Biomedical subjects

D M Stern

Publications and source records attributed to D M Stern.

At least 73 records · Page 4Linked to original sources

PubMed1998

Expression of the 150-kd oxygen-regulated protein in human breast cancer.

Tumor cells subjected to environmental stress, such as oxygen deprivation followed by reoxygenation, redirect biosynthetic pathways to express oxygen-regulated proteins (ORPs) and heat-shock proteins (HSPs). The 150-kd oxygen-regulated protein (ORP150) is a novel endoplasmic reticulum-associated polypeptide in the HSP70 family. In view of links between expression of HSPs/ORPs and tumor properties, especially tumor invasiveness and resistance to therapeutic regimens, expression of ORP150 in human breast cancers was examined. Western and Northern blotting demonstrated elevated expression of ORP150 in breast cancer, regardless of estrogen receptor status, compared with normal breast tissue. Immunohistochemical and in situ hybridization techniques revealed that infiltrating cancer cells in the stroma expressed ORP150 more strongly than large nests of cancer cells. Furthermore, pancreatic and thyroid carcinomas also displayed greater ORP150 expression. These results suggest that ORP150 is up-regulated in tumors and, in breast tumors, may be associated with tumor invasiveness.

Adenocarcinoma, Mucinous↗

PubMed1997

Role of NFkappaB in the mortality of sepsis.

Binding activity for nuclear factor kappa B (NFkappaB) consensus probes was studied in nuclear extracts from peripheral blood mononuclear cells of 15 septic patients (10 surviving and 5 not surviving). Nonsurvivors could be distinguished from survivors by an increase in NFkappaB binding activity during the observation period (P < 0.001). The increase in NFkappaB binding activity was comparable to the APACHE-II score as a predictor of outcome. Intravenous somatic gene transfer with an expression plasmid coding for IkappaBalpha was used to investigate the role of members of the NFkappaB family in a mouse model of endotoxemia. In this model, increased NFkappaB binding activity was present after injection of LPS. Intravenous somatic gene transfer with IkappaBalpha given before LPS attenuated renal NFkappaB binding activity and increased survival. Endothelial cells and monocytes/macrophages were the major target cells for somatic gene transfer, transfected with an average transfection efficiency of 20-35%. Tissue factor, a gene under regulatory control of NFkappaB, was induced by LPS. Somatic gene transfer with a reporter plasmid containing the functional tissue factor promoter demonstrated NFkappaB-dependent stimulation by LPS. Intravenous somatic gene transfer with IkappaBalpha reduced LPS-induced renal tissue factor expression, activation of the plasmatic coagulation system (decrease of thrombin-antithrombin III complexes) and renal fibrin/fibrinogen deposition. Somatic gene transfer with an expression plasmid with tissue factor cDNA in the antisense direction (in contrast to sense or vector alone) also increased survival. Furthermore, antisense tissue factor decreased renal tissue factor expression and the activation of the plasmatic coagulation system.

Adult↗

PubMed1997

Cloning of a putative vesicle transport-related protein, RA410, from cultured rat astrocytes and its expression in ischemic rat brain.

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was evaluated in cultured rat astrocytes subjected to hypoxia/reoxygenation. Using differential display, a novel putative vesicle transport-related factor (RA410) was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed RA410 to be composed of domains common to vesicle transport-related proteins of the Sec1/Unc18 family, including Sly1p and Sec1p (yeast), Rop (Drosophila), Unc18 (Caenorhabditis elegans), and Munc18 (mammalian), suggesting its possible role in vesicular transport. Northern analysis of normal rat tissues showed the highest expression of RA410 transcripts in testis. When astrocyte cultures were subjected to a period of hypoxia followed by reoxygenation, induction of RA410 mRNA was observed within 15 min of reoxygenation, reaching a maximum by 60 min. At the start of reoxygenation, the addition of diphenyl iodonium, an NADPH oxidase inhibitor, blocked in parallel astrocyte generation of reactive oxygen intermediates and expression of RA410 message. In contrast, cycloheximide did not affect RA410 mRNA levels, indicating that RA410 is an immediate-early gene in the setting of reoxygenation. Using polyclonal antibody raised against an RA410-derived synthetic peptide, Western blotting of lysates from reoxygenated astrocytes displayed an immunoreactive band of approximately 70 kDa, the expression of which followed induction of the mRNA. Fractionation of astrocyte lysates on sucrose gradients showed RA410 antigen to be predominantly in the plasma membrane. Immunoelectron microscopic analysis demonstrated RA410 in large vesicles associated with the Golgi, but not in the Golgi apparatus itself, consistent with its participation in post-Golgi transport. Consistent with these in vitro data, RA410 expression was observed in rat brain astrocytes following transient occlusion of the middle cerebral artery. These data provide insight into a new protein (RA410) that participates in the ischemia-related stress response in astrocytes.

Amino Acid Sequence↗

PubMed1997

Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation.

Clinical conditions associated with local or systemic hypoxemia can lead to prothrombotic diatheses. This study was undertaken to establish a model of whole-animal hypoxia wherein oxygen deprivation by itself would be sufficient to trigger tissue thrombosis. Furthermore, this model was used to test the hypothesis that hypoxia-induced mononuclear phagocyte (MP) recruitment and tissue factor (TF) expression may trigger the local deposition of fibrin which occurs in response to oxygen deprivation. Using an environmental chamber in which inhaled oxygen tension was lowered to 6%, hypoxic induction of thrombosis was demonstrated in murine pulmonary vasculature by 8 h based upon: (a) immunohistologic evidence of fibrin formation in hypoxic lung tissue using an antifibrin antibody, confirmed by 22.5-nm strand periodicity by electron microscopy; (b) immunoblots revealing fibrin gamma-gamma chain dimers in lungs from hypoxic but not normoxic mice or hypoxic mice treated with hirudin; (c) accelerated deposition of 125I-fibrin/fibrinogen and 111In-labeled platelets in the lung tissue of hypoxic compared with normoxic animals; (d) reduction of tissue 125I-fibrin/fibrinogen accumulation in animals which had either been treated with hirudin or depleted of platelets before hypoxic exposure. Because immunohistochemical analysis of hypoxic pulmonary tissue revealed strong MP staining for TF, confirmed by increased TF RNA in hypoxic lungs, and because 111In-labeled murine MPs accumulated in hypoxic pulmonary tissue, we evaluated whether recruited MPs might be responsible for initiation of hypoxia-induced thrombosis. This hypothesis was supported by several lines of evidence: (a) MP depletion before hypoxia reduced thrombosis, as measured by reduced 125I-fibrin/fibrinogen deposition and reduced accumulation of cross-linked fibrin by immunoblot; (b) isolated murine MPs demonstrated increased TF immunostaining when exposed to hypoxia; and (c) administration of an anti-rabbit TF antibody that cross-reacts with murine TF decreased 125I-fibrin/fibrinogen accumulation and cross-linked fibrin accumulation in response to hypoxia in vivo. In summary, these studies using a novel in vivo model suggest that MP accumulation and TF expression may promote hypoxia-induced thrombosis.

Animals↗

PubMed1997

Hypoxia-induced modulation of endothelial cell properties: regulation of barrier function and expression of interleukin-6.

The endothelial cell response to hypoxia involves a range of adaptive mechanisms that reflect an active response of the cell's biosynthetic and metabolic apparatus. Hypoxia-mediated suppression of endothelial barrier function, resulting in increased vascular leakage, is likely to contribute to pulmonary and cerebral edema associated with high altitude and is closely associated with a fall in intracellular cyclic AMP levels. Buttressing of this second messenger pathway in the endothelium using membrane permeant cyclic AMP analogs prevents increased vascular leakage due to hypoxia. Application of this principle to organ preservation has shown that supplementation with cyclic AMP analogs or inhibition of endogenous cAMP metabolism enables extension of the time a harvested organ can remain extracorporeally, after which transplantation is successful. The underlying mechanism through which cyclic AMP exerts its effects appears to be maintenance of vascular homeostasis in the graft. A distinct adaptive mechanism triggered in the endothelium by hypoxia is expression of the cytokine interleukin-6 (IL-6) by a novel mechanism involving transcription driven by the nuclear factor IL-6 (NF-IL-6) DNA binding site in the promoter. IL-6 may exert protective effects on vascular function, thereby limiting vascular injury by a different mechanism than those recruited by elevated cAMP levels. These studies provide insights into tow independent mechanisms through which endothelium responds to oxygen deprivation, and suggest possible new approaches to attentuate vascular injury associated with ischemia.

Animals↗

PubMed1996

Intravenous somatic gene transfer with antisense tissue factor restores blood flow by reducing tumor necrosis factor-induced tissue factor expression and fibrin deposition in mouse meth-A sarcoma.

Fibrin is deposited on the endothelial cell surface in the vasculature of murine methylcholanthrene A-induced sarcomas after injection of tumor necrosis factor (TNF). Capillary endothelial cells of the tumor vascular bed become positive for tissue factor after TNF injection, based on immunocytochemistry and in situ hybridization. Intravascular clot formation was not dependent on tissue factor derived from tumor cells, since in vessels of tumors not expressing tissue factor, TNF also induced fibrin/fibrinogen deposition. However, the time course of fibrin/fibrinogen deposition after TNF differed in tumors expressing no, little, or greater amounts of tissue factor. Fibrin/fibrinogen deposition was more rapid in tumors in which the neoplastic cells expressed tissue factor than in tumors not expressing tissue factor. In the tumors not expressing tissue factor, activation of coagulation was dependent on TNF-induced synthesis of tissue factor by host cells, i.e., endothelium or monocytes/macrophages. Intravenous somatic gene transfer with tissue factor cDNA in the antisense orientation (but not sense or vector alone) reduced intravascular fibrin/fibrinogen deposition and restored blood flow to the tumor, showing that de novo tissue factor expression is central in TNF-induced activation of the coagulation mechanism.

Animals↗

PubMed1996

Purification and characterization of a novel stress protein, the 150-kDa oxygen-regulated protein (ORP150), from cultured rat astrocytes and its expression in ischemic mouse brain.

As the most abundant cell type in the central nervous system, astrocytes are positioned to nurture and sustain neurons, especially in response to cellular stresses, which occur in ischemic cerebrovascular disease. In a previous study (Hori, O., Matsumoto, M., Kuwabara, K., Maeda, M., Ueda, H., Ohtsuki, T., Kinoshita, T., Ogawa, S., Kamada, T., and Stern, D. (1996) J. Neurochem., in press), we identified five polypeptide bands on SDS-polyacrylamide gel electrophoresis, corresponding to molecular masses of about 28, 33, 78, 94, and 150 kDa, whose expression was induced/enhanced in astrocytes exposed to hypoxia or hypoxia followed by replacement into the ambient atmosphere (reoxygenation). In the current study, the approximately 150-kDa polypeptide has been characterized. Chromatography of lysates from cultured rat astrocytes on fast protein liquid chromatography Mono Q followed by preparative SDS-polyacrylamide gel electrophoresis led to isolation of a approximately 150-kDa band only observed in hypoxic cells and which had a unique N-terminal sequence of 15 amino acids. Antisera raised to either the purified approximately 150-kDa band in polyacrylamide gels or to a synthetic peptide comprising the N-terminal sequence detected the same polypeptide in extracts of cultured rat astrocytes exposed to hypoxia; expression was not observed in normoxia but was induced by hypoxia within 24 h, augmented further during early reoxygenation, and thereafter decreased to the base line by 24 h in normoxia. ORP150 expression in hypoxic astrocytes resulted from de novo protein synthesis, as shown by inhibition in the presence of cycloheximide. In contrast to hypoxia-mediated induction of the approximately 150-kDa polypeptide, neither heat shock nor a range of other stimuli, including hydrogen peroxide, cobalt chloride, 2-deoxyglucose, or tunicamycin, led to its expression, suggesting selectivity for production of ORP150 in response to oxygen deprivation, i.e. it was an oxygen-regulated protein (ORP150). Northern and nuclear run-off analysis confirmed the apparent selectivity for ORP150 mRNA induction in hypoxia. Subcellular localization studies showed ORP150 to be present intracellularly within endoplasmic reticulum and only in hypoxic astrocytes, not cultured microglia, endothelial cells, or neurons subject to hypoxia. Consistent with these in vitro results, induction of cerebral ischemia in mice resulted in expression of ORP150 (the latter was not observed in normoxic brain). These data suggest that astroglia respond to oxygen deprivation by redirection of protein synthesis with the appearance of a novel stress protein, ORP150. This polypeptide, selectively expressed by astrocytes, may contribute to their adaptive response to ischemic stress, thereby ultimately contributing to enhanced survival of neurons.

Aerobiosis↗

PubMed1996

Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation.

The period of hypoxia is an important priming event for the vascular dysfunction that accompanies reperfusion, with endothelial cells (ECs) and neutrophils (PMNs) playing a central role. We hypothesized that EC Weibel-Palade (WP) body exocytosis during the hypoxic/ischemic period during organ preservation permits brisk PMN recruitment into postischemic tissue, a process further amplified in an oxidant-rich milieu. Exposure of human umbilical vein ECs to a hypoxic environment (pO2 approximately 20 torr) stimulated release of von Willebrand factor (vWF), stored in EC WP bodies, as well as increased expression of the WP body-derived PMN adhesion molecule P-selectin at the EC surface. Increased binding of 111In-labeled PMNs to hypoxic EC monolayers (compared with normoxic controls) was blocked with a blocking antibody to P-selectin, but was not affected by a nonblocking control antibody. Although increased P-selectin expression and vWF release were also noted during reoxygenation, hypoxia alone (even in the presence of antioxidants) was sufficient to increase WP body exocytosis. To determine the relevance of these observations to hypothermic cardiac preservation, during which the pO2 within the cardiac vasculature declines to similarly low levels, experiments were performed in a rodent (rat and mouse) cardiac preservation/transplantation model. Immunodepletion of recipient PMNs or administration of a blocking anti-P-selectin antibody before transplantation resulted in reduced graft neutrophil infiltration and improved graft survival, compared with identically preserved hearts transplanted into control recipients. To establish the important role of endothelial P-selectin expression on the donor vasculature, murine cardiac transplants were performed using homozygous P-selectin deficient and wild-type control donor hearts flushed free of blood/platelets before preservation/transplantation. P-selectin-null hearts transplanted into wild-type recipients demonstrated a marked (13-fold) reduction in graft neutrophil infiltration and increased graft survival compared with wild-type hearts transplanted into wild-type recipients. To determine whether coronary endothelial WP exocytosis may occur during cardiac preservation in humans, the release of vWF into the coronary sinus (CS) was measured in 32 patients during open heart surgery. CS samples obtained at the start and conclusion of the ischemic period demonstrated an increase in CS vWF antigen (by ELISA) consisting of predominantly high molecular weight multimers (by immunoelectrophoresis). These data suggest that EC WP exocytosis occurs during hypothermic cardiac preservation, priming the vasculature to recruit PMNs rapidly during reperfusion.

Animals↗

PubMed1996

Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis of stroke.

Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P < 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P < 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P < 0.005), a 35% increase in survival (P < 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P < 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.

Animals↗

PubMed1996

Exposure of astrocytes to hypoxia/reoxygenation enhances expression of glucose-regulated protein 78 facilitating astrocyte release of the neuroprotective cytokine interleukin 6.

Astrocytes exposed to hypoxia (H) or hypoxia/ reoxygenation (H/R) maintain cell viability and display changes in protein biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocytes exposed to H showed induction of an approximately 78-kDa polypeptide that demonstrated sequence identity with glucose-regulated protein (GRP) 78. Cell lysates from H/R astrocytes displayed induction of neuroprotective interleukin (IL) 6, which was present in a high-molecular-weight complex also containing GRP78, suggesting that GRP78 might be functioning as a chaperone during cellular stress consequent on H/R. Introduction of antisense oligonucleotide to GRP78 into astrocytes prevented expression of the protein and suppressed H/R-induced astrocyte release of IL-6 by approximately 50%. These data indicate that modulation of astrocyte properties during oxygen deprivation results, in part, from intracellular glucose depletion and subsequent expression of GRP78, which sustains generation of neuroprotective IL-6 under the stress of H/R.

Animals↗

PubMed1996

Receptors for advance glycation end-products (AGE) - expression by endothelial cells in non-diabetic uraemic patients.

BACKGROUND: Cellular actions of advanced glycation end-products (AGE) are mediated by a receptor for AGE (RAGE), a novel integral membrane protein. Immunohistochemical studies show only low-level RAGE antigen expression in endothelial cells. Design. It was the purposes of the study to compare expression of RAGE antigen by endothelial cells in non-diabetic uraemic patients (n=8) with non-uraemic controls (n=11). Samples of arterial tissue were obtained at the time of renal transplantation (in uraemic patients) and abdominal surgery (in controls). RAGE antigen was visualized using guinea-pig anti-RAGE IgG and PAP technique. RESULTS: Marked staining for RAGE was noted in endothelial cells, both arterial endothelium and endothelium of vasa vasorum of normoglycaemic uraemic patients, but was not demonstrable in endothelial cells of large arteries and only faintly expressed in vasa vasorum of non-uraemic individuals. CONCLUSION: Normal endothelial cells do not constitually express RAGE antigen; in contrast it is expressed by arterial and capillary endothelial cells of uraemic patients. The observation is of note in view of the putative role of AGE of causing atherosclerotic and non-atherosclerotic vascular lesions.

Adult↗

PubMed1996

Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia.

The recent availability of transgenic mice has led to a burgeoning number of reports describing the effects of specific gene products on the pathophysiology of stroke. Although focal cerebral ischemia models in rats have been well described, descriptions of a murine model of middle cerebral artery occlusion are scant and sources of potential experimental variability remain undefined. We hypothesized that slight technical modifications would produce widely discrepant results in a murine model of stroke and that controlling surgical and procedural conditions could lead to reproducible physiological and anatomic stroke outcomes. To test this hypothesis, we established a murine model that would permit either permanent or transient focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery. This study provides a detailed description of the surgical technique and reveals important differences among strains commonly used in the production of transgenic mice. In addition to strain-related differences, infarct volume, neurological outcome, and cerebral blood flow appear to be importantly affected by temperature during the ischemic and postischemic periods, mouse size, and the size of the suture that obstructs the vascular lumen. When these variables were kept constant, there was remarkable uniformity of stroke outcome. These data emphasize the protective effects of hypothermia in stroke and might help to standardize techniques among different laboratories to provide a cohesive framework for evaluating the results of future studies in transgenic animals.

Animals↗

PubMed1995

Complement-mediated regulation of tissue factor activity in endothelium.

Inflammation and immunity may be associated with endothelial cell (EC) injury and thrombus formation. We explored the mechanisms through which a humoral immune response directed against the endothelium might promote coagulation. Using the interaction of anti-EC antibodies and complement (C) with cultured EC as a model, we studied the expression and function of tissue factor, a cofactor for factor VIIa-mediated conversion of factor X to Xa. Exposure of EC to anti-EC antibodies and C in sublytic amounts stimulated the synthesis of tissue factor over a period of 16-42 h. Cell surface expression of tissue factor activity required activation of C and assembly of the membrane attack complex, because expression was inhibited by soluble CR1 and was not detected in the absence of C8. Elaboration of tissue factor messenger RNA was observed over a period of 8-30 h and required protein synthesis. Expression of tissue factor was not a direct consequence of the action of C on the EC but was a secondary response that required as an intermediate step the release of interleukin 1 alpha, an early product of the EC response to C activation. These findings suggest that, after the assembly of membrane attack complex on EC, the production of tissue factor and initiation of coagulation in a blood vessel depend on the production of interleukin 1 alpha and on its availability to stimulate affected EC.

Amino Acid Sequence↗

PubMed1995

Gene disruptions using P transposable elements: an integral component of the Drosophila genome project.

Biologists require genetic as well as molecular tools to decipher genomic information and ultimately to understand gene function. The Berkeley Drosophila Genome Project is addressing these needs with a massive gene disruption project that uses individual, genetically engineered P transposable elements to target open reading frames throughout the Drosophila genome. DNA flanking the insertions is sequenced, thereby placing an extensive series of genetic markers on the physical genomic map and associating insertions with specific open reading frames and genes. Insertions from the collection now lie within or near most Drosophila genes, greatly reducing the time required to identify new mutations and analyze gene functions. Information revealed from these studies about P element site specificity is being used to target the remaining open reading frames.

Animals↗

PubMed1995

Ischemia-induced interleukin-8 release after human heart transplantation. A potential role for endothelial cells.

BACKGROUND: Interleukin-8 (IL-8) secreted from endothelial cells is a powerful neutrophil chemoattractant and activator. We hypothesized that human endothelial cells deprived of oxygen would secrete IL-8, which might translate into elevated IL-8 production after cardiac ischemia. Furthermore, we hypothesized that coronary sinus (CS) IL-8 levels would be particularly high after cardiac preservation for transplantation, due to extended ischemic times. METHODS AND RESULTS: Human saphenous vein endothelial cells exposed to a hypoxic environment (PO2 < 20 mm Hg) demonstrated a time-dependent release of IL-8 (measured by ELISA) into the culture supernatant as early as 4 hours after exposure. To determine whether cardiac preservation in humans was associated with IL-8 production, we obtained CS blood samples 5 minutes after reperfusion in a consecutive series of patients after they underwent cardiac transplantation (CTX, n = 20) or elective cardiac surgery (non-CTX, n = 21). CTX patients demonstrated significantly higher CS IL-8 levels than non-CTX patients (325 +/- 123 versus 50 +/- 17 ng/mL, respectively, P < .05). Further analysis of the CS samples revealed that a biochemical marker of myocyte injury (myoglobin) was similarly elevated in the CTX patients compared with the non-CTX patients (3340 +/- 625 versus 1151 +/- 525 ng/mL, respectively, P < .05). CONCLUSIONS: These differences may reflect the longer ischemic times of CTX compared with non-CTX hearts (161 +/- 10 versus 80 +/- 6 minutes, P < .0001) and suggest that the neutrophil chemoattractant/activator IL-8 may contribute to myocyte injury after prolonged hypothermic cardiac ischemia, as occurs during human cardiac transplantation.

Aged↗

PubMed1995

Hypoxia and modification of the endothelium: implications for regulation of vascular homeostatic properties.

Hypoxia is a common denominator of ischemic microenvironments. Endothelium subjected to oxygen deprivation maintains cell viability and basic biosynthetic mechanisms, but displays multiple changes in properties relevant to vascular homeostasis, including suppression of the anticoagulant cofactor thrombomodulin, decreased barrier function, and generation of proinflammatory cytokines. Diminished intracellular cAMP during the period of hypoxia and lowered nitric oxide/cGMP in the subsequent reperfusion period are proposed as fundamental mechanisms driving vascular dysfunction impacting on coagulation, permeability, vasomotor tone and leukocyte adhesivity. The period of organ preservation for transplantation, recognized to be associated with hypoxia, primes mechanisms leading to subsequent vascular dysfunction which can be ameliorated by buttressing cAMP and nitric oxide/cGMP intra- and intercellular second messenger systems. A mechanism likely to contribute to hypoxia-mediated generation of cytokines, such as interleukin 6, is activation of the transcription factor NF-IL-6, which occurs in oxygen deprivation. These data indicate that study of cellular mechanisms of endothelial perturbation in hypoxia is likely to provide insights ultimately applicable to ischemia-induced vascular damage.

Animals↗

PubMed1995

Inhaled nitric oxide fails to confer the pulmonary protection provided by distal stimulation of the nitric oxide pathway at the level of cyclic guanosine monophosphate.

It has been suggested that inhaled nitric oxide gas may be beneficial after lung transplantation, because endogenous levels of pulmonary nitric oxide decline rapidly after reperfusion. However theoretical concerns remain about the formation of highly toxic oxidants during the quenching of nitric oxide by superoxide. To determine whether distal stimulation of the nitric oxide-cyclic guanosine monophosphate pathway at the level of cyclic guanosine monophosphate might confer the beneficial vascular effects of nitric oxide without its potential toxicities, we studied an orthotopic rat left lung transplant model. In this model, hemodynamic and survival measurements can be obtained independent of the native right lung. Lungs were preserved for 6 hours at 4 degrees C in Euro-Collins solution alone (control, n = 6) or supplemented with the cyclic guanosine monophosphate analog, 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic guanosine monophosphate (cGMP, n = 4). In additional experiments in which lungs were preserved with Euro-Collins solution alone, inhaled nitric oxide was administered during reperfusion (NO, n = 12). Thirty minutes after transplantation and ligation of the native right pulmonary artery, pulmonary vascular resistance, arterial oxygenation, graft neutrophil infiltration (myeloperoxidase activity), and recipient survival were evaluated. Cyclic guanosine monophosphate decreased pulmonary vascular resistance (1.1 +/- 0.2 vs 12.1 +/- 6.3 mm Hg/ml/min, p < 0.05), improved oxygen tension (369 +/- 56 vs 82.8 +/- 48 mm Hg, p < 0.05), reduced myeloperoxidase activity (1.7 +/- 0.3 vs 3.1 +/- 0.9 delta Abs 460 nm/min, p < 0.05), and improved recipient survival (100% vs 0%, p < 0.005) compared with Euro-Collins solution alone (control group). Animals receiving inhaled nitric oxide during reperfusion did not differ from control animals with respect to any of these parameters. These data suggest that distal stimulation of the nitric oxide-cyclic guanosine monophosphate pathway at the level of cyclic guanosine monophosphate has a protective effect that is not seen with inhaled nitric oxide in the immediate pulmonary reperfusion period.

Administration, Inhalation↗

PubMed1995

Nitroglycerin maintains graft vascular homeostasis and enhances preservation in an orthotopic rat lung transplant model.

Transplanted lungs often fail during the peritransplantation period for poorly understood reasons. Because the nitric oxide pathway regulates pulmonary vascular tone, helps to maintain the integrity of the endothelial barrier, and modulates neutrophil adhesivity and activation, we hypothesized that perturbation of this pathway during the preservation and reperfusion of transplanted lungs might play a critical role in mediating early graft failure. To evaluate whether supplementing the preservation solution with the nitric oxide donor nitroglycerin enhances lung preservation for transplantation, we obtained hemodynamic measurements in a model of orthotopic left lung transplantation in the rat after ligation of the native right pulmonary artery. In these experiments, recipient survival and hemodynamics depended solely on the transplanted lung. The left lung was harvested from 22 rats, flushed with either lactated Ringer's solution alone (control, n = 11) or Ringer's solution supplemented with nitroglycerin (0.1 mg/ml, n = 11), preserved for 4 hours at 4 degrees C, and then transplanted using a rapid cuff technique for bronchial and vascular anastomoses. Nitroglycerin significantly improved arterial blood oxygenation (339 +/- 66 versus 130 +/- 12 mm Hg, p < 0.05), increased pulmonary arterial flow (7.6 +/- 1.9 versus 0.9 +/- 0.2 ml/min, p < 0.005), decreased pulmonary vascular resistance (1.7 +/- 0.4 versus 6.6 +/- 1.9 x 10(3) Wood units, p < 0.05), and enhanced recipient survival (64% versus 0%, p < 0.05). Control grafts had significantly greater neutrophil accumulation (50% greater as quantified by myeloperoxidase activity, p < 0.05) than grafts preserved in the presence of nitroglycerin. These studies show that supplementation of the preservation solution with the nitric oxide donor nitroglycerin maintains graft vascular homeostasis and significantly improves pulmonary function and recipient survival after transplantation.

Animals↗