Despite the opioid overdose crisis, cocaine remains a widely abused illicit drug by the public and by veterans. While opioid overdose primarily causes respiratory failure, cocaine abuse is mainly associated with cardiovascular (CV) toxicities, which include hypertension (HTN), aortic stiffness, and atherosclerosis. Indeed, cocaine abuse represents a significant CV risk for the general population and for veterans. It is known that cocaine stimulates the sympathetic nervous system (SNS) by inhibiting norepinephrine (NE) reuptake at nerve terminals; however, recent evidence suggests that inhibition of NE reuptake may not be the major driver of cocaine-induced HTN. As such, the mechanisms mediating the effects of cocaine on the CV system remain largely unknown. To that end, we recently performed small RNA and RNA sequencing in the aortas from mice treated with cocaine, cocaine methiodide (CM, which does not enter the central nervous system), or saline to identify potential microRNA (miR)—mRNA pathways that mediate the CV effects of cocaine. Nine miR—mRNA pathways were implicated. We prioritized and identified two miR- mRNA axes based on their levels of expression changes and relevance to CV physiology. They are: 1) the ↑miR-30c—↓Malic Enzyme 1 (ME1)—↑reactive oxygen species (ROS) activity, which is crucial in HTN and vascular aging (aortic stiffness); and 2) the ↓miR-423—↑Cacna2d2 (encoding the α2δ-2 subunit of voltage-dependent calcium channels) —↑calcium influx resulting in increased intracellular calcium concentration ([Ca2+]i) which is critical in controlling vascular smooth muscle cell (SMC) contractility and blood pressure (BP). We thoroughly investigated the miR-30c pathway and recently published our findings in the journal Hypertension. In preliminary studies, we showed that cocaine- and CM-induced silencing of miR-423-5p expression and subsequent upregulation of Cacna2d2 led to increased [Ca2+]i, which augmented contractility in cultured SMCs. Furthermore, miR-423-5p overexpression ameliorated cocaine-induced BP elevation in vivo. Interestingly, miR-423-5p has been associated with heart failure and coronary artery disease. Its role in the pathogenesis of HTN remains unknown. Based on our published work and preliminary studies, we hypothesize that the miR-423—Cacna2d2 axis plays an important role in cocaine-induced HTN by regulating calcium influx and intracellular calcium concentrations ([Ca2+]i) in vascular cells. In addition, recent studies support a strong crosstalk between these two biological processes—Ca2+ signaling and ROS. We have pilot data showing that modification of both miR axes largely abrogated cocaine-induced SMC contraction. Therefore, we further hypothesize that these two pathways work synergistically to mediate cocaine-induced CV consequences. We will thoroughly characterize the ↓miR-423-5p— ↑Cacna2d2—↑ [Ca2+]i axis and its interaction with the ↑miR-30c—↓ME1—↑ROS pathway in mediating the effects of cocaine on the CV system...