Lonafarnib and everolimus reduce pathology in iPSC-derived tissue engineered blood vessel model of Hutchinson-Gilford Progeria Syndrome

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, fatal genetic disease that accelerates atherosclerosis. With a limited pool of HGPS patients, clinical trials face unique challenges and require reliable preclinical testing. We previously reported a 3D tissue engineered blood vessel (TEBV) microphysiological system fabricated with iPSC-derived vascular cells from HGPS patients. HGPS TEBVs exhibit features of HGPS atherosclerosis including loss of smooth muscle cells, reduced vasoactivity, excess extracellular matrix (ECM) deposition, inflammatory marker expression, and calcification. We tested the effects of HGPS therapeutics Lonafarnib and Everolimus separately and together, currently in Phase I/II clinical trial, on HGPS TEBVs. Everolimus decreased reactive oxygen species levels, increased proliferation, reduced DNA damage in HGPS vascular cells, and improved vasoconstriction in HGPS TEBVs. Lonafarnib improved shear stress response of HGPS iPSC-derived endothelial cells (viECs) and reduced ECM deposition, inflammation, and calcification in HGPS TEBVs. Combination treatment with Lonafarnib and Everolimus produced additional benefits such as improved endothelial and smooth muscle marker expression and reduced apoptosis, as well as increased TEBV vasoconstriction and vasodilation. These results suggest that a combined trial of both drugs may provide cardiovascular benefits beyond Lonafarnib, if the Everolimus dose can be tolerated.

. Cell viability with Lonafarnib and Everolimus treatment.
Representative images (a, c) and quantification (b, d) of calcein-AM/EthD-1 staining on HGPS 167 CL2 viSMCs (a, b) and viECs (c, d) treated with Lonafarnib (LF) and Everolimus (Ev). 'V' indicates DMSO vehicle control. Scale bar 200 µm in a and 100 µm in c. Arrows in c denote EthD-1 to identify dead cells. Mean ± SD, N = 5 per group for viSMCs and N=2 for viECs. Data analyzed by ANOVA followed by post hoc Tukey text.

Figure S2. Entire Western Blots of Progerin Levels for Different Everolimus Concentrations
Images of the entire western blots for quantification of progerin levels in Figure 1. Each blot was cut and regions noted separately stained for GAPDH and progerin. Images of the Western blots for quantification of protein levels in Figure S3. Each blot was cut and regions noted separately stained for GAPDH, progerin, lamin A, and lamin C.

Figure S3. Effect of Everolimus on levels of progerin, lamin A and lamin C in HGPS viECs and viSMCs.
The viSMCs and viECs were incubated for 7 days in media containing 0.1 µM Everolimus as in Figure 1. Western blot was performed using an antibody to lamin A/C for viSMC (a) and viECs (b) which were quantified by densitometry (b, d). Data analyzed by ANOVA followed by post hoc Tukey text.

Figure S4. Misshapen HGPS nuclei
(a) Images of normal and misshapen Hoechst-stained HGPS nuclei. Images of Hoechst-stained HGPS viSMC (b) and viEC (c) nuclei after exposure to the indicated concentrations of Everolimus (Ev) for 7 days, scale bar = 50 µm. Images were sharpened in ImageJ to enhance contrast.

Figure S5. Everolimus reduces ROS levels, increases proliferation, and decreases DNA damage in HGPS 003 CL1D viSMCs and viECs.
Healthy viSMCs (a-d) and viECs (e-h) and HGPS 003 CL1D viSMCs and viECs treated with different combinations of Lonafarnib (LF) and Everolimus (Ev) for: fold change of DCFDA mean fluorescence intensity compared with healthy in viSMCs (a) and viECs (e); percent positive Ki67 nuclei in viSMCs (b) and viECs (f); percent nuclei with ɣH2A.X foci in viSMCs (c) and viECs (g); and number of ɣH2A.X foci per total cells in viSMCs (d) and viECs (h). V' indicates DMSO vehicle control. Healthy bars are shown in gray and HGPS bars are shown in white. Data presented as mean ± SD. N = 3 experiments per group. Data were analyzed by ANOVA followed by a post hoc Tukey test. Groups connected by the same letter are not significantly different from each other (p>0.05). Groups labeled with different letters are significantly different from each other (p<0.05). Exact p-values for significant differences are provided in Table S3.    Representative images of DAF-FM diacetate in HGPS 167 (a) and HGPS 003 (b) viECs exposed to 12 dynes/cm 2 shear stress for 24 hours, with and without 7-day LF and Ev treatment. Scale bar 100 µm. Quantification is shown in Fig. 3a, b.