HEPARIN MODULATION OF VEGF BINDING TO FIBRONECTIN

肝素调节 VEGF 与纤连蛋白的结合

• 批准号: 7369292
• 负责人: MATTHEW A NUGENT
• 金额: $ 2.15万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369292
• 关键词: HEPARIN; MODULATION; VEGF; BINDING; FIBRONECTIN

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It is known that the action of vascular endothelial growth factor (VEGF) is regulated through its interactions with components of the extracellular matrix, such as fibronectin and heparan sulfate. One of the mechanisms by which fibronectin exerts its regulatory activity could be by directly binding to VEGF. We conducted a detailed study of the interactions between VEGF and fibronectin and investigated how heparin modulates this interaction using a combination of in vitro binding assays and atomic force microscopy. Plasma fibronectin was adsorbed either on hydrophobic or hydrophilic surfaces in the presence and absence of heparin and equilibrium binding of soluble 125I-VEGF165 was measured. The fibronectin bound VEGF was released into two successive fractions by incubations with a high ionic strength solution and a NaOH solution respectively, which represent distinct populations of VEGF binding sites on fibronectin. We found two VEGF binding sites per fibronectin dimer, mainly ionic in nature, with a Kd value in the range of 1-6*10-7 M. One of these binding sites is pH sensitive and interacts with VEGF only at low pH, a condition encountered in hypoxic tissues. The pH sensitive binding of VEGF to fibronectin may be reflective of a regulatory mechanism by which fibronectin directs VEGF to hypoxic areas in order to promote angiogenesis. The other binding site is not sensitive to pH, but is highly affected by fibronectin conformation in that it becomes available only under conditions where fibronectin adopts an extended conformation. Heparin stabilizes the extended conformation and consequently increases VEGF binding to this site on fibronectin. The effect of heparin potentially reflects a process whereby heparan sulfate within the extracellular matrix can act as a docking site for fibronectin to coordinate its interactions with VEGF. Consistent with this possible role, adsorption of fibronectin to hydrophilic surfaces, which also favors the extended conformation, increased VEGF binding. The effects of heparin on fibronectin conformation were studied by atomic force microscopy with fibronectin adsorbed on mica in the presence or absence of heparin. Under these conditions, three structural populations of fibronectin molecules were identified: compact molecules of an ellipsoid shape and molecules of a more extended configuration, containing either two or three distinct domains. The length of the molecules belonging to the two- and three-domain categories was significantly increased in the presence of heparin. Changes in the diameter and height of the individual domains that accompany this increase in length suggest that heparin triggers a partial unfolding of fibronectin, exposing various binding sites on the surface of the molecule. This effect is dependent on the particular chemical composition and size of heparin, as other glycosaminoglycans and certain chemical derivatives and fragments of heparin do not act in a similar way. Our data suggest a potential role for heparan sulfate proteoglycans to regulate angiogenesis by modulating the interactions between VEGF and fibronectin within the extracellular matri

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。众所周知，血管内皮生长因子（VEGF）的作用通过其与细胞外基质的成分（例如纤连蛋白和硫酸乙酰肝素）的相互作用来调节。纤连蛋白施加其调节活性的机制之一可以通过直接与VEGF结合。我们对VEGF和纤连蛋白之间的相互作用进行了详细研究，并研究了肝素如何使用体外结合测定和原子力显微镜的组合来调节这种相互作用。在存在和不存在肝素的情况下，将血浆纤连蛋白吸附在疏水或亲水表面上，并测量可溶性125I-VEGF165的平衡结合。通过与高离子强度溶液和NaOH溶液孵育，纤连蛋白结合的VEGF分别释放为两个连续的部分，该溶液分别代表了纤连蛋白上VEGF结合位点的不同种群。我们发现每个纤连蛋白二聚体的两个VEGF结合位点，主要是离子本质上，其KD值在1-6*10-7 M的范围内。这些结合位点之一是pH敏感的，并且仅在低pH下与VEGF相互作用，在低氧组织中遇到的条件。 VEGF与纤连蛋白的pH敏感结合可能反映了一种调节机制，纤连蛋白将VEGF引导到低氧区域以促进血管生成。另一个结合位点对pH不敏感，但受纤连蛋白构象的高度影响，因为它仅在纤连蛋白采用扩展构象的条件下可用。肝素稳定了扩展的构象，因此增加了纤连蛋白上的VEGF与该位点的结合。肝素的作用有可能反映出一个过程，在该过程中，细胞外基质中硫酸盐可以充当纤连蛋白的对接位点，以协调其与VEGF的相互作用。与这种可能的作用一致，将纤连蛋白吸附到亲水性表面，这也有利于扩展构象，增加了VEGF结合。在存在或不存在肝素的情况下，用原子力显微镜用吸附在云母上的原子力显微镜研究了肝素对纤连蛋白构象的影响。在这些条件下，鉴定了三个结构性群群的结构群：椭圆形形状的紧凑分子和更扩展的构型的分子，其中包含两个或三个不同的域。在存在肝素的情况下，属于两域和三域类别的分子的长度显着增加。伴随该长度增加的单个结构域的直径和高度的变化表明，肝素会触发纤连蛋白的部分展开，暴露于分子表面上的各种结合位点。这种作用取决于肝素的特定化学成分和大小，因为其他糖胺聚糖和肝素的某些化学衍生物和碎片也不以类似的方式起作用。我们的数据表明，通过调节细胞外基质中VEGF和纤连蛋白之间的相互作用，乙酰硫酸盐蛋白聚糖在调节血管生成中的潜在作用