Appraisal of hepatic lipase and lipoprotein lipase activities in mice.

A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required.

Angiogenic murine endothelial progenitor cells are derived from a myeloid bone marrow fraction and can be identified by endothelial NO synthase expression.

OBJECTIVE: Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization and are therefore of great interest for autologous cell therapies to treat ischemic vascular disease. However, the origin and functional properties of these EPCs are still in debate. METHODS AND RESULTS: Here, ex vivo expanded murine EPCs were characterized in terms of phenotype, lineage potential, differentiation from bone marrow (BM) precursors, and their functional properties using endothelial NO synthase (eNOS)-green fluorescent protein transgenic mice. Despite high phenotypic overlap with macrophages and dendritic cells, EPCs displayed unique eNOS expression, endothelial lineage potential in colony assays, and angiogenic characteristics, but also immunologic properties such as interleukin-12p70 production and low levels of T-cell stimulation. The majority of EPCs developed from an immature, CD31(+)Ly6C+ myeloid progenitor fraction in the BM. Addition of myeloid growth factors such as macrophage-colony-stimulating factor (M-CSF) and granulocyte/macrophage (GM)-CSF stimulated the expansion of spleen-derived EPCs but not BM-derived EPCs. CONCLUSIONS: The close relationship between EPCs and other myeloid lineages may add to the complexity of using them in cell therapy. Our mouse model could be a highly useful tool to characterize EPCs functionally and phenotypically, to explore the origin and optimize the isolation of EPC fractions for therapeutic neovascularization.

Control of myocardial oxygen consumption in transgenic mice overexpressing vascular eNOS.

Our objective was to investigate the potential role of selective endothelial nitric oxide (NO) synthase (eNOS) overexpression in coronary blood vessels in the control of myocardial oxygen consumption (MVO2). Transgenic (Tg) eNOS-overexpressing mice (eNOS Tg) (n=22) and wild-type (WT) mice (n=24) were studied. Western blot analysis indicated greater than sixfold increase of eNOS in cardiac tissue. Echocardiography in awake mice indicated no difference in cardiac function between WT and eNOS Tg; however, systolic pressure in eNOS Tg mice decreased significantly (126 +/- 2.3 to 109 +/- 2.3 mmHg; P <0.05), whereas heart rate (HR) was not different. Total peripheral resistance (TPR) was also decreased (9.8 +/- 0.8 to 7.6 +/- 0.4 4 mmHg.ml(-1).min; P <0.05) in eNOS Tg. Furthermore, female eNOS Tg mice showed even lower TPR (7.2 +/- 0.4 mmHg.ml(-1).min) compared with male eNOS mice (8.6 +/- 0.5, mmHg.ml.min(-1); P <0.05). Left ventricular slices were isolated from WT and eNOS Tg mice. With the use of a Clark-type oxygen electrode in an airtight bath, MVO2 was determined as the percent decrease during increasing doses (10(-10) to 10(-4) mol/l) of bradykinin (BK), carbachol (CCh), forskolin (10(-12) to 10(-6) mol/l), or S-nitroso-N-acetyl penicillamine (SNAP; 10(-7) to 10(-4) mol/l). Baseline MVO2 was not different between WT (181 +/- 13 nmol.g(-1).min(-1)) and eNOS Tg (188 +/- 14 nmol.g(-1).min(-1)). BK decreased MVO2 (10(-4) mol/l) in WT by 17% +/- 1.1 and 33% +/- 2.7 in eNOS Tg (P < 0.05). CCh also decreased MVO2, 10(-4) mol/l, in WT by 20% +/- 1.7 and 31% +/- 2.0 in eNOS Tg (P <0.05). Forskolin (10(-6) mol/l) or SNAP (10(-4) mol/l) also decreased MVO2 in WT by 24% +/- 2.8 and 36% +/- 1.8 versus eNOS 31% +/- 1.8 and 37% +/- 3.5, respectively. N-nitro-L-arginine methyl ester (10(-3) mol/l) inhibited the MVO2 reduction to BK, CCh, and forskolin by a similar degree (P <0.05), but not to SNAP. Thus selective overexpression of eNOS in cardiac blood vessels in mice enhances the control of MVO2 by eNOS-derived NO.

Evaluation of phospholipid transfer protein and cholesteryl ester transfer protein as contributors to the generation of pre beta-high-density lipoproteins.

High-density lipoproteins (HDLs) are considered anti-atherogenic because they mediate peripheral cell cholesterol transport to the liver for excretion and degradation. An important step in this reverse cholesterol-transport pathway is the uptake of cellular cholesterol by a specific subclass of small, lipid-poor apolipoprotein A-I particles designated pre beta-HDL. The two lipid-transfer proteins present in human plasma, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), have both been implicated in the formation of pre beta-HDL. In order to investigate the relative contribution of each of these proteins, we used transgenic mouse models. Comparisons were made between human CETP transgenic mice (huCETPtg), human PLTP transgenic mice (huPLTPtg) and mice transgenic for both lipid-transfer proteins (huCETPtg/huPLTPtg). These animals showed elevated plasma levels of CETP activity, PLTP activity or both activities, respectively. We evaluated the generation of pre beta-HDL in mouse plasma by immunoblotting and crossed immuno-electrophoresis. Generation of pre beta-HDL was equal in huCETPtg and wild-type mice. In contrast, in huPLTPtg and huCETPtg/huPLTPtg mice, pre beta-HDL generation was 3-fold higher than in plasma from either wild-type or huCETPtg mice. Our findings demonstrate that, of the two plasma lipid-transfer proteins, PLTP rather than CETP is responsible for the generation of pre beta-HDL. These data support the hypothesis of a role for PLTP in the initial stage of reverse cholesterol transport.

Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice.

Plasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress human PLTP were generated. Compared with wild-type mice, these mice show a 2.5- to 4.5-fold increase in PLTP activity in plasma. This results in a 30% to 40% decrease of plasma levels of HDL cholesterol. Incubation of plasma from transgenic animals at 37 degrees C reveals a 2- to 3-fold increase in the formation of pre-beta-HDL compared with plasma from wild-type mice. Although pre-beta-HDL is normally a minor subfraction of HDL, it is known to be a very efficient acceptor of peripheral cell cholesterol and a key mediator in reverse cholesterol transport. Further experiments show that plasma from transgenic animals is much more efficient in preventing the accumulation of intracellular cholesterol in macrophages than plasma from wild-type mice, despite lower total HDL concentrations. It is concluded that PLTP can act as an antiatherogenic factor preventing cellular cholesterol overload by generation of pre-beta-HDL.

Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver.

We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization: it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed.

The murine CYLN2 gene: genomic organization, chromosome localization, and comparison to the human gene that is located within the 7q11.23 Williams syndrome critical region.

Cytoplasmic linker proteins (CLIPs) have been proposed to mediate the interaction between specific membranous organelles and microtubules. We have recently characterized a novel member of this family, called CLIP-115. This protein is most abundantly expressed in the brain and was found to associate both with microtubules and with an organelle called the dendritic lamellar body. CLIP-115 is highly homologous to CLIP-170, or restin, which is a protein involved in the binding of endosomes to microtubules. Using the rat cDNA as a probe we have isolated overlapping cosmids containing the complete murine and part of the human CYLN2 (cytoplasmic linker-2) genes, which encode CLIP-115. The murine gene spans 60 kb and consists of 17 exons, and its promoter is embedded in a CpG island. Murine CYLN2 maps to the telomeric end of mouse chromosome 5. The human CYLN2 gene is localized to a syntenic region on chromosome 7q11.23, which is commonly deleted in Williams syndrome. It spans at least 140 kb at the 3' end of the deletion. Human CYLN2 is very likely identical to the previously characterized, incomplete WSCR4 and WSCR3 transcription units.

The human prostate-specific transglutaminase gene (TGM4): genomic organization, tissue-specific expression, and promoter characterization.

Human prostate-specific transglutaminase (hTGP) is a cross-linking enzyme secreted by the prostate. In this study, we performed dot blot analysis of 50 normal human tissues to demonstrate unambiguously the prostate-specific expression of hTGP. Furthermore, we elucidated the genomic organization of the TGM4 gene, the gene encoding hTGP. The structure of this gene displays striking similarity to that of other transglutaminase (TGase) genes. The TGM4 gene spans approximately 35 kb of genomic DNA and consists of 13 exons and 12 introns. The main transcription initiation site is located 52 bp upstream of the translational start codon. A hTGP splice variant of intron 1 was detected. This splice variant contains an in-frame antisense Alu element insertion. The TGM4 promoter was analyzed by sequencing and transfection experiments. At positions -1276 to -563, the promoter harbors a cyclophilin pseudogene with 94% similarity to the cyclophilin A cDNA. Deletion mapping of the TGM4 promoter in the transiently transfected human prostate cancer cell line PC346C showed comparable activity of 2.1-, 1.5-, and 0.5-kb promoter fragments.

Structural relation between HDL-binding proteins in porcine liver.

We have found strong evidence for a relation between three high-density lipoprotein (HDL)-binding proteins of 90, 110, and 180 kDa in porcine liver that were detected by ligand blotting. Because HDL-binding proteins with identical molecular masses were detected in human liver, all subsequent experiments were performed with porcine liver proteins. An antiserum raised against a highly purified preparation of the 90-kDa HDL-binding protein, designated 90-PC, showed cross-immunoreactivity with the 110- and 180-kDa HDL-binding proteins. Purified protein preparations of the 90-, 110-, and 180-kDa HDL-binding proteins were obtained and analyzed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. Under nonreducing conditions these preparations showed protein bands with the expected molecular masses. Reduction of these preparations resulted in protein bands of 90 kDa. Ligand blotting experiments with 125I-HDL showed protein bands of 90, 110, and 180 kDa under nonreducing conditions and a 90-kDa protein band in all three preparations under reducing conditions. Immunoblotting experiments with 90-PC antiserum resulted in a similar pattern. The three protein preparations were then subjected to cyanogen bromide cleavage and the resulting peptides separated on gel. Immunoblotting with the 90-PC antibody revealed a pattern of protein bands that was remarkably similar in all three protein preparations. Immunohistochemical localization studies with the 90-PC antibody showed that the HDL-binding proteins were present both at the borders of the sinusoids as well as within the hepatocellular plates.(ABSTRACT TRUNCATED AT 250 WORDS)

High density lipoprotein-binding proteins in porcine liver. Isolation and histological localization.

The antiatherogenic properties of high density lipoproteins (HDLs) are thought to reside in their involvement in the reverse cholesterol transport pathway. Specific HDL-binding proteins could play a key role in this process. Two HDL-binding proteins of approximately 90 and 180 kd were identified in porcine liver by ligand blotting and were purified to apparent homogeneity by a combination of protein extraction, DEAE-cellulose chromatography, Con A-Sepharose chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding of 125I-HDL by these proteins could be actively competed for by unlabeled HDL but not by low density lipoprotein. Polyclonal antisera have been raised against these two proteins. Each antiserum recognized only one of the HDL-binding proteins, indicating that they are not immunologically related. Moreover, striking differences in localization were observed in immunohistochemical studies. The 90-kd protein is located within the hepatocellular plates, while the 180-kd protein is present along the lining of the sinusoids. These results suggest functional differences between the two HDL-binding proteins described.

Monoclonal antibodies in detection of choroidal melanoma.

Three monoclonal antibodies (MoAbs) prepared against cutaneous melanomas were tested against one group of 12 choroidal melanomas with indirect immunofluorescence in frozen sections. A fourth MoAb was tested in paraffin sections of a second group of 47 choroidal melanomas. One MoAb (NKI-M7) did not react with choroidal melanoma, even though it had a high sensitivity for cutaneous melanoma. A second MoAb (NKI-M6) showed a positive reaction with only 2/12 choroidal melanomas. The third MoAb (NKI/beteb) reacted with all choroidal melanomas, regardless of the cell type. MoAb NKI/C-3 was positive with 38/47 (81%) choroidal melanomas. We conclude that NKI/C-3 and NKI/beteb have a high sensitivity for both cutaneous and choroidal melanomas in frozen sections. Of these two antibodies NKI/beteb was the most specific for cutaneous naevi and melanomas.