Identifying molecular and functional similarities and differences between human primary cardiac valve interstitial cells and ventricular fibroblasts.

Introduction: Fibroblasts are mesenchymal cells that predominantly produce and maintain the extracellular matrix (ECM) and are critical mediators of injury response. In the heart, valve interstitial cells (VICs) are a population of fibroblasts responsible for maintaining the structure and function of heart valves. These cells are regionally distinct from myocardial fibroblasts, including left ventricular cardiac fibroblasts (LVCFBs), which are located in the myocardium in close vicinity to cardiomyocytes. Here, we hypothesize these subpopulations of fibroblasts are transcriptionally and functionally distinct. Methods: To compare these fibroblast subtypes, we collected patient-matched samples of human primary VICs and LVCFBs and performed bulk RNA sequencing, extracellular matrix profiling, and functional contraction and calcification assays. Results: Here, we identified combined expression of SUSD2 on a protein-level, and MEOX2, EBF2 and RHOU at a transcript-level to be differentially expressed in VICs compared to LVCFBs and demonstrated that expression of these genes can be used to distinguish between the two subpopulations. We found both VICs and LVCFBs expressed similar activation and contraction potential in vitro, but VICs showed an increase in ALP activity when activated and higher expression in matricellular proteins, including cartilage oligomeric protein and alpha 2-Heremans-Schmid glycoprotein, both of which are reported to be linked to calcification, compared to LVCFBs. Conclusion: These comparative transcriptomic, proteomic, and functional studies shed novel insight into the similarities and differences between valve interstitial cells and left ventricular cardiac fibroblasts and will aid in understanding region-specific cardiac pathologies, distinguishing between primary subpopulations of fibroblasts, and generating region-specific stem-cell derived cardiac fibroblasts.

Angiogenic Secretion Profile of Valvular Interstitial Cells Varies With Cellular Sex and Phenotype.

Angiogenesis is a hallmark of fibrocalcific aortic valve disease (CAVD). An imbalance of pro- and anti-angiogenic factors is thought to play a role in driving this disease process, and valvular interstitial cells (VICs) may act as a significant source of these factors. CAVD is also known to exhibit sexual dimorphism in its presentation, and previous work suggested that VICs may exhibit cellular-scale sex differences in the context of angiogenesis. The current study sought to investigate the production of angiogenesis-related factors by male and female VICs possessing quiescent (qVIC) or activated (aVIC) phenotypes. Production of several pro-angiogenic growth factors was elevated in porcine aVICs relative to qVICs, with sex differences found in both the total amounts secreted and their distribution across media vs. lysate. Porcine valvular endothelial cells (VECs) were also sex-separated in culture and found to behave similarly with respect to metabolic activity, viability, and tubulogenesis, but male VECs exhibited higher proliferation rates than female VECs. VECs responded to sex-matched media conditioned by VICs with increased tubulogenesis, but decreased proliferation, particularly upon treatment with aVIC-derived media. It is likely that this attenuation of proliferation resulted from a combination of decreased basic fibroblast growth factor and increased thrombospondin-2 (TSP2) secreted by aVICs. Overall, this study indicates that VICs regulate angiogenic VEC behavior via an array of paracrine molecules, whose secretion and sequestration are affected by both VIC phenotype and sex. Moreover, strong sex differences in TSP2 secretion by VICs may have implications for understanding sexual dimorphism in valve fibrosis, as TSP2 is also a powerful regulator of fibrosis.

Engineered Collagen Matrices.

Collagen is the most abundant protein in mammals, accounting for approximately one-third of the total protein in the human body. Thus, it is a logical choice for the creation of biomimetic environments, and there is a long history of using collagen matrices for various tissue engineering applications. However, from a biomaterial perspective, the use of collagen-only scaffolds is associated with many challenges. Namely, the mechanical properties of collagen matrices can be difficult to tune across a wide range of values, and collagen itself is not highly amenable to direct chemical modification without affecting its architecture or bioactivity. Thus, many approaches have been pursued to design scaffold environments that display critical features of collagen but enable improved tunability of physical and biological characteristics. This paper provides a brief overview of approaches that have been employed to create such engineered collagen matrices. Specifically, these approaches include blending of collagen with other natural or synthetic polymers, chemical modifications of denatured collagen, de novo creation of collagen-mimetic chains, and reductionist methods to incorporate collagen moieties into other materials. These advancements in the creation of tunable, engineered collagen matrices will continue to enable the interrogation of novel and increasingly complex biological questions.