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1.  Angiopoietin-2 Is Critical for Cytokine-Induced Vascular Leakage 
PLoS ONE  2013;8(8):e70459.
Genetic experiments (loss-of-function and gain-of-function) have established the role of Angiopoietin/Tie ligand/receptor tyrosine kinase system as a regulator of vessel maturation and quiescence. Angiopoietin-2 (Ang-2) acts on Tie2-expressing resting endothelial cells as an antagonistic ligand to negatively interfere with the vessel stabilizing effects of constitutive Ang-1/Tie-2 signaling. Ang-2 thereby controls the vascular response to inflammation-inducing as well as angiogenesis-inducing cytokines. This study was aimed at assessing the role of Ang-2 as an autocrine (i.e. endothelial-derived) regulator of rapid vascular responses (within minutes) caused by permeability-inducing agents. Employing two independent in vivo assays to quantitatively assess vascular leakage (tracheal microsphere assay, 1–5 min and Miles assay, 20 min), the immediate vascular response to histamine, bradykinin and VEGF was analyzed in Ang-2-deficient (Ang-2−/−) mice. In comparison to the wild type control mice, the Ang2−/− mice demonstrated a significantly attenuated response. The Ang-2−/− phenotype was rescued by systemic administration (paracrine) of an adenovirus encoding Ang-2. Furthermore, cytokine-induced intracellular calcium influx was impaired in Ang-2−/− endothelioma cells, consistent with reduced phospholipase activation in vivo. Additionally, recombinant human Ang-2 (rhAng-2) alone was unable to induce vascular leakage. In summary, we report here in a definite genetic setting that Ang-2 is critical for multiple vascular permeability-inducing cytokines.
doi:10.1371/journal.pone.0070459
PMCID: PMC3734283  PMID: 23940579
2.  Measurement of Angiogenic Phenotype by Use of Two-Dimensional Mesenteric Angiogenesis Assay 
Successful therapeutic angiogenesis requires an understanding of how the milieu of growth factors available combine to form a mature vascular bed. This requires a model in which multiple physiological and cell biological parameters can be identified. The adenoviral-mediated mesenteric angiogenesis assay as described here is ideal for that purpose. Adenoviruses expressing growth factors (vascular endothelial growth factor [VEGF] and angiopoietin 1 [Ang-1]) were injected into the mesenteric fat pad of adult male Wistar rats. The clear, thin, and relatively avascular mesenteric panel was used to measure increased vessel perfusion by intravital microscopy. In addition, high-powered microvessel analysis was carried out by immunostaining of features essential for the study of angiogenesis (endothelium, pericyte, smooth muscle cell area, and proliferation), allowing functional data to be obtained in conjunction with high-power microvessel ultrastructural analysis. A combination of individual growth factors resulted in a distinct vascular phenotype from either factor alone, with all treatments increasing the functional vessel area. VEGF produced shorter, narrow, highly branched, and sprouting vessels with normal pericyte coverage. Ang-1 induced broader, longer neovessels with no apparent increase in branching or sprouting. However, Ang-1-induced blood vessels displayed a significantly higher pericyte ensheathment. Combined treatment resulted in higher perfusion, larger and less-branched vessels, with normal pericyte coverage, suggesting them to be more mature. This model can be used to show that Ang-1 and VEGF use different physiological mechanisms to enhance vascularisation of relatively avascular tissue.
PMCID: PMC2879323  PMID: 19301676
Angiogenesis; angiopoietin 1; pericyte; VEGF
3.  Critical Role of Tissue Kallikrein in Vessel Formation and Maturation 
Objective
Human Tissue Kallikrein (hKLK1) overexpression promotes an enduring neovascularization of ischemic tissue, yet the cellular mechanisms of hKLK1-induced arteriogenesis remain unknown. Furthermore, no previous study has compared the angiogenic potency of hKLK1, with its loss of function polymorphic variant, rs5515 (R53H), which possesses reduced kinin-forming activity.
Methods and Results
Here, we demonstrate that tissue kallikrein knockout mice (KLK1−/−) show impaired muscle neovascularization in response to hindlimb ischemia. Gene-transfer of wild-type Ad.hKLK1 but not Ad.R53H-hKLK1 was able to rescue this defect. Similarly, in the rat mesenteric assay, Ad.hKLK1 induced a mature neovasculature with increased vessel diameter through kinin-B2 receptor-mediated recruitment of pericytes and vascular smooth muscle cells, whereas Ad.R53H-hKLK1 was ineffective. Moreover, hKLK1 but not R53H-hKLK1 overexpression in the zebrafish induced endothelial precursor cell migration and vascular remodeling. Furthermore, Ad.hKLK1 activates metalloproteinase (MMP) activity in normoperfused muscle and fails to promote reparative neovascularization in ischemic MMP9−/− mice, whereas its proarteriogenic action was preserved in ApoE−/− mice, an atherosclerotic model of impaired angiogenesis.
Conclusions
These results demonstrate the fundamental role of endogenous Tissue Kallikrein in vascular repair and provide novel information on the cellular and molecular mechanisms responsible for the robust arterialization induced by hKLK1 overexpression.
doi:10.1161/ATVBAHA.108.182139
PMCID: PMC2827862  PMID: 19164804
angiogenesis; gene therapy; gene mutations; metalloproteinases; tissue kallikrein
4.  Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice 
The Journal of Clinical Investigation  2009;119(8):2359-2365.
Hemangiomas are the most common type of tumor in infants. As they are endothelial cell–derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T–transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/–, and Ang2–/– mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/– cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/– cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function.
doi:10.1172/JCI33877
PMCID: PMC2719922  PMID: 19620773
5.  VEGF-C induced angiogenesis preferentially occurs at a distance from lymphangiogenesis 
Cardiovascular research  2007;78(2):315-323.
Aims
Vascular endothelial growth factor-C (VEGF-C) has been shown to stimulate both angiogenesis and lymphangiogenesis in some but not all models where VEGF-C is over-expressed. Our aim was to investigate the interaction between lymphangiogenesis and angiogenesis in adult tissues regulated by VEGF-C and identify evidence of polarized growth of lymphatics driven by specialized cells at the tip of the growing sprout.
Methods and results
We used an adult model of lymphangiogenesis in the rat mesentery. The angiogenic effect of VEGF-C was markedly attenuated in the presence of a growing lymphatic network. Furthermore, we show that this growth of lymphatic vessels can occur both by recruitment of isolated lymphatic islands to a connected network and by filopodial sprouting. The latter is independent of polarized tip cell differentiation that can be generated all along lymphatic capillaries, independently of the proliferation status of the lymphatic endothelial cells.
Conclusion
These results both demonstrate a dependence of VEGF-C-mediated angiogenesis on lymphatic vascular networks and indicate that the mechanism of VEGF-C-mediated lymphangiogenesis is different from that of classical angiogenic mechanisms.
doi:10.1093/cvr/cvm094
PMCID: PMC2613351  PMID: 18065770
Angiogenesis; Lymphangiogenesis; VEGF-C; Sprouting
6.  Arteriolar Genesis and Angiogenesis Induced by Endothelial Nitric Oxide Synthase Overexpression Results in a Mature Vasculature 
Background
Generation of physiologically active vascular beds by delivery of combinations of growth factors offers promise for vascular gene therapy.
Methods and Results
In a mesenteric model of physiological angiogenesis, combining endothelial nitric oxide synthase (eNOS) (and hence NO production) with VEGF and angiopoietin-1 overexpression resulted in a more functional vascular phenotype than growth factor administration alone. eNOS gene delivery upregulated eNOS, VEGF, and Ang-1 to similar levels as gene transfer with VEGF or Ang-1. eNOS overexpression resulted in neovascularization to a similar extent as VEGF and Ang-1 combined, but not by sprouting angiogenesis. Whereas combining Ang-1 and VEGF increased both exchange vessels and conduit vessels, neither growth factor nor eNOS alone resulted in vessels with smooth muscle cell (SMC) coverage. In contrast, combining all three generated microvessels with SMCs (arteriolar genesis) and further increased functional vessels. Use of a vasodilator, prazosin, in combination with Ang1 and VEGF, but not alone, also generated SMC-positive vessels.
Conclusion
Coexpression of eNOS, VEGF, and Ang-1 results in a more mature vascularization of connective tissue, and generates new arterioles as well as new capillaries, and provides a more physiological therapeutic approach than single growth factor administration, by combining hemodynamic forces with growth factors.
doi:10.1161/ATVBAHA.108.169375
PMCID: PMC2566940  PMID: 18497305
angiogenesis; arteriogenesis; VEGF; Ang-1; eNOS; pericyte; vascular smooth muscle

Results 1-6 (6)