3.2.1. Apoptosis Ratio

MMP secretion is closely connected with cell death mechanisms. Therefore, the experimental setup used for zymography was also used to perform cell nuclei staining to evaluate apoptosis. The cells were treated with different compounds and cultivated on cover slips. After stimulation of VSMC with IL-1α for 48 h, apoptosis was increased, as demonstrated by an apoptotic ratio (Figure 3). More condensed small cell nuclei with irregular form (blebbing) as well as more disintegrating cells were detected (Figure 3A). IL-1α stimulation increased the number of apoptotic cell nuclei as compared with control. JWH-133 and rimonabant partially mitigated this effect. Rimonabant increased the ratio of normal cell nuclei to apoptotic cell nuclei by 2.5-fold and JWH-133 by 2.0-fold compared to IL-1α-treated cells (Figure 3B). Treatment with ACEA and AM630 did not have a similar effect; apoptosis levels in those two groups were comparable to that after IL-1α stimulation (Figure 3B; IL-1α = 1.0).

The same experimental setup was performed in H9c2 cells. The proapoptotic effect of the IL-1α stimulation was less pronounced in comparison to the IL-1α stimulation in VSMC, hence the effect of the treatment also showed smaller effects. IL-1α treatment increased the number of apoptotic cells 1.2-fold as compared to the control. The CB2R agonist JWH-133 ameliorated this increase 1.1-fold and rimonabant 1.3-fold, while AM 630 treatment showed no difference to the IL-1α group and ACEA showed even more apoptotic cell nuclei than the IL-1α-stimulated H9c2 cells (Figure S2).

**Figure 3.** (**A**). Effect of rimonabant, AM630, ACEA and JWH-133 on IL-1α-induced apoptosis in VSMCs. Magnification ×10. More condensed small cell nuclei with irregular form (blebbing) as well as more disintegrating cells were detected after IL-1α stimulation (**B**). (**a**) The ratio of normal cell nuclei to apoptotic cell nuclei in VSMC. The higher the bar, the more normal VSMC could be found in the treatment group. (**b**) The relation of apoptotic ratio in the treatment group to the ratio of the IL-1α-stimulated group. The resulting number expresses the factor of increased normal cell nuclei in comparison to the IL-1α group.

#### 3.2.2. Regulation of Caspase-3, FasL and TGF-Beta1

We further investigated the expression of apoptotic markers caspase-3 and FasL as well TGF-beta1 by fluorescence staining in VSMC. The intensity of caspase-3 expression in different groups is shown in Figure 4A. caspase-3 staining was localized intracellularly in VSMCs. IL-1α showed similar levels of caspase-3 signal compared with the control group. Rimonabant increased caspase-3 expression (2.3-fold), and the CB1R antagonist ACEA showed an even higher caspase-3 signal (2.8-fold) as compared with IL-1α group. The CB2R antagonist AM 630 showed an increased caspase-3 signal (1.5-fold), whereas JWH-133 decreased caspase-3 expression under the IL-1α level (by 43.2%) as well as under the control level. Thus, it appears that CB2R activation reduced apoptosis via caspase-3 signaling.

**Figure 4.** *Cont*.

**Figure 4.** (**A**) (**a**) Caspase-3 expression signal in VSMCs, 48 h after treatment with rimonabant, AM 630, ACEA and JWH-133; fluorescent images obtained with Biorevo BZ 900 Microscope using 10× magnification. Exposure time was equal in all observed groups. (**b**) Intensity of caspase-3 expression signal. The results were attained by measuring three areas of interest of the fluorescent image and subtracting the background signal (*n* = 3). (**c**) Representative Western blots of caspase-3 in VSMCs, 48 h after stimulation with IL-1<sup>α</sup>. (**B**) (**a**) FasL expression signal in VSMCs, 48 h after treatment with rimonabant, AM 630, ACEA and JWH-133; fluorescent images obtained with Biorevo BZ 900 Microscope using 10× magnification. Exposure time was equal in all observed groups. (**b**) Intensity of FasL expression signal. The results were attained by measuring three areas of interest of the fluorescent image and subtracting the background signal (*n* = 3). (**c**) Intensity of TGF-beta1 expression signal in VSMCs, 48 h after treatment with rimonabant, AM 630, ACEA, and JWH-133, attained by measuring three areas of interest of the fluorescent image and subtracting the background signal. The measurements were repeated three times per image. Statistical testing was performed using unpaired *t*-tests. Significance was expressed when *p* < 0.05; (\* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001).

FasL staining was strongly increased by 75.5% after IL-1α stimulation compared with the control (Figure 4B). CB1R blockage with rimonabant showed a decrease of 57.0% in FasL signal compared to IL-1<sup>α</sup>, while IL-1α plus ACEA showed the highest expression of all treatment compounds (Figure 4B(a,b)). Treatment with JWH-133 decreased FasL fluorescence signal, 4.5-fold compared to IL-1<sup>α</sup>, even under control group levels (Figure 4B(a,b)). This result suggests an antiapoptotic effect mitigated via CB2R activation.

Interestingly, TGF-beta1-expression staining showed opposing results compared to FasL expression (Figure 4C). The involvement of TGF-beta1 in the regulation of cell apoptosis has long been a point of discussion, since it contributes to a plethora of processes in the cell. Our results showed a 3.4-fold downregulation of TGF-beta1 in the IL-1α group compared to control (*p* < 0.001) (Figure 4C). Rimonabant and JWH-133 (*p* < 0.005) reduced this decrease (Figure 4C), while treatment with AM 630 and ACEA even decreased TGF-beta1 as compared to the IL-1α-stimulated group (19.1%, and 11.0%, respectively, *p* < 0.001).

#### *3.3. Regulation of Cell Proliferation: IncuCyte Live-Cell Analysis*

IncuCyte live-cell analysis enables the visualization and quantification of cell behavior over time, thus providing insight into cell proliferation and cell death dynamics.

We performed live-cell analysis using a DMEM cell medium containing either 1% FBS or 10% FBS. In line with our previous experiments for zymography analysis, 1% FBS cell medium was used initially. However, such experimental conditions provoked stagnating confluence due to cell death in all groups including the control. Growth rates without IL-1α stimulation in the first 24 h and 48 h were extremely low in all treatment groups (0.1–5.6%). A minimal cell growth rate was observed in the ACEA group (+0% in 24 h, + 3% after 48 h), being in line with the results obtained in the apoptosis analysis.

In order to improve growth conditions, repeated experiments were performed using 10% FBS medium (Figure 5A). A 10% FBS medium increased the growth rates of the control cells in comparison with 1% FBS (14.1% vs. 5.6% after 24 h and 28.0% vs. 5.1% after 48 h). The growth rate of the VSMC after 24 h and 48 h after using 10% FBS DMEM are presented in Figure 5A(a). IL-1α increased the growth rate in comparison to the control in 24 h (*p* < 0.001). Growth rates equalized after 48 h and showed no significant differences between cannabinoid treatment groups without IL-1α stimulation and the control group (Figure S3).

**Figure 5.** *Cont*.

**Figure 5.** (**A**) (**a**) VSMC, cell confluence difference (cell growth) at 24 h (confluence at 24 h-confluence at 0 h) and the confluence at 48 h (confluence at 48 h-confluence at 0 h) in the treatment groups. The experiment was performed using 10% FBS medium with and without IL-1α stimulation in all treatment groups. (**b**) VSMC, cell growth in dynamic. Representative graph obtained from IncuCyte Live-Cell Analysis System. Cell proliferation was monitored by analyzing the occupied area (% confluence) of cell images over 100 h. Analysis of the IncuCyte images was performed with Incucyte® Analysis Software. The experiment was performed using 10% FBS medium with and without IL-1α stimulation in all treatment groups. (**B**) The growth rate (confluence difference) of VSMCs estimated by IncuCyte live-cell analysis after treatment with compounds after IL-1α stimulation in 24 h (**a**) and 48 h (**b**). Statistical testing was performed using unpaired *t*-tests. Significance was expressed when *p* < 0.05; *n* = 6–32 (\* *p* < 0.05; \*\*\* *p* < 0.001).

We repeated the experiment using IL-1α stimulation in cannabinoid treatment groups. IL-1α increased cell growth rate after 24 h compared to the control group (Figure 5B(b)), and JWH-133 mitigated this increase (Figure 5B(b); *p* < 0.05). After 48 h, only the control compound ECEG decreased the cell growth (Figure 5B(b); *p* < 0.05) as compared with IL-1<sup>α</sup>.

Repeating the experimental setup using the cardiac H9c2 cell line, we achieved differing results. H9c2 as a secondary cell line showed an increased growth rate. After treatment, similar growth rates leading to full confluence in all groups were observed with and without IL-1α stimulation (Figure S4).

#### *3.4. Regulation of Glucose, Lactate and Electrolytes*

In order to ascertain if the treatment compounds influenced cell metabolics, we measured glucose, lactate and electrolytes concentrations in the supernatant. IL-1α stimulation decreased glucose concentration in the supernatant of VSMCs (Figure 6a; *p* < 0.01). JWH-133 and rimonabant normalized glucose levels, as compared to IL-1α stimulation, up to the control levels (Figure 6a; *p* < 0.05). ACEA and AM 630, in contrast, showed no significant effects (Figure 6a).

**Figure 6.** Concentration of glucose (**a**) and lactate (**b**) in the supernatant of VSMC after 48 h of treatment with compounds and IL-1α stimulation. Concentration of glucose (**c**) and lactate (**d**) in the supernatant of H9c2 cells after 48 h of treatment with compounds and IL-1α stimulation. The values are expressed as mean ± SD (VSMC, *n* = 3–9; H9C2 cells, *n* = 3). Statistical testing was performed using unpaired *t*-tests. (\* *p* < 0.05; \*\* *p* < 0.01).

Concomitantly, lactate concentration was increased after the IL-1α stimulation (Figure 6b; 2.2-fold). Rimonabant (by 46.5%) and JWH-133 (by 52.7%) reduced this increase (Figure 6b). The correlation analysis confirmed a negative correlation between glucose levels and lactate levels in the JWH-133 group (r = −0.99; *p* < 0.05) and AM630 group (r = −0.99; *p* < 0.01).

Similar measurements were performed in the supernatant of treated H9c2 cells (Figure 6c,d). JWH-133 also reduced the decrease in glucose concentration after IL-1α stimulation (Figure 6c; *p* < 0.05). In H9c2 cells, lactate levels showed less scattering and similar levels in all treatment groups (Figure 6d). The electrolytes Na, K and Cl were not affected by the different treatments, neither in VSMCs (Figure 7a–c) nor in H9c2 (Figure S5).

**Figure 7.** The concentrations of potassium (**a**), sodium (**b**) and chloride (**c**), measured in cell supernatant of VSMCs 48 h after treatments with compounds and IL-1α stimulation.

A comparison of the CB1 and CB2 receptor agonists and antagonists on cytokineinduced MMPs secretion, apoptosis, glucose uptake and cell proliferation in VSMCs and cardiac H9c2 cells is presented in Table 1.

**Table 1.** Comparison of the effects of the CB1 and CB2 receptor agonists and antagonists on cytokineinduced MMPs secretion, apoptosis, glucose uptake and cell proliferation in VSMCs and cardiac H9c2 cells. ↑—increase; ↓—decrease.



**Table 1.** *Cont.*
