Background: Endothelial dysfunction is central to PAH. In this study, we simultaneously analysed circulating levels of endothelial microvesicles (EMVs) and progenitor cells (PCs) in PAH and in controls, as biomarkers of pulmonary endothelial integrity and evaluated differences among PAH subtypes and as a response to treatment.

Methods: Forty-seven controls and 144 patients with PAH (52 idiopathic, 9 heritable, 31 associated with systemic sclerosis, 15 associated with other connective tissue diseases, 20 associated with HIV and 17 associated with portal hypertension) were evaluated. Forty-four patients with scleroderma and 22 with HIV infection, but without PAH, were also studied. Circulating levels of EMVs, total (CD31⁺CD42b^-) and activated (CD31⁺CD42b^-CD62E⁺), as well as circulating PCs (CD34⁺CD133⁺CD45^low) were measured by flow cytometry and the EMVs/PCs ratio was computed. In treatment-naïve patients, measurements were repeated after 3 months of PAH therapy.

Results: Patients with PAH showed higher numbers of EMVs and a lower percentage of PCs, compared with healthy controls. The EMV/PC ratio was increased in PAH patients, and in patients with SSc or HIV without PAH. After starting PAH therapy, individual changes in EMVs and PCs were variable, without significant differences being observed as a group. Conclusion: PAH patients present disturbed vascular homeostasis, reflected in changes in circulating EMV and PC levels, which are not restored with PAH targeted therapy. Combined measurement of circulating EMVs and PCs could be foreseen as a potential biomarker of endothelial dysfunction in PAH.

Keywords: PAH-specific treatment; biomarkers; endothelial dysfunction; endothelial extracellular vesicles; progenitor cells; pulmonary arterial hypertension.

The experimental aim of this study was to determine, in vitro, the effects of glucose-induced EMPs on endothelial cell expression of E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 and platelet cell adhesion molecule-1 (PECAM-1). Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 10⁵ cells/condition. HUVECs were incubated with media containing either 25 mM d-glucose (concentration representing a hyperglycemic state) or 5 mM d-glucose (normoglycemic condition) for 48 h to generate EMPs. EMP identification (CD144⁺) and concentration were determined by flow cytometry. HUVECs (3 × 10⁶ cells/condition) were treated with either high glucose-derived EMPs (hgEMPs) or normal glucose-derived (ngEMPs) for 24 h and surface expression of E-selectin (CD62E-PE), ICAM-1 (CD54-FITC), VCAM-1 (CD106-APC) and PECAM-1 (CD31-BV) was assessed by flow cytometry and reported as mean fluorescent intensity (MFI). Hyperglycemic-derived EMPs induced significantly higher surface expression of E-selectin (2614 ± 132 vs. 2010 ± 204 MFI), ICAM-1 (2110 ± 81 vs. 1688 ± 152 MFI), VCAM-1 (3589 ± 431 vs. 2134 ± 386) and PECAM-1 (4237 ± 395 vs. 2525 ± 269 MFI) on endothelial cells than EMPs from normoglycemic conditions. Microparticle-induced cell adhesion molecule expression provides potential novel mechanistic insight regarding the accelerated risk of atherosclerotic vascular disease associated with hyperglycemia.

OBJECTIVE

E-selectin (CD62E) is an adhesion molecule that participates in the binding of leukocytes to activated blood vascular endothelium. The present study was undertaken to characterise the pattern of E-selectin expression on epithelial cells of the gingival crevice and oral epithelium.

METHODS

A panel of six anti-E-selectin monoclonal antibodies was reacted with cryosections of human gingiva and cytospins of cultured gingival keratinocytes.

RESULTS

Three antibodies raised against leukocyte binding epitopes of the molecule (H4/18, H18/7, 1.2B6) were reactive with gingival keratinocytes, three were negative (4D10, BBA1, BBA2), while all six reacted with endothelial cells. Staining with H18/7 and 1.2B6 was weaker and more variable than with H4/18. In cell culture, levels of E-selectin expression reduced slowly, and were only modestly increased by treatment with exogenous TNF alpha, a known inducer of E-selectin on endothelium.

CONCLUSIONS

E-selectin epitopes are expressed on gingival keratinocytes. These binding epitopes may be involved in the traffic of leukocytes in this tissue compartment.

In this study we have investigated the effect of GM-CSF and IL3 on Human Umbilical Vein Endothelial Cells (HUVEC). We studied the adhesion properties of HUVEC for non stimulated human elutriated monocytes, as well as the transendothelial migration of these cells. We analysed the expression of adhesion molecules (VLA4/CDw49d, VCAM1/CD106, LFA1/CD11a, ICAM1/CD54, CD18, L-selectin/CD62L, PeCAMl/CD31, ELAM1/CD62E) induced in monocyte adhesion and transmigration. Optimal conditions of HUVEC stimulation with IL3 and GM-CSF were obtained with 100 U/ml of each cytokine. IL3 and GM-CSF were found to induce HUVEC proliferation, more than twofold at day 7 of the culture compared to controls. HUVEC proliferation was not stimulated by IL1α, a slight inhibitory effect was observed at 250 and 500 U/ml. We showed that GM-CSF, IL3 and their combination mimic on activation like status that on which is expressed by an enhancement of adhesion and migration of non stimulated monocytes to and across cytokines activated HUVEC monolayers. After 6 hours activation with IL3 or GM-CSF, more than 60% of the monocytes are adherent to HUVEC after a contact of 30 minutes (vs 30.8 ± 4.6% for untreated control HUVEC). This percentage increased to 80% after a 7 days culture period in presence of the same cytokines (vs 40 ± 5.1% for untreated control HUVEC). IL3 was very effective at inducing monocyte transendothelial migration. The potency of IL3 is seen to be 2 to 3 fold higher than GM-CSF in this system. GM-CSF and IL3 modulate on HUVEC the expression of adhesion molecules induced in monocyte adhesion and transendothelial migration processes. We showed that anti-ELAMl inhibit in part monocyte migration (8.5 ± 3% vs 46.33 ± 4.03% without MoAb; vs 5.1 ± 2% with ICAM1, ELAM1 and VCAMI MoAbs).

Costimulatory molecules play critical roles during cell-mediated immune responses. We undertook this study to determine whether CD154-CD40 interactions induced human endothelial cell (EC) activation via the nuclear factor (NF)-kappaB pathway, and whether the upregulation of monocyte-derived CD40 and CD80 is NF-kappaB pathway dependent. A CD154-expressing D1.1 cell-EC coculture with or without the NF-kappaB inhibitor BAY11-7082 was established to examine EC activation as indicated by CD62E expression. Peripheral blood mononuclear cell (PBMC)-EC cocultures were performed in the presence or absence of BAY11-7082; the expression of CD40 and CD80 on monocytes was analyzed by FACS. Allogeneic mixed lymphocyte-EC reaction (MLER) was performed to determine the inhibitory effects of BAY11-7082 to prevent lymphocyte proliferation. FACS demonstrated upregulation of EC-derived CD62E expression induced by CD154 expressing D1.1 cells. BAY11-7082 pretreated EC failed to upregulate CD62E after interaction with D1.1 cells. Monocytes upregulated CD40 and CD80 expression during PBMC-HEC interaction, and BAY11-7082 suppressed monocyte-derived CD40 and CD80 expression in a dose-dependent manner. The monocyte-derived CD86 expression was downregulated by NF-kappaB inhibitor. BAY11-7082 demonstrated inhibition of lymphocyte proliferation of allogeneic MLER. This study demonstrated that the NF-kappaB inhibitor BAY11-7082 prevented CD154-CD40 interaction-induced EC activation, suggesting that the activation of EC by T-cell-derived CD154 is via NF-kappaB pathway. The NF-kappaB inhibitor suppressed upregulation of monocytederived CD40 and CD80. Additionally, BAY11-7082 suppressed lymphocyte proliferation in response to allogeneic EC. These data indicated that NF-kappaB plays an important role in regulating costimulatory molecules in allogeneic immune responses, and strengthens the rationale for the use of NF-kappaB-directed therapy in allotransplantation.

OBJECTIVE

To study the effect of PF4 and relative peptide PF4 17-70 on the chemoattract ability, the expression of adhesion molecules and CXCR4 on the flesh cord blood CD34+ cells.

METHODS

CD34+ cells were separated from the cord blood using MACS immune magnetic beads, the chemoattract ability was assayed using the Transwell board, the expression of adhesion molecules and CXCR4 was measured by FACS.

RESULTS

(1) PF4 and PF4 17-70 increased the migration of the CD34+ cells, the chemoattract percentage of PF4 was 157.43% +/- 50.06% (P < 0.05) and PF4 17-70 was 187.02% +/- 10.69% (P < 0.05). (2) The expression of CD49d and CXCR4 on the CD34+ cells increased after PF4 incubated, but the expressions of other adherent molecules including CD31, CD44, CD11a, CD62p, CD62E did not change.

CONCLUSIONS

PF4 has the chemoattract ability on the umbilical blood CD34+ cells by promoting the expression of integrin CD49d and CXCR4, PF4 may help the cord stem cells homing.

: To evaluate the plasma levels of soluble endothelial cell molecules in patients with venous thromboembolism (VTE) out of the acute phase as compared with healthy individuals. We also investigated the possible associations of the soluble endothelial cell molecules among them, as well as with other clinical and laboratory data, including the numbers of circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP), and CEC expressing activation-related [cluster of differentiation (CD)54 and CD62E] and procoagulant (CD142) markers. In total, 15 patients with VTE and 20 normal individuals were studied. The CEC and CEP were quantified and characterized by flow cytometry. The soluble molecules studied included P-selectin, E-selectin, intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1 and tissue factor (ELISA), and von Willebrand factor antigen (immunoturbidimetry). VTE patients had significantly higher levels of vascular cell adhesion molecule 1 and von Willebrand factor antigen and lower levels of soluble E-selectin than controls. They also showed significantly higher numbers of CEC, as of activated/procoagulant CEC and lower numbers of CEP, compared with controls. We did not find any correlation between the levels of soluble molecules and the numbers of endothelial cell in circulation, but there was with several clinical and laboratory data in VTE patients. Our results would suggest that in VTE patients, the endothelium remains activated and in some hypercoagulable state. The levels of soluble endothelial cell molecules did not seem to be directly related to the numbers of CEC and CEP neither reflected the number of activated CEC, which may be because of the different function that surface and soluble molecules may have.

The aim of this work was to evaluate contribution of released membrane particles (RMP) to the development of systemic inflammatory response (SIR) after aortocoronary bypass grafting (ACBG). The number of RMP carrying surface adhesion molecules, CD62L, CD62P, CD62E, was shown to increase in the early postoperative period in parallel with the enhancement of lymphocyte plasma membrane blebbing and elevation of cytokine levels in peripheral blood. It is concluded that (1) activation of plasma membrane blebbing in peripheral blood cells underlies the appearance of RMP in circulation; (2) increased number of RMP expressing CD62L, CD62P, CD62E is a marker of intercellular communication associated with the development of SIR and suggests new mechanisms of RMP involvement in the reaction of organism to massive surgical injury.

OBJECTIVE

To investigate the effects of microparticles (Mps) from different people on the vascular endothelial cell function.

METHODS

Third generation human umbilical vein endothelial cells (HUVECs) were cultured with Mps-containing plasma samples from 5 systemic lupus erythematosus (SLE) patients undergoing heavy steroid treatment, 5 patients with steroid-induced avascular necrosis of femoral head (ANFH), 4 patients with alcohol induced ANFH, and 4 healthy persons for 12 h, 24 h, and 48 h respectively. Plasma samples from the above persons with the Mps filtered were used as experimental controls. HUVECs cultured with blank culture fluid were used as blank controls. Inverse phase contrast microscopy was used to observe the morphology of the HUVECs. PE-CD31 and PEcy5-CD62E were added into the flow cytometric test tubes and flow cytometry (FC) was used to count the number of CD62E +/ CD31 + Mps in the medium. RT-PCR was used to measure the mRNA expression of the apoptotic gene fas (fas/beta-actin).

RESULTS

Microscopy showed no distinct difference between the morphology of the HUVECs among the different groups. FC showed that the number of CD62E +/CD31 + Mps 48 hours after the 20% Mp stimulation of the steroid-treated SLE group was significantly lower than that of the control group (P = 0.035). RT-PCR showed that 48 hours after the stimulation the levels of mRNA fas/beta-actin of the steroid-treated SLE group and steroid induced ANFH group were 0.914 +/- 0.226 and 0.776 +/- 0.230 respectively, both significantly higher than those of the control groups (0.832 +/- 0. 200 and 0.669 +/- 0.148 respectively, P = 0.005 and P = 0.006).

CONCLUSIONS

The Mps from the steroid treated patients aggravate the apoptosis of HUVECs, and the Mps from the steroid induced ANFH patients augment the production of apoptosis gene in HUVECs. The Mps from healthy people and alcohol induced ANFH patients have no relationship with HUVEC apoptosis.

Endothelial dysfunction is a common feature of early complications of hemato-oncologic therapy. The aim of our study was to assess the profile of endothelial function at diagnosis time, then during initial treatment phase of acute lymphoblastic leukemia (ALL), and to verify the presence of its correlation with early clinical outcome (ECO). 28 ALL children and 18 healthy age-matched control ones were recruited. Study group was examined at baseline and at 33rd and 78th day of treatment. At each protocol step the endothelial function was assessed by measurement of sP-selectin (CD62-P), PAI-1(serpinE1), sE-selectin (CD62E), sICAM-1(sCD54), sVCAM-1(sCD106), and VEGF concentrations. Higher baseline sICAM-1 and sVCAM-1 levels and lower sP-selectin and VEGF were observed in children with ALL. sICAM-1, sVCAM-1, and sE-selectin levels were decreasing following the treatment with protocol I. Higher sE-selectin and lower baseline sICAM-1 levels were observed in children treated unsuccessfully. Lower PAI-1 levels were observed in children who survived. Higher baseline sE-selectin levels and lower sICAM-1 and VEGF were observed in children treated unsuccessfully. A decrease in sE-selectin and lower PAI-1 at the 78th day of therapy were associated with better ECO. High baseline VEGF and sE-selectin levels, significant increase in PAI-1, and low initial sICAM-1 levels are prognostics for poorer prognosis in the ALL children.

Many strategies for redirecting the tropism of murine Moloney leukemia virus (MMLV) have been described. Preformed virion-liposome complexes, termed virosomes, have been reported to be relatively stable. Virosomes mediate envelope-independent transduction that allows efficient superinfection of resistant cell lines; however, virosome-mediated transduction behaves in a non-target-specific manner. We developed a novel method using antibodies to direct MMLV to vascular endothelium. We have given the term immunovirosomes to the complexes formed between viruses, liposomes, and antibodies. These immunovirosomes improve the transduction efficiency of the viruses and alter their tropism. We have shown improved transduction when immunovirosomes were targeted at the endocytic receptors CD71 and CD62E/P and rather less good delivery when targeted at CD106. The enhancement of the transduction efficiency was transient, however, suggesting that rerouting the entry pathway of viruses alters the expression properties of the viruses.

No detailed information currently exists about the immune phenotypic profiles of peripheral blood stem cells (PBSCs) obtained by different mobilization regimens. The effects of these profiles on the outcome of transplantation are largely unknown. In this prospective study, the surface immune phenotypic features (CD11a, CD18, CD31, CD38, CD44, CD62e, CD62L, CD90, CD117, CD135 and CD184 expression) of sorted PBSCs that had been mobilized by growth factor with (group I and group II) or without (group III) disease-specific chemotherapies were investigated. The immune phenotypic features on mobilized PBSCs in groups I, II and III were not significantly different. The CD31 (platelet endothelial cell adhesion molecule-1) positivity ratio on PBSCs inversely correlated with both the duration of neutrophil (r=-0.32, p=0.03) and platelet (r=-0.36, p=0.02) engraftment. No relationship was found between the engraftment (neutrophil and platelet) durations and CD184 (chemokine receptor CXC motif receptor 4 [CXCR4]) expression on PBSCs. We demonstrated that the surface immune phenotypic profiles on PBSCs obtained by several mobilization regimens were not different. To our knowledge, this is the first study to demonstrate that CD31 expression on human PBSCs may positively affect both neutrophil and platelet engraftment. Contrary to our expectations, CD184 (CXCR4) expression on PBSCs has no effect on neutrophil or platelet engraftment. Considered together, our results suggest that additional surface antigens (such as CD31) may be more effective in the homing process.

OBJECTIVE

To explore the relationship between the alterations of circulating cell-derived microparticles (MPs) and large-dose glucocorticosteroid application.

METHODS

Peripheral blood samples were collected from 33 patients with history of large-dose glucocorticosteroid application (glucocorticosteroid group) and 24 age-, sex-, and race-matched healthy controls (healthy control group). Platelet-poor plasma was obtained by centrifugation. Plasma microparticles were labeled with monoclonal antibodies of PE-conjugated mouse anti-human CD31, CD54, and FITC-conjugated mouse anti-human CD42b, CD45, CD51/61, CD105, and PE-Cy5-conjugated mouse anti-human CD62E. Cell-derived microparticles were measured by three-color flow cytometry.

RESULTS

The mean ranks of CD31+ MPs, CD45+ MPs, CD51/61 MPs, CD31+ CD42b+ MPs, and CD31+ CD45+ MPs of the glucocorticosteroid group were: 24.1, 25.5, 25.6, 21.6, and 24.8 respectively, all significantly lower than those of the control group (35.7, 33.8, 33.7, 39.3, and 34.8 respectively, P = 0.009, 0.019, 0.045, 0.000, and 0.009).

CONCLUSIONS

High-dose glucocorticosteroid application remarkably reduces plasma MPs that may be responsible for microcirculation disturbance.

OBJECTIVE

To study the effects on adherence of hematopoietic stem/progenitor cells, PF4 was assessed alone or in combination with IL-3 for effects on the total adherence and various kinds of adhesion molecules of KG1a cells as well as actin polymerization in KG1a cells.

METHODS

The total adherence was assayed by crystal violet dye staining. The adhesion molecule expression was determined by FACS analysis. These adhesion molecule monoclonal antibodies individually blocked total adherence by MTT. F-actin content was monitored by fluorospectrophotometry.

RESULTS

100 ng/ml PF4 could increase the total adherence of KG1a cells by 80%. 20 ng/ml IL-3 could increase the total adherence of KG1a cells by 96%. When PF4 and IL-3 were combined, the total adherence could be promoted by 97%. Exposure of 1 x 10(6) cells/ml of KG1a cells to 100 ng/ml PF4 the increased total adherence of KG1a cells was mediated by PECAM-1 (CD31), CD44, LFA-1 (CD11a) and Mac-1 (CD11b) but not by P-selectin (CD62P) and E-selectin (CD62E). These adhesion molecule monoclonal antibodies could individually block total adherence for 34%-43%. Similar phenomenon was observed when IL-3 was added onto KG1a cells. Further study found that PF4 induced actin polymerization of KG1a cells.

CONCLUSIONS

Our study indicated that PF4 promoted total adherence, as well as several adhesion molecule expression and actin polymerization of KG1a cells. The results suggest that PF4 may have therapeutic utility along with other cytokines by enhancing the total adhesion of hematopoietic stem/progenitor cells to promote the homing.

Mesenchymal cells are successfully used to create cell-loaded devices in tissue engineering. Molecular properties of the cells and interaction with polymer scaffolds regulate the development of desired tissues. The present study compared the molecular markers in mesenchymal pleuripotent C3H10T1/2 and osteogenic MBA-15 cells. The cells express transcription factors (TF) of chondro-ostegenic pathway (cbfa-1 and c-fos) and MyoD - TF of muscle differentiation pathway, but not myogenin. Analyzed cells expressed receptors for glucocorticoids, growth hormone, prolactin, and PTH, which indicates their potential responsiveness to systemic signals. Analysis of mRNA encoding for receptors of TGFbeta, TNF, and various interleukins revealed differential expression of IL-2r and TGFbeta-1r receptors, which were expressed by MBA-15 but not by C3H10T1/2 cells. Expression of functional genes indicates differences in the stages of cell differentiation: ALK was present in MBA-15 only, while both cell types expressed collagen-I. Furthermore, we evaluated the expression of adhesion proteins that mediate cell-polymer interactions by flow cytometry analysis. Cell adhesion molecules (CAMs) analyzed were integrinalpha-M (CD11b), selectin-E (CD62E), and PECAM-1 (CD31), which have shown differential expression on cells cultured on plastic, poly(L-lactic acid) (PLLA) or poly(DL-lactide-glycolide acid) (PDLGA) polymer films. Detailed molecular characterization of mesenchymal cells will enable optimization of culture conditions for successful creation of implantable cell-loaded constructs.

OBJECTIVE

To explore the effect of hemangiopoietin (HAPO) on the adhesive properties of human umbilical vein endothelial cells (HUVEC).

METHODS

The adhesion of HUVEC and the expressions of CD54, CD102, CD106, CD31, CD62E, and CD62P were measured by adhesion assay, flow cytometry, and semi-quantitative RT-PCR.

RESULTS

HAPO enhanced the total adherence of HUVEC in a concentration-dependent manner. Flow cytometry analysis revealed that the treatment of HAPO resulted in a significantly increased expression of CD106 and CD62E on HUVEC in a time-dependent manner. When HUVEC were incubated with HAPO for 6 h, the percentage of CD106 + HUVEC and CD62E HUVEC increased about 2.10 folds and 5.84 folds, respectively, compared with control. The time-course of adhesive molecules mRNA expression indicated that the expression of CD106 and CD62E reached at the maximum 1.86 folds and 6.16 folds, respectively, compared with control.

CONCLUSIONS

HAPO may facilitate the homing of hematopoietic stem/progenitor cells.

OBJECTIVE

To study the effect of PF4 on the adherence of leukemic CD34+ KG1a cell to human umbilical vein endothelial cell line ECV-304 cell and on the expression of adhesive molecules.

METHODS

Adhesion assay and adhesion blocking assay were respectively applied to measure the effect of PF4 and/or adhesion molecule monoclonal antibodies on the adhesion property of KG1a. The expressions of adhesion molecules were determined by RT-PCR and FACS analysis.

RESULTS

The adhesion of KG1a cells to ECV-304 was significantly enhanced in the presence of PF4. Such enhancement was also observed when KG1a or ECV-304 cells were separately treated with PF4 before interaction. The adhesion capacity of KG1a cells was reduced when cells were co-incubated with the blocking monoclonal antibodies (MoAbs) against CD49d, CD106, CD54, respectively. In contrast, MoAbs against CD62L, CD62P and CD62E had no such effect. During a period of 3 hours when KG1a or ECV-304 cells were respectively incubated with PF4, the mRNA expressions of CD49 d, CD54 were up-regulated. Furthermore, when KG1a or ECV-304 cells were incubated with PF4 for 2 hours, respectively, the percentages of CD49d+ KG1a cells and CD54+ ECV-304 were increased significantly.

CONCLUSIONS

PF4 can enhance KG1a cell adhesive capacity by increasing the expressions of adhesion molecules.

Endothelial cell activation is thought to play an important role in xenograft rejection through cell retraction and expression of pro-coagulant and pro-inflammatory factors. Identification of antibodies recognizing porcine endothelial molecules would be useful to study and manipulate the inflammatory response to a xenograft. The aim of this study was to investigate the cross-reactivity of antibodies directed against human adhesion molecules and von Willebrand factor (vWF). Binding of monoclonal antibodies (mAbs) directed against human CD3 1, CD44, CD49, CD54, CD62E, CD102, and CD106 was evaluated on resting and activated endothelial cells from human and pig by flow cytometry. Among 30 antibodies tested, 4 were shown to react with pig cells. Two of them, directed against human CD62E (E-selectin) and rabbit CD106 (VCAM-1) reacted strongly with activated and/or resting pig cells, whereas two others, directed to human CD31 (PECAM) and CD44 (H-CAM), bound weakly to pig cells. In addition, we analyzed the cross-reactivity of five polyclonal or monoclonal antibodies to human or pig vWF with human, baboon, rhesus, pig, and rat vWF. Binding of antibodies was tested by ELISA by using platelet lysates as source of vWF from the different species. Four anti-human or porcine vWF antibodies exhibited a broad reactivity with vWF from all species, whereas one anti-human vWF antibody was specific for primate vWF. In this study, we identified a small number of cross-reacting antibodies that may prove useful to study in vitro and in vivo xenogeneic responses. However, the weak antibody cross-reactivity observed with most porcine molecules points out the necessity of producing species-specific antibodies to study the immune response to xenografts or for use as specific immunosuppressive therapeutic reagents.

New findings: What is the central question of this study? What are the cellular and molecular determinants of increased risk for cardiovascular disease from prolonged sitting? What is the main finding and its importance? Prolonged sitting, independent of calf raise interruption strategies, decreases microparticle counts linked to endothelial activation and apoptosis. An acute bout of prolonged sitting appears to promote paradoxical decreases in microparticle counts, but the implications are not yet clear.

Abstract: Repeated exposure to prolonged sitting increases the risk for cardiovascular disease. However, the cellular links by which repeated exposure to prolonged sitting lead to increased cardiovascular risk have not been fully elucidated, with markers of vascular damage and repair such as microparticles (MPs) and circulating angiogenic cell (CACs) being promising targets. The objective of the study was to examine the effects of 3 h of sitting with or without intermittent calf raises on annexin V⁺ /CD34⁺ , annexin V⁺ /CD62E⁺ , and annexin V⁺ /CD31⁺ /42b^- MP populations linked to CAC paracrine activity, endothelial activation and apoptosis, respectively, as well as CD14⁺ /31⁺ , CD3⁺ /31⁺ , and CD34⁺ CACs, which are linked to endothelial repair. In a random order, 20 sedentary participants (14 females, 22 ± 3 years) remained seated for 180 min with or without performing 10 calf raises every 10 min. Blood samples were obtained after 20 min of quiet rest in the supine position before and after sitting. Overall, sitting decreased annexin V⁺ /CD34⁺ MPs (-12 ± 5 events µl^-1 , P < 0.01), annexin V⁺ /CD62E⁺ MPs (-17 ± 4 events µl^-1 , P < 0.001), and annexin V⁺ /CD31⁺ /42b^- MPs (-22 ± 6 events µl^-1 , P < 0.001) regardless of condition. There were no differences in endothelin-1 plasma concentration, CD14⁺ /31⁺ , CD34⁺ or CD3⁺ /31⁺ CAC frequencies. Sitting did not alter CAC number, but decreased MPs linked to endothelial activation, apoptosis and CAC paracrine activity in a manner that was independent of muscle contraction. These findings support changes in markers of endothelial activation and apoptosis with sedentary behaviour and provide new insights into altered intercellular communication with physical inactivity such as prolonged sitting.

Keywords: physical inactivity; sitting; vascular function.

Recent findings have suggested that the primary factors for development of chronic venous disease (CVD), which commonly manifests as varicose veins (VV), are due to structural and biochemical modifications of the vessel wall. The aim of this exploratory study was to characterize by flow cytometry the endothelial cells (EC) mechanically extracted from the varicose saphenous veins (VSV) segments of patients submitted to VV surgery, and to compare the expression of cell surface molecules in these EC with that observed in the EC from the graft SV (GSV) of patients undergoing bypass surgery. EC were isolated from distal- (varicose trunk) and from proximal- (nearly normal) VSV segments of 30 patients submitted to VV surgery, and from proximal GSV segments of 20 patients submitted to bypass surgery (control group), using a mechanical method, and their immunophenotype was characterized by flow cytometry. EC were identified as being CD45^negCD146^brightCD31^bright, and analyzed for expression of activation-related (CD54, CD62E, CD106), procoagulant (CD142), and cell junction (CD31, CD146) molecules, and for the scavenger receptor, CD36. The EC harvested from the SV segments of CVD patients had lower expression of all the molecules evaluated, in comparison to controls; these differences were more evident for the EC isolated from the distal-VSV. The EC extracted from the proximal- and distal-VSV segments of the CVD patients also differ from each other, the first having lower levels of CD62E, CD106, CD142 and CD36. Groups did not match for gender and controls were heterogeneous concerning the underlying pathologies, which may have a confounding effect. Our study revealed that the EC isolated from varicose (distal) and nearly normal (proximal) VSV segments of the CVD patients differ phenotypically from each other, and from the EC of the control group. The VSV segments more affected by the CVD have the lowest expression of the studied markers. We hypothesize that CVD is associated with a decrease on the EC surface molecules, causing EC dysfunctionality. Further studies with a large number of gender-matched participants are needed, to confirm the results obtained in this exploratory study.