Canonical and Non-Canonical Functions of Erythropoietin and Its Receptor in Mature Nucleated Erythrocytes of Western Clawed Frog, Xenopus tropicalis.

Enucleated erythrocytes are characteristic of adult mammals. In contrast, fish, amphibians, reptiles, birds, and fetal mammals possess nucleated erythrocytes in their circulation. Erythroid maturation is regulated by erythropoietin (EPO) and its receptor (EPOR), which are conserved among vertebrates. In mammals, EPOR on the erythroid progenitor membrane disappears after terminal differentiation. However, in western clawed frog, Xenopus tropicalis, mature erythrocytes maintain EPOR expression, suggesting that they have non-canonical functions of the EPO-EPOR axis rather than proliferation and differentiation. In this study, we investigated the non-canonical functions of EPOR in Xenopus mature erythrocytes. EPO stimulation of peripheral erythrocytes did not induce proliferation but induced phosphorylation of intracellular proteins, including signal transducer and activator of transcription 5 (STAT5). RNA-Seq analysis of EPO-stimulated peripheral erythrocytes identified 45 differentially expressed genes (DEGs), including cytokine inducible SH2 containing protein gene (cish) and suppressor of cytokine signaling 3 gene (socs3), negative regulators of the EPOR-Janus kinase (JAK)-STAT pathway. These phosphorylation studies and pathway analysis demonstrated the activation of the JAK-STAT pathway through EPO-EPOR signaling in erythrocytes. Through comparison with EPO-responsive genes in mouse erythroid progenitors obtained from a public database, we identified 31 novel EPO-responsive genes indicating non-canonical functions. Among these, we focused on ornithine decarboxylase 1 gene (odc1), which is the rate-limiting enzyme in polyamine synthesis and affects hematopoietic progenitor differentiation and the endothelial cell response to hypoxic stress. An EPO-supplemented culture of erythrocytes showed increased odc1 expression followed by a decrease in polyamine-rich erythrocytes, suggesting EPO-responsive polyamine excretion. These findings will advance our knowledge of the unknown regulatory systems under the EPO-EPOR axis and functional differences between vertebrates' nucleated and enucleated erythrocytes.

The Selective Serotonin 2A Receptor Antagonist Sarpogrelate Prevents Cardiac Hypertrophy and Systolic Dysfunction via Inhibition of the ERK1/2-GATA4 Signaling Pathway.

Drug repositioning has recently emerged as a strategy for developing new treatments at low cost. In this study, we used a library of approved drugs to screen for compounds that suppress cardiomyocyte hypertrophy. We identified the antiplatelet drug sarpogrelate, a selective serotonin-2A (5-HT2A) receptor antagonist, and investigated the drug's anti-hypertrophic effect in cultured cardiomyocytes and its effect on heart failure in vivo. Primary cultured cardiomyocytes pretreated with sarpogrelate were stimulated with angiotensin II, endothelin-1, or phenylephrine. Immunofluorescence staining showed that sarpogrelate suppressed the cardiomyocyte hypertrophy induced by each of the stimuli. Western blotting analysis revealed that 5-HT2A receptor level was not changed by phenylephrine, and that sarpogrelate suppressed phenylephrine-induced phosphorylation of ERK1/2 and GATA4. C57BL/6J male mice were subjected to transverse aortic constriction (TAC) surgery followed by daily oral administration of sarpogrelate for 8 weeks. Echocardiography showed that 5 mg/kg of sarpogrelate suppressed TAC-induced cardiac hypertrophy and systolic dysfunction. Western blotting revealed that sarpogrelate suppressed TAC-induced phosphorylation of ERK1/2 and GATA4. These results indicate that sarpogrelate suppresses the development of heart failure and that it does so at least in part by inhibiting the ERK1/2-GATA4 signaling pathway.

Investigation of the effects of urea cycle amino acids on the expression of ALB and CEBPB in the human hepatocellular carcinoma cell line FLC-4.

Cell lines are powerful tools for research into liver function at the molecular level. However, they are generally unsuitable for rigorously assessing the effects of amino acid composition, because many lines require serum-containing medium for their maintenance. Here, we aimed to investigate the effects of ornithine and arginine, which are included in the characteristic metabolic process in hepatocyte, on a human hepatoma-derived cell line (FLC-4) that can be cultured in serum-free medium. FLC-4 cells were cultured under the following three conditions: + ornithine/ - arginine, - ornithine/ - arginine, and -ornithine/ + arginine. Albumin expression evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay and showed no obvious differences based on the presence of ornithine or arginine. However, the mRNA levels of two liver-enriched transcription factors (CEBPB and HNF1A), which are involved in regulating albumin expression, were significantly higher in cells grown in medium-containing arginine than that in cells grown in ornithine-containing medium. Western blotting showed that the levels both activating and inhibitory C/EBPß isoforms were significantly increased in cells grown in arginine medium. Furthermore, we have found that depletion of both ornithine and arginine, the polyamine sources, in the medium did not cause polyamine deficiency. When ornithine and arginine were depleted, albumin production was significantly reduced at the mRNA level, CEBPB mRNA levels were increased, and the level of activating form of C/EBPß was increased. The results of this study suggest that in hepatocyte, these two amino acids might have different functions, and because of which they elicit disparate cellular responses.

Novel biallelic splice-site BBS1 variants in Bardet-Biedle syndrome: a case report of the first Japanese patient.

PURPOSE: To report the clinical and genetic features of a 9-year-old female Japanese patient with Bardet-Biedl syndrome (BBS). METHODS: Genetic analysis using whole-exome sequencing (WES) was performed for the patient and her parents to identify disease-causing variants. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to investigate the impact of splice-site variants. Comprehensive ophthalmic and systemic examinations, including electroretinography (ERG), were performed. RESULTS: In the patient, WES identified novel compound heterozygous splice-site variants (c.124+2T>G and c.723+2T>G) in the BBS1 gene, and RT-PCR revealed skipping of exons 2 and 8 (p.N17AfsX56 and p.T198_K241del). Each parent had one of the variants. Ophthalmologically, the patient's decimal best-corrected visual acuity was 0.6 in the right eye and 0.4 in the left eye. Funduscopy revealed no apparent retinal degeneration or narrowed blood vessels in the periphery, but macular abnormalities were found on fundus autofluorescence imaging and optical coherence tomography images. Unexpectedly, non-recordable responses in rod ERG were found, with a non-recordable response of the right eye and an extremely reduced and delayed a-wave of the left eye in standard ERG, non-recordable responses in cone ERG, and extremely decreased responses in 30 Hz flicker ERG. Finally, the patient fulfilled four primary features of BBS diagnostic criteria: rod-cone dystrophy, polydactyly, central obesity, and learning disabilities, being diagnosed with BBS. CONCLUSIONS: This is the first report of a BBS patient with biallelic splice-site BBS1 variants in the Japanese population. Disparity between funduscopic and ERG findings may be a feature of BBS1-associated rod-cone dystrophy.

Compound heterozygous splice site variants in the SCLT1 gene highlight an additional candidate locus for Senior-Løken syndrome.

Senior Løken syndrome (SLS) is a heterogeneous disorder characterized by severe retinal degenerations and juvenile-onset nephronophthisis. Genetic variants in ten different genes have been reported as the causes of SLS. Clinical evaluation of a patient with SLS and her unaffected parents revealed that the patient had infantile-onset retinal dystrophy and juvenile-onset nephronophthisis. Other systemic abnormalities included hepatic dysfunction, megacystis, mild learning disability, autism, obesity, and hyperinsulinemia. Whole-exome sequencing identified compound heterozygous SCLT1 variants (c.1218 + 3insT and c.1631A > G) in the patient. The unaffected parents were heterozygous for each variant. Transcript analysis using reverse transcription PCR demonstrated that the c.1218 + 3insT variant leads to exon 14 skipping (p.V383_M406del), while the other variant (c.1631A > G) primarily leads to exon 17 skipping (p.D480EfsX11) as well as minor amounts of two transcripts (6 bps deletion in the last of exon 17 [p.V543_K544del] and exons 17 and 18 skipping [p.D480E, S481_K610del]). Immunohistochemical analysis demonstrated that the Sclt1 protein was localized to the distal appendage of the photoreceptor basal body, indicating a ciliary protein. In conclusion, we identified compound heterozygous splice site variants of SCLT1 in a patient with a new form of ciliopathies that exhibits clinical features of SLS.

Polyamine flux suppresses histone lysine demethylases and enhances ID1 expression in cancer stem cells.

Cancer stem cells (CSCs) exhibit tumorigenic potential and can generate resistance to chemotherapy and radiotherapy. A labeled ornithine decarboxylase (ODC, a rate-limiting enzyme involved in polyamine [PA] biosynthesis) degradation motif (degron) system allows visualization of a fraction of CSC-like cells in heterogeneous tumor populations. A labeled ODC degradation motif system allowed visualization of a fraction of CSC-like cells in heterogeneous tumor populations. Using this system, analysis of polyamine flux indicated that polyamine metabolism is active in CSCs. The results showed that intracellular polyamines inhibited the activity of histone lysine 4 demethylase enzymes, including lysine-specific demethylase-1 (LSD1). Chromatin immunoprecipitation with Pol II antibody followed by massively parallel DNA sequencing, revealed the global enrichment of Pol II in transcription start sites in CSCs. Increase of polyamines within cells resulted in an enhancement of ID1 gene expression. The results of this study reveal details of metabolic pathways that drive epigenetic control of cancer cell stemness and determine effective therapeutic targets in CSCs.

Novel ubiquitin-independent nucleolar c-Myc degradation pathway mediated by antizyme 2.

The proto-oncogene c-Myc encodes a short-lived protein c-Myc that regulates various cellular processes including cell growth, differentiation and apoptosis. Degradation of c-Myc is catalyzed by the proteasome and requires phosphorylation of Thr-58 for ubiquitination by E3 ubiquitin ligase, Fbxw7/ FBW7. Here we show that a polyamine regulatory protein, antizyme 2 (AZ2), interacts with c-Myc in the nucleus and nucleolus, to accelerate proteasome-mediated c-Myc degradation without ubiquitination or Thr-58 phosphorylation. Polyamines, the inducer of AZ2, also destabilize c-Myc in an AZ2-dependent manner. Knockdown of AZ2 by small interfering RNA (siRNA) increases nucleolar c-Myc and also cellular pre-rRNA whose synthesis is promoted by c-Myc. AZ2-dependent c-Myc degradation likely operates under specific conditions such as glucose deprivation or hypoxia. These findings reveal the targeting mechanism for nucleolar ubiquitin-independent c-Myc degradation.

Multisite Lymphaticovenular Bypass Using Supermicrosurgery Technique for Lymphedema Management in Lower Lymphedema Cases.

BACKGROUND: The impact of lymphaticovenous anastomosis on lymphedema has yet to be defined. The authors investigated the clinical evidence regarding the effectiveness of lymphaticovenous anastomosis in lower limb lymphedema. METHODS: Eighty-four patients (162 limbs; 73 female and 11 male patients) with lower limb lymphedema who underwent multisite lymphaticovenous anastomosis in the authors' clinic between August of 2010 and May of 2014 were included in this retrospective study. Lymphedema was diagnosed using lymphoscintigraphy and indocyanine green lymphography. All lymphaticovenous anastomoses were performed under local anesthesia. The lymphatic vessels that were identified were classified using the normal, ectasis, contraction, and sclerosis type (NECST) classification. Limb circumference, subjective symptoms, and frequency of cellulitis were evaluated. RESULTS: The average patient age was 60 years (range, 24 to 94 years); mean postoperative follow-up period was 18.3 months (range, 6 to 51 months). The postoperative change rate in limb circumference indicated that 67 limbs (47.7 percent) were classified as improved, 35 (27.3 percent) were classified as stable, and 32 (25 percent) were classified as worse. Postoperative interview revealed improvement in subjective symptoms in 67 limbs (61.5 percent), no change in 38 (34.9 percent), and exacerbation in four (3.7 percent). The postoperative mean occurrence of cellulitis was decreased to 0.13 times per year compared with 0.89 preoperatively, which was statistically significant (p = 0.00084). Multiple regression analysis using the postanastomosis limb circumference and NECST classification confirmed the following results: change rate (percent) = -0.40 + (0.30 × N) + (-0.84 × E) + (0.22 × C) + (-0.61 × S). CONCLUSION: Lymphaticovenous anastomosis is effective for lower limb lymphedema, in point of limb circumference, subjective symptoms, and the frequency of cellulitis. CLINCAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Polyamine regulating protein antizyme binds to ATP citrate lyase to accelerate acetyl-CoA production in cancer cells.

Antizyme (AZ) regulates cellular polyamines (i.e., putrescine, spermidine, and spermine) through binding to ornithine decarboxylase and subsequent ubiquitin-independent degradation of the enzyme protein by the 26S proteasome. Screening for AZ-binding proteins using a yeast two-hybrid system identified ATP citrate lyase (ACLY), a cytosolic enzyme which catalyzes the production of acetyl-CoA that is used for lipid anabolism or acetylation of cellular components. We confirmed that both AZ1 and AZ2 bind to ACLY and AZ colocalizes with ACLY to the cytoplasm. Unexpectedly, neither AZ1 nor AZ2 accelerated ACLY degradation. Additionally, purified AZ, particularly AZ1, increased the activity of purified ACLY in a dose-dependent manner in vitro, suggesting that AZ activates ACLY through protein-protein interaction. Polyamines themselves had no effect on the ACLY activity in vitro. Knockdown of AZ1 and/or AZ2 in human cancer cells significantly decreased the ACLY activity as well as cellular levels of acetyl-CoA and cholesterol. Our results are the first to show the crosstalk between polyamine and acetyl-CoA metabolism. We hypothesize that AZ may promote acetyl-CoA synthesis to downregulate spermidine and spermine through acetylation.

Combined conservative treatment and lymphatic venous anastomosis for severe lower limb lymphedema with recurrent cellulitis.

BACKGROUND: Lymphedema may be treated either conservatively or surgically. Although conservative therapy is the first-line treatment, some patients are refractory to it and repeat severe cellulitis. We usually perform lymphaticovenous anastomosis (LVA) for lymphedema patients, and LVA can reduce the frequency of cellulitis. CASE REPORT: A 67-year-old woman who had undergone a radical hysterectomy, pelvic lymphadenectomy, and postoperative radiotherapy for cervical cancer at the age 50 years. She developed lymphedema in both legs, and high-pressure compression stockings caused lymphorrhea in the groin and thigh, resulting in recurrent episodes of cellulitis. Lymphoscintigraphy revealed dilation of the lymphatic vessels in both legs. Results of an indocyanine green test revealed dermal backflow throughout the lower body. After wearing low-pressure stocking, we performed LVA to reduce cellulitis. After confirming the result of LVA, the patients started wearing high-pressure stocking. The patient underwent a subsequent LVA, 3 months after the first, to further improve edema. The lymphorrhea resolved, and cellulitis did not recur. CONCLUSIONS: The combination of surgical treatment and conservative treatment is important for severe lymphedema treatment. Although conservative treatment is usually said to be the first-line treatment, LVA can antecede in cases refractory to conservative treatment.

MicroRNA-21 is a candidate driver gene for 17q23-25 amplification in ovarian clear cell carcinoma.

BACKGROUND: Epithelial ovarian cancer (EOC) is the most common cause of gynecological malignancy-related mortality. Ovarian clear cell carcinoma (CCC) has unique clinical characteristics and behaviors that differ from other histological types of EOC, including a frequent association with endometriosis and a highly chemoresistant nature, resulting in poor prognosis. However, factors underlying its malignant behavior are still poorly understood. Aberrant expression of microRNAs has been shown to be involved in oncogenesis, and microRNA-21 (miR-21) is frequently overexpressed in many types of cancers. The aim of this study was to investigate the role of miR-21 in 17q23-25 amplification associated with CCC oncogenesis. METHODS: We identified 17q23-25 copy number aberrations among 28 primary CCC tumors by using a comparative genomic hybridization method. Next, we measured expression levels of the candidate target genes, miR-21 and PPM1D, for 17q23-25 amplification by real-time RT-PCR analysis and compared those data with copy number status and clinicopathological features. In addition, immunohistochemical analysis of PTEN (a potential target of miR-21) was performed using the same primary CCC cases. We investigated the biological significance of miR-21 overexpression in CCC using a loss-of-function antisense approach. RESULTS: 17q23-25 amplification with both miR-21 overexpression and PTEN protein loss was detected in 4/28 CCC cases (14.2%). The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively). A significant correlation was observed between miR-21 overexpression and endometriosis. Both PTEN mRNA and PTEN protein expression were increased by miR-21 knockdown in CCC cells. We also confirmed that miR-21 directly bound to the 3'-untranslated region of PTEN mRNA using a dual-luciferase reporter assay. CONCLUSIONS: MiR-21 is a possible driver gene other than PPM1D for 17q23-25 amplification in CCC. Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene.

Rho-kinase regulation of TNF-α-induced nuclear translocation of NF-κB RelA/p65 and M-CSF expression via p38 MAPK in mesangial cells.

The small GTPase Rho and its downstream effector, Rho-associated coiled-coil containing protein kinase (Rho-kinase), regulate a number of cellular processes, including organization of the actin cytoskeleton, cell adhesion, and migration. While pharmacological inhibitors of Rho-kinase signaling are known to block renal inflammation, the molecular basis for this effect is unclear. Here, we provide evidence that proinflammatory TNF-α promotes mesangial expression of macrophage colony-stimulating factor (M-CSF), a key regulator for the growth and differentiation of mononuclear phagocytes, in a Rho-kinase-dependent manner. Consistent with this observation, TNF-α-mediated renal expression of M-CSF in insulin-resistant db/db mice was downregulated by Rho-kinase inhibition. Small interfering RNA-facilitated knockdown of Rho-kinase isoforms ROCK1 and ROCK2 indicated that both isoforms make comparable contributions to regulation of M-CSF expression in mesangial cells. From a mechanistic standpoint, Western blotting and EMSA showed that Rho-kinase and its downstream target p38 MAPK regulate nuclear translocation of NF-κB RelA/p65 and subsequent DNA binding activity, with no significant effects on IκBα degradation and RelA/p65 phosphorylation. Moreover, we showed that Rho-kinase-mediated cytoskeletal organization is required for the nuclear uptake of RelA/p65. Collectively, these findings identify Rho-kinase as a critical regulator of chemokine expression and macrophage proliferation.

Predictive lymphatic mapping: a method for mapping lymphatic channels in patients with advanced unilateral lymphedema using indocyanine green lymphography.

In severe lymphedema, indocyanine green lymphography cannot be used to map lymphatic channels before lymphaticovenular anastomosis (LVA) because linear lymphatics cannot be detected in a severely affected leg. Here, we describe a new method, which we refer to as predictive lymphatic mapping, to predict the location of lymphatics for anastomosis in unilateral lymphedema, thereby improving surgical accuracy and efficiency. The approach consists of marking anatomical landmarks and joining selected landmarks with fixed lines. The distance from these fixed lines to lymphatic channels mapped by indocyanine green lymphography in the unaffected leg is then measured, scaled up based on the difference in circumference between the legs, and transposed to the affected leg. To date, we have used this method in 5 cases of unilateral or asymmetric lymphedema of the lower extremities. In no cases have we failed to find a lymphatic channel suitable for LVA within a 2-cm incision. These results suggest that predictive lymphatic mapping is a useful additional tool for surgeons performing LVA under local anesthesia, which will help to improve the accuracy of incisions and the efficiency of surgery.

High-accuracy diagnosis and regional classification of lymphedema using indocyanine green fluorescent lymphography after gynecologic cancer treatment.

BACKGROUND: Secondary lymphedema is defined as swelling of the limbs caused by retention of lymph after cancer therapy. We diagnosed lymphedema using indocyanine green (ICG) fluorescent lymphography and developed a classification based on 12 regional types of edema in the lower bodies, with the goal of improved understanding of the pathology. METHODS: The subjects were 72 consecutive female patients aged 25 to 88 years (mean, 54.5 years) with secondary lymphedema of the lower extremities and abdominal area. The traditional diagnosis of lymphedema was stages 0, 1, 2, 3 and 4 in 5, 11, 19, 24, and 13 patients, respectively. All patients were examined by ICG lymphography. RESULTS: Features of dermal backflow were noted in most patients after cancer therapy, and the incidence was particularly high after radiotherapy. Regional analysis of lymphedema was classified into 12 types (A to L, definitions are given for major categories). The number of patients (number receiving radiation therapy in parentheses) in each type were A, 1 (0); B, 3 (1); C, 13 (1); D, 1 (0); E, 2 (0); F, 0 (0); G, 1 (0); H, 7 (3); I, 13 (3); J, 6 (2); K, 20 (3); and L, 5 (2). CONCLUSIONS: The ICG test permits definite diagnosis of lymphedema at a very early stage and in mild cases. The regional analysis enables establishment of policies for conservative or surgical treatment (for example, lymphaticovenous anastomosis) for individual regions, thereby facilitating more effective lymphedema treatment.

Multiple forms of mouse antizyme inhibitor 1 mRNA differentially regulated by polyamines.

Antizyme inhibitor 1 (Azin1), a positive regulator of cellular polyamines, is induced by various proliferative stimuli and repressed by polyamines. It has been reported that the translational repression of Azin1 by polyamines involves an upstream open reading frame on the mRNA, but little has been known about polyamine effect on its transcription or splicing. We found multiple forms of Azin1 transcripts formed by alternative splicing and initiation of transcription from putative alternative start sites. One of the novel splice variants, Azin1-X, has a premature termination codon on 5' extension of exon 7, encodes a C-terminal truncated form of protein (Azin1ΔC), and is subject to nonsense-mediated mRNA decay. 2-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, increased both transcription from the canonical transcription start site and the ratio of the full-length mRNA to Azin1-X mRNA, whereas polyamines show the opposite effect. Thus, polyamines regulate two novel steps of Azin1 expression, namely the transcription and a particular splicing pattern, both of which may affect the level of mRNA encoding the full-length active Azin1 protein.

The effect of lymphatico-venous anastomosis for an intractable ulcer at the lower leg in a marked obese patient.

Secondary lymphedema occurs after trauma, cancer surgery, or obesity, and wounds in lymphedema can easily become intractable. We report positive results using lymphatico-venous anastomosis (LVA) to treat a post-traumatic lymph fistula and an intractable ulcer in a severely obese patient. A 41-year-old male (BMI 51.8), one year prior, had a traffic injury, and had an 18-cm contusion in his right leg. Six months later, lymph leakage in a 14 cm × 8 cm region and a 5 cm × 3 cm skin ulcer occurred in the center of the wound. We made a diagnosis of lymphedema resulting from obesity, accompanied with lymphorrhea and intractable ulcer. He was unable to reach his legs owing to obesity, making complex physical therapy impossible. We performed LVA under local anesthesia. The lymphorrhea healed 2 weeks after the operation and had not recurred 3 months after the operation. The leg lymphedema improved after the surgery without the compression therapy. In cases of intractable ulcers, suspected of being caused by lymphostasis, treatments indicated for lymphedema, for example LVA, may possibly allow satisfactory wound healing. © 2013 Wiley Periodicals, Inc. Microsurgery 34:64-67, 2014.

Rho-kinase inhibition prevents the progression of diabetic nephropathy by downregulating hypoxia-inducible factor 1α.

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic nephropathy. Accumulating evidence shows that hypoxia-inducible factor-1α (HIF-1α) is a key regulator of renal sclerosis under diabetic conditions. However, the interactions of Rho-kinase and HIF-1α in the development of renal dysfunction have not been defined. Here, we assessed whether Rho-kinase blockade attenuates HIF-1α induction and the subsequent fibrotic response using type 2 diabetic mice and cultured mesangial cells. Fasudil, a Rho-kinase inhibitor, reduced urinary albumin excretion, mesangial matrix expansion, and the expression of fibrotic mediators in db/db mice. Mechanistically, HIF-1α accumulation and the expression of its target genes that contribute to diabetic glomerulosclerosis were also prevented by fasudil in the renal cortex. In mesangial cells, Rho/Rho-kinase signaling was activated under hypoxic conditions. Further in vitro studies showed that pharmacological and genetic inhibition of Rho-kinase promoted proteasomal HIF-1α degradation, which subsequently suppressed HIF-1-dependent profibrotic gene expression by upregulation of prolyl hydroxylase 2. Thus, we found a previously unrecognized renoprotective mechanism for the effects of Rho-kinase inhibition and this could be a potential therapeutic target for the treatment of diabetic nephropathy.

[A case of early gastric cancer responding to adjuvant chemotherapy for treatment of colon cancer].

A 63-year-old man underwent colonoscopy owing to a positive fecal occult blood test, and he was diagnosed as having advanced sigmoid colon cancer. Esophagogastroduodenoscopy( EGD), performed as a preoperative examination, revealed an 8 mm early gastric cancer in the lower body of his stomach. We performed laparoscopic sigmoid colon resection and D3 lymphadenectomy first because the patient had advanced sigmoid colon cancer. The histopathological diagnosis was Stage II; however, vascular invasion was apparent. Some authors have reported that chemotherapy with 5-fluorouracil (FU) is effective against early gastric cancer; therefore, we administered postoperative adjuvant chemotherapy comprising uracil and tegafur( UFT)+Leucovorin( LV) tablets before ESD for early gastric cancer. Two months later, follow-up EGD showed that the gastric cancer had become flat and small. Five months later, it resembled a scar, and examination of a biopsy showed no malignant finding. We continued to administer chemotherapy to the patient for 6 months. Nine months after the discontinuation of chemotherapy, EGD showed only a scar and biopsy revealed no malignant finding.

Indocyanine green lymphography is superior to lymphoscintigraphy in imaging diagnosis of secondary lymphedema of the lower limbs.

OBJECTIVE: Lymphedema is commonly viewed as difficult to treat, but lymphaticovenous anastomosis applied early after onset can be curative in some cases. Therefore, early diagnosis of cancer-related lymphedema is important. Lymphoscintigraphy is currently the most common method used for imaging diagnosis of lymphedema, but indocyanine green fluorescence lymphangiography (ICG lymphography) is also increasingly used for this purpose. The goal of this study was to compare the accuracy of these methods for diagnosis of lymphedema. METHODS: This was a prospective comparative study, conducted at a general hospital in Japan. The subjects were 29 consecutive patients (all female; age range, 32-79 years) with lymphedema (58 limbs, including healthy ones) after gynecologic cancer care who underwent lymphedema treatment at The University of Tokyo and Saiseikai Kawaguchi General Hospital between April 2011 and December 2011. All subjects were referred to our department for lower extremity lymphoscintigraphy and ICG lymphography. The sensitivity and specificity of lymphoscintigraphy and ICG lymphography were calculated for all limbs and for diagnosis of early lymphedema in affected limbs (International Society of Lymphology stages 0 and I). In each analysis, receiver-operating characteristic curves were prepared to compare the accuracy of the two methods. RESULTS: In receiver-operating characteristic analysis of 58 limbs, the area under the curve was 0.72642 for lymphoscintigraphy and 0.90943 for ICG lymphography. In 34 limbs with early lymphedema, the area under the curve was 0.55882 for lymphoscintigraphy and 0.81471 for ICG lymphography. CONCLUSIONS: ICG lymphography was more accurate than lymphoscintigraphy for detecting lymphedema and was particularly useful for diagnosis of early lymphedema. This is clinically important since early diagnosis may permit curative treatment of lymphedema.

Low-invasive lymphatic surgery and lymphatic imaging for completely healed intractable pudendal lymphorrhea after gynecologic cancer treatment.

Lower limb lymphedema and an accompanying lymphatic fistula (lymphorrhea) occur as complications after gynecologic surgery to treat cancer. Herein, we report the case of a 68-year-old woman who underwent resection and radiotherapy because of uterine cervical cancer (stage 2a) 20 years previously. Left lower limb and pudendal lymphedema and continuous lymphorrhea developed soon after surgery. Conservative treatment was administered; however, the edema increased, and a pudendal lymphatic fistula and cellulitis developed repeatedly. Lymphovascular anastomosis (LVA) and lymph vessel ligation were performed after preoperative evaluation via lymphoscintigraphy and indocyanine green (ICG) lymphography. A radioisotope injected into the first interdigit pedal region flowed into the pudendal region via the inguinal lymph nodes at preoperative lymphoscintigraphy. Linear patterns were observed up to the half level of the crus, and stardust patterns occurred over the lower abdominal and pudendal regions at ICG lymphography. During surgery, ICG lymphography was also used to identify the site of the fistula. With the patient under local anesthesia, LVA was applied in the half crus and left inguinal regions, followed by ligation and division of lymph vessels flowing into the fistula. The region around the fistula was excised as a 1 × 3-cm tissue block. As of 5 months after surgery, no recurrence of lymphatic fistula or exacerbation of lymphedema has occurred. This case shows the effectiveness of preoperative ICG lymphography and lymphoscintigraphy followed by treatment via lymph vessel ligation and LVA for curative resolution of a lymphatic fistula.