• Projects NeoCoronary and CardioRegen

Coronary heart disease is one of the most common causes of death globally. Therefore, we need novel agents that stimulate angiogenesis to restore blood flow in patients with ischemic diseases.
CCBE1 is a secreted protein emerged as a crucial regulator of VEGFC signaling. More recently, we have unveiled that CCBE1 is also crucial for proper coronary vasculature formation. In both processes, CCBE1 exerts its function by enhancing VEGFC signaling, being required for the maturation of VEGFC into its active form.
Besides its role in coronary vasculature formation, we demonstrated that CCBE1 is required for cardiac mesoderm formation and multipotent cardiovascular progenitors’ differentiation in mouse ESCs. Using our knockout mouse models, we observed that loss of Ccbe1 leads to defect in coronary plexus development and thinner ventricular compact myocardium.

The main aims of this project are:

• • Examine the role of CCBE1 during mouse coronary vasculature development
  • Understand if CCBE1 directs coronary vessels (arteries and veins) formation from hPS cells.
  • Validate the potential of CCBE1 in the formation of new vessels and in the remodeling/repair of injured cardiac tissue both by using 3D cell-based models as well as using mouse models of myocardial infarction.
  • Determine the role of CCBE1 in myocardial growth and in CM proliferation in the compact layer, suggesting that Ccbe1 is required for proper myocardial compaction.

• Project CardioProD

Cardiovascular disease is responsible for unhealthy ageing, impaired quality of life for the patient and caretakers, and high health resources consumption. This project is an integrated approach to study and develop new therapeutic approaches for heart diseases, accounting for the increase of morbidity and decrease in life quality.
Our laboratory demonstrated that DAND5 is the only member of the Cerberus/DAN family of secreted antagonists of Nodal and Wnt signaling cascades with expression in the heart. By binding to the signaling ligands, DAND5 prevents agonist-receptor interaction inhibiting the signaling cascade events.
We have been recently driving a study with a cohort of human patients with congenital heart disease from which we identified a variation in the Cerl2 human homologue, DAND5. We are now using the iPS cell technology after generating iPS cell lines for some of these patients and performing studies of patient specific disease modeling on the study of cardiomyocyte formation and most importantly, proliferation.
We verified an increase in the proliferation index of CMs by modulating DAND5 levels. Our preliminary data indicate that reduced levels of DAND5 lead to increased cardiovascular progenitors’ numbers and, in addition, extend their progenitor state for a longer period.

The main aims of this project are:

• • Make noticeable progress in pluripotent stem cell-derived cardiomyocyte maturity toward a more clinically relevant model for cardiac regeneration.
  • Define the signals that promote cardiomyocyte maturation by DAND5 modulation.
  • Understand the endogenous proliferative ability of heart myocytes to enhance cardiac muscle mass and function recovery.
  • Identify potential therapeutic molecular targets that will stimulate cardiomyocyte proliferation after heart injury.

• NeoCoronary: Role of CCBE1 during coronary vasculature development and neo-angiogenesis upon myocardial infarction, through stimulation of VEGF-C signaling - 02/SAICT/2017/029590 (2018/2021). Part. Inst.: IBET/UNL.
• CardioProD: Control of cardiomyocyte proliferation in disease and regenerative medicine - PTDC/BIM-MED/3363/2014 (2016/2019). Part. Inst.: Univ. Coimbra.
• CardioRegen: Integrative studies on the role of Ccbe1 in cardiogenesis: from the embryo to scalable multipronged generation of hESC-derived myocardial cells for heart tissue repair. - HMSP-ICT/0039/2013 (2014/2019). Part. Inst.: Harvard Medical School, IBET/UNL.
• "Role of the novel ccbe1 growth factor in the generation of cardiac tissue from ES cells" - PTDC/SAU-ENB/121095/2010 (2012/2015)
  
  

As project co-leader & collaborator

• NETDIAMOND: NEw Targets in DIAstolic heart failure: from coMOrbidities to persoNalizeD medicine - PAC/2015 (2016/2021). Part. Inst.: Univ. Coimbra, Univ. Nova Lisboa, Univ. Lisboa, Univ. Aveiro, Univ. Porto.
• The impact of alternative splicing on structure and function of molecular networks during stem cell differentiation. - PTDC/BIA-GEN/116519/2010 (2012/2016). Part. Inst.: Univ. Algarve.
  
  

José Manuel Inácio, João von Gilsa Lopes, Ana Mafalda Silva, Fernando Cristo, Sara Marques, Matthias E. Futschik, José António Belo (2021). DAND5 inactivation promotes cardiac differentiation in mouse embryonic stem cells. Front. Cell Dev. Biol. doi: 10.3389/fcell.2021.629430.

José M. Inácio, Micael Almeida, Fernando Cristo and José A. Belo (2019). Generation of gene-corrected human induced pluripotent stem cell line derived from a patient with laterality defects and congenital heart anomalies with a c.455G>A alteration in DAND5. Stem Cell Research. doi.org/10.1016/j.scr.2019.101677.

Bover O, Justo T, Pereira PNG, Facucho-Oliveira J, Inácio JM, Ramalho JS, Domian IJ, Belo JA (2018). Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells. PLoS One. 2018 Oct 3;13(10):e0205108. doi: 10.1371/journal.pone.0205108. eCollection 2018.

Tavares AT, Jacinto A and Belo JA. (2018). Identification of chick Lefty2 asymmetric enhancer. Matters Select. 2018 Jul 19. doi: 10.19185/matters.201807000006

Bonet F, Pereira PNG, Bover O, Marques S, Inácio JM, Belo JA (2018). CCBE1 is required for coronary vessel development and proper coronary artery stem formation in the mouse heart. Dev Dyn. 247:1135–1145. 2018 Sep 11. doi: 10.1002/dvdy.24670.

Silva MM, Gomes-Alves P, Rosa S, Simão D, Inácio JM, Peixoto C, Serra M, Belo JA, Alves PM. (2018). Full-length human CCBE1 production and purification: leveraging bioprocess development for high quality glycosylation attributes and functionality. J Biotechnol. 2018 Aug 27;285:6-14. doi: 10.1016/j.jbiotec.2018.08.015.

Selin Pars, Fernando Cristo, José M. Inácio, Graça Rosas, Isabel Marques Carreira, Joana Barbosa Melo, Luís Pereira de Almeida, Patrícia Mendes, Duarte Saraiva Martins, José Maio, Rui Anjos and José A. Belo (2018). Generation and characterization of a human iPS cell line from a patient-related control to study disease mechanisms associated with DAND5 p.R152H alteration. Stem Cell Research 29:202-206. DOI: 0.1016/j.scr.2018.04.015.

José A. Belo, Sara Marques and José M. Inácio (2017). The Role of Cerl2 in the Establishment of Left-Right Asymmetries during Axis Formation and Heart Development. J. Cardiovasc. Dev. Dis. 2017, 4, 23; Doi:10.3390/jcdd4040023.

Fernando Cristo, José M. Inácio, Graça Rosas, Isabel Marques Carreira, Joana Barbosa Melo, Luís Pereira de Almeida, Patrícia Mendes, Duarte Saraiva Martins, José Maio, Rui Anjos and José A. Belo (2017). Generation of human iPSC line from a patient with laterality defects and associated congenital heart anomalies carrying a DAND5 missense alteration. Stem Cell Research 25:152-156. DOI: 10.1016/j.scr.2017.10.19.

Furtado J, Bento M, Correia E, Inácio JM, Belo JA (2014) Expression and Function of Ccbe1 in the Chick Early Cardiogenic Regions Are Required for Correct Heart Development. PLoS ONE 9(12): e115481. doi:10.1371/journal.pone.0115481

Ana Carolina Araújo, Sara Marques and José António Belo (2014). Target inactivation of cerl-2 leads to left ventricular cardiac hyperplasia and systolic dysfunction in mouse. PLoS One. 9(7):e102716. DOI: 10.1371/journal.pone.0102716).

Ana Rubina Perestrelo, Ana Grenha, Ana M. Rosa da Costa and José António Belo (2014). Locust Bean Gum as an alternative polymeric coating for embryonic stem cell culture. Mater Sci Eng C Mater Biol Appl. 40:336-44. doi: 10.1016/j.msec.2014.04.022.

Ana Rubina Perestrelo, Fouzi Mouffouk, Ana M. Rosa da Costa and José António Belo (2013). Novel triblock copolymer nanofiber system as an alternative support for embryonic stem cells growth and pluripotency. T. Eng. Reg. Med. 2016 Oct;10(10):E467-E476. DOI: 10.1002/term.1838.

Vinhas M, Araújo AC, Ribeiro S, Brás Rosário L and Belo JA (2013). Transthoracic echocardiography reference values in juvenile and adult 129/Sv mice. Cardiovascular Ultrasound 11:12 (doi:10.1186/1476-7120-11-12).

Inácio JM, Marques S, Nakamura T, Shinohara K, Meno C, Hamada H, and Belo JA (2013). The Dynamic Right

• Fernando Cristo, (...) and José A. Belo (2017). "Functional and disease modeling study of DAND5 variant in patients with Congenital Heart Disease and laterality defects." 21.ª Reunião Anual Sociedade Portuguesa de Genética Humana, Almada, Portugal. Honorable mention, Poster presentation.
• Prémio APIFARMA para a mobilidade, José A. Belo (2006).
• Prémio Sociedade Portuguesa de Genética Humana – 2005, atribuído ao melhor artigo publicado nesta área numa revista cientifica internacional (Marques et al, 2004).
• Fundação Luso Americana para o Desenvolvimento (FLAD) fellowship to attend the meeting “Mouse Molecular Genetics”, 2004, at Cold Spring Harbor Laboratory, NY, USA
• Fundação Calouste Gulbenkian (FCG) fellowship to attend the The 5th EMBL “Mouse Molecular Genetics Meeting”, 2003, at the EMBL, Heidelberg, Germany.

• Stefano Piccolo, Padova University, Italy
• Hiroshi Hamada, Osaka University, Osaka, Japan
• Margarida Serra, Animal Cell Technology Unit, ITQB-NOVA and iBET, Oeiras, Portugal
• Ibrahim Domian, Harvard Medical School, Cambridge, USA
• Paula Alves, Animal Cell Technology Unit, ITQB-NOVA and iBET, Oeiras, Portugal
• Abel Oliva, ITQB-NOVA and iBET, Oeiras, Portugal
• Rui Anjos, Hospital Santa Cruz, Lisboa, Portugal
• Matthias Futschik, FMCB, University of Algarve, Portugal
• José Maria Pérez-Pomares, Andalusian Center for Nanomedicine & Biotechnology, University of Malaga, Spain