Liwu Zhou

Harmful Effects of Leukocyte-Rich Platelet-Rich Plasma on Rabbit Tendon Stem Cells In Vitro:

Background: Platelet-rich plasma (PRP) is now widely used as a promising treatment for patients with tendinopathy. However, the efficacy of PRP treatment for tendinopathy is controversial mainly because of inconsistent results from human clinical trials and particularly because the concentration and effect of leukocytes in PRP remain largely unknown. Hypothesis: Leukocyte-rich PRP (L-PRP) inhibits growth factor release, decreases proliferation, and induces nontenocyte differentiation of tendon stem cells (TSCs); increases catabolic cytokine concentrations; and causes inflammation and apoptosis. Thus, L-PRP has a detrimental effect on tendon stem/progenitor cells, which impairs injured tendon healing. Study Design: Controlled laboratory study. Methods: Pure PRP (P-PRP) and L-PRP were prepared from the same individual rabbit blood, and platelet numbers in each PRP product were adjusted to reach the same level. The leukocyte level in L-PRP was 4 and 8 times higher than those in whole blood and P-PRP, respectively. The growth factors in both P-PRP and L-PRP were measured by enzyme-linked immunosorbent assay kits. The morphology, stemness, proliferation, and differentiation of TSCs grown in L-PRP and P-PRP were examined by microscopy, immunocytochemistry, population doubling time, quantitative real-time polymerase chain reaction, and histological analysis. Results: L-PRP produced lower levels of growth factors, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factor (TGF)–β1, and platelet-derived growth factor (PDGF), than did P-PRP. TSC proliferation was significantly decreased in L-PRP in a concentration-dependent manner. Furthermore, TSCs cultured in P-PRP produced more collagen and formed tendon-like tissue; however, TSCs grown in L-PRP differentiated into nontenocytes and produced more inflammatory factors such as membrane-associated prostaglandin synthase (mPGES) and interleukin (IL)–1β. Moreover, L-PRP was associated with increased apoptosis. Conclusion: L-PRP has harmful effects on TSCs. Clinical Relevance: This study revealed the direct effects of different compositions of PRP on TSCs and provided basic scientific data to help understand the cellular and molecular mechanisms of the efficacy of PRP treatment in clinical use.

Sox4 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cell via activation of long noncoding RNA DANCR

Mesenchymal stem cells (MSCs) have several features that make them an attractive option for potentiating cartilage repair. Synovium-derived (SMSCs) have been recently recognized as an excellent source. SRY-related HMG-box (Sox) family plays an important role in the proliferation and differentiation of SMSCs. However, the role of Sox4 in human SMSCs remains elusive. In the present study, we investigated the role of Sox4 in SMSCs through gain-of-function studies and found that Sox4 promoted cell proliferation and chondrogenesis. Furthermore, Sox4 could directly bind to the promoter of long noncoding RNA DANCR and increased its expression. Finally, knockdown of DANCR could reverse the stimulative effect of Sox4 on the proliferation and chondrogenesis of SMSCs. Taken together, our data highlights the pivotal role of Sox4 in the proliferation and differentiation of SMSCs.

Long Noncoding RNA DANCR Is a Positive Regulator of Proliferation and Chondrogenic Differentiation in Human Synovium-Derived Stem Cells

Cartilage tissues have limited capacity for repair after damage and then cause osteoarthritis, so finding alternative treatment is ongoing. Mesenchymal stem cells (MSCs) have become a promising therapy for cartilage damage and diseases due to the advantages of easy separation, high proliferative potentiality, and genetic stability. Synovium-derived MSCs (SMSCs) have been recognized as an ideal source for cartilage repair. In our previous study, we found that Sox4 promoted proliferation and chondrogenesis of SMSCs through upregulation of long noncoding RNA (lncRNA) DANCR. However, the exact molecular mechanism by which DANCR promotes proliferation and chondrogenesis of SMSCs remains unknown. In the present study, we investigated the effect of lncRNA DANCR on the proliferation and chondrogenesis of SMSCs. We found that overexpression of DANCR could promote proliferation and chondrogenesis of SMSCs, while knockdown of DANCR had the opposite effect. Moreover, our data demonstrated that DANCR directly interacted with myc, Smad3, and STAT3 mRNA to regulate their stability. Finally, we found that the promotion of SMSC proliferation induced by DANCR depended on myc. Also, DANCR activated chondrogenesis of SMSCs via upregulation of Smad3 and STAT3 expression. Our growing knowledge of the role of DANCR is pointing toward its potential use as a novel therapeutic approach for cartilage damage and diseases.

Long noncoding RNA DANCR regulates miR-1305-Smad 4 axis to promote chondrogenic differentiation of human synovium-derived stem cells.

miRNAs have been reported to regulate cellular differentiation by modulating multiple signaling pathways. Long noncoding RNA (lnc RNA) DANCR was previously identified to be critical for the chondrogenesis of human synovium-derived mesenchymal stem cells (SMSC), however, the underlying molecular mechanism requires better understanding. Here, miRNA expression profiling in DANCR overexpressed in SMSCs identified significant down-regulation of miR-1305, which serves as a downstream target of DANCR. Notably, miR-1305 overexpression reversed DANCR-induced cell proliferation and chondrogenic differentiation of SMSCs, which suggested that miR-1305 antagonized the function of DANCR. Mechanistically, highly expressed miR-1305 resulted in the decreased expression of the TGF-β pathway member Smad4, and inhibition of miR-1305 enhanced the expression level of Smad4. Depletion of Smad4 suppressed the promotion of DANCR in cell proliferation and chondrogenesis of SMSCs. Collectively, our results characterized miR-1305-Smad4 axis as a major downstream functional mechanism of lncRNA DANCR in promoting the chondrogenesis in SMSCs.

Free fatty acids are responsible for the hidden blood loss in total hip and knee arthroplasty

Hidden blood loss (HBL), commonly seen post total knee or hip arthroplasty, causes postoperative anemia even after reinfusion or blood transfusion based on the visible blood loss volume. The mechanism of HBL remains unclear although more than one theory had tried to explain it. Free fatty acids, metabolites of fatty emboli that are generated during TKA, THA and other surgery manipulating the medullary canal of femur, had been demonstrated to stimulate the neutrophils in producing reactive oxygen species such (ROS) as hydrogen peroxide and chlorous peroxide. Erythrocytes injury was also shown in parasitic infection, chronic renal disease and paroxysmal nocturnal hemoglobinuria in a mechanism of oxidation of membrane polyunsaturated fatty acids and cytosolic hemoglobin by ROS. Based on these results we hypothesize that free fatty acids generated from fatty emboli in blood circulation are responsible for the hidden blood loss through peroxidating injury of membrane molecules of RBC and hemoglobin. Antioxidants administered intra- or post-operatively are predicted to play a protective role in erythrocytes oxidation and potentially reduce the volume of hidden blood loss after arthroplasty.

TheBDKRB2 +9/-9 Polymorphisms Influence Pro-Inflammatory Cytokine Levels in Knee Osteoarthritis by Altering TLR-2 Expression: Clinical and in Vitro Studies

Background/Aims: The bradykinin B2 receptor (BDKRB2) +9/-9 gene polymorphisms have been shown to be associated with the susceptibility and severity of osteoarthritis (OA); however, the underlying mechanisms are unclear. In this study, we investigated the correlation between the BDKRB2 +9/-9 polymorphisms and pro-inflammatory cytokine levels in OA and the molecular mechanisms involved. Methods: A total of 156 patients with primary knee OA and 121 healthy controls were enrolled. The BDKRB2 +9/-9 polymorphisms were genotyped. The tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 levels were determined using Enzyme-linked immunosorbent assay (ELISA). The toll-like receptor (TLR)-2 and TLR-4 mRNA levels were determined by quantitative real-time PCR. The basal and bradykinin-stimulated pro-inflammatory cytokine secretion in human OA synoviocytes and the involvement of TLR-2 and mitogen-activated protein kinases (MAPKs) were investigated. Results: The presence of -9 bp genotype is associated with higher TNF-α, IL-6, and IL-8 levels and higher TLR-2 expression in OA patients. The basal and bradykinin-induced TLR-2 expressions in human OA synoviocytes were significantly reduced by specific inhibitors of p38, JNK1/2, and ERK1/2. Both the B2 receptor antagonist MEN16132 and TLR-2 silencing inhibited IL-6 and IL-8 secretion in human OA synoviocytes. Conclusion: The data suggested that the BDKRB2 +9/-9 polymorphisms influence pro-inflammatory cytokine levels in knee osteoarthritis by altering TLR-2 expression.

miR-338-5p Regulates the Viability, Proliferation, Apoptosis and Migration of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Targeting NFAT5

Background/Aims: MicroRNAs (miRNAs) have been reported to be involved in Rheumatoid arthritis (RA) pathogenesis and prognosis. However, little is known about the disease mechanism in RA. Here, we aim to investigate the potential association between miR-338-5p and NFAT5 in RA. Methods: Aberrant expression of miR-338-5p in RA tissues and rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) compared to the normal were determined by RT-qPCR. Cell viability was determined using the CCK-8 assay, and cell apoptosis was analyzed via Annexin V-FITC/PI double staining and was detected using flow cytometry. The targeted relationship was determined by TargetScan database and dual luciferase reporter gene assay. Results: Upregulation of miR-338-5p facilitated the proliferation, migration, invasion and induced G0/G1 arrest of RAFLSs while miR-338-5p inhibitor functioned oppositely. Nuclear factor of activated T-cells 5 (NFAT5) was confirmed as a downstream target of miR-338-5p which expression was directly suppressed by miR-338-5p. Overexpression of NFAT5 attenuated the proliferation and metastasis of RAFLSs and those changes could be rescued by co-transfection of miR-338-5p. Conclusion: miR-338-5p promotes RAFLS’s viability and proliferation, migration by targeting NFAT5, suggesting a novel therapeutic strategy for RA.

BDKRB2 +9/-9 bp polymorphisms influence BDKRB2 expression levels and NO production in knee osteoarthritis.

The bradykinin B2 receptor (BDKRB2) plays a key role in the inflammation process of osteoarthritis. Nitric oxide has also long been considered to be a catabolic factor that contributes to inflammatory response and the osteoarthritis disease pathology. Several studies have reported that the BDKRB2 +9/−9 bp polymorphisms are associated with transcription of the receptor. However, the roles of BDKRB2 polymorphisms in inflammation in osteoarthritis remain unclear. This study enrolled 156 subjects with primary knee osteoarthritis and 58 healthy volunteers. BDKRB2 polymorphisms were genotyped, and the mRNA and protein levels of BDKRB2 in synovial tissues from osteoarthritis patients were measured by quantitative real-time polymerase chain reaction and western blot analysis, respectively. Nitric oxide production in serum from patients with osteoarthritis was measured using a nitric oxide assay kit. We found that the mean BDKRB2 mRNA levels were significantly higher in Kallgren-Lawrence grade-4 osteoarthritis patients than patients with lower grade osteoarthritis. The +9/−9 bp polymorphisms significantly affected the BDKRB2 mRNA and protein expression levels in synovial tissues from osteoarthritis subjects. Osteoarthritis patients with +9/−9 and −9/−9 genotypes had higher BDKRB2 expression levels in synovial tissue and nitric oxide production in serum. Moreover, positive correlation was found between the BDKRB2 levels in synovial tissue and nitric oxide production. Compared with health controls, significant increases of nitric oxide production in osteoarthritis were detected which were associated with increasing severity of osteoarthritis. Multiple linear regression analysis (adjusted for gender and age) showed serum nitric oxide level was positively associated with BDKRB2 polymorphism and Kallgren-Lawrence grade and was inversely associated with obesity. Our findings showed that the BDKRB2 +9/−9 bp polymorphisms affected the gene expression and nitric oxide production, which were associated with radiographic severity of osteoarthritis, suggesting that the BDKRB2 +9/−9 bp polymorphisms may act as a genetic modulator of osteoarthritis, and play an essential role in inflammatory process in osteoarthritis.