TGF-β1 Promotes Lymphangiogenesis during Peritoneal Fibrosis

Peritoneal fibrosis (PF) causes ultrafiltration failure (UFF) and is a complicating factor in long-term peritoneal dialysis. Lymphatic reabsorption also may contribute to UFF, but little is known about lymphangiogenesis in patients with UFF and peritonitis. We studied the role of the lymphangiogenesis mediator vascular endothelial growth factor-C (VEGF-C) in human dialysate effluents, peritoneal tissues, and peritoneal mesothelial cells (HPMCs). Dialysate VEGF-C concentration correlated positively with the dialysate-to-plasma ratio of creatinine (D/P Cr) and the dialysate TGF-β1 concentration. Peritoneal tissue from patients with UFF expressed higher levels of VEGF-C, lymphatic endothelial hyaluronan receptor-1 (LYVE-1), and podoplanin mRNA and contained more lymphatic vessels than tissue from patients without UFF. Furthermore, mesothelial cell and macrophage expression of VEGF-C increased in the peritoneal membranes of patients with UFF and peritonitis. In cultured mesothelial cells, TGF-β1 upregulated the expression of VEGF-C mRNA and protein, and this upregulation was suppressed by a TGF-β type I receptor (TGFβR-I) inhibitor. TGF-β1–induced upregulation of VEGF-C mRNA expression in cultured HPMCs correlated with the D/P Cr of the patient from whom the HPMCs were derived (P<0.001). Moreover, treatment with a TGFβR-I inhibitor suppressed the enhanced lymphangiogenesis and VEGF-C expression associated with fibrosis in a rat model of PF. These results suggest that lymphangiogenesis associates with fibrosis through the TGF-β–VEGF-C pathway.The decrease in ultrafiltration capacity that is associated with the high peritoneal solute transport that is observed after prolonged peritoneal dialysis (PD) treatment is a major reason for its discontinuation.^1–4 Several studies have shown that a higher peritoneal solute transport rate is associated with reduced survival of PD patients.^1,2,5 The characteristic features of chronic peritoneal damage in PD treatment are associated with submesothelial fibrosis and neoangiogenesis.^6,7 Analyses of the surface peritoneum showed no significant changes in vessel density with duration of PD.^6,8 In addition, the vessel density in patients with ultrafiltration failure (UFF) was significantly higher than the vessel density in normal individuals or non-PD patients, but it was not higher than the vessel density in patients undergoing PD.⁶ These findings suggest that factors other than increased vascular density may be involved in disease states associated with increased transport of peritoneal membranes. In addition, the relationship between peritoneal fibrosis and UFF remains obscure.Blood capillaries have a continuous basal lamina with tight interendothelial junctions and are supported by pericytes and smooth muscle cells. In contrast, lymphatic capillaries are thin-walled with a wide lumen and do not contain pericytes or basement membrane. The structures of lymphatic vessels are suitable for the removal of tissue fluid, cells, and macromolecules from the interstitium.^9–11 If lymphangiogenesis develops in the peritoneal membrane, absorption of the PD fluid could be increased and lead to UFF. An increase in the number of lymphatic vessels has recently been reported in several disease conditions, including tumor metastasis,^12–15 chronic respiratory inflammatory diseases,^16–18 wound healing,¹⁹ and renal transplant rejection.^20,21 We recently reported that lymphangiogenesis had developed in tubulointerstitial fibrosis of human renal biopsy specimens,²² and we also reported the mechanisms of lymphangiogenesis in rat unilateral ureteral obstruction models.²³The lymphatic absorption rate, which is measured by the rate at which intraperitoneally administered radioactive serum albumin or macromolecule dextran 70 disappears, is significantly higher in patients with UFF, and lymphatic reabsorption is considered to be one of the causes of UFF.^24–27 However, the results from these clinical approaches have been controversial.^28,29 In addition, little is known about the pathology and the process of lymphangiogenesis in patients with UFF and peritonitis.In this study, we investigated lymphangiogenesis and the expression of vascular endothelial growth factor-C (VEGF-C), which is a potentially important mediator of lymphangiogenesis, in human peritoneal tissues, PD effluent, and peritoneal mesothelial cells. We also explored VEGF-C induction by TGF-β1 in the human mesothelial cell line (Met-5A) and cultured human peritoneal mesothelial cells (HPMCs) from the spent PD effluent of patients with varying rates of peritoneal transport. Finally, we explored the relationship between peritoneal fibrosis and lymphangiogenesis in rats that were administered chlorhexidine gluconate (CG) into the abdominal cavity, which provides a model of chemically induced peritoneal inflammation/fibrosis.^30–32 This work is the first report to show that lymphangiogenesis is linked to the peritoneal fibrosis that is often associated with a high peritoneal transport rate.     

Platelet‐rich plasma protects rotator cuff‐derived cells from the deleterious effects of triamcinolone acetonide

Triamcinolone acetonide (TA) injections are widely used to treat enthesopathy, but they may induce adverse effects such as tendon impairment and rupture. Platelet‐rich plasma (PRP) is a blood fraction containing high platelet concentrations and various growth factors that play a role in tissue repair processes. The purpose of this study is to investigate whether TA has deleterious effects on human rotator cuff‐derived cells, and if PRP can protect these cells from the effects of TA. Human rotator cuff‐derived cells were cultured with and without TA and PRP, and the culture without any additive served as the control. Cell morphology was assessed at days 7 and 21. Cell viability was evaluated at days 1, 7, 14, and 21 by a water‐soluble tetrazolium salt assay. Induction of apoptosis was measured by immunofluorescence staining and flow cytometry at day 7. Induction of cleaved caspase‐3 was measured by immunofluorescence staining at day 7. The cells cultured with TA had a flattened and polygonal shape at day 7. The cells cultured with both TA and PRP were similar in appearance to control cells. Exposure to TA also significantly decreased cell viability, but cell viability did not decrease when PRP was added along with TA. The number of apoptotic cells increased with TA exposure, while addition of PRP prevented cell apoptosis. In conclusion, the deleterious effect of TA was prevented by PRP, which can be used as a protective agent for patients receiving local TA injections. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 976–982, 2013     

<Emphasis Type="Italic">KRAS</Emphasis> and <Emphasis Type="Italic">BRAF</Emphasis> Mutations in 203 Esophageal Squamous Cell Carcinomas: Pyrosequencing Technology and Literature Review

Background

Epidermal growth factor receptor (EGFR) signaling is one of the most promising targets for molecular-targeted therapies in esophageal squamous cell carcinoma (ESCC). Thus, the molecular diagnosis of KRAS and BRAF mutations is clinically important in therapeutic decision making. However, the frequency of KRAS and BRAF mutations in ESCCs remains inconclusive because of the limited sample sizes of previous studies (all N ≤ 80). Pyrosequencing is a nonelectrophoretic nucleotide extension sequencing technology that can be used for mutation testing.

Methods

The frequency of KRAS and BRAF mutations was examined using a nonbiased database of 203 resected ESCCs and a high-throughput pyrosequencing assay.

Results

The validity of the KRAS pyrosequencing method was initially demonstrated by detection of all 4 types of KRAS mutations [c.35G>T (codon 12 GGT>GTT), c.35G>A (codon 12 GGT>GAT), c.34G>T (codon 12 GGT>TGT), c.38G>A mutation (codon 13 GGC>GAC)], which had been previously diagnosed using Scorpion-ARMS technology, in 9 colon cancer tissues (9 of 9; 100 %). Similar results were demonstrated for BRAF mutational status in 3 colon cancer cell lines (HCT116, Colo201, and HT29), which were validated by Sanger dideoxy sequencing. Subsequently, the KRAS mutation was found to be extremely rare (1 of 203; 0.5 %), and the BRAF mutation was absent (0 of 203; 0 %), in the dataset of 203 ESCCs.

Conclusions

These results suggest that KRAS and BRAF mutations play a limited role in the development of ESCC and that mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in ESCC.

     

Bisphenol A induces a shift in sex differentiation gene expression with testis-ova or sex reversal in Japanese medaka (Oryzias latipes)

Bisphenol A (BPA), a very important raw material in the plastics industry, is an endocrine-disrupting chemical in teleost fish. Although BPA induces testis-ova and sex reversal in teleost fish species, the molecular mechanism remains unclear. We evaluated the effects of BPA (measured concentrations: 45, 92, 326, 1030 and 3406 μg/L) on Japanese medaka (Oryzias latipes) using OECD TG234 (2011, Fish Sexual Development Test, OECD Guidelines for the Testing of Chemicals, Section 2). BPA at 1030 and 3406 μg/L induced testis-ova and sex reversal with female-type secondary sexual characteristics in XY males at 30 and 60 days posthatching (dph). Then we examined the BPA effect on the expression of sex differentiation genes related to the testis-ova and sex reversal in XY medaka. BPA exposure (1030 and 3406 μg/L) suppressed gsdf mRNA expression and increased cyp19a1a mRNA expression in XY individuals at stage 38 and 30 dph, although foxl2 mRNA expression showed no change. Interestingly, the concentration of BPA that suppressed gsdf mRNA expression at the larval stage was consistent with that needed to induce testis-ova and sex reversal. These results suggest that the gsdf gene at the embryonic stage can be used as a useful biomarker for predicting the impact of estrogenic endocrine-disrupting chemicals on sexual differentiation in Japanese medaka.     

Surgical Apgar Score Predicted Postoperative Morbidity After Esophagectomy for Esophageal Cancer

Background

Recently, a simple and easy complication prediction system, the Surgical Apgar Sore (SAS) calculated by three intraoperative parameters (estimated blood loss, lowest mean arterial pressure, and lowest heart rate), has been proposed for general surgery. This study aimed to determine if the SAS could accurately predict perioperative morbidity in patients undergoing esophagectomy for esophageal cancer.

Methods

We investigated 399 patients who underwent esophagectomy at the Kumamoto University Hospital between April 2007 and March 2015. Clinical data, including intraoperative parameters, were collected retrospectively. Patients had postoperative morbidities classified as Clavien–Dindo grade III or more. Univariate and multivariate analyses were performed to elucidate factors that affected the development of complications.

Results

The mean age of the study population was 65.7 years, 357 patients (89.5 %) were male. The frequency of any morbidity was 32.3 %. Univariate analyses showed that the SAS as well as preoperative chemotherapy, volume of bleeding, and reconstruction of organs were associated with morbidities. Multivariate analysis showed that a SAS < 5 was found to be an independent risk factor for morbidities.

Conclusion

The SAS is considered to be useful for predicting the development of postoperative morbidities after esophagectomy for esophageal cancer.