Bacterial lipopolysaccharide or endotoxin induces actin reorganization, increased paracellular permeability, and endothelial cell detachment from the underlying extracellular matrix in vitro. We studied the effect of endotoxin on transendothelial albumin flux and detachment of endothelial cells cultured on gelatin-impregnated filters. The endotoxin-induced changes in endothelial barrier function and detachment occurred at doses and times that were compatible with endotoxininduced apoptosis. Since the actin cytoskeleton and cellcell and cell-matrix adhesion all participate in the regulation of the paracellular pathway and cell-matrix interactions, we studied whether protein components of the actin-linked adherens junctions were modified in response to endotoxin. Components of cell-cell (␤-and ␥-catenin) and cell-matrix (focal adhesion kinase and p130Cas ) adherens junctions were cleaved by caspases activated during apoptosis with dose and time requirements that paralleled those seen for barrier dysfunction and detachment. Cleavage of focal adhesion kinase led to its dissociation from the focal adhesion-associated signaling protein, paxillin, resulting in reduced paxillin tyrosine phosphorylation. Inhibition of caspase-mediated cleavage of these proteins protected against detachment but not opening of the paracellular pathway. Therefore, endotoxin-induced disruption of endothelial monolayer integrity and survival signaling events is mediated, in part, through caspase cleavage of adherens junction proteins.

The NADPH oxidase of phagocytes generates microbicidal oxidants in response to a variety of stimuli. Its activation and assembly involve multiple SH3 domain interactions among several oxidase components. Here we present evidence that the cytosolic oxidase-associated protein, p40(phox), mediates down-regulation of NADPH oxidase through interactions with its SH3 domain. Recombinant p40(phox) was produced in several eukaryotic expression systems (insect, mammalian, and yeast) to explore its role in oxidase function in relation to domains involved in interactions with other factors, p47(phox) and p67(phox). p40(phox) inhibited oxidase activity in vitro when added to neutrophil membranes and recombinant p47(phox), p67(phox), and p21rac. Co-transfection of p40(phox) into K562 cells resulted in significant decreases ( approximately 40%) in whole cell oxidase activity. Furthermore, the isolated SH3 domain of p40(phox) was even more effective in inhibiting whole cell oxidase activity, consistent with experiments showing that this domain binds to the same proline-rich target in p47(phox) (residues 358-390) that interacts with p67(phox). In contrast, deletion of the carboxyl-terminal domain of p40(phox) that binds to p67(phox) did not relieve its oxidase inhibitory effects. Thus, p40(phox) appears to down-regulate oxidase function by competing with an SH3 domain interaction between other essential oxidase components.

Human amniotic membrane (AM) has a long history of clinical use for wound treatment. AM serves as a wound protective barrier maintaining proper moisture. AM is anti-inflammatory, anti-microbial and antifibrotic, and supports angiogenesis, granulation tissue formation and wound re-epithelialization. These properties of AM are attributed to its native extracellular matrix, growth factors, and endogenous cells including mesenchymal stem cells. Advances in tissue preservation have helped to overcome the short shelf life of fresh AM and led to the development of AM products for clinical use. Viable cryopreserved amnion (VCAM), which retains all native components of fresh AM, has shown positive outcomes in clinical trials for wound management. However, cryopreservation requires ultra-low temperature storage and shipment that limits widespread use of VCAM. We have developed a lyopreservation technique to allow for ambient storage of living tissues. Here, we compared the structural, molecular, and functional properties of a viable lyopreserved human amniotic membrane (VLAM) with properties of VCAM using in vitro and in vivo wound models. We found that the structure, growth factors, and cell viability of VLAM is similar to that of VCAM and fresh AM. Both, VCAM and VLAM inhibited TNF-α secretion and upregulated VEGF expression in vitro under conditions designed to mimic inflammation and hypoxia in a wound microenvironment, and resulted in wound closure in a diabetic mouse chronic wound model. Taken together, these data demonstrate that VLAM structural and functional properties are equivalent to VCAM but without the constraints of ultra-low temperature storage.

Post-operative adhesions, a common complication of surgery, cause pain, impair organ functionality, and often require additional surgical interventions. Control of inflammation, protection of injured tissue, and rapid tissue repair are critical for adhesion prevention. Adhesion barriers are biomaterials used to prevent adhesions by physical separation of opposing injured tissues. Current adhesion barriers have poor anti-inflammatory and tissue regenerative properties. Umbilical cord tissue (UT), a part of the placenta, is inherently soft, conforming, biocompatible, and biodegradable, with antimicrobial, anti-inflammatory, and antifibrotic properties, making it an attractive alternative to currently available adhesion barriers. While use of fresh tissue is preferable, availability and short storage time limit its clinical use. A viable cryopreserved UT (vCUT) “point of care” allograft has recently become available. vCUT retains the extracellular matrix, growth factors, and native viable cells with the added advantage of a long shelf life at −80 °C. In this study, vCUT's anti-adhesion property was evaluated in a rabbit abdominal adhesion model. The cecum was abraded on two opposing sides, and vCUT was sutured to the abdominal wall on the treatment side; whereas the contralateral side of the abdomen served as an internal untreated control. Gross and histological evaluation was performed at 7, 28, and 67 days post-surgery. No adhesions were detectable on the vCUT treated side at all time points. Histological scores for adhesion, inflammation, and fibrosis were lower on the vCUT treated side as compared to the control side. In conclusion, the data supports the use of vCUT as an adhesion barrier in surgical procedures.

Objective: Wound healing is a complex process involving the dynamic interplay of various types of cells and bioactive factors. Impaired wound healing is characterized by a loss in synchronization of the process, resulting in non-healing chronic wounds. Human amniotic membrane (AM) has been shown to be effective in the management of chronic wounds. Recently, a viable lyopreserved AM (VLAM) has been developed. The VLAM retains the structural, molecular, and functional properties of fresh AM with the advantage of a long shelf life for living tissue at ambient temperatures. The objective of this study was to evaluate the effects of VLAM on the impaired wound microenvironment and wound closure in db/db mice. Approach: VLAM or saline gel (control) was applied weekly to 7-mm excisional wounds in diabetic (db/db) mice. Wound appearance and size were assessed weekly. Inflammation and redox state in wounds were tested by cytokine gene and protein expression, and by catalase and glutathione peroxidase activities, respectively. Wound tissue granulation and neovascularization were assessed histologically. Results: Diabetic wounds treated with VLAM closed faster than control wounds. On an average, VLAM-treated wounds closed 4 days faster than the control wounds, with a significantly faster rate of closure at days 7 and 14 as compared with control wounds. The faster closure correlated with a decrease in the expression of proinflammatory factors and oxidative stress, and an increase in angiogenesis and dermal thickness. Innovation: Effects of VLAM on a chronic wound microenvironment and underlying molecular mechanisms were investigated for the first time. Conclusion: VLAM accelerates wound closure in db/db mice by decreasing inflammation and oxidative stress and supporting wound tissue granulation, neovascularization, and re-epithelialization.

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Objective: Clinical studies have demonstrated that the use of cryopreserved amnion or trophoblast (TR)-free chorion, containing viable cells, in the treatment of chronic wounds results in high rate of wound closure. Recently, a new lyopreservation method has been developed for preservation of amnion that also retains the endogenous viable cells. The objective of this study was to use this method for lyopreservation of TR-free chorionic membrane (viable lyopreserved chorionic membrane [VLCM]) and compare it with the viable cryopreserved chorionic membrane (VCCM). A second objective was to investigate the immunogenicity of chorion, an important question that has not been fully addressed. Approach: Chorion immunogenicity was tested in vitro in a mixed lymphocyte reaction and lipopolysaccharide (LPS) challenge assay, and in vivo in a mouse subcutaneous pocket implantation model. VLCM tissue structure was assessed histologically, growth factor content by multiplex assay, and cell viability by LIVE/DEAD cell fluorescent staining. Inhibition of tumor necrosis factor α secretion by LPS-activated THP-1 cells and endothelial cell tubule formation assays were performed to evaluate the anti-inflammatory and proangiogenic properties, respectively. An in vivo rabbit abdominal adhesion model was used to evaluate the antifibrotic properties. Results: Chorionic membrane without trophoblast (CM) was shown to be nonimmunogenic. Tissue architecture, growth factors, and cell viability of fresh CM were maintained in VLCM and VCCM. In vitro studies showed that anti-inflammatory and angiogenic properties were retained in VLCM. Furthermore, VLCM prevents formation of postsurgical adhesions in a rabbit abdominal surgical adhesion model. Innovation: Characterization of structural and functional properties of VLCM is reported for the first time. Conclusion: Similar to VCCM, VLCM retains native components of fresh CM, including collagen-rich extracellular matrix, growth factors, and viable cells. In vitro and in vivo models demonstrate that VLCM is anti-inflammatory, proangiogenic and antifibrotic. Results of this study support the structural and functional equivalency between VLCM and VCCM.

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