Molecular mechanisms and functional consequence of cannabidiol-induced heme oxygenase-1 and -2 expression in human endothelial cells
Final Report Abstract
The heme oxygenase (HO) system, consisting of an inducible HO‐1 and a constitutive HO‐2 isoform, has attracted attention in context of vascular diseases. HO enzymes regulate heme homeostasis via rate limitation of oxidative heme breakdown, thereby removing potential cytotoxic heme and producing cytoprotective biliverdin and carbon monoxide. HO‐1 has been reported to confer antiatherogenic properties in endothelial cells, including inhibition of reactive oxygen species (ROS) formation and apoptosis, and induction of protective autophagy. In addition, studies have identified HO‐1 to play a role in protecting against disease‐related vascular smooth muscle cell (VSMC) properties such as proliferation and migration. On the other hand, reports also suggest that excessive and sustained HO‐1 expression has negative effects on cell function and survival, with the cytotoxic component attributed to the release of iron or high levels of CO, an inhibitor of the respiratory chain. The studies for this project focused on the HO‐1 system in different cell types and here on modulation by cannabinoids, which are increasingly the focus of research as potential pharmacotherapeutic approaches for various indications. The project mainly concerned the phytocannabinoid cannabidiol (CBD), which, in contrast to Δ9‐tetrahydrocannabinol (THC), has no psychoactivity. CBD is approved in combination with THC as Nabiximols for the treatment of spasticity associated with multiple sclerosis and as Epidyolex for the treatment of rare childhood forms of epilepsy. As a result of our investigations, a concentration‐dependent increase of HO‐1 expression by CBD in human umbilical vein endothelial cells (HUVEC; Böckmann and Hinz. Cells 2020, 9, 1703), human umbilical artery smooth muscle cells (HUASMC; Schwartz et al. Oncotarget 2018, 9, 34595‐34616) and adipose tissue‐derived mesenchymal stem cells (ADMSCs; Bublitz et al. Cells 2020, 9, 2298) independent of cannabinoid receptor activation could be demonstrated. Instead, increased HO‐1 expression by CBD was inhibited in HUVEC and HUASMC when ROS formation was simultaneously suppressed. 8 In HUVEC, 6 µM CBD resulted in increased metabolic activity, while 10 µM CBD caused a corresponding decrease as well as increased cleavage of the apoptosis marker caspase‐3. In addition, CBD triggered a concentration‐dependent increase in the autophagy marker LC3A/B‐II. Inhibition of autophagy by bafilomycin A1 resulted in apoptosis induction by 6 µM CBD and further enhancement of the proapoptotic effect of 10 µM CBD. On the other hand, inhibition of HO‐1 activity with tin protoporphyrin IX (SnPPIX), inhibition of HO‐1 expression by ROS scavenging or knockdown of HO‐1 expression by siRNA against Nrf2 (HO‐1‐regulatory transcription factor) was associated with a decrease in CBD‐mediated autophagy and apoptosis. In summary, this data revealed for the first time ROS‐ mediated HO‐1 expression in endothelial cells as a mechanism by which CBD mediates protective autophagy but fails to prevent apoptosis‐inducing cell death at higher CBD concentrations. For HUASMC, CBD‐induced HO‐1 expression was associated with inhibition of growth factor‐mediated proliferation and migration. However, neither inhibition of HO‐1 activity nor knockdown of HO‐1 protein attenuated the CBD‐mediated anti‐proliferative and anti‐migratory effects. Rather, inhibition or depletion of HO‐1 induced apoptosis and enhanced CBD‐mediated effects on proliferation and migration. Overall, this work provided the first evidence for CBD‐mediated enhancement of HO‐1 in VSMC and possible protective effects of this cannabinoid against abnormal VSMC proliferation and migration, which are closely linked to the development and progression of cardiovascular and cancer diseases. On the other hand, our data argue against a role for HO‐1 in CBD‐mediated inhibition of proliferation and migration, while supporting its anti‐apoptotic role in oxidative stress‐mediated cell fate. This was followed by studies with ADMSCs, which are a promising therapeutic option after myocardial ischaemia or myocardial infarction, but whose potential is limited due to high cell mortality after transplantation, probably caused by ROS and mitogen‐deficient microenvironments. To identify protective strategies for ADMSCs, the influence of CBD and the endocannabinoid analogue R(+)‐ methanandamide (MA) on the induction of HO‐1 and autophagy was investigated under serum‐free conditions. Only CBD showed an HO‐1 induction, whereas time‐ and concentration‐dependent increases in autophagy and metabolic activity could be detected for cannabinoids. However, knockdown of HO‐1 by siRNA or SnPPIX had no effect on CBD‐induced autophagy and metabolic activity. On the other hand, inhibition of autophagy by bafilomycin A1 resulted in a significant decrease in cannabinoid‐induced metabolic activity and an increase in apoptosis. Under these circumstances, a significant induction of HO‐1 expression was also demonstrated for MA. Remarkably, suppression of HO‐1 by SnPPIX under conditions of autophagy deficit resulted in significant inhibition of apoptosis in cannabinoid‐treated cells. In conclusion, the cannabinoids studied thus enhance the metabolic viability of ADMSCs under serum‐free conditions by inducing HO‐1‐independent autophagy, but contribute to apoptosis under conditions of additional autophagy deficit via an HO‐1‐dependent pathway.
Publications
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SB202190 inhibits endothelial cell apoptosis via induction of autophagy and heme oxygenase‐1. Oncotarget 2018, 9, 23149‐23163
Schwartz M, Böckmann S, Borchert P, Hinz B
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Up‐regulation of heme oxygenase‐1 expression and inhibition of disease‐associated features by cannabidiol in vascular smooth muscle cells. Oncotarget 2018, 9, 34595‐34616
Schwartz M, Böckmann S, Hinz B
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Cannabidiol Promotes Endothelial Cell Survival by Heme Oxygenase‐1‐ Mediated Autophagy. Cells 2020, 9, 1703
Böckmann S, Hinz B
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Cannabinoid‐Induced Autophagy and Heme Oxygenase‐1 Determine the Fate of Adipose Tissue‐Derived Mesenchymal Stem Cells under Stressful Conditions. Cells 2020, 9, 2298
Bublitz K, Böckmann S, Peters K, Hinz B
