Glycosphingolipids Mediate Diabetic Wound Healing Impairment

 描述（由申请人提供）：伤口愈合不良是胰岛素抵抗型糖尿病的主要健康问题。需要提高对伤口病理学的理解，包括角质形成细胞（KC）在上皮再形成中的功能，以及对受损伤口愈合的新干预措施。神经节苷脂GM 3是一种在膜水平调节受体信号传导的鞘糖脂。我们已经发现GM 3和GM 3合酶（GM 3S）在人类和小鼠糖尿病皮肤中增加。此外，在KC中，TNF增加和过量葡萄糖诱导的胰岛素抵抗与GM 3增加相关，并被GM 3消耗逆转，表明GM 3介导糖尿病伤口愈合障碍。在饮食诱导的肥胖（DIO）GM 3合酶敲除（GM 3S-/-）小鼠中，伤口正常愈合，与DIO野生型（WT）同窝出生的小鼠中的延迟愈合相反。此外，我们通过用局部施用的GM 3S siRNA球形核酸（SNA）纳米颗粒缀合物消耗GM 3，在WT DIO糖尿病小鼠中复制了这种伤口愈合改善的逆转。通过GM 3S SNA或GZ 161（一种葡萄糖神经酰胺合成酶抑制剂）消耗GM 3，通过激活胰岛素样生长因子-1受体（IGF 1 R）和Rac 1信号传导加速2D角质形成细胞培养物中的迁移。GM 3的增加，包括用过量葡萄糖或慢性低剂量TNF α治疗，抑制IGF 1 R信号传导并抑制KC迁移。我们的长期目标是测试皮肤中的GM 3消耗作为一种新的手段来逆转糖尿病患者受损的伤口愈合，并了解GM 3如何抑制IGF 1 R激活和抑制角质形成细胞迁移。我们的第一个目的是通过比较局部GM 3S siRNA SNA和局部或口服GZ 161促进愈合的能力来优化基于GM 3消耗的干预。我们将在具有荧光感觉神经的DIO小鼠中测试这些治疗选择，以跟踪治疗对皮肤神经支配的影响。然后将在具有更多慢性伤口的糖尿病模型（生物膜激发的db/db小鼠）中研究最有效的疗法。使用正常和糖尿病3D共培养伤口模型，我们将验证我们在2D KC中的观察结果，并将研究GM 3调节对胰岛素/IGF-1信号传导轴的影响（Aim 2）。最后，作为我们的第三个目标，并专注于IGF 1 R，我们将阐明GM 3在调节胰岛素/IGF-1信号传导的膜定位，动力学和分子相互作用中的作用。我们将询问IGF 1 R与GM 3的直接相互作用，并检验我们的假设，即GM 3的增加会阻止IGF 1 R网格蛋白介导的内吞作用和信号传导。最后，我们将使用FLIM来确定GM 3消耗是否增加IR-IGF 1 R杂合受体以增强IGF-1反应。这些拟议的研究将增加我们对影响皮肤中胰岛素/IGF-1信号传导的膜动力学的理解。此外，伤口愈合的加速，无论是通过基因抑制的纳米递送还是GM 3合成的小分子抑制，都可以快速跟踪转化应用，作为糖尿病伤口的新治疗方法。

 DESCRIPTION (provided by applicant): Poor wound healing is a major health issue in insulin-resistant diabetes. Improved understanding of wound pathology, including the function of keratinocytes (KCs) in re-epithelialization, and new interventions for impaired wound healing are needed. Ganglioside GM3 is a glycosphingolipid that regulates receptor signaling at the membrane level. We have found that GM3 and GM3 synthase (GM3S) are increased in human and mouse diabetic skin. In addition, TNF and excess glucose-induced insulin resistance in KCs is associated with increased GM3 and reversed by GM3 depletion, suggesting that GM3 mediates diabetic wound healing impairment. Wounds heal normally in diet-induced obese (DIO) GM3 synthase knockout (GM3S-/-) mice, in contrast to delayed healing in DIO wildtype (WT) littermates. Furthermore, we have replicated reversal of this wound healing improvement in WT DIO diabetic mice by depleting GM3 with topically applied GM3S siRNA spherical nuclei acid (SNA) nanoparticle conjugates. Depleting GM3 by either GM3S SNA or GZ 161, a glucosylceramide synthase inhibitor, accelerates migration in 2D keratinocyte cultures by activating insulin-like growth factor-1 receptor (IGF1R) and Rac1 signaling. Increases in GM3, including by treatment with excess glucose or chronic, low-dose TNF, suppress IGF1R signaling and inhibit KC migration. Our long-term goals are to test GM3 depletion in skin as a novel means to reverse the impaired wound healing in diabetics and understand how GM3 inhibits IGF1R activation and suppresses keratinocyte migration. Our first aim is to optimize GM3 depletion-based intervention by comparing the ability of topical GM3S siRNA SNA and topical or oral GZ 161 to promote healing. We will test these therapeutic options in a DIO mouse with fluorescent sensory nerves to track the impact of treatment on cutaneous innervation. The most efficacious therapy will then be studied in a diabetic model with more chronic wounds, the biofilm-challenged db/db mouse. Using normal and diabetic 3D co-culture wound models, we will validate our observations in 2D KCs and will investigate the effects of GM3 modulation on the insulin/IGF-1 signaling axis (Aim 2). Finally, as our third aim and with a focus on IGF1R, we will elucidate the role of GM3 in regulating the membrane-based localization, dynamics, and molecular interactions of insulin/IGF-1 signaling. We will interrogate the direct interactions of IGF1R with GM3 and test our hypothesis that increased GM3 prevents IGF1R clathrin-mediated endocytosis and signaling. Finally, we will use FLIM to determine if GM3 depletion increases IR-IGF1R hybrid receptors to boost IGF-1 responses. These proposed studies will increase our understanding of the membrane-based dynamics that impact insulin/IGF-1 signaling in skin. Furthermore, acceleration of wound healing, whether by nano-delivery of gene suppression or small molecule inhibition of GM3 synthesis, could be fast-tracked towards translational application as a new treatment approach for diabetic wounds.