Standardization of preoperative stoma site marking and its utility for preventing stoma leakage: a retrospective study of 519 patients who underwent laparoscopic/robotic rectal cancer surgery.

PURPOSE: Stoma site marking is an important preoperative intervention for preventing various stoma-associated complications. In our institution, standardized stoma site marking is routinely performed before rectal cancer surgery with stoma creation, and various stoma-associated factors are recorded in the ostomy-record template. The present study investigated risk factors for stoma leakage. METHODS: Our stoma site marking is standardized so that it can be performed by non-stoma specialists. To identify risk factors of stoma leakage at 3 months after surgery, various preoperative factors associated with stoma site marking in our ostomy-record template were retrospectively analyzed in 519 patients who underwent rectal cancer surgery with stoma creation from 2015 to 2020. RESULTS: Stoma leakage was seen in 35 of the 519 patients (6.7%). The distance between the stoma site marking and the umbilicus was less than 60 mm in 27 of the 35 patients (77%) who experienced stoma leakage, so a distance of less than 60 mm was identified as an independent risk factor for stoma leakage. Aside from preoperative factors, stoma leakage was also caused by postoperative skin wrinkles or surgical scars near the stoma site in 8 of 35 patients (23%). CONCLUSION: Preoperative standardized stoma site marking is necessary to achieve reliable marking that is easy to perform. To reduce the risk of stoma leakage, a distance of 60 mm or more between the stoma site marking and the umbilicus is ideal, and surgeons need to contrive ways to keep surgical scars away from the stoma site.

Immunogenomic profiles associated with response to neoadjuvant chemoradiotherapy in patients with rectal cancer.

BACKGROUND: Accumulating evidence suggests that radiotherapy success has an immune-associated component. The immunogenomic profiles associated with responses to chemoradiotherapy (CRT) were assessed in patients with locally advanced rectal cancer in this study. METHODS: CD8+ tumour-infiltrating lymphocyte (TIL) and stromal lymphocyte densities were assessed by immunohistochemistry using pretreatment biopsies from patients with advanced rectal cancer who had preoperative CRT. Whole-exome sequencing and gene expression microarray analysis were conducted to investigate the genomic properties associated with the response to CRT and CD8+ TIL density. Response to CRT was determined based on Dworak tumour regression grade (TRG); tumours with complete (TRG 4) or near-complete (TRG 3) regression were grouped as good responders, and those with TRG 1 as non-responders. RESULTS: Immunohistochemical examinations (275 patients) showed that pre-CRT CD8+ TIL density was associated with better response to CRT and improved recurrence-free survival, whereas pre-CRT stromal CD8+ cell density was not associated with either response to CRT or recurrence-free survival. Whole-exome sequencing (74 patients) showed that the numbers of single-nucleotide variations (SNVs) and neoantigens predicted from SNVs were higher in good responders than in non-responders, and these correlated positively with CD8+ TIL density (rS = 0·315 and rS = 0·334 respectively). Gene expression microarray (90 patients) showed that CD8A expression correlated positively with the expression of programmed cell death 1 (PDCD1) (rS = 0·264) and lymphocyte-activation gene 3 (LAG3) (rS = 0·507). CONCLUSION: Pre-CRT neoantigen-specific CD8+ T cell priming may be a key event in CRT responses where immune checkpoint molecules could be useful targets to enhance tumour regression.

ANTECEDENTES: Las evidencias existentes sugieren que el éxito de la radioterapia tiene un componente asociado con el sistema inmunitario. En este estudio se evaluaron los perfiles inmunogenómicos asociados con la respuesta a la quimiorradioterapia (chemoradiotherapy, CRT) en pacientes con cáncer de recto localmente avanzado. MÉTODOS: Las densidades de los linfocitos infiltrantes de tumor CD8+ (tumour-infiltrating lymphocyte, TIL) y de los linfocitos del estroma se evaluaron por inmunohistoquímicas en las biopsias antes del tratamiento de pacientes con cáncer de recto localmente avanzado que recibieron CRT preoperatoria. Se realizó secuenciación de todo el exoma, así como microarrays de expresión génica, para investigar las propiedades genómicas asociadas con la respuesta a la CRT y a la densidad de los TIL CD8+. La respuesta a la CRT se determinó según el grado de regresión del tumor de Dworak (tumour regression grade, TRG), agrupándose como buenos respondedores los casos de regresión tumoral completa (TRG4) o casi completa (TRG3) y como no respondedores, los casos de grado TRG1. RESULTADOS: Los exámenes inmunohistoquímicos (n = 275) mostraron que la densidad pre-CRT de TIL CD8+ se asoció con una mejor respuesta a la CRT y una mejor supervivencia libre de recidiva, aunque la densidad de células CD8+ del estroma previa a la CRT no se asoció con la respuesta a la CRT ni con la supervivencia libre de recidiva. La secuenciación de todo el exoma (n = 74) mostró que el número de variaciones de nucleótidos únicos (single nucleotide variations, SNVs) y los neoantígenos predichos a partir de los SNVs fueron mayores en los que respondieron bien que en los que no respondieron, y éstos se correlacionaron positivamente con la densidad de los TIL CD8+ (Spearman r = 0,315 y r = 0,334 respectivamente). Los microarrays de expresión génica (n = 90) mostraron que la expresión CD8A se correlacionó positivamente con la expresión del ligando de muerte programada-1 (r = 0,264) y con el antígeno linfocitario del gen 3 (r = 0,507). CONCLUSIÓN: La activación de células T CD8+ específicas para neoantígenos previa a la CRT puede ser un evento clave en la respuesta a la misma donde las moléculas del punto de control inmunitario podrían ser dianas útiles para intensificar la regresión del tumor.

Randomized clinical trial of oral and intravenous versus intravenous antibiotic prophylaxis for laparoscopic colorectal resection.

BACKGROUND: The use of oral prophylactic antibiotics for the prevention of surgical-site infection (SSI) in patients undergoing laparoscopic surgery for colorectal cancer is controversial. The aim of this RCT was to evaluate whether intravenous perioperative antibiotics are inferior to combined preoperative oral and perioperative intravenous antibiotics in this setting. METHODS: Patients undergoing elective laparoscopic colorectal resection in a single cancer centre were assigned randomly to combined preoperative oral antibiotics (metronidazole and kanamycin) and perioperative intravenous antibiotics (cefmetazole) (oral/IV group) or to perioperative intravenous antibiotics (cefmetazole) alone (IV-only group). Patients were stratified for the analyses based on type of operation (colonic surgery, anterior resection or abdominoperineal resection), preoperative use of mechanical bowel preparation, preoperative chemoradiotherapy and the presence of diabetes mellitus. The primary endpoint was the overall rate of SSI. Secondary endpoints were the rates of incisional site infection, organ/space infection, anastomotic leakage, intra-abdominal abscess, adverse events and postoperative complications. RESULTS: Of 540 patients offered participation in the trial in 2013-2014, 515 agreed to take part and were randomized. Some 256 patients in the IV-only group and 255 in the oral/IV group completed the treatment per protocol. The overall rate of SSI was 7·8 per cent (20 of 256) in the IV-only group and 7·8 per cent (20 of 255) in the oral/IV group, confirming that perioperative administration of intravenous antibiotics alone was not inferior to the combined regimen (P = 0·017). There were no differences in rates of incisional site infection (5·5 versus 5·9 per cent respectively), organ/space infection (2·3 versus 2·0 per cent) or other secondary endpoints between the two groups. CONCLUSION: Intravenous perioperative antimicrobial prophylaxis alone is not inferior to combined preoperative oral and intravenous perioperative prophylaxis with regard to SSI in patients with colorectal cancer undergoing elective laparoscopic resection. Registration number: UMIN000019339 ( http://www.umin.ac.jp/ctr/).

Laparoscopic salvage lateral pelvic lymph node dissection for locally recurrent rectal cancer.

AIM: The lateral pelvic lymph nodes are one of the major sites and sources of local recurrence (LR) after surgery for rectal cancer. Salvage lateral pelvic lymph node dissection (LPLD) is potentially curative, but the value of laparoscopic surgery in such cases is unknown. Our aim was to report the technical details of laparoscopic salvage LPLD for LR at these nodes after rectal cancer surgery. METHOD: The study was based on nine patients who underwent laparoscopic salvage LPLD for LR at the lateral pelvic lymph nodes after surgery for rectal cancer. The safety and feasibility of this procedure were determined. RESULTS: The median operation time was 381 min and the median estimated blood loss was 130 ml. There were no conversions. Adjacent structures removed en bloc were the pelvic plexus in four patients, the internal iliac artery in seven patients and the seminal vesicle in one patient. The median number of metastatic lymph nodes was 1 (range 1-11). CONCLUSION: Our novel technique of laparoscopic salvage LPLD for LR at the lateral pelvic lymph nodes is safe and feasible.

Supermicrovascular anastomosis training using chicken wings and colored water.

Plastic surgeons require experience in supermicroscopic vascular anastomosis. Herein, we report a simple, rapid, and cost-effective training method using chicken wings and colored water. The avian ventral metacarpal artery was selected for dissection and anastomosis to mimic supermicrosurgery. Over 14 weeks (one anastomosis per day), the ulnar artery in 100 chicken wings was exposed by dissection, cut proximally, and injected with blue food dye-colored water by an inexperienced surgeon. After ligating the artery branches, it was cut and subjected to end-to-end anastomosis. Next, colored water was injected into the ulnar artery to check for suture sufficiency. The vessel was re-dissected to inspect the lumen and sutures qualitatively. Of the 100 wings, the first and last 20 wings' ventral metacarpal artery dissection, anastomosis times, and leakage frequency were compared. Avian ventral metacarpal artery diameter was recorded, and the cumulative anastomosis time where individual anastomosis times started decreasing was determined. Leakage rates before and after this point were compared. The avian ventral metacarpal artery diameter was 0.7-0.8 mm. The last 20 wings had significantly shorter median dissection times (12:27 vs. 17:45 min), anastomosis times (9:02 vs. 12:29 min), and leakage rates (15% vs. 70%); more even stitching and parallel ligature points; and less vessel layer inversion than the first 20 wings. After a cumulative anastomosis time of 10 h 26 min, individual times sharply decreased, and the leakage rate decreased significantly (58.3% vs. 23.8%). The proposed method significantly improved supermicrosurgical anastomosis. Thus, we believe that this method will help surgeons improve their supermicrosurgical skills.

Efflux transporter mRNA expression profiles in differentiating JEG-3 human choriocarcinoma cells as a placental transport model.

The kinetics of drug transport across the trophoblast layer is determined by several factors. Human choriocarcinoma cell lines like BeWo and JEG-3 have been used as models of the trophoblast layer to examine the placental transport of drugs. Previously, the drugs examined in these models have been readily transported across the trophoblast layer via cellular gap junctions. These backgrounds enabled us to establish the differentiating JEG-3 cell (DJEG) layer model, which suppresses paracellular drug transport, as an evaluation system of placental drug transport. The efflux transporters on the trophoblast layer assume the meaningful role of protecting the fetus from xenobiotic substances. In order to clarify the usefulness of our DJEG placental drug transport model, this study examined the mRNA expression profiles of the efflux transporters MRPs, MDR1, and BCRP in JEG-3 cells and compared them with those of BeWo cells and their known placental expression. We suggest that the mRNA of efflux transporters MRP 1-8 and BCRP are expressed widely in JEG-3 cells; however, expression levels of MDR1 mRNA were undetectable. It was also indicated that polymorphisms of BCRP C421A in both the BeWo and JEG-3 cells are of the wild-type. We demonstrated the efflux transporters' expression profiles, as well as those of the BeWo cells, was demonstrated in the DJEG placental drug transport evaluating model as well as the BeWo cells, in the DJEG placental drug transport evaluation model. Based on these findings, we hope that the DJEG model will be adequate for use in evaluating placental drug transport in relation to the transporter proteins.

IL-8 in cerebrospinal fluid from children with acute encephalopathy is higher than in that from children with febrile seizure.

We identify possible differences in the cytokine/chemokine profiles in cerebrospinal fluid (CSF) from children with encephalopathy and febrile seizure. Interleukin (IL)-1beta, 2, 4, 5, 6, 7, 8, 10, 12, 13, 17, interferon-gamma, tumour necrosis factor-alpha, granulocyte colony-stimulating factor, granulocyte monocyte colony-stimulating factor, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1beta were measured simultaneously in CSF supernatants from children with encephalopathy (n = 8), febrile seizure (n = 16) and fever without neurological complications (n = 8). IL-8 in CSF from children with encephalopathy was significantly elevated compared to that in CSF from children with febrile seizure and fever without neurological complications. IL-8 in CSF was also higher than serum IL-8, suggesting that increased IL-8 was generated from glia cells or astrocytes, not by leakage from serum. Increased IL-8 in CSF in encephalopathy may protect against severe brain damage.

Correlation of autophagy type in podocytes with histopathological diagnosis of IgA nephropathy.

OBJECTIVE: IgA nephropathy (IgA-N) frequently leads to progressive renal failure, thus estimation of the degree of progression is important for patient management. Autophagy is a mechanism that facilitates clearance of waste products to preserve renal function. The aim of this study was to assess autophagy in podocytes in children with progressive IgA-N at initial diagnosis by electron microscopy and investigate the relationship between the types of autophagy and severity of the disease. METHODS: Renal biopsies from 16 children with established progressive IgA-N were examined by light and transmission electron microscopy with reference to autophagy types in the podocytes and histopathological diagnosis of IgA-N. RESULTS: Two autophagy types were found. Type I rarely transformed to autophagic vacuoles and did not dissolve, thus possibly impairing cell function. However, type II frequently transformed to autophagosomes and autophagic vacuoles thus facilitating protein and lipid clearance. Of the 16 children studied, 8 (50%) with type I autophagy at initial diagnosis showed focal proliferative glomerulosclerosis (GN) of mild type (3 cases, 37.5%), mild/moderate type (2 cases, 25%) and moderate type (3 cases, 37.5%). In contrast, the remaining 8 children with type II autophagy at initial diagnosis showed focal proliferative GN of mild type in 7 (87.5%) and mild/moderate type in 1 (12.5%) case. CONCLUSION: In IgA-N children, the occurrence of type I autophagy is correlated with histopathologically more progressive disease, possibly reflecting a tendency to a poorer prognosis.

Prognostic impact of residual lateral lymph node metastasis after neoadjuvant (chemo)radiotherapy in patients with advanced low rectal cancer.

Background: There is a lack of large studies focusing on the prognostic significance of lateral lymph node (LLN) metastasis following LLN dissection (LLND) in rectal cancer. The aim of this study was to evaluate the prognostic impact of LLN metastases on survival of patients with advanced low rectal cancer. Methods: Consecutive patients with locally advanced, but not metastatic, extraperitoneal rectal cancer treated with neoadjuvant (chemo)radiotherapy plus total mesorectal excision between 2004 and 2015 were included in the study. LLND was performed when pretreatment imaging documented enlarged LLNs (7 mm or greater in size). Localization of nodal metastases and long-term outcomes were analysed. Kaplan-Meier analysis was used to compare the survival of patients with ypN0 disease with that of patients with mesorectal ypN+/LLN- status and patients with positive LLNs. The Cox proportional hazards model was used to evaluate predictors of disease-free survival (DFS) and local recurrence. Results: A total of 613 patients were included in the study; LLND was performed in 212 patients (34·6 per cent) and 57 (9·3 per cent) had LLN metastasis. Patients with LLN metastasis had improved DFS and local recurrence cumulative incidence rates compared with patients with mesorectal ypN2+/LLN- disease (DFS: P = 0·014; local recurrence: P = 0·006). Although the DFS rate of patients with LLN metastasis was worse than that of patients with ypN0 disease (P < 0·001), the cumulative incidence of local recurrence was similar (P = 0·491). In multivariable analysis, residual LLN metastasis was not an independent predictor of worse DFS or local recurrence. Conclusion: LLN metastasis is not an independent predictor of local recurrence or survival. Survival of patients presenting with LLN metastasis after (chemo)radiotherapy was intermediate between that of patients with ypN0 status and those with mesorectal ypN2 positivity.

Antecedentes: No existen en la literatura grandes estudios dirigidos a investigar la importancia pronóstica de las metástasis en los ganglios linfáticos laterales (lateral lymph nodes, LLN) después de la disección de los mismos (LLN dissection, LLND) en pacientes con cáncer de recto. El objetivo de este estudio fue evaluar el impacto pronóstico de las metástasis en los LLN sobre la supervivencia de los pacientes con cáncer de recto. Métodos: Se analizaron 613 pacientes consecutivos con cáncer de recto localmente avanzado extraperitoneal y no metastásico tratados con (quimio)radioterapia neoadyuvante seguida de resección total del mesorrecto (total mesorectal excision, TME) entre 2004 y 2015. Se realizó una LLND cuando el estudio mediante pruebas de imagen previo el tratamiento mostró LLN aumentados de tamaño ≥ 7 mm. Se analizó la localización de las metástasis ganglionares y los resultados a largo plazo. El análisis de supervivencia se realizó mediante el método de Kaplan­Meier para comparar las supervivencias de los pacientes ypN0 frente a los pacientes ypN con positividad mesorrectal/LLN negativos y frente a los pacientes LLN positivos. Se utilizó el modelo de riesgo proporcional de Cox para evaluar los factores predictivos de supervivencia libre de enfermedad y de recidiva local. Resultados: Se realizó una LLND en 212 (34,6%) pacientes, y 57 (9,3%) pacientes presentaban metástasis en los LLN. Los pacientes con metástasis en los LLN presentaron mejores curvas de incidencia acumulada de recidiva local y de supervivencia libre de enfermedad en comparación con los pacientes con ganglios mesorrectales ypN2 positivos/LLN negativos (respectivamente, P = 0,0135 y P = 0,0060). Aunque la curva de la supervivencia libre de enfermedad de los pacientes con metástasis en los LLN fue peor que la de los pacientes ypN0 (P < 0,0001), la incidencia acumulada de recidiva local fue similar (P = 0,4905). En el análisis multivariable, la metástasis residual en los LLN no fue un factor predictivo independiente de peor supervivencia libre de enfermedad ni de recidiva local. Conclusión: Las metástasis en los LLN no es un factor predictivo independiente de recidiva local o supervivencia. Los pacientes que presentaron metástasis en los LLN después de (quimio)radioterapia mostraron características de supervivencia intermedias entre ypN0 y pacientes con ganglios mesorrectales ypN2 positivos.

Laparoscopic gastrectomy for gastric cancer: experience with more than 600 cases.

BACKGROUND: Among the less invasive operations noted in recent years, laparoscopic gastrectomy for gastric cancer has become popular because of advances in surgical techniques. The authors performed laparoscopic gastrectomy with regional lymph node dissection for 612 cases of gastric malignancies between March 1998 and August 2006. The technique and results of laparoscopic gastrectomy for gastric cancer are presented. METHODS: Of the 612 gastric malignancy cases, distal gastrectomy was performed in 485 cases, proximal gastrectomy in 42 cases, and total gastrectomy in 85 cases. In all the cases, D1 or D2 lymph node dissection was performed according to the general rule of the Japanese Gastric Cancer Association. RESULTS: Quicker recovery was observed in the laparoscopic gastrectomy cases than in the open cases. The postoperative complications with this technique were within a permissible range. No statistical difference was seen in the survival curve after surgery between the laparoscopic group of advanced cases preoperatively diagnosed as surgical T2N1 or lower and the open group. CONCLUSION: The laparoscopic technique is not only less invasive, but also similarly safe and curative compared with open gastrectomy.

Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways.

Here we report that synaptic and extrasynaptic NMDA (N-methyl-D-aspartate) receptors have opposite effects on CREB (cAMP response element binding protein) function, gene regulation and neuron survival. Calcium entry through synaptic NMDA receptors induced CREB activity and brain-derived neurotrophic factor (BDNF) gene expression as strongly as did stimulation of L-type calcium channels. In contrast, calcium entry through extrasynaptic NMDA receptors, triggered by bath glutamate exposure or hypoxic/ischemic conditions, activated a general and dominant CREB shut-off pathway that blocked induction of BDNF expression. Synaptic NMDA receptors have anti-apoptotic activity, whereas stimulation of extrasynaptic NMDA receptors caused loss of mitochondrial membrane potential (an early marker for glutamate-induced neuronal damage) and cell death. Specific blockade of extrasynaptic NMDA receptors may effectively prevent neuron loss following stroke and other neuropathological conditions associated with glutamate toxicity.

Continuous infusion of 5-fluorouracil with versus without low-dose, consecutive administration of cisplatin in advanced colorectal cancer. A prospective randomized phase II study.

Recently, the treatment of advanced gastric cancer by continuous infusion of 5-fluorouracil (5-FU) with low-dose cisplatin (CDDP) has improved efficacy without severe toxicities. The possible effectiveness of 5-FU+low-dose CDDP for colorectal cancer (CRC) is intriguing. One hundred fifty-five patients with far-advanced CRC including at least one measurable lesion were enrolled in a prospective randomized clinical trial funded by the Japanese Foundation for Multidisciplinary Treatment of Cancer. These patients were assigned to the two arms to assess the value of low-dose CDDP when added to a continuous intravenous infusion of 5-FU at a dose of 300 mg/m(2)/24 hrs in a one-week cycle consisting of 5 days of treatment and 2 days of rest for at least 12 weeks. CD-DP was given intravenously at a dose of 3 mg/m(2) on days 1-5 and days 8-12, and then at a dose of 7 mg/m(2) twice a week. Three patients were excluded from the trial. The response rate in the 5-FU+low-dose CDDP arm (n=75) was significantly higher than that in the 5-FU arm (n=77) (25.3% vs. 11.7%; P = 0.037). There was no significant difference in the median overall survival time between the 5-FU+low-dose CDDP arm and the 5-FU arm (479 and 491 days, respectively). Grades 3/4 toxicities occurred infrequently in both arms. The quality of life was almost the same between the arms. Low-dose CDDP improved the response rate while keeping toxicities within clinically acceptable limits. However, this combined treatment did not confer a survival advantage over treatment with continuous infusion of 5-FU alone for patients with far-advanced CRC; that might be attributable to the short CDDP administration setting of 12 weeks.

Triangulating stapling technique for reconstruction after colectomy.

BACKGROUND/AIMS: To retrospectively compare the triangulating stapling technique for colocolonic anastomosis with hand-sewn anastomosis and functional end-to-end anastomosis. METHODOLOGY: Data from 646 patients who underwent colectomy for cancer from 1993 to 2004 were extracted by chart review. Patients were divided into three groups based on the type of anastomosis: handsewn (n=233), functional end-to-end (n=71), and the triangulating stapling method (n=346). Demographic data and clinical characteristics of the three groups were similar. RESULTS: Anastomotic leakage was significantly more common in the hand-sewn group than the triangular stapling group (hand-sewn; 3.0%, functional end-to-end; 2.8%, triangulating, 0.6%) (P < 0.05). No patient developed bleeding or stenosis at the anastomosis, and the incidence of wound infection was equivalent among the three groups. One death due to anastomotic failure occurred in each of the functional end-to-end and triangulating stapling groups. The cost of triangulating stapling was approximately Yen 36,000 lower than the cost of the functional end-to-end anastomosis. CONCLUSIONS: The triangulating stapling technique is an attractive alternative to other methods for creating a colocolonic anastomosis.

Combined histochemical and biochemical analysis of sex hormone dependence of ferric nitrilotriacetate-induced renal lipid peroxidation in ddY mice.

Ferric nitrilotriacetate (Fe-NTA) induces renal proximal tubular necrosis, a consequence of lipid peroxidation, that finally leads to a high incidence of renal adenocarcinoma in rats and mice. Male animals are much more susceptible than female animals to both effects. Moreover, the distribution of the susceptible proximal tubules is different between male and female animals. The present study investigated the effects of castration and sex hormones on Fe-NTA-induced renal lipid peroxidation. Male and female ddY mice were either left untreated, castrated, and/or treated with testosterone or estriol. Histochemical (reactivity to cold Schiff's reagent) and biochemical (thiobarbituric acid-reactive substance) evaluations were performed 1 h after the i.p. injection of Fe-NTA (5 mg iron/kg). Testosterone treatment and/or oophorectomy increased the Schiff positivity of the renal cortical proximal tubules and the amount of thiobarbituric acid-reactive substance (testosterone-treated female greater than intact female, P less than 0.005; castrated female greater than intact female, P less than 0.1; castrated and testosterone-treated female greater than intact female, P less than 0.005). In contrast, estriol treatment and/or orchiectomy decreased the Schiff positivity of the renal cortical proximal tubules and the amount of thiobarbituric acid-reactive substance (estriol-treated male less than intact male, P less than 0.01; castrated male less than intact male, P less than 0.01; castrated and estriol-treated male less than intact male, P less than 0.005). Estradiol treatment produced similar results to estriol treatment (estradiol-treated male less than intact male, P less than 0.005). Castration and/or administration of the opposite sex hormone reversed the sex difference in the distribution of proximal tubules susceptible to lipid peroxidation. However, the i.v. injection to male mice, 5 min prior to the Fe-NTA treatment, of conjugated estrogen that is promptly excreted via the urine produced no significant effect. Thus, altered metabolic pathways rather than the direct scavenging activity of estrogens seem to be involved in the sex hormone-dependent difference of lipid peroxidation. Genetically determined sex hormone status appears to have influenced the incidence of Fe-NTA-induced renal adenocarcinoma in intact animals.

Synthesis of a fluorogenic mucopolysaccharide by chondrocytes in cell culture with 4-methylumbelliferyl beta-D-xyloside.

Culture of chondrocytes in the presence of 4-methylumbelliferyl beta-D-xyloside resulted in a synthesis of protein-free, fluorogenic chondroitin sulfate which was heterogeneous on DEAE-cellulose chromatography. Degradation of the major chromatographic fraction with chondroitinase-ABC yielded, in addition to a large quantity of delta4-glucuronic acid-containing disaccharides, two fluorogenic oligosaccharides of different size. Quantitative analysis showed that delta4-glucuronic acid, galactose, xylose, and 4-methylumbelliferone were present in the small oligosaccharide fragment in a molar ratio of 1:2:1:1. Since these analytical data are analogous to those reported for glycopeptides derivedfrom proteochondroitin sulfates, it may be suggested that 4-methyl-umbelliferyl beta-D-xyloside replaces the need for xylosyl protein core in the normal synthesis of proteochondroitin sulfate with a resultant production of the unusual polysaccharide bearing the added xyloside at the reducing end.

Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide.

BACKGROUND: Vein graft disease limits the late results of coronary revascularization. C-type natriuretic peptide (CNP) inhibits the growth of vascular smooth muscle cells. Given the effects of CNP on cGMP cascade, we hypothesized that transfected CNP genes modulate endothelial repair and thrombogenicity in the vein graft. METHODS AND RESULTS: Autologous rabbit jugular vein grafts were incubated ex vivo in a solution of adenovirus vectors containing CNP gene (Ad.CNP) or Escherichia coli lac Z gene (Ad.LacZ) and then interposed in the carotid artery. Reendothelialization, mural thrombi formation, and intima/media ratio were evaluated on the 14th and 28th postoperative days. More reendothelialization was seen in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts both at 14 days (0.81+/-0.05 versus 0.30+/-0.14, P<0.01) and at 28 days (0.96+/-0.01 versus 0.45+/-0.08, P<0.001). The mural thrombus area was smaller in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts. Neointimal thickening was significantly suppressed in the Ad.CNP group. The in vitro wound assay with human coronary artery endothelial cells revealed significant potentiation of the wound repair process by CNP and atrial natriuretic peptide administration. CONCLUSIONS: Infected Ad.CNP accelerated reendothelialization and suppressed thrombosis and neointimal hyperplasia. The method may potentially prevent vein graft disease in patients undergoing coronary artery revascularization.

Inhibition of rho-associated kinase results in suppression of neointimal formation of balloon-injured arteries.

BACKGROUND: Rho-associated kinase (ROCK), an effector of small GTPase Rho, regulates vascular tone via a calcium sensitization mechanism and plays a key role in the pathogenesis of hypertension. However, its role in vascular growth remains unclear. METHODS AND RESULTS: Y-27632, a specific ROCK inhibitor, and the overexpression of dominant-negative ROCK suppressed the mitogen-induced DNA synthesis of cultured vascular smooth muscle cells (VSMCs), which indicates the essential role of ROCK in the control of VSMC proliferation in vitro. Y-27632 also suppressed the chemotaxis of VSMCs. Male Wistar rats were systemically given Y-27632 (35 to 70 mg. kg(-1). day(-1)) through an intraperitoneal infusion. The neointimal formation of balloon-injured carotid arteries was significantly suppressed in Y-27632-treated rats (intima/media ratio, 0.22+/-0.02) compared with vehicle-treated rats (intima/media ratio, 0.92+/-0.21) or hydralazine-treated rats with a similar blood pressure decrease (intima/media ratio, 1.03+/-0.15). The phosphorylation of myosin phosphatase and myosin light chain was elevated in injured arteries in a Y-27632-sensitive manner, indicating the augmentation of ROCK activity in neointimal formation. The downregulation of the cyclin-dependent kinase inhibitor p27(kip1) in injured vessels was reversed by Y-27632 treatment, reflecting the antiproliferative effect of ROCK inhibition in vivo. CONCLUSIONS: We conclude that ROCK plays a key role in the process of neointimal formation after balloon injury. Thus, the inhibition of ROCK may be a potential therapeutic strategy for treating vascular proliferative disorders and hypertension.