Coronary vessel development

and artery differentiation

Distinct molecular pathways of multiple progenitors

Given that multiple sources of coronary vessels exist, we questioned whether the sinus venosus and endocardium grow in response to similar or distinct molecular pathways. Our experiments have identified two ligands required specifically for sinus venosus-derived coronary development, the growth factor VEGF-C and the peptide hormone ELABELA. In contrast, they are not required for endocardium angiogenesis (Chen et al., 2014b; Sharma et al., 2017). Vegfc and Elabela are expressed by the epicardium on the surface of the heart where sinus venosus sprouts emerge, emphasizing the importance of epicardial cells in stimulating sinus venosus sprouting. Sinus venosus sprouting also specifically requires, CCBE1, an extracellular matrix protein that facilitates VEGF-C processing, and the ELABELA receptor, APJ (Sharma et al., 2017). It is relevant to note that the endocardial activators are known for being hypoxia-inducible, while the SV activators are not, and only endocardial sprouts first emerge into tissues labeled with probes that bind hypoxic tissues (Sharma et al., 2017).

Biological advantages of multiple progenitors

Compensation: One hypothesis for why coronaries have multiple origins is that it could provide an alternative progenitor pool if one source is disrupted. Performing endocardial lineage tracing in APJ mutants revealed that endocardial-derived vessels expand when sinus venosus angiogenesis is deficient (Sharma et al., 2017).

Support of rapid tissue growth and ventricular compaction: Another hypothesis for the importance of multiple coronary progenitors is that it allows for faster vascularization that could better support rapid growth of the heart wall. Coincident with the onset of coronary angiogenesis, the ventricular heart wall begins transforming from loosely packed trabeculae to thick, compact muscle. This transformation is called ventricular compaction, and, when defective in humans, causes a cardiomyopathy termed Left Ventricular Non-Compaction. We discovered that endothelial-specific deletion of the chromatin remodeler Ino80 causes ventricular non-compaction in mice due to defective coronary angiogenesis from both the sinus venosus and endocardium. This study (Rhee et al., 2018) revealed that angiogenesis from both coronary progenitor pools is required to promote optimal expansion of the compact heart wall through production of a paracrine factor.

Single cell analysis of coronary development

Using the power of single-cell RNA sequencing, we discovered that a population of single cells within the immature vessel plexus are pre-specified during mid-gestation and subsequently go on to build the majority of the coronary arteries. Analyzing the vein to artery cell fate switch at single cell resolution revealed two novel and unexpected features: (1) The vein to artery conversion is gradual and overlapping until a transcriptional threshold is reached forming pre-specified artery cells, and (2) This arterial pre-specification threshold is actively inhibited by cell cycle activation induced by the vein specifying transcription factor, COUP-TF2. Understanding artery development at this new level of detail has implications for strategizing coronary artery regeneration in the diseased or injured heart.

Responses to mechanical signals

Blood flow-guided endothelial cell behaviors

We found that the transcription factor, DACH1, allows endothelial cells to polarize, align, and migrate against the direction of flow. Accordingly, Dach1-deficient mice have small coronary arteries and suppressed adult heart function (Chang et al., 2017). DACH1 induces the expression of the chemokine Cxcl12, which is required for migration against the direction of fluid flow. Interestingly, not only does DACH1 facilitate blood flow guided migration and alignment, but mature artery-type blood flow (uniform laminar) specifically downregulates its expression. Our data leads to a model where DACH1 stimulates coronary artery growth by activating endothelial cell migration against blood flow via Cxcl12 expression. Arteries then shut off this growth response after mechanically sensing that they have reached a mature morphology (i.e. acquired uniform laminar flow). Thus, we identified how specific mechanical signals modulate an artery growth signal to shape the arterial tree.

Injury Responses and Regeneration

Developing organisms create tissues de novo, and the underlying instructions could inform organ regeneration. With this mindset, we study coronary arteries, which bring blood flow to heart muscle, in hopes of eventually treating coronary artery disease (CAD), the number one killer worldwide. We have discovered how mouse coronary arteries are built, and reinstated developmental pathways in adults to aid recovery following cardiac injury.

One benefit of studying developmental biology is that one learns step-wise instructions for building a tissue, which could be utilized to re-build organs as a regenerative therapy. In two studies, we have demonstrated that learning basic developmental concepts can inform new regeneration strategies in the heart (Das et al., 2019; Raftrey et al., 2020). First, we discovered that the developing neonatal mouse heart has the unique ability to quickly and robustly grow collateral arteries in response to experimental myocardial infarction. Collateral arteries are arteries that bridge two conventional arterial branches and provide blood flow to occluded vessels (i.e. natural bypasses)(Bardot and Dubois, 2019; Das et al., 2019). We found that myocardial infarction caused tissue hypoxia, which stimulated the expression of the migratory cue CXCL12 by capillaries situated between injured and healthy arteries. These capillaries become a reparative niche by attracting artery cells that express the CXCL12 receptor, CXCR4, and supporting their transformation into collateral arteries. Remarkably, the neonatal heart can fully regenerate, which is lost in adulthood, and our work showed that its capacity to form collateral arteries is one reason why. We injected CXCL12 into injured adult hearts and found that this re-activated collateral development, presenting the exciting possibility that knowledge of developmental processes could identify reparative pathways.

Second, we turned to Dach1, a transcription factor showed was required for coronary artery growth during development (Chang et al., 2017). When we genetically induced expression of Dach1 in adult mice, we found increased animal survival and heart function after myocardial infarction (Raftrey et al., 2020). Dach1 functioned by inducing pre-artery specification in capillary endothelial cells, which facilitated their migration against the direction of flow where they extended artery branching.