Adipose-derived stromal cell secreted factors induce the elastogenesis cascade within 3D aortic smooth muscle cell constructs

Abstract

Objective Elastogenesis within the medial layer of the aortic wall involves a cascade of events orchestrated primarily by smooth muscle cells, including transcription of elastin and a cadre of elastin chaperone matricellular proteins, deposition and cross-linking of tropoelastin coacervates, and maturation of extracellular matrix fiber structures to form mechanically competent vascular tissue. Elastic fiber disruption is associated with aortic aneurysm; in aneurysmal disease a thin and weakened wall leads to a high risk of rupture if left untreated, and non-surgical treatments for small aortic aneurysms are currently limited. This study analyzed the effect of adipose-derived stromal cell secreted factors on each step of the smooth muscle cell elastogenesis cascade within a three-dimensional fibrin gel culture platform. Approach and results We demonstrate that adipose-derived stromal cell secreted factors induce an increase in smooth muscle cell transcription of tropoelastin, fibrillin-1, and chaperone proteins fibulin-5, lysyl oxidase, and lysyl oxidase-like 1, formation of extracellular elastic fibers, insoluble elastin and collagen protein fractions in dynamically-active 30-day constructs, and a mechanically competent matrix after 30 days in culture. Conclusion Our results reveal a potential avenue for an elastin-targeted small aortic aneurysm therapeutic, acting as a supplement to the currently employed passive monitoring strategy. Additionally, the elastogenesis analysis workflow explored here could guide future mechanistic studies of elastin formation, which in turn could lead to new non-surgical treatment strategies.

Extracellular Vesicles Derived from Primary Adipose Stromal Cells Induce Elastin and Collagen Deposition by Smooth Muscle Cells within 3D Fibrin Gel Culture

“…This study utilizes a 3D culture system to demonstrate for the first time that EVs derived from ASC conditioned media with characteristics of exosomes and microvesicles (Figure 1) can be used to induce production of two important vascular ECM components by SMCs, specifically elastin and fibrillar collagen. While gene expression analysis after 30 days only reflected a prolonged elevation of fibrillin-1 transcription by media supplementation with 3x EV treatment (Figure 2), the elastin and collagen ECM contents of both discs and strands was elevated by supplementation of 1x or 3x EVs, similar to what has been previously reported by our group for CM [15] (Figure 3). The uniaxial elastic moduli of strand constructs, however, were not affected by EV treatment (Figure 4).…”

“…SMC-seeded fibrin gel constructs were formed as previously described [ 15 ] in “disc” and “strand” configurations using 3.7 mg/mL bovine fibrinogen type I (Sigma Aldrich #F8630, St. Louis, MI, USA), 0.21 U/mL bovine thrombin (Sigma Aldrich #605157, St. Louis, MI, USA), and 5 × 10 5 SMCs/mL. Constructs were allowed to polymerize for one hour before the addition of SBM for 24 h prior to initial treatment.…”

“…After pre-heating template (65 °C, 5 min), synthesis of first-strand cDNA used random hexamer primers and the SuperScript IV First-Strand Synthesis System (Invitrogen #18091050, Waltham, MA, USA) (23 °C/10 min, 55 °C/10 min, 80 °C/10 min). RT-qPCR was performed using KiCqStart SYBR Green ReadyMix with ROX (Sigma-Aldrich #KCQS02) and previously published primers [ 15 ]. Post-amplification melt curves validated proper amplification.…”

“…As established previously [ 15 , 23 ], base hydrolysis and subsequent centrifugation (3000× g ) were used to separate insoluble elastin protein from soluble non-elastin protein (0.1M NaOH, 1 h at 98 °C). Acid hydrolysis (6N HCl, 24 h at 110 °C) and assay quantification on both soluble and insoluble fractions (ninhydrin-based for elastin, hydroxyproline-based for collagen) was used to quantify ECM content within each 3D construct.…”

“…We have previously demonstrated that delivering the secretome of adipose stromal cells (ASCs) to vascular SMCs in 3D gel culture increases the deposition of both elastin and collagen [ 15 ]. Others have shown a potent effect of mesenchymal stem cells [ 16 ] and their extracellular vesicles (EVs) [ 17 ] on reducing aneurysm growth, furthering the premise of this study.…”