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Supplementary Data from Activated Platelets Provide a Functional Microenvironment for the Antiangiogenic Fragment of Histidine-Rich Glycoprotein

The angiogenesis inhibitor histidine-rich glycoprotein (HRG) constitutes one of several examples of molecules regulating both angiogenesis and hemostasis. The antiangiogenic properties of HRG are mediated via its proteolytically released histidine- and proline-rich (His/Pro-rich) domain. Using a combination of immunohistochemistry and mass spectrometry, we here provide biochemical evidence for the presence of a proteolytic peptide, corresponding to the antiangiogenic domain of HRG, in vivo in human tissue. This finding supports a role for HRG as an endogenous regulator of angiogenesis. Interestingly, the His/Pro-rich peptide bound to the vessel wall in tissue from cancer patients but not to the vasculature in tissue from healthy persons. Moreover, the His/Pro-rich peptide was found in close association with platelets. Relesate from in vitro–activated platelets promoted binding of the His/Pro-rich domain of HRG to endothelial cells, an effect mediated by Zn2+. Previous studies have shown that zinc-dependent binding of the His/Pro-rich domain of HRG to heparan sulfate on endothelial cells is required for inhibition of angiogenesis. We describe a novel mechanism to increase the local concentration and activity of an angiogenesis inhibitor, which may reflect a host response to counteract angiogenesis during pathologic conditions. Our finding that tumor angiogenesis is elevated in HRG-deficient mice supports this conclusion. (Mol Cancer Res 2009;7(11):1792–802)

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This paper presents a new design of a microfluidic device combining hydrodynamic trapping and impedance spectroscopy measurements of single cells. Four microelectrodes integrated within cell traps enable impedance measurements in varying electrode pair configurations. To improve the impedance response of the microelectrodes, a modification using electrodeposition of a conductive polymer was applied. A considerable decrease of impedance magnitude was observed, and thus a significant enhancement of the useful frequency range was obtained. After electrode modification, the 12 × 22 μm² electrodes were sensitive for frequencies ranging from 10 KHz to 2–5 MHz. Impedance measurements were carried out on single mouse oocytes with and without the surrounding glycoprotein matrix, called zona pellucida. Higher impedance values were obtained for zona pellucida-free than for zona pellucida-intact oocytes, reflecting the known high electrical conductivity of the zona pellucida.

Background Hereditary deficiency of adenine phosphoribosyltransferase causes 2,8-dihydroxyadenine (2,8-DHA) nephropathy, a rare condition characterized by formation of 2,8-DHA crystals within renal tubules. Clinical relevance of rodent models of 2,8-DHA crystal nephropathy induced by excessive adenine intake is unknown. Methods Using animal models and patient kidney biopsies, we assessed the pathogenic sequelae of 2,8-DHA crystal-induced kidney damage. We also used knockout mice to investigate the role of TNF receptors 1 and 2 (TNFR1 and TNFR2), CD44, or alpha2-HS glycoprotein (AHSG), all of which are involved in the pathogenesis of other types of crystal-induced nephropathies. Results Adenine-enriched diet in mice induced 2,8-DHA nephropathy, leading to progressive kidney disease, characterized by crystal deposits, tubular injury, inflammation, and fibrosis. Kidney injury depended on crystal size. The smallest crystals were endocytosed by tubular epithelial cells. Crystals of variable size were excreted in urine. Large crystals obstructed whole tubules. Medium-sized crystals induced a particular reparative process that we term extratubulation. In this process, tubular cells, in coordination with macrophages, overgrew and translocated crystals into the interstitium, restoring the tubular luminal patency; this was followed by degradation of interstitial crystals by granulomatous inflammation. Patients with adenine phosphoribosyltransferase deficiency showed similar histopathological findings regarding crystal morphology, crystal clearance, and renal injury. In mice, deletion of Tnfr1 significantly reduced tubular CD44 and annexin two expression, as well as inflammation, thereby ameliorating the disease course. In contrast, genetic deletion of Tnfr2, Cd44, or Ahsg had no effect on the manifestations of 2,8-DHA nephropathy. Conclusions Rodent models of the cellular and molecular mechanisms of 2,8-DHA nephropathy and crystal clearance have clinical relevance and offer insight into potential future targets for therapeutic interventions.

ObjectiveCalcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic target that could be influenced by dietary of parenteral supplementation.Approach and ResultsWe studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the TRPM6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with dietary phosphate restriction, magnesium supplementation, or by parenteral administration of fetuin-A or pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement.ConclusionsWe show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, pyrophosphate, and magnesium. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, pyrophosphate and magnesium levels.Subject CodesAnimal Models of Human Disease, Fibrosis, Inflammation, Proteomics, Peripheral Vascular Disease

Calcification is a pathological accumulation of calcium phosphate in soft tissues including blood vessels [1] where it can cause cardiovascular morbidity and mortality e.g. heart attack [2] . Mixing blood serum with supersaturated calcium and phosphate results in colloidal calcium protein particles (CPPs) undergoing a typical ripening process. The conversion time can be optically determined by light scatter measurements of smaller primary and larger secondary CPPs (Fig 1). The half-maximal conversion time T 50 is a measure of the calcification risk in the patient. Here we report the miniaturization of the experimental setup on a polymer microchip fabricated by ultrasonic processing. Compared to the usual measurement of light scatter in a nephelometer, the analysis was accelerated by increasing the temperature. Furthermore, better reproducible results were obtained by using a staggered herringbone mixer to mix the reactants [3] . A Peltier element is employed to heat the chip. Using this setup, the analysis was shortened to 16.1 ± 1.1 min compared to 279 ± 9 min obtained in a nephelometer by increasing the temperature from 38 °C to 52 °C (Fig. 4).

Current directions in biomedical engineering 4(1), 367-370 (2018). doi:10.1515/cdbme-2018-0088, Published by De Gruyter, Berlin

Many polycation-based gene delivery vectors show high transfection in vitro, but their cationic nature generally leads to significant toxicity and poor in vivo performance which significantly hampers their clinical applicability. Unlike conventional polycation-based systems, decationized polyplexes are based on hydrophilic and neutral polymers. They are obtained by a 3-step process: charge-driven condensation followed by disulfide crosslinking stabilization and finally polyplex decationization. They consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). In the present paper the applicability of decationized polyplexes for systemic administration was evaluated. Cy5-labeled decationized polyplexes were evaluated for stability in plasma by fluorescence single particle tracking (fSPT), which technique showed stable size distribution for 48 h unlike its cationic counterpart. Upon the incubation of the polymers used for the formation of polyplexes with HUVEC cells, MTT assay showed excellent cytocompatibility of the neutral polymers. The safety was further demonstrated by a remarkable low teratogenicity and mortality activity of the polymers in a zebrafish assay, in great contrast with their cationic counterpart. Near infrared (NIR) dye-labeled polyplexes were evaluated for biodistribution and tumor accumulation by noninvasive optical imaging when administered systemically in tumor bearing mice. Decationized polyplexes exhibited an increased circulation time and higher tumor accumulation, when compared to their cationic precursors. Histology of tumors sections showed that decationized polyplexes induced reporter transgene expression in vivo. In conclusion, decationized polyplexes are a platform for safer polymeric vectors with improved biodistribution properties when systemically administered.

Background and objectives Patients receiving hemodialysis are at risk of cardiovascular events. A novel blood test ( T 50 test) determines the individual calcification propensity of blood. Design, setting, participants, & measurements T 50 was determined in 2785 baseline serum samples of patients receiving hemodialysis enrolled in the Evaluation of Cinacalcet Therapy to Lower Cardiovascular Events (EVOLVE) trial and the T 50 results were related to patient outcomes. Results Serum albumin, bicarbonate, HDL cholesterol, and creatinine were the main factors positively/directly and phosphate was the main factor negatively/inversely associated with T 50 . The primary composite end point (all-cause mortality, myocardial infarction [MI], hospitalization for unstable angina, heart failure, or peripheral vascular event [PVE]) was reached in 1350 patients after a median follow-up time of 619 days. After adjustments for confounding, a lower T 50 was independently associated with a higher risk of the primary composite end point as a continuous measure (hazard ratio [HR] per 1 SD lower T 50 , 1.15; 95% confidence interval [95% CI], 1.08 to 1.22; P T 50 was associated with a higher risk in all-cause mortality (HR per 1 SD lower T 50 , 1.10; 95% CI, 1.02 to 1.17; P =0.001), MI (HR per 1 SD lower T 50 , 1.38; 95% CI, 1.19 to 1.60; P T 50 , 1.22; 95% CI, 1.05 to 1.42; P =0.01). T 50 improved risk prediction (integrated discrimination improvement and net reclassification improvement, P P =0.001) of the primary composite end point. Conclusions Blood calcification propensity was independently associated with the primary composite end point, all-cause mortality, MI, and PVE in the EVOLVE study and improved risk prediction. Prospective trials should clarify whether T 50 -guided therapies improve outcomes.

Histidine-rich glycoprotein (HRG) is a 75-kDa heparin-binding plasma protein implicated in the regulation of tumor growth and vascularization. In this study, we show that hrg−/− mice challenged with fibrosarcoma or pancreatic carcinoma grow larger tumors with increased metastatic properties. Compared with wild-type mice, fibrosarcomas in hrg−/− mice were more hypoxic, necrotic, and less perfused, indicating enhanced vessel abnormalization. HRG deficiency was associated with a suppressed antitumor immune response, with both increased infiltration of M2 marker–expressing macrophages and decreased infiltration of dendritic cells and cytotoxic T cells. Analysis of transcript expression in tumor-associated as well as peritoneal macrophages from hrg−/− mice revealed an increased expression of genes associated with a proangiogenic and immunoinhibitory phenotype. In accordance, expression arrays conducted on HRG-treated peritoneal macrophages showed induction of genes involved in extracellular matrix biology and immune responsiveness. In conclusion, our findings show that macrophages are a direct target of HRG. HRG loss influences macrophage gene regulation, leading to excessive stimulation of tumor angiogenesis, suppression of tumor immune response, and increased tumor growth and metastatic spread. Cancer Res; 72(8); 1953–63. ©2012 AACR.

Advances in high-throughput screening (HTS) instrumentation have led to enormous reduction of costs (e.g., of pipetting stations) and to the development of smaller instruments for automation of day-to-day routines in small research laboratories. In the biomaterials community, there has been an increasing interest for standardized screening protocols to identify cell type-specific cytocompatible biomaterials suitable for tissue engineering (TE) applications. In this study, the authors established a multiplexed assay protocol for toxicity screening of biomaterials using a low- to medium-throughput robotic liquid handling station (LHS). The protocol contains analysis of viability, cytotoxicity, and apoptosis combined in one assay. This study includes performance results of a side-by-side comparison of the EpMotion 5070 LHS and conventional pipetting/dispensing systems. Critical parameters were optimized each for a given platform. Higher accuracy and reproducibility were achieved for LHS compared to manually treated samples. The practicability and accuracy of the method in a typical small laboratory setting were tested by running daily routine tasks by trained and untrained laboratory staff. In addition, advantages and disadvantages as well as the step-by-step application protocol are reported. The approach described provides a potential utility in screening biomaterials toxicity, allowing researchers to meet the needs of low- and medium-throughput laboratories.

The angiogenesis inhibitor histidine-rich glycoprotein (HRG) constitutes one of several examples of molecules regulating both angiogenesis and hemostasis. The antiangiogenic properties of HRG are mediated via its proteolytically released histidine- and proline-rich (His/Pro-rich) domain. Using a combination of immunohistochemistry and mass spectrometry, we here provide biochemical evidence for the presence of a proteolytic peptide, corresponding to the antiangiogenic domain of HRG, in vivo in human tissue. This finding supports a role for HRG as an endogenous regulator of angiogenesis. Interestingly, the His/Pro-rich peptide bound to the vessel wall in tissue from cancer patients but not to the vasculature in tissue from healthy persons. Moreover, the His/Pro-rich peptide was found in close association with platelets. Relesate from in vitro–activated platelets promoted binding of the His/Pro-rich domain of HRG to endothelial cells, an effect mediated by Zn2+. Previous studies have shown that zinc-dependent binding of the His/Pro-rich domain of HRG to heparan sulfate on endothelial cells is required for inhibition of angiogenesis. We describe a novel mechanism to increase the local concentration and activity of an angiogenesis inhibitor, which may reflect a host response to counteract angiogenesis during pathologic conditions. Our finding that tumor angiogenesis is elevated in HRG-deficient mice supports this conclusion. (Mol Cancer Res 2009;7(11):1792–802)

Therapeutic benefits of autologous nerve grafting in repair of peripheral nerve lesions have not been reached using any alternative nerve guide. Nevertheless, issues of co-morbidity and limited availability of donor nerves urgently ask for a need of bioartificial nerve guides which could either replace or complement autologous nerve grafts. It is increasingly appreciated that optimal nerve guides comprise both physical and molecular cues in support of peripheral axon regeneration. Now, we present a collagen-based microstructured 3D nerve guide containing numerous longitudinal guidance channels with dimensions resembling natural endoneurial tubes. Moreover, these nerve guides could be functionalized by Schwann cell (SC) seeding. Viable SCs did not only adhere to the nerve guide, but also migrated throughout the guidance channels. Of particular importance was the observation that SCs within the guidance channels formed cellular columns reminiscent of "Bands of Bungner", which are crucial structures in the natural process of peripheral nerve regeneration during the Wallerian degeneration. We, therefore, conclude that our orientated 3D nerve guides (decorated with SCs) with their physical and molecular properties may hold great promise in the repair of peripheral nerve lesion and serve as a basis for future experimental regeneration studies.

Over the last decade, bioprinting technologies have begun providing important tissue engineering strategies for regenerative medicine and organ transplantation. The major drawback of past approaches has been poor or inadequate material-printing device and substrate combinations, as well as the relatively small size of the printed construct. Here, we hypothesise that cell-laden hydrogels can be printed when submerged in perfluorotributylamine (C(12)F(27)N), a hydrophobic high-density fluid, and that these cells placed within three-dimensional constructs remain viable allowing for cell proliferation and production of extracellular matrix. Human mesenchymal stem cells and MG-63 cells were encapsulated into agarose hydrogels, and subsequently printed in high aspect ratio in three dimensional structures that were supported in high density fluorocarbon. Three-dimensional structures with various shapes and sizes were manufactured and remained stable for more than six months. Live/dead and DAPI stainings showed viable cells 24 h after the printing process, as well as after 21 days in culture. Histological and immunohistochemical analyses after 14 and 21 days revealed viable cells with marked matrix production and signs of proliferation. The compressive strength values of the printed gels consequently increased during the two weeks in culture, revealing encouraging results for future applications in regenerative medicine.