Investigating the crosstalk between cardiomyocytes and endothelial cells to understand the poor angiogenic capacity of the adult heart
Problem or question being addressed:
The adult heart is not able to regenerate or revascularize itself after ischemic damage. Despite advances in pharmacological and surgical interventions, no effective therapy exists to regenerate the heart. This project aims to investigate which are the molecular interactions between cardiomyocytes (CMs) and cardiac endothelial cells (ECs) that block cardiac revascularization and regeneration.
Rationale for your approach:
Heart failure is a major health problem worldwide. To emphasize the burden of this disease, COronaVIrus Disease-19 (COVID19) caused 6 million deaths from its beginning 3 years ago, representing just over one third of all deaths caused by heart failure in 1 year. Globally, approximately 18 million people die of myocardial infarction and other cardiovascular diseases every year (WHO). Among these deaths, 85% is due to anatomical or functional disorders of the coronary arteries (Wong 2014; Malakar et al. 2019; Kumar et al. 2019). In the last 20 years numerous therapeutic approaches have been attempted to promote CM proliferation (Tzahor et al. 2017; Ciucci G, Colliva A, Vuerich R et al. 2022). However, none of these reached or passed the clinical phase. Beside CM replacement, revascularization of the damaged myocardium results essential for cardiac regeneration (Singh et al 2022; de Wit et al 2020; Wu et al. 2021). In the neonatal heart as well, ECs form new vessels inside the damaged area well before CMs start proliferating, indicating that cardiac remuscularization is tightly coupled with angiogenesis (Oka et al. 2014; Marin-Juez et al. 2016; Ingason et al. 2018). However, contrary to the neonatal heart, the adult heart is not able to form new vessels after ischemic damage neither upon stimulation with pro-angiogenic stimuli (Kocijan et al. 2020). The mechanisms behind the poor angiogenic potential of adult heart are still missing, in particular very little is known about CM-EC interaction (Colliva et al. 2019). Although some EC molecules have been described to have an important role during normal cardiac development and homeostasis, the mechanisms and the signals by which CMs modulate EC biology are still poorly investigated (Colliva et al. 2019). This project aims to discover whether CMs, which lose their proliferative potential during the first week after birth, send inhibitory signals that in turn might inhibit adult ECs to form new vessels.
Preliminary data indicate that adult CMs inhibit the proliferation and tube formation capacity of ECs when they are seeded in co-culture, while neonatal CMs do not. Moreover, ECs are able to engraft and form vascular structures when injected in neonatal heart, while very few ECs survive in the adult heart after injection. These results suggest the presence of some inhibitory signals in the adult cardiac environment, probably produced by CMs.
Details of suggested approach:
To explore whether CMs express membrane or secreted proteins that inhibit EC proliferation and angiogenesis, I will build an interactome that connects putative ligands produced by adult CMs with receptors expressed by cardiac ECs using RNA sequencing data. For this analysis, I will exclude the genes that are similarly upregulated in both adult and neonatal CMs. After pairing ligands with their receptors, I will confirm the activation of receptor-downstream signaling molecules using NicheNet. Then, the functional role of the candidate molecules produced by adult CMs will be validated using two complementary approaches of loss and gain of function. On one hand, in the loss of function approach I will silence the interactors in ECs using the corresponding siRNA delivered by lipids (RNAiMAX). After 48 hours, the ECs will be cultured together with adult CMs to identify the receptors involved in the inhibition of EC proliferation. We expect that the depletion of anti-angiogenic interactions will result in increased EC proliferation (assessed by EdU incorporation), despite the presence of adult CMs in co-culture. On the other hand,
I will overexpress the candidate molecules produced by adult CMs in neonatal ones using expression plasmids or mRNA coding for the corresponding genes. Thus, neonatal CMs will acquire the anti-angiogenic phenotype of the adult ones. One day after the transfection, ECs will be seeded on the top of neonatal CMs and the day after, EdU will be added for 48 hours to assess EC proliferation. We expect that the expression of anti-angiogenic molecules will result in decreased EC proliferation (assessed by EdU incorporation).
Complementary approaches of loss and gain of function studies will lead to identification of anti-angiogenic interactors, that will then be silenced in vivo to stimulate the angiogenic response, either after myocardial infarction or upon stimulation with pro-angiogenic factors. Precisely, I will select the three most potent anti-angiogenic interactors produced by adult CMs to generate AAV9 vectors to express specific shRNAs for these targets. Alternatively, I will take advantage of MHC-CAS9 mice, in which CAS9 is expressed only in CMs. This model enables specific knock-out of the target genes using sgRNAs. The sgRNAs will be delivered to CMs using either AAV9 vector or lipid nanoparticles. Then, to stimulate the angiogenic response we will either induce myocardial infarction through coronary artery ligation or overexpress VEGF-A using AAV9-VEGF vector. We expect that the downregulation of selected anti-angiogenic factors will promote cardiac EC proliferation and formation of new vessels, through either angiogenesis or arteriogenesis.
How it will affect the broader field:
The understanding of the molecular mechanisms by which ECs and CMs communicate stands as a crucial step towards the understanding why the adult heart is not able to form new vessels and thus might ease the development of innovative and effective strategies to promote cardiac regeneration by the inhibition of anti-angiogenic interactions. On the other hand, the identification of the anti-angiogenic molecules produced by adult CMs might pave the way for the usage of these factors in the inhibition of tumor angiogenesis.
· Aurora AB, Porrello ER, Tan W, Mahmoud AI, Hi JA, Bassel-Duby R, et al. Mac- rophages are required for neonatal heart regeneration. J Clin Invest. 2014;124: 1382-92.
· Browaeys, R., Saelens, W. & Saeys, Y. NicheNet: modeling intercellular communication by linking ligands to target genes. Nat. Methods 17, 159-162 (2019).
· Ciucci, Giulio et al. “Biologics and cardiac disease: challenges and opportunities.” Trends in pharmacological sciences, S0165-6147(22)00128-6. 29 Jun. 2022, doi:10.1016/j.tips.2022.06.001
· Colliva A et al. “Endothelial cell-cardiomyocyte crosstalk in heart development and disease.” The Journal of physiology vol. 598,14 (2020): 2923-2939. doi:10.1113/JP276758
· de Wit, Lousanne et al. “Cellular and Molecular Mechanism of Cardiac Regeneration: A Comparison of Newts, Zebrafish, and Mammals.” Biomolecules vol. 10,9 1204. 19 Aug. 2020, doi:10.3390/biom10091204
· Ingason, Arnar B et al. "Angiogenesis precedes cardiomyocyte migration in regenerating mammalian hearts." The Journal of thoracic and cardiovascular surgery vol. 155,3 (2018): 1118-1127.e1. doi:10.1016/j.jtcvs.2017.08.127
· Kocijan T et al. Genetic lineage tracing reveals poor angiogenic potential of cardiac endothelial cells. Cardiovasc Res. 2020. doi:10.1093/cvr/cvaa012
· Kumar, Santosh, Gang Wang, Na Zheng, Wanwen Cheng, Kunfu Ouyang, Hairuo Lin, Yulin Liao, and Jie Liu. 2019. “HIMF (Hypoxia-Induced Mitogenic Factor)-IL (Interleukin)-6 Signaling Mediates Cardiomyocyte-Fibroblast Crosstalk to Promote Cardiac Hypertrophy and Fibrosis.” Hypertension (Dallas, Tex.: 1979) 73 (5): 1058–70.
· Malakar, Arup Kr., Debashree Choudhury, Binata Halder, Prosenjit Paul, Arif Uddin, and Supriyo Chakraborty. 2019. “A Review on Coronary Artery Disease, Its Risk Factors, and Therapeutics.” Journal of Cellular Physiology 234 (10): 16812–23. https://doi.org/10.1002/jcp.28350.