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Trabecular meshwork endothelial (TME) cells secrete a number of factors, such as enzymes and cytokines, which modulate the functions of the cells and the extracellular matrix of the conventional aqueous outflow pathway. TME cells usually secrete these factors in response to stimuli such as mechanical stretching, laser irradiation and pro-inflammatory cytokines. Here, we report that cultured human TME cells isolated from two non-glaucomatous individuals secrete significant quantities of the chemotactic cytokines IL8, CXCL6 and MCP1 in the absence of any stimulation. The secretion of these chemokines was augmented by treatment with the pro-inflammatory cytokines TNFalpha and IL1beta. By way of comparison, there was little or very low production of the three chemokines by human non-pigmented ciliary epithelial cells in the absence of stimulation. Our findings provide support to our recent observations that monocytes, presumably under the influence of chemotactic signals, circulate through the trabecular meshwork in the normal state and also that cytokines regulate the permeability of Schlemm's canal endothelial cells. In addition, the fact that normal TME cells constitutively secrete chemotactic cytokines strengthens the notion that cytokines play a key role in the homeostasis of the outflow of the aqueous humor and, possibly, in the pathogenesis of glaucoma.

More than 3 decades ago, it was predicted that a laser instrument capable of emitting nanosecond pulses of green light would “activate” irradiated cells due to thermal effects induced by the photothermolysis of melanin granules.1 Only a decade later, the neodymium-doped yttrium aluminum garnet (Nd-YAG) laser instrument became available, as well as the selective laser trabeculoplasty (SLT) procedure.2 Soon thereafter, it was shown that melanin granules in SLT-irradiated TMEs do become depigmented as they undergo “selective” photothermolysis.3,4 Later, it was also learned that SLT-irradiated trabecular meshwork cells (TMEs) and Schlemm's canal endothelial cells (SCEs) undergo the upregulation of numerous genes at the genomic-wide level as they become activated.5–7 On learning that Argon laser-irradiated TMEs secrete IL-1α, IL-1β, and TNF-α,8 we established that SLT-irradiated TMEs and SCEs also secrete the same three cytokines.5,6 Moreover, we also learned that adding selected recombinant cytokines onto SCE monolayers induced marked increases in fluid flow.5,6 Cytokines have also been identified in the aqueous humor of glaucoma patients,9 and certain chemokines are constitutively secreted by TMEs and may maintain a population of resident mononuclear-cells in the conventional aqueous outflow pathway (CAOP).10 Moving forward, using Affymetrix microarrays, we have now identified 28 upregulated and 18 downregulated cytokine genes after exposing TMEs and SCEs to either SLT irradiation or to media conditioned by SLT-irradiated TMEs or SCEs. In addition, we identified six cytokine-receptor genes upregulated or downregulated in TMEs or SCEs after at least one of these treatments. Further, using ELISA and protein antibody arrays, we profiled at least eight upregulated cytokines in SLT-irradiated CAOP explants ex vivo representing an extensive cytokine-driven signaling pathway.

The liver X receptor (LXR) agonist TO901317 inhibited the synthesis of apolipoprotein A1 (apo A1) by human liver-derived cells, including the formation of lipid-poor, prebeta-migrating high-density lipoprotein (HDL). Despite activation of the lipid transporter ABCA1 under these conditions, cellular efflux of PL and cholesterol from liver cells was also reduced. By assaying transcription from full-length and truncated promoters and by site-directed mutagenesis, the effect of LXR and its ligand was localized to a binding site for hepatic nuclear factor-4 (HNF4) in the proximal apo A1 promoter (-132/-119 bp). Chromatin immunoprecipitation analysis of apo A1 transcription complexes from control and ligand-activated cells showed an increase in the binding of reported apo A1 transcriptional inhibitor COUP-TF, which competes with HNF4 for DNA binding. It also identified LXR in the apo A1 transcription complex of TO901317-treated cells. Displacement of HNF4 from the -132/-119 bp promoter DNA sequence in the presence of TO901317 was confirmed by gel shift analysis. These data indicate that LXR can be a significant negative regulator of apo A1 transcription and HDL synthesis.

In human vascular smooth muscle cells, inhibitors of protein kinase C activity reduced serine phosphorylation of caveolin-1 and increased sterol binding by this protein. This was measured after immunoprecipitation of caveolin-1 from cells labeled with tritiated cholesterol or the photoactivable cholesterol analogue FCBP [Fielding et al. (2002) Biochemistry 41, 4929-4937]. At the same time cellular sterol efflux was inhibited. Mutagenesis within a caveolin-1 central domain (residues 80-104) suggested a major role for serine-80 in mediating both of these effects. To perturb sterol binding, platelet-derived growth factor was added to the cells, leading to a transient loss of caveolin-1-associated sterol. Under these conditions, sterol efflux was stimulated, and caveolin-1 phosphorylation at tyrosine(14), assayed with a selective antibody, was substantially increased above baseline levels. These changes were also blocked by inhibitors of protein kinase C activity. Selective inhibitors of the platelet-derived growth factor receptor and downstream kinases were used to show that loss of sterol from caveolin-1 preceded tyrosine phosphorylation, but relipidation was dependent on phosphotyrosine hydrolysis.

Chylomicrons (CM) can bind endotoxin (lipopolysaccharide [LPS]), forming CM-LPS complexes, and protect against endotoxic shock and death in rodent models of gram-negative sepsis. The liver appears to play a central role in this process, as demonstrated by the increased uptake of LPS by this organ. We examined the effect of CM on the uptake and cellular response to injected 125I-LPS by hepatocytes and hepatic nonparenchymal cells. Whereas CM increased the uptake of LPS by both hepatocytes and Kupffer cells, the increase was proportionately greater in hepatocytes than Kupffer cells. Importantly, CM-LPS complexes inhibited inducible nitric oxide synthase (iNOS) mRNA expression and NO production in Kupffer cells and endothelial cells, reducing mRNA levels by 45% to 50% as compared with LPS alone. CM-bound LPS also reduced NO production by hepatocytes in response to cytokine stimulation. Lastly, CM-LPS complexes yielded a concentration-dependent inhibition of LPS-induced tumor necrosis factor alpha (TNF-alpha) production by Kupffer cells in vitro. These data indicate that the mechanism by which CM protect against endotoxicity may involve an increased uptake of LPS by hepatocytes. Moreover, uptake of CM-bound LPS by liver cells attenuates the capacity of these cells to respond to proinflammatory stimulation. These results highlight important anti-inflammatory properties of CM.

Apolipoprotein A1 (apo A1), the major protein of high-density lipoprotein, is secreted as a proprotein and then cleaved by an uncharacterized metalloproteinase. Here this enzyme is identified as C-terminal procollagen endoproteinase/bone morphogenetic protein-1 (BMP-1). Studies with recombinant BMP-1, BMP-1 antibody, and BMP-1 siRNA establish this proteinase as the major or only apo A1-converting activity secreted by human liver-derived (HepG2) cells and CHO cells stably expressing human apo A1. BMP-1 stimulates the conversion of newly secreted proapo A1 to its phospholipid- (PL-) binding form. In this way it promotes formation of functional HDL and reverse cholesterol transport, while inhibiting filtration and clearance of uncleaved proprotein. Alpha2-macroglobulin, a protease inhibitor secreted as part of the innate immune response, inhibits BMP-1 activity and blocks the maturation of proapo A1. The decrease in circulating apo A1 levels that is characteristic of the response to inflammation and infection may be mediated, at least in part, via BMP-1 by this novel mechanism.

The mechanism of formation of functional high-density lipoprotein (HDL) from secreted lipid-free apolipoprotein A1 (apo A1) was determined using human liver-derived (HepG2) cells, human intestine-derived (CaCO2) cells, and CHO cells stably expressing full-length human apo A1 (CHO-A1 cells). In each cell line, a significant proportion of secreted apo A1 had a Stokes radius of 2.6 nm and was inactive in binding phospholipids (PL) or free cholesterol (FC). Extracellularly, in a reaction dependent on membrane transporter ABCA1, prealpha-migrating 2.6 nm apo A1 was converted to a prebeta-migrating product that was able to bind PL. Both forms were reactive with mAb55201, a monoclonal antibody specific for native plasma lipid-poor (prebeta1) HDL [Nakamura, Y., et al. (2004) Biochemistry 43, 14311-14318]. The physical properties of precursor and product apo A1 suggested that both are monomers, with Stokes radii of 2.6 and 3.6 nm, respectively, consistent with the absence of intermolecular cross-linking of apo A1 in lipid-poor HDL, reported previously. Product but not precursor apo A1 promoted reverse cholesterol transport (RCT) from human aortic smooth muscle cells. These studies suggest an important contribution of secreted lipid-free apo A1 to HDL formation.

Background.There is a high incidence of perioperative morbidity and mortality in patients with obstructive jaundice. The absence of bile in the gastrointestinal tract promotes bacterial overgrowth and the increased translocation of bacteria and endotoxin to the liver which has been postulated to inhibit Kupffer cell function in these patients. But, biliary tract obstruction can directly damage liver cells and thus alter their function. Thus, we hypothesized that obstructive jaundice alone alters Kupffer cell function independent of the effects of bacterial translocation. This study was designed to evaluate the contribution of bacterial translocation to the altered Kupffer cell function observed in patients with obstructive jaundice. Methods.Sprague–Dawley rats were randomized to three groups of six animals each. Group 1 underwent common bile duct ligation with intestinal bile salt replacement (sodium taurocholate 100 mg/kg/day) via gastrostomy and an implantable osmotic pump (CBDL + bile salts), Group 2 underwent common bile duct ligation with normal saline replacement (CBDL + saline), and Group 3 underwent a sham operation (sham control). After 7 days, tissue and blood were collected for bacterial translocation and biochemical analyses. Examination of cultured Kupffer cell function included measuring the phagocytosis of heat-killedCandida albicansand endotoxin (LPS)-induced TNFα and nitric oxide production. Results.While bacterial translocation and cecal bacterial counts were significantly increased in the CBDL + saline group, these parameters were both reduced to control levels following intestinal bile salt replacement (CBDL + bile salts). Altered Kupffer cell function, as measured by the increased phagocytosis ofC. albicansand LPS-induced NO production, and decreased LPS-induced TNFα production were observed in all animals with obstructive jaundice regardless of bile salt replacement. Conclusion.Kupffer cell function appears to be differentially affected by obstructive jaundice and these altered functions can occur independent of bacterial translocation.