Hyperhomocysteinemia-Induced miR-30d-5p Regulates TIMP3 and MMP9, Enhancing Atherosclerosis Progression

Background and Aims: Elevated homocysteine (Hcy) levels are a risk factor for cardiovascular diseases, including atherosclerosis. MicroRNAs (miRNAs) influence genes linked to atherosclerosis, but their role in hyperhomocysteinemia (HHcy) remains unclear. miR-30d-5p is up-regulated in the plasma of patients with HHcy and deep venous thrombosis (DVT), with TIMP3 identified as a potential target. In this study, we aimed to investigate whether HHcy-induced miR-30d-5p regulates TIMP3 and its downstream effects on metalloproteinases MMP2 and MMP9, thereby playing a role in the pathophysiology of atherosclerosis. Methods: Mouse and human macrophages were treated with Hcy to assess gene and protein expression. Transfection experiments and dual luciferase reporter assays confirmed miRNA-target interactions. ApoE-/- mice received Hcy supplementation (1.8 g/L DL-Hcy) to induce atherosclerosis, with analyses of lesions, CD68, TIMP3 immunostaining, and in situ metalloproteinase activity. Results: miR-30d-5p was up-regulated in macrophages treated with Hcy, while TIMP3 expression decreased. TIMP3 was validated as a direct miR-30d-5p target through luciferase assays. miR-30d-5p inhibition in macrophages restored TIMP3 expression, whereas miR-30d-5p overexpression increased MMP9 and MMP2 protein levels and activity. In vivo, HHcy-induced atherosclerosis in ApoE-/- mice showed larger aortic lesions, higher macrophage and miR-30d-5p content, reduced TIMP3 levels, and elevated metalloproteinase activity compared to controls. Conclusions: These results provide evidence that miR-30d-5p downregulates TIMP3 and increases the activity of MMP9 in vitro and in vivo, suggesting another potential mechanism by which Hcy affects atherogenicity.