Receptor Heparan Sulfate Proteoglycan Research Articles

Atherogenesis and plaque rupture, surface/interface-related phenomena

In atherogenesis, free oxygen radicals cause a lipid peroxidation of apoB100-containing lipoproteins in the blood, at the blood–endothelium-interface and in the subendothelial space. These lipoproteins easily aggregate, bind to their receptor heparan sulfate proteoglycan and calcify to arteriosclerotic nanoplaques (ternary complexes). Nanoplaque formation was measured by ellipsometry, both in vitro on an HS-PG coated hydrophobic silica surface and also in vivo on living human coronary endothelial cells, which had overgrown the silica surface. Reversely, we show with the same techniques that, in dependence on the degree of peroxidation and epitope in concern, oxLDL attacks its molecular receptor and thus can induce degradation of arteriosclerotic plaques and, in a combined action with inflammatory processes, even a plaque rupture.In a previous work, we had found PML-NB, fibrous cap (collagens, proteoglycans) and HSBGF binding sites (e.g., TGFβ1) up-regulated and NFκB down-regulated. With this background knowledge we created a molecular feedback control circuit model where PML-NB functions as regulation centre, fibrous cap as controlled variable, HSBGF binding sites as receptor and NFκB as effector. Since NFκB is inhibited by one reaction strand in this model and inhibits itself collagen and proteoglycan synthesis in the fibrous cap of the plaque, this double check (disinhibition) causes a stabilization of the fibrous cap through a specially strong collagen and proteoglycan production, which in addition is supported by circulating TGFβ. TGFβ furthers also calcification, so that fibrous cap tensile strength and resistance to shear stress are imparted. This way, a plaque rupture may possibly be averted.

Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape

Endosomal escape after endocytosis is a critical step for protein-based agents to exhibit their effects in the cytosol of cells. However, antibodies internalized into cells by endocytosis cannot reach the cytosol due to their inability to escape from endosomes. Here, we report a unique endosomal escape mechanism of the IgG-format TMab4 antibody, which can reach the cytosol of living cells after internalization. Dissociation of TMab4 from its cell surface receptor heparan sulfate proteoglycan by activated heparanase in acidified early endosomes and then local structural changes of the endosomal escape motif of TMab4 in response to the acidified endosomal pH were critical for the formation of membrane pores through which TMab4 escaped into the cytosol. Identification of structural determinants of endosomal escape led us to generate a TMab4 variant with ~3-fold improved endosomal escape efficiency. Our finding of the endosomal escape mechanism of the cytosol-penetrating antibody and its improvement will establish a platform technology that enables a full-length IgG antibody to directly target cytosolic proteins.

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis

Cell surface targeting of recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to modify AAV's natural tropism. As modification of the capsid surface is likely to affect the mechanism of vector internalization and consequently the vector's intracellular fate, we investigated early steps in cell transduction of rAAV capsid insertion mutants. Mutants displaying peptides with neutral overall charge at position 587 transduced cells independently of AAV2's primary receptor heparan sulfate proteoglycan (HSPG), whereas mutants carrying positively charged insertions were capable of HSPG binding with affinities correlating with their net positive charge. Whereas rAAV2 is internalized via an HSPG- and clathrin-dependent pathway, HSPG-binding mutants used a clathrin- and caveolin-independent mechanism. Surprisingly, although this pathway was as efficient in mediating vector entry as the one used by rAAV2, successful cell transduction was hampered at a post-entry step, presumably caused by inefficient endosomal escape. In contrast, HSPG-independent, clathrin-dependent internalization used by non-HSPG-binding mutants correlated with efficient nuclear delivery of vector genomes and robust transgene expression. These findings indicate that cell surface targeting strategies should direct uptake of rAAV targeting vectors to clathrin-mediated endocytosis, the naturally evolved entry route of AAV, to promote successful intracellular processing and re-targeting of rAAV's tropism.

Reduction of Arteriosclerotic Nanoplaque Formation and Size by n-3 Fatty Acids in Patients after Valvular Defect Operation

Background/Methods: Coating a silica surface with the isolated lipoprotein receptor heparan sulfate proteoglycan (HS-PG) from arterial endothelium and vascular matrices, we could observe the very earliest stages of arteriosclerotic plaque development by ellipsometric techniques in vitro (patent EP 0 946 876). This so-called nanoplaque formation is represented by the ternary aggregational complex of the HS-PG receptor, lipoprotein particles and calcium ions. The model was validated in several clinical studies on statins in cardiovascular high-risk patients applying their native blood lipoprotein fractions. Results: In 7 patients who had undergone a valvular defect operation, the reduction of arteriosclerotic nanoplaque formation in normal Krebs solution amounted to 6.1 ± 2.3% (p < 0.0156) and of nanoplaque size to 37.5 ± 13.2% (p < 0.0312), respectively, after a 3-month therapy with n-3 fatty acids (3 ··3 g daily, Ameu® 500 mg). Additionally, the quotient oxLDL/LDL was lowered by 6.8 ± 2.1% (p < 0.0166), the MDA concentration remained unchanged and the lipoprotein(a) concentration decreased by 15.8 ± 5.6% (p < 0.0469) in the patients’ blood. The concentration of the nanoplaque promoting particles VLDL and total triglycerides was diminished by 34.1 ± 11.6% (p < 0.0469) and 26.7 ± 10.8% (p < 0.0156), respectively. Furthermore, the ratio of the strongly atherogenic small dense to the total LDL cholesterol (LDL5+LDL6)/LDLtot decreased by 9.9 ± 3.0% (p < 0.0174). Conclusions: A combinatorial regression analysis revealed a basis for a mechanistic explanation of nanoplaque reduction under n-3 fatty acid treatment. This effect was possibly due to the beneficial changes in lipid concentrations and an attenuation of the risk factors oxLDL/LDL and (LDL5+LDL6)/LDLtot.

Target Cell Cyclophilins Facilitate Human Papillomavirus Type 16 Infection

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV–induced diseases.

Improved cardiac gene transfer by transcriptional and transductional targeting of adeno-associated viral vectors

Vectors based on recombinant adeno-associated virus 2 (AAV-2) are a promising tool for cardiac gene transfer. However, potential therapeutic applications need to consider the predominant transduction of the liver once AAV-2 vectors enter the systemic circulation. We therefore aimed to increase efficiency and specificity of cardiac vector delivery by combining transcriptional and cell surface targeting. For analysis of transcriptional targeting, recombinant AAV vectors were generated harboring a luciferase reporter gene under control of the cytomegalovirus (CMV) promoter or the 1.5-kb cardiac myosin light chain promoter fused to the CMV immediate-early enhancer (CMV(enh)/MLC1.5). Luciferase activities were determined in representative organs three weeks after intravenous injection of the vector into adult mice. Transductional targeting was studied using luciferase-reporter constructs crosspackaged into capsids of AAV serotypes 1 to 6 and modified AAV-2 capsids devoid of binding their primary receptor heparan sulfate proteoglycan. Intravenous injections of AAV-2 vectors harboring the CMV(enh)/MLC1.5 promoter enabled a specific and 50-fold higher reporter gene expression in left ventricular myocardium of adult mice compared to vectors containing the CMV promoter. Comparison of AAV-2 vector genomes crosspackaged into capsids of AAV-1 to -6 showed that AAV-1, -4, -5, and -6 capsids increased cardiac transduction efficiency by about 10-fold. However, transduction of other organs such as the liver was also increased after systemic administration. In contrast, AAV-2-based vectors with ablated binding to their primary receptor heparan sulfate proteoglycan enabled a significantly increased efficiency of cardiac gene transfer and reduced transduction of the liver. Combining transcriptional targeting by the CMV(enh)/MLC1.5 promoter and AAV vectors devoid of binding the AAV-2 primary receptor results in an efficient cardiac gene transfer with a significantly reduced hepatic transduction.

95. Retargeting AAV-2 to Angiogenic Endothelial Cells with Tumor Vasculature Specific Capsid Variants

A promising target for cancer gene therapy is the tumor endothelium, which forms the inner wall of tumor vessels and is therefore essential for the oxygen and nutrient supply of the tumor. On these tumor vessels specific surface molecules are upregulated or specific isoforms are expressed. The development of targeting technologies allows the generation of viral vectors specifically tailored to bind to such molecules. Genetic incorporation of receptor specific ligands into the capsid position I-587 (Girod et al., 1999) seems to be the most promising approach for the generation of retargeting vectors based on the adeno-associated virus (AAV2). Therefore, we used this position to insert previously described tumor vasculature specific peptides into the capsid of AAV2 vectors. These targeting vectors could be generated with titers comparable to wild type AAV2. We could demonstrate that infections of these new retargeting vectors were mediated by the inserted peptides and that the same insertions interfered with the usage of AAV2s primary receptor heparan sulfate proteoglycan. Furthermore, two of the four peptides successfully shifted the viral tropism towards angiogenic HUVEC, an in vitro model for tumor vasculature.

The Heparan Sulfate-Fibroblast Growth Factor Family: Diversity of Structure and Function

The fibroblast growth factor (FGF) receptor complex is a ubiquitous regulator of development and adult tissue homeostasis that bridges the peri-cellular matrix and the intracellular environment. Diverse members of the FGF polypeptide family, the FGF receptor tyrosine kinase (FGFRTK) family and the FGF receptor heparan sulfate proteoglycan (FGFRHS) family combine to result in active and specific FGFR signal transduction complexes. Regulated alternate splicing and combination of variant subdomains give rise to diversity of FGFRTK monomers. Divalent cations cooperate with the FGFRHS to conformationally restrict FGFRTK trans-phosphorylation, which causes depression of kinase activity and facilitates appropriate activation of the FGFR complex by FGF. Diffusional and conformational molecular models of the oligomeric FGFR complex are presented to explain how different point mutations in the FGFRTK commonly cause craniofacial and skeletal abnormalities of graded severity by graded increases in FGF-independent activity of total FGFR complexes. The role of the FGF family in liver growth and function and in prostate tumor progression is discussed.