Circulating endothelial and endothelial progenitor cells in patients with severe sepsis

Elevated circulating endothelial cell (CEC) and circulating endothelial progenitor cell (CEPC) counts may indicate vascular damage and disease status, but data on these cell populations in patients with severe sepsis are limited. This study compared CEC and CEPC counts in patients with and without severe sepsis following intensive care unit (ICU) admission. Venous blood samples were collected within 24 h, 48-72 h, and 120-144 h. Baseline demographics, 28-day mortality, ICU and hospital days, and Sequential Organ Failure Assessment (SOFA) scores were recorded. Patients with (n = 18) and without (n = 28) severe sepsis were balanced for mean age (63.7 and 61.3 years, respectively) and gender. There were no differences in 28-day mortality, ICU days, or hospital days. Baseline SOFA scores were higher in the sepsis group. At 48-72 h, patients with severe sepsis had significantly higher median CEC counts (51.5 vs. 28.0 cells/4 ml of blood, P = 0.02). CEC values for all ICU patients were significantly (P < 0.05) higher than in healthy volunteers. CEPC counts in both cohorts ranged from 0 to > 21 colonies/4 ml blood (mean = 1.13 ± 2.25; median = 0) without significant differences at any time point. This study demonstrates the ability to quantify CECs and CEPCs using consensus methodology. Understanding the relationship between CEC/CEPC counts and outcomes may provide insight into the mechanisms of endothelial cell changes in severe sepsis.     

Circulating endothelial progenitor cells during human pregnancy

The precise molecular and cellular mechanisms that regulate maternal vascular development during gestation are largely unknown. Endothelial progenitor cells (EPCs), which play an important role in vascular homeostasis, have been discovered in the circulation. We examined the level of circulating EPCs throughout uncomplicated pregnancies (n = 20) and assessed the correlation between serum estradiol levels and the number of EPCs. The number of circulating EPCs increased gradually and paralleled the progression of gestational age. In addition, the number of EPCs correlated significantly with the level of serum estradiol. The present study suggests that EPCs may play an important role in the regulation and maintenance of the placental development and vascular integrity during pregnancy.     

Circulating endothelial progenitor cells correlate with erectile function in patients with coronary heart disease.

AIMS: The aim of the study was to determine the influence of endothelial progenitor cells (EPC) on erectile dysfunction (ED). EPC play a major role in repair mechanisms of the endothelial monolayer, but the role of EPC in ED is unclear. METHODS AND RESULTS: Circulating levels of CD34(+)/KDR(+) and CD133(+) EPC were determined in 119 patients with known coronary artery disease. ED was evaluated with an ED-score generated from the KEED questionnaire. Prevalence of ED was 59.7%. In univariate analysis, age, hypertension, reduced left ventricular ejection fraction (LVEF), diabetes, and circulating levels of CD133(+) EPC, but not cardiovascular drug treatment were associated with ED. Body mass index (BMI) was positively (r = 0.319, P=0.003) and high-density lipoprotein was negatively (r=-0.246, P=0.034) correlated with ED. Adjustment for age, diabetes, hypertension, BMI, smoking, LVEF, use of statins and lower urinary tract symptoms, and prior coronary intervention revealed low levels of circulating immature CD133(+) EPC as independent risk factor for ED (95% CI -11.183 to -1.7371, P=0.008). CONCLUSION: Reduced levels of circulating CD133(+) EPC are an independent risk factor for ED. Thus, EPC may be a link between cardiovascular risk factors, endothelial dysfunction, and ED.     

Circulating endothelial progenitor cells in patients with unstable angina: association with systemic inflammation.

BACKGROUND: Endothelial progenitor cells (EPC) are present in peripheral blood and can develop a functional endothelial phenotype. The number and function of circulating EPCs are altered in atherosclerosis, diabetes, and after myocardial infarction and EPCs have been shown to promote postnatal angiogenesis and vasculogenesis. We investigated the number and adhesive properties of EPCs from patients with unstable angina and no evidence of cardiac necrosis. METHODS AND RESULTS: Patients were selected with unstable angina (n=29) and no evidence of cardiac necrosis, and controls with stable angina (n=12) and atherosclerotic risk factors, medication use, and coronary vessel involvement similar to patients. Circulating EPC numbers were determined by colony-forming unit assay and their adhesive properties were evaluated by EPC capacity to bind immobilised fibronectin. High-sensitivity C-reactive protein (hsCRP) was determined in all patients. Circulating EPCs were significantly increased in patients with unstable as compared with stable angina (24.5+/-2.6 vs. 13.3+/-2.9, respectively). Seven unstable angina patients followed up for 3 months after clinical stabilisation exhibited a reduction of close to 50% in circulating EPC numbers. The adhesive capacity of EPCs from patients with unstable and stable angina did not differ. A positive correlation was found between systemic CRP levels and circulating EPC numbers, but not their adhesive capacity. CONCLUSION: Patients with unstable angina and no evidence of cardiac necrosis exhibited increased circulating EPCs. Systemic inflammation, in addition to recognised growth factors, could play a role in the peripheral mobilisation of EPCs in patients with anginal syndromes.     

Circulating endothelial progenitor cells and coronary collaterals in patients with non-ST segment elevation myocardial infarction

BACKGROUND: Endothelial progenitor cells (EPCs) are bone marrow-derived cells that are augmented in response to ischemia and incorporated into neovascularization sites. We sought to determine whether circulating EPCs are related to collateral formation following non-ST segment elevation myocardial infarction (NSTEMI). METHODS: Twenty patients who underwent percutaneous coronary intervention (PCI) within a week of NSTEMI were divided into two groups: patients without collaterals (coll-, n=10) and patients with Rentrop grade 3--4 collaterals (coll+, n=10). Blood samples were drawn before PCI and 24+/- 2 h after PCI. EPC colonies were grown from peripheral blood mononuclear cells, characterized, and counted. Using flow cytometry the percentage of cells co expressing vascular endothelial growth factor receptor-2 and CD 133 was determined. RESULTS: The coll+ group had higher degree of culprit vessel stenosis and lower initial thrombolysis in myocardial infarction flow grade. The relative number of EPCs before PCI was significantly higher in the coll+ group than in the coll- group (1.49 +/- 0.9% vs. 0.77+/- 0.4%, p= 0.045). There were no significant intergroup differences in the number of EPC colony-forming cells. The number of EPC colonies increased in the coll- group after PCI (9.5 +/- 4.8 to 14.0 +/- 5.9/10(6) cells, p=0.01). CONCLUSIONS: This study supports an association between circulating EPC levels and collateral formation in patients with an NSTEMI.     

Circulating endothelial progenitor cells in patients with dysfunctional versus normally functioning congenitally bicuspid aortic valves

Patients with bicuspid aortic valve (BAV) may gradually develop significant valve dysfunction, whereas others remain free of dysfunction. Factors that determine the prognosis of BAV remain unclear. Because endothelial progenitor cells (EPCs) have a role in the repair of endothelial surfaces after injury, we hypothesized that EPCs may also be involved in preventing BAV degeneration. Accordingly, we compared EPC level and function in patients with BAV with versus without valve dysfunction. The study group included 22 patients with BAV and significant valve dysfunction (at least moderate aortic regurgitation and/or at least moderate aortic stenosis). The control group included 28 patients with BAV without valve dysfunction. All patients had 1 blood sample taken. Proportion of peripheral mononuclear cells expressing vascular endothelial growth factor receptor 2, CD133 and CD34 was evaluated by flow cytometry. EPC colony-forming units (CFUs) were grown from peripheral mononuclear cells, characterized, and counted after 7 days of culture. The 2 groups had similar clinical characteristics except for higher prevalence of hypertension in the dysfunctional valve group. Number of EPC CFUs was smaller in the dysfunctional valve group (32 CFUs/plate, 15 to 42.5, vs 48 CFUs/plate, 30 to 62.5, respectively, p = 0.01), and the migratory capacity of the cells in this group was decreased. In addition, the proportion of cells coexpressing vascular endothelial growth factor receptor 2, CD133, and CD34 tended to be smaller in the dysfunctional valve group. In conclusion, patients with BAV and significant valve dysfunction appear to have circulating EPCs with impaired functional properties. These findings require validation by further studies.     

Circulating progenitor cells and their interaction with platelets in patients with an acute coronary syndrome

CD34⁺ cells expressing KDR (CD34⁺/KDR⁺) represent a small proportion of circulating progenitor cells that have the capacity to interact with platelets and to differentiate into mature endothelial cells, thus contributing to vascular homeostasis and regeneration as well as to re-endothelialization. We investigated the levels of CD34⁺ and CD34⁺/KDR⁺ progenitor cells as well as their interaction with platelets in acute coronary syndrome (ACS) patients before the initiation (baseline) of their treatment with a P2Y₁₂ receptor antagonist, and at 5-days post-treatment (follow-up). Sixty-seven consecutive ACS patients and thirty healthy subjects (controls) participated in the study. On admission, all patients received 325 mg aspirin, followed by 100 mg/day and then were loaded either with 600 mg clopidogrel or 180 mg ticagrelor, followed by 75 mg/day (n = 36) or 90 mg × 2/day (n = 31), respectively. The levels of circulating CD34⁺ and CD34⁺/KDR⁺ progenitor cells, as well as their interaction with platelets, were determined by flow cytometry, before and after activation with ADP, in vitro. The circulating levels of CD34⁺ and CD34⁺/KDR⁺ cells in both patient groups at baseline were lower compared with controls while they were significantly increased at 5-days of follow-up in both groups, this increase being more pronounced in the ticagrelor group. The platelet/CD34⁺ (CD61⁺/CD34⁺) conjugates were higher at baseline and reduced at follow-up while the platelet/KDR⁺ (CD61⁺/KDR⁺) conjugates were lower at baseline and increased at follow-up, both changes being more pronounced in the ticagrelor group. ADP activation of control samples significantly increased the KDR expression by CD34⁺ cells and the CD61⁺/KDR⁺ conjugates, these parameters being unaffected in patients at baseline but increased at follow-up. Short-term dual antiplatelet therapy in ACS patients restores the low platelet/KDR⁺ conjugates and CD34⁺ cell levels and improves the low membrane expression levels of KDR in these cells, an effect being more pronounced in ticagrelor-treated patients. This may represent a pleiotropic effect of antiplatelet therapy towards vascular endothelial regeneration.     

Effect of simvastatin on endothelial function in cardiac syndrome X patients

Patients with cardiac syndrome X with mild hypercholesterolemia were randomized to placebo (n = 20) or simvastatin 20 mg/day (n = 20). In the simvastatin group, there was a significant (26%; p < 0.0001) decrease in total cholesterol, a 38% (p < 0.0001) decrease in low-density lipoprotein cholesterol levels, and 7% a (p < 0.0001) increase in high-density lipoprotein cholesterol levels, without significant changes in triglyceride levels. Brachial artery flow-mediated dilation increased significantly (52% relative increase, p < 0.0001), and the time to > 1-mm ST-segment depression during stress testing was longer by the end of the study (p < 0.0001).     

Circulating endothelial cells in patients with septic shock

The vascular endothelium has a central role in the control of microvascular tone, and it has been proposed that vascular endothelial damage occurs in septic shock, producing multiorgan failure. We have developed a method of detecting circulating endothelial cells (EC) that provides direct evidence of EC shedding in human sepsis. Human umbilical vein endothelial cells (HUVEC) were seeded in whole blood and recovered by isopycnic centrifugation to validate the technique. Blood samples were subsequently taken from 11 healthy volunteers, nine ventilated intensive care unit (ICU) control patients without sepsis, eight patients with sepsis but without shock, and 15 patients with septic shock. EC were identified by indirect immunofluorescence, using antibodies to von Willebrand factor (vWf) and the vascular endothelial growth factor receptor KDR. Mean HUVEC recovery was 86% for 20 to 100 seeded cells/ml of blood. vWf-positive EC counts per milliliter were significantly higher (analysis of variance [ANOVA], p < 0.0001) in patients with sepsis (16.1 +/- 2.7 [mean +/- SEM]) and septic shock (30.1 +/- 3.3) than in healthy (1.9 +/- 0.5) or ICU controls (2.6 +/- 0.6). KDR-positive EC counts per milliliter were also significantly higher (ANOVA, p < 0.0001) in patients with sepsis (4.2 +/- 1.1/ml) and septic shock (10.4 +/- 1.2/ml) than in healthy (0.7 +/- 0.3/ml) or ICU controls (0.5 +/- 0.2/ml). Cell counts made with anti-vWf antibody were consistently higher than those made with anti KDR antibody, but correlation between the two counts was high (r(2) = 0.93). The number of circulating KDR-positive EC was significantly higher in patients who died of septic shock than in survivors (12.0 +/- 1.6/ml versus 7.1 +/- 1.2/ml, p = 0.026). An increase in circulating EC can be identified during sepsis and septic shock. This supports the hypothesis that endothelial damage occurs in human sepsis.     

Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells

Circulating endothelial progenitor cells (EPC) are incorporated into newly formed capillaries, enhance neovascularization after hind limb ischemia and improve cardiac function after ischemic injury. Incorporated progenitor cells may also promote neovascularization and cardiac regeneration by releasing factors, which act in a paracrine manner to support local angiogenesis and mobilize tissue residing progenitor cells. Therefore, we analyzed the expression profile of cytokines in human peripheral blood-derived EPC as opposed to human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), and CD14(+) monocytes by microarray technology. A gene tree analysis revealed a distinct expression pattern of angiogenic growth factors in EPC, mature endothelial cells, and CD14(+) monocytes. VEGF-A, VEGF-B, SDF-1, and IGF-1 mRNA levels were higher in EPC as compared to HUVEC or HMVEC. The enhanced mRNA expression was paralleled by a significant release of VEGF, SDF-1, and IGF-1 protein into the cell culture supernatant of EPC. Moreover, immunohistological analysis of ischemic limbs from nude rats revealed that VEGF is also released from recruited human EPC in vivo. As a functional consequence, conditioned medium of EPC induced a strong migratory response of mature endothelial cells, which was significantly inhibited by VEGF and SDF-1 neutralizing antibodies. Finally, conditioned medium of EPC significantly stimulated the migration of cardiac resident c-kit(+) progenitor cells in vitro. Taken together, EPC exhibit a high expression of angiogenic growth factors, which enhanced migration of mature endothelial cells and tissue resident cardiac progenitor cells. In addition to the physical contribution of EPC to newly formed vessels, the enhanced expression of cytokines may be a supportive mechanism to improve blood vessel formation and cardiac regeneration after cell therapy.