Bioactive VEGF-C from E. coli.

Vascular endothelial growth factor-C (VEGF-C) stimulates lymphatic vessel growth in transgenic models, via viral gene delivery, and as a recombinant protein. Expressing eukaryotic proteins like VEGF-C in bacterial cells has limitations, as these cells lack specific posttranslational modifications and provisions for disulfide bond formation. However, given the cost and time savings associated with bacterial expression systems, there is considerable value in expressing VEGF-C using bacterial cells. We identified two approaches that result in biologically active Escherichia coli-derived VEGF-C. Expectedly, VEGF-C expressed from a truncated cDNA became bioactive after in vitro folding from inclusion bodies. Given that VEGF-C is one of the cysteine-richest growth factors in humans, it was unclear whether known methods to facilitate correct cysteine bond formation allow for the direct expression of bioactive VEGF-C in the cytoplasm. By fusing VEGF-C to maltose-binding protein and expressing these fusions in the redox-modified cytoplasm of the Origami (DE3) strain, we could recover biological activity for deletion mutants lacking the propeptides of VEGF-C. This is the first report of a bioactive VEGF growth factor obtained from E. coli cells circumventing in-vitro folding.

KLK3 in the Regulation of Angiogenesis-Tumorigenic or Not?

In this focused review, we address the role of the kallikrein-related peptidase 3 (KLK3), also known as prostate-specific antigen (PSA), in the regulation of angiogenesis. Early studies suggest that KLK3 is able to inhibit angiogenic processes, which is most likely dependent on its proteolytic activity. However, more recent evidence suggests that KLK3 may also have an opposite role, mediated by the ability of KLK3 to activate the (lymph)angiogenic vascular endothelial growth factors VEGF-C and VEGF-D, further discussed in the review.

Outside in and brakes off for lymphatic growth.

In this issue of Science Signaling, Kataru et al. did two simple but powerful tweaks to the typical studies that aim to advance our understanding of proangiogenic interventions. They shifted the focus from the outside of the endothelial cell to the inside, and they chose not to deliver an angiogenic signal, but instead to release the brakes from an already existing signal.

Proteolytic Cleavages in the VEGF Family: Generating Diversity among Angiogenic VEGFs, Essential for the Activation of Lymphangiogenic VEGFs.

Specific proteolytic cleavages turn on, modify, or turn off the activity of vascular endothelial growth factors (VEGFs). Proteolysis is most prominent among the lymph-angiogenic VEGF-C and VEGF-D, which are synthesized as precursors that need to undergo enzymatic removal of their C- and N-terminal propeptides before they can activate their receptors. At least five different proteases mediate the activating cleavage of VEGF-C: plasmin, ADAMTS3, prostate-specific antigen, cathepsin D, and thrombin. All of these proteases except for ADAMTS3 can also activate VEGF-D. Processing by different proteases results in distinct forms of the "mature" growth factors, which differ in affinity and receptor activation potential. The "default" VEGF-C-activating enzyme ADAMTS3 does not activate VEGF-D, and therefore, VEGF-C and VEGF-D do function in different contexts. VEGF-C itself is also regulated in different contexts by distinct proteases. During embryonic development, ADAMTS3 activates VEGF-C. The other activating proteases are likely important for non-developmental lymphangiogenesis during, e.g., tissue regeneration, inflammation, immune response, and pathological tumor-associated lymphangiogenesis. The better we understand these events at the molecular level, the greater our chances of developing successful therapies targeting VEGF-C and VEGF-D for diseases involving the lymphatics such as lymphedema or cancer.

Cleavage of the Drosophila screw prodomain is critical for a dynamic BMP morphogen gradient in embryogenesis.

Dorsoventral patterning of the Drosophila embryo is regulated by graded distribution of bone morphogenetic proteins (BMPs) composed of two ligands, decapentaplegic (Dpp) a BMP2/4 ortholog and screw (Scw) a BMP5/6/7/8 family member. scw(E1) encodes an unusual allele that was isolated as a dominant enhancer of partial loss-of-function mutations in dpp. However, the molecular mechanisms that underlie this genetic interaction remain to be addressed. Here we show that scw(E1) contains a mutation at the furin cleavage site within the prodomain that is crucial for ligand production. Furthermore, our data show that Scw(E1) preferentially forms heterodimers with Dpp rather than homotypic dimers, providing a possible explanation for the dominant negative phenotype of scw(E1) alleles. The unprocessed prodomain of Scw(E1) remains in a complex with the Dpp:Scw heterodimer, and thus could interfere with interaction of the ligand with the extracellular matrix, or the kinetics of processing/secretion of the ligand in vivo. These data reveal novel mechanisms by which post-translational regulation of Scw can modulate Dpp signaling activity.

Evolutional imprints on the sequences of BMP2/4/DPP type proteins.

Decapentaplegic (DPP) and bone morphogenetic protein (BMP)-2 and -4 type ligands form a branch of the transforming growth factor-beta (TGFbeta) superfamily. They play prominent roles in metazoan developmental processes as diverse as cell proliferation, apoptosis, differentiation and cell-fate determination. Maturation of the BMP2/4/DPP type proteins requires proteolytic cleavage of their proproteins by furin-type proprotein convertases (PCs). Even though cleavage of the prodomain is critical for signaling, much less attention has been paid to the role of proteolytic processing. Our studies suggest that the cleavage sites of BMP2/4/DPP type proteins have been diversified and can be categorized into at least four different types. These findings indicate that the cleavage sites in BMP2/4/DPP prodomains are tolerant to mutations acquired through evolution and have adapted to diversified environments among species. These results provide insights for further studies of evolution and molecular diversity. In this Extra View we discuss the evolutional variation seen in the sequences of BMP2/4/DPP type proteins.

The Drosophila DPP signal is produced by cleavage of its proprotein at evolutionary diversified furin-recognition sites.

Maturation of bone morphogenetic proteins (BMPs) requires cleavage of their precursor proteins by furin-type proprotein convertases. Here, we find that cleavage sites of the BMP2/4/decapentaplegic (DPP) subfamily have been evolutionary diversified and can be categorized into 4 different types. Cnidaria BMP2/4/DPP is considered to be a prototype containing only 1 furin site. Bilateria BMP2/4/DPP acquired an additional cleavage site with either the combination of minimal-optimal or optimal-optimal furin sites. DPPs belonging to Diptera, such as Drosophila and mosquito, and Lepidoptera of silkworm contain a third cleavage site between the 2 optimal furin sites. We studied how the 3 furin sites (FSI-III) of Drosophila DPP coordinate maturation of ligands and contribute to signals in vivo. Combining mutational analysis of furin-recognition sites and RNAi experiments, we found that the Drosophila DPP precursor is initially cleaved at an upstream furin-recognition site (FSII), with consequent cleavages at 2 furin sites (FSI and FSIII). Both Dfurin1 and Dfurin2 are involved in the processing of DPP proproteins. Biochemical and genetic analyses using cleavage mutants of DPP suggest the first cleavage at FSII to be critical and sufficient for long-range DPP signaling. Our data suggest that the Drosophila DPP precursor is cleaved in a different manner from vertebrate BMP4 even though they are functional orthologs. This indicates that the furin-cleavage sites in BMP2/4/DPP precursors are tolerant to mutations acquired through evolution and have adapted to different systems in diversified species.

Stabilized HIF-1alpha is superior to VEGF for angiogenesis in skeletal muscle via adeno-associated virus gene transfer.

Therapeutic angiogenesis provides a potential alternative for the treatment of cardiovascular ischemic diseases. Vascular endothelial growth factor (VEGF) is an important component of the angiogenic response to ischemia. Here we used adeno-associated virus (AAV) gene delivery to skeletal muscle to examine the effects of VEGF vs. a stabilized form of hypoxia-inducible factor-1alpha (HIF-1alpha). The recombinant AAVs were injected into mouse tibialis anterior muscle, and their effects were analyzed by immunohistochemistry and functional assays. These analyses showed that stabilized HIF-1alpha markedly increase capillary sprouting and proliferation, whereas VEGF164 or VEGF120 induced only proliferation of endothelial cells without formation of proper capillary structures. The Evans Blue permeability assay indicated that, unlike VEGF, HIF-1alpha overexpression did not increase vascular leakiness in the transduced muscle. Doppler ultrasound imaging showed that vascular perfusion in the HIF-1alpha treated muscles was significantly enhanced when compared to the controls and not further improved by co-expression of the arteriogenic growth factors angiopoietin-1 or platelet-derived growth factor-B. Our results show that AAV-mediated transduction of a stabilized form of HIF-1alpha can circumvent the problems associated with overexpression of individual angiogenic growth factors. HIF-1alpha should thus offer a potent alternative for pro-angiogenic gene therapy.