Introduction
Children with Discrete Sub-Aortic Stenosis (DSS) experience a narrowing of the left ventricular outflow tract (LVOT), caused by the formation of a fibro membranous tissue. This blockage increases the pressure in the LVOT, which is located just below the aortic valve [1,2]. DSS is an uncommon occurrence in children with congenital heart defects, with a prevalence rate of around 6% [3~6]. The pathogenesis of DSS is hypothesized to begin during the first 10 years of life [6]. Symptoms include chest pain, heart failure, syncope, with about 50% of patients having a heart murmur that grows with age [5,7]. The morphology of the lesions associated with DSS typically consists of a fibromuscular tissue ring or a localized protrusion of fibrous tissue [8]. It is believed that the condition is initiated by geometric abnormalities in the LVOT, which generates turbulent blood flow and altered shear forces that trigger a fibrotic response [9~14]. Although surgical resection of the membranes provides relief, there is a high risk of recurrence (20-30%) in what is defined as an aggressive DSS phenotype, with female sex being a risk factor [15~17]. Without Intervention, DSS can lead to left ventricle hypertrophy, aortic regurgitation, endocarditis, arrhythmias, and in extreme cases, death [18~22].
Surgical correction of the membrane has been demonstrated to be a viable option for eliminating obstruction. The membrane will either be removed via myomectomy, or just the fibrous tissue ring resected [123]. However, it has the potential to pose numerous risks, such as those associated with anesthesia, sternotomy, and heart bypass [4]. In extreme cases of aggressive DSS, open-heart surgery is a higher risk, thus patients undergo percutaneous balloon dilation [24]. This procedure lasts months to a few years, and then surgery must be performed [5]. These surgical approaches don’t last, and most patients will require reoperation, due to regrowth of the membrane, and long-term follow up care [1, 5, 23, 25].
It has been observed that DSS lesions are composed of 5 layers of endothelial and fibroblast cells [26, 27]. Researchers have hypothesized that the increased blood pressure, or turbulent jet due to DSS lesions on the LVOT, narrows the subaortic tract, causing a disturbance in cellular function in these layers [6, 28]. Observations have shown that endothelial cells release growth factors when subjected to shear stress above normal levels, inducing proliferation of other cells, such as fibroblasts, that comprise the blood vessel layers and influencing the mechanical properties of blood vessels [1, 14, 29, 30, 31]. In a similar but separate study conducted showed that endothelial cells, when exposed to shear stress, induce the release of basic fibroblast growth factor (bFGF) that stimulates the proliferation of fibroblasts [30]. Current studies are still limited in understanding a consistent pattern of pathogenesis of DSS that can be targeted for therapies and diagnostic options for pediatric patients with this disease.
Although 2D cell culture studies allow researchers to understand the biology and morphology of cells and tissues, it is limited in the accuracy of how cells interact with the extracellular environment and different cell types [32~34]. 3D models allow for a more accurate study by mimicking disease conditions [35, 36]. With 3D models we can reproduce the authentic characteristics of the heart’s tissue organization and microenvironment [34~38]. As previously determined, DSS is hypothesized to be caused by interactions between 2 different cell types, EECs and Fibroblasts [39,40]. Conventional cell culture systems and models are centered around a single cell type, which is vastly different from the natural environment of the cell [41, 42]. Thus, this limitation makes it difficult to understand the mechanistic and biological properties of the disease. Creation of a 3D co-culture system that eliminates the disadvantages of a traditional culture system that is restricted in mimicking the natural environment and interactions between cells and extracellular components [42~45].
We aim to replicate the functional response of fibroblasts in DSS patients and transition from a monolayer 2D culture to a 3D co-culture system. To gain an understanding of DSS progression, several aspects of the tissue must be considered. First, the tissue is 3D in nature, i.e., the fibroblasts are organized within a complex 3D ECM matrix. Second, the matrix will become stiffer over time, i.e., dynamically changing stiffness. Third, the relative spatial positions of the EECs and the fibroblasts are more complex than can be replicated in simple 2D monolayer cultures. In this article we will show the development and optimization of a 3D co-culture system with porcine cardiac fibroblasts and endocardial endothelial cells and subject the model to shear stress conditions as observed in DSS.