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Cannabinoid Receptor Type 2 (CB2) Dependent and Independent Effects of WIN55,212-2 on Atherosclerosis in Ldlr-null MiceNetherland-Van Dyke, Courtney, Rodgers, Ward, Fulmer, Makenzie, Lahr, Zachary, Thewke, Douglas 01 July 2015 (has links)
PURPOSE: WIN55,212-2, a potent synthetic agonist of cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2), reduces atherosclerosis in apolipoprotein E (ApoE) null mice. Although pharmacologic evidence suggests the anti-atherosclerotic effects of WIN55,212-2 are mediated CB2, this remains to be confirmed by genetic studies. Therefore, in this study, we investigated the effects of WIN55,212-2 on development of atherosclerosis in low-density lipoprotein receptor (Ldlr) null mice with and without homozygous deletion of the CB2 gene. METHODS: After 6 weeks on an atherogenic diet, groups of CB2 and CB2 Ldlr-null mice received a daily intraperitoneal injection of WIN55,212-2 or vehicle. After two weeks, plasma lipid levels and atherosclerosis in the aortic root were quantified. RESULTS: Plasma cholesterol and triglyceride levels did not differ between CB2 and CB2 mice and WIN55,212-2 had no effect on total cholesterol levels in either genotype. However, triglyceride levels in both CB2 and CB2 mice were significantly lowered by WIN55,212-2. The size of aortic root lesions did not differ significantly between CB2 and CB2 mice with or without WIN55,212-2 treatment. However, WIN55,212-2 treatment significantly lowered lesional macrophage accumulation in CB2 mice, and lesional smooth muscle content in both CB2 and CB2 mice. Lesional apoptosis was also greater in CB2 mice compared to CB2mice, and only reduced by WIN55,212-2 in CB2 mice. Collagen content and elastin fiber fragmentation were unaffected by genotype or WIN55,212-2. CONCLUSIONS: WIN55,212-2 treatment does not alter lesion size in Ldlr null-mice, but does modify lesion cellularity CB2-dependent and CB2-independent mechanisms.
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The effect of synthetic cannabinoids and their combination with TGF-β3 on wound healing of cell cultured human bone cell monolayers and 3D models : the role of synthetic cannabinoid HU308 and HU308/TGF-β3 combinations on cellular adhesion, proliferation, wound healing, nitric oxide, MMP-2 and ECM protein regulation of MG-63 osteoblast monolayers and 3D modelsGenedy, Mohamed January 2013 (has links)
Despite the ongoing political debate regarding the legality of medical marijuana, clinical investigations of the therapeutic use of cannabinoids are now more prevalent than at any time in history. Cannabinoids have been shown to have analgesic, anti-spasmodic, anticonvulsant, anti-tremor, anti-psychotic, anti-inflammatory, anti-oxidant, anti-emetic and appetite-stimulant properties. There are mainly two well-known cannabinoid receptors, CB1 and CB2, located in the central (CB1) and peripheral (CB2) nervous systems as well as the immune system. More recently, endocannabinoids (ligands) and their receptors have also been found in the skeleton which appear as the main body system and physiologically regulated by CB2. This study aimed to examine the effect of both CB1 and CB2 receptor stimulation on wound closure response of MG-63 osteoblast bone cell monolayers using different treatments with cannabinoid such as Winn55,212-2, URB602 and HU308. Also, cell adhesion, cell proliferation and cell length was investigated. The study also aimed to examine the effect of HU308 treatments in combination with TGF-β3 (transforming growth factor beta -3) on wound healing, cell adhesion and extracellular matrix up regulation (collagen type I, fibronectin and protien S-100A6) as well as other biological factors such as secretion of matrix metalloproteinase (MMP-2) and nitric oxide (NO). Finally, this study investigated HU308/TGF-β3 combination treatment on the regulation of extracellular matrix (collagen type I, fibronectin and protien S-100A6) in a 3D multilayer system of MG-63 osteoblast bone cells. Wound healing assays of MG-63 monolayers revealed accelerated wound repair as well as increased cell proliferation mainly regulated through CB2 receptors, and that treatments with HU308 and HU308/TGF-β3 achieved minimum closure timings compared with control groups (P<0.05). Our finding suggested that proliferation rate with 500nM HU308 was significantly higher than control and TGF-β3/HU308 combination groups (P<0.05). Interestingly, percentage of wound remained open after 15 hours for combination groups was 17.6%±1.32 whereas treatment with 500nM HU308 had 20%±2.25 indicating that the combination groups took the lead throughout wound healing. It was also observed that bridge formation in all treatment groups was taking place between 15 to 20 hour periods whereas within control treatments bridge formation started to take place after 25 hours. Cell surface attachment was examined via the trypsinization assay in which the time taken to trypsinize cells from the surface provided a means of assessing the strength of attachment. The results indicated that higher concentrations of HU308 (2μM), induced significant force of cell attachment compared with control and concentrations of 500nM and 1μM (P<0.05). However, groups treated with TGF-β3 and combination HU308/TGF-β3 indicated reduced cell surface attachment compared with control groups, indicating enhanced cell migration. Immunofluorescence staining as well as Elisa based semi-quantification technique indicated that both collagen type I and fibronectin were unregulated using higher concentrations of HU308 with decreased cell proliferation compared to lower concentrations. Nevertheless, protein S-100A6 was up-regulated in treatments with HU308, TGF-β3 and their combination HU308/TGF-β3 (P<0.05), indicating the positive role of these treatments in promoting cell differentiation. MMP-2 levels in the current study were also shown to be concentration-dependent, i.e. higher concentrations of HU308 significantly reduced MMP-2 secretion leading to decreased cell migration, while HU308/TGF-β3 combination treatment increased MMP-2 levels, indicating an increase in cell migration. The current study also examined levels of nitric oxide synthesis in relation to different treatments with HU308, TGF-β3 and HU308/TGF-β3 combination. It was found that nitric oxide up-regulation influences rate of MG-63 osteoblast wound healing in a concentration dependent manner. Lastly, UpCell culture dishes proved to have efficacy in obtaining a multilayer model of MG-63 osteoblast system in-vitro through changes in cell morphology. It was also found that treatments with HU308, TGF-β3 and HU308/TGF-β3 combination influenced collagen type I, fibronecton and protein S-100A6 secretion. These findings supported the earlier Elisa based semi-quantification results obtained for monolayer cultures.
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The effect of synthetic cannabinoids and their combination with TGF-β3 on wound healing of cell cultured human bone cell monolayers and 3D models. The role of synthetic cannabinoid HU308 and HU308/TGF-β3 combinations on cellular adhesion, proliferation, wound healing, nitric oxide, MMP-2 and ECM protein regulation of MG-63 osteoblast monolayers and 3D modelsGenedy, Mohamed A. January 2013 (has links)
Despite the ongoing political debate regarding the legality of medical marijuana, clinical
investigations of the therapeutic use of cannabinoids are now more prevalent than at any
time in history. Cannabinoids have been shown to have analgesic, anti-spasmodic, anticonvulsant,
anti-tremor, anti-psychotic, anti-inflammatory, anti-oxidant, anti-emetic and
appetite-stimulant properties. There are mainly two well-known cannabinoid receptors,
CB1 and CB2, located in the central (CB1) and peripheral (CB2) nervous systems as well
as the immune system. More recently, endocannabinoids (ligands) and their receptors
have also been found in the skeleton which appear as the main body system and
physiologically regulated by CB2.
This study aimed to examine the effect of both CB1 and CB2 receptor stimulation on
wound closure response of MG-63 osteoblast bone cell monolayers using different
treatments with cannabinoid such as Winn55,212-2, URB602 and HU308. Also, cell
adhesion, cell proliferation and cell length was investigated. The study also aimed to
examine the effect of HU308 treatments in combination with TGF-β3 (transforming
growth factor beta -3) on wound healing, cell adhesion and extracellular matrix up
regulation (collagen type I, fibronectin and protien S-100A6) as well as other biological
factors such as secretion of matrix metalloproteinase (MMP-2) and nitric oxide (NO).
Finally, this study investigated HU308/TGF-β3 combination treatment on the regulation
of extracellular matrix (collagen type I, fibronectin and protien S-100A6) in a 3D
multilayer system of MG-63 osteoblast bone cells.
Wound healing assays of MG-63 monolayers revealed accelerated wound repair as well
as increased cell proliferation mainly regulated through CB2 receptors, and that
treatments with HU308 and HU308/TGF-β3 achieved minimum closure timings
compared with control groups (P<0.05). Our finding suggested that proliferation rate
with 500nM HU308 was significantly higher than control and TGF-β3/HU308
combination groups (P<0.05). Interestingly, percentage of wound remained open after
15 hours for combination groups was 17.6%±1.32 whereas treatment with 500nM
HU308 had 20%±2.25 indicating that the combination groups took the lead throughout wound healing. It was also observed that bridge formation in all treatment groups was
taking place between 15 to 20 hour periods whereas within control treatments bridge
formation started to take place after 25 hours.
Cell surface attachment was examined via the trypsinization assay in which the time
taken to trypsinize cells from the surface provided a means of assessing the strength of
attachment. The results indicated that higher concentrations of HU308 (2μM), induced
significant force of cell attachment compared with control and concentrations of 500nM
and 1μM (P<0.05). However, groups treated with TGF-β3 and combination
HU308/TGF-β3 indicated reduced cell surface attachment compared with control
groups, indicating enhanced cell migration.
Immunofluorescence staining as well as Elisa based semi-quantification technique
indicated that both collagen type I and fibronectin were unregulated using higher
concentrations of HU308 with decreased cell proliferation compared to lower
concentrations. Nevertheless, protein S-100A6 was up-regulated in treatments with
HU308, TGF-β3 and their combination HU308/TGF-β3 (P<0.05), indicating the
positive role of these treatments in promoting cell differentiation. MMP-2 levels in the
current study were also shown to be concentration-dependent, i.e. higher concentrations
of HU308 significantly reduced MMP-2 secretion leading to decreased cell migration,
while HU308/TGF-β3 combination treatment increased MMP-2 levels, indicating an
increase in cell migration. The current study also examined levels of nitric oxide
synthesis in relation to different treatments with HU308, TGF-β3 and HU308/TGF-β3
combination. It was found that nitric oxide up-regulation influences rate of MG-63
osteoblast wound healing in a concentration dependent manner.
Lastly, UpCell culture dishes proved to have efficacy in obtaining a multilayer model of
MG-63 osteoblast system in-vitro through changes in cell morphology. It was also
found that treatments with HU308, TGF-β3 and HU308/TGF-β3 combination
influenced collagen type I, fibronecton and protein S-100A6 secretion. These findings
supported the earlier Elisa based semi-quantification results obtained for monolayer
cultures.
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The effect of synthetic cannabinoids on wound healing of chondrocyte monolayers and pseudo 3D cartilage tissue : effect of different concentrations of synthetic cannabinoids WIN55, 212-2, URB602 and HU-308 with and without their antagonists on wound healing of chondrocyte monolayers and pseudo 3D cartilage tissueAbdeldayem, Ali Ibrahim Al January 2013 (has links)
Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of cannabinoids and also their potentials for cartilage repair and regeneration. Various wound healing techniques can be used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissue constructs. The effect of different concentrations of the synthetic cannabinoids WIN55, 212-2 (WIN-2), URB602 and HU-308 with and without their antagonists on the wound healing of chondrocyte monolayers was investigated using a simple scratch assay model. The three cannabinoids were found to increase wound healing of chondrocyte monolayers, but at different rates. WIN55, 212-2 at a concentration of 1μM had the highest effect of increasing both migration and proliferation of chondrocytes cultured in a chondrogenic media, which increased the rate of wound closure. It was also found that treating the cells with 2μM of any of the cannabinoids lead to a decrease in cell proliferation and the rate of wound closure. These findings were further investigated, by studying the effect of WIN-2 on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by wounded chondrocyte monolayers. Moreover, expression of collagen type-I, collagen type-II, fibronectin and S100 proteins were detected using immunofluorescence and verified quantitatively using ELISA based techniques, following treatment with 1μM and 2μM of WIN-2, for both 2D monolayers and 3D sheets. Treating chondrocytes with 1μM of WIN-2 significantly increased collagen type-II, fibronectin and S100, and significantly reduced collagen type-I compared to control groups in monolayers and chondrocyte cell sheets. On the other hand, both concentrations of WIN-2 significantly reduced the expression of the inflammation markers NO, and MMP-2, in a dose dependent manner. These findings highlight the potential use of the synthetic cannabinoid for improving the rate of wound closure as well as acting as an antiinflammatory agent, which could be used to enhance tissue engineering protocols aimed at cartilage repair.
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The effect of synthetic cannabinoids on wound healing of chondrocytes monolayers and pseudo 3D cartilage tissue. Effect of different concentrations of synthetic cannabinoids WIN55, 212-2, URB602 and HU-308 with and without their antagonists on wound healing of chondrocyte monolayers and pseudo 3D cartilage tissue.Abdeldayem, Ali I.A. January 2013 (has links)
Studies have been conducted to highlight the anti-inflammatory and immunosuppressive
properties of cannabinoids and also their potentials for cartilage repair and regeneration.
Various wound healing techniques can be used to investigate the mechanisms of
chondrocyte repair in monolayers or three dimensional tissue constructs. The effect of
different concentrations of the synthetic cannabinoids WIN55, 212-2 (WIN-2), URB602
and HU-308 with and without their antagonists on the wound healing of chondrocyte
monolayers was investigated using a simple scratch assay model. The three
cannabinoids were found to increase wound healing of chondrocyte monolayers, but at
different rates. WIN55, 212-2 at a concentration of 1μM had the highest effect of
increasing both migration and proliferation of chondrocytes cultured in a chondrogenic
media, which increased the rate of wound closure. It was also found that treating the
cells with 2μM of any of the cannabinoids lead to a decrease in cell proliferation and the
rate of wound closure. These findings were further investigated, by studying the effect
of WIN-2 on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2) expressed by
wounded chondrocyte monolayers. Moreover, expression of collagen type-I, collagen
type-II, fibronectin and S100 proteins were detected using immunofluorescence and
verified quantitatively using ELISA based techniques, following treatment with 1μM
and 2μM of WIN-2, for both 2D monolayers and 3D sheets. Treating chondrocytes with
1μM of WIN-2 significantly increased collagen type-II, fibronectin and S100, and
significantly reduced collagen type-I compared to control groups in monolayers and
chondrocyte cell sheets. On the other hand, both concentrations of WIN-2 significantly
reduced the expression of the inflammation markers NO, and MMP-2, in a dose
dependent manner. These findings highlight the potential use of the synthetic
cannabinoid for improving the rate of wound closure as well as acting as an antiinflammatory
agent, which could be used to enhance tissue engineering protocols aimed
at cartilage repair. / Egyptian Government
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The effect of WIN55, 212-2 on protein S100, matrix metalloproteinase-2 and nitric oxide expression of chondrocyte monolayerAbdeldayum, Ali I.A., Youseffi, Mansour, Sefat, Farshid, Genedy, Mohamed A., Abdul Jamil, M.M., Javid, F. 06 January 2017 (has links)
Yes / Studies have been conducted to highlight the anti-inflammatory and immunosuppressive properties of
synthetic cannabinoids as well as their potential for cartilage repair. Various wound healing techniques can be
used to investigate the mechanisms of chondrocyte repair in monolayers or three dimensional tissues constructs.
In this work the effect of WIN55, 212-2 (WIN-2) on nitric oxide (NO) and matrix metalloproteinase-2 (MMP-2)
expressed by wounded chondrocyte monolayers was investigated. Moreover, expression of collagen type-I and
type-II, fibronectin and S100 proteins were detected using immunofluorescence and quantitatively verified using
ELISA based techniques following treatment with 1 μM and 2 μM of WIN-2. Treating chondrocytes with 1 μM
of WIN-2 significantly increased expression of collagen type-II, fibronectin and S100, and significantly reduced
collagen type-I expressions as compared to the control groups. On the other hand, both concentrations of WIN-2
significantly reduced the expression of the inflammation markers NO and MMP-2 in a dose dependent manner.
These findings highlight the potential use of the synthetic cannabinoids for improving cartilage healing properties
as well as acting as an anti-inflammatory agent which could be used to enhance tissue engineering protocols
aimed at cartilage repair.
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COMBINED ANTIPROLIFERATIVE EFFECTS OF THE AMINOALKYLINDOLE WIN55,212-2 AND RADIATION IN BREAST CANCER CELLSEmery, Sean 10 January 2014 (has links)
The potential antitumor activity of mixed CB1/CB2 cannabinoid receptor agonists, such as the aminoalkylindole WIN55,212-2 (WIN2), has been extensively studied, but little information is available as to their potential interaction with conventional cancer therapies, such as ionizing radiation (IR). In the present work, we investigated the effects of WIN2 on the antiproliferative effects of radiation in human (MCF-7 and MDA-MB-231) and murine (4T1) breast cancer cells, as well as an immortalized human breast epithelial cell line (MCF-10A). WIN2 or radiation alone inhibited breast tumor growth, while the combination of WIN2 and radiation was more effective than either agent alone in breast cancer cells. WIN2 showed lower potency in MCF-10A cells than MCF-7 cells, but was still able to augment the effects of radiation at higher doses. The stereoisomer of WIN2, WIN55,212-3 (WIN3) failed to inhibit growth or potentiate the growth-inhibitory effects of radiation, indicating stereospecificity in all cell lines tested. The combination of WIN2 and IR was examined in vivo but the results were inconclusive. Interestingly, while other aminoalkylindoles, pravadoline and JWH-015, enhanced the antiproliferative effects of radiation, this was not the case for other synthetic cannabinoids (i.e., nabilone, CP55,940 and methanandamide) or phytocannabinoids (i.e., ∆9-tetrahydrocannabinol and cannabidiol). The antiproliferative actions of WIN2 were not ameliorated by CB1, CB2, TRPV1, or PPAR receptor antagonists, suggesting the possibility of a novel site of action. Studies utilizing sphingosine-1-phosphate (S1P) agonists and estradiol suggest that WIN2 interferes with S1P signaling in cell proliferation, but agonist stimulated [³⁵S]GTPγS binding assays show that this antagonism is not occurring at the level of S1P receptors. In addition, WIN2 did not alter radiation-induced DNA damage or the rate of DNA repair based on γH2AX staining. Treatment with WIN2 and radiation promoted both autophagy and senescence, but not apoptosis or necrosis. Time course studies combined with senescence and cell death data suggest that radiation-induced senescence, while WIN2 induced classical growth arrest and the WIN2/IR combination produced parallel mechanisms of both senescent growth arrest and classical growth arrest. Taken together, these findings raise the possibility that aminoalkylindole compounds targeting a novel site of action represents a potential strategy to augment the effectiveness of radiation treatment in breast cancer.
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