Pharmacological inhibition of the F1 -ATPase/P2Y1 pathway suppresses the effect of apolipoprotein A1 on endothelial nitric oxide synthesis and vasorelaxation.

AIM: The contribution of apolipoprotein A1 (APOA1), the major apolipoprotein of high-density lipoprotein (HDL), to endothelium-dependent vasodilatation is unclear, and there is little information regarding endothelial receptors involved in this effect. Ecto-F1 -ATPase is a receptor for APOA1, and its activity in endothelial cells is coupled to adenosine diphosphate (ADP)-sensitive P2Y receptors (P2Y ADP receptors). Ecto-F1 -ATPase is involved in APOA1-mediated cell proliferation and HDL transcytosis. Here, we investigated the effect of lipid-free APOA1 and the involvement of ecto-F1 -ATPase and P2Y ADP receptors on nitric oxide (NO) synthesis and the regulation of vascular tone. METHOD: Nitric oxide synthesis was assessed in human endothelial cells from umbilical veins (HUVECs) and isolated mouse aortas. Changes in vascular tone were evaluated by isometric force measurements in isolated human umbilical and placental veins and by assessing femoral artery blood flow in conscious mice. RESULTS: Physiological concentrations of lipid-free APOA1 enhanced endothelial NO synthesis, which was abolished by inhibitors of endothelial nitric oxide synthase (eNOS) and of the ecto-F1 -ATPase/P2Y1 axis. Accordingly, APOA1 inhibited vasoconstriction induced by thromboxane A2 receptor agonist and increased femoral artery blood flow in mice. These effects were blunted by inhibitors of eNOS, ecto-F1 -ATPase and P2Y1 receptor. CONCLUSIONS: Using a pharmacological approach, we thus found that APOA1 promotes endothelial NO production and thereby controls vascular tone in a process that requires activation of the ecto-F1 -ATPase/P2Y1 pathway by APOA1. Pharmacological targeting of this pathway with respect to vascular diseases should be explored.

Endothelium trans differentiated from Wharton's jelly mesenchymal cells promote tissue regeneration: potential role of soluble pro-angiogenic factors.

BACKGROUND: Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton's jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo. METHODS: Mesenchymal stem cells were isolated from human Wharton's jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO). RESULTS: hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures. CONCLUSION: These results demonstrate that mesenchymal stem cells isolated from Wharton's jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.

Role of lectin-like oxidized low density lipoprotein-1 in fetoplacental vascular dysfunction in preeclampsia.

The bioavailability of nitric oxide (NO) represents a key marker in vascular health. A decrease in NO induces a pathological condition denominated endothelial dysfunction, syndrome observed in different pathologies, such as obesity, diabetes, kidney disease, cardiovascular disease, and preeclampsia (PE). PE is one of the major risks for maternal death and fetal loss. Recent studies suggest that the placenta of pregnant women with PE express high levels of lectin-like oxidized LDL receptor-1 (LOX-1), which induces endothelial dysfunction by increasing reactive oxygen species (ROS) and decreasing intracellular NO. Besides LOX-1 activation induces changes in migration and apoptosis of syncytiotrophoblast cells. However, the role of this receptor in placental tissue is still unknown. In this review we will describes the physiological roles of LOX-1 in normal placenta development and the potential involvement of this receptor in the pathophysiology of PE.

L-arginine transport and nitric oxide synthesis in human endothelial progenitor cells.

Nitric oxide (NO) is an endogenous vasodilator molecule synthetized from L-arginine by a family of nitric oxide synthases. In differentiated human endothelial cells, it is well known that L-arginine uptake via cationic amino acid transporters (y(+)/CAT) or system y(+)L is required for the NO synthesis via endothelial nitric oxide synthase, but there are no reports in human endothelial progenitor cell (hEPC). Therefore, we isolated hEPCs from peripheral blood of healthy donors and cultured them for either 3 (hEPC-3d) or 14 days (hEPC-14d) to characterize the L-arginine transport and NO synthesis in those cells. L-arginine transport and NO synthesis were analyzed in the presence or absence of N-ethylmaleimide or L-nitroarginine methyl ester, as inhibitors of y(+)/CAT system and nitric oxide synthases, respectively. The results showed that L-arginine uptake is higher in hEPC-14d than in hEPC-3d. Kinetic parameters for L-arginine transport showed the existence of at least 2 transporter systems in hEPC: a high affinity transporter system (K(m)= 4.8 ± 1.1 µM for hEPC-3d and 6.1 ± 2.4 µM for hEPC-14d) and a medium affinity transporter system (K(m) = 85.1 ± 4.0 µM for hEPC-3d and 95.1 ± 8 µM for hEPC-14d). Accordingly, hEPC expressed mRNA and protein for CAT-1 (ie, system y(+)) and mRNA for 2 subunits of y(+)L system, yLAT1, and 4F2hc. Higher L-citruline production and NO bioavailability (4-fold), and endothelial nitric oxide synthase expression (both mRNA and protein) were observed in hEPC-14d compared with hEPC-3d. Finally, the high L-citruline formation observed in hEPC-14d was blocked by N-ethylmaleimide. In conclusion, this study allowed to identity a functional L-arginine/NO pathway in two hEPC differentiation stages, which improves the understanding of the physiology of these precursor cells.

Adenosine A2A and A3 receptors are involved in the human endothelial progenitor cells migration.

Human endothelial progenitor cells (hEPC) are recruited to sites of neovascularization where they differentiate into endothelial cells. The signals/factors responsible for hEPC migration and adhesion to sites of injury are not well understood. Elevated levels of adenosine are known to increase mature endothelial cell migration in response to tissue injury. However, the understanding of the role of adenosine in the physiology of hEPC is very limited. Using quantitative polymerase chain reaction and western blot analyses, we detected the expression of the adenosine receptors A2A, A2B, and A3 in hEPC. Stimulation of adenosine receptors using adenosine or the nonselective agonist adenosine-5'-N-ethylcarboxamide (NECA) increased hEPC migration in 1.4-fold and 2.1-fold (P < 0.01), respectively. Stimulation of hEPC using the A2A-specific agonist CGS-21680 resembled the effect observed in migration when using adenosine or NECA. Consequently, NECA and CGS-21680-stimulated migration of hEPC were reverted using the A2A receptor antagonist ZM-241385. NECA-stimulated migration was inhibited in dose-dependent manner using MRS-1523 (Ki of 147 ± 0.016 nM), MRS-1754 (Ki of 1900 ± 0.02 nM), or ZM-241385 (Ki of 0.2 ± 0.01 nM). In conclusion, adenosine stimulates hEPC migration by activating A2A and A3 but not A2B receptors and provides evidence to support a role of adenosine in modulating angiogenic capacity of hEPC.

Fetal endothelium dysfunction is associated with circulating maternal levels of sE-selectin, sVCAM1, and sFlt-1 during pre-eclampsia.

OBJECTIVES: To evaluate the association between endothelial activation markers in the maternal circulation with nitric oxide (NO) synthesis in human umbilical endothelial cells. STUDY DESIGN: This is a case-control study of normal and pre-eclamptic pregnancies. The levels of sE-selectin, soluble vascular cell adhesion molecule 1 (sVCAM-1), and soluble fms-like tyrosine kinase 1 (sFlt-1) were measured by enzyme-linked immunosorbent assay, and histamine-induced NO synthesis was detected by fluorometric examination of the human umbilical vein endothelial cells (HUVECs) isolated from normal and pathological pregnancies. RESULTS: Mothers with severe pre-eclamptic pregnancies have premature and smaller babies than mothers with normal pregnancies (P < 0.05); they also have high maternal plasma levels of sVCAM-1 (∼2-fold), sFlt-1 (∼2.5-fold), and lower (∼70%) histamine-stimulated NO synthesis in HUVECs. A positive relationship between systolic blood pressure (SBP) and plasma levels of sE-selectin, sVCAM-1, and sFlt-1 was demonstrated. Moreover, levels of sE-selectin, sVCAM-1, and sFlt-1 were negatively associated with newborn weight (NBW), gestational age at delivery, and NO synthesis. Women with high E-selectin (>63 ng/ml), VCAM-1 (>752 ng/ml), and sFlt-1 (>15204 pg/ml) showed high risk (∼2-fold) for preterm delivery and very preterm delivery, or fetal weight <1500 g (∼1.5-fold) compared with women with low levels. CONCLUSIONS: High circulating levels of maternal endothelial dysfunction markers present in pre-eclampsia are associated with decreased NO synthesis in fetal endothelium.