Human Umbilical Artery Endothelial Cells Research Articles

Nrf2 pre-recruitment at Enhancer 2 is a hallmark of H2 O2 -induced epigenetic transcriptional memory in the HMOX1 gene in human umbilical artery endothelial cells.

Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity.

Protective role of CXCR7 activation in neonatal hyperoxia-induced systemic vascular remodeling and cardiovascular dysfunction in juvenile rats

Neonatal hyperoxia induces long-term systemic vascular stiffness and cardiovascular remodeling, but the mechanisms are unclear. Chemokine receptor 7 (CXCR7) represents a key regulator of vascular homeostasis and repair by modulating TGF-β1 signaling. This study investigated whether pharmacological CXCR7 agonism prevents neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction in juvenile rats. Newborn Sprague Dawley rat pups assigned to room air or hyperoxia (85% oxygen), received CXCR7 agonist, TC14012 or placebo for 3 weeks. These rat pups were maintained in room air until 6 weeks when aortic pulse wave velocity doppler, cardiac echocardiography, aortic and left ventricular (LV) fibrosis were assessed. Neonatal hyperoxia induced systemic vascular stiffness and cardiac dysfunction in 6-week-old rats. This was associated with decreased aortic and LV CXCR7 expression. Early treatment with TC14012, partially protected against neonatal hyperoxia-induced systemic vascular stiffness and improved LV dysfunction and fibrosis in juvenile rats by decreasing TGF-β1 expression. In vitro, hyperoxia-exposed human umbilical arterial endothelial cells and coronary artery endothelial cells had increased TGF-β1 levels. However, treatment with TC14012 significantly reduced the TGF-β1 levels. These results suggest that dysregulation of endothelial CXCR7 signaling may contribute to neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction.

#6062 EFFECT OF KIDNEY TRANSPLANTATION ON ARTERIOLAR MOLECULAR PATHWAYS IN CHILDREN WITH PRECEDING PERITONEAL DIALYSIS

Abstract Background and Aims Patients on peritoneal dialysis suffer from progressive peritoneal membrane transformation and accelerated vascular disease, underlying molecular machineries have recently been described. Molecular effects of kidney transplantation (KTx) on vascular and peritoneal pathophysiology following PD are hardly understood. Method Fat surrounded omental arterioles (i.e. not directly exposed to PD fluids) from age-matched (median 6,5 years) children with chronic kidney disease (CKD5, n = 8), 5 children on PD [neutral pH, low glucose degradation product content, median PD duration 22 (5,50) months] and 6 children, who underwent successful KTx 4-5 weeks after a median 22 (12, 36) months of PD were analyzed by digital histomorphometry. Following microdissection from fat tissue, arterioles underwent transcriptome and proteome analyses, followed by Gene Set Enrichment Analysis (GSEA), weighted gene co-expression network analysis (WGCNA), gene ontology (GO) and protein interaction analysis (string-db.org). Key pathways were validated in independent, matched cohorts by quantitative immunohistochemistry (n = 15/group) and in vitro. Results 5000 most variable arteriolar transcripts (cut off p-value&amp;lt;0.05 and |r| &amp;gt;0.5) were grouped into 23 modules by WGCNA, of which 8 significantly differed between CKD5, PD and KTx. Seven were specifically related to KTx and one module to PD. Four out of seven KTx specific modules resulted in significant GO terms summarized as fatty acid biosynthesis, negative regulation of RNA metabolism, cell cycle arrest, and apoptosis. The PD specific module was associated with muscle cell proliferation, detoxification and complement activation (with thrombospondin as most interconnected gene). Multi-omics analysis of KTx vs. PD demonstrated concordant upregulation of lipid and fatty acid biosynthesis with the hub gene fatty acid synthase (FASN) and downregulation of positive cell cycle regulation. In independent, matched cohorts, arteriolar abundance of thrombospondin and terminal complement complex were higher in children on PD, arterioles from children after KTx had levels comparable to arterioles from children with CKD5. Key drivers of fibrotic process, arteriolar TGF-ß and pSMAD2/3, were also higher in PD arterioles compared to CKD5 and persistently high after KTx. Arteriolar cell cycle arrest marker p16 and apoptosis marker cleaved Casp3 were higher after KTx as compared to PD and CKD5. FASN was abundant in intima and media layers of all arterioles, and three-fold higher after KTx then in children with PD and CKD5, respectively. Neutral lipids (oil red staining) were present in all three groups, more abundant in the media than in the intima, and two-fold increased in the media of children after KTx compared to children on PD (p = 0.018) and with CKD5 (p = 0.002). In vitro, methylprednisolone and tacrolimus, but not mycophenolate mofetil increased FASN abundance and activity in human umbilical arterial endothelial and vascular smooth muscle cells compared to media controls. Conclusion We for the first time comprehensively describe molecular pathways activated after KTx in children following a standard immunosuppressive regime. After KTx, arteriolar fatty acid biosynthesis, apoptosis and cell cycle arrest markers are increased as compared to arterioles from children with PD and CKD5; profibrotic pathways induced by PD are persistently activated. Arteriolar hubgene FASN is three-fold more abundant after KTx and neutral lipid deposition is increased, presumably due to the induction of FASN activity by methylprednisolone and tacrolimus.

Endothelial basement membrane laminins - new players in mouse and human myoendothelial junctions and shear stress communication

Basement membranes (BMs) are critical but frequently ignored components of the vascular system. Using high-resolution confocal imaging of whole-mount-stained mesenteric arteries, we identify integrins, vinculin, focal adhesion kinase (FAK) and several BM proteins including laminins as novel components of myoendothelial junctions (MEJs), anatomical microdomains that are emerging as regulators of cross-talk between endothelium and smooth muscle cells (SMCs). Electron microscopy revealed multiple layers of the endothelial BM that surround endothelial projections into the smooth muscle layer as structural characteristics of MEJs. The shear-responsive calcium channel TRPV4 is broadly distributed in endothelial cells and occurs in a proportion of MEJs where it localizes to the tips of the endothelial projections that are in contact with the underlying SMCs. In mice lacking the major endothelial laminin isoform, laminin 411 (Lama4−/−), which we have previously shown over-dilate in response to shear and exhibit a compensatory laminin 511 upregulation, localization of TRPV4 at the endothelial-SMC interface in MEJs was increased. Endothelial laminins do not affect TRPV4 expression, rather in vitro electrophysiology studies using human umbilical cord arterial endothelial cells revealed enhanced TRPV4 signalling upon culturing on an RGD-motif containing domain of laminin 511. Hence, integrin-mediated interactions with laminin 511 in MEJ structures unique to resistance arteries modulate TRPV4 localization at the endothelial-smooth muscle interface in MEJs and signalling over this shear-response molecule.

Generation of Rh D-negative blood using CRISPR/Cas9.

Blood supply shortages, especially the shortage of rare blood types, threaten the current medical system. Research on stem cells has shed light on in vitro blood cell manufacturing. The in vitro production of universal red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has become the focus of transfusion medicine. To obtain O-type Rh D-negative blood, we developed O-type Rh D-negative human (h)iPSCs using homology-directed repair (HDR)-based CRISPR/Cas9. HuAiPSCs derived from human umbilical arterial endothelial cells and showing haematopoietic differentiation preferences were selected for gene modification. Guide RNAs (gRNAs) were selected, and a donor template flanked by gRNA-directed homologous arms was set to introduce a premature stop code to RHD exon 2. CRISPR/Cas9 gene editing has resulted in the successful generation of an RHD knockout cell line. The HuAiPSC-A1-RHD-/- cell line was differentiated into haematopoietic stem/progenitor cells and subsequently into erythrocytes in the oxygen concentration-optimized differentiation scheme. HuAiPSC-A1-RHD-/- derived erythrocytes remained positive for the RBC markers CD71 and CD235a. These erythrocytes did not express D antigen and did not agglutinate in the presence of anti-Rh D reagents. In conclusion, taking the priority of haematopoietic preference hiPSCs, the HDR-based CRISPR/Cas9 system and optimizing the erythroid-lineage differentiation protocol, we first generated O-type Rh D-negative universal erythrocytes from RHD knockout HuAiPSCs. Its production is highly efficient and shows great potential for clinical applications.

Preferential Hematopoietic Differentiation in Induced Pluripotent Stem Cells Derived From Human Umbilical Cord Arterial Endothelial Cells.

The major obstacle for applications of human induced pluripotent stem cells (hiPSCs) is efficient and controlled lineage-specific differentiation. Hence, a deeper understanding of the initial populations of hiPSCs is required to instruct proficient lineage commitment. hiPSCs were generated from somatic cells by transduction of 4 human transcription factors (OCT4, SOX2, KLF4, and C-MYC) using Sendai virus vectors. Genome-wide DNA methylation analysis and transcriptional analysis were performed to evaluate the pluripotent capacity and somatic memory state of hiPSCs. Flow cytometric analysis and colony assays were performed to assess the hematopoietic differentiation capacity of hiPSCs. Here, we reveal human umbilical arterial endothelial cell-derived induced pluripotent stem cells (HuA-iPSCs) exhibit indistinguishable pluripotency in comparison with human embryonic stem cells and hiPSCs derived from other tissues of origin (umbilical vein endothelial cells, cord blood, foreskin fibroblasts, and fetal skin fibroblasts). However, HuA-iPSCs retain a transcriptional memory typical of the parental human umbilical cord arterial endothelial cells, together with a strikingly similar DNA methylation signature to umbilical cord blood-derived induced pluripotent stem cells that distinguishes them from other human pluripotent stem cells. Ultimately, HuA-iPSCs are most efficient in targeted differentiation toward hematopoietic lineage among all human pluripotent stem cells based on the functional and quantitative evaluation of both flow cytometric analysis and colony assays. Application of the Rho-kinase activator significantly reduces the effects of preferential hematopoietic differentiation in HuA-iPSCs, reflected in CD34+ cell percentage of day 7, hematopoietic/endothelial-associated gene expression, and even colony-forming unit numbers. Collectively, our data suggest that somatic cell memory may predispose HuA-iPSCs to differentiate more amenably into hematopoietic fate, bringing us closer to generating hematopoietic cell types in vitro from nonhematopoietic tissue for therapeutic applications.

Endothelial PTP4A1 mitigates vascular inflammation via USF1/A20 axis-mediated NF-κB inactivation.

The nuclear factor-κB (NF-κB) signalling pathway plays a critical role in the pathogenesis of multiple vascular diseases. However, in endothelial cells (ECs), the molecular mechanisms responsible for the negative regulation of the NF-κB pathway are poorly understood. In this study, we investigated a novel role for protein tyrosine phosphatase type IVA1 (PTP4A1) in NF-κB signalling in ECs. In human tissues, human umbilical artery ECs, and mouse models for loss of function and gain of function of PTP4A1, we conducted histological analysis, immunostaining, laser-captured microdissection assay, lentiviral infection, small interfering RNA transfection, quantitative real-time PCR and reverse transcription-PCR, as well as luciferase reporter gene and chromatin immunoprecipitation assays. Short hairpin RNA-mediated knockdown of PTP4A1 and overexpression of PTP4A1 in ECs indicated that PTP4A1 is critical for inhibiting the expression of cell adhesion molecules (CAMs). PTP4A1 increased the transcriptional activity of upstream stimulatory factor 1 (USF1) by dephosphorylating its S309 residue and subsequently inducing the transcription of tumour necrosis factor-alpha-induced protein 3 (TNFAIP3/A20) and the inhibition of NF-κB activity. Studies on Ptp4a1 knockout or transgenic mice demonstrated that PTP4A1 potently regulates the interleukin 1β-induced expression of CAMs in vivo. In addition, we verified that PTP4A1 deficiency in apolipoprotein E knockout mice exacerbated high-fat high-cholesterol diet-induced atherogenesis with upregulated expression of CAMs. Our data indicate that PTP4A1 is a novel negative regulator of vascular inflammation by inducing USF1/A20 axis-mediated NF-κB inactivation. Therefore, the expression and/or activation of PTP4A1 in ECs might be useful for the treatment of vascular inflammatory diseases.

Transforming growth factor-β-induced secretion of extracellular vesicles from oral cancer cells evokes endothelial barrier instability via endothelial-mesenchymal transition

BackgroundDuring metastasis, cancer cells undergo epithelial-mesenchymal transition (EMT) in response to transforming growth factor-β (TGF-β), which is abundant in the tumor microenvironment, and acquire invasive and metastatic potentials. Metastasis to distant organs requires intravascular invasion and extravasation of cancer cells, which is accompanied by the disruption of the adhesion between vascular endothelial cells. Cancer cell-derived extracellular vesicles (EVs) have been suggested to induce the destabilization of normal blood vessels at the metastatic sites. However, the roles of EVs secreted from cancer cells that have undergone EMT in the destabilization of blood vessels remain to be elucidated. In the present study, we characterized EVs secreted by oral cancer cells undergoing TGF-β-induced EMT and elucidated their effects on the characteristics of vascular endothelial cells.MethodsInduction of EMT by TGF-β in human oral cancer cells was assessed using quantitative RT-PCR (qRT-PCR) and immunocytochemistry. Oral cancer cell-derived EVs were isolated from the conditioned media of oral cancer cells that were treated with or without TGF-β using ultracentrifugation, and characterized using nanoparticle tracking analysis and immunoblotting. The effects of EVs on human umbilical artery endothelial cells were examined by qRT-PCR, cellular staining, and permeability assay. The significant differences between means were determined using a t-test or one-way analysis of variance with Tukey’s multiple comparisons test.ResultsOral cancer cells underwent EMT in response to TGF-β as revealed by changes in the expression of epithelial and mesenchymal cell markers at both the RNA and protein levels. Oral cancer cells treated with TGF-β showed increased EV production and altered EV composition when compared with untreated cells. The EVs that originated from cells that underwent EMT by TGF-β induced endothelial-mesenchymal transition, which was characterized by the decreased and increased expression of endothelial and mesenchymal cell markers, respectively. EVs derived from oral cancer cells also induced intercellular gap formation which led to the loss of endothelial cell barrier stability.ConclusionsEVs released from oral cancer cells that underwent TGF-β-induced EMT target endothelial cells to induce vascular destabilization. Detailed characterization of oral cancer-derived EVs and factors responsible for EV-mediated vascular instability will lead to the development of agents targeting metastasis.

Growth State-Dependent Expression of Arachidonate Lipoxygenases in the Human Endothelial Cell Line EA.hy926.

Endothelial cells regulate vascular homeostasis through the secretion of various paracrine molecules, including bioactive lipids, but little is known regarding the enzymes responsible for generating these lipids under either physiological or pathophysiological conditions. Arachidonate lipoxygenase (ALOX) expression was therefore investigated in confluent and nonconfluent EA.h926 endothelial cells, which represent the normal quiescent and proliferative states, respectively. mRNAs for ALOX15, ALOX15B, and ALOXE3 were detected in EA.hy926 cells, with the highest levels present in confluent cells compared to nonconfluent cells. In contrast, ALOX5, ALOX12, and ALOX12B mRNAs were not detected. At the protein level, only ALOX15B and ALOXE3 were detected but only in confluent cells. ALOXE3 was also observed in confluent human umbilical artery endothelial cells (HUAEC), indicating that its expression, although previously unreported, may be a general feature of endothelial cells. Exposure to laminar flow further increased ALOXE3 levels in EA.hy926 cells and HUAECs. The evidence obtained in this study indicates that proliferative status and shear stress are both important factors that mediate endothelial ALOX gene expression. The presence of ALOX15B and ALOXE3 exclusively in quiescent human endothelial cells suggests their activity likely contributes to the maintenance of a healthy endothelium.

Correlation between decreased plasma miR-29a and vascular endothelial injury induced by hyperlipidemia.

Hyperlipidemia is amajor risk factor for vascular endothelial injury and atherosclerosis leading to cardiovascular diseases. Early diagnosis of vascular endothelial injury is important for the prevention and prognosis of cardiovascular diseases. This study aimed to investigate sensitive circulating microRNA (miRNA) as apotential diagnostic biomarker of vascular endothelial injury in ahyperlipidemic rat model. The miRNA expression profile was detected by miRNA microarray. The hyperlipidemic rat model was established by intraperitoneal injection of vitamin D3 combined with ahigh-fat diet. Plasma miRNA levels were measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). No significant difference was found in the types of highly expressed miRNAs between human umbilical artery endothelial cells (HUAEC) and human umbilical vein endothelial cells (HUVEC). Atotal of 10highly expressed miRNAs in endothelial cells were selected as candidate miRNAs, including miR-21, miR-126, let-7a, miR-23a, miR-221, miR-125b, miR-26a, miR-29a, miR-16, and miR-100. The plasma levels of let-7a, miR-126, miR-21, and miR-26a were significantly elevated in hyperlipidemic rats at 30and 50days after modeling, while the plasma level of miR-29a was significantly decreased. No significant change was found in the plasma levels of miR-125b, miR-23a, miR-221, miR-100, and miR-16. Interestingly, a significant reduction in plasma miR-29 level was detected as early as 20days after modeling, which was earlier than for soluble intercellular adhesion molecule‑1 (sICAM-1). The plasma levels of endothelial cell-enriched miRNAs were correlated with vascular endothelial injury induced by hyperlipidemia. miR-29a might serve as apotential early diagnostic biomarker of endothelial injury-related diseases.

Celastrol: A new potential therapeutic option in pulmonary hypertension associated to bronchopulmonary dysplasia

Bronchopulmonary dysplasia is a common pathology of premature newborns caused by oxidative stress and mechanical ventilation, resulting in hypoalveolization and impaired angiogenesis. It can be complicated by pulmonary hypertension (HTP-DBP) with excess mortality. Celastrol has anti-inflammatory and antioxidant properties. So, we addressed its potential therapeutic interest in HTP-DBP. Different concentrations of celastrol were used in a murine model of hyperoxic HTP-DBP (14 days with 90% O 2 ) and we evaluated hemodynamic parameters with echocardiography and lung remodeling with histological methods. Pulmonary arterial (PA) reactivity was addressed with myograph. Cytoplasmic calcium (Ca2 + i) response to endothelin-1 (ET-1) was evaluated in human fetal PA smooth muscle cells (fPASMC) with spectrophotometry in normoxic (21% O 2 ) and hyperoxic conditions (48 h with 60% O 2 ). Inflammation was addressed with IL6 ELISA assay in fPASMC. Celastrol reduces mortality at doses of 1 and 0.5 mg/kg/d compared to the hyperoxic control group. At 1 mg/kg/d, it normalizes PAAT (PA acceleration time) ( P < 0.001) and reduces vascular hyperreactivity to phenylephrine and ET-1 ( P < 0.001) with no effect on the alteration of the response to acetylcholine induced by hyperoxia. At doses of 0.1, 0.5 and 1 mg/kg/d, it decreases right heart hypertrophy and vascular remodeling (wall thickness) but has no effect on alveolization and vascular density (vWF staining). Finally, although celastrol (0.1 and 0.3 μM) decreases the increased Ca2 + i response to ET-1 in hyperoxia in fPASMC, expression of ET-1 receptors (Western blotting and qPCR for ET-A and ET-B) was unchanged. However, celastrol 0.5 μM decreases ET-1 concentration measured with an ELISA assay in human umbilical arterial endothelial cells. An increased concentration of IL6 was also observed in fPASMC in hyperoxia and was decreased by celastrol 0.5 μM. Celastrol has a preventive dose-dependent effect on some hallmarks of HTP induced by hyperoxia and could thus be considered as an interesting new potential therapeutic option for HTP-DBP.

MO465: Molecular Mechanisms of Vascular Ageing in Children With Chronic Kidney Disease

Abstract BACKGROUND AND AIMS Paediatric patients with chronic kidney disease (CKD) develop significant atherosclerosis and vascular calcifications until early adulthood. Largely devoid of confounding lifestyle related factors and underlying disease mostly limited to congenital abnormalities of the kidneys and urinary tract, these patients provide highly sensitive and specific information on early CKD-induced molecular mechanisms of vascular disease and on putative therapeutic targets. METHOD Standardized omental and parietal peritoneal tissue samples from 95 non-CKD individuals [median age 9.2 (interquartile range, IQR 15), 110 children with CKD5 (median age 8.6 (IQR 12)] underwent digital histomorphometry. Omental arterioles microdissected from surrounding fat tissue underwent whole-exome and proteome analyses, followed by gene set enrichment and Ingenuity pathway analysis. Vascular calcification pathway analysis was performed for 370 biological processes and molecular functions associated with vascular calcification extracted from Gene Ontology database. Key regulated pathways were validated by quantitative immunostaining. The effects of uraemic toxins on endothelial integrity were studied in vitro in human umbilical arterial and vein endothelial cells in Transwells. RESULTS Lumen to vessel diameter (L/V) ratio was reduced in patients with CKD5. Parietal peritoneal arteriole L/V ratio was 0.54 (0.2) versus 0.63 (0.1) in non-CKD controls, omental arteriole L/V ratio 0.58 (0.1) versus 0.76 (0.1) in controls (both P &amp;lt; 0.001), indicating significant CKD5 related vascular disease. These findings were independent of underlying disease entities and gender. The parietal peritoneal submesothelial space exhibited infiltration of single CD45 positive lymphocytes, mesothelial cells which had undergone epithelial-to-mesenchymal-transition, and isolated peritoneal fibrin deposits. Submesothelial TGF-ß induced pSMAD was 4-fold increased and IL-17A 2-fold, while VEGF was not different compared to non-CKD controls. Gene set enrichment analysis of omental arteriolar multi-omics identified enrichment of pathways including telomere extension by telomerase, chromatin histone methylation, actin cytoskeleton, integrin- and tight junction signaling, and focal adhesion in children with CKD5 children compared to controls (P &amp;lt; 0.05). Vascular calcification pathway analysis identified 16/370 pathways significantly enriched on arteriolar transcriptome (P &amp;lt; 0.01), related to Wnt signalling, extracellular matrix organization, complement activation, autophagy and ossification. Applying the same threshold on proteome level, 10 calcification-related arteriolar pathways were identified and included DNA damage, fatty acid metabolism, calcium ion binding, extracellular matrix organization and complement activation. In independent age-matched cohorts, CKD5 children had shorter endothelial telomere and less endothelial methylated histone 3. The endothelial complement system was activated and arteriolar actin cytoskeleton interacting proteins gamma actin and profilin-1 were reduced in CKD5, cofilin-1 remained unchanged. In vitro, methylglyoxal and 3.4-di-deoxyglucosone-3-ene reduced transendothelial resistance, increased endothelial monolayer permeability and induced cytoskeleton disassembly (zonula occludens-1 and F-actin). These effects were prevented by co-incubation with anserine, 3-methylhistidine and alanyl-glutamine, but not by carnosine, L-histidine, 1-methylhistidine and methyl-alanyl. CONCLUSION CKD5 results in major vascular ageing already in early childhood. Multi-omics analysis of omental arterioles identified specific mechanisms of CKD-induced vascular ageing and of vascular calcification. Endothelial cell barrier integrity is impaired, and in vitro reversed by specific dipeptides.

Effect of celastrol on pulmonary hypertension associated to bronchopulmonary dysplasia

Bronchopulmonary dysplasia is a common pathology of premature newborns caused by oxidative stress and mechanical ventilation, resulting in hypoalveolization and impaired angiogenesis. It can be complicated by pulmonary hypertension (HTP-DBP) with excess mortality. Celastrol has anti-inflammatory and antioxidant properties and we addressed its potential therapeutic interest in HTP-DBP. Different concentrations of celastrol were used in a murine model of hyperoxic HTP-DBP (14 days with 90% O2) and we evaluated hemodynamic parameters with echocardiography and lung remodeling with histological methods. Pulmonary arterial (PA) reactivity was addressed with myograph. Cytoplasmic calcium (Ca2 + i) response to endothelin-1 (ET-1) was evaluated in human fetal PA smooth muscle cells (fPASMC) with spectrophotometry in normoxic (21% O2) and hyperoxic conditions (48 h with 60% O2). Inflammation was addressed with IL6 ELISA assay in fPASMC. Celastrol reduces mortality at doses of 1 and 0.5 mg/kg/d compared to the hyperoxic control group. At 1 mg/kg/d, it normalizes PAAT (PA acceleration time) ( P < 0.001) and reduces vascular hyperreactivity to phenylephrine ( P < 0.001) with no effect on the alteration of the response to acetylcholine induced by hyperoxia. At doses of 0.1, 0.5 and 1 mg/kg/d, it decreases right heart hypertrophy and vascular remodeling (wall thickness) but has no effect on alveolization and vascular density (vWF staining). Finally, although celastrol (0.1 and 0.3 μM) decreases the increased Ca2 + i response to ET-1 in hyperoxia in fPASMC, expression of ET-1 receptors (Western blotting and qPCR for ET-A and ET-B) was unchanged. However, celastrol 0.5 μM decreases ET-1 concentration measured with an ELISA assay in human umbilical arterial endothelial cells. An increased concentration of IL6 was also observed in fPASMC in hyperoxia and was decreased by celastrol 0.5 μM. Celastrol has a preventive dose-dependent effect on some hallmarks of HTP induced by hyperoxia and could thus be considered as an interesting new potential therapeutic option for HTP-DBP.

Beneficial effects of PCSK9 inhibition with alirocumab in familial hypercholesterolemia involve modulation of new immune players.

Familial hypercholesterolemia (FH) is associated with low-grade systemic inflammation, a key driver of premature atherosclerosis. We investigated the effects of inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) function on inflammatory state, endothelial dysfunction and cardiovascular outcomes in patients with FH. Fourteen patients with FH were evaluated before and 8 weeks after administration of a PCSK9 blocking monoclonal antibody (alirocumab, 150 mg/subcutaneous/14 days). In vivo and ex vivo analysis revealed that alirocumab blunted the attachment of leukocytes to TNFα-stimulated human umbilical arterial endothelial cells (HUAEC) and suppressed the activation of platelets and most leukocyte subsets, which was accompanied by the diminished expression of CX3CR1, CXCR6 and CCR2 on several leukocyte subpopulations. By contrast, T-regulatory cell activation was enhanced by alirocumab treatment, which also elevated anti-inflammatory IL-10 plasma levels and lowered circulating pro-inflammatory cytokines. Plasma levels of IFNγ positively correlated with levels of total and LDL-cholesterol, whereas circulating IL-10 levels negatively correlated with these key lipid parameters. In vitro analysis revealed that TNFα stimulation of HUAEC increased the expression of PCSK9, whereas endothelial PCSK9 silencing reduced TNFα-induced mononuclear cell adhesion mediated by Nox5 up-regulation and p38-MAPK/NFκB activation, concomitant with reduced SREBP2 expression. PCSK9 silencing also decreased endothelial CX3CL1 and CXCL16 expression and chemokine generation. In conclusion, PCSK9 inhibition impairs systemic inflammation and endothelial dysfunction by constraining leukocyte-endothelium interactions. PCSK9 blockade may constitute a new therapeutic approach to control the inflammatory state associated with FH, preventing further cardiovascular events in this cardiometabolic disorder.

Activation of the Constitutive Androstane Receptor Inhibits Leukocyte Adhesiveness to Dysfunctional Endothelium.

Leukocyte cell recruitment into the vascular subendothelium constitutes an early event in the atherogenic process. As the effect of the constitutive androstane receptor (CAR) on leukocyte recruitment and endothelial dysfunction is poorly understood, this study investigated whether the role of CAR activation can affect this response and the underlying mechanisms involved. Under physiological flow conditions, TNFα-induced endothelial adhesion of human leukocyte cells was concentration-dependently inhibited by preincubation of human umbilical arterial endothelial cells with the selective human CAR ligand CITCO. CAR agonism also prevented TNFα induced VCAM-1 expression, as well as MCP-1/CCL-2 and RANTES/CCL-5 release in endothelial cells. Suppression of CAR expression with a small interfering RNA abrogated the inhibitory effects of CITCO on these responses. Furthermore, CITCO increased interaction of CAR with Retinoid X Receptor (RXR) and reduced TNFα-induced p38-MAPK/NF-κB activation. In vivo, using intravital microscopy in the mouse cremasteric microcirculation treatment with the selective mouse CAR ligand TCPOBOP inhibited TNFα-induced leukocyte rolling flux, adhesion, and emigration and decreased VCAM-1 in endothelium. These results reveal that CAR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps in the leukocyte recruitment cascade. Therefore, CAR agonists may constitute a new therapeutic tool in controlling cardiovascular disease-associated inflammatory processes.

Flow-through isolation of human first trimester umbilical cord endothelial cells

Human umbilical vein and artery endothelial cells (HUVEC; HUAEC), placental endothelial cells (fpAEC), and endothelial colony-forming cells (ECFC) from cord blood are a widely used model for researching placental vascular development, fetal and placental endothelial function, and the effect of adverse conditions in pregnancy thereon. However, placental vascular development and angiogenesis start in the first weeks of gestation, and adverse conditions in pregnancy may also affect endothelial function before term, suggesting that endothelial cells from early pregnancy may respond differently. Thus, we established a novel, gentle flow-through method to isolate pure human umbilical endothelial cells from first trimester (FTUEC). FTUEC were characterized and their phenotype was compared to the umbilical endothelium in situ as well as to other fetal endothelial cell models from term of gestation, i.e. HUVEC, fpAEC, ECFC. FTUEC possess a CD34-positive, juvenile endothelial phenotype, and can be expanded and passaged. We regard FTUEC as a valuable tool to study developmental processes as well as the effect of adverse insults in pregnancy in vitro.

Specific arterio-venous transcriptomic and ncRNA-RNA interactions in human umbilical endothelial cells: A meta-analysis

SummaryWhether arterial-venous differences of primary endothelial cells commonly used for vascular research are preserved in vitro remains under debate. To address this issue, a meta-analysis of Affymetrix transcriptomic data sets from human umbilical artery (HUAECs) and vein (HUVEC) endothelial cells was performed. The meta-analysis showed 2,742 transcripts differentially expressed (false discovery rate <0.05), of which 78% were downregulated in HUVECs. Comparisons with RNA-seq data sets showed high levels of agreement and correlation (p < 0.0001), identifying 84 arterial-venous identity markers. Functional analysis revealed enrichment of key vascular processes in HUAECs/HUVECs, including nitric oxide- (NO) and hypoxia-related genes, as well as differences in miRNA- and ncRNA-mRNA interaction profiles. A proof of concept of these findings in primary cells exposed to hypoxia in vitro and in vivo confirmed the arterial-venous differences in NO-related genes and miRNAs. Altogether, these data defined a cross-platform arterial-venous transcript profile for cultured HUAEC-HUVEC and support a preserved identity involving key vascular pathways post-transcriptionally regulated in vitro.

Bazedoxifene increases the proliferation of human arterial endothelial cells but does not affect the expression of cyclins A, B, and D1 and of p27Kip1

Objective Endothelial dysfunction and denudation are considered a first step in atherosclerosis. Endothelial proliferation is key for cellular repair. The effect of bazedoxifene on the vascular endothelium has not been explored. We investigated the effect of bazedoxifene on endothelial cell proliferation. Methods Primary cultures from human umbilical artery endothelial cells were used in dose-response experiments (0.1, 1.0, and 10.0 EC50 dose) with bazedoxifene, estradiol, raloxifene and a combination of bazedoxifene and estradiol. Proliferation was assessed with the XTT colorimetric cell-proliferation assay. The possible participation of cyclins A, B, D1 and p27Kip1 was analyzed by the measurement of their expression at both the protein and the gene levels. Results A significant increase of similar size for cell proliferation was obtained with bazedoxifene, estradiol and raloxifene, but no significant change was observed for the association of bazedoxifene and estradiol. The impact was detected at the first 0.1 EC50 dose and was not dose-dependent. Estradiol achieved a significant increase in the protein expression of cyclin A and p27Kip1, but no change was detected for the other compounds at either the gene or protein level. Conclusion Bazedoxifene demonstrated a proliferative effect of similar size to estradiol in cultured human umbilical artery endothelial cells. The molecular mechanisms need further investigation.

Venous and Arterial Endothelial Cells from Human Umbilical Cords: Potential Cell Sources for Cardiovascular Research.

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.

The effect of hydroxychloroquine on platelet activation in model experiments.

Hydroxychloroquine (HCQ) is an antimalarial agent with pleiotropic effects and now represents a cornerstone in the management of patients with autoimmune conditions. While clinical series suggest anti-thrombotic properties, the way in which HCQ exerts this effect remains to be fully explained. Following a 24-h incubation of human umbilical vein endothelial cells (HUVEC) and human umbilical arterial endothelial cells (HUAEC) with HCQ (concentration 500, 1000 and 2000ng/ml), these cells were then stimulated for an hour with tumor necrosis factor alpha (TNF-α) and were subsequently incubated in direct contact with thrombin-activated platelets. The expression of CD40L on platelets was measured by flow cytometry. The expression of CD40L on platelets significantly increased after direct incubation with 1000ng/ml and 2000ng/ml concentrations of HCQ. In contrast, after pre-incubation of HUAECs with 1000ng/ml HCQ and following stimulation with platelets the expression of CD40L was significantly reduced also after stimulation with thrombin and TNF-α activated platelets. It was shown that the expression of CD40L on the platelets was not significantly reduced by different HCQ concentrations after contact with HCQ pre-incubated HUVECs. HCQ reduces the stimulatory effect of thrombin and TNF-α on platelet activation in the presence of endothelial cells. Our experiments suggest that HCQ pre-incubated HUAEC cells result in a reduced platelets activation measured by means of CD40L expression. Further, our results show that direct HCQ incubation of platelets (without the presence of EC) increased the expression of CD40L suggesting that the observed effect of HCQ on platelet activation may be EC mediated.