Heparin-resistance in AL amyloidosis: a case report.

BACKGROUND: Non-AT-III mediated heparin-resistance during CPB occurs by complex-forming with heparin-binding proteins. Currently, there are no specific recommendations for non-AT-III mediated heparin-resistance. CASE PRESENTATION: We present a fatal case of a 70-yr-old male-patient undergoing cardiac-surgery in which refractory heparin-resistance was observed. The massive AL amyloidosis found at autopsy is thought to be responsible and illustrates that awareness and knowledge of the etiology and perioperative strategies of non-AT-III mediated heparin-resistance is important. CONCLUSION: For anticoagulation during cardiopulmonary bypass surgery in case of a non-AT-III medicated heparin resistance, we refer to the decision tree added to this manuscript and if necessary to consider direct thrombin inhibitors, such as bivalirudin or argatroban, as it bypasses the complexing pathway.

A collagen plug with shape memory to seal iatrogenic fetal membrane defects after fetoscopic surgery.

Iatrogenic preterm premature rupture of fetal membranes (iPPROM) after fetal surgery remains a strong trigger for premature birth. As fetal membrane defects do not heal spontaneously and amniotic fluid leakage and chorioamniotic membrane separation may occur, we developed a biocompatible, fetoscopically-applicable collagen plug with shape memory to prevent leakage. This plug expands directly upon employment and seals fetal membranes, hence preventing amniotic fluid leakage and potentially iPPROM. Lyophilized type I collagen plugs were given shape memory and crimped to fit through a fetoscopic cannula (Ø 3 mm). Expansion of the plug was examined in phosphate buffered saline (PBS). Its sealing capacity was studied ex vivo using human fetal membranes, and in situ in a porcine bladder model. The crimped plug with shape memory expanded and tripled in diameter within 1 min when placed into PBS, whereas a crimped plug without shape memory did not. In both human fetal membranes and porcine bladder, the plug expanded in the defect, secured itself and sealed the defect without membrane rupture. In conclusion, collagen plugs with shape memory are promising as medical device for rapid sealing of fetoscopic defects in fetal membranes at the endoscopic entry point.

Initial Steps towards Spatiotemporal Signaling through Biomaterials Using Click-to-Release Chemistry.

The process of wound healing is a tightly controlled cascade of events, where severe skin wounds are resolved via scar tissue. This fibrotic response may be diminished by applying anti-fibrotic factors to the wound, thereby stimulating regeneration over scarring. The development of tunable biomaterials that enable spatiotemporal control over the release of anti-fibrotics would greatly benefit wound healing. Herein, harnessing the power of click-to-release chemistry for regenerative medicine, we demonstrate the feasibility of such an approach. For this purpose, one side of a bis-N-hydroxysuccinimide-trans-cyclooctene (TCO) linker was functionalized with human epidermal growth factor (hEGF), an important regulator during wound healing, whereas on the other side a carrier protein was conjugated-either type I collagen scaffolds or bovine serum albumin (BSA). Mass spectrometry demonstrated the coupling of hEGF-TCO and indicated a release following exposure to dimethyl-tetrazine. Type I collagen scaffolds could be functionalized with the hEGF-TCO complex as demonstrated by immunofluorescence staining and Western blotting. The hEGF-TCO complex was also successfully ligated to BSA and the partial release of hEGF upon dimethyl-tetrazine exposure was observed through Western blotting. This work establishes the potential of click-to-release chemistry for the development of pro-regenerative biomaterials.

Biomaterial-Mediated Protein Expression Induced by Peptide-mRNA Nanoparticles Embedded in Lyophilized Collagen Scaffolds.

In our aging society, the number of patients suffering from poorly healing bone defects increases. Bone morphogenetic proteins (BMPs) are used in the clinic to promote bone regeneration. However, poor control of BMP delivery and thus activity necessitates high doses, resulting in adverse effects and increased costs. It has been demonstrated that messenger RNA (mRNA) provides a superior alternative to protein delivery due to local uptake and prolonged expression restricted to the site of action. Here, we present the development of porous collagen scaffolds incorporating peptide-mRNA nanoparticles (NPs). Nanoparticles were generated by simply mixing aqueous solutions of the cationic cell-penetrating peptide PepFect14 (PF14) and mRNA. Peptide-mRNA complexes were uniformly distributed throughout the scaffolds, and matrices fully preserved cell attachment and viability. There was a clear dependence of protein expression on the incorporated amount of mRNA. Importantly, after lyophilization, the mRNA formulation in the collagen scaffolds retained activity also at 4 °C over two weeks. Overall, our results demonstrate that collagen scaffolds incorporating peptide-mRNA complexes hold promise as off-the-shelf functional biomaterials for applications in regenerative medicine and constitute a viable alternative to lipid-based mRNA formulations.

Construction and evaluation of an antibody phage display library targeting heparan sulfate.

Heparan sulfate (HS) is a linear polysaccharide with high structural diversity. Different HS epitopes have been detected and localized using single chain variable fragment (scFv) antibodies from a 'single pot' phage display library containing a randomized complementarity determining region of the heavy chain (CDR3). In this study, we created a new library containing anti-HS scFvs that all harbor a dp-38 heavy chain segment where the CDR3 region was engineered to contain the XBBXBX heparin binding consensus site (X = any amino acid, B = R, K or H). The library contained ~1.73 × 106 unique antibodies and was biopanned against HS from several sources. The selected antibodies were sequenced and chemically/immunohistologically characterized. A number of 67 anti-HS scFv antibodies were selected, of which 31 contained a XBBXBX CDR3 sequence. There was a clear preference for glycine at the first and proline at the fourth position of the CDR3. The sequence GZZP(R/K)X (Z = R, K or H, but may also contain N, S, or Q) was unusually overrepresented. Selected antibodies reacted with HS/heparin, but not with other glycosaminoglycans. Antibodies reacted differentially with respect to N-, 2-O, or 6-O-desulfated heparin preparations, and showed distinct topologies of HS epitopes in rat kidney sections. The library may be instrumental in the selection of a large pool of HS epitope-specific antibodies, and - since all antibodies differ only in their 6 amino acid CDR region - may be a tool for a rational design of antibodies recognizing specific HS sulfation patterns.

A versatile salt-based method to immobilize glycosaminoglycans and create growth factor gradients.

Glycosaminoglycans (GAGs) are known to play pivotal roles in physiological processes and pathological conditions. To study interactions of GAGs with proteins, immobilization of GAGs is often required. Current methodologies for immobilization involve modification of GAGs and/or surfaces, which can be time-consuming and may involve specialized equipment. Here, we use an efficient and low-cost method to immobilize GAGs without any (chemical) modification using highly concentrated salt solutions. A number of salts from the Hofmeister series were probed for their capacity to immobilize heparin and chondroitin-6-sulfate on microtiter plates applying single chain antibodies against GAGs for detection (ELISA). From all salts tested, the cosmotropic salt ammonium sulfate was most efficient, especially at high concentrations (80-100% (v/v) saturation). Immobilized GAGs were bioavailable as judged by their binding of FGF2 and VEGF, and by their susceptibility towards GAG lyases (heparinase I, II and III, chondroitinase ABC). Using 80% (v/v) saturated ammonium sulfate, block and continuous gradients of heparin were established and a gradient of FGF2 was created using a heparin block gradient as a template. In conclusion, high concentrations of ammonium sulfate are effective for immobilization of GAGs and for the establishment of gradients of both GAGs and GAG-binding molecules, which enables the study to the biological roles of GAGs.

Bladder Regeneration Using Multiple Acellular Scaffolds with Growth Factors in a Bladder.

INTRODUCTION: Tissue engineering may become an alternative to current bladder augmentation techniques. Large scaffolds are needed for clinically significant augmentation, but can result in fibrosis and graft shrinkage. The purpose of this study was to investigate the use of multiple scaffolds instead of one large scaffold, to enhance bladder tissue regeneration and bladder capacity. Second, acellular collagen, collagen-heparin, and collagen-heparin scaffolds with growth factors (GFs) were used and the biological activity of the different scaffolds was compared in a large animal model. MATERIALS AND METHODS: Scaffolds were made of bovine type I collagen with or without heparin (Ø = 3.2 cm). Collagen-heparin scaffolds were loaded with GFs, vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and heparin-binding epidermal growth factor (HB-EGF). Three identical scaffolds prepared from collagen (COL-group), collagen with heparin (COLHEP-group), or collagen-heparin with growth factors (COLHEPGF-group) were implanted in one porcine bladder. The outcome was compared with sham-operated animals (Sham-group), in which no scaffold was used. Urodynamic evaluation was performed before surgery and 3 months after bladder reconstruction, together with histological evaluation. RESULTS: Survival rate was 92%, 12 animals completed the study, 3 of every group, 1 animal developed peritonitis due to urine leakage and was sacrificed. The regenerated area was largest in the COLHEP-group, and least in the COL-group (p = 0.002). Histological evaluation revealed a normal urothelial layer and good angiogenesis in all groups, and comparable ingrowth of smooth muscle cells. Urodynamics showed no statistically significant differences in bladder capacity and compliance between groups. Bladder capacity and compliance was very high in this animal model, which made it impossible to study the increase due to augmentation. CONCLUSIONS: Implantation of multiple collagen-heparin scaffolds in one bladder is feasible in a porcine model, resulting in tissue almost indistinguishable from native tissue involving all cell layers of the bladder. Collagen scaffolds with heparin incorporated resulted in a larger area of regenerated tissue. To reach clinically significant augmentation, multiple larger collagen-heparin scaffolds, with or without GFs, need to be tested to study the largest possible diameter of scaffold and number of used scaffolds still resulting in well-vascularized tissue.

Sequencing of glycosaminoglycans with potential to interrogate sequence-specific interactions.

Technologies to sequence nucleic acids/proteins are widely available, but straightforward methodologies to sequence complex polysaccharides are lacking. We here put forward a strategy to sequence glycosaminoglycans, long linear polysaccharides involved in many biochemical processes. The method is based on the covalent immobilization and (immuno)chemical characterization of only those size-separated saccharides that harbor the original reducing end of the full-length chain. Using this methodology, the saccharide sequence of the chondroitin sulfate chain of the proteoglycan bikunin was determined. The method can be performed in any standard biochemical lab and opens studies to the interaction of complex saccharide sequences with other biomolecules.

Visualisation of newly synthesised collagen in vitro and in vivo.

Identifying collagen produced de novo by cells in a background of purified collagenous biomaterials poses a major problem in for example the evaluation of tissue-engineered constructs and cell biological studies to tumor dissemination. We have developed a universal strategy to detect and localize newly deposited collagen based on its inherent association with dermatan sulfate. The method is applicable irrespective of host species and collagen source.

Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology.

Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-ß), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis.

Biodistribution of size-selected lyophilisomes in mice.

Lyophilisomes are a novel class of proteinaceous biodegradable nano/microparticle capsules developed for tumor drug delivery. The in vivo characteristics of lyophilisomes are unknown and, therefore, the time course of biodistribution of sized albumin-based lyophilisomes in CD1 mice after intravenous administration was studied. Lyophilisomes, prepared from Dylight680-labeled albumin, were sized using a sucrose gradient centrifugation methodology and four fractions with a mean size of approximately 200nm, 400nm, 550nm, and 650nm were pooled for in/ex vivo localization, (immuno)histochemistry and biochemical analysis. Lyophilisomes were rapidly taken out of the circulation by the liver and spleen. Immunohistochemistry revealed that lyophilisomes were taken up in the liver by F4/80 positive macrophages, and in the spleen by Sign-R1 positive macrophages specifically located in the marginal zones. Lyophilisomes were most likely degraded by the liver and spleen and subsequently excreted via the urine, as high levels of degraded Dylight680-labeled albumin were detected in the urine. This was corroborated by electron microscopy of the spleen, which showed intact lyophilisomes in the marginal zone 5 and 30min after injection, but not after 2h. In conclusion, IV injected lyophilisomes are rapidly entrapped by liver and splenic macrophages, biodegraded, and excreted in the urine.

'Immunosequencing' of heparan sulfate from human cell lines and rat kidney: the (GlcNS6S-IdoA2S)3 motif, recognized by antibody NS4F5, is located towards the non-reducing end.

HS (heparan sulfate) is a long linear polysaccharide, variably modified by epimerization and sulfation reactions, and is organized into different domains defined by the extent of modification. To further elucidate HS structural organization, the relative position of different HS structures, identified by a set of phage-display-derived anti-HS antibodies, was established. Two strategies were employed: inhibition of HS biosynthesis using 4-deoxy-GlcNAc, followed by resynthesis, and limited degradation of HS using heparinases. Using both approaches, information about the position of antibody-defined HS structures was identified. The HS structure recognized by the antibody NS4F5, rigorously identified as (GlcN6S-IdoA2S)3, was found towards the non-reducing end of the HS chain.

Extraction and structural analysis of glycosaminoglycans from formalin-fixed, paraffin-embedded tissues.

Glycosaminoglycans (GAGs) are long, anionic polysaccharides involved in many basic aspects of mammalian physiology and pathology. Here we describe a method to extract GAGs from formalin-fixed, paraffin-embedded tissues and found that they are structurally comparable with GAGs extracted from frozen tissues. We employed this method to structurally characterize GAGs in tissues, including laser-dissected layers of skin and pathological specimens. This method enables the use of the archival paraffin-embedded material for detailed (structural) analysis of GAGs.

Construction of a microstructured collagen membrane mimicking the papillary dermis architecture and guiding keratinocyte morphology and gene expression.

A papillary-structured collagen fibril membrane is created, mimicking the 3D-architecture of the human papillary dermis. Primary human keratinocytes cultured to confluency on papillar-structured films are compared to keratinocytes cultured on flat membranes. Microscopical evaluation reveals the presence of morphologically distinct cells at the base of the papillar structures that are not observed on flat membranes. Gene expression microarrays and RT-qPCR indicate that these cells are in a more proliferative/migrational state, whereas cells on flat membranes have a more differentiated expression profile. Immunohistochemical stainings confirm these results. In conclusion, specific collagen architecture can direct keratinocyte behavior, and this may be used to further improve skin regeneration.

A comparison of seven methods to analyze heparin in biomaterials: quantification, location, and anticoagulant activity.

Glycosaminoglycans, like heparin, are frequently incorporated in biomaterials because of their capacity to bind and store growth factors and because of their hydrating properties. Heparin is also often used in biomaterials for its anticoagulant activity. Analysis of biomaterial-bound heparin is challenging because most assays are based on heparin in solution. In this study, seven different methods were probed to analyze heparin covalently attached to collagen scaffolds. For each method, the basic mechanism and the advantages and disadvantages are given. An analysis by the factor Xa assay and the Farndale assay clearly indicated that the amount of immobilized heparin cannot be determined correctly when the scaffold is intact. Scaffolds had to be proteolytically digested or acid treated to obtain reliable measurements. Methods used to quantify the amount of bound heparin included a hexosamine assay, an uronic acid assay, a Farndale assay, agarose gel electrophoresis, and immuno-dot blot analysis. Location and semiquantification of heparin were accomplished by immunofluorescence. Although all assays had their advantages and disadvantages, the hexosamine assay turned out to be the most robust and is recommended as the preferred assay to quantify the amount of heparin bound to scaffolds. It is applicable to all scaffolds that are acid hydrolyzable. This study may allow researchers in the field to select the most appropriate method to analyze glycosaminoglycans in biomaterials.

Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.

Aberrant fibrillin-1 expression in early emphysematous human lung: a proposed predisposition for emphysema.

Parenchymal destruction, airspace enlargement, and loss of elasticity are hallmarks of pulmonary emphysema. Although the basic mechanism is unknown, there is a consensus that malfunctioning of the extracellular matrix is a major contributor to the pathogenesis of emphysema. In this study, we analyzed the expression of the elastic fiber protein fibrillin-1 in a large number (n=69) of human lung specimens with early-onset emphysema. Specimens were morphologically characterized by the Destructive Index, the Mean Linear Intercept, and the Panel Grading. We observed a strong correlation (P<0.001) of aberrant fibrillin-1 staining with the degree of destruction of lung parenchyma (r=0.71), airspace enlargement (r=0.47), and emphysema-related morphological abnormalities (r=0.69). There were no obvious correlations with age and smoking behavior. Staining for three other extracellular matrix components (type I collagen, type IV collagen, and laminin) was not affected. The aberrant fibrillin-1 staining observed in this study is similar to that observed in Marfan syndrome, a syndrome caused by mutations in the gene encoding fibrillin-1. Strikingly, emphysema is noticed in a number of Marfan patients. This, together with the notion that disruption of the fibrillin-1 gene in mice results in emphysematous lesions, makes fibrillin-1 a strong candidate to be involved in the etiology and pathogenesis of emphysema.

3-O-sulfated oligosaccharide structures are recognized by anti-heparan sulfate antibody HS4C3.

Antibodies against heparan sulfate (HS) are useful tools to study the structural diversity of HS. They demonstrate the large sequential variation within HS and show the distribution of HS oligosaccharide sequences within their natural environment. We analyzed the distribution and the structural characteristics of the oligosaccharide epitope recognized by anti-HS antibody HS4C3. Biosynthetic and synthetic heparin-related oligosaccharide libraries were used in affinity chromatography, immunoprecipitation, and enzyme-linked immunosorbent assay to identify this epitope as a 3-O-sulfated motif with antithrombin binding capacity. The antibody binds weakly to any N-sulfated, 2-O- and 6-O-sulfated hexa- to octasaccharide fragment but strongly to the corresponding oligosaccharide when there is a 3-O-sulfated glucosamine residue present in the sequence. This difference was highlighted by affinity interaction and immunohistochemistry at salt concentrations from 500 mm. At physiological salt conditions the antibody strongly recognized basal lamina of epithelia and endothelia. At 500 mm salt conditions, when 3-O sulfation is required for binding, antibody recognition was more restricted and selective. Antibody HS4C3 bound similar tissue structures as antithrombin in rat kidney. Furthermore, antithrombin and antibody HS4C3 could compete with one another for binding to heparin. Antibody HS4C3 was also able to inhibit the anti-coagulant activities of heparin and Arixtra as demonstrated using the activated partial thromboplastin time clotting and the anti-factor Xa assays. In summary, antibody HS4C3 selectively detects 3-O-sulfated HS structures and interferes with the coagulation activities of heparin by association with the anti-thrombin binding pentasaccharide sequence.

Short-term variability of biomarkers of proteinase activity in patients with emphysema associated with type Z alpha-1-antitrypsin deficiency.

BACKGROUND: The burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longitudinal variability of these biomarkers is unknown but desirable for clinical studies with proteinase inhibitors. METHODS: We measured three different types of biomarkers, including desmosines, elastase-formed fibrinogen fragments and heparan sulfate epitope JM403, in plasma and urine for a period of 7 weeks in a group of 12 patients who participated in a placebo-controlled study to assess the safety of a single inhalation of hyaluronic acid. RESULTS: Effect of study medication on any of the biomarkers was not seen. Baseline desmosines in plasma and urine correlated with baseline CO diffusion capacity (R = 0.81, p = 0.01 and R = 0.65, p = 0.05). Mean coefficient of variation within patients (CVi) for plasma and urine desmosines was 18.7 to 13.5%, respectively. Change in urinary desmosine levels correlated significantly with change in plasma desmosine levels (R = 0.84, p < 0.01). Mean CVi for fibrinogen fragments in plasma was 20.5% and for JM403 in urine was 27.8%. No correlations were found between fibrinogen fragments or JM403 epitope and desmosines. CONCLUSION: We found acceptable variability in our study parameters, indicating the feasibility of their use in an evaluation of biochemical efficacy of alpha-1-antitrypsin augmentation therapy in Pi Z subjects.

Hypoxia-induced dysfunction of rat diaphragm: role of peroxynitrite.

Oxidants may play a role in hypoxia-induced respiratory muscle dysfunction. In the present study we hypothesized that hypoxia-induced impairment in diaphragm contractility is associated with elevated peroxynitrite generation. In addition, we hypothesized that strenuous contractility of the diaphragm increases peroxynitrite formation. In vitro force-frequency relationship, isotonic fatigability, and nitrotyrosine levels were assessed under hypoxic (Po(2) approximately 6.5 kPa) and hyperoxic (Po(2) approximately 88.2 kPa) control conditions and also in the presence of authentic peroxynitrite (60 min), ebselen (60 min), and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine acetate (L-NMMA) (90 min). A hypoxia-induced downward shift of the force-frequency relationship was associated with elevated nitrotyrosine level in the diaphragm. During hypoxia, both ebselen and L-NMMA decreased nitrotyrosine levels but did not affect force generation. Strenuous contractions impaired force generation but did not affect nitrotyrosine levels in the diaphragm during hypoxia. But under hyperoxic conditions, fatiguing contractions were associated with elevated diaphragm nitrotyrosine levels. Under hyperoxic conditions exogenous peroxynitrite impaired force generation and increased nitrotyrosine level. These studies show that hypoxia-induced impairment in diaphragm contractility is associated with increased diaphragm protein nitration, but no causal relationship was found between diaphragm nitrotyrosine formation and in vitro force generation.