Lyn M. Wise

Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3.

Vascular endothelial growth factor receptor‐3 (VEGFR‐3/Flt4) binds two known members of the VEGF ligand family, VEGF‐C and VEGF‐D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR‐3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR‐3. We show that VEGFR‐3 stimulation alone protects the lymphatic endothelial cells from serum deprivation‐induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C‐dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin‐sensitive induction of Akt phosphorylation. These results define the critical role of VEGF‐C/VEGFR‐3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.

Viral Vascular Endothelial Growth Factors Vary Extensively in Amino Acid Sequence, Receptor-binding Specificities, and the Ability to Induce Vascular Permeability yet Are Uniformly Active Mitogens

Infections of humans and ungulates by parapoxviruses result in skin lesions characterized by extensive vascular changes that have been linked to viral-encoded homologues of vascular endothelial growth factor (VEGF). VEGF acts via a family of receptors (VEGFRs) to mediate endothelial cell proliferation, vascular permeability, and angiogenesis. The VEGF genes from independent parapoxvirus isolates show an extraordinary degree of inter-strain sequence variation. We conducted functional comparisons of five representatives of the divergent viral VEGFs. These revealed that despite the sequence divergence, all were equally active mitogens, stimulating proliferation of human endothelial cells in vitro and vascularization of sheep skin in vivo with potencies equivalent to VEGF. This was achieved even though the viral VEGFs bound VEGFR-2 less avidly than did VEGF. Surprisingly the viral VEGFs varied in their ability to cross-link VEGFR-2, induce vascular permeability and bind neuropilin-1. Correlations between these three activities were detected. In addition it was possible to correlate these functional variations with certain sequence and structural motifs specific to the viral VEGFs. In contrast to the conserved ability to bind human VEGFR-2, the viral growth factors did not bind either VEGFR-1 or VEGFR-3. We propose that the extensive sequence divergence seen in the viral VEGFs was generated primarily by selection against VEGFR-1 binding.

Sequence and Functional Analysis of a Homolog of Interleukin-10 Encoded by the Parapoxvirus Orf Virus

Orf virus is a large DNA virus and is the type species of the Parapoxvirus genus of the family Poxviridae. Orf virus infects the epithelium of sheep and goats and is transmissible to humans. Recently we discovered a gene in orf virus that encodes a polypeptide with remarkable homology to mammalian interleukin (IL-10) and viral encoded IL-10s of herpes viruses. The predicted polypeptide sequence shows high levels of amino acid identity to IL-10 of sheep (80%), cattle (75%), humans (67%) and mice (64%), as well as IL-10-like proteins of Epstein-Barr virus (63%) and equine herpes virus (67%). The C-terminal region, comprising two-thirds of the orf virus protein, is identical to ovine IL-10 which suggests that this gene has been captured from its host sheep during the evolution of orf virus. In contrast the N-terminal region shows little homology with cellular IL-10s and in this respect resemble other viral IL-10s. IL-10 is a pleiotrophic cytokine that can exert either immunostimulatory or immunosuppressive effects on many cell types. IL-10 is a potent anti-inflammatory cytokine with inhibitory effects on non-specific immunity in particular macrophage function and Th1 effector function. Our studies so far, indicate, that the functional activities of orf virus IL-10 are the same as ovine IL-10. Orf virus IL-10 stimulates mouse thymocyte proliferation and inhibits cytokine synthesis in lipopolysaccharide-activated ovine macrophages, peripheral blood monocytes and keratinocytes. Infection of sheep with an IL-10 deletion mutant of orf virus has shown that interferon-γ levels are higher in tissue infected with the mutant virus than the parent virus. The functional activities of IL-10 and our data on orf virus IL-10 suggest a role in immune evasion.

Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved, functionally relevant structure

The first report of a vascular endothelial growth factor (VEGF)-like gene in Orf virus included the surprising observation that the genes from two isolates (NZ2 and NZ7) shared only 41.1% amino acid sequence identity. We have examined this sequence disparity by determining the VEGF gene sequence of 21 isolates of Orf virus derived from diverse sources. Most isolates carried NZ2-like VEGF genes but their predicted amino acid sequences varied by up to 30.8% with an average amino acid identity between pairs of NZ2-like sequences of 86.1%. This high rate of sequence variation is more similar to interspecies than intraspecies variability. In contrast, only three isolates carried an NZ7-like VEGF gene and these varied from the NZ7 sequence by no more than a single nucleotide. The VEGF family are ligands for a set of tyrosine kinase receptors. The viral VEGFs are unique among the family in that they recognize VEGF receptor 2 (VEGFR-2) but not VEGFR-1 or VEGFR-3. Comparisons of the viral VEGFs with other family members revealed some correlations between conserved residues and the ability to recognize specific VEGF receptors. Despite the sequence variations, structural predictions for the viral VEGFs were very similar to each other and to the structure determined by X-ray crystallography for human VEGF-A. Structural modelling also revealed that a groove seen in the VEGF-A homodimer and believed to play a role in its binding to VEGFR-1 is blocked in the viral VEGFs. This may contribute to the inability of the viral VEGFs to bind VEGFR-1.

The vascular endothelial growth factor (VEGF)-E encoded by orf virus regulates keratinocyte proliferation and migration and promotes epidermal regeneration.

Vascular endothelial growth factor (VEGF)‐A, a key regulator of cutaneous blood vessel formation, appears to have an additional role during wound healing, enhancing re‐epithelialization. Orf virus, a zoonotic parapoxvirus, induces proliferative skin lesions that initiate in wounds and are characterized by extensive blood vessel formation, epidermal hyperplasia and rete ridge formation. The vascular changes beneath the lesion are largely due to viral‐expressed VEGF‐E. This study investigated using mouse skin models whether VEGF‐E can induce epidermal changes such as that seen in the viral lesion. Injection of VEGF‐E into normal skin increased the number of endothelial cells and blood vessels within the dermis and increased epidermal thickening and keratinocyte number. Injection of VEGF‐E into wounded skin, which more closely mimics orf virus lesions, increased neo‐epidermal thickness and area, promoted rete ridge formation, and enhanced wound re‐epithelialization. Quantitative RT‐PCR analysis showed that VEGF‐E did not induce expression of epidermal‐specific growth factors within the wound, but did increase matrix metalloproteinase (MMP)‐2 and MMP‐9 expression. In cell‐based assays, VEGF‐E induced keratinocyte migration and proliferation, responses that were inhibited by a neutralizing antibody against VEGF receptor (VEGFR)‐2. These findings demonstrate that VEGF‐E, both directly and indirectly, regulates keratinocyte function, thereby promoting epidermal regeneration.

Molecular Genetic Analysis of Orf Virus: A Poxvirus That Has Adapted to Skin

Orf virus is the type species of the Parapoxvirus genus of the family Poxviridae. It induces acute pustular skin lesions in sheep and goats and is transmissible to humans. The genome is G+C rich, 138 kbp and encodes 132 genes. It shares many essential genes with vaccinia virus that are required for survival but encodes a number of unique factors that allow it to replicate in the highly specific immune environment of skin. Phylogenetic analysis suggests that both viral interleukin-10 and vascular endothelial growth factor genes have been “captured” from their host during the evolution of the parapoxviruses. Genes such as a chemokine binding protein and a protein that binds granulocyte-macrophage colony-stimulating factor and interleukin-2 appear to have evolved from a common poxvirus ancestral gene while three parapoxvirus nuclear factor (NF)-κB signalling pathway inhibitors have no homology to other known NF-κB inhibitors. A homologue of an anaphase-promoting complex subunit that is believed to manipulate the cell cycle and enhance viral DNA synthesis appears to be a specific adaptation for viral-replication in keratinocytes. The review focuses on the unique genes of orf virus, discusses their evolutionary origins and their role in allowing viral-replication in the skin epidermis.

The chemokine-binding protein encoded by the poxvirus orf virus inhibits recruitment of dendritic cells to sites of skin inflammation and migration to peripheral lymph nodes

Orf virus (ORFV) is a zoonotic parapoxvirus that induces acute pustular skin lesions in sheep and humans. ORFV can reinfect its host and the discovery of several secreted immune modulatory factors that include a chemokine‐binding protein (CBP) may explain this phenomenon. Dendritic cells (DC) are professional antigen presenting cells that induce adaptive immunity and their recruitment to sites of infection in skin and migration to peripheral lymph nodes is critically dependent on inflammatory and constitutive chemokine gradients respectively. Here we examined whether ORFV‐CBP could disable these gradients using mouse models. Previously we established that ORFV‐CBP bound murine inflammatory chemokines with high affinity and here we show that this binding spectrum extends to constitutive chemokines CCL19 and CCL21. Using cell‐based chemotaxis assays, ORFV‐CBP inhibited the movement of both immature and mature DC in response to these inflammatory and constitutive chemokines respectively. Moreover in C57BL/6 mice, intradermally injected CBP potently inhibited the recruitment of blood‐derived DC to lipopolysaccharide‐induced sites of skin inflammation and inhibited the migration of ex vivo CpG‐activated DC to inguinal lymph nodes. Finally we showed that ORFV‐CBP completely inhibited T responsiveness in the inguinal lymph nodes using intradermally injected DC pulsed with ovalbumin peptide and transfused transgenic T cells.

Orf virus-encoded chemokine-binding protein is a potent inhibitor of inflammatory monocyte recruitment in a mouse skin model.

The parapoxvirus orf virus causes pustular dermatitis in sheep and is transmissible to humans. The virus encodes a secreted chemokine-binding protein (CBP). We examined the ability of this protein to inhibit migration of murine monocytes in response to CC inflammatory chemokines, using chemotaxis assays, and its effects on monocyte recruitment into the skin, using a mouse model in which inflammation was induced with bacterial lipopolysaccharide. CBP was shown to bind murine chemokines CCL2, CCL3 and CCL5 with high affinity by surface plasmon resonance and it completely inhibited chemokine-induced migration of monocytes at a CBP:chemokine molar ratio of 4:1. In the mouse, low levels of CBP potently inhibited the recruitment of Gr-1+/CD11b+ monocytes to the site of inflammation in the skin but had little effect on neutrophil recruitment, suggesting that this factor plays a role in disrupting chemokine-induced recruitment of specific immune cell types to infection sites.

Orf virus IL‐10 accelerates wound healing while limiting inflammation and scarring

Interleukin (IL)‐10 plays a critical role in controlling wound inflammation and scar formation. Orf virus, a zoonotic parapoxvirus, induces proliferative skin lesions that resolve with minimal scarring. Orf virus encodes a range of factors that subvert the hosts response to infection, including a homolog of IL‐10. This study investigated, using a murine full‐thickness wound model, whether purified orf virus IL‐10 (ovIL‐10) can regulate skin repair and scarring. Repeat injections of ovIL‐10 into wounded skin accelerated wound closure. Histological analyses of wound sections revealed that treatment with ovIL‐10 accelerated wound reepithelialization, granulation tissue coverage of the wound bed, and improved wound revascularization. In addition, wounds treated with ovIL‐10 showed a reduction in macrophage infiltration, myofibroblast differentiation, and wound contraction. Treatment of wounds with ovIL‐10 also resulted in a reduction in visible scarring that was consistent with the extent of scar tissue formed. Quantitative polymerase chain reaction analysis confirmed that ovIL‐10 reduced the expression of key mediators of inflammation and granulation tissue formation. These findings show that ovIL‐10, like mammalian IL‐10, limits inflammation and scar tissue formation and reveal a new role for both mammalian and viral IL‐10 in mediating tissue repair.

Effect of a Broad-Specificity Chemokine-Binding Protein on Brain Leukocyte Infiltration and Infarct Development

Background and Purpose— Expression of numerous chemokine-related genes is increased in the brain after ischemic stroke. Here, we tested whether post-stroke administration of a chemokine-binding protein (CBP), derived from the parapoxvirus bovine papular stomatitis virus, might reduce infiltration of leukocytes into the brain and consequently limit infarct development. Methods— The binding spectrum of the CBP was evaluated in chemokine ELISAs, and binding affinity was determined using surface plasmon resonance. Focal stroke was induced in C57Bl/6 mice by middle cerebral artery occlusion for 1 hour followed by reperfusion for 23 or 47 hours. Mice were treated intravenously with either bovine serum albumin (10 &mgr;g) or CBP (10 &mgr;g) at the commencement of reperfusion. At 24 or 48 hours, we assessed plasma levels of the chemokines CCL2/MCP-1 and CXCL2/MIP-2, as well as neurological deficit, brain leukocyte infiltration, and infarct volume. Results— The CBP interacted with a broad spectrum of CC, CXC, and XC chemokines and bound CCL2/MCP-1 and CXCL2/MIP-2 with high affinity (pM range). Stroke markedly increased plasma levels of CCL2/MCP-1 and CXCL2/MIP-2, as well as numbers of microglia and infiltrating leukocytes in the brain. Increases in plasma chemokines were blocked in mice treated with CBP, in which there was reduced neurological deficit, fewer brain-infiltrating leukocytes, and ≈50% smaller infarcts at 24 hours compared with bovine serum albumin–treated mice. However, CBP treatment was no longer protective at 48 hours. Conclusions— Post-stroke administration of CBP can reduce plasma chemokine levels in association with temporary atten uation of brain inflammation and infarct volume development.