Secretome of Hypoxia-Preconditioned Mesenchymal Stem Cells Promotes Liver Regeneration and Anti-Fibrotic Effect in Liver Fibrosis Animal Model.

<b>Background and Objective:</b> Liver fibrosis (LF) is a most common pathological process characterized by the activation of hepatocytes leading to the accumulation of extracellular matrix (ECM). Hypoxia precondition treated in MSCs (H-MSCs) could enhance their immunomodulatory and regeneration capability, through expressing robust anti-inflammatory cytokines and growth factors, known as H-MSCs secretome (SH-MSCs) that are critical for the improvement of liver fibrosis. However, the study regarding the efficacy and mechanism of action of SH-MSCs in ameliorating liver fibrosis is still inconclusive. In this study, the therapeutic potential and underlying mechanism for SH-MSCs in the treatment of liver fibrosis were investigated. <b>Materials and Methods:</b> A rat model with liver fibrosis induced by CCl₄ was created and maintained for 8 weeks. The rats received intravenous doses of SH-MSCs and secretome derived from normoxia MSCs (SN-MSCs), filtered using a tangential flow filtration (TFF) system with different molecular weight cut-off categories, both at a dosage of 0.5 mL. The ELISA assay was employed to examine the cytokines and growth factors present in both SH-MSCs and SN-MSCs. On the ninth day, the rats were euthanized and liver tissues were collected for subsequent histological examination and analysis of mRNA expression. <b>Results:</b> The ELISA test revealed that SH-MSCs exhibited higher levels of VEGF, PDGF, bFGF, IL-10, TGF-ß and IL-6 compared to SN-MSCs. <i>In vivo</i>, administration of SH-MSCs notably decreased mortality rates. It also demonstrated a reduction in liver fibrosis, collagen fiber areas, α-SMA positive staining and relative mRNA expression of TGF-ß. Conversely, SN-MSCs also contributed to liver fibrosis improvement, although SH-MSCs demonstrated more favorable outcomes. <b>Conclusion:</b> Current findings suggested that SH-MSCs could improve CCl₄-induced liver fibrosis and decrease α-SMA and TGF-ß expression.

Meta-Analysis on Efficacy and Complications of Bone Morphogenetic Protein-2 for Posterior Fusion of Cervical Spine.

OBJECTIVE: Recombinant human bone morphogenetic protein-2 (rhBMP-2) is effective for promoting robust fusion for long-level cervical deformity and revision surgeries. However, only a few studies have reported its efficacy and complications in posterior cervical fusion (PCF). METHODS: Therefore we evaluated the efficacy and complications of rhBMP-2 application in PCF surgery by searching 3 electronic databases (PubMed, Cochrane Database, and EMBASE) for studies that evaluated the use of rhBMP-2 in PCF. Five studies (1 prospective and 4 retrospective) were included in the meta-analysis. RESULTS: The quality of each study was assessed, and data on pseudarthrosis, wound infection, neurologic, and immediate medical complications were extracted and analyzed. We found that the use of rhBMP-2 in PCF showed significant benefits in terms of pseudarthrosis and no significant increases in the risk for neurologic and immediate medical complications regardless of the dose. However, high-dose (>2.1 mg/level) rhBMP-2 was a risk factor for wound infection after PCF. CONCLUSIONS: Our meta-analysis of the currently available literature suggests that patients with PCF may benefit from BMP-2 usage without increasing the risk of complications. However, dose control and containment are important to ensure a low risk of complications.

Crocetin ameliorative effect on diabetic nephropathy in rats through a decrease in transforming growth factor-ß and an increase in glyoxalase-I activity.

BACKGROUND & AIMS: Glycation, oxidative stress, and inflammation due to the elevation of transforming growth factor-ß1 (TGF-ß1) participate in diabetic nephropathy (DN). Thus, we investigated for the first time the effect of crocetin (Crt) on the renal histopathological parameters, TGF-ß1 and glycation, oxidative stress, as well as inflammatory markers in the DN rat model. METHODS: Forty male Wistar rats were randomly divided into 4 equal groups: normal (N), N + Crt, DN, and DN + Crt. DN was induced in rats with a combination of nephrectomy and streptozotocin. Treated groups received 100 mg/kg of Crt via intraperitoneal injection monthly for 3 months. Different glycation (glycated albumin, glycated LDL, Methylglyoxal, and pentosidine), oxidative stress (advanced oxidation protein products, malondialdehyde, glutathione, and paraoxonase-I (PON-1)), and inflammatory markers (tumor necrosis factor-α, myeloperoxidase, and TGF-ß1), blood glucose, insulin, lipid profile, creatinine in the serum, and proteinuria, as well as the glyoxalase-1 (GLO-1) activity, was determined. RESULTS: Crt decreased renal biochemical (Cre and PU) and histopathological (glomerulosclerosis) renal dysfunction parameters, diverse glycation, oxidative stress, and inflammatory markers in the DN rats. Furthermore, the treatment corrected glycemia, insulin resistance, and dyslipidemia as well as induced the activities of GLO-1 and PON-1. Over and above, the treatment decreased TGF-ß1 in their serum (p > 0.001). CONCLUSIONS: Crocetin improved DN owing to an advantageous effect on metabolic profile. Further, the treatment with a reducing effect on TGF-ß1, oxidative stress, glycation, and inflammation markers along with an increase in Glo-1 activity showed multiple protective effects on kidney tissue.

Puerarin alleviates chronic renal failure-induced pyroptosis in renal tubular epithelial cells by targeting miR-342-3p/TGF-ß/SMAD axis.

BACKGROUND: Chronic renal failure (CRF) is the result of kidney damage. Puerarin is a flavonoid with specific nephroprotective effect, but its effect on CRF needs further research. This study explored the effect of puerarin on CRF and the potential molecular mechanism. METHODS: Adenine was used to establish an in vivo CRF model in rats, and rats were intragastrically administered with puerarin at a dose of 400 mg/kg body weight once a day from day 1 to day 28. Hematoxylin and eosin (HE) and Masson staining were used to observe the morphology and fibrosis of kidney tissue. Lipopolysaccharide (LPS) (400 ng/mL)/H2O2 (200 µM) was applied to human kidney 2 (HK-2) cells to construct an in vitro CRF model. Enzyme-linked immunosorbent assay (ELISA) was performed to validate interleukin (IL)-1ß and IL-18 levels. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to detect microRNA (miR)-342-3p levels. Transforming growth factor beta (TGF-ß)1, SMAD2, SMAD3, and pyroptosis marker proteins were detected by Western blot. The interaction between miR-342-3p and TGF-ß/SMAD was determined by a dual-luciferase reporter gene assay. Cell Counting Kit-8 (CCK-8) assay was utilized to determine cell viability. RESULTS: In the CRF model, puerarin alleviated renal injury and fibrosis and reduced creatinine (Cr) and blood urea nitrogen (BUN) levels. At the same time, miR-342-3p was downregulated, while the TGF-ß/SMAD axis was activated and levels of IL-1ß and IL-18 were increased. After treatment of CRF rats with puerarin, the expression level of miR-342-3p was increased, the TGF-ß/SMAD axis was inhibited, and the secretion of IL-1ß and IL-18 was decreased. MiR-342-3p directly bound to and negatively regulated the expression of TGF-ß1, SMAD2, and SMAD3. In the in vitro CRF model, miR-342-3p inhibited HK-2 cell pyroptosis by inhibiting the TGF-ß/SMAD axis. CONCLUSION: Puerarin reduced renal injury and pyroptosis in CRF rats by targeting the miR-342-3p/TGF-ß/SMAD axis.

Application of rhBMP in spinal fusion surgery: any correlation of cancer incidence? A systematic review and meta-analysis.

PURPOSE: Safety concerns regarding the application of bone morphogenetic proteins (BMPs) have been highlighted in recent years. It is noted that both BMP and their receptors being identified as a trigger for cancer growth. Here, we aimed to determine the safety and efficacy of BMP for spinal fusion surgery. METHODS: We conducted this systematic review on topics of spinal fusion surgery with rhBMP application from three database (PubMed, EuropePMC, and Clinicaltrials.gov) with MeSH phrases such as "rh-BMP," "rhBMP," "spine surgery," "spinal arthrodesis," and "spinal fusion" were searched (using the Boolean operators "and" and "or"). Our research includes all articles, as long as published in English language. In the face of disagreement between the two reviewers, we discussed it together until all authors reached a consensus. The primary key outcome of our study is the incidence of cancer following rhBMP implantation. RESULTS: Our study included a total of 8 unique studies (n = 37,682). The mean follow-up varies among all studies, with the longest follow-up is 66 months. Our meta-analysis showed that exposure to rhBMP in spinal surgery did increase the risk of cancers (RR 1.85, 95%CI [1.05, 3.24], p = 0.03). CONCLUSIONS: Our study found that rhBMP was not associated with the increased risk of cancer incidence within the rhBMP cohort. Still, we did face several limitations, in which further studies are needed to confirm the result of our meta-analysis.

Evaluation of the efficacy and safety of Escherichia coli-derived recombinant human bone morphogenetic protein-2 in transforaminal lumbar interbody fusion to treat degenerative spinal disease: a protocol of prospective, randomized controlled, assessor-blinded, open-label, multicenter trial.

BACKGROUND: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been widely used as an alternative bone graft in spine fusion surgery. However, clinical outcome such as effects and complications has not yet been revealed for transforaminal lumbar interbody fusion (TLIF). Although previous studies have reported some results, the evidence is weak. Therefore, the purpose of this trial is to evaluate the effectiveness and safety of Escherichia coli-derived rhBMP-2 combined with hydroxyapatite (HA) in TLIF. METHODS: This trial is designed as a prospective, assessor-blinded, open-label, multicenter, randomized controlled study. Participants will be recruited from six tertiary teaching hospitals. All randomized participants will be undergoing one- or two-level TLIF with rhBMP-2 (77 participants) as the active experimental group or with an auto-iliac bone graft (77 participants) as the control group. The primary interbody fusion rate outcome will be evaluated using computed tomography (CT) 12 months after surgery. The secondary outcomes will be as follows: clinical outcomes (visual analog scale score, EuroQol-5-dimensions-5-level score, Oswestry Disability Index score, and some surgery-related variables) and adverse effects (radiculitis, heterotrophic ossification, endplate resorption, and osteolysis). Radiological outcomes will be evaluated using simple radiography or CT. All outcomes will be measured, collected, and evaluated before surgery and at 12, 24, and 52 weeks postoperatively. DISCUSSION: This study will be the primary of its kind to evaluate the effectiveness and safety of E. coli-derived rhBMP-2 with HA in one- or two-level TLIF. It is designed to evaluate the equivalence of the results between rhBMP-2 with HA and auto-iliac bone graft using an appropriate sample size, assessor-blinded analyses, and prospective registration to avoid bias. This study will set up clear conclusions for using E. coli-derived rhBMP-2 with HA in TLIF. TRIAL REGISTRATION: This study protocol was registered at Korea Clinical Research Information Service ( https://cris.nih.go.kr ; number identifier: KCT0005610) on 19 November 2020. And protocol version is v1.1, January 2022.

Activin B promotes the initiation and progression of liver fibrosis.

The role of activin B, a transforming growth factor ß (TGFß) superfamily cytokine, in liver health and disease is largely unknown. We aimed to investigate whether activin B modulates liver fibrogenesis. Liver and serum activin B, along with its analog activin A, were analyzed in patients with liver fibrosis from different etiologies and in mouse acute and chronic liver injury models. Activin B, activin A, or both was immunologically neutralized in mice with progressive or established carbon tetrachloride (CCl4 )-induced liver fibrosis. Hepatic and circulating activin B was increased in human patients with liver fibrosis caused by several liver diseases. In mice, hepatic and circulating activin B exhibited persistent elevation following the onset of several types of liver injury, whereas activin A displayed transient increases. The results revealed a close correlation of activin B with liver injury regardless of etiology and species. Injured hepatocytes produced excessive activin B. Neutralizing activin B largely prevented, as well as improved, CCl4 -induced liver fibrosis, which was augmented by co-neutralizing activin A. Mechanistically, activin B mediated the activation of c-Jun-N-terminal kinase (JNK), the induction of inducible nitric oxide synthase (iNOS) expression, and the maintenance of poly (ADP-ribose) polymerase 1 (PARP1) expression in injured livers. Moreover, activin B directly induced a profibrotic expression profile in hepatic stellate cells (HSCs) and stimulated these cells to form a septa structure. Conclusions: We demonstrate that activin B, cooperating with activin A, mediates the activation or expression of JNK, iNOS, and PARP1 and the activation of HSCs, driving the initiation and progression of liver fibrosis.

Prevention of urethral fibrosis induced by transforming growth factor beta 1 using selective Wnt/ß-catenin signaling inhibitors in a rat model.

OBJECTIVES: To determine the anti-fibrotic effects of Wnt/ß-catenin signaling inhibitors on urethral stricture. METHODS: Human fibroblasts were exposed to transforming growth factor beta 1 combined with various concentrations of Wnt/ß-catenin inhibitors (ICG-001, IWR-1, and PRI-724), and cell proliferation and migration were evaluated. Urethral fibrosis was induced in male Sprague-Dawley rats by urethral injection of transforming growth factor beta 1 and co-treatement with inhibitors. Urethral tissues were harvested 2 weeks after the injection. The messenger ribonucleic acid and protein expression was examined for fibrosis markers Axin-1, collagen type 1, alpha smooth muscle actin, and ß-catenin. Histological analysis of fibrosis and collagen deposition was also performed. RESULTS: Cell migration was ameliorated by ICG-001 and PRI-724. Protein and messenger ribonucleic acid expression of collagen type 1 and alpha smooth muscle actin in transforming growth factor beta 1-treated fibroblasts decreased in a concentration-dependent manner with the ICG-001 and PRI-724 treatments (P < 0.05). However, there were no significant changes with the IWR-1 treatment. Collagen type I and alpha smooth muscle actin messenger ribonucleic acid and protein expression were both significantly increased in the urethral tissues of rats with transforming growth factor beta 1-induced urethral fibrosis. Rats co-treated with ICG-001 or PRI-724 showed relatively mild fibrosis and significantly reduced collagen type I and alpha smooth muscle actin messenger ribonucleic acid and protein expression (P < 0.05). CONCLUSIONS: ICG-001 and PRI-724 significantly ameliorated urethral fibrosis induced by transforming growth factor beta 1 in rats. These results suggest that ICG-001 and PRI-724 can be developed as therapeutics for treating urethral stricture.

Genome-wide CRISPR Screening to Identify Drivers of TGF-ß-Induced Liver Fibrosis in Human Hepatic Stellate Cells.

Liver fibrosis progression in chronic liver disease leads to cirrhosis, liver failure, or hepatocellular carcinoma and often ends in liver transplantation. Even with an increased understanding of liver fibrogenesis and many attempts to generate therapeutics specifically targeting fibrosis, there is no approved treatment for liver fibrosis. To further understand and characterize the driving mechanisms of liver fibrosis, we developed a high-throughput genome-wide CRISPR/Cas9 screening platform to identify hepatic stellate cell (HSC)-derived mediators of transforming growth factor (TGF)-ß-induced liver fibrosis. The functional genomics phenotypic screening platform described here revealed the novel biology of TGF-ß-induced fibrogenesis and potential drug targets for liver fibrosis.

LOXL2 Inhibitor Attenuates Angiotensin II-Induced Atrial Fibrosis and Vulnerability to Atrial Fibrillation through Inhibition of Transforming Growth Factor Beta-1 Smad2/3 Pathway.

OBJECTIVES: Angiotensin II (Ang II)-induced atrial fibrosis plays a vital role in the development of atrial fibrillation (AF). Lysyl oxidase-like 2 (LOXL2) plays an essential role in matrix remodeling and fibrogenesis, indicating it may involve fibrosis-associated diseases. This study aims to elucidate the role of LOXL2 in AF, and its specific inhibitor can suppress Ang II-induced inflammatory atrial fibrosis and attenuate the enhanced vulnerability to AF. METHODS: Male mice C57BL/6 were subcutaneously infused with either saline or Ang II (2 mg/kg/day) for 4 weeks. DMSO or LOXL2 inhibitor LOXL2-IN-1 hydrochloride (LOXL2-IN-1) at a dose of 100 µg/kg/day were intraperitoneally injected once daily for 4 weeks. Morphological, histological, and biochemical analyses were performed. AF was induced by transesophageal burst pacing in vivo. RESULTS: Expression of LOXL2 was increased in serum of AF patients and Ang II-treated mice. LOXL2-IN-1 significantly attenuated Ang II-induced AF vulnerability, cardiac hypertrophy, atrial inflammation, and fibrosis. LOXL2-IN-1 suppressed Ang II-induced expression of transforming growth factor beta-1 (TGF-ß1) and collagen I and phosphorylation of Smad2/3 in atrial tissue. CONCLUSIONS: LOXL2 is a target of AF, and its inhibitor prevents atrial fibrosis and attenuated enhanced vulnerability to AF potentially through the TGF-ß/Smad pathway.

Dose Adjustment Associated Complications of Bone Morphogenetic Protein: A Longitudinal Assessment.

OBJECTIVE: Bone morphogenetic protein (BMP) is a growth factor that aids in osteoinduction and promotes bone fusion. There is a lack of literature regarding recombinant human BMP-2 (rhBMP-2) dosage in different spine surgeries. This study aims to investigate the trends in rhBMP-2 dosage and the associated complications in spinal arthrodesis. METHODS: A retrospective study was conducted investigating spinal arthrodesis using rhBMP-2. Variables including age, procedure type, rhBMP-2 size, complications, and postoperative imaging were collected. Cases were grouped into the following surgical procedures: anterior lumbar interbody fusion/extreme lateral interbody fusion (ALIF/XLIF), posterior lumbar interbody fusion/transforaminal lumbar interbody fusion (PLIF/TLIF), posterolateral fusion (PLF), anterior cervical discectomy and fusion (ACDF), and posterior cervical fusion (PCF). RESULTS: A total of 1209 patients who received rhBMP-2 from 2006 to 2020 were studied. Of these, 230 were categorized as ALIF/XLIF, 336 as PLIF/TLIF, 243 as PLF, 203 as ACDF, and 197 as PCF. PCF (P < 0.001), PLIF/TLIF (P < 0.001), and PLF (P < 0.001) demonstrated a significant decrease in the rhBMP-2 dose used per level, with major transitions seen in 2018, 2011, and 2013, respectively. In our sample, 129 complications following spinal arthrodesis were noted. A significant relation between rhBMP-2 size and complication rates (χ2= 73.73, P = 0.0029) was noted. rhBMP-2 dosage per level was a predictor of complication following spinal arthrodesis (odds ratio = 1.302 [1.05-1.55], P < 0.001). CONCLUSIONS: BMP is an effective compound in fusing adjacent spine segments. However, it carries some regional complications. We demonstrate a decreasing trend in the dose/vertebral level. A decrease rhBMP-2 dose per level correlated with a decrease in complication rates.

MicroRNA­375 prevents TGF­ß­dependent transdifferentiation of lung fibroblasts via the MAP2K6/P38 pathway.

Transdifferentiation of lung fibroblasts to myofibroblasts is a crucial pathophysiological process in pulmonary fibrosis. MicroRNA­375 (miR­375) was initially identified as a tumor­suppressive factor, and its expression was negatively associated with the severity of lung cancer; however, its role and potential mechanism in myofibroblast transdifferentiation and pulmonary fibrosis remain unclear. In the present study, human lung fibroblasts were stimulated with transforming growth factor­ß (TGF­ß) to induce myofibroblast transdifferentiation. A mimic and inhibitor of miR­375, and their negative controls, were used to overexpress or suppress miR­375 in lung fibroblasts, respectively. The mRNA expression levels of fibrotic markers, and protein expression of α­smooth muscle actin and periostin, were subsequently detected by reverse transcription­quantitative PCR and western blotting, to assess myofibroblast transdifferentiation. miR­375 was markedly upregulated in human lung fibroblasts after TGF­ß stimulation. The miR­375 mimic alleviated, whereas the miR­375 inhibitor aggravated TGF­ß­dependent transdifferentiation of lung fibroblasts. Mechanistically, miR­375 prevented myofibroblast transdifferentiation and collagen synthesis by blocking the P38 mitogen­activated protein kinases (P38) pathway, and P38 suppression abrogated the deleterious effect of the miR­375 inhibitor on myofibroblast transdifferentiation. Furthermore, the present study revealed that mitogen­activated protein kinase kinase 6 was involved in P38 inactivation by miR­375. In conclusion, miR­375 was implicated in modulating TGF­ß­dependent transdifferentiation of lung fibroblasts, and targeting miR­375 expression may help to develop therapeutic approaches for treating pulmonary fibrosis.

Loss of NF-E2 expression contributes to the induction of profibrotic signaling in diabetic kidneys.

AIMS: This study aimed to examine the anti-fibrotic role of Nuclear Factor-Erythroid derived 2 (NF-E2) in human renal tubule (HK-11) cells and in type 1 and type 2 diabetic (T1D, T2D) mouse kidneys. MAIN METHODS: Anti-fibrotic effects of NF-E2 were examined in transforming growth factor-ß (TGF-ß) treated HK-11 cells by over-expressing/silencing NF-E2 expression and determining its effects on profibrotic signaling. NF-E2 proteasomal degradation was confirmed by proteasome inhibition in HK-11 cells and diabetic mice. Clinical relevance of changes in NF-E2 expression to fibrotic changes in the kidney were assessed in T1D and T2D mouse kidneys. KEY FINDINGS: NF-E2 expression was significantly decreased in TGF-ß treated HK-11 cells and in kidneys of diabetic mice with concurrent increase in expression of fibrotic proteins. TGF-ß treatment of HK-11 cells did not inhibit NF-E2 mRNA expression, suggesting that the post-translational changes may contribute to NF-E2 protein degradation. The down-regulation of NF-E2 expression was attributed to its proteasomal degradation, as TGF-ß- and diabetes-induced NF-E2 down regulation was prevented by proteasome inhibitor treatment. In HK-11 cells TGF-ß treatment decreased E-cadherin expression and induced pSer82Hsp27/NF-E2 association, likely to promote NF-E2 degradation, as Hsp27 can target proteins to the proteasome. A critical role for NF-E2 in regulation of renal fibrosis was demonstrated as over-expression of NF-E2 or silencing NF-E2 expression, decreased or increased profibrotic proteins in TGF-ß-treated HK-11 cells, respectively. SIGNIFICANCE: NF-E2, a novel anti-fibrotic protein, is down-regulated in diabetic kidneys. Preserving/inducing NF-E2 expression in diabetic kidneys may provide a therapeutic potential to combat DN.

Subacute TGFß Exposure Drives Airway Hyperresponsiveness in Cystic Fibrosis Mice through the PI3K Pathway.

Cystic fibrosis (CF) is a lethal genetic disease characterized by progressive lung damage and airway obstruction. The majority of patients demonstrate airway hyperresponsiveness (AHR), which is associated with more rapid lung function decline. Recent studies in the neonatal CF pig demonstrated airway smooth muscle (ASM) dysfunction. These findings, combined with observed CF transmembrane conductance regulator (CFTR) expression in ASM, suggest that a fundamental defect in ASM function contributes to lung function decline in CF. One established driver of AHR and ASM dysfunction is transforming growth factor (TGF) ß1, a genetic modifier of CF lung disease. Prior studies demonstrated that TGFß exposure in CF mice drives features of CF lung disease, including goblet cell hyperplasia and abnormal lung mechanics. CF mice displayed aberrant responses to pulmonary TGFß, with elevated PI3K signaling and greater increases in lung resistance compared with controls. Here, we show that TGFß drives abnormalities in CF ASM structure and function through PI3K signaling that is enhanced in CFTR-deficient lungs. CF and non-CF mice were exposed intratracheally to an adenoviral vector containing the TGFß1 cDNA, empty vector, or PBS only. We assessed methacholine-induced AHR, bronchodilator response, and ASM area in control and CF mice. Notably, CF mice demonstrated enhanced AHR and bronchodilator response with greater ASM area increases compared with non-CF mice. Furthermore, therapeutic inhibition of PI3K signaling mitigated the TGFß-induced AHR and goblet cell hyperplasia in CF mice. These results highlight a latent AHR phenotype in CFTR deficiency that is enhanced through TGFß-induced PI3K signaling.

Assessment of effects of rhBMP-2 on interbody fusion with a novel rat model.

BACKGROUND CONTEXT: The effects of using off-label recombinant human bone morphogenetic protein (rhBMP)-2 for interbody fusion are controversial. Although animal models of posterolateral fusion are well-established, establishing animal models to validate the safety and efficacy of interbody fusion is difficult, which may contribute to the inconsistent clinical results. PURPOSE: To develop a novel animal model of interbody fusion in rat coccygeal vertebrae without destroying bony endplates. STUDY DESIGN: An experimental animal study. METHODS: Forty-five male Sprague-Dawley rats underwent coccygeal interbody fusion without violating vertebral endplates. The animals were divided into three different groups based on the materials that were implanted into the interbody space (1) allogeneic iliac bone (IB) alone (IB group), (2) IB and 3 µg of rhBMP-2 (BMP low-dose group), or (3) IB and 10 µg of rhBMP-2 (BMP high-dose group). Fusion rates were investigated using microcomputed tomography 6 weeks after the operation. The incidence of adverse events, including soft-tissue swelling, delayed wound healing, osteolysis, and ectopic bone formation were evaluated. The total number of adverse events (using the adverse event score) in each group and the swelling ratio (calculated using the surgical site tissue volume [TV; TV on postoperative day 1/preoperative TV]) were also evaluated. RESULTS: The fusion rates in the BMP low- and high-dose groups (33.3% and 46.7%) were not significantly different, but both were significantly higher than that in the IB group (0%) (p=.042 and .006, respectively). Significant differences in the incidence of osteolysis, adverse event scores, and swelling ratios were observed only between the BMP high-dose and IB groups (p=.043, .006 and .014, respectively). CONCLUSIONS: We developed a novel rat model of interbody fusion in which the vertebral endplates were not violated, reflecting the normal clinical setting. rhBMP-2 use increased the fusion rate, but a higher dose of rhBMP-2 did not lead to a higher fusion rate than that for low-dose rhBMP-2; conversely, it led to an increase in the occurrence of adverse events. CLINICAL SIGNIFICANCE: This novel rat model of coccygeal interbody fusion that preserved bony endplates has clinical significance for validating the effectiveness of biologics or bone graft substitutes before clinical trial.

The Effect of Enteral Nutrition Support Rich in TGF-ß in the Treatment of Inflammatory Bowel Disease in Childhood.

Background and Objective: Malnutrition is a major complication of inflammatory bowel disease (IBD). Our aim of the study was to examine the effects of Modulen IBD supplementation, which was administered to IBD patients without limiting their daily diet in addition to medical treatment, on the clinical, laboratory, anthropometric values, and disease activities of these patients. Materials and Methods: Seventy three children with IBD were evaluated retrospectively. The cases were classified as those who had Crohn disease receiving (CD-M; n = 16) or not receiving Modulen IBD (CD; n = 19) and those who had ulcerative colitis receiving (UC-M; n = 13) or not receiving Modulen IBD (UC; n = 25). Disease activities, laboratory values, remission rates, and anthropometric measurements of the groups were compared. In addition to IBD treatment, Modulen IBD in which half of the daily calorie requirement was provided was given for eight weeks. Results: In the third month of treatment, 14 (88%) patients were in remission in CD-M group and eight (42%) patients were in remission in CD group. The height and weight z scores, which were low at the time of diagnosis, improved in the first week in CD-M group. Inflammatory parameters (UC) were significantly lower in the UC-M group compared to the UC group in first and third months. In the third month, eight (62%) patients in the UC-M group and four (16%) in the UC group were remitted clinically and in terms of laboratory values. Conclusions: TGF-ß-rich enteral nutrition support in children with IBD is an easy, effective, and reliable approach. It was shown that TGF-ß-rich enteral nutritional supplementation enabled the disease to enter the remission earlier, and contributed to the early recovery of weight and height scores.

PARP-1/2 Inhibitor Olaparib Prevents or Partially Reverts EMT Induced by TGF-ß in NMuMG Cells.

Poly- adenosine diphosphate (ADP)-ribose (PAR) is a polymer synthesized as a posttranslational modification by some poly (ADP-ribose) polymerases (PARPs), namely PARP-1, PARP-2, tankyrase-1, and tankyrase-2 (TNKS-1/2). PARP-1 is nuclear and has also been detected in extracellular vesicles. PARP-2 and TNKS-1/2 are distributed in nuclei and cytoplasm. PARP or PAR alterations have been described in tumors, and in particular by influencing the Epithelial- Mesenchymal Transition (EMT), which influences cell migration and drug resistance in cancer cells. Pro-EMT and anti-EMT effects of PARP-1 have been reported while whether PAR changes occur specifically during EMT is currently unknown. The PARP-1/2 inhibitor Olaparib (OLA) is approved by FDA to treat certain patients harboring cancers with impaired homologous recombination. Here, we studied PAR changes and OLA effects on EMT. Total and nuclear PAR increased in EMT while PAR belts were disassembled. OLA prevented EMT, according to: (i) molecular markers evaluated by immuno-cytofluorescence/image quantification, Western blots, and RNA quantitation, (ii) morphological changes expressed as anisotropy, and (iii) migration capacity in the scratch assay. OLA also partially reversed EMT. OLA might work through unconventional mechanisms of action (different from synthetic lethality), even in non-BRCA (breast cancer 1 gene) mutated cancers.

Inhibition of BRD4 attenuates transverse aortic constriction- and TGF-ß-induced endothelial-mesenchymal transition and cardiac fibrosis.

Cardiac fibrosis (CF), a process characterized by potentiated proliferation of cardiac fibroblasts and excessive secretion and deposition of extracellular matrix (ECM) from the cells, contributes strongly to the pathogenesis of a series of cardiovascular (CV) diseases, including AMI, heart failure and atrial fibrillation. Endothelial-mesenchymal transition (EndMT), one of the sources of transformed cardiac fibroblasts, has been reported as a key factor involved in CF. However, the molecular basis of EndMT has not been thoroughly elucidated to date. At the posttranscriptional level, of the three epigenetic regulators, writer and eraser are reported to be involved in EndMT, but the role of reader in the process is still unknown. In this study, we aimed to explore the role of Bromodomain-containing protein 4 (BRD4), an acetyl-lysine reader protein, in EndMT-induced CF and related mechanisms. We found that BRD4 was upregulated in endothelial cells (ECs) in the pressure-overload mouse heart and that its functional inhibitor JQ1 potently attenuated the TAC-induced CF and preserved cardiac function. In umbilical vein endothelial cells (HUVECs) and mouse aortic endothelial cells (MAECs), bothJQ1 and shRNA-mediated silencing of BRD4 blocked TGF-ß-induced EC migration, EndMT and ECM synthesis and preserved the EC sprouting behavior, possibly through the downregulation of a group of transcription factors specific for EndMT (Snail, Twist and Slug), the Smads pathway and TGF-ß receptor I. In the absence of TGF-ß stimulation, ectopic expression of BRD4 alone could facilitate EndMT, accelerate migration and increase the synthesis of ECM. In vivo, JQ1 also attenuated TAC-induced EndMT and CF, which was consistent with JQ1's intracellular mechanisms of action. Our results showed that BRD4 plays a critical role in EndMT-induced CF and that targeting BRD4 might be a novel therapeutic option for CF.

Postoperative Seroma Formation After Posterior Cervical Fusion with Use of RhBMP-2: A Report of Two Cases.

CASE: We present 2 cases of postoperative seroma formation following posterior cervical fusion with the use of recombinant human bone morphogenetic protein-2 (rhBMP-2). CONCLUSION: Although some who advocate for the off-label use of rhBMP-2 in patients undergoing posterior cervical spine fusion believe it to be safe, relatively little has been published regarding complication rates. We believe that rhBMP-2 carries a risk of seroma formation in patients who undergo posterior cervical fusion, which necessitates the use of a postoperative drain. Surgeons should have a low threshold for obtaining postoperative magnetic resonance imaging in a symptomatic patient.

Pseudo-Pedicle Heterotopic Ossification From Use of Recombinant Human Bone Morphogenetic Protein 2 (rhBMP-2) in Transforaminal Lumbar Interbody Fusion Cages.

We conducted a study to determine the common characteristics of patients who developed radiculopathy symptoms and corresponding heterotopic ossification (HO) from transforaminal lumbar interbody fusions (TLIF) using recombinant human bone morphogenetic protein 2 (rhBMP-2). HO can arise from a disk space with rhBMP-2 use in TLIF. Formation of bone around nerve roots or the thecal sac can cause a radiculopathy with a consistent pattern of symptoms. We identified 38 patients (26 males, 12 females) with a mean (SD) age of 50.8 (7.5) years who developed radiculopathy symptoms and corresponding HO from TLIF with rhBMP-2 in the disk space between 2002 and 2015. To document this complication and improve its recognition, we recorded common patterns of symptom development and radiologic findings: specifically, time from implantation of rhBMP-2 to symptom development, consistency with side of TLIF placement, and radiologic findings. Radicular pain generally developed a mean (SD) of 3.8 (1.0) months after TLIF with rhBMP-2. Development of radiculopathy symptoms corresponded to consistent "pseudo-pedicle"-like HO. In all 38 patients, HO arising from the annulotomy site showed a distinct pseudo-pedicle pattern encompassing nerve roots and the thecal sac. In addition, development of radiculopathy symptoms and corresponding HO appear to be independent of amount of rhBMP-2. HO resulting from TLIF with rhBMP-2 in the disk space is a pain generator and a recognizable complication that can be diagnosed by assessment of symptoms and computed tomography characteristics.