Long-Term Quality of Life Assessment After Successful Endoscopic Vacuum Therapy of Defects in the Upper Gastrointestinal Tract Quality of Life After EVT.

BACKGROUND: Accumulating evidence indicates that anastomotic leakages and perforations of the upper gastrointestinal tract (uGIT) can be treated successfully with endoscopic vacuum therapy (EVT). So far, no data is available regarding the long-term quality of life (QoL) after successful EVT of defects in the uGIT. METHODS: We present a prospective survey on long-term Qol of 52 patients treated by EVT for defects of the uGIT. Results are compared with 63 of 221 patients treated by esophagectomy without anastomotic insufficiency (w/o EVT) between 12/2011 and 12/2015. The Gastrointestinal Quality of Life-Index (GIQLI) score was determined by a 36-item questionnaire of 25 respondents with EVT and 50 respondents w/o EVT. RESULTS: The response rate was 78.95% (75/95) including 25 survey respondents who were treated with EVT for anastomotic insufficiency secondary to esophagectomy or gastrectomy (n = 19), iatrogenic esophageal perforation (n = 4), and Boerhaave syndrome (n = 2) and 50 respondents with complication-free esophagectomy w/o EVT. The median follow-up was 19 months for EVT patients and 21 months for patients w/o EVT. Except for "social function" (p = 0.009) in favor for patients w/o EVT, the median GIQLI score did not differ significantly between both study groups concerning the categories 'symptoms', 'emotions', 'physical functions', and 'medical treatment' resulting in a total median GIQLI score of 83 in EVT versus 96.5 in patients w/o EVT (p = 0.185). Spearman Rho analysis revealed that a high GIQLI score correlated with a low ASA score (p < 0.001), a benign pathology (p = 0.001), and a hospital stay less than 21 days (p < 0.001). CONCLUSION: EVT in the uGIT is well tolerated by the patients and accompanied by a satisfactory long-term QoL.

Endoscopic Vacuum Therapy (EVT)-a New Concept for Complication Management in Bariatric Surgery.

BACKGROUND: Bariatric surgery is the most efficient therapy for morbid obesity. Staple line and anastomotic leakage are the most feared postoperative complications after Roux-en-Y gastric bypass and laparoscopic sleeve gastrectomy (LSG). Traditional treatment options like revisional surgery and endoscopic stent placement are associated with high morbidity and mortality as well as variable success rates. Endoscopic vacuum therapy (EVT) has shown to be a new successful and feasible treatment option for leaks of different etiology after major gastro-esophageal surgery. METHOD: We report a case of the EVT principle being applied in a patient with three major leaks located apart from each other within the gastric staple line after LSG for morbid obesity (BMI 62.7). EVT was initiated on postoperative day 8. RESULTS: In total, 18 endoscopic interventions were performed in 72 days, the vacuum sponge being replaced endoscopically every 4 days. Hospital length of stay was 106 days. No relevant procedure related complications were observed during the course of therapy and during the follow up. CONCLUSION: EVT of postoperative leaks in the upper GI tract has been shown to be feasible and safe. It combines defect closure and effective drainage and allows a periodic inspection of the wound cavity. In case of therapeutic failure, it does not jeopardize surgical repair or stent placement. Even though the techniques and materials used in EVT still vary considerably according to local expertise, EVT has the potential to succeed as a nonsurgical, feasible, safe, and effective treatment option for postoperative leaks in bariatric surgery.

Pre-emptive endoscopic vacuum therapy for treatment of anastomotic ischemia after esophageal resections.

Background and study aims Endoscopic vacuum therapy (EVT) is a promising new approach for the treatment of anastomotic leakage in the gastrointestinal tract. Here, we present the first case series demonstrating successful use of EVT for the treatment of post-esophagectomy anastomotic ischemia prior to development of leakage. Patients and methods Between 2012 and 2015, intraluminal EVT was performed in eight patients with anastomotic ischemia following esophagectomy. The primary outcome measure was successful mucosal recovery. Secondary outcome measures were duration of treatment, number of sponge changes, septic course, and associated complications. Results Complete mucosal recovery was achieved in six patients (75 %) with different degrees of anastomotic ischemia. In two patients (25 %), small anastomotic leaks developed, which resolved by continuing the EVT treatment. Median duration of EVT treatment until mucosal recovery was 16 days (range 6 - 35), with a median of 5 sponge changes per patient (range 2 - 11). No EVT-associated complications were noted. Three patients developed anastomotic stenoses, which were treated by endoscopic dilation therapy. Conclusion This is the first case series to demonstrate that the early use of EVT potentially modulates clinical outcomes and infection parameters in patients with anastomotic ischemia following esophagectomy. Further studies are needed to define the indications and patients who are most likely to benefit from early EVT.

Successful closure of defects in the upper gastrointestinal tract by endoscopic vacuum therapy (EVT): a prospective cohort study.

BACKGROUND: Perforations and anastomotic leakages of the upper gastrointestinal (GI) tract cause a high morbidity and mortality rate. Only limited data exist for endoscopic vacuum therapy (EVT) in the upper GI tract. METHODS: Fifty-two patients (37 men and 15 women, ages 41-94 years) were treated (12/2011-12/2015) with EVT for anastomotic insufficiency secondary to esophagectomy or gastrectomy (n = 39), iatrogenic esophageal perforation (n = 9) and Boerhaave syndrome (n = 4). After diagnosis, polyurethane sponges were endoscopically positioned with a total of 390 interventions and continuous negative pressure of 125 mm of mercury (mmHg) was applied to the EVT-system. Sponges were changed endoscopically twice per week. Clinical and therapy-related data and mortality were analyzed. RESULTS: After 1-25 changes of the sponge at intervals of 3-5 days with a mean of 6 sponge changes and a mean duration of therapy of 22 days, the defects were healed in 94.2 % of all patients without revision surgery. In three patients (6 %), EVT failed. Two of these patients died due to hemorrhage related to EVT. Four postinterventional strictures were observed during the follow-up of up to 4 years. CONCLUSION: Esophageal wall defects of different etiology in the upper gastrointestinal tract can be treated successfully with EVT, considering that indication for EVT should be weighed carefully. EVT can be regarded as a novel life-saving therapeutic tool.

Comparison of Endoscopic Vacuum Therapy Versus Stent for Anastomotic Leak After Esophagectomy.

BACKGROUND: Endoscopic vacuum therapy is a novel option for the management of esophageal leaks. This study compares endoscopic vacuum therapy versus placement of covered stents for anastomotic leaks after esophagectomy. METHODS: N = 45 consecutive patients with anastomotic leaks following esophagectomy (including patients referred to our center from other hospitals for complication management) were managed by endoscopic therapy at our institution from January 2009 to February 2015. Outcomes of stent and endoscopic vacuum therapy were analyzed retrospectively. RESULTS: Thirty patients received endoscopic stent placement and 15 endoscopic vacuum therapy. In the stent group, seven patients were switched to endoscopic vacuum and four to surgery. Classified by type of initial endoscopic therapy, the success rate (anastomotic healing, patient recovered) was higher for endoscopic vacuum therapy (endoscopic vacuum 93.3%, stent 63.3 %; p = 0.038). Classified by final endoscopic therapy (after switches in therapy), success rates were 86.4 and 60.9% (p = 0.091), respectively. There was no difference observed in mortality, duration of therapy, and length of hospital stay between the study groups. CONCLUSIONS: Endoscopic vacuum therapy might be more effective than endoscopic stent placement in the management of esophageal anastomotic leaks.

A two-step multidisciplinary approach to treat recurrent esophageal strictures in children with epidermolysis bullosa dystrophica.

In children with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), esophageal scarring leads to esophageal strictures with dysphagia, followed by malnutrition and delayed development. We describe a two-step multidisciplinary therapeutic approach to overcome malnutrition and growth retardation. In Step 1, under general anesthesia, orthograde balloon dilation of the esophagus is followed by gastrostomy creation using a direct puncture technique. In Step 2, further esophageal strictures are treated by retrograde dilation via the established gastrostomy; this step requires only a short sedation period. A total of 12 patients (median age 7.8 years, range 6 weeks to 17 years) underwent successful orthograde balloon dilation of esophageal strictures combined with direct puncture gastrostomy. After 12 and 24 months in 11 children, a substantial improvement of growth and nutrition was achieved (body mass index [BMI] standard deviation score [SDS] + 0.59 and + 0.61, respectively). In one child, gastrostomy was removed because of skin ulcerations after 10 days. Recurrent esophageal strictures were treated successfully in five children. The combined approach of balloon dilation and gastrostomy is technically safe in children with RDEB, and helps to promote catch-up growth and body weight. In addition, recurrent esophageal strictures can be treated successfully without general anesthesia in a retrograde manner via the established gastrostomy.

Novel treatment options for perforations of the upper gastrointestinal tract: endoscopic vacuum therapy and over-the-scope clips.

Endoscopic management of leakages and perforations of the upper gastrointestinal tract has gained great importance as it avoids the morbidity and mortality of surgical intervention. In the past years, covered self-expanding metal stents were the mainstay of endoscopic therapy. However, two new techniques are now available that enlarge the possibilities of defect closure: endoscopic vacuum therapy (EVT), and over-the-scope clip (OTSC). EVT is performed by mounting a polyurethane sponge on a gastric tube and placing it into the leakage. Continuous suction is applied via the tube resulting in effective drainage of the cavity and the induction of wound healing, comparable to the application of vacuum therapy in cutaneous wounds. The system is changed every 3-5 d. The overall success rate of EVT in the literature ranges from 84% to 100%, with a mean of 90%; only few complications have been reported. OTSCs are loaded on a transparent cap which is mounted on the tip of a standard endoscope. By bringing the edges of the perforation into the cap, by suction or by dedicated devices, such as anchor or twin grasper, the OTSC can be placed to close the perforation. For acute endoscopy associated perforations, the mean success rate is 90% (range: 70%-100%). For other types of perforations (postoperative, other chronic leaks and fistulas) success rates are somewhat lower (68%, and 59%, respectively). Only few complications have been reported. Although first reports are promising, further studies are needed to define the exact role of EVT and OTSC in treatment algorithms of upper gastrointestinal perforations.

Endoscopic closure of postoperative gastrointestinal leakages and fistulas with the Over-the-Scope Clip (OTSC).

BACKGROUND: In contrast to conventional Through-the-Scope Clips, the novel Over-the-Scope Clip (OTSC(®)) allows endoscopic full thickness closure of gastrointestinal leakages. The purpose of this study was to evaluate the efficacy and safety of the OTSC for the management of postoperative gastrointestinal leakages and fistulas. METHODS: We retrospectively reviewed a series of 14 consecutive patients with postoperative gastrointestinal leakages and fistulas who were treated by OTSC application. RESULTS: Nine OTSCs were used for upper GI tract leakages; five were used for colorectal leakages. Seventy-nine percent (11/14) of leakages were chronic (treated by OTSC later than postoperative day 14). In nine patients, other therapies preceded OTSC application. Median follow-up time was 5.5 months (range, 0.25-17). Primary technical success was achieved in all (14/14) patients. No adverse events related to the use of the OTSC device were noted. Three early recurrences were observed (two colonic fistulas, one esophageal anastomotic leakage), leading to a long-term success rate of 79 % (11/14). Leakage closure finally was achieved in these three patients by surgery or endoscopic vacuum therapy. CONCLUSIONS: The OTSC system is an effective and safe method for the management of postoperative leakages and fistulas of the gastrointestinal tract. Its exact place in treatment algorithms of postoperative leakages will have to be determined.

Intestinal cancer risk in Crohn's disease: a meta-analysis.

AIM OF THE STUDY: To clarify the intestinal cancer risk in Crohn's disease (CD). METHODS: 20 clinical studies (1965-2008) with a total of 40,547 patients with Crohn's disease-associated cancer (CDAC) were included in the meta-analysis ("inverse variance weighted" method). RESULTS: The incidence of CDAC in any CD patient was 0.8/1,000 person years duration (pyd) (CI, 0.6-1.0). The incidences of different carcinomas were: colorectal cancer 0.5/1,000 pyd (CI, 0.3-0.6), small bowel carcinoma 0.3/1,000 pyd (CI, 0.1-0.5), and cancers arising from CD-associated fistulae 0.2/1,000 pyd (CI, 0.0-0.4). Compared to the incidence in an age-matched standard population, the risk of colorectal cancer was increased by factor 2-3 and of small bowel cancer by factor 18.75, respectively. Mean patient age at diagnosis of CD-associated colorectal cancer was 51.5 years, thus 20 years earlier than in a standard population. The mean duration of CD until diagnosis of CDAC was 18.3 years. Duration of CD, age at diagnosis of CD, and anatomical area of CD involvement had no significant influence on cancer incidence. CONCLUSIONS: CD is a risk factor for colorectal cancer, small bowel cancer, and fistula cancer; however, compared to ulcerative colitis, cancer risk is moderate.

Interferon-gamma regulates intestinal epithelial homeostasis through converging beta-catenin signaling pathways.

Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-gamma (IFN-gamma) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-beta-catenin and Wingless-Int (Wnt)-beta-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-gamma resulted in activation of beta-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-gamma treatment and reduced proliferation through depletion of the Wnt coreceptor LRP6. These effects were enhanced by tumor necrosis factor-alpha (TNF-alpha), suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased beta-catenin-T cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-gamma-deficient mice. Thus, we propose that IFN-gamma regulates intestinal epithelial homeostasis by sequential regulation of converging beta-catenin signaling pathways.

Annexin A1 regulates intestinal mucosal injury, inflammation, and repair.

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (-/-) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (-/-) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.

Role of the intestinal barrier in inflammatory bowel disease.

A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the interstitium. The single layer of intestinal epithelial cells (IECs) serves as a dynamic interface between the host and its environment. Cell polarity and structural properties of the epithelium is complex and is important in the development of epithelial barrier function. Epithelial cells associate with each other via a series of intercellular junctions. The apical most intercellular junctional complex referred to as the Apical Junction Complex (AJC) is important in not only cell-cell recognition, but also in the regulation of paracellular movement of fluid and solutes. Defects in the intestinal epithelial barrier function have been observed in a number of intestinal disorders such as inflammatory bowel disease (IBD). It is now becoming evident that an aberrant epithelial barrier function plays a central role in the pathophysiology of IBD. Thus, a better understanding of the intestinal epithelial barrier structure and function in healthy and disease states such as IBD will foster new ideas for the development of therapies for such chronic disorders.

Formyl peptide receptor-1 activation enhances intestinal epithelial cell restitution through phosphatidylinositol 3-kinase-dependent activation of Rac1 and Cdc42.

Inflammatory disorders of the gastrointestinal tract result in the breakdown of the intestinal epithelial barrier in the form of erosion and ulceration. To reestablish the epithelial barrier, the epithelium must efficiently migrate to reseal wounds. Numerous signaling cascades are involved in the induction and regulation of this complex process. N-formyl peptide receptors comprise a group of Gi-coupled receptors that regulate innate immune responses. Previously, we identified the expression of functional N-formyl peptide receptors in model SK-CO15 intestinal epithelial cells and observed a role for activation of these receptors in regulating cellular invasive behavior. In these studies, we performed formyl peptide receptor-1 (FPR) localization and evaluated its role in regulating intestinal epithelial cell wound closure. Immunolocalization studies using a recently developed specific monoclonal anti-FPR Ab demonstrated its localization along the lateral membrane of crypt epithelial cells in normal human colonic epithelium. In vitro studies using the classical FPR agonist fMLF showed that FPR activation significantly enhances model intestinal epithelial cell restitution and that FPR localized along actin filaments in lamellipodial and filopodial extrusions. The increase in cell migration was associated with activation of PI3K, Rac1, and Cdc42. Pharmacologic inhibition of PI3K activity abrogated the fMLF-induced increase in wound closure and activation of both Rac1 and Cdc42. Inhibition of Rac1 and Cdc42 using pharmacologic inhibitors and dominant negative mutants also inhibited the fMLF-induced increase in cell migration. Taken together, theses results support a novel role for FPR stimulation in enhancing intestinal epithelial cell restitution through PI3K-dependent activation of Rac1 and Cdc42.

JAM-A regulates permeability and inflammation in the intestine in vivo.

Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A-deficient (JAM-A(-/-)) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A(-/-) mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A(-/-) mice. The in vivo observations were epithelial specific, because monolayers of JAM-A(-/-) epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A(-/-) mice and in JAM-A small interfering RNA-treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A(-/-) mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A(-/-) animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.

Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium.

Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.

PECAM-1 (CD 31) mediates transendothelial leukocyte migration in experimental colitis.

Transendothelial migration of circulating leukocytes into the colonic wall is a key step in the development of the inflammatory infiltrate in inflammatory bowel disease (IBD). The platelet-endothelial cell adhesion molecule-1 PECAM-1 (CD31) is expressed in the tight junction area of endothelial cells, where it is supposed to support the transmigration process. The aim of this study was to determine the role of PECAM-1 in experimental IBD and to show whether blockade of PECAM-1 has therapeutic effects. Chronic colitis was induced in female BALB/c mice by cyclic oral administration of dextran sodium sulfate (DSS) 3% (wt/vol). Expression of PECAM-1 was visualized by immunohistochemistry. In the treatment group animals received 1 mg/kg anti-PECAM-1 (2H8) ip daily starting on day 26. On day 30 leukocyte adhesion and migration was measured during N(2)O-isoflurane anesthesia in the distal colon by intravital microscopy. Disease activity index (DAI), histology, and MPO levels were compared with healthy and diseased controls. PECAM-1 was expressed in colitic mice. Chronic DSS colitis was characterized by a marked increase in rolling, adherent, and transmigrated leukocytes compared with healthy controls. Immunoblockade of PECAM-1 reduced leukocyte transmigration significantly and also diminished leukocyte rolling and sticking in an indirect manner. It also resulted in a significantly diminished DAI and MPO levels, as well as an amelioration of the histological inflammation score. PECAM-1 plays an important role in transendothelial leukocyte migration in DSS colitis. PECAM-1 could be a novel target for antibody-based treatment in IBD.

Intestinal cancer in patients with Crohn's disease.

BACKGROUND: Surveillance of intestinal cancer in Crohn's disease (CD) has often been advocated. To date, no clear evidence exists whether CD patients are at special risk for intestinal cancer. An increased incidence of small bowel adenocarcinoma is suggested. However, recent figures also suggest an increased risk of CD associated colorectal cancer. We report our experience with 10 cases of CD complicated by intestinal adenocarcinoma. MATERIALS AND METHODS: Our institutional database included 330 patients treated for CD between 1988-2005. Data of patients that developed carcinoma within Crohn's lesions of either small or large bowel were analyzed. RESULTS: Ten patients were diagnosed with CD complicated by carcinoma. In nine patients, cancer was present in the colorectum and in one, in Crohn's ileitis. Tumors were in conjunction with fistulae in three and developed within strictures in five patients. Mean age at the time of diagnosis of CD was 43 years. Mean duration of CD until diagnosis of cancer was 14 years. Only five patients were diagnosed for cancer preoperatively. Staging revealed advanced tumors in almost all patients. Mean survival after surgery was 29 months (2-149 months). CONCLUSIONS: Cancer risk in CD and especially in Crohn's colitis may still be underestimated. Delayed diagnosis resulted in a poor prognosis. The value of colonoscopy as surveillance tool is questioned by the fact that in our patients, carcinoma was diagnosed in some patients preoperatively by routine colonoscopy. Therefore, additional markers should be identified to detect CD patients at risk.

Organized migration of epithelial cells requires control of adhesion and protrusion through Rho kinase effectors.

Migration of epithelial cell sheets, a process involving F-actin restructuring through Rho family GTPases, is both physiologically and pathophysiologically important. Our objective was to clarify the mechanisms whereby the downstream RhoA effector Rho-associated coil-coil-forming kinase (ROCK) influences coordinated epithelial cell motility. Although cells exposed to a pharmacological ROCK inhibitor (Y-27632) exhibited increased spreading in wound closure assays, they failed to migrate in a cohesive manner. Two main phenomena were implicated: the formation of aberrant protrusions at the migrating front and the basal accumulation of F-actin aggregates. Aggregates reflected increased membrane affiliation and detergent insolubility of the actin-binding protein ezrin and enhanced coassociation of ezrin with the membrane protein CD44. While F-actin aggregation following ROCK inhibition was recapitulated by inhibiting myosin light chain (MLC) phosphorylation with the MLC kinase inhibitor ML-7, the latter did not influence protrusiveness and, in fact, significantly decreased cell migration. Our results suggest that excessive protrusiveness downstream of ROCK inhibition reflects an influence of ROCK on F-actin stability via LIM kinase 1 (LIMK-1), which phosphorylates and inactivates cofilin. Y-27632 reduced the levels of both active LIMK-1 and inactive cofilin (phospho forms), and expression of a dominant negative LIMK-1 mutant stimulated leading edge protrusiveness. Furthermore, Y-27632-induced protrusions were partially reversed by overexpression of LIMK-1 to restore cofilin phosphorylation. In summary, our results provide new evidence suggesting that adhesive and protrusive events involved in organized epithelial motility downstream of ROCK are separately coordinated through the phosphorylation of (respectively) MLC and cofilin.