Organ-on-a-chip for studying immune cell adhesion to liver sinusoidal endothelial cells: the potential for testing immunotherapies and cell therapy trafficking.

Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with advanced tumours, yet the majority remain resistant to this therapy and urgent approaches are needed to boost the efficacy of these agents. Targeting the liver endothelial cells, as the orchestrators of immune cell recruitment, within the tumour microenvironment of this highly vascular cancer could potentially boost immune cell infiltration. We demonstrate the successful culture of primary human liver endothelial cells in organ-on-a-chip technology followed by perfusion of peripheral blood mononuclear cells. We confirm, with confocal and multiphoton imaging, the capture and adhesion of immune cells in response to pro-inflammatory cytokines in this model. This multicellular platform sets the foundation for testing the efficacy of new therapies in promoting leukocyte infiltration across liver endothelium as well as a model for testing cell therapy, such as chimeric antigen receptor (CAR)-T cell, capture and migration across human liver endothelium.

Metformin ameliorates ROS-p53-collagen axis of fibrosis and dyslipidemia in type 2 diabetes mellitus-induced left ventricular injury.

BACKGROUND: The link between oxidative stress (ROS), apoptosis (p53) and fibrosis (collagen) in type 2 diabetes mellitus (T2DM)-induced cardiac injury in the presence and absence of the antidiabetic drug, metformin has not been investigated before. MATERIAL AND METHODS: T2DM was induced in rats by a combination of high carbohydrate and fat diets (HCFD) and streptozotocin (50 mg/kg) injection. The protection group started metformin (200 mg/kg) treatment 14 days prior to the induction of diabetes and continued on metformin and HCFD until being sacrificed at week 12. RESULTS: Diabetes significantly induced blood levels of ROS and left ventricular p53 and collagen expression that was inhibited by metformin. Metformin also significantly reduced glycated haemoglobin and dyslipidemia induced by diabetes. In addition, a significant correlation between ROS-p53-collagen axis and glycaemia and hyperlipidaemia was observed. CONCLUSIONS: These findings show that metformin provides substantial protection against diabetic cardiomyopathy-induced ROS-p53 mediated fibrosis and dyslipidemia.

Hyperglycaemia up-regulates placental growth factor (PlGF) expression and secretion in endothelial cells via suppression of PI3 kinase-Akt signalling and activation of FOXO1.

Placenta growth factor (PlGF) is a pro-inflammatory angiogenic mediator that promotes many pathologies including diabetic complications and atherosclerosis. Widespread endothelial dysfunction precedes the onset of these conditions. As very little is known of the mechanism(s) controlling PlGF expression in pathology we investigated the role of hyperglycaemia in the regulation of PlGF production in endothelial cells. Hyperglycaemia stimulated PlGF secretion in cultured primary endothelial cells, which was suppressed by IGF-1-mediated PI3K/Akt activation. Inhibition of PI3K activity resulted in significant PlGF mRNA up-regulation and protein secretion. Similarly, loss or inhibition of Akt activity significantly increased basal PlGF expression and prevented any further PlGF secretion in hyperglycaemia. Conversely, constitutive Akt activation blocked PlGF secretion irrespective of upstream PI3K activity demonstrating that Akt is a central regulator of PlGF expression. Knock-down of the Forkhead box O-1 (FOXO1) transcription factor, which is negatively regulated by Akt, suppressed both basal and hyperglycaemia-induced PlGF secretion, whilst FOXO1 gain-of-function up-regulated PlGF in vitro and in vivo. FOXO1 association to a FOXO binding sequence identified in the PlGF promoter also increased in hyperglycaemia. This study identifies the PI3K/Akt/FOXO1 signalling axis as a key regulator of PlGF expression and unifying pathway by which PlGF may contribute to common disorders characterised by endothelial dysfunction, providing a target for therapy.

Suppression of type 2 diabetes mellitus-induced aortic ultrastructural alterations in rats by insulin: an association of vascular injury biomarkers.

Diabetes represents a major public health problem and an estimated 70% of people with diabetes die of cardiovascular complications. The protective effect of insulin treatment against ultrastructural damage to the tunica intima and tunica media of the aorta induced by type 2 diabetes mellitus (T2DM) has not been investigated before using transmission electron microscopy (TEM). Therefore, we induced T2DM in rats using high fat diet and streptozotocin (50 mg/kg) and administered insulin daily by i.v injection for 8 weeks to the treatment group. Whereas, the T2DM control group were left untreated for the duration of the experiment. A comparison was also made between the effect of insulin on aortic tissue and the blood level of biomarkers of vascular injury, inflammation, and oxidative stress. T2DM induced profound ultrastructural damage to the aortic endothelium and vascular smooth muscle cells, which were substantially protected with insulin. Furthermore, insulin returned blood sugar to a control level and significantly (p < .05) inhibited diabetic up-regulation of endothelial and leukocyte intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), endothelial cell adhesion molecules, P-selectin and E-selectin, tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), and malondialdehyde (MDA). Furthermore, insulin augmented the blood level of the anti-oxidant enzyme superoxide dismutase (SOD). We conclude that in a rat model of T2DM, insulin treatment substantially reduces aortic injury secondary to T2DM for a period of 8 weeks, possibly due to the inhibition of hyperglycemia, vascular activation, inflammation, and oxidative stress.

Isolation and Culture of Human Endothelial Cells from Micro- and Macro-vessels.

The endothelium from different vascular beds exhibits a high degree of phenotypic heterogeneity. Endothelial cells (EC) can be harvested easily from large vessels by mechanical removal or collagenase digestion. In particular, the human umbilical vein has been used due to its wide availability, and the study of ECs derived from it has undoubtedly greatly advanced our knowledge of vascular biology. However, the majority of the body's endothelium (>95 %) forms the microvasculature, and it is these cells providing the interface between the blood and tissues that play a critical role in the development of new blood vessels. This has led to the establishment of techniques for the isolation of microvascular ECs (MEC) from different tissues to provide more physiologically relevant in vitro models of angiogenesis and EC function.In this chapter the use of superparamagnetic beads (Dynabeads) coated with anti-PECAM-1 (CD31) antibodies (PECA-beads) to culture MECs from human adipose tissue is described along with the standard methods used to characterize them. Adipose tissue is an ideal source of MECs as it is composed mainly of adipocytes with a very rich microvasculature and is easy to disaggregate. Furthermore, it can be obtained in large quantities during plastic surgery procedures. Adipose obtained at reduction mammoplasty or abdominoplasty is first dissected free of the connective tissue, minced finely, and subjected to collagenase type II digestion. The adipocytes are removed by centrifugation to obtain a microvessel rich pellet, which is further disaggregated with trypsin/EDTA solution. Following filtration to remove fragments of the connective tissue, the pellet is incubated with PECA-beads and microvessel fragments/ECs and washed and harvested using a magnet. In addition, the adaptation of this basic technique for the isolation of the human lung and stomach MECs is also described along with common methods for the preparation of large vessel endothelial cells.

Carbon monoxide inhibits sprouting angiogenesis and vascular endothelial growth factor receptor-2 phosphorylation.

Carbon monoxide (CO) is a gaseous autacoid known to positively regulate vascular tone; however, its role in angiogenesis is unknown. The aim of this study was to investigate the effect of CO on angiogenesis and vascular endothelial growth factor (VEGF) receptor-2 phosphorylation. Human umbilical vein endothelial cells (HUVECs) were cultured on growth factor-reduced Matrigel and treated with a CO-releasing molecule (CORM-2) or exposed to CO gas (250 ppm). Here, we report the surprising finding that exposure to CO inhibits vascular endothelial growth factor (VEGF)-induced endothelial cell actin reorganisation, cell proliferation, migration and capillary-like tube formation. Similarly, CO suppressed VEGF-mediated phosphorylation of VEGFR-2 at tyrosine residue 1175 and 1214 and basic fibroblast growth factor- (FGF-2) and VEGF-mediated Akt phosphorylation. Consistent with these data, mice exposed to 250 ppm CO (1h/day for 14 days) exhibited a marked decrease in FGF-2-induced Matrigel plug angiogenesis (p<0.05). These data establish a new biological function for CO in angiogenesis and point to a potential therapeutic use for CO as an anti-angiogenic agent in tumour suppression.

The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis.

VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.

Loss of Akt activity increases circulating soluble endoglin release in preeclampsia: identification of inter-dependency between Akt-1 and heme oxygenase-1.

AIMS: Endothelial dysfunction is a hallmark of preeclampsia. Desensitization of the phosphoinositide 3-kinase (PI3K)/Akt pathway underlies endothelial dysfunction and haeme oxygenase-1 (HO-1) is decreased in preeclampsia. To identify therapeutic targets, we sought to assess whether these two regulators act to suppress soluble endoglin (sEng), an antagonist of transforming growth factor-ß (TGF-ß) signalling, which is known to be elevated in preeclampsia. METHODS AND RESULTS: Vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), angiopoietin-1 (Ang-1), and insulin, which all activate the PI3K/Akt pathway, inhibited the release of sEng from endothelial cells. Inhibition of the PI3K/Akt pathway, by overexpression of phosphatase and tensin homolog (PTEN) or a dominant-negative isoform of Akt (Akt(dn)) induced sEng release from endothelial cells and prevented the inhibitory effect of VEGF-A. Conversely, overexpression of a constitutively active Akt (Akt(myr)) inhibited PTEN and cytokine-induced sEng release. Systemic delivery of Akt(myr) to mice significantly reduced circulating sEng, whereas Akt(dn) promoted sEng release. Phosphorylation of Akt was reduced in preeclamptic placenta and this correlated with the elevated level of circulating sEng. Knock-down of Akt using siRNA prevented HO-1-mediated inhibition of sEng release and reduced HO-1 expression. Furthermore, HO-1 null mice have reduced phosphorylated Akt in their organs and overexpression of Akt(myr) failed to suppress the elevated levels of sEng detected in HO-1 null mice, indicating that HO-1 is required for the Akt-mediated inhibition of sEng. CONCLUSION: The loss of PI3K/Akt and/or HO-1 activity promotes sEng release and positive manipulation of these pathways offers a strategy to circumvent endothelial dysfunction.

Autocrine activity of soluble Flt-1 controls endothelial cell function and angiogenesis.

BACKGROUND: The negative feedback system is an important physiological regulatory mechanism controlling angiogenesis. Soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1), acts as a potent endogenous soluble inhibitor of VEGF- and placenta growth factor (PlGF)-mediated biological function and can also form dominant-negative complexes with competent full-length VEGF receptors. METHODS AND RESULTS: Systemic overexpression of VEGF-A in mice resulted in significantly elevated circulating sFlt-1. In addition, stimulation of human umbilical vein endothelial cells (HUVEC) with VEGF-A, induced a five-fold increase in sFlt-1 mRNA, a time-dependent significant increase in the release of sFlt-1 into the culture medium and activation of the flt-1 gene promoter. This response was dependent on VEGF receptor-2 (VEGFR-2) and phosphoinositide-3'-kinase signalling. siRNA-mediated knockdown of sFlt-1 in HUVEC stimulated the activation of endothelial nitric oxide synthase, increased basal and VEGF-induced cell migration and enhanced endothelial tube formation on growth factor reduced Matrigel. In contrast, adenoviral overexpression of sFlt-1 suppressed phosphorylation of VEGFR-2 at tyrosine 951 and ERK-1/-2 MAPK and reduced HUVEC proliferation. Preeclampsia is associated with elevated placental and systemic sFlt-1. Phosphorylation of VEGFR-2 tyrosine 951 was greatly reduced in placenta from preeclamptic patients compared to gestationally-matched normal placenta. CONCLUSION: These results show that endothelial sFlt-1 expression is regulated by VEGF and acts as an autocrine regulator of endothelial cell function.

Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells.

The proteinase-activated receptor 2 (PAR-2) expression is increased in endothelial cells derived from women with preeclampsia, characterized by widespread maternal endothelial damage, which occurs as a consequence of elevated soluble vascular endothelial growth factor receptor-1 (sVEGFR-1; commonly known as sFlt-1) in the maternal circulation. Because PAR-2 is upregulated by proinflammatory cytokines and activated by blood coagulation serine proteinases, we investigated whether activation of PAR-2 contributed to sVEGFR-1 release. PAR-2-activating peptides (SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2)) and factor Xa increased the expression and release of sVEGFR-1 from human umbilical vein endothelial cells. Enzyme-specific, dominant-negative mutants and small interfering RNA were used to demonstrate that PAR-2-mediated sVEGFR-1 release depended on protein kinase C-beta(1) and protein kinase C-epsilon, which required intracellular transactivation of epidermal growth factor receptor 1, leading to mitogen-activated protein kinase activation. Overexpression of heme oxygenase 1 and its gaseous product, carbon monoxide, decreased PAR-2-stimulated sVEGFR-1 release from human umbilical vein endothelial cells. Simvastatin, which upregulates heme oxygenase 1, also suppressed PAR-2-mediated sVEGFR-1 release. These results show that endothelial PAR-2 activation leading to increased sVEGFR-1 release may contribute to the maternal vascular dysfunction observed in preeclampsia and highlights the PAR-2 pathway as a potential therapeutic target for the treatment of preeclampsia.

Angiopoietin-2 confers Atheroprotection in apoE-/- mice by inhibiting LDL oxidation via nitric oxide.

Atherosclerosis is promoted by a combination of hypercholesterolemia and vascular inflammation. The function of Angiopoietin (Ang)-2, a key regulator of angiogenesis, in the maintenance of large vessels is unknown. A single systemic administration of Ang-2 adenovirus (AdAng-2) to apoE(-/-) mice fed a Western diet significantly reduced atherosclerotic lesion size ( approximately 40%) and oxidized LDL and macrophage content of the plaques. These beneficial effects were abolished by the inhibition of nitric oxide synthase (NOS). In endothelial cells, endothelial NOS activation per se inhibited LDL oxidation and Ang-2 stimulated NO release in a Tie2-dependent manner to decrease LDL oxidation. These findings demonstrate a novel atheroprotective role for Ang-2 when endothelial cell function is compromised and suggest that growth factors, which stimulate NO release without inducing inflammation, could offer atheroprotection.

Negative regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1.

BACKGROUND: Preeclampsia is characterized clinically by hypertension and proteinuria. Soluble Flt-1 (sFlt-1; also known as soluble vascular endothelial growth factor receptor-1 [VEGFR-1]) and soluble endoglin (sEng) are elevated in preeclampsia, and their administration to pregnant rats elicits preeclampsia-like symptoms. Heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO) exert protective effects against oxidative stimuli. Thus, we hypothesized that HO-1 upregulation may offer protection against preeclampsia by inhibiting sFlt-1 and sEng release. METHODS AND RESULTS: Preeclamptic villous explants secreted high levels of sFlt-1 and sEng. Adenoviral overexpression of HO-1 in endothelial cells inhibited VEGF-mediated sFlt-1 release and interferon-gamma- and tumor necrosis factor-alpha-induced sEng release, whereas HO-1 inhibition potentiated sFlt-1 and sEng production from endothelial cells and placental villous explants. Consistent with these findings, mice lacking HO-1 produced higher levels of sFlt-1 and sEng compared with wild-type mice. Using selective ligands (VEGF-E and placental growth factor) and a receptor-specific inhibitor (SU-1498), we demonstrated that VEGF-induced sFlt-1 release was VEGFR-2 dependent. Furthermore, CO-releasing molecule-2 (CORM-2) or CO decreased sFlt-1 release and inhibited VEGFR-2 phosphorylation. Treatment of endothelial cells with statins upregulated HO-1 and inhibited the release of sFlt-1, whereas vitamins C and E had no effect. CONCLUSIONS: The present study demonstrates that the HO-1/CO pathway inhibits sFlt-1 and sEng release, providing compelling evidence for a protective role of HO-1 in pregnancy, and identifies HO-1 as a novel target for the treatment of preeclampsia.

Angiotensin II induces soluble fms-Like tyrosine kinase-1 release via calcineurin signaling pathway in pregnancy.

Maternal endothelial dysfunction in preeclampsia is associated with increased soluble fms-like tyrosine kinase-1 (sFlt-1), a circulating antagonist of vascular endothelial growth factor and placental growth factor. Angiotensin II (Ang II) is a potent vasoconstrictor that increases concomitant with sFlt-1 during pregnancy. Therefore, we speculated that Ang II may promote the expression of sFlt-1 in pregnancy. Here we report that infusion of Ang II significantly increases circulating levels of sFlt-1 in pregnant mice, thereby demonstrating that Ang II is a regulator of sFlt-1 secretion in vivo. Furthermore, Ang II stimulated sFlt-1 production in a dose- and time-dependent manner from human villous explants and cultured trophoblasts but not from endothelial cells, suggesting that trophoblasts are the primary source of sFlt-1 during pregnancy. As expected, Ang II-induced sFlt-1 secretion resulted in the inhibition of endothelial cell migration and in vitro tube formation. In vitro and in vivo studies with losartan, small interfering RNA specific for calcineurin and FK506 demonstrated that Ang II-mediated sFlt-1 release was via Ang II type 1 receptor activation and calcineurin signaling, respectively. These findings reveal a previously unrecognized regulatory role for Ang II on sFlt-1 expression in murine and human pregnancy and suggest that elevated sFlt-1 levels in preeclampsia may be caused by a dysregulation of the local renin/angiotensin system.

Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis.

Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase Cgamma1. Consistent with these findings, the VEGFR-1-specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2-activated phospholipase Cgamma1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to Ets binding sites.

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma.

The angiogenic factor vascular endothelial growth factor-D (VEGF-D) isa ligand for VEGF receptor-3 (VEGFR-3/Flt-4) and receptor-2 (VEGFR-2/KDR)and is implicated in the development of lymphatic vessels and promotion of lymphatic metastases. We assessed the expression of VEGF-D and VEGFR-3 in relation to microvessel density (MVD) in colorectal carcinomas (CRC), adenomas, and adjacent normal tissue by immunohistochemistry on consecutive archival sections. VEGF-D was detected in malignant and benign epithelium and in some smooth muscle of the colorectum. High-grade VEGF-D expression was observed frequently (74%) in CRC compared with adenomas (0%) and adjacent normal mucosa (22%). High-grade VEGF-D expression was not correlated with MVD, Dukes' stage (A to C), or tumor differentiation, but was associated with lymphatic involvement and patient survival. By multivariate analysis, VEGF-D expression was found to be an independent prognostic factor for both disease-free and overall survival. VEGFR-3 expression was detected in a subset of vessels, typically thin-walled and devoid of RBCs, in 89% of CRC cases examined. VEGFR-3-positive vessel densities increased progressively from normal mucosa to adenomas and carcinomas and were correlated with MVD, but not with Dukes' stage (A to C), tumor differentiation, or VEGF-D expression. VEGFR-3 expression was spatially associated with macrophage-rich inflammatory infiltrates, which were significantly more frequent among VEGFR-3-positive cases. We conclude that VEGF-D expression, but not that of its receptor VEGFR-3, is an independent prognostic indicator in CRC. VEGF-D expression may be associated with disease outcome through the promotion of lymphatic involvement/metastases.