Biomedical subjects

Xiangdong Wang

Publications and source records attributed to Xiangdong Wang.

At least 37 records · Page 2Linked to original sources

PubMed2005

Potential role of reactive oxygen species in pancreatitis-associated multiple organ dysfunction.

BACKGROUND: Severe acute pancreatitis is still associated with substantial morbidity and mortality. Experimental and clinical studies have demonstrated that reactive oxygen species (ROS) represent early occurring inflammatory mediators contributing to cell dysfunction, both locally in the pancreas and remote organs. METHOD: A systematic literature review was conducted to investigate the potential roles of intra- and intercellular, as well as interorgan signaling of ROS in the development of pancreatitis-associated multiple organ dysfunction syndrome (MODS). A text word search of the Medline, PubMed and Cochrane databases, and a manual search of the citations from these references, was performed. RESULTS: ROS directly compromise cellular damage and regulate intercellular signals in pancreatitis-associated MODS. ROS are involved in leukocyte activation, production of cytokines, endothelial barrier dysfunction, and microcirculatory barrier dysfunction in acute pancreatitis. Beside effects on intercellular signaling, ROS also affect intracellular events and activate the transcription factor nuclear factor kappa B that regulates inflammatory cytokine expression. CONCLUSION: ROS is a critical factor responsible for the development of pancreatitis-induced remote organ dysfunction via intercellular and interorgan signaling. The role of antioxidant treatment, included as a part of multimodal management, remains to be investigated.

Humans↗

PubMed2005

Severity of pancreatitis-associated gut barrier dysfunction is reduced following treatment with the PAF inhibitor lexipafant.

The aim of the present study was to investigate the potential effect of treatment with a platelet-activating factor (PAF) antagonist, lexipafant (BB-882), on gut endothelial and epithelial barrier dysfunction and leukocyte recruitment in rats with acute pancreatitis. Severe acute pancreatitis was induced by the intraductal administration of 5% sodium taurodeoxycholate and pancreatitis-associated gut barrier dysfunction was characterized by increased exudation of radiolabelled albumin into the interstitium and alterations in bidirectional (over both the endothelial and epithelial barrier components) permeability of the intestine at the early stage of bile salt-induced acute pancreatitis. Levels of interleukin 1beta and 6, ileal and colonic myeloperoxidase (MPO) content, clearance of radiolabelled albumin from blood to the gut lumen or gut lumen to blood, and leakage of radiolabelled albumin to the ileum or colon were measured 3 and 12h after induction of acute pancreatitis. Treatment with lexipafant 30 min and 6h after pancreatitis reduced severity of pancreatitis-associated intestinal dysfunction, associated with a diminish in systemic concentrations of IL-1 and local leukocyte recruitment. The findings imply that PAF plays a critical role in the development of pancreatitis-associated gut barrier dysfunction and that PAF antagonist in some forms may represent potential candidates for future therapeutic intervention.

Acute Disease↗

PubMed2005

Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage.

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.

ADAM Proteins↗

PubMed2005

Angiogenesis, endothelial cell functions, and tumor cell growth in biodegradable and nonbiodegradable devices.

Development of tissue engineering creates multiple potentials for clinical treatment and scientific research. Biodegradable collagen matrices have been found to support simultaneous autotransplantation of hepatocytes after major liver resection. Dynamic angiogenesis in biodegradable devices (BDD) and nondegradable devices (NDD) transplanted into the renal subcapsule and subcutaneous tissue was measured by the distribution of radiolabeled red blood cells and serum albumin. The circulation, microvascular integrity, and capacities of endothelial cells (adhesion, proliferation, and migration) were investigated within 2 weeks after subcutaneous transplantation of both devices. Patterns of tumor cell growth in both devices were morphologically studied. After subcutaneous transplantation, significant angiogenesis was noted at 1 week in BDD implants and from 2 weeks and on in NDD implants, with an increase in implant blood and plasma volumes. Leakage index of radiolabeled albumin in NDD implants was significantly higher than in BDD implants, while the leakage index 2 weeks after BDD implant was similar to that in subcutaneous tissues. Adhesion, proliferation and migration rates of endothelial cells isolated from both devices were higher than from subcutaneous tissues. Endothelial proliferation and migration rates in BDD implants were significantly higher at 1 week, while in NDD at 2 weeks. Tumor cells migrated and grew on the top surface of NDD with a flattened shape, while growing within the BDD forming a round mass. Endothelial capacities, angiogenetic procedure, and biological and physical characteristics of the device contribute to patterns of tumor cell growth in the device. Biodegradable collagen matrix with three-dimensional structure is suitable for simultaneous transplantation with cells without prevascularization.

Absorbable Implants↗

PubMed2005

Ethanol binging exacerbates sinusoidal endothelial and parenchymal injury elicited by acetaminophen.

BACKGROUND/AIMS: The pathophysiology of binge drinking of ethanol and its potentiation of acetaminophen (APAP) toxicity has received very little attention. To evaluate if ethanol binging sensitizes hepatic sinusoidal endothelial cells (SEC) and liver to APAP toxicity. METHODS: The histopathological responses to APAP were evaluated in the livers of mice gavaged with APAP alone, following a single, week-end type ethanol binge (4 g/kg every 12 h x 5 doses) or three weekly binges. RESULTS: Six hours after APAP, 600 mg/kg elicited severe centrilobular necrosis together with hemorrhagic congestion and infiltration of erythrocytes into the Space of Disse through large gaps that had formed in SEC. There was no evidence of parenchymal injury at 2 h, but gaps already were formed through the cytoplasm of the SEC by coalescence of fenestrae. A single binge followed by 300 mg/kg APAP elicited SEC and parenchymal injury equivalent to 600 mg/kg APAP alone at 2 and 6 h. The responses were exacerbated following three binges. Lower glutathione levels in the liver were shown in ethanol-binged animals. CONCLUSIONS: Ethanol binging increases APAP hepatotoxicity. SEC are an early target for APAP-induced injury and ethanol binging enhances the SEC injury prior to evidence of parenchymal cell injury.

Acetaminophen↗

PubMed2005

Effects of budesonide and N-acetylcysteine on acute lung hyperinflation, inflammation and injury in rats.

Leukocyte activation and production of inflammatory mediators and reactive oxygen species are important in the pathogenesis of lipopolysaccharide (LPS)-induced acute lung injury. The present study investigated acute lung hyperinflation, edema, and lung inflammation 4 h after an intratracheal instillation of LPS (0.5, 2.5, 5, 10, 50, 100, 500, 1000, and 5000 microg/ml/kg). Effects of budesonide, an inhaled anti-inflammatory corticosteroids, and N-acetylcysteine (NAC), an antioxidant, were evaluated in Wistar rats receiving either low (2.5 microg/ml/kg) or high (50 microg/ml/kg) concentrations of LPS. This study demonstrates that LPS in a concentration-dependent pattern induces acute lung hyperinflation measured by excised lung gas volume (25-45% above control), lung injury indicated by increased lung weight (10-60%), and lung inflammation characterized by the infiltration of leukocytes (40-14000%) and neutrophils (80-17000%) and the production of cytokines (up to 2700%) and chemokines (up to 350%) in bronchoalveolar lavage fluid (BALF). Pretreatment with NAC partially prevented tumor necrosis factor alpha (TNFalpha) production induced by the low concentration of LPS, while pretreatment with budesonide totally prevented the increased production of TNFalpha, interleukin (IL)-1beta, IL-6, and monocyte chemoattractive protein (MCP)-1 after LPS challenge at both low and high concentrations. Budesonide failed to prevent BALF levels of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant 1 (GRO/CINC-1) as well as lung hyperinflation induced by both low and high concentrations of LPS. Pretreatment with budesonide totally prevented the formation of lung edema at the low concentration of LPS and had partial effects on acute lung injury and leukocyte influx at the high concentrations. Thus, our data indicate that therapeutic effects of budesonide and NAC are dependent upon the severity of the disease.

Acetylcysteine↗

PubMed2005

Role of nuclear factor-kappaB, reactive oxygen species and cellular signaling in the early phase of acute pancreatitis.

OBJECTIVE: Increased knowledge of regulation of signaling proteins in acute pancreatitis (AP) could potentially contribute to the development of novel agents targeted at regulation of cellular signaling. MATERIAL AND METHODS: Severe AP was induced by administration of 5% sodium taurodeoxycholate in rats. Thirty minutes prior to induction of AP, the animals had an intraperitoneal injection of the antioxidant, N-acetylcysteine (NAC; 10 mg/kg), the NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC; 100 mg/kg), the ERK inhibitor, PD-98059 (1 mg/kg), or the tyrosine kinase inhibitor, Genistein (1 mg/kg). Plasma levels of IL-6 and IL-10 were determined by ELISA and myeloperoxidase (MPO) levels were measured in the pancreas and lungs 3 and 6 h after sham operation or induction of AP. RESULTS: AP results in significant increases in plasma levels of IL-6 at 6 h and IL-10 at 3 and 6 h. Plasma levels of IL-6 were significantly decreased after administration of NAC. NAC pretreatment also increased the ratio of IL-10/IL-6. MPO levels in the pancreas (at 3 and 6 h) and lungs (3 h) were significantly increased in animals with pancreatitis. Pretreatment with NAC, PDTC, PD98059 or Genistein significantly decreased MPO levels in the pancreas at 3 and 6 h and following the administration of PD-98059 or NAC at 6 h. Pretreatment with NAC significantly decreased MPO levels in the lungs at 3 h. CONCLUSIONS: Pretreatment with NAC could regulate the pro-and anti-inflammatory cytokine balance, probably through NF-kappaB and ROS signaling pathways. The regulation of signaling pathways during the sequestration of neutrophils in acute pancreatitis seems to vary between organs.

Acute Disease↗

PubMed2004

A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: impact on GH signaling and GHR proteolysis.

The GH receptor (GHR) mediates metabolic and somatogenic actions of GH. Its extracellular domain (ECD; residues 1-246) has two subdomains, each with seven beta strands organized into two antiparallel beta sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, whereas subdomain 2 harbors a dimerization interface between GHR dimers that alters conformation in response to GH. A regulated GHR metalloprotease cleavage site is in the membrane-proximal stem region of subdomain 2. We have identified a monoclonal anti-ECD antibody, anti-GHR(ext-mAb), which recognizes the rabbit and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHR(ext-mAb) recognized a glutathione-S-transferase (GST) fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHR(ext-mAb) failed to recognize by immunoprecipitation a previously characterized dimerization interface mutant GHR that is incompetent for signaling. In signaling experiments, brief pretreatment of GH-responsive human fibrosarcoma cells with anti-GHR(ext-mAb) dramatically inhibited GH-induced Janus kinase 2 and signal transducer and activator of transcription 5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHR(ext-mAb) only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Furthermore, anti-GHR(ext-mAb) prevented phorbol ester-stimulated GHR proteolysis, but GHR cleavage site mutants were normally recognized by the antibody, indicating that the stem region cleavage site is not a direct epitope. A Fab fragment of anti-GHR(ext-mAb) inhibited GH-induced GHR disulfide linkage and signaling, as well as phorbol ester-induced GHR proteolysis, in a fashion similar to the intact antibody. Thus, our findings suggest that anti-GHR(ext-mAb) has promise as a GH antagonist and as a tool in studies of conformational changes required for GHR activation.

Animals↗

PubMed2004

Rat liver sinusoidal endothelial cell phenotype is maintained by paracrine and autocrine regulation.

The phenotypic features of liver sinusoidal endothelial cells (SEC), open fenestrae in sieve plates and lack of a basement membrane, are lost with capillarization. The current study examines localization of CD31 as a marker for the dedifferentiated, nonfenestrated SEC and examines regulation of SEC phenotype in vitro. CD31 localization in SEC was examined by confocal microscopy and immunogold-scanning electron microscopy. SEC cultured for 1 day express CD31 in the cytoplasm, whereas after 3 days, CD31 is also expressed on cell-cell junctions. Immunogold-scanning electron microscopy confirmed the absence of CD31 surface expression on fenestrated SEC 1 day after isolation and demonstrated the appearance of CD31 surface expression on SEC that had lost fenestration after 3 days in culture. SEC isolated from fibrotic liver do show increased expression of CD31 on the cell surface. Coculture with either hepatocytes or stellate cells prevents CD31 surface expression, and this effect does not require heterotypic contact. The paracrine effect of hepatocytes or stellate cells on SEC phenotype is abolished with anti-VEGF antibody and is reproduced by addition of VEGF to SEC cultured alone. VEGF stimulates SEC production of nitric oxide. NG-nitro-L-arginine methyl ester blocked the paracrine effect of hepatocytes or stellate cells on SEC phenotype and blocked the ability of VEGF to preserve the phenotype of SEC cultured alone. In conclusion, surface expression of CD31 is a marker of a dedifferentiated, nonfenestrated SEC. The VEGF-mediated paracrine effect of hepatocytes or stellate cells on maintenance of SEC phenotype requires autocrine production of nitric oxide by SEC.

Animals↗

PubMed2004

Growth hormone alters epidermal growth factor receptor binding affinity via activation of extracellular signal-regulated kinases in 3T3-F442A cells.

Epidermal growth factor receptor (EGFR) is a transmembrane protein that binds EGF in its extracellular domain and initiates signaling via intrinsic tyrosine kinase activity in its cytoplasmic domain. EGFR is important in development, cellular proliferation, and cancer. GH is a critical growthpromoting and metabolic regulatory hormone that binds the GH receptor, thereby engaging various signaling pathways, including ERKs. Prior studies suggest cross-talk between the GH receptor and EGFR signaling systems. Using the GH- and EGF-responsive 3T3-F442A preadipocyte, we previously observed that GH, in addition to causing EGFR tyrosine phosphorylation, also induced EGFR phosphorylation that was detected by PTP101, an antibody reactive with ERK consensus phosphorylation sites. This latter phosphorylation was prevented by pretreatment with MAPK kinase (MEK)1 inhibitors, suggesting ERK pathway dependence. Furthermore, GH cotreatment with EGF markedly slowed EGF-induced EGFR degradation and down-regulation, thereby potentiating EGF-induced EGFR signaling. These effects were also MEK1 dependent and suggested ERK pathway-dependent influence of GH on EGF-induced EGFR postendocytic trafficking and signaling. We now explore the impact of GH on cell surface binding of EGF in 3T3-F442A cells. We found that GH pretreatment caused transient, but substantial, lessening of (125)I-EGF binding. Competitive binding experiments revealed that the decreased binding was primarily due to decreased affinity, rather than a change in the number of EGF binding sites. The effect of GH on EGF binding was concentration dependent and temporally correlated with GH-induced ERK activation and EGFR PTP101-reactive phosphorylation. Blockade of the MEK1/ERK but not the protein kinase C pathway, prevented GH's effects on EGF binding, and our results indicate that the mechanisms of GH- and phorbol-12-myristate-13-acetateinduced inhibition of EGF binding differ substantially. Overall, our findings suggest that GH can modulate both EGF binding kinetics and the EGFR's postbinding signaling itinerary in a MEK1/ERK pathway-dependent fashion.

3T3 Cells↗

PubMed2004

Lung inflammatory responses and hyperinflation induced by an intratracheal exposure to lipopolysaccharide in rats.

Exposure of the respiratory tract to lipopolysaccharide (LPS) induces acute local inflammation and tissue injury associated with the various deliveries of LPS. To determine potential association of local inflammatory responses with respiratory tract dysfunction, infiltration of inflammatory cells, production of inflammatory mediators, lung hyperinflation and edema were measured in Wister rats 2, 4, and 24 h after an intratracheal administration of LPS at different doses (5, 50, 500 and 5000 microg/ml/kg). Lung hyperinflation determined by an increased excised lung gas volume was significantly increased 2 and 4 h after LPS instillation and lung edema occurred from 2 h onward. Peak BAL levels of TNFalpha appeared at 2 h, MCP-1 at 4 h, and IL-6 at 2 and 4 h, while BAL levels of IL-1beta were increased during 24 h after the intratracheal instillation of LPS. Neutrophilia in BAL fluid was noted from 2 h post-challenge. Our results demonstrate a clear dose-related change in the lung weight at 4 and 24 h, in the BAL levels of MCP-1 at 4 h, and IL-6 and IL-1beta at 2 and 4 h. It seems important to understand polymorphisms of LPS-induced lung hyperinflation and inflammation. Lung hyperinflation and inflammation may be independent during the development of acute lung injury.

Animals↗

PubMed2003

Fis stabilizes the interaction between RNA polymerase and the ribosomal promoter rrnB P1, leading to transcriptional activation.

It has been shown that Fis activates transcription of the ribosomal promoter rrnB P1; however, the mechanism by which Fis activates rrnB P1 transcription is not fully understood. Paradoxically, although Fis activates transcription of rrnB P1 in vitro, transcription from the promoter containing Fis sites (as measured from rrnB P1-lacZ fusions) is not reduced in a fis null mutant strain. In this study, we further investigated the mechanism by which Fis activates transcription of the rrnB P1 promoter and the role of Fis in rRNA synthesis and cell growth in Escherichia coli. Like all other stringent promoters investigated so far, open complex of rrnB P1 has been shown to be intrinsically unstable, making open complex stability a potential regulatory step in transcription of this class of promoters. Our results show that Fis acts at this regulatory step by stabilizing the interaction between RNA polymerase and rrnB P1 in the absence of NTPs. Mutational analysis of the Fis protein demonstrates that there is a complete correlation between Fis-mediated transcriptional activation of rrnB P1 and Fis-mediated stabilization of preinitiation complexes of the promoter. Thus, our study indicates that Fis-mediated stabilization of RNA polymerase-rrnB P1 preinitiation complexes, presumably at the open complex step, contributes prominently to transcriptional activation. Furthermore, our in vivo results show that rRNA synthesis from the P1 promoters of several rRNA operons are reduced 2-fold in a fis null mutant compared with the wild type strain, indicating that Fis plays an important role in the establishment of robust rRNA synthesis when E. coli cells are emerging from a growth-arrested phase to a rapid growth phase. Thus, our results resolve an apparent paradox of the role of Fis in vitro and in vivo in the field.

Cell Division↗

PubMed2003

Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling.

GH signaling depends on functional interaction of the GH receptor (GHR) and the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), which possesses a C-terminal kinase domain, a catalytically inactive pseudokinase domain just N-terminal to the kinase domain, and an N-terminal half shown by us and others to harbor elements for GHR association. Computational analyses indicate that JAKs contain in their N termini ( approximately 450 residues) divergent FERM domains. FERM domains (or subdomains within them) in JAKS may be important for associations with cytokine receptors. For some cytokine receptors, JAK interaction may be required for receptor surface expression. We previously demonstrated that a JAK2 mutant devoid of its N-terminal 239 residues (JAK2-Delta1-239) did not associate with GHR and could not mediate GH- induced signaling. In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. While JAK2 expression is not required for detectable surface GHR expression, an increased JAK2 level increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptor's efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.

Animals↗

PubMed2003

Reduced proteolysis of rabbit growth hormone (GH) receptor substituted with mouse GH receptor cleavage site.

GH binding protein (GHBP) is a circulating form of the GH receptor (GHR) extracellular domain, which derives by alternative splicing of the GHR gene (in mice and rats) and by metalloprotease-mediated GHR proteolysis with shedding of the extracellular domain as GHBP (in rabbits, humans, and other species). Inducible proteolysis of either mouse (m) or rabbit (rb) GHR is detected in cell culture in response to phorbol ester and other stimuli, yielding a cell-associated GHR remnant (comprised of the cytoplasmic and transmembrane domains and a small portion of the proximal extracellular domain) and down-regulating GH signaling. In this report, we map the mGHR cleavage site by adenoviral overexpression of a membrane-anchored mGHR mutant lacking its cytoplasmic domain and purification and N-terminal sequencing of the phorbol 12-myristate 13-acetate-induced remnant protein. The sequence obtained was LEACEEDI, which matches the mGHR extracellular domain stem region sequence L265EACEEDI272, indicating that mGHR cleavage occurs in the extracellular domain nine residues outside of the transmembrane domain, in the same region (but at different residues) as the rbGHR cleavage site we recently mapped. We studied the effects on receptor proteolysis and GHBP shedding of replacing rbGHR cleavage site residues with those corresponding to the mGHR cleavage site. We analyzed five separate rodentized rbGHR mutants incorporating mGHR amino acids either at or surrounding the cleavage site. Each mutant was normally processed, displayed at the cell surface, and responded to GH stimulation by undergoing tyrosine phosphorylation. Only the mutants replaced with mGHR cleavage site residues, rather than surrounding residues, exhibited deficient inducible proteolysis and GHBP shedding. These findings suggested that the GHR cleavage sites in the two species differ in their susceptibility to cleavage. This difference may underlie interspecies variation in utilization of proteolysis to generate GHBP.

Amino Acid Sequence↗

PubMed2003

Embolization by sinusoidal lining cells obstructs the microcirculation in rat sinusoidal obstruction syndrome.

Mechanisms leading to the obstruction of the microcirculation in sinusoidal obstruction syndrome (SOS) have been unclear. Because this occurs at the onset of disease, this is a potential key target for therapeutic intervention. Rats were treated with monocrotaline with or without continuous intraportal infusion of glutathione and were studied at 0.5, 1, 2, 4, 6, and 10 days after monocrotaline treatment with the use of in vivo microscopy and transmission electron microscopy. Sinusoidal perfusion decreased from days 1 through 10 with a nadir on day 4. At 12 h, numerous swollen sinusoidal endothelial cells (SECs) were observed. Subsequently, red blood cells penetrated into the space of Disse through gaps between and through swollen SEC and dissected the sinusoidal lining away from the parenchymal cells. Sinusoidal blood flow was obstructed by an embolism of aggregates of sinusoidal lining cells, red blood cells, and adherent monocytes. All changes were prevented by glutathione infusion, notably the initial swelling of SEC. SOS is initiated by changes in SEC. Microcirculatory obstruction is due to dissection of the sinusoidal lining, followed by embolization of the sinusoid by sinusoidal lining cells, compounded by aggregates of monocytes adherent in the sinusoids. Glutathione prevents SOS by preserving an intact sinusoidal barrier.

Animals↗

PubMed2003

Sinusoidal obstruction syndrome (veno-occlusive disease) in the rat is prevented by matrix metalloproteinase inhibition.

BACKGROUND & AIMS: The mechanical origins of the obstruction in sinusoidal obstruction syndrome are initiated by dehiscence of sinusoidal endothelial cells from the space of Disse. The biochemical changes that permit the dehiscence of the sinusoidal endothelial cells were investigated. METHODS: In vitro and in vivo studies examined changes induced by monocrotaline, a pyrrolizidine alkaloid that induces sinusoidal obstruction syndrome in both humans and experimental animals. RESULTS: In the monocrotaline-induced rat model of sinusoidal obstruction syndrome, there was an early increase of matrix metalloproteinase-9 and a later, lower-magnitude increase of matrix metalloproteinase-2 in the liver. In vitro studies of sinusoidal endothelial cells, hepatocytes, stellate cells, and Kupffer cells showed that sinusoidal endothelial cells are the major source of both basal and monocrotaline-induced matrix metalloproteinase-9/matrix metalloproteinase-2 activity. Monocrotaline caused depolymerization of F-actin in sinusoidal endothelial cells, and blocking of F-actin depolymerization prevented the increase in matrix metalloproteinase activity. Administration of matrix metalloproteinase inhibitors prevented the signs and histological changes associated with sinusoidal obstruction syndrome. CONCLUSIONS: Monocrotaline causes depolymerization of F-actin in sinusoidal endothelial cells, which leads to increased expression of metalloproteinase-9 and matrix metalloproteinase-2 by sinusoidal endothelial cells. Inhibition of matrix metalloproteinase-9 and matrix metalloproteinase-2 prevents the development of sinusoidal obstruction syndrome, establishing that matrix metalloproteinase inhibitors may be a therapeutically viable strategy for prevention.

Actins↗

PubMed2003

Decreased hepatic nitric oxide production contributes to the development of rat sinusoidal obstruction syndrome.

This study examined the role of decreased nitric oxide (NO) in the microcirculatory obstruction of hepatic sinusoidal obstruction syndrome (SOS). SOS was induced in rats with monocrotaline. Monocrotaline caused hepatic vein NO to decrease by 30% at 24 hours and by 70% at 72 hours; this decrease persisted throughout late SOS. N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, exacerbated monocrotaline toxicity, whereas V-PYRRO/NO, a liver-selective NO donor prodrug, restored NO levels, preserved sinusoidal endothelial cell (SEC) integrity and sinusoidal perfusion as assessed by in vivo microscopy and electron microscopy, and prevented clinical and histologic evidence of SOS. NO production in vitro by SEC and Kupffer cells, the 2 major liver cell sources of NO, decreases largely in parallel with loss of cell viability after exposure to monocrotaline. Increased matrix metalloproteinase (MMP) activity increases early on in SOS and this increase in activity has been implicated in initiating SOS. Infusion of V-PYRRO-NO prevented the monocrotaline-induced increase in MMP-9. In conclusion, decreased hepatic NO production contributes to the development of SOS. Infusion of an NO donor preserves SEC integrity and prevents development of SOS. These findings show that a decrease in NO contributes to SOS by allowing up-regulation of MMP activity, loss of sinusoidal integrity, and subsequent disruption of sinusoidal perfusion.

Animals↗

PubMed2002

Metalloprotease-mediated GH receptor proteolysis and GHBP shedding. Determination of extracellular domain stem region cleavage site.

Growth hormone-binding protein (GHBP) is complexed to a substantial fraction of circulating GH. In humans, rabbits, and other species, GHBP derives from proteolytic shedding of the GH receptor (GHR) extracellular domain. In cell culture studies, stimuli such as phorbol ester, platelet-derived growth factor, or serum induce GHR proteolysis, which concomitantly yields shed GHBP in cell supernatants and a cell-associated cytoplasmic domain-containing GHR remnant. This process is sensitive to metalloprotease inhibition, and genetic reconstitution studies identify tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17), a transmembrane metalloprotease, as a GHR sheddase. Stimuli that induce GHR proteolysis render cells less responsive to GH, but the mechanism(s) of this desensitization is not yet understood. In this study, we mapped the rabbit (rb) GHR cleavage site. We adenovirally expressed a C-terminal epitope-tagged rbGHR lacking most of its cytoplasmic domain, purified the remnant protein induced by the phorbol ester, PMA, and derived the cleavage site by N-terminal sequencing of the purified remnant. The N-terminal sequence, (239)FTCEEDFR(246), matched perfectly the rbGHR and suggests that cleavage occurs eight residues from the membrane in the proximal extracellular domain stem region. Deletion and alanine substitution mutagenesis indicated that, similar to other TACE substrates, the spacing of residues in this region, more than their identity, influences GHR cleavage susceptibility. Further, we determined that PMA pretreatment desensitized a cleavage-sensitive GHR mutant, but not a cleavage-insensitive mutant, to GH-induced JAK2 activation. These results suggest that inducible GHR proteolysis can regulate GH signaling.

Amino Acid Sequence↗