Fig 2: A schematic figure of phenotypes for progeria endothelial cells (ECs) and vascular smooth muscle cell (VSMCs). Both HGPS-ECs and HGPS-VSMCs exhibit increased inflammatory cytokine expression, impaired shelterin complex proteins, shortened telomeres, increased DNA damage, and greater DNA accessibility. However, DNA accessibility and telomere shortening are far more severe in HGPS-ECs, suggesting an important role in the initiation and progression of the arterial occlusive disease in progeria.

Fig 3: Alterations in the expression and interaction of shelterin protein complexes. (A) A schematic figure of the shelterin protein complex. (B and C) Western blot for six shelterin proteins and quantification in endothelial cells and vascular smooth muscle cells from both control and Hutchinson–Gilford progeria syndrome; n = 4–6. (D) Co-immunoprecipitation of γH2A.X and TIN2 with RAP1 in both endothelial cells and vascular smooth muscle cells from control and Hutchinson–Gilford progeria syndrome; n = 3 (E) Co-immunoprecipitation of TRF2 with lamin A in both endothelial cells and vascular smooth muscle cells from control and Hutchinson–Gilford progeria syndrome; n = 3. C, control; HGPS, Hutchinson–Gilford progeria syndrome. Statistical analysis was performed by an unpaired t-test when comparing two groups. A value of P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: DNA damage, inflammation, and DNA accessibility in endothelial cells vs. vascular smooth muscle cells. (A) Immunofluorescence staining for DNA damage stained nuclei (DAPI, blue), γH2A.X (green), and 53BP1 (red) (scale bar: 20 μm), and quantification in (B) showing significantly increased colocalization of γH2A.X and 53BP1 in HGPS-ECs, in which HGPS-VSMCs not as strong; n = 30, replicated three times. (C) Quantitative reverse transcription polymerase chain reaction for inflammatory cytokines expression in both endothelial cells and vascular smooth muscle cells; n = 3–7. (D) Immunofluorescence staining for nuclei (DAPI, blue), lamin A (green), and histone H1 (red), and quantification in (E), showing a decrease of histone H1 in both HGPS-ECs and HGPS-VSMCs (scale bar: 20 μm). The white arrows represent cells lacking the histone H1 staining; n = 35. (F) Immunofluorescence staining for nuclei (DAPI, blue), lamin A (green), and HP1α (red), and quantification in (G), showing a decrease of HP1α in both HGPS-ECs and HGPS-VSMCs (scale bar: 20 μm). The white arrows represent cells that barely had HP1α staining; n = 44. (H–J) Western blot and quantification for histone H1 and HP1α showed a decreased expression in both HGPS-ECs and HGPS-VSMCs; n = 3. C, control; HGPS, Hutchinson–Gilford progeria syndrome. Statistical analysis was performed by an unpaired t-test when comparing two groups. A value of P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: Aging is more accelerated in HGPS-ECs. (A) ß-gal staining for control-ECs and HGPS-ECs on Days 8, 14, 21, and 28, showing an increasing trend over time (scale bar: 300 µm). (B) Quantification of ß-gal staining comparing HGPS-ECs with HGPS-VSMCs at corresponding timepoints. Timepoints 1, 2, 3, and 4 represent in endothelial cells as Days 8, 14, 21, and 28 and in vascular smooth muscle cells as Days 30, 37, 44, and 51; n = 4. (C) Telomere probe intensities were normalized with nucleus area for control and HGPS-ECs on Days 8, 14, 21, and 28, N=30 for each group. (D) Proliferation of endothelial cells within 10 days from both control and Hutchinson–Gilford progeria syndrome; n = 3. (E) Proliferation of vascular smooth muscle cells within 10 days from both control and Hutchinson–Gilford progeria syndrome; n = 3. (F) Quantitative reverse transcription polymerase chain reaction for inflammatory cytokines IL-1ß, IL-6, TNF-a, and ICAM1 expression showed increased inflammatory cytokine expression in endothelial cells over time; n = 3–6. (G) Quantitative reverse transcription polymerase chain reaction for progerin expression over time on Days 7, 14, 21, and 28, showed a significant increase of progerin expression over time in HGPS-ECs compared with its control. n = 4–6. Statistical analysis was performed by an unpaired t-test when comparing two groups. For =3 groups, statistical analysis was performed using one-way analysis of variance followed by a post hoc Tukey’s test. A value of P<0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.