阅读说明：本技术 使Sirt7基因表达的作用剂及其用途 (Agent for expressing Sirt7 gene and use thereof ) 是由 刘宝华 孙世民 唐小龙 于 2019-10-12 设计创作，主要内容包括：本发明提供一种使Sirt7基因表达的作用剂,特别是使血管内皮(VE)特异性Sirt7基因表达的重组腺相关病毒(rAAV)及其用途。本发明还提供了一种利用该作用剂,尤其是rAAV,来改善新生血管、改善衰老特征、延长寿命和治疗年龄相关疾病的方法。(The invention provides an agent for expressing Sirt7 gene, in particular a recombinant adeno-associated virus (rAAV) for expressing Sirt7 gene specific to Vascular Endothelial (VE) and application thereof. The invention also provides a method of using the agent, particularly rAAV, to improve neovascularization, improve aging characteristics, prolong life, and treat age-related disorders.)

1. An agent that causes the expression of Sirt7 gene.

2. The agent of claim 1, wherein the agent is a carrier.

3. The agent of claim 2 wherein the vector is a plasmid and/or viral vector.

4. The agent according to claim 3, wherein the viral vector is a recombinant adeno-associated virus (rAAV), in particular rAAV serotype 1.

5. The agent of any one of claims 1-4 wherein Sirt7 gene expression is Vascular Endothelial (VE) -specific expression; in particular, Sirt7 gene expression was driven by the ICAM2 promoter.

6. Use of an agent according to any one of claims 1-5 in the manufacture of a medicament for improving neovascularization, improving aging characteristics, preventing aging, prolonging life, and/or treating hakinson-gilford progeria syndrome (HGPS) and/or an age-related disorder.

7. The use according to claim 6, wherein the aging, HGPS and/or age-related disease is characterized by Vascular Endothelial (VE) dysfunction.

8. Use according to claim 6 or 7, wherein the age-related disease is cardiovascular disease (CAD), arthritis, muscular atrophy and/or osteoporosis.

9. Use according to claim 8, wherein the cardiovascular disease is heart failure and/or atherosclerosis.

10. A method for improving neovascularization, improving aging characteristics, preventing aging, prolonging lifespan, and/or treating hakinson-gilford progeria syndrome (HGPS) and/or an age-related disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of an agent as described in any one of claims 1-5.

Technical Field

The present invention relates to gene-targeted therapies. In particular, the present invention relates to agents that express the Sirt7 gene that are useful for revitalizing blood vessels, prolonging life, and treating age-related diseases.

Technical Field

Aging is the greatest risk factor for many age-related diseases, such as vascular dysfunction and cardiovascular disease (CVD) (1). The blood vessels consist of the intima (consisting of Endothelial Cells (ECs)), the media (consisting of Vascular Smooth Muscle Cells (VSMCs)) and the adventitia (consisting of connective tissue) (2). The endothelium separates the vessel wall from the blood stream and has an irreplaceable role in regulating vascular tone and homeostasis (3, 4). Age-related functional decline in endothelial cells and vascular smooth muscle cells is a major cause of cardiovascular disease (4-6). Endothelial cells secrete a variety of vasodilators and vasoconstrictors, which act on vascular smooth muscle cells and induce vasoconstriction and dilation (7). For example, Nitric Oxide (NO) is synthesized by L-arginine via endothelial NO synthase (eNOS) and released onto vascular smooth muscle cells to induce vasodilation (8). When endothelial cells age or function is dysregulated, vasoconstrictor, procoagulant and proinflammatory cytokines are released; this effect decreases the bioavailability of NO, which in turn increases intimal permeability and endothelial cell migration (9). Despite advances in understanding the mechanisms of endothelial dysfunction, it is unclear whether it directly causes aging in the body.

More and more data indicate that the mechanism of normal aging is similar to the hachnson-gilford progeria syndrome (HGPS), a syndrome of premature aging in which affected patients often die of cardiovascular disease (10-15). The premature senility syndrome is mainly composed of c.1824C in LMNA gene>T, p.g.g608g mutation, which activates the alternative splicing event and produces a truncated form of laminA of 50 amino acids, called progerin presenilin (10). Murine Lmna^G609GLMNA equivalent to human^G608GThis results in an aging phenotype similar to that of the premature aging syndrome (16). Progerin has been shown to target smooth muscle cells and cause vascular calcification and atherosclerosis (17-22). Two recent groups of work have shown that smooth muscle cell-specific progerin knockin mice are healthy and normal in lifespan, but when crossed with Apoe-/-mice, vascular calcification, atherosclerosis and shortened lifespan occurred (23; 24). Vascular endothelium contribution to systemic/body senescence compared to smooth muscle cellsThe effect is elusive. It is still pressing to study the effects of dysfunction on systemic aging and the targeting potential of HGPS clinical therapies.

Disclosure of Invention

We generated an Lmna with pathogenic HGPS^G609GKnock-in mouse model of mutations, called progerin. We will use Lmna^f/fMice were crossed with Tie2-Cre line to obtain Lmna^f/f(ii) a TC mice, which exhibit defective microvascular and neovascularisation, accelerated aging and shortened lifespan. Single cell transcriptomic analysis of Mouse Lung Endothelial Cells (MLECs) showed a significant up-regulation of the inflammatory response. At the molecular level, progerin interacts with and destroys the NAD + -dependent deacylase Sirt 7; aberrant expression of Sirt7 reduced the inflammatory response induced by progerin in human umbilical vein endothelial cells. Most notably, vascular endothelial targeted Sirt7 gene therapy driven by the ICAM2 promoter in rAAV1 vectors improved neovascularization, improved senescence characteristics and reduced Lmna^f/f(ii) a The life span of TC mice is prolonged by more than 75%. These data support that endothelial dysfunction is a major trigger of systemic aging and emphasize that gene therapy is a potential strategy for clinical treatment of HGPS and age-related vascular dysfunction.

In a first aspect, the present invention provides an agent that causes the expression of the Sirt7 gene.

In a second aspect, the present invention provides the use of an agent according to the first aspect in the manufacture of a medicament for improving neovascularization, improving aging characteristics, preventing aging, prolonging life, and/or treating the hakinson-gilford progeria syndrome (HGPS) and/or age-related disorders.

In a third aspect, the present invention provides a method for improving neovascularization, improving aging characteristics, preventing aging, prolonging lifespan, and/or treating hakinson-gilford progeria syndrome (HGPS) and/or an age-related disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of an agent according to the first aspect.

Drawings

FIG. 1 shows CD31⁺Single cell transcriptome profile of mouse lung endothelial cells. (A) Sequencing of CD31 by FACS⁺Mouse lung endothelial cells were analyzed for purity. (B) CD31⁺The t-SNE projection of the cells shows four clusters: endothelial Cells (EC), B lymphocytes (B-like), T lymphocytes (T-like), and macrophages: (Sample) was obtained. (C) Marker gene expression in four clusters: endothelial cells (Cd31, Cd34, Cdh5), B-like (Ly6d, Cd22, Cd81), T-like (Cd3d, Cd3e, Cd28) andand (Cd14, Cd68, Cd 282). (D) Display Lmna^G609G/G609G(G609G) and Lmna^f/f(Flox) heat map of marker gene expression levels in mice.

FIG. 2 shows that single cell transcriptomics analysis indicates the presence of inflammatory responses and cardiac dysfunction in presenile endothelial cells. (A) Lmna from transcriptome data^G609G/G609G(G609G, green) and Lmna^f/f(Flox, orange) CD31⁺t-SNE projection of mouse lung endothelial cells. (B-D) GO and KEGG pathway enrichment for differentially expressed genes between G609G and Flox cells. Lmna^G609G/G609GMouse lung endothelial cells show an enrichment of genes that regulate the inflammatory response (C) and genes associated with cardiac dysfunction (D). (E) Quantitative PCR analysis of altered genes observed in human umbilical vein endothelial cells abnormally expressing progerin or wild-type LMNA in (C) and (D). Data represent mean ± s.e.m. P<0.05，*P<0.01，*P<0.001 (student's t-test).

Figure 3 shows endothelial specific dysfunction in premature aging mice. (A, B) derived from (A) Lmna^f/f(ii) a TC mice and (B) Lmna^G609G/G609GAnd Lmna^f/fThoracic aortic section of control mice H&E staining, showing intimal-medial thickening. Scale bar, 20 μm. (C) Lmna^f/f(ii) a TC mice and Lmna^f/fAcetylcholine (ACh) -induced thoracic aortic vasodilation in control mice. P<0.01。(D)Lmna^G609G/G609GAcetylcholine-induced chest dominance in mice and control miceThe arterial vasodilation. P<0.01. (E) Sodium Nitroprusside (SNP) -induced Lmna^G609G/G609GThoracic aortic vasodilation in mice and control mice. (F) From Lmna^{f /f}(ii) a TC and eNOS levels in thoracic aortic sections of control mice. Scale bar, 20 μm.

All data represent mean ± s.e.m. P values were calculated by student t-test.

FIG. 4 shows a decrease in capillary density and defect in neovascularization. (A) Lmna^f/f(ii) a TC and Lmna^f/fCD31 in mouse⁺Immunofluorescence staining of gastrocnemius (left) and quantification (right). Scale bar, 50 μm. (B) Lmna^f/f(ii) a TC and Lmna^f/fImmunofluorescent staining of CD31 in mouse liver. Scale bar, 50 μm. (C) Lmna^f/f(ii) a TC and Lmna^f/fRepresentative microcirculation images (left) and quantification of blood flow recovery (right) after hind limb ischemia in mice. (D) 14 days after femoral artery ligation CD31⁺Representative cross-sections and quantification of gastrocnemius. Scale bar, 50 μm. All data represent mean ± s.e.m. P values were calculated by student t-test.

FIG. 5 shows Lmna^f/f(ii) a Systemic senescence phenotype of TC mice. (A-C) Masson trichrome staining showed Lmna^f/f(ii) a Atherosclerotic plaques in aorta (a), smooth muscle cell loss (B), and cardiac fibrosis (C) of TC mice. Scale bar, 20 μm. (D) Heart weight and echocardiographic parameters including heart rate, cardiac output, Left Ventricular (LV) ejection fraction, and LV ejection shortening. P<0.05，Lmna^f/f(ii) a TC vs Lmna^f/fA mouse. (E) Lmna^f/f(ii) a The running endurance of TC mice decreased. P<0.001. (F) micro-CT analysis shows that in Lmna^f/f(ii) a Reduction in trabecular bone volume/tissue volume (BV/TV), trabecular number (tb.n) and trabecular thickness (tb.th), and increase in trabecular detachment (tb.sp) in TC mice. P<0.05，Lmna^f/f(ii) a TC vs Lmna^f/fA mouse. (G) Lmna^G609G/G609G、Lmna^G609G/+、Lmna^f/f(ii) a TC and Lmna^f/fLife span of mice. (H) Male Lmna^G609G/G609G、Lmna^G609G/+、Lmna^f/f(ii) a TC and Lmna^f/fBody of mouseAnd (4) heavy. P<0.05，Lmna^f/f(ii) a TC vs Lmna^f/fA mouse; p<0.001，Lmna^G609G/G609GComparison with Lmna^f/fA mouse. All data represent mean ± s.e.m. P values were calculated by student t test, except that statistical comparison of survival data was by Log-rank test.

FIG. 6 shows that accumulation of progerin destabilizes Sirt 7. (A) Sirt7^-/-And Sirt7^+/+Quantification of blood flow recovery after hind limb ischemia in mice. (B) Representative immunoblots showing indicated protein levels in human umbilical vein endothelial cells treated with si-SIRT7 or scarmble (Scam). (C) Real-time PCR analysis of indicated gene expression in human umbilical vein endothelial cells treated with si-SIRT7 or Scam. P<0.05, siRNA vs Scam. (D) Representative immunoblots, showing indicated sirtuin (Sirt1, Sirt6, and Sirt7) protein levels in FACS sorted mouse lung endothelial cells. Note Lmna^f/f(ii) a Sirt7 was down-regulated in TC mouse lung endothelial cells, whereas Sirt6 was up-regulated and Sirt1 was hardly changed. (E) Co-immunoprecipitation (Co-IP) experiments showed HA-SIRT7 in anti-FLAG-laminA and anti-FLAG-progerin immunoprecipitates. (F) Representative immunoblots, showed that polyubiquinated SIRT7 was up-regulated in the presence of progerin, but down-regulated in the presence of laminA. (G) Representative immunoblots showing SIRT7 protein levels in the presence of laminA or progerin in HEK293 cells treated with Cycloheximide (CHX) and/or MG132 (M). All data represent mean ± s.e.m. P values were calculated by student t-test.

Figure 7 shows that vascular endothelium-targeted Sirt7 therapy revitalizes the microvasculature and prolongs lifespan of the premature aging mice. (A) Real-time PCR analysis of the abnormally up-regulated gene after SIRT7 overexpression in human umbilical vein endothelial cells with progerin overexpression was performed. P<0.05，**P<0.01，***P<0.001. (B) Lmna with hindlimb ischemia with/without IS7O particle treatment^f/f(ii) a Determination of neovascularization in TC mice. P<0.05. (C) Immunofluorescence microscopy of FLAG-SIRT7 and CD31 expression in gastrocnemius 14 days after femoral artery ligation. Scale bar, 25 μm. (D) Lmna treated/untreated with IS7O particles^f/f(ii) a CD31 in TC mice⁺Percentage of endothelial cellsAnd (4) the ratio. P<0.001. (E) Lmna after IS7O treatment^f/f(ii) a Representative immunofluorescence images of TC mice liver, aorta and muscle showing CD31 with FLAG-SIRT7 expression⁺Endothelial cells. Scale bar, 50 μm. (F) Representative immunoblots, showing expression of FLAG-SIRT7 in aorta and Whole Bone Marrow Cells (WBMC). It was noted that FLAG-SIRT7 was detected only in whole bone marrow cells. (G) Lmna treated and untreated by IS7O^f/f(ii) a TC mice and Lmna^G609G/+Life span of mice. (H) Lmna treated and untreated by IS7O^f/f(ii) a TC mice and Lmna^f/fBody weight of the mice. All data represent mean ± s.e.m. P values were calculated by student t test, except that statistical comparison of survival data was by Log-rank test.

FIG. 8 shows Lmna^f/fProduction of mice and Lmna^G609G/G609GPhenotypic analysis of mice. (A) Carry Lmna^G609GMutation (Lmna 1827C>Lmna of T)^f/fSchematic drawing of the knock-in strategy for mice. (B) Lmna^G609G/G609GMouse and Lmna^f/fRepresentative photographs of control mice. (C) Representative immunoblots showed Lmna^G609G/+、Lmna^G609G/G609GAnd Lmna^+/+Lamin, Progerin and Lamin A expression in control mice. (D) Lmna^G609G/+、Lmna^G609G/G609GAnd Lmna^+/+Mouse longevity determination.

FIG. 9 shows CD31⁺Single cell transcriptomics analysis of mouse lung endothelial cells. (A) Lmna^G609G/G609G(G609G) and Lmna^f/f(Flox)CD31⁺P21 in mouse Lung endothelial cells^Cip/Waf1The mRNA level. p21^Cip/Waf1Endothelial cells isolated from G609G mouse andthe specificity is increased in the sample cells. (B) G609G and Flox CD31⁺Cd45 and Tie2 levels in mouse lung endothelial cells. Cd45 expression is absent in endothelial cells, and Tie2 expression is endothelial cell specific.

FIG. 10 shows vascular endothelial specific progerin expression. (A-B) examination by immunofluorescence stainingMeasuring Lmna^f/f(ii) a TC and Lmna^f/fProgerin and CD31 expression in aortic (a) and muscle (B) tissues of mice. Scale bar, 50 μm.

FIG. 11 shows Lmna^G609G/+Vasodilation analysis of mice. Lmna^G609G/+And Lmna^+/+Acetylcholine (ACh) -induced (left) and Sodium Nitroprusside (SNP) -induced (right) vasodilation in control mice. P<0.01。

FIG. 12 shows the expression of atherosclerosis-related and osteoporosis-related genes in mouse lung endothelial cell transcriptome.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention provides an agent that causes Sirt7 gene expression; preferably, the agent is a carrier; more preferably, the vector is a plasmid and/or viral vector; most preferably, the viral vector is a recombinant adeno-associated virus (rAAV), particularly a rAAV serotype 1.

In a specific embodiment, Sirt7 gene expression is vascular endothelial-specific expression; in particular, Sirt7 gene expression was driven by the ICAM2 promoter.

The invention also provides the use of the agent in the manufacture of a medicament for improving neovascularization, improving aging characteristics, preventing aging, prolonging life, and/or treating hakinson-gilford progeria syndrome (HGPS) and/or an age-related disease. Preferably, the age-related disease is cardiovascular disease (CAD), arthritis, muscle wasting and/or osteoporosis. More preferably, the cardiovascular disease is heart failure and/or atherosclerosis.

In a specific embodiment, the aging, HGPS and/or age-related disease is characterized by vascular endothelial dysfunction.

The invention also provides a method for improving neovascularization, improving aging characteristics, preventing aging, prolonging lifespan, and/or treating hakinson-gilford progeria syndrome and/or an age-related disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of an agent. Preferably, the age-related disease is cardiovascular disease, arthritis, muscle wasting and/or osteoporosis. More preferably, the cardiovascular disease is heart failure and/or atherosclerosis.

In a specific embodiment, the aging, HGPS and/or age-related disease is characterized by vascular endothelial dysfunction.

The present invention will be further illustrated by the following experimental procedures and examples, which are for illustrative purposes only and do not limit the scope of the present invention.

Experimental procedure

Animal(s) production

Lmna^f/fAllele (Lmna flanked by 2 loxP sites^G609G) Produced by the national society of biotechnology limited. Briefly, 5 'and 3' homology arms were amplified from BAC clones RP23-21K15 and RP23-174J9, respectively. The G609G (GGC to GGT) mutation was introduced in exon 11 of the 3' homology arm. C57BL/6 embryonic stem cells are used for gene targeting. To obtain a generalized progerin expression (Lmna)^G609G/G609G) Then, Lmna^f/fMice were housed with E2A-Cre mice. Lmna for obtaining vascular endothelial specific progerin expression^f/fMice were housed with Tie2-Cre mice. Mice were housed and treated according to protocols approved by the committee for the use of live animals in the teaching and research of shenzhen university, china.

Hind limb ischemia

Male mice of 4 months old were anesthetized with 4% chloral hydrate (0.20ml/20g) by intraperitoneal injection. Hindlimb ischemia was performed by unilateral femoral artery ligation and resection as previously described (48). Briefly, the neurovascular pedicles were observed under an optical microscope after a 1 cm incision on the skin of the left hind limb. Ligation was performed in the left femoral artery proximal to the superficial abdominal artery branch and anterior to the great saphenous artery. Then, the attached branch between the femoral artery and the ligation is excised. The skin was sutured using 4-0 suture and erythromycin ointment was applied to prevent post-operative wound infection. The dynamic microcirculation imaging system (Shenzhen shengqiang, China) is used for evaluating the recovery condition of blood flow before and after the operation. Relative blood flow recovery is expressed as the ratio of ischemic to non-ischemic. At least three mice were included in each experimental group.

Cell culture

HEK293 cells and human umbilical vein endothelial cells were purchased from ATCC. HEK293 cells in 10% Fetal Bovine Serum (FBS) supplementedDMEM (Life technologies, USA) at 37 deg.C and 5% CO₂Culturing under the conditions of (1). Human umbilical vein endothelial cells in a medium containing 15% FBS, 50. mu.g/ml endothelial growth supplement (ECGS) and 100. mu.g/ml heparinM199 (Life technologies, USA) 5% CO at 37 deg.C₂Culturing under the condition. All cell lines used were validated by Short Tandem Repeat (STR) profiling and were mycoplasma free.

RNA isolation and quantitative PCR (Q-PCR) analysis

According to manufacturer's instructions, useThe reagent RNAioso Plus (Takara, Japan) extracts total RNA from cells or mouse tissues and transcribes it into cDNA using 5 XPrimescript RT Master Mix (Takara, Japan). mRNA levels were determined by quantitative PCR using SYBR Premix Ex Taq II (Takara, Japan) detected on a CFX ligation real-time PCR detection system (Bio-Rad).

Protein extraction and western blotting

For protein extraction, cells were suspended in SDS lysis buffer and boiled. Then, the lysate was centrifuged at 12,000 Xg for 2 minutes, and the supernatant was collected. For western blotting, protein samples were separated on SDS-polyacrylamide gels, transferred to PVDF membranes (Millipore, usa), blocked with 5% skim milk, and incubated with relevant antibodies. Images were acquired on a Bio-Rad system.

Immunofluorescence staining

From Lmna^G609G/G609G、Lmna^+/+、Lmna^f/f(ii) a TC and Lmna^f/fMice collected aortic, skeletal and liver tissues.Frozen sections were prepared and fixed with 4% PFA, permeabilized with 0.3% Triton X-100, blocked with 5% BSA and 1% goat serum, and then incubated with primary antibody for 2 hours at room temperature or overnight at 4 ℃. After 3 washes with PBST, sections were incubated with secondary antibody for 1 hour at room temperature and then stained with DAPI anti-fade mounting medium. Images were captured under a zeiss LSM880 confocal microscope.

Masson trichrome stain

Paraffin-embedded sections of PFA-fixed tissue were deparaffinized and hydrated. Staining was then performed using Masson trichrome staining kit (bi yun day, china). Briefly, sections were soaked in Bouin buffer at 37 ℃ for 2 hours, and then stained with a celestine blue stain, a hematoxylin stain, a ponceau stain, and a aniline blue solution for 3 minutes in this order. After 3 times of ethanol dehydration, the solid-liquid (BBI Life sciences, China) sealing piece is sealed by neutral balsam. Images were captured under a zeiss LSM880 confocal microscope.

Fluorescence activated cell sorting

Mice were sacrificed by decapitation. The lungs were then harvested, cut into small pieces, and digested with collagenase I (200U/ml) and neutral protease (0.565mg/ml) for 1 hour at 37 ℃. The isolated cells were incubated with PE-conjugated anti-CD 31 antibody for 1 hour at 4 ℃ and then with 7-AAD (1:100) for 5 minutes. CD31 positive and 7-AAD negative cells were sorted on a flow cytometer (BD biosciences, USA).

Electromyography

Male mice 4 months old were anesthetized with 4% chloral hydrate by intraperitoneal injection. The thoracic aorta was collected, rinsed in ice-cold Krebs solution and cut into 2 mm long rings. Each aortic annulus was immersed in 5ml of oxygenated (95% O) solution at 37 deg.C in a myograph chamber (620M, Danish Myo Technology)₂And 5% CO₂) Krebs solution for 30 minutes. Each loop was stretched in a stepwise fashion to optimal resting tension (thoracic aorta to-9 mN) and equilibrated for 30 min. Then, 100mM K + Krebs solution was added to the chamber to cause the reference contraction, and then rinsed with Krebs solution at 37 ℃ until the baseline was reached. Vasodilation induced by acetylcholine (ACh) or Sodium Nitroprusside (SNP) (1nM to 100 μm) was recorded in the 5-HT (2 μm) systolic loop. Data ofExpressed as a percentage of force reduction and peak K + induced contraction. At least three mice were included in each experimental group.

Mouse/human cytokine antibody array

Samples of mice or humans according to the manufacturer's instructionsCytokine assays were performed. Briefly, membranes were incubated in blocking buffer for 30 minutes at room temperature. Samples prepared from serum or cell lysate were added to each membrane and incubated for 4 hours at room temperature. After 3 washes with buffer 1 and 2 washes with buffer 2, the membrane was reacted with the biotinylated antibody mixture at 4 ℃ overnight. After 2 hours incubation with 1000 × HRP-streptavidin, the membrane was washed again 3 times with buffer 1 and twice with buffer 2 before visualization using the Bio-Rad detection system. At least three mice were included in each experimental group.

Echocardiography

Male mice 7-8 months old were anesthetized by isoflurane gas inhalation and then subjected to transthoracic echocardiography (IU22, Philips). The parameters obtained include heart rate, cardiac output, left ventricular posterior wall size (LVPWD), left ventricular end-diastolic size (LVEDD), left ventricular end-systolic diameter (LVEDD), LV ejection fraction, and LV shortening fraction. At least three mice were included in each experimental group.

Bone Density analysis

Male mice 7-8 months old were sacrificed by decapitation. The femur was fixed in 4% PFA overnight at 4 ℃. Relevant data were collected by micro-CT (Scanco Medical, μ CT 100). At least three mice were included in each experimental group.

Endurance run test

Fatigue resistance was monitored using a Rota-Rod treadmill (YLS-4C, Jinan-Yankee research, China). Briefly, mice were placed on a rotating runway and the rotation speed was gradually increased to 40 r/min. When mice become exhausted, they will safely fall off the rotating runway and the latency to fall is recorded. At least three mice were included in each experimental group.

10 XGenomics Single cell RNA sequencing

CD31 isolated from murine lungs by FACS⁺Cells (a)>90% survival) was used for single cell RNA sequencing. A sequence library was constructed according to the Chromium Single Cell Instrument library protocol (49). Briefly, Chromium was used^TMSingle cell 3' transcriptome kit v2 version barcoded and reverse transcribed single cell RNA, followed by fragmentation and amplification to generate cDNA. cDNA was quantified using an agilent bioanalyzer DNA chip and the library was sequenced using Illumina Hiseq PE150, assigning approximately 10-30M of raw data to each cell. Reads were mapped to the mouse mm9 genome and analyzed using STAR:>90% of the reads map reliably to genomic regions,>50% mapped to exon regions. Cell range 2.1.0 was used to align reads, generate feature barcode matrices, and perform clustering and gene expression analysis. Each cell obtains>80,000 mean reads and 900 median genes. UMI (unique molecular identifier) counts are used to quantify gene expression levels and the t-SNE algorithm is used for dimensionality reduction. The cell population was then clustered by k-means clustering (k-4). Log2FoldChange is the ratio of gene expression of one cluster to that of all other cells. The p-value was calculated using a negative binomial test, and the error discovery rate was determined by the Benjamini-Hochberg program. GO and KEGG enrichment assays were performed in DAVID version 6.8 (50).

Statistical analysis

Statistical significance was determined using the two-tailed student t-test, but statistical comparison of survival data was performed by the log-rank test. All data are expressed as mean ± s.d. or mean ± s.e.m. as shown, and p-values <0.05 are considered statistically significant.

Example 1

Single cell transcriptomics analysis showed CD31⁺There are four major clusters of cells in Mouse Lung Endothelial Cells (MLEC)

To investigate the aging mechanism of vascular endothelium, we generated a mouse model of conditional progerin knockin, where Lmna^G609GThe mutation is flanked by loxP sites, i.e., Lmna^f/fMice (fig. 8A). Lmna is^f/fMice were crossed with E2A-Cre mice in which Cre recombinase was ubiquitously expressed, including germ cells, to produce Lmna^G609G/G609GA mouse. Progerin in these Lmna^G609G/G609GIt is ubiquitously expressed in mice, and it recapitulates many of the presenile features found in the premature aging syndrome, including growth retardation and shortened lifespan, etc. (FIGS. 8B-D).

To understand the major changes in VE, we used FACS (fig. 1A) from three pairs of Lmna^G609G/G609G(G609G) and Lmna^f/f(Flox) control mice in which CD31 was isolated⁺Mouse lung endothelial cells (25) were subjected to 10 × Genomics single cell RNA sequencing. We recovered 6,004 cells (4,137 from G609G, 1,867 from Flox mice) and used the k-means clustering algorithm to group the cells into four groups (fig. 1B). As expected, one group showed high Cd31, Cd34, and Cdh5 expression, and thus represented mouse lung endothelial cells to a large extent. By FACS with CD31⁺The other three groups, co-purified with mouse lung endothelial cells, showed relatively low Cd31 expression (more than 10-fold lower than mouse lung endothelial cells) but higher Cd45 expression (fig. 9). Further analysis showed that these clusters most likely contained: b lymphocytes with high Cd22, Cd81, and Ly6d expression (B-like); t lymphocytes with high Cd3d, Cd3e, and Cd28 expression (T-like); and macrophages with high Cd22, Cd81, and Ly6d expression (Sample) (fig. 1C). Most marker gene expression levels were comparable between G609G and Flox mice, except Cd34 and Icam1, which were significantly elevated in G609GECs, and in G609GIncreased Cd14 and Vcam1 in the sample cells (fig. 1D). Notably, Icam1 and Vcam1 are among the most conserved markers of endothelial senescence and atherosclerosis. Thus, we have established Lmna^f/fA conditional progerin KI mouse model and reveals a unique endothelial cell population for mechanistic studies.

Example 2

Presenile endothelial cells exhibit a systemic inflammatory response

In four CDs 31⁺In the mouse lung endothelial cell cluster, endothelial cells andthe sample cells showed high levels of p21^{Cip1 /Waf1}(FIG. 9A), this is a typical aging marker. This finding suggests that these cells are the main target of progerin in the context of aging. Interestingly, one previous study reported that Lmna was obtained by combining Lmna with Lmna^f/+Obtained by crossing to Lyz-Cre mouseThe senescence phenotype caused by specific progerin was minimal (23), which means thatMay play only a minor role in body aging. Therefore, we focused on endothelial cells for further analysis. We recovered 899 and 445 endothelial cells from E2A and Flox mice, respectively (fig. 2A). Selection of these mice for changes in expression>1.5 times of the genes were analyzed for GO and KEGG. We observed a significant abundance of pathways that regulate chemotaxis, the immune response to malaria and trypanosomiasis, inflammatory bowel disease and rheumatoid arthritis, and pathways critical to cardiac function (fig. 2B-D). To confirm this observation and to exclude paracrine effects of other cell types, we overexpressed progerin in Human Umbilical Vein Endothelial Cells (HUVECs) and analyzed representative genes by quantitative PCR. Most of the genes examined, including IL6, IL8, IL15, CXCL1 and IL1 α, were significantly upregulated upon aberrant progerin expression (fig. 2E). Taken together, these data suggest that progerin may cause an inflammatory response in endothelial cells, leading to systemic aging of various organs.

Example 3

Vasodilation deficiency of senilism mice caused by vascular endothelial dysfunction

To verify whether vascular endothelial dysfunction plays an important role in systemic aging, we will Lmna^f/fMice were crossed with Tie2-Cre line to produce Lmna^f/f(ii) a TC mice in which Cre recombinase expression is driven by the promoter/enhancer of the endothelial specific Tie2 gene (26). Single cell transcriptome analysis confirmed that Tie2 was detected predominantly in endothelial cells (fig. 9B). Consistently, in Lmna^f/f(ii) a Progerin was observed in the vascular endothelium of TC mice, but in Lmna^f/fNot observed in vascular endothelium of control mice or other tissues (fig. 10). Vascular endothelial specific progerin induced Lmna^f/f(ii) a Thickening of the inner membrane in TC mice in a manner consistent with that of total KI mice, i.e., Lmna^G609G/G609GMice were similar (FIGS. 4A-B). These were followed by functional analyses of the vascular endothelium based on acetylcholine (ACh) modulated vasodilation. Acetylcholine-induced thoracic aortic relaxation at Lmna^f/f(ii) a Significant impairment in TC mice (fig. 4C). In Lmna^G609G/G609GAnd Lmna^G609G/+Similar defects were observed in mice (fig. 4D and 11), where progerin was expressed in both endothelial cells and smooth muscle cells (23). To gain more evidence of supporting vascular endothelial-specific dysfunction, we examined thoracic aortic relaxation induced by Sodium Nitroprusside (SNP), a smooth muscle cell-dependent vasodilator. And Lmna^f/fComparison in Lmna in control mice^G609G/G609GAnd Lmna^G609G/+There was little difference in thoracic aortic vasodilation observed (fig. 4E and fig. 11), supporting that vascular endothelial dysfunction is a key factor in the vasodilation deficiency in the premature aging mice. Since NO is the most potent vasodilator, we examined Lmna^f/f(ii) a TC and Lmna^f/feNOS levels in the thoracic aorta of control mice. As expected, with Lmna^f/fLmna as compared to control mice^f/f(ii) a The eNOS levels of TC mice were significantly reduced (fig. 4F). Thus, this data confers vascular endothelial-specific dysfunction in premature aging mice.

Example 4

Defect of neovascularization after ischemia in presenile mice

Both reduced capillary density and the ability to neovascularization are characteristic of endothelial dysfunction (1). We examined Lmna by immunofluorescence staining^f/f(ii) a TC mice eachMicrovasculature in seed tissue. We observed Lmna compared to control^f/f(ii) a CD31 in TC mice⁺Significant loss of endothelial cells (fig. 5A-B). We further examined Lmna^f/f(ii) a TC mice ability to ischemia-induced neovascularization following femoral artery ligation. In fact, Lmna compared to the control group^f/f(ii) a Limb perfusion was significantly attenuated following TC mouse ischemia (fig. 5C). Histological analysis confirmed that Lmna^f/f(ii) a Defects in the restoration of blood flow in TC mice reflect an impaired ability to form new blood vessels in the ischemic area (fig. 5D). In summary, Lmna^f/f(ii) a TC mice are characterized by endothelial cell loss, decreased capillary density, and a defect in their ability to form new blood vessels.

Example 5

Endothelial dysfunction is one of the causes of systemic aging

Single cell transcriptome and Lmna^G609G/G609GCardiac dysfunction in mice was associated (figure 2). In Lmna^G609G/G609GThe association of genetic changes associated with atherosclerosis and osteoporosis was evident in endothelial cells (online human mendelian genetic database) (fig. 12). Therefore, we conclude that endothelial-specific dysfunction may be sufficient to trigger systemic senescence. Remarkably, atherosclerosis is in Lmna^f/f(ii) a Prominent in TC mice (FIG. 5A; atherosclerotic aortic plaques observed in all eight tested mice), and severe fibrosis in arteries and heart (FIGS. 5B-C); both are typical characteristics of aging. In addition, with Lmna^f/fLmna as compared to control mice^f/f(ii) a The heart/body weight ratio of TC was significantly increased (fig. 5D). Echocardiography confirmed, with Lmna^f/fLmna 7-8 months old compared to control mice^f/f(ii) a The heart rate, cardiac output, Left Ventricular Ejection Fraction (LVEF) and fractional shortening (LVFS) of TC decreased significantly. Lmna^f/f(ii) a Running endurance in TC mice was largely affected (fig. 5E), which may be a reflection of muscle atrophy and/or cardiac dysfunction. In addition, micro-computed tomography (micro-CT) found Lmna^f/f(ii) a Trabecular bone volume/tissue volume (BV/TV), trabecular number (tb.n) and trabecular thickness (tb.th) were reduced in TC mice, but trabecular separationIncrease (tb.sp) (fig. 5F), indicating osteoporosis, an important hallmark of systemic senescence (27). Most notably, vascular endothelial-specific dysfunction not only accelerated aging of various tissues/organs, but also shortened Lmna^f/f(ii) a Median lifespan of TC mice (2 weeks), degree and Lmna^G609G/G609GMice (21 weeks) were similar (fig. 5G). Interestingly, Lmna^G609G/G609GMice began losing weight approximately from 8 weeks of age, while Lmna^f/f(ii) a The TC mice lost only slightly in body weight (fig. 5H), indicating that weight loss itself is less likely to be the primary cause of premature aging compared to endothelial dysfunction. Taken together, these results indicate that endothelial dysfunction, at least in premature aging, is a causal factor in systemic aging.

Example 6

Accumulation of progerin destabilizes SIRT7

The absence of Sirt7, an NAD + dependent deacylase, leads to cardiac dysfunction with systemic inflammation and accelerated aging (28, 29). We noted that Sirt7 KO mice were deficient in neovascularization (fig. 6A). Knock-out Sirt7 upregulated the levels of IL1 β and IL6 in human umbilical vein endothelial cells as determined by western blotting and real-time PCR (fig. 6B-C). Notably, in Lmna^f/f(ii) a In TC mouse lung endothelial cells, Sirt7 protein levels were reduced by nearly 50% (fig. 6D). In contrast, in Lmna^f/f(ii) a There was little reduction in Sirt6 and Sirt1 levels in TC mouse lung endothelial cells. Furthermore, Co-immunoprecipitation (Co-IP) showed that laminA interacted with Sirt7, which was significantly enhanced in the case of progerin (fig. 6E). HA-SIRT7 was polyubiquitinated, and it was enhanced in the presence of progerin compared to lamINA (FIG. 6F). Aberrant expression of progerin in human umbilical vein endothelial cells accelerated SIRT7 protein degradation, which was inhibited by MG132 (fig. 6E). These data indicate that accumulation of progerin destabilizes Sirt7 via a proteasome pathway in progeria cells.

Example 7

Vascular endothelium-specific expression of Sirt7 improves senescence profile and prolongs lifespan

We conclude that Sirt7 may be the basis for vascular endothelial dysfunction in senilism miceA foundation. To test this hypothesis, we first examined whether abnormal Sirt7 could restore an exacerbated inflammatory response in human umbilical vein endothelial cells. As shown, overexpression of Sirt7 significantly down-regulated the expression of various inflammatory genes, such as IL1 β (fig. 7A). To test the function of Sirt7 in defective neovascularization in vivo, we generated a recombinant AAV serotype 1(rAAV1) kit in which Sirt7 gene expression was driven by a synthetic ICAM2 promoter (IS7O), which ensured vascular endothelial-specific expression (30, 31). As shown, at 1.25 × 10¹⁰In situ injection of IS7O at a dose of 50 μ l/viral genome-containing particle (vg) significantly improved Lmna^f/f(ii) a Vascularization of TC mice (fig. 7B). Aberrant expression of Sirt7 and an increase in CD 31-labeled endothelial cells were confirmed in neovascular endothelial cells by fluorescence confocal microscopy (fig. 7C-D).

We next raised the question of whether IS7O could ameliorate premature aging and extend life. For this reason, IS7O pellets were injected through the tail vein starting at 21 weeks of age when premature mice began to die. The injection is repeated every other week at a concentration of 5 × 10¹⁰vg/200. mu.l/mouse. Although all untreated mice died before 34 weeks of age, most mice receiving 5 IS7O treatments survived when sacrificed at 44 weeks of age for histological analysis. Abnormal expression of FLAG-SIRT7 was observed in endothelial cells of the liver, muscle and aorta, but not in bone marrow cells (WBMC), as determined by fluorescence microscopy and/or western blotting (fig. 7E-F). Most notably, the median lifetime is extended by 76% -from 25 weeks to>For 44 weeks (fig. 7G). In Lmna^f/f(ii) a Age-related weight loss was slightly restored in TC mice after being treated with IS7O (fig. 7H). These data indicate that progerin-induced vascular endothelial dysfunction and systemic aging is due in part, if not in whole, to a decrease in Sirt 7.

Discussion of the related Art

Increasing evidence supports endothelial dysfunction as a significant hallmark of vascular aging and cardiovascular disease (32-34). However, the fundamental question remains whether vascular endothelial dysfunction can trigger systemic aging. Thin blood vesselThe heterogeneity of cells and their close association with blood flow make it difficult to understand the major functions of the vascular endothelium. Murine Lmna^G609GThe mutation corresponded to L Zmpste24 found in humans with HGPS^-/-MNA^G608GLeading to a premature aging phenotype in various tissues/organs, thus providing an ideal model for studying aging mechanisms at the tissue and organism level. From Lmna^G609GModel data indicate that smooth muscle cells are the major cause of vascular disease such as atherosclerosis (17-22). A recent study showed that Lmna^G609GSpecific expression in smooth muscle cells leads to atherosclerosis and shortens the lifespan of Apoe-/-mice predisposed to atherosclerosis (23). We used Tie2-Cre mice to generate vascular endothelial specific Lmna^G609GMouse model. Lmna^f/f(ii) a TC mice display systemic Lmna^G609GModels similar vascular dysfunction, accelerated aging and shortened lifespan. Tie2 expression has been reported not only in endothelial cells but also in hematopoietic lineages (35). Our single cell transcriptomics data were mainly on mouse lung endothelial cells rather than B, T orTie2 transcripts were identified in the sample cells. When the synthetic ICAM2 promoter was used to drive abnormal expression of FLAG-SIRT7 in recovery experiments, abnormal FLAG-SIRT7 was successfully detected in endothelial cells of aorta, muscle and liver, but hardly in whole bone marrow cells. Thus, Tie 2-driven progerin expression, combined with synthetic ICAM 2-driven SIRT7 recovery, largely ensured a specific contribution of endothelial cells in systemic senescence. Notably, although HSC number and function are in another model of progeria, Zmpste24^-/-Mice (15) declined, but healthy hematopoietic progenitors were transplanted to Zmpste24 in the context of systemic senescence^-/-Little effect was observed in mice. Recently, Hamczyk et al found Lmna in macrophages^G609GKnock-in of alleles mediated by LysM-Cre only affects senescence and longevity (23). Therefore, our data advantageously demonstrate, as the largest secretory organ (3,4), bloodThe endothelium plays a key role in regulating systemic aging and longevity. Our findings are supported by Foisner et al that expression of progerin driven by the vascular endothelial-cadherin promoter in transgenic lines leads to cardiovascular abnormalities and shortened life span (36).

One limitation to the understanding of the mechanisms of vascular endothelial dysfunction is vascular cell heterogeneity and the lack of a suitable endothelial cell system in vitro. Here, we used single cell RNA sequencing technology to analyze the transcriptome of mouse lung endothelial cells. Surprisingly, despite FACS achievement>Mouse lung endothelial cells of 95% purity, but isolated by CD 31-immunofluorescence labeling, were a mixture of cells, including endothelial cells, T-like, B-like, anda sample cell. Despite FACS enrichment, these non-endothelial cells express low levels of CD31 mRNA, increasing cell surface proteins such as CD 31T-like, B-like andpossibility of cell-like cells being obtained from adjacent endothelial cells by intercellular protein transfer (37). Nevertheless, these findings indicate that CD31 cannot be purified alone⁺Cells and bring them together for mechanistic studies, otherwise misleading conclusions may be drawn. We compared the expression of genes associated with atherosclerosis, arthritis, heart failure, osteoporosis or amyotrophic lateral sclerosis (online human mendelian genetic database) between premature failure and control in all four clusters. Mainly in endothelial cells andsignificant changes in these genes/pathways were observed in the sample cells (fig. S7). At this stage, it is difficult to distinguish between cell autonomous and paracrine effects between different cell populations. In the future, it is worth in Lmna^f/f(ii) a Similar analysis was performed in TC mouse lung endothelial cells. These data will help to study the paracrine effects of endothelial cells on other cell populations.

Since the causal relationship between LMNA G608G mutation and HGPS was identified, efforts have been made to develop HGPS therapeutics. FTI (39, 40), resveratrol and N-acetylcysteine (NAC) (15) treatment reduced the premature aging characteristics and extended the lifespan of the mouse model of premature aging. Incubation of rapamycin (41) and metformin (42) restored senescence in HGPS cells. Based on these observations, HGPS patients taking FTI-lonafarnib in clinical trials showed significant improvement in health status, reduction in mortality and potential prolongation of life (about 1-2 years) (43-45). With gene therapy and the dispensable effects of laminA, morpholino oligos (16) and CRISPR-Cas 9 design (46,47), prevention of laminA/progerin production, alleviation of aging characteristics and prolongation of longevity from 25% to 40% in premature aging mice. However, given the indispensable function of laminA in humans, these genome modification strategies require further experimentation prior to potential clinical applications. Here, applying different strategies, we found that rAAV1-SIRT7(IS7O), against dysfunctional VE, can improve the premature aging characteristics to a large extent and almost double the median lifetime (from 25 weeks to >44 weeks). To our knowledge, this is the most significant recovery of premature aging in mouse models by gene therapy.

In general, it has been found that vascular endothelial dysfunction is a trigger for systemic aging and is a risk factor for age-related diseases such as atherosclerosis, heart failure and osteoporosis. This suggests that many clinically used VE-targeting drugs and molecules may be good candidates for the treatment of age-related diseases other than cardiovascular diseases. Also, findings in endothelial progenitor cells suggest a great potential for stem cell-based therapeutic strategies in premature aging and anti-aging applications.

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