A J van Zonneveld

Cloning of a cDNA Encoding Chitotriosidase, a Human Chitinase Produced by Macrophages

We have recently observed that chitotriosidase, a chitinolytic enzyme, is secreted by activated human macrophages and is markedly elevated in plasma of Gaucher disease patients (Hollak, C. E. M., van Weely, S., van Oers, M. H. J., and Aerts, J. M. F. G.(1994) J. Clin. Invest. 93, 1288-1292). Here, we report on the cloning of the corresponding cDNA. The nucleotide sequence of the cloned cDNA predicts a protein with amino acid sequences identical to those established for purified chitotriosidase. Secretion of active chitotriosidase was obtained after transient transfection of COS-1 cells with the cloned cDNA, confirming its identity as chitotriosidase cDNA. Chitotriosidase contains several regions with high homology to those present in chitinases from different species belonging to family 18 of glycosyl hydrolases. Northern blot analysis shows that expression of chitotriosidase mRNA occurs only at a late stage of differentiation of monocytes to activated macrophages in culture. Our results show that, in contrast to previous beliefs, human macrophages can synthesize a functional chitinase, a highly conserved enzyme with a strongly regulated expression. This enzyme may play a role in the degradation of chitin-containing pathogens and can be used as a marker for specific disease states.

Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family.

A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full‐length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full‐length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk‐ cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti‐plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk‐ cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre‐PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine‐linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1‐proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)

The Light Chain of Factor VIII Comprises a Binding Site for Low Density Lipoprotein Receptor-related Protein

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K d ≈ 60 nm). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC50 ≈ 1 nm). In addition, a 12-fold molar excess of the physiological carrier of FVIII,i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of125I-labeled FVIII by LRP-expressing cells (≈ 8 fmol/105 cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k on≈ 5 × 104 m −1s−1; k off ≈ 2.5 × 10−3 s−1; K d ≈ 50 nm). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on thein vivo survival of FVIII.

THE SECOND AND FOURTH CLUSTER OF CLASS A CYSTEINE-RICH REPEATS OF THE LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN SHARE LIGAND-BINDING PROPERTIES

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated α2-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA·plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody α2MRα3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3–C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.

Ligand-receptor interactions of the low density lipoprotein receptor-related protein, a multi-ligand endocytic receptor

Summary The low density lipoprotein receptor-related protein (LRP) is a large membrane glycoprotein that is a member of the low density lipoprotein (LDL) receptor family of endocytic receptors. In contrast to the restricted ligand specificity of the LDL receptor, LRP can bind and internalize a remarkable spectrum of structurally-unrelated classes of ligands suggesting a role for the receptor in diverse physiological and patho-physiological processes ranging from lipoprotein metabolism, cell growth and cell migration to atherosclerosis and Alzheimers disease. In this review we will summarize the current insights in the biology of LRP and particularly focus on the recent progress in our understanding of the molecular mechanisms that enable LRP to interact specifically with such a multitude of different ligands.

Microvascular Damage in Type 1 Diabetic Patients Is Reversed in the First Year After Simultaneous Pancreas–Kidney Transplantation

Simultaneous pancreas–kidney transplantation (SPK) is an advanced treatment option for type 1 diabetes mellitus (DM) patients with microvascular disease including nephropathy. Sidestreamdarkfield (SDF) imaging has emerged as a noninvasive tool to visualize the human microcirculation. This study assessed the effect of SPK in diabetic nephropathy (DN) patients on microvascular alterations using SDF and correlated this with markers for endothelial dysfunction. Microvascular morphology was visualized using SDF of the oral mucosa in DN (n = 26) and SPK patients (n = 38), healthy controls (n = 20), DM1 patients (n = 15, DM ≥ 40 mL/min) and DN patients with a kidney transplant (KTx, n = 15). Furthermore, 21 DN patients were studied longitudinally up to 12 months after SPK. Circulating levels of angiopoietin‐1 (Ang‐1), angiopoietin‐2 (Ang‐2) and soluble thrombomodulin (sTM) were measured using ELISA. Capillary tortuosity in the DN (1.83 ± 0.42) and DM ≥ 40 mL/min (1.55 ± 0.1) group was increased and showed reversal after SPK (1.31 ± 0.3, p < 0.001), but not after KTx (1.64 ± 0.1). sTM levels were increased in DN patients and reduced in SPK and KTx recipients (p < 0.05), while the Ang‐2/Ang‐1 ratio was normalized after SPK and not after KTx alone (from 0.16 ± 0.04 to 0.08 ± 0.02, p < 0.05). Interestingly, in the longitudinal study, reversal of capillary tortuosity and decrease in Ang‐2/Ang‐1 ratio and sTM was observed within 12 months after SPK. SPK is effective in reversing the systemic microvascular structural abnormalities in DN patients in the first year after transplantation.

Gene Transfer into Specific Vascular Cells

Gene transfer therapy in vascular surgery is on the horizon and will include the insertion of genes for anti-clotting proteins into the endothelial lining of vascular grafts and for genes controlling the proliferation of smooth muscle cells after endovascular intervention. Here we address the possibility of targeting genes to specific vascular cells using non-infectious methods, DEAE-dextran or lipofectin complexes of reporter genes, to aid transfection of endothelial cells, smooth muscle cells and fibroblasts cultured from human umbilical veins or arteries. For these studies we used the firefly luciferase gene under control of several different promoters including those for the Rous sarcoma virus (RSV) and for tissue plasminogen activator type 1 (PAI-1). DEAE-dextran mediated transfections resulted in low level, transient (2-5 days) expression of RSV-luciferase in all three cell types. Lipofectin mediated transfections resulted in a four-to-five-fold higher expression of RSV-luciferase in endothelial and smooth muscle cells, expression remaining fairly stable for up to 14 days. One particular PAI-1 promoter construct of 800 bp was only half as effective as the RSV promoter in the expression of luciferase from smooth muscle cells, 82 +/- 9 and 35 +/- 11 ng mg-1 respectively (p less than 0.02). In contrast these two promoters resulted in very similar expression of luciferase in endothelial cells, 64 +/- 8 and 67 +/- 10 ng mg-1 respectively. These experiments demonstrate the possibilities of augmenting cultured vascular cells with foreign genes using lipofectin, a cationic lipid, for insertions into endothelial and smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial plasminogen activator inhibitor: A new member of the serpin gene family

A human endothelial cDNA expression library, based on the Escherichia coli plasmid pUC9, was screened with a heterologous antibody raised against purified bovine aortic endothelial plasminogen activator inhibitor (PAI). A synthetic oligonucleotide, derived from a partial PAI cDNA expression clone, was used to select a full-length PAI cDNA, the size of which coincides with the length of PAI mRNA (approximately 2350 nucleotides) as determined by Northern blot analysis. The authenticity of full-length PAI cDNA is demonstrated by the expression of biologically active PAI both in lysates of transformed E. coli cells and in conditioned media of mouse Ltk- cells, transfected with PAI cDNA inserted into vector pSV2. Analysis of the de novo synthesized anti-plasminogen activator activity, employing reverse fibrin autography, shows that transfected mouse Ltk- cells synthesize a polypeptide with a mol. wt identical to that of the native PAI glycoprotein (Mr 52,000), whereas in E. coli an unglycosylated, active product with a mol. wt of 43,000 is made. The amino acid sequence, derived from the determined nucleotide sequence, shows that pre-PAI consists of 402 amino acids. It is proposed that the mature PAI is preceded by a signal peptide of 23 amino acid residues. The amino acid sequence of mature PAI includes three potential asparagine-linked glycosylation sites and lacks cysteine residues. The predicted amino acid sequence reveals significant homology with members of the serine protease inhibitor (Serpin) family, e.g. alpha 1-proteinase inhibitor and antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet density per monocyte predicts adverse events in patients after percutaneous coronary intervention

Monocyte recruitment to damaged endothelium is enhanced by platelet binding to monocytes and contributes to vascular repair. Therefore, we studied whether the number of platelets per monocyte affects the recurrence of adverse events in patients after percutaneous coronary intervention (PCI). Platelet-monocytes complexes with high and low median fluorescence intensities (MFI) of the platelet marker CD42b were isolated using cell sorting. Microscopic analysis revealed that a high platelet marker MFI on monocytes corresponded with a high platelet density per monocyte while a low platelet marker MFI corresponded with a low platelet density per monocyte (3.4 ± 0.7 vs 1.4 ± 0.1 platelets per monocyte, P=0.01). Using real-time video microscopy, we observed increased recruitment of high platelet density monocytes to endothelial cells as compared with low platelet density monocytes (P=0.01). Next, we classified PCI scheduled patients (N=263) into groups with high, medium and low platelet densities per monocyte and assessed the recurrence of adverse events. After multivariate adjustment for potential confounders, we observed a 2.5-fold reduction in the recurrence of adverse events in patients with a high platelet density per monocyte as compared with a low platelet density per monocyte [hazard ratio=0.4 (95% confidence interval, 0.2-0.8), P=0.01]. We show that a high platelet density per monocyte increases monocyte recruitment to endothelial cells and predicts a reduction in the recurrence of adverse events in patients after PCI. These findings may imply that a high platelet density per monocyte protects against recurrence of adverse events.

The second and fourth cluster of class a cysteinerich repeats of the low density lipoprotein receptor-related protein (LRP) share ligandbinding properties

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated alpha(2)-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA.plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody alpha(2)MRalpha3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.