Phosphorylation of pericyte FAK-Y861 affects tumour cell apoptosis and tumour blood vessel regression.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in many cancer types and in vivo studies have shown that vascular endothelial cell FAK expression and FAK-phosphorylation at tyrosine (Y) 397, and subsequently FAK-Y861, are important in tumour angiogenesis. Pericytes also play a vital role in regulating tumour blood vessel stabilisation, but the specific involvement of pericyte FAK-Y397 and FAK-Y861 phosphorylation in tumour blood vessels is unknown. Using PdgfrßCre + ;FAKWT/WT, PdgfrßCre + ;FAKY397F/Y397F and PdgfrßCre + ;FAKY861F/Y861F mice, our data demonstrate that Lewis lung carcinoma tumour growth, tumour blood vessel density, blood vessel perfusion and pericyte coverage were affected only in late stage tumours in PdgfrßCre + ;FAKY861F/Y861F but not PdgfrßCre + ;FAKY397F/Y397F mice. Further examination indicates a dual role for pericyte FAK-Y861 phosphorylation in the regulation of tumour vessel regression and also in the control of pericyte derived signals that influence apoptosis in cancer cells. Overall this study identifies the role of pericyte FAK-Y861 in the regulation of tumour vessel regression and tumour growth control and that non-phosphorylatable FAK-Y861F in pericytes reduces tumour growth and blood vessel density.

Tumor Angiogenesis Is Differentially Regulated by Phosphorylation of Endothelial Cell Focal Adhesion Kinase Tyrosines-397 and -861.

Expression of focal adhesion kinase (FAK) in endothelial cells (EC) is essential for angiogenesis, but how FAK phosphorylation at tyrosine-(Y)397 and Y861 regulate tumor angiogenesis in vivo is unknown. Here, we show that tumor growth and angiogenesis are constitutively reduced in inducible, ECCre+;FAKY397F/Y397F -mutant mice. Conversely, ECCre+;FAKY861F/Y861F mice exhibit normal tumor growth with an initial reduction in angiogenesis that recovered in end-stage tumors. Mechanistically, FAK-Y397F ECs exhibit increased Tie2 expression, reduced Vegfr2 expression, decreased ß1 integrin activation, and disrupted downstream FAK/Src/PI3K(p55)/Akt signaling. In contrast, FAK-Y861F ECs showed decreased Vegfr2 and Tie2 expression with an enhancement in ß1 integrin activation. This corresponds with a decrease in Vegfa-stimulated response, but an increase in Vegfa+Ang2- or conditioned medium from tumor cell-stimulated cellular/angiogenic responses, mimicking responses in end-stage tumors with elevated Ang2 levels. Mechanistically, FAK-Y861F, but not FAK-Y397F ECs showed enhanced p190RhoGEF/P130Cas-dependent signaling that is required for the elevated responses to Vegfa+Ang2. This study establishes the differential requirements of EC-FAK-Y397 and EC-FAK-Y861 phosphorylation in the regulation of EC signaling and tumor angiogenesis in vivo. SIGNIFICANCE: Distinct motifs of the focal adhesion kinase differentially regulate tumor blood vessel formation and remodeling.

Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium-specific FAK-kinase dead mice.

Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase-dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb-iCreert -driven endothelial cell (EC)-specific, tamoxifen-inducible homozygous mutant mouse lines: FAK wild-type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAKWT/WT , ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAKWT/WT and ECCre-;FAKWT/WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice, as compared with Cre - littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice. Furthermore, although ECCre+;FAKKD/KD mice showed reduced blood vessel leakage, ECCre+;FAKDM/DM and ECCre-;FAKDM/DM mice showed no difference in leakage. Mechanistically, fibronectin-stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD/KD ECs as compared with Cre+;FAKWT/WT cells, with no change in phosphorylation of the known Src targets FAK-Y577, FAK-Y861, FAK-Y925, paxillin-Y118, p130Cas-Y410. Cre+;FAKDM/DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE-cadherin-pY658 levels in Cre+;FAKKD/KD ECs were rescued in Cre+FAKDM/DM ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAKDM/DM mice. We show that EC-specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAKDM/DM mice restored the KD-dependent tumour vascular leakage observed in ECCre+;FAKKD/KD mice in vivo. This study opens new fields in in vivo FAK signalling. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Haematopoietic focal adhesion kinase deficiency alters haematopoietic homeostasis to drive tumour metastasis.

Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis.

Endothelial-cell FAK targeting sensitizes tumours to DNA-damaging therapy.

Chemoresistance is a serious limitation of cancer treatment. Until recently, almost all the work done to study this limitation has been restricted to tumour cells. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.

FAK-heterozygous mice display enhanced tumour angiogenesis.

Genetic ablation of endothelial focal adhesion kinase (FAK) can inhibit pathological angiogenesis, suggesting that loss of endothelial FAK is sufficient to reduce neovascularization. Here we show that reduced stromal FAK expression in FAK-heterozygous mice unexpectedly enhances both B16F0 and CMT19T tumour growth and angiogenesis. We further demonstrate that cell proliferation and microvessel sprouting, but not migration, are increased in serum-stimulated FAK-heterozygous endothelial cells. FAK-heterozygous endothelial cells display an imbalance in FAK phosphorylation at pY397 and pY861 without changes in Pyk2 or Erk1/2 activity. By contrast, serum-stimulated phosphorylation of Akt is enhanced in FAK-heterozygous endothelial cells and these cells are more sensitive to Akt inhibition. Additionally, low doses of a pharmacological FAK inhibitor, although too low to affect FAK autophosphorylation in vitro, can enhance angiogenesis ex vivo and tumour growth in vivo. Our results highlight a potential novel role for FAK as a nonlinear, dose-dependent regulator of angiogenesis where heterozygous levels of FAK enhance angiogenesis.

Stromal Claudin14-heterozygosity, but not deletion, increases tumour blood leakage without affecting tumour growth.

The maintenance of endothelial cell-cell junctions is vital for the control of blood vessel leakage and is known to be important in the growth and maturation of new blood vessels during angiogenesis. Here we have investigated the role of a tight junction molecule, Claudin 14, in tumour blood vessel leakage, angiogenesis and tumour growth. Using syngeneic tumour models our results showed that genetic ablation of Claudin 14 was not sufficient to affect tumour blood vessel morphology or function. However, and surprisingly, Claudin 14-heterozygous mice displayed several blood vessel-related phenotypes including: disruption of ZO-1-positive cell-cell junctions in tumour blood vessels; abnormal distribution of basement membrane laminin around tumour blood vessels; increased intratumoural leakage and decreased intratumoural hypoxia. Additionally, although total numbers of tumour blood vessels were increased in Claudin 14-heterozygous mice, and in VEGF-stimulated angiogenesis ex vivo, the number of lumenated vessels was not changed between genotypes and this correlated with no difference in syngeneic tumour growth between wild-type, Claudin 14-heterozygous and Claudin 14-null mice. Lastly, Claudin 14-heterozygosity, but not complete deficiency, also enhanced endothelial cell proliferation significantly. These data establish a new role for Claudin 14 in the regulation of tumour blood vessel integrity and angiogenesis that is evident only after the partial loss of this molecule in Claudin 14-heterozyous mice but not in Claudin 14-null mice.

Regulation of vascular endothelial function by procyanidin-rich foods and beverages.

Flavonoid-rich diets are associated with a lower mortality from cardiovascular disease. This has been linked to improvements in endothelial function. However, the specific flavonoids, or biologically active metabolites, conferring these beneficial effects have yet to be fully defined. In this experimental study of the effect of flavonoids on endothelial function cultured endothelial cells have been used as a bioassay with endothelin-1 (ET-1) synthesis being measured an index of the response. Evaluation of the relative effects of extracts of cranberry juice compared to apple, cocoa, red wine, and green tea showed inhibition of ET-1 synthesis was dependent primarily on their oligomeric procyanidin content. Procyanidin-rich extracts of cranberry juice triggered morphological changes in endothelial cells with reorganization of the actin cytoskeleton and increased immunostaining for phosphotyrosine residues. These actions were independent of antioxidant activity. Comparison of the effects of apple procyanidin monomers through heptamer showed a clear structure-activity relationship. Although monomer, dimer, and trimer had little effect on ET-1 synthesis, procyanidin tetramer, pentamer, hexamer, and heptamer produced concentration-dependent decreases with IC(50) values of 5.4, 1.6, 0.9, and 0.7 microM, respectively. Levels of ET-1 mRNA showed a similar pattern of decreases, which were inversely correlated with increased expression of Kruppel-like factor 2 (KLF2), a key endothelial transcription factor with a broad range of antiatherosclerotic actions including suppression of ET-1 synthesis. Future investigations of procyanidin-rich products should assess the role KLF2 induction plays in the beneficial vascular effects of high flavonoid consumption.

Existence of multiple isoforms of HS1-associated protein X-1 in murine and human tissues.

To date, the literature concerning the HS1 (haematopoietic cell-specific protein 1)-associated protein X-1 (HAX1) protein has reported considerable variation regarding its function in mammalian cells, subcellular localisation and binding partners. We show here that HAX1 comprises a family of proteins. Murine tissues express three mRNA variants, encoded by two genes on chromosomes 2 and 3. The chromosome 2 gene is intronless and would encode a protein 100% identical with that encoded by chromosome 3. In humans, alternative splice variants, encoded by the chromosome 1 gene, produce a family of transcripts composed of up to eight members. Based on the sequences published in GenBank and Ensembl, we designed specific primers and detected by PCR three mRNA species in murine tissues and eight variants in human cells. We screened a panel of 19 human cell lines as well as primary fibroblasts, oral keratinocytes and freshly isolated peripheral blood mononuclear cells. All human cells studied expressed at least six of the possible HAX1 mRNA variants. In silico analysis of the variants revealed an open reading frame in all of them, suggesting that murine and human tissues can express two and eight HAX1 proteins, respectively. Analysis of human protein lysates by Western blotting with the use of a monoclonal anti-HAX1 antibody revealed multiple bands. These bands were decreased after treatment of cells with a single small interfering RNA duplex targeting a region common to six of the variants, confirming their identity as HAX1 proteins. Comparison of the human variants with the six HAX1 homologues described to date in the chimpanzee (Pan troglodytes) and the four homologues described in macaque (Macaca mulatta) revealed very high conservation with only one amino acid substitution between human and chimpanzee homologues. Moreover, a number of additional products were amplified and sequenced, which indicated that further human isoforms are likely to exist. These findings are likely to explain the current confusion concerning putative HAX1 function.

Homocysteine-induced endothelin-1 release is dependent on hyperglycaemia and reactive oxygen species production in bovine aortic endothelial cells.

BACKGROUND: Elevated plasma homocysteine (Hcy) is a risk factor for coronary disease. The objective of this study was to investigate whether Hcy either alone or in high glucose conditions induces endothelin-1 (ET-1) synthesis via the production of reactive oxygen species (ROS). METHODS: Bovine aortic endothelial cells were grown in high (25 mmol/l) and low (5 mmol/l) glucose medium. RESULTS: In high glucose, Hcy caused a time-dependent increase in ET-1 release, which was greatest with 50 micromol/l Hcy at 24 h (p < 0.01). This effect was not seen in low glucose conditions. In high glucose and 50 micromol/l Hcy, ET-1 mRNA levels were maximal after 1 h (p < 0.05). Tissue factor mRNA levels were raised at 4 h (p < 0.05) and functional activity was raised at 6 h (p < 0.01). Intracellular ROS production was increased by 50 micromol/l Hcy after 24 h (p < 0.05) but only in high glucose. To investigate the role of mitochondrial metabolism in ROS production, cells were incubated with thenoyltrifluoroacetone (inhibitor of complex II) or carbonyl cyanide m-chlorophenylhydrazone (uncoupler of oxidative phosphorylation). Both compounds abolished the Hcy-induced increase in ROS production and ET-1 release. There was an alteration in intracellular glutathione (GSH) levels with Hcy treatment with more oxidised GSH present. CONCLUSION: The combined metabolic burden of Hcy and high glucose stimulates ET-1 synthesis in bovine aortic endothelial cells via a mechanism dependent on the production of mitochondrial ROS, but may not be generalisable to all types of endothelial cells.

The differential contribution of tumour necrosis factor to thermal and mechanical hyperalgesia during chronic inflammation.

Therapies directed against tumour necrosis factor (TNF) are effective for the treatment of rheumatoid arthritis and reduce pain scores in this condition. In this study, we sought to explore mechanisms by which TNF contributes to inflammatory pain in an experimental model of arthritis. The effects of an anti-TNF agent, etanercept, on behavioural pain responses arising from rat monoarthritis induced by complete Freund's adjuvant were assessed and compared with expression of TNF receptors (TNFRs) by dorsal root ganglion (DRG) cells at corresponding time points. Etanercept had no effect on evoked pain responses in normal animals but exerted a differential effect on the thermal and mechanical hyperalgesia associated with rat arthritis induced by complete Freund's adjuvant (CFA). Joint inflammation was associated with increased TNFR1 and TNFR2 expression on DRG cells, which was maintained throughout the time course of the model. TNFR1 expression was increased in neuronal cells of the DRG bilaterally after arthritis induction. In contrast, TNFR2 expression occurred exclusively on non-neuronal cells of the macrophage-monocyte lineage, with cell numbers increasing in a TNF-dependent fashion during CFA-induced arthritis. A strong correlation was observed between numbers of macrophages and the development of mechanical hyperalgesia in CFA-induced arthritis. These results highlight the potential for TNF to play a vital role in inflammatory hyperalgesia, both by a direct action on neurons via TNFR1 and by facilitating the accumulation of macrophages in the DRG via a TNFR2-mediated pathway.

Factor VIIa stimulates endothelin-1 synthesis in TNF-primed endothelial cells by activation of protease-activated receptor 2.

The mechanisms linking prothrombotic changes to endothelial dysfunction and accelerated atheroma formation have yet to be fully defined. Expression of TF (tissue factor) on the endothelium is potentially an initiating event as binding and activation of FVII (factor VII) can result in thrombosis. Although PAR2 (protease-activated receptor-2) is expressed on vascular endothelium, its precise physiological significance and mechanism of activation have yet to be defined. In the present study, we investigated whether PAR2 can be activated by FVIIa (activated FVII) and induce ET-1 (endothelin-1) synthesis. In bovine aortic endothelial cells pretreated with TNF (tumour necrosis factor-alpha) to increase TF expression, FVIIa stimulated ET-1 synthesis via activation of PAR2. Although FX (factor X) alone was inactive, this response was enhanced by using FVII and FX in combination. Inhibition of the proteolytic activity of FVIIa abolished the response. The PAR2 agonist peptide SLIGKV also enhanced ET-1 release on TNF-pretreated cells. The response to FVIIa was inhibited by a PAR2 antagonist peptide FSLLRY. Inhibition of the p38 MAPK (mitogen-activated protein kinase) reduced PAR2 expression and the ET-1 response. In summary, FVIIa can stimulate ET-1 synthesis in endothelial cells by activating PAR2, demonstrating a potential link between thrombotic processes and endothelial cell dysfunction.

The procyanidin-induced pseudo laminar shear stress response: a new concept for the reversal of endothelial dysfunction.

Reduced endothelium-dependent vasodilator responses with increased synthesis of ET-1 (endothelin-1) are characteristics of endothelial dysfunction in heart failure and are predictive of mortality. Identification of treatments that correct these abnormalities may have particular benefit for patients who become refractory to current regimens. Hawthorn preparations have a long history in the treatment of heart failure. Therefore we tested their inhibitory effects on ET-1 synthesis by cultured endothelial cells. These actions were compared with that of GSE (grape seed extract), as the vasoactive components of both these herbal remedies are mainly oligomeric flavan-3-ols called procyanidins. This showed extracts of hawthorn and grape seed were equipotent as inhibitors of ET-1 synthesis. GSE also produced a potent endothelium-dependent vasodilator response on preparations of isolated aorta. Suppression of ET-1 synthesis at the same time as induction of endothelium-dependent vasodilation is a similar response to that triggered by laminar shear stress. Based on these results and previous findings, we hypothesize that through their pharmacological properties procyanidins stimulate a pseudo laminar shear stress response in endothelial cells, which helps restore endothelial function and underlies the benefit from treatment with hawthorn extract in heart failure.

A role for increased mRNA stability in the induction of endothelin-1 synthesis by lipopolysaccharide.

An association exists between infection and cardiovascular diseases, including atherosclerosis, stroke and myocardial infarction. This may involve endothelin-1 (ET-1) which has been implicated in these and other vascular pathologies. ET-1 synthesis is controlled primarily by the level of its mRNA and numerous stimuli, including infection, lead to elevated ET-1 levels. Here, we have investigated the regulation of ET-1 release and preproET-1 (ppET-1) mRNA in bovine aortic endothelial cells by lipopolysaccharide (LPS). ET-1 release from bovine aortic endothelial cells was stimulated by LPS and reporter gene assays implicated LPS-induced ppET-1 transcription. However, changes in transcription were modest compared to increases in ET-1 synthesis. Therefore, ppET-1 mRNA levels were measured by real-time reverse transcription-polymerase chain reaction. The effect of LPS on ppET-1 mRNA levels was more marked than on transcription (1.2-fold increase in transcription vs. 5.5-fold increase in ppET-1 mRNA). Analysis of ppET-1 mRNA stability by real-time reverse transcription-polymerase chain reaction showed that LPS increased its half-life by approximately 2-fold. Thus, upregulated ppET-1 mRNA and hence increased ET-1 synthesis may be due to both increased transcription and reduced mRNA degradation. These effects of LPS on mRNA stability may be a key mechanism in vascular pathologies through which many proteins are induced in response to infection.

Comparison of red wine extract and polyphenol constituents on endothelin-1 synthesis by cultured endothelial cells.

Regular consumption of red wine reduces mortality from coronary heart disease. This observation has been attributed to the anti-thrombotic effects of ethanol and to the antioxidant properties of polyphenolic compounds present in red wine. Here we show that an extract of red wine polyphenols causes a concentration-dependent inhibition of endothelin-1 synthesis in cultured bovine aortic endothelial cells. This action was associated with modifications in phosphotyrosine staining, indicating that the active components of red wine cause specific modifications of tyrosine kinase signalling. Thus inhibition of endothelin-1 synthesis by red wine may reduce the development of atherosclerosis, and hence decrease coronary heart disease.

Inhibitory effect of adrenomedullin on basal and tumour necrosis factor alpha-stimulated endothelin-1 synthesis in bovine aortic endothelial cells is independent of cyclic AMP.

Adrenomedullin (ADM) is a potent vasodilator and reverses the vasoconstrictor action of endothelin-1 (ET-1). These studies aimed to determine the effect of ADM on ET-1 synthesis in bovine aortic endothelial cells (BAEC) and to identify the possible mechanisms involved. In this cell model, ADM increased cyclic AMP production by BAEC with threshold concentrations of 100 pM and an EC(50) of 1 nM. This effect was not blocked by co-treatment with the CGRP type 1 receptor antagonist CGRP(8--37). ADM caused a potent concentration-dependent inhibition of ET-1 release that was correlated with reduced preproET-1 mRNA levels. This reached a maximal reduction of 70% compared to basal levels after 2 and 6 hr exposure of BAEC to 1 nM ADM, with significant decreases at concentrations as low as 10 pM. However, a 100-fold discrepancy between the threshold ADM concentration for cyclic AMP production and inhibition of ET-1 release was observed. Treatment of BAEC with tumour necrosis factor alpha (TNFalpha; 10 ng/mL) caused a 2-fold increase over basal ET-1 release. ADM caused a more marked reduction in stimulated ET-1 synthesis with a threshold of 1 pM, and suppression of ET-1 release to basal levels at 100 nM. 8-Bromo cyclic AMP, showed no concentration-dependent inhibition of ET-1 release, yet caused a 50% reduction in TNFalpha-stimulated intercellular adhesion molecule-1 (ICAM-1) mRNA levels. Thus, physiological ADM concentrations inhibit ET-1 synthesis independently of cyclic AMP in BAEC at the level of preproET-1 mRNA expression. The high sensitivity of this inhibition implicates ADM as an important physiological regulator of endothelial ET-1 production.