Patent Application
20220031762
The present invention relates to endothelial progenitor cells (EPCs) and their role in preventing aging, extending lifespan and treating age-related diseases. In particular, the present invention relates to use of endothelial progenitor cells in clinical progeria treatment.
The statements herein only provide background information related to the present application, and do not necessarily constitute prior art.
Aging represents the largest risk factor for many age-related diseases, as exemplified by vascular dysfunction and cardiovascular diseases (CVDs). The blood vessel consists of the tunica intima (composed of endothelial cells; ECs), the tunica media (composed of vascular smooth muscle cells; VSMCs) and the tunica adventitia (consisting of connective tissue). The endothelium separates the vessel wall from the blood flow and has an irreplaceable role in regulating vascular tone and homeostasis. Age-related functional decline in ECs and VSMCs is a main cause of CVDs. ECs secrete various vasodilators and vasoconstrictors that act on VSMCs and induce blood-vessel contraction and relaxation. For instance, nitric oxide (NO) is synthesized from L-arginine by endothelial NO synthase (eNOS) in ECs and is released on VSMCs to induce blood-vessel relaxation. When ECs become senescent or dysfunctional, vasoconstrictive, pro-coagulative and pro-inflammatory cytokines are released; this effect reduces NO bioavailability and in turn increases vascular intimal permeability and EC migration. Despite advances in the understanding of the mechanisms of endothelial dysfunction, it is unclear whether it directly triggers organismal aging.
Accumulating data suggest that the mechanisms underlying normal aging are similar to those governing Hutchinson-Gilford progeria syndrome (HGPS)—a premature aging syndrome in which affected patients typically succumb to CVDs. HGPS is predominantly caused by an a c. 1824 C>T, p. G608G mutation in LMNA gene, which activates an alternate splicing event and generates a 50-amino-acid-truncated form of lamin A, referred to as progerin. The murine LmnaG609G, which is equivalent to LMNAG608G in humans, causes aging phenotypes resembling HGPS. It has been shown that progerin targets SMCs and causes blood vessel calcification and atherosclerosis. Recent work by two groups showed that SMC-specific progerin knock-in mice are healthy and have a normal lifespan, but suffer from blood-vessel calcification, atherosclerosis and shortened lifespan when crossed to Apoe−/− mice. In contrast to SMCs, the contributing roles of the vascular endothelium (VE) to systemic/organismal aging are elusive.
Endothelial progenitor cells (EPCs) mainly exist in the bone marrow. Upon VE injury, cytokines and growth factors, such as VEGF, SDF-1, G-CSF and estrogen, mobilize EPCs to the peripheral circulation. The EPCs then seed at the injury site and promote repair via neovascularization. An age-related decline in the number and function of EPCs is a main reason for decreased VE repair capacity. Progeria models exhibit depleted stem cells, including mesenchymal stem cells (MSCs), epithelial stem cells, muscular stem cells and hematopoietic stem cells (HSCs). Questions remain as to whether EPCs also decline in progeria and if so, whether this decline causally accelerates aging. To address these issues, we generated a conditional progerin (LmnaG609G) knock-in (KI) model, i.e. Lmnaf/f mice. In combination with E2A-Cre and Tie2-Cre mice, we aimed to investigate the roles of the VE dysfunction and the EPCs to systemic aging.
Vascular dysfunction is one of the typical characteristics of aging, but its contributing roles to systemic aging is lacking experimental evidence. Accumulating data suggest that mechanisms underlying aging are similar to those governing Hutchinson-Gilford progeria syndrome (HGPS), a premature aging syndrome in which affected patients typically succumb to cardiovascular diseases (CVDs). Here, we generated a knock-in mouse model with the causative HGPS LmnaG609G mutation. Using the Lmnaf/f and Tie2-Cre mice, we showed that endothelial-specific dysfunction compromises the microvasculature and neovascularization and accelerates aging in multiple tissues/organs. Most importantly, endothelial-specific dysfunction shortens lifespan in Lmnaf/f; TC mice. Mechanistically, single-cell transcriptomic analysis of murine lung endothelial cells (MLECs) revealed a significant up-regulation of genes that regulate inflammation including Il6, Il8, Il15, Cxcl1 and Il1α etc. Further determined by FACS analysis and neovascularization assay, we observed that the number and function of EPCs in the bone marrow decline in Lmnaf/f; TC mice compared to Lmnaf/f control mice. Replenishing wild-type EPCs rejuvenates neovascularization capacity, ameliorates aging features and extends lifespan in progeria mice. These data reveal that endothelial dysfunction triggers systemic aging and highlight EPC therapy as a potential anti-aging strategy and clinical progeria treatment.
In one aspect, the present invention provides use of endothelial progenitor cells (EPCs) in the manufacture of a medicament for rejuvenating neovascularization capacity, ameliorating aging features, preventing aging, extending lifespan, and/or treating progeria and/or age-related diseases. Preferably, the age-related diseases are cardiovascular diseases and/or osteoporosis. More preferably, the cardiovascular diseases are atherosclerosis and/or heart failure.
In another aspect, the present invention provides a method for rejuvenating neovascularization capacity, ameliorating aging features, preventing aging, extending lifespan, and/or treating progeria and/or age-related diseases, comprising administering a pharmaceutically effective amount of EPCs to a subject in need thereof. Preferably, the age-related diseases are cardiovascular diseases and/or osteoporosis. More preferably, the cardiovascular diseases are atherosclerosis and/or heart failure.
The present invention provides use of endothelial progenitor cells (EPCs) in the manufacture of a medicament for rejuvenating neovascularization capacity, ameliorating aging features, preventing aging, extending lifespan, and/or treating progeria and/or age-related diseases, more preferably atherosclerosis and/or heart failure.
In a specific embodiment, the EPCs are CD133+ EPCs.
In a specific embodiment, the age-related diseases are characterized by vascular endothelium (VE) dysfunction.
In particular, the VE dysfunction includes a loss of endothelial cells, reduced capillary density and defective neovascularization capacity.
More particularly, the VE dysfunction is caused by progerin.
The present invention also provides a method for rejuvenating neovascularization capacity, ameliorating aging features, preventing aging, extending lifespan, and/or treating progeria and/or age-related diseases, comprising administering a pharmaceutically effective amount of EPCs to a subject in need thereof; preferably, the age-related diseases are cardiovascular diseases and/or osteoporosis, more preferably atherosclerosis and/or heart failure.
In a specific embodiment, the EPCs are CD133+ EPCs.
In a specific embodiment, the age-related diseases are characterized by vascular endothelium (VE) dysfunction.
In particular, the VE dysfunction includes a loss of endothelial cells, reduced capillary density and defective neovascularization capacity.
More particularly, the VE dysfunction is caused by progerin.
The present invention will be further demonstrated by the following experimental procedures and examples, which are used only for illustration purpose, but not limiting the scope of the present invention.
Experimental Procedures
Animals
Lmnaf/f alleles (LmnaG609G flanked by 2 loxP sites) were generated accordingly. The 5′ and 3′ homology arms were amplified from BAC clones RP23-21K15 and RP23-174J9, respectively. The G609G (GGC to GGT) mutation was introduced into exon 11 in the 3′ homology arm. C57BL/6 embryonic stem cells were used for gene targeting. To obtain ubiquitous progerin expression (LmnaG609G/G609G) Lmnaf/f mice were bred with E2A-Cre mice. To obtain VE-specific progerin expression, Lmnaf/f mice were bred with 71e2-cre mice. Mice were purchased from Cyagen Biosciences Inc., China, housed and handled in accordance with protocols approved by the Committee on the Use of Live Animals in Teaching and Research of Shenzhen University, China.
Hind Limb Ischemia
Four months old male mice were anesthetized with 4% chloral hydrate (0.20 ml/20 g) by intraperitoneal injection. Hind limb ischemia was performed by unilateral femoral artery ligation and excision, as previously described. In brief, the neurovascular pedicle was visualized under a light microscope following a 1-cm incision in the skin of the left hind limb. Ligations were made in the left femoral artery proximal to the superficial epigastric artery branch and anterior to the saphenous artery. Then, the femoral artery and the attached branches between ligations were excised. The skin was closed using a 4-0 suture line and erythromycin ointment was applied to prevent wound infection after surgery. Recovery of the blood flow was evaluated before and after surgery using a dynamic microcirculation imaging system (Teksgray, Shenzhen, China). Relative blood flow recovery is expressed as the ischemia to non-ischemia ratio. At least three mice were included in each experimental group.
Cell Culture
HEK293 cells and human umbilical vein endothelial cells (HUVECs) were purchased from ATCC. HEK293 cells were cultured in Gibco® DMEM (Life Technologies, USA) supplemented with 10% fetal bovine serum (FBS) at 37° C., 5% CO2. HUVECs were cultured in Gibco® M199 (Life Technologies, USA) supplemented with 15% FBS, 50 μg/ml endothelial cell growth supplement (ECGS) and 100 μg/ml heparin at 37° C., 5% CO2. All cell lines used were authenticated by short tandem repeat (STR) profile analysis and were mycoplasma free.
RNA Isolation and Quantitative PCR (Q-PCR) Analysis
Total RNA was extracted from cells or mouse tissues using Trizol® reagent RNAiso Plus (Takara, Japan) and transcribed into cDNA using 5× Primescript RT Master Mix (Takara, Japan), following the manufacturer's instructions. The mRNA levels were determined by quantitative PCR with SYBR Premix Ex Taq 11 (Takara, Japan) detected on a CFX Connected Real-Time PCR Detection System (Bio-Rad). All primer sequences are listed in Table 1.
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×g for 2 min 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% non-fat milk and incubated with the relevant antibodies. Images were acquired on a Bio-Rad system. All antibodies are listed in Table 2.
Immunofluorescence Staining
Aorta, skeletal muscle and liver tissues were collected from LmnaG609G/G609G Lmna+/+, Lmnaf/f; TC and Lmnaf/f mice. Frozen sections were prepared and fixed in 4% PFA, permeabilized with 0.3% Triton X-100, blocked with 5% BSA and 1% goat serum, and then incubated with primary antibodies at room temperature for 2 h or at 4° C. overnight. After three washes with PBST, the sections were incubated with secondary antibodies for 1 h at room temperature and then stained with DAPI anti-fade mounting medium. Images were captured under a Zeiss LSM880 confocal microscope. All antibodies are listed in Table 2.
Masson Trichrome Staining
Paraffin-embedded sections of PFA-fixed tissues were dewaxed and hydrated. Staining was then performed using a Masson trichrome staining kit (Beyotime, China). In brief, the sections were dipped in Bouin buffer for 2 h at 37° C., and then successively stained with Celestite blue staining solution, Hematoxylin staining solution, Ponceau's staining solution and Aniline blue solution for 3 minutes. After dehydrating with ethyl alcohol three times, the sections were mounted with Neutral Balsam Mounting Medium (BBI Life Science, China). Images were captured under a Zeiss LSM880 confocal microscope.
Fluorescence Activated Cell Sorting (FACS)
Mice were sacrificed by decapitation. The lungs were then collected, cut into small pieces and then digested with collagenase I (200 U/ml) and neutral protease (0.565 mg/ml) for 1 h at 37° C. The isolated cells were incubated with PE-conjugated anti CD31 antibody for 1 h at 4° C. and then 7-AAD (1:100) for 5 min. CD31-positive and 7-AAD-negative cells were sorted on a flow cytometer (BD biosciences, USA).
Myography
Four months old male mice were anesthetized with 4% chloral hydrate by intraperitoneal injection. Thoracic aortas were collected, rinsed in ice-cold Krebs solution and cut into 2 mm-length rings. Each aorta ring was bathed in 5 ml oxygenated (95% O2 and 5% CO2) Krebs solution at 37° C. for 30 min in a myograph chamber (620M, Danish Myo Technology). Each ring was stretched in a stepwise fashion to the optimal resting tension (thoracic aortas to ˜9 mN) and equilibrated for 30 min. Then, 100 mM K+ Krebs solution was added to the chambers to elicit a reference contraction and then washed out with Krebs solution at 37° C. until achieved a baseline. Vasodilatation induced by acetylcholine (Ach) or sodium nitroprusside (SNP) (1 nM to 100 μM) was recorded in 5-HT (2 μM) contracted rings. Data are represented as a percentage of force reduction and the peak of K+-induced contraction. At least three mice were included in each experimental group.
Mice/Human Cytokine Antibody Array
A cytokine assay for mice or human samples (RayBio®) was performed according to the manufacturer's instructions. Briefly, membranes were incubated in blocking buffer for 30 min at room temperature. The samples prepared from serum or cell lysates were added to each membrane and incubated for 4 h at room temperature. After three washes with buffer 1 and two washes with buffer 2, the membranes were reacted with a biotinylated antibody cocktail at 4° C. overnight. After incubation with 1000×HRP-Streptavidin for 2 h, the membranes were again washed three times with buffer 1 and two times with buffer 2 and then visualized using a Bio-Rad detection system. At least three mice were included in each experimental group.
Echocardiography
7-8 months old male mice were anesthetized by isoflurane gas inhalation and then subjected to transthoracic echocardiography (IU22, Philips). Parameters, including heart rate, cardiac output, left ventricular posterior wall dimension (LVPWD), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic diameter (LVESD), LV ejection fraction and LV fractional shortening were acquired. At least three mice were included in each experimental group.
Bone Density Analysis
7-8 months old male mice were sacrificed by decapitation. The thigh bone was fixed in 4% PFA at 4° C. overnight. The relevant data were collected by micro-CT (Scanco Medical, pCT100). At least three mice were included in each experimental group.
Endurance Running Test
A Rota-Rod Treadmill (YLS-4C, Jinan Yiyan Scientific Research Company, China) was used to monitor fatigue resistance. Briefly, mice were placed on the rotating lane and the speed of the rotations gradually increased to 40 r/min. When the mice were exhausted, they were safely dropped from the rotating lane and the latency to fall was recorded. At least three mice were included in each experimental group.
10× Genomics Single-Cell-RNA-Sequencing
CD31+ cells isolated from murine lung by FACS (>90% viability) were used for single-cell RNA sequencing. A sequence library was built according to the Chromium Single Cell Instrument library protocol. Briefly, single-cell RNAs were barcoded and reverse-transcribed using Chromium™ Single Cell 3′ Reagent Kits v2, then fragmented and amplified to generate cDNAs. The cDNAs were quantified using an Agilent Bioanalyzer 2100 DNA Chip, and the library was sequenced using an Illumina Hiseq PE150 with ˜10-30M raw data assigned for each cell. The reads were mapped to the mouse mm9 genome and analyzed using STAR: >90% reads mapped confidently to genomic regions and >50% mapped to exonic regions. Cell Ranger 2.1.0 was employed to align reads, generate feature-barcode matrices and perform clustering and gene expression analysis. >80,000 mean reads and 900 median genes per cell were obtained. The UMI (unique molecular identifier) counts were used to quantify the gene expression levels and the t-SNE algorithm was used for dimensionality reduction. The cell population was then clustered by k-means clustering (k=4). The Log2FoldChange was the ratio of gene expression of one cluster to that of all other cells. The p-value was calculated using the negative binomial test and the false discovery rate was determined by Benjamini-Hochberg procedure. GO and KEGG enrichment analysis were performed in DAVID version 6.8.
Isolation of CD133+ Progenitor Cells
3 months old male mice were sacrificed by decapitation. The femora and tibiae were separated and placed in a 0.5 ml micro-centrifuge tube which had a hole drilled in the bottom. A 1.5 ml micro-centrifuge tube was used to nest the 0.5 ml tube and the pair of tubes was centrifuged at 10,000×g for 15 sec. The bone marrow was suspended in 1 ml red blood cell lysis buffer at room temperature for 5 min, and the suspension was strained successively through a 75-μm and then 40-μm cell strainer (FALCON®, USA). After centrifugation at 300×g at 4° C. for 5 min, the cells were suspended in 500 μl MACS buffer and incubated with 5 μl anti-CD133 antibody (Miltenyi Biotec, Germany) for 10 min. After incubating with 20 μl beads (Miltenyi Biotec, Germany) in 80 μl MACS buffer, CD133+ progenitor cells were obtained by magnetic selection. At least three mice were included in each experimental group.
Statistical Analysis
A two-tailed Student's t-test was used to determine statistical significance. All data are presented as the means±s.d. or means±s.e.m. as indicated, and a p value <0.05 was considered statistically significant.
An outstanding question in the field of aging is whether endothelial dysfunction causally triggers systemic aging. The heterogeneity of vascular cells and their close communication with the blood stream, however, renders it difficult to understand the primary function of the VE. The murine LmnaG609G mutation, which is equivalent to human LMNAG608G, causes aging phenotypes in various tissues resembling HGPS. To examine the contributing roles of the VE to systemic aging, we generated a mouse model of conditional progerin knock-in, in which the LmnaG609G mutation was flanked with loxP sites, i.e. Lmnaf/f mice (
To understand the primary alterations in the VE, we isolated CD31+ MLECs from three pairs of LmnaG609G/G609G (G609G) and Lmnaf/f (Flox) control mice by FACS (
Of the four clusters of CD31+ MLECs, ECs and Mφ-like cells showed high levels of p21Cip1/Waf1 (
Our single-cell transcriptomic analysis in MLECs and quantitative PCR in HUVECs suggest that the VE have essential roles in systemic aging. To confirm these findings, we crossed the Lmnaf/f mice to a Tie2-Cre line, in which Cre recombinase expression is driven by the promoter/enhancer of endothelial-specific Tie2 gene, to generate Lmnaf/f; TC mice. Single-cell transcriptome analysis confirmed that Tie2 gene was mainly detected in ECs (
Reduced capillary density and neovascularization capacity are both characteristics of vascular aging. We thus examined the microvasculature in various tissues of Lmnaf/f; TC mice by immunofluorescence staining. We observed a significant loss in CD31+ ECs in Lmnaf/f; TC mice compared to controls (
Our single-cell transcriptomic data implicated heart dysfunction in LmnaG609G/G609G mice (
The VE-specific dysfunction not only accelerated aging in various tissues/organs, but also shortened the median lifespan in Lmnaf/f; TC mice (24 weeks), to a similar extent as LmnaG609G/G609G mice (21 weeks) (
CD133+ mononuclear cells are enriched in the bone marrow and are potential EPCs that are essential for vascular hemostasis. We purified CD133+ EPCs from Lmnaf/f; TC mice and Lmnaf/f control mice by FACS and studied the functional relevance in VE dysfunction and aging. Here, we found that >30% of the freshly isolated EPCs were positive for low density lipoproteins (as indicated by Dil-acLDL labeling) and Ulex europaeus agglutinin 1 (UEA-1) (
We then asked whether EPCs have a causal role in accelerating aging and shortening lifespan in progeria mice. To this end, we injected (via the tail vein) 1×106 MACS (Magnetic-activated cell sorting)-purified EPCs from rosa26-rainbow mice into LmnaG609G/G609G mice. EPCs were administered from 15 weeks before the earliest death event in LmnaG609G/G609G mice and repeated every week. Two of the EPC-treated mice were still alive at 27 weeks-of-age and were sacrificed for histological analysis. Donor-derived ECs were detected by fluorescence microscopy in the liver, muscle, aorta and lung (
Mounting evidence supports that endothelial dysfunction is a conspicuous marker for vascular aging and CVDs. Whether endothelial dysfunction primarily triggers organismal aging, however, is elusive. The murine LmnaG609G mutation, equivalent to the LMNAG608G found in humans with HGPS, causes premature aging phenotypes in various tissues/organs, thus providing an ideal model for studying aging mechanisms at both the tissue and organismal level. Data from the LmnaG609G model have suggested that SMCs are the primary cause of vascular diseases, such as atherosclerosis. Interestingly, a recent study showed that the specific expression of LmnaG609G in SMCs only causes atherosclerosis and shortens lifespan in atherosclerosis-prone apolipoprotein E-deficient (Apoe−/−) mice. The researchers also found that macrophage-specific LmnaG609G knock-in mediated by Lyz-Cre merely affects aging and lifespan. Here, we used Tie2-Cre mice to generate a VE-specific LmnaG609G model. These mice exhibited vascular dysfunction, accelerated aging and a shortened lifespan to a similar extent to the whole body LmnaG609G model. In support of our findings, Foisner et al. recently reported that the VE-cadherin promoter-driven endothelial-specific expression of progerin in a transgenic line causes cardiovascular abnormalities and shortened lifespan. The data from both our study and that of Foisner strongly suggest that, as the largest secretory organ, the VE is pivotal in regulating systemic aging and lifespan.
One limitation in the understanding of mechanisms of VE dysfunction is the vascular cell heterogeneity and the lack of appropriate in vitro system for ECs. Here, we took advantage of single-cell RNA sequencing technique to analyze the transcriptomes of MLECs. Surprisingly, although >95% purity was achieved by FACS, MLECs isolated by CD31-immunofluorescence labeling turned out to be a mixture of cells, including ECs, T-like, B-like and Mφ-like cells. It is unclear whether these cells are T cells, B cells and Mφ cells that express low level of CD31, or are transdifferentiated from ECs. Nevertheless, this finding suggests that one can't just purify CD31+ cells and pool them together for further mechanistic study, otherwise might get misleading conclusion. Indeed, we compared the expression of genes that are associated with atherosclerosis, arthritis, heart failure, osteoporosis or amyotrophy (the Online Mendelian Inheritance in Man database) between progeroid and control in all four clusters. An obvious alteration of these genes/pathway was observed mainly in ECs and Mφ-like cells (
The stem-cell theory of aging dictates that the number and functional decline of stem cells directly leads to defective tissue regeneration and consequently organismal aging. EPCs, MSCs and HSCs represent 3 stem-cell populations found in the bone marrow, of which the latter two have implicated clinical potential. We previously showed that the number and function of MSCs and HSCs decline in another progeria mouse model, Zmpste24−/− mice; however, we didn't observe any beneficial effect when MSCs from healthy donors was transplanted into Zmpste24−/− mice by tail vein injection. Consistent with the rapid decline of HSCs and MSCs, we found that the number and function of EPCs, represented by CD133+ mononuclear cells, significantly declined in progeria mice compared to healthy controls. Remarkably, EPC transplantation via tail vein injection improved the microvasculature, attenuated body-weight loss, and extended lifespan in progeria mice. To our knowledge, this study provides the first evidence to support the potential of stem-cell therapy in progeria treatment. We thus consider it worthwhile to optimize the conditions of this therapy to maximize the rescue effect elicited by EPCs, and to screen for chemicals that increase the number, improve the function and promote the mobilization of EPCs. Indeed, various drugs clinically used to treat CVDs, such as statins and PPARγ agonists, can mobilize EPCs from the bone marrow to peripheral circulation and enhance endothelial repair. Thus, further investigation is warranted to examine whether these drugs can slow down aging and promote longevity.
Collectively, we reveal that VE dysfunction is a trigger of systemic aging and is also a risk factor for age-related diseases like atherosclerosis, heart failure and osteoporosis. It suggests that many clinically used drugs and molecules that target VE might serve as good candidates in the treatment of age-related diseases other than CVDs. Likewise, the findings in EPCs implicate great potentials of stem-cell-based therapeutic strategy for progeria as well as in anti-aging applications.
This application is a continuation-in-part of International Patent Application No. PCT/CN2019/076488 with a filing date of Feb. 28, 2019, designating the United States, now pending. The content of the aforementioned application is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2019/076488 | Feb 2019 | US |
| Child | 17461094 | US |
