Platelet rich plasma (PRP) induces chondroprotection via increasing autophagy, anti-inflammatory markers, and decreasing apoptosis in human osteoarthritic cartilage.

OBJECTIVES: Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration. METHODS: Osteoarthritic chondrocytes were co-cultured with an increasing concentration of PRP obtained from healthy donors. The effect of PRP on the proliferation of chondrocytes was performed using cell counting and WST8 proliferation assays. Autophagy, apoptosis and intracellular level of IL-4, IL-10, and IL-13 were determined using flow cytometry analyses. Autophagy markers BECLIN and LC3II were also determined using quantitative polymerase chain reaction (qPCR). qPCR and ELISA were used to measure the expression of ADAMDTS-5, MMP3, MMP13, TIMP-1-2-3, aggregan, Collagen type 2, TGF-ß, Cox-2, Il-6, FOXO1, FOXO3, and HIF-1 in tissues and co-cultured media. RESULTS: PRP increased significantly the proliferation of chondrocytes, decreased apoptosis and increased autophagy and its markers along with its regulators FOXO1, FOXO3 and HIF-1 in osteoarthritic chondrocytes. Furthermore, PRP caused a dose-dependent significant decrease in MMP3, MMP13, and ADAMTS-5, IL-6 and COX-2 while increasing TGF-ß, aggregan, and collagen type 2, TIMPs and intracellular IL-4, IL-10, IL-13. CONCLUSION: These results suggest that PRP could be a potential therapeutic tool for the treatment of OA.

Molecular targeted therapy: A new avenue in glioblastoma treatment.

Glioblastoma, also referred to as glioblastoma multiforme (GBM), is grade IV astrocytoma characterized by being fast-growing and the most aggressive brain tumor. In adults, it is the most prevalent type of malignant brain tumor. Despite the advancements in both diagnosis tools and therapeutic treatments, GBM is still associated with poor survival rate without any statistically significant improvement in the past three decades. Patient's genome signature is one of the key factors causing the development of this tumor, in addition to previous radiation exposure and other environmental factors. Researchers have identified genomic and subsequent molecular alterations affecting core pathways that trigger the malignant phenotype of this tumor. Targeting intrinsically altered molecules and pathways is seen as a novel avenue in GBM treatment. The present review shed light on signaling pathways and intrinsically altered molecules implicated in GBM development. It discussed the main challenges impeding successful GBM treatment, such as the blood brain barrier and tumor microenvironment (TME), the plasticity and heterogeneity of both GBM and TME and the glioblastoma stem cells. The present review also presented current advancements in GBM molecular targeted therapy in clinical trials. Profound and comprehensive understanding of molecular participants opens doors for innovative, more targeted and personalized GBM therapeutic modalities.

Activation of autophagy following [HuArgI (Co)-PEG5000]-induced arginine deprivation mediates cell death in colon cancer cells.

Deregulating cellular energetics by reprogramming metabolic pathways, including arginine metabolism, is critical for cancer cell onset and survival. Drugs that target the specific metabolic requirements of cancer cells have emerged as promising targeted cancer therapeutics. In this study, we investigate the therapeutic potential of targeting colon cancer cells using arginine deprivation induced by a pegylated cobalt-substituted recombinant human Arginase I [HuArgI (Co)-PEG5000]. Four colon cancer cell lines were tested for their sensitivity to [HuArgI (Co)-PEG5000] as well as for their mechanism of cell death following arginine deprivation. All four cell lines were sensitive to arginine deprivation induced by [HuArgI (Co)-PEG5000]. All cells expressed ASS1 and were rescued from arginine deprivation-induced cytotoxicity by the addition of excess L-citrulline, indicating they are partially auxotrophic for arginine. Mechanistically, cells treated with [HuArgI (Co)-PEG5000] were negative for AnnexinV and lacked caspase activation. Further investigation revealed that arginine deprivation leads to a marked and prolonged activation of autophagy in both Caco-2 and T84 cell lines. Finally, we show that [HuArgI (Co)-PEG5000] causes cell death by sustained activation of autophagy as evidenced by the decrease in cell cytotoxicity upon treatment with chloroquine, an autophagy inhibitor. Altogether, these data demonstrate that colon cancer cells are partially auxotrophic for arginine and sensitive to [HuArgI (Co)-PEG5000]-induced arginine deprivation. They also show that the activation of autophagy does not play protective roles but rather, induces cytotoxicity and leads to cell death.

Dysregulation of Rho GTPases in orofacial cleft patients-derived primary cells leads to impaired cell migration, a potential cause of cleft/lip palate development.

Cleft lip and/or palate are a split in the lip, the palate or both. This results from the inability of lip buds and palatal shelves to properly migrate and assemble during embryogenesis. By extracting primary cells from a cleft patient, we aimed at offering a better understanding of the signaling mechanisms and interacting molecules involved in the lip and palate formation and fusion. With Rho GTPases being indirectly associated with cleft occurrence, we investigated the role of the latter in both. First, whole exome sequencing was conducted in a patient with cleft lip and palate. Primary fibroblastic cells originating from the upper right gingiva region were extracted and distinct cellular populations from two individuals were obtained: a control with no cleft phenotype and a patient with a cleft lip and palate. The genetic data showed three candidate variables in ARHGEF18, EPDR1, and CUL7. Next, the molecular data showed no significant change in proliferation rates between healthy patient cells and CL/P patient cells. However, CL/P patient cells showed decreased migration, increased adhesion and presented with a more elongated phenotype. Additionally, RhoA activity was upregulated in these cells, whereas Cdc42 activity was downregulated, resulting in loss of polarity. Our results are suggestive of a possible correlation between a dysregulation of Rho GTPases and the observed phenotype of cleft lip and palate patient cells. This insight into the intramolecular aspect of this disorder helps link the genetic defect with the observed phenotype and offers a possible mechanism by which CL/P occurs.

Altered regulation of cell migration in IRF6-mutated orofacial cleft patients-derived primary cells reveals a novel role of Rho GTPases in cleft/lip palate development.

Orofacial clefts are the most common congenital craniofacial birth defects. They occur from a failure in cell proliferation and fusion of neural crest cells of the lip buds and/or palatal shelves. In this study, we investigate the genetic basis and molecular mechanisms in primary cells derived from a cleft and lip palate patient presenting van der Woude syndrome (VWS). Since mutations in the integrin genes are widely correlated with VWS, Interferon Regulatory Factor 6 (IRF6) screening was conducted in a cohort of 200 participants presenting with orofacial anomalies. Primary fibroblastic cells derived from the upper right gingiva and palatal regions were isolated and two cellular populations from two participants were obtained: a control with no cleft phenotype and a patient with a cleft phenotype typical of van der Woude syndrome (VWS). IRF6 targeted sequencing revealed mutations in two distinct families. Our results showed no alteration in the viability of the CLP/VWS patient cells, suggesting the phenotype associate with the disease is not secondary to a defect in cell proliferation. We did however detect a significant decrease in the migratory ability of the CLP with Van der Woude syndrome (CLP/VWS) patient cells, which could account for the phenotype. When compared to normal cells, patient cells showed a lack of polarization, which would account for their lack of mobility. Patient cells showed protrusions all around the cells and a lack of defined leading edge. This was reflected with actin staining, WAVE2 and Arp2 around the cell, and correlated with an increase in Rac1 activation. Consistently with the increase in Rac1 activation, patient cells showed a loss in the maturation of focal adhesions needed for contractility, which also accounts for the lack in cell migration. Our findings give increased understanding of the molecular mechanisms of VWS and expands the knowledge of van der Woude syndrome (VWS) occurrence by providing a strong molecular evidence that CLP with Van der Woude syndrome (CLP/VWS) phenotype is caused by a defect in normal physiological processes of cells.

RHOG Activates RAC1 through CDC42 Leading to Tube Formation in Vascular Endothelial Cells.

Angiogenesis is a hallmark of cancer cell malignancy. The role of the RHO family GTPase RHOG in angiogenesis in vascular endothelial cells has recently been elucidated. However, the regulation of RHOG during this process, as well as its cross-talk with other RHO GTPases, have yet to be fully examined. In this study, we found that siRNA-mediated depletion of RHOG strongly inhibits tube formation in vascular endothelial cells (ECV cells), an effect reversed by transfecting dominant active constructs of CDC42 or RAC1 in the RHOG-depleted cells. We also found CDC42 to be upstream from RAC1 in these cells. Inhibiting either Phosphatidyl inositol (3) kinase (PI3K) with Wortmannin or the mitogen-activated protein kinase extracellular-regulated kinase (MAPK ERK) with U0126 leads to the inhibition of tube formation. While knocking down either RHO, GTPase did not affect p-AKT levels, and p-ERK decreased in response to the knocking down of RHOG, CDC42 or RAC1. Recovering active RHO GTPases in U0126-treated cells also did not reverse the inhibition of tube formation, placing ERK downstream from PI3K-RHOG-CDC42-RAC1 in vascular endothelial cells. Finally, RHOA and the Rho activated protein kinases ROCK1 and ROCK2 positively regulated tube formation independently of ERK, while RHOC seemed to inhibit the process. Collectively, our data confirmed the essential role of RHOG in angiogenesis, shedding light on a potential new therapeutic target for cancer malignancy and metastasis.

Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis.

AIM: To investigate the effect of adipose-derived mesenchymal stem cells (ADMSCs) and their conditioned media (CM) on hepatocellular carcinoma (HCC) cell tumorigenesis. METHODS: The proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit 8 (commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays. RESULTS: Our data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3. CONCLUSION: These findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.

Rheumatoid Synovial Fluids Regulate the Immunomodulatory Potential of Adipose-Derived Mesenchymal Stem Cells Through a TNF/NF-κB-Dependent Mechanism.

Introduction: Adipose-derived mesenchymal stem cells (ADSC) have been shown to have remarkable immune-modulating effects. However, their efficacy in clinical trials has yet to be fully demonstrated. This could be due to a lack of a proper inflammatory environment in vivo that primes ADSC. Here, we define how the articular microenvironment of rheumatoid arthritis (RA) patients modulates the therapeutic efficiency of ADSC. Methods: Synovial fluids (SF) were collected from 8 RA patients, 2 Spondyloarthritis patients and one control synovial fluid from a patient undergoing traumatic-related surgery. SF inflammatory status was determined by routine analysis and quantification of pro-inflammatory cytokines. ADSC were first treated with SF and ADSC proliferation and gene expression of immunomodulatory factors was evaluated. In order to determine the mechanisms underlying the effect of SF on ADSC, tumor necrosis factor (TNF), interleukin-6 (IL-6), and NF-κB neutralization assays were performed. To evaluate the effect of SF on ADSC functions, ADSC were pre-treated with SF and then co-cultured with either macrophages or T cells. The modulation of their phenotype was assessed by flow cytometry. Results: Pro-inflammatory RASF maintained the proliferative capacity of ADSC and upregulated the gene expression of cyclooxygenase-2 (COX2), indoleamine-1,2-dioxygenase (IDO), interleukin-6 (IL-6), tumor-necrosis factor stimulated gene 6 (TSG6), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and programmed death-ligand 1 (PD-L1), all factors involved in ADSC immunomodulatory potential. The RASF-induced gene expression was mainly mediated by TNF alone or in combination with IL-6 and signaled through the NF-κB pathway. Conditioning ADSC with pro-inflammatory RASF enhanced their ability to induce CD4+Foxp3+CD25high regulatory T cells (Tregs) and inhibit pro-inflammatory markers CD40 and CD80 in activated macrophages. Conclusions: Inflammatory synovial fluids from RA patients had the capacity to modulate ADSC response, to induce Tregs and modulate the phenotype of macrophages. The clinical use of ADSC in affected joints should take into account the influence of the local articular environment on their potential. Having a sufficient pro-inflammatory microenvironment will determine whether optimal immunoregulatory response should be expected. Direct ADSC intra-articular delivery to patients could be a potential strategy to properly prime their immunomodulatory potential and enhance their clinical benefits.

Corrigendum: Rheumatoid Synovial Fluids Regulate the Immunomodulatory Potential of Adipose-Derived Mesenchymal Stem Cells Through a TNF/NF-κB-Dependent Mechanism.

[This corrects the article DOI: 10.3389/fimmu.2019.01482.].

Effect of age and body mass index on the yield of stromal vascular fraction.

OBJECTIVE: The aim of this study was to determine the influence of age, body mass index, and site of liposuction on the cell yield of SVF. METHODS: A prospective study was performed on 58 patients. The average age was 39 years old, with BMI ≤ 25 or BMI ≥ 25. Fat tissue was harvested from the abdominal region, flanks, or thighs and SVF was isolated. RESULTS: The yield of viable SVF was evaluated by trypan blue, and the markers of stem cells were evaluated by flow cytometry. The cells were positive for stem cells markers, the age, sex of the patient had no impact on SVF cell yield with an average of 1.17 × 10^8. However, the BMI > 25 had resulted in higher cell numbers, and the harvest site had a significant impact on cell yield with abdomen being the site of interest. CONCLUSION: These data demonstrate that the age of the person does not affect the cell yield of SVF; nevertheless, the donor site and BMI might be important factors in affecting cell number.

Metformin Treatment Inhibits Motility and Invasion of Glioblastoma Cancer Cells.

Glioblastoma multiforme (GBM) is one of the most common and deadliest cancers of the central nervous system (CNS). GBMs high ability to infiltrate healthy brain tissues makes it difficult to remove surgically and account for its fatal outcomes. To improve the chances of survival, it is critical to screen for GBM-targeted anticancer agents with anti-invasive and antimigratory potential. Metformin, a commonly used drug for the treatment of diabetes, has recently emerged as a promising anticancer molecule. This prompted us, to investigate the anticancer potential of metformin against GBMs, specifically its effects on cell motility and invasion. The results show a significant decrease in the survival of SF268 cancer cells in response to treatment with metformin. Furthermore, metformin's efficiency in inhibiting 2D cell motility and cell invasion in addition to increasing cellular adhesion was also demonstrated in SF268 and U87 cells. Finally, AKT inactivation by downregulation of the phosphorylation level upon metformin treatment was also evidenced. In conclusion, this study provides insights into the anti-invasive antimetastatic potential of metformin as well as its underlying mechanism of action.

An Evaluation of the Stemness, Paracrine, and Tumorigenic Characteristics of Highly Expanded, Minimally Passaged Adipose-Derived Stem Cells.

The use of adipose-derived stem cells (ADSC) in regenerative medicine is rising due to their plasticity, capacity of differentiation and paracrine and trophic effects. Despite the large number of cells obtained from adipose tissue, it is usually not enough for therapeutic purposes for many diseases or cosmetic procedures. Thus, there is the need for culturing and expanding cells in-vitro for several weeks remain. Our aim is to investigate if long- term proliferation with minimal passaging will affect the stemness, paracrine secretions and carcinogenesis markers of ADSC. The immunophenotypic properties and aldehyde dehydrogenase (ALDH) activity of the initial stromal vascular fraction (SVF) and serially passaged ADSC were observed by flow cytometry. In parallel, the telomerase activity and the relative expression of oncogenes and tumor suppressor genes were assessed by q-PCR. We also assessed the cytokine secretion profile of passaged ADSC by an ELISA. The expanded ADSC retain their morphological and phenotypical characteristics. These cells maintained in culture for up to 12 weeks until P4, possessed stable telomerase and ALDH activity, without having a TP53 mutation. Furthermore, the relative expression levels of TP53, RB, and MDM2 were not affected while the relative expression of c-Myc decreased significantly. Finally, the levels of the secretions of PGE2, STC1, and TIMP2 were not affected but the levels of IL-6, VEGF, and TIMP 1 significantly decreased at P2. Our results suggest that the expansion of passaged ADSC does not affect the differentiation capacity of stem cells and does not confer a cancerous state or capacity in vitro to the cells.