Development of a universal, oriented antibody immobilization method to functionalize vascular prostheses for enhanced endothelialization for potential clinical application.

BACKGROUND: Thrombosis is a common cause of vascular prosthesis failure. Antibody coating of prostheses to capture circulating endothelial progenitor cells to aid endothelialization on the device surface appears a promising solution to prevent thrombus formation. Compared with random antibody immobilization, oriented antibody coating (OAC) increases antibody-antigen binding capacity and reduces antibody immunogenicity in vivo. Currently, few OAC methods have been documented, with none possessing clinical application potential. RESULTS: Dopamine and the linker amino-PEG8-hydrazide-t-boc were successfully deposited on the surface of cobalt chromium (CC) discs, CC stents and expanded polytetrafluoroethylene (ePTFE) grafts under a slightly basic condition. CD34 antibodies were immobilized through the reaction between aldehydes in the Fc region created by oxidation and hydrazides in the linker after t-boc removal. CD34 antibody-coated surfaces were integral and smooth as shown by scanning electron microscopy (SEM), had significantly reduced or no substrate-specific signals as revealed by X-ray photoelectron spectroscopy, were hospitable for HUVEC growth as demonstrated by cell proliferation assay, and specifically bound CD34 + cells as shown by cell binding testing. CD34 antibody coating turned hydrophobic property of ePTFE grafts to hydrophilic. In a porcine carotid artery interposition model, a confluent monolayer of cobblestone-shaped CD31 + endothelial cells on the luminal surface of the CD34 antibody coated ePTFE graft were observed. In contrast, thrombi and fibrin fibers on the bare graft, and sporadic cells on the graft coated by chemicals without antibodies were seen. CONCLUSION: A universal, OAC method was developed. Our in vitro and in vivo data suggest that the method can be potentially translated into clinical application, e.g., modifying ePTFE grafts to mitigate their thrombotic propensity and possibly provide for improved long-term patency for small-diameter grafts.

Antibody functionalized intravascular devices combined with genetically engineered endothelial colony-forming cells for targeted drug delivery: A proof-of-concept study.

This study was designed to test the ability of ex vivo antibody-coated intravascular devices to capture genetically engineered pig endothelial colony-forming cells (ECFCs) as proof of concept for their potential for in vivo targeted drug delivery. Human α-calcitonin gene-related peptide (α-CGRP) was chosen as the therapeutic molecule as it is unsuitable for systemic administration due to its potent peripheral arterial vasodilatory effect and short half-life in blood, requiring local delivery to yield therapeutic benefit in a particular vascular bed. H-2Kk, a murine leukocyte surface antigen, served as the selection marker for genetically modified ECFCs. H-2Kk antibody was immobilized on electropolished cobalt-chromium (CC) discs, CC stents and ePTFE grafts through dopamine self-polymerization. The functionalized surface was integral and smooth, lacked or had significantly reduced chemical signals specific for substrates. Pig bone marrow-derived ECFCs transfected with a plasmid constructed for H-2Kk and α-CGRP expression produced H-2Kk on cell surface and biologically active α-CGRP in culture medium. H-2Kk antibody-coated substrates bound H-2Kk ECFCs but not control ECFCs in vitro. Bare or only dopamine-coated substrates did not bind H-2Kk ECFCs. These data suggest that implantation of antibody functionalized devices combined with injection of genetically modified ECFCs could be potentially applied for targeted drug delivery.

The predicted collagen-binding domains of Drosophila SPARC are essential for survival and for collagen IV distribution and assembly into basement membranes.

The assembly of basement membranes (BMs) into tissue-specific morphoregulatory structures requires non-core BM components. Work in Drosophila indicates a principal role of collagen-binding matricellular glycoprotein SPARC (Secreted Protein, Acidic, Rich in Cysteine) in larval fat body BM assembly. We report that SPARC and collagen IV (Col(IV)) first colocalize in the trans-Golgi of hemocyte-like cell lines. Mutating the collagen-binding domains of Drosophila SPARC led to the loss of colocalization with Col(IV), a fibrotic-like BM, and 2nd instar larval lethality, indicating that SPARC binding to Col(IV) is essential for survival. Analysis of this mutant at 2nd instar reveals increased Col(IV) puncta within adipocytes, reflecting a disruption in the intracellular chaperone-like activity of SPARC. Removal of the disulfide bridge in the C-terminal EF-hand2 of SPARC, which is known to enhance Col(IV) binding, did not lead to larval lethality; however, a less intense fat body phenotype was observed. Additionally, both SPARC mutants exhibited altered fat body BM pore topography. Wing imaginal disc-derived SPARC did not localize within Col(IV)-rich matrices. This raises the possibility that SPARC interaction with Col(IV) requires initial intracellular interaction to colocalize at the BM or that wing-derived SPARC undergoes differential post-translational modifications that impacts its function. Collectively, these data provide evidence that the chaperone-like activity of SPARC on Col(IV) begins just prior to their co-secretion and demonstrate for the first time that the Col(IV) chaperone-like activity of SPARC is necessary for Drosophila development beyond the 2nd instar.

Persistently elevated early warning scores and lactate identifies patients at high risk of mortality in suspected sepsis.

OBJECTIVE: In the UK, the National Early Warning Score (NEWS) is recommended as part of screening for suspicion of sepsis. Is a change in NEWS a better predictor of mortality than an isolated score when screening for suspicion of sepsis?. METHODS: A prospectively gathered cohort of 1233 adults brought in by ambulance to two UK nonspecialist hospitals, with suspicion of sepsis at emergency department (ED) triage (2015-2017) was analysed. Associations with 30-day mortality and ICU admission rate were compared between groups with an isolated NEWS ≥5 points prehospital and those with persistently elevated NEWS prehospital, in ED and at ward admission. The effect of adding the ED (venous or arterial) lactate was also assessed. RESULTS: Mortality increased if the NEWS persisted ≥5 at ED arrival 22.1% vs. 10.2% [odds ratio (OR) 2.5 (1.6-4.0); P < 0.001]. Adding an ED lactate ≥2 mmol/L was associated with an increase in mortality greater than for NEWS alone [32.2% vs. 13.3%, OR 3.1 (2.2-4.1); P < 0.001], and increased ICU admission [13.9% vs. 3.7%, OR 3.1 (2.2-4.3); P < 0.001]. If NEWS remained ≥5 at ward admission (predominantly within 4 h of ED arrival), mortality was 32.1% vs. 14.3%, [OR 2.8 (2.1-3.9); P < 0.001] and still higher if accompanied by an elevated ED lactate [42.1% vs. 16.4%, OR 3.7 (2.6-5.3); P < 0.001]. CONCLUSION: Persistently elevated NEWS, from prehospital through the ED to the time of ward admission, combined with an elevated ED lactate identifies patients with suspicion of sepsis at highest risk of in-hospital mortality.

Collagen IV trafficking: The inside-out and beyond story.

Collagen IV networks endow basement membranes (BMs) with remarkable tensile strength and function as morphoregulatory substrata for diverse tissue-specific developmental events. A complex repertoire of intracellular and extracellular molecular interactions are required for collagen IV secretion and supramolecular assembly into BMs. These include intracellular chaperones such as Heat shock protein 47 (Hsp47) and the chaperone-binding trafficking protein Transport and Golgi organization protein 1 (Tango1). Mutations in these proteins lead to compromised collagen IV protomer stability and secretion, leading to defective BM assembly and function. In addition to intracellular chaperones, a role for extracellular chaperones orchestrating the transport, supramolecular assembly, and architecture of collagen IV in BM is emerging. We present evidence derived from evolutionarily distant model organisms that supports an extracellular collagen IV chaperone-like activity for the matricellular protein SPARC (Secreted Protein, Acidic, Rich in Cysteine). Loss of SPARC disrupts BM homeostasis and compromises tissue biomechanics and physiological function. Thus, the combined contributions of intracellular and extracellular collagen IV-associated chaperones and chaperone-like proteins are critical to ensure proper secretion and stereotypic assembly of collagen IV networks in BMs.