Introduction/Purpose: Diabetes and dyslipidemia induce metabolic stress (MS), a key driver of atherosclerosis through chronic inflammation, monocyte accumulation and endothelial dysfunction. The mechanisms by which MS modulates monocyte-endothelial cell interactions remain poorly characterized. Therefore, we investigated the how MS influences monocyte diapedesis via the Osteopontin/αvβ3/CD44v6 axis.
Methods: Human coronary artery endothelial cells (HCAEC) were stimulated with medium containing serum of hyperlipidaemia (HL) patients mimicking MS (hyperglycemia+oxLDL). Expression levels of Osteopontin (OPN) were measured via FACS and rt-qPCR. Cleavage was detected via ELISA. Additionally, CD14++ monocytes obtained from healthy donors were preconditioned with oxLDL or recombinant human OPN (rhOPN). We investigated the expression of OPN-coreceptors, CD51 and CD44v6, post-stimulation. Functional analyses were carried out using phase contrast microscopy under physiological flow conditions (flow assays). Firstly, we conditioned CD14++ monocytes in control or MS or oxLDL medium and allowed them to interact to an EC monolayer. Secondly, we exposed EC to oxLDL prior assay. OPN-coreceptors were blocked by additional rhOPN treatment of the cells. Cell-cell interactions during flow assay were tracked.
Results: EC surface OPN was significantly upregulated by HL serum (43% enhancement, p=0.01), whereas the OPN levels in the serum itself did not differ between non-HL and HL. Surface expression of OPN was increased by EC preconditioning in MS (47% increase, p =0.045) and oxLDL (61% increase, p=0.009).
Monocyte stimulation with MS significantly induced CD44v6 (1.3-fold, p=0.042), CD51 (2.2-fold, p=0.042) and CD61 (1.63-fold, p-value=0.042), which was consistent with oxLDL alone. Additional exposition to rhOPN did not alter the expression of monocytic adhesion molecules. Our study revealed a 3-fold enhanced adhesion of MS-conditioned monocytes to an EC monolayer. This effect was reversed by exposure to rhOPN prior to the flow assay, resulting in adhesion rates comparable to those of unstimulated monocytes. Furthermore, a significant delay in the initiation of transendothelial migration (TEM) after MS conditioning was uncovered (2.2-fold delay, p =0.048). TEM itself appears to be faster in the presence of MS (1.7-times faster TEM, p=0.024). The observed effect of MS preconditioning on accelerated TEM was significantly diminished when monocytes were exposed to rhOPN (1.8-times slower TEM, p=0.024). The TEM speed in MS/OPN group was comparable to that of unstimulated monocytes. In contrast, exposure of EC to oxLDL did not significantly alter monocytic adhesion rates to the EC-monolayer. Interestingly, the preconditioning of EC with oxLDL did not affect the initiation of TEM. Despite that, the TEM phase was significantly shorter, when EC were conditioned in an oxLDL environment (1.5-times faster TEM, p-value<0.0001). This effect was lost, when monocytes were also exposed to oxLDL.
Conclusions: Our study reveals that MS excerts biphasic control over monocyte trafficking. MS appears to increase monocytic adhesiveness, while primarily affecting TEM processes by altering the EC OPN/CD44v6 axis. These findings provide new insights into MS-mediated vascular inflammation and highlight potential therapeutic targets for atherosclerosis. Further research is needed to elucidate the underlying molecular mechanisms.
