Macrovascular Endothelial Cells Research Articles

Isolation, purification, and conditional immortalization of murine glomerular endothelial cells of microvascular phenotype

Glomerular endothelial cells (GEnC) are a specialized microvascular subset of endothelial cells that, when injured, result in many types of diseases within the kidney. Thus, techniques to study GEnC in a cell culture system are important to investigate mechanisms of GEnC injury. Studies of endothelial cell function in culture have predominately relied on using macrovascular endothelial cells from vascular areas other than the glomerulus. Over the last 15 years, glomerular endothelial cells lines have been created but were isolated by targeting cells expressing CD31. Some studies identified endothelial cells isolated from the microvasculature do not express CD31 and some suggest that CD31+ cells are phenotypically different than endothelial cells found in capillaries. Here we detail our method of isolation, purification, and conditional immortalization of mouse glomerular endothelial cells targeting endothelial cells that do not express CD31.•This method allows for isolation, purification, and conditional immortalization of glomerular endothelial cells for continued passage of GEnCs beyond that of primary cell culture.•This method can be used in genetically modified mice to investigate how a modification of a specific gene or protein affects the glomerular endothelium at the cellular level.

Macrovascular Endothelial Cells Enhance the Motility of Liver Cancer Cells by Up-regulation of MMP-3, Activation of Integrin/FAK Signaling Pathway and Induction of Non-classical Epithelial-mesenchymal Transition.

Background: Liver cancer with portal vein tumor thrombus (PVTT) indicates a serious prognosis. The molecular mechanism of PVTT formation is not totally clarified, the invasion of blood vessels by liver cancer cells is the key step and portal vein endothelial cells plays critical role.Methods: Conditioned medium (CM) of human umbilical vein endothelial cells (HUVEC) were used to culture liver cancer cells and prostate cancer cells for cell motility and viability analysis for the purpose of simulating the role of macrovascular endothelial cells in the development of liver cancer.Results: HUVEC-CM caused long spindle-shaped changes in liver cancer cells; the invasion and migration ability of Bel-7402 and MHCC-LM3 (cultured in HUVEC-CM) increased significantly. Integrins/FAK (focal adhesion kinase) signaling pathway was activated and MMP-3 was up-regulated. However, classical epithelial-mesenchymal transition (EMT) did not involve. HUVEC-CM caused a decrease of cell population in G1- and S-phase of Bel-7402, it also caused an accumulation of cell population in G1 phase and a decrease of cell population in S-phase of MHCC-LM3, MHCC-97L and DU-145. HUVEC-CM promotes apoptosis of Bel-7402 and MHCC-97L and the nude mouse tumorigenic experiment did not find that the HUVEC-CM increase the tumorigenic ability of liver cancer cells.Conclusion: HUVEC may provide an easy-to-adhere roadbed for liver cancer cells invasion of blood vessels by altering extracellular matrix (ECM), activating integrins/FAK pathway and inducing non-classical EMT. The effect of HUVEC-CM on cell viability was cancer cell type dependent. It is a meaningful glance at the mechsanism of PVTT.

TNF-α induces acyl-CoA synthetase 3 to promote lipid droplet formation in human endothelial cells

Chronic inflammation contributes to cardiovascular disease. Increased levels of the inflammatory cytokine, TNF-α, are often present in conditions associated with cardiovascular disease risk, and TNF-α induces a number of pro-atherogenic effects in macrovascular endothelial cells, including expression of adhesion molecules and chemokines, and lipoprotein uptake and transcytosis to the subendothelial tissue. However, little is known about the roles of acyl-CoA synthetases (ACSLs), enzymes that esterify free fatty acids into their acyl-CoA derivatives, or about the effects of TNF-α on ACSLs in endothelial cells. Therefore, we investigated the effects of TNF-α on ACSLs and downstream lipids in cultured human coronary artery endothelial cells and human umbilical vein endothelial cells. We demonstrated that TNF-α induces ACSL1, ACSL3, and ACSL5, but not ACSL4, in both cell types. TNF-α also increased oleoyl-CoA levels, consistent with the increased ACSL3 expression. RNA-sequencing demonstrated that knockdown of ACSL3 had no marked effects on the TNF-α transcriptome. Instead, ACSL3 was required for TNF-α-induced lipid droplet formation in cells exposed to oleic acid. These results demonstrate that increased acyl-CoA synthesis as a result of ACSL3 induction is part of the TNF-α response in human macrovascular endothelial cells.

Capsid Engineering Overcomes Barriers Toward Adeno-Associated Virus Vector-Mediated Transduction of Endothelial Cells

Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-VEC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.

Endothelial activation and injury by microparticles in patients with systemic lupus erythematosus and rheumatoid arthritis

BackgroundEndothelial activation and damage is commonly observed in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and is related to development of atherosclerosis and cardiovascular diseases. Different components of the immune system seem to participate in the endothelial injury, such as generation of autoantibodies and formation of immune complexes (ICs). Microparticles (MPs) and their immune complexes (MPs-ICs) are increased in the circulation of patients with SLE and RA; therefore, we propose these extracellular vesicles could interact and modulate the function of endothelial cells. Hence, the effect of MPs and MPs-ICs from patients with SLE and RA in endothelial cells was evaluated.MethodsMacrovascular and microvascular endothelial cells were exposed to MPs and MPs-ICs from healthy donors and patients with SLE and RA. Vesicles uptake/binding, expression of adhesion molecules, cytokine and chemokine production, monocyte adherence, and alterations of endothelial monolayer were evaluated by flow cytometry and fluorescence microscopy.ResultsEndothelial cells internalized MPs and MPs-ICs and increased CD54 and CD102 expression and CCL2, CCL5, and IL-6 production after the treatment with these extracellular vesicles, which led to an increase in the adherence of classic monocytes. These vesicles also induced low expression of VE-cadherin in membrane, depolymerization of actin filaments, and formation of intercellular spaces, which led to endothelial death and increased permeability after MPs and MPs-ICs exposure.ConclusionsMPs and MPs-ICs from patients with SLE and RA increase adhesion molecules expression, chemokine production, and structural alterations in macrovascular and microvascular endothelial cells. Therefore, high counts of these vesicles in patients would promote endothelial alterations and secondary tissue leukocyte infiltration.

Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition.

The endothelial-to-mesenchymal transition (EndMT) is a biological process where endothelial cells (ECs) acquire a fibroblastic phenotype after concomitant loss of the apical-basal polarity and intercellular junction proteins. This process is critical to embryonic development and is involved in diseases such as fibrosis and tumor progression. The signaling pathway of the transforming growth factor β (TGF-β) is an important molecular route responsible for EndMT activation. However, it is unclear whether the anatomic location of endothelial cells influences the activation of molecular pathways responsible for EndMT induction. Our study investigated the molecular mechanisms and signaling pathways involved in EndMT induced by TGF-β2 in macrovascular ECs obtained from different sources. For this purpose, we used four types of endothelial cells (coronary artery endothelial cells, CAECs; primary aortic endothelial cells PAECs; human umbilical vein endothelia cells, HUVECs; and human pulmonary artery endothelial cells, HPAECs) and stimulated with 10 ng/mL of TGF-β2. We observed that among the ECs analyzed in this study, PAECs showed the best response to the TGF-β2 treatment, displaying phenotypic changes such as loss of endothelial marker and acquisition of mesenchymal markers, which are consistent with the EndMT activation. Moreover, the PAECs phenotypic transition was probably triggered by the extracellular signal–regulated kinases 1/2 (ERK1/2) signaling pathway activation. Therefore, the anatomical origin of ECs influences their ability to undergo EndMT and the selective inhibition of the ERK pathway may suppress or reverse the progression of diseases caused or aggravated by the involvement EndMT activation.

Heme Drives Susceptibility of Glomerular Endothelium to Complement Overactivation Due to Inefficient Upregulation of Heme Oxygenase-1.

Atypical hemolytic uremic syndrome (aHUS) is a severe disease characterized by microvascular endothelial cell (EC) lesions leading to thrombi formation, mechanical hemolysis and organ failure, predominantly renal. Complement system overactivation is a hallmark of aHUS. To investigate this selective susceptibility of the microvascular renal endothelium to complement attack and thrombotic microangiopathic lesions, we compared complement and cyto-protection markers on EC, from different vascular beds, in in vitro and in vivo models as well as in patients. No difference was observed for complement deposits or expression of complement and coagulation regulators between macrovascular and microvascular EC, either at resting state or after inflammatory challenge. After prolonged exposure to hemolysis-derived heme, higher C3 deposits were found on glomerular EC, in vitro and in vivo, compared with other EC in culture and in mice organs (liver, skin, brain, lungs and heart). This could be explained by a reduced complement regulation capacity due to weaker binding of Factor H and inefficient upregulation of thrombomodulin (TM). Microvascular EC also failed to upregulate the cytoprotective heme-degrading enzyme heme-oxygenase 1 (HO-1), normally induced by hemolysis products. Only HUVEC (Human Umbilical Vein EC) developed adaptation to heme, which was lost after inhibition of HO-1 activity. Interestingly, the expression of KLF2 and KLF4—known transcription factors of TM, also described as possible transcription modulators of HO-1- was weaker in micro than macrovascular EC under hemolytic conditions. Our results show that the microvascular EC, and especially glomerular EC, fail to adapt to the stress imposed by hemolysis and acquire a pro-coagulant and complement-activating phenotype. Together, these findings indicate that the vulnerability of glomerular EC to hemolysis is a key factor in aHUS, amplifying complement overactivation and thrombotic microangiopathic lesions.

A769662 Inhibits Insulin-Stimulated Akt Activation in Human Macrovascular Endothelial Cells Independent of AMP-Activated Protein Kinase.

Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound 991 and AICAR had no substantial inhibitory effect on insulin-stimulated Akt phosphorylation in ECs. Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation.

Lymphatic endothelial cells promote productive and latent HIV infection in resting CD4+ T cells

BackgroundAn HIV cure has not yet been achieved because latent viral reservoirs persist, particularly in resting CD4+ T lymphocytes. In vitro, it is difficult to infect resting CD4+ T cells with HIV-1, but infections readily occur in vivo. Endothelial cells (EC) line the lymphatic vessels in the lymphoid tissues and regularly interact with resting CD4+ T cells in vivo. Others and we have shown that EC promoted productive and latent HIV infection of resting CD4+ T cells. However, the EC used in previous studies were from human umbilical cords (HUVEC), which are macrovascular; whereas EC residing in the lymphoid tissues are microvascular.MethodsIn this study, we investigated the effects of microvascular EC stimulation of resting CD4+ T cells in establishing viral infection and latency. Human resting and activated CD4+ T cells were cultured alone or with endothelial cells and infected with a pseudotyped virus. Infection levels, indicated by green fluorescent protein expression, were measured with flow cytometry and data was analyzed using Flowing Software and Excel.ResultsWe confirmed that EC from lymphatic tissue (LEC) were able to promote HIV infection and latency formation in resting CD4+ T cells while keeping them in resting phenotype, and that IL-6 was involved in LEC stimulation of CD4+ T cells. However, there are some differences between stimulation by LEC and HUVEC. Unlike HUVEC stimulation, we demonstrated that LEC stimulation of resting memory T cells does not depend on major histocompatibility complex class II (MHC II) interactions with T cell receptors (TCR) and that CD2-CD58 interactions were not involved in LEC stimulation of resting T cells. LEC also secreted lower levels of IL-6 than HUVEC. We also found that LEC stimulation increases HIV infection rates in activated CD4+ T cells.ConclusionsWhile differences in T cell stimulation between lymphatic EC and HUVEC were observed, we confirmed that similar to macrovascular EC stimulation, microvascular EC stimulation promotes direct HIV infection and latency formation in resting CD4+ T cells without T cell activation. LEC stimulation also increased infection rates in activated CD4+ T cells. Additionally, the present study established a physiologically more relevant model of EC interactions with resting CD4+ T cells and further highlighted the importance of investigating the roles of EC in HIV infection and latency in both resting and activated CD4+ T cells.

CCL20, a direct-acting pro-angiogenic chemokine induced by hepatitis C virus (HCV): Potential role in HCV-related liver cancer

The CCL20/CCR6 chemokine/receptor axis has previously been shown to contribute to the initiation and progression of hepatocellular carcinoma (HCC) through the recruitment of CCR6-positive leukocytes to the tumor microenvironment. In particular, high serum levels of CCL20 are reported in patients with HCC induced by the hepatitis C virus (HCV). A potential non-immune role for the CCL20/CCR6 axis in HCC development has not yet been investigated.Microarray analysis (Benkheil et al., paper submitted for publication), revealed that CCL20 is highly upregulated in hepatoma cells infected with HCV compared with non-infected hepatoma cells. To determine the role of the CCL20/CCR6 axis in HCV-related HCC, we first explored which cell populations express CCR6 in human liver tissue with chronic disease or HCC. Immunohistochemical (IHC) analysis revealed that CCR6 is present on endothelial cells (ECs) of portal blood vessels in livers with chronic HCV infection and in HCV- and alcoholic-HCC tissue. In addition, we found CCR6 to be expressed on primary macrovascular (HUVECs) and microvascular ECs (HMVEC-ds) where it co-expressed with the endothelial marker CD31. In vitro angiogenesis experiments revealed that CCL20 is a direct pro-angiogenic molecule that induces EC invasion, sprouting and migration through CCR6. Moreover, using the angiogenesis matrigel plug assay in immunodeficient NMRI-nu mice, we clearly showed that CCL20 induces blood vessel formation, by attracting CCR6-positive ECs. Finally, we demonstrated that HCV-induced CCL20 protein expression and secretion in hepatoma cells could be abolished by antiviral treatment, indicating that CCL20 expression is dependent on HCV replication. In contrast to HCV, HBV-infection resulted in a decreased expression of CCL20, implying a virus-specific effect.Taken together, we identified HCV-induced CCL20 as a direct pro-angiogenic factor that acts on endothelial CCR6. These results suggest that the CCL20/CCR6 axis contributes to hepatic angiogenesis, promoting the hypervascular state of HCV-HCC.

Diversity in human placental microvascular endothelial cells and macrovascular endothelial cells

Angiogenesis is fundamental to normal placental development, and aberrant angiogenesis contributes substantially to placental pathologies. Placental angiogenesis is a pivotal process that plays a key mechanistic role in the elaboration of the placental villous tree, which is mainly taken by human placental microvascular endothelial cells (HPMECs), present in the fetal capillaries of chorionic villi, and macrovascular human umbilical vein endothelial cells (HUVECs) also play a role in this process. These are the two types of endothelial cells that form the placenta and differ in morphology and function. The placental vasculature represents a distinct territory that is highly specialized in structure and function. To distinguish the differences between HPMECs and HUVECs, we isolated HPMECs by paramagnetic particle separation and HUVECs through trypsinization and validated their characteristics. Then, we examined their response to fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), as well as the underlying signaling mechanisms and their transcriptomes. We found that cultured HPMECs and HUVECs took up DiI-Ac-LDL and formed capillary-like tube structures on Matrigel. HPMECs and HUVECs had different expressions of eNOS, PROKR1 and PROKR2, and these characteristics substantiate the endothelial nature of cultured cells. FGF2 and VEGF stimulated the proliferation and migration of HPMECs and HUVECs via activation of PI3K/AKT1 and MEK1/MEK2/ERK1/ERK2. Interestingly, EG-VEGF increased the proliferation and migration of HPMECs via only MEK1/MEK2/ERK1/ERK2 and not PI3K/AKT1. Microarray analysis showed that there were some differentially expressed genes between HPMECs and HUVECs. Gene ontology analysis indicated that the differentially expressed genes were highly related to G-protein coupled receptor signaling pathway, angiogenesis, L-lysine transmembrane transport and blood vessel remodeling. These data provided evidence of heterogeneity between microvascular HPMECs and macrovascular HUVECs that most likely reflected significant differences in endothelial cell function in the two different cellular environments.

Anti-angiogenic effect of adiponectin in human primary microvascular and macrovascular endothelial cells

Neovascularization in retina and choroid involves interplay of many cytokines and growth factors. Vascular endothelial growth factor (VEGF) being a pro-angiogenic molecule has been found to be high in aqueous and vitreous humour of patients with proliferative diabetic retinopathy (PDR). VEGF is also found in the fibroblast and retinal pigment epithelial cells (RPE) of choroidal neovascular (CNV) membranes isolated from patients. Though anti-VEGF agents cause regression of clinically visible new vessels, there is evidence that they increase the occurrence of retinal tractional detachment and other adverse effects in PDR and CNV treatments. Adiponectin (APN) is a cytokine, found to be involved in the pathobiology of PDR. It is unclear whether APN plays a reparative or pathological role in the disease condition. In this study, we explored the effect of APN on tube formation in the primary culture of human umbilical vein macrovascular endothelial cells (HUVEC), human retinal microvascular endothelial cells (hREC) and human choroidal endothelial cells (hCEC). Anti-VEGF agent, bevacizumab (avastin) was used as a control. Full-length pAc-APN transfected in HUVEC, hRECs and hCECs inhibited basal tube formation and migration comparable to bevacizumab (Avastin™). In hRECs, full length pAc-APN reduced VEGF or PDR vitreous mediated migration. In a similar way, rAPN significantly disrupted VEGF and PDR vitreous induced tube formation in HUVEC and hREC. Moreover, rAPN significantly reduced VEGF influenced proliferation and phosphorylation of ERK1/2 in hREC. Altogether, our study suggests that APN may be effective in the treatment of retinal neovascularization.

Endothelialization and Anticoagulation Potential of Surface-Modified PET Intended for Vascular Applications.

In vascular tissue engineering, great attention is paid to the immobilization of biomolecules onto synthetic grafts to increase bio- and hemocompatibility-two critical milestones in the field. The surface modification field of poly(ethylene terephthalate) (PET), a well-known vascular-graft material, is matured and oversaturated. Nevertheless, most developed methods are laborious multistep procedures generally accompanied by coating instability or toxicity issues. Herein, a straightforward surface modification procedure is presented engineered to simultaneously promote surface endothelialization and anticoagulation properties via the covalent immobilization of gelatin through a photoactivated azide derivative. A complete physicochemical characterization and biological study including cytotoxicity and endotoxin testing are performed. In addition, biocompatibility toward small (diameter ≤ 6 mm) and/or large caliber (diameter ≥ 6 mm) vessels is assessed by micro- and macrovascular endothelial cell assays. Superior bio- and hemocompatibility properties are seen for the gelatin-covalently modified PET surfaces compared to the conventional surface-modification procedures based on physisorption.

Acute promyelocytic leukemia cell adhesion to vascular endothelium is reduced by heparins.

Adhesion of acute promyelocytic leukemia (APL) cells to endothelial cells (EC) is among the mechanisms of the APL-associated coagulopathy, responsible for early hemorrhagic deaths in affected patients. We compared the effects of dalteparin and enoxaparin, two low-molecular-weight heparins (LMWH), and unfractionated heparin (UFH), on APL NB4 adhesion to micro- (HMEC-1) and macro-vascular EC (HUVEC), in resting and interleukin-1β (IL-1β)-stimulated conditions. The heparin effect on EC adhesion molecule (ICAM-1, VCAM-1, E-selectin) expression was also assessed. In HMEC-1, dalteparin inhibited IL1β-induced NB4 adhesion by 80%, enoxaparin by 52%, and UFH by 44%. Similar results were obtained in HUVEC. This was associated with a significant decrease of VCAM-1 and ICAM-1 expression. In conclusion, we show that LMWH significantly counteract APL cell adhesion to the vessel wall, by modulating EC adhesion molecule expression. This property of heparins may represent one approach for hampering excess clotting activation and microthrombi deposition in APL.

Endothelial GPCRs Activate p38 MAPK Inflammatory Signaling Via Non‐canonical TAB1, 2 and 3‐dependent Pathways

Vasculature disease is a leading cause of morbidity and mortality worldwide. A hallmark of vascular inflammation is endothelial cell dysfunction, which is induced by vascular inflammatory mediators, many of which act through G protein‐coupled receptors (GPCRs). We previously showed that thrombin stimulation of endothelial protease‐activated receptor‐1 (PAR1) activates p38 MAPK through a non‐canonical pathway mediated by TAB1 (transforming growth factor‐β‐activated kinase binding protein‐1). TAB1 binds directly to p38α inducing a conformational change resulting in autophosphorylation and activation bypassing the requirement for upstream MAP2Ks. We also found that non‐canonical p38 activation induced by thrombin/PAR1 promotes endothelial barrier permeability in vitro and further showed that p38 is required for PAR1‐stimulated vascular leakage in vivo. While non‐canonical p38 activation induced by PAR1 is established, it remains unclear if other endothelial GPCRs also promote p38 activation through a TAB1‐dependent pathway and was examined. Here we report that multiple GPCRs agonists including histamine, PGE2, ADP and thrombin induce p38 activation via the non‐canonical TAB‐dependent pathway to promote cytokine production. We assessed GPCR‐induced p38 activation in macrovascular and microvascular endothelial cell lines including HUVEC, EA.hy926 and HDMECs. Using immunoblotting and qPCR, the expression of p38, TAB1, 2, 3 and MKK3,6 were quantified and appear to be comparable across the endothelial cell lines. To determine if p38 activation induced by GPCRs is mediated by autophosphorylation, we used the p38α/β SB203580 inhibitor which blocks p38 autophosphorylation but not phosphorylation mediated by upstream MAP2Ks. In HUVEC, EA.hy926 and HDMEC pretreated with SB203580, all four agonists including histamine, PGE2, ADP and thrombin failed to promote p38 phosphorylation, suggesting that endothelial GPCR‐induced p38 activation occurs through autophosphorylation. Next, we used combinations of TAB1, 2 and 3‐specific siRNAs to deplete endothelial cells of TAB proteins and found that all GPCR agonists require TAB expression to induce maximal p38 activation in endothelial cells. In addition, endothelial GPCR‐induced cytokine production was also dependent on p38 signaling and TAB expression. Taken together, these data support a critical role for TAB1‐dependent activation of p38 MAPK induced by endothelial GPCRs in inflammatory signaling.Support or Funding InformationThis work is funded by NIH/NIGMS R01 GM090689, R01 GM116597 and UC Tobacco‐related Disease Research Program High Impact Pilot Award 26IP‐0050This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fatty acid transporter expression and regulation is impaired in placental macrovascular endothelial cells in obese women

Objective: Fetal fatty acid (FA) delivery is ultimately controlled by placental transport. Focus has been the maternal-placental interface, but regulation at the feto-placental interface is unknown.Methods: Placental macrovascular endothelial cells (EC) (n = 4/group) and trophoblasts (TB) (n = 5/group) were isolated from lean (pregravid BMI <25 kg/m2) and obese (body mass index (BMI) > 30) women. Fatty acid transporters FAT/CD36, FABPpm, FATP4, FABP 3, 4 and 5, PLIN2 and PPARα, δ, γ expression, was measured in EC and TB. Transporter response to 24 h palmitate (PA) was assessed.Results: mRNA expression of FABP3, 4, 5 and PPARγ was 2- to 3-fold reduced in EC of obese versus lean women (p < .03), but not in TB. Protein level of FABPpm was 20% lower in obese (p < .05). Palmitate (PA) up-regulated CD36, FABP3, FABP4, and PLIN2 gene expression by 3- to 4-fold in lean but not obese EC (p < .05), while PA increased FABP4 and PLIN2 in lean and obese TB, and FABP5 in lean (p < .05) EC. PA exposure up-regulated peroxisome proliferator activated receptors (PPARs) 2-fold in lean and obese EC (p < .05), but not in TB.Conclusions: In obese women, FA transporter expression is lower in placental EC, but not TB, and less sensitive to saturated FA, compared to lean women. FA transport may be regulated at the feto-placental interface.

Neutrophil Extracellular Traps Drive Endothelial-to-Mesenchymal Transition.

An excessive release and impaired degradation of neutrophil extracellular traps (NETs) leads to the continuous exposure of NETs to the endothelium in a variety of hematologic and autoimmune disorders, including lupus nephritis. This study aims to unravel the mechanisms through which NETs jeopardize vascular integrity. Microvascular and macrovascular endothelial cells were exposed to NETs, and subsequent effects on endothelial integrity and function were determined in vitro and in vivo. We found that endothelial cells have a limited capacity to internalize NETs via the receptor for advanced glycation endproducts. An overflow of the phagocytic capacity of endothelial cells for NETs resulted in the persistent extracellular presence of NETs, which rapidly altered endothelial cell-cell contacts and induced vascular leakage and transendothelial albumin passage through elastase-mediated proteolysis of the intercellular junction protein VE-cadherin. Furthermore, NET-associated elastase promoted the nuclear translocation of junctional β-catenin and induced endothelial-to-mesenchymal transition in cultured endothelial cells. In vivo, NETs could be identified in kidney samples of diseased MRL/lpr mice and patients with lupus nephritis, in whom the glomerular presence of NETs correlated with the severity of proteinuria and with glomerular endothelial-to-mesenchymal transition. These results indicate that an excess of NETs exceeds the phagocytic capacity of endothelial cells for NETs and promotes vascular leakage and endothelial-to-mesenchymal transition through the degradation of VE-cadherin and the subsequent activation of β-catenin signaling. Our data designate NET-associated elastase as a potential therapeutic target in the prevention of endothelial alterations in diseases characterized by aberrant NET release.

Development and evaluation of injectable nanosized drug delivery systems for apigenin

The purpose of this study was to develop different injectable nanosized drug delivery systems (NDDSs) i.e. liposome, lipid nanocapsule (LNC) and polymeric nanocapsule (PNC) encapsulating apigenin (AG) and compare their characteristics to identify the nanovector(s) that can deliver the largest quantity of AG while being biocompatible. Two liposomes with different surface characteristics (cationic and anionic), a LNC and a PNC were prepared. A novel tocopherol modified poly(ethylene glycol)-b-polyphosphate block-copolymer was used for the first time for the PNC preparation. The NDDSs were compared by their physicochemical characteristics, AG release, storage stability, stability in serum, complement consumption and toxicity against a human macrovascular endothelial cell line (EAhy926). The diameter and surface charge of the NDDSs were comparable with previously reported injectable nanocarriers. The NDDSs showed good encapsulation efficiency and drug loading. Moreover, the NDDSs were stable during storage and in fetal bovine serum for extended periods, showed low complement consumption and were non-toxic to EAhy926 cells up to high concentrations. Therefore, they can be considered as potential injectable nanocarriers of AG. Due to less pronounced burst effect and extended release characteristics, the nanocapsules could be favorable approaches for achieving prolonged pharmacological activity of AG using injectable NDDS.

Culture of human cells in experimental units for spaceflight impacts on their behavior.

Because space missions produce pathophysiological alterations such as cardiovascular disorders and bone demineralization which are very common on Earth, biomedical research in space is a frontier that holds important promises not only to counterbalance space-associated disorders in astronauts but also to ameliorate the health of Earth-bound population. Experiments in space are complex to design. Cells must be cultured in closed cell culture systems (from now defined experimental units (EUs)), which are biocompatible, functional, safe to minimize any potential hazard to the crew, and with a high degree of automation. Therefore, to perform experiments in orbit, it is relevant to know how closely culture in the EUs reflects cellular behavior under normal growth conditions. We compared the performances in these units of three different human cell types, which were recently space flown, i.e. bone mesenchymal stem cells, micro- and macrovascular endothelial cells. Endothelial cells are only slightly and transiently affected by culture in the EUs, whereas these devices accelerate mesenchymal stem cell reprogramming toward osteogenic differentiation, in part by increasing the amounts of reactive oxygen species. We conclude that cell culture conditions in the EUs do not exactly mimic what happens in a culture dish and that more efforts are necessary to optimize these devices for biomedical experiments in space. Impact statement Cell cultures represent valuable preclinical models to decipher pathogenic circuitries. This is true also for biomedical research in space. A lot has been learnt about cell adaptation and reaction from the experiments performed on many different cell types flown to space. Obviously, cell culture in space has to meet specific requirements for the safety of the crew and to comply with the unique environmental challenges. For these reasons, specific devices for cell culture in space have been developed. It is important to clarify whether these alternative culture systems impact on cell performances to allow a correct interpretation of the data.

High glucose facilitated endothelial heparanase transfer to the cardiomyocyte modifies its cell death signature.

The secretion of enzymatically active heparanase (HepA) has been implicated as an essential metabolic adaptation in the heart following diabetes. However, the regulation and function of the enzymatically inactive heparanase (HepL) remain poorly understood. We hypothesized that in response to high glucose (HG) and secretion of HepL from the endothelial cell (EC), HepL uptake and function can protect the cardiomyocyte by modifying its cell death signature. HG promoted both HepL and HepA secretion from microvascular (rat heart micro vessel endothelial cells, RHMEC) and macrovascular (rat aortic endothelial cells, RAOEC) EC. However, only RAOEC were capable of HepL reuptake. This occurred through a low-density lipoprotein receptor-related protein 1 (LRP1) dependent mechanism, as LRP1 inhibition using small interfering RNA (siRNA), receptor-associated protein, or an LRP1 neutralizing antibody significantly reduced uptake. In cardiomyocytes, which have a negligible amount of heparanase gene expression, LRP1 also participated in the uptake of HepL. Exogenous addition of HepL to rat cardiomyocytes produced a dramatically altered expression of apoptosis-related genes, and protection against HG and H2O2 induced cell death. Cardiomyocytes from acutely diabetic rats demonstrated a robust increase in LRP1 expression and levels of heparanase, a pro-survival gene signature, and limited evidence of cell death, observations that were not apparent following chronic and progressive diabetes. Our results highlight EC-to-cardiomyocyte transfer of heparanase to modulate the cardiomyocyte cell death signature. This mechanism was observed in the acutely diabetic heart, and its interruption following chronic diabetes may contribute towards the development of diabetic cardiomyopathy.