Regulation of hypoxia-induced angiogenesis: a chaperone escorts VEGF to the dance.
Regulation of hypoxia-induced angiogenesis: a chaperone escorts VEGF to the dance.
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DOI：
10.1172/jci13374
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发表时间：
2001-07
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通讯作者：
G. Semenza
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作者：
G. Semenza
与无脊椎动物相比，氧气可以通过扩散充分输送到所有细胞，而脊椎动物由于体型较大，需要专门用于氧气输送的循环系统。当全身供氧不足时，心输出量增加，红细胞生成增加。然而，当氧气输送局部受损时，例如在灌注因损伤或血管狭窄而中断的组织中，主要反应是血管生成，即新血管的萌芽。当组织内的细胞通过增加 VEGF 的产生来应对缺氧时，血管生成的复杂过程（参见参考文献 1-3）就开始了。 VEGF 被分泌并与位于血管内皮细胞表面的同源受体酪氨酸激酶（VEGFR1 和 VEGFR2）结合。受体连接触发一系列细胞内信号传导途径，从而启动血管生成。这种生物过程的临床重要性在过去十年中变得越来越明显，血管生成现在代表了预防和治疗多种疾病（尤其是缺血性心血管疾病和癌症）的新治疗方法的主要焦点 (1-3)。如果血管生成是盛大的舞蹈，那么 VEGF 就是舞会上的美女，数百名研究人员现在正在研究其表达的调节和生物学后果（参见参考文献 4）。尽管 VEGF 表达仅代表复杂的血管生成反应的一个组成部分，并且单独施用 VEGF 可能不足以影响治疗性血管生成，但 VEGF 仍然可以被认为是血管内皮细胞的关键激活剂。因此，建立调节 VEGF 活性的分子机制对于了解血管生成及其临床靶向方式至关重要。当细胞缺氧时，由于 mRNA 转录增加和 mRNA 降解减少，VEGF mRNA 表达被诱导 (5-7)。尽管缺氧细胞中翻译起始速率整体降低，但 VEGF mRNA 含有内部核糖体进入位点 (IRES)，可确保缺氧条件下有效的 VEGF 蛋白合成 (8)。此外，VEGFR1 (FLT-1) mRNA 的表达是缺氧诱导的 (9)，表明内皮必须感知缺氧才能最大限度地接受 VEGF 刺激。 VEGFR2 (KDR) 蛋白的内皮细胞表达也通过旁分泌机制在缺血组织中诱导 (10)。最后，胎盘生长因子 (PlGF) 与 VEGFR1 结合 (4)，在许多组织中由缺氧诱导，与 VEGF 结合对血管生成具有协同作用 (11)。尽管已知参与 VEGF 信号传导的缺氧分子反应非常复杂，但我们对这一过程的理解仍在不断加深。在本期 JCI 中，Ozawa 等人。 (12) 报道研究表明，伴侣蛋白 ORP150（150 kDa 的氧调节蛋白）是 VEGF 蛋白从内质网到高尔基体随后分泌的细胞内转运所必需的。 ORP150 首次被鉴定和纯化为在暴露于缺氧的星形胶质细胞原代培养物中表达的蛋白质 (13)，并且与 VEGF 一样，ORP150 mRNA 表达也是缺氧诱导的 (12)。为了证实他们的研究结果的生理和临床相关性，Ozawa 等人。证明 ORP150 和 VEGF 在人类伤口巨噬细胞中共表达。将编码 ORP150 的腺病毒给予糖尿病小鼠的伤口可加速体内新血管形成和修复。 ORP150过表达增加了缺氧组织培养细胞中VEGF蛋白的分泌，而ORP150反义RNA的表达导致VEGF在内质网内积累。数据表明，ORP150是VEGF分泌所必需的，并且在缺氧细胞中，VEGF水平增加需要ORP150表达相应增加。如果这是真的，那么人们就会预期 ORP150 在缺血性和肿瘤细胞中与 VEGF 共表达。刺激或抑制 ORP150 表达或活性将对 VEGF 和血管生成产生相应的影响，这些假设无疑将在不久的将来得到检验。总而言之，过去六年进行的研究结果描绘了组织氧浓度对 VEGF 活性的复杂调节（表 （表 1）.1）。这种反应是如何在分子水平上协调的？缺氧诱导因子 1 (HIF-1) 通过与位于转录起始位点 5' 1 kb 处的缺氧反应元件结合，负责缺氧细胞中 VEGF 基因的转录激活 (14, 15)。转录后，VEGF mRNA 会与 HuR 和其他蛋白质结合，在缺氧条件下抑制其降解 (16)。目前尚不清楚是否存在识别 IRES 的特定翻译起始因子以及该因子的表达是否由缺氧诱导。 HIF-1 还与缺氧内皮细胞中 FLT-1 mRNA 表达的诱导有关 (9)。由于 ORP150 mRNA 表达是由缺氧诱导的，因此出现了 HIF-1 是否激活 ORP150 转录的问题。事实上，HIF-1 可以在多个水平上协调调节 VEGF 信号传导（表 （表 1），1），类似于它对编码葡萄糖转运蛋白和糖酵解酶的十多个基因的协调调节，这些基因提供对缺氧的代谢适应（15）。最近发现脑源性神经营养因子是缺氧神经元中 ORP150 调节的蛋白 (17)，这也表明 ORP150 可能作为其他 HIF-1 调节和/或缺氧诱导生长因子（例如血管生成素 2、促红细胞生成素和 IGF-2）的伴侣。除了描绘分子调节的优雅程序之外，测试这些假设还可以提供关于哪些分子最适合诱导治疗性血管生成的信息，假设调节控制因素可能具有最大的多效性作用，从而最好地概括缺氧引起的复杂生理反应。表 1 缺氧/缺血组织中 VEGF 信号传导的调节 Ozawa 等人。 (12) 选择伤口愈合作为临床条件，以证明 ORP150 作为体内 VEGF 分泌介质的相关性。这是一个有趣的选择，因为实验和临床数据表明早期伤口并不缺氧 (18)，而高氧会刺激伤口血管生成和愈合 (19)。伤口巨噬细胞的 VEGF 转录可能会受到活化中性粒细胞产生的活性氧的刺激 (20)。介导这种反应的转录因子尚未确定。然而，HIF-1 活性是由炎症细胞因子（如 IL-1 和 TNF-α）诱导的 (21)，这些细胞因子在早期伤口中高水平产生 (18)，从而为非缺氧伤口内由 HIF-1 介导的 VEGF 产生提供了机制 (22)。这些观察结果表明，涉及 HIF-1 和/或其他因子的共享转录机制具有潜在的重要性，无论刺激的性质如何，这些转录机制都会耦合 VEGF 和 ORP150 的表达。因此，虽然 NCBI PubMed 数据库中列出的 4700 多篇出版物表明 VEGF 已经成熟，但 Ozawa 等人。已经表明它仍然需要一个陪伴者。
In contrast to invertebrate species, in which oxygen can be adequately transported to all cells by diffusion, vertebrates, with their larger body size, require a circulatory system that is specialized for oxygen delivery. When systemic oxygen delivery is inadequate, cardiac output increases and red blood cell production is augmented. However, when oxygen delivery is impaired locally, as in a tissue in which perfusion has been interrupted by injury or vascular stenosis, a primary response is angiogenesis, the sprouting of new blood vessels. The complex process of angiogenesis (reviewed in refs. 1–3) begins when cells within a tissue respond to hypoxia by increasing their production of VEGF. VEGF is secreted and binds to cognate receptor tyrosine kinases (VEGFR1 and VEGFR2) located on the surface of vascular endothelial cells. Receptor ligation triggers a cascade of intracellular signaling pathways that initiate angiogenesis. The clinical importance of this biological process has become increasingly apparent over the last decade, and angiogenesis now represents a major focus for novel therapeutic approaches to the prevention and treatment of multiple diseases, most notably ischemic cardiovascular disease and cancer (1–3). If angiogenesis is the big dance, then VEGF is the belle of the ball, and hundreds of investigators are now studying the regulation and biological consequences of its expression (reviewed in ref. 4). Although VEGF expression represents only one component of the complex angiogenic response and VEGF administration alone may not be sufficient to effect therapeutic angiogenesis, VEGF can nevertheless be considered a critical activator of vascular endothelial cells. Establishing the molecular mechanisms regulating VEGF activity is therefore essential for understanding angiogenesis and how it may be targeted clinically. When cells are subjected to hypoxia, VEGF mRNA expression is induced as a result of both increased mRNA transcription and decreased mRNA degradation (5–7). Whereas the rate of translation initiation is globally decreased in hypoxic cells, VEGF mRNAs contain an internal ribosome entry site (IRES) that ensures efficient VEGF protein synthesis under hypoxic conditions (8). In addition, expression of VEGFR1 (FLT-1) mRNA is hypoxia-inducible (9), indicating that the endothelium must sense hypoxia in order to be maximally receptive to VEGF stimulation. Endothelial cell expression of VEGFR2 (KDR) protein is also induced in ischemic tissue via a paracrine mechanism (10). Finally, placental growth factor (PlGF), which binds to VEGFR1 (4), is induced by hypoxia in many tissues and in combination with VEGF has synergistic effects on angiogenesis (11). Despite the impressive complexity of the molecular responses to hypoxia that are known to be involved in VEGF signaling, our understanding of this process continues to grow. In this issue of the JCI, Ozawa et al. (12) report studies indicating that the chaperone protein ORP150 (oxygen-regulated protein of 150 kDa) is required for the intracellular transport of VEGF protein from the endoplasmic reticulum to the Golgi apparatus for subsequent secretion. ORP150 was first identified and purified as a protein expressed in primary cultures of astrocytes exposed to hypoxia (13), and, like VEGF, ORP150 mRNA expression is also hypoxia-inducible (12). To confirm the physiological and clinical relevance of their findings, Ozawa et al. demonstrate coexpression of ORP150 and VEGF in human wound macrophages. Administration of adenovirus encoding ORP150 to wounds of diabetic mice accelerated neovascularization and repair in vivo. ORP150 overexpression increased VEGF protein secretion in hypoxic tissue culture cells, whereas expression of ORP150 antisense RNA resulted in accumulation of VEGF within the endoplasmic reticulum. The data indicate that ORP150 is required for VEGF secretion and that in hypoxic cells increased levels of VEGF necessitate a corresponding increase in ORP150 expression. If this is true, then one would expect ORP150 to be coexpressed with VEGF in ischemic and neoplastic cells. Stimulation or inhibition of ORP150 expression or activity would then have a corresponding effect on VEGF and angiogenesis, hypotheses that will undoubtedly be tested in the near future. Taken together, the results of studies conducted over the last six years have delineated complex regulation of VEGF activity by tissue oxygen concentrations (Table (Table1).1). How is this response orchestrated at the molecular level? Hypoxia-inducible factor 1 (HIF-1) is responsible for the transcriptional activation of the VEGF gene in hypoxic cells by binding to a hypoxia response element located 1 kb 5′ to the transcription initiation site (14, 15). Once transcribed, VEGF mRNA is bound by HuR and other proteins that inhibit its degradation under hypoxic conditions (16). It is not known whether a specific translation initiation factor exists that recognizes the IRES and whether expression of this factor is induced by hypoxia. HIF-1 has also been implicated in the induction of FLT-1 mRNA expression in hypoxic endothelial cells (9). Since ORP150 mRNA expression is induced by hypoxia, the question arises whether HIF-1 activates ORP150 transcription. Indeed, HIF-1 may coordinately regulate VEGF signaling at multiple levels (Table (Table1),1), similar to its coordinate regulation of more than a dozen genes encoding the glucose transporters and glycolytic enzymes that provide metabolic adaptation to hypoxia (15). The recent finding that brain-derived neurotrophic factor is an ORP150-regulated protein in hypoxic neurons (17) also suggests that ORP150 may function as a chaperone for other HIF-1–regulated and/or hypoxia-inducible growth factors such as angiopoietin 2, erythropoietin, and IGF-2. In addition to delineating elegant programs of molecular regulation, testing these hypotheses may provide information as to which molecules are best suited to induce therapeutic angiogenesis, with the assumption being that regulatory control factors may have the most pleiotropic effects and thus best recapitulate the complex physiologic responses evoked by hypoxia. Table 1 Regulation of VEGF signaling in hypoxic/ischemic tissue Ozawa et al. (12) chose wound healing as the clinical condition in which to demonstrate the relevance of ORP150 as a mediator of VEGF secretion in vivo. This is an interesting choice because experimental and clinical data suggest that the early wound is not hypoxic (18) and that hyperoxia stimulates wound angiogenesis and healing (19). VEGF transcription by wound macrophages may be stimulated by reactive oxygen species that are generated by activated neutrophils (20). The transcription factor mediating this response has not been identified. However, HIF-1 activity is induced by inflammatory cytokines such as IL-1 and TNF-α (21) which are generated at high levels in early wounds (18), thus providing a mechanism for VEGF production mediated by HIF-1 within nonhypoxic wounds (22). These observations point to the potential importance of shared transcriptional mechanisms, involving HIF-1 and/or other factors, that would couple VEGF and ORP150 expression regardless of the nature of the inciting stimulus. Thus, while the more than 4700 publications listed in the NCBI PubMed database suggest that VEGF has come of age, Ozawa et al. have shown that it still requires a chaperone.