Role of Matrix Proteins in Hypoxia and Angiogenesis

Principal Investigator: Stéphane GERMAIN, DR1 Inserm

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 (EC) 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 (Fig 1).

Germain - Figure 1

Figure 1: The filopodia are guided on the interstitial fibronectin network (black strings). The provisional extracellular matrix (ECM) is remodeled at the front of the tip cell (gray network) and the basement membrane (black line) is deposited at the back of the tip cell as well as by stalk cells (white) and surrounding pericytes (light gray).

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We are using a multidisciplinary approach combining gene discovery approach for complex human diseases, vascular cell biology (endothelial and smooth muscle cells) cell cultures 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, Angiopoietin-like 4 and Lysysl Oxidase like 2 in regulating angiogenesis and vascular integrity.

By gain-of-function and loss-of-function approaches, both in vitro and in vivo, we are also studying ECM composition, deposition, posttranslational modifications and rearrangement by ECs. Indeed, hypoxia-driven vascular remodeling is dynamically regulated through activities of ECM modifying enzyme, such as Lysysl Oxidase like 2 or Transgluaminase-2 that eventually regulate both matricellular proteins and growth factor availability, that eventually affect angiogenesis (Figure 2&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.

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.


Figure 2: Confocal images of Tg(fli1:egfp)y1 zebrafish embryos at 24 and 48 hpf, injected with either mismatch (ms-MO) or loxl2a morpholinos (loxl2a-MO1). Bar: 100 µm.

Germain Capillary formation

Selected publications 2004-2017

- Ferratge, S., Ha, G., Carpentier, G., Arouche, N., Bascetin, R., Muller, L., Germain, S. & Uzan, G. (2017), Initial clonogenic potential of human endothelial progenitor cells is predictive of their further properties and establishes a functional hierarchy related to immaturity. Stem Cell Res 21, 148–159. 

- Gomez Perdiguero E, Liabotis-Fontugne A, Durand M, Faye C, Ricard-Blum S, Simonutti M, Augustin S, Robb BM, Paques M, Valenzuela DM, Murphy AJ, Yancopoulos GD, Thurston G, Galaup A, Monnot C & Germain S. (2016), ANGPTL4-αvβ3 integrin interaction counteracts hypoxia-induced vascular permeability by modulating Src signaling downstream of VEGFR2. J Pathol.

- Chevalier, N.R., Gazguez, E., Bidault, L., Guilbert, T., Vias, C., Vian, E., Watanabe, Y., Muller, L., Germain, S., Bondurand, N., Dufour, S. & Fleury, V. (2016), How Tissue Mechanical Properties Affect Enteric Neural Crest Cell Migration. Sci Rep, Feb 18;6:20927.

- Gorin, C., Rochefort, G.Y., Bascetin, R., Ying, H., Lesieur, J., Sadoine, J., Beckouche, N., Berndt, S., Novais, A., Lesage, M., Hosten, B., Vercellino, L., Merlet, P., Le-Denmat, D., Marchiol, C., Letourneur, D., Nicoletti, A., Vital, S.O., Poliard, A., Salmon, B., Muller, L., Chaussain, C. & Germain, S. (2016), Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion. Stem Cells Transl Med. Mar;5(3):392-404.


Online Cover This week features a Research Article that shows that transglutaminase-2 prevents heparan sulfate from potentiating signaling by a specific VEGF isoform, thereby attenuating blood vessel formation. The image shows retinal vascularization in a mouse deficient in transglutaminase-2.

[Image: Thomas Mathivet/CIRB, CNRS, and INSERM, Paris, France]

- Beckouche N., Bignon M., Lelarge V., Mathivet T., Pichol-Thievend C., Berndt S., Hardouin J., Garand M., Ardidie-Robouant C., Barret A., Melino G., Lortat-Jacob H., Muller L., Monnot C. & Germain S. (2015). The interaction of heparan sulfate proteoglycans with endothelial transglutaminase - 2 limits VEGF165 - induced angiogenesis. Science Signaling 8, ra70.

- Bouleti, C., Mewton, N., Germain, S. (2015), The no-reflow phenomenon: State of the art. Arch Cardiovasc Dis, Dec;108(12): 661–674.

- Bouleti C., Mathivet T., Serfaty J.-M., Vignolles N., Berland E., Monnot C., Cluzel P., Steg P.G., Montalescot G. & Germain S. (2015a). Angiopoietin-like 4 serum levels on admission for acute myocardial infarction are associated with no-reflow. Int. J. Cardiol. 187, 511–516.

- Varna M., Gapihan G., FeugeasJ.P., Ratajczak P., Tan S., Ferreira I., Leboeuf C., Setterblad N., Duval A., Verine J., Germain S., Mongiat-Artus P., Janin A. & Bousquet G. (2015), Stem cells increase in numbers in perinecrotic areas in human renal cancer. Clin Cancer Res. Feb 15;21(4):916-24.

- Bouleti C., Mathivet T., Lapergue B., Monnot C. & Germain S. (2014), Protection of vascular integrity in reperfusion during stroke, Med Sci (Paris), Jun-Jul;30(6-7):608-10.

- Besnier M., Galaup A., Nicol L., Henry J.-P., Coquerel D., Gueret A., Mulder P., Brakenhielm E., Thuillez C., Germain S., Richard V. & Ouvrard-Pascaud A. (2014), Enhanced angiogenesis and increased cardiac perfusion after myocardial infarction in protein tyrosine phosphatase 1B-deficient mice. FASEB J, 13.

- Le Gal L., Alonso F., Wagner C., Germain S., Nardelli Haefliger D., Meda P. & Haefliger J.A. (2014), Restoration of Connexin 40 in renin-producing cells reduces the hypertension of Cx40 null mice. Hypertension Jun;63(6):1198-204.

- Gauvrit S., Philippe J., Lesage M., Tjwa M., Godin I. & Germain S. (2014), The role of RNA interference in the developmental separation of blood and lymphatic vasculature. Vascular cell, 6;9.

- Bouleti C., Mathivet T., Coqueran B., Serfaty J.-M., Lesage M., Berland E., Ardidie- Robouant C., Kauffenstein G., Henrion D., Lapergue B., Mazighi M., Duyckaerts C., Thurston G., Valenzuela D.M., Murphy A.J., Yancopoulos G.D., Monnot C., Margaill I. & Germain S. (2013), Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischemic stroke. European Heart Journal, May 14.

- 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.

- Castanares-Zapatero D., Bouleti C., Sommereyns C., Gerber B., Lecut C., Mathivet T., Horckmans M., Communi D., Foretz M., Vanoverschelde J.L., Germain S., Bertrand L., Laterre P.F., Oury C., Viollet B., Horman S. & Beauloye C. (2013), Connection Between Cardiac Vascular Permeability, Myocardial Edema, and Inflammation During Sepsis: Role of the α1AMP-Activated Protein Kinase Isoform. Crit Care Medicine. Aug 20.

- 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.

- Perdiguero E.G., 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. Oct 21;286(42):36841-51.
* 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.


Group leader:
Germain Stéphane, DR2 INSERM

Senior researchers:
Corvol Pierre, Pr. Collège de France
Monnot Catherine, CR1 INSERM
Muller Laurent, CR1 INSERM

Bouleti Claire, PH AP-HP
Bréchot Nicolas, PH AP-HP

Postdoctoral fellows & PhD Students:
Besbes Samaher, Postdoctoral fellow
Atlas Yoann, PhD student
Liabotis Athanasia, PhD student
Marchand Marion, PhD student
Mauroux Adèle, PhD student
Bousseau Simon, PhD student

Technical staff:
Lesage Matthieu, CDF
Ardidie-Robouant Corinne, CDF