Exosomal miR-21-5p derived from multiple myeloma cells promote renal epithelial-mesenchymal transition through targeting TGF-ß/SMAD7 signalling pathway.

The prognosis of multiple myeloma (MM) patients combined with renal insufficiency is poor. Renal fibrosis is an important pathological cause for MM patients combined with renal insufficiency. It is reported that epithelial-mesenchymal transition (EMT) of renal proximal tubular epithelial cells is an important mechanism in renal fibrosis. We speculated that EMT might play an important role in the renal insufficiency of MM with unclear mechanism. MM cells derived exosomes could affect the function of targeted cells by delivering microRNAs (miRNAs). Literature has shown that the expression of miR-21 is closely related to EMT. In this research, we found that co-culture of HK-2 cells (human renal proximal tubular epithelial cells) and exosomes derived from MM cells promoted the EMT of HK-2 cells, resulting in the down-regulation of epithelial-related marker (E-cadherin), and up-regulation of stroma-related marker (Vimentin). Meanwhile, the expression of SMAD7, one of the downstream targets in the TGF-ß signalling pathway, was suppressed and the expression of TGF-ß was increased. After transfecting the inhibitor of miR-21 in MM cells, the expression of miR-21 in exosomes secreted by MM cells was significantly decreased, and the co-culture of these treated exosomes and HK-2 cells inhibited the EMT of HK-2 cells. In conclusion, these findings showed that exosomal miR-21 derived from MM cells could promote renal EMT through targeting TGF-ß/SMAD7 signalling pathway.

Low UVB Fluences Augment Microvesicle Particle Generation in Keratinocytes.

Microvesicle particles (MVP) are bioactive subcellular particles which have been recently implicated in the keratinocyte response to many environmental stressors including ultraviolet B radiation (UVB). Previous studies have demonstrated that UVB generates high levels of MVP in a process involving the platelet-activating factor receptor (PAFR) and the enzyme acid sphingomyelinase (aSMase). Yet the fluences of UVB needed to generate MVP are usually above those commonly encountered. Using models including human epithelial cell lines in vitro, human skin explants ex vivo and murine studies in vivo, the present studies indicate that pretreatment of epithelial cells/skin with PAFR agonist/phorbol ester can synergize with low fluences of UVB to generate high levels of MVP. These studies indicate the possibility that MVP could play a role in combinatorial pathologic processes involving UVB.

Evidence for Systemic Reactive Oxygen Species in UVB-mediated Microvesicle Formation.

Recent studies have implicated subcellular microvesicle particles (MVP) in the ability of ultraviolet B radiation to exert both local and systemic effects. Indeed, UVB generates MVP (UVB-MVP) in human skin and systemically following phototherapy. The current studies were designed to test the hypothesis that the ability of UVB to generate MVP was dependent upon reactive oxygen species (ROS). To that end, we tested urine samples from subjects undergoing UVB phototherapy for the presence of isoprostanes as well as the oxidized guanosine derivative 8OHdG. We also conducted a clinical study in which volar forearms of subjects were treated with localized UVB and erythema/MVP measured. The same cohort was then treated with 7 days of vitamin C (2 g day-1 ) and vitamin E (1000 IU day-1 ), and UVB-induced MVPs tested on the contralateral forearm. Urine specimens from subjects undergoing phototherapy were found to have increased levels of isoprostanes and 8OHdG, with maximal levels noted 8-16 h post-treatment. Treatment with antioxidant vitamins resulted in diminished UVB-generated skin MVP to baseline levels. These studies suggest that whole-body UVB generates a systemic pro-oxidative response, and that antioxidants can attenuate localized skin UVB-MVPs.

Acute ethanol exposure stimulates microvesicle particle generation in keratinocytes.

Ethanol has been demonstrated to exert profound effects upon cells and tissues via multiple mechanisms. One recently appreciated means by which cells can communicate with other cells is via the production and release of extracellular vesicles. Though smaller exosomes have been demonstrated to be released in response to ethanol exposure, the ability of ethanol to modulate the generation and release of larger microvesicle particles (MVP) is lesser studied. The present studies examined the ability of exogenous ethanol to generate MVP with a focus on skin cells. Acute ethanol exposure resulted in augmented MVP release in keratinocytes and in the skin and blood of mice. Unlike other stimuli such as ultraviolet B radiation or thermal burn injury, ethanol-mediated MVP release was independent of the Platelet-activating Factor receptor (PAFR). However, ethanol pretreatment was found to augment thermal burn injury-induced MVP in a PAFR-dependent manner. These studies provide a novel mechanism for ethanol-mediated effects, that could be relevant in the significant toxicity associated with thermal burn injury in the setting of alcohol intoxication.

4R-cembranoid protects neuronal cells from oxygen-glucose deprivation by modulating microglial cell activation.

As major immune responsive cells in the central nervous system (CNS), activated microglia can present pro-inflammatory M1 phenotype aggravating the neuronal injury or anti-inflammatory M2 phenotype providing neuroprotection and promoting neuronal survival in neurodegenerative diseases. In this study, we demonstrated that a compound, 4R-cembranoid (4R, 1S, 2E, 4R, 6R,-7E, 11E-2, 7, 11-cembratriene-4, 6-diol cembranoids) promoted M2 phenotype while attenuated M1 phenotype in N9 cells, a microglial cell line. Following Lipopolysaccharides (LPS) or Oxygen-glucose deprivation (OGD) treatment, the N9 cells treated by 1 µM 4R showed an increased Arginase-1 (Arg1, a M2 marker) expression and a reduced inducible nitric oxide synthase (iNOS, M1 marker) expression. In addition, the conditioned medium of 4R-treated post-OGD N9 cells protected neuro2a cells, a neuronal cell line, from OGD-induced injury. The viability of neuro2a cells in OGD condition was increased by 54.5% after treated with the conditioned medium of 4R-treated post-OGD N9 cells. Furthermore, we demonstrated the protective mechanism of 4R was associated with a decreased TNF-α release and an increased IL-10 release from N9 cells. In conclusion, our study demonstrated that the neuroprotective effects of 4R were through the regulation of microglial activation by promoting the protective M2 activation and inhibiting the damaging M1 activation. Therefore, the findings of this study suggest that 4R could be a promising lead structure for the development of drugs for the treatment of ischemic stroke and other neurodegenerative diseases with an inflammatory component involved.

Keratinocyte-derived microvesicle particles mediate ultraviolet B radiation-induced systemic immunosuppression.

A complete carcinogen, ultraviolet B (UVB) radiation (290-320 nm), is the major cause of skin cancer. UVB-induced systemic immunosuppression that contributes to photocarcinogenesis is due to the glycerophosphocholine-derived lipid mediator platelet-activating factor (PAF). A major question in photobiology is how UVB radiation, which only absorbs appreciably in the epidermal layers of skin, can generate systemic effects. UVB exposure and PAF receptor (PAFR) activation in keratinocytes induce the release of large numbers of microvesicle particles (MVPs; extracellular vesicles ranging from 100 to 1000 nm in size). MVPs released from skin keratinocytes in vitro in response to UVB (UVB-MVPs) are dependent on the keratinocyte PAFR. Here, we used both pharmacologic and genetic approaches in cells and mice to show that both the PAFR and enzyme acid sphingomyelinase (aSMase) were necessary for UVB-MVP generation. Our discovery that the calcium-sensing receptor is a keratinocyte-selective MVP marker allowed us to determine that UVB-MVPs leaving the keratinocyte can be found systemically in mice and humans following UVB exposure. Moreover, we found that UVB-MVPs contained bioactive contents including PAFR agonists that allowed them to serve as effectors for UVB downstream effects, in particular UVB-mediated systemic immunosuppression.

C6-ceramide treatment inhibits the proangiogenic activity of multiple myeloma exosomes via the miR-29b/Akt pathway.

BACKGROUND: The increased bone marrow angiogenesis is involved in the progression of multiple myeloma (MM) with the underlying mechanism poorly understood. Cancer-released exosomes could play an important role in the pathological angiogenesis through exosomal microRNAs (miRs) delivery. It is reported that miR-29b played an important role in regulating the tumor angiogenesis. METHODS: In this study, we explored the role of C6-ceramide (C6-cer, a Ceramide pathway activator) in the angiogenic effect of MM exosomes and its potential mechanism. MM cells (OPM2 and RPMI-8226) treated with C6-cer were studied for its effects on the endothelial cell (EC) functions. RESULTS: Our results showed that exosomes released from MM cells treated by C6-cer (ExoC6-cer) significantly inhibited the proliferation, migration and tube formation of ECs. For mechanism studies, we found that the level of miR-29b was increased in ECs treated by ExoC6-cer, while mRNA and protein expressions of Akt3, PI3K and VEGFA were decreased in ECs, indicating the involvement of Akt pathway. Furthermore, downregulation of miR-29b by inhibitor administration could prevent the ExoC6-cer-induced cell proliferation, migration and angiogenesis of ECs, accompanied with the increased expressions of Akt3, PI3K and VEGFA. CONCLUSIONS: Collectively, our data suggest that ExoC6-cer-mediated miR-29b expression participates in the progression of MM through suppressing the proliferation, migration and angiogenesis of ECs by targeting Akt signal pathway.

Thermal Burn Injury Generates Bioactive Microvesicles: Evidence for a Novel Transport Mechanism for the Lipid Mediator Platelet-Activating Factor (PAF) That Involves Subcellular Particles and the PAF Receptor.

Thermal burn injuries are an important environmental stressor that can result in considerable morbidity and mortality. The exact mechanism by which an environmental stimulus to skin results in local and systemic effects is an area of active research. One potential mechanism to allow skin keratinocytes to disperse bioactive substances is via microvesicle particles, which are subcellular bodies released directly from cellular membranes. Our previous studies have indicated that thermal burn injury of the skin keratinocyte in vitro results in the production of the lipid mediator platelet-activating factor (PAF). The present studies demonstrate that thermal burn injury to keratinocytes in vitro and human skin explants ex vivo, and mice in vivo generate microvesicle particles. Use of pharmacologic and genetic tools indicates that the optimal release of microvesicles is dependent upon the PAF receptor. Of note, burn injury-stimulated microvesicle particles do not carry appreciable protein cytokines yet contain high levels of PAF. These studies describe a novel mechanism involving microvesicle particles by which a metabolically labile bioactive lipid can travel from cells in response to environmental stimuli.

Gemcitabine Induces Microvesicle Particle Release in a Platelet-Activating Factor-Receptor-Dependent Manner via Modulation of the MAPK Pathway in Pancreatic Cancer Cells.

Studies, including ours, have shown that pro-oxidative stressors, such as chemotherapeutic agents, generate oxidized lipids with agonistic platelet-activating factor (PAF) activity. Importantly, recent reports have implicated that these PAF-agonists are transported extracellularly via microvesicle particles (MVPs). While the role of PAF-receptor (PAF-R) has been implicated in mediating chemotherapy effects, its significance in chemotherapy-mediated MVP release in pancreatic cancer has not been studied. The current studies determined the functional significance of PAF-R in gemcitabine chemotherapy-mediated MVP release in human pancreatic cancer cells. Using PAF-R-expressing (PANC-1) and PAF-R-deficient (Hs766T) cells, we demonstrate that gemcitabine induces MVP release in a PAF-R-dependent manner. Blocking of PAF-R via PAF-R antagonist or inhibition of MVP generation via inhibitor of acid sphingomyelinase (aSMase) enzyme, significantly attenuated gemcitabine-mediated MVP release from PANC-1 cells, however, exerted no effects in Hs766T cells. Notably, MVPs from gemcitabine-treated PANC-1 cells, contained a measurable amount of PAF-agonists. Mechanistically, pretreatment with ERK1/2 or p38 inhibitors significantly abrogated gemcitabine-mediated MVP release, indicating the involvement of mitogen-activated protein kinase (MAPK) pathway in PAF-R-dependent gemcitabine-mediated MVP release. These findings demonstrate the significance of PAF-R in gemcitabine-mediated MVP release, as well as the rationale of evaluating PAF-R targeting agents with gemcitabine against pancreatic cancer.

Systemic Platelet-Activating Factor-Receptor Agonism Enhances Non-Melanoma Skin Cancer Growth.

Platelet-activating factor-receptor (PAF-R) agonists are pleiotropic lipid factors that influence multiple biological processes, including the induction and resolution of inflammation as well as immunosuppression. PAF-R agonists have been shown to modulate tumorigenesis and/or tumor growth in various skin cancer models by suppressing either cutaneous inflammation and/or anti-tumoral adaptive immunity. We have previously shown that a chronic systemic PAF-R agonist administration of mice enhances the growth of subcutaneously implanted melanoma tumors. Conversely, chronic topical applications of a PAF-R agonist suppressed non-melanoma skin cancer (NMSC) in a topical chemical carcinogenesis model (dimethylbenz[a]anthracene/phorbol 12-myristate 13-acetate (DMBA/PMA)) in-part via anti-inflammatory effects. These results indicate that the context of PAF-R agonist exposure via either chronic cutaneous or systemic administration, result in seemingly disparate effects on tumor promotion. To further dissect the contextual role of PAF-R agonism on tumorigenesis, we chronically administered systemic PAF-R agonist, carbamoyl-PAF (CPAF) to mice under a cutaneous chemical carcinogenesis protocol, recently characterized to initiate both NMSC and melanocytic nevus formation that can progress to malignant melanoma. Our results showed that while systemic CPAF did not modulate melanocytic nevus formation, it enhanced the growth of NMSC tumors.

UVB-generated Microvesicle Particles: A Novel Pathway by Which a Skin-specific Stimulus Could Exert Systemic Effects.

Ultraviolet B radiation (UVB) exerts profound effects on human skin. Much is known regarding the ability of UVB to generate a plethora of bioactive agents ranging from cytokines and other bioactive proteins, lipid mediators and microRNAs. It is presumed that these agents are in large part responsible for the effects of UVB, which is only absorbed appreciably in the epidermis. However, the exact mechanism by which these bioactive agents can leave the epidermis are as yet unclear. This review addresses the potential role of microvesicle particles (MVP) as UVB signaling agents through transmitting biologic mediators. New data are provided that UVB treatment of human skin explants also generates MVP production. We hypothesize that UVB production of MVPs (UVB-MVP) could serve this important function of transmitting keratinocyte-derived bioactive agents. Moreover, we propose that UVB-MVP formation involves the lipid mediator platelet-activating factor. This novel pathway has the potential to be exploited pharmacologically to modulate UVB effects.

Angiotensin-(1-7) counteracts angiotensin II-induced dysfunction in cerebral endothelial cells via modulating Nox2/ROS and PI3K/NO pathways.

Angiotensin (Ang) II, the main effector of the renin-angiotensin system, has been implicated in the pathogenesis of vascular diseases. Ang-(1-7) binds to the G protein-coupled Mas receptor (MasR) and can exert vasoprotective effects. We investigated the effects and underlying mechanisms of Ang-(1-7) on Ang II-induced dysfunction and oxidative stress in human brain microvascular endothelial cells (HbmECs). The pro-apoptotic activity, reactive oxygen species (ROS) and nitric oxide (NO) productions in HbmECs were measured. The protein expressions of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2), serine/threonine kinase (Akt), endothelial nitric oxide synthase (eNOS) and their phosphorylated forms (p-Akt and p-eNOS) were examined by western blot. MasR antagonist and phosphatidylinositol-3-kinase (PI3K) inhibitor were used for receptor/pathway verification. We found that Ang-(1-7) suppressed Ang II-induced pro-apoptotic activity, ROS over-production and NO reduction in HbmECs, which were abolished by MasR antagonist. In addition, Ang-(1-7) down-regulated the expression of Nox2, and up-regulated the ratios of p-Akt/Akt and its downstream p-eNOS/eNOS in HbmECs. Exposure to PI3K inhibitor partially abrogated Ang-(1-7)-mediated protective effects in HbmECs. Our data suggests that Ang-(1-7)/MasR axis protects HbmECs from Ang II-induced dysfunction and oxidative stress via inhibition of Nox2/ROS and activation of PI3K/NO pathways.

Cellular Membrane Microparticles: Potential Targets of Combinational Therapy for Vascular Disease.

Vascular disease constitutes the leading health problem throughout the entire world. Current therapies for vascular disease mainly rely on comprehensive strategies including control of risk factors, vascular interventions and conventional supportive treatments. To improve the preventive and therapeutic efficacies of current approaches, novel combinational therapies are required. Microparticles (MPs) are small membrane vesicles derived from cells undergoing stress, activation or apoptosis. They carry the characteristics of their parent cells, enabling them to serve as potential biomarkers for various diseases. Of note, MPs also have been shown to mediate cell communications through transferring membrane proteins, phospholipids and RNAs from their parent cells to recipient cells. Recent novel approaches have started to reveal the functions of MPs. In this review, we summarize the general concepts and the latest research progress in MPs. And the potential of MPs as novel targets of combinational therapy for vascular disease will be discussed.