Glycosphingolipids Mediate Diabetic Wound Healing Impairment

 描述(申请人提供)：伤口愈合不良是胰岛素抵抗糖尿病患者的主要健康问题。需要更好地了解伤口病理，包括角质形成细胞(KCs)在再上皮化中的作用，以及对受损的伤口愈合进行新的干预。神经节苷脂GM3是一种糖鞘糖脂，在膜水平调节受体信号。我们发现GM3和GM3合成酶(GM3S)在人和小鼠糖尿病皮肤中增加。此外，肿瘤坏死因子和过量葡萄糖诱导的KCs胰岛素抵抗与Gm3增加有关，并被Gm3耗尽所逆转，提示Gm3介导了糖尿病创面愈合损害。饮食诱导肥胖(DIO)GM3合酶基因敲除(GM3S-/-)小鼠的伤口愈合正常，而DIO野生型(WT)小鼠伤口愈合延迟。此外，我们通过外用GM3S siRNA球形核酸(SNA)纳米颗粒结合物耗尽GM3，在WT DIO糖尿病小鼠身上复制了这种伤口愈合改善的逆转。用GM3S SNA或葡萄糖神经酰胺合成酶抑制剂GZ 161耗尽GM3，通过激活胰岛素样生长因子-1受体(IGF1R)和rac1信号通路，加速2D角质形成细胞的迁移。GM3的增加，包括通过高糖或长期、低剂量的肿瘤坏死因子治疗，抑制IGF1R信号和抑制KC迁移。我们的长期目标是测试GM3在皮肤中的耗竭，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GM3如何抑制IGF1R的激活和抑制角质形成细胞的迁移。我们的第一个目标是通过比较外用GM3S siRNA SNA和外用或口服GZ 161促进愈合的能力来优化基于GM3耗竭的干预措施。我们将在带有荧光感觉神经的DIO小鼠身上测试这些治疗方案，以跟踪治疗对皮肤神经的影响。然后，最有效的治疗方法将在有更多慢性伤口的糖尿病模型中进行研究，即生物膜挑战的db/db小鼠。使用正常和糖尿病3D共培养创伤模型，我们将在2D KCs中验证我们的观察结果，并将研究GM3调节对胰岛素/IGF-1信号轴的影响(目标2)。最后，作为我们的第三个目标，并以IGF1R为重点，我们将阐明GM3在调节胰岛素/IGF-1信号的膜定位、动力学和分子相互作用中的作用。我们将询问IGF1R与GM3的直接相互作用，并检验我们的假设，即增加GM3可阻止IGF1R网状蛋白介导的内吞作用和信号转导。最后，我们将使用FOLIM来确定GM3的缺失是否会增加IR-IGF1R杂交受体来促进IGF-1的反应。这些拟议的研究将增加我们对影响皮肤中胰岛素/IGF-1信号转导的基于膜的动力学的理解。此外，无论是通过纳米基因抑制还是小分子抑制GM3合成来加速伤口愈合，都可以快速应用于翻译应用，作为糖尿病伤口的一种新的治疗方法。

 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.