In Vivo Matrigel Plug Assay as a Potent Method to Investigate Specific Individual Contribution of Angiogenesis to Blood Flow Recovery in Mice.

Neovascularization restores blood flow recovery after ischemia in peripheral arterial disease. The main two components of neovascularization are angiogenesis and arteriogenesis. Both of these processes contribute to functional improvements of blood flow after occlusion. However, discriminating between the specific contribution of each process is difficult. A frequently used model for investigating neovascularization is the murine hind limb ischemia model (HLI). With this model, it is difficult to determine the role of angiogenesis, because usually the timing for the sacrifice of the mice is chosen to be optimal for the analysis of arteriogenesis. More importantly, the occurring angiogenesis in the distal calf muscles is probably affected by the proximally occurring arteriogenesis. Therefore, to understand and subsequently intervene in the process of angiogenesis, a model is needed which investigates angiogenesis without the influence of arteriogenesis. In this study we evaluated the in vivo Matrigel plug assay in genetic deficient mice to investigate angiogenesis. Mice deficient for interferon regulatory factor (IRF)3, IRF7, RadioProtective 105 (RP105), Chemokine CC receptor CCR7, and p300/CBP-associated factor (PCAF) underwent the in vivo Matrigel model. Histological analysis of the Matrigel plugs showed an increased angiogenesis in mice deficient of IRF3, IRF7, and RP105, and a decreased angiogenesis in PCAF deficient mice. Our results also suggest an involvement of CCR7 in angiogenesis. Comparing our results with results of the HLI model found in the literature suggests that the in vivo Matrigel plug assay is superior in evaluating the angiogenic response after ischemia.

Variations in Surgical Procedures for Inducing Hind Limb Ischemia in Mice and the Impact of These Variations on Neovascularization Assessment.

Mouse hind limb ischemia is the most common used preclinical model for peripheral arterial disease and critical limb ischemia. This model is used to investigate the mechanisms of neovascularization and to develop new therapeutic agents. The literature shows many variations in the model, including the method of occlusion, the number of occlusions, and the position at which the occlusions are made to induce hind limb ischemia. Furthermore, predefined end points and the histopathological and radiological analysis vary. These differences hamper the correlation of results between different studies. In this review, variations in surgical methods of inducing hind limb ischemia in mice are described, and the consequences of these variations on perfusion restoration and vascular remodeling are discussed. This study aims at providing the reader with a comprehensive overview of the methods so far described, and proposing uniformity in research of hind limb ischemia in a mouse model.

CCR7-CCL19/CCL21 Axis is Essential for Effective Arteriogenesis in a Murine Model of Hindlimb Ischemia.

BACKGROUND: In order to identify factors that stimulate arteriogenesis after ischemia, we followed gene expression profiles in two extreme models for collateral artery formation over 28 days after hindlimb ischemia, namely "good-responding" C57BL/6 mice and "poor-responding" BALB/c mice. METHODS AND RESULTS: Although BALB/c mice show very poor blood flow recovery after ischemia, most known proarteriogenic genes were upregulated more excessively and for a longer period than in C57BL/6 mice. In clear contrast, chemokine genes Ccl19, Ccl21a, and Ccl21c and the chemokine receptor CCR7 were upregulated in C57BL/6 mice 1 day after hindlimb ischemia, but not in BALB/C mice. CCL19 and CCL21 regulate migration and homing of T lymphocytes via CCR7. When subjecting CCR7-/-/LDLR-/- mice to hindlimb ischemia, we observed a 20% reduction in blood flow recovery compared with that in LDLR-/- mice. Equal numbers of α-smooth muscle actin-positive collateral arteries were found in the adductor muscles of both mouse strains, but collateral diameters were smaller in the CCR7-/-/LDLR-/-. Fluorescence-activated cell sorter analyses showed that numbers of CCR7+ T lymphocytes (both CD4+ and CD8+) were decreased in the spleen and increased in the blood at day 1 after hindlimb ischemia in LDLR-/- mice. At day 1 after hindlimb ischemia, however, numbers of activated CD4+ T lymphocytes were decreased in the draining lymph nodes of LDLR-/- mice compared with CCR7-/-/LDLR-/- mice. CONCLUSIONS: These data show that CCR7-CCL19/CCL21 axis facilitates retention CD4+ T lymphocytes at the site of collateral artery remodeling, which is essential for effective arteriogenesis.

A small molecule approach to engineering vascularized tissue.

The repertoire of growth factors determines the biological engagement of human mesenchymal stromal cells (hMSCs) in processes such as immunomodulation and tissue repair. Hypoxia is a strong modulator of the secretome and well known stimuli to increase the secretion of pro-angiogenic molecules. In this manuscript, we employed a high throughput screening assay on an hMSCs cell line in order to identify small molecules that mimic hypoxia. Importantly, we show that the effect of these small molecules was cell type/species dependent, but we identified phenanthroline as a robust hit in several cell types. We show that phenanthroline induces high expression of hypoxia-target genes in hMSCs when compared with desferoxamine (DFO) (a known hypoxia mimic) and hypoxia incubator (2% O(2)). Interestingly, our microarray and proteomics analysis show that only phenanthroline induced high expression and secretion of another angiogenic cytokine, interleukin-8, suggesting that the mechanism of phenanthroline-induced hypoxia is distinct from DFO and hypoxia and involves the activation of other signaling pathways. We showed that phenanthroline alone was sufficient to induce blood vessel formation in a Matrigel plug assay in vivo paving the way to its application in ischeamic-related diseases.