Methodological Approach to Use Fresh and Cryopreserved Vessels as Tools to Analyze Pharmacological Modulation of the Angiogenic Growth.

The sprouting of new vessels is greatly influenced by the procedure chosen. We sought to optimize the experimental conditions of the angiogenic growth of fresh and cryopreserved vessels cultured in Matrigel with the aim to use this system to analyze the pharmacological modulation of the process. Segments of second-order branches of rat mesenteric resistance arteries, thoracic aorta of rat or mouse, and cryopreserved rat aorta and human femoral arteries were cultured in Matrigel for 7-21 days in different mediums, as well as in the absence of endothelial or adventitia layer. Quantification of the angiogenic growth was performed by either direct measurement of the mean length of the neovessels or by calcein AM staining and determination of fluorescence intensity and area. Fresh and cryopreserved arterial rings incubated in Matrigel exhibited a spontaneous angiogenic response that was strongly accelerated by fetal calf serum. Addition of vascular endothelial growth factor, fibroblast growth factor, endothelial growth factor, or recombinant insulin-like growth factor failed to increase aortic sprouting, unless all were added together. Removal of adventitia, but not the endothelial layer, abrogated the angiogenic response of aortic rings. Determination of the mean neovessel length is an easy and accurate method to quantify the angiogenic growth devoid of confounding factors, such as inclusion of other cellular types surrounding the neovessels. Activity of a α1-adrenoceptor agonist (phenylephrine) and its inhibition by a selective antagonist (prazosin) were analyzed to prove the usefulness of the Matrigel system to evaluate the pharmacological modulation of the angiogenic growth.

Investigation of new 2-aryl substituted Benzothiopyrano[4,3-d]pyrimidines as kinase inhibitors targeting vascular endothelial growth factor receptor 2.

Vascular Endothelial Growth Factor (VEGF) pathway has emerged as one of the most important positive modulators of Angiogenesis, a central process implicated in tumour growth and metastatic dissemination. This led to the design and development of anti-VEGF monoclonal antibodies and small-molecule ATP-competitive VEGFR-inhibitors. In this study, we describe the synthesis and the biological evaluation of novel 2-aryl substituted benzothiopyrano-fused pyrimidines 1a-i, 2a-i and 3a-i. The ability of the compounds to target the VEGF pathway was determined in vitro exploiting the compounds' antiproliferative efficacy against HUVEC cells. The VEGFR-2 inhibition was confirmed by enzymatic assays on recombinant human kinase insert domain receptor (KDR), by cell-based phospho-VEGFR-2 inhibition assays, and by ex vivo rat aortic ring tests. The selectivity profile of the best performing derivatives belonging to series 2 was further explored combining modeling studies and additional assays in a panel of human cell lines and other kinases.

Medicinal chemistry of indolylglyoxylamide GABAA/BzR high affinity ligands: identification of novel anxiolytic/non sedative agents.

The classical benzodiazepines (Bz) constitute a well-known class of therapeutics displaying hypnotic, anxiolytic and anticonvulsant effects acting upon a specific binding site (BzR) belonging to the GABAA receptor complex. Their usefulness, however, is limited by a broad range of side effects; consequently the fact that the action of GABA with the receptor complex could be allosterically modulated by a wide variety of chemical entities, made the Bz binding site, from late eighties to nowdays, the target of extensive research programmes directed to the identification of new ligands displaying varying degrees of affinity- and efficacy-selectivity for the different GABAA/BzR-subtypes. The principal aim has been to discover ideal sedative-hypnotic agents (selective 1 agonists), anxiolytic agents (selective 2/ 3 agonists), or cognitive enhancers (selective 5 inverse agonists). In this connection, an important contribution in the field of GABAA/BzR ligands was made by the research group directed by Professor Antonio Da Settimo at the University of Pisa. The purpose of this review is therefore to describe the studies, performed from early '80s, on the several classes of BzR ligands developed featuring the indol-3-ylglyoxyl scaffold. All the compounds reported have been summarized on the basis of their main chemical structural features, focusing attention on their SARs, which determined the affinity profiles or efficacy-selectivity. Moreover, the biological studies performed within each class of compounds allowed the identification of new derivatives exhibiting an anxiolytic/nonsedative profile, either in vitro (full 2 agonism and 1 partial agonism/ antagonism) and in vivo (anxiolytic/nonsedative activity in mice).