and rats, isolating mononuclear cells from peripheral blood by Ficoll density gradient centrifugation, and characterizing the blood-derived EPC based on morphology, immunophenotyping, gene expression and other functional analyses. These EPC showed clonal growth similar to that of human umbilical vein endothelial cells when cultured in complete EGM-2 medium on collagen I-precoated culture plates. The EPC exhibited a typical cobblestone-like morphology and were relatively homogeneous by the third passage. The cells expressed the typical endothelial marker CD31 based on flow cytometry and fluorescence microscopy, formed capillary-like structures when cultured in Matrigel, internalized DiI-acetylated low-density lipoprotein, bound Ulex europaeus agglutinin-1, and expressed CD31 and several other endothelial markers (VEGFR2, VE-cadherin, Tie-2, eNOS, vWF) at significantly higher levels than bone marrow-derived mesenchymal stem cells. These results suggest that the combination of GCSF and AMD3100 can efficiently release stem cells into peripheral circulation and generate EPC that show the desired morphological, immunophenotypic and functional characteristics. This minimally invasive approach may be useful for autologous cell transplantation for postnatal neovasculogenesis and tissue repair.

CD34+/CD144+ circulating endothelial cells as an indicator of carotid atherosclerosis.

BACKGROUND: The relationships between the number of circulating endothelial cells (CECs) or endothelial progenitor cells (EPCs) and indicators of carotid atherosclerosis, such as the intima-media thickness (IMT) and plaque score are not well characterized in patients with chronic ischemic stroke. The objective of this study was to investigate these relationships in patients with chronic ischemic stroke and in patients with risk factors for stroke. METHODS: A total of 58 patients (69.6 ± 10.0 years, 21 females) with chronic ischemic stroke or with risk factors for stroke were included in this study. IMT was measured using an IntimaScope, and the numbers of CECs and EPCs were measured using flow cytometry. CECs and EPCs were defined as CD34+/CD144+ and CD34+/CD133+ cells, respectively. RESULTS: The number of CECs in patients with large artery atherosclerosis was higher than that in patients with cardioembolism or small vessel occlusion (P < .05). In contrast, there were no significant differences in the number of EPCs between groups. A positive correlation was also observed between the plaque score and the number of CECs (r(2) = .139, P < .05, n = 36). Moreover, the number of CECs in patients with moderate and severe atherosclerosis (.32 ± .11/µL, n = 22) was higher than that in patients with no plaque and mild atherosclerosis (.25 ± .07/µL, n = 34, P < .05). CONCLUSIONS: The number of CECs was high in patients with large artery atherosclerosis who experienced chronic ischemic stroke. And this number may reflect severity of carotid atherosclerosis.

The association between circulating CD34+CD133+ endothelial progenitor cells and reduced risk of Alzheimer's disease in the Framingham Heart Study.

Aim: Endothelial dysfunction has been associated with both cerebrovascular pathology and Alzheimer's disease (AD). However, the connection between circulating endothelial cells and the risk of AD remains uncertain. The objective was to leverage data from the Framingham Heart Study to investigate various circulating endothelial subtypes and their potential correlations with the risk of AD. Methods: The study conducted data analyses using Cox proportional hazard regression and linear regression methods. Additionally, genome-wide association study (GWAS) was carried out to further explore the data. Results: Among the eleven distinct circulating endothelial subtypes, only circulating endothelial progenitor cells (EPCs) expressing CD34+CD133+ were found to be negatively and dose-dependently associated with reduced AD risk. This association persisted even after adjusting for age, sex, years of education, apolipoprotein E (APOE) ε4 status, and various vascular diseases. Particularly noteworthy was the significant association observed in individuals with hypertension and cerebral microbleeds. Consistently, positive associations were identified between CD34+CD133+ EPCs and specific brain regions, such as higher proportions of circulating CD34+CD133+ cells correlating with increased volumes of white matter and the hippocampus. Additionally, a GWAS study unveiled that CD34+CD133+ cells influenced AD risk specifically in individuals with homozygous genotypes for variants in two stem cell-related genes: kirre like nephrin family adhesion molecule 3 (KIRREL3, rs580382 CC and rs4144611 TT) and exocyst complex component 6B (EXOC6B, rs61619102 CC). Conclusions: The findings suggest that circulating CD34+CD133+ EPCs possess a protective effect and may offer a new therapeutic avenue for AD, especially in individuals with vascular pathology and those carrying specific genotypes of KIRREL3 and EXOC6B genes.

Endothelial progenitor cells in patients with chronic obstructive pulmonary disease.

The pathogenesis of chronic obstructive pulmonary disease is not fully understood. The objective of this study was to compare circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease to age, sex, and cigarette smoking matched healthy controls. Patients with chronic obstructive pulmonary disease (n = 37) and healthy controls (n = 19) were matched by age, sex, and smoking status. Circulating hematopoietic progenitor cells (CD34(+) or CD133(+) mononuclear cells) and endothelial progenitor cells (CD34(+)KDR(+) or CD34(+)CD133(+)KDR(+) mononuclear cells) were quantified by flow cytometry. Endothelial cell-colony forming units from peripheral blood mononuclear cells were quantified in vitro and phenotypic analysis carried out using immunocytochemistry. Patients with chronic obstructive pulmonary disease had more circulating mononuclear cells compared with controls (8.4 ± 0.6 vs. 5.9 ± 0.4 × 10(9) cells/l; P = 0.02). CD34(+) hematopoietic progenitor cells were reduced as a proportion of mononuclear cells in patients compared with controls (0.99 ± 0.12 vs. 1.9 ± 0.12%; P = 0.02); however, there were no differences in the absolute number of CD34(+), CD34(+)KDR(+), or CD34(+)CD133(+)KDR(+) cells (P > 0.05 for all). Endothelial cell-colony forming units were increased in patients with chronic obstructive pulmonary disease compared with controls (13.7 ± 5.2 vs. 2.7 ± 0.9 colonies; P = 0.048). In contrast to previous studies, the number of circulating progenitor cells was not reduced in patients with chronic obstructive pulmonary disease compared with carefully matched controls. It seems unlikely that circulating endothelial progenitor cells or failure of angiogenesis plays a central role in the development of emphysema.

Levels of circulating endothelial progenitor cells inversely correlate with manic and positive symptom severity in patients with bipolar disorder.

OBJECTIVES: Patients with bipolar disorder (BPD) are at high risk of cardiovascular diseases (CVDs) that are attributed to endothelial dysfunction. Circulating endothelial progenitor cells (cEPCs) are proposed as indicators of endothelial dysfunction. This study examined the relationship of cEPC numbers with BPD diagnosis and its clinical symptoms in patients with BPD. METHODS: We recruited 48 patients with BPD and 50 healthy controls (HCs). All the patients had scores of <13 on the Young Mania Rating Scale (YMRS). In addition to the YMRS and Clinical Global Impression for BPD (CGI-BP), bipolar patients were assessed using relevant measurements for their depression, anxiety, general psychopathology, cognitive dysfunction and deficit, somatic symptoms, quality of life, and level of disability. cEPC counts were measured using flow cytometry. RESULTS: The numbers of immature and mature cEPCs in the bipolar patients did not significantly differ from those in the HCs. After correction for multiple comparisons and controlling for body mass index and smokers, the number of immature cEPCs was observed to be inversely correlated with CGI-BP (corrected p [pcorr ] = .00018) and positive scores in the positive and negative syndrome scale (PANSS-P; pcorr  = .0049). The number of mature cEPCs was inversely correlated with YMRS (pcorr  = .014), CGI-BP (pcorr  = .00022), and PANSS-P (pcorr  = .0049) scores. In multivariate stepwise analysis, numbers of both types of cEPCs were associated with CGI-BP. CONCLUSIONS: cEPC levels, an indicator of endothelial dysfunction and risk of CVDs, may be associated manic and positive symptom severities in patients with BPD.

Endothelial Progenitor Cell CD34+ and CD133+ Concentrations and Soluble HLA-G Concentrations During Pregnancy and in Cord Blood in Undifferentiated Connective Tissue Diseases Compared to Controls.

The objective of this study is to evaluate endothelial progenitor cells (EPCs) CD34+ CD133- and CD34+ CD133+ and soluble HLA-G (sHLA-G) concentrations among undifferentiated connective tissue disease (UCTD) subjects, compared to controls, during pregnancy and in cord blood. This is a case-control study including 29 controls and 29 UCTDs. CD34+ CD133-, CD34+ CD133+, and sHLA-G concentrations were detected in maternal plasma and in cord blood. This study was approved by the Medical-Ethical Committee of our Institution (Current Research Project N. 901-rcr2017i-23 of IRCCS Foundation Policlinico San Matteo of Pavia). Circulating CD34+ CD133- and CD34+ CD133+ counts and sHLA-G (soluble human leucocyte antigen G) concentrations in maternal peripherical blood were higher in UCTD compared to those in controls in first and third trimester of pregnancy and at delivery (p < 0.001). Maternal CD34+ CD133- and CD34+ CD133+ counts were strongly and significantly correlated in UCTD (Spearman's rho = 0.79, p < 0.0001) but not in controls (Spearman's rho = 0.10, p = 0.35). Cord blood CD34+ CD133- and CD34+ CD133+ median counts and median sHLA-G concentrations were higher among UCTD subjects than in controls (p < 0.001). Cord blood CD34+ and CD133+ counts were inversely and significantly correlated with sHLA-G concentrations among UCTDs, but not in controls. Early UCTD is characterized by increased EPC levels in maternal plasma and in cord blood and higher levels of sHLA-G, compared to controls. Data suggest that fetoplacental unit plays an independent role in the EPC response to a systemic autoimmune disease.

Current concepts on endothelial stem cells definition, location, and markers.

Ischemic vascular disease is a major cause of mortality and morbidity worldwide, and regeneration of blood vessels in perfusion-deficient tissues is a worthwhile therapeutic goal. The idea of delivering endothelial stem/progenitor cells to repair damaged vasculature, reperfuse hypoxic tissue, prevent cell death, and consequently diminish tissue inflammation and fibrosis has a strong scientific basis and clinical value. Various labs have proposed endothelial stem/progenitor cell candidates. This has created confusion, as there are profound differences between these cell definitions based on isolation methodology, characterization, and reparative biology. Here, a stricter definition based on stem cell biology principles is proposed. Although preclinical studies have often been promising, results from clinical trials have been highly contradictory and served to highlight multiple challenges associated with disappointing therapeutic benefit. This article reviews recent accomplishments in the field and discusses current difficulties when developing endothelial stem cell therapies. Emerging evidence that disputes the classic view of the bone marrow as the source for these cells and supports the vascular wall as the niche for these tissue-resident endothelial stem cells is considered. In addition, novel markers to identify endothelial stem cells, including CD157, EPCR, and CD31low VEGFR2low IL33+ Sox9+ , are described.

Safety and efficacy of intrarenal arterial autologous CD34+ cell transfusion in patients with chronic kidney disease: A randomized, open-label, controlled phase II clinical trial.

BACKGROUND: This was a randomized, open-label, controlled phase II clinical trial to investigate the safety, efficacy, and outcomes of intrarenal artery infusion of autologous peripheral-blood-derived CD34+ cells for patients with chronic kidney disease (CKD; ie, stage III or IV). MATERIALS AND METHODS: Between October 2016 and July 2018, 52 consecutive patients with CKD at stage III or IV were randomly allocated into a treatment group (TG; 2.5 × 107 cells for each intrarenal artery; n = 26) and a control group (CG; standardized pharmacotherapy only; n = 26). The primary endpoints included safety and change of creatinine level/creatinine clearance. The secondary endpoints were 12-month combined unfavorable clinical outcomes (defined as dialysis or death), improvement in proteinuria, and CD34+ cell-related adverse events. RESULTS: All patients were uneventfully discharged after CD34+ cell therapy. The baseline endothelial progenitor cell (EPC) populations did not differ between TG and CG (P > .5). Flow cytometric analysis showed increases in circulating EPC (ie, CD34+KDR+CD45dim / CD34+CD133+CD45dim /CD31+CD133+CD45dim /CD34+CD133+KDR+/CD133+) and hematopoietic stem cell (CD34+) populations after granulocyte-colony stimulating factor treatment (all P < .001). Besides, Matrigel assay of angiogenesis was also significantly enhanced (all P < .001). Renal-venous blood samplings (ie, at 0, 5, 10, and 30 minutes after CD34+ cell infusion) demonstrated significant progressive increases in EPC level (P for trend <.001) among the TG patients. One-year combined unfavorable clinical outcomes were significantly lower in TG than those in CG (0% [0] vs 13.3% [4], P = .038). By 12 months after CD34+ cell therapy, circulating creatinine level, ratio of urine protein to urine creatinine, and creatinine clearance showed no difference between TG and CG (all P > .1). CONCLUSION: CD34+ cell therapy was safe and improved 1-year outcome.

Anti-angiogenic efficacy in invasive breast carcinoma patients depends on clinicopathological determinants.

PURPOSE: The biggest problem with the occurrence of breast cancer is late diagnosis, which is associated with high mortality rates. The aim of the study was to appraise the number of circulating endothelial precursors and the concentration of vascular endothelial growth factor A (VEGF-A) and the soluble forms of its receptors, sVEGFR1 and sVEGFR2, in breast cancer patients with respect to clinicopathological features. MATERIAL AND METHODS: The study involved 85 women of Caucasian ethnicity aged 45-66 with primary breast cancer without distant metastases (M0). Inclusion criteria were as follows: histopathological examination confirming the diagnosis of primary breast cancer, without previous radiotherapy and chemotherapy. Immunohistochemistry evaluation of oestrogen and progesterone receptors, human epidermal growth factor receptor 2, Ki67 expression was made in all cases. In the EDTA-plasma, the concentrations of VEGF-A and its soluble receptors, sVEGFR1 and sVEGFR2, were measured applying immunoassay techniques. Circulating endothelial progenitor cells (EPCs) were identified with the immune-phenotype CD45-, CD34+, CD133+, CD31+ using flow cytometry. RESULTS: Older women with breast cancer had significantly higher concentrations of VEGF-A as well as sVEGFR2 compared with their younger counterparts. A significantly higher concentration of the soluble form of VEGF receptor type 1 in patients with T1 breast cancer in relation to T2 cases was noted. Also, negative correlations between circulating EPCs and histological grading as well as a soluble form of VEGFR2 with histological grading of breast cancer according to the Elston-Ellis classification were observed. CONCLUSIONS: Anti-angiogenic potential is divergent in relation to the clinicopathological determinants.

Low Pre-Treatment Count of Circulating Endothelial Progenitors as a Prognostic Biomarker of the High Risk of Breast Cancer Recurrence.

Neoangiogenesis is mediated by circulating bone marrow-derived endothelial progenitors (circulating EPCs). The aim of the study was the quantification of circulating EPCs from the peripheral blood mononuclear cells of invasive breast cancer (IBrC) patients by flow cytometry, before and after cancer adjuvant treatment. A total of 88 stage IA-IIB primary IBrC patients were enrolled prospectively. Circulating EPCs with the immune-phenotype CD45-CD34+CD133+CD31+ were assessed. Treatment significantly reduced the number of EPCs/µL in the general IBrC cohort. However, there was a relevant elevation in the number of circulating EPCs after nine months of adjuvant treatment in the group of patients aged ≥ 55 years, of T2 clinical type, with nodal involvement (N1) and Ki67 expression > 15%. Follow-up revealed a significantly higher incidence of disease relapse in breast cancer patients with low pre-treatment circulating EPCs levels compared with those with a high baseline circulating EPCs count. The receiver-operating characteristic curve identified a tumour diameter of 2.1 cm as the best cut-off value to discriminate between disease-relapse subjects and non-relapse disease cases. Our study strongly indicates that, next to tumour diameter and Ki67 expression, circulating bone marrow-derived EPCs may serve as useful markers for predicting therapeutic outcomes as well as a future prognosis.

The endothelial status reflected by circulating endothelial cells, circulating endothelial progenitor cells and soluble thrombomodulin in patients with mild and resistant hypertension.

BACKGROUND: The aim of the present study was to evaluate endothelial status by measuring the concentration of novel markers of endothelial dysfunction (ED): a number of circulating endothelial cells (CECs), circulating endothelial progenitor cells (CEPCs) and their ratio (CEPCs/CECs) as well as a traditional parameter - soluble thrombomodulin (sTM) in patients with resistant (RH) and mild hypertension (MH). MATERIALS AND METHODS: Thirty patients with MH and thirty subjects with RH were involved in the study. The control group included thirty-three age and sex-matched normotensive volunteers. We used multicolor flow cytometry for CECs and CEPCs analysis and the commercial human sTM ELISA kit to measure plasma sTM concentration. RESULTS: An elevated CECs number and a decreased CEPCs/CECs ratio was found in MH as well as in RH patients in comparison with normotensive volunteers. CECs correlated positively with an increased triglycerides in MH patients and an elevated LDL-cholesterol and hsCRP in RH group. Positive correlation between CEPCs and LDL-cholesterol level was observed in both types of hypertension. CONCLUSIONS: The results of the present study suggest that an endothelial alteration accompanies hypertension. The number of CECs reflecting the extent of endothelial damage does not appear to be related to the severity of disease. The drastically decreased ratio between CEPCs and CECs observed in both groups of patients suggests an inadequate process of endothelial regeneration. Among analyzed factors inflammation and lipid abnormalities may have significant contribution in endothelial pathology in hypertension.

Diagnostic and Prognostic Relevance of Red Blood Cell Distribution Width for Vascular Aging and Cardiovascular Diseases.

Evidence suggests association of red blood cell distribution width (RDW) with cardiovascular diseases (CVDs). On the contrary, we underline that the sole RDW values cannot represent a valid CVD biomarker. High RDW values are expression of biological effects of a lot of both endogenous and exogenous factors (i.e., age, sex, genetic background, inflammation, hormones, drugs, diet, exercise, hematological analyzers, and ranges of values), modulating the biology and physiology of erythrocytes. Thus, the singular monitoring of RDW cannot be used to predict cardiovascular disorders. Accordingly, we have reviewed the evidence for potential relationship of RDW values with alterations in the cardiovascular system (i.e., regenerative capacity, endothelial turnover, and senescence of cardiovascular cells), associated with vascular aging and disease. In addition, we highlight the inevitable impact of biases in clinical application of RDW related to CVDs. Based on our thorough review of literature, we suggest a combined evaluation of RDW with other emerging biomarkers related to vascular aging and the diagnosis and prognosis of CVDs, including telomere length of leukocytes, circulating nucleated red blood cells (nRBCs) and endothelial progenitor cells (EPCs) in future large scale studies.

High levels of circulating endothelial progenitor cells in patients with diabetic retinopathy are positively associated with ARHGAP22 expression.

Diabetic retinopathy (DR) is a common microvascular complication of diabetes. Circulating endothelial progenitor cells (EPCs) are derived from bone marrow and are characterized by pathological retinal neovascularization. Rho GTPase Activating Protein 22 (ARHGAP22) is a DR susceptibility gene that interacts with its downstream regulatory protein ras-related C3 botulinum toxin substrate 1 (Rac1), to assist in endothelial cell angiogenesis and increasing capillary permeability. The aim of this study was to elucidate the relationship between ARHGAP22 expression and EPC levels in type 2 diabetes (T2D) patients with DR. Fifty T2D patients with DR were recruited. Circulating EPCs were characterized as CD31+/vascular endothelial growth factor-2+/CD45dim/CD133+ and were quantified using triple staining flow cytometry. Real-time polymerase chain reaction tests were used to quantify ARHGAP22 expression. We found that T2D patients with proliferative DR had significantly lower EPC levels than those with non-proliferative DR (P = 0.028). T2D patients with EPC levels above the median value (> 4 cells/105 events) had higher levels of ARHGAP22 expression (P = 0.002). EPC levels were positively correlated with ARHGAP22 expression (r = 0.364, P = 0.009). Among T2D patients with DR, a higher expression of ARHGAP22 was associated with higher levels of EPCs. ARHGAP22 may be involved in the mobilization or active circulation of EPCs, thus contributing to neovascularization during DR development.

Atorvastatin improves coronary flow and endothelial function in patients with coronary slow flow.

The underlying mechanisms behind the effect of atorvastatin on patients with coronary slow flow (CSF) remain largely unknown. To investigate the possible underlying molecular mechanisms 108 patients were divided into atorvastatin group and control group. Coronary flow was quantified according to corrected TIMI frame count (CTFC). Serum high sensitivity C-reactive protein (hs-CRP), lipids, ET-1, interleukin (IL)-6, NO, circulating endothelial progenitor cell (cEPC) count, adhesion, migration and proliferation were measured in pretreatment and post-treatment. After respective treatment, the atorvastatin group had significantly decreased levels of TC, TG, LDL-C, hs-CRP, ET-1 and IL-6 and increased NO compared to the control group. The atorvastatin group had a more significant improvement of CTFC, effective rate, cEPC number, EPC adhesion, migration and proliferation compared to the control group. In conclusion, atorvastatin can be used in treatment of CSF by suppressing inflammation and improving endothelial function.

New arguments for beneficial effects of alpha-lipoic acid on the cardiovascular system in the course of type 2 diabetes.

PURPOSE: Alpha-lipoic acid (ALA), widely known as an antioxidant, modifies also serum levels of angiogenic factors in type 2 diabetic patients. These pharmacological activities may influence the status of the cardiovascular system. Taking into consideration that diabetes is related to the increased cardiovascular risk we investigated several effects of ALA on angiogenic factors in the myocardium and in the aortal wall using a rat model of type 2 diabetes. METHODS: Diabetes was induced in Wistar rats by a fat-rich diet and by intraperitoneal injection of a small dose of streptozotocin (30 mg/kg). Animals were divided into 3 groups: ALA-treated type 2 diabetes rat model, placebo-treated type 2 diabetes rat model and placebo-treated non-diabetic rats. ALA was administered orally once a day, 20 mg/kg, for 8 consecutive weeks. mRNA VEGF, VEGF-R1 and VEGF-R2 expression was measured in the myocardium and the aortal wall, simultaneously with circulating VEGF and circulating endothelial cells (cEC) and endothelial progenitor cells (cEPC). RESULTS: ALA induced pro-angiogenic effect in the myocardium of rats with diabetes increasing mRNA VEGF expression and decreasing mRNA VEGFR-1 expression, while in the aortal wall ALA increased mRNA VEGFR-2 and VEGFR-1 expression. cVEGF in the ALA-treated group was higher comparing to both control groups. It was revealed that cEC percentage in the ALA-treated group was decreased with no effect on the percentage of cEPC. CONCLUSIONS: In summary, the current data provide novel findings about potential beneficial effects of ALA on angiogenic factors in the cardiovascular system, especially on myocardium, in the course of type 2 diabetes.