Department of Surgery, University of Colorado Denver, Aurora, CO 80045.
Department of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Int J Biol Sci. 2020 Oct 3;16(15):3062-3074. doi: 10.7150/ijbs.49332. eCollection 2020.
Chronic valvular inflammation associated with monocyte infiltration promotes calcific aortic valve disease (CAVD) progression. Further, innate immunity in aortic valve interstitial cells (AVICs), mediated by Toll-like receptors (TLRs), up-regulates cellular inflammatory, fibrogenic and osteogenic activities. Currently, the pro-inflammatory communication between monocytes and AVICs and the underlying mechanism are unclear. We hypothesized that monocytes up-regulate AVIC inflammatory activity. This study sought to characterize the interaction between monocytes and AVICs and to elucidate the mechanism underlying cell-to-cell communication. AVICs, monocytes and co-cultures were exposed to a low concentration of TLR2 activator Pam3CSK4 (0.03 µg/ml). The TLR2 activator at this dose induced a marked increase in AVIC production of ICAM-1 and VCAM-1 only when co-cultured with monocytes. Adding conditioned medium from Pam3CSK4-treated monocytes (Pam3 CM, containing 0.1 µg/ml of Pam3CSK4) to AVIC culture (30% vol/vol; diluting Pam3CSK4 to 0.03 µg/ml) greatly increased the expression of adhesion molecules while adding conditioned medium from untreated monocytes (control CM) had no effect. Inhibition or knockdown of TLR2 in AVICs markedly reduced ICAM-1 and VCAM-1 expression induced by Pam3 CM. Further, Pam3 CM increased TLR2 levels in AVICs. Multiplex-ELISA analysis of Pam3 CM identified greater levels of TNF-α. Neutralization of TNF-α abolished the effect of Pam3 CM on AVIC TLR2 levels, resulting in marked attenuation of its potency in the induction of adhesion molecule expression. This study demonstrates that activated monocytes use paracrine signaling to sensitize AVICs for inflammatory responses to a low level of TLR2 activator. The mechanism of sensitization involves up-regulation of AVIC TLR2 levels by TNF-α from monocytes. Infiltrated monocytes in aortic valve tissue may exacerbate valvular inflammation by rendering AVICs hypersensitive to TLR2 activators.
慢性瓣膜炎症伴单核细胞浸润促进了钙化性主动脉瓣疾病（CAVD）的进展。此外，主动脉瓣间质细胞（AVICs）中的固有免疫通过 Toll 样受体（TLRs）上调细胞炎症、纤维化和成骨活性。目前，单核细胞与 AVICs 之间的促炎通讯及其潜在机制尚不清楚。我们假设单核细胞上调了 AVIC 的炎症活性。本研究旨在描述单核细胞与 AVIC 之间的相互作用，并阐明细胞间通讯的潜在机制。AVICs、单核细胞和共培养物被暴露于低浓度的 TLR2 激活剂 Pam3CSK4（0.03 µg/ml）。在与单核细胞共培养时，该剂量的 TLR2 激活剂仅诱导 AVIC 产生 ICAM-1 和 VCAM-1 的显著增加。将来自 Pam3CSK4 处理的单核细胞的条件培养基（Pam3 CM，含有 0.1 µg/ml 的 Pam3CSK4）添加到 AVIC 培养物中（30%体积/体积；将 Pam3CSK4 稀释至 0.03 µg/ml）可大大增加粘附分子的表达，而添加未经处理的单核细胞的条件培养基（对照 CM）则没有影响。AVIC 中的 TLR2 抑制或敲低显著降低了 Pam3 CM 诱导的 ICAM-1 和 VCAM-1 表达。此外，Pam3 CM 增加了 AVIC 中的 TLR2 水平。Pam3 CM 的多重 ELISA 分析确定了更高水平的 TNF-α。TNF-α 的中和消除了 Pam3 CM 对 AVIC TLR2 水平的影响，导致其诱导粘附分子表达的效力显著减弱。本研究表明，激活的单核细胞使用旁分泌信号来使 AVIC 对低水平的 TLR2 激活剂产生炎症反应敏感。敏化的机制涉及 TNF-α 从单核细胞上调 AVIC 的 TLR2 水平。主动脉瓣组织中浸润的单核细胞可能通过使 AVIC 对 TLR2 激活剂过度敏感而加剧瓣膜炎症。
