Hai-Qiang Wang

Deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral disc degeneration by targeting FADD and caspase-3.

The role of apoptosis in the pathogenesis of intervertebral disc degeneration (IDD) remains enigmatic. Accumulating evidence has shown that the apoptotic machinery is regulated by miRNAs. We hypothesized that miRNAs might contribute to apoptosis in IDD. We have found that 29 miRNAs were differentially expressed and miR‐155 was down‐regulated in degenerative nucleus pulposus (NP). The deregulation of miR‐155 was further verified using real‐time PCR (0.56 fold, p < 0.05). Bioinformatics target prediction identified FADD and caspase‐3 as putative targets of miR‐155. Furthermore, miR‐155 inhibited FADD and caspase‐3 expression by directly targeting their 3′‐UTRs, which was abolished by mutation of the miR‐155 binding sites. In vitro up‐regulation of miR‐155 in human NP cells by transfection with lentiviral pre‐miR‐155 resulted in repression of FADD and caspase‐3; whereas knockdown of miR‐155 with lentiviral antigomiR‐155 led to over‐expression of FADD and caspase‐3. Also, Fas‐mediated apoptosis was increased when antagonizing miR‐155 and decreased when using pre‐miR‐155 in human NP cells. In addition, we presented direct evidence of NP cells undergoing apoptosis in IDD tissues using transmission electron microscopy analysis. Moreover, a combination of in situ hybridization (ISH) and immunohistochemistry (IHC) revealed that miR‐155 expressed in the cytoplasm of human NP cells with reverse correlation with FADD and caspase‐3. In summary, this is the first study addressing the underlying mechanisms of IDD in terms of apoptosis and miRNAs. Furthermore, caspase‐3 is identified as a novel target of miR‐155. Our results suggest that deregulated miR‐155 promotes Fas‐mediated apoptosis in human IDD by targeting FADD and caspase‐3, implicating an aetiological and therapeutic role of miR‐155 in IDD. Copyright

Aberrantly expressed long noncoding RNAs in human intervertebral disc degeneration: a microarray related study.

IntroductionIn addition to the well-known short noncoding RNAs such as microRNAs (miRNAs), increasing evidence suggests that long noncoding RNAs (lncRNAs) act as key regulators in a wide aspect of biologic processes. Dysregulated expression of lncRNAs has been demonstrated being implicated in a variety of human diseases. However, little is known regarding the role of lncRNAs with regards to intervertebral disc degeneration (IDD). In the present study we aimed to determine whether lncRNAs are differentially expressed in IDD.MethodsAn lncRNA-mRNA microarray analysis of human nucleus pulposus (NP) was employed. Bioinformatics prediction was also applied to delineate the functional roles of the differentially expressed lncRNAs. Several lncRNAs and mRNAs were chosen for quantitative real-time PCR (qRT-PCR) validation.ResultsMicroarray data profiling indicated that 116 lncRNAs (67 up and 49 down) and 260 mRNAs were highly differentially expressed with an absolute fold change greater than ten. Moreover, 1,052 lncRNAs and 1,314 mRNAs were differentially expressed in the same direction in at least four of the five degenerative samples with fold change greater than two. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for the differentially expressed mRNAs indicated a number of pathways, such as extracellular matrix (ECM)-receptor interaction. A coding-noncoding gene co-expression (CNC) network was constructed for the ten most significantly changed lncRNAs. Annotation terms of the coexpressed mRNAs were related to several known degenerative alterations, such as chondrocyte differentiation. Moreover, lncRNAs belonging to a particular subgroup were identified. Functional annotation for the corresponding nearby coding genes showed that these lncRNAs were mainly associated with cell migration and phosphorylation. Interestingly, we found that Fas-associated protein factor-1 (FAF1), which potentiates the Fas-mediated apoptosis and its nearby enhancer-like lncRNA RP11-296A18.3, were highly expressed in the degenerative discs. Subsequent qRT-PCR results confirmed the changes.ConclusionsThis is the first study to demonstrate that aberrantly expressed lncRNAs play a role in the development of IDD. Our study noted that up-regulated RP11-296A18.3 highly likely induced the over-expression of FAF1, which eventually promoted the aberrant apoptosis of disc cells. Such findings further broaden the understanding of the etiology of IDD.

''Spring-back'' closure associated with open-door cervical laminoplasty

BACKGROUND CONTEXTnSpring-back complication after open-door laminoplasty as described by Hirabayashi is a well-known risk, but its definition, incidence, and associated neurologic outcome remain unclear.nnnOBJECTIVEnTo investigate the incidence and the neurologic consequence of spring-back closure after open-door laminoplasty.nnnSTUDY DESIGNnA retrospective radiographic and clinical review.nnnOUTCOME MEASURESnLateral cervical spine X-rays were evaluated. Anteroposterior diameters (APD) of the vertebral canal of C3-C7 were measured. Spring-back was defined as loss of APD on follow-up in comparison to immediate postoperative canal expansion. The loss of the end-on lamina silhouette with consequent reappearance of the lateral profile of the spinous processes was also assessed to verify the presence of spring-back. Spring-back closure was classified based on whether the collapse was total or partial, and whether all the operated levels or only a subset had collapsed (ie, complete vs. partial closure, segmental closure vs. total-construct closure). Neurologic status was documented using the Japanese Orthopaedic Association (JOA) score.nnnMETHODSnThirty consecutive patients who underwent open-door laminoplasty from 1995 to 2005 at a single institution with a minimum follow-up of 2 years were assessed. They were all operated on using the classic Hirabayashi technique. Radiographic outcomes were assessed independently by two individuals.nnnRESULTSnSixteen men and 14 women with an average follow-up of 5 years (range, 2-12 years) were included. Of these patients, 24 had cervical spondylotic myelopathy and six had ossification of the posterior longitudinal ligament. Spring-back closure was found in three patients (10%) and 7 of 117 laminae (6%) within 6 months of the operation, which was further confirmed by computed tomography and magnetic resonance imaging. All spring-back closures were partial segmental closures. Gender and age were not significant factors related to spring back (p>.05). The mean JOA score on follow-up was 12.5, with a recovery rate of 40%. All patients with spring back and available JOA data exhibited postoperative neurologic deterioration. Of the three patients with spring back, two patients underwent revision surgery, whereas one declined.nnnCONCLUSIONSnSpring-back closure occurred in 10% of our patients at or before 6 months after surgery. The incidence of spring-back by level (ie, 117 laminae) was 6%, mainly occurring at the lower cervical spine. All spring-back closures were partial segmental closures, most commonly involving C5 and C6. Postoperative neurologic deficit was associated with spring-back closure; therefore, surgeons should adopt preemptive surgical measures to prevent the occurrence of such a complication.

Novel diagnostic and prognostic methods for disc degeneration and low back pain

Low back pain (LBP) is the world’s leading debilitating condition [1]. It is estimated that 80% of the general population in the United States will develop LBP at one point in time [2,3]. Such pain can lead to diminished daily function and quality of life and work disability [4,5]. Not surprisingly, spine surgery to address LBP is one of the top five surgeries performed in the United States [6] where approximately 90-billion US dollars in health-care expenses are used annually to treat LBP [7]. As data indicate, LBP is clearly related with detrimental socioeconomic and health-care consequences that motivate efforts to identify LPB risk factors to develop improved prevention and treatment strategies. n nAlthough the etiology of LBP is multifaceted, disc degeneration (DD) has been suggested to be one of the most prominent risk factors [8–11]. However, whether DD is synonymous with LBP continues to be a topic of immense controversy. In this article, we review the current data regarding pain generating pathways and epidemiological evidence associating DD with LBP. In further support of this association, we review novel disc imaging techniques that may increase LBP diagnostic sensitivity and specificity. Finally, we discuss relevant aspects of the field of pain genes that may shed further light as to the links between DD and pain.

Down-Regulated CK8 Expression in Human Intervertebral Disc Degeneration

As an intermediate filament protein, cytokeratin 8 (CK8) exerts multiple cellular functions. Moreover, it has been identified as a marker of notochord cells, which play essential roles in human nucleus pulposus (NP). However, the distribution of CK8 positive cells in human NP and their relationship with intervertebral disc degeneration (IDD) have not been clarified until now. Here, we found the percentage of CK8 positive cells in IDD (25.7±4.14%) was significantly lower than that in normal and scoliosis NP (51.9±9.73% and 47.8±5.51%, respectively, p<0.05). Western blotting and qRT-PCR results confirmed the down-regulation of CK8 expression in IDD on both of protein and mRNA levels. Furthermore, approximately 37.4% of cell clusters were CK8 positive in IDD. Taken together, this is the first study to show a down-regulated CK8 expression and the percentage of CK8 positive cell clusters in IDD based upon multiple lines of evidence. Consequently, CK8 positive cells might be considered as a potential option in the development of cellular treatment strategies for NP repair.

FasL Expression on Human Nucleus Pulposus Cells Contributes to the Immune Privilege of Intervertebral Disc by Interacting with Immunocytes

The mechanisms of immune privilege in human nucleus pulposus (NP) remain unclear. Accumulating evidence indicates that Fas ligand (FasL) might play an important role in the immune privilege of the disc. We aimed for addressing the role of FasL expression in human intervertebral disc degeneration (IDD) and immune privilege in terms of the interaction between NP cells and immunocytes via the FasL-Fas machinery. We collected NP specimens from 20 patients with IDD as degenerative group and 8 normal cadaveric donors as control. FasL expression was detected by qRT-PCR, western blotting and flow cytometry (FCM). We also collected macrophages and CD8+ T cells from the peripheral blood of patients with IDD for co-cultures with NP cells. And macrophages and CD8+ T cells were harvested for apoptosis analysis by FCM after 2 days of co-cultures. We found that FasL expression in mRNA, protein and cellular resolutions demonstrated a significant decrease in degenerative group compared with normal control (p<0.05). FCM analysis found that human NP cells with increased FasL expression resulted in significantly increased apoptosis ratio of macrophages and CD8+ T cells. Our study demonstrated that FasL expression tends to decrease in degenerated discs and FasL plays an important role in human disc immune privilege, which might provide a novel target for the treatment strategies for IDD.

Insights into the Hallmarks of Human Nucleus Pulposus Cells with Particular Reference to Cell Viability, Phagocytic Potential and Long Process Formation

Objective: As a main cellular component within the disc, nucleus pulposus (NP) cells play important roles in disc physiology. However, little is known on the biologic hallmarks of human NP cells. Therefore, the present study aimed to address the features of human NP cells. Methods: Human NP samples were collected from normal cadavers, patients with scoliosis and disc degeneration as normal, disease control and degenerative NP, respectively. The NP samples were studied using transmission electron microscopy and TUNEL assay. Pre-digested NP samples were studied using flow cytometry with PI/Annexin V staining. Results: Both control and degenerative human NP consisted of mainly viable cells with a variety of morphology. Both necrosis and apoptosis were noted in human NP as forms of cell death with increased apoptosis in degenerative NP, which was further confirmed by the TUNEL assay. Phagocytic NP cells had the hallmarks of both stationary macrophages with lysosomes and NP cells with the endoplasmic reticulum. Annulus fibrosus cells have similar morphologic characteristics with NP cells in terms of cell nest, phagocytosis and intracellular organs. Moreover, NP cells with long processes existed in degenerative and scoliotic NP rather than normal NP. When cultured in glucose-free medium, NP cells developed long and thin processes. Conclusion: Human degenerative NP consists of primarily viable cells. We present direct and in vivo evidence that both human annulus fibrosus and NP cells have phagocytic potential. Moreover, NP cells with long processes exist in both scoliotic and degenerative NP with lack of glucose as one of the possible underlying mechanisms.

Impact of direct cell co-cultures on human adipose-derived stromal cells and nucleus pulposus cells.

Biologic and cellular treatment strategies aiming for curing intervertebral disc degeneration (IDD) have been proposed recently. Given the convenient availability and expansion potential, adipose‐derived stromal cells (ADSCs) might be an ideal cell candidate. However, the interaction between ADSCs and nucleus pulposus (NP) cells still remains ambiguous, especially in direct co‐cultures of the two types of cells. Nevertheless, NP markers in ADSCs after co‐cultures were unidentified. Here, we addressed the interaction of human ADSCs and NP cells in a direct co‐culture system for the first time. As a result, ADSCs could differentiate to the NP cell phenotype with a significant up‐regulated expression of multiple genes and proteins in extracellular matrix (ECM) (SOX9, COL2A1, ACAN, and COL6A2), relative NP markers (FOXF1, PAX1, CA12, and HBB) and pertinent growth factors (CDMP‐1, TGF‐β1, IGF‐1, and CTGF). Moreover, the gene expression of COL2A1, ACAN, and COL6A2 of degenerate NP cells was also up‐regulated. Collectively, these results suggest that direct co‐cultures of ADSCs and NP cells may exert a reciprocal impact, that is, both stimulating ADSCs differentiation to the NP cell phenotype and inducing NP cells to regain functional phenotype. Accordingly, ADSCs might be a potential candidate in the development of cellular treatment strategies for IDD.

Landscape of RNAs in human lumbar disc degeneration

Accumulating evidence indicates noncoding RNAs (ncRNAs) fine-tune gene expression with mysterious machinery. We conducted a combination of mRNA, miRNA, circRNA, LncRNA microarray analyses on 10 adults lumbar discs. Moreover, we performed additional global exploration on RNA interacting machinery in terms of in silico computational pipeline. Here we show the landscape of RNAs in human lumbar discs. In general, the RNA-abundant landscape comprises 14,635 mRNAs (37.93%), 2,059 miRNAs (5.34%), 18,995 LncRNAs (49.23%) and 2,894 (7.5%) circRNAs. Chromosome 1 contributes for RNA transcription at most (10%). Bi-directional transcription contributes evenly for RNA biogenesis, in terms of 5′ to 3′ and 3′ to 5′. Despite the majority of circRNAs are exonic, antisense (1.49%), intergenic (0.035%), intragenic (1.69%), and intronic (6.29%) circRNAs should not be ignored. A single miRNA could interact with a multitude of circRNAs. Notably, CDR1as or ciRS-7 harbors 66 consecutive binding sites for miR-7-5p (previous miR-7), evidencing our pipeline. The majority of binding sites are perfect-matched (78.95%). Collectively, global landscape of RNAs sheds novel insights on RNA interacting mechanisms in human intervertebral disc degeneration.

Integration of current identity-based district-varied health insurance schemes in China: implications and challenges

With China’s great efforts to improve public health insurance, clear progress has been achieved toward the ambitious full health insurance coverage strategy for all. The current health insurance schemes in China fall into three categories: urban employee basic health insurance scheme, urban resident scheme, and new rural cooperative medical system. Despite their phasic success, these substantially identity-based, district-varied health insurance schemes have separate operation mechanisms, various administrative institutions, and consequently poor connections. On the other hand, the establishment and implementation of various health insurance schemes provide the preconditioning of more sophisticated social health insurance schemes, the increase in the income of urban and rural people, and the great importance attached by the government. Moreover, the reform of the “Hukou” (household register) system provides economical, official, and institutional bases. Therefore, the establishment of an urban-rural integrated, citizen-based, and nationwide-universal health insurance scheme by the government is critically important to attain equality and national connection. Accordingly, the differences between urban and rural areas should be minimized. In addition, the current schemes, administrative institutions, and networks should be integrated and interconnected. Moreover, more expenditure on health insurance might be essential for the integration despite the settings of global financial crisis. Regardless of the possible challenges in implementation, the proposed new scheme is promising and may be applied in the near future for the benefit of the Chinese people and global health.