Vitamin D Metabolism and Function in the Cornea and Anterior Segment

维生素D（Vit D）不足和缺乏是一个重要的健康问题。我们的实验室一直在进行开创性的研究 检查维生素D在眼睛中的作用，重点是角膜。令人兴奋的新数据指出了治疗潜力 维生素D在治疗眼部疾病中的作用。一般的假设是，维生素D缺乏或不足可导致 由原发性损伤或疾病引起的加重的前段相关眼科问题。 具体目标1。检查维生素D缺乏对糖尿病小鼠角膜上皮伤口愈合的影响。 理由。糖尿病是许多病理状况中的一个重要的并发因素， 影响了大约70%的糖尿病患者。目标1的假设，基于使用 糖尿病VDR基因敲除小鼠，是维生素D缺乏会导致角膜伤口明显加剧赤字 治愈糖尿病小鼠。 approach.将在对照和维生素D受体（VDR）敲除糖尿病小鼠中制造角膜上皮伤口。 将比较愈合率，并检查角膜的形态差异。维生素D的影响 将确定代谢物对这些VDR敲除糖尿病小鼠中伤口愈合的影响。 具体目标2。检查24，25D对角膜上皮细胞功能的影响。 理由。我们以前证明，24，25 D的浓度是最高的所有维生素D代谢物在眼睛。 最近的研究表明，24，25 D不仅仅是一种无活性的代谢物。我们的初步研究表明，24，25 D可能 在角膜上皮的功能中起重要作用。 approach.将使用细胞培养来研究24，25D对角膜上皮细胞增殖和迁移的影响。 24，25 D对VDR表达和关键的维生素D代谢相关酶CYP27 B和CYP24 A1的影响将被研究。 使用qPCR和蛋白质印迹法测量。将使用VDR敲除小鼠检查通过VDR的24，25D信号传导 和细胞培养。结合目的1和3，我们将分别检查24，25 D对角膜上皮细胞的影响， 伤口愈合沿着间隙连接功能和桥粒。 具体目标3。确定维生素D如何影响角膜上皮细胞间连接。 理由。我们以前证明维生素D影响角膜上皮间隙连接和紧密连接的功能， 交叉点基于我们的初步研究，我们将检验我们报道的差距连接功能效应 与特定间隙连接蛋白的密度和分布直接相关， 我们在VDR敲除小鼠中观察到的粘附与桥粒蛋白密度和分布的变化有关。 approach.确定VDR敲除对角膜上皮mRNA水平、密度和间隙分布的影响。 连接和桥粒特异性蛋白。我们还将研究VDR基因敲除对角膜缘细胞的影响 使用我们最近描述的FRAP方法计算间隙结扩散系数。我们还将确定维生素D 使用细胞培养物刮取负载和FRAP测量，补充影响间隙连接性。

Vitamin D (Vit D) insufficiency and deficiency is a significant health issue. Our lab has been conducting pioneering studies examining the role of Vit D in the eye, with a focus on the cornea. Exciting emerging data point to the therapeutic potential of Vit D in treating ocular pathologies. The general hypothesis is that Vit D deficiency or insufficiency can lead to exacerbated anterior segment-related ophthalmic problems brought on by a primary insult or disease. Specific Aim 1. Examine the influence of Vit D deficiency on corneal epithelial wound healing in diabetic mice. Rationale. Diabetes is a significant complicating factor in any number of pathological conditions, and diabetic keratopathy effects approximately 70% of all diabetics. The hypothesis for Aim 1, based on novel preliminary data collected using diabetic VDR knockout mice, is that Vit D deficiency will lead to a significantly exacerbated deficit in the corneal wound healing of diabetic mice. Approach. Cornea epithelial wounds will be made in control and vitamin D receptor (VDR) knockout diabetic mice. Healing rates will be compared and corneas will be examined for morphological differences. The influence of Vit D metabolites on wound healing in these VDR knockout diabetic mice will be determined. Specific Aim 2. Examine effects of 24,25D on corneal epithelial cell function. Rationale. We previously demonstrated that the concentration of 24,25D is the highest of all Vit D metabolites in the eye. Recent studies indicate that 24,25D is not just an inactive metabolite. Our preliminary studies demonstrate that 24,25D may play a significant role in the function of the corneal epithelium. Approach. The influence of 24,25D on corneal epithelial cell proliferation and migration will be studied using cell culture. 24,25D effects on the expression of VDR and the key Vit D metabolic-related enzymes CYP27B and CYP24A1 will be measured using qPCR and western blotting. 24,25D signaling through VDR will be examined using VDR knockout mice and cell culture. Integrated with Aims 1 and 3, respectively, we will examine the influence of 24,25D on corneal epithelial wound healing along with gap junction function and desmosomes. Specific Aim 3. Determine how Vit D influences corneal epithelial intercellular junctions. Rationale. We previously demonstrated that Vit D influences the function of corneal epithelial gap junctions and tight junctions. We will test the hypothesis, based on our preliminary studies, that the gap junction functional effects we reported are directly related to modified density and distribution of specific gap junction proteins and that the weak epithelial attachment we observe in VDR knockout mice is related to changes in desmosome protein density and distribution. Approach. Determine the influence of VDR knockout on corneal epithelial mRNA levels, density, and distribution of gap junction- and desmosome-specific proteins. We will also examine the influence of VDR knockout on corneal limbal cell gap junction diffusion coefficients using the FRAP method we recently described. We will also determine if Vit D supplementation affects gap junction connectivity using cell culture scrape loading and FRAP measurements.