Arterial stiffening and SMC mechanobiology in Hutchinson-Guilford Progeria Syndrome

总结 Hutchinson-Gilford早衰综合征（HGPS）是一种罕见的遗传性早衰疾病， LMNA的常染色体显性突变，LMNA是编码核纤层蛋白A的基因。突变蛋白LmnAG608G已经被 名为"早老素。"患有HGPS的儿童通常在青少年时期死于CVD （动脉粥样硬化、心肌梗塞和/或中风）。值得注意的是，心血管疾病和死亡发生在缺乏 高胆固醇，但HGPS患者的动脉异常僵硬，动脉僵硬度一直是 被认为是CVD的胆固醇非依赖性风险因素。此外，我们以前表明，在体内， 抑制动脉硬化可减少apoE缺失小鼠的动脉粥样硬化。因为早老素是异常的 法尼基化，HGPS的治疗集中在法尼基转移酶抑制剂（FTI），但这些研究没有 直接解决被认为是引发HGPS早期死亡的心血管病理学。 我们最近获得了LMNAG609G小鼠，其对应于人HGPS中的LMNAG608G突变， 这只老鼠表现出了与人类疾病相似的动脉硬化现象。 动脉切片的免疫染色和ECM表达阵列已经确定了两个主要候选人。 动脉过早硬化，我们会在这里进行研究我们还发现HGPS动脉缺乏 他们对血管收缩剂的反应，我们的初步结果将这种收缩性缺陷与一种惊人的 两个成熟的平滑肌分化/CArG基因的解偶联：平滑肌的表达 肌球蛋白重链（SM-MHC）在HGPS中减少，而平滑肌肌动蛋白（SMA）水平相对减少。 正常重要的是，我们已经能够概括这种非偶联SM-MHC与SMA的体内表型 在来自WT和HGPS动脉瘤的原代平滑肌细胞（SMC）中的表达。SM-MHC是最常见的 在分化的SMC中的高收缩性状态的重要调节剂。因此，这些发现和相关的 牵引力显微镜（TFM）实验的初步研究，使我们的一个新的模型，过早 HGPS中的动脉硬化：突变LaminA（早老蛋白）的表达导致优先下调 这将HGPS SMC锁定在一种新的中间张力状态，在这种状态下，它们的行为更加 就像去分化的SMC，产生ECM蛋白和ECM重塑酶， 加速动脉硬化。目的1将使用年龄匹配的WT和HGPS小鼠来测试因果关系。 i）ECM重塑事件和过早动脉硬化与ii）SM-MHC表达之间的关系 和动脉ECM重塑。目标2将使用分离的SMC来鉴定分子机制和因果关系。 HGPS、SM-MHC表达、细胞收缩性和ECM重塑之间的关系。它还将 建立早老蛋白和可能的WT LaminA表达影响SM-MHC的分子机制 基因表达。目标3将测试动脉硬化、ECM重塑和血管生成之间的相似性和差异。 和SM-MHC/SMA表达在正常老化与HGPS。

SUMMARY Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disease of premature aging caused by an autosomal dominant mutation in LMNA, the gene encoding laminA. The mutant protein, LmnAG608G has been named "Progerin." Children with HGPS typically die in their teenage years as a consequence of CVD (atherosclerosis, myocardial infarction and/or stroke). Remarkably, CVD and death occurs in the absence of high cholesterol, but the arteries of HGPS patients are abnormally stiff, and arterial stiffness has been identified as a cholesterol-independent risk factor for CVD. Moreover, we previously showed that in vivo inhibition of arterial stiffening reduces atherosclerosis in apoE-null mice. Because Progerin is aberrantly farnesylated, therapies for HGPS have focused farnesyltransferase inhibitors (FTIs), but those studies do not directly address the cardiovascular pathology that is thought to trigger early death in HGPS. We recently obtained the LMNAG609G mouse that corresponds to the LMNAG608G mutation in human HGPS and show here that this mouse phenocopies the human disease in showing premature arterial stiffening. Immunostaining of arterial sections and an ECM expression array have identified two lead candidates for this premature arterial stiffening, and those will be studied here. We also found that HGPS arteries are deficient in their response to vasoconstrictors, and our preliminary results link this contractility defect to a striking uncoupling of two well established smooth muscle differentiation/CArG genes: expression of smooth muscle myosin heavy chain (SM-MHC) is reduced in HGPS while smooth muscle actin (SMA) levels are relatively normal. Importantly, we have been able to recapitulate this in vivo phenotype of uncoupled SM-MHC vs. SMA expression in primary smooth muscle cells (SMCs) from WT and HGPS aortas. SM-MHC is among the most important regulators of the high contractility state in differentiated SMCs. Thus, these findings, and related traction force microscopy (TFM) experiments in Preliminary Studies, lead us to a new model for premature arterial stiffening in HGPS: the expression of mutant LaminA (Progerin) leads to a preferential downregulation of SM-MHC, and this locks HGPS SMCs into a novel intermediate tensional state in which they behave more like a de-differentiated SMC, producing ECM proteins and ECM remodeling enzymes that lead to an acceleration of arterial stiffening. Aim 1 will use age-matched WT and HGPS mice to test for causal relationships between i) ECM remodeling events and premature arterial stiffening and ii) SM-MHC expression and arterial ECM remodeling. Aim 2 will use isolated SMCs to identify molecular mechanisms and causal relationships between HGPS, SM-MHC expression, cellular contractility, and ECM remodeling. It will also establish molecular mechanisms by which expression of Progerin, and possibly WT LaminA, affects SM-MHC gene expression. Aim 3 will test for similarities and differences between arterial stiffening, ECM remodeling and SM-MHC/SMA expression in normal aging vs. HGPS.