Modulation of miR29a and ADAM12 to improve peripheral arterial disease

调节 miR29a 和 ADAM12 以改善外周动脉疾病

• 批准号: 10094744
• 负责人: Ayotunde Oluropo Dokun
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094744
• 关键词: 3-Dimensional; Affect; American; Amputation; Ankle; Atherosclerosis; Biological Assay; Biopsy; Blood Vessels; Blood flow; C57BL/6 Mouse; Cell Proliferation; Cell Survival; Cessation of life; Chromosome 7; Clinical; Complication; Data; Diabetes Mellitus; Disease Outcome; Endothelial Cells; Endothelium; Exposure to; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic; Genetic Polymorphism; Human; Hypoxia; Immunoprecipitation; Impairment; In Vitro; Inbred BALB C Mice; Individual; Ischemia; Limb structure; Lower Extremity; Manipulative Therapies; Measures; Mediating; Medical; Messenger RNA; MicroRNAs; Mouse Strains; Mus; Muscle; Necrosis; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Pathway interactions; Patients; Perfusion; Peripheral arterial disease; Play; Pre-Clinical Model; Quantitative Trait Loci; RNA; Receptor Activation; Receptor Protein-Tyrosine Kinases; Recovery; Regulation; Risk; Rodent; Role; Severities; Severity of illness; Single Nucleotide Polymorphism; Skeletal Muscle; TIE-2 Receptor; Testing; Therapeutic; Therapeutic Effect; Time; Tissues; Tube; Up-Regulation; Walking; Work; angiogenesis; arm; base; biobank; critical limb Ischemia; differential expression; genomic locus; high risk; improved; in vivo; indexing; knock-down; limb ischemia; overexpression; pre-clinical; prevent; progenitor; protein expression; public health relevance

 DESCRIPTION (provided by applicant):¨Peripheral arterial disease (PAD) is a major complication of systemic atherosclerosis which affects approximately 12 million Americans and puts them at risk for lower extremity amputation and death. The presence of diabetes (DM) in individuals with PAD further increases the risk of amputation and death compared to PAD alone. Current medical treatments target systemic atherosclerosis but are not able to improve perfusion to the ischemic limb and directly treat the problem in PAD. There is a pressing clinical need for therapeutic approaches for PAD. Using an established preclinical model of PAD (hind limb ischemia), we previously showed that a quantitative trait locus on the short arm of mouse chromosome 7 (termed LSQ-1) determined perfusion recovery outcomes. Here we have further refined this locus to a region containing 25 genes. Through gene expression profiling we identified 5 genes that are differentially expressed in the ischemic hind limbs of the 2 mouse strains (C57BL/6 and Balb/c) that were used in the original QTL identification. Additionally, we identified ADAM12 as the most differentially expressed of the 5 genes and explored its role in perfusion recovery. We find that ADAM12 is : a) up-regulated in ischemic mouse hind limbs in C57BL/6 mice and in endothelial cells (ECs) exposed to simulated ischemia, b) a knock-down of ADAM12 in vivo in C57BL/6 mice was sufficient to impair perfusion recovery and in vitro decreased EC proliferation, tube formation and survival in simulated ischemia, c) over-expression of ADAM12 in vivo in the Balb/c mouse strain that had poor up-regulation of ADAM12 was sufficient to improve its perfusion recovery following HLI and in vitro increased EC proliferation and survival in hypoxia. These effects of ADAM12 were at least in part, through activation of the receptor tyrosine kinase Tie2. Additionally, we find that in humans with PAD a single nucleotide polymorphism (SNP) in ADAM12 is associated with critical limb ischemia, a severe form of PAD. This data suggests that ADAM12 may play a central role in PAD outcomes. To better understand ADAM12 regulation in ischemia we explored the role of miR29a, a microRNA shown to suppress ADAM12 expression in fibroblast. We find that miR29a overexpression can suppress endothelial cell ADAM12 up-regulation in ischemia. Additionally, we and others have shown that the same C57BL/6 strain that upregulates ADAM12 robustly and recovers favorable following HLI, recovers poorly when DM1 or DM2 is superimposed. Therefore we analyzed miR29a and ADAM12 expression in ischemic hind limbs of C57BL/6 mice with DM1 or DM2 compared to DM C57BL/6 without DM. We found in C57BL/6 mice with DM1 or DM2 miR29a expression is not suppressed and the expression of ADAM12 is blunted. Consistent with these preclinical findings, we also found human skeletal muscle biopsy from PAD patients with DM show elevated levels of miR29a compared to those with PAD alone. Taken together this results we form the central hypothesis that in the absence of DM, PAD/HLI suppresses miR29a expression, resulting in up-regulation of ADAM12. Dysregulation of this pathway in DM contributes to poorer PAD outcomes. Aim 1 Determine whether miR29a negatively regulates ADAM12 expression in the ischemic hind limbs and whether this is sufficient to impair perfusion recovery and increase tissue loss following HLI. Aim 2 will investigate the therapeutic potential of systemic delivery of anti-miR29a compared to systemic delivery of ADAM12 gene on perfusion recovery and tissue loss in mice with type 1 or type 2 DM following HLI. We will establish the effects of these therapeutic strategies on arteriogenesis in the abductor muscles, angiogenesis and circulating progenitors in the ischemic hind limbs. Aim 3 will use an existing human skeletal muscle bio-bank to determine whether a) miR29a and ADAM12 protein expression differs in skeletal muscles of individuals with PAD versus controls without PAD. b) In individuals with PAD we will establish whether miR29a and ADAM12 protein expression in the lower extremity skeletal muscles differ dependent on DM status. c) Across all subjects with PAD, determine whether lower extremity skeletal muscle miR29a and ADAM12 expression will correlate with clinical measures of PAD severity (ankle brachial index and peak walking time).

 描述（由申请人提供）： 外周动脉疾病 (PAD) 是全身动脉粥样硬化的主要并发症，影响约 1200 万美国人，使他们面临下肢截肢和死亡的风险。 PAD 患者存在糖尿病 (DM)。与单独的 PAD 治疗相比，进一步增加了截肢和死亡的风险，但无法直接改善缺血肢体的灌注。临床上迫切需要治疗 PAD 的方法，利用已建立的 PAD（后肢缺血）模型，我们之前发现小鼠 7 号染色体短臂上有一个数量性状位点（称为 LSQ）。 -1) 确定灌注恢复结果。在这里，我们进一步将该基因座细化为包含 25 个基因的区域。通过基因表达谱，我们鉴定了 2 个缺血后肢中差异表达的 5 个基因。此外，我们将 ADAM12 确定为 5 个基因中表达差异最大的基因，并探讨了其在灌注恢复中的作用。在 C57BL/6 小鼠的缺血小鼠后肢和暴露于模拟缺血的内皮细胞 (EC) 中上调，b) ADAM12 在体内的敲低C57BL/6 小鼠足以损害灌注恢复，并在体外降低模拟缺血中的 EC 增殖、管形成和存活，c) ADAM12 上调较差的 Balb/c 小鼠品系体内 ADAM12 过表达ADAM12 的这些作用至少部分是通过激活受体酪氨酸激酶 Tie2 实现的。此外，我们发现在患有 PAD 的人类中，ADAM12 中的单核苷酸多态性 (SNP) 与严重肢体缺血（一种严重的 PAD）相关。该数据表明 ADAM12 可能在 PAD 结局中发挥核心作用。在缺血中，我们探索了 miR29a 的作用，这是一种能够抑制成纤维细胞中 ADAM12 表达的 microRNA，我们发现 miR29a 过度表达可以抑制内皮细胞。 ADAM12 在缺血中上调。我们和其他人已经证明，在 HLI 后强劲上调 ADAM12 并恢复良好的相同 C57BL/6 菌株，在叠加 DM1 或 DM2 时恢复效果也较差，因此我们分析了缺血后的 miR29a 和 ADAM12 表达。我们发现，患有 DM1 或 DM2 的 C57BL/6 小鼠的四肢与没有 DM 的 DM C57BL/6 小鼠相比。在具有 DM1 或 DM2 的 C57BL/6 小鼠中，miR29a 表达未受到抑制，ADAM12 表达减弱，与这些临床前发现一致，我们还发现，与 PAD 患者相比，来自 DM 患者的骨骼肌活检显示 miR29a 水平升高。单独综合这些结果，我们形成了中心假设：在没有 DM 的情况下，PAD/HLI 抑制 miR29a 表达，从而导致ADAM12 的上调。目标 1 确定 miR29a 是否负向调节缺血后肢中的 ADAM12 表达，以及这是否足以损害 HLI 后的灌注恢复并增加组织损失。将研究系统性递送抗 miR29a 与系统性递送 ADAM12 基因相比，对 2 型小鼠的灌注恢复和组织损失的治疗潜力我们将确定这些治疗策略对 HLI 后的 1 型或 2 型 DM 的外展肌动脉生成、血管生成和缺血后肢循环祖细胞的影响。 ) 患有 PAD 的个体与未患有 PAD 的对照组的骨骼肌中 miR29a 和 ADAM12 蛋白表达存在差异 b) 在患有 PAD 的个体中，我们将确定是否存在差异。下肢骨骼肌中 miR29a 和 ADAM12 蛋白表达的差异取决于 DM 状态 c) 在所有患有 PAD 的受试者中，确定下肢骨骼肌 miR29a 和 ADAM12 表达是否与 PAD 严重程度的临床指标（踝臂指数和步行峰值）相关。时间）。

项目成果

microRNA-29a Regulates ADAM12 Through Direct Interaction With ADAM12 mRNA and Modulates Postischemic Perfusion Recovery.

• DOI: 10.1161/jaha.122.025727
• 发表时间: 2022-08-16
• 期刊: JOURNAL OF THE AMERICAN HEART ASSOCIATION
• 影响因子: 5.4
• 作者: Lamin, Victor;Verry, Joseph;Dokun, Olumayowa S.;Kronemberger, Ana;Wong, Thomas;Lira, Vitor A.;Dokun, Ayotunde O.
• 通讯作者: Dokun, Ayotunde O.

Individuals with Peripheral Artery Disease (PAD) and Type 1 Diabetes Are More Likely to Undergo Limb Amputation than Those with PAD and Type 2 Diabetes.

• DOI: 10.3390/jcm9092809
• 发表时间: 2020-08-31
• 期刊: Journal of clinical medicine
• 影响因子: 3.9
• 作者: Jain N;Agarwal MA;Jalal D;Dokun AO
• 通讯作者: Dokun AO

Dual specificity phosphatase 5 regulates perfusion recovery in experimental peripheral artery disease.

• DOI: 10.1177/1358863x19866254
• 发表时间: 2019-10
• 期刊: Vascular medicine (London, England)
• 作者: Alleboina S;Ayalew D;Peravali R;Chen L;Wong T;Dokun AO
• 通讯作者: Dokun AO

BAG3 Attenuates Ischemia-Induced Skeletal Muscle Necroptosis in Diabetic Experimental Peripheral Artery Disease.

• DOI: 10.3390/ijms231810715
• 发表时间: 2022-09-14
• 期刊: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
• 影响因子: 5.6
• 作者: Mani, Arul M.;Dhanabalan, Karthik;Lamin, Victor;Wong, Thomas;Singh, Madhu, V;Dokun, Ayotunde O.
• 通讯作者: Dokun, Ayotunde O.