Effects on nitric oxide production of urolithins, gut-derived ellagitannin metabolites, in human aortic endothelial cells

The consumption of foodstuffs yielding circulating compounds able to maintain endothelial function by improving nitric oxide (NO) bioavailability can be considered as an effective strategy for cardiovascular disease prevention. This work assessed the in vitro effects of urolithin A, urolithin B, and urolithin B-glucuronide, ellagitannin-derived metabolites of colonic origin, on NO release and endothelial NO synthase (eNOS) activation in primary human aortic endothelial cells (HAECs). Urolithins were tested both individually at 15 μM and as a mixture of 5 μM each, at different time points. The biotransformation of these molecules in cell media due to cell metabolism was also evaluated by UHPLC-MSn . The mix of urolithins at 5 μM significantly increased nitrite/nitrate levels following 24 h of incubation, while single urolithins at 15 μM did not modify NO bioavailability. Both the mix of urolithins at 5 μM and urolithin B-glucuronide at 15 μM activated eNOS expression. All urolithins underwent metabolic reactions, but these were limited to conjugation with sulfate moieties. This study represents a step forward in the understanding of cardiovascular health benefits of ellagitannin-rich foodstuffs and backs the idea that peripheral cells may contribute to urolithin metabolism

Stearic acid at physiologic concentrations induces in vitro lipotoxicity in circulating angiogenic cells

Background and aims Saturated free fatty acids (SFAs) can induce lipotoxicity in different cells. No studies have investigated the effects of SFA in circulating angiogenic cells (CACs), which play a key role in endothelial repair processes. The aim of the study was to assess the effects of SFAs, specifically stearic acid (SA), on viability and function of CACs and to investigate potential underlying molecular mechanisms. Methods CACs were isolated from healthy subjects by established methods. CACs were incubated with BSA-complexed stearate (100 μM) to assess the time course (from 8 to 24 h exposure) of the effects on viability and apoptosis (activation of caspases 3/7), angiogenic function (tube formation assay), pro-inflammatory cytokine (IL-1β, IL-6, IL-8, MCP-1 and TNFα) gene expression (qPCR) and secretion (ELISA), activation of MAPK (JNK, p38 and Erk1/2) by Western blot and endoplasmic reticulum (ER) stress marker (CHOP, BIP, ATF4, XBP-1 and sXBP-1) gene expression by qPCR. Results Stearic acid activates effector caspases in CACs in a dose- and time-dependent manner. SA also impairs CAC function and increases pro-inflammatory molecule (IL-1β, IL-6, IL-8, MCP-1 and TNFα) gene expression and secretion in CACs starting from 3 h of incubation. The activation of JNK by SA mediates pro-inflammatory response, but it may be not necessary for apoptosis. Moreover, SA induces the expression of ER stress markers across the three branches of the ER stress response. Conclusions In humans, both function and viability of CACs are exquisitely vulnerable to physiologic concentrations of stearate; lipotoxic impairment of endothelial repair processes may be implicated in vascular damage caused by SFAs.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Pioglitazone Improves In Vitro Viability and Function of Endothelial Progenitor Cells from Individuals with Impaired Glucose Tolerance

BACKGROUND: Evidence suggests that the PPARγ-agonist insulin sensitizer pioglitazone, may provide potential beneficial cardiovascular (CV) effects beyond its anti-hyperglycaemic function. A reduced endothelial progenitor cell (EPC) number is associated with impaired glucose tolerance (IGT) or diabetes, conditions characterised by increased CV risk. AIM: To evaluate whether pioglitazone can provide benefit in vitro in EPCs obtained from IGT subjects. MATERIALS AND METHODS: Early and late-outgrowth EPCs were obtained from peripheral blood mononuclear cells of 14 IGT subjects. The in vitro effect of pioglitazone (10 µM) with/without PPARγ-antagonist GW9662 (1 µM) was assessed on EPC viability, apoptosis, ability to form tubular-like structures and pro-inflammatory molecule expression. RESULTS: Pioglitazone increased early and late-outgrowth EPC viability, with negligible effects on apoptosis. The capacity of EPCs to form tubular-like structures was improved by pioglitazone in early (mean increase 28%; p=0.005) and late-outgrowth (mean increase 30%; p=0.037) EPCs. Pioglitazone reduced ICAM-1 and VCAM-1 adhesion molecule expression in both early (p=0.001 and p=0.012 respectively) and late-outgrowth (p=0.047 and p=0.048, respectively) EPCs. Similarly, pioglitazone reduced TNFα gene and protein expression in both early (p=0.034;p=0.022) and late-outgrowth (p=0.026;p=0.017) EPCs compared to control. These effects were prevented by incubation with the PPARγ-antagonist GW9662. CONCLUSION: Pioglitazone exerts beneficial effects in vitro on EPCs isolated from IGT subjects, supporting the potential implication of pioglitazone as a CV protective agents

Vildagliptin, but not glibenclamide, increases circulating endothelial progenitor cell number: A 12-month randomized controlled trial in patients with type 2 diabetes

Background: Fewer circulating endothelial progenitor cells (EPCs) and increased plasma (C-term) stromal cell-derived factor 1α (SDF-1α), a substrate of DPP-4, are biomarkers, and perhaps mediators, of cardiovascular risk and mortality. Short-term/acute treatment with DPP-4 inhibitors improve EPC bioavailability; however, long-term effects of DPP-4i on EPCs bioavailability/plasma (C-term) SDF-1α are unknown. Methods: Randomized (2:1) open-label trial to compare the effects of vildagliptin (V) (100 mg/day) vs glibenclamide (G) (2.5 mg bid to a maximal dose of 5 mg bid) on circulating EPC levels at 4 and 12 months of treatment in 64 patients with type 2 diabetes in metformin failure. At baseline, and after 4 and 12 months, main clinical/biohumoral parameters, inflammatory biomarkers, concomitant therapies, EPC number (CD34+/CD133+/KDR+/106 cytometric events) and plasma (C-term) SDF-1α (R&D system) were assessed. Results: Baseline characteristics were comparable in the two groups. V and G similarly and significantly (p < 0.0001) improved glucose control. At 12 months, V significantly increased EPC number (p < 0.05) and significantly reduced (C-term) SDF-1α plasma levels (p < 0.01) compared to G, with no differences in inflammatory biomarkers. Conclusions: V exerts a long-term favorable effect on EPC and (C-term) SDF-1α levels at glucose equipoise, thereby implying a putative beneficial effect on vascular integrity. Trial registration Clinical Trials number: NCT01822548; name: Effect of Vildagliptin vs. Glibenclamide on Circulating Endothelial Progenitor Cell Number Type 2 Diabetes. Registered 28 March, 2013

N-3 PUFA increase bioavailability and function of endothelial progenitor cells

BACKGROUND AND AIMS: Recent data suggest that n-3 PUFA exert beneficial effects on endothelial progenitor cell (EPC) biology. We sought to investigate whether these effects might be mediated by enhanced EPC in vitro function and/or in vivo bioavailability. METHODS AND RESULTS: CACs and late-outgrowth EPCs were isolated from peripheral blood mononuclear cells obtained from 12 donor buffy-coats. The effect of n-3 PUFA (EPA : DHA = 0.9 : 1.5; 9 μM EPA plus 15 μM DHA) was tested on CAC/EPC viability, function (tube-formation) and pro-inflammatory molecule expression. Circulating EPC (cells positive for CD34, CD133 and kinase insert domain receptor - KDR cell-surface antigens by flow cytometry) number was evaluated in 20 healthy subjects (10 F/10 M, 32 ± 5 years), randomized to receive 4 mackerel or sardine portions per week for 6 weeks followed by a 6 week free-diet period. N-3 PUFA improved CAC and late-outgrowth EPC viability (p < 0.05) and the capacity to form tube-like structures in CACs (+38%; p < 0.05) and late-outgrowth EPCs (+15%; p < 0.05). ICAM-1 expression was reduced in both CACs (p < 0.05) and late-outgrowth EPCs (p < 0.05) and VCAM-1 in late-outgrowth EPCs (p < 0.005). N-3 PUFA significantly decreased TNF-α and MCP-1 expression in CACs and IL-8, TNF-α and MCP-1 in late-outgrowth EPCs (p < 0.05). Circulating EPC number significantly improved after 6 weeks of a fish-enriched diet (p < 0.01) and returned to baseline levels after a 6 week free-diet period (p < 0.01). Plasma EPA levels were independently and positively associated with EPC levels (p < 0.005). CONCLUSION: Our findings support the case of a beneficiary role played by n-3 PUFA in EPC function and bioavailability

Pancreatic β-cell protection from inflammatory stress by the endoplasmic reticulum proteins thrombospondin 1 and mesencephalic astrocyte-derived neutrotrophic factor (MANF)

Cytokine-induced endoplasmic reticulum (ER) stress is one of the molecular mechanisms underlying pancreatic β-cell demise in type 1 diabetes. Thrombospondin 1 (THBS1) was recently shown to promote β-cell survival during lipotoxic stress. Here we show that ER-localized THBS1 is cytoprotective to rat, mouse, andhumanβ-cells exposed to cytokines or thapsigargininduced ER stress. THBS1 confers cytoprotection by maintaining expression of mesencephalic astrocyte-derived neutrotrophic factor (MANF) in β-cells and thereby prevents the BH3-only protein BIM (BCL2-interacting mediator of cell death)-dependent triggering of the mitochondrial pathway of apoptosis. Prolonged exposure ofβ-cells to cytokines or thapsigargin leads to THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in β-cells should be explored as a cytoprotective strategy in type 1 diabetes

Characterization of late-outgrowth EPCs.

<p>Morphology and phenotype of late-outgrowth EPC derived from human peripheral blood mononuclear cells. Colonies of late-outgrowth EPCs with a cobblestone-like morphology (A). Representative flow cytometry analysis of late-outgrowth EPCs (red) compared to early EPCs (purple) for the expression of CD31, KDR, CD146 and CD14 (B). Immunofluorescent staining of late-outgrowth EPCs for CD34, Ve-cad (vascular endothelial cadherin) and vWF (von Willebrand factor) (C), immunofluorescence staining of late-outgrowth EPC for DiI-LDL (red), lectin (green) and merge (bar = 50 µm) (D). eNOS and GAPDH gene expression in late EPCs (E) (bp = base pairs; eNOS = endothelial nitric oxide synthase; GAPDH = Glyceraldehyde 3-phosphate dehydrogenase).</p

Effect of pioglitazone on EPC function.

<p>Effect of pioglitazone (10 µM), pioglitazone + GW9662 (1 µM) and vehicle culture conditions on early and late-outgrowth EPC tube formation capacity expressed as total tube length (A) and as number of closed circles formed by tube-like structures (B); representation of early and late-outgrowth EPC tube formation assay showing the network formed by EPCs plus HUVEC on Matrigel (EPCs are red stained with Dil) in the presence of pioglitazone and vehicle (C); (*p<0.05 vs vehicle) (Pio = pioglitazone).</p

Pioglitazone effect on EPC pro-inflammatory molecule expression.

<p>Effect of pioglitazone on PPARγ, adhesion molecule and TNFα expression in EPCs. PPARγ gene expression in early and late-outgrowth EPC exposed to pioglitazone (10 µM) and pioglitazone + GW9662 (1 µM) (A) Effect of pioglitazone in modulating ICAM-1 and VCAM-1 expression by early and late-outgrowth EPCs (cytofluorimetric analyses). Results are reported as delta % of MFI (mean fluorescence intensity) with vehicle condition (B). Effects of pioglitazone on TNFα gene (C) and protein (D) expression in early and late-outgrowth EPC. (*p<0.05 vs vehicle; **p<0.01 vs vehicle). Real time PCR data are expressed as −ΔΔCt and represent the relative gene expression of EPC cultured in the presence of pioglitazone 10 µM (with or without GW9662 1 µM) in relation to vehicle, normalized for the endogenous control GAPDH. Protein expression is measured with ELISA assay from culture supernatants. Results from five independent experiments performed in duplicate are shown (*p<0.05 vs vehicle) (Pio = pioglitazone).</p