Tetrahydrobiopterin is a secretory product of murine vascular endothelial cells

Tetrahydrobiopterin (BH4) is an obligatory cofactor of nitric oxide synthase and an essential regulator of its activity. The murine vascular endothelial cell line send1 constitutively secretes large amounts of BH4 as well as scant amounts of neopterin, an oxidized intermediate in the de novo biosynthesis of BH4. Further enhancement of BH4 and neopterin secretion is achieved by activation with endotoxin (LPS) and interferon-gamma. This finding is in accordance with previous described BH4 secretion by human endothelial cells. It supports the view that endothelial cells are the source of BH4 serving vascular needs in vivo. In septic conditions, BH4 released by endothelial cells in large amounts could serve induced nitric oxide synthase in smooth muscle cells, thereby acting as another endothelium-derived relaxing factor mediating vasodilatation.     

Immunoregulatory effects of interferon-beta and interacting cytokines on human vascular endothelial cells. Implications for multiple sclerosis autoimmune diseases

BACKGROUND AND PURPOSE: Radiolabeled immunoglobulin therapy (RIT) can be a selective, effective, low-toxicity outpatient cancer therapy. A consensus on the best approach for the preclinical and clinical development of RIT reagents needs to be developed. We report the M.D. Anderson Cancer Center prior experience in translating RIT from laboratory to clinic for the treatment of Hodgkin's disease and propose a flow diagram for the development of RIT for other malignancies. MATERIAL AND METHODS: Three different animal models are described: nude mice bearing human tumor xenografts, normal beagle dogs, and normal rhesus monkeys. We produced and purified antibodies and prepared chelate-immunoconjugates reactive with six different human tumor-associated antigens. The Igs used were derived from rabbits, mice, and humans (human-derived RIT reagents being less immunogenic in human patients). Eighty patients with refractory Hodgkin's disease were treated with radiolabeled antiferritin. RESULTS: We recommend a two-injection scheme using, (1) an indium-111-labeled radioimmunoconjugate for diagnosis, pharmacokinetic studies, and dosimetry, and (2) a yttrium-90-labeled radioimmunoconjugate for therapy. The animal models provide useful data on tumor targeting, radiotoxicology, and undesirable biodistributions. A 70% response rate is obtained in patients with advanced recurrent Hodgkin's disease. More extensive preclinical testing allows for safer and more effective clinical RIT studies. CONCLUSIONS: We recommend, (1) preclinical optimization of chelation chemistry, Ig size, Ig origin, route of administration, and fractionation, (2) new clinical Phase I-III studies more appropriate for RIT development than the classical Phase I-III studies used for the development of chemotherapeutic agents, and (3) more extensive preclinical testing of RIT reagents.     

Human luteinizing hormone and chorionic gonadotropin are targeted to a regulated secretory pathway in GH3 cells

LH is a dimeric glycoprotein hormone that is stored in the anterior pituitary and is released in response to GnRH, while the placental hormone, human CG (hCG), sharing the same alpha-subunit and a related beta-subunit, is secreted constitutively. In search of a determinant that allows sorting of LH into a regulated secretory pathway, the genes encoding the common alpha- and LH/CG beta-subunits were expressed in the GH3 rat pituitary tumor cell line, which contains a regulated secretory pathway. Steady state labeling and subsequent chase experiments showed that not only LH but also hCG can be sorted to a regulated secretory pathway; after an initial period of constitutive secretion, the mature forms of both hormones containing processed oligosaccharides were stored intracellularly, and their release was stimulated by either forskolin or KCl depolarization. In Chinese hamster ovary cells, which lack a regulated pathway and are devoid of storage granules, only hormones containing unprocessed N-linked oligosaccharides were found. In GH3 cells the LH beta-subunit was partially retained in an endoglycosidase H-sensitive form, presumably in the endoplasmic reticulum; the enzyme-resistant fraction was secreted through a regulated secretory pathway. A large fraction of the hCG beta-subunit was released constitutively, although some mature hCG beta-subunit accumulated in secretory granules and was released by forskolin. The common alpha-subunit was secreted constitutively with little intracellular accumulation of the mature forms. We conclude that the LH beta-subunit contains sufficient information to direct LH to a regulated pathway, and alpha:LH beta assembly is not a prerequisite for this targeting. The sorting of hCG to a regulated pathway in GH3 cells presumably reflects a structural similarity between LH and hCG. In addition, we have shown that GH3 cells can recognize the N-linked oligosaccharides on the gonadotropin subunits as substrates for sulfation.     

cAMP stimulates osteoblast-like differentiation of calcifying vascular cells. Potential signaling pathway for vascular calcification

The role of the cAMP signaling pathway in vascular calcification was investigated using calcifying vascular cells (CVC) derived from primary aortic medial cell cultures. We previously showed that CVC have fibroblastic morphology and express several osteoblastic differentiation markers. After confluency, they aggregate into cellular condensations, which later mature into nodules where mineralization is localized. Here, we investigated the effects of cAMP on CVC differentiation because it plays a role in both osteoblastic differentiation and vascular disease. Dibutyryl-cAMP or forskolin treatment of CVC for 3 days induced osteoblast-like "cuboidal" morphology, inhibited proliferation, and enhanced alkaline phosphatase activity, all early markers of osteoblastic differentiation. Isobutylmethylxanthine and cholera toxin had the same effects. Treatment of CVC with pertussis toxin, however, did not induce the morphological change or increase alkaline phosphatase activity, although it inhibited CVC proliferation to a similar extent. cAMP also increased type I procollagen production and gene expression of matrix gamma-carboxyglutamic acid protein, recently shown to play a role in in vivo vascular calcification. cAMP inhibited the expression of osteopontin but did not affect the expression of osteocalcin and core binding factor. Prolonged cAMP treatment enhanced matrix calcium-mineral incorporation but inhibited the condensations resulting in diffuse mineralization throughout the monolayer of cells. Treatment of CVC with a protein kinase A-specific inhibitor, KT5720, inhibited alkaline phosphatase activity and mineralization during spontaneous CVC differentiation. These results suggest that the cAMP pathway promotes in vitro vascular calcification by enhancing osteoblast-like differentiation of CVC.     

Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development

The subendothelial aggregation and retention of low density lipoprotein (LDL) are key events in atherogenesis, but the mechanisms in vivo are not known. Previous studies have shown that treatment of LDL with bacterial sphingomyelinase (SMase) in vitro leads to the formation of lesion-like LDL aggregates that become retained on extracellular matrix and stimulate macrophage foam cell formation. In addition, aggregated human lesional LDL, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an arterial wall SMase in vivo, and several arterial wall cell types secrete a SMase (S-SMase). S-SMase, however, has a sharp acid pH optimum using a standard in vitro SM-micelle assay. Thus, a critical issue regarding the potential role of S-SMase in atherogenesis is whether the enzyme can hydrolyze lipoprotein-SM, particularly at neutral pH. We now show that S-SMase can hydrolyze and aggregate native plasma LDL at pH 5.5 but not at pH 7.4. Remarkably, LDL modified by oxidation, treatment with phospholipase A2, or enrichment with apolipoprotein CIII, which are modifications associated with increased atherogenesis, is hydrolyzed readily by S-SMase at pH 7.4. In addition, lipoproteins from the plasma of apolipoprotein E knock-out mice, which develop extensive atherosclerosis, are highly susceptible to hydrolysis and aggregation by S-SMase at pH 7.4; a high SM:PC ratio in these lipoproteins appears to be an important factor in their susceptibility to S-SMase. Most importantly, LDL extracted from human atherosclerotic lesions, which is enriched in sphingomyelin compared with plasma LDL, is hydrolyzed by S-SMase at pH 7.4 10-fold more than same donor plasma LDL, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. In summary, atherogenic lipoproteins are excellent substrates for S-SMase, even at neutral pH, making this enzyme a leading candidate for the arterial wall SMase that hydrolyzes LDL-SM and causes subendothelial LDL aggregation.     

Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2-3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind approximately 100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.     

Expression of vascular cell adhesion molecule-1 by vascular endothelial cells in immune and nonimmune inflammatory reactions in the skin

Expression of VCAM-1 was compared with that of E-selectin in cytokine-induced lesions and in delayed-type hypersensitivity reactions to tuberculin purified protein derivative (PPD) in pig skin. Lumenally expressed Ags were quantified by measuring localization in skin of i.v. injected (111)In-mAb 10.2C7 (anti-vascular cell adhesion molecule-1 (anti-VCAM-1), (125)I-mAb 1.2B6 (anti-E-selectin), and (99m)Tc-MOPC21 (control IgG1). Anti-VCAM-1 mAb uptake was greater following intradermal (i.d.) injection of TNF-alpha than following injection of IL-1, while the two cytokines induced similar uptake of anti-E-selectin. In immunologically naive pigs there was no detectable increase in anti-VCAM-1 after i.d. injection of PPD, although anti-E-selectin uptake was increased at 3 and 6 h. In contrast, i.d. injection of PPD in sensitized pigs led to increased uptake of both anti-VCAM-1 and anti-E-selectin at 6, 8, 24, and 48 h, each of which was significantly greater than the uptake of control IgG1 into the same lesions (each p < 0.01). Anti-TNF-alpha mAb abolished the increased uptake of anti-VCAM-1 3 and 8 h following i.d. injection of PPD in sensitized pigs and significantly inhibited uptake at 24 h (p = 0.0025), but did not significantly reduce uptake of anti-E-selectin. We conclude that in this delayed-type hypersensitivity model 1) E-selectin expression by endothelial cells follows sequential Ag nonspecific and immune-specific phases, 2) increased VCAM-1 expression by endothelial cells is only seen in sensitized animals, and 3) expression of VCAM-1 appears to be relatively more dependent on TNF-alpha than E-selectin. Differential expression of E-selectin and VCAM-1 may influence the leukocytic infiltrate during the course of nonspecific and immune-specific inflammatory reactions.     

Synovial fibroblasts and the sphingomyelinase pathway: sphingomyelin turnover and ceramide generation are not signaling mechanisms for the actions of tumor necrosis factor-alpha

PURPOSE: To test the hypothesis that magnetic resonance (MR) digital subtraction angiography is superior to two-dimensional time-of-flight (TOF) MR angiography for demonstration of patent arteries in the distal lower extremity. MATERIALS AND METHODS: Thirty-seven lower extremities in 23 consecutive patients were imaged with two-dimensional TOF MR angiography and two-dimensional MR digital subtraction angiography. Images were interpreted in a randomized and blinded manner. Each lower extremity was subdivided into seven potential arterial segments. The number of digital arteries visualized was also determined. Overall image quality of MR digital subtraction and TOF angiograms was compared. The relative ability of MR digital subtraction angiography and TOF MR angiography to demonstrate patent arterial segments was assessed. RESULTS: MR digital subtraction angiography was significantly superior to TOF MR angiography for demonstration of patent arterial segments and digital arteries (P < .001). MR digital subtraction angiographic images were qualitatively superior to TOF images (P < .001). CONCLUSION: Two-dimensional MR digital subtraction angiography is superior to two-dimensional TOF MR angiography for help in identifying patent segments in the distal lower extremity.     

Identification of genes differentially expressed in vascular smooth muscle cells following benzo[a]pyrene challenge: implications for chemical atherogenesis

We have previously shown that serial passage of vascular smooth muscle cells (vSMCs) treated with a single low dose of benzo[a]pyrene (BaP) induces acquisition of highly proliferative (i.e. atherogenic) phenotypes. To define the molecular basis of this response, differential display polymerase chain reaction was used to identify early target genes in murine vSMCs challenged with 3 microM BaP for 8 hr. Of 170 differentially expressed cDNAs, 111 were re-amplified, and 64 examined for homology to known genes. Aac11 apoptosis inhibitor, aldose reductase, GalNAc transferase, TCP-1 chaperonin gene, and mouse mitochondrial gene, were downregulated in vSMCs treated with BaP. In contrast, enhanced expression of unique retrotransposon cDNAs were found in BaP-treated cells. This is the first report showing that BaP modulates the expression of these genes in mammalian cells. Of particular interest is the modulation of retrotransposon mRNAs which coupled to other genetic events, may play a significant role in the atherogenic response to this carcinogen.     

Human endothelial cell storage granules: a novel intracellular site for isoforms of the endothelin-converting enzyme

The participation of oxidative mechanisms in major histocompatibility complex (MHC) class II-restricted antigen presentation was studied in vitro. In general, antigen processing is inhibited when peritoneal macrophages (MO) are incubated with scavengers of reactive oxygen intermediates (ROI): mannitol (an.OH scavenger), dimethylurea (DMTU, which reacts with H2O2 and HOCl) and NCO-700 (an epoxysuccinic acid derivative which inhibits oxidant production by activated phagocytes and can scavenge reactive oxygen species in both NaOCl and hypoxanthine (XOD) systems). However, neither rotenone and antimycins (inhibitors of O-2 production at the NADH dehydrogenase and ubiquinone-cytochrome b regions, respectively) nor aminoguanidine (an inducible nitric oxide synthase inhibitor) impaired antigen presentation, thus indirectly discarding the participation of mitochondrial oxidation and reactive nitrogen intermediates (RNI) in antigen processing. ROI scavengers do not inhibit the MHC class II-restricted presentation of antigens that need processing but have their disulphide bonds reduced. It can be shown that oxidation of protein antigens (either by chlorination or performic acid treatment) allow protein unfolding and enhance both processing and exposure of immunogenic epitopes to specific T cells.     

Thrombopoietin and its receptor, c-mpl, are constitutively expressed by mouse liver endothelial cells: evidence of thrombopoietin as a growth factor for liver endothelial cells

Present data suggest that the primary site of thrombopoietin (TPO) mRNA is the liver. Previously, we reported that specific murine liver endothelial cells (LEC-1) located in the hepatic sinusoids support in vitro megakaryocytopoiesis from murine hematopoietic stem cells suggesting that these cells may be a source of TPO. We report here that TPO and its receptor, c-mpl, are coexpressed on cloned LEC-1. Enzyme-linked immunosorbent assay (ELISA), biological assay, and flow cytometry studies confirmed the expression of both TPO and its receptor, respectively, at the protein level. TPO activity was enhanced in supernatants from LEC-1 treated with tumor necrosis factor (TNF)-alpha and gamma-interferon (INF). Our results show that TPO through its receptor stimulated the growth of LEC-1 in vitro. These observations establish LEC-1 as a novel source of TPO in the liver. To our knowledge, this is the first report that liver endothelial cells express both TPO and its receptor, c-mpl, and our findings indicate that this cytokine constitutes a growth factor for liver endothelial cells in vitro.     

Comparison of adenovirus gene transfer to vascular endothelial cells in cell culture, organ culture, and in vivo

A replication-defective adenovirus 5 vector carrying the beta-galactosidase reporter gene was tested for its efficiency for gene delivery to vascular endothelial cells in various situations. Both porcine and human primary vascular endothelial cell cultures were very efficiently infected (>90%) at adenovirus concentrations of 10(10) pfu/ml or higher. Cultured rat fibroblasts and keratinocytes were even more readily infected, with >90% infection with adenovirus titers of 10(8) pfu/ml or higher. However, nondividing vascular endothelium in situ was very poorly transduced. Pieces of aorta from adult pigs, sheep, rabbit and rat, and pieces of human umbilical artery and vein were studied in organ culture. These showed only occasional positive vascular endothelial cells when exposed to the adenovirus vector at concentrations up to 5x10(11) pfu/ml. Kidney perfusion studies in rats and pigs gave similar results. The only exception to the above findings was in very young (3-4 day old) piglets, which showed excellent (>90%) infection of vascular endothelium with the adenovirus vector at titers of 10(10) pfu/ml. Our data suggest that adenovirus vectors will not be of value for gene delivery to uninjured vascular endothelium in situ, and are therefore unsuited for ex vivo genetic manipulation of vascular endothelium in organs for transplantation.     

Immunotargeting of liposomes to activated vascular endothelial cells: a strategy for site-selective delivery in the cardiovascular system

Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1beta (IL-1beta). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1beta-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2-4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.     

Defensin stimulates the binding of lipoprotein (a) to human vascular endothelial and smooth muscle cells

There is evidence to suggest that elevated plasma levels of lipoprotein (a) [Lp(a)] represent a risk factor for the development of atherosclerotic vascular disease, but the mechanism by which this lipoprotein localizes to involved vessels is only partially understood. In view of studies suggesting a link between inflammation and atherosclerosis and our previous finding that leukocyte defensin modulates the interaction of plasminogen and tissue-type plasminogen activator with cultured human endothelial cells, we examined the effect of this peptide on the binding of Lp(a) to cultured vascular endothelium and vascular smooth muscle cells. Defensin increased the binding of Lp(a) to endothelial cells approximately fourfold and to smooth muscle cells approximately sixfold. Defensin caused a comparable increase in the amount of Lp(a) internalized by each cell type, but Lp(a) internalized as a consequence of defensin being present was not degraded, resulting in a marked increase in the total amount of cell-associated lipoprotein. Abundant defensin was found in endothelium and in intimal smooth muscle cells of atherosclerotic human cerebral arteries, regions also invested with Lp(a). These studies suggest that defensin released from activated or senescent neutrophils may contribute to the localization and persistence of Lp(a) in human vessels and thereby predispose to the development of atherosclerosis.     

Regulated exocytosis in vascular endothelial cells can be triggered by intracellular guanine nucleotides and requires a hydrophobic, thiol-sensitive component. Studies of regulated von Willebrand factor secretion from digitonin permeabilized endothelial cells

To study the intracellular events leading to regulated exocytosis in human umbilical vein endothelial cells (HUVEC) the plasma membrane of HUVEC was selectively permeabilized with digitonin while retaining secretory function. Fusion of Weibel-Palade bodies, the secretory organelle of HUVEC, with the plasma membrane was detected by assaying the media for von Willebrand factor (vWF). The secretion from permeabilized cells faithfully reflects that in intact cells by a number of criteria. First, in the presence of calcium, permeabilized HUVEC secreted vWF with the same kinetics and to the same extent as intact cells stimulated with secretagogue. In addition, the vWF secreted by permeabilized cells after stimulus was exclusively the processed mature form found in Weibel-Palade bodies. Release required micromolar levels of calcium. In addition, GTPgammaS could also stimulate release by a parallel pathway. Both calcium- and GTPgammaS-stimulated secretion required a thiol-sensitive component. The hydrophobic thiol alkylating agent U73122 inhibited calcium-dependent and GTPgammaS-stimulated secretion. Surprisingly, N-ethylmaleimide, a hydrophilic alkylating agent, did not inhibit secretion. The N-ethylmaleimide-sensitive fusion protein (NSF), a protein implicated in a variety of vesicle fusion events, did not appear to be the target of U73122. These data strongly suggests the participation of a non-NSF, membrane-associated protein in regulated secretion in endothelial cells. Further, there appear to be two parallel pathways leading to secretion in HUVEC, one stimulated by elevated levels of calcium and the other mediated by a GTP-binding protein.     

A dual role for endothelial cells in cytomegalovirus infection? A study of cytomegalovirus infection in a series of rat endothelial cell lines

Several clinical findings point to the involvement of microvascular endothelial cells in cytomegalovirus-related pathology. In this study the interactions of cytomegalovirus (CMV) with microvascular endothelial cells was investigated in an in vitro rat model. A series of rat endothelial cell lines, considered representative for the heterogeneity of heart microvascular endothelium in vivo, were infected with rat CMV (RCMV). The course of infection and production of infectious virus were examined using immunofluorescence staining and plaque titration assays, and was compared with infection of fully permissive rat fibroblasts. These endothelial cell lines displayed differences in susceptibility to CMV infection. Two endothelial cell lines (RHEC 50 and 191) were practically non-permissive, while four endothelial cell lines (RHEC 3, 10, 11 and 116) were partly permissive for CMV infection. In contrast to CMV infection in fibroblasts, only limited infection of the permissive endothelial cell lines was observed without spreading of CMV infection through the monolayer, although infectious virus was produced. Detachment of infected endothelial cells and recovery of the monolayer with time was observed. The detached endothelial cells were able to transmit CMV infection to fibroblast monolayers, but not to endothelial monolayers. Our in vitro results demonstrate differences in permissiveness for RCMV between the series of rat endothelial cell lines, which is suggestive for endothelial heterogeneity to CMV infection in vivo. Our findings indicate that endothelial cells are relatively resistant to CMV infection and that, upon infection, the endothelial monolayer may dispose of the virus via detachment of the infected cells. This points to a dual role for the endothelium in CMV infection in vivo: a barrier for CMV infection (by the endothelial monolayer) on the one hand and spreading of CMV infection (by detached infected cells) on the other hand.     

Processing of chimeric antisense oligonucleotides by human vascular smooth muscle cells and human atherosclerotic plaque. Implications for antisense therapy of restenosis after angioplasty

BACKGROUND: Antisense oligonucleotides have been used in animals to inhibit the accumulation of vascular smooth muscle cells (VSMCs) after arterial injury. This has raised prospects for an oligonucleotide-mediated approach to prevent restenosis in patients undergoing angioplasty. However, little is known about the processing of oligonucleotides by human VSMCs or their bioavailability in human atherosclerotic tissue. METHODS AND RESULTS: Oligonucleotides were synthesized with a mixture of unmodified and sulfur-modified linkages (S-chimeric oligonucleotides). These were more stable than unmodified oligonucleotides and could be recovered from within human VSMCs after 36 hours. Oligonucleotide antisense to human proliferating cell nuclear antigen mRNA specifically reduced DNA synthesis (P < .01) and proliferating cell nuclear antigen protein content (P < .05) in human VSMCs. Confocal microscopy of both live and fixed cells showed modest oligonucleotide uptake that was primarily nuclear. Surprisingly, cationic liposomes did not enhance nuclear uptake but led to extensive, punctated cytoplasmic loading without an enhanced antisense effect. Oligonucleotides incubated with human coronary atherosclerosis fragments associated with cells within 1 hour, despite the presence of abundant extracellular matrix. CONCLUSIONS: S-chimeric oligonucleotides are stable and can specifically inhibit gene expression in human VSMCs. Nuclear transport is a feature of oligonucleotide processing by human VSMCs, indicating a potential influence at the nuclear level rather than with cytoplasmic mRNA. Cationic liposomes increased oligonucleotide uptake but not intracellular bioavailability, and S-chimeric oligonucleotides can be incorporated into cells within human atherosclerotic plaque, despite the presence of a dense extracellular matrix.     

Apolipoprotein E containing high density lipoprotein stimulates endothelial production of heparan sulfate rich in biologically active heparin-like domains. A potential mechanism for the anti-atherogenic actions of vascular apolipoprotein e

Reduced heparin and heparan sulfate (HS) proteoglycans (PG) have been observed in both inflammation and atherosclerosis. Methods to increase endogenous heparin and heparan sulfate are not known. We found that incubation of endothelial cells with 500-1,000 micrograms/ml high density lipoprotein (HDL) increased 35SO4 incorporation into PG by 1.5-2.5-fold. A major portion of this increase was in HS and was the result of increased synthesis. Total PG core proteins were not altered by HDL; however, the ratio of 35SO4 to [3H]glucosamine was increased by HDL, suggesting increased sulfation of glycosaminoglycans. In addition, HDL increased the amount of highly sulfated heparin-like HS in the subendothelial matrix. HS from HDL-treated cells bound 40 +/- 5% more 125I-antithrombin III (requires 3-O sulfated HS) and 49 +/- 3% fewer monocytes. Moreover, the HS isolated from HDL-treated cells inhibited smooth muscle cell proliferation (by 83 +/- 5%) better than control HS (56 +/- 6%) and heparin (42 +/- 6%). HDL isolated from apolipoprotein E (apoE)-null mice did not stimulate HS production unless apoE was added. ApoE also stimulated HS production in the absence of HDL. ApoE did not increase 35SO4 incorporation in macrophages and fibroblasts, suggesting that this is an endothelial cell-specific process. Receptor-associated protein inhibited apoE-mediated stimulation of HS only at higher (20 micrograms/ml) doses, suggesting the involvement of a receptor-associated protein-sensitive pathway in mediating apoE actions. In summary, our data identify a novel mechanism by which apoE and apoE-containing HDL can be anti-atherogenic. Identification of specific apoE peptides that stimulate endothelial heparin/HS production may have important therapeutic applications.     

Human diploid fibroblast cell culture medium contains a factor that increases cytosolic Ca2+ and stimulates prostaglandin synthesis by cultured bovine aortic endothelial cells

Endothelin-1 (ET-1), a vascular endothelium-derived peptide, regulates microcirculation by modulating Ca2+ ion channels. Intravitreally injected ET-1 constricts retinal vessels and reduces blood flow in the optic nerve capillaries. We examined the antagonistic effect of a calcium-ion channel blocker, nicardipine, on ET-1 effects on visual evoked potential (VEP). ET-1 (10(-6); 10 microL) was injected into the posterior vitreous of rabbit eyes. Intravenous nicardipine (20 micrograms/kg) was also given, and VEP was monitored for 2 hours following injection. Thirty minutes after injection, ET-1 had reduced VEP amplitude to 42.6% of the baseline level. The reduction effect continued for the remainder of the study. Nicardipine suppressed the ET-1-induced reduction of VEP amplitude (P < 0.05, Scheffe). The vasospasm produced by ET-1, which reduces the VEP amplitude, involves the CA2+ ion channel. Since nicardipine interferes with the activity of ET-1, we believe that Ca2+ channel blockers can be useful in the treatment of ischemic retinal and optic nerve disorders that are related to abnormal ET-1 production.