Primary Cultures Of Endothelial Cells Research Articles

Production and control of coagulation proteins for factor X activation in human endothelial cells and fibroblasts

Human endothelial cells (ECs) synthesize, store, and secrete von Willebrand factor multimeric strings and coagulation factor (F) VIII. It is not currently known if ECs produce other coagulation factors for active participation in coagulation. We found that 3 different types of human ECs in primary culture produce clotting factors necessary for FX activation via the intrinsic (FVIII-FIX) and extrinsic (tissue factor [TF]-FVII) coagulation pathways, as well as prothrombin. Human dermal fibroblasts were used as comparator cells. TF, FVII, FIX, FX, and prothrombin were detected in ECs, and TF, FVII, FIX, and FX were detected in fibroblasts. In addition, FVII, FIX, FX, and prothrombin were detected by fluorescent microscopy in EC cytoplasm (associated with endoplasmic reticulum and Golgi proteins). FX activation occurred on human umbilical vein EC surfaces without the addition of external coagulation proteins, proteolytic enzymes, or phospholipids. Tumour necrosis factor, which suppresses the generation of activated protein C and increases TF, augmented FX activation. Fibroblasts also produced TF, but (in contrast to ECs) were incapable of activating FX without the exogenous addition of FX and had a marked increase in FX activation following the addition of both FX and FVII. We conclude that human ECs produce their own coagulation factors that can activate cell surface FX without the addition of exogenous proteins or phospholipids.

Endothelial Protein C Receptor Could Contribute to Experimental Malaria-Associated Acute Respiratory Distress Syndrome.

The severity of Plasmodium falciparum malaria is associated with parasite cytoadherence, but there is limited knowledge about the effect of parasite cytoadherence in malaria-associated acute respiratory distress syndrome (ARDS). Our objective was to evaluate the cytoadherence of infected red blood cells (iRBCs) in a murine model of ARDS and to appraise a potential function of endothelial protein C receptor (EPCR) in ARDS pathogenesis. DBA/2 mice infected with P. berghei ANKA were classified as ARDS- or hyperparasitemia- (HP-) developing mice according to respiratory parameters and parasitemia. Lungs, blood, and bronchoalveolar lavage were collected for gene expression or protein analyses. Primary cultures of microvascular lung endothelial cells from DBA/2 mice were analyzed for iRBC interactions. Lungs from ARDS-developing mice showed evidence of iRBC accumulation along with an increase in EPCR and TNF concentrations. Furthermore, TNF increased iRBC adherence in vitro. Dexamethasone-treated infected mice showed low levels of TNF and EPCR mRNA expression and, finally, decreased vascular permeability, thus protecting mice from ARDS. In conclusion, we identified that increased iRBC cytoadherence in the lungs underlies malaria-associated ARDS in DBA/2-infected mice and that inflammation increased cytoadherence capacity, suggesting a participation of EPCR and a conceivable target for drug development.

11-β-hydroxysterols as possible endogenous stimulators of mitochondrial biogenesis as inferred from epicatechin molecular mimicry

Currently, there is great interest in identifying endogenous (i.e. physiological) stimulators of mitochondrial biogenesis (MB), in particular, those that may mediate the effects of exercise. The molecular size of the cacao flavanols (epicatechin and catechin) highly resembles that of sterols and epicatechin has been reported to activate cells surface receptors leading to the stimulation of MB in endothelial and skeletal muscle cells translating into enhanced exercise capacity. We therefore hypothesize, that epicatechin may be acting as a structural mimic of an as yet unknown sterol capable of stimulating MB. We developed a new synthetic process for obtaining enantiomerically pure preparations of (-)-epicatechin and (+)-epicatechin. Applying spatial analytics and molecular modeling, we found that the two isoforms of epicatechin, (-) and (+), have a structural resemblance to 11-β-hydroxypregnenolone, a sterol with no previously described biological activity. As reported in this proof-of-concept study performed in primary cultures of endothelial and muscle cells, 11-β-hydroxypregnenolone is one of the most potent inducers of MB as significant activity can be detected at femtomolar levels. The relative potency of (-)/(+)-epicatechin isoforms and on inducing MB correlates with their degree of spatial homology towards the 11-β-hydroxypregnenolone. On the basis of these results, the detailed in vivo characterization of the potential for these sterols to act as endogenous modulators of MB is warranted.

Lytic cycle of Besnoitia besnoiti tachyzoites displays similar features in primary bovine endothelial cells and fibroblasts

BackgroundBovine besnoitiosis, caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that continues to spread in Europe in the absence of control tools. In this scenario, in vitro culture systems are valuable tools to carry out drug screenings and to unravel host-parasite interactions. However, studies performed in bovine target cells are scarce.MethodsThe objective of the present study was to obtain primary bovine aortic endothelial cells (BAECs) and fibroblast cell cultures, target cells during the acute and the chronic stage of the disease, respectively, from healthy bovine donors. Afterwards, expression of surface (CD31, CD34 and CD44) and intracellular markers (vimentin and cytokeratin) was studied to characterize cell populations by flow cytometry. Next, the lytic cycle of B. besnoiti tachyzoites was studied in both target cells. Invasion rates (IRs) were determined by immunofluorescence at several time points post-infection, and proliferation kinetics were studied by quantitative PCR (qPCR). Finally, the influence of bovine viral diarrhea virus (BVDV) co-infection on the host cell machinery, and consequently on B. besnoiti invasion and proliferation, was investigated in BAECs.ResultsMorphology and cytometry results confirmed the endothelial and fibroblast origins. CD31 was the surface marker that best discriminated between BAECs and fibroblasts, since fibroblasts lacked CD31 labelling. Expression of CD34 was weak in low-passage BAECs and absent in high-passage BAECs and fibroblasts. Positive labelling for CD44, vimentin and cytokeratin was observed in both BAECs and fibroblasts. Regarding the lytic cycle of the parasite, although low invasion rates (approximately 3–4%) were found in both cell culture systems, more invasion was observed in BAECs at 24 and 72 hpi. The proliferation kinetics did not differ between BAECs and fibroblasts. BVDV infection favoured early Besnoitia invasion but there was no difference in tachyzoite yields observed in BVDV-BAECs compared to BAECs.ConclusionsWe have generated and characterized two novel standardized in vitro models for Besnoitia besnoiti infection based on bovine primary target BAECs and fibroblasts, and have shown the relevance of BVDV coinfections, which should be considered in further studies with other cattle pathogens.

The effects of Vitis vinifera L. phenolic compounds on a blood-brain barrier culture model: Expression of leptin receptors and protection against cytokine-induced damage.

The medicinal properties of grapes (Vitis vinifera L.) are well known since ancient times. Ethnobotanical grape preparations, like the Ayurvedic Darakchasava are used as cardiotonic and for the treatment of cardiovascular diseases. Dried grape products are also applied in Iranian traditional medicine for memory problems, which are linked to the pathology of brain microvessels, a special part of the cardiovascular system. The anti-inflammatory and protective effects of these traditional preparations on the cardiovascular system are related to their bioactive phenolic compounds. The blood-brain barrier (BBB), formed by brain capillaries, is not only involved in inflammatory and other diseases of the central nervous system, but also in many systemic diseases with an inflammatory component. Dietary obesity is a systemic chronic inflammatory condition in which the peripheral and central vascular system is affected. Among the cerebrovascular changes in obesity defective leptin transport across the BBB related to central leptin resistance is observed. Our aim was to study the protective effects of grape phenolic compounds epicatechin (EC), gallic acid (GA) and resveratrol (RSV) and grape-seed proanthocyanidin-rich extract (GSPE) on a cytokine-induced vascular endothelial inflammation model. Using a culture model of the BBB we investigated cytokine-induced endothelial damage and changes in the expression of leptin receptors and leptin transfer. For the BBB model, primary cultures of rat brain endothelial cells, glial cells and pericytes were used in co-culture. Cells were treated by tumor necrosis factor-α (TNF-α) and interleukin-1 β (IL-1β) (10 ng/ml each) to induce damage. Cell toxicity was evaluated by the measurement of impedance. The expression of leptin receptors was assessed by RT-qPCR and western blot. The production of reactive oxygen species (ROS) and nitric oxide (NO) were detected by fluorescent probes. GSPE (10 μg/ml), EC (10 μM), GA (1 μM) or RSV (10 μM) did not change the viability of brain endothelial cells. The gene expression of the short leptin receptor isoform, Ob-Ra, was up-regulated by GSPE, EC and RSV, while the mRNA levels of Lrp2 and clusterin, clu/ApoJ were not affected. The tested compounds did not change the expression of the long leptin receptor isoform, Ob-Rb. RSV protected against the cytokine-induced increase in albumin permeability of the BBB model. GSPE and EC exerted an antioxidant effect and GSPE increased NO both alone and in the presence of cytokines. The cytokine-induced nuclear translocation of transcription factor NF-κB was blocked by GSPE, GA and RSV. Cytokines increased the mRNA expression of Lrp2 which was inhibited by EC. RSV increased Ob-Ra and Clu in the presence of cytokines. Cytokines elevated leptin transfer across the BBB model, which was not modified by GSPE or RSV. Our results obtained on cell culture models confirm that natural grape compounds protect vascular endothelial cells against inflammatory damage in accordance with the ethnopharmacological use of grape preparations in cardiovascular diseases. Furthermore, grape compounds and GSPE, by exerting a beneficial effect on the BBB, may also be considered in the treatment of obesity after validation in clinical trials.

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (<10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% ± 1.2%, 94.4% ± 1.1%, and 92.4% ± 1.7% (mean ± SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Isolation, Culture and Functional Characterization of Glia and Endothelial Cells From Adult Pig Brain.

Primary cultures of glial and endothelial cells are important tools for basic and translational neuroscience research. Primary cell cultures are usually generated from rodent brain although considerable differences exist between human and rodent glia and endothelial cells. Because many translational research projects aim to identify mechanisms that eventually lead to diagnostic and therapeutic approaches to target human diseases, glia, and endothelial cultures are needed that better reflect the human central nervous system (CNS). Pig brain is easily accessible and, in many aspects, close to the human brain. We established an easy and cost-effective method to isolate and culture different primary glial and endothelial cells from adult pig brain. Oligodendrocyte, microglia, astrocyte, and endothelial primary cell cultures were generated from the same brain tissue and grown for up to 8 weeks. Primary cells showed lineage-specific morphology and expressed specific markers with a purity ranging from 60 to 95%. Cultured oligodendrocytes myelinated neurons and microglia secreted tumor necrosis factor alpha when induced with lipopolysaccharide. Endothelial cells showed typical tube formation when grown on Matrigel. Astrocytes enhanced survival of co-cultured neurons and were killed by Aquaporin-4 antibody positive sera from patients with Neuromyelitis optica. In summary, we established a new method for primary oligodendrocyte, microglia, endothelial and astrocyte cell cultures from pig brain that provide a tool for translational research on human CNS diseases.

Neonates from women with pregestational maternal obesity show reduced umbilical vein endothelial response to insulin

ObjectivePregestational maternal obesity (PGMO) associates with foetoplacental vascular endothelial dysfunction and higher risk for insulin resistance in the neonate. We characterised the PGMO consequences on the insulin response of the human foetoplacental vasculature. MethodsUmbilical veins were from pregnancies where the mother was with PGMO (body mass index 30–42.3 kg/m2, n = 33) or normal pregestational weight (PGMN) (body mass index 19.5–24.4 kg/m2, n = 21) with total gestational weight gain within the physiological range. Umbilical vein ring segments were mounted in a myograph for isometric force measurements. Primary cultures of human umbilical vein endothelial cells were used in passage 3. Vessel rings and cells were exposed to 1 nmol/L insulin (20 min) in the absence or presence of 100 μmol/L NG-nitro-l-arginine methyl ester (inhibitor of nitric oxide synthase, NOS). ResultsVessel rings from PGMO showed reduced nitric oxide synthase-activity dependent dilation to insulin or calcitonin-gene related peptide compared with PGMN. PGMO associated with higher inhibitor phosphorylation of the insulin receptor substrate 1 (IRS-1) and lower activator phosphorylation of protein kinase B/Akt (Akt). Cells from PGMO also showed lower nitric oxide level and reduced activator serine1177 but increased inhibitor threonine495 phosphorylation of endothelial nitric oxide synthase (eNOS) and saturable transport of l-arginine. HUVECs from PGMO were not responsive to insulin. ConclusionThe lack of response to insulin by the foetoplacental endothelium may result from reduced IRS-1/Akt/eNOS signalling in PGMO. These findings may result in higher risk of insulin resistance in neonates to PGMO pregnancies.

AKAP12 deficiency impairs VEGF-induced endothelial cell migration and sprouting.

AimProtein kinase (PK) A anchoring protein (AKAP) 12 is a scaffolding protein that anchors PKA to compartmentalize cyclic AMP signalling. This study assessed the consequences of the downregulation or deletion of AKAP12 on endothelial cell migration and angiogenesis.MethodsThe consequences of siRNA‐mediated downregulation AKAP12 were studied in primary cultures of human endothelial cells as well as in endothelial cells and retinas from wild‐type versus AKAP12−/− mice. Molecular interactions were investigated using a combination of immunoprecipitation and mass spectrometry.ResultsAKAP12 was expressed at low levels in confluent endothelial cells but its expression was increased in actively migrating cells, where it localized to lamellipodia. In the postnatal retina, AKAP12 was expressed by actively migrating tip cells at the angiogenic front, and its deletion resulted in defective extension of the vascular plexus. In migrating endothelial cells, AKAP12 was co‐localized with the PKA type II‐α regulatory subunit as well as multiple key regulators of actin dynamics and actin filament‐based movement; including components of the Arp2/3 complex and the vasodilator‐stimulated phosphoprotein (VASP). Fitting with the evidence of a physical VASP/AKAP12/PKA complex, it was possible to demonstrate that the VEGF‐stimulated and PKA‐dependent phosphorylation of VASP was dependent on AKAP12. Indeed, AKAP12 colocalized with phospho‐Ser157 VASP at the leading edge of migrating endothelial cells.ConclusionThe results suggest that compartmentalized AKAP12/PKA signalling mediates VASP phosphorylation at the leading edge of migrating endothelial cells to translate angiogenic stimuli into altered actin dynamics and cell movement.

Interactions between carboxypeptidase M and kinin B1 receptor in endothelial cells.

Carboxypeptidase M (CPM) is a glycosylphosphatidylinositol anchored enzyme that plays an important role in the kallikrein-kinin system (KKS). CPM catalytic domain hydrolyzes Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating des-Arg-kinins, the agonists of B1 receptor (B1R). It is known that CPM and kinin B1R are co-localized in the plasma membrane microdomains, where they interact with each other, facilitating receptor signaling. We hypothesized here that this CPM-B1R interaction could also affect the activity of the enzyme. Thus, in this work, we evaluated the impact of B1R presence or absence on CPM activity and expression, using primary culture of microvascular endothelial cells from wild-type, kinin B1R knockout mice (B 1 -/- ), and transgenic rats overexpressing B1 receptor exclusively in the endothelium. In addition, HEK293T cells, as wells as B 1 -/- primary culture of endothelial cells, both transfected with B1R, were also used. CPM expression and activity were downregulated in cells of knockout mice compared to control and this reduction was rescued after B1R transfection. Cells overexpressing B1R presented higher levels of CPM mRNA, protein, and activity. This profile was reverted by pre-incubation with the B1R antagonist, R715, in highly expressing receptor cells. Our data show that kinin B1R positively modulates both CPM expression and activity, suggesting that CPM-B1R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among different components of KKS and contributes to a better understanding of its patho-physiological role.

15-epi-lipoxin A4 inhibits TNF-α-induced tissue factor expression via the PI3K/AKT/ NF-κB axis in human umbilical vein endothelial cells

Inflammation and coagulation are two important processes implicated in venous thromboembolism (VTE). 15-epi-lipoxin A4 (15-epi-LXA4) is the epimer of LXA4, a small lipid molecule, is known to play a key role in the resolution of inflammation. This study aimed to demonstrate whether 15-epi-LXA4 could suppress the inflammatory factor tumor necrosis factor-alpha (TNF-α)-induced upregulation of tissue factor (TF), an important regulator of the blood coagulation cascade, and evaluated the possible underlying mechanisms. We found that 15-epi-LXA4 not only reduced the up-regulation of mRNA and antigens, but also lowered the activity of TF (elevated by TNF-α) in primary culture of human umbilical vein endothelial cells (pc-HUVECs). In addition, 15-epi-LXA4 suppressed the activation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, induced by TNF-α, in pc-HUVECs. 15-epi-LXA4 also inhibited the binding of NF-κB on the TF promoter, which is otherwise enhanced by TNF-α. The role of 15-epi-LXA4 in regulating TNF-α-induced effects was enhanced by the PI3K inhibitor and prevented by the PI3K activator. In conclusion, 15-epi-LXA4 lowered the TNF-α-induced high TF expression and activity by suppressing PI3K/AKT signaling activation, thereby reducing the binding capacity of NF-κB on the TF promoter in pc-HUVECs.

In Vitro Anti-Inflammatory Effect of Salvia sagittata Ethanolic Extract on Primary Cultures of Porcine Aortic Endothelial Cells.

The aim of the present research was to study the effects of an ethanolic extract of Salvia sagittata Ruiz & Pav (SSEE), an endemic Ecuadorian plant traditionally used to treat inflammation and different intestinal affections, on primary cultures of porcine aortic endothelial cells (pAECs). pAECs were cultured in the presence of different concentrations (1-200 μg/mL) of SSEE for 24 h, and cytotoxicity was evaluated by the MTT assay. SSEE did not negatively affect cellular viability at any concentration tested. Cell cycle was analyzed and no significant change was observed. Then, the anti-inflammatory effects of SSEE on pAECs were analyzed using a lipopolysaccharide (LPS) as the inflammatory stimulus. Different markers involved in the inflammatory process, such as cytokines and protective molecules, were evaluated by real-time quantitative PCR and Western blot. SSEE showed the ability to restore pAEC physiological conditions reducing interleukin-6 and increasing Heme Oxygenase-1 protein levels. The phytochemical composition of SSEE was also evaluated via HPLC-DAD and spectrophotometric assays. The presence of different phenolic acids and flavonoids was revealed, with rosmarinic acid as the most abundant component. SSEE possesses an interesting antioxidant activity, as assessed through both the Oxygen Radical Absorbance Capacity (ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. In conclusion, results suggest that SSEE is endowed with an in vitro anti-inflammatory effect. This represents the initial step in finding a possible scientific support for the traditional therapeutic use of this plant.

Geniposide protects against hypoxia/reperfusion-induced blood-brain barrier impairment by increasing tight junction protein expression and decreasing inflammation, oxidative stress, and apoptosis in an in vitro system

The blood-brain barrier (BBB) is involved in the pathogeneses of ischemic stroke (IS). Geniposide (GEN), an iridoid glycoside isolated from Gardenia jasminoides Ellis, has been used for the treatment of IS. However, the effects of GEN on the BBB are poorly understood. In vitro disease models of the BBB could be very helpful for the elucidation of underlying mechanisms and the development of novel therapeutic strategies. Therefore, we established an in vitro BBB model composed of primary cultures of brain microvascular endothelial cells and astrocytes. We then used this in vitro model to investigate the effect of GEN on the function of the BBB. Oxygen glucose deprivation and reoxygenation (OGD/R) significantly increased permeability and cell apoptosis in this in vitro BBB model. Notably, GEN pretreatment effectively improved the BBB function by decreasing the permeability of the BBB, promoting expression of tight junction proteins (zonula occludens-1, claudin-5, and occludin) and gamma-glutamyl transpeptidase, increasing transendothelial electrical resistance, mitigating oxidative stress damage and the release of inflammatory cytokines, downregulating the expression levels of matrix metallopeptidases-9 (MMP-9) and MMP-2, and increasing the release of brain derived neurotrophic factor and glial cell derived neurotrophic factor. Therefore, GEN can ameliorate the BBB dysfunction induced by OGD/R conditions through multiple protective mechanisms. The findings suggest that GEN may be an appropriate drug for restoring the barrier function of the BBB.

Platelet Lysate Inhibits NF-κB Activation and Induces Proliferation and an Alert State in Quiescent Human Umbilical Vein Endothelial Cells Retaining Their Differentiation Capability.

Injured blood vessel repair and blood circulation re-establishment are crucial events for tissue repair. We investigated in primary cultures of human umbilical vein endothelial cells (HUVEC), the effects of platelet lysate (PL), a cocktail of factors released by activated platelets following blood vessel disruption and involved in the wound-healing process triggering. PL exerted a protective effect on HUVEC in an inflammatory milieu by inhibiting IL-1α-activated NF-κB pathway and by inducing the secretion of PGE2, a pro-resolving molecule in the wound microenvironment. Moreover, PL enhanced HUVEC proliferation, without affecting their capability of forming tube-like structures on matrigel, and activated resting quiescent cells to re-enter cell cycle. In agreement with these findings, proliferation-related pathways Akt and ERK1/2 were activated. The expression of the cell-cycle activator Cyclin D1 was also enhanced, as well as the expression of the High Mobility Group Box-1 (HMGB1), a protein of the alarmin group involved in tissue homeostasis, repair, and remodeling. These in vitro data suggest a possible in vivo contribution of PL to new vessel formation after a wound by activation of cells resident in vessel walls. Our biochemical study provides a rationale for the clinical use of PL in the treatment of wound healing-related pathologies.

Endothelium‐Specific Acid Ceramidase Gene Deletion Enhances Exosome Secretion and Release of NLRP3 Inflammasome Products during Hyperglycemia

Exosomes have been demonstrated to be one of the mechanisms mediating the release of a variety of intracellular signaling molecules to mediate cell‐to‐cell communication. However, it remains unknown whether exosomes mediate the release of Nod‐like receptor pyrin domain 3 (NLRP3) inflammasome products such as IL‐1β from endothelial cells and how exosome‐mediated‐inflammatory signaling is regulated. The present study hypothesized that lysosomal acid ceramidase (AC) determines the fate of the multivesicular body (MVB) to control the exosome‐mediated release of NLRP3 inflammasome products during hyperglycemia. Using streptozotocin (STZ)‐induced mouse model of diabetes mellitus (DM), we found that endothelium‐specific AC gene knock‐out mice (Asah1fl/fl/ECcre) had significantly enhanced formation and activation of NLRP3 inflammasomes including IL‐1β production in the coronary arterial endothelium. These mice also had increased thickening of the coronary arterial wall and reduced expression of tight junction protein (ZO‐1 and ZO‐2) compared to their wild‐type (WT/WT) littermates. Interestingly, we observed that the expression of exosome markers such as CD63 and alkaline phosphatase (ALP) was also augmented in STZ‐treated Asah1fl/fl/ECcre mice compared to WT/WT mice, which was accompanied by an increased IL‐1β release of exosomes into the blood. In the primary cultures of coronary endothelial cells (CECs) from WT/WT and Asah1fl/fl/ECcre mice, we demonstrated that the AC deficiency markedly enhanced the formation and activation of the NLRP3 inflammasomes, but significantly down‐regulated tight junction proteins when CECs were exposed to high levels of glucose. The CECs from Asah1fl/fl/ECcre mice also had decreased MVBs and increased exosome release in response to high glucose stimulation and these exosomes contained increased IL‐1β. These results from our in vivo and in vitro experiments suggest that AC importantly controls exosome secretion and release of NLRP3 inflammasome products in CECs and its deficiency enhances this exosome‐mediated release of NLRP3 inflammasome products to activate coronary arterial inflammatory response during hyperglycemia.Support or Funding InformationHL057244, HL075316, and DK120491This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of Histone Deacetylase 6 (HDAC6) in the diabetic retina and in retinal endothelial cells exposed to glucidic stress, promotes oxidative stress through suppression of the thioredoxin system

Retinal microvascular dysfunction characterizes the pathophysiology of diabetic retinopathy (DR), a common complication of diabetes and a leading cause of blindness in the World. Oxidative stress and inflammation have been implicated as major pathogenic hubs for the induction and progression of DR. Reduced endogenous antioxidant activities within the retinal microvasculature can significantly contribute to oxidative stress in the diabetic retina. We have previously shown that diabetes inhibits the thioredoxin system (TrxS) and others have shown that the cytosolic histone deacetylase 6 (HDAC6) can promote oxidative stress in a not yet understood mechanism. Here we have investigated the role of HDAC6 in regulating the TrxS in the diabetic retinal microvasculature. HDAC6 expression and activity were assessed by immunoblotting and fluorimetric assay (respectively) in retinas of streptozotocin‐induced diabetic rats (STZ‐rats, 8 weeks of diabetes) and in primary culture of human retinal endothelial cells (HuREC) exposed to disease‐relevant glucose levels (HG=25mM D‐glucose) for 48 hours. Changes in TrxS were monitored by assessing the expression and activity of its functional unit, thioredoxin‐1 (Trx‐1), by immunoblotting and a commercially available fluorimetric assay, in STZ‐rats retinas and in HuREC exposed to HG. Some experiments in HuREC were conducted also in presence of 5μM of the specific HDAC6 inhibitor tubastatin A. CellROX assay and dot‐blot assessing 5‐nitrotyrosine (5‐NT) and 4 hydroxynonenal (4‐HNE) were used to measure oxidative stress in our experimental groups. HDAC6 expression and activity were found significantly elevated in retinas of STZ‐rats compared to normal age‐matched controls and in HuREC exposed to HG, but not to normal glucose (5mM D‐glucose) or the osmotic control L‐glucose (25mM). At the same time Trx‐1 expression was increased in diabetic retinas and in HuREC exposed to HG, whereas its activity was significantly decreased. Parallel to loss of Trx‐1 activity and increased HDAC6 expression and activity, we also found that oxidative stress parameters were also elevated. In HuREC, tubastatin A was able to block HG‐induced up‐regulation of oxidative stress parameters while up‐regulating Trx‐1 activity and normalizing its expression. In summary, our data confirm previous observations on the role of HDAC6 in promoting oxidative stress and disclose HDAC6 role in suppressing Trx‐1 activity. Therefore, our results, support HDAC6 pathogenic role in promoting oxidative stress in the diabetic retinal microvasculature and its implication in the pathogenesis of diabetic retinopathy.Support or Funding InformationEY022416This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phytochemical Sulforaphane Provides Cerebrovascular Protection in a Large Animal Model of Brain Oxidative Stress Injury Caused by Prolonged Neonatal Asphyxia

Prolonged asphyxia in newborn infants may lead to cerebrovascular dysfunction and neurological complications. The neonatal brain is compromised by asphyxia/reventilation via a mechanism that involves oxidative stress. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, such as brussels sprouts and broccoli, has antioxidant and cytoprotective properties. We tested the hypothesis that SFN exhibits protective properties in neonatal cerebral circulation during prolonged asphyxia. Newborn pigs provide a relevant model of neonatal cerebrovascular regulation. In the first series of experiments, we investigated whether SFN exhibited vasoactive properties in neonatal cerebral circulation by detecting responses of pial arterioles to topical and systemic administration of a synthetic D, L‐SFN. Intravital microscopy using the cranial window technique was implemented to detect these responses. Topical application of SFN (1 μM – 1 mM) to the cerebral surface produced dilation of pial arterioles up to 40% above the baseline. Systemic administration of SFN (0.4 mg/kg i.p.) also produced long lasting cerebral vasodilation 20–30% above the baseline, suggesting that SFN is a brainpermeable drug. In the second series of the experiments, we tested the effects of SFN on the cerebral vascular outcome of asphyxia. We used a model of prolonged asphyxia (50 minutes) that combined severe hypoxia (PaO2, 20–30 mm Hg), hypercapnia (PaCO2, 70–80 mm Hg), and acidosis (pH, 6.9–7.0) while avoiding cerebral ischemia. Cerebral vascular functions were tested in saline‐ and SFN‐treated newborn pigs 48 hours after asphyxia. Prolonged asphyxia followed by reventilation with room air caused injury to endothelial and astrocyte components of the neurovascular unit leading to cerebral vascular dysfunction. SFN (0.4 mg/kg, i.p.) administered 30 minutes before asphyxia prevented loss of cerebral vascular responses to endothelium‐ and astrocyte‐dependent stimuli. SFN administered 20 minutes after the onset of asphyxia also preserved cerebral vascular dilator functions. We then tested the anti‐apoptotic effects of SFN in the in vitro model of oxidative stress injury in primary cultures of cerebral endothelial cells and cortical astrocytes from newborn pigs. SFN (1–5 μM) effectively prevented apoptotic death of endothelial cells and astrocytes caused by oxidative stress induced by xanthine/xanthine oxidase, glutamate, and TNF‐alpha. Overall, SFN exhibited vasodilator and cerebroprotective properties in neonatal cerebral circulation and protected the neurovascular unit from oxidative stress injury caused by prolonged asphyxia/reventilation. We propose that SFN can be used as a novel therapeutic agent for preventing of hypoxic‐ischemic encephalopathy caused by prolonged neonatal asphyxia.Support or Funding InformationThe study was supported by National Institutes of Health: RO1HL42851 (CWL), RO1NS101717 (HP), and R01NS105655 (HP).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract A094: Characterization of pancreatic cancer endothelial cells: Approach to enhance immune cell infiltration for immunotherapy

Abstract The hurdles in realizing immunotherapy success for cure stem from the fact that cancer patients are either refractory to immune response and/or develop resistance. We previously proposed that these phenomena are due, in part, to the deployment of tumor-associated antigens, or employment of tumor-associated endothelium acting as a gatekeeper for immune cell infiltration into the cancer tissue (Nakajima et al, Cancer Res 2017;77:5441-4). Here, an extensive study unveiled functional/molecular differences of endothelium derived from pancreatic cancer and normal pancreas. They were isolated from fresh surgical specimen by magnet-based selection. The primary culture of tumor-associated endothelial cells was confirmed by double positive expressions of endothelial markers, CD31 and ERG1. They showed the short vessel formations and the narrow area of capillary network, indicating the low potential of angiogenesis. Further, peripheral blood–derived lymphocytes were less adhering to the tumor-associated endothelial cells. To find the molecular differences, microarray analysis was performed, and identified 2748 molecules distinct from endothelial cells of noncancerous tissues (p&amp;lt;0.05). Then, a bioinformatic approach, i.e., ingenuity pathway analysis (IPA) reconstructed 47 pathways (p&amp;lt;0.05). Among them, TNFR1 signaling appeared as a highly possible pathway, which is involved in immune cell infiltration: (p=0.005, z score=2.00) with aberrantly expressed upstream molecules of the pathway. As for the downstream molecules, ICAM1 was highly expressed, whereas VCAM1 and E-selectin were generally suppressed on pathologic specimens of pancreatic cancer (n=20) as well as primary cultured tumor-associated endothelial cells. The unfulfilled expressions may be causes of noninflamed immune microenvironment of pancreatic cancer. Thus, our study highlights possible molecular candidates of tumor-associated endothelium shaping tumor microenvironment to enhance immune cell infiltration for successful immunotherapy. Citation Format: Kosei Nakajima, Yashunori Ino, Toshimitsu Iwasaki, Nobuyoshi Hiraoka. Characterization of pancreatic cancer endothelial cells: Approach to enhance immune cell infiltration for immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A094.

Abstract WMP77: Ischemic Preconditioning Improves Long-Term Outcomes and Preserves Blood-Brain Barrier After Ischemic Stroke via Oxidative Signaling and Nrf2 Activation

Introduction: Ischemic preconditioning (IPC) protects the brain against subsequent ischemic injuries. However, its protective mechanisms and long-term impact on stroke outcomes are unknown. IPC requires mild oxidative stress and may activate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Endogenous lipid electrophiles serve as robust Nrf2 activators. This study tests long-term effects of IPC and explores the mechanisms of IPC-mediated ischemic tolerance, focusing on lipid electrophiles and Nrf2 pathway. Methods: Wildtype (WT) and Nrf2 knockout (KO) mice were subjected to 60-min middle cerebral artery occlusion (MCAO) 3 d after IPC (12-min MCAO). Tissue loss, blood-brain barrier (BBB) damage, and neurobehavioral outcomes were assessed up to 35 d post stroke. The molecular mechanisms were explored by primary endothelial cell (EC) cultures with plasmid transfection and molecular biological approaches. Results: IPC reduced sensorimotor and cognitive deficits and tissue loss up to 35 d post stroke in WT mice, while Nrf2 KO abolished IPC-mediated protection. IPC led to mild oxidative stress, lipid electrophile generation and Nrf2 pathway activation. Prominent Nrf2 activation was seen in ECs and a selected group of astrocytes in tight association with microvessels, both of which are BBB components. As expected, IPC reduced BBB leakage 48 h post stroke and increased expressions of junctional proteins Claudin-5 and VE-Cadherin. Moreover, Nrf2 directly regulated Claudin-5 and VE-Cadherin promoter activities. Finally, a novel mechanism for electrophiles to activate Nrf2 was identified-through direct inhibition of glycogen synthase kinase 3β (GSK3β) activity via GSK3β-C199 residue. Conclusions: IPC preserves the BBB and provides long-term neuroprotection against stroke. Mechanistically, mild oxidative stress in IPC generates a pool of electrophiles, which then activates Nrf2 pathway through direct inhibition GSK3β-dependent Nrf2 degradation.