VCE-005.1, an hypoxia mimetic betulinic acid derivative, induces angiogenesis and shows efficacy in a murine model of traumatic brain injury.

One of the main global causes of mortality and morbidity is traumatic brain injury (TBI). Neuroinflammation and brain-blood barrier (BBB) disruption play a pivotal role in the pathogenesis of acute and chronic TBI onset. The activation of the hypoxia pathway is a promising approach for CNS neurodegenerative diseases, including TBI. Herein, we have studied the efficacy of VCE-005.1, a betulinic acid hydroxamate, against acute neuroinflammation in vitro and on a TBI mouse model. The effect of VCE-005.1 on the HIF pathway in endothelial vascular cells was assessed by western blot, gene expression, in vitro angiogenesis, confocal analysis and MTT assays. In vivo angiogenesis was evaluated through a Matrigel plug model and a mouse model of TBI induced by a controlled cortical impact (CCI) was used to assess VCE-005.1 efficacy. VCE-005.1 stabilized HIF-1α through a mechanism that involved AMPK and stimulated the expression of HIF-dependent genes. VCE-005.1 protected vascular endothelial cells under prooxidant and pro-inflammatory conditions by enhancing TJ protein expression and induced angiogenesis both in vitro and in vivo. Furthermore, in CCI model, VCE-005.1 greatly improved locomotor coordination, increased neovascularization and preserved BBB integrity that paralleled with a large reduction of peripheral immune cells infiltration, recovering AMPK expression and reducing apoptosis in neuronal cells. Taken together, our results demonstrate that VCE-005.1 is a multitarget compound that shows anti-inflammatory and neuroprotective effects mainly by preventing BBB disruption and has the potential to be further developed pharmacologically in TBI and maybe other neurological conditions that concur with neuroinflammation and BBB disruption.

A cannabidiol aminoquinone derivative activates the PP2A/B55α/HIF pathway and shows protective effects in a murine model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is characterized by a primary mechanical injury and a secondary injury associated with neuroinflammation, blood-brain barrier (BBB) disruption and neurodegeneration. We have developed a novel cannabidiol aminoquinone derivative, VCE-004.8, which is a dual PPARγ/CB2 agonist that also activates the hypoxia inducible factor (HIF) pathway. VCE-004.8 shows potent antifibrotic, anti-inflammatory and neuroprotective activities and it is now in Phase II clinical trials for systemic sclerosis and multiple sclerosis. Herein, we investigated the mechanism of action of VCE-004.8 in the HIF pathway and explored its efficacy in a preclinical model of TBI. METHODS: Using a phosphoproteomic approach, we investigated the effects of VCE-004.8 on prolyl hydroxylase domain-containing protein 2 (PHD2) posttranslational modifications. The potential role of PP2A/B55α in HIF activation was analyzed using siRNA for B55α. To evaluate the angiogenic response to the treatment with VCE-004.8 we performed a Matrigel plug in vivo assay. Transendothelial electrical resistance (TEER) as well as vascular cell adhesion molecule 1 (VCAM), and zonula occludens 1 (ZO-1) tight junction protein expression were studied in brain microvascular endothelial cells. The efficacy of VCE-004.8 in vivo was evaluated in a controlled cortical impact (CCI) murine model of TBI. RESULTS: Herein we provide evidence that VCE-004.8 inhibits PHD2 Ser125 phosphorylation and activates HIF through a PP2A/B55α pathway. VCE-004.8 induces angiogenesis in vivo increasing the formation of functional vessel (CD31/α-SMA) and prevents in vitro blood-brain barrier (BBB) disruption ameliorating the loss of ZO-1 expression under proinflammatory conditions. In CCI model VCE-004.8 treatment ameliorates early motor deficits after TBI and attenuates cerebral edema preserving BBB integrity. Histopathological analysis revealed that VCE-004.8 treatment induces neovascularization in pericontusional area and prevented immune cell infiltration to the brain parenchyma. In addition, VCE-004.8 attenuates neuroinflammation and reduces neuronal death and apoptosis in the damaged area. CONCLUSIONS: This study provides new insight about the mechanism of action of VCE-004.8 regulating the PP2A/B55α/PHD2/HIF pathway. Furthermore, we show the potential efficacy for TBI treatment by preventing BBB disruption, enhancing angiogenesis, and ameliorating neuroinflammation and neurodegeneration after brain injury.

Detrimental Effect of Cannabidiol on the Early Onset of Diabetic Nephropathy in Male Mice.

Anti-inflammatory and antidiabetogenic properties have been ascribed to cannabidiol (CBD). CBD-based medicinal drugs have been approved for over a lustrum, and a boom in the commercialization of CBD products started in parallel. Herein, we explored the efficacy of CBD in streptozotocin (STZ)-induced diabetic mice to prevent diabetic nephropathy at onset. Eight-to-ten-week-old C57BL6J male mice were treated daily intraperitoneally with 10 mg/kg of CBD or vehicle for 14 days. After 8 days of treatment, mice were challenged with STZ or vehicle (healthy-control). At the end of the study, non-fasting blood glucose (FBG) level was 276 ± 42 mg/dL in vehicle-STZ-treated compared to 147 ± 9 mg/dL (p ≤ 0.01) in healthy-control mice. FBG was 114 ± 8 mg/dL in vehicle-STZ-treated compared to 89 ± 4 mg/dL in healthy-control mice (p ≤ 0.05). CBD treatment did not prevent STZ-induced hyperglycemia, and non-FBG and FBG levels were 341 ± 40 and 133 ± 26 mg/dL, respectively. Additionally, treatment with CBD did not avert STZ-induced glucose intolerance or pancreatic beta cell mass loss compared to vehicle-STZ-treated mice. Anatomopathological examination showed that kidneys from vehicle-STZ-treated mice had a 35% increase of glomerular size compared to healthy-control mice (p ≤ 0.001) and presented lesions with a 43% increase in fibrosis and T cell infiltration (p ≤ 0.001). Although treatment with CBD prevented glomerular hypertrophy and reduced T cell infiltration, it significantly worsened overall renal damage (p ≤ 0.05 compared to vehicle-STZ mice), leading to a more severe renal dysfunction than STZ alone. In conclusion, we showed that CBD could be detrimental for patients with type 1 diabetes, particularly those undergoing complications such as diabetic nephropathy.

EHP-101 alleviates angiotensin II-induced fibrosis and inflammation in mice.

Some cannabinoids showed anti-inflammatory and antifibrotic activities. EHP-101 is an oral lipidic formulation of the novel non-psychotropic cannabidiol aminoquinone VCE-004.8, which showed antifibrotic activity in murine models of systemic sclerosis induced by bleomycin. We herein examined the effect of EHP-101 on cardiac and other organ fibrosis in a mouse model induced by Angiotensin II. VCE-004.8 inhibited TGFß- and Ang II-induced myofibroblast differentiation in cardiac fibroblasts detected by α-SMA expression. VCE-004.8 also inhibited Ang II-induced ERK 1 + 2 phosphorylation, NFAT activation and mRNA expression of IL1ß, IL6, Col1A2 and CCL2 in cardiac fibroblasts. Mice infused with Ang II resulted in collagen accumulation in left ventricle, aortic, dermal, renal and pulmonary tissues; oral administration of EHP-101, Ajulemic acid and Losartan improved these phenotypes. In myocardial tissue, Ang II induced infiltration of T cells and macrophages together with the accumulation of collagen and Tenascin C; those were all reduced by either EHP-101 or Losartan treatment. Cardiac tissue RNA-Seq analyses revealed a similar transcriptomic signature for both treatments for inflammatory and fibrotic pathways. However, the gene set enrichment analysis comparing data from EHP-101 vs Losartan showed specific hallmarks modified only by EHP-101. Specifically, EHP-101 inhibited the expression of genes such as CDK1, TOP2A and MKi67 that are regulated to the E2 factor family of transcription factors. This study suggests that the oral administration of EHP-101 prevents and inhibits cardiac inflammation and fibrosis. Furthermore, EHP-101 inhibits renal, pulmonary and dermal fibrosis. EHP-101 could offer new opportunities in the treatment of cardiac fibrosis and other fibrotic diseases.

Cannabidiol and Other Cannabinoids in Demyelinating Diseases.

A growing body of preclinical evidence indicates that certain cannabinoids, including cannabidiol (CBD) and synthetic derivatives, may play a role in the myelinating processes and are promising small molecules to be developed as drug candidates for management of demyelinating diseases such as multiple sclerosis (MS), stroke and traumatic brain injury (TBI), which are three of the most prevalent demyelinating disorders. Thanks to the properties described for CBD and its interesting profile in humans, both the phytocannabinoid and derivatives could be considered as potential candidates for clinical use. In this review we will summarize current advances in the use of CBD and other cannabinoids as future potential treatments. While new research is accelerating the process for the generation of novel drug candidates and identification of druggable targets, the collaboration of key players such as basic researchers, clinicians and pharmaceutical companies is required to bring novel therapies to the patients.

Neuroprotection with the cannabigerol quinone derivative VCE-003.2 and its analogs CBGA-Q and CBGA-Q-Salt in Parkinson's disease using 6-hydroxydopamine-lesioned mice.

The quinone derivative of the non-psychotropic cannabinoid cannabigerol (CBG), so-called VCE-003.2, has been recently investigated for its neuroprotective properties in inflammatory models of Parkinson's disease (PD) in mice. Such potential derives from its activity at the peroxisome proliferator-activated receptor-γ (PPAR-γ). In the present study, we investigated the neuroprotective properties of VCE-003.2 against the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA), in comparison with two new CBG-related derivatives, the cannabigerolic acid quinone (CBGA-Q) and its sodium salt CBGA-Q-Salt, which, similarly to VCE-003.2, were found to be active at the PPAR-γ receptor, but not at the cannabinoid CB1 and CB2 receptors. First, we investigated their cytoprotective properties in vitro by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-003.2 at a concentration of 20 µM, which was not reversed by the blockade of PPAR-γ receptors with GW9662, supporting its activity at an alternative site (non-sensitive to classic antagonists) in this receptor. We also found CBGA-Q and CBGA-Q-Salt being cytoprotective in this cell assay, but their effects were completely eliminated by GW9662, thus indicating that they are active at the canonical site in the PPAR-γ receptor. Then, we moved to in vivo testing using mice unilaterally lesioned with 6-OHDA. Our data confirmed that VCE-003.2 administered orally (20 mg/kg) preserved tyrosine hydroxylase (TH)-positive nigral neurons against 6-OHDA-induced damage, whereas it completely attenuated the astroglial (GFAP) and microglial (CD68) reactivity found in the substantia nigra of lesioned mice. Such neuroprotective effects caused an important recovery in the motor deficiencies displayed by 6-OHDA-lesioned mice in the pole test and the cylinder rearing test. We also investigated CBGA-Q, given orally (20 mg/kg) or intraperitoneally (10 mg/kg, i.p.), having similar benefits compared to VCE-003.2 against the loss of TH-positive nigral neurons, glial reactivity and motor defects caused by 6-OHDA. Lastly, the sodium salt of CBGA-Q, given orally (40 mg/kg) to 6-OHDA-lesioned mice, also showed benefits at behavioral and histopathological levels, but to a lower extent compared to the other two compounds. In contrast, when given i.p., CBGA-Q-Salt (10 mg/kg) was poorly active. We also analyzed the concentrations of dopamine and its metabolite DOPAC in the striatum of 6-OHDA-lesioned mice after the treatment with the different compounds, but recovery in the contents of both dopamine and DOPAC was only found after the treatment with VCE-003.2. In summary, our data confirmed the neuroprotective potential of VCE-003.2 in 6-OHDA-lesioned mice, which adds to its previous activity found in an inflammatory model of PD (LPS-lesioned mice). Additional phytocannabinoid derivatives, CBGA-Q and CBGA-Q-Salt, also afforded neuroprotection in 6-OHDA-lesioned mice, but their effects were lower compared to VCE-003.2, in particular in the case of CBGA-Q-Salt. In vitro studies confirmed the relevance of PPAR-γ receptors for these effects.

Δ9-Tetrahydrocannabinolic Acid markedly alleviates liver fibrosis and inflammation in mice.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the Western world, and it is closely associated to obesity, type 2 diabetes mellitus, and dyslipidemia. Medicinal cannabis and some neutral cannabinoids have been suggested as a potential therapy for liver diseases. HYPOTHESIS: Δ9-tetrahydrocannabinolic acid (Δ9-THCA), the non-psychotropic precursor of Δ9-THC, is one of the most abundant cannabinoids presents in Cannabis Sativa. However, its biological activities have been poorly investigated. Herein, we studied the antifibrotic and antiinflammatory activities of Δ9-THCA in two different animal models of liver injury, providing a rationale for additional studies on the medicinal use of this cannabinoid in the treatment of liver fibrosis and the management of NAFLD. STUDY DESIGN: The antifibrotic activity of Δ9-THCA in vitro was investigated in the cell lines LX-2 and NIH-3T3-Col1A2-luc. Non-alcoholic liver fibrosis was induced in mice by CCl4 treatment or, alternatively, by 23-week high fat diet (HFD) feeding. Δ9-THCA was administered daily intraperitoneally during the CCl4 treatment or during the last 3 weeks in HFD-fed mice. METHODS: TGFß-induced profibrotic gene expression was analyzed by luciferase and qPCR assays. Liver fibrosis and inflammation were assessed by immunochemistry and qPCR. Blood glucose, insulin, leptin and triglyceride levels were measured in HFD mice. RESULTS: Δ9-THCA inhibited the expression of Tenascin C (TNC) and Col3A1 induced by TGFß in LX-2 cells and the transcriptional activity of the Col1A2 promoter in fibroblasts. Δ9-THCA significantly attenuated CCl4-induced liver fibrosis and inflammation and reduced T cell and macrophage infiltration. Mice fed HFD for 23 weeks developed severe obesity (DIO), fatty liver and marked liver fibrosis, accompanied by immune cell infiltration. Δ9-THCA, significantly reduced body weight and adiposity, improved glucose tolerance, and drastically attenuated DIO-induced liver fibrosis and immune cell infiltration. CONCLUSIONS: Δ9-THCA prevents TGFß-induced fibrotic markers in vitro and liver inflammation and fibrogenesis in vivo, providing a rationale for additional studies on the medicinal use of this cannabinoid, as well as cannabis preparations containing it, for the treatment of liver fibrosis and the management of NAFLD.

Betulinic acid hydroxamate prevents colonic inflammation and fibrosis in murine models of inflammatory bowel disease.

Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) and is defined as an excessive accumulation of scar tissue in the intestinal wall. Intestinal fibrosis occurs in both forms of IBD: ulcerative colitis and Crohn's disease. Small-molecule inhibitors targeting hypoxia-inducing factor (HIF) prolyl-hydroxylases are promising for the development of novel antifibrotic therapies in IBD. Herein, we evaluated the therapeutic efficacy of hydroxamate of betulinic acid (BHA), a hypoxia mimetic derivative of betulinic acid, against IBD in vitro and in vivo. We showed that BAH (5-20 µM) dose-dependently enhanced collagen gel contraction and activated the HIF pathway in NIH-3T3 fibroblasts; BAH treatment also prevented the loss of trans-epithelial electrical resistance induced by proinflammatory cytokines in Caco-2 cells. In two different murine models (TNBS- and DSS-induced IBD) that cause colon fibrosis, oral administration of BAH (20, 50 mg/kg·d, for 17 days) prevented colon inflammation and fibrosis, as detected using immunohistochemistry and qPCR assays. BAH-treated animals showed a significant reduction of fibrotic markers (Tnc, Col1a2, Col3a1, Timp-1, α-SMA) and inflammatory markers (F4/80+, CD3+, Il-1ß, Ccl3) in colon tissue, as well as an improvement in epithelial barrier integrity and wound healing. BHA displayed promising oral bioavailability, no significant activity against a panel of 68 potential pharmacological targets and was devoid of genotoxicity and cardiotoxicity. Taken together, our results provide evidence that oral administration of BAH can alleviate colon inflammation and colitis-associated fibrosis, identifying the enhancement of colon barrier integrity as a possible mechanism of action, and providing a solid rationale for additional clinical studies.

Cannabinoids in Metabolic Syndrome and Cardiac Fibrosis.

PURPOSE OF REVIEW: This article provides a concise overview of how cannabinoids and the endocannabinoid system (ECS) have significant implications for the prevention and treatment of metabolic syndrome (MetS) and for the treatment of cardiovascular disorders, including cardiac fibrosis. RECENT FINDINGS: Over the past few years, the ECS has emerged as a pivotal component of the homeostatic mechanisms for the regulation of many bodily functions, including inflammation, digestion, and energy metabolism. Therefore, the pharmacological modulation of the ECS by cannabinoids represents a novel strategy for the management of many diseases. Specifically, increasing evidence from preclinical research studies has opened new avenues for the development of cannabinoid-based therapies for the management and potential treatment of MetS and cardiovascular diseases. Current information indicates that modulation of the ECS can help maintain overall health and well-being due to its homeostatic function. From a therapeutic perspective, cannabinoids and the ECS have also been shown to play a key role in modulating pathophysiological states such as inflammatory, neurodegenerative, gastrointestinal, metabolic, and cardiovascular diseases, as well as cancer and pain. Thus, targeting and modulating the ECS with cannabinoids or cannabinoid derivatives may represent a major disease-modifying medical advancement to achieve successful treatment for MetS and certain cardiovascular diseases.

Effects of EHP-101 on inflammation and remyelination in murine models of Multiple sclerosis.

Multiple Sclerosis (MS) is characterized by a combination of inflammatory and neurodegenerative processes in the spinal cord and the brain. Natural and synthetic cannabinoids such as VCE-004.8 have been studied in preclinical models of MS and represent promising candidates for drug development. VCE-004.8 is a multitarget synthetic cannabidiol (CBD) derivative acting as a dual Peroxisome proliferator-activated receptor-gamma/Cannabinoid receptor type 2 (PPARγ/CB2) ligand agonist that also activates the Hypoxia-inducible factor (HIF) pathway. EHP-101 is an oral lipidic formulation of VCE-004.8 that has shown efficacy in several preclinical models of autoimmune, inflammatory, fibrotic, and neurodegenerative diseases. EHP-101 alleviated clinical symptomatology in EAE and transcriptomic analysis demonstrated that EHP-101 prevented the expression of many inflammatory genes closely associated with MS pathophysiology in the spinal cord. EHP-101 normalized the expression of several genes associated with oligodendrocyte function such as Teneurin 4 (Tenm4) and Gap junction gamma-3 (Gjc3) that were downregulated in EAE. EHP-101 treatment prevented microglia activation and demyelination in both the spinal cord and the brain. Moreover, EAE was associated with a loss in the expression of Oligodendrocyte transcription factor 2 (Olig2) in the corpus callosum, a marker for oligodendrocyte differentiation, which was restored by EHP-101 treatment. In addition, EHP-101 enhanced the expression of glutathione S-transferase pi (GSTpi), a marker for mature oligodendrocytes in the brain. We also found that a diet containing 0.2% cuprizone for six weeks induced a clear loss of myelin in the brain measured by Cryomyelin staining and Myelin basic protein (MBP) expression. Moreover, EHP-101 also prevented cuprizone-induced microglial activation, astrogliosis and reduced axonal damage. Our results provide evidence that EHP-101 showed potent anti-inflammatory activity, prevented demyelination, and enhanced remyelination. Therefore, EHP-101 represents a promising drug candidate for the potential treatment of different forms of MS.

Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1.

Cannabidiol (CBD) is a major non-psychotropic phytocannabinoid that attracted a great attention for its therapeutic potential against different pathologies including skin diseases. However, although the efficacy in preclinical models and the clinical benefits of CBD in humans have been extensively demonstrated, the molecular mechanism(s) and targets responsible for these effects are as yet unknown. Herein we characterized at the molecular level the effects of CBD on primary human keratinocytes using a combination of RNA sequencing (RNA-Seq) and sequential window acquisition of all theoretical mass spectrometry (SWATH-MS). Functional analysis revealed that CBD regulated pathways involved in keratinocyte differentiation, skin development and epidermal cell differentiation among other processes. In addition, CBD induced the expression of several NRF2 target genes, with heme oxygenase 1 (HMOX1) being the gene and the protein most upregulated by CBD. CRISPR/Cas9-mediated genome editing, RNA interference and biochemical studies demonstrated that the induction of HMOX1 mediated by CBD, involved nuclear export and proteasomal degradation of the transcriptional repressor BACH1. Notably, we showed that the effect of BACH1 on HMOX1 expression in keratinocytes is independent of NRF2. In vivo studies showed that topical CBD increased the levels of HMOX1 and of the proliferation and wound-repair associated keratins 16 and 17 in the skin of mice. Altogether, our study identifies BACH1 as a molecular target for CBD in keratinocytes and sets the basis for the use of topical CBD for the treatment of different skin diseases including atopic dermatitis and keratin disorders.

Cannabinoid derivatives acting as dual PPARγ/CB2 agonists as therapeutic agents for systemic sclerosis.

The endocannabinoid system (ECS) may play a role in the pathophysiology of systemic sclerosis (SSc). Cannabinoids acting as dual PPARγ/CB2 agonists, such as VCE-004.8 and Ajulemic acid (AjA), have been shown to alleviate skin fibrosis and inflammation in SSc models. Since both compounds are being tested in humans, we compared their activities in the bleomycin (BLM) SSc model. Specifically, the pharmacotranscriptomic signature of the compounds was determined by RNA-Seq changes in the skin of BLM mice treated orally with AjA or EHP-101, a lipidic formulation of VCE-004.8. While both compounds down-regulated the expression of genes involved in the inflammatory and fibrotic components of the disease and the pharmacotranscriptomic signatures were similar for both compounds in some pathways, we found key differences between the compounds in vasculogenesis. Additionally, we found 28 specific genes with translation potential by comparing with a list of human scleroderma genes. Immunohistochemical analysis revealed that both compounds prevented fibrosis, collagen accumulation and Tenascin C (TNC) expression. The endothelial CD31+/CD34+ cells and telocytes were reduced in BLM mice and restored only by EHP-101 treatment. Finally, differences were found in plasmatic biomarker analysis; EHP-101, but not AjA, enhanced the expression of some factors related to angiogenesis and vasculogenesis. Altogether the results indicate that dual PPARγ/CB2 agonists qualify as a novel therapeutic approach for the treatment of SSc and other fibrotic diseases. EHP-101 demonstrated unique mechanisms of action related to the pathophysiology of SSc that could be beneficial in the treatment of this complex disease without current therapeutic options.

EHP-101, an oral formulation of the cannabidiol aminoquinone VCE-004.8, alleviates bleomycin-induced skin and lung fibrosis.

Systemic sclerosis (SSc) or scleroderma is a chronic multi-organ autoimmune disease characterized by vascular, immunological, and fibrotic abnormalities. The etiology of SSc is unknown, but there is growing evidence that dysfunction of the endocannabinoid system (ECS) plays a critical role in its development. Since the semi-synthetic cannabinoquinoid VCE-004.8 could alleviate bleomycin (BLM)-induced skin fibrosis, we have investigated an oral lipid formulation (EHP-101) of this dual PPARγ/CB2 receptors activator for the prevention of skin- and lung fibrosis and of collagen accumulation in BLM challenged mice. Immunohistochemistry analysis of the skin showed that EHP-101 could prevent macrophage infiltration as well as the expression of Tenascin C (TNC), vascular cell adhesion molecule 1 (VCAM1), and the α-smooth muscle actin (SMA). EHP-101 could also prevent the reduced expression of vascular CD31 typical of skin fibrosis. RNAseq analysis of skin biopsies showed a clear effect of EHP-101 in the inflammatory and epithelial-mesenchymal transition transcriptomic signatures. TGF-ß-regulated genes [matrix metalloproteinase-3 (Mmp3), cytochrome b-245 heavy chain (Cybb), lymphocyte antigen 6E (Ly6e), vascular cell adhesion molecule-1 (Vcam1) and Integrin alpha-5 (Itga5)] were induced in BLM mice and repressed by EHP-101 treatment. By intersecting differentially expressed genes in EHP-101-treated mice with a dataset of human scleroderma intrinsic genes, 53 overlapped genes were discovered, including biomarkers of SSc like the C-C motif chemokine 2 (Ccl2) and the interleukin 13 receptor subunit alpha 1 (IL-13Ra1) genes. Taken together, these data provide a rationale for further developing VCE-004.8 as an orally active agent to alleviate scleroderma and, possibly, other fibrotic diseases as well.

VCE-004.3, a cannabidiol aminoquinone derivative, prevents bleomycin-induced skin fibrosis and inflammation through PPARγ- and CB2 receptor-dependent pathways.

BACKGROUND AND PURPOSE: The endocannabinoid system and PPARγ are important targets for the development of novel compounds against fibrotic diseases such as systemic sclerosis (SSc), also called scleroderma. The aim of this study was to characterize VCE-004.3, a novel cannabidiol derivative, and study its anti-inflammatory and anti-fibrotic activities. EXPERIMENTAL APPROACH: The binding of VCE-004.3 to CB1 and CB2 receptors and PPARγ and its effect on their functional activities were studied in vitro and in silico. Anti-fibrotic effects of VCE-004.3 were investigated in NIH-3T3 fibroblasts and human dermal fibroblasts. To assess its anti-inflammatory and anti-fibrotic efficacy in vivo, we used two complementary models of bleomycin-induced fibrosis. Its effect on ERK1/2 phosphorylation induced by IgG from SSc patients and PDGF was also investigated. KEY RESULTS: VCE-004.3 bound to and activated PPARγ and CB2 receptors and antagonized CB1 receptors. VCE-004.3 bound to an alternative site at the PPARγ ligand binding pocket. VCE-004.3 inhibited collagen gene transcription and synthesis and prevented TGFß-induced fibroblast migration and differentiation to myofibroblasts. It prevented skin fibrosis, myofibroblast differentiation and ERK1/2 phosphorylation in bleomycin-induced skin fibrosis. Furthermore, it reduced mast cell degranulation, macrophage activation, T-lymphocyte infiltration, and the expression of inflammatory and profibrotic factors. Topical application of VCE-004.3 also alleviated skin fibrosis. Finally, VCE-004.3 inhibited PDGF-BB- and SSc IgG-induced ERK1/2 activation in fibroblasts. CONCLUSIONS AND IMPLICATIONS: VCE-004.3 is a novel semisynthetic cannabidiol derivative that behaves as a dual PPARγ/CB2 agonist and CB1 receptor modulator that could be considered for the development of novel therapies against different forms of scleroderma.

Identical COL71A1 heterozygous mutations resulting in different dystrophic epidermolysis bullosa phenotypes.

Dystrophic epidermolysis bullosa is a rare blistering condition caused by mutations in the COL7A1 gene. Different clinical variants have been described, with dominant and recessive inheritance, but no consistent findings have been elucidated to establish a genotype-phenotype correlation. We present three unrelated patients with two identical pathogenic compound heterozygous mutations in the COL7A1 gene that developed different clinical forms of dystrophic epidermolysis bullosa-epidermolysis bullosa pruriginosa and mild recessive non-Hallopeau-Siemens-raising the possibility of other genetic or environmental modifying factors responsible for the phenotype of the disease.

The adipokine lipocalin-2 in the context of the osteoarthritic osteochondral junction.

Obesity and osteoarthritis (OA) form a vicious circle in which obesity contributes to cartilage destruction in OA, and OA-associated sedentary behaviour promotes weight gain. Lipocalin-2 (LCN2), a novel adipokine with catabolic activities in OA joints, contributes to the obesity and OA pathologies and is associated with other OA risk factors. LCN2 is highly induced in osteoblasts in the absence of mechanical loading, but its role in osteoblast metabolism is unclear. Therefore, because osteochondral junctions play a major role in OA development, we investigated the expression and role of LCN2 in osteoblasts and chondrocytes in the OA osteochondral junction environment. Our results showed that LCN2 expression in human osteoblasts and chondrocytes decreased throughout osteoblast differentiation and was induced by catabolic and inflammatory factors; however, TGF-ß1 and IGF-1 reversed this induction. LCN2 reduced osteoblast viability in the presence of iron and enhanced the activity of MMP-9 released by osteoblasts. Moreover, pre-stimulated human osteoblasts induced LCN2 expression in human chondrocytes, but the inverse was not observed. Thus, LCN2 is an important catabolic adipokine in osteoblast and chondrocyte metabolism that is regulated by differentiation, inflammation and catabolic and anabolic stimuli, and LCN2 expression in chondrocytes is regulated in a paracrine manner after osteoblast stimulation.

Inhibition of the canonical Wnt pathway by high glucose can be reversed by parathyroid hormone-related protein in osteoblastic cells.

Recent in vivo findings suggest that the bone sparing effect of parathyroid hormone-related protein (PTHrP) in diabetic mice might occur at least in part through targeting a suppressed Wnt/ß-catenin pathway in osteoblasts. We here aimed to examine the inhibitory action of a high glucose environment on specific components of the canonical Wnt pathway, and the putative compensatory effects of PTHrP, in osteoblastic cell cultures. Mouse osteoblastic MC3T3-E1 cells and primary cultures of fetal mouse calvaria were exposed to normal (5.5 mM) or high (25 mM) D-glucose (HG), with or without PTHrP (1-36) or PTHrP (107-139) for different times. In some experiments, MC3T3-E1 cells were incubated with the Wnt pathway activators Wnt3a and LiCl, or were transfected with plasmids encoding either a mutated ß-catenin that cannot be targeted for degradation or a human PTHrP (-36/+139) cDNA, or the corresponding empty plasmid, in the presence or absence of HG. The gene expression of Wnt3a and low density receptor-like proteins (LRP)-5 and 6, as well as ß-catenin protein stabilization and ß-catenin-dependent transcription activity were evaluated. Oxidative stress status under HG condition was also assessed. The present data demonstrate that HG can target different components of the canonical Wnt pathway, while ß-catenin degradation appears to be a key event leading to inhibition of Wnt/ß-catenin signaling in mouse osteoblastic cells. Both PTHrP peptides tested were able to counteract this deleterious action of HG. These in vitro findings also provide new clues to understand the underlying mechanisms whereby PTHrP can increase bone formation.

Src kinases mediate VEGFR2 transactivation by the osteostatin domain of PTHrP to modulate osteoblastic function.

Parathyroid hormone-related protein (PTHrP) stimulates osteoblastic function through its N- and C-terminal domains. Since the osteogenic action of the latter domain appears to depend at least in part on its interaction with the vascular endothelial growth factor (VEGF) system, we aimed to explore the putative mechanism underlying this interaction in osteoblasts. Using native conditions for protein extraction and immunoblotting, we found that both PTHrP (107-139) and the shorter PTHrP (107-111) peptide (known as osteostatin), at 100 nM, promoted the appearance of a VEGF receptor (VEGFR) 2 protein band of apparent Mr. wt. 230 kDa, which likely represents its activation by dimer formation, in mouse osteoblastic MC3T3-E1 cells. Moreover, osteostatin (100 nM) maximally increased VEGFR2 phosphorylation at Tyr-1059 within 5-10 min in both MC3T3-E1 and rat osteoblastic osteosarcoma UMR-106 cells. This phosphorylation elicited by osteostatin appears to be VEGF-independent, but prevented by the VEGFR2 activation inhibitor SU1498 and also by the Src kinase inhibitors SU6656 and PP1. Furthermore, osteostatin induced phosphorylation of Src, extracellular signal-regulated kinase (ERK) and Akt with a similar time course to that observed for VEGFR2 activation in these osteoblastic cells. This osteostatin-dependent induction of ERK and Akt activation was abrogated by SU6656. Up-regulation of VEGF and osteoprotegerin gene expression as well as the pro-survival effect induced by osteostatin treatment were all prevented by both SU1498 and SU6656 in these osteoblastic cells. Collectively, these findings demonstrate that the osteostatin domain of C-terminal PTHrP phosphorylates VEGFR2 through Src activation, which represents a mechanism for modulating osteoblastic function.