Gonzalez Azuah L, Youwakim Cristina M, Leake Brenda F, Fuller Kristin K, Rahman S M Jamshedur, Dungan Matthew M, Gu Katherine, Bonin Jesse L, Cavnar Ashley B, Michell Danielle L, Davison Lindsay M, Cutchins Calliope, Chu Yunli E, Yuan Shuai, Yurdagul Arif, Traylor James G, Orr A Wayne, Kohutek Zachary A, Linton MacRae F, MacNamara Katherine C, Ferrell P Brent, Vickers Kasey C, Madhur Meena S, Brown Jonathan D, Doran Amanda C
Department of Pathology, Microbiology, and Immunology (A.L.G., M.M.D., A.B.C., K.C.V., A.C.D.), Vanderbilt University, Nashville, TN.
Department of Medicine (C.M.Y., B.F.L., K.K.F., S.M.J.R., K.G., D.L.M., L.M.D., C.C., Y.E.C., M.F.L., P.B.F., K.C.V., J.D.B., A.C.D.), Vanderbilt University Medical Center, Nashville, TN.
Arterioscler Thromb Vasc Biol. 2025 Jul;45(7):e286-e306. doi: 10.1161/ATVBAHA.125.322530. Epub 2025 May 8.
Chronic inflammation is a major driver of atherosclerotic cardiovascular disease, and therapeutics that target inflammation reduce cardiac events beyond levels seen with strategies targeting cholesterol alone. RNA sequencing revealed increased expression of CaMK4 (calcium/calmodulin-dependent protein kinase IV) in advanced/unstable human carotid artery plaque. We validated this finding in mouse and human atherosclerotic lesions, demonstrating increased CaMK4 in plaque macrophages. Therefore, we hypothesized that CaMK4 would promote inflammation and impair resolution in atherosclerosis.
We obtained mice in which exon 3 within the kinase domain of CaMK4 is deleted, leading to degradation and deletion of the gene (). Control and mice were injected with a gain-of-function AAV (adeno-associated virus) 8-PCSK9 (proprotein convertase subtilisin/kexin type 9) virus, rendering them hypercholesterolemic, and fed a high-fat/high-cholesterol diet for 12 weeks.
Hypercholesterolemic mice developed smaller and more stable lesions compared with control mice. Surprisingly, mice had a peripheral monocytosis with skewing of monocyte populations toward the nonclassical Ly6c subset, suggesting a less inflammatory monocyte population. Silencing or inhibition of CaMK4 in human monocytes recapitulated this phenotype. In response to hypercholesterolemia, which promotes myelopoiesis, mice had markedly more myeloid progenitors. monocytes expressed higher levels of genes associated with myeloid differentiation and recruitment of ATF6 (activating transcription factor 6) to conserved binding sites. In addition, monocytes expressed higher levels of , which promotes conversion of Ly6c to Ly6c monocytes. monocytes failed to efficiently traffic in vitro and in vivo. Bone marrow-derived macrophages generated from marrow had a more proreparative phenotype than control macrophages, consistent with our in vivo observations in the plaque.
These findings suggest that CaMK4 is an important regulator of the myelopoietic response to hypercholesterolemia through ATF6-mediated transcriptional regulation and that loss of functional CaMK4 promotes a proreparative phenotype in myeloid cells. Therefore, targeting CaMK4 may offer a unique way to target the progression of atherosclerosis.
慢性炎症是动脉粥样硬化性心血管疾病的主要驱动因素，针对炎症的治疗方法可降低心脏事件的发生率，其效果超过单纯针对胆固醇的治疗策略。RNA测序显示，在晚期/不稳定的人类颈动脉斑块中，CaMK4（钙/钙调蛋白依赖性蛋白激酶IV）的表达增加。我们在小鼠和人类动脉粥样硬化病变中验证了这一发现，表明斑块巨噬细胞中的CaMK4增加。因此，我们推测CaMK4会促进动脉粥样硬化中的炎症并损害炎症消退。
我们获得了CaMK4激酶结构域内第3外显子缺失的小鼠，导致该基因降解和缺失。对照小鼠和CaMK4基因敲除小鼠注射功能获得性腺相关病毒8-PCSK9（前蛋白转化酶枯草杆菌蛋白酶/kexin 9型）病毒，使其发生高胆固醇血症，并给予高脂/高胆固醇饮食12周。
与对照小鼠相比，高胆固醇血症的CaMK4基因敲除小鼠形成的病变更小且更稳定。令人惊讶的是，CaMK4基因敲除小鼠出现外周血单核细胞增多，单核细胞群体向非经典Ly6c亚群倾斜，提示炎症性较低的单核细胞群体。在人类单核细胞中沉默或抑制CaMK4可重现这一表型。在促进骨髓生成的高胆固醇血症反应中，CaMK4基因敲除小鼠的骨髓祖细胞明显更多。CaMK4基因敲除单核细胞表达与骨髓分化相关的基因水平更高，且激活转录因子6（ATF6）募集到保守结合位点。此外，CaMK4基因敲除单核细胞表达更高水平的促进Ly6c单核细胞向Ly6c单核细胞转化的基因。CaMK4基因敲除单核细胞在体外和体内均不能有效地迁移。由CaMK4基因敲除骨髓产生的骨髓来源巨噬细胞比对照巨噬细胞具有更具修复性的表型，这与我们在斑块中的体内观察结果一致。
这些发现表明，CaMK4是通过ATF6介导的转录调控对高胆固醇血症骨髓反应的重要调节因子，功能性CaMK4的缺失促进了骨髓细胞中的促修复表型。因此，靶向CaMK4可能为靶向动脉粥样硬化进展提供一种独特的方法。
