TGF beta and AKT signal-driven pathogenesis in keratoconus

圆锥角膜中 TGF β 和 AKT 信号驱动的发病机制

• 批准号: 9282779
• 负责人: Shukti Chakravarti
• 金额: $ 57.96万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-10 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9282779
• 关键词: Affect; Age; Alleles; Apoptosis; Astigmatism; Autophagocytosis; Biochemistry; Bioinformatics; Biological Assay; Biology; Blindness; CRISPR/Cas technology; Candidate Disease Gene; Cell Culture System; Cell Culture Techniques; Cells; Cellular Stress; Cellular biology; Cicatrix; Cluster Analysis; Collagen; Complement; Connective Tissue; Connective Tissue Diseases; Contact Lenses; Cornea; Data Set; Diagnostic; Disease; Dizygotic Twins; Down Syndrome; Economic Burden; Ehlers-Danlos Syndrome; Extracellular Matrix; FOXO1A gene; Family; Gene Expression; Gene Proteins; Gene Targeting; Genes; Genetic; Genomics; Hereditary Disease; Heritability; Heterogeneity; Human; In Situ Hybridization; Incidence; Individual; Institutes; International; Keratoconus; Laboratories; Lead; Ligands; Long-Term Effects; Mediating; Medicine; Modeling; Molecular; Mutation; Organoids; Oxidative Stress; PI3K/AKT; Pathogenesis; Pathogenicity; Pathway interactions; Patient Recruitments; Process; Production; Productivity; Protein Isoforms; Proteins; Proteome; Proteomics; Proto-Oncogene Proteins c-akt; Quality of life; Recording of previous events; Regulation; Repression; Research; Signal Transduction; Small Interfering RNA; Specificity; Stromal Cells; Suggestion; Susceptibility Gene; Testing; Therapeutic Intervention; Thick; Thinness; Transcript; Transcription Alteration; Transforming Growth Factor beta; Transplantation; Validation; Variant; base; biological adaptation to stress; cell growth regulation; cohort; crosslink; curative treatments; cytokine; deep sequencing; differential expression; disorder subtype; exome; exome sequencing; experience; genome wide association study; health economics; insight; knock-down; loss of function; novel; nutrient deprivation; phosphoproteomics; public health relevance; receptor; response; trait; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

 DESCRIPTION (provided by applicant) Keratoconus (KC), with an incidence of ~1/1,000, is a degenerative weakening and thinning of the corneal connective tissue extracellular matrix (ECM), causing high astigmatism, scarring, and vision loss in severe cases. There are no curative treatments: the disorder is managed by hard contact lens use and UV cross-linking of collagens (CXL) to temporarily strengthen the connective tissue, at early stages, and cornea transplants, at advanced stages. However, the long-term effects of CXL are unknown and as KC onsets at a young age, it impacts ones productivity and quality of life tremendously. We hypothesize that dysregulated stress response in keratocytes, the specialized cells of the cornea and disruptions in the stromal ECM they produce are major disease underpinnings. KC is multifactorial with a strong genetic component: 18% of cases have a positive family history, concordance is significantly higher in monozygotic than dizygotic twins, and KC is positively associated with heritable traits like central corneal thickness and genetic disorders like trisomy 21 and Ehlers- Danlos syndrome types. Past linkage, candidate gene sequencing and genome-wide association studies (GWAS) have led to suggestive clues but have not identified causal genes. Consequently, we propose a functionally- complemented exome sequencing study that relies on three major advances from our laboratory: (1) We have performed proteomics and preliminary RNA-Seq screens of KC and control corneas to identify the universe of known and novel corneal RNAs and proteins within which genetic defects occur. (2) We have established a 'disease-in-a-dish' model using keratocytes from individual donor (DN) and KC corneas, where KC cells mimic degenerative features of the cornea. (3) Our expression and functional studies of KC indicate a dysregulated network involving cytokines, TGFb and AKT, as major effectors of cellular stress and ECM perturbations in KC. Aim I will investigate TGFb and PI3K/AKT signal transductions in cellular stress and ECM/collagen disruptions in KC cell cultures. Aim II will identify transcriptional alterations in individual KC and DN corneas by RNA-sequencing. Aim III will identify KC susceptibility genes by exome sequencing of familial and isolated cases. We have established an active Keratoconus Research Center at Johns Hopkins Wilmer Institute for long-term patient recruitment, locally and through national/international collaborators, with an experienced research team in ocular medicine (Walter Stark, Albert Jun), bioinformatics, genomics and proteomics (Steven Salzberg, Aravinda Chakravarti, Akhilesh Pandey) and ECM cell biology and biochemistry (Shukti Chakravarti). Our findings will elucidate keratoconus pathogenesis at a molecular level and lead to strategies for therapeutic interventions.

 描述（由申请人提供） 圆锥角膜（KC）的发病率约为1/1，000，是角膜结缔组织细胞外基质（ECM）的退行性弱化和变薄，严重时会导致高度散光、瘢痕形成和视力丧失。目前还没有治愈性治疗方法：这种疾病的治疗方法是在早期阶段使用硬接触透镜和紫外线交联胶原蛋白（CXL）来暂时增强结缔组织，在晚期阶段进行角膜移植。然而，CXL的长期影响是未知的，由于KC在年轻时发病，它极大地影响了人们的生产力和生活质量。我们假设，角膜细胞（角膜的特化细胞）的应激反应失调以及它们产生的基质ECM的破坏是主要的疾病基础。KC是多因素的，具有很强的遗传成分：18%的病例具有阳性家族史，单卵双胞胎的一致性显著高于双卵双胞胎，并且KC与可遗传性状如中央角膜厚度和遗传疾病如21三体和Ehlers-Danlos综合征类型正相关。过去的连锁，候选基因测序和全基因组关联研究（GWAS）已经导致了暗示性线索，但尚未确定致病基因。因此，我们提出了一种功能互补的外显子组测序研究，其依赖于我们实验室的三个主要进展：（1）我们已经对KC和对照角膜进行了蛋白质组学和初步RNA-Seq筛选，以鉴定其中发生遗传缺陷的已知和新型角膜RNA和蛋白质的范围。(2)我们已经建立了一个“disease-in-a-dish”模型，使用来自个体供体（DN）的角膜细胞和KC角膜，其中KC细胞模拟角膜的退行性特征。(3)我们的KC的表达和功能研究表明，一个失调的网络，涉及细胞因子，TGF β和AKT，作为细胞应激和ECM扰动KC的主要效应。目的研究KC细胞在细胞应激和ECM/胶原破坏中TGF β和PI 3 K/AKT信号转导的作用。目的II将通过RNA测序鉴定单个KC和DN角膜中的转录改变。目的通过对家族性和孤立病例的外显子组测序，确定KC易感基因。我们在约翰霍普金斯威尔默研究所建立了一个活跃的圆锥角膜研究中心，在当地和通过国家/国际合作者招募长期患者，拥有一支经验丰富的眼科医学（Walter Stark，Albert Jun），生物信息学，基因组学和蛋白质组学（Steven Salzberg，Aravinda Chakravarti，Akhilesh Pandey）以及ECM细胞生物学和生物化学（Shukti Chakravarti）的研究团队。我们的研究结果将阐明圆锥角膜的发病机制在分子水平上，并导致治疗干预的策略。