Research ArticleHEALTH AND MEDICINE

Vascular endothelium–targeted Sirt7 gene therapy rejuvenates blood vessels and extends life span in a Hutchinson-Gilford progeria model

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Science Advances  19 Feb 2020:
Vol. 6, no. 8, eaay5556
DOI: 10.1126/sciadv.aay5556
  • Fig. 1 Single-cell transcriptomic profiles of CD31+ MLECs.

    (A) Purity analysis of sorted CD31+ MLECs by FACS. SSC, side scatter; FSC, forward scatter; PE, phycoerythrin. (B) t-Distributed stochastic neighbor embedding (t-SNE) projection of CD31+ cells revealed four clusters: ECs (green), B lymphocytes (B-like; orange), T lymphocytes (T-like; blue), and macrophages (Mφ-like; red). (C) Marker gene expression in the four clusters: ECs (Cd31, Cd34, and Cdh5), B-like (Ly6d, Cd22, and Cd81), T-like (Cd3d, Cd3e, and Cd28), and Mφ-like (Cd14, Cd68, and Cd282). (D) Heatmap showing marker gene expression levels in LmnaG609G/G609G (G609G) and Lmnaf/f (Flox) mice.

  • Fig. 2 Single-cell transcriptomic analysis indicates an inflammatory response and cardiac dysfunction in progeroid ECs.

    (A) t-SNE projection of LmnaG609G/G609G (G609G; green) and Lmnaf/f (Flox; orange) CD31+ MLECs according to transcriptomic data. (B to D) GO and KEGG pathway enrichment of differentially expressed genes between G609G and Flox cells. LmnaG609G/G609G MLECs show enrichment in genes that regulate the inflammatory response (C) and genes related to heart dysfunction (D). FC, fold change; FDR, false discovery rate. (E) Quantitative PCR analysis of altered genes observed in (C) and (D) in HUVECs with ectopic expression of progerin or wild-type LMNA. Data represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test).

  • Fig. 3 Endothelial-specific dysfunction and defective neovascularization in progeria mice.

    (A and B) Hematoxylin and eosin staining of thoracic aorta sections from (A) Lmnaf/f;TC and (B) LmnaG609G/G609G and Lmnaf/f control mice showing intima-media thickening. Scale bar, 20 μm. (C) ACh-induced thoracic aorta vasodilation in Lmnaf/f;TC and Lmnaf/f control mice. **P < 0.01. 5-HT, 5-hydroxytryptamine. (D) ACh-induced thoracic aorta vasodilation in LmnaG609G/G609G and control mice. **P < 0.01. (E) SNP-induced thoracic aorta vasodilation in LmnaG609G/G609G and control mice. (F) eNOS level in thoracic aorta sections from Lmnaf/f;TC and control mice. Scale bar, 20 μm. (G) Immunofluorescence staining (left) and quantification (right) of CD31+ gastrocnemius muscle in Lmnaf/f;TC and Lmnaf/f mice. Scale bar, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (H) CD31 immunofluorescence staining in Lmnaf/f;TC and Lmnaf/f liver. Scale bar, 50 μm. (I) Representative microcirculation images (left) and quantification of blood flow recovery (right) following hindlimb ischemia in Lmnaf/f;TC and Lmnaf/f mice. (J) Representative transverse sections and quantification of CD31+ gastrocnemius muscle 14 days after femoral artery ligation. Scale bar, 50 μm. All data represent means ± SEM. P values were calculated by Student’s t test. Photo credits: Shimin Sun, School of Life Sciences, Shandong University of Technology; Medical Research Center, Shenzhen University (A, B, F, H, and J); Weifeng Qin, Medical Research Center, Shenzhen University (G and I).

  • Fig. 4 Systemic aging phenotypes in Lmnaf/f;TC mice.

    (A to C) Masson trichrome staining showing an atheromatous plaque in the aorta (A), SMC loss (B), and cardiac fibrosis (C) in Lmnaf/f;TC mice. Scale bar, 20 μm. (D) Heart weight and echocardiographic parameters, including heart rate, cardiac output, left ventricular (LV) ejection fraction (LVEF), and left ventricular ejection shortening (LVFS). *P < 0.05, Lmnaf/f;TC versus Lmnaf/f mice. (E) Decreased running endurance in Lmnaf/f;TC mice. ***P < 0.001. (F) Micro-CT analysis showing a decrease in trabecular bone volume/tissue volume (BV/TV), trabecular number, and trabecular thickness and an increase in trabecular separation in Lmnaf/f;TC mice. *P < 0.05, Lmnaf/f;TC versus Lmnaf/f mice. (G) Life span of LmnaG609G/G609G, LmnaG609G/+, Lmnaf/f;TC, and Lmnaf/f mice. (H) Body weight of male LmnaG609G/G609G, LmnaG609G/+, Lmnaf/f;TC, and Lmnaf/f mice. *P < 0.05, Lmnaf/f;TC versus Lmnaf/f mice; ***P < 0.001, LmnaG609G/G609G versus Lmnaf/f mice. All data represent means ± SEM. P values were calculated by Student’s t test, except that statistical comparison of the survival data was performed by log-rank test. Photo credits: Weifeng Qin, Medical Research Center, Shenzhen University (A and B); Shimin Sun, School of Life Sciences, Shandong University of Technology; Medical Research Center, Shenzhen University (C).

  • Fig. 5 Accumulation of progerin destabilizes Sirt7.

    (A) Quantification of blood flow recovery following hindlimb ischemia in Sirt7−/− and Sirt7+/+ mice. (B) Left: Representative immunoblots showing indicated protein levels in HUVECs treated with si-SIRT7 or scramble (Scram). Right: Quantification of relative protein levels. *P < 0.05 and **P < 0.01, small interfering RNA (siRNA) versus Scram. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Real-time PCR analysis of the indicated gene expression in HUVECs treated with si-SIRT7 or Scram. *P < 0.05, siRNA versus Scram. (D) Left: Representative immunoblots showing indicated sirtuin protein levels in FACS-sorted MLECs. Right: Quantification of relative protein levels. *P < 0.05. Note that down-regulated Sirt7 but rather up-regulated Sirt6 and hardly changed SIRT1 in Lmnaf/f;TC MLECs. (E) Left: Co-immunoprecipitation (IP) experiments showing hemagglutinin (HA)–SIRT7 in anti–FLAG–Lamin A and anti–FLAG-progerin immunoprecipitates. Right: Quantification of relative protein levels. *P < 0.05. (F) Left: Representative immunoblots showing polyubiquitinated SIRT7, which was up-regulated in the presence of progerin but rather down-regulated in the presence of Lamin A. Right: Quantification of relative protein levels. *P < 0.05. (G) Representative immunoblots showing SIRT7 protein levels in the presence of Lamin A or progerin in HEK293 cells treated with cycloheximide (CHX) and/or MG132 (M). Quantification of relative SIRT7 protein levels was shown. *P < 0.05, progerin versus Lamin A. All data represent means ± SEM. P values were calculated by Student’s t test. Photo credit: Xiaolong Tang, Medical Research Center, Shenzhen University (B, D, E, F, and G).

  • Fig. 6 VE-targeted Sirt7 therapy rejuvenates the microvasculature and extends life span in progeria mice.

    (A) Real-time PCR analysis of genes that are aberrantly up-regulated in progerin-overexpressing HUVECs upon overexpression of SIRT7. *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Neovascularization assay in Lmnaf/f;TC mice with hindlimb ischemia, treated with or without IS7O particles. **P < 0.01. (C) Immunofluorescence microscopy analysis of FLAG-SIRT7 and CD31 expression in gastrocnemius muscle 14 days after femoral artery ligation. Scale bar, 25 μm. (D) Percent CD31+ ECs in Lmnaf/f;TC mice treated with or without IS7O particles. ***P < 0.001. (E) Representative immunofluorescence images of the liver, aorta, and muscle of Lmnaf/f;TC mice after IS7O therapy, showing CD31+ ECs with FLAG-SIRT7 expression. Scale bar, 50 μm. (F) Representative immunoblots showing expression of FLAG-SIRT7 in aorta and WBMCs. Note that FLAG-SIRT7 was merely detected in WBMCs. (G) Life span of IS7O-treated and untreated Lmnaf/f;TC and LmnaG609G/+ mice. (H) Body weight of IS7O-treated and untreated Lmnaf/f;TC and Lmnaf/f mice. All data represent means ± SEM. P values were calculated by Student’s t test, except that the statistical comparison of survival data was performed by log-rank test. Photo credits: Shimin Sun, School of Life Sciences, Shandong University of Technology; Medical Research Center, Shenzhen University (C and E); Xiaolong Tang, Medical Research Center, Shenzhen University (F).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/8/eaay5556/DC1

    Fig. S1. Generation of Lmnaf/f mice and phenotypic analysis of LmnaG609G/G609G mice.

    Fig. S2. Single-cell transcriptomic analysis of CD31+ MLECs.

    Fig. S3. VE-specific progerin expression.

    Fig. S4. Vasodilation analysis of LmnaG609G/+ mice.

    Fig. S5. Expression of atherosclerosis- and osteoporosis-associated genes in MLEC transcriptomes.

    Table S1. List of primer sequences.

    Table S2. List of antibodies.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Generation of Lmnaf/f mice and phenotypic analysis of LmnaG609G/G609G mice.
    • Fig. S2. Single-cell transcriptomic analysis of CD31+ MLECs.
    • Fig. S3. VE-specific progerin expression.
    • Fig. S4. Vasodilation analysis of LmnaG609G/+ mice.
    • Fig. S5. Expression of atherosclerosis- and osteoporosis-associated genes in MLEC transcriptomes.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). List of primer sequences.
    • Table S2 (Microsoft Excel format). List of antibodies.

    Files in this Data Supplement:

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