Cecoltan Sergiu, Ciortan Letitia, Macarie Razvan D, Vadana Mihaela, Mihaila Andreea C, Tucureanu Monica, Vlad Mihaela-Loredana, Droc Ionel, Gherghiceanu Mihaela, Simionescu Agneta, Simionescu Dan Teodor, Butoi Elena, Manduteanu Ileana
Biopathology and Therapy of Inflammation, Institute of Cellular Biology and Pathology "Nicolae Simionescu", Bucharest, Romania.
Cardiovascular Surgery Department, Central Military Hospital, Bucharest, Romania.
Front Cardiovasc Med. 2021 Aug 12;8:714573. doi: 10.3389/fcvm.2021.714573. eCollection 2021.
Valvular endothelial cells (VEC) have key roles in maintaining valvular integrity and homeostasis, and dysfunctional VEC are the initiators and major contributors to aortic valve disease in diabetes. Previous studies have shown that HG stimulated an inflammatory phenotype in VEC. Inflammation was shown to induce endothelial to mesenchymal transition (EndMT), a process extensively involved in many pathologies, including calcification of the aortic valve. However, the effect of HG on EndMT in VEC is not known. In addition, there is evidence that endothelin (ET) is a proinflammatory agent in early diabetes and was detected in aortic stenosis, but it is not known whether HG induces ET and endothelin receptors and whether endothelin modulates HG-dependent inflammation in VEC. This study aims to evaluate HG effects on EndMT, on endothelin and endothelin receptors induction in VEC and their role in HG induced VEC inflammation. We developed a new 3D model of the aortic valve consisting of a hydrogel derived from a decellularized extracellular cell matrix obtained from porcine aortic root and human valvular cells. VEC were cultured on the hydrogel surface and VIC within the hydrogel, and the resulted 3D construct was exposed to high glucose (HG) conditions. VEC from the 3D construct exposed to HG exhibited: attenuated intercellular junctions and an abundance of intermediate filaments (ultrastructural analysis), decreased expression of endothelial markers CD31 and VE-cadherin and increased expression of the mesenchymal markers α-SMA and vimentin (qPCR and immunocytochemistry), increased expression of inflammatory molecules ET-1 and its receptors ET-A and ET-B, ICAM-1, VCAM-1 (qPCR and Immunocytochemistry) and augmented adhesiveness. Blockade of ET-1 receptors, ET-A and ET-B reduced secretion of inflammatory biomarkers IL-1β and MCP-1 (ELISA assay). This study demonstrates that HG induces EndMT in VEC and indicates endothelin as a possible target to reduce HG-induced inflammation in VEC.
瓣膜内皮细胞（VEC）在维持瓣膜完整性和内环境稳定方面发挥着关键作用，功能失调的VEC是糖尿病患者主动脉瓣疾病的起始因素和主要促成因素。先前的研究表明，高糖（HG）刺激VEC产生炎症表型。炎症被证明可诱导内皮向间充质转化（EndMT），这一过程广泛参与包括主动脉瓣钙化在内的多种病理过程。然而，HG对VEC中EndMT的影响尚不清楚。此外，有证据表明内皮素（ET）是早期糖尿病中的一种促炎因子，在主动脉狭窄中也可检测到，但尚不清楚HG是否诱导ET及其受体，以及内皮素是否调节HG依赖的VEC炎症。本研究旨在评估HG对VEC中EndMT、内皮素及其受体诱导的影响，以及它们在HG诱导的VEC炎症中的作用。我们构建了一种新的主动脉瓣三维模型，该模型由源自猪主动脉根部脱细胞细胞外基质和人瓣膜细胞的水凝胶组成。将VEC培养在水凝胶表面，将瓣膜间质细胞（VIC）培养在水凝胶内部，然后将所得的三维构建体暴露于高糖（HG）条件下。暴露于HG的三维构建体中的VEC表现出：细胞间连接减弱和大量中间丝（超微结构分析），内皮标志物CD31和VE-钙黏蛋白表达降低，间充质标志物α-平滑肌肌动蛋白（α-SMA）和波形蛋白表达增加（定量聚合酶链反应和免疫细胞化学），炎症分子ET-1及其受体ET-A和ET-B、细胞间黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）表达增加（定量聚合酶链反应和免疫细胞化学）以及黏附性增强。阻断ET-1受体ET-A和ET-B可减少炎症生物标志物白细胞介素-1β（IL-1β）和单核细胞趋化蛋白-1（MCP-1）的分泌（酶联免疫吸附测定）。本研究表明，HG诱导VEC发生EndMT，并表明内皮素可能是减少HG诱导的VEC炎症的一个靶点。
