**1. Introduction**

Recent studies have demonstrated hemodynamic and cardiometabolic effects of cannabinoids [1–5]. The cannabinoid system modulates the extracellular matrix turnover in the heart and blood vessels [6,7]. These findings have led to interest in the biochemical bases of their action. Endocannabinoids exert their effects, at least in part, by stimulating two main receptor subtypes, CB1 and CB2 (CB1R and CB2R), which belong to a group of seven transmembrane-spanning receptors. They are coupled to Gi/o-proteins and act via an inhibition of adenylylcyclase and subsequently the reduction of cAMP [8,9]. Other than the adenylylcyclase/cAMP pathways, several other intracellular pathways are influenced, such as p38, JNK and ERK [10]. Cannabinoid receptor ligands are divided into endogenous cannabinoids, such as 2-AG and AEA, and exogenous cannabinoids, such as derivatives from the cannabis sativa plant or synthetic cannabinoids [10]. Currently, novel modulators of the cannabinoid system are under investigations. New compounds

**Citation:** Greiner, B.; Sommerfeld, M.; Kintscher, U.; Unger, T.; Kappert, K.; Kaschina, E. Differential Regulation of MMPs, Apoptosis and Cell Proliferation by the Cannabinoid Receptors CB1 and CB2 in Vascular Smooth Muscle Cells and Cardiac Myocytes. *Biomedicines* **2022**, *10*, 3271. https://doi.org/10.3390/ biomedicines10123271

Academic Editor: Wesley M. Raup-Konsavage

Received: 21 November 2022 Accepted: 13 December 2022 Published: 16 December 2022

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are able to bind to CB receptors in the low nanomolar range with a marked selectivity towards the receptors [11]. Moreover, multitargeting G-protein-coupled receptors is also a promising strategy, as shown for antinociception by bivalent agonists for the opioid and cannabinoid receptors [12,13]. CB1R stimulation elicits bradycardia, negative inotropy and hypotension [14]. The CB1R is also implicated in inflammation, apoptosis and oxidative stress in the heart [15], whereas the CB2R may play a protective anti-inflammatory, antioxidative and antiatherogenic role [15–17]. We have previously shown that blockade of the CB1R with rimonabant decreased collagen accumulation and prevented upregulation of the profibrotic protein TGF-β1 in the heart and aorta in a myocardial infarction model [7]. Moreover, CB1R blockade also reduced the activity of the matrix metalloprotease 9 (MMP-9) in cardiac fibroblasts [7]. On the other hand, genetic deletion of the CB2R increased TGF-β1 and collagen production in a heart failure model [18], pointing to opposite effects of CB1R and CB2R. Recent studies provided further findings on extracellular matrix regulation by cannabinoids in the heart and vessels. For example, CB2R knockout models showed an increase in atherosclerotic vascular changes as well as an increase in MMP-9 expression [19]. In patients with high plaque instability of the carotid artery, decreased CB2R expression was correlated with a MMP-9 increase [20]. Based on these findings, we hypothesized that cannabinoids may influence proteolytic processes in the vessels directly *via* CB1R and CB2R, which are known to be localized on vascular smooth muscle cells (VSMCs) [21,22]. Therefore, we aimed to investigate whether cannabinoids regulate the activity of MMPs in the cells of rat vascular (VSMCs) and cardiac origin (H9c2). Given that the gelatinase A (MMP2) and gelatinase B (MMP9) are capable of degrading components of the extracellular matrix [23], we have focused on their regulation. By using various cannabinoid receptor ligands, we also intended to explore which receptor subtype is implicated in proteolysis. In our study, we used four different synthetic compounds that activate or block cannabinoid receptors: arachidonyl-2-chloroethylamide (ACEA), a CB1R agonist; rimonabant, a CB1R antagonist; John W. Huffman (JWH133), a CB2R agonist; and 6-Iodopravadoline (AM630), a CB2R antagonist. Since MMPs secretion is closely connected to cell proliferation, apoptosis and glucose metabolism, we analyzed their regulation.

#### **2. Materials and Methods**
