IL-1ß promotes transendothelial migration of PBMCs by upregulation of the FN/α5ß1 signalling pathway in immortalised human brain microvascular endothelial cells.

Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain. The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the ß1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether ß1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1ß (IL-1ß)-induced inflammatory in vitro BBB model. We demonstrate that IL-1ß increases cell-matrix adhesion and induces a redistribution of active ß1 integrins to the basal surface. In particular, binding of α5ß1 integrin to its ligand fibronectin is enhanced and α5ß1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5ß1 integrin reduces the IL-1ß-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs). These data imply that IL-1ß-induced inflammation not only destabilizes tight junctions but also increases α5ß1 integrin-dependent cell-matrix adhesion to fibronectin.

Inositol-C2-PAF acts as a biological response modifier and antagonizes cancer-relevant processes in mammary carcinoma cells.

PURPOSE: Previous studies have identified alkyl-phospholipids as promising compounds for cancer therapy by targeting constituents of the cell membrane and different signaling pathways. We previously showed that the alkylphospholipid Inositol-C2-PAF inhibits the proliferation and migration of immortalized keratinocytes and the squamous carcinoma-derived cell line SCC-25. Here, we investigated the effect of this compound on growth and motility as well as its mode of action in mammary carcinoma-derived cell lines. METHODS: Using BrdU incorporation and haptotactic cell migration assays, we assessed the effects of Inositol-C2-PAF on MCF-7 and MBA-MB-231 cell proliferation and migration. The phosphorylation status of signaling molecules was investigated by Western blotting as well as indirect immunofluorescence analysis and capillary isoelectric focusing. RESULTS: We found that Inositol-C2-PAF inhibited the growth as well as the migration in MCF-7 and MBA-MB-231 cells. Furthermore, we found that this compound inhibited phosphorylation of the protein kinase Akt at serine residue 473, but had no impact on phosphorylation at threonine 308. Phosphorylation of other kinases, such as Erk1/2, FAK and Src, which are targeted by Inositol-C2-PAF in other cells, remained unaffected by the compound in the mammary carcinoma-derived cell lines tested. In MCF-7 cells, we found that IGF-1-induced growth, as well as phosphorylation of AktS473, mTOR and the tumor suppressor pRB, was inhibited in the presence of Inositol-C2-PAF. Moreover, we found that in these cells IGF-1 had no impact on migration and did not seem to be linked to full Akt activity. Therefore, MCF-7 cell migration appears to be inhibited by Ino-C2-PAF in an Akt-independent manner. CONCLUSION: The antagonistic effects of Inositol-C2-PAF on cell migration and proliferation are indicative for its potential for breast cancer therapy, alone or in combination with other cytostatic drugs.

Inositoylated platelet-activating factor (Ino-C2-PAF) modulates dynamic lymphocyte-endothelial cell interactions and alleviates psoriasis-like skin inflammation in two complementary mouse models.

Psoriasis, a tumor necrosis factor alpha (TNFα)-governed inflammatory disorder with prominent dysregulation of cutaneous vascular functions, has evolved into a model disorder for studying anti-inflammatory therapies. We present experimental in vitro and in vivo data on 1-O-octadecyl-2-O-(2-(myo-inositolyl)-ethyl)-sn-glycero-3-(R/S)-phosphatidyl-choline (Ino-C2-PAF), the lead compound of a class of synthetic glycosylated phospholipids, in anti-inflammatory therapy. Ino-C2-PAF strongly induced apoptosis only in TNFα-stimulated, but not in untreated human vascular endothelial cells. Moreover, TNFα-induced endothelial adhesion molecules that mediated the rolling and firm adhesion of leukocytes (vascular cell adhesion protein-1 (VCAM-1), E-selectin, and ICAM-1) were selectively downregulated by Ino-C2-PAF. Similarly, expression of L-selectin, VCAM-1 receptor α4ß1 integrin , and lymphocyte function-associated antigen-1 on human peripheral blood mononuclear cells was reduced without induction of apoptosis. Functionally, these changes were accompanied by significant impairment of rolling and adhesion of human peripheral blood lymphocytes on TNFα-activated endothelial cells in a dynamic flow chamber system. When the therapeutic potential of Ino-C2-PAF was assessed in two complementary mouse models of psoriasis, K5.hTGFß1 transgenic and JunB/c-Jun-deficient mice, Ino-C2-PAF led to significant alleviation of the clinical symptoms and normalized the pathological cutaneous changes including vascularization. There were no overt adverse effects. These findings suggested that Ino-C2-PAF is a potential candidate in the therapy of inflammatory skin diseases that include abnormal vascular functions.

Glycosidated phospholipids - a promising group of anti-tumour lipids.

Synthetic alkylphospholipids (APLs), exhibit similarity to the platelet-activating factor (PAF). These compounds have antiproliferative effects on tumour cells and can therefore be regarded as a new class of drugs. Unlike classic cytostatic agents, synthetic alkylphospholipids do not interfere with the DNA or the mitotic spindle apparatus. Instead, due to their aliphatic character, alkylphospholipids accumulate in cell membranes, where they have an impact on lipid metabolism and lipid-dependent signalling pathways which leads to inhibition of proliferation and induction of apoptosis in malignant cells. Normal cells remain unaffected by these compounds. Glycosidated phospholipids, are a novel class of alkylphospholipids, in which carbohydrates or carbohydrate-related molecules are introduced in the chemical lead of PAF. These hybrid alkylphospholipids also exhibit anti-proliferative capacity. Furthermore, members of this subfamily also modulate cell adhesion, differentiation, apoptosis and migration of tumour cells. Among the members of this group, Inositol-C2-platelet-activating factor (Ino-C2-PAF) is the most effective compound developed so far. Recently, we also showed that Ino-C2-PAF exhibited the strongest impact on the gene expression levels of immortalised keratinocytes in comparison to edelfosine and another glycosidated alkylphospholipid, Glucose-platelet-activating factor (Glc-PAF). Furthermore, Ino-C2-PAF reduced the expression of genes encoding proteins associated with inflammation and the innate and acquired immune responses.

Inositol-C2-PAF down-regulates components of the antigen presentation machinery in a 2D-model of epidermal inflammation.

In cutaneous inflammatory diseases, such as psoriasis, atopic dermatitis and allergic contact dermatitis, skin-infiltrating T lymphocytes and dendritic cells modulate keratinocyte function via the secretion of pro-inflammatory cytokines. Keratinocytes then produce mediators that recruit and activate immune cells and amplify the inflammatory response. These pathophysiological tissue changes are caused by altered gene expression and the proliferation and maturation of dermal and epidermal cells. We recently demonstrated that the glycosidated phospholipid Ino-C2-PAF down-regulates a plethora of gene products associated with innate and acquired immune responses and inflammation in the HaCaT keratinocyte cell line. To further evaluate the influence of Ino-C2-PAF we established an in vitro 2D-model of epidermal inflammation. The induction of inflammation and the impact of Ino-C2-PAF were assessed in this system using a genome-wide microarray analysis. In addition, the expression of selected genes was validated using qRT-PCR and flow cytometry. Treatment of the keratinocytes with a mix of proinflammatory cytokines resulted in transcriptional effects on a variety of genes involved in cutaneous inflammation and immunity, while additional treatment with Ino-C2-PAF counteracted the induction of many of these genes. Remarkably, Ino-C2-PAF suppressed the expression of a group of targets that are implicated in antigen processing and presentation, including MHC molecules. Thus, it is conceivable that Ino-C2-PAF possess therapeutic potential for inflammatory skin disorders, such as psoriasis and allergic contact dermatitis.

The novel synthetic ether lipid inositol-C2-PAF inhibits phosphorylation of the tyrosine kinases Src and FAK independent of integrin activation in transformed skin cells.

New alkyl-phospholipids that are structurally derived from platelet-activating factor are promising candidates for anticancer treatment. The mechanism of action of derivatives of the platelet-activating factor is distinctly different from that of known DNA- or tubulin-targeting anticancer agents because they are incorporated into cell membranes, where they accumulate and interfere with a wide variety of key enzymes. We recently presented evidence of a novel group of alkyl-phospholipids, glycosidated phospholipids that efficiently inhibit cell proliferation. One member of this group, inositol-C2-PAF (Ino-C2-PAF), displays high efficacy and low cytotoxicity in HaCaT-cells, an immortalized non-tumorigenic skin keratinocyte cell line. Here, we show that Ino-C2-PAF also inhibits the motility of the skin-derived transformed cell lines HaCaT and squamous cell carcinoma (SCC)-25. This decrease in motility is accompanied by an altered F-actin cytoskeleton, increased clustering of integrins, and increased cell-matrix adhesion. Despite enhanced integrin clustering and matrix adhesion, we observed less phosphorylation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and Src, key regulators of cellular motility, at focal adhesion sites. Transient transfection of constitutively active variants of FAK and Src could at least in part bybass this inhibitory effect of Ino-C2-PAF. This fact indicates that Ino-C2-PAF interferes with the fine-tuned balance between adhesion and migration. Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines.