Role of HIF-1 in Intervertebral Disc Function

描述（由申请人提供）：在椎间盘中，髓核（NP）细胞在独特的环境生态位中发挥作用，其特征在于非常有限的血管供应，对椎间盘细胞和富含蛋白多糖的细胞外基质施加代谢限制。我们已经表明，NP的氧张力非常低，与所有其他组织不同，NP的细胞表现出HIF-1a的组成型表达。我们提出了一个新的假设，即HIF-1a的组成性表达有助于NP细胞适应其无血管环境，HIF-1活性的变化是其生存和功能所必需的。第一个具体目的是检验高组成性HIF-1a表达是缺氧椎间盘中NP细胞存活所必需的假设;研究调节HIF-1a组成性表达和活性的机制;使用来自人退变椎间盘的组织将NP HIF-1活性与退变和凋亡易感性相关联。我们将短暂和稳定地抑制HIF-1a，并评估HIF-1a下调对细胞存活和死亡的影响。然后，我们将恢复受抑制细胞中的HIF-1a表达，并评估存活率、凋亡和细胞表型。我们还将确定pVHL，FIH和PHDs表达的变化是否调节NP细胞中HIF-1a的表达和转录活性。我们将使用从临床人类椎间盘样本中分离的细胞，具有不同程度的退变，以确定对促肾上腺皮质激素治疗的敏感性是否与内源性HIF-1a活性和退变水平有关。第二个具体目的是检验组成性HIF-1a表达调节蛋白聚糖合成和GAG链形成和硫酸化的假设;在人类退行性椎间盘样本中将HIF-1活性与蛋白聚糖合成相关联。我们将确定组成型HIF-1a在聚集蛋白聚糖核心蛋白（一种具有HIF-1应答启动子元件的分子）的表达和合成中的作用。此外，我们将评估HIF-1a在调节GAG合成（葡萄糖醛酸基转移酶I; GlcAT-I）和软骨素硫酸化（软骨素-4-O-磺基转移酶2; C4 ST-2）所需的酶表达中的作用。我们将研究从临床人类椎间盘样本中分离的细胞的蛋白聚糖合成是否与HIF-1a活性相关。最后一个具体目标是使用慢病毒HIF-1a-SiRNA在NP细胞中开发抑制HIF-1a的兔模型;了解HIF-1a的失活是否促进细胞结构的丧失、蛋白聚糖合成的减少和椎间盘的退变。为了补充体内研究，并研究NP退变中的早期细胞事件，我们将使用凋亡的HIF-1a小鼠进行椎间盘器官培养研究。将通过注射表达cre重组酶的腺病毒实现器官培养盘中HIF-1a基因的失活。拟议研究提供的信息将为NP细胞早期退行性变化的机制提供见解，并为制定预防退行性椎间盘疾病的干预策略提供基础。公共卫生相关性腰痛的发病率非常高，通常与椎间盘的退行性变化有关，美国医疗保健行业每年的成本超过330亿美元。目前没有一种疗法可以完全恢复退行性椎间盘的功能，从而防止受损脊柱的进一步恶化。拟议的研究将提供对椎间盘早期退行性变化机制的深入了解，并可能允许制定预防退行性疾病的干预策略。

DESCRIPTION (provided by applicant): In the intervertebral disc, the cells of the nucleus pulposus (NP) function in a unique environmental niche, characterized by a very limited vascular supply that imposes metabolic constraints on the disc cells and a proteoglycan-rich extracellular matrix. We have shown that the oxygen tension of the NP is very low and unlike all other tissues, cells of the NP exhibit constitutive expression of HIF-1a. We advance the new hypothesis that constitutive expression of HIF-1a serves to adapt the NP cells to their avascular environment and that changes in HIF-1 activity is required for their survival and function. The first Specific Aims is to test the hypotheses that high constitutive HIF-1a expression is required for survival of the NP cells in the hypoxic disc; to investigate mechanisms regulating constitutive expression and activity of HIF-1a; using tissue derived from human degenerative discs to correlate NP HIF-1 activity with degeneration and susceptibility to apoptosis. We will transiently and stably suppress HIF-1a and assess the impact of HIF-1a downregulation on cell survival and death. We will then restore HIF-1a expression in suppressed cells and evaluate survival, apoptosis and cell phenotype. We will also determine if changes in expression of pVHL, FIH and PHDs regulate HIF-1a expression and transcriptional activity in NP cells. We will use cells isolated form clinical human disc samples, with varying degree of degeneration, to determine if susceptibility to apoptogen treatment is linked to endogenous HIF-1a activity and to the level of degeneration. The second specific aim is to test the hypothesis that constitutive HIF-1a expression regulates proteoglycan synthesis and GAG chain formation and sulfation; to correlate HIF-1 activity to proteoglycan synthesis in human degenerative disc samples. We will determine the role of constitutive HIF-1a in the expression and synthesis of aggrecan core protein, a molecule that has a HIF-1 responsive promoter element. In addition, we will evaluate the role of HIF-1a in regulating expression of enzymes required for GAG synthesis (glucuronosyltransferase I; GlcAT-I) and chondroitin sulfation (chondroitin-4-O-sulfosyl transferase 2; C4ST-2). We will investigate if proteoglycan synthesis by cells isolated form clinical human disc samples is linked to HIF-1a activity. The last Specific Aim is to develop a rabbit model with suppression of HIF-1a in NP cells using lentiviral HIF-1a-SiRNA; to learn if inactivation of HIF-1a promotes a loss of cellularity, a decrease in proteoglycan synthesis and degeneration of the intervertebral disc. To complement the in vivo studies, and to investigate early cellular events in NP degeneration, we will perform disc organ culture studies using floxed-HIF-1a mouse. Inactivation of the HIF-1a gene in organ cultured discs will be achieved by injecting adenovirus expressing cre recombinase. The information provided by proposed studies will provide insights into mechanisms of early degenerative changes in the NP cells and provide foundation for development of interventional strategies to prevent degenerative disc disease. PUBLIC HEALTH RELEVANCE The incidence of low back pain, which is often linked to degenerative changes in the intervertebral disc, is extraordinary high with annual costs to the US health care industry exceeding 33 billion dollars. None of the current therapies can completely restore the function of the degenerative intervertebral disc and thereby prevent further deterioration of the compromised spine. The proposed studies will provide insights into mechanisms of early degenerative changes in the disc and may permit development of interventional strategies to prevent degenerative disease.