Saporiti Federica, Piacentini Luca, Alfieri Valentina, Bono Elisa, Ferrari Fabrizio, Chiesa Mattia, Colombo Gualtiero I
Unit of Immunology and Functional Genomics, Centro Cardiologico Monzino IRCCS, Milan, Italy.
Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy.
Cell Physiol Biochem. 2019;52(6):1339-1360. doi: 10.33594/000000094.
BACKGROUND/AIMS: Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration.
We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²⁺ mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis.
We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²⁺ signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes.
Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries.
背景/目的：黑皮质素受体（MCRs）属于一种具有多种稳态和保护作用的激素信号通路。微血管和脐静脉内皮细胞（ECs）表达黑皮质素系统的成分，包括1型受体（MC1R），其在调节炎症和血管张力中发挥作用。由于内皮细胞表现出显著的异质性，我们研究了人动脉内皮细胞是否表达任何功能性MCR，以及其激活是否影响细胞迁移。
我们使用逆转录实时PCR检测原代人动脉内皮细胞中黑皮质素系统成分的表达。我们通过蛋白质印迹、免疫组织化学、cAMP生成和细胞内Ca²⁺动员试验评估MC1R蛋白表达和活性。我们进行了间隙闭合和划痕试验，以检测用α-黑素细胞刺激素（α-MSH）刺激后细胞的迁移，α-MSH是该受体亲和力最高的天然配体。我们通过微阵列分析评估迁移细胞中不同时间依赖性的转录变化。
我们发现人主动脉内皮细胞（HAoECs）表达功能性活性MC1R。与微血管内皮细胞不同，动脉细胞不表达α-MSH前体阿片促黑素皮质素原，也不产生该激素。MC1R与单次脉冲的α-MSH结合在定向迁移试验和划痕伤口愈合试验中均加速了HAoEC的迁移。这与Ca²⁺信号增强和cAMP升高受抑制有关。对进行定向迁移的HAoECs进行的全基因组时间进程表达分析允许识别参与细胞外基质-受体相互作用、囊泡介导的运输和金属传感的基因的动态协同调节——这些对内皮细胞运动性都有既定的影响——而不影响促凝血和抗凝血基因之间的平衡。
我们的工作拓宽了对外周表达的MC1R的认识。这些结果表明该受体由动脉内皮细胞组成性表达，并为MC1R的一种新的稳态功能提供了证据，其激活可能参与预防/修复大血管动脉中的内皮功能障碍或剥脱。
