Integrin-dependent Akt1 activation regulates PGC-1 expression and fatty acid oxidation.

BACKGROUND: Poly-N-acetyl glucosamine nanofibers derived from a marine diatom have been used to increase cutaneous wound healing. These nanofibers exert their activity by specifically activating integrins, which makes them a useful tool for dissecting integrin-mediated pathways. We have shown that short-fiber poly-N-acetyl glucosamine nanofiber (sNAG) treatment of endothelial cells results in increased cell motility and metabolic rate in the absence of increased cell proliferation. RESULTS: Using a Seahorse Bioanalyzer to measure oxygen consumption in real time, we show that sNAG treatment increases oxygen consumption rates, correlated with an integrin-dependent activation of Akt1. Akt1 activation leads to an increase in the expression of the transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). This is not due to increased mitochondrial biogenesis, but is associated with an increase in the expression of pyruvate dehydrogenase kinase 4 (PDK4), suggesting regulation of fatty acid oxidation. Blockade of fatty acid oxidation with etomoxir, an O-carnitine palmitoyltransferase-1 inhibitor, blocks the sNAG-dependent increased oxygen consumption. (3)H-palmitate uptake experiments indicate a PDK4-dependent increase in fatty acid oxidation, which is required for nanofiber-induced cell motility. CONCLUSIONS: Our findings imply a linear pathway whereby an integrin-dependent activation of Akt1 leads to increased PGC-1α and PDK4 expression resulting in increased energy production by fatty acid oxidation.

mRDH bandage for surgery and trauma: data summary and comparative review.

BACKGROUND: Bleeding often poses significant life-threatening situations to surgeons. After trauma, a one-third of civilian casualties and one-half of combat casualties die as a result of exsanguination. Recent advances have provided promising new hemostatic dressings that are applied directly to severely bleeding wounds in the pre-hospital period. METHODS: The modified Rapid Deployment Hemostat (mRDH) trauma/surgery bandage, containing fully acetylated, diatom-derived, poly-N-acetyl-glucosamine fibers, has a unique multifactorial hemostatic action that incorporates vasoconstriction, erythrocyte agglutination, and platelet and RBC activation. RESULTS: Animal studies have shown that the mRDH bandage quickly and completely stops both venous and arterial bleeding, even in the presence of a coagulopathy. A prospective study in humans is in accord with these findings. CONCLUSION: The mRDH trauma/surgery bandage was able to increase survival of patients after high-grade liver trauma with an associated coagulopathy. Additional clinical studies support this result.

Poly-N-acetyl glucosamine fibers activate bone regeneration in a rabbit femur injury model.

BACKGROUND: The purpose of this study was to evaluate the ability of a membrane material, consisting only of short poly-N-acetyl glucosamine (sNAG) nanofibers, to regenerate bone tissue after implantation into circular holes in the rabbit femur. METHODS: Three circular holes were created in the femurs of five male New Zealand white rabbits. The holes were ∼ 2.0 mm in diameter. Three holes in the left femur were implanted with the comparative control substance (Bone Wax; Ethicon, Inc.); three holes in the right femur were implanted with the sNAG membrane test article. Animals were killed 4 weeks after surgery, and macroscopic evaluation of the implant sites was made. Hematoxylin and eosin histology was performed on both control and test sites. RESULTS: All control (bone wax) sites had visible holes (defects) at the 28-day end point of the study and showed no evidence of new bone formation. All the 15 sNAG test sites were found to have new bone tissue present in the bone hole defects. Hematoxylin and eosin histology of the sNAG-treated test sites showed the presence of osteoblasts, osteocytes, and trabecula of new bone formation at the 28-day end point of the study. CONCLUSIONS: The sNAG membrane test material activated the regeneration of new bone tissue in a rabbit femur bone model after 28 days of implantation, whereas the control bone wax material did not.

Poly-N-acetyl glucosamine gel matrix as a non-viral delivery vector for DNA-based vaccination.

Intramuscular administration of plasmid DNA vaccines is one of the main delivery approaches that can generate antigen specific T cell responses. However, major limitations of the intramuscular delivery strategy are the low level of myocyte transfection, resulting in a minimal level of protein expression; the inability to directly target antigen presenting cells, in particular dendritic cells, which are critical for establishment of efficacious antigen-specific immune responses. Although several viral vectors have been designed to improve plasmid DNA delivery, they have limitations, including the generation of neutralizing antibodies in addition to lacking the simplicity and versatility required for universal clinical application. We have developed an inexpensive non-viral delivery vector based on the polysaccharide polymer poly-N-acetyl glucosamine with the capability to target dendritic cells. This vector is fully biocompatible, biodegradable, and nontoxic. The advantage of the application of this delivery system relative to other approaches is discussed.

Poly-N-acetyl glucosamine nanofibers: a new bioactive material to enhance diabetic wound healing by cell migration and angiogenesis.

INTRODUCTION: In several fields of surgery, the treatment of complicated tissue defects is an unsolved clinical problem. In particular, the use of tissue scaffolds has been limited by poor revascularization and integration. In this study, we developed a polymer, poly-N-acetyl-glucosamine (sNAG), with bioactive properties that may be useful to overcome these limitations. OBJECTIVE: To develop a scaffold-like membrane with bioactive properties and test the biologic effects in vitro and in vivo in diabetic wound healing. METHODS: In vitro, cells-nanofibers interactions were tested by cell metabolism and migration assays. In vivo, full thickness wounds in diabetic mice (n = 15 per group) were treated either with sNAG scaffolds, with a cellulosic control material, or were left untreated. Wound healing kinetics, including wound reepithelialization and wound contraction as well as microscopic metrics such as tissue growth, cell proliferation (Ki67), angiogenesis (PECAM-1), cell migration (MAP-Kinase), and keratinocyte migration (p 63) were monitored over a period of 28 days. Messenger RNA levels related to migration (uPAR), angiogenesis (VEGF), inflammatory response (IL-1beta), and extracellular matrix remodeling (MMP3 and 9) were measured in wound tissues. RESULTS: sNAG fibers stimulated cell metabolism and the in vitro migratory activity of endothelial cells and fibroblasts. sNAG membranes profoundly accelerated wound closure mainly by reepithelialization and increased keratinocyte migration (7.5-fold), granulation tissue formation (2.8-fold), cell proliferation (4-fold), and vascularization (2.7-fold) compared with control wounds. Expression of markers of angiogenesis (VEGF), cell migration (uPAR) and ECM remodeling (MMP3, MMP9) were up-regulated in sNAG treated wounds compared with controls. CONCLUSIONS: The key mechanism of the bioactive membranes is the cell-nanofiber stimulatory interaction. Engineering of bioactive materials may represent the clinical solution for a number of complex tissue defects.

The design and testing of a dual fiber textile matrix for accelerating surface hemostasis.

The standard treatment for severe traumatic injury is frequently compression and application of gauze dressing to the site of hemorrhage. However, while able to rapidly absorb pools of shed blood, gauze fails to provide strong surface (topical) hemostasis. The result can be excess hemorrhage-related morbidity and mortality. We hypothesized that cost-effective materials (based on widespread availability of bulk fibers for other commercial uses) could be designed based on fundamental hemostatic principles to partially emulate the wicking properties of gauze while concurrently stimulating superior hemostasis. A panel of readily available textile fibers was screened for the ability to activate platelets and the intrinsic coagulation cascade in vitro. Type E continuous filament glass and a specialty rayon fiber were identified from the material panel as accelerators of hemostatic reactions and were custom woven to produce a dual fiber textile bandage. The glass component strongly activated platelets while the specialty rayon agglutinated red blood cells. In comparison with gauze in vitro, the dual fiber textile significantly enhanced the rate of thrombin generation, clot generation as measured by thromboelastography, adhesive protein adsorption and cellular attachment and activation. These results indicate that hemostatic textiles can be designed that mimic gauze in form but surpass gauze in ability to accelerate hemostatic reactions.

Differential effect of materials for surface hemostasis on red blood cell morphology.

The design of devices for surface (topical) hemostasis has been based on maximizing activation of platelets and accelerating coagulation pathways. The studies reported herein examine another aspect of blood contact with topical hemostasis materials, i.e., surface binding of red blood cells (RBCs) and related alterations in RBC morphology. Whole blood was allowed to contact poly-N-acetyl glucosamine (pGlcNAc) containing materials: pGlcNAc nanofibers with parallel polymer alignment (beta-pGlcNAc), chitin, and chitosan. The effect on RBC morphology and function via contact with the artificial surfaces on the cell's morphology was examined with scanning and transmission electron microscopy (TEM). beta-pGlcNAc was found to densely bind RBCs and induce a stomatocytic-like morphology. Chitin and chitosan also bound RBCs, but with approximately 10-fold lower levels and with less distinct general morphologies. beta-pGlcNAc is thus unique in the nature of its interaction with RBCs. These studies indicate that the differential ability of various materials to bind and alter the morphology of RBCs at the artificial surface interface with blood is an important consideration in the design of devices for surface hemostasis.

Non-classical processes in surface hemostasis: mechanisms for the poly-N-acetyl glucosamine-induced alteration of red blood cell morphology and surface prothrombogenicity.

It is well established that platelets and the intrinsic plasma coagulation pathway can be activated when blood contacts artificial surfaces. Experiments were performed to assess the effect of hemostatic poly-N-acetyl glucosamine (pGlcNAc) nanofibers on red blood cells. The pGlcNAc nanofibers, isolated from a marine diatom, interact with red blood cells (RBCs) to produce stomatocytes. The stomatocytes could be converted to echinocytes by treatment with echinocytic reagents, as measured by electron microscopy. Electrophoretic and Western blot analysis of RBC surface proteins demonstrated that pGlcNAc fibers were bound to band 3 of the RBC. An important and unique result of the interaction of RBCs with pGlcNAc fibers was the activation of the intrinsic coagulation cascade. This prothrombotic effect was associated with the presentation of phosphatidylserine on the outer layer of the surface membrane of nanofiber bound RBCs. The results demonstrate that RBCs can play a direct and important role in achieving surface hemostasis by accelerating the generation of thrombin, and add to the growing body of evidence that RBCs can strongly interact with hemostatic systems.

Effects of poly-N-acetyl glucosamine (pGlcNAc) patch on wound healing in db/db mouse.

BACKGROUND: Poly-N-acetyl glucosamine (pGlcNAc) nanofiber-based materials, produced by a marine microalga, have been characterized as effective hemostatic agents. In this study, we hypothesized that a pGlcNAc fiber patch may enhance wound healing in the db/db mouse. METHODS: pGlcNAc patches were applied on 1-cm, full-thickness, skin wounds in the db/db mouse model. Wounds (n = 15 per group) were dressed with a pGlcNAc nanofiber patch for 1 hour, 24 hours, or left untreated. After the application time, patches were removed and wounds were allowed to heal spontaneously. The rate of wound closure was evaluated by digital analysis of unclosed wound area as a function of time. At day 10, wounds (n = 7 per group) were harvested and quantified with immunohistochemical markers of proliferation (Ki-67) and vascularization (platelet endothelial cell adhesion molecule). RESULTS: Wounds dressed with pGlcNAc patches for 1 hour closed faster than control wounds, reaching 90% closure in 16.6 days, 9 days faster than untreated wounds. Granulation tissue showed higher levels of proliferation and vascularization after 1-hour treatment than the 24-hour and left-untreated groups. Foreign body reaction to the material was not noted in applications up to 24 hours. DISCUSSION: In addition to its hemostatic properties, the pGlcNAc material also appears to accelerate wound closure in healing-impaired genetically diabetic mice. This material, with its combination of hemostatic and wound healing properties, has the potential to be effective agent for the treatment of complicated wounds.

Poly-N-acetyl glucosamine nanofibers regulate endothelial cell movement and angiogenesis: dependency on integrin activation of Ets1.

Poly-N-acetyl glucosamine (pGlcNAc) nanofiber-derived materials effectively achieve hemostasis during surgical procedures. Treatment of cutaneous wounds with pGlcNAc in a diabetic mouse animal model causes marked increases in cell proliferation and angiogenesis. We sought to understand the effect of the pGlcNAc fibers on primary endothelial cells (EC) in culture and found that pGlcNAc induces EC motility. Cell motility induced by pGlcNAc fibers is blocked by antibodies directed against alphaVbeta3 and alpha5beta1 integrins, both known to play important roles in the regulation of EC motility, in vitroand in vivo. pGlcNAc treatment activates mitogen-activated protein kinase and increases Ets1, vascular endothelial growth factor (VEGF) and interleukin 1 (IL-1) expression. pGlcNAc activity is not secondary to its induction of VEGF; inhibition of the VEGF receptor does not inhibit the pGlcNAc-induced expression of Ets1 nor does pGlcNAc cause the activation of VEGF receptor. Both dominant negative and RNA interference inhibition of Ets1 blocks pGlcNAc-induced EC motility. Antibody blockade of integrin results in the inhibition of pGlcNAc-induced Ets1 expression. These findings support the hypothesis that pGlcNAc fibers induce integrin activation which results in the regulation of EC motility and thus in angiogenesis via a pathway dependent on the Ets1 transcription factor and demonstrate that Ets1 is a downstream mediator of integrin activation.

Hemostatic properties of glucosamine-based materials.

Glucosamine- and N-acetyl glucosamine-containing polymers are being used in an increasing number of biomedical applications, including in products for surface (topical) hemostasis. The studies presented here investigate the relationship between the structure (conformation) and function (activation of hemostasis) of glucosamine-based materials. Several polymer systems were studied, including fibers isolated from a microalgal source containing poly-N-acetyl glucosamine polymers that are organized in a parallel, hydrogen-bonded tertiary structure and can be chemically modified to an antiparallel orientation; and gel formulation derivatives of the microalgal fibers consisting of partially deacetylated (F2 gel) and fully deacetylated (F3 gel) polymers. Comparison of the properties of the poly-N-acetyl glucosamine fiber-derived materials with chitin, chitosan, and commercial chitosan-based products are presented. Several studies were performed with the glucosamine-based materials, including (1) an analysis of the ability of materials to activate platelets and turnover of the intrinsic coagulation cascade, (2) an examination of the viscoelastic properties of mixtures of platelet-rich plasma and the glucosamine-based materials via thromboelastography, and (3) scanning electron microscopic studies to examine the morphology of the glucosamine-based materials. The results presented demonstrate that hemostatic responses to the glucosamine-based materials studied are highly dependent on their chemical nature and tertiary/quaternary structure. The unique natural microalgal fibers were found to have strongly prohemostatic activity compared to the other materials studied.

Review: novel nonviral delivery approaches for interleukin-12 protein and gene systems: curbing toxicity and enhancing adjuvant activity.

It has become increasingly apparent that the ability to generate an optimal host immune response requires effective cross talk between the innate and adaptive components of the immune system. Pro-inflammatory cytokines, in particular those that can induce a danger signal, often called signal 3, are crucial in this role of initiating and augmenting the presentation of exogenous antigen to T cells by dendritic cells. Interleukin-12 (IL-12) in particular has been defined as a "signal 3" cytokine required for the antigen cross priming. Given this unique interactive function, a significant amount of work has been performed to define possible therapeutic applications for IL-12. Systemic IL-12 administration can clearly act as a potent adjuvant for postvaccination T cell responses in a variety of diseases. As an example, in the cancer setting, systemic IL-12 is capable of suppressing tumor growth, metastasis, and angiogenesis in vivo. IL-12, however, has been associated with significant dose- and schedule-dependent toxicity in early clinical trials, results that have proven to be a major obstacle to its clinical application. Recent research has focused on decreasing the toxicity of IL-12 using different delivery approaches, including virus-based and gene-modified cell-based delivery. Although effective, these approaches also have limitations, including the generation of neutralizing antibodies, in addition to lacking the simplicity and versatility required for universal clinical application. Thus, there is a significant interest in the development of alternative delivery approaches for IL-12 administration that can overcome these issues. Several nonviral delivery approaches for IL-12 protein or gene expression vectors are being defined, including alum, liposomes, and polymer-based delivery. These developing approaches have shown promising adjuvant effects with significantly lessened systemic toxicity. This article discusses the potential capabilities of these nonvirus-based IL-12 delivery systems in different disease settings, including allergy, infection, and cancer.

Arterial embolization using poly-N-acetyl glucosamine gel in a rat kidney model.

Aqueous solutions of poly-N-acetyl glucosamine (p-GlcNAc) exhibit a liquid-gel transition at physiological pH and temperature. This feature inspired the authors to conduct a study to evaluate the macro- and histological changes of rat kidneys after embolization using either p-GlcNAc gel injection into the renal artery or ligation of the renal artery. The procedures were performed in 46 rats through open abdominal surgeries. Animals were sacrificed at 3 days and at 1, 3, 5, and 8 weeks postoperatively. The results of both macro-observation and histological study showed that p-GlcNAc gels were effective in causing necrosis and subsequent fibrosis in all embolized kidneys. The data indicate that p-GlcNAc gel may have promise as an effective agent for therapeutic embolization.

Synergistic platelet integrin signaling and factor XII activation in poly-N-acetyl glucosamine fiber-mediated hemostasis.

The polymer poly-N-acetylglucosamine (pGlcNAc) containing fiber material is becoming increasingly important as a topical agent for hemostasis at wound sites. The pGlcNAc polymeric fiber provides hemostasis through redundant mechanisms that include platelet activation for fibrin network formation. The research presented here better defines the mechanism for the effect of pGlcNAc containing fibers on platelet-mediated processes. Adsorption experiments demonstrated that pGlcNAc fibers tightly bind most major plasma proteins and a specific sub-set of platelet surface proteins, including the integrin beta(3) subunit (CD61) and the von Willebrand receptor GP1b (CD42b). The result of this interaction is a platelet-dependent acceleration of fibrin gel formation. Accelerated fibrin polymerization is sensitive to factor XII inhibition by corn trypsin inhibitor and integrin inactivation with integrilin. Confocal microscopy studies show that when platelet integrins contact plasma protein-saturated pGlcNAc fibers, an increase in intracellular free calcium for platelet activation occurs to drive surface expression of phosphatidyl serine (PS). Thus, a catalytic surface for thrombin generation and accelerated fibrin clot formation results from the interaction of platelets with pGlcNAc. These findings, when considered with the observation that pGlcNAc fibers also induce red blood cell agglutination and vasoconstriction, provides an explanation for the ability of the pGlcNAc material to provide hemostasis in a wide variety of clinical applications.

Isolation, purification, and characterization of poly-N-acetyl glucosamine use as a hemostatic agent.

BACKGROUND: A new polymeric material, poly-N-acetyl glucosamine (p-GlcNAc) fiber, has been identified and is effective in achieving hemostasis in surgical procedures and trauma. The p-GlcNAc material is purified from large-scale cultures of a marine microalga. METHODS: Poly-N-acetyl glucosamine materials have been formulated as films, sponges, gels, and microspheres. The polymer's structure has been characterized by chemical composition, carbohydrate analysis, spectroscopic techniques, intrinsic viscosity, and electron microscopy. RESULTS: Carbohydrate analyses indicate that the primary sugar present in p-GlcNAc is N-acetyl glucosamine. Elemental analyses yield percentage values for carbon, nitrogen, and hydrogen that support that the polymer is fully acetylated. Molecular weight determinations indicate that the polymer has a molecular weight of 2.0 x 10(6) Da. Fourier transform infrared, nuclear magnetic resonance, and circular dichroism spectral data have defined a unique tertiary structure. Biologic testing demonstrated that p-GlcNAc materials are fully biocompatible. CONCLUSION: The p-GlcNAc fiber has a unique beta-tertiary structure.

Paracrine release of IL-12 stimulates IFN-gamma production and dramatically enhances the antigen-specific T cell response after vaccination with a novel peptide-based cancer vaccine.

Interleukin-12 can act as a potent adjuvant for T cell vaccines, but its clinical use is limited by toxicity. Paracrine administration of IL-12 could significantly enhance the response to such vaccines without the toxicity associated with systemic administration. We have developed a novel vaccine delivery system (designated F2 gel matrix) composed of poly-N-acetyl glucosamine that has the dual properties of a sustained-release delivery system and a potent adjuvant. To test the efficacy of paracrine IL-12, we incorporated this cytokine into F2 gel matrix and monitored the response of OT-1 T cells in an adoptive transfer model. Recipient mice were vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12 (paracrine IL-12), or F2 gel/SIINFEKL plus systemic IL-12 (systemic IL-12). Systemic levels of IL-12 were lower in paracrine IL-12-treated mice, suggesting that paracrine administration of IL-12 may be associated with less toxicity. However, paracrine administration of IL-12 was associated with an enhanced Ag-specific T cell proliferative and functional response. Furthermore, paracrine IL-12 promoted the generation of a stable, functional memory T cell population and was associated with protection from tumor challenge. To study the mechanisms underlying this enhanced response, wild-type and gene-deficient mice were used. The enhanced immune response was significantly reduced in IFN-gamma(-/-) and IL-12R beta 2(-/-) recipient mice suggesting that the role of IL-12 is mediated, at least in part, by host cells. Collectively, the results support the potential of F2 gel matrix as a vaccine delivery system and suggest that sustained paracrine release of IL-12 has potential clinical application.

The RDH bandage: hemostasis and survival in a lethal aortotomy hemorrhage model.

BACKGROUND: The Rapid Deployment Hemostat (RDH) Bandage has been designed in collaboration with the Office of Naval Research for the treatment of bleeding because of extremity trauma. It is intended as both a battlefield and civilian severe trauma wound dressing. It consists of a specific formulation of Marine Polymer Technologies' proprietary hemostatic polymer poly-N-acetyl glucosamine, and has received FDA clearance. This study compares the hemostatic capabilities of the RDH Bandage with the standard U.S Army First Aid Field Bandage (AFAFB), utilizing a controlled lethal aortotomy model of hemorrhage. MATERIALS AND METHODS: Aortic punch wounds 4 mm in diameter were made in the abdominal aortas of female Yorkshire White swine, and were allowed to bleed for 5 s before application of test materials. Test hemostats were applied to the wound with manual compression for 10 min. Total loss of blood was determined in each experiment. Bandages were removed at the end of 2 h, for those animals that survived, and the onset of re-bleeding was observed. Animals were monitored for an additional 30 min to assess survival following bandage removal. Hemostatic efficacy was judged by the total loss of blood, and the survival of the animals. RESULTS: Eighty percent of the animals treated with the RDH Bandage survived the study through the entire protocol, whereas only 40% of those treated with the Army First Aid Field Bandage survived the removal of manual compression step, and none survived following the removal of bandage after the 2 h observation/monitoring period. The average blood loss for the RDH Bandage treated animals was 234 ml, and the average blood loss for the Army First Aid Field Bandage treated animals was 1071 ml, through the observation/monitoring period. CONCLUSIONS: The RDH Bandage is significantly superior to the standard issue U.S. Army First Aid Field Bandage in the control of hemorrhage in a lethal swine abdominal aortotomy hemorrhage model, resulting in decreased blood loss and increased survival.

Mechanisms of enhanced antigen-specific T cell response following vaccination with a novel peptide-based cancer vaccine and systemic interleukin-2 (IL-2).

Systemic interleukin-2 (IL-2) therapy has been shown to enhance the clinical efficacy of peptide-based cancer vaccines. However, the mechanisms involved in this complex response remain poorly defined. IL-2 is known to be a potent T cell growth factor, but recent studies suggest that IL-2 is also involved in the regulation of T cell immune responses by increasing the susceptibility of proliferating T cells to apoptosis. Using an adoptive transfer model, we demonstrate that the administration of systemic IL-2 significantly enhances the primary and memory immune responses following peptide-based vaccination. In order to define the mechanisms of IL-2 therapy on the antigen-specific T cell response, the kinetics of T cell proliferation, apoptosis, and trafficking were explored. Systemic IL-2 therapy did not appear to alter the kinetics of T cell proliferation immediately following vaccination, but did prolong the proliferative response. Furthermore, IL-2 therapy did not significantly influence apoptosis of proliferating T cells. Such therapy did, however, potentiate L-selectin (CD62L) downregulation on activated antigen-specific T cells, and altered their trafficking confirming their potential therapeutic value. Our findings support the use of systemic IL-2 following peptide-based vaccination, and suggest that IL-2 therapy enhances the primary and memory immune responses by prolonging the proliferative response and altering the trafficking of antigen-specific T cells.

Vascular effects of poly-N-acetylglucosamine in isolated rat aortic rings.

BACKGROUND: [corrected] Poly-N-acetylglucosamine (p-GlcNAc) is a secretion of marine diatoms that is known to be useful in controlling bleeding. As a component of promoting hemostasis, p-GlcNAc is thought to exert vasoconstrictor effects in arteries. The present study was undertaken to determine whether p-GlcNAc induced a significant vasoconstrictor effect and, if so, what the mechanism of this effect might be. MATERIALS AND METHODS: We examined vascular effects of p-GlcNAc on isolated aortic rings obtained from Sprague-Dawley rats. The rings were suspended in organ baths and precontracted with U46619, a thromboxane A2 mimetic. RESULTS: p-GlcNAc produced a concentration-dependent vasoconstriction over the range of 14 to 100 microg/ml. At a concentration of 100 microg/ml, p-GlcNAc significantly contracted aortic rings by 133 +/- 20 mg of developed force (P < 0.01). Neither a deacetylated derivative of p-GlcNAc nor a structurally related macromolecule, chitin, contracted rat aortic rings, indicating a specificity for p-GlcNAc. The vasoconstriction to p-GlcNAc was totally abolished in deendothelialized rat aortic rings, suggesting that an endothelial component is essential to the vasoconstriction. Pretreatment with the endothelin ET(A) receptor antagonist, JKC-301 (0.5 and 1 microM), significantly diminished p-GlcNAc-induced vasoconstriction by 57 to 61% (P < 0.01). However, p-GlcNAc did not significantly diminish nitric oxide release from rat aortic endothelium. CONCLUSION: These results provide evidence that p-GlcNAc significantly contracts isolated rat aortic rings via an endothelium-dependent mechanism, partly via enhancement of endothelin-1 release from endothelial cells.