Human Omental Microvascular Endothelial Cells Research Articles

Cathepsin L-induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma

BackgroundNew treatment options for metastasised high-grade serous carcinoma (HGSC) are urgently needed. HGSC frequently metastasises to the omentum, inducing angiogenesis in the local omental microvasculature to facilitate tumour growth. We previously showed that HGSC-secreted cathepsin L (CathL) induces pro-angiogenic changes in disease relevant human omental microvascular endothelial cells (HOMECs), suggesting a role in tumour angiogenesis. Here we investigate whether CathL acts by inducing local production of the carbohydrate-binding protein galectin-1 (Gal1), which has been reported to be involved in tumourigenesis in other tumours.MethodsHOMECs were used for all experiments. Gal1 mRNA and protein levels were measured by RT-PCR and ELISA respectively. Gal1-induced cell proliferation was assessed using WST-1 assay, migration using a transwell assay and in vivo Gal1 expression by immunohistochemistry.ResultsCathL transcriptionally regulated HOMEC production and secretion of Gal1 via activation of NFκB (significantly inhibited by sulfasalazine). Gal1 significantly enhanced HOMEC migration (p < 0.001) and proliferation (p < 0.001), suggesting an autocrine action. The latter was significantly reduced by the MEK/ERK1/2 inhibitors U0126 and PD98059 suggesting downstream activation of this pathway. Immunohistochemical analysis of omenta from HGSC patients with or without metastatic disease demonstrated a positive correlation between Gal1 expression and number of microvessels (r = 0.8702, p < 0.001), and area of vessels (r = 0.7283, p < 0.001), supporting a proangiogenic role for Gal1 in omental metastases.ConclusionHOMEC Gal1 transcription and release in response to CathL secreted from metastasising HGSC acts in an autocrine manner on the local microvasculature to induce pro-angiogenic changes, highlighting a potential new therapeutic target.

Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways

Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum.

T26: Tumour secreted factors cathepsins D and L induce pro-angiogenic changes in human omental microvascular endothelial cells (HOMECs) in ovarian cancer metastasis

Background Epithelial ovarian cancer frequently metastasizes to the omentum, a process that requires pro-angiogenic activation of HOMECs by tumour -secreted factors in their microenvironment. We have previously shown that ovarian cancer cells secrete a range of factors with possible roles in metastatic angiogenesis including the lysosomal proteases cathepsin D (CD) and cathepsin L (CL). However, the role of these proteases in ovarian cancer metastasis to the omentum is not fully understood. Aim To investigate whether proliferative effects of CD and CL are dependent on their catalytic activity in HOMECs. To investigate the intracellular signalling kinases activated by CD and CL. Method HOMEC proliferation was assessed by using a colorimetric (WST1) assay. Potential signalling pathways were examined by phosphokinase array and ELISAs. pH experiments were carried out examine whether the observed effects were due to the catalytic activity of CD and CL. Result CD and CL (50 ng/ml) significantly increased HOMEC proliferation to 141 ± 27% ( p = 0.001, n = 50) and 151% ± 34% ( p = 0.001, n = 45) respectively vs. control (100%) 72 h post-treatment. Inhibitors of CD and CL enzyme activity had no effect on HOMEC proliferation and subsequent pH data suggest a nonproteolytic mitogenic activity of these cathepsins. Both proteins induced phosphorylation of ERK1/2, AKT and p38 α to ∼2, ∼1.5 and ∼1.5 folds respectively relative to total levels (compared to control). Conclusion CD and CL induced proliferation in HOMECs. CD and CL may non- proteolytically contribute to pro-angiogenic responses of the omental microvasculature. Induction of phosphorylation of proliferative kinases ERK1/2, AKT and p38 α suggest possible downstream signalling cascades of these proteins.

Epithelial Ovarian Cancer-Induced Angiogenic Phenotype of Human Omental Microvascular Endothelial Cells May Occur Independently of VEGF Signaling

Epithelial ovarian cancer (EOC) metastasizes transcoelomically to the peritoneum and omentum, and despite surgery and chemotherapy, recurrent disease is likely. Metastasis requires the induction of proangiogenic changes in the omental microenvironment and EOC-induced omental angiogenesis is currently a key therapeutic target. In particular, antiangiogenic therapies targeting the vascular endothelial growth factor A (VEGFA) pathway are commonly used, although, with limited effects. Here, using human omental microvascular endothelial cells (HOMECs) and ovarian cancer cell lines as an in vitro model, we show that factors secreted from EOC cells increased proliferation, migration, and tube-like structure formation in HOMECs. However, EOC-induced angiogenic tube-like formation and migration were unaffected by inhibition of tyrosine kinase activity of VEGF receptors 1 and 2 (Semaxanib; SU5416) or neutralization of VEGFA (neutralizing anti-VEGFA antibody), although VEGFA165-induced HOMEC migration and tube-like structure formation were abolished. Proteomic investigation of the EOC secretome identified several alternative angiogenesis-related proteins. We screened these for their ability to induce an angiogenic phenotype in HOMECs, i.e., proliferation, migration, and tube-like structure formation. Hepatocyte growth factor (HGF) and insulin-like growth factor binding protein 7 (IGFBP-7) increased all three parameters, and cathepsin L (CL) increased migration and tubule formation. Further investigation confirmed expression of the HGF receptor c-Met in HOMECs. HGF- and EOC-induced proliferation and angiogenic tube structure formation were blocked by the c-Met inhibitor PF04217903. Our results highlight key alternative angiogenic mediators for metastatic EOC, namely, HGF, CL, and IGFBP-7, suggesting that effective antiangiogenic therapeutic strategies for this disease require inhibition of multiple angiogenic pathways.

Therapeutic Angiogenesis by Implantation of a Capillary Structure Constituted of Human Adipose Tissue Microvascular Endothelial Cells

We previously reported a novel technology for the engineering of a capillary network using an optical lithographic technique. To apply this technology to the therapy of ischemic diseases, we tested human omental microvascular endothelial cells (HOMECs) as an autologous cell source and decellularized human amniotic membranes (DC-AMs) as a pathogen-free and low immunogenic transplantation scaffold. Human umbilical vein endothelial cells were aligned on a patterned glass substrate and formed a capillary structure when transferred onto an amniotic membrane (AM). In contrast, HOMECs were scattered and did not form a capillary structure on AMs. Treatment of HOMECs with sphingosine 1-phosphate (S1P) inhibited HOMEC migration and enabled HOMEC formation of a capillary structure on AMs. Using quantitative RT-PCR and Western blot analyses, we demonstrated that the main S1P receptor in HOMECs is S1P(2), which is lacking in human umbilical vein endothelial cells, and that inhibition of cell migration by S1P is mediated through an S1P(2)-Rho-Rho-associated kinase signaling pathway. Implantation of capillaries engineered on DC-AMs into a hindlimb ischemic nude mouse model significantly increased blood perfusion compared with controls. A capillary network consisting of HOMECs on DC-AMs can be engineered ex vivo using printing technology and S1P treatment. This method for regeneration of a capillary network may have therapeutic potential for ischemic diseases.

Ets-1 regulates angiogenesis by inducing the expression of urokinase-type plasminogen activator and matrix metalloproteinase-1 and the migration of vascular endothelial cells.

The coordinate induction of protease activities and cell migration is a principal feature of endothelial cells (ECs) invading the interstitial space in the initial step of angiogenesis. However, the molecular mechanisms of these events are not fully characterized. Ets-1 is a member of the ets gene family of transcription factors, which binds to the Ets binding motif in the cis-acting elements and regulates the expression of certain genes. Four typical angiogenic growth factors, aFGF, bFGF, VEGF, and EGF, induced the expression of ets-1 mRNA in either human umbilical vein endothelial cells (HUVECs), ECV-304 cells (immortalized HUVECs), or human omental microvascular endothelial cells (HOMECs). The expression of ets-1 reached its maximum at 2 hr after factor addition and then decreased to the basal level by 12 hr. For characterization of the role of Ets-1 in angiogenesis, ets-1 antisense and sense oligodeoxynucleotides (ODNs) were constructed. The ets-1 antisense ODN but not sense ODN efficiently blocked the synthesis of Ets-1 protein by human ECs in response to angiogenic growth factors. Moreover, the ets-1 antisense ODN but not sense ODN almost completely abolished the binding of endothelial cell extract to DNA containing the Ets binding motif. The expression of urokinase-type plasminogen activator and matrix metalloproteinase-1 and the migration of ECs in response to growth factors were significantly inhibited by ets-1 antisense ODN but not by sense ODN. Tube formation by HOMECs in type 1 collagen gel stimulated with EGF was abrogated by ets-1 antisense ODN. Finally, the expression of Ets-1 protein in ECs during angiogenesis in vivo was confirmed by an immunohistochemical analysis using a murine angiogenesis model. These results indicate that the induction of ets-1 mRNA is a mutual phenomenon in ECs stimulated with angiogenic growth factors. Ets-1 appears to play an important role in angiogenesis, regulating the expression of proteases and the migration of ECs.

Comparison of prostanoid synthesis in cultured human vascular endothelial cells derived from omentum and umbilical vein

Endothelial seeding of vascular grafts may reduce thrombogenicity and significantly improve graft patency. For clinical studies endothelial cells derived from human omentum may be the ideal source of cells as they are obtainable in large numbers. This study was designed to assess their ability to produce the antithrombogenic substance prostacyclin (PGI2). Human omental microvascular endothelial cells (HOTMECs) were grown and their ability to produce prostacyclin (PGI2), PGE2, PGF2 alpha and thromboxane A2 (TXA2) in response to a range of agonists was measured by radioimmunoassay. Control experiments were performed using human umbilical vein endothelial cells (HUVECs). Both HUVECs and HOTMECs synthesised similar quantities of PGI2 basally and both increased production after adding A23187 (calcium ionophore), arachidonic acid, sodium fluoride and phorbol ester. In contrast, both thrombin and histamine were potent stimulators of PGI2 release in HUVECs but were without effect on HOTMECs. In both cell types serotonin, carbachol and noradrenaline were without effect. The pattern of release of PGE2, PGF2 alpha and TXA2 were identical to those of PGI2 in both cell types but the quantities released were lower. These results show that HOTMECs can produce the antithrombogenic agent PGI2 in significant quantities and that they do so by mechanisms similar to those of large vessel endothelium. This supports the proposal that they would be a suitable cell source for vascular graft seeding.

Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.

Evidence of endoplasmic reticulum-related Ca2+ ATPase in human microvascular endothelial cells

We have demonstrated by immunological and molecular methods the presence of a reticulum endoplasmic-related Ca2+-ATPase in human omental microvascular endothelial cells (HOME cells). HOME cells reacted positively with a previously characterized sarcoplasmic reticulum Ca2+-ATPase antibody as demonstrated by indirect immunofluorescence. Western blotting revealed that the antibody recognized a 95-100 kDa protein. 35S-Metabolic labeling led to the detection of a similar protein with which the purified sarcoplasmic reticulum Ca2+-ATPase competed. Dot-blotting experiments indicated that a substantial amount of Ca2+-ATPase was present in HOME cell membranes. In addition, Northern blot analysis using a cDNA probe from cardiac sarcoplasmic reticulum showed the presence of mRNA species of 4-kb. As these experiments were conducted in comparison with cell types with well-defined Ca2+-ATPase in HOME cells.

Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells

The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells ( K D of 42.0 ± 3.8 p M and 70,526 ± 6121 binding sites/cell for the high-affinity sites, K D of 0.933 ± 0.27 n M and 630,252 ± 172,459 sites/cell for low-affinity binding sites). The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely 125I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound 125I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At 37 °C, 30% of the cell-associated 125I-bFGF became resistant to the acidic wash after 90 min, suggesting that this fraction of bound 125I-bFGF was internalized. At this temperature, degradation of the internalized ligand was followed after 1 h by the appearance of three major bands of 15,000, 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.

Vascular origin determines plasminogen activator expression in human endothelial cells: Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney.Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/105 cells/24 h and 50.42 ng/105 cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/105 cells/24 h and 2.17 ng/105 cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.

Binding of heparin to human microvascular endothelial cells and the effect on proliferation

We have studied the binding of 125I-heparin to human omental microvascular endothelial cells (HOME cells) and investigated its effect on cell proliferation. At 20 °C, the binding reached a steady state from 4 hours onwards. Saturation of 125I-heparin binding occured at 200 nM. Scatchard analysis indicated one class of binding sites (KD = 0.023 μM, 2 × 10 −6 sites/cell). Using fractionated heparin and other sulfated polysaccharides it was demonstrated that the binding was dependent on the charge and the molecular weight of the compounds. The binding was followed by the partial internalization of the bound ligand (23.8 %). Heparin and Stipocus Japonicus mucopolysaccharide (SJAMP) inhibited the proliferation of exponentially growing HOME cells and human umbilical vein endothelial cells (HUVE cells). However, serum-deprived HOME cells were not inhibited by heparin.

STIMULATION OF ENDOTHELIAL CELL GROWTH BY SERA FROM DIABETIC PATIENTS WITH RETINOPATHY

An in-vitro proliferation assay has shown that sera from patients with diabetic retinopathy, particularly those with the proliferative form, are two to four times more effective than sera from non-diabetics at stimulating 3H-thymidine incorporation into both human umbilical vein and human omental microvascular endothelial cells, but not at stimulating incorporation into human dermal fibroblasts or 3T3 cells. The factor(s) is heat stable and of molecular weight < 15 000, and its presence is unrelated to metabolic control. It is not present in patients with other forms of diabetic vascular disease, which suggests that it is not related to carbohydrate or lipid metabolism. These results provide evidence against the hypothesis that metabolic disturbances are central to the development of diabetic microvascular disease, and raise possibilities of novel forms of therapeutic intervention.