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

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. In HREC/HRPC cultures, TGF-ß in media increased, decreasing in triple cultures. High VEGF levels in triple cultures witnessed the establishment of a strongly in vitro angiogenic environment. Y-79 induced in HREC an increase in c- and iPLA2, phospho-cPLA2, inducible COX-2 protein expressions, PLA2 activities and prostaglandin E2 (PGE2) release. These effects were attenuated when HRPC were introduced in triple culture. Moreover, antibody silencing of TGF-ß demonstrated a strong correlation between the signalling pathway triggered by TGF-ß of pericytal origin and the phospholipase activation and the modulation of PGE2 release. Inhibiting VEGFA effect, the HRPC loss in triple culture decreased, showing its modulatory effect on their survival. Relying on the data here presented, sustaining the pericytal survival in a tumour retinal environment could ensure the integrity of microvessels and the TGF-ß supply, essential for controlling aberrant endothelial pruning and angiogenesis.

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). On the contrary, a significant increase in the frequency of the GG homozygous genotype of rs248793 (SRD5A1) was observed in the PAD patient group in comparison to controls (p=0.01). These data confirm that the GG genotype of rs248793 in the SRD5A1 gene is significantly associated with symptomatic PAD and show a trend towards a stronger association with the non-diabetic status.

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. In EC, C6 induced an increase in iPLA2, cPLA2 and COX-2 total protein expression. Moreover, the increase in endothelial cPLA2 phosphorylation was attenuated by kinase inhibitors. Both EC proliferation and signal protein phosphorylation were attenuated when PC were in triple culture. In EC/C6 supernatants, and, in a lesser extent, in EC/PC co-cultures, an enhancement in prostaglandins E2 (PGE2) was found. The presence of PC in triple-cultures caused a decrease in production of PGE2 respect to EC/C6 double-cultures. In all systems, AACOCF3 and BEL significantly reduced PGE2 secretion. In Matrigel-based assays, emerging branch points from EC cell bodies and tubule-like structures were observed. C6 conditioned EC/PC co-cultures in constituting poorly organized tubules. Transfection of EC with c- and iPLA2 siRNA strongly reduced in vitro tubulogenesis. Data here reported indicate that PKCα, ERK kinase phosphorylation, PLA2s and COX-2 activation, and PGE2 production in EC stimulated by tumor cells are coincident phenomena and could represent therapeutic targets in chemoprevention of glioma. Moreover, PC exhibited an important "modulating" role in the initial stages of angiogenesis driven by a brain tumor.

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. PLA2 activity, VEGF and PGE2 levels and cell proliferation were measured, with or without PLA2 inhibition. Diabetes was induced in rats by STZ injection and PLA2 activity along with VEGF, TNFα and ICAM-1 levels were measured in retina. High or fluctuating glucose induced BRB breakdown, and increased PLA2 activity, PGE2 and VEGF in EC/PC co-cultures; inhibition of PLA2 in mono- or co-cultures treated with high or fluctuating glucose dampened PGE2 and VEGF production down to the levels of controls. High or fluctuating glucose increased EC number and reduced PC number in co-cultures; these effects were reversed after transfecting EC with small interfering RNA targeted to PLA2. PLA2 and COX-2 protein expressions were significantly increased in microvessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNFα that were reduced by treatment with a cPLA2 inhibitor. In conclusion, the present findings indicate that PLA2 upregulation represents an early step in glucose-induced alteration of BRB, possibly upstream of VEGF; thus, PLA2 may be an interesting target in managing diabetic retinopathy.

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. In supernatants of E. coli-stimulated co-cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated co-cultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR-1 but not VEGFR-2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR-1, but not VEGFR-2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reduced permeability and BRPC loss were found. These data suggest that VEGFR-1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.

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. The physicochemical characterization of PHMS and control glass surface by time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy, pointed to the main role of the polymer hydrophobilicy to explain the observed cellular behavior. Moreover, enhanced intercellular cross-talk was evidenced by the up-regulation and activation of cytoplasmic and Ca(2+)-independent phospholipase A(2) (cPLA(2) and iPLA(2)) expression and cPLA(2) phosphorylation, leading to the cell proliferation and microcapillary formation on the PHMS surface, as evidenced by confocal microscopy analyses. Co-cultures, established with growth-arrested PCs by treatment with mitomycin C, showed an increase in EC proliferation on PHMS. AACOCF(3) or co-transfection with cPLA(2) and iPLA(2)siRNA reduced cell proliferation. The results highlight the major role played by EC/PC cross-talk as well as the hydrophobic character of the substrate surface, to promote microcapillary formation. Our findings suggest an attractive strategy for vascular tissue engineering and provide new details on the interplay of artificial substrates and capillary formation.

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. The traversal of bacteria did not determine trans-endothelial electrical resistance (TEER) and ZO-1 expression changes and was reduced by PLA(2)s siRNA. cPLA(2) and iPLA(2) enzyme activities and cPLA(2) phosphorylation were stimulated after E. coli incubation and were attenuated by PLA(2), PI3-K, ERK 1/2 inhibitors. Our results demonstrate the role of PKCα/ERK/MAPK signaling pathways in governing the E. coli penetration into the brain.

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. The primary sequences of such proteins are conserved from invertebrates, such as worms and flies that lack discernible vascular systems compared to mammals, but all these systems have sophisticated neuronal wiring involving such molecules. Ancestral VEGFs and receptors (VEGFRs) could have been used to develop and maintain the nervous system in primitive eukaryotes. During evolution, the demands of increased morphological complexity required systems for transporting molecules and cells, i.e., biological conductive tubes. We propose that the VEGF-VEGFR axis was subverted by evolution to mediate the formation of biological tubes necessary for transport of fluids, e.g., blood. Increasingly, there is evidence that aberrant VEGF-mediated responses are also linked to neuronal dysfunctions ranging from motor neuron disease, stroke, Parkinson's disease, Alzheimer's disease, ischemic brain disease, epilepsy, multiple sclerosis, and neuronal repair after injury, as well as common vascular diseases (e.g., retinal disease). Manipulation and correction of the VEGF response in different neural tissues could be an effective strategy to treat different neurological diseases.

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. The glioma CM effect was significantly reduced by cytosolic (cPLA(2)) and Ca(++)-independent (iPLA(2)) phospholipase A(2), cyclooxygenase-2, and protein kinase inhibitors. In GP8.3 ECs, cPLA(2) and iPLA(2) enzyme activities and phosphorylation of cPLA(2), significantly stimulated after 24h CM co-incubation, were attenuated by PLA(2), PI3-K, MEK-1, and ERK1/2 inhibitors. By confocal microscopy, in glioma CM-stimulated ECs, enhancement of fluorescence signals for phospho-cPLA(2), phospho-ERK1/2, phospho-PKCα, COX-2, and iPLA(2) was in parallel observed. Electroporation of anti-iPLA(2) and cPLA(2) antibodies and siRNAs directed against iPLA(2) and cPLA(2) significantly inhibited cell proliferation and migration. Incubation of CM- or VEGF peptide-stimulated ECs with antibodies against VEGF or VEGFR-1/-2 receptors strongly reduced mitotic rate, cell migration, and phospho-cPLA(2) and iPLA(2) protein levels. The findings suggest that PLA(2) activities are involved in stimulating EC migration and proliferation in the presence of glioma CM and that cPLA(2) is positively regulated upstream by PI3-K, PKCα, and ERK1/2 signal cascades. Our work provides new insights in understanding EC metabolism and signaling during tumor angiogenesis.

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. This review will summarize recent findings on the presence and possible role attributed to PLA(2)s in a variety of EC lines grown alone, as well as in isolated cancer cell lines, or in co-cultures in which signal transduction and cross-talk mechanisms between tumor cells and ECs, largely undefined, begin to be unravelled.

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. Inhibitors such as wortmannin and LY294002 (vs. PI3 kinase activity), AACOCF(3), (vs. cPLA(2) and iPLA(2)), PD98059 (vs. ERK1/2 activity) and NS-398 (vs. cyclooxygenase-2 activity, COX-2) were all able to block cell proliferation and motility determined using a scratch wound healing assay in melanoma CMs-stimulated EC monolayers. These media also support the enhanced cell proliferation of primary ECs derived from rat brain (BBEC). Electroporation of anti-cPLA(2) antibody into ECs markedly inhibited the EC proliferation in response to CMs. With both CMs, phosphorylation of cPLA(2), PKCalpha, ERK1/2, protein and mRNA expression of cPLA(2) and iPLA(2), and COX-2 protein expression were significantly stimulated after 24 h coincubation, and attenuated by specific inhibitors. By confocal microscopy, activation of cPLA(2), ERK1/2, PKCalpha and COX-2 in perinuclear and membrane regions of ECs grown in CM-stimulated cultures were clearly observed. Thus MEK-PKCalpha-ERK1/2 and PI3-K/Akt survival pathways are activated in EC cultures during the interaction with CM from both melanoma cell lines, providing new insight in understanding EC metabolism and signaling. These pathways represent potential therapeutic targets to inhibit or enhance tumor angiogenesis.

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. The most pronounced overexpression of iPLA(2) and COX-1 was found in two melanoma-derived cells, M14 and COLO38. Normal human melanocytes and the M10 melanoma cell line displayed no COX-2 expression. Using subcellular fractionation, Western blot and confocal microcopy analyses, in paradigmatic SK-MEL28 and SK-MEL93 cells we showed that iPLA(2), COX-1 and even cPLA(2) were equally located in the cytosol, membrane structures and perinuclear region while COX-2 was preferentially associated with the cytosol. Specific inhibitors of these three enzymes significantly reduced the basal proliferation rate either in melanocytes or in melanoma cell lines. These results, coupled with the inhibition of the cell proliferation by electroporation of melanoma cells with cPLA(2) or COX-2 antibodies, demonstrate that a possible correlation between PLA(2)-COX expression and tumor cell proliferation in the melanocytic system does exist. In addition, the high expression level of both PLA(2)s and COXs suggests that eicosanoids modulate cell proliferation and tumor invasiveness.

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. UV-O(3) treatment did not induce significant roughness changes (under 1 nm) as shown by atomic force spectroscopy measurements (AFM). The EC adhesion and spreading onto untreated and modified surfaces were investigated both before and after immobilization of collagen (CA) and fibronectin (FN) adlayers. AFM analyses showed an open-weave protein layer on both untreated polymers which became a tight-woven net after UV-O(3) irradiation of underlying films. On day 5 after seeding, cell count analyses on irradiated PET surfaces, CA/FN-coated or not, showed EC adhesion and proliferation significantly greater than those on untreated polymers, indicating that UV-O(3) irradiation promoted fast endothelialization. A less pronounced EC spreading behaviour on treated PHMS was observed. In ECs grown on irradiated and CA- or FN-coated PET, the levels of phospho-protein kinase Calpha (p-PKCalpha, phospho-ERK1/2, and phospho-cytosolic phospholipase A(2) (p-cPLA(2)), all enzymes taken as signaling markers of cell adhesion and proliferation, decreased in comparison to those in CA- or FN-coated untreated PET. In contrast, in ECs grown on UV-O(3)-treated PHMS, Western blot analyses showed increased levels of p-PKCalpha, p-ERK1/2 and p-cPLA(2) in comparison with cells grown onto untreated polymer. The growth response of ECs to the substrates was related to the changes of polarity properties of UV-O(3)-treated polymer films, from hydrophobic/neutral towards hydrophilic/charged layers, and the signaling pathway remodelling to the cell proliferation degree.

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. In ECs, the protein level of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 was also enhanced significantly, a finding not observed in cocultured pericytes. The expression of protein kinase C-alpha (PKCalpha) and its phosphorylated form was also enhanced in ECs. Wortmannin, LY294002, and PD98059, used as inhibitors of upstream kinases (the PI3-kinase/Akt/PDK1 or MEK-1 pathway) in cultures, markedly attenuated AA release and the expression of phosphorylated forms of endothelial cPLA(2), PKCalpha, and ERK1/2. By confocal microscopy, activation of PKCalpha in perinuclear regions of ECs grown in coculture as well as strong activation of cPLA(2) in ECs taken from a model of mixed culture were clearly observed. However, no increased expression of both enzymes was found in cocultured pericytes. Our findings indicate that a sequential activation of PKCalpha contributes to endothelial ERK1/2 and cPLA(2) phosphorylation induced by either soluble factors or direct cell-to-cell contact, and that the PKCalpha-cPLA(2) pathway appears to play a key role in the early phase of EC-pericyte interactions regulating blood retina or blood-brain barrier maturation.

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. Here, we studied the contribution of Ca(2+)-independent phospholipase A(2) (iPLA(2)), and the role of the MAP kinase family as well as both cPLA(2) and iPLA(2) mRNA expression by RT-PCR in oxLDL toxicity to GP8.39 cells in vitro. The activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH(2)-terminal kinase (JNK) was assessed with Western blotting and kinase activity assays. iPLA(2) activity, which was found as a membrane-associated enzyme, was more stimulated by oxLDL compared with native LDL. The phosphorylation of ERK1/2, p38 and JNKs was also significantly enhanced in a dose-dependent manner. PD98059, an ERK inhibitor, SB203580, a p38 inhibitor, and SP600125, an JNK inhibitor, abolished the stimulation of all three members of the MAPK family by oxLDL. Confocal microscopy analysis and subcellular fractionation confirmed either an increase in phosphorylated form of ERKs, p38 and JNKs, or their nuclear translocation upon activation. A strong inhibition of MAPK activation was also observed when endothelial cells were treated with GF109203X, a PKC inhibitor, indicating the important role of both PKC and all three MAPKs in mediating the maximal oxLDL response. Finally, compared with samples untreated or treated with native LDL, treatment with oxLDL (100 muM hydroperoxides) for 24 h significantly increased the levels of constitutively expressed iPLA(2) protein (by 5.1-fold) and mRNA (by 3.1-fold), as well as cPLA(2) protein (by 4.4-fold) and mRNA (by 1.5-fold). Together, these data link the stimulation of PKC-ERK-p38-JNK pathways and PLA(2) activity by oxLDL to the prooxidant mechanism of the lipoprotein complex, which may initially stimulate the endothelial cell reaction against noxious stimuli as well as metabolic repair, such as during inflammation and atherosclerosis.

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. Abeta(25-35) peptide evoked AA release as well as stimulated phosphorylation of ERK1/2, p38 MAPKs and cPLA(2), but not c-Jun N-terminal kinase (JNK/SAPK). PD98059, an inhibitor of ERK-activating kinase MEK-1, and SB203580, an inhibitor of p38 protein kinase, abolished the stimulation of AA release and MAPK activities. In cells stimulated by Abeta(25-35) peptide, Western blotting and confocal microscopy analyses confirmed either an increase in the phosphorylated form of ERKs and p38 or their nuclear translocation. A complete inhibition of MAPK activation and AA release was also observed when pericytes were treated with GF109203X, a general PKC inhibitor, indicating the important role of both PKC and the two MAPKs in mediating the Abeta peptide response. Compared with samples untreated or treated with reverse Abeta(35-25) peptide, pretreatment with 50 microM Abeta(25-35) for 24 h significantly increased the level of constitutively expressed iPLA(2) mRNA by 25%, which seems to depend on the activation of kinases. By contrast, the level of cPLA(2) mRNA remained unchanged. Together, these data link either the stimulation of PKC-ERK-p38 cascades or PLA(2) activity by Abeta peptide to prooxidant mechanism induced by amyloid, which may initially stimulate the cell reaction as well as metabolic repair, such as during inflammation.

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. Exposure to both Abeta peptides had no significant effect on cPLA(2) protein content as tested by Western immunoblot analysis. The addition of Abetas to quiescent pericytes was followed by phosphorylation of cPLA(2) and arachidonic acid release. Treatment with inhibitors (AACOCF(3), staurosporine and cycloheximide) resulted in a sharp decrease in basal and stimulated cPLA(2) activity. Inactivating effects of bromoenol lactone (BEL), inhibitor of iPLA(2), demonstrated that the stimulation of total PLA(2) activity by Abetas was mediated by both PLA(2) enzymes. Taken together with our previous observations that both Abeta peptides may induce hydrolysis of phosphatidylcholine, the present results provide evidence that this process is cooperatively mediated by cPLA(2) activation/translocation and iPLA(2) activation. The effect is very likely triggered by a mild prooxidant mechanism which was not able to divert the cell to degeneration. The data confirm the hypothesis that pericytes could be a target of potential vascular damage and reactivity during processes involving amyloid accumulation.

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. On the other hand, the adhesion and proliferation of GP8.39 endothelial cells (EC), which are strongly associated with pericytes in microvasculature, were very scarce onto both untreated and surface-modified PHMS. The surface-selective pericytal response was related to changes of physicochemical properties of PHMS film, from hydrophobic/neutral towards hydrophilic/negatively charged polymer layers, as well as to short- and long-time events of cell-surface interaction. We propose that surface properties can mediate and modulate cell-polymer matrix adhesion through the establishment of stereospecific chemical interactions and/or electrostatic repulsion, which can also explain the different behavior of pericytes compared to EC.

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 +/- 1.2 nmol/mg protein), and the EDTA-sensitive phosphodiesterase activity was also found to be markedly high (9.80 +/- 1.5 nmol/min/mg protein) compared to that estimated in liver and brain (1-3 nmol/min/mg protein) or in renal epithelial cell culture (0.27 nmol/min/mg protein). The reaction conditions were in general agreement with those found earlier in brain and other tissues. The majority of the enzyme specific activity was located in the plasma membrane, whereas a minor part was present in the microsomal fraction. The physiological significance of the high catabolic phosphodiesterase activity in these cells may be related to the transfer, followed by deacylation, of lysophosphatidylcholine from the bloodstream to nervous tissue. In addition, capillary pericytes in culture were able to incorporate 3H-choline rapidly into choline-containing soluble phosphorylated intermediates and into phosphatidylcholine. To find a positive and negative effector on phosphatidylcholine formation, adenosine, an important intercellular mediator in the retina in response to alterations in oxygen delivery, and endothelin-1, a potent paracrine mediator present at the blood-brain and blood-retina barrier, were tested. The cells cultured for 1 or 24 h in a medium containing adenosine at concentrations of 10(-6) and 10(-4) M showed significant reduction in 3H-choline incorporation compared to control cultures, whereas endothelin-1, at a concentration of 10 and 100 nM, caused stimulation of phosphatidylcholine biosynthesis. These findings provide evidence that both agonists may modulate phosphatidylcholine metabolism in pericytes.

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 (1.5 mg/dish; up to 350-450 nmol hydroperoxides/mg protein) for two temporally distinct phases (short incubation period up to 1 h, or long incubation period spanning 24 h). Peroxidation parameters (conjugated dienes, MDA, hydroperoxides and LDH release) and arachidonic acid (AA) release were determined. Cell lysates and subcellular fractions were assayed for cPLA(2) while the cytotoxic effect and apoptosis were monitored by morphological changes, trypan blue dye exclusion, MTT reduction test, caspase-3 activity, COMET and laser confocal fluorescence microscopy (LCFM) analyses. Effects of alpha-tocopherol and 85-kDa PLA(2) inhibitor (AACOCF(3)), alone or in combination, were also tested. Immunoblot analysis of cPLA(2) was carried out on cell fraction proteins. After incubation for 1 or 24 h, oxLDL (100-200 microM hydroperoxides), but not nLDL, markedly increased lipid peroxidation, cPLA(2) activity and AA release in a dose-dependent manner. AACOCF(3) and antioxidant alpha-tocopherol (1 mM) strongly inhibited the prooxidant-stimulated AA release. Long-term exposure (24 h) to oxLDL (100 microM) had no effect on the cPLA(2) protein content as tested by Western immunoblot analysis, while showing a sharp cytotoxic effect on the cells. Caspase-3 activity and LCFM analysis indicated that oxLDL (100/200 microM) were able to trigger an apoptotic process. The results suggest that (i) ECs may be the target of extensive oxidative damage caused by oxLDL; (ii) activation of cPLA(2) mediates liberation of AA; (iii) cPLA(2) expression was not stimulated by long-term exposure to oxLDL; (iv) oxidized specific constituents of oxLDL, acting as regulatory signals, increase the ability of ECs to degrade membrane phospholipids, end products of which are linked to the development of atherosclerotic lesions.