Functional genomics of vascular endothelial cells

Angiogenesis, the formation of new blood vessels from preexisting ones, is a process involved in normal development as well as in several pathological conditions, such as cancer, ischemic heart disease, wound healing and certain retinal complications. Antiangiogenic targeting is therefore a promising new therapeutic principle. However, few blood vessel-specific drug targets have been identified, and information is still limited about endothelial cell (EC)-specific molecular processes. Here we aimed at identifying novel key players and signaling pathways during angiogenesis, and to determine the EC-specific core transcriptome in vivo.

During angiogenesis, specialized endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of vascular endothelial growth factor (VEGF)-A. We found that Delta-like 4 (DLL4)/Notch1 signaling regulated the formation of appropriate numbers of tip cells to control vessel sprouting and branching in the developing postnatal retina. Inhibition of Notch-signaling led to excessive tip cell formation, and increased vascular density. Conversely, activation of Notch-signaling led to fewer tip cells and reduced vessel density. DLL4/Notch1-signaling between ECs therefore restricts tip cell formation in response to VEGF, leading to correct sprouting and branching patterns. We also found that blocking VEGF receptor 3 (VEGFR-3) signaling with antibodies resulted in decreased sprouting, vascular density, vessel branching, and EC proliferation. Antibodies against VEGFR-3 and VEGFR-2 in combination had additive effects. Notch inhibition led to endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3. These findings suggest that Notch and VEGFR-3 signaling may constitute new targets for anti-angiogenic therapy.

In order to identify additional candidate vascular drug targets, we combined publicly available gene expression data with own transcriptional profiles of mouse microvasculature. In this way we identified 58 gene transcripts with broad and specific expression in microvascular endothelium, of which 32 presently lack known functions in vascular biology. 7 of the 32 genes showed considerably enriched expression in the microvasculature, namely: Eltd1, Gpr116, Ramp2, Slc9a3r2, Slc43a3, and NM_023516. The 32 gene products were all predicted to be cell surface expressed, or implicated in cell signaling processes, and are therefore interesting as putative microvascular drug targets. We also identified yet another set of new candidate vascular targets by combining reverse- and chemical genetics. In the reverse genetics screen, 50 genes were knocked down in zebrafish and 16 of these were found to be necessary for developmental angiogenesis. In the chemical genetics screen, 28 compounds targeting 69 proteins selectively inhibited endothelial sprouting. The reverse- and chemical genetics screens identified an overlap of three members of a superfamily of serine/threonine (S/T) protein phosphatases, Ppp1ca, Ppp1cc and Ppp4c, and one compound, Endothall, targeting that family. Treatment of zebrafish with Endothall led to a dose-dependent effect on lumen formation, similar to that seen in zebrafish knockdowns of the identified S/T protein phosphatases.

The discoveries made in this study span from detailed insights into specific endothelial signaling pathways to global effects on endothelial gene expression, representing different angles of angiogenesis and vascular biology research. Overall, the results in this study contribute to the understanding of the vasculature and its transcriptome.

List of scientific papers

I. Hellström M, Phng LK, Hofmann JJ, Wallgard E, Coultas L, Lindblom P, Alva J, Nilsson AK, Karlsson L, Gaiano N, Yoon K, Rossant J, Iruela-Arispe ML, Kalén M, Gerhardt H, Betsholtz C (2007). Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature. 445(7129): 776-80. Epub 2007 Jan 28
https://pubmed.ncbi.nlm.nih.gov/17259973

II. Tammela T, Zarkada G, Wallgard E, Murtomäki A, Suchting S, Wirzenius M, Waltari M, Hellström M, Schomber T, Peltonen R, Freitas C, Duarte A, Isoniemi H, Laakkonen P, Christofori G, Ylä-Herttuala S, Shibuya M, Pytowski B, Eichmann A, Betsholtz C, Alitalo K (2008). Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature. 454(7204): 656-60. Epub 2008 Jun 25
https://pubmed.ncbi.nlm.nih.gov/18594512

III. Wallgard E, Larsson E, He L, Hellström M, Armulik A, Nisancioglu MH, Genove G, Lindahl P, Betsholtz C (2008). Identification of a core set of 58 gene transcripts with broad and specific expression in the microvasculature. Arterioscler Thromb Vasc Biol. 28(8): 1469-76. Epub 2008 May 15
https://pubmed.ncbi.nlm.nih.gov/18483404

IV. Kalén M, Wallgard E, Asker N, Nasevicius A, Billgren E, Larson JD, Wadman SA, Clark KJ, He L, Karlsson-Lindahl L, Häger AK, Samuelsson T, Essner JJ, Hackett PB, Hellström M (2008). Combination of reverse- and chemical genetic screens reveals novel angiogenesis inhibitors and targets. [Manuscript]