Targeting cellular metabolism inhibits oxLDL-induced transdifferentiation of HCSMCs to macrophage-like cells

Background:
Atherosclerosis is the leading driver of cardiovascular disease-related mortality and morbidity in developed countries. Lineage tracing studies in atherogenesis could show that vascular smooth muscle cells (VSMCs) undergo transdifferentiation and exhibit phenotypic characteristics of distinct cell types like macrophages, thereby promoting instability of atherosclerotic lesions. Here, we investigated the molecular and metabolic changes occurring in VSMCs during oxidized LDL(oxLDL)-induced transdifferentiation to a macrophage-like phenotype. We also demonstrated that the transdifferentiation of VSMCs can be blocked via re-programming of cellular metabolism, which may offer a therapeutic approach to reduce atherosclerosis.

Methods:
To induce transdifferentiation, cultivated Human Coronary Smooth Muscle Cells (HCSMCs) were incubated with oxLDL (15 μg/ml) for 48 h in the presence or absence of specific metabolic inhibitors. Transdifferentiation was assessed by use of RT-qPCR, FACS and confocal microscopy. Expression of metabolic genes and cellular metabolic rate were evaluated using RT-qPCR and Agilent Seahorse XF, respectively. Lactate production and glucose consumption were measured using standardized commercial kits.

Results:
OxLDL-induced transdifferentiation of HCSMCs was characterized by downregulation of mRNA levels of smooth muscle cell specific markers SM22α and α-SMA accompanied by an upregulation of mRNA levels of the macrophage-specific marker CD68. The transdifferentiation was orchestrated by a downregulation of the transcriptional coactivator Myocardin and upregulation of the transcription factor Kruppel-like factor 4 (KLF4). Metabolic characterization revealed that transdifferentiated HCSMCs displayed a reduced basal oxygen consumption rate (OCR) as well as a significantly reduced maximum respiratory capacity (MRC) equivalent to a decrease in oxidative phosphorylation (OXPHOS). Accordingly, the lactate concentration was significantly increased in transdifferentiated HCSMCs proving a metabolic switch from OXPHOS to glycolysis. On the transcriptional level, the metabolic switch was characterized by a significantly increased expression of glucose transporter 1 (GLUT1) and of the key regulatory enzymes hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3). Interestingly, the phenotypic switch of HCSMCs was prevented by inhibition of the glycolytic pathway on different levels. Treatment of the cells with 2-Deoxy-D-glucose (2-DG, a glucose analog blocking glycolysis at the level of HK2), 3PO (inhibitor of PFKFB3) and KC7F2 (inhibitor of HIF1α) inhibited the oxLDL-induced transdifferentiation by enhancing the expression of SM22α and α-SMA (partially above the baseline level) while reducing the expression of CD68. In line with this, an increase in basal OCR and MRC of HCSMCs treated with the aforementioned inhibitors was detectable. Intriguingly, blocking of the mitochondrial pyruvate carrier by UK-5099 resulting in impaired OXPHOS also prevented downregulation of SM22α and α-SMA while it did not affect the upregulation of CD68 suggesting that SMC transdifferentiation is dependent on a complex interplay between glycolysis and OXPHOS.

Conclusion:
We could show that oxLDL-induced transdifferentiation of HCSMCs to a macrophage-like phenotype can be prevented by targeting the glycolytic pathway. This represents an innovative approach of targeting atherosclerosis by metabolic manipulation.