**3. Results**

#### *3.1. Regulation of MMP-2 and MMP-9*

MMPs' enzymatic activity levels in conditioned media were demonstrated by gelatin zymography. Experiments were performed in different passages of VSMC (P4-P12, *n* = 10–16). The results are presented in Figure 1.

MMP-9 showed a greater upregulation by IL-1α stimulation (Figure 1a; 92.1%, *p* < 0.0001) than proMMP-2 (Figure 1b; 39.7%, *p* < 0.001) and MMP-2 (Figure 1c; 13.6%, n.s.). In comparison to the IL-1α group, treatment with JWH-133 reduced MMP-9 activity by 30.4% (Figure 1a; *p* < 0.0001). Rimonabant, ACEA and AM630 reduced MMP-9 activity slightly but not significantly. Even though less upregulated by IL-1<sup>α</sup>, proMMP-2 was also reduced by JWH-133 treatment (Figure 1b; 27.6%; *p* < 0.05). Rimonabant reduced proMMP-2 by 14.2%, while AM 630 and ACEA decreased proMMP-2 only by 7.7% and 7.9%, respectively. These changes were statistically not significant.

MMP-2, while showing only a minor upregulation after IL-1α stimulation, was also reduced by JWH-133 treatment (Figure 1c; 13.2%; *p* < 0,05). Rimonabant reduced MMP-2 by 10.1%, AM 630 by 11.2% and ACEA only by 6.3%, showing no significant effects. Representative zymographies are shown in Figure 1d,e.

The same experimental setup was used to evaluate MMP activity in H9c2 cardiac cells after treatment with the cannabinoid receptor agonists and antagonists after IL-1α stimulation. Similar to VSMC, MMP activity was more affected by the treatment with the CB2R agonist JWH-133, which decreased MMP-9 by 12.6% and proMMP-2 by 29.9% (n.s., Figure S1).

MMP-2,9 protein expression analysis is presented in Figure 2. Apart from the proMMP-9 (92 kDa) and MMP-9 (72 kDa) bands, three bands in the area of 45 to 60 kDa were detected (Figure 2a). ProMMP-9 expression increased by 48.4% after stimulation with IL-1α compared to control (Figure 2b), and the CB2R agonist JWH-133 augmented the increase by 35.5%, whereas the CB2R antagonist AM 630 increased the expression of proMMP-9 by 50.8%. Moreover, proMMP-9 expression was increased by rimonabant (+4.5%) and CB1R agonist ACEA (+13.0%). The bands in the area of 45–60 kDa, which were not expressed in the control group, showed a higher degree of regulation than the proMMP-9 and MMP-9 bands (Figure 2b). The CB2R agonist JWH-133 reduced the MMP expression of the three bands by 60.7%, and the CB1R antagonist rimonabant reduced MMP expression by 25.7%.

The correlating agonists/antagonists increased MMP activity at this molecular weight, AM630 by (+46.2%) and ACEA by (+40.9%).

**Figure 1.** Effect of rimonabant (Rimona), AM630, ACEA and JWH-133 on IL-1α-induced secretion of MMP-9 (**a**), proMMP-2 (**b**) and MMP-2 (**c**) in VSMCs, 48 h after treatment. The *graphs* represent the *densitometric* analysis (mean ± SD; *n* = 11). Statistical analysis performed with *t*-test with Welch's correction, \* *p* < 0.05; \*\*\*\* *p* < 0.0001. (**d**) MMP-9 activity, gelatin zymography, VSMCs, representative zymogram. (**e**) ProMMP-2, MMP-2 activity VSMCs, representative zymogram.

**Figure 2.** (**a**) Representative Western blots of MMP-9 in VSMCs, 48 h after stimulation with IL-1<sup>α</sup>. (**b**) Densiometric analysis of proMMP-9. (**c**) Densiometric analysis of MMP-9. (**d**) Densiometric analysis of MMP (45–60 kDa).

#### *3.2. Regulation of Apoptosis*
