Inhibition of ribosome biogenesis induces apoptosis in p53-/- cells

The nucleolus is an important region of the nucleus that acts as the main site of rRNA transcription by RNA polymerase I (Pol-I), and one of the most prominent morphological markers of proliferative and invasive cancers is increased nucleolar size. Increases in Pol-I transcription leads to increased levels of ribosome biogenesis that are able to fuel rapid cell growth and proliferation due to increased ribosome numbers. Therefore Pol-I transcription seems a viable target for the development of anticancer therapeutics as abrogation of Pol-I transcription leads to cessation of cell growth and eventual cell death. We have confirmed that ellipticine compound, 9-Hydroxyellipticine (9HE), is an efficient inhibitor of Pol-I transcription in vitro and in p53+/+ and -/- cell lines in vivo. Short-term treatments (≤24 h) with micromolar concentrations of 9HE leads to decreased cell viability and proliferation, and leads to activation of caspases 3, 8 and 9, indicating that both intrinsic and extrinsic cell death mechanisms are activated upon Pol-I inhibition. Reactive oxygen species levels were also studied following short and long term treatments with 9HE and there was a 2/3-fold increase in cellular ROS levels. Long-term 9HE treatment (≥24 h) leads to cellular senescence as indicated by increased cellular morphology and senescence associated β-galactosidase staining, and this senescence is not accompanied by induction of autophagy. Following 24 h treatment there is also accumulation of cells in the G0/G1 phase of the cell cycle and qPCR analysis of cell cycle regulators shows down-regulation of G1/S transition associated cyclins. These data show that Pol-I transcription is a viable target for the development of novel chemotherapeutics, although further delineation of the cell death pathways remains. To further elucidate the mechanism, the role of mitochondrial death signals will be investigated by determination of cytochrome c release and regulation of Bcl2 family member proteins.

The role of immune-related myeloid cells in angiogenesis

Macrophage function is not restricted to the innate and adaptive immune responses, but also includes host defence, wound healing, angiogenesis and homeostatic processes. Within the spectrum of macrophage activation there are two extremes: M1 classically activated macrophages which have a pro-inflammatory phenotype, and M2 alternatively activated macrophages which are pro-angiogenic and anti-inflammatory. An important property of macrophages is their plasticity to switch from one phenotype to the other and they can be defined in their polarisation state at any point between the two extremes. In order to determine what stage of activation macrophages are in, it is essential to profile various phenotypic markers for their identification. This review describes the angiogenic role for myeloid cells: circulating monocytes, Tie-2 expressing monocytes (TEMs), myeloid-derived suppressor cells (MDSCs), tumour associated macrophages (TAMs), and neutrophils. Each cell type is discussed by phenotype, roles within angiogenesis and possible targets as a cell therapy. In addition, we also refer to our own research on myeloid angiogenic cells (MACs), outlining their ability to induce angiogenesis and their similarities to alternatively activated M2 macrophages. MACs significantly contribute to vascular repair through paracrine mechanisms as they lack the capacity to differentiate into endothelial cells. Since MACs also retain plasticity, phenotypic changes can occur according to disease states and the surrounding microenvironment. This pro-angiogenic potential of MACs could be harnessed as a novel cellular therapy for the treatment of ischaemic diseases, such as diabetic retinopathy, hind limb ischaemia and myocardial infarction; however, caution needs to be taken when MACs are delivered into an inflammatory milieu.

A review of patents relating to therapeutic angiogenesis using endothelial progenitors and other vasculogenesis-related cell types

Stem and progenitor cells have generated considerable scientific and commercial interest in recent years due to their potential for novel cell therapy for a variety of medical conditions. A highly active research area in the field of regenerative medicine is vascular biology. Blood vessel repair and angiogenesis are key processes with endothelial progenitor cells (EPCs) playing a central role. Clinical trials for ischemic conditions, such as myocardial infarction and peripheral arterial disease, have suggested cell therapies to be feasible, safe, and potentially beneficial. Development of efficient methodologies to deliver EPC-based cytotherapies offers new hope for millions of patients with ischemic conditions. Evidence indicates that EPCs, depending on the subtype, mediate angiogenesis through different mechanisms. Differentiation into endothelium and complete integration into damaged vasculature was the first EPC mechanism to be proposed. However, many studies have demonstrated that vasoregulatory paracrine factor secretion by transplanted cells is also important. Many EPC subsets enhance angiogenesis and promote tissue repair by cytokine release without incorporating into the damaged vasculature. Whatever the mechanism, vascular repair and therapeutic angiogenesis using EPCs represent a realistic treatment option and also provides many commercialization opportunities. This review discusses recent advances in the EPC field whilst recounting relevant patents.

Experimental Autoimmune Uveoretinitis (EAU)-Related Tissue Damage and Angiogenesis Is Reduced in CCL2−/−CX3CR1gfp/gfp Mice

Purpose: To investigate the roles of the CCL2-CCR2 and CX3CL1-CX3CR1 pathways in experimental autoimmune uveoretinitis (EAU)-mediated retinal tissue damage and angiogenesis.

Methods: The C57BL/6J wild-type (WT) and CCL2−/−CX3CR1gfp/gfp (double knockout [DKO]) mice were immunized with IRBP1-20. Retinal inflammation and tissue damage were evaluated clinically and histologically at different days postimmunization (p.i.). Retinal neovascular membranes were evaluated by confocal microscopy of retinal flat mounts, and immune cell infiltration by flow cytometry.

Results: At day 25 p.i., DKO mice had lower clinical and histological scores and fewer CD45highCD11b+ infiltrating cells compared with WT mice. The F4/80+macrophages constitute 40% and 21% and CD11b+Gr-1+Ly6G+ neutrophils constitute 10% and 22% of retinal infiltrating cells in WT and DKO mice, respectively. At the late stages of EAU (day 60–90 p.i.), DKO and WT mice had similar levels of inflammatory score. However, less structural damage and reduced angiogenesis were detected in DKO mice. Neutrophils were rarely detected in the inflamed retina in both WT and DKO mice. Macrophages and myeloid-derived suppressor cells (MDSCs) accounted for 8% and 3% in DKO EAU retina, and 19% and 10% in WT EAU retina; 71% of infiltrating cells were T/B-lymphocytes in DKO EAU retina and 50% in WT EAU retina.

Conclusions: Experimental autoimmune uveoretinitis–mediated retinal tissue damage and angiogenesis is reduced in CCL2−/−CX3CR1gfp/gfp mice. Retinal inflammation is dominated by neutrophils at the acute stage and lymphocytes at the chronic stage in these mice. Our results suggest that CCR2+ and CX3CR1+monocytes are both involved in tissue damage and angiogenesis in EAU.

A simple inhibition coefficient for quantifying potency of biocontrol agents against plant-pathogenic fungi

Microbial interactions depend on a range of biotic and environmental variables, and are both dynamic and unpredictable. For some purposes, and under defined conditions, it is nevertheless imperative to evaluate the inhibitory efficacy of microbes, such as those with potential as biocontrol agents. We selected six, phylogenetically diverse microbes to determine their ability to inhibit the ascomycete Fusarium
coeruleum, a soil-dwelling pathogen of potato tubers that causes the storage disease dry rot. Interaction assays, where colony development was quantified (for both fungal pathogen and potential control agents), were therefore carried out on solid media. The key parameters that contributed to, and were indicative of, inhibitory efficacy were identified as: fungal growth-rates (i) prior to contact with the biocontrol
agent and (ii) if/once contact with the biocontrol agent was established (i.e. in the zone of mixed
culture), and (iii) the ultimate distance traveled by the fungal mycelium. It was clear that there was no correlation between zones of fungal inhibition and the overall reduction in the extent of fungal colony development. An inhibition coefficient was devised which incorporated the potential contributions of distal inhibition of fungal growth-rate; prevention of mycelium development in the vicinity of the biocontrol
agent; and ability to inhibit plant-pathogen growth-rate in the zone of mixed culture (in a ratio of 2:2:1). The values derived were 84.2 for Bacillus subtilis (QST 713), 74.0 for Bacillus sp. (JC12GB42), 30.7 for Pichia anomala (J121), 19.3 for Pantoea agglomerans (JC12GB34), 13.9 for Pantoea sp. (S09:T:12), and
21.9 (indicating a promotion of fungal growth) for bacterial strain (JC12GB54). This inhibition coefficient, with a theoretical maximum of 100, was consistent with the extent of F. coeruleum-colony development (i.e. area, in cm2) and assays of these biocontrol agents carried out previously against Fusarium
spp., and other fungi. These findings are discussed in relation to the dynamics and inherent complexity of natural ecosystems, and the need to adapt models for use under specific sets of conditions.

MicroRNA-199b modulates vascular cell fate during iPS cell differentiation by targeting the notch ligand jagged1 and enhancing VEGF signaling

Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalised medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation towards ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration.

Methods and Results: In this study we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen-coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR-199b is involved in EC differentiation. A step-wise increase in expression of miR-199 was detected during EC differentiation. Notably, miR-199b targeted the Notch ligand JAG1, resulting in VEGF transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA-mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR-199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR-199b-mediated inhibition of iPS cell differentiation towards SMCs. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR-199b also regulated VEGF expression and angiogenesis.

Conclusions: This study indicates a novel role for miR-199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

Stabilisation of MAD2 expression through inhibition of miR-433; A novel mechanism for restoring chemo-responsiveness in ovarian tumours

Epithelial ovarian carcinoma (EOC) is characterised by late diagnosis and recurrences, both of which contribute to the high morbidity and mortality of this cancer. Unfortunately, EOC has an innate susceptibility to become chemo-resistant. Specifically, up to 30% of patients may not respond to current standard chemotherapy (paclitaxel and platinum in combination) and of those who have an initial response, some patients relapse within a few months. Therefore, in order to improve patient outcome it is crucial to establish what factors influence a patients' individualised response to chemotherapy. We analysed MAD2 protein expression in a patient cohort of 35 ovarian tumours and a panel of 5 ovarian cancer cell lines. We have demonstrated that low nuclear MAD2 expression intensity was significantly associated with chemo-resistant ovarian tumours (p=0.0136). Moreover, in vitro studies of the 5 ovarian cancer cell lines revealed that reduced MAD2 expression was associated with paclitaxel resistance. In silico analysis identified a putative miR-433 binding domain in the MAD2 3′UTR and expression profiling of miR-433 in the ovarian cancer cell lines showed that low MAD2 protein expression was associated with high miR-433 levels. In vitro over-expression of miR-433 attenuated MAD2 protein expression with a concomitant increase in cellular resistance to paclitaxel. Over-expression of a morpholino oligonucleotide that blocks miR-433 binding to MAD2 3′UTR stabilised MAD2 protein expression and protects from miR-433 induced degradation. Furthermore, miR-433 expression analysis in 35 ovarian tumour samples revealed that high miR-433 expression was associated with advanced stage presentations (p=0.0236). In conclusion, ovarian tumours that display low nuclear MAD2 intensity are chemo-resistant and stabilising MAD2 expression by antagonising miR-433 activity is a potential mechanism for restoring chemo-responsiveness in these tumours.

FKBPL Is a Critical Antiangiogenic Regulator of Developmental and Pathological Angiogenesis

OBJECTIVE: The antitumor effects of FK506-binding protein like (FKBPL) and its extracellular role in angiogenesis are well characterized; however, its role in physiological/developmental angiogenesis and the effect of FKBPL ablation has not been evaluated. This is important as effects of some angiogenic proteins are dosage dependent. Here we evaluate the regulation of FKBPL secretion under angiogenic stimuli, as well as the effect of FKBPL ablation in angiogenesis using mouse and zebrafish models.

APPROACH AND RESULTS: FKBPL is secreted maximally by human microvascular endothelial cells and fibroblasts, and this was specifically downregulated by proangiogenic hypoxic signals, but not by the angiogenic cytokines, VEGF or IL8. FKBPL's critical role in angiogenesis was supported by our inability to generate an Fkbpl knockout mouse, with embryonic lethality occurring before E8.5. However, whilst Fkbpl heterozygotic embryos showed some vasculature irregularities, the mice developed normally. In murine angiogenesis models, including the ex vivo aortic ring assay, in vivo sponge assay, and tumor growth assay, Fkbpl(+/-) mice exhibited increased sprouting, enhanced vessel recruitment, and faster tumor growth, respectively, supporting the antiangiogenic function of FKBPL. In zebrafish, knockdown of zFkbpl using morpholinos disrupted the vasculature, and the phenotype was rescued with hFKBPL. Interestingly, this vessel disruption was ineffective when zcd44 was knocked-down, supporting the dependency of zFkbpl on zCd44 in zebrafish.

CONCLUSIONS: FKBPL is an important regulator of angiogenesis, having an essential role in murine and zebrafish blood vessel development. Mouse models of angiogenesis demonstrated a proangiogenic phenotype in Fkbpl heterozygotes.

HDAC Inhibition Overcomes Acute Resistance to MEK Inhibition in BRAF-Mutant Colorectal Cancer by Downregulation of c-FLIPL

Purpose: Activating mutations in the BRAF oncogene are found in 8% to 15% of colorectal cancer patients and have been associated with poor survival. In contrast with BRAF-mutant (MT) melanoma, inhibition of the MAPK pathway is ineffective in the majority of BRAFMT colorectal cancer patients. Therefore, identification of novel therapies for BRAFMT colorectal cancer is urgently needed.

Experimental Design: BRAFMT and wild-type (WT) colorectal cancer models were assessed in vitro and in vivo. Small-molecule inhibitors of MEK1/2, MET, and HDAC were used, overexpression and siRNA approaches were applied, and cell death was assessed by flow cytometry, Western blotting, cell viability, and caspase activity assays.

Results: Increased c-MET-STAT3 signaling was identified as a novel adaptive resistance mechanism to MEK inhibitors (MEKi) in BRAFMT colorectal cancer models in vitro and in vivo. Moreover, MEKi treatment resulted in acute increases in transcription of the endogenous caspase-8 inhibitor c-FLIPL in BRAFMT cells, but not in BRAFWT cells, and inhibition of STAT3 activity abrogated MEKi-induced c-FLIPL expression. In addition, treatment with c-FLIP–specific siRNA or HDAC inhibitors abrogated MEKi-induced upregulation of c-FLIPL expression and resulted in significant increases in MEKi-induced cell death in BRAFMT colorectal cancer cells. Notably, combined HDAC inhibitor/MEKi treatment resulted in dramatically attenuated tumor growth in BRAFMT xenografts.

Conclusions: Our findings indicate that c-MET/STAT3-dependent upregulation of c-FLIPL expression is an important escape mechanism following MEKi treatment in BRAFMT colorectal cancer. Thus, combinations of MEKi with inhibitors of c-MET or c-FLIP (e.g., HDAC inhibitors) could be potential novel treatment strategies for BRAFMT colorectal cancer.