Regulation of Vascular Smooth Muscle Cell Phenotype in Three-Dimensional Coculture System by Jagged1-Selective Notch3 Signaling

Abstract

The modulation of vascular smooth muscle cell (VSMC) phenotype is an essential element to fabricate engineered conduits of clinical relevance. In vivo, owing to their close proximity, endothelial cells (ECs) play a role in VSMC phenotype switching. Although considerable progress has been made in vascular tissue engineering, significant knowledge gaps exist on how the contractile VSMC phenotype is induced at the conclusion of the tissue fabrication process. The objectives of this study were as follows: (1) to establish ligand presentation modes on transcriptional activation of VSMC-specific genes, (2) to develop a three-dimensional (3D) coculture model using human coronary artery smooth muscle cells (HCASMCs) and human coronary artery endothelial cells (HCAECs) on porous synthetic scaffolds and, (3) to investigate EC-mediated Notch signaling in 3D cultures and the induction of the HCASMC contractile phenotype. Whereas transcriptional activation of VSMC-specific genes was not induced by presenting soluble Jagged1 and Jagged1 bound to protein G beads, a direct link between HCAEC-bound Jagged1 and HCASMC differentiation genes was observed. Our 3D culture results showed that HCASMCs seeded to scaffolds and cultured for up to 16 days readily attached, infiltrated the scaffold, proliferated, and formed dense confluent layers. HCAECs, seeded on top of an HCASMC layer, formed a distinct, separate monolayer with cell-type partitioning, suggesting that HCAEC growth was contact inhibited. While we observed EC monolayer formation with 200,000 HCAECs/scaffold, seeding 400,000 HCAECs/scaffold revealed the formation of cord-like structures akin to angiogenesis. Western blot analyses showed that 3D coculture induced an upregulation of Notch3 receptor in HCASMCs and its ligand Jagged1 in HCAECs. This was accompanied by a corresponding induction of the contractile HCASMC phenotype as demonstrated by increased expression of smooth muscle-α-actin (SM-α-actin) and calponin. Knockdown of Jagged1 with siRNA showed a reduction in SM-α-actin and calponin in cocultures, identifying a link between Jagged1 and the expression of contractile proteins in 3D cocultures. We therefore conclude that the Notch3 signaling pathway is an important regulator of VSMC phenotype and could be targeted when fabricating engineered vascular tissues.

Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress

“…Our observations are in agreement with previous findings that in co-culture, ECs inhibited SMC proliferation, migration, and collagen synthesis, and supported SMC contractile phenotype [28][29][30]. Furthermore, it has been shown that in co-culture, HCAECs could increase the expression of SM-22α and calponin in HCASMCs [12], suggesting a regulatory role of intercellular contacts with HCAECs in HCASMC differentiation. Moreover, when cocultured on the opposite sides of Transwell membranes, ECs and SMCs were shown to form myoendothelial gap junctions [9,28], through which ECs could directly communicate with SMCs and regulate their functional activity [31].…”

“…Myoendothelial gap junctions can be mimicked in vitro by co-culturing ECs and SMCs on the opposite sides of a porous membrane; this model have been widely used to study the mechanisms of cell-cell interaction in the vasculature. Thus, it has been reported that in human coronary artery SMCs (HCASMCs) co-cultured with human coronary ECs (HCAECs), differentiation markers were significantly induced, indicating a regulatory role of EC-SMC intercellular contacts in SMC phenotypic switching [12]. Furthermore, in a static ECs-SMCs co-culture model using Transwell inserts, myoendothelial gap junctions have been shown to modulate the differentiation of pulmonary arterial SMCs [13,14].…”

“…In addition, endothelial Cx43 and Cx40, but not Cx37, demonstrated mechanosensitivity to shear stress [19,20]; thus, Cx43 distribution on the endothelium surface was changed in response to shear stress [21]. A recent study revealed that shear stress at low levels (4 dyn/cm 2 ), but not at high levels (12 ), upregulated Cx43 expression in human umbilical vein ECs (HUVECs) co-cultured with HUVSMCs, and induced HUVEC inflammatory phenotype [22]. Overall, these findings suggest that the exposure to shear stress of ECs co-cultured with SMCs may regulate SMC phenotype switching, and the formation of myoendothelial gap junctions may play a pivotal role in the process.…”

Jagged1/Notch3 Signaling Modulates Hemangioma-Derived Pericyte Proliferation and Maturation

“…Interestingly, studies investigating the function of Notch signaling in pericyte/vSMC have yielded conflicting results, describing both anti-proliferation and pro-proliferation functions as well as both anti-maturation and pro-maturation functions for Notch [26,27]. These discrepancies have been speculated to be a result of the highly context-dependent nature and tight spatio-temporal regulation of Notch pathway components during vascular development [9].…”

Nitric Oxide Stimulates Matrix Synthesis and Deposition by Adult Human Aortic Smooth Muscle Cells Within Three-Dimensional Cocultures

“…More recently, Bhattacharya et al developed a coculture of human coronary artery cells in 3D poly(carbonate urethane) scaffolds to investigate EC-mediated Notch signaling and induction of SMC contractile phenotype. 50 These studies led to a critical understanding of the benefits of cocultures, mechanical stimuli, and 3D scaffolds to mimic physiological conditions, and investigated biological mechanisms under more controlled environments. Our current experimental setup differs from these previous studies as follows: (1) collagen gels were used in this study compared with synthetic polymeric scaffolds tested in previous studies; (2) human aortic ECs were cultured within 3D scaffolds in our study instead of a confluent monolayer, to enable paracrine signaling between ECs and SMCs; and (3) the microfluidic setup developed here would allow diffusion of paracrine signals and exogenous molecules under controlled static or dynamic flow conditions, depending on the experimental need.…”