Wang, Wei

Elevated levels of follistatin‐like protein 1 (FSTL1) have been found both in mouse models for human rheumatoid arthritis (RA) and collagen‐induced arthritis (CIA). In this study, we elucidated the potential mechanisms by which FSTL1 contributes to the pathogenesis of RA. Fibroblast‐like synoviocytes (FLSs) were established from synovial tissues of RA patients and stimulated with human recombinant FSTL1. Protein and mRNA expression levels of select matrix metalloproteinases (i.e., MMP1, MMP3, MMP13) in FLS were measured by, respectively, real‐time RT‐qPCR and ELISA. Activation of MAPK and other pathways that affect MMPs were evaluated by Western blotting. We also compared concentrations of MMPs in plasma in RA patients versus healthy controls (HC). Expression levels of MMP1, MMP3, and MMP13 were clearly stimulated by FSTL1 in vitro. FSTL1 activated the inflammation‐related NF‐κB signaling pathway, as well as all three mitogen‐activated protein kinase (MAPK) pathways and the JAK/STAT3 pathway. Moreover, select chemical inhibitors that target p38 (SB203580), Erk1/2 (SP600125), JNK (SCH772984), STAT3 (AG490), and NF‐κB (BAY 11–7082) significantly attenuated MMP expression. Inhibition of Toll‐like receptor 4 by compound TAK‐242 significantly abolished those effects of FSTL1. Importantly, elevated plasma concentrations of MMP3 were found to correlate with plasma FSTL1 levels in RA patients. These findings suggest that FSTL1 accelerates RA progression by activating MAPK, JAK/STAT3, and NF‐κB pathways to enhance secretion of different MMPs and this enhancement is via TLR4. Targeting FSTL1 may provide a promising pharmacological drug therapy to ameliorate RA symptoms and perhaps reverse disease progression.

ABSTRACTDegenerative disk disease of the spine is a major cause of back pain and disability. Optimization of regenerative medical therapies for degenerative disk disease requires a deep mechanistic understanding of the factors controlling the structural integrity of spinal tissues. In this investigation, we sought to identify candidate regulatory genes controlling extracellular matrix synthesis in spinal tissues. To achieve this goal we performed high throughput next generation RNA sequencing on 39 annulus fibrosus and 21 nucleus pulposus human tissue samples. Specimens were collected from patients undergoing surgical discectomy for the treatment of degenerative disk disease. Our studies identified associations between extracellular matrix genes, growth factors, and other important regulatory molecules. The fibrous matrix characteristic of annulus fibrosus was associated with expression of the growth factors platelet derived growth factor beta (PDGFB), vascular endothelial growth factor C (VEGFC), and fibroblast growth factor 9 (FGF9). Additionally we observed high expression of multiple signaling proteins involved in the NOTCH and WNT signaling cascades. Nucleus pulposus extracellular matrix related genes were associated with the expression of numerous diffusible growth factors largely associated with the transforming growth signaling cascade, including transforming factor alpha (TGFA), inhibin alpha (INHA), inhibin beta A (INHBA), bone morphogenetic proteins (BMP2, BMP6), and others. Clinical significance: this investigation provides important data on extracellular matrix gene regulatory networks in disk tissues. This information can be used to optimize pharmacologic, stem cell, and tissue engineering strategies for regeneration of the intervertebral disk and the treatment of back pain. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1356–1369, 2018.

ABSTRACTPreservation of osteochondral allografts used for transplantation is critical to ensure favorable outcomes for patients after surgical treatment of cartilage defects. To study the biological effects of protocols currently used for cartilage storage, we investigated differences in gene expression between stored allograft cartilage and fresh cartilage from living donors using high throughput molecular screening strategies. We applied next generation RNA sequencing (RNA‐seq) and real‐time reverse transcription quantitative polymerase chain reaction (RT‐qPCR) to assess genome‐wide differences in mRNA expression between stored allograft cartilage and fresh cartilage tissue from living donors. Gene ontology analysis was used to characterize biological pathways associated with differentially expressed genes. Our studies establish reduced levels of mRNAs encoding cartilage related extracellular matrix (ECM) proteins (i.e., COL1A1, COL2A1, COL10A1, ACAN, DCN, HAPLN1, TNC, and COMP) in stored cartilage. These changes occur concomitantly with increased expression of “early response genes” that encode transcription factors mediating stress/cytoprotective responses (i.e., EGR1, EGR2, EGR3, MYC, FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND). The elevated expression of “early response genes” and reduced levels of ECM‐related mRNAs in stored cartilage allografts suggests that tissue viability may be maintained by a cytoprotective program that reduces cell metabolic activity. These findings have potential implications for future studies focused on quality assessment and clinical optimization of osteochondral allografts used for cartilage transplantation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1950–1959, 2016.

Rationale : Semaphorin 3A ( SEMA3A )-encoded semaphorin is a chemorepellent that disrupts neural patterning in the nervous and cardiac systems. In addition, SEMA3A has an amino acid motif that is analogous to hanatoxin, an inhibitor of voltage-gated K + channels. SEMA3A -knockout mice exhibit an abnormal ECG pattern and are prone to ventricular arrhythmias and sudden cardiac death. Objective : Our aim was to determine whether SEMA3A is a naturally occurring protein inhibitor of K v 4.3 (I to ) channels and its potential contribution to Brugada syndrome. Methods and Results : K v 4.3, Na v 1.5, Ca v 1.2, or K v 4.2 were coexpressed or perfused with SEMA3A in HEK293 cells, and electrophysiological properties were examined via whole-cell patch clamp technique. SEMA3A selectively altered K v 4.3 by significantly reducing peak current density without perturbing K v 4.3 cell surface protein expression. SEMA3A also reduced I to current density in cardiomyocytes derived from human-induced pluripotent stem cells. Disruption of a putative toxin binding domain on K v 4.3 was used to assess physical interactions between SEMA3A and K v 4.3. These findings in combination with coimmunoprecipitations of SEMA3A and K v 4.3 revealed a potential direct binding interaction between these proteins. Comprehensive mutational analysis of SEMA3A was performed on 198 unrelated SCN5A genotype–negative patients with Brugada syndrome, and 2 rare SEMA3A missense mutations were identified. The SEMA3A mutations disrupted SEMA3A’s ability to inhibit K v 4.3 channels, resulting in a significant gain of K v 4.3 current compared with wild-type SEMA3A. Conclusions : This study is the first to demonstrate SEMA3A as a naturally occurring protein that selectively inhibits K v 4.3 and SEMA3A as a possible Brugada syndrome susceptibility gene through a K v 4.3 gain-of-function mechanism.