Endoglin (Eng) is a co-receptor of the transforming growth factor β superfamily playing an important role in endothelial dysfunction. TRC105 (carotuximab) is a monoclonal antibody that blocks Eng and its downstream Smad signaling pathway. Here we have investigated for the first time the effects of TRC105 treatment on the development of endothelial dysfunction induced by 7-ketocholesterol (7K) or high glucose (HG), focusing on Eng expression, signaling, and function. In the hypercholesterolemia study, human aortic endothelial cells (HAoECs) were treated with TRC105 (300 μg/ml) for 1 h, followed by the addition of 7K (10 μg/ml) for another 12 h. In the hyperglycemia study, HAoECs were exposed to HG (45 mM) for 60 h, followed by the addition of TRC105 for another 12 h, and cells treated with 5mM glucose and 40 mM mannitol served as control. Protein levels, adhesion, and transmigration of monocytes were assessed by flow cytometry, mRNA expression was measured by qRT-PCR. 7K and HG treatment increased protein levels of NF-κB and Eng and adhesion and transmigration of monocytes through HAoECs monolayer. TRC105 pretreatment reduced the 7K- or HG-induced Eng protein levels and pSmad1/5 and pSmad2/3 signaling. Despite increased protein levels of P-selectin and VCAM-1, TRC105 mediated blockage of Eng prevented 7K- and HG-induced adhesion and transmigration of monocytes through endothelial monolayers. These results suggest that TRC105-mediated Eng blockage can counteract the hypercholesterolemia- and hyperglycemia-induced endothelial dysfunction in HAoECs, suggesting that Eng might be a potential therapeutic target in disorders associated with elevated cholesterol and glucose levels.

Centro de Investigaciones Biológicas Margarita Salas Consejo Superior de Investigaciones Científicas Madrid Spain

Department of Biological and Medical Sciences Faculty of Pharmacy in Hradec Kralove Charles University Hradec Kralove Czechia

Department of Medical Biochemistry Faculty of Medicine in Hradec Kralove Charles University Hradec Kralove Czechia

Tracon Pharmaceuticals Inc San Diego CA United States

Zobrazit více v PubMed

Vicen M, Igreja Sá IC, Tripská K, Vitverová B, Najmanová I, Eissazadeh S, et al. Membrane and soluble endoglin role in cardiovascular and metabolic disorders related to metabolic syndrome. Cell Mol Life Sci. (2021) 78:2405–18. 10.1007/s00018-020-03701-w PubMed DOI PMC

Luchetti F, Canonico B, Cesarini E, Betti M, Galluzzi L, Galli L, et al. 7-Ketocholesterol and 5,6-secosterol induce human endothelial cell dysfunction by differential mechanisms. Steroids. (2015) 99:204–11. 10.1016/j.steroids.2015.02.008 PubMed DOI

Wielkoszyński T, Zalejska-Fiolka J, Strzelczyk JK, Owczarek AJ, Cholewka A, Furmański M, et al. Oxysterols increase inflammation, lipid marker levels and reflect accelerated endothelial dysfunction in experimental animals. Mediators Inflamm. (2018) 2018:2784701. 10.1155/2018/2784701 PubMed DOI PMC

Levitan I, Volkov S, Subbaiah PV. Oxidized LDL: diversity, patterns of recognition, and pathophysiology. Antioxid Redox Signal. (2010) 13:39–75. 10.1089/ars.2009.2733 PubMed DOI PMC

Chalubinski M, Zemanek K, Skowron W, Wojdan K, Gorzelak P, Broncel M. The effect of 7-ketocholesterol and 25-hydroxycholesterol on the integrity of the human aortic endothelial and intestinal epithelial barriers. Inflamm Res. (2013) 62:1015–23. 10.1007/s00011-013-0660-x PubMed DOI PMC

Alt A, Miguel-Romero L, Donderis J, Aristorena M, Blanco FJ, Round A, et al. Structural and functional insights into endoglin ligand recognition and binding. PLoS One. (2012) 7:e29948. 10.1371/journal.pone.0029948 PubMed DOI PMC

Lopez-Novoa JM, Bernabeu C. The physiological role of endoglin in the cardiovascular system. Am J Physiol Heart Circ Physiol. (2010) 299:H959–74. 10.1152/ajpheart.01251.2009 PubMed DOI

Bot PTG, Hoefer IE, Sluijter JPG, Vliet PV, Smits AM, Lebrin F, et al. Increased expression of the transforming growth factor-β signaling pathway, endoglin, and early growth response-1 in stable plaques. Stroke. (2009) 40:439–47. 10.1161/STROKEAHA.108.522284 PubMed DOI

St-Jacques S, Forte M, Lye SJ, Letarte M. Localization of endoglin, a transforming growth factor-beta binding protein, and of CD44 and integrins in placenta during the first trimester of pregnancy. Biol Reprod. (1994) 51:405–13. 10.1095/biolreprod51.3.405 PubMed DOI

Meurer S, Wimmer AE, van de Leur E, Weiskirchen R. Endoglin trafficking/exosomal targeting in liver cells depends on n-glycosylation. Cells. (2019) 8:997. 10.3390/cells8090997 PubMed DOI PMC

Lastres P, Bellon T, Cabanas C, Sanchez-Madrid F, Acevedo A, Gougos A, et al. Regulated expression on human macrophages of endoglin, an Arg-Gly-Asp-containing surface antigen. Eur J Immunol. (1992) 22:393–7. 10.1002/eji.1830220216 PubMed DOI

Aristorena M, Gallardo-Vara E, Vicen M, de Las Casas-Engel M, Ojeda-Fernandez L, Nieto C, et al. MMP-12, secreted by pro-inflammatory macrophages, targets endoglin in human macrophages and endothelial cells. Int J Mol Sci. (2019) 20:3107. 10.3390/ijms20123107 PubMed DOI PMC

Hawinkels LJ, Kuiper P, Wiercinska E, Verspaget HW, Liu Z, Pardali E, et al. Matrix metalloproteinase-14 (MT1-MMP)-mediated endoglin shedding inhibits tumor angiogenesis. Cancer Res. (2010) 70:4141–50. 10.1158/0008-5472.CAN-09-4466 PubMed DOI

Blazquez-Medela AM, Garcia-Ortiz L, Gomez-Marcos MA, Recio-Rodriguez JI, Sanchez-Rodriguez A, Lopez-Novoa JM, et al. Increased plasma soluble endoglin levels as an indicator of cardiovascular alterations in hypertensive and diabetic patients. BMC Med. (2010) 8:86. 10.1186/1741-7015-8-86 PubMed DOI PMC

Igreja SÁIC, Tripska K, Hroch M, Hyspler R, Ticha A, Lastuvkova H, et al. Soluble endoglin as a potential biomarker of nonalcoholic steatohepatitis (NASH) development, participating in aggravation of NASH-Related changes in mouse liver. Int J Mol Sci. (2020) 21:9021. 10.3390/ijms21239021 PubMed DOI PMC

Blaha M, Cermanova M, Blaha V, Jarolim P, Andrys C, Blazek M, et al. Elevated serum soluble endoglin (sCD105) decreased during extracorporeal elimination therapy for familial hypercholesterolemia. Atherosclerosis. (2008) 197:264–70. 10.1016/j.atherosclerosis.2007.04.022 PubMed DOI

Visek J, Blaha M, Blaha V, Lasticova M, Lanska M, Andrys C, et al. Monitoring of up to 15 years effects of lipoprotein apheresis on lipids, biomarkers of inflammation, and soluble endoglin in familial hypercholesterolemia patients. Orphanet J Rare Dis. (2021) 16:110. 10.1186/s13023-021-01749-w PubMed DOI PMC

Leanos-Miranda A, Navarro-Romero CS, Sillas-Pardo LJ, Ramirez-Valenzuela KL, Isordia-Salas I, Jimenez-Trejo LM. Soluble endoglin as a marker for preeclampsia. Its severity, and the occurrence of adverse outcomes. Hypertension. (2019) 74:991–7. 10.1161/HYPERTENSIONAHA.119.13348 PubMed DOI

Blann AD, Wang JM, Wilson PB, Kumar S. Serum levels of the TGF-beta receptor are increased in atherosclerosis. Atherosclerosis. (1996) 120:221–6. 10.1016/0021-9150(95)05713-7 PubMed DOI

Bilir B, Ekiz Bilir B, Yilmaz I, Soysal Atile N, Yildirim T, Kara SP, et al. Association of apelin, endoglin and endocan with diabetic peripheral neuropathy in type 2 diabetic patients. Eur Rev Med Pharmacol Sci. (2016) 20:892–8. PubMed

Blazquez-Medela AM, Garcia-Ortiz L, Gomez-Marcos MA, Recio-Rodríguez JI, Sánchez-Rodríguez A, López-Novoa JM, et al. Increased plasma soluble endoglin levels as an indicator of cardiovascular alterations in hypertensive and diabetic patients. BMC Med. (2010) 8:86. 10.1186/1741-7015-8-86 PubMed DOI PMC

Doghish AS, Bassyouni AA, Mahfouz MH, Abd El-Aziz HG, Zakaria RY. Plasma endoglin in Type2 diabetic patients with nephropathy. Diabetes Metab Syndr. (2019) 13:764–8. 10.1016/j.dsx.2018.11.058 PubMed DOI

Ekiz-Bilir B, Bilir B, Aydin M, Soysal-Atile N. Evaluation of endocan and endoglin levels in chronic kidney disease due to diabetes mellitus. Arch Med Sci. (2019) 15:86–91. 10.5114/aoms.2018.79488 PubMed DOI PMC

Hiden U, Lassance L, Tabrizi NG, Miedl H, Tam-Amersdorfer C, Cetin I, et al. Fetal insulin and IGF-II contribute to gestational diabetes mellitus (GDM)-associated up-regulation of membrane-type matrix metalloproteinase 1 (MT1-MMP) in the human feto-placental endothelium. J Clin Endocrinol Metab. (2012) 97:3613–21. 10.1210/jc.2012-1212 PubMed DOI

Ceriello A, La Sala L, De Nigris V, Pujadas G, Testa R, Uccellatore A, et al. GLP-1 reduces metalloproteinase-14 and soluble endoglin induced by both hyperglycemia and hypoglycemia in type 1 diabetes. Endocrine. (2015) 50:508–11. 10.1007/s12020-015-0565-2 PubMed DOI

Uzui H, Hayashi H, Nakae I, Matsumoto T, Uenishi H, Hayasaki H, et al. Pitavastatin decreases serum LOX-1 ligand levels and MT1-MMP expression in CD14-positive mononuclear cells in hypercholesterolemic patients. Int J Cardiol. (2014) 176:1230–2. 10.1016/j.ijcard.2014.07.213 PubMed DOI

Ruiz-Llorente L, Gallardo-Vara E, Rossi E, Smadja DM, Botella LM, Bernabeu C. Endoglin and alk1 as therapeutic targets for hereditary hemorrhagic telangiectasia. Expert Opin Ther Targets. (2017) 21:933–47. 10.1080/14728222.2017.1365839 PubMed DOI

Tillet E, Ouarné M, Desroches-Castan A, Mallet C, Subileau M, Didier R, et al. A heterodimer formed by bone morphogenetic protein 9 (BMP9) and BMP10 provides most BMP biological activity in plasma. J Biol Chem. (2018) 293:10963–74. 10.1074/jbc.RA118.002968 PubMed DOI PMC

Santibañez JF, Quintanilla M, Bernabeu C. TGF-β/TGF-β receptor system and its role in physiological and pathological conditions. Clin Sci. (2011) 121:233–51. PubMed

Lebrin F, Goumans MJ, Jonker L, Carvalho RL, Valdimarsdottir G, Thorikay M, et al. Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction. EMBO J. (2004) 23:4018–28. PubMed PMC

Munoz-Felix JM, Gonzalez-Nunez M, Lopez-Novoa JM. ALK1-Smad1/5 signaling pathway in fibrosis development: friend or foe? Cytokine Growth Factor Rev. (2013) 24:523–37. PubMed

Rossi E, Sanz-Rodriguez F, Eleno N, Duwell A, Blanco FJ, Langa C, et al. Endothelial endoglin is involved in inflammation: role in leukocyte adhesion and transmigration. Blood. (2013) 121:403–15. PubMed

Vicen M, Vitverova B, Havelek R, Blazickova K, Machacek M, Rathouska J, et al. Regulation and role of endoglin in cholesterol-induced endothelial and vascular dysfunction in vivo and in vitro. FASEB J. (2019) 33:6099–114. 10.1096/fj.201802245R PubMed DOI

Ceriello A, Quagliaro L, Piconi L, Assaloni R, Da Ros R, Maier A, et al. Effect of postprandial hypertriglyceridemia and hyperglycemia on circulating adhesion molecules and oxidative stress generation and the possible role of simvastatin treatment. Diabetes. (2004) 53:701–10. 10.2337/diabetes.53.3.701 PubMed DOI

La Sala L, Pujadas G, De Nigris V, Canivell S, Novials A, Genovese S, et al. Oscillating glucose and constant high glucose induce endoglin expression in endothelial cells: the role of oxidative stress. Acta Diabetol. (2015) 52:505–12. 10.1007/s00592-014-0670-3 PubMed DOI

Botella LM, Sanchez-Elsner T, Sanz-Rodriguez F, Kojima S, Shimada J, Guerrero-Esteo M, et al. Transcriptional activation of endoglin and transforming growth factor-beta signaling components by cooperative interaction between Sp1 and KLF6: their potential role in the response to vascular injury. Blood. (2002) 100:4001–10. 10.1182/blood.V100.12.4001 PubMed DOI

Henry-Berger J, Mouzat K, Baron S, Bernabeu C, Marceau G, Saru JP, et al. Endoglin (CD105) expression is regulated by the liver X receptor alpha (NR1H3) in human trophoblast cell line JAR. Biol Reprod. (2008) 78:968–75. 10.1095/biolreprod.107.066498 PubMed DOI

Valbuena-Diez AC, Blanco FJ, Oujo B, Langa C, Gonzalez-Nunez M, Llano E, et al. Oxysterol-induced soluble endoglin release and its involvement in hypertension. Circulation. (2012) 126:2612–24. 10.1161/CIRCULATIONAHA.112.101261 PubMed DOI

Liu Y, Paauwe M, Nixon AB, Hawinkels L. Endoglin targeting: lessons learned and questions that remain. Int J Mol Sci. (2020) 22:147. 10.3390/ijms22010147 PubMed DOI PMC

Rosen LS, Gordon MS, Robert F, Matei DE. Endoglin for targeted cancer treatment. Curr Oncol Rep. (2014) 16:365. PubMed

Liu Y, Tian H, Blobe GC, Theuer CP, Hurwitz HI, Nixon AB. Effects of the combination of TRC105 and bevacizumab on endothelial cell biology. Invest New Drugs. (2014) 32:851–9. PubMed PMC

Madonna R, Montebello E, Lazzerini G, Zurro M, De Caterina R. NA+/H+ exchanger 1- and aquaporin-1-dependent hyperosmolarity changes decrease nitric oxide production and induce VCAM-1 expression in endothelial cells exposed to high glucose. Int J Immunopathol Pharmacol. (2010) 23:755–65. 10.1177/039463201002300309 PubMed DOI

Brcakova E, Fuksa L, Cermanova J, Kolouchova G, Hroch M, Hirsova P, et al. Alteration of methotrexate biliary and renal elimination during extrahepatic and intrahepatic cholestasis in rats. Biol Pharm Bull. (2009) 32:1978–85. 10.1248/bpb.32.1978 PubMed DOI

Puig-Kroger A, Relloso M, Fernandez-Capetillo O, Zubiaga A, Silva A, Bernabeu C, et al. Extracellular signal-regulated protein kinase signaling pathway negatively regulates the phenotypic and functional maturation of monocyte-derived human dendritic cells. Blood. (2001) 98:2175–82. 10.1182/blood.v98.7.2175 PubMed DOI

Liu R, Shen H, Wang T, Ma J, Yuan M, Huang J, et al. TRAF6 mediates high glucose-induced endothelial dysfunction. Exp Cell Res. (2018) 370:490–7. PubMed

Quagliaro L, Piconi L, Assaloni R, Da Ros R, Maier A, Zuodar G, et al. Intermittent high glucose enhances ICAM-1, VCAM-1 and E-selectin expression in human umbilical vein endothelial cells in culture: the distinct role of protein kinase C and mitochondrial superoxide production. Atherosclerosis. (2005) 183:259–67. 10.1016/j.atherosclerosis.2005.03.015 PubMed DOI

Hemling P, Zibrova D, Strutz J, Sohrabi Y, Desoye G, Schulten H, et al. Hyperglycemia-induced endothelial dysfunction is alleviated by thioredoxin mimetic peptides through the restoration of VEGFR-2-induced responses and improved cell survival. Int J Cardiol. (2020) 308:73–81. 10.1016/j.ijcard.2019.12.065 PubMed DOI

Lv ZH, Phuong TA, Jin SJ, Li XX, Xu M. Protection by simvastatin on hyperglycemia-induced endothelial dysfunction through inhibiting NLRP3 inflammasomes. Oncotarget. (2017) 8:91291–305. 10.18632/oncotarget.20443 PubMed DOI PMC

Sanchez-Elsner T, Botella LM, Velasco B, Langa C, Bernabeu C. Endoglin expression is regulated by transcriptional cooperation between the hypoxia and transforming growth factor-beta pathways. J Biol Chem. (2002) 277:43799–808. 10.1074/jbc.M207160200 PubMed DOI

van Uden P, Kenneth NS, Rocha S. Regulation of hypoxia-inducible factor-1alpha by NF-kappaB. Biochem J. (2008) 412:477–84. PubMed PMC

Liu Y, Starr MD, Brady JC, Dellinger A, Pang H, Adams B, et al. Modulation of circulating protein biomarkers following TRC105 (anti-endoglin antibody) treatment in patients with advanced cancer. Cancer Med. (2014) 3:580–91. 10.1002/cam4.207 PubMed DOI PMC

Kumar S, Pan CC, Bloodworth JC, Nixon AB, Theuer C, Hoyt DG, et al. Antibody-directed coupling of endoglin and MMP-14 is a key mechanism for endoglin shedding and deregulation of TGF-β signaling. Oncogene. (2014) 33:3970–9. 10.1038/onc.2013.386 PubMed DOI PMC

Blankenberg S, Barbaux S, Tiret L. Adhesion molecules and atherosclerosis. Atherosclerosis. (2003) 170:191–203. PubMed

Rossi E, Bernabeu C, Smadja DM. Endoglin as an adhesion molecule in mature and progenitor endothelial cells: a function beyond TGF-β. Front Med. (2019) 6:10. 10.3389/fmed.2019.00010 PubMed DOI PMC