**4. Discussion**

CB receptors are implicated in cardiovascular patho/physiological processes [1–3,5], in particular, in the degradation of the extracellular matrix (ECM) [6,7]. The vascular and cardiac cells could be regulated by these receptors, affecting cell metabolism, proteolytic processes, cell death and proliferation.

In the present study, by using various CB receptor ligands, we intended to find out which receptor subtype is implicated in proteolysis in VSMC. We demonstrated that both CB receptors are involved in the regulation of MMPs, although the CB2R subtype plays a more important role. In our study, the stimulation of the CB2R in the VSMCs by the agonist JWH-133 reduced MMP-9 secretion in the supernatant and decreased proMMP-9 protein expression in the cells. In contrast, the CB2R antagonist AM630 increased MMP-9 expression.

Gelatinase MMP-2 is known to be involved in the degradation of extracellular matrix components and angiogenesis [23]. JWH-133 induced a reduction of both proMMP-2 and MMP-2. The same tendency was demonstrated in H9c2 cells of cardiac origin. Thus, we provide evidence that CB2R stimulation prevents the cytokine-induced MMP-9 and MMP-2 secretion. Our finding is in line with a study from [20] in neutrophils, which showed that treatment with JWH-133 reduced the release of TNF-α-induced MMP-9 via ERK1/2 phosphorylation. Moreover, JWH-133 exhibited antiproteolytic effects against MMP-1 and MMP-3 in human tenon fibroblasts [26].

CB1R inhibition with rimonabant in the present study also tended to decrease MMP-9 secretion, confirming our previous data obtained in cardiac fibroblasts [7]. Despite this, an opposite regulation by the CB1R agonist ACEA could not be shown.

In summary, CB2R stimulation decreased proteolytic activity in VSMC, mainly by downregulation of MMP-9.

Given the multiple roles of MMPs in cell death and especially in apoptosis, we decided to gain further insights in the effects of the CB agonists and antagonists on apoptosis. The CB2R agonist JWH-133 as well as the CB1R antagonist rimonabant mitigated the celldamaging apoptotic effect of cytokine stimulation in VSMC, as demonstrated by nucleus staining. Further, we could show that the CB2R agonist JWH-133 reduced the expression of apoptotic markers caspase-3 and FasL, whereas the CB2R antagonist showed increased caspase-3 expression.

Our results concerning the role of the CB2R in apoptosis are in harmony with studies on cell survival performed in cardiac myocytes and fibroblasts [15] and in the heart ischemia–reperfusion model [27]. Moreover, our data on the CB1R are also in line with the findings that deletion of CB1R or treatment of diabetic mice with CB1R antagonist SR141716 prevented retinal cell death [28].

Nevertheless, the experiments in the H9c2 cell line failed to demonstrate a regulation of apoptosis via CB receptors. Given that these cells showed a decreased reactivity to the IL-1 α stimulation, another protocol of apoptosis induction should be tested in future investigations.

TGF-beta1 is an important multifunctional cytokine, which is implied in extracellular matrix remodeling, cell proliferation and cell apoptosis [29]. Interestingly, TGF-beta1 expression was decreased after IL-1 α stimulation, in contrast to MMP-9, MMP-2 regulation and the apoptosis rate. These findings are in agreemen<sup>t</sup> with Risinger G.M. et al. [30], who showed that TGF-beta1 suppresses the upregulation of MMP-2 by VSMCs. Notably, the CB1R antagonist rimonabant and the CB2R agonist JWH-133 normalized TGF-beta1 expression in the VSMCs up to the control levels.

VSMC proliferation is known to be important for vascular wall remodeling in response to injury. Given that the amount of secreted MMPs depends on cell number, the effect of the treatment protocols on cell proliferation was studied. Therefore, IncuCyte cell life analysis was used to obtain data on the dynamics of cell proliferation and cell death. Neither the CB2R agonist JWH-133 nor the CB1R antagonist rimonabant significantly influenced cell proliferation under the given experimental conditions, suggesting that MMP secretion and apoptosis are regulated by the CB receptors. Nevertheless, at higher FBS concentrations (10%), JWH-133 at one time point 24 h showed antiproliferative properties. Our results may partly explain the controversy from previous studies showing pro-proliferative [22] and antiproliferative [16,31] effects on CB2R stimulation. Interestingly, we also found a strong antiproliferative effect of ECEG that has been used as a control substance in our experimental setting. Thus, further investigations on the role of the CB2R as well as ECEG in the atherosclerosis, angiogenesis and tumor growth would be important.

Since cell metabolism is an essential link between apoptosis and cell proliferation [32], we also addressed the regulation of glucose, lactate and electrolytes after treatment. The electrolytes sodium, potassium and chloride were not affected by treatment with CB1R and CB2R agonists and antagonists. IL-1 α stimulation strongly decreased glucose concentration in the supernatant in comparison to the control group. Such decrease can be explained by an increase in glucose uptake into the cell due to activation of glucose transporters GLUT1/4, which are predominant transporters in VSMCs [33,34]. CB2R stimulation with JWH-133 as well as CB1R inhibition with rimonabant reduced the decrease in glucose levels of the supernatant, pointing to a possible interaction of the CB receptors with glucose transporters. Concomitantly, the concentration of lactate was increased after IL-1 α stimulation, and rimonabant and JWH-133 also reduced this increase. The effects of JWH-133 on glucose levels were confirmed in cardiac H9c2 cells.

Whether glucose regulation by CB receptors is primary to MMPs secretion requires further investigation. Metabolic changes in VSMC not only contribute to the regulation of cell proliferation, apoptosis and proteolysis but also regulate a switch from the "contractile" phenotype to the proliferative "synthetic" VSMC phenotype [35], thereby influencing the progression of vascular diseases. Therefore, the involvement of the CB receptors in the regulation of glucose metabolism is of relevance in the context of several vascular diseases, including atherosclerosis, diabetes, hypertension and aneurysms.

In summary, the CB1R and the CB2R exert opposite effects on the regulation of cell glucose metabolism, proteolysis and apoptosis in VSMCs and cardiac H9c2 cells.

The stimulation of the CB2R reduced the cytokine-activated secretion of proMMP-2, MMP-2 and MMP-9, reduced FasL and caspase-3 mediated apoptosis, normalized the expression of TGF-beta 1 and prevented cytokine-induced increase in glucose uptake into the cell. CB1R inhibition showed similar protective properties but to a lesser extent. These

findings may pave the way to new approaches to treat cardiovascular diseases, especially those associated with extracellular matrix degradation.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines10123271/s1, Figure S1: Effect of Rimonabant, AM630, ACEA and JWH-133 on IL-1α induced secretion of MMP-9 (a), proMMP-2 (b) and MMP-2 (c) in H9c2 cells, 48h after treatment. The graphs represent the densitometric analysis (mean ± SD; *n* = 5–9 for MMP-9 and MMP-2, pro-MMP-2 (Rimonabant *n* =1; Control, IL1-alpha *n* = 2; ACEA, JWH-133, AM 630 *n* = 3). Statistical analysis performed with *t*-test with Welchs correction, \* *p* < 0.05; Figure S2: (a) The ratio of non-apoptotic normal cell nuclei to apoptotic cell nuclei in H9c2 cells 48 h after stimulation with IL-1α (b) The non-apoptotic/apoptotic cell nuclei ratio in relation to IL-1α stimulation. Treatment groups with a ratio over 1.0 have more normal cell nuclei than those in the IL-1α group; Figure S3: The growth rate (confluence difference) of VSMCs estimated by IncuCyte live-cell analysis after treatment with compounds without IL-1α stimulation in the treatment groups in 24 h (a) and 48 h (b). Statistical testing was performed using unpaired *t*-tests between treatment and control group. Significance was expressed when *p* < 0.05; *n* = 5–32 (\* *p* < 0.05; \*\*\* *p* < 0,01; \*\*\*\* *p* < 0.001); Figure S4: The growth rate (confluence difference) of H9c2 cells estimated by IncuCyte live-cell analysis after treatment with compounds without IL-1α stimulation in 24 h (a) and 48 h (b) and with IL1-alpha stimulation in 24 h (c) and 48 h (d). (e) and (f) H9c2, cell growth in dynamic. Representative graph obtained from IncuCyte Live Cell Analysis System. Cell proliferation was monitored by analysing the occupied area (% confluence) of cell images over 48 h. Analysis of the IncuCyte images was performed with Incucyte®Analysis Software. The experiment was performed using 10% FBS medium with (f) and without (e) IL-1α stimulation in all treatment groups; Figure S5: The concentrations of potassium (a), sodium (b) and chloride (c), measured in cell supernatant of H9c2 cells 48 h after treatments with compounds and IL-1α stimulation.

**Author Contributions:** B.G.: conception and design, performing experiments, collection and assembly of data, data analysis and interpretation, manuscript writing; M.S.: collection and assembly of data; U.K., T.U.: administrative support and advising on manuscript writing; K.K.: conception and design, administrative support, data analysis and interpretation, advising on manuscript writing; E.K.: conception and design, provision of study material, data analysis and interpretation, manuscript writing, final approval of the manuscript. U.K. received research grants/speaker honoraria from Bayer; speaker honoraria from Berlin Chemie, Boehringer Ingelheim, Daiichi Sankyo, Novartis, Sanofi Servier; and participated in advisory boards of Berlin Chemie, Boehringer Ingelheim, Novartis and Sanofi. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Charité Universitätsmedizin Berlin, Germany, and by the "Vascular Network" between CARIM—School for Cardiovascular Diseases, Maastricht University, The Netherlands, and Charité—Universitätsmedizin Berlin, Institute of Pharmacology, Germany. U.K. is supported by the DZHK; BER 5.4 PR, the Deutsche Forschungsgemeinschaft (DFG—KI 712/10-1; SFB-1470-A09); and the Einstein Foundation/Foundation Charité (EVF-BIH-2018-440). K.K. is supported by the Deutsche Forschungsgemeinschaft (DFG—KA 1820/9-1; KA 1820/10-1).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.
