Targeting, Quantifying, and Isolating Heterogeneous Populations of Senescent Cells from Tissues via Cell Surface and Secreted Proteomes

通过细胞表面和分泌的蛋白质组靶向、定量和分离组织中的异质衰老细胞群

基本信息

批准号：
10688781
负责人：
Nathan Basisty
金额：
$ 11.54万
依托单位：
NATIONAL INSTITUTE ON AGING
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10688781
关键词：
Adipocytes Adipose tissue Aging Atlases Baltimore Biological Markers Biology Blood Vessels Cardiac Cell Aging Cell Culture Techniques Cell Surface Proteins Cell surface Cells Cohort Studies Collaborations Data Databases Disease Excision Fatty acid glycerol esters Fibroblasts Flow Cytometry Glycoproteins Hospitals Human Immunofluorescence Immunologic Individual Institutional Review Boards Kidney Link Longevity Longitudinal Studies Lung Mass Spectrum Analysis Membrane Proteins Metabolic Methods Modernization Mus Musculoskeletal Neurologic Phenotype Plasma Population Population Heterogeneity Proteins Proteome Proteomics Resources Saints Smooth Muscle Myocytes Specimen Spectrometry Technology Testing Therapeutic Tissues Translations Validation Vascular Endothelial Cell Vascular Smooth Muscle Work age related aged base candidate marker cell type experimental study frailty human tissue immunocytochemistry in vivo insight molecular marker monocyte obese person personalized approach phenotypic biomarker pre-clinical predictive signature senescence subcutaneous targeted biomarker targeted treatment therapy development

项目摘要

In our first objective, substantial progress has been made in refining the list of SASP biomarker candidates based on human cohorts studies. In early collaborative studies with Drs. Luigi Ferrucci and Toshiko Tanaka, we identified a subset of the senescence-associated secretory phenotype (SASP) that are associated with aging in human plasma in the Baltimore Longitudinal Study of Aging. Over the last year we have expanded proteomic profiling of senescence into monocytes, which has yielded a largely distinct list of senescence-associated proteins from earlier studies. To test whether monocyte-senescence specific signatures are predictive of aging, frailty, and disease status in human populations, we are continuing to collaborate with NIA human cohort studies. Toward our second objective, we have collected early data on the cell-surface proteome (surfaceome) of senescent cells, from which we will identify and prioritize the most specific surfaceome candidates for targeting senescent cells. To identify the most specific surface proteins, we have compared the cell surface proteins we identified on senescent fibroblasts with the cell-surface protein atlas database (Baush-Fluck et al. 2015. PLOS One) and rank our candidates based on the number of cell-types they are known to be expressed in. To expand and validate our list of senescent cell surface markers, we have also collected senescent and non-senescent cell surface proteins using an additional method termed glyco-cell surface capture, which isolates cell surface N-linked glycoproteins. Over the past year we have optimized mass spectrometry acquisisiton methods for the analysis of surfaceome studies. We have additionally performed pilot surfaceome studies using a new optimized glyco-cell surface capture approach in several cell types: fibroblasts, monocytes, pre-adipocytes, vascular smooth muscle cells, and vascular endothelial cells. We are now performing comprehensive quantitative comparison of cell surface changes in senescent versus non-senescent cells of each cell type. In early studies we have identified 4 candidate senescence-specific cell surface proteins for validation in human tissues. To validate senescent markers in vivo in a tissue type that matches the cell culture experiments used for the initial discovery of the surfaceome, we have initiated collaborations to obtain monocytes and adipose tissues from humans. To validate cell surface markers in monocytes, our colleagues at the BLSA will share monocyte tissues from young and aged individuals. These specimens will be probed for candidate surfaceome proteins by immunofluorescence. Additionally, to validate the pre-adipocytes surfaceome, we will obtain fat tissue. To this end we have submitted an IRB proposal with Dr. Steven Cunningham (Ascension Saint Agnes Hospital, Baltimore) to collect omental and subcutaneous fat for the validation of senescence in young, old and obese individuals in vivo. Going forward we will validate surfaceome markers in both tissue in vivo using immunocytochemistry and flow cytometry.

在我们的第一个目标中，基于人类人群研究的SASP生物标志物候选者列表已经取得了重大进展。在与博士的早期合作研究中。 Luigi Ferrucci和Toshiko Tanaka，我们确定了与衰老相关的分泌表型（SASP）的一部分，这些分泌表型（SASP）与巴尔的摩衰老纵向研究中的人血浆中的衰老有关。在过去的一年中，我们将衰老的蛋白质组学分析扩展到单核细胞中，这在早期研究中产生了与衰老相关的蛋白质的很大不同清单。为了测试单核细胞年份特定特定的特定特定特定特定的特定标志，可以预测人口中的衰老，脆弱和疾病状况，我们正在继续与NIA人类同伴研究合作。朝向第二个目标，我们收集了有关衰老细胞细胞表面蛋白质组（表面体）的早期数据，我们将从中识别并优先考虑最特定的Surfaceome候选者来靶向衰老细胞。为了确定最特定的表面蛋白质，我们已经比较了我们在衰老成纤维细胞上鉴定的细胞表面蛋白与细胞表面蛋白质图集数据库（Baush-Fluck等人，2015年。PLOS。一个），并根据细胞类型的数量来表达我们的列表，以扩展我们的表面和遗物，以使我们的列表分布。细胞表面蛋白使用另一种方法称为Glyco细胞表面捕获，该方法分离了细胞表面N-连接的糖蛋白。在过去的一年中，我们已经优化了质谱学获取方法，用于分析表面研究。我们还使用了几种细胞类型的新的优化的Glyco细胞表面捕获方法进行了前曲面表面研究：成纤维细胞，单核细胞，脂肪细胞，血管平滑肌细胞和血管内皮细胞。现在，我们正在对每种细胞类型的衰老与非年代细胞的细胞表面变化进行全面的定量比较。在早期研究中，我们已经确定了4种候选衰老特异性细胞表面蛋白，以在人体组织中进行验证。为了验证与用于最初发现Surfaceome的细胞培养实验相匹配的组织类型中的衰老标记，我们已经开始合作以获取人类的单核细胞和脂肪组织。为了验证单核细胞中的细胞表面标记，我们在BLSA的同事将分享来自年轻人和老年人的单核细胞组织。这些标本将通过免疫荧光探测候选表面蛋白。此外，为了验证脂肪细胞的表面体，我们将获得脂肪组织。为此，我们已经向IRB提出了一项IRB提案，并向史蒂文·坎宁安（Steven Cunningham）（巴尔的摩的升天圣艾格尼丝医院）提出了一项提议，以收集胶质和皮下脂肪，以确认体内年轻，老年人和肥胖的人的衰老。展望未来，我们将使用免疫细胞化学和流式细胞仪来验证两个组织中的表面标记。