Formation of new blood vessels in the heart can be studied in cell cultures.

Controlled induction of the formation of new microvessels, i.e., therapeutic angiogenesis, may be used one day to treat patients that for example had suffered a myocardial infarction. Experimental models of angiogenesis in the heart in vivo substantially stress the animal. We therefore developed a model of angiogenesis of the heart in vitro, where mouse and rat heart pieces are stimulated under controlled conditions in a three dimensional matrix. Capillary-like sprouts emerging in these cultures represent early to midterm steps of angiogenesis and can be quantified to study potential angiogenic compounds and underlying mechanisms.

A versatile in vitro assay for investigating angiogenesis of the heart.

Neovascularization in the heart is usually investigated with models of angiogenesis in vivo. Here we present a simple model that allows investigating heart angiogenesis in mice and rats in vitro. Small pieces of left ventricular myocardium were cultured in three-dimensional fibrin gels for 10 days. A single mouse heart allowed assessing 24 conditions, each tested in octuplicates. Rat recombinant VEGF164, human recombinant bFGF, and human recombinant PDGF-BB were used under normoxia (21% O2) and hypoxia (3% O2), and outgrowth of endothelial sprouts from heart pieces was quantified. In 4-week-old OF1 mice, endothelial sprouts formed spontaneously. In contrast, in 12-week-old adult mice, virtually no sprouts formed under normoxia. Under hypoxia, sprout formation increased substantially. Different growth factors induced formation of distinct patterns of sprouts and unorganized single cells. Sprouts were composed of endothelial cells with smooth muscle cells or pericytes interacting with them, as assessed by immunohistochemistry. Taken together, our model is suited for investigation of angiogenesis of the heart in vitro. It may allow performing extensive series of experiments in vitro including rapid screening of pharmacological compounds and assessment of mechanisms of heart angiogenesis in transgenic animals in an easy straightforward manner.

Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR)-dependent signaling.

Angiogenesis and vascular cell proliferation are pivotal in physiological and pathological processes including atherogenesis, restenosis, wound healing, and cancer development. Here we show that mammalian target of rapamycin (mTOR) signaling plays a key role in hypoxia-triggered smooth muscle and endothelial proliferation and angiogenesis in vitro. Hypoxia significantly increased DNA synthesis and proliferative responses to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) in rat and human smooth muscle and endothelial cells. In an in vitro 3-dimensional model of angiogenesis, hypoxia increased PDGF- and FGF-stimulated sprout formation from rat and mouse aortas. Hypoxia did not modulate PDGF receptor mRNA, protein, or phosphorylation. PI3K activity was essential for cell proliferation under normoxic and hypoxic conditions. Activities of PI3K-downstream target PKB under hypoxia and normoxia were comparable. However, mTOR inhibition by rapamycin specifically abrogated hypoxia-mediated amplification of proliferation and angiogenesis, but was without effect on proliferation under normoxia. Accordingly, hypoxia-mediated amplification of proliferation was further augmented in mTOR-overexpressing endothelial cells. Thus, signaling via mTOR may represent a novel mechanism whereby hypoxia augments mitogen-stimulated vascular cell proliferation and angiogenesis.

Tracheal development in Drosophila melanogaster as a model system for studying the development of a branched organ.

The development of the tracheal system of Drosophila melanogaster represents a paradigm for studying the molecular mechanisms involved in the formation of a branched tubular network. Tracheogenesis has been characterized at the morphological, cellular and genetic level and a series of successive, but linked events have been described as the basis for the formation of the complex network of tubules which extend over the entire organism. Tracheal cells stop to divide early in the process of tracheogenesis and the formation of the interconnected network requires highly controlled cell migration events and cell shape changes. A number of genes involved in these two processes have been identified but in order to obtain a more complete view of branching morphogenesis, many more genes carrying essential functions have to be isolated and characterized. Here, we provide a progress report on our attempts to identify further genes expressed in the tracheal system. We show that empty spiracles (ems), a head gap gene, is required for the formation of a specific tracheal branch, the visceral branch. We also identified a Sulfotransferase and a Multiple Inositol Polyphosphate phosphatase that are strongly upregulated in tracheal cells and discuss their possible involvement in tracheal development.

Inhibition of NO biosynthesis, but not elevated blood pressure, reduces angiogenesis in rat models of secondary hypertension.

Arterial hypertension (AH) is characterized by reduced nitric oxide (NO) biosynthesis, vasoconstriction, and reduced microvascular density. In this study we asked whether AH also reduces the number of microvessels by impairing angiogenesis. AH was induced in Dahl salt-sensitive rats (DSS) with a salt diet and in Wistar-Kyoto rats by inhibiting NO formation with Nomega-nitro-L-arginine (NNA). Three weeks after induction of AH, two wound chambers containing collagen I (Vitrogen) were sutured into the mesenteric cavity of each animal. After additional 14 days, wound chamber neovascularization and the extent of vascularized connective tissue ingrowth were quantified. In NNA-induced AH, the number of newly formed vessels and the ingrowth of vascularized connective tissue into the wound chamber decreased as compared to controls. However, the number of newly formed vessels and the ingrowth of vascularized connective tissue did not change with increasing blood pressure in salt-fed DSS rats as compared to those fed a normal diet. Inhibition of NO biosynthesis, but not necessarily elevating blood pressure, reduces angiogenesis. Microvascular rarefaction in AH may be partially due to reduced angiogenesis because of impaired NO biosynthesis.