Cannabidiol (CBD) Protects Adipose-Derived Mesenchymal Stem Cells (ASCs) against Endoplasmic Reticulum Stress Development and Its Complications.

BACKGROUND: Recent studies suggested that individuals with metabolic disorders have altered function of adipocytes and adipose stem cell subpopulations, which impairs tissue homeostasis, promoting insulin resistance and diabetes development. The non-psychoactive phytocannabinoid CBD was found to modulate adipose tissue metabolism, however, its exact role in controlling ASCs' fate is still poorly understood. OBJECTIVES: This investigation aimed to elucidate whether pretreatment of ASCs with CBD can protect against ER stress development and maintain the cytophysiological properties of cells. METHODS: Human ASCs were cultured under control and adipogenic conditions. Prior to the experiments, cells in the experimental group were pretreated with CBD following the addition of an ER stress inducer-tunicamycin. After the experiments, the cells were subsequently tested for expression of the apoptotic, ER stress, and anti-inflammatory-related genes using RT-qPCR. Oxidative stress was analysed with flow cytometric assays. RESULTS: Cells pretreated with CBD displayed decreased apoptosis and enhanced proliferation rate. Additionally, the expression of pro-inflammatory cytokines and miRNAs was significantly reduced. The obtained results also demonstrated an obvious reduction in intracellular accumulated ROS and NO, as well as mitigated ER stress through the down-regulation of IRE-1, PERK, CHOP, and ATF6 transcripts upon CBD treatment. CONCLUSION: The presented data provide the evidence that CBD protects ASCs against ER stress development and its complications and, thus, offers new insights for the management of obesity through the regulation of adipose tissue dynamics.

Aminopropyltriethoxysilane (APTES)-Modified Nanohydroxyapatite (nHAp) Incorporated with Iron Oxide (IO) Nanoparticles Promotes Early Osteogenesis, Reduces Inflammation and Inhibits Osteoclast Activity.

Due to its increased prevalence, osteoporosis (OP) represents a great challenge to health care systems and brings an economic burden. To overcome these issues, treatment plans that suit the need of patients should be developed. One of the approaches focuses on the fabrication of personalized biomaterials, which can restore the balance and homeostasis of disease-affected bone. In the presented study, we fabricated nanometer crystalline hydroxyapatite (nHAp) and iron oxide (IO) nanoparticles stabilized with APTES and investigated whether they can modulate bone cell metabolism and be useful in the fabrication of personalized materials for OP patients. Using a wide range of molecular techniques, we have shown that obtained nHAp@APTES promotes viability and RUNX-2 expression in osteoblasts, as well as reducing activity of critical proinflammatory cytokines while inhibiting osteoclast activity. Materials with APTES modified with nHAp incorporated with IO nanoparticles can be applied to support the healing of osteoporotic bone fractures as they enhance metabolic activity of osteoblasts and diminish osteoclasts' metabolism and inflammation.

Artificial Mitochondrial Transfer (AMT) for the Management of Age-related Musculoskeletal Degenerative Disorders: An Emerging Avenue for Bone and Cartilage Metabolism Regulation.

Musculoskeletal system disorders are among the most common age-related conditions worldwide. All associated with a degeneration of the supporting tissues under pro-inflammatory micro- and macro-environments, the erosion of cartilage and later of bones, are the main hallmarks of these pathologies. Affected chondrocytes, osteoblasts and synoviocytes, that are all critical actors in the bone and cartilage defects exhibit mitochondrial dysfunction that develops immediately following cartilage and bone injury, and leads to tissue residing specific cell death, cartilage degeneration, bone erosion, and ultimately post-traumatic musculoskeletal degeneration. Herein, we would like to introduce a novel concept for bone and cartilage related defects treatment based on artificial transfer of exogeneous functional mitochondria (AMT). Particularly, we believe that because mitochondrial failure critically contributes to degenerative disorders onset and progression, replacing malfunctioning mitochondria with their healthy and functional counterparts can represent a novel, and effective therapeutic solution for the management of bone and cartilage related degenerative diseases. Artificial mitochondrial transfer (AMT) may reverse the failed metabolic status of musculoskeletal tissues cells and reduce bone and cartilage tissues defects by restoring mitochondrial bioenergetics.

The Hepatic Stellate Cells (HSTCs) and Adipose-derived Mesenchymal Stem Cells (ASCs) Axis as a Potential Major Driver of Metabolic Syndrome - Novel Concept and Therapeutic Implications.

Herein, we would like to introduce a novel concept for the prevention and treatment of metabolic syndrome, which is based on molecular relationship between liver and adipose tissue. Particularly, we believe, that unravelling the molecular crosstalk between hepatokines and adipokines will allow to better understand the pathophysiology of metabolic diseases and allow to develop novel, effective therapeutic solutions against obesity and metabolic syndrome. Inter-organ communication on the level of stem progenitor cells-hepatic stellate cells (HSTCs) and adipose-derived progenitors (ASCs) could represents a key mechanism involved in controlling glucose tolerance as well as insulin sensitivity.

Novel Nanohydroxyapatite (nHAp)-Based Scaffold Doped with Iron Oxide Nanoparticles (IO), Functionalized with Small Non-Coding RNA (miR-21/124) Modulates Expression of Runt-Related Transcriptional Factor 2 and Osteopontin, Promoting Regeneration of Osteoporotic Bone in Bilateral Cranial Defects in a Senescence-Accelerated Mouse Model (SAM/P6). PART 2.

PURPOSE: Healing of osteoporotic defects is challenging and requires innovative approaches to elicit molecular mechanisms promoting osteoblasts-osteoclasts coupling and bone homeostasis. METHODS: Cytocompatibility and biocompatibility of previously characterised nanocomposites, i.e Ca5(PO4)3OH/Fe3O4 (later called nHAp/IO) functionalised with microRNAs (nHAp/IO@miR-21/124) was tested. In vitro studies were performed using a direct co-culture system of MC3T3-E1 pre-osteoblast and 4B12 pre-osteoclasts. The analysis included determination of nanocomposite influence on cultures morphology (confocal imaging), viability and metabolic activity (Alamar Blue assay). Pro-osteogenic signals were identified at mRNA, miRNA and protein level with RT-qPCR, Western blotting and immunocytochemistry. Biocompatibility of biomaterials was tested using bilateral cranial defect performed on a senescence-accelerated mouse model, ie SAM/P6 and Balb/c. The effect of biomaterial on the process of bone healing was monitored using microcomputed tomography. RESULTS: The nanocomposites promoted survival and metabolism of bone cells, as well as enhanced functional differentiation of pre-osteoblasts MC3T3-E1 in co-cultures with pre-osteoclasts. Differentiation of MC3T3-E1 driven by nHAp/IO@miR-21/124 nanocomposite was manifested by improved extracellular matrix differentiation and up-regulation of pro-osteogenic transcripts, ie late osteogenesis markers. The nanocomposite triggered bone healing in a cranial defect model in SAM/P6 mice and was replaced by functional bone in Balb/c mice. CONCLUSION: This study demonstrates that the novel nanocomposite nHAp/IO can serve as a platform for therapeutic miRNA delivery. Obtained nanocomposite elicit pro-osteogenic signals, decreasing osteoclasts differentiation, simultaneously improving osteoblasts metabolism and their transition toward pre-osteocytes and bone mineralisation. The proposed scaffold can be an effective interface for in situ regeneration of osteoporotic bone, especially in elderly patients.

Laurus nobilis ethanolic extract attenuates hyperglycemia and hyperinsulinemia-induced insulin resistance in HepG2 cell line through the reduction of oxidative stress and improvement of mitochondrial biogenesis - Possible implication in pharmacotherapy.

The aim of this study was to establish the potential effect of Laurus nobilis ethanolic extract on improving insulin sensitivity and protecting liver cells from apoptosis, mitochondrial dysfunction, oxidative stress (OS), and inflammation; all of which considered as major alterations occurring during insulin resistance (IR) as well as diabetes onset, in hyperinsulinemic and hyperglycemic-induced HepG2 cell line. Thereby, L. nobilis ethanolic extract has been first chemically characterized using LC-MS/MS technique. Subsequently, HepG2 cells were pre-treated with an optimal concentration of L. nobilis ethanolic extract for 24 h, and then, subjected to 30 mM D-glucose and 500 nM insulin mixture for another 24 h in order to induce hyperinsulinemia and hyperglycaemia (HI/HG) status. Several parameters such as biocompatibility, hepatotoxicity, reactive oxygen species (ROS), mitochondrial transmembrane potential, dynamics, and metabolism, multicaspase activity, glucose uptake, in addition to genes and proteins expression levels were investigated. The obtained results showed that the bioactive extract of Laurus nobilis increased the number of living cells and their proliferation rate, significantly attenuated apoptosis by modulating pro-apoptotic pathways (p21, p53 and Bax genes), allowed a relative normalization of caspases-activity, and decreased the expression of inflammatory markers including c-Jun, NF-κB and Tlr4 transcripts. L. Nobilis ethanolic extract reduced considerably total intracellular ROS levels in challenged HepG2 cells, and regulated the mitochondrial OXPHOS pathway, demonstrating the potential antioxidant effect of the plant. Ethanolic plant extract increased insulin sensitivity, since an elevated expression of master transcripts responsible for insulin sensitivity including IRS1, IRS2, INSR was found. Taken together, obtained data suggest that L. nobilis ethanolic extract offers new insights in the development of potential antioxidant, insulin sensitizing as well as hepatoprotective drugs.

Hyoscyamus albus nortropane alkaloids reduce hyperglycemia and hyperinsulinemia induced in HepG2 cells through the regulation of SIRT1/NF-kB/JNK pathway.

BACKGROUND: Chronic superphysiological glucose and insulin concentrations are known to trigger several tissue and organ failures, including insulin resistance, oxidative stress and chronic low-grade inflammation. Hence, the screening for molecules that may counteract such conditions is essential in current existing therapeutic strategies, thereby the use of medicinal plant derivatives represents a promising axis in this regard. METHODS: In this study, the effect of a selected traditional medicinal plant, Hyoscyamus albus from which, calystegines have been isolated, was investigated in an experimental model of hyperinsulinemia and hyperglycemia induced on HepG2 cells. The mRNA and protein expression levels of different insulin signaling, gluconeogenic and inflammatory pathway- related molecules were examined. Additionally, cell viability and apoptosis, oxidative stress extent and mitochondrial dysfunctions were assayed using flow cytometric and qRT-PCR techniques. RESULTS: Treatment of IR HepG2 cells with calystegines strongly protected the injured cells from apoptosis, oxidative stress and mitochondrial integrity loss. Interestingly, nortropane alkaloids efficiently regulated the impaired glucose metabolism in IR HepG2 cells, through the stimulation of glucose uptake and the modulation of SIRT1/Foxo1/G6PC/mTOR pathway, which is governing the hepatic gluconeogenesis. Furthermore, the alkaloidal extract restored the defective insulin signaling pathway, mainly by promoting the expression of Insr at the mRNA and protein levels. What is more, treated cells exhibited significant mitigated inflammatory response, as evidenced by the modulation and the regulation of the NF- κB/JNK/TLR4 axis and the downstream proinflammatory cytokines recruitment. CONCLUSION: Overall, the present investigation demonstrates that calystegines from Hyoscyamus albus provide cytoprotection to the HepG2 cells against insulin/glucose induced insulin resistance and apoptosis due to the regulation of SIRT1/Foxo1/G6PC/mTOR and NF-κB/JNK/TLR4 signaling pathways. Video Abstract.

Sex Hormone Binding Globulin (SHBG) Mitigates ER Stress in Hepatocytes In Vitro and Ex Vivo.

Despite multiple research studies regarding metabolic syndrome and diabetes, the full picture of their molecular background and pathogenies remains elusive. The latest studies revealed that sex hormone-binding globulin (SHBG)-a serum protein released mainly by the liver-may participate in metabolic dysregulation, as its low serum level correlates with a risk for obesity, metabolic syndrome, and diabetes. Yet, the molecular phenomenon linking SHBG with these disorders remains unclear. In the presented study, we investigate how exogenous SHBG affects metabolically impaired hepatocytes with special attention to endoplasmic reticulum stress (ER stress) and lipid metabolism both in vitro and ex vivo. For that reason, palmitate-treated HepG2 cells and liver tissue samples collected post mortem were cultured in the presence of 50 nM and 100 nM SHBG. We found that SHBG protects against ER stress development and its progression. We have found that SHBG decreased the expression levels of inositol-requiring enzyme 1 (IRE1α), activating transcription factor 6 (ATF6), DNA damage-inducible transcript 3 (CHOP), and immunoglobulin heavy chain-binding protein (BIP). Furthermore, we have shown that it regulates lipolytic gene expression ex vivo. Additionally, herein, we deliver a novel large-animal model to study SHBG in translational research. Our data provide new insights into the cellular and molecular mechanisms by which SHBG modulates hepatocyte metabolism and offer a new experimental approach to study SHBG in human diseases.

SIRT1+ Adipose Derived Mesenchymal Stromal Stem Cells (ASCs) Suspended in Alginate Hydrogel for the Treatment of Subchondral Bone Cyst in Medial Femoral Condyle in the Horse. Clinical Report.

Stem cell based therapy are now commonly applied in human and veterinary medical practice especially in orthopaedics. Mesenchymal stromal stem cells isolated from adipose tissue (ASC) are first choice option due to relatively non-invasive and safe procedure of tissue harvesting. However, ASC therapeutic potential strongly rely on patients general health condition, age and life-style. For that reason, to enhance therapeutic potential of cells, they are modified in vitro using different approaches. Previous studies have shown, that ASC treated with resveratrol, herein called SIRT+, are characterised by decreased senescence, increased proliferation rate and improved clinical outcome in autologous therapies. Herein, SIRT + cells in alginate hydrogel were applied to 5 years old warm breed mare was clinically evaluated due to the left hind lameness due to subchondral bone cyst. The therapeutic effect was assessed by the analysis of lameness score and radiological evaluation. This case report demonstrates the therapeutic potential of SIRT + cells in the treatment of orthopaedics disorders in horses as complete bone remodelling occurred after therapy and horse came back to training.

Age-dependent impairment of adipose-derived stem cells isolated from horses.

BACKGROUND: Progressive loss of cell functionality caused by an age-related impairment in cell metabolism concerns not only mature specialized cells but also its progenitors, which significantly reduces their regenerative potential. Adipose-derived stem cells (ASCs) are most commonly used in veterinary medicine as an alternative treatment option in ligaments and cartilage injuries, especially in case of high-value sport horses. Therefore, the main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young (< 5), middle-aged (5-15), and old (> 15 years old). METHODS: ASCs were isolated from three age-matched horse groups using an enzymatic method. Molecular changes were assessed using qRT-PCR, ELISA and western blot methods, flow cytometry-based system, and confocal and scanning electron microscopy. RESULTS: Our findings showed that ASCs derived from the middle-aged and old groups exhibited a typical senescence phenotype, such as increased percentage of G1/G0-arrested cells, binucleation, enhanced ß-galactosidase activity, and accumulation of γH2AX foci, as well as a reduction in cell proliferation. Moreover, aged ASCs were characterized by increased gene expression of pro-inflammatory cytokines and miRNAs (interleukin 8 (IL-8), IL-1ß, tumor necrosis factor α (TNF-α), miR-203b-5p, and miR-16-5p), as well as apoptosis markers (p21, p53, caspase-3, caspase-9). In addition, our study revealed that the protein level of mitofusin 1 (MFN1) markedly decreased with increasing age. Aged ASCs also displayed a reduction in mRNA levels of genes involved in stem cell homeostasis and homing, like TET-3, TET-3 (TET family), and C-X-C chemokine receptor type 4 (CXCR4), as well as protein expression of DNA methyltransferase (DNMT1) and octamer transcription factor 3/4 (Oct 3/4). Furthermore, we observed a higher splicing ratio of XBP1 (X-box binding protein 1) mRNA, indicating elevated inositol-requiring enzyme 1 (IRE-1) activity and, consequently, increased endoplasmic reticulum (ER) stress. We also observed reduced levels of glucose transporter 4 (GLUT-4) and insulin receptor (INSR) which indicated impaired insulin sensitivity. CONCLUSIONS: Obtained data suggest that ASCs derived from horses older than 5 years old exhibited several molecular alternations which markedly limit their regenerative capacity. The results provide valuable information that allows for a better understanding of the molecular events occurring in ASCs in the course of aging and may help to identify new potential drug targets to restore their regenerative potential.

Promotion through external magnetic field of osteogenic differentiation potential in adipose-derived mesenchymal stem cells: Design of polyurethane/poly(lactic) acid sponges doped with iron oxide nanoparticles.

Recently, iron oxide nanoparticles (IONPs) have gathered special attention in regenerative medicine. Owing to their magnetic and bioactive properties, IONPs are utilized in the fabrication of novel biomaterials. Yet, there was no report regarding thermoplastic polyurethane (TPU) and poly(lactic acid) (PLA) polymer doped with IONPs on osteogenic differentiation of mesenchymal stem cells. Thus the objectives of presented study was to: (a) fabricate magnetic TPU + PLA sponges doped with iron (III) oxide Fe2 O3 nanoparticles; (b) investigate the effects of biomaterial and its exposition to static magnetic field (MF) on osteogenic differentiation, proliferation, and apoptosis in adipose-derived mesenchymal stem cells (ASCs). TPU + PLA sponges were prepared using solvent casting technique while incorporation of the Fe2 O3 nanoparticles was performed with solution cast method. RT-PCR was applied to evaluate expression of osteogenic-related genes and integrin's in cells cultured on fabricated materials with or without the stimulation of static MF. MF stimulation enhanced the expression of osteopontin and collagen type I while decreased expression of bone morphogenetic protein 2 in tested magnetic materials-TPU + PLA/1% Fe2 O3 and TPU + PLA/5% Fe2 O3 . Therefore, TPU + PLA sponges doped with IONPs and exposure to MF resulted in improved osteogenic differentiation of ASC.

Microvesicles isolated from 5-azacytidine-and-resveratrol-treated mesenchymal stem cells for the treatment of suspensory ligament injury in horse-a case report.

BACKGROUND: In athlete horses, suspensory ligament (SL) injuries are the most common cause of lameness. Healing of SL injury is still problematic, and even proper rehabilitation and pharmacological therapy do not guarantee returning to the initial performance level. In our previous studies, we have shown that a combination of 5-azacytidine (AZA) and resveratrol (RES) exerts beneficial, rejuvenating effects on metabolic syndrome derived adipose-derived stem cells (ASCs). Thus, in the presented research, we investigate whether not only rejuvenated ASC but also microvesicles (MVsAZA/RES) secreted by them possess enhanced regenerative properties in SL injury. METHODS: In the presented study, a 6-year-old Dutch Warmblood gelding, working in jumping, was diagnosed with SL injury using ultrasonography, Doppler, real-time elastography and thermography. As a therapeutic strategy, the affected animal was treated with extracellular microvesicles derived from ASC treated with the combination of 5-azacytydine (AZA) and resveratrol (RES) (MVsAZA/RES). RESULTS: First, anti-apoptotic effects of MVsAZA/RES were tested in co-culture with metabolic syndrome derived ASC. The proliferation of cells and expression of pro-apoptotic genes were investigated. Then, MVsAZA/RES were injected directly into the injured SL of the Dutch Warmblood gelding. In vitro assays revealed that MVsAZA/RES enhance the proliferation of ASC and exert an anti-apoptotic effect. In the affected horse, the application of MVsAZA/RES resulted in increased lesion filling and improvement of angiogenesis and elasticity in injured tissue. CONCLUSIONS: As MVsAZA/RES mimic several of the biological actions exerted by ASC, they have become an alternative for stem cell-based therapies and can be effectively applied for the treatment of SL injury in horses.