Heparan Sulfate Proteoglycans in Human Hematopoiesis

硫酸乙酰肝素蛋白多糖在人体造血中的作用

• 批准号: 8139532
• 负责人: Pankaj GUPTA
• 金额: --
• 依托单位: MINNEAPOLIS VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8139532
• 关键词: Adult; Allogenic; Autologous; Binding; Binding Sites; Bone Marrow Stem Cell; Bone Morphogenetic Proteins; Cell Count; Cell Maintenance; Cell Proliferation; Cell Survival; Cell surface; Cells; Development; Diabetic Nephropathy; Differentiation and Growth; Diffusion; Disease; Embryo; Embryonic Development; Erinaceidae; Extracellular Matrix; Functional disorder; Funding; Future; Grant; Health; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Purging; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Heparan Sulfate Biosynthesis; Heparan Sulfate Proteoglycan; Heparin; Heparin Binding; Heparitin Sulfate; Human; In Vitro; Inorganic Sulfates; Kinetics; Lung diseases; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Mus; Mutant Strains Mice; Non obese; Non-Malignant; Osteoblasts; Osteopenia; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Play; Population; Regulation; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; Role; SCID Mice; Secondary to; Signal Pathway; Signal Transduction; Source; Stem cells; Structure; Supplementation; Time; Transplantation; Umbilical Cord Blood; Umbilical Cord Blood Transplantation; Undifferentiated; Unspecified or Sulfate Ion Sulfates; Veterans; Work; autocrine; cancer cell; chemokine; chordin; clinical application; cytokine; diabetic; extracellular; hematopoietic stem cell fate; improved; in vivo; inhibitor/antagonist; macromolecule; mimetics; morphogens; notch protein; novel; paracrine; progenitor; stem cell biology; stem cell niche

DESCRIPTION (provided by applicant): Background: Hematopoietic stem cell (HSC) transplantation can cure several malignancies and non-malignant hematopoietic conditions. Umbilical cord blood (UCB) is an easily available source of HSC, but its use for transplanting adults is limited by insufficient HSC numbers. An improved understanding of factors required for human HSC expansion is essential for developing conditions that induce long-term in vitro survival and proliferation of HSC. Although short-term expansion in culture has been achieved by cytokines and morphogens, factors and conditions required for long-term in vitro survival and expansion of transplantable UCB HSC are unknown. In vivo, HSC reside in highly specialized "stem cell niches," likely formed by osteoblasts, wherein specific combinations of cytokines and matrix/cell surface macromolecules localize to regulate stem cell pool size and govern controlled proliferation and differentiation. With MERIT grant funding, we showed that one such matrix macromolecule, stromal heparan sulfate (HS), is a critical component required for recapitulation of the human HSC niche in vitro, wherein HS co-localize and modulate the activity of cytokines, chemokines, morphogens (including bone morphogenetic proteins: BMPs) and matrix components on HSC. Our group also showed that HS support ex vivo, cytokine-mediated modest short-term expansion of human UCB derived repopulating stem cells (NOD/SCID mouse repopulating cells) that can be transferred to secondary recipients. Repopulating HSC were lost when cultured in cytokines alone (without HS). More recently, we found that several BMPs, including those inhibitory to HSC maintenance, are produced by UCB progenitors themselves. Consistent with this, antagonism of endogenous autocrine/paracrine BMP signaling by exogenously provided BMP antagonist Gremlin1 further augmented long-term maintenance and modest expansion of primitive hematopoietic progenitors. Gremlin1 is an extracellular heparin-binding BMP inhibitor that plays a critical role in growth, differentiation and embryonic development and in various diseases. It is produced by osteoblasts and many other cells, and modulates several morphogens and pathways (e.g. Wnts, Sonic hedgehog and Notch) known to influence HSC fate. Because others and we have shown that HS regulates the diffusion, local concentration and activity of other heparin-binding BMP antagonists, we speculate that may modulate the effects of Gremlin1 secreted by osteoblasts in the stem cell niche. Gremlin1 and HS may thus contribute to the regulation of HSC survival and proliferation. Hypothesis: Gremlin1 is a functionally relevant component of the stem cell niche, where its activity on stem cells is modulated by binding to and interacting with osteoblast HS. Specific Aims: (1) Perform a structural and functional analysis of interactions between HS and Gremlin1: determine binding kinetics, identify binding domains on each molecule, and examine the effect of binding interactions on Gremlin1 function. (2) Examine the functional role of Gremlin1 in the stem cell niche: Using mutant mice that over-express Gremlin1 in osteoblasts, and mice deficient in HS biosynthesis, determine if and how osteoblast Gremlin1 influences (a) endogenous hematopoiesis, (b) the stem-cell supportive function of osteoblasts in vitro, and (c) HSC fate following transplantation. (d) Examine how endogenous osteoblast HS influences Gremlin1-induced HSC survival and proliferation. Significance, relevance to veterans< health: Understanding the interactions of Gremlin1 with HS, and the mechanism(s) of their action on HSC will improve our understanding of stem cell biology and may thus help develop conditions that support long-term stem cell survival and proliferation in vitro. The structure-function studies on HS-binding sites in Gremlin1 may also improve our understanding of disease pathophysiology, and inform future development of peptide-mimetics/novel drugs for the treatment of several disorders prevalent in veterans in which Gremlin1 plays a pathophysiological role, including diabetic nephropathy, vitreoretinopathy, osteopenia, lung disease and cancer. PUBLIC HEALTH RELEVANCE: Insufficient hematopoietic stem cell (HSC) numbers limit the use of preferable transplantation strategies such as transplantation of human umbilical cord blood HSC, or autologous HSC purged of cancer cells, for adult Veterans. Our preliminary findings indicate that the multi-functional molecule Gremlin1, together with heparan sulfate (to which Gremlin1 appears to bind), contributes to regulation of HSC in the bone marrow stem cell niche. Understanding the structural aspects and functional consequences of binding interactions of Gremlin1 with heparan sulfate, and the role of Gremlin1 in the stem cell niche, will enhance our understanding of HSC biology, and may help develop better conditions for inducing HSC expansion for transplantation. The structure- function studies on HS-binding sites in Gremlin1 may also inform future development of novel drugs for other disorders prevalent in Veterans in which Gremlin1 plays a pathophysiological role, such as diabetic nephropathy, vitreo-retinopathy, osteopenia, lung disease and cancer.

描述（由申请人提供）： 背景：造血干细胞（HSC）移植可以治疗多种恶性肿瘤和非恶性造血系统疾病。脐带血（UCB）是一种很容易获得的HSC来源，但由于HSC数量不足，其用于移植成人受到限制。对人类HSC扩增所需因素的进一步了解对于开发诱导HSC长期体外存活和增殖的条件至关重要。虽然细胞因子和形态发生素已经实现了培养中的短期扩增，但可移植UCB HSC的长期体外存活和扩增所需的因素和条件尚不清楚。在体内，HSC存在于高度特化的"干细胞龛"中，可能由成骨细胞形成，其中细胞因子和基质/细胞表面大分子的特定组合定位以调节干细胞池大小并控制受控的增殖和分化。在MERIT的资助下，我们发现了一种这样的基质大分子，基质硫酸乙酰肝素（HS），是体外重现人HSC生态位所需的关键组分，其中HS共定位和调节HSC上细胞因子，趋化因子，形态发生蛋白（包括骨形态发生蛋白：BMP）和基质组分的活性。我们的研究小组还表明，HS支持离体的、由精氨酸介导的人UCB衍生的再生干细胞（NOD/SCID小鼠再生细胞）的适度短期扩增，所述再生干细胞可以转移到第二受体。当在单独的细胞因子（没有HS）中培养时，再生HSC丢失。最近，我们发现几种BMP，包括抑制HSC维持的BMP，是由UCB祖细胞本身产生的。与此一致，外源性BMP拮抗剂Gremlin 1对内源性自分泌/旁分泌BMP信号传导的拮抗作用进一步增强了原始造血祖细胞的长期维持和适度扩增。Gremlin 1是一种细胞外肝素结合BMP抑制剂，在生长、分化和胚胎发育以及各种疾病中发挥关键作用。它由成骨细胞和许多其他细胞产生，并调节已知影响HSC命运的几种形态发生素和途径（例如Wnts，Sonic hedgehog和Notch）。因为其他人和我们已经表明，HS调节其他肝素结合BMP拮抗剂的扩散，局部浓度和活性，我们推测可能调节干细胞龛中成骨细胞分泌的Gremlin 1的作用。Gremlin1和HS可能因此有助于调节HSC的存活和增殖。假设：Gremlin 1是干细胞龛的功能相关组分，其对干细胞的活性通过与成骨细胞HS结合并相互作用来调节。具体目标：（1）对HS和Gremlin 1之间的相互作用进行结构和功能分析：确定结合动力学，鉴定每个分子上的结合结构域，并检查结合相互作用对Gremlin 1功能的影响。(2)检查Gremlin 1在干细胞生态位中的功能作用：使用在成骨细胞中过表达Gremlin 1的突变小鼠和HS生物合成缺陷的小鼠，确定成骨细胞Gremlin 1是否以及如何影响（a）内源性造血，（B）体外成骨细胞的干细胞支持功能，以及（c）移植后HSC的命运。(d)检查内源性成骨细胞HS如何影响Gremlin 1诱导的HSC存活和增殖。意义，与退伍军人健康的相关性：了解Gremlin 1与HS的相互作用，以及它们对HSC的作用机制，将提高我们对干细胞生物学的理解，从而有助于开发支持体外长期干细胞存活和增殖的条件。对Gremlin1中HS结合位点的结构-功能研究也可以提高我们对疾病病理生理学的理解，并为将来开发用于治疗退伍军人中流行的几种疾病的肽模拟物/新药提供信息，其中Gremlin1在其中发挥病理生理学作用，包括糖尿病肾病，玻璃体视网膜病变，骨质减少，肺病和癌症。 公共卫生相关性： 造血干细胞（HSC）数量不足限制了优选的移植策略的使用，例如对成年退伍军人移植人脐带血HSC或清除癌细胞的自体HSC。我们的初步研究结果表明，多功能分子Gremlin 1与硫酸乙酰肝素（Gremlin 1似乎与硫酸乙酰肝素结合）一起有助于骨髓干细胞龛中HSC的调节。了解Gremlin 1与硫酸乙酰肝素结合相互作用的结构方面和功能后果，以及Gremlin 1在干细胞生态位中的作用，将增强我们对HSC生物学的理解，并可能有助于开发更好的条件诱导HSC扩增用于移植。对Gremlin 1中HS结合位点的结构-功能研究也可以为将来开发用于退伍军人中流行的其他疾病的新药提供信息，其中Gremlin 1在其中发挥病理生理作用，例如糖尿病肾病，玻璃体视网膜病变，骨质减少，肺病和癌症。