Role of Ajap1 in Cardiovascular Development
Authors: Poles Asha , Gorski Grzegorz , Caskey Elizabeth , McEldrew James , Ottman Patrick , Hatcher Cathy J. 
Published: November 2023

Coronary vessels supply oxygen and nutrients to the contractile myocardium to maintain embryonic and adult cardiac tissue homeostasis. Formation of these vessels is controlled by a network of molecular signals that determine progenitor cell fate and function. Most coronary vascular smooth muscle cells are derived from the epicardium that descends from the proepicardium (PE). However, coronary vascular endothelial cells are derived from multiple progenitors. The transitory PE gives rise to nearly 20% of embryonic coronary arterial and capillary endothelial cells via epicardial‐derived cells (EPDCs) arising from the PE. Coronary vascular progeny arise from EPDCs through a series of cellular events tightly regulated by transcription factors. Expression of the Tbx5 transcription factor in the PE contributes to epicardium and coronary vessel formation. PE‐specific deletion of Tbx5 impairs epicardium and coronary vessel formation in the embryonic mouse heart, and leads to reduced epicardial Ajap1 mRNA expression.

Study Objective

Ajap1 plays a prominent role in various tissues as a cell adhesion molecule, but its role in the heart is undetermined. We sought to determine the contribution of Ajap1 to cardiovascular development.

Methods

First, we examined the ability of TBX5 to regulate AJAP1 transcription. A luciferase reporter plasmid containing a putative human AJAP1 promoter was transfected into human lung epithelial cells that stably express human TBX5. The resultant luciferase activity was measured. Second, we assessed the spatiotemporal expression pattern of Ajap1 in the embryonic and adult mouse hearts. Sectioned mouse hearts were immunostained for Ajap1 and co‐stained with various antibodies to mark specific cell lineages within the developing heart. Third, we analyzed the cellular function of AJAP1. A human mammary epithelial cell line that appropriately models the specialized epithelial cell behavior of epicardial cells was subjected to in vitro AJAP1 gene silencing with a pool of AJAP1‐specific small interfering RNAs to prevent translation of AJAP1 protein. We performed a variety of cell function‐based assays on these AJAP1‐silenced cells to assess alterations to in vitro cell behavior.

Results

Our data indicate that TBX5 regulates AJAP1 transcription. Expression analysis of Ajap1 reveals its presence in the developing mouse heart by embryonic day (E) 9.5 where it is found in the PE and newly formed epicardial cells. Ajap1 is expressed in the epicardium and nascent coronary vessels by E13.5. It becomes localized to coronary vessels in late‐stage embryos. Our functional analysis of AJAP1‐silenced cells provides some mechanistic insight into how AJAP1 contributes to cardiovascular development.

Conclusions

We demonstrate that TBX5 regulates AJAP1 transcription. Furthermore, we postulate that Ajap1 plays a vital role in formation and maturation of the coronary vascular plexus during embryonic development.

Support or Funding Information

PCOM Center for Chronic Disorders of Aging