The Effect of Combining Hyaluronic Acid and Human Dermal Fibroblasts on Tendon Healing.

BACKGROUND: The incidence of rotator cuff tears is rapidly increasing, and operative techniques for rotator cuff repair have been developed. However, the rates of postoperative retear remain high. PURPOSE/HYPOTHESIS: The purpose was to determine the effects of human dermal fibroblasts (HDFs) with hyaluronic acid (HA) on tendon-to-bone healing in a rabbit model of chronic rotator cuff tear injury. It was hypothesized that HA would enhance HDF proliferation and that a combination of HA and HDFs would produce a synergistic effect on the healing of repaired rotator cuff tendons of rabbits. STUDY DESIGN: Controlled laboratory study. METHODS: For in vitro study, HDFs were plated on a 24-well plate. After 1 day, 2 wells were designated as the test group and treated with 0.75% HA in phenol red-free Dulbecco's modified Eagle medium (DMEM). An other 2 wells served as control groups and were treated with the same volume of phenol red-free DMEM without HA. Each group was duplicated, resulting in a total of 4 wells, with 2 wells in each group for replication purposes. The cells were incubated for 24 hours, followed by 72-hour cultivation. Absorbance ratios at 96 and 24 hours were compared to evaluate cell proliferation. For the in vivo study, a total of 24 rabbits were randomly allocated to groups A, B, and C (n = 8 each). Supraspinatus tendons were detached bilaterally and left for 6 weeks to establish a chronic rotator tear model. Torn tendons were subsequently repaired using the following injections: group A, 0.5 × 106 HDFs with HA; group B, HA only; and group C, saline only. At 12 weeks after repair, biomechanical tests and histological evaluation were performed. RESULTS: In vitro study showed that HDF proliferation significantly increased with HA (HDFs with HA vs HDFs without HA; 3.96 ± 0.09 vs 2.53 ± 0.15; P < .01). In vivo, group A showed significantly higher load-to-failure values than the other groups (53.8 ± 6.9 N/kg for group A, 30.6 ± 6.4 N/kg for group B, and 24.3 ± 7.6 N/kg for group C; P < .001). Histological evaluation confirmed that group A showed higher collagen fiber density and better collagen fiber continuity, tendon-to-bone interface maturation, and nuclear shape than the other groups (all P < .05). CONCLUSION: This controlled laboratory study verified the potential of the combination of HDFs and HA in enhancing healing in a chronic rotator cuff tear rabbit model. CLINICAL RELEVANCE: A potential synergistic effect on rotator cuff tendon healing may be expected from a combination of HDFs and HA.

Allogeneic Dermal Fibroblasts Improve Tendon-to-Bone Healing in a Rabbit Model of Chronic Rotator Cuff Tear Compared With Platelet-Rich Plasma.

PURPOSE: To compare the effects of allogeneic dermal fibroblasts (ADFs) and platelet-rich plasma (PRP) on tendon-to-bone healing in a rabbit model of chronic rotator cuff tear. METHODS: Thirty-two rabbits were divided into 4 groups (8 per group). In 2 groups, the supraspinatus tendon was detached and was left as such for 6 weeks. At 6 weeks after creating the tear model, we performed transosseous repair with 5 × 106 ADFs plus fibrin injection in the left shoulder and PRP plus fibrin in the right shoulder. The relative expression of the COL1, COL3, BMP2, SCX, SOX9, and ACAN genes was assessed at 4 weeks (group A) and 12 weeks (group B) after repair. Histologic and biomechanical evaluations of tendon-to-bone healing at 12 weeks were performed with ADF injection in both shoulders in group C and PRP injection in group D. RESULTS: At 4 weeks, COL1 and BMP2 messenger RNA expression was higher in ADF-injected shoulders (1.6 ± 0.8 and 1.0 ± 0.3, respectively) than in PRP-injected shoulders (1.0 ± 0.3 and 0.6 ± 0.3, respectively) (P = .019 and P = .013, respectively); there were no differences in all genes in ADF- and PRP-injected shoulders at 12 weeks (P > .05). Collagen continuity, orientation, and maturation of the tendon-to-bone interface were better in group C than in group D (P = .024, P = .012, and P = .013, respectively) at 12 weeks, and mean load to failure was 37.4 ± 6.2 N/kg and 24.4 ± 5.2 N/kg in group C and group D, respectively (P = .015). CONCLUSIONS: ADFs caused higher COL1 and BMP2 expression than PRP at 4 weeks and showed better histologic and biomechanical findings at 12 weeks after rotator cuff repair of the rabbit model. ADFs enhanced healing better than PRP in the rabbit model. CLINICAL RELEVANCE: This study could serve as a transitional study to show the effectiveness of ADFs in achieving tendon-to-bone healing after repair of chronic rotator cuff tears in humans.

Safety and Efficacy of Autologous Dermal Fibroblast Injection to Enhance Healing After Full-Thickness Rotator Cuff Repair: First-in-Human Pilot Study.

BACKGROUND: There is growing interest in various biological supplements to improve tendon healing in patients after arthroscopic rotator cuff repair. The ideal biological supplement to strengthen rotator cuff remains unknown. PURPOSE: To assess the safety and efficacy of autologous cultured dermal fibroblast (ADF) injection on tendon-to-bone healing in patients after arthroscopic rotator cuff repair. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Included were 6 patients who underwent arthroscopic rotator cuff repair between June 2018 and March 2020; all patients had a full-thickness rotator cuff tear (>2 cm) involving the supraspinatus and infraspinatus tendons. The patients were injected with ADF between the repaired tendon and footprint during arthroscopic rotator cuff repair using the suture bridge technique. The safety of ADFs and the procedure was evaluated at 5 weeks postoperatively, and the anatomical healing of the repaired tendon was accessed at 6 months postoperatively using magnetic resonance imaging and at 12 months using ultrasonography. Outcomes including shoulder range of motion (ROM), visual analog scale (VAS) for pain, and functional scores were measured at 6 and 12 months postoperatively. RESULTS: Adverse reactions to ADF injection were not observed at 6 months after surgery. VAS and functional scores at 6 and 12 months postoperatively were significantly improved compared with preoperative scores (all P < .05). However, there was no significant difference on any ROM variable between preoperative and postoperative measurements at 6 and 12 months (all P > .05). No healing failure was found at 6 and 12 months postoperatively. CONCLUSION: There was no adverse reaction to ADF injection, and all patients had successful healing after rotator cuff repair. A simple and easily accessible ADF injection may be a novel treatment option for increasing the healing capacity of torn rotator cuff tendons. Further clinical research is needed to verify the study results.

Rotator Cuff Tendon Healing Using Human Dermal Fibroblasts: Histological and Biomechanical Analyses in a Rabbit Model of Chronic Rotator Cuff Tears.

BACKGROUND: Tenocytes derived from tendons have been reported to be effective in the treatment of rotator cuff tears through the expression of extracellular matrix proteins. Human dermal fibroblasts, known to express collagen types I and III as tenocytes do, may likely be substitutes for tenocytes to enhance healing rotator cuff tears. PURPOSE: To demonstrate the capability of human dermal fibroblasts to enhance healing of rotator cuff tears. STUDY DESIGN: Controlled laboratory study. METHODS: The cellular properties and expression profiles of growth factors were compared between human dermal fibroblasts and tenocytes. In both cell types, a series of extracellular matrix proteins were analyzed along with matrix metalloproteinases and tissue inhibitors of metalloproteinases involved in the collagenolytic system. A total of 35 rabbits were divided into 5 groups: normal (n = 2), saline control (n = 9), fibrin control (n = 9), low dose of human fibroblasts (HF-LD; n = 9), and high dose of human fibroblasts (HF-HD; n = 6). Cells were injected into the sutured lesions at 6 weeks after creation of bilateral rotator cuff tears, followed by histological and biomechanical analyses at 12 weeks. RESULTS: Human dermal fibroblasts exhibited a protein expression pattern similar to that of tenocytes. More specifically, the expression levels of collagen types I and III were comparable between fibroblasts and tenocytes. The histological analysis of 30 surviving rabbits showed that collagen fibers were more continuous and better oriented with a more mature interface between the tendon and bone in the sutured lesions in the HF-LD and HF-HD groups. Most importantly, biomechanical strength, measured using the load to failure at the injection site, was 58.8 ± 8.9 N/kg in the HF-HD group, increasing by approximately 2-fold (P = .0003) over the saline control group. CONCLUSION: Human dermal fibroblasts, showing cellular properties comparable with tenocytes, effectively enhanced healing of chronic rotator cuff tears in rabbits. CLINICAL RELEVANCE: Human dermal fibroblasts can be used in place of tenocytes to enhance healing of rotator cuff tears.

Identification of XAF1-MT2A mutual antagonism as a molecular switch in cell-fate decisions under stressful conditions.

XIAP-associated factor 1 (XAF1) is a tumor suppressor that is commonly inactivated in multiple human neoplasms. However, the molecular mechanism underlying its proapoptotic function remains largely undefined. Here, we report that XAF1 induction by heavy metals triggers an apoptotic switch of stress response by destabilizing metallothionein 2A (MT2A). XAF1 directly interacts with MT2A and facilitates its lysosomal degradation, resulting in the elevation of the free intercellular zinc level and subsequent activation of p53 and inactivation of XIAP. Intriguingly, XAF1 is activated as a unique transcription target of metal-regulatory transcription factor-1 (MTF-1) in signaling apoptosis, and its protein is destabilized via the lysosomal pathway by MTF-1-induced MT2A under cytostatic stress conditions, indicating the presence of mutual antagonism between XAF1 and MT2A. The antagonistic interplay between XAF1 and MT2A acts as a key molecular switch in MTF-1-mediated cell-fate decisions and also plays an important role in cell response to various apoptotic and survival factors. Wild-type (WT) XAF1 but not MT2A binding-deficient mutant XAF1 increases the free intracellular zinc level and accelerates WT folding of p53 and degradation of XIAP. Consistently, XAF1 evokes a more drastic apoptotic effect in p53+/+ versus isogenic p53-/- cells. Clinically, expression levels of XAF1 and MT2A are inversely correlated in primary colon tumors and multiple cancer cell lines. XAF1-depleted xenograft tumors display an increased growth rate and a decreased apoptotic response to cytotoxic heavy metals with strong MT2A expression. Collectively, this study uncovers an important role for XAF1-MT2A antagonism as a linchpin to govern cell fate under various stressful conditions including heavy metal exposure.

XAF1 directs apoptotic switch of p53 signaling through activation of HIPK2 and ZNF313.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a tumor suppressor that is frequently inactivated in many human cancers. However, the molecular mechanism underlying its growth-inhibitory function remains largely unknown. Here, we report that XAF1 forms a positive feedback loop with p53 and acts as a molecular switch in p53-mediated cell-fate decisions favoring apoptosis over cell-cycle arrest. XAF1 binds directly to the N-terminal proline-rich domain of p53 and thus interferes with E3 ubiquitin ligase MDM2 binding and ubiquitination of p53. XAF1 stimulates homeodomain-interacting protein kinase 2 (HIPK2)-mediated Ser-46 phosphorylation of p53 by blocking E3 ubiquitin ligase Siah2 interaction with and ubiquitination of HIPK2. XAF1 also steps up the termination of p53-mediated cell-cycle arrest by activating zinc finger protein 313 (ZNF313), a p21(WAF1)-targeting ubiquitin E3 ligase. XAF1 interacts with p53, Siah2, and ZNF313 through the zinc finger domains 5, 6, and 7, respectively, and truncated XAF1 isoforms preferentially expressed in cancer cells fail to form a feedback loop with p53. Together, this study uncovers a novel role for XAF1 in p53 stress response, adding a new layer of complexity to the mechanisms by which p53 determines cell-fate decisions.

PPARδ promotes oncogenic redirection of TGF-ß1 signaling through the activation of the ABCA1-Cav1 pathway.

TGF-ß1 plays biphasic functions in prostate tumorigenesis, inhibiting cell growth at early stages but promoting malignant progression at later stages. However, the molecular basis for the oncogenic conversion of TGF-ß1 function remains largely undefined. Here, we demonstrate that PPARδ is a direct transcription target of TGF-ß1 and plays a critical role in oncogenic redirection of TGF-ß1 signaling. Blockade of PPARδ induction enhances tumor cell response to TGF-ß1-mediated growth inhibition, while its activation promotes TGF-ß1-induced tumor growth, migration and invasion. PPARδ-mediated switch of TGF-ß1 function is associated with down- and upregulation of Smad and ERK signaling, respectively, and tightly linked to its function to activate ABCA1 cholesterol transporter followed by caveolin-1 (Cav1) induction. Intriguingly, TGF-ß1 activation of the PPARδ-ABCA1-Cav1 pathway facilitates degradation of TGF-ß receptors (TßRs) and attenuates Smad but enhances ERK response to TGF-ß1. Expression of PPARδ and Cav1 is tightly correlated in both prostate tissues and cell lines and significantly higher in cancer vs. normal tissues. Collectively, our study shows that PPARδ is a transcription target of TGF-ß1 and contributes to the oncogenic conversion of TGF-ß1 function through activation of the ABCA1-Cav1-TßR signaling axis.

Caveolin-1 increases aerobic glycolysis in colorectal cancers by stimulating HMGA1-mediated GLUT3 transcription.

Caveolin-1 (CAV1) acts as a growth suppressor in various human malignancies, but its expression is elevated in many advanced cancers, suggesting the oncogenic switch of its role during tumor progression. To understand the molecular basis for the growth-promoting function of CAV1, we characterized its expression status, differential roles for tumor growth, and effect on glucose metabolism in colorectal cancers. Abnormal elevation of CAV1 was detected in a substantial fraction of primary tumors and cell lines and tightly correlated with promoter CpG sites hypomethylation. Depletion of elevated CAV1 led to AMPK activation followed by a p53-dependent G1 cell-cycle arrest and autophagy, suggesting that elevated CAV1 may contribute to ATP generation. Furthermore, CAV1 depletion downregulated glucose uptake, lactate accumulation, and intracellular ATP level, supporting that aerobic glycolysis is enhanced by CAV1. Consistently, CAV1 was shown to stimulate GLUT3 transcription via an HMGA1-binding site within the GLUT3 promoter. HMGA1 was found to interact with and activate the GLUT3 promoter and CAV1 increased the HMGA1 activity by enhancing its nuclear localization. Ectopic expression of HMGA1 increased glucose uptake, whereas its knockdown caused AMPK activation. In addition, GLUT3 expression was strongly induced by cotransfection of CAV1 and HMGA1, and its overexpression was observed predominantly in tumors harboring high levels of CAV1 and HMGA1. Together, these data show that elevated CAV1 upregulates glucose uptake and ATP production through HMGA1-mediated GLUT3 transcription, suggesting that CAV1 may render tumor cells growth advantages by enhancing aerobic glycolysis.

Epigenetic alteration of PRKCDBP in colorectal cancers and its implication in tumor cell resistance to TNFα-induced apoptosis.

PURPOSE: PRKCDBP is a putative tumor suppressor in which alteration has been observed in several human cancers. We investigated expression and function of PRKCDBP in colorectal cells and tissues to explore its candidacy as a suppressor in colorectal tumorigenesis. EXPERIMENTAL DESIGN: Expression and methylation status of PRKCDBP and its effect on tumor growth were evaluated. Transcriptional regulation by NF-κB signaling was defined by luciferase reporter and chromatin immunoprecipitation assays. RESULTS: PRKCDBP expression was hardly detectable in 29 of 80 (36%) primary tumors and 11 of 19 (58%) cell lines, and its alteration correlated with tumor stage and grade. Promoter hypermethylation was commonly found in cancers. PRKCDBP expression induced the G(1) cell-cycle arrest and increased cellular sensitivity to various apoptotic stresses. PRKCDBP was induced by TNFα, and its level correlated with tumor cell sensitivity to TNFα-induced apoptosis. PRKCDBP induction by TNFα was disrupted by blocking NF-κB signaling while it was enhanced by RelA transfection. The PRKCDBP promoter activity was increased in response to TNFα, and this response was abolished by disruption of a κB site in the promoter. PRKCDBP delayed the formation and growth of xenograft tumors and improved tumor response to TNFα-induced apoptosis. CONCLUSIONS: PRKCDBP is a proapoptotic tumor suppressor which is commonly altered in colorectal cancer by promoter hypermethylation, and its gene transcription is directly activated by NF-κB in response to TNFα. This suggests that PRKCDBP inactivation may contribute to tumor progression by reducing cellular sensitivity to TNFα and other stresses, particularly under chronic inflammatory microenvironment.

NF-kappaB activates transcription of the RNA-binding factor HuR, via PI3K-AKT signaling, to promote gastric tumorigenesis.

BACKGROUND & AIMS: HuR is a RNA-binding factor whose expression is commonly upregulated in some human tumor types. We explored the molecular mechanism underlying HuR elevation and its role in gastric cancer tumorigenesis. METHODS: HuR expression and subcellular localization were determined by polymerase chain reaction, immunoblot, and immunohistochemical analyses. Its effect on tumor growth was characterized using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and soft agar analyses. Luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were used to measure transcriptional activation by nuclear factor kappaB (NF-kappaB) signaling. RESULTS: Compared with normal gastric tissues, HuR was expressed at higher levels in gastric tumors, particularly in advanced versus early tumors; this increase was associated with enhanced cytoplasmic translocation of HuR. HuR overexpression increased proliferation of tumor cells, activating the G(1) to S transition of the cell cycle, DNA synthesis, and anchorage-independent growth. Small interfering RNA-mediated knockdown of HuR expression reduced tumor cell proliferation and response to apoptotic stimuli. No genetic or epigenetic alterations of HuR were observed in gastric tumor cell lines or primary tumors; overexpression depended on phosphatidylinositol 3-kinase/AKT signaling and NF-kappaB activity. AKT activation increased p65/RelA binding to a putative NF-kappaB binding site in the HuR promoter, the stability of HuR target transcripts, and the cytoplasmic import of HuR. CONCLUSIONS: HuR is a direct transcription target of NF-kappaB; its activation in gastric cancer cell lines depends on phosphatidylinositol 3-kinase/AKT signaling. HuR activation by this pathway has proliferative and antiapoptotic effects on gastric cancer cells.

Frequent alteration of XAF1 in human colorectal cancers: implication for tumor cell resistance to apoptotic stresses.

BACKGROUND & AIMS: X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) is a candidate tumor suppressor located at the chromosome 17p13 region, but the molecular basis underlying its inactivation in human tumors and growth-inhibiting function has not been well defined. We explored the candidacy of XAF1 as a suppressor in colorectal tumorigenesis. METHODS: XAF1 expression was characterized by polymerase chain reaction-based cloning, isoform-specific polymerase chain reaction, ribonuclease protection, and immunoblot assays. Allelic loss of the gene was evaluated by loss of heterozygosity (LOH) assay, and promoter CG dinucleotide (CpG) site methylation was determined using bisulfite sequencing. The effect of XAF1 on tumor growth was examined using flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, colony formation, and viability assays. RESULTS: Expression of 5 XAF1 variants including 2 novel transcripts was down-regulated concomitantly in 11 of 20 (55%) cell lines and 26 of 65 (40%) primary tumors. XAF1 reduction was tumor-specific and showed a correlation with advanced stage and high grade of tumor. LOH of the gene was found in 12 of 33 (36%) tumors. Promoter CpG site methylation was observed frequently in both cell lines and tumor tissues including many LOH tumors, suggesting that biallelic inactivation of XAF1 might be common in colorectal cancers. XAF1 expression suppressed tumor cell growth and enhanced cellular response to various apoptotic stimuli, such as 5-fluorouracil, etoposide, H(2)O(2), gamma-irradiation, ultraviolet, and tumor necrosis factor-alpha, whereas knockdown of its expression protected cells from the stresses. CONCLUSIONS: Genetic and epigenetic alteration of XAF1 is a common event in colorectal tumorigenesis and contributes to the malignant tumor progression by providing survival advantages for tumor cells under various stress conditions.

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L.

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.