Rafael Moreno-Luna

Olive oil polyphenols decrease blood pressure and improve endothelial function in young women with mild hypertension.

BACKGROUND Olive oil polyphenols have been associated with several cardiovascular health benefits. This study aims to examine the influence of a polyphenol-rich olive oil on blood pressure (BP) and endothelial function in 24 young women with high-normal BP or stage 1 essential hypertension. METHODS We conducted a double-blind, randomized, crossover dietary-intervention study. After a run-in period of 4 months (baseline values), two diets were used, one with polyphenol-rich olive oil (∼30 mg/day), the other with polyphenol-free olive oil. Each dietary period lasted 2 months with a 4-week washout between diets. Systolic and diastolic BP, serum or plasma biomarkers of endothelial function, oxidative stress, and inflammation, and ischemia-induced hyperemia in the forearm were measured. RESULTS When compared to baseline values, only the polyphenol-rich olive oil diet led to a significant (P < 0.01) decrease of 7.91 mm Hg in systolic and 6.65 mm Hg of diastolic BP. A similar finding was found for serum asymmetric dimethylarginine (ADMA) (-0.09 ± 0.01 µmol/l, P < 0.01), oxidized low-density lipoprotein (ox-LDL) (-28.2 ± 28.5 µg/l, P < 0.01), and plasma C-reactive protein (CRP) (-1.9 ± 1.3 mg/l, P < 0.001). The polyphenol-rich olive oil diet also elicited an increase in plasma nitrites/nitrates (+4.7 ± 6.6 µmol/l, P < 0.001) and hyperemic area after ischemia (+345 ± 386 perfusion units (PU)/sec, P < 0.001). CONCLUSIONS We concluded that the consumption of a diet containing polyphenol-rich olive oil can decrease BP and improve endothelial function in young women with high-normal BP or stage 1 essential hypertension.

Equal modulation of endothelial cell function by four distinct tissue-specific mesenchymal stem cells

Mesenchymal stem cells (MSCs) can generate multiple end-stage mesenchymal cell types and constitute a promising population of cells for regenerative therapies. Additionally, there is increasing evidence supporting other trophic activities of MSCs, including the ability to enable formation of vasculature in vivo. Although MSCs were originally isolated from the bone marrow, the presence of these cells in the stromal vascular fraction of multiple adult tissues has been recently recognized. However, it is unknown whether the capacity to modulate vasculogenesis is ubiquitous to all MSCs regardless of their tissue of origin. Here, we demonstrated that tissue-resident MSCs isolated from four distinct tissues have equal capacity to modulate endothelial cell function, including formation of vascular networks in vivo. MSCs were isolated from four murine tissues, including bone marrow, white adipose tissue, skeletal muscle, and myocardium. In culture, all four MSC populations secreted a plethora of pro-angiogenic factors that unequivocally induced proliferation, migration, and tube formation of endothelial colony-forming cells (ECFCs). In vivo, co-implantation of MSCs with ECFCs into mice generated an extensive network of blood vessels with ECFCs specifically lining the lumens and MSCs occupying perivascular positions. Importantly, there were no differences among all four MSCs evaluated. Our studies suggest that the capacity to modulate the formation of vasculature is a ubiquitous property of all MSCs, irrespective of their original anatomical location. These results validate multiple tissues as potential sources of MSCs for future cell-based vascular therapies.

Transdermal regulation of vascular network bioengineering using a photopolymerizable methacrylated gelatin hydrogel.

The search for hydrogel materials compatible with vascular morphogenesis is an active area of investigation in tissue engineering. One candidate material is methacrylated gelatin (GelMA), a UV-photocrosslinkable hydrogel that is synthesized by adding methacrylate groups to the amine-containing side-groups of gelatin. GelMA hydrogels containing human endothelial colony-forming cells (ECFCs) and mesenchymal stem cells (MSCs) can be photopolymerized ex vivo and then surgically transplanted in vivo as a means to generate vascular networks. However, the full clinical potential of GelMA will be best captured by enabling minimally invasive implantation and in situ polymerization. In this study, we demonstrated the feasibility of bioengineering human vascular networks inside GelMA constructs that were first subcutaneously injected into immunodeficient mice while in liquid form, and then rapidly crosslinked via transdermal exposure to UV light. These bioengineered vascular networks developed within 7 days, formed functional anastomoses with the host vasculature, and were uniformly distributed throughout the constructs. Most notably, we demonstrated that the vascularization process can be directly modulated by adjusting the initial exposure time to UV light (15-45 s range), with constructs displaying progressively less vascular density and smaller average lumen size as the degree of GelMA crosslinking was increased. Our studies support the use of GelMA in its injectable form, followed by in situ transdermal photopolymerization, as a preferable means to deliver cells in applications that require the formation of vascular networks in vivo.

Human endothelial colony-forming cells serve as trophic mediators for mesenchymal stem cell engraftment via paracrine signaling

Significance Endothelial colony-forming cells (ECFCs) circulate in peripheral blood and contribute to the formation of new vasculature. Here, we demonstrate that ECFCs can also function as paracrine mediators prior to the establishment of blood perfusion, modulating the regenerative potential of human mesenchymal stem cells (MSCs). We show that MSCs are highly dependable on platelet-derived growth factor BB and that ECFCs provide critical angiocrine factors needed to preserve MSCs as viable. This early angiocrine support ultimately enables extensive engraftment and long-term differentiation of transplanted MSCs. This study provides new insights into the biological attributes of ECFCs that may expand their therapeutic potential. We foresee the use of ECFCs as a means to improve the outcome of MSC transplantation. Endothelial colony-forming cells (ECFCs) are endothelial precursors that circulate in peripheral blood. Studies have demonstrated that human ECFCs have robust vasculogenic properties. However, whether ECFCs can exert trophic functions in support of specific stem cells in vivo remains largely unknown. Here, we sought to determine whether human ECFCs can function as paracrine mediators before the establishment of blood perfusion. We used two xenograft models of human mesenchymal stem cell (MSC) transplantation and studied how the presence of ECFCs modulates MSC engraftment and regenerative capacity in vivo. Human MSCs were isolated from white adipose tissue and bone marrow aspirates and were s.c. implanted into immunodeficient mice in the presence or absence of cord blood-derived ECFCs. MSC engraftment was regulated by ECFC-derived paracrine factors via platelet-derived growth factor BB (PDGF-BB)/platelet-derived growth factor receptor (PDGFR)-β signaling. Cotransplanting ECFCs significantly enhanced MSC engraftment by reducing early apoptosis and preserving stemness-related properties of PDGFR-β+ MSCs, including the ability to repopulate secondary grafts. MSC engraftment was negligible in the absence of ECFCs and completely impaired in the presence of Tyrphostin AG1296, an inhibitor of PDGFR kinase. Additionally, transplanted MSCs displayed fate-restricted potential in vivo, with adipose tissue-derived and bone marrow-derived MSCs contributing exclusive differentiation along adipogenic and osteogenic lineages, respectively. This work demonstrates that blood-derived ECFCs can serve as paracrine mediators and regulate the regenerative potential of MSCs via PDGF-BB/PDGFR-β signaling. Our data suggest the systematic use of ECFCs as a means to improve MSC transplantation.

Decreased Level of Cord Blood Circulating Endothelial Colony–Forming Cells in Preeclampsia

Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony–forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=15) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation, and migration toward vascular endothelial growth factor-A and fibroblast growth factor-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies (P=0.04), a reduction that was independent of other obstetric factors. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events.

Reduced sTWEAK and Increased sCD163 Levels in HIV-Infected Patients: Modulation by Antiretroviral Treatment, HIV Replication and HCV Co-Infection

Background Patients infected with the human immunodeficiency virus (HIV) have an increased risk of cardiovascular disease due to increased inflammation and persistent immune activation. CD163 is a macrophage scavenger receptor that is involved in monocyte-macrophage activation in HIV-infected patients. CD163 interacts with TWEAK, a member of the TNF superfamily. Circulating levels of sTWEAK and sCD163 have been previously associated with cardiovascular disease, but no previous studies have fully analyzed their association with HIV. Objective The aim of this study was to analyze circulating levels of sTWEAK and sCD163 as well as other known markers of inflammation (hsCRP, IL-6 and sTNFRII) and endothelial dysfunction (sVCAM-1 and ADMA) in 26 patients with HIV before and after 48 weeks of antiretroviral treatment (ART) and 23 healthy subjects. Results Patients with HIV had reduced sTWEAK levels and increased sCD163, sVCAM-1, ADMA, hsCRP, IL-6 and sTNFRII plasma concentrations, as well as increased sCD163/sTWEAK ratio, compared with healthy subjects. Antiretroviral treatment significantly reduced the concentrations of sCD163, sVCAM-1, hsCRP and sTNFRII, although they remained elevated when compared with healthy subjects. Antiretroviral treatment had no effect on the concentrations of ADMA and sTWEAK, biomarkers associated with endothelial function. The use of protease inhibitors as part of antiretroviral therapy and the presence of HCV-HIV co-infection and/or active HIV replication attenuated the ART-mediated decrease in sCD163 plasma concentrations. Conclusion HIV-infected patients showed a proatherogenic profile characterized by increased inflammatory, immune-activation and endothelial-dysfunction biomarkers that partially improved after ART. HCV-HIV co-infection and/or active HIV replication enhanced immune activation despite ART.

Role of Circulating Cell-free DNA Levels in Patients With Severe Preeclampsia and HELLP Syndrome

BACKGROUND Increased plasma levels of circulating cell-free DNA (c-f DNA) have been recently described in diseases related to ischemia and/or hypoxia. Preeclampsia (PCL) is a hypertensive disorder of pregnancy, of unknown origin, where a defective placentation resulting in placental ischemia plays an important role. HELLP syndrome (haemolysis, elevated liver enzymes, and low platelet count) is the most serious form of PCL. The origin of the disease is unknown, and there are no markers to help us to make an early diagnosis of disease or to predict patients who are at risk of suffering serious complications. METHODS We measured circulating c-f DNA levels in a group of control pregnant women (n = 20), patients with mild PCL (n = 9), patients with severe PCL (n = 24), and patients with HELLP syndrome (n = 8). RESULTS Values of circulating c-f DNA were 333.59 ± 64.3 ng/ml in control subjects; 635.11 ± 111.7 ng/ml in patients with mild PCL; 1,264.63 ± 127.1 ng/ml in patients with severe PCL, and 1,595.95 ± 269.8 ng/ml in patients with HELPP syndrome. (P < 0.0001). Values of c-f DNA >950 ng/ml had a sensitivity and specificity for detecting severe PCL and/or HELLLP syndrome of 0.71 and 0.93, respectively. CONCLUSIONS As far as we know, this is the first report of increased c-f DNA levels in HELLP syndrome. In this preliminary report, we have observed a gradual and strong relation between c-f DNA levels and range of severity of PCL, with it the highest in patients with HELLP syndrome. Further studies are needed for evaluating the utility of this technique in hypertensive disorders of pregnancy and, particularly, in HELLP syndrome.

Triglyceride-Rich Lipoprotein Regulates APOB48 Receptor Gene Expression in Human THP-1 Monocytes and Macrophages

The postprandial metabolism of dietary fats implies that the production of TG-rich lipoproteins (TRL) contributes to the progression of plaque development. TRL and their remnants cause rapid receptor-mediated monocyte/macrophage lipid engorgement via the cell surface apoB48 receptor (apoB48R). However, the mechanistic basis for apoB48 receptor (APOB48R) regulation by postprandial TRL in monocytes and macrophages is not well established. In this study, we investigated the effects of postprandial TRL from healthy volunteers on the expression of APOB48R mRNA and lipid uptake in human THP-1 monocytes and THP-1-derived macrophages. The expression of APOB48R mRNA was upregulated in THP-1 monocytes, but downregulated in THP-1-derived macrophages when treated with postprandial TRL (P < 0.05), in a dose- and time-dependent manner. TG and free cholesterol were dramatically increased in THP-1-derived macrophages (140 and 50%, respectively; P < 0.05) and in THP-1 monocytes (160 and 95%, respectively; P < 0.05). This lipid accumulation was severely decreased (~50%; P < 0.05) in THP-1-derived macrophages by small interfering RNA (siRNA) targeting of APOB48R. Using PPAR and retinoid X receptor (RXR) agonists, antagonists, and siRNA, our data indicate that PPARα, PPARγ, and RXRα are involved in postprandial TRL-induced APOB48R transcriptional regulation. Co-incubation with acyl-CoA synthetase or acyl-CoA:cholesterol acyltransferase inhibitors potentiated the effects of postprandial TRL on the expression of APOB48R mRNA in THP-1 monocytes and THP-1-derived macrophages. Our findings collectively suggest that APOB48R represents a molecular target of postprandial TRL via PPAR-dependent pathways in human THP-1 monocytes and macrophages and advance a potentially important link between postprandial metabolism of dietary fats and atherogenesis.

Human white adipose tissue vasculature contains endothelial colony-forming cells with robust in vivo vasculogenic potential

Blood-derived endothelial colony-forming cells (ECFCs) have robust vasculogenic potential that can be exploited to bioengineer long-lasting human vascular networks in vivo. However, circulating ECFCs are exceedingly rare in adult peripheral blood. Because the mechanism by which ECFCs are mobilized into circulation is currently unknown, the reliability of peripheral blood as a clinical source of ECFCs remains a concern. Thus, there is a need to find alternative sources of autologous ECFCs. Here we aimed to determine whether ECFCs reside in the vasculature of human white adipose tissue (WAT) and to evaluate if WAT-derived ECFCs have equal clinical potential to blood-derived ECFCs. We isolated the complete endothelial cell (EC) population from intact biopsies of normal human subcutaneous WAT by enzymatic digestion and selection of CD31+ cells. Subsequently, we extensively compared WAT-derived EC phenotype and functionality to bonafide ECFCs derived from both umbilical cord blood and adult peripheral blood. We demonstrated that human WAT is indeed a dependable source of ECFCs with indistinguishable properties to adult peripheral blood ECFCs, including hierarchical clonogenic ability, large expansion potential, stable endothelial phenotype, and robust in vivo blood vessel-forming capacity. Considering the unreliability and low rate of occurrence of ECFCs in adult blood and that biopsies of WAT can be obtained with minimal intervention in an ambulatory setting, our results indicate WAT as a more practical alternative to obtain large amounts of readily available autologous ECFCs for future vascular cell therapies.

Which parameter is better to define endothelial dysfunction in a test of postocclusive hyperemia measured by laser-Doppler flowmetry?

Background and objectiveEndothelial function can be measured by the level of reactive vasodilation due to a transient ischemia caused by a blood pressure cuff on the arm, measured using Laser-Doppler flowmetry. This device has software that provides various parameters that can measure the magnitude of this response, but there are no general agreements with regard to which of them is the best to use. In this study, we analyze which of the parameters obtained using this technique is better to discriminate between patients with coronary artery disease (CAD) and healthy controls. MethodsWe analyzed 40 patients with proven CAD and 60 healthy controls. We studied the hyperemic response to the ischemia in the forearm using a Laser-Doppler flowmeter. ResultsThe most important differences between patients and controls were determined considering the area of hyperemia, which was 2.6 times higher in healthy controls than that in patients (754.9±566.4 vs 1981.3±1156.3 perfusion units per second, P<0.001). To diagnose the disease, a cutoff point of 860 perfusion units per second had a sensitivity of 0.82 and a specificity of 0.97. This is probably because the area of hyperemia measures at the same time speed, intensity, and duration of the hyperemic response. ConclusionThe area of hyperemia was the parameter with a higher sensitivity and specificity for identification of patients with CAD. Nevertheless, further studies are needed to confirm the usefulness of this parameter, obtained using a noninvasive test, to assess the presence of subclinical coronary heart disease.