Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, Oxford, MS 38677, USA.
Cells. 2021 Mar 4;10(3):557. doi: 10.3390/cells10030557.
Cardiovascular disease, specifically heart failure, is a common complication for individuals with type 2 diabetes mellitus. Heart failure can arise with stiffening of the left ventricle, which can be caused by "active" cardiac fibroblasts (i.e., myofibroblasts) remodeling the extracellular matrix (ECM). Differentiation of fibroblasts to myofibroblasts has been demonstrated to be an outcome of AGE/RAGE signaling. Hyperglycemia causes advanced glycated end products (AGEs) to accumulate within the body, and this process is greatly accelerated under chronic diabetic conditions. AGEs can bind and activate their receptor (RAGE) to trigger multiple downstream outcomes, such as altering ECM remodeling, inflammation, and oxidative stress. Previously, our lab has identified a small GTPase, Rap1a, that possibly overlaps the AGE/RAGE signaling cascade to affect the downstream outcomes. Rap1a acts as a molecular switch connecting extracellular signals to intracellular responses. Therefore, we hypothesized that Rap1a crosses the AGE/RAGE cascade to alter the expression of AGE/RAGE associated signaling proteins in cardiac fibroblasts in type 2 diabetic mice. To delineate this cascade, we used genetically different cardiac fibroblasts from non-diabetic, diabetic, non-diabetic RAGE knockout, diabetic RAGE knockout, and Rap1a knockout mice and treated them with pharmacological modifiers (exogenous AGEs, EPAC, Rap1a siRNA, and pseudosubstrate PKC-ζ). We examined changes in expression of proteins implicated as markers for myofibroblasts (α-SMA) and inflammation/oxidative stress (NF-κB and SOD-1). In addition, oxidative stress was also assessed by measuring hydrogen peroxide concentration. Our results indicated that Rap1a connects to the AGE/RAGE cascade to promote and maintain α-SMA expression in cardiac fibroblasts. Moreover, Rap1a, in conjunction with activation of the AGE/RAGE cascade, increased NF-κB expression as well as hydrogen peroxide concentration, indicating a possible oxidative stress response. Additionally, knocking down Rap1a expression resulted in an increase in SOD-1 expression suggesting that Rap1a can affect oxidative stress markers independently of the AGE/RAGE signaling cascade. These results demonstrated that Rap1a contributes to the myofibroblast population within the heart via AGE/RAGE signaling as well as promotes possible oxidative stress. This study offers a new potential therapeutic target that could possibly reduce the risk for developing diabetic cardiovascular complications attributed to AGE/RAGE signaling.
心血管疾病，特别是心力衰竭，是 2 型糖尿病患者的常见并发症。心力衰竭可能源于左心室僵硬，这可能是由“活跃”的心肌成纤维细胞（即肌成纤维细胞）重塑细胞外基质（ECM）引起的。已经证明，成纤维细胞向肌成纤维细胞的分化是 AGE/RAGE 信号的结果。高血糖导致体内积累晚期糖基化终产物（AGEs），而在慢性糖尿病条件下，这一过程大大加速。AGEs 可以与它们的受体（RAGE）结合并激活，从而触发多种下游结果，例如改变 ECM 重塑、炎症和氧化应激。以前，我们的实验室已经鉴定出一种小 GTPase，Rap1a，它可能与 AGE/RAGE 信号级联重叠，以影响心肌成纤维细胞的下游结果。Rap1a 充当将细胞外信号连接到细胞内反应的分子开关。因此，我们假设 Rap1a 通过 AGE/RAGE 级联改变 2 型糖尿病小鼠心肌成纤维细胞中与 AGE/RAGE 相关的信号蛋白的表达。为了描绘这个级联，我们使用了来自非糖尿病、糖尿病、非糖尿病 RAGE 敲除、糖尿病 RAGE 敲除和 Rap1a 敲除小鼠的遗传上不同的心肌成纤维细胞，并对其进行了药理学修饰（外源性 AGEs、EPAC、Rap1a siRNA 和假底物 PKC-ζ）。我们检测了作为肌成纤维细胞标志物（α-SMA）和炎症/氧化应激标志物（NF-κB 和 SOD-1）的蛋白质表达的变化。此外，还通过测量过氧化氢浓度来评估氧化应激。我们的结果表明，Rap1a 连接到 AGE/RAGE 级联，以促进和维持心肌成纤维细胞中 α-SMA 的表达。此外，Rap1a 与 AGE/RAGE 级联的激活一起增加了 NF-κB 的表达和过氧化氢浓度，表明可能存在氧化应激反应。此外，敲低 Rap1a 表达会导致 SOD-1 表达增加，这表明 Rap1a 可以独立于 AGE/RAGE 信号级联影响氧化应激标志物。这些结果表明，Rap1a 通过 AGE/RAGE 信号促进心脏中的肌成纤维细胞群，并可能促进潜在的氧化应激。这项研究提供了一个新的潜在治疗靶点，可能会降低归因于 AGE/RAGE 信号的糖尿病心血管并发症的风险。
