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）移植可以治愈多种恶性肿瘤和非恶性造血疾病。脐带血 (UCB) 是一种容易获得的 HSC 来源，但由于 HSC 数量不足，其在成人移植中的使用受到限制。更好地了解人类 HSC 扩增所需的因素对于开发诱导 HSC 体外长期存活和增殖的条件至关重要。尽管细胞因子和形态发生素已在培养物中实现了短期扩增，但可移植 UCB HSC 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“干细胞生态位”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库的大小并控制受控的增殖和分化。在 MERIT 资助下，我们证明了这样一种基质大分子，基质硫酸乙酰肝素 (HS)，是体外重现人类 HSC 生态位所需的关键成分，其中 HS 共定位并调节细胞因子、趋化因子、形态发生素的活性（包括骨形态发生蛋白：BMP）和 HSC 上的基质成分。我们的小组还表明，HS 支持离体、细胞因子介导的人 UCB 衍生的再增殖干细胞（NOD/SCID 小鼠再增殖细胞）的适度短期扩增，这些细胞可以转移到第二受体。当仅用细胞因子（不含 HS）培养时，重新增殖的 HSC 会丢失。最近，我们发现一些 BMP，包括那些抑制 HSC 维持的 BMP，是由 UCB 祖细胞本身产生的。与此一致的是，外源提供的 BMP 拮抗剂 Gremlin1 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张。 Gremlin1 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着关键作用。它由成骨细胞和许多其他细胞产生，并调节已知影响 HSC 命运的多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，我们推测它可能调节干细胞微环境中成骨细胞分泌的 Gremlin1 的作用。 Gremlin1 和 HS 可能因此有助于 HSC 存活和增殖的调节。假设：Gremlin1 是干细胞生态位的功能相关成分，其对干细胞的活性通过与成骨细胞 HS 的结合和相互作用来调节。具体目标： (1) 对 HS 和 Gremlin1 之间的相互作用进行结构和功能分析：确定结合动力学，识别每个分子上的结合域，并检查结合相互作用对 Gremlin1 功能的影响。 (2) 检查 Gremlin1 在干细胞生态位中的功能作用：使用在成骨细胞中过度表达 Gremlin1 的突变小鼠和 HS 生物合成缺陷的小鼠，确定成骨细胞 Gremlin1 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 Gremlin1 诱导的 HSC 存活和增殖。意义、与退伍军人健康的相关性：了解 Gremlin1 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，从而可能有助于开发支持长期干细胞存活和生存的条件。体外增殖。 Gremlin1 HS结合位点的结构功能研究也可能提高我们对疾病病理生理学的理解，并为未来开发肽模拟物/新药提供信息，用于治疗退伍军人中常见的几种疾病，其中Gremlin1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) 数量不足限制了成年退伍军人优选移植策略的使用，例如人脐带血 HSC 移植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 Gremlin1 与硫酸乙酰肝素（Gremlin1 似乎与其结合）一起有助于调节骨髓干细胞生态位中的 HSC。了解 Gremlin1 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 Gremlin1 在干细胞生态位中的作用，将增强我们对 HSC 生物学的理解，并可能有助于为移植诱导 HSC 扩增开发更好的条件。 Gremlin1 中 HS 结合位点的结构功能研究也可能为未来开发治疗退伍军人中普遍存在的 Gremlin1 发挥病理生理作用的其他疾病的新药提供信息，例如糖尿病肾病、玻璃体视网膜病、骨质减少、肺病和癌症。