Establishment of conditionally immortalized, sex-specific human atrial cardiac microvascular endothelial cell lines for physiologically relevant in vitro modeling

Abstract

The cardiac microvasculature plays a critical role in maintaining myocardial homeostasis, yet it remains one of the least understood components of the cardiovascular system. Existing in vitro endothelial models typically rely on primary cells, human umbilical vein endothelial cells, or other non–tissue-specific endothelial lines to approximate cardiac microvascular function. These models, however, suffer from limited reproducibility and scalability or fail to accurately recapitulate the phenotype of cardiac microvascular endothelial cells, limiting the development of physiologically relevant platforms for studying cardiac biology and endothelial–cardiomyocyte interactions.

To overcome these challenges, we established conditionally immortalized human atrial cardiac microvascular endothelial cell lines derived from both male and female donors, engineered to retain key phenotypic characteristics of the cardiac microvasculature. Primary endothelial cells were isolated from adult human left atrial appendage tissue by enzymatic dissociation followed by CD31-based magnetic-activated cell sorting. The isolated cells were subsequently transduced with a doxycycline-inducible lentiviral vector encoding CDK4 and Cyclin D1, together with a constitutively expressed human telomerase construct. The resulting cell lines demonstrated robust, doxycycline-dependent proliferative capacity, and their endothelial identity was confirmed by expression of the canonical markers CD31, VE-cadherin, and von Willebrand factor.

Functionally, these immortalized endothelial cells modulated electrophysiological behavior in a transwell co-culture with atrial myocytes, reducing action potential duration and demonstrating physiologically relevant endothelial–cardiomyocyte interactions. Ongoing validation studies are assessing long-term stability, scalability, and functional performance of the lines.

The establishment of stable, sex-specific human cardiac microvascular endothelial cell lines provides a much-needed resource for reproducible, tissue-relevant experimentation and has the potential to significantly enhance the predictive power of cardiovascular research platforms.