Mechanisms of endothelial cell dysfunction in cystic fibrosis

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF

Association of the 1q25 diabetes-specific coronary heart disease locus with slterations of the γ-glutamyl cycle and increased methylglyoxal levels in endothelial cells

A chromosome 1q25 variant (rs10911021) has been associated with coronary heart disease (CHD) in type 2 diabetes. In human umbilical vein endothelial cells (HUVECs), the risk allele "C" is associated with lower expression of the adjacent gene GLUL encoding glutamine synthase, converting glutamic acid to glutamine. To further investigate the mechanisms through which this locus affects CHD risk, we measured 35 intracellular metabolites involved in glutamic acid metabolism and the γ-glutamyl cycle in 62 HUVEC strains carrying different rs10911021 genotypes. Eight metabolites were positively associated with the risk allele (17-58% increase/allele copy, P = 0.046-0.002), including five γ-glutamyl amino acids, β-citryl-glutamate, N-acetyl-aspartyl-glutamate, and ophthalmate-a marker of γ-glutamyl cycle malfunction. Consistent with these findings, the risk allele was also associated with decreased glutathione-to-glutamate ratio (-9%, P = 0.012), decreased S-lactoylglutathione (-41%, P = 0.019), and reduced detoxification of the atherogenic compound methylglyoxal (+54%, P = 0.008). GLUL downregulation by shRNA caused a 40% increase in the methylglyoxal level, which was completely prevented by glutamine supplementation. In summary, we have identified intracellular metabolic traits associated with the 1q25 risk allele in HUVECs, including impairments of the γ-glutamyl cycle and methylglyoxal detoxification. Glutamine supplementation abolishes the latter abnormality, suggesting that such treatment may prevent CHD in 1q25 risk allele carriers

Calcitonin-Induced Effects on Amniotic Fluid-Derived Mesenchymal Stem Cells

Background/Aims: Mesenchymal stem cells from human amniotic fluid (huAFMSCs) can differentiate into multiple lineages and are not tumorigenic after transplantation, making them good candidates for therapeutic purposes. The aim was to determine the effects of calcitonin on these huAFMSCs during osteogenic differentiation, in terms of the physiological role of calcitonin in bone homeostasis. Methods: For huAFMSCs cultured under different conditions, we assayed: expression of the calcitonin receptor, using immunolabelling techniques; proliferation and osteogenesis, using colorimetric and enzymatic assays; intracellular Ca2+ and cAMP levels, using videomicroscopy and spectrophotometry. Results: The calcitonin receptor was expressed in proliferating and osteo-differentiated huAFMSCs. Calcitonin triggered intracellular Ca2+ increases and cAMP production. Its presence in cell medium also induced dose-dependent inhibitory effects on proliferation and increased osteogenic differentiation of huAFMSCs, as also indicated by enhancement of specific markers and alkaline phosphatase activity. Conclusions: These data show that huAFMSCs represent a potential osteogenic model to study in-vitro cell responses to calcitonin (and other members of the calcitonin family). This leads the way to the opening of new lines of research that will add new insight both in cell therapies and in the pharmacological use of these molecules

Calcitonin-Induced Effects on Amniotic Fluid-Derived Mesenchymal Stem Cells

Background/Aims: Mesenchymal stem cells from human amniotic fluid (huAFMSCs) can differentiate into multiple lineages and are not tumorigenic after transplantation, making them good candidates for therapeutic purposes. The aim was to determine the effects of calcitonin on these huAFMSCs during osteogenic differentiation, in terms of the physiological role of calcitonin in bone homeostasis. Methods: For huAFMSCs cultured under different conditions, we assayed: expression of the calcitonin receptor, using immunolabelling techniques; proliferation and osteogenesis, using colorimetric and enzymatic assays; intracellular Ca2+ and cAMP levels, using videomicroscopy and spectrophotometry. Results: The calcitonin receptor was expressed in proliferating and osteo-differentiated huAFMSCs. Calcitonin triggered intracellular Ca2+ increases and cAMP production. Its presence in cell medium also induced dose-dependent inhibitory effects on proliferation and increased osteogenic differentiation of huAFMSCs, as also indicated by enhancement of specific markers and alkaline phosphatase activity. Conclusions: These data show that huAFMSCs represent a potential osteogenic model to study in-vitro cell responses to calcitonin (and other members of the calcitonin family). This leads the way to the opening of new lines of research that will add new insight both in cell therapies and in the pharmacological use of these molecules

Oleanolic acid rescues critical features of umbilical vein endothelial cells permanently affected by hyperglycemia

Skin wound healing is a physiological process that involves several cell types. Among them, endothelial cells are required for inflammation resolution and neo‐angiogenesis, both necessary for tissue restoration after injury. Primary human umbilical vein endothelial cells (C‐HUVECs) are derived from the umbilical cord. When women develop gestational diabetes, chronic exposure to hyperglycemia induces epigenetic modifications in these cells (GD‐HUVECs), leading to a permanent pro‐inflammatory phenotype and impaired angiogenesis in contrast to control cells. Oleanolic acid (OA) is a bioactive triterpenoid known for its epithelial cell migration promotion stimulation and higher tensile strength of wounds. However, the potentially anti‐inflammatory and pro‐angiogenic properties of OA are still under investigation. We tested OA on C‐ and GD‐HUVECs under inflammatory conditions induced by low levels of the inflammatory cytokine TNF-α. Reduced expression of adhesion molecules VCAM1, ICAM1, and SELE was obtained in OA‐pre‐treated C‐ and GD‐HUVECs. Additionally, protein VCAM1 levels were also decreased by OA. Coherently, monocyte adhesion assays showed that a lower number of monocytes adhered to GD‐HUVEC endothelium under OA pre‐treatment when compared to untreated ones. It is noteworthy that OA improved angiogenesis parameters in both phenotypes, being especially remarkable in the case of GD‐HUVECs, since OA strongly rescued their poor tube formation behavior. Moreover, endothelial cell migration was improved in C‐ and GD‐HUVECs in scratch assays, an effect that was further confirmed by focal adhesion (FA) remodeling, revealed by paxillin staining on immunocytochemistry assays. Altogether, these results suggest that OA could be an emergent wound healing agent due to its capacity to rescue endothelial malfunction caused by hyperglycemia.Medicin

Plasma from pre-pubertal obese children impairs insulin stimulated Nitric Oxide (NO) bioavailability in endothelial cells: Role of ER stress.

Childhood obesity is commonly associated with early signs of endothelial dysfunction, characterized by impairment of insulin signaling and vascular Nitric Oxide (NO) availability. However, the underlying mechanisms remain to be established. Hence, we tested the hypothesis that endothelial insulin-stimulated NO production and availability was impaired and related to Endoplasmic Reticulum (ER) in human umbilical vein endothelial cells (HUVECs) cultured with plasma obtained from pre-pubertal obese (OB) children. OB children (N = 28, age: 8.8 ± 2.2; BMI z-score: 2.15 ± 0.39) showed impaired fasting glucose, insulin and HOMA-IR than normal weight children (CTRL; N = 28, age: 8.8 ± 1.7; BMI z-score: 0.17 ± 0.96). The in vitro experiments showed that OB-plasma significantly impaired endothelial insulin-stimulated NO production and bioavailability compared to CTRL-plasma. In parallel, in HUVECs OB-plasma increased GRP78 and activated PERK, eIF2α, IkBα and ATF6 (all ER stress markers). Moreover, OB-plasma increased NF-κB activation and its nuclear translocation. Notably, all these effects proved to be significantly restored by using PBA and TUDCA, known ER stress inhibitors. Our study demonstrate for the first time that plasma from obese children is able to induce in vitro endothelial insulin resistance, which is characterized by reduced insulin-stimulated NO production and bioavailability, endothelial ER stress and increased NF-κB activation

Cancer mortality in Common Mental Disorders: A 10-year retrospective cohort study

Purpose: Individuals with Common Mental Disorders (CMDs) may have a higher cancer mortality. The purpose of this study was to examine cancer-related mortality among patients with CMDs and verify which cancer types are predominantly involved. Methods: We used the Regional Mental Health Registry of the Emilia-Romagna region, in Northern Italy to identify patients aged ≥ 18 years who received an ICD 9-CM diagnosis of CMDs (i.e., depressive and neurotic disorders) over a 10 year period (2008-2017). Information on cause of death was retrieved from the Regional Cause of Death Registry. Comparisons were made with data from the regional population without CMDs. Results: Among 101,487 patients suffering from CMDs (55.7% depression; 44.3% neurotic disorders), 3,087 (37.8%) died from neoplasms. The total standardized mortality ratio (SMR) was 1.82 (95% CI 1.78-1.86) while the SMR for all neoplasms was 2.08 (95% CI 2.01-2.16). Individuals of both genders, with both depressive and neurotic disorders had a higher risk of death from almost all cancers compared with the regional population. Conclusion: Patients with CMDs have considerably higher cancer mortality risk than the general population. Higher mortality was observed for a broad range of cancers associated with different aetiologies. It is imperative to promote cancer awareness, prevention and treatment for people with CMDs

Oleanolic acid rescues critical features of umbilical vein endothelial cells permanently affected by hyperglycemia

Skin wound healing is a physiological process that involves several cell types. Among them, endothelial cells are required for inflammation resolution and neo‐angiogenesis, both necessary for tissue restoration after injury. Primary human umbilical vein endothelial cells (C‐HUVECs) are derived from the umbilical cord. When women develop gestational diabetes, chronic exposure to hyperglycemia induces epigenetic modifications in these cells (GD‐HUVECs), leading to a permanent pro‐inflammatory phenotype and impaired angiogenesis in contrast to control cells. Oleanolic acid (OA) is a bioactive triterpenoid known for its epithelial cell migration promotion stimulation and higher tensile strength of wounds. However, the potentially anti‐inflammatory and pro‐angiogenic properties of OA are still under investigation. We tested OA on C‐ and GD‐HUVECs under inflammatory conditions induced by low levels of the inflammatory cytokine TNF-α. Reduced expression of adhesion molecules VCAM1, ICAM1, and SELE was obtained in OA‐pre‐treated C‐ and GD‐HUVECs. Additionally, protein VCAM1 levels were also decreased by OA. Coherently, monocyte adhesion assays showed that a lower number of monocytes adhered to GD‐HUVEC endothelium under OA pre‐treatment when compared to untreated ones. It is noteworthy that OA improved angiogenesis parameters in both phenotypes, being especially remarkable in the case of GD‐HUVECs, since OA strongly rescued their poor tube formation behavior. Moreover, endothelial cell migration was improved in C‐ and GD‐HUVECs in scratch assays, an effect that was further confirmed by focal adhesion (FA) remodeling, revealed by paxillin staining on immunocytochemistry assays. Altogether, these results suggest that OA could be an emergent wound healing agent due to its capacity to rescue endothelial malfunction caused by hyperglycemia