Arterial stiffening and SMC mechanobiology in Hutchinson-Guilford Progeria Syndrome

摘要 Hutchinson-Gilford Progeria综合征(HGPS)是一种罕见的遗传性早衰疾病，由 LMNA基因常染色体显性突变，LMNA是一种常染色体显性突变。突变蛋白LmnAG608G已经被 名为“Progerin”患有HGPS的儿童通常在十几岁时死于心血管疾病 (动脉粥样硬化、心肌梗死和/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，而且动脉僵硬 被确认为心血管疾病的胆固醇非依赖性危险因素。此外，我们之前在活体内证明了 抑制动脉硬化可减少载脂蛋白E缺失小鼠的动脉粥样硬化。因为普罗杰林异常 HGP的法尼化治疗集中在法尼基转移酶抑制剂(FTI)上，但这些研究没有 直接解决被认为会导致HGPS早期死亡的心血管病理。 我们最近获得了对应于人类HGPS中LMNAG608G突变的LMNAG609G小鼠，并 在这里显示，这只小鼠表现出人类疾病的表现，表现为动脉过早僵硬。 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对象 动脉过早僵硬，这些将在这里进行研究。我们还发现，HGPS动脉缺乏 他们对血管收缩药的反应，我们的初步结果将这种收缩缺陷与一种引人注目的 两个成熟的平滑肌分化/CAPG基因的解偶联：平滑肌的表达 HGPS患者肌球蛋白重链(SM-MHC)减少，而平滑肌肌动蛋白(SMA)水平相对较低 很正常。重要的是，我们已经能够总结出未偶联的SM-MHC与SMA的体内表型 在WT和HGPS主动脉的原代培养的平滑肌细胞中的表达。SM-MHC是最大的 分化的SMC高收缩状态的重要调节因子。因此，这些发现和相关的 牵引力显微镜(TFM)实验的初步研究，引导我们找到了一种新的早产模型 HGPS中的动脉硬化：突变层(Progerin)的表达导致优先下调 SM-MHC，这将HGPS SMC锁定到一种新的中间拉伸状态，在这种状态下它们的行为 就像去分化的SMC一样，产生ECM蛋白和ECM重塑酶，从而导致 动脉硬化加速。目标1将使用年龄匹配的WT和HGPS小鼠来测试原因 I)ECM重塑事件与动脉过早硬化和II)SM-MHC表达的关系 和动脉ECM重塑。AIM 2将使用分离的SMC来确定分子机制和原因 HGPS、SM-MHC表达与细胞收缩和细胞外基质重塑的关系它还将 建立Progerin表达，可能还有WT膜影响SM-MHC的分子机制 基因表达。目标3将测试动脉硬化、ECM重塑 SM-MHC/SMA在正常增龄组和HGPS组中的表达。

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.