A combination of low-dose bevacizumab and imatinib enhances vascular normalisation without inducing extracellular matrix deposition.

BACKGROUND: Vascular endothelial growth factor (VEGF)-targeting drugs normalise the tumour vasculature and improve access for chemotherapy. However, excessive VEGF inhibition fails to improve clinical outcome, and successive treatment cycles lead to incremental extracellular matrix (ECM) deposition, which limits perfusion and drug delivery. We show here, that low-dose VEGF inhibition augmented with PDGF-R inhibition leads to superior vascular normalisation without incremental ECM deposition thus maintaining access for therapy. METHODS: Collagen IV expression was analysed in response to VEGF inhibition in liver metastasis of colorectal cancer (CRC) patients, in syngeneic (Panc02) and xenograft tumours of human colorectal cancer cells (LS174T). The xenograft tumours were treated with low (0.5 mg kg-1 body weight) or high (5 mg kg-1 body weight) doses of the anti-VEGF antibody bevacizumab with or without the tyrosine kinase inhibitor imatinib. Changes in tumour growth, and vascular parameters, including microvessel density, pericyte coverage, leakiness, hypoxia, perfusion, fraction of vessels with an open lumen, and type IV collagen deposition were compared. RESULTS: ECM deposition was increased after standard VEGF inhibition in patients and tumour models. In contrast, treatment with low-dose bevacizumab and imatinib produced similar growth inhibition without inducing detrimental collagen IV deposition, leading to superior vascular normalisation, reduced leakiness, improved oxygenation, more open vessels that permit perfusion and access for therapy. CONCLUSIONS: Low-dose bevacizumab augmented by imatinib selects a mature, highly normalised and well perfused tumour vasculature without inducing incremental ECM deposition that normally limits the effectiveness of VEGF targeting drugs.

Dual targeting of Angiopoetin-2 and VEGF potentiates effective vascular normalisation without inducing empty basement membrane sleeves in xenograft tumours.

BACKGROUND: Effective vascular normalisation following vascular endothelial growth factor (VEGF) inhibition is associated with endothelial cell regression leaving empty basement membrane sleeves (BMS). These long-lived BMS permit the rapid regrowth of tumour vasculature upon treatment cessation and promote resistance to VEGF-targeting drugs. Previous attempts at removing BMS have failed. Angiopoietin-2 (Ang2) is a vascular destabilizing factor that antagonises normalisation. We hypothesised that Ang2 inhibition could permit vascular normalisation at significantly reduced doses of VEGF inhibition, avoiding excessive vessel regression and the formation of empty BMS. METHODS: Mice xenografted with human colorectal cancer cells (LS174T) were treated with low (0.5 mg kg(-1)) or high (5 mg kg(-1)) doses of the VEGF-targeting antibody bevacizumab with or without an Ang2 blocking peptibody L1-10. Tumour growth, BMS formation and normalisation parameters were examined including vessel density, pericyte coverage, adherence junctions, leakiness, perfusion, hypoxia and proliferation. RESULTS: Dual targeting of VEGF and Ang2 achieved effective normalisation at only one-tenth of the dose required with bevacizumab alone. Pericyte coverage, vascular integrity, adherence junctions and perfusion as prerequisites for improved access of chemotherapy were improved without inducing empty BMS that facilitate rapid vascular regrowth. CONCLUSIONS: Dual targeting of VEGF and Ang2 can potentiate the effectiveness of VEGF inhibitors and avoid the formation of empty BMS.

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis.

Cell surface targeting of recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to modify AAV's natural tropism. As modification of the capsid surface is likely to affect the mechanism of vector internalization and consequently the vector's intracellular fate, we investigated early steps in cell transduction of rAAV capsid insertion mutants. Mutants displaying peptides with neutral overall charge at position 587 transduced cells independently of AAV2's primary receptor heparan sulfate proteoglycan (HSPG), whereas mutants carrying positively charged insertions were capable of HSPG binding with affinities correlating with their net positive charge. Whereas rAAV2 is internalized via an HSPG- and clathrin-dependent pathway, HSPG-binding mutants used a clathrin- and caveolin-independent mechanism. Surprisingly, although this pathway was as efficient in mediating vector entry as the one used by rAAV2, successful cell transduction was hampered at a post-entry step, presumably caused by inefficient endosomal escape. In contrast, HSPG-independent, clathrin-dependent internalization used by non-HSPG-binding mutants correlated with efficient nuclear delivery of vector genomes and robust transgene expression. These findings indicate that cell surface targeting strategies should direct uptake of rAAV targeting vectors to clathrin-mediated endocytosis, the naturally evolved entry route of AAV, to promote successful intracellular processing and re-targeting of rAAV's tropism.

Elevated XIAP expression alone does not confer chemoresistance.

BACKGROUND: In various tumour types, elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) has been observed and XIAP targeting in diverse tumour entities enhanced the susceptibility to chemotherapeutic agents. Therefore, XIAP has been described and reviewed repeatedly as a chemoresistance factor in different tumour entities. However, rather than being an adverse prognostic marker, recent data suggest that elevated XIAP expression may be associated with a favourable clinical outcome. These somewhat conflicting findings, and the fact that in early studies XIAP suppressed apoptosis only when expressed transiently at levels far in excess of its physiological concentration, argue that the function of XIAP as an anti-apoptotic factor in tumour cells is both more complex and diverse than previously appreciated. METHODS: To better understand the impact of long-term elevated XIAP expression on resistance to chemotherapy, we generated cell lines stably overexpressing XIAP. The role of mitochondria was examined by stable expression of Bcl2 or stable knockdown of second mitochondria-derived activator of caspase (SMAC) in combination with up- or downregulation of XIAP expression. RESULTS: Our data show that long-term expression of XIAP at concentrations comparable to that in tumour cells (two- to five-fold increase) resulted in little or no resistance towards chemotherapeutic drugs. The XIAP overexpression only in conjunction with stable knockdown of a single XIAP-antagonising factor such as SMAC resulted in severe resistance to cytostatic agents demonstrating XIAP as a potent chemoresistance factor only in cells lacking functional XIAP regulatory circuits. CONCLUSION: Our results demonstrated that elevated XIAP expression alone cannot serve as a predictive marker of chemoresistance. Our data suggest that in order to predict the impact of XIAP on chemosusceptibility for a given tumour entity, the expression levels and functional states of all XIAP modulators need to be taken into account.

Identification of serum angiopoietin-2 as a biomarker for clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy.

BACKGROUND: The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC. METHODS: Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification. RESULTS: Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31%; P<0.01), a prolonged median progression-free survival (14.1 vs 8.5 months; P<0.01) and a reduction of 91% in the hazard of death (P<0.05). CONCLUSION: Serum Ang-2 is a candidate biomarker for outcome of patients with metastatic CRC treated with bevacizumab-containing therapy, and it should be further validated to customise combined chemotherapeutic and anti-angiogenic treatment.

Mitochondrial respiration controls neoangiogenesis during wound healing and tumour growth.

The vasculature represents a highly plastic compartment, capable of switching from a quiescent to an active proliferative state during angiogenesis. Metabolic reprogramming in endothelial cells (ECs) thereby is crucial to cover the increasing cellular energy demand under growth conditions. Here we assess the impact of mitochondrial bioenergetics on neovascularisation, by deleting cox10 gene encoding an assembly factor of cytochrome c oxidase (COX) specifically in mouse ECs, providing a model for vasculature-restricted respiratory deficiency. We show that EC-specific cox10 ablation results in deficient vascular development causing embryonic lethality. In adult mice induction of EC-specific cox10 gene deletion produces no overt phenotype. However, the angiogenic capacity of COX-deficient ECs is severely compromised under energetically demanding conditions, as revealed by significantly delayed wound-healing and impaired tumour growth. We provide genetic evidence for a requirement of mitochondrial respiration in vascular endothelial cells for neoangiogenesis during development, tissue repair and cancer.

The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu.

The human gene for the neural cell adhesion molecule L1 is located on Xq28 between the ALD and MeCP2 loci. Mutations in the L1 gene are associated with four related neurological disorders, X-linked hydrocephalus, spastic paraplegia (SPG1), MASA syndrome, and X-linked corpus callosum agenesis. The clinical relevance of L1 has led us to sequence the L1 gene in human and to investigate its conservation in the vertebrate model genome of the pufferfish, Fugu rubripes (Fugu), a species with a compact genome of around 40Mb. For this purpose we have sequenced a human and a Fugu cosmid clone containing the corresponding L1 genes. For comparison, we have also amplified and sequenced the complete Fugu L1 cDNA. We find that the genomic structure of L1 is conserved. The human and Fugu L1 gene both have 28 exons of nearly identical size. Differential splicing of exons 2 and 27 is conserved over 430 million years, the evolutionary time span between the teleost Fugu and the human L1 gene. In contrast to previously published Fugu genes, many introns are larger in the Fugu L1 gene, making it slightly larger in size despite the compact nature of the Fugu genome. Homology at the amino acid and the nucleotide level with 40% and 51%, respectively, is lower than that of any previously reported Fugu gene. At the level of protein structure, both human and Fugu L1 molecules are composed of six immunoglobulin (Ig)-like domains and five fibronectin (Fn) type III domains, followed by a transmembrane domain and a short cytoplasmic domain. Only the transmembrane and the cytoplasmic domains are significantly conserved in Fugu, supporting their proposed function in intracellular signalling and interaction with cytoskeletal elements in the process of neurite outgrowth and fascicle formation. Our results show that the cytoplasmic domain can be further subdivided into a conserved and a variable region, which may correspond to different functions. Most pathological missense mutations in human L1 affect conserved residues. Fifteen out of 22 reported missense mutations alter amino acids that are identical in both species.

The expression of Myf5 in the developing mouse embryo is controlled by discrete and dispersed enhancers specific for particular populations of skeletal muscle precursors.

The development of skeletal muscle in vertebrate embryos is controlled by a transcriptional cascade that includes the four myogenic regulatory factors Myf5, Myogenin, MRF4 and MyoD. In the mouse embryo, Myf5 is the first of these factors to be expressed and mutational analyses suggest that this protein acts early in the process of commitment to the skeletal muscle fate. We have therefore analysed the regulation of Myf5 gene expression using transgenic technology and find that its control is markedly different from that of the other two myogenic regulatory factor genes previously analysed, Myogenin and MyoD. We show that Myf5 is regulated through a number of distinct and discrete enhancers, dispersed throughout 14 kb spanning the MRF4/Myf5 locus, each of which drives reporter gene expression in a particular subset of skeletal muscle precursors. This region includes four separate enhancers controlling expression in the epaxial muscle precursors of the body, some hypaxial precursors of the body, some facial muscles and the central nervous system. These elements separately or together are unable to drive expression in the cells that migrate to the limb buds and in some other muscle subsets and to correctly maintain expression at late times. We suggest that this complex mechanism of control has evolved because different inductive signals operate in each population of muscle precursors and thus distinct enhancers, and cognate transcription factors, are required to interpret them.

Hedgehog signalling is required for maintenance of myf5 and myoD expression and timely terminal differentiation in zebrafish adaxial myogenesis.

Hedgehog proteins have been implicated in the control of myogenesis in the medial vertebrate somite. In the mouse, normal epaxial expression of the myogenic transcription factor gene myf5 is dependent on Sonic hedgehog. Here we examine in zebrafish the interaction between Hedgehog signals, the expression of myoD family genes, including the newly cloned zebrafish myf5, and slow myogenesis. We show that Sonic hedgehog is necessary for normal expression of both myf5 and myoD in adaxial slow muscle precursors, but not in lateral paraxial mesoderm. Expression of both genes is initiated normally in rostral presomitic mesoderm in sonic you mutants, which lack all Sonic hedgehog. Similar initiation continues during tailbud outgrowth when the cells forming caudal somites are generated. However, adaxial cells in sonic you embryos are delayed in terminal differentiation and caudal adaxial cells fail to maintain myogenic regulatory factor expression. Despite these defects, other signals are able to maintain, or reinitiate, some slow muscle development in sonic you mutants. In the cyclops mutant, the absence of floorplate-derived Tiggywinkle hedgehog and Sonic hedgehog has no discernible effect on slow adaxial myogenesis. Similarly, the absence of notochord-derived Sonic hedgehog and Echidna hedgehog in mutants lacking notochord delays, but does not prevent, adaxial slow muscle development. In contrast, removal of both Sonic hedgehog and a floorplate signal, probably Tiggywinkle hedgehog, from the embryonic midline in cyclops;sonic you double mutants essentially abolishes slow myogenesis. We conclude that several midline signals, likely to be various Hedgehogs, collaborate to maintain adaxial slow myogenesis in the zebrafish embryo. Moreover, the data demonstrate that, in the absence of this required Hedgehog signalling, expression of myf5 and myoD is insufficient to commit cells to adaxial myogenesis.