(I-328) Inclusion of an adventitial layer to better model Hutchinson-Gilford Progeria Syndrome in tissue-engineered blood vessels

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare and fatal disease characterized by premature aging¹. A mutation in the LMNA gene produces a truncated form of lamin A called progerin which leads to severe atherosclerosis and premature death^2-5. There is a need for models of HGPS due to the low number of patients worldwide able to participate in clinical trials for new therapeutics⁶. Current in vitro models rely on two-dimensional, static culture of HGPS patient-derived cells and assess different cell types individually⁷. In vivo mouse models of HGPS display many similarities with human disease pathology⁸. However, therapies that have proven effective in mouse models have shown only modest benefits in patient trials⁹. Models that can utilize human cells and an improved three-dimensional, dynamic culture system that incorporates multiple affected cell types could more accurately simulate the human condition and potentially be better predictors of drug efficacy. We have previously developed a tissue-engineered blood vessel (TEBV) model of HGPS using smooth muscle (SMCs) and endothelial cells (ECs) derived from patient induced pluripotent stem cells (iPSC)¹⁰. This model exhibited some of the vascular disease characteristics. However, it does not contain an adventitial layer with fibroblasts. Adventitial thickening is a critical characteristic of HGPS vascular pathology and may increase vascular stiffness, exacerbating adverse cardiovascular events. We hypothesize that adventitial fibroblasts are the primary contributors to vascular fibrosis, calcification, and stiffening in HGPS and their inclusion will result in a more physiologically relevant model for studying disease pathology and drug discovery.

We obtained iPSCs and dermal fibroblasts (DFs) from healthy or HGPS patients through the Progeria Research Foundation. The iPSCs were differentiated into SMCs and ECs using previously established protocols^10,11. The three cell types (DFs, SMCs, and ECs) were used to fabricate healthy and HGPS TEBVs. SMCs were encapsulated in a 7 mg/mL type I collagen solution and injected into a cylindrical mold containing a smaller cylindrical insert. The solution was polymerized at 37C and then dehydrated to increase collagen density and strength. DFs were encapsulated in a similar collagen solution and injected through a cylindrical mold on top of the SMC gel layer. The solution was polymerized at 37C and dehydrated. The cylindrical insert was removed, revealing an open lumen, and ECs were injected into the TEBV and incubated for one hour with rotation to allow uniform attachment. TEBVs were perfused for three weeks at a flow rate that produced physiological shear stresses of about 5 dynes/cm². After perfusion, healthy and HGPS TEBVs were evaluated for the common vascular disease characteristics of ECM accumulation and inflammation. Collagen IV and fibronectin expression were assessed through Western blot and fluorescence imaging. Secretion of inflammatory cytokines IL-6 and IL-1β were measured using ELISA while expression of the vascular cell adhesion molecule 1 (VCAM1) was evaluated using Western blot.

HGPS TEBVs expressed significantly more collagen IV (p< 0.02) compared to healthy TEBVs [Figure 1A]. Fibronectin expression was also higher in HGPS TEBVs, however, this did not reach significance (p=0.087) [Figure 1B]. This increase in ECM accumulation is a hallmark of HGPS vascular disease and was quantitatively confirmed using immunofluorescence imaging [Figure 1C]. HGPS TEBVs also exhibited elevated inflammation compared to healthy TEBVs, with significantly increased VCAM1 expression (p< 0.02) [Figure 2A] as well as increased IL-6 (p< 0.02) and IL-1β (p< 0.02) secretion [Figure 2B-2C]. An elevated, chronic inflammation is associated with atherosclerosis and has been shown in vessels from HGPS patients^12,13. Our HGPS TEBVs exhibit relevant pathological characteristics, supporting their utility as a model of the disease. Future studies could investigate other features critical to HGPS and vascular health, such as SMC content, adventitial thickening, and vessel calcification and stiffness. The inclusion of the adventitial layer enables the investigation of the role of fibroblasts on progression of HGPS in vessels and provides a means to study the specific effects of new therapeutics on the three major cell types within the vasculature.

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