An in vitro retinoblastoma human triple culture model of angiogenesis: a modulatory effect of TGF-β.

Retinoblastoma is the most common intraocular tumour in children. In view of understanding the molecular mechanisms through which angiogenic switch on happens in the early phases of reciprocal interaction between tumour and cells constituting retinal microvessel, Transwell co-cultures constituted by human retinal endothelial cells (HREC), pericytes (HRPC), and human retinoblastoma cell line Y-79 were performed. Y-79 enhanced HREC proliferation, reduced by the introduction of HRPC in triple culture.

ICAM-1 and SRD5A1 gene polymorphisms in symptomatic peripheral artery disease.

The genotype distribution of two gene polymorphisms, previously associated with peripheral artery disease (PAD), has been evaluated in a population of diabetic (DPAD) and non-diabetic (NDPAD) patients affected by symptomatic PAD (stages II-IV). A decreased frequency of the AA genotype of rs5498 (ICAM-1) was observed in the PAD subjects compared to controls but this result did not reach statistical significance (p=0.06 by chi-squared test).

Endothelial PKCα-MAPK/ERK-phospholipase A2 pathway activation as a response of glioma in a triple culture model. A new role for pericytes?

In view of understanding the molecular mechanisms through which angiogenic switch on happens in the early phases of reciprocal interaction between tumor and cells constituting microvessel, a triple culture model in which endothelial cells (EC), pericytes (PC) and glioma C6 cells were cultured together. In the present work, we observed that C6 enhanced EC proliferation. This effect was reduced by cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2), cyclooxygenase-2 (COX-2), PI3-K, MEK-1, and ERK1/2 inhibitors and by siRNAs against both PLA2s.

Role of phospholipases A2 in diabetic retinopathy: in vitro and in vivo studies.

Diabetic retinopathy is one of the leading causes of blindness and the most common complication of diabetes with no cure available. We investigated the role of phospholipases A2 (PLA2) in diabetic retinopathy using an in vitro blood-retinal barrier model (BRB) and an in vivo streptozotocin (STZ)-induced diabetic model. Mono- and co-cultures of endothelial cells (EC) and pericytes (PC), treated with high or fluctuating concentrations of glucose, to mimic the diabetic condition, were used.

VEGF receptor-1 involvement in pericyte loss induced by Escherichia coli in an in vitro model of blood brain barrier.

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood-brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co-culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co-culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2 ) enzyme activities.

Microcapillary-like structures prompted by phospholipase A2 activation in endothelial cells and pericytes co-cultures on a polyhydroxymethylsiloxane thin film.

A thin film of poly(hydroxymethylsiloxane) (PHMS) has been deposited on glass dishes and tested as artificial support material for vascularization from mixed cultures of endothelial cells (EC) and pericytes (PC). The EC/PC co-cultures adhered massively on PHMS, with the formation of net-like microcapillary structures. Such evidence was not found on control glass substrates in the same co-culture conditions neither on PHMS for EC and PC in monocultures.

Involvement of PKCα-MAPK/ERK-phospholipase A(2) pathway in the Escherichia coli invasion of brain microvascular endothelial cells.

Escherichia coli K1 is the most common Gram-negative organism that causes neonatal meningitis following penetration of the blood-brain barrier. In the present study we demonstrated the involvement of cytosolic (cPLA(2)) and calcium-independent phospholipase A(2) (iPLA(2)) and the contribution of cyclooxygenase-2 products in E. coli invasion of microvascular endothelial cells.

Evolution of the VEGF-regulated vascular network from a neural guidance system.

The vascular network is closely linked to the neural system, and an interdependence is displayed in healthy and in pathophysiological responses. How has close apposition of two such functionally different systems occurred? Here, we present a hypothesis for the evolution of the vascular network from an ancestral neural guidance system. Biological cornerstones of this hypothesis are the vascular endothelial growth factor (VEGF) protein family and cognate receptors.

Cytosolic and calcium-independent phospholipase A(2) mediate glioma-enhanced proangiogenic activity of brain endothelial cells.

Glioma is characterized by an active production of proangiogenic molecules. We observed that conditioned medium (CM) from C6 glioma significantly enhanced proliferation and migration of immortalized rat brain GP8.3 endothelial cells (ECs) and primary bovine brain microvascular ECs.

Phospholipase A(2): new lessons from endothelial cells.

The investigation of intracellular phospholipase A(2) (PLA(2)) enzymes in endothelial cells (ECs) seems interesting because it may contribute to unveil the mechanisms of biological processes such as angiogenesis, adhesion and transmigration of inflammatory cells, atherogenesis, blood brain barrier and tumor progression. To date, limited information is available regarding the function and regulation of three well characterized phospholipases, Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)) and secretory PLA(2) (sPLA(2)) along the intracellular signaling pathways in quiescent and proliferating ECs. PLA(2)s could be potentially involved in signaling cascades by which ECs promote the highly organized multicellular complexes consisting of either an endothelium, brain pericytes and astrocytes, or cellular constituents of the tumor microvasculature.

PKCalpha-MAPK/ERK-phospholipase A2 signaling is required for human melanoma-enhanced brain endothelial cell proliferation and motility.

The largely undefined signal transduction mechanisms and cross-talk between human melanoma cell (HMC) lines and brain endothelial cells (ECs) involved in tumor cell interaction and adhesion were investigated. In immortalized rat brain GP8.3 EC cultures, conditioned media (CM) prepared from SK-MEL28 and OCM-1 melanoma cells significantly enhanced arachidonic acid release, cytosolic phospholipase A(2) (cPLA(2)) and Ca(+)-independent phospholipase A(2) (iPLA(2)) specific activities, and cell growth by 24 h.

Expression of Ca(2+)-independent and Ca(2+)-dependent phospholipases A(2) and cyclooxygenases in human melanocytes and malignant melanoma cell lines.

We provide novel evidence that human melanoma cell lines (M10, M14, SK-MEL28, SK-MEL93, 243MEL, 1074MEL, OCM-1, and COLO38) expressed, at mRNA and protein levels, either Ca(2+)-independent phospholipase A(2) (iPLA(2)) or cytosolic phospholipase A(2) (cPLA(2)) and its phosphorylated form. Normal human melanocytes contained the lowest levels of both PLA(2)s. Cyclooxygenase-1 and -2 (COX-1 and COX-2) were also expressed in cultured tumor cells as measured by Western blots.

UV-O3-treated and protein-coated polymer surfaces facilitate endothelial cell adhesion and proliferation mediated by the PKCalpha/ERK/cPLA2 pathway.

We examined the adhesion and proliferation of immortalized endothelial cells GP8.39 (ECs) onto polyethyleneterephtalate (PET) and polyhydroxymethylsiloxane (PHMS) thin films, functionalized by UV-O(3) treatment and/or protein immobilization. The modified surface topography showed partial oxidation for both polymers, a slight increase in wettability and monopolar basic character for PET, and a hydrophilic bipolar acid-base behaviour for PHMS.

Endothelial cell-pericyte cocultures induce PLA2 protein expression through activation of PKCalpha and the MAPK/ERK cascade.

Little is known about the regulatory mechanisms of endothelial cell (EC) proliferation by retinal pericytes and vice versa. In a model of coculture with bovine retinal pericytes lasting for 24 h, rat brain ECs showed an increase in arachidonic acid (AA) release, whereas Western blot and RT-PCR analyses revealed that ECs activated the protein expression of cytosolic phospholipase A(2) (cPLA(2)) and its phosphorylated form and calcium-independent intracellular phospholipase A(2) (iPLA(2)). No activation of the same enzymes was seen in companion pericytes.

Activation of phospholipase A(2) and MAP kinases by oxidized low-density lipoproteins in immortalized GP8.39 endothelial cells.

In immortalized rat brain endothelial cells (GP8.39), we have previously shown that oxidized LDL (oxLDL), after 24-h treatment, stimulates arachidonic acid release and phosphatidylcholine hydrolysis by activation of cytosolic phospholipase A(2) (cPLA(2)). A putative role for MAPKs in this process has emerged.

MAPKs mediate the activation of cytosolic phospholipase A2 by amyloid beta(25-35) peptide in bovine retina pericytes.

We have previously shown that, in bovine retina pericytes, amyloid beta(1-42) and its truncated form containing amino acids 25-35, after 24 h treatment, stimulate arachidonic acid (AA) release and phosphatidylcholine hydrolysis, by activation of both cytosolic (cPLA(2)) and Ca(2+)-independent (iPLA(2)) phospholipase A(2). A putative role for MAP kinases in this process emerged. Here we studied the role of the MAP-kinase family as well as both cPLA(2) and iPLA(2) mRNA expression by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the same sublethal model of amyloid-beta (Abeta) damage to pericytes in vitro.

Amyloid beta(1-42) and its beta(25-35) fragment induce activation and membrane translocation of cytosolic phospholipase A2 in bovine retina capillary pericytes.

We investigated changes in cytosolic phospholipase A(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) activities in bovine retina capillary pericytes after stimulation with 50 microM amyloid-beta (Abeta) (1-42) and its (25-35) fragment, over 24 h (mild, sublethal model of cell damage). In the presence of Abeta peptides, we found that cPLA(2) activity was increased and translocated from the cytosolic fraction to the membrane system, particularly in the nuclear region. Reversed-sequence Abeta(35-25) peptide did not stimulate or induce cPLA(2) translocation.

Pericyte adhesion and growth onto polyhydroxymethylsiloxane surfaces nanostructured by plasma treatment and ion irradiation.

The study deals with the adhesion and proliferation of bovine retina pericytes onto surfaces of poly(hydroxymethylsiloxane) (PHMS) modified either by cold plasma or by low-energy ion beams. The surface treatment was able to convert the original polymer matrix into SiO2-like phases for O2-plasma or ion-mixed SiCxOy(Hz) phases for ion irradiation, respectively, with different modification levels of the surface free energy (SFE) and related surface wettability. Pericytes exhibited a negligible adhesion and proliferation onto untreated PHMS, an enhanced adhesion but not proliferation on plasma-treated PHMS, and great adhesion and proliferation to full confluence on ion-irradiated PHMS, as measured by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), quartz crystal microbalance, and optical microscopy.

Characterization of glycerophosphocholine phosphodiesterase activity and phosphatidylcholine biosynthesis in cultured retinal microcapillary pericytes. Effect of adenosine and endothelin-1.

In pericytes from bovine retina, the enzyme glycerophosphocholine phosphodiesterase, catalyzing the hydrolysis of sn-glycero-3-phosphocholine to glycero-3-phosphate and choline, has been characterized with respect to pH optimum, metal ion dependence, Km, inhibitors, and subcellular localization. In these cells, the natural substrate sn-glycero-3-phosphocholine was present at relatively high concentration (6.4 +/- 1.

Cytosolic phospholipase A2 mediates arachidonoyl phospholipid hydrolysis in immortalized rat brain endothelial cells stimulated by oxidized LDL.

We tested the hypothesis that oxidized low-density lipoprotein (oxLDL), administered in sublethal doses to the culture medium of immortalized rat brain endothelial cells (ECs, GP8.39), acts as a prooxidant signal to stimulate peroxidation processes and membrane phospholipid hydrolysis. ECs were grown at confluence in a medium with or without native LDL (nLDL) or oxLDL (1.

Effects of aging and amyloid-beta peptides on choline acetyltransferase activity in rat brain.

Choline acetyltransferase (ChAT, acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.