Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in mice, now allowing its further characterization in vivo. We also illustrate that modulation of the EPCR-FVIIa interaction may lead to improved FVIIa therapeutics.
The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off‐target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin‐containing TCP‐1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A‐induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.
The purpose of this study was to describe the cellular architecture of normal human peripapillary sclera (PPS) and evaluate surface topography's role in fibroblast behavior. METHODS.PPS cryosections from nonglaucomatous eyes were labelled for nuclei, fibrillar actin (FA), and alpha smooth muscle actin (αSMA) and imaged. Collagen fibrils were imaged using second harmonic generation. Nuclear density and aspect ratio of the internal PPS (iPPS), outer PPS (oPPS), and peripheral sclera were determined. FA and αSMA fibril alignment with collagen extracellular matrix (ECM) was determined. PPS fibroblasts were cultured on smooth or patterned membranes under mechanical strain and in the presence of TGFβ1 and 2. RESULTS.The iPPS (7.1 ± 2.0 × 10 −4 , P < 0.0001) and oPPS (5.3 ± 1.4 × 10 −4 , P = 0.0013) had greater nuclei density (nuclei/μm 2 ) than peripheral sclera (2.5 ± 0.8 × 10 −4 ). The iPPS (2.0 ± 0.3, P = 0.002) but not oPPS (2.4 ± 0.4, P = 0.45) nuclei had smaller aspect ratios than peripheral (2.7 ± 0.5) nuclei. FA was present throughout the scleral stroma and was more aligned with oPPS collagen (9.6 ± 1.9 degrees) than in the peripheral sclera (15.9 ± 3.9 degrees, P =0.002). The αSMA fibers in the peripheral sclera were less aligned with collagen fibrils (26.4 ± 4.8 degrees) than were FA (15.9 ± 3.9 degrees, P = 0.0002). PPS fibroblasts cultured on smooth membranes shifted to an orientation perpendicular to the direction of cyclic uniaxial strain (1 Hz, 5% strain, 42.2 ± 7.1 degrees versus 62.0 ± 8.5 degrees, P < 0.0001), whereas aligned fibroblasts on patterned membranes were resistant to strain-induced reorientation (5.9 ± 1.4 degrees versus 10 ± 3.3 degrees, P = 0.21). Resistance to re-orientation was reduced by TGFβ treatment (10 ± 3.3 degrees without TGFβ1 compared to 23.1 ± 4.5 degrees with TGFβ1, P < 0.0001). CONCLUSIONS.Regions of the posterior sclera differ in cellular density and nuclear morphology. Topography alters the cellular response to mechanical strain.
Biomaterials are implanted in millions of individuals worldwide each year. Both naturally derived and synthetic biomaterials induce a foreign body reaction that often culminates in fibrotic encapsulation and reduced functional lifespan. In ophthalmology, glaucoma drainage implants (GDIs) are implanted in the eye to reduce intraocular pressure (IOP) in order to prevent glaucoma progression and vision loss. Despite recent efforts towards miniaturization and surface chemistry modification, clinically available GDIs are susceptible to high rates of fibrosis and surgical failure. Here, we describe the development of synthetic, nanofiber‐based GDIs with partially degradable inner cores. We evaluated GDIs with nanofiber or smooth surfaces to investigate the effect of surface topography on implant performance. We observed in vitro that nanofiber surfaces supported fibroblast integration and quiescence, even in the presence of pro‐fibrotic signals, compared to smooth surfaces. In rabbit eyes, GDIs with a nanofiber architecture were biocompatible, prevented hypotony, and provided a volumetric aqueous outflow comparable to commercially available GDIs, though with significantly reduced fibrotic encapsulation and expression of key fibrotic markers in the surrounding tissue. We propose that the physical cues provided by the surface of the nanofiber‐based GDIs mimic healthy extracellular matrix structure, mitigating fibroblast activation and potentially extending functional GDI lifespan.
The purpose of these studies was to evaluate clinical, functional, and histopathological features of glaucoma drainage implants (GDIs) fabricated from novel, customtailored expanded polytetrafluoroethylene (ePTFE). Implants of matching footprints were fabricated from silicone (Control) and novel, bilayered ePTFE. ePTFE implants included: (a) one that inflated with aqueous humor (AH) (High), (b) one that inflated with a lower profile (Low), (c) an uninflated implant not connected to the anterior chamber (Flat), and (d) one filled with material that did not allow AH flow (Filled). All implants were placed in adult New Zealand White rabbits and followed over 1-3 months with clinical exams and intraocular pressure. The permeability of tissue capsules surrounding GDIs was assessed using constant-flow perfusion with fluoresceinated saline at physiologic flow rates. After sacrifice, quantitative histopathological measures of capsule thickness were compared among devices, along with qualitative assessment of cellular infiltration and inflammation. Capsular thickness was significantly reduced in blebs over ePTFE (61.4 ± 53 μm) versus silicone implants (193.6 ± 53 μm, p = .0086). AH exposure did not significantly alter capsular thickness, as there was no significant difference between High and Filled (50.9 ± 29, p = .34) implants. Capsules around ePTFE implants demonstrated permeability with steady-state pressure: flow relationships at physiologic flow rates and rapid pressure decay with flow cessation, while pressure in control blebs increased even at low flow rates and showed little decay. Perfused fluorescein dye appeared beyond the plate border only in ePTFE implants. ePTFE implants are associated with thinner, more permeable capsules compared to silicone implants simulating presently used devices.
APOBEC3 (A3) enzymes deaminate DNA cytosine bases and are implicated as the cause of a prevalent mutational signature found in cancer genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes. A3 enzymes normally function as part of the innate immune system by deaminating viral genomes to restrict infection. Basal expression of A3 enzymes is low in healthy tissues, but is increased by type I interferon signaling. Aside from interferon induction, the mechanisms regulating expression and activity of A3 enzymes are unknown. Thus the circumstances that allow for off-target activity of A3 on cellular DNA, leading to mutations and genome instability, cannot be predicted. We sought to determine protein interactors of A3 enzymes to identify potential regulators of expression and/or activity. Using a doxycycline-inducible expression system, we introduced A3 genes into isogenic cell lines. Each A3 enzyme was immunoprecipitated (IP) and lysates were analyzed by mass spectrometry (MS). MS analysis of A3A interactors revealed all 8 subunits of the TriC/CCT (TCP-1 ring complex or chaperonin containing TCP1) complex. The CCT complex is a molecular chaperone that assists in folding of many newly-synthesized proteins, preventing aggregation and facilitating protein function. Validation of MS results by A3A IP followed by immunoblotting confirmed interaction with CCT complex subunits. Endogenous A3A levels are increased significantly by interferon in peripheral blood mononuclear cells (PBMCs). We performed IP of CCT in PBMCs followed by immunoblot for A3A, which demonstrated an interaction between endogenous A3A and the CCT complex. To evaluate the impact of the CCT-A3A interaction, we used siRNA to knock down the CCT complex. Although CCT is an essential chaperonin, transient knockdown of individual subunits resulted in minimally decreased cell viability. However, knockdown of CCT subunits combined with induction of A3A expression led to significant decrease in cell proliferation and increase in cell death. To determine the mechanism by which CCT knockdown with A3A expression results in cytotoxicity, we performed an in vitro deamination assay on lysates from cells that were transfected with siRNA targeting CCT subunits and/or treated with doxycycline to induce A3A expression. Quantification of deamination activity demonstrated that CCT knockdown resulted in increased in A3A activity relative to expression levels. Finally, we evaluated cancer genome sequences in TCGA and found an enrichment of the mutational signature attributed to A3 deamination in cancers with deleterious mutations in CCT subunit genes. Together, these data suggest that the CCT complex regulates A3A activity, and disruption of CCT function results in increased A3A mutational activity. Citation Format: Abby M. Green, Ariel S. Dineen, Katarzyna Kulej, Julia H. Szeto, Matthew D. Weitzman. The CCT chaperonin is a regulator of mutagenic APOBEC3A activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3495.
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