Role of Matrix Proteins in Hypoxia and Angiogenesis

Stéphane Germain

The sequence of biological events that permits an organism to maintain tissue viability in hypoxia remains poorly understood. Variations in oxygen concentration lead to respiratory, metabolic and vascular adaptations in tissues. How hypoxic endothelial cells integrate chemical signals with mechanical cues from their local tissue microenvironment in order to produce functional capillary networks that exhibit specialized form remains an open question. A key role of hypoxia in the regulation of many endothelial functions is nevertheless well established and growing evidence show that angiogenesis, defined as the events leading to blood vessels formation by sprouting or growth of preexisting vessels, can be triggered by hypoxia, both during development and in pathological conditions, such as cardiovascular ischemia and tumors. Our team is interested in understanding how angiogenesis and vascular integrity are regulated in hypoxic conditions

Schematic drawing of a filopodial-extending tip cell (dark gray) migrating toward the hypoxic area (gray gradient)

We are using a multidisciplinary approach combining gene discovery approach for complex human diseases, vascular cell biology (endothelial and smooth muscle cells cell cultured in 2-D and 3-D, biochemistry, molecular biology, various imaging approaches (confocal analyses, videomicroscopy), gene expresion analyses, protein production, and preclinical models of human diseases.

Our efforts have recently been focused on characterizing the role of Thrombospondin-1 and Angiopoietin-like 4 in regulating angiogenesis and vascular integrity.

The complementary technical expertise of the members of the team together with the established collaborations with clinicians (Pathology, Urology, Cancer and Biochemistry departments, Hopital Saint-Louis) and in association with the tumor bank of Hopital Saint-Louis, led to the definition of angptl4 mRNA as an accurate marker for primary ccRCC diagnosis. Moreover, angptl4 mRNA expression allows to discriminate the renal origin of metastases from clear-cell carcinomas arising from various organs.

By gain-of-function and loss-of-function approaches, we are also studying ECM composition, deposition, posttranslational modifications and rearrangement in vitro and in vivo. Indeed, hypoxia-driven vascular remodeling is dynamically regulated through modulation of ECM modifying enzyme activities that eventually affect both matricellular proteins and growth factor availability, that eventually affect angiogenesis (Figure 3).
Altogether, our studies aimed at better understanding of the complex interplay between ECs and soluble growth factors and mechanical factors from the ECM will certainly have significant implications for understanding the regulation of developmental and pathological angiogenesis driven by hypoxia.

This assay combines cells grown on Cytodex microcarrier beads with fibrin to provide an easy, rapid, and reliable method for evaluating and measuring angiogenic activity.

Selected publications 2004-2011

Galaup A., Gomez E., Souktani R., Durand M., Cazes A., Monnot C., Teillon J., Le Jan S., Bouleti C., Briois G., Philippe J., Pons S., Martin V., Assaly R., Bonnin P., Ratajczak P., Janin A., Thurston G., Valenzuela D.M., Murphy A.J., Yancopoulos G.D., Tissier R., Berdeaux A., Ghaleh B. & Germain S. (2012), Protection against myocardial infarction and no-reflow through preservation of vascular integrity by angiopoietin-like 4. Circulation, Jan 3;125(1):140-9.

- Flamant M., Bollée G., Schordan S., Fligny C., Rumpel E., Milon M., Schordan E., Sabaa N., Vandermeersch S., Galaup A., Rodenas A., Casal I., Sunnarborg S.W., Salant D.J., Kopp J.B., Threadgill D.W., Quaggin S.E., Dussaule J.C., Germain S., Mesnard L., Endlich K., Boucheix C., Belenfant C., Callard P., Endlich N. & Tharaux P.L. (2011), The Epidermal Growth Factor Receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis; the identification of possible therapy. Nature Medicine, Sep 25;17(10): 1242-50.

- Gomez Perdiguero E., Galaup A.*, Durand M.*, Teillon J., Philippe J., Valenzuela D.M., Murphy A.J., Yancopoulos G.D., Thurston G. & Germain S. (2011), Alteration of developmental and pathological retinal angiogenesis in angptl4-deficient mice. J. Biol. Chem.
* both authors contributed equally

- Bignon M.*, Pichol-Thievend C.*, Hardouin J., Malbouyres M., Bréchot N., Nasciutti L., Barret A., Teillon J., Etienne E., Caron M., Joubert-Caron R., Monnot C., Ruggiero F., Muller L. & Germain S. (2011), Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane. Blood, Oct 6;118(14):3979-89.
* both authors contributed equally

- Desch M., Harlander S., Neubauer B., Gerl M., Germain S., Castrop H., Todorov V.T. (2011), cAMP target sequences enhCRE and CNRE sense low-salt intake to increase human renin gene expression in vivo. Pflugers Arch. May;461(5):567-77.

- Germain S. & Eichmann A. (2010), VEGF and ephrin-B2: a bloody duo. Nat Med. Jul;16(7):752-4.

- Smadja D.M., d'Audigier C., Weiswald L.B., Badoual C., Dangles-Marie V., Mauge L., Evrard S., Laurendeau I., Lallemand F., Germain S., Grelac F., Dizier B., Vidaud M., Bièche I, Gaussem P. (2010), The Wnt antagonist Dickkopf-1 increases endothelial progenitor cell angiogenic potential. Arterioscler Thromb Vasc Biol. Dec;30(12):2544-52.

- Verine J., Lehmann-Che J., Soliman H., Feugeas J.P., Vidal J.S., Mongiat-Artus P., Belhadj S., Philippe J., Lesage M., Wittmer E., Chanel S., Couvelard A., Ferlicot S., Rioux-Leclercq N., Vignaud J.M., Janin A. & Germain S. (2010), Determination of angptl4 mRNA as a diagnostic marker of primary and metastatic clear cell renal-cell carcinoma. PLoS One. Apr 29;5(4):e10421.

- Germain S., Monnot C., Muller L. & Eichmann A. (2010), Hypoxia-driven angiogenesis: role of tip cells and extracellular matrix scaffolding. Curr Opin Hematol. May;17(3):245-51.

- Le Dall J., Ho-Tin-Noé B., Louedec L., Meilhac O., Roncal C., Carmeliet P., Germain S., Michel J.B. & Houard X. (2010), Immaturity of microvessels in haemorrhagic plaques is associated with proteolytic degradation of angiogenic factors. Cardiovasc Res. Jan 1;85(1):184-93.

- Chomel C., Cazes A., Faye C., Bignon M., Gomez E., Ardidie-Robouant C., Barret A., Ricard-Blum S., Muller L., Germain S. & Monnot C. (2009), Interaction of the coiled-coil domain with glycosaminoglycans protects angiopoietin-like 4 from proteolysis and regulates its antiangiogenic activity. FASEB J Mar;23(3):940-9.

- Abou-Khalil R., Le Grand F., Pallafacchina G., Valable S., Authier F.J., Rudnicki M.A., Gherardi R.K., Germain S., Chretien F., Sotiropoulos A., Lafuste P., Montarras D. & Chazaud B. (2009), Autocrine and paracrine angiopoietin 1/Tie-2 signaling promotes muscle satellite cell self-renewal. Cell Stem Cell, Sep 4;5(3):298-309.

- Bréchot N., Gomez E., Bignon M., Khallou-Laschet J., Dussiot M., Cazes A., Alanio-Bréchot C., Durand M., Philippe J., Silvestre J.S., Van Rooijen N., Corvol P., Nicoletti A., Chazaud B. & Germain S. (2008), Modulation of macrophage activation state protects tissue from necrosis during critical limb ischemia in thrombospondin-1-deficient mice. PloS ONE, 3(12):e3950.

- Alliouachene S., Tuttle R.L., Boumard S., Lapointe T., Berissi S., Germain S., Jaubert F. & Tosh M.D. (2008), Constitutively active Akt1 expression in mouse pancreas requires S6 kinase 1 for insulinoma formation. J Clin Invest, 118(11):3629-38.

- Sabaa N., de Franceschi L., Bonnin P., Castier Y., Malpeli G., Debbabi H., Galaup A., Maier-Redelsperger M., Vandermeersch S., Scarpa A., Janin A., Levy B., Girot R., Beuzard Y., Leboeuf C., Henri A., Germain S., Dussaule J.C. & Tharaux P.L. (2008), Endothelin receptor antagonism prevents hypoxia-induced mortality and morbidity in a mouse model of sickle-cell disease. J Clin Invest, May;118(5):1924-33.

- Magnon C., Opolon P., Ricard M., Connault E., Ardouin P., Galaup A., Métivier D., Bidart J.M., Germain S., Perricaudet M. & Schlumberger M. (2007), Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1alpha-mediated tumor apoptotic switch. J Clin Invest, Jul;117(7):1844-55.

- Galaup A., Cazes A., Le Jan S., Philippe J., Connault E., Le Coz E., Mekid E., Mir L.M., Opolon P., Corvol P., Monnot C. & Germain S. (2006), Angiopoietin-like 4 prevents metastasis through inhibition of vascular permeability and tumor cell motility and invasiveness. Proc Natl Acad Sci, Dec 5;103(49):18721-6.

- Cazes A., Galaup A., Chomel C., Bignon M., Brechot N., Le Jan S., Weber H., Corvol P., Muller L., Germain S. & Monnot C. (2006), Extracellular matrix-bound angiopoietin-like 4 inhibits endothelial cell adhesion, migration, and sprouting and alters actin cytoskeleton. Circ Res, Nov 24;99(11):1207-15.

- Le Jan S., Le Meur N., Cazes A., Philippe J., Le Cunff M., Léger J., Corvol P. & Germain S. (2006), Characterization of the expression of the hypoxia-induced genes neuritin, TXNIP and IGFBP3 in cancer. FEBS Letters, Jun 12;580(14):3395-400.

- Kermorvant-Duchemin E., Sennlaub F., Sirinyan M., Brault S., Andelfinger G., Kooli A, Germain S., Ong H., d’Orleans-Juste P., Gobeil Jr F., Zhu T., Boisvert C., Hardy P., Jain K., Falck J.R., Balazy M. & Chemtob S. (2005), Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1-dependent microvascular degeneration. Nature Medicine, Dec;11(12):1339-45.

People

Director :
Germain Stéphane, DR2 INSERM

Senior researchers :
Monnot Catherine, CR1 INSERM
Muller Laurent, CR1 INSERM

Clinicians :
Cazes Aurélie, MCU AP-HP
Verine Jérôme, AHU AP-HP

Post-doctoral fellows :
Galaup Ariane, CDD chercheurs confirmés INSERM
Gauvrit Sébastien

Doctorants :
Pichol-Thievend Cathy
Beckouche Nathan
Bouleti Claire
Briois Gaëlle

Technical staff :
Barret Alain, CNRS
Lesage Mathieu, CDF
Robouant Ardidie Corinne, CDF