Early Adhesion Formation Research Articles

Nascent Adhesoins that Form on all Substrates by Recruiting Unliganded Integrins and are Important for Mechanotransduction

Integrin adhesions assemble and mature in response to ligand binding and mechanical factors, but the molecular-level organization is not known. We report that ∼100-nm clusters of ∼50 β3-activated integrins form very early adhesions under a wide variety of conditions on RGD surfaces. These adhesions form similarly on fluid and rigid substrates, but most adhesions are transient on rigid substrates. Without talin or actin polymerization, few early adhesions form, but expression of either the talin head or rod domain in talin-depleted cells restores early adhesion formation. Mutation of the integrin binding site in the talin rod decreases cluster size. We suggest that the integrin clusters constitute universal early adhesions and that they are the modular units of cell matrix adhesions. They require the association of activated integrins with cytoplasmic proteins, in particular talin and actin, and cytoskeletal contraction on them causes adhesion maturation for cell motility and growth. Using gold nano-patterning we observe that these clusters grow using unliganded but activated integrins. This indicates a mechanism to assemble the clusters rapidly around an activated and liganded integrin. Integrins are not enzymes hence they would need the aid of other enzymes for mechanotransduction. Nascent adhesions have distinct functions on compliant and rigid substrates wherein, the epidermal growth factor receptor is recruited to these clusters only on the rigid substrates, in absence of EGF. This receptor acts as a mechanoenzyme that is required for mechanotransduction within fibroblasts. Taken together our studies show that nascent adhesions form on substrates of vastly varying rigidities. These modular adhesions are formed by a cohort of unliganded integrins recruited to adhesions sites by cytoplasmic factors and they are central to bring about different mechano-transduction on different substrates.

Nascent Integrin Adhesions Form on All Matrix Rigidities after Integrin Activation

Integrin adhesions assemble and mature in response to ligand binding and mechanical factors, but the molecular-level organization is not known. We report that ∼100-nm clusters of ∼50 β3-activated integrins form very early adhesions under a wide variety of conditions on RGD surfaces. These adhesions form similarly on fluid and rigid substrates, but most adhesions are transient on rigid substrates. Without talin or actin polymerization, few early adhesions form, but expression of either the talin head or rod domain in talin-depleted cells restores early adhesion formation. Mutation of the integrin binding site in the talin rod decreases cluster size. We suggest that the integrin clusters constitute universal early adhesions and that they are the modular units of cell matrix adhesions. They require the association of activated integrins with cytoplasmic proteins, in particular talin and actin, and cytoskeletal contraction on them causes adhesion maturation for cell motility and growth.

Effect of intraperitoneal cetuximab administration on colonic anastomosis and early postoperative adhesion formation in a rat model.

We aimed to evaluate the effect of intraperitoneal cetuximab administration on the healing of anastomosis and development of early adhesion formation in a rat model. Twenty-four female rats were used. A colon segment was resected and end-to-end anastomosis was performed. The rats were randomized into three groups after the performance of colonic anastomosis and received 10 mL of intraperitoneal solution including study drugs after closure of abdominal cavity: normal saline was administered to the normal saline group (n=8), cetuximab (400 mg/m(2)) was administered to the postoperative 1 group (n=8) 1 day after surgery, and cetuximab (400 mg/m(2)) was administered to the peroperative group (n=8) during surgery. The mean adhesion grade was 2.63±0.92, and 0.50±0.76 and 0.63±0.74 for control and test groups, respectively. Cetuximab reduced adhesion formation in test groups (p<0.05). When all groups were compared, it was found that vascular endothelial growth factor levels decreased significantly only in the abdomen (p<0.05). Hydroxyproline levels and anastomosis bursting pressure were examined, and a statistical difference was found between groups (hydroxyproline p<0.05, bursting pressure p<0.05). However, when postoperative 1 day group was compared with the control group, it was found that there was no difference between groups according to these parameters (p>0.05), but when peroperative group was compared with the control group a significant decrease was observed in both parameters. Histopathological healing score was also evaluated. No statistical difference between groups was found. Twenty-four hours later from the operation, intraperitoneal cetuximab therapy may be a safe and feasible treatment for metastatic colorectal patients.

Down-regulating ERK1/2 and SMAD2/3 phosphorylation by physical barrier of celecoxib-loaded electrospun fibrous membranes prevents tendon adhesions

Peritendinous adhesions, as a major problem in hand surgery, may be due to the proliferation of fibroblasts and excessive collagen synthesis, in which ERK1/2 and SMAD2/3 plays crucial roles. In this study, we hypothesized that the complication progression could be inhibited by down-regulating ERK1/2 and SMAD2/3 phosphorylation of exogenous fibroblasts with celecoxib. Celecoxib was incorporated in poly(l-lactic acid)-polyethylene glycol (PELA) diblock copolymer fibrous membranes via electrospinning. Results of an in vitro drug release study showed celecoxib-loaded membrane had excellent continuous drug release capability. It was found that celecoxib-loaded PELA membranes were not favorable for the rabbit fibroblast and tenocyte adhesion and proliferation. In a rabbit tendon repair model, we first identified ERK1/2 and SMAD2/3 phosphorylation as a critical driver of early adhesion formation progression. Celecoxib released from PELA membrane was found to down-regulate ERK1/2 and SMAD2/3 phosphorylation, leading to reduced collagen I and collagen Ⅲ expression, inflammation reaction, and fibroblast proliferation. Importantly, the celecoxib-loaded PELA membranes successfully prevented tissue adhesion compared with control treatment and unloaded membranes treatment. This approach offers a novel barrier strategy to block tendon adhesion through targeted down-regulating of ERK1/2 and SMAD2/3 phosphorylation directly within peritendinous adhesion tissue.

Inhibition of Contractility and RhoA Deactivation Trigger Podosome Formation

Development of integrin-mediated cell-matrix adhesions is important in the regulation of cell growth and differentiation. Activated integrin clusters are sites of actin polymerization, and the physical properties of extracellular matrix ligands play critical roles in the development of adhesion structures. Previously, we have utilized RGD-membranes, continuous supported bilayers with freely diffusing lipid-anchored RGD ligands, to activate integrins and investigate force dependence of initial integrin clustering during early adhesion formation (Yu et al., PNAS 2011). At later times (>45 minutes) on RGD-membranes, we found that fibroblasts natively form podosomes as adhesion structures when cell-matrix traction force cannot be stabilized. Podosome core assembly was initiated by Arp2/3-mediated actin polymerization locally within integrin clustering sites that were characterized by integrin-associated proteins, such as vinculin, paxillin, and talin. In addition, myosin-1E/1F and CARMIL1 were found as new markers at podosome cores. We utilized a FRET-based RhoA biosensor to measured differential RhoA activities in cells forming podosomes on RGD-membranes versus focal adhesions on RGD-glass. RhoA-GTP levels were indeed low in cells forming podosomes on RGD-membranes, whereas RhoA-GTP levels were high in the cells on RGD-glass. Constitutively active RhoA-Q63L mutant blocked podosome formation on RGD-membranes. Furthermore, fibroblasts on RGD-membranes with dense nano-partitions (1μm-pitch line partitions) in RGD-membranes provided mechanical barriers that enabled adhesion maturation that was not possible with wider line-partitions (4μm) in RGD-membrane. When a single cell adhered over both regions of high and low density, podosomes formed over low-density regions only. From our data, we suggest that development of podosomes as adhesion structures requires inhibition of traction forces between integrin receptors and matrix ligands. Activation of myosin contractility at integrin clusters activates focal adhesion formation and inhibits podosome formation at integrin clusters, giving rise to a mutually exclusive behavior of focal adhesions and podosomes.

Force at Early Adhesion Formation: Integrin Activation Triggers Contractile Movement and Subsequent Outward Translocation of Integrin-RGD Clusters

Integrin-mediated adhesions play important roles in many biology events, including stem cell differentiation, cancer metastasis, and the immune response. However, The mechanism of early adhesion formation remains unclear. Using mobile RGD ligands on lipid bilayers with nano-fabricated physical barriers, we observe surprising long-range lateral movements of ligated integrins during the process of cell spreading. At first, RGD-activated integrin clusters stimulate actin polymerization that radiates from the clusters. Myosin II contraction of actin from adjacent clusters produces contractile pairs that move toward each other against barriers. Force generated by myosin II stimulates a Src kinase-dependent lamellipodial extension and outward movement of clusters. Subsequent retraction by myosin II causes inward movement of clusters. The final cell spread area increases with the density of periodic barriers. Early integrin clustering recruits adhesion proteins, talin, paxillin and focal adhesion kinase, irrespective of force generation. However, recruitment of vinculin is only observed upon contraction. Thus, we suggest that integrin activation and early clustering are independent of lateral forces. Src-dependent actin polymerization on integrin clusters and subsequent contraction stimulate active spreading with outward forces from actin polymerization followed by a second wave of contraction. These early mechanical steps provide new targets to control integrin-dependent adhesion and motility.

Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation

Integrin-mediated adhesions are critical for stem cell differentiation, cancer metastasis, and the immune response [Hynes RO (2009) Science 326:1216-1219]. However, the mechanisms of early adhesion formation remain unclear, especially the effects of lateral clustering of integrins and the role of the Src family kinases. Using mobile Arg-Gly-Asp (RGD) peptide ligands on lipid bilayers with nano-fabricated physical barriers [Salaita K, et al. (2010) Science 327:1380-1385], we observe surprising long-range lateral movements of ligated integrins during the process of cell spreading. Initially, RGD-activated integrin clusters stimulate actin polymerization that radiates from the clusters. Myosin II contraction of actin from adjacent clusters produces contractile pairs that move toward each other against barriers. Force generated by myosin II stimulates a Src kinase-dependent lamellipodial extension and outward movement of clusters. Subsequent retraction by myosin II causes inward movement of clusters. The final cell spread area increases with the density of periodic barriers. Early integrin clustering recruits adhesion proteins, talin, paxillin, and FAK, irrespective of force generation. However, recruitment of vinculin is only observed upon contraction. Thus, we suggest that integrin activation and early clustering are independent of lateral forces. Clustering activates Src-dependent actin polymerization from clusters. Myosin contraction of clusters to lines stimulates active spreading with outward forces from actin polymerization followed by a second wave of contraction. Many of these early mechanical steps are not evident in cells spreading on immobilized matrices perhaps because of the low forces involved. These observations can provide new targets to control integrin-dependent adhesion and motility.

Tc-99m-PEG-Liposomes Target Both Adhesions and Abscesses and Their Reduction by Hyaluronate in Rats With Fecal Peritonitis

Abdominal adhesions and abscesses are a major source of morbidity and mortality after abdominal surgery and peritonitis. Adhesions are hard to detect with standard imaging techniques. Liposomes, coated with polyethylene glycol (PEG), represent an agent developed for infection imaging. This study investigated the capacity of 99mTc-PEG-liposomes to localize early adhesion formation after peritonitis. Additionally, the value of 99mTc-PEG-liposomes for therapy evaluation of hyaluronan solution, which reduces adhesion and abscess formation in experimental peritonitis, was assessed. In 24 rats, a bacterial peritonitis was induced by performing a cecal ligation and puncture procedure. The animals were treated with sodium chloride solution or 0.4% hyaluronan solution intra-abdominally. One week later, scintigraphy was performed using 99mTc-PEG-liposomes, and abnormal focal uptake in the abdomen was scored. Thereafter, autopsy was performed and adhesions and abscesses were scored. A significant correlation was found between the total adhesion score and the scintigraphic score (P < 0.01, r = 0.65). Treatment with hyaluronan significantly reduced the total adhesion score (P = 0.01). The size of abscesses significantly correlated with the scintigraphic score (P < 0.01, r = 0.65). Treatment with hyaluronan reduced the size of abscesses (P < 0.05). 99mTc-PEG-liposomes are able to detect early adhesions and abscesses and may be used for therapy evaluation of agents that reduce adhesions and abscesses.

Analysis of Sodium Carboxymethylcellulose Administration and Related Factors Associated with Postoperative Colic and Survival in Horses with Small Intestinal Disease

To analyze the effect of the intraoperative use of sodium carboxymethylcellulose (CBMC) and related perioperative factors on postoperative colic and survival in horses that had abdominal surgery for colic. Retrospective study. Horses (n=203) that had surgery for small intestinal disease; 33 horses had intraoperative administration of CBMC. Information was obtained from medical records for 170 horses that had surgery for colic before use of CBMC and 33 horses that had intraoperative CBMC. Kaplan-Meier survival curves were used to estimate median survival time and a Cox proportional hazards model was used to estimate the hazard ratio for the effect of CBMC and other perioperative variables on survival. Seventy-five percent of horses administered CBMC survived to 180 days, whereas 75% of untreated horses survived 8 days (median survival time=18 days). Horses not administered CBMC were twice as likely to die compared with horses administered CBMC. Horses that had postoperative ileus (POI) were 1.4 times more likely to die than horses without ileus. Similarly, horses with signs of colic after surgery were 1.3 times more likely to die than horses without postoperative signs of colic. CBMC administration is seemingly protective against death and prolongs survival when used intraoperatively in horses with small intestine disease, particularly horses with postoperative colic or POI. Both POI and colic increased risk of death after surgery. Intraoperative administration of CBMC in horses that have surgery for small intestinal disease may improve survival, possibly by reducing early adhesion formation.

Fibrin Sealant (Tissucol) Enhances Tissue Integration of Condensed Polytetrafluoroethylene Meshes and Reduces Early Adhesion Formation in Experimental Intraabdominal Peritoneal Onlay Mesh Repair

The laparoscopic intraabdominal peritoneal onlay mesh repair (IPOM) is a common technique for the reinforcement of multiple ventral hernias or defined defects after laparotomies. However, the placement of synthetic meshes in the intraabdominal cavity can be associated with severe complications. Adhesions frequently originate from the implant and protruding parts of fixation devices, presenting a serious clinical problem with potentially detrimental consequences. This study was designed to assess the impact of fibrin sealing with Tissucol (FS; Baxter, Vienna, Austria) on adhesion formation to condensed polytetrafluoroethylene meshes (Motif Meshes, MM; Proxy Biomedical, Galway, Ireland) as well as on tissue integration of these implants in experimental IPOM repair in rats. It was tested whether FS application allowed the reduction of sutures for mesh fixation without increasing the risk of mesh dislocation. Sixteen rats were assigned to the implantation of MM with four nonresorbable sutures (Synthofil; Ethicon, Norderstedt, Germany) with additional fibrin coating with 0.2 mL FS or to MM fixation with six nonresorbable sutures without FS (n = 8 per group). MM with 2 cm in diameter were implanted in open IPOM by a laparatomy. The observation period of 17 days ensured assessment of adhesions after the full degradation of FS. Adhesions were rated with the score suggested by Vandendael. Histology was performed. All eight MMs without FS sealing elicited severe (grade III) adhesions, whereas fibrin-sealed MM were rated mild in 1, moderate in 5, and severe in 2 cases. The superior finding in the FS group was statistically significant. Impaired integration of sutured-only MM was observed in four cases, whereas all FS-sealed MM were well integrated. FS improves the tissue integration, reduces early adhesion formation to cPTFE implants, and allows reduction of perforating fixation devices in experimental IPOM repair.