The Tie2-agonist Vasculotide rescues mice from influenza virus infection.

Seasonal influenza virus infections cause hundreds of thousands of deaths annually while viral mutation raises the threat of a novel pandemic strain. Antiviral drugs exhibit limited efficacy unless administered early and may induce viral resistance. Thus, targeting the host response directly has been proposed as a novel therapeutic strategy with the added potential benefit of not eliciting viral resistance. Severe influenza virus infections are complicated by respiratory failure due to the development of lung microvascular leak and acute lung injury. We hypothesized that enhancing lung endothelial barrier integrity could improve the outcome. Here we demonstrate that the Tie2-agonist tetrameric peptide Vasculotide improves survival in murine models of severe influenza, even if administered as late as 72 hours after infection; the benefit was observed using three strains of the virus and two strains of mice. The effect required Tie2, was independent of viral replication and did not impair lung neutrophil recruitment. Administration of the drug decreased lung edema, arterial hypoxemia and lung endothelial apoptosis; importantly, Vasculotide is inexpensive to produce, is chemically stable and is unrelated to any Tie2 ligands. Thus, Vasculotide may represent a novel and practical therapy for severe infections with influenza.

Effects of a synthetic PEG-ylated Tie-2 agonist peptide on endotoxemic lung injury and mortality.

A synthetic 7-mer, HHHRHSF, was recently identified by screening a phage display library for binding to the Tie-2 receptor. A polyethylene-oxide clustered version of this peptide, termed vasculotide (VT), was reported to activate Tie-2 and promote angiogenesis in a mouse model of diabetic ulcer. We hypothesized that VT administration would defend endothelial barrier function against sepsis-associated mediators of permeability, prevent lung vascular leakage arising in endotoxemia, and improve mortality in endotoxemic mice. In confluent human microvascular endothelial cells, VT prevented endotoxin-induced (lipopolysaccharides, LPS O111:B4) gap formation, loss of monolayer resistance, and translocation of labeled albumin. In 8-wk-old male C57Bl6/J mice given a ∼70% lethal dose of endotoxin (15 mg/kg ip), VT prevented lung vascular leakage and reversed the attenuation of lung vascular endothelial cadherin induced by endotoxemia. These protective effects of VT were associated with activation of Tie-2 and its downstream mediator, Akt. Echocardiographic studies showed only a nonsignificant trend toward improved myocardial performance associated with VT. Finally, we evaluated survival in this mouse model. Pretreatment with VT improved survival by 41.4% (n = 15/group, P = 0.02) and post-LPS administration of VT improved survival by 33.3% (n = 15/group, P = 0.051). VT-mediated protection from LPS lethality was lost in Tie-2 heterozygous mice, in agreement with VT's proposed receptor specificity. We conclude that this synthetic Tie-2 agonist, completely unrelated to endogenous Tie-2 ligands, is sufficient to activate the receptor and its downstream pathways in vivo and that the Tie-2 receptor may be an important target for therapeutic evaluation in conditions of pathological vascular leakage.

Experimental assessment of pro-lymphangiogenic growth factors in the treatment of post-surgical lymphedema following lymphadenectomy.

INTRODUCTION: Lymphedema is a frequent consequence of lymph node excision during breast cancer surgery. Current treatment options are limited mainly to external compression therapies to limit edema development. We investigated previously, post-surgical lymphedema in a sheep model following the removal of a single lymph node and determined that autologous lymph node transplantation has the potential to reduce or prevent edema development. In this report, we examine the potential of lymphangiogenic therapy to restore lymphatic function and reduce post-surgical lymphedema. METHODS: Lymphangiogenic growth factors (vascular endothelial growth factor-C (VEGF-C) and angiopoitein-2 (ANG-2)) were loaded into a gel-based drug delivery system (HAMC; a blend of hyaluronan and methylcellulose). Drug release rates and lymphangiogenic signaling in target endothelial cells were assessed in vitro and vascular permeability biocompatibility tests were examined in vivo. Following, the removal of a single popliteal lymph node, HAMC with the growth factors was injected into the excision site. Six weeks later, lymphatic functionality was assessed by injecting (125)Iodoine radiolabelled bovine serum albumin ((125)I-BSA) into prenodal vessels and measuring its recovery in plasma. Circumferential leg measurements were plotted over time and areas under the curves used to quantify edema formation. RESULTS: The growth factors were released over a two-week period in vitro by diffusion from HAMC, with 50% being released in the first 24 hours. The system induced lymphangiogenic signaling in target endothelial cells, while inducing only a minimal inflammatory response in sheep. Removal of the node significantly reduced lymphatic functionality (Nodectomy 1.9 ± 0.9, HAMC alone 1.7 ± 0.8) compared with intact groups (3.2 ± 0.7). There was no significant difference between the growth factor treatment group (2.3 ± 0.73) and the intact group. An increase in the number of regenerated lymphatic vessels at treatment sites was observed with fluoroscopy. Groups receiving HAMC plus growth factors displayed significantly reduced edema (107.4 ± 51.3) compared with non-treated groups (nodectomy 219.8 ± 118.7, and HAMC alone 162.6 ± 141). CONCLUSIONS: Growth factor therapy has the potential to increase lymphatic function and reduce edema magnitude in an animal model of lymphedema. The application of this concept to lymphedema patients warrants further examination.

Experimental assessment of autologous lymph node transplantation as treatment of postsurgical lymphedema.

BACKGROUND: The authors' objective was to test whether the transplantation of an autologous lymph node into a nodal excision site in sheep would restore lymphatic transport function and reduce the magnitude of postsurgical lymphedema. METHODS: As a measure of lymph transport, iodine-125 human serum albumin was injected into prenodal vessels at 8 and 12 weeks after surgery, and plasma levels of the protein were used to calculate the transport rate of the tracer to blood (percent injected per hour). Edema was quantified from the circumferential measurement of the hind limbs. RESULTS: The transplantation of avascular lymph nodes at 8 (n = 6) and 12 weeks (n = 6) produced lymphatic function levels of 12.3 +/- 0.5 and 12.6 +/- 0.8, respectively. These values were significantly less (p < 0.001) than those measured at similar times in the animals receiving sham surgical procedures (16.6 +/- 0.7, n = 6; and 16.1 +/- 0.7, n = 6, respectively). When vascularized transplants were performed, lymphatic function was similar to the sham controls and significantly greater (p < 0.001) than that of the avascular group (8 weeks, 15.8 +/- 0.9, n = 8; 12 weeks, 15.7 +/- 1.0, n = 10). Lymph transport correlated significantly with the health of the transplanted nodes (scaled with histologic analysis) (p < 0.0001). The vascularized node transplants (n = 18) were associated with the greatest clinical improvement, with the magnitude of edema in these limbs exhibiting significantly lower levels of edema (p = 0.039) than nontreated limbs (n = 18). CONCLUSIONS: The successful reimplantation of a lymph node into a nodal excision site has the potential to restore lymphatic function and facilitate edema resolution. This result has important conceptual implications in the treatment of postsurgical lymphedema.

Lymphedema development and lymphatic function following lymph node excision in sheep.

BACKGROUND: Our objective was to develop an animal model of postsurgical lymphedema that would permit quantitation of edema and lymphatic function after the removal of a single popliteal lymph node in sheep. METHODS: As a measure of lymph transport, (125)I-human serum albumin was injected into prenodal vessels at 8, 12 and 16 weeks after nodal excision, and plasma levels of the protein tracer were used to calculate the transport rate of the tracer to blood (percent injected per hour). Edema was quantified from the circumferential measurement of the hind limbs. RESULTS AND CONCLUSIONS: Following nodal excision, the limbs became progressively more edematous up to 3 days after nodectomy. After this, the swelling decreased but had not resolved even at 16 weeks after surgery. Compared with control limbs (17.2 +/- 0.6; n = 7), lymphatic function was depressed at 8 weeks after surgery (10.6 +/- 1.5; n = 7). At 12 (14.4 +/- 1.0; n = 7) and 16 weeks (13.9 +/- 1.0; n = 6), regeneration of lymphatic vessels at the excision site helped to restore about 80% of lymphatic capacity. These techniques may be helpful in understanding the pathophysiology associated with cancer-related postsurgical lymphedema and may facilitate the development of new strategies to treat or prevent this condition.

Acceleration of diabetic wound healing by an angiopoietin peptide mimetic.

Angiopathies are one of the leading underlying causes of morbidity in diabetic patients. Poorly managed blood glucose levels contribute to vascular defects that manifest themselves in numerous different clinical conditions, including diabetic retinopathy, nephropathy, peripheral artery disease, and compromised wound healing. The angiopoietin family (Angs 1-4) has been shown to play a critical role in the growth and maintenance of vasculature. Here we evaluate the efficacy of a new Ang-based peptidomimetic compound, Vasculotide, on diabetic-related wound healing. Stimulation of endothelial cells (ECs) with Vasculotide results in activation of the Ang receptor, Tie 2, and its associated signaling pathways. This activation promoted biological responses such as EC survival, migration, and matrix metalloproteinase 2 (MMP2) production. We show that Vasculotide alone and in combination with vascular endothelial growth factor (VEGF) results in the production of well-arborized vessels supported by myogenic cells. Using an excisional skin-wound model produced on the back of diabetic B6.Cg-m(+/+)Lepr(db)/J (db/db) mice, we found that Vasculotide-treated wounds presented with decreased wound closure times (p < 0.05) and dramatic increases in granulation tissue (p < 0.01). Although the potential of this novel proangiogenic compound in treating microvascular dysfunction is not strictly limited to topical administration, we provide mechanistic evidence as a proof of principle in support of its efficacious use in diabetic wound healing.

The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis.

Nutrients and bioenergetics are prerequisites for proliferation and survival of mammalian cells. We present evidence that the cyclin-dependent kinase inhibitor p27(Kip1), is phosphorylated at Thr 198 downstream of the Peutz-Jeghers syndrome protein-AMP-activated protein kinase (LKB1-AMPK) energy-sensing pathway, thereby increasing p27 stability and directly linking sensing of nutrient concentration and bioenergetics to cell-cycle progression. Ectopic expression of wild-type and phosphomimetic Thr 198 to Asp 198 (T198D), but not unstable Thr 198 to Ala 198 (p27(T198A)) is sufficient to induce autophagy. Under stress conditions that activate the LKB1-AMPK pathway with subsequent induction of autophagy, p27 knockdown results in apoptosis. Thus LKB1-AMPK pathway-dependent phosphorylation of p27 at Thr 198 stabilizes p27 and permits cells to survive growth factor withdrawal and metabolic stress through autophagy. This may contribute to tumour-cell survival under conditions of growth factor deprivation, disrupted nutrient and energy metabolism, or during stress of chemotherapy.

Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice.

Hemangioma of infancy is the most common neoplasm of childhood. While hemangiomas are classic examples of angiogenesis, the angiogenic factors responsible for hemangiomas are not fully understood. Previously, we demonstrated that malignant endothelial tumors arise in the setting of autocrine loops involving vascular endothelial growth factor (VEGF) and its major mitogenic receptor vascular endothelial growth factor receptor 2. Hemangiomas of infancy differ from malignant endothelial tumors in that they usually regress, or can be induced to regress by pharmacologic means, suggesting that angiogenesis in hemangiomas differs fundamentally from that of malignant endothelial tumors. Here, we demonstrate constitutive activation of the endothelial tie-2 receptor in human hemangioma of infancy and, using a murine model of hemangioma, bEnd.3 cells; we show that bEnd.3 hemangiomas produce both angiopoietin-2 (ang-2) and its receptor, tie-2, in vivo. We also demonstrate that inhibition of tie-2 signaling with a soluble tie-2 receptor decreases bEnd.3 hemangioma growth in vivo. The efficacy of tie-2 blockade suggests that either tie-2 activation or ang-2 may be required for in vivo growth. To address this issue, we used tie-2-deficient bEnd.3 hemangioma cells, which, surprisingly, were fully proficient in in vivo growth. Previous studies from our laboratory and others have implicated reactive oxygen-generating nox enzymes in the angiogenic switch, so we examined the effect of nox inhibitors on ang-2 production in vitro and on bEnd.3 tumor growth in vivo. We then inhibited ang-2 production pharmacologically using novel inhibitors of nox enzymes and found that this treatment nearly abolished bEnd.3 hemangioma growth in vivo. Signal-transduction blockade targeting ang-2 production may be useful in the treatment of human hemangiomas in vivo.

The roles of versican V1 and V2 isoforms in cell proliferation and apoptosis.

Versican is a large chondroitin sulfate proteoglycan belonging to the lectican family. Alternative splicing of versican generates at least four isoforms named V0, V1, V2, and V3. We have shown that the versican V1 isoform not only enhanced cell proliferation, but also modulated cell cycle progression and protected the cells from apoptosis. Futhermore, the V1 isoform was able to not only activate proto-oncogene EGFR expression and modulate its downstream signaling pathway, but also induce p27 degradation and enhance CDK2 kinase activity. As well, the V1 isoform down-regulated the expression of the proapoptotic protein Bad. By contrast, the V2 isoform exhibited opposite biological activities by inhibiting cell proliferation and down-regulated the expression of EGFR and cyclin A. Furthermore, V2 did not contribute apoptotic resistance to the cells. In light of these results, we are reporting opposite functions for the two versican isoforms whose expression is differentially regulated. Our studies suggest that the roles of these two isoforms are associated with the subdomains CSbeta and CSalpha, respectively. These results were confirmed by silencing the expression of versican V1 with small interfering RNA (siRNA), which abolished V1-enhanced cell proliferation and V1-induced reduction of apoptosis.