Essential Oils from Mediterranean Plants Inhibit In Vitro Monocyte Adhesion to Endothelial Cells from Umbilical Cords of Females with Gestational Diabetes Mellitus.

Essential oils (EOs) are mixtures of volatile compounds belonging to several chemical classes derived from aromatic plants using different distillation techniques. Recent studies suggest that the consumption of Mediterranean plants, such as anise and laurel, contributes to improving the lipid and glycemic profile of patients with diabetes mellitus (DM). Hence, the aim of the present study was to investigate the potential anti-inflammatory effect of anise and laurel EOs (AEO and LEO) on endothelial cells isolated from the umbilical cord vein of females with gestational diabetes mellitus (GDM-HUVEC), which is a suitable in vitro model to reproduce the pro-inflammatory phenotype of a diabetic endothelium. For this purpose, the Gas Chromatographic/Mass Spectrometric (GC-MS) chemical profiles of AEO and LEO were first analyzed. Thus, GDM-HUVEC and related controls (C-HUVEC) were pre-treated for 24 h with AEO and LEO at 0.025% v/v, a concentration chosen among others (cell viability by MTT assay), and then stimulated with TNF-α (1 ng/mL). From the GC-MS analysis, trans-anethole (88.5%) and 1,8-cineole (53.9%) resulted as the major components of AEO and LEO, respectively. The results in C- and GDM-HUVEC showed that the treatment with both EOs significantly reduced: (i) the adhesion of the U937 monocyte to HUVEC; (ii) vascular adhesion molecule-1 (VCAM-1) protein and gene expression; (iii) Nuclear Factor-kappa B (NF-κB) p65 nuclear translocation. Taken together, these data suggest the anti-inflammatory efficacy of AEO and LEO in our in vitro model and lay the groundwork for further preclinical and clinical studies to study their potential use as supplements to mitigate vascular endothelial dysfunction associated with DM.

Endothelial cells from umbilical cord of women affected by gestational diabetes: A suitable in vitro model to study mechanisms of early vascular senescence in diabetes.

Human umbilical cord endothelial cells (HUVECs) obtained from women affected by gestational diabetes (GD-HUVECs) display durable pro-atherogenic modifications and might be considered a valid in vitro model for studying chronic hyperglycemia effects on early endothelial senescence. Here, we demonstrated that GD- compared to C-HUVECs (controls) exhibited oxidative stress, altered both mitochondrial membrane potential and antioxidant response, significant increase of senescent cells characterized by a reduced NAD-dependent deacetylase sirtuin-1 (SIRT1) activity together with an increase in cyclin-dependent kinase inhibitor-2A (P16), cyclin-dependent kinase inhibitor-1 (P21), and tumor protein p53 (P53) acetylation. This was associated with the p300 activation, and its silencing significantly reduced the GD-HUVECs increased protein levels of P300 and Ac-P53 thus indicating a persistent endothelial senescence via SIRT1/P300/P53/P21 pathway. Overall, our data suggest that GD-HUVECs can represent an "endothelial hyperglycemic memory" model to investigate in vitro the early endothelium senescence in cells chronically exposed to hyperglycemia in vivo.

Osteogenic transdifferentiation of vascular smooth muscle cells isolated from spontaneously hypertensive rats and potential menaquinone-4 inhibiting effect.

Vascular calcification (VC) is an active and cell-mediated process that shares many common features with osteogenesis. Knowledge demonstrates that in the presence of risk factors, such as hypertension, vascular smooth muscle cells (vSMCs) lose their contractile phenotype and transdifferentiate into osteoblastic-like cells, contributing to VC development. Recently, menaquinones (MKs), also known as Vitamin K2 family, has been revealed to play an important role in cardiovascular health by decreasing VC. However, the MKs' effects and mechanisms potentially involved in vSMCs osteoblastic transdifferentiation are still unknown. The aim of this study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of MKs family, in the modulation of the vSMCs phenotype. To achieve this, vascular cells from spontaneously hypertensive rats (SHR) were used as an in vitro model of cell vascular dysfunction. vSMCs from Wistar Kyoto normotensive rats were used as control condition. The results showed that MK-4 preserves the contractile phenotype both in control and SHR-vSMCs through a γ-glutamyl carboxylase-dependent pathway, highlighting its capability to inhibit one of the mechanisms underlying VC process. Therefore, MK-4 may have an important role in the prevention of vascular dysfunction and atherosclerosis, encouraging further in-depth studies to confirm its use as a natural food supplement.

Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: An in vitro study.

Mesenchymal stromal cells (MSCs) are considered to be an excellent source in regenerative medicine. They contain several cell subtypes, including multipotent stem cells. MSCs are of particular interest as they are currently being tested using cell and gene therapies for a number of human diseases. They represent a rare population in tissues; for this reason, they require, before being transplanted, an in vitro amplification. This process may induce replicative senescence, thus affecting differentiation and proliferative capacities. Increasing evidence suggests that MSCs from fetal tissues are significantly more plastic and grow faster than MSCs from bone marrow. Here, we compare amniotic fluid mesenchymal stromal cells (AF-MSCs) and bone marrow mesenchymal stromal cells (BM-MSCs) in terms of cell proliferation, surface markers, multidifferentiation potential, senescence, and DNA repair capacity. Our study shows that AF-MSCs are less prone to senescence with respect to BM-MSCs. Moreover, both cell models activate the same repair system after DNA damage, but AF-MSCs are able to return to the basal condition more efficiently with respect to BM-MSCs. Indeed, AF-MSCs are better able to cope with genotoxic stress that may occur either during in vitro cultivation or following transplantation in patients. Our findings suggest that AF-MSCs may represent a valid alternative to BM-MSCs in regenerative medicine, and, of great relevance, the investigation of the mechanisms involved in DNA repair capacity of both AF-MSCs and BM-MSCs may pave the way to their rational use in the medical field.

Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.

The proresolution lipid mediator lipoxin (LX)A4 bestows protective bioactions on endothelial cells. We examined the impact of LXA4 on transcellular endothelial signaling via microRNA (miR)-containing microvesicles. We report LXA4 inhibition of MV release by TNF-α-treated HUVECs, associated with the down-regulation of 18 miR in endothelial microvesicles (EMVs) and the up-regulation of miR-126-5p, both in HUVECs and in EMVs. LXA4 up-regulated miR-126-5p by ∼5-fold in HUVECs and promoted a release of microvesicles (LXA4-EMVs) that enhanced miR-126-5p by ∼7-fold in recipient HUVECs. In these cells, LXA4-EMVs abrogated the up-regulation of VCAM-1, induced in recipient HUVECs by EMVs released by untreated or TNF-α-treated HUVECs. LXA4-EMVs also reduced by ∼40% the expression of SPRED1, which we validated as an miR-126-5p target, whereas they stimulated monolayer repair in an in vitro wound assay. This effect was lost when the EMVs were depleted of miR-126-5p. These results provide evidence that changes in miR expression and microvesicle packaging and transfer represent a mechanism of action of LXA4, which may be relevant in vascular biology and inflammation.-Codagnone, M., Recchiuti, A., Lanuti, P., Pierdomenico, A. M., Cianci, E., Patruno, S., Mari, V. C., Simiele, F., Di Tomo, P., Pandolfi, A., Romano, M. Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.

Liraglutide mitigates TNF-α induced pro-atherogenic changes and microvesicle release in HUVEC from diabetic women.

BACKGROUND: To evaluate whether exposure to GLP-1 receptor agonist Liraglutide could modulate pro-atherogenic alterations previously observed in endothelial cells obtained by women affected by gestational diabetes (GD), thus exposed in vivo to hyperglycemia, oxidative stress, and inflammation and to evaluate endothelial microvesicle (EMV) release, a new reliable biomarker of vascular stress/damage. METHODS: We studied Liraglutide effects and its plausible molecular mechanisms on monocyte cell adhesion and adhesion molecule expression and membrane exposure in control (C-) human umbilical vein endothelial cells (HUVEC) as well as in HUVEC of women affected by GD exposed in vitro to TNF-α. In the same model, we also investigated Liraglutide effects on EMV release. RESULTS: In response to TNF-α, endothelial monocyte adhesion and VCAM-1 and ICAM-1 expression and exposure on plasma membrane was greater in GD-HUVEC than C-HUVEC. This was the case also for EMV release. In GD-HUVEC, Liraglutide exposure significantly reduced TNF-α induced endothelial monocyte adhesion as well as VCAM-1 and ICAM-1 expression and exposure on plasma membrane. In the same cells, Liraglutide exposure also reduced MAPK/NF-kB activation, peroxynitrite levels, and EMV release. CONCLUSIONS: TNF-α induced pro-atherogenic alterations are amplified in endothelial cells chronically exposed to hyperglycemia in vivo. Liraglutide mitigates TNF-α effects and reduces cell stress/damage indicators, such as endothelial microvesicle (EMV) release. These results foster the notion that Liraglutide could exert a protective effect against hyperglycemia and inflammation triggered endothelial dysfunction.

Nitric oxide synthetic pathway and cGMP levels are altered in red blood cells from end-stage renal disease patients.

Red blood cells (RBCs) enzymatically produce nitric oxide (NO) by a functional RBC-nitric oxide synthase (RBC-NOS). NO is a vascular key regulatory molecule. In RBCs its generation is complex and influenced by several factors, including insulin, acetylcholine, and calcium. NO availability is reduced in end-stage renal disease (ESRD) and associated with endothelial dysfunction. We previously demonstrated that, through increased phosphatidylserine membrane exposure, ESRD-RBCs augmented their adhesion to human cultured endothelium, in which NO bioavailability decreased. Since RBC-NOS-dependent NO production in ESRD is unknown, this study aimed to investigate RBC-NOS levels/activation, NO production/bioavailability in RBCs from healthy control subjects (C, N = 18) and ESRD patients (N = 27). Although RBC-NOS expression was lower in ESRD-RBCs, NO, cyclic guanosine monophosphate (cGMP), RBC-NOS Serine1177 phosphorylation level and eNOS/Calmodulin (CaM)/Heat Shock Protein-90 (HSP90) interaction levels were higher in ESRD-RBCs, indicating increased enzyme activation. Conversely, following RBCs stimulation with insulin or ionomycin, NO and cGMP levels were significantly lower in ESRD- than in C-RBCs, suggesting that uremia might reduce the RBC-NOS response to further stimuli. Additionally, the activity of multidrug-resistance-associated protein-4 (MRP4; cGMP-membrane transporter) was significantly lower in ESRD-RBCs, suggesting a possible compromised efflux of cGMP across the ESRD-RBCs membrane. This study for the first time showed highest basal RBC-NOS activation in ESRD-RBCs, possibly to reduce the negative impact of decreased NOS expression. It is further conceivable that high NO production only partially affects cell function of ESRD-RBCs maybe because in vivo they are unable to respond to physiologic stimuli, such as calcium and/or insulin.

Diabetes and Its Cardiovascular Complications: Potential Role of the Acetyltransferase p300.

Diabetes has been shown to accelerate vascular senescence, which is associated with chronic inflammation and oxidative stress, both implicated in the development of endothelial dysfunction. This condition represents the initial alteration linking diabetes to related cardiovascular (CV) complications. Recently, it has been hypothesised that the acetyltransferase, p300, may contribute to establishing an early vascular senescent phenotype, playing a relevant role in diabetes-associated inflammation and oxidative stress, which drive endothelial dysfunction. Specifically, p300 can modulate vascular inflammation through epigenetic mechanisms and transcription factors acetylation. Indeed, it regulates the inflammatory pathway by interacting with nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) or by inducing its acetylation, suggesting a crucial role of p300 as a bridge between NF-κB p65 and the transcriptional machinery. Additionally, p300-mediated epigenetic modifications could be upstream of the activation of inflammatory cytokines, and they may induce oxidative stress by affecting the production of reactive oxygen species (ROS). Because several in vitro and in vivo studies shed light on the potential use of acetyltransferase inhibitors, a better understanding of the mechanisms underlying the role of p300 in diabetic vascular dysfunction could help in finding new strategies for the clinical management of CV diseases related to diabetes.

Effect of the Human Amniotic Membrane on the Umbilical Vein Endothelial Cells of Gestational Diabetic Mothers: New Insight on Inflammation and Angiogenesis.

One of the most relevant diabetes complications is impaired wound healing, mainly characterized by reduced peripheral blood flow and diminished neovascularization together with increased inflammation and oxidative stress. Unfortunately, effective therapies are currently lacking. Recently, the amniotic membrane (AM) has shown promising results in wound management. Here, the potential role of AM on endothelial cells isolated from the umbilical cord vein of gestational diabetes-affected women (GD-HUVECs), has been investigated. Indeed, GD-HUVECs in vivo exposed to chronic hyperglycemia during pregnancy compared to control cells (C-HUVECs) have shown molecular modifications of cellular homeostasis ultimately impacting oxidative and nitro-oxidative stress, inflammatory phenotype, nitric oxide (NO) synthesis, and bioavailability, thus representing a useful model for studying the mechanisms potentially supporting the role of AM in chronic non-healing wounds. In this study, the anti-inflammatory properties of AM have been assessed using a monocyte-endothelium interaction assay in cells pre-stimulated with tumor necrosis factor-α (TNF-α) and through vascular adhesion molecule expression and membrane exposure, together with the AM impact on the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway and NO bioavailability. Moreover, GD-HUVEC migration and tube formation ability were evaluated in the presence of AM. The results showed that AM significantly reduced TNF-α-stimulated monocyte-endothelium interaction and the membrane exposure of the endothelial vascular and intracellular adhesion molecules (VCAM-1 and ICAM-1, respectively) in both C- and GD-HUVECs. Strikingly, AM treatment significantly improved vessel formation in GD-HUVECs and cell migration in both C- and GD-HUVECs. These collective results suggest that AM positively affects various critical pathways in inflammation and angiogenesis, thus providing further validation for ongoing clinical trials in diabetic foot ulcers.

Estrogen Receptor 1 (ESR1) and the Wnt/ß-Catenin Pathway Mediate the Effect of the Coumarin Derivative Umbelliferon on Bone Mineralization.

Bone physiology is regulated by osteoblast and osteoclast activities, both involved in the bone remodeling process, through deposition and resorption mechanisms, respectively. The imbalance between these two phenomena contributes to the onset of bone diseases. Among these, osteoporosis is the most common metabolic bone disorder. The therapies currently used for its treatment include antiresorptive and anabolic agents associated with side effects. Therefore, alternative therapeutic approaches, including natural molecules such as coumarin and their derivatives, have recently shown positive results. Thus, our proposal was to investigate the effect of the coumarin derivative umbelliferon (UF) using an interesting model of human osteoblasts (hOBs) isolated from osteoporotic patients. UF significantly improved the activity of osteoporotic-patient-derived hOBs via estrogen receptor 1 (ESR1) and the downstream activation of ß-catenin pathway. Additionally, hOBs were co-cultured in microgravity with human osteoclasts (hOCs) using a 3D system bioreactor, able to reproduce the bone remodeling unit in bone loss conditions in vitro. Notably, UF exerted its anabolic role by reducing the multinucleated cells. Overall, our study confirms the potential efficacy of UF in bone health, and identified, for the first time, a prospective alternative natural compound useful to prevent/treat bone loss diseases such as osteoporosis.

L-carnitine is an osmotic agent suitable for peritoneal dialysis.

Excessive intraperitoneal absorption of glucose during peritoneal dialysis has both local cytotoxic and systemic metabolic effects. Here we evaluate peritoneal dialysis solutions containing L-carnitine, an osmotically active compound that induces fluid flow across the peritoneum. In rats, L-carnitine in the peritoneal cavity had a dose-dependent osmotic effect similar to glucose. Analogous ultrafiltration and small solute transport characteristics were found for dialysates containing 3.86% glucose, equimolar L-carnitine, or combinations of both osmotic agents in mice. About half of the ultrafiltration generated by L-carnitine reflected facilitated water transport by aquaporin-1 (AQP1) water channels of endothelial cells. Nocturnal exchanges with 1.5% glucose and 0.25% L-carnitine in four patients receiving continuous ambulatory peritoneal dialysis were well tolerated and associated with higher net ultrafiltration than that achieved with 2.5% glucose solutions, despite the lower osmolarity of the carnitine-containing solution. Addition of L-carnitine to endothelial cells in culture increased the expression of AQP1, significantly improved viability, and prevented glucose-induced apoptosis. In a standard toxicity test, the addition of L-carnitine to peritoneal dialysis solution improved the viability of L929 fibroblasts. Thus, our studies support the use of L-carnitine as an alternative osmotic agent in peritoneal dialysis.

Myoinositol Reduces Inflammation and Oxidative Stress in Human Endothelial Cells Exposed In Vivo to Chronic Hyperglycemia.

Myo-inositol (Myo) improves insulin resistance, glucose metabolism, and helps gestational diabetes (GDM) management. GDM is associated with a pro-inflammatory state and increased oxidative stress, which are both involved in vascular damage in diabetes. Our aim was to study Myo anti-inflammatory/antioxidant potential effects on an in vitro model of human umbilical vein endothelial cells (HUVECs). To this end, monocyte cell adhesion to HUVECs, adhesion molecule membrane exposure, and oxidative stress levels were determined in cells from control (C-) and GDM women treated during pregnancy either with diet only (GD-) or with diet plus Myo (GD+Myo). To deeply study the vascular effects of Myo, the same evaluations were performed in C- and GD-HUVECs following 48 h in vitro stimulation with Myo. Notably, we first observed that GD-HUVECs obtained from women assuming Myo supplementation exhibited a significantly decreased number of monocytes that adhered to endothelial cells, less adhesion molecule exposure, and lower intracellular reactive oxygen species (ROS) levels in the basal state as compared to GD-HUVECs obtained from women treated by diet only. This Myo anti-inflammatory/antioxidant effect was confirmed by 48 h in vitro stimulation of GD-HUVECs as compared to controls. Altogether, these results strongly suggest that Myo may exert protective actions against chronic inflammation induced by endothelial dysfunction in diabetes.

Cytotoxic and Genotoxic Effects of Composite Resins on Cultured Human Gingival Fibroblasts.

The aim of the study was to evaluate the cytotoxic and genotoxic potential of five commercially available dental composite resins (CRs), investigating the effect of their quantifiable bisphenol-A-glycidyl-methacrylate (Bis-GMA) and/or triethylene glycol dimethacrylate (TEGDMA) release. Experiments were performed using the method of soaking extracts, which were derived from the immersion of the following CRs in the culture medium: Clearfil-Majesty-ES-2, GrandioSO, and Enamel-plus-HRi (Bis-GMA-based); Enamel-BioFunction and VenusDiamond (Bis-GMA-free). Human Gingival Fibroblasts (hGDFs) were employed as the cellular model to mimic in vitro the oral cavity milieu, where CRs simultaneously release various components. Cell metabolic activity, oxidative stress, and genotoxicity were used as cellular outcomes. Results showed that only VenusDiamond and Enamel-plus-HRi significantly affected the hGDF cell metabolic activity. In accordance with this, although no CR-derived extract induced a significantly detectable oxidative stress, only VenusDiamond and Enamel-plus-HRi induced significant genotoxicity. Our findings showed, for the CRs employed, a cytotoxic and genotoxic potential that did not seem to depend only on the actual Bis-GMA or TEGDMA content. Enamel-BioFunction appeared optimal in terms of cytotoxicity, and similar findings were observed for Clearfil-Majesty-ES-2 despite their different Bis-GMA/TEGDMA release patterns. This suggested that simply excluding one specific monomer from the CR formulation might not steadily turn out as a successful approach for improving their biocompatibility.

Air and surface measurements of SARS-CoV-2 inside a bus during normal operation.

Transmission pathways of SARS-CoV-2 are aerosol, droplet and touching infected material. The diffusion of the virus contagion among people is easier in indoor location, but direct detection of SARS-CoV-2 in air or on surfaces is quite sparse, especially regarding public transport, while it would be important to know how and if it is safe to use them. To answer these questions we analysed the air and the surfaces most usually touched by passengers inside a city bus during normal operation, in order to understand the possible spreading of the virus and the effectiveness of the protective measures. The measurements were carried out across the last week of the lockdown and the first week when, gradually, all the travel restrictions were removed. The air and surface samples were analysed with the RT-PCR for the detection of SARS-CoV-2 virus. After two weeks of measurements and more than 1100 passenger travelling on the bus the virus was never detected both on surfaces and on air, suggesting that the precautions adopted on public transportation are effective in reducing the COVID-19 spreading.

Anti-inflammatory Role of Carotenoids in Endothelial Cells Derived from Umbilical Cord of Women Affected by Gestational Diabetes Mellitus.

Diabetes is associated with vascular inflammation, endothelial dysfunction, and oxidative stress, promoting the development of cardiovascular diseases (CVD). Several studies showed that a carotenoid-rich diet is associated to a reduced cardiovascular risk in healthy and diabetic subjects, although the mechanisms of action are still unknown. Here, the potential role of ß-carotene (BC) and lycopene (Lyc) in human endothelial cells isolated from human umbilical cord vein (HUVECs) of women with gestational diabetes (GD) and respective controls (C) has been investigated. Results showed that BC and Lyc reduced the tumor necrosis factor alpha- (TNF-α-) stimulated monocyte-endothelium interaction (adhesion assay), membrane exposure (flow cytometry), and total expression levels (Western blot) of VCAM-1 and ICAM-1 in both cell types. Moreover, the treatment with BC and Lyc reduced the TNF-α-induced nuclear translocation of NF-κB (image flow cytometry) by preserving bioavailability of nitric oxide (NO, flow cytometry, and cGMP EIA kit assay), a key vasoactive molecule. Notably, BC and Lyc pretreatment significantly reduced peroxynitrite levels (flow cytometry), contributing to the redox balance protection. These results suggest a new mechanism of action of carotenoids which exert vascular protective action in diabetic condition, thus reinforcing the importance of a carotenoid-rich diet in the prevention of diabetes cardiovascular complications.

Identification and Characterization of a Stem Cell-Like Population in Bovine Milk: A Potential New Source for Regenerative Medicine in Veterinary.

Milk is a complex fluid required for development, nutrition and immunological protection to the newborn offspring. Interestingly, latest finding proved the presence of novel stem cell population in human milk with multilineage differentiation potential. Given that little is known about cellular milk content in other mammalian species such as bovine, the purpose of our study was to isolate and characterize a potential stem cell-like population in bovine milk. In detail, we first analyzed the phenotype of the isolated cells able to grow in plastic adherence and then their capability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bovine milk stem cells (bMSCs) resulted plastic adherent and showed a heterogeneous population with epithelial and spindle-shaped cells. Successively, their immunophenotype indicated that bovine milk cells were positive for the typical epithelial markers E-cadherin, cytokeratin-14, cytokeratin-18, and smooth muscle actin. Notably, a subset (30%-40%), constantly observed in purified milk cells, showed the typical mesenchymal surface antigens CD90, CD73, and CD105. Furthermore, the same percentage of bMSCs expressing CD90, CD73, and CD105 presented the stemness markers SOX2 and OCT4 translocated in their nuclei. Finally, our data showed that bMSCs were able to differentiate into osteoblasts, chondroblasts, and adipocytes. In addition, the flow cytometry analysis revealed the presence of a subpopulation of events characterized by typical extracellular vesicles (EVs, size 0.1-1 µm), which did not contain nuclei and were positive for the same markers identified on the surface of bMSCs (CD73, CD90, and CD105), and thus might be considered milk cell-derived EVs. In conclusion, our data suggest that bovine milk is an easily available source of multipotent stem cells able to differentiate into multiple cell lineages. These features can open new possibilities for development biology and regenerative medicine in veterinary area to improving animal health.

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment.

Anti-Inflammatory Activity of Marine Ovothiol A in an In Vitro Model of Endothelial Dysfunction Induced by Hyperglycemia.

Chronic hyperglycemia is associated with oxidative stress and vascular inflammation, both leading to endothelial dysfunction and cardiovascular disease that can be weakened by antioxidant/anti-inflammatory molecules in both healthy and diabetic subjects. Among natural molecules, ovothiol A, produced in sea urchin eggs to protect eggs/embryos from the oxidative burst at fertilization and during development, has been receiving increasing interest for its use as an antioxidant. Here, we evaluated the potential antioxidative/anti-inflammatory effect of purified ovothiol A in an in vitro cellular model of hyperglycemia-induced endothelial dysfunction employing human umbilical vein endothelial cells (HUVECs) from women affected by gestational diabetes (GD) and from healthy mothers. Ovothiol A was rapidly taken up by both cellular systems, resulting in increased glutathione values in GD-HUVECs, likely due to the formation of reduced ovothiol A. In tumor necrosis factor-α-stimulated cells, ovothiol A induced a downregulation of adhesion molecule expression and decrease in monocyte-HUVEC interaction. This was associated with a reduction in reactive oxygen and nitrogen species and an increase in nitric oxide bioavailability. These results point to the potential antiatherogenic properties of the natural antioxidant ovothiol A and support its therapeutic potential in pathologies related to cardiovascular diseases associated with oxidative/inflammatory stress and endothelial dysfunction.

Calcimimetic R-568 vasodilatory effect on mesenteric vascular beds from normotensive (WKY) and spontaneously hypertensive (SHR) rats. Potential involvement of vascular smooth muscle cells (vSMCs).

The potential role of calcimimetics as vasculotropic agents has been suggested since the discovery that calcium sensing receptors (CaSRs) are expressed in cardiovascular tissues. However, whether this effect is CaSR-dependent or -independent is still unclear. In the present study the vascular activity of calcimimetic R-568 was investigated in mesenteric vascular beds (MVBs) isolated from Spontaneously Hypertensive rats (SHR) and the relative age-matched Wistar-Kyoto (WKY) control rats. Pre-constricted MBVs were perfused with increasing concentrations of R-568 (10 nM- 30 µM) resulting in a rapid dose-dependent vasodilatation. However, in MVBs from SHR this was preceded by a small but significant vasoconstriction at lowest nanomolar concentrations used (10-300 nM). Pre-treatment with pharmacological inhibitors of nitric oxide (NO) synthase (NOS, L-NAME), KCa channels (CTX), cyclo-oxygenase (INDO) and CaSR (Calhex) or the endothelium removal suggest that NO, CaSR and the endothelium itself contribute to the R-568 vasodilatory/vasoconstrictor effects observed respectively in WKY/SHR MVBs. Conversely, the vasodilatory effects resulted by highest R-568 concentration were independent of these factors. Then, the ability of lower R-568 doses (0.1-1 µM) to activate endothelial-NOS (eNOS) pathway in MVBs homogenates was evaluated. The Akt and eNOS phosphorylation levels resulted increased in WKY homogenates and Calhex significantly blocked this effect. Notably, this did not occur in the SHR. Similarly, vascular smooth muscle cells (vSMCs) stimulation with lower R-568 doses resulted in Akt activation and increased NO production in WKY but not in SHR cells. Interestingly, in these cells this was associated with the absence of the biologically active dimeric form of the CaSR thus potentially contributing to explain the impaired vasorelaxant effect observed in response to R-568 in MVB from SHR compared to WKY. Overall, these findings provide new insight on the mechanisms of action of the calcimimetic R-568 in modulating vascular tone both in physiological and pathological conditions such as hypertension.

Mechanisms of uremic erythrocyte-induced adhesion of human monocytes to cultured endothelial cells.

In end-stage renal disease (ESRD) endothelium may represent a key target for the action of circulating elements, such as modified erythrocytes (RBC) and/or plasmatic factors, that may facilitate inflammation and the vasculopathy associated with uremia. We have previously demonstrated that phosphatidylserine (PS) exposure on the surface of RBC from ESRD patients increases RBC-human umbilical vein endothelial cell (HUVEC) interactions and causes decreased nitric oxide (NO) production. We postulated that, besides the pro-inflammatory effects due to decreased NO bio-availability, enhanced ESRD-RBC-HUVEC interactions might directly stimulate pro-inflammatory pathways leading to increased vascular adhesion molecule expression. ESRD-RBC-endothelial cell interactions induced a time-dependent up-regulation of VCAM-1 and ICAM-1 (measured by Western blot (WB) and real-time PCR), associated with mitogen-activated protein kinase (MAPK) activation and impairment of the Akt/endothelial nitric oxide synthase (eNOS) signaling cascade, measured by WB. In reconstitution experiments, normal RBC incubated with uremic plasma showed increased PS exposure and significantly increased VCAM-1 and ICAM-1 mRNA levels when incubated on HUVEC. Interestingly, ESRD-RBC induced increased expression of adhesion molecules was prevented by Annexin-V (AnV, able to mask PS on RBC surface), anti-integrin-alpha(v)beta3, anti-thrombospondin-1 (TSP-1), and PD98059 (a selective inhibitor of MAPK phosphorylation). Moreover, AnV reversed the ESRD-RBC effects on MAPK and Akt/eNOS signaling pathways. Our data demonstrate that, possibly via a direct interaction with the endothelial thrombospondin-(alpha(v)beta3) integrin complex, ESRD-RBC-HUVEC adhesion induces a vascular inflammatory phenotype. Thus, intervention targeting ESRD-RBC increased adhesion to endothelium and/or MAPK and Akt/eNOS pathways may have the potential to prevent vascular lesions under uremic conditions.