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Endocannabinoids have been shown to play a complex role in the pathophysiology of a number of cardiovascular disorders. In the present study, the effects of the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were investigated in human coronary artery smooth muscle cells (HCASMC) and human coronary artery endothelial cells (HCAEC) with regard to potential atheroprotective and anti-inflammatory effects. In HCASMC, AEA showed an inhibitory effect on platelet-derived growth factor-induced migration, but not proliferation, independent of major cannabinoid-activatable receptors (CB 1 , CB 2 , TRPV1), while 2-AG left both responses unaffected. In HCAEC, AEA at concentrations of 6 and 10 µM significantly inhibited the interleukin (IL)-1β- and lipopolysaccharide (LPS)-stimulated expression of vascular cell adhesion molecule-1 (VCAM-1) and LPS-induced intercellular adhesion molecule-1 (ICAM-1), again independently of the abovementioned receptors. Corresponding effects were observed to a lesser extent in the presence of 2-AG, in most cases not significantly. The detection of activated phosphoproteins as well as experiments with inhibitors of corresponding signaling pathways suggest that AEA interferes with IL-1β-induced VCAM-1 expression via inhibition of protein kinase B/Akt and Src kinase activation and attenuates LPS-induced VCAM-1 and ICAM-1 expression via inhibition of signal transducer and activator of transcription 3 (STAT3) phosphorylation. As expected, AEA also led to a significant inhibition of monocyte adhesion to IL-1β- and LPS-stimulated HCAEC, with siRNA experiments confirming the functional role of VCAM-1 and ICAM-1 in this assay. 2-AG showed a comparatively weaker but, in the case of LPS stimulation, still significant inhibition of adhesion. In summary, the results emphasize the potential of AEA as a protective regulator of atherosclerotic and inflammation-related changes in HCASMC and HCAEC and encourage further corresponding preclinical studies with this endocannabinoid.

Polycystic kidney disease (PKD) is characterized by extensive cyst formation and progressive fibrosis. However, the molecular mechanisms whereby the loss/loss-of-function of Polycystin 1 or 2 (PC1/2) provokes fibrosis are largely unknown. The small GTPase RhoA has been recently implicated in cystogenesis , and we identified the RhoA/cytoskeleton/myocardin-related transcription factor (MRTF) pathway as an emerging mediator of epithelium-induced fibrogenesis. Therefore, we hypothesized that MRTF is activated by PC1/2 loss and plays a critical role in the fibrogenic reprogramming of the epithelium. The loss of PC1 or PC2, induced by siRNA in vitro, activated RhoA and caused cytoskeletal remodeling and robust nuclear MRTF translocation and overexpression. These phenomena were also manifested in PKD1 (RC/RC) and PKD2 (WS25/-) mice, with MRTF translocation and overexpression occurring predominantly in dilated tubules and the cyst-lining epithelium, respectively. In epithelial cells, a large cohort of PC1/PC2 downregulation-induced genes was MRTF-dependent, including cytoskeletal, integrin-related, and matricellular/fibrogenic proteins. Epithelial MRTF was necessary for the paracrine priming of the fibroblast-myofibroblast transition. Thus, MRTF acts as a prime inducer of epithelial fibrogenesis in PKD. We propose that RhoA is a common upstream inducer of both histological hallmarks of PKD: cystogenesis and fibrosis.

Cardiac fibrosis is a key aspect of heart failure, leading to reduced ventricular compliance and impaired electrical conduction in the myocardium. Various pathophysiologic conditions can lead to fibrosis in the left ventricle (LV) and/or right ventricle (RV). Despite growing evidence to support the transcriptomic heterogeneity of cardiac fibroblasts (CFs) in healthy and diseased states, there have been no direct comparisons of CFs in the LV and RV. Given the distinct natures of the ventricles, we hypothesized that LV- and RV-derived CFs would display baseline transcriptomic differences that influence their proliferation and differentiation following injury. Bulk RNA sequencing of CFs isolated from healthy murine left and right ventricles indicated that LV-derived CFs may be further along the myofibroblast transdifferentiation trajectory than cells isolated from the RV. Single-cell RNA-sequencing analysis of the two populations confirmed that Postn + CFs were more enriched in the LV, whereas Igfbp3 + CFs were enriched in the RV at baseline. Notably, following pressure overload injury, the LV developed a larger subpopulation of pro-fibrotic Thbs4 +/ Cthrc1 + injury-induced CFs, while the RV showed a unique expansion of two less-well-characterized CF subpopulations ( Igfbp3 + and Inmt +). These findings demonstrate that LV- and RV-derived CFs display baseline subpopulation differences that may dictate their diverging responses to pressure overload injury. Further study of these subpopulations will elucidate their role in the development of fibrosis and inform on whether LV and RV fibrosis require distinct treatments.

Skin cancer is the most common malignant disease worldwide and, therefore, also poses a challenge from a pharmacotherapeutic perspective. Derivatives of indirubin are an interesting option in this context. In the present study, the effects of 3-[3'-oxo-benzo[ b ]thiophen-2'-( Z )-ylidene]-1-(β-d-glucopyranosyl)-oxindole (KD87), a thia-analogous indirubin N -glycoside, on the viability and mitochondrial properties of melanoma (A375) and squamous cell carcinoma cells (A431) of the skin were investigated. In both cell lines, KD87 caused decreased viability, the activation of caspases-3 and -7, and the inhibition of colony formation. At the mitochondrial level, a concentration-dependent decrease in both the basal and ATP-linked oxygen consumption rate and in the reserve capacity of oxidative respiration were registered in the presence of KD87. These changes were accompanied by morphological alterations in the mitochondria, a release of mitochondrial cytochrome c into the cytosol and significant reductions in succinate dehydrogenase complex subunit B (SDHB, subunit of complex II) in A375 and A431 cells and NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8, subunit of complex I) in A375 cells. The effect of KD87 was accompanied by a significant upregulation of the enzyme heme oxygenase-1, whose inhibition led to a partial but significant reduction in the metabolic-activity-reducing effect of KD87. In summary, our data show a mitochondria-targeting effect of KD87 as part of the cytotoxic effect of this compound on skin cancer cells, which should be considered in future studies with this class of compounds.

Atherosclerosis is associated with vascular smooth muscle cell proliferation, chronic vascular inflammation, and leukocyte adhesion. In view of the cardioprotective effects of cannabinoids described in recent years, the present study investigated the impact of the non-psychoactive phytocannabinoids cannabidiol (CBD) and tetrahydrocannabivarin (THCV) on proliferation and migration of human coronary artery smooth muscle cells (HCASMC) and on inflammatory markers in human coronary artery endothelial cells (HCAEC). In HCASMC, CBD and THCV at nontoxic concentrations exhibited inhibitory effects on platelet-derived growth factor-triggered proliferation (CBD) and migration (CBD, THCV). When interleukin (IL)-1β- and lipopolysaccharide (LPS)-stimulated HCAEC were examined, both cannabinoids showed a concentration-dependent decrease in the expression of vascular cell adhesion molecule-1 (VCAM-1), which was mediated independently of classical cannabinoid receptors and was not accompanied by a comparable inhibition of intercellular adhesion molecule-1. Further inhibitor experiments demonstrated that reactive oxygen species, p38 mitogen-activated protein kinase activation, histone deacetylase, and nuclear factor κB (NF-κB) underlie IL-1β- and LPS-induced expression of VCAM-1. In this context, CBD and THCV were shown to inhibit phosphorylation of NF-κB regulators in LPS- but not IL-1β-stimulated HCAEC. Stimulation of HCAEC with IL-1β and LPS was associated with increased adhesion of monocytes, which, however, could not be significantly abolished by CBD and THCV. In summary, the results highlight the potential of the non-psychoactive cannabinoids CBD and THCV to regulate inflammation-related changes in HCASMC and HCAEC. Considering their effect on both cell types studied, further preclinical studies could address the use of CBD and THCV in drug-eluting stents for coronary interventions.

JZL184, an inhibitor of monoacylglycerol lipase (MAGL) and thus of the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG), mediates various anticancer effects in preclinical studies. However, studies on the effect of this or other MAGL inhibitors under hypoxia, an important factor in tumor biology and response to cancer therapy, have not yet been performed in cancer cells. In the present study, the impact of the conditioned media (CM) of A549 and H358 lung cancer cells incubated with JZL184 under hypoxic conditions on the angiogenic properties of human umbilical vein endothelial cells (HUVECs) was investigated. Treatment of HUVECs with CM derived from cancer cells cultured for 48 h under hypoxic conditions was associated with a substantial increase in migration and tube formation compared with unconditioned medium, which was inhibited when cancer cells were incubated with JZL184. In this process, JZL184 led to a significant increase in 2-AG levels in both cell lines. Analysis of a panel of proangiogenic factors revealed inhibition of hypoxia-induced vascular endothelial growth factor (VEGF) expression by JZL184. Antiangiogenic and VEGF-lowering effects were also demonstrated for the MAGL inhibitor MJN110. Receptor antagonist experiments suggest partial involvement of the cannabinoid receptors CB 1 and CB 2 in the antiangiogenic and VEGF-lowering effects induced by JZL184. The functional importance of VEGF for angiogenesis in the selected system is supported by observations showing inhibition of VEGF receptor 2 (VEGFR2) phosphorylation in HUVECs by CM from hypoxic cancer cells treated with JZL184 or when hypoxic cancer cell-derived CM was spiked with a neutralizing VEGF antibody. On the other hand, JZL184 did not exert a direct effect on VEGFR2 activation induced by recombinant VEGF, so there seems to be no downstream effect on already released VEGF. In conclusion, these results reveal a novel mechanism of antiangiogenic action of JZL184 under conditions of hypoxic tumor-endothelial communication.

Despite the well-described anticarcinogenic effects of endocannabinoids, the influence of the endocannabinoid system on tumor angiogenesis is still debated. In the present study, conditioned medium (CM) from A549 and H358 lung cancer cells treated with ascending concentrations of the monoacylglycerol lipase (MAGL) inhibitor JZL184 and 2-arachidonoylglycerol (2-AG), a prominent MAGL substrate, caused a concentration-dependent reduction in human umbilical vein endothelial cell (HUVEC) migration and tube formation compared with CM from vehicle-treated cancer cells. Comparative experiments with MAGL inhibitors JW651 and MJN110 showed the same results. On the other hand, the angiogenic properties of HUVECs were not significantly altered by direct stimulation with JZL184 or 2-AG or by exposure to CM of JZL184- or 2-AG-treated non-cancerous bronchial epithelial cells (BEAS-2B). Inhibition of HUVEC migration and tube formation by CM of JZL184- and 2-AG-treated A549 cells was abolished in the presence of the CB 1 antagonist AM-251. Increased release of tissue inhibitor of metalloproteinase-1 (TIMP-1) from JZL184- or 2-AG-stimulated A549 or H358 cells was shown to exert an antiangiogenic effect on HUVECs, as confirmed by siRNA experiments. In addition, JZL184 caused a dose-dependent regression of A549 tumor xenografts in athymic nude mice, which was associated with a decreased number of CD31-positive cells and upregulation of TIMP-1-positive cells in xenograft tissue. In conclusion, our data suggest that elevation of 2-AG by MAGL inhibition leads to increased release of TIMP-1 from lung cancer cells, which mediates an antiangiogenic effect on endothelial cells.

The endocannabinoid system has been shown to be involved in various skin functions, such as melanogenesis and the maintenance of redox balance in skin cells exposed to UV radiation, as well as barrier functions, sebaceous gland activity, wound healing and the skin's immune response. In addition to the potential use of cannabinoids in the treatment and prevention of skin cancer, cannabinoid compounds and derivatives are of interest as potential systemic and topical applications for the treatment of various inflammatory, fibrotic and pruritic skin conditions. In this context, cannabinoid compounds have been successfully tested as a therapeutic option for the treatment of androgenetic alopecia, atopic and seborrhoeic dermatitis, dermatomyositis, asteatotic and atopic eczema, uraemic pruritis, scalp psoriasis, systemic sclerosis and venous leg ulcers. This review provides an insight into the current literature on cannabinoid compounds as potential medicines for the treatment of skin diseases.

Body implants and implantable medical devices have dramatically improved and prolonged the life of countless patients. However, our body repair mechanisms have evolved to isolate, reject, or destroy any object that is recognized as foreign to the organism and inevitably mounts a foreign body reaction (FBR). Depending on its severity and chronicity, the FBR can impair implant performance or create severe clinical complications that will require surgical removal and/or replacement of the faulty device. The number of review articles discussing the FBR seems to be proportional to the number of different implant materials and clinical applications and one wonders, what else is there to tell? We will here take the position of a fibrosis researcher (which, coincidentally, we are) to elaborate similarities and differences between the FBR, normal wound healing, and chronic healing conditions that result in the development of peri-implant fibrosis. After giving credit to macrophages in the inflammatory phase of the FBR, we will mainly focus on the activation of fibroblastic cells into matrix-producing and highly contractile myofibroblasts. While fibrosis has been discussed to be a consequence of the disturbed and chronic inflammatory milieu in the FBR, direct activation of myofibroblasts at the implant surface is less commonly considered. Thus, we will provide a perspective how physical properties of the implant surface control myofibroblast actions and accumulation of stiff scar tissue. Because formation of scar tissue at the surface and around implant materials is a major reason for device failure and extraction surgeries, providing implant surfaces with myofibroblast-suppressing features is a first step to enhance implant acceptance and functional lifetime. Alternative therapeutic targets are elements of the myofibroblast mechanotransduction and contractile machinery and we will end with a brief overview on such targets that are considered for the treatment of other organ fibroses.

We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-β1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

The administration of adipose tissue-derived mesenchymal stem cells (ADMSCs) represents a promising therapeutic option after myocardial ischemia or myocardial infarction. However, their potential is reduced due to the high post-transplant cell mortality probably caused by oxidative stress and mitogen-deficient microenvironments. To identify protection strategies for ADMSCs, this study investigated the influence of the non-psychoactive phytocannabinoid cannabidiol (CBD) and the endocannabinoid analogue R(+)-methanandamide (MA) on the induction of heme oxygenase-1 (HO-1) and autophagy under serum-free conditions. At a concentration of 3 µM, CBD induced an upregulation of HO-1 mRNA and protein within 6 h, whereas for MA only a late and comparatively lower increase in the HO-1 protein could be detected after 48 h. In addition, both cannabinoids induced time- and concentration-dependent increases in LC3A/B-II protein, a marker of autophagy, and in metabolic activity. A participation of several cannabinoid-binding receptors in the effect on metabolic activity and HO-1 was excluded. Similarly, knockdown of HO-1 by siRNA or inhibition of HO-1 activity by tin protoporphyrin IX (SnPPIX) had no effect on CBD-induced autophagy and metabolic activity. On the other hand, the inhibition of autophagy by bafilomycin A 1 led to a significant decrease in cannabinoid-induced metabolic activity and to an increase in apoptosis. Under these circumstances, a significant induction of HO-1 expression after 24 h could also be demonstrated for MA. Remarkably, inhibition of HO-1 by SnPPIX under conditions of autophagy deficit led to a significant reversal of apoptosis in cannabinoid-treated cells. In conclusion, the investigated cannabinoids increase 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.

Cannabidiol (CBD), a non-psychoactive cannabinoid, has been reported to mediate antioxidant, anti-inflammatory, and anti-angiogenic effects in endothelial cells. This study investigated the influence of CBD on the expression of heme oxygenase-1 (HO-1) and its functional role in regulating metabolic, autophagic, and apoptotic processes of human umbilical vein endothelial cells (HUVEC). Concentrations up to 10 µM CBD showed a concentration-dependent increase of HO-1 mRNA and protein and an increase of the HO-1-regulating transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). CBD-induced HO-1 expression was not decreased by antagonists of cannabinoid-activated receptors (CB 1 , CB 2 , transient receptor potential vanilloid 1), but by the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC). The incubation of HUVEC with 6 µM CBD resulted in increased metabolic activity, while 10 µM CBD caused decreased metabolic activity and an induction of apoptosis, as demonstrated by enhanced caspase-3 cleavage. In addition, CBD triggered a concentration-dependent increase of the autophagy marker LC3A/B-II. Both CBD-induced LC3A/B-II levels and caspase-3 cleavage were reduced by NAC. The inhibition of autophagy by bafilomycin A 1 led to apoptosis induction by 6 µM CBD and a further increase of the proapoptotic effect of 10 µM CBD. On the other hand, the inhibition of HO-1 activity with tin protoporphyrin IX (SnPPIX) or knockdown of HO-1 expression by Nrf2 siRNA was associated with a decrease in CBD-mediated autophagy and apoptosis. In summary, our data show for the first time ROS-mediated HO-1 expression in endothelial cells as a mechanism by which CBD mediates protective autophagy, which at higher CBD concentrations, however, can no longer prevent cell death inducing apoptosis.

Human pregnancy-specific glycoproteins (PSGs) serve immunomodulatory and pro-angiogenic functions during pregnancy and are mainly expressed by syncytiotrophoblast cells. While PSG mRNA expression in extravillous trophoblasts (EVTs) was reported, the proteins were not previously detected. By immunohistochemistry and immunoblotting, we show that PSGs are expressed by invasive EVTs and co-localize with integrin 5. In addition, we determined that native and recombinant PSG1, the most highly expressed member of the family, binds to 51 and induces the formation of focal adhesion structures resulting in adhesion of primary EVTs and EVT-like cell lines under 21% oxygen and 1% oxygen conditions. Furthermore, we found that PSG1 can simultaneously bind to heparan sulfate in the extracellular matrix and to 51 on the cell membrane. Wound healing assays and single-cell movement tracking showed that immobilized PSG1 enhances EVT migration. Although PSG1 did not affect EVT invasion in the in vitro assays employed, we found that the serum PSG1 concentration is lower in African-American women diagnosed with early-onset and late-onset preeclampsia, a pregnancy pathology characterized by shallow trophoblast invasion, than in their respective healthy controls only when the fetus was a male; therefore, the reduced expression of this molecule should be considered in the context of preeclampsia as a potential therapy., Competing Interests: The authors report no financial or other conflict of interest relevant to this study. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.