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Introduction: The introduction of-or the transition to-a new electronic health record system (EHR) places an unrecognized burden on health care providers in our ever-changing health care environment of increased mandates, increased overhead, and reduced reimbursement to practice medicine., Purpose: The purpose of this study was to track the non-reimbursed time investment required for 6 providers from an independent spine surgery practice after a hospital system independently decided to transition to a new EHR system., Results: Between the 6 providers of the practice, 266 hours of required classroom time, in-person training, and video and phone call teaching sessions had to be completed to become "proficient" so that clinical utilization of the system for in-patient care was certified by the hospital trainers and the EHR company., Conclusions: The burdens associated with the introduction of mandatory EHRs are putting tremendous pressure on providers from a time commitment perspective. This detracts from patient care during that time and deserves to be compensated for by the EHR companies that extract billions of dollars from both federal and private insurers' health care budgets., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The accurate and safe positioning of cervical pedicle screws is crucial. While augmented reality (AR) use in spine surgery has previously demonstrated clinical utility in the thoracolumbar spine, its technical feasibility in the cervical spine remains less explored., Purpose: The objective of this study was to assess the precision and safety of AR-assisted pedicle screw placement in the cervical spine., Study Design: In this experimental study, 5 cadaveric cervical spine models were instrumented from C3 to C7 by 5 different spine surgeons. The navigation accuracy and clinical screw accuracy were evaluated., Methods: Postprocedural CT scans were evaluated for clinical accuracy by 2 independent neuroradiologists using the Gertzbein-Robbins scale. Technical precision was assessed by calculating the angular trajectory (°) and linear screw tip (mm) deviations in the axial and sagittal planes from the virtual pedicle screw position as recorded by the AR-guided platform during the procedure compared to the actual pedicle screw position derived from postprocedural imaging., Results: A total of forty-one pedicle screws were placed in 5 cervical cadavers, with each of the 5 surgeons navigating at least 7 screws. Gertzbein-Robbins grade of A or B was achieved in 100% of cases. The mean values for tip and trajectory errors in the axial and sagittal planes between the virtual versus actual position of the screws was less than 3 mm and 30°, respectively (p<.05). None of the cervical screws violated the cortex by more than 2 mm or displaced neurovascular structures., Conclusions: AR-assisted cervical pedicle screw placement in cadavers demonstrated clinical accuracy comparable to existing literature values for image-guided navigation methods for the cervical spine., Clinical Significance: This study provides technical and clinical accuracy data that supports clinical trialing of AR-assisted subaxial cervical pedicle screw placement., Competing Interests: Declarations of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Biomaterial-associated infection (BAI) is considered a unique infection due to the presence of a biomaterial yielding frustrated immune-cells, ineffective in clearing local micro-organisms. The involvement of surface-adherent/surface-adapted micro-organisms in BAI, logically points to biomaterial surface-modifications for BAI-control. Biomaterial surface-modification is most suitable for prevention before adhering bacteria have grown into a mature biofilm, while BAI-treatment is virtually impossible through surface-modification. Hundreds of different surface-modifications have been proposed for BAI-control but few have passed clinical trials due to the statistical near-impossibility of benefit-demonstration. Yet, no biomaterial surface-modification forwarded, is clinically embraced. Collectively, this leads us to conclude that surface-modification is a dead-end road. Accepting that BAI is, like most human infections, due to surface-adherent biofilms (though not always to a foreign material), and regarding BAI as a common infection, opens a more-generally-applicable and therewith easier-to-validate road. Pre-clinical models have shown that stimuli-responsive nano-antimicrobials and antibiotic-loaded nanocarriers exhibit prolonged blood-circulation times and can respond to a biofilm's micro-environment to penetrate and accumulate within biofilms, prompt ROS-generation and synergistic killing with antibiotics of antibiotic-resistant pathogens without inducing further antimicrobial-resistance. Moreover, they can boost frustrated immune-cells around a biomaterial reducing the importance of this unique BAI-feature. Time to start exploring the nano-road for BAI-control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Necrotizing enterocolitis (NEC) is a life-threatening inflammatory disease. Its onset might be triggered by Toll-Like Receptor 4 (TLR4) activation via bacterial lipopolysaccharide (LPS). We hypothesize that a deficiency of intestinal alkaline phosphatase (IAP), an enzyme secreted by enterocytes that dephosphorylates LPS, may contribute to NEC development., Methods: In this prospective pilot study, we analyzed intestinal resection specimens from surgical NEC patients, and from patients undergoing Roux-Y reconstruction for hepatobiliary disease as controls. We assessed IAP activity via enzymatic stainings and assays and explored IAP and TLR4 co-localization through immunofluorescence., Results: The study population consisted of five NEC patients (two Bell's stage IIb and three-stage IIIb, median (IQR) gestational age 25 (24-28) weeks, postmenstrual age at diagnosis 28 (26-31) weeks) and 11 controls (unknown age). There was significantly lower IAP staining in NEC resection specimens [49 (41-50) U/g of protein] compared to controls [115 (76-144), P  = 0.03]. LPS-dephosphorylating activity was also lower in NEC patients [0.06 (0-0.1)] than in controls [0.3 (0.2-0.5), P  = 0.003]. Furthermore, we observed colocalization of IAP and TLR4 in NEC resection specimens., Conclusion: This study suggests a significantly lower IAP level in resection specimens of NEC patients compared to controls. This lower IAP activity suggests a potential role of IAP as a protective agent in the gut, which needs further confirmation in larger cohorts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Martins, Hulscher, Timmer, Kooi and Poelstra.)

Necrotizing enterocolitis (NEC) is a devastating neonatal disease that affects neonates worldwide and often leads to high morbidity and mortality rates. Despite extensive research, the cause of NEC remains unclear, and current treatment options are limited. An important novel finding is the potential role of intestinal Alkaline Phosphatase (IAP) in both pathogenesis and treatment of NEC. IAP can play a vital role in detoxifying liposaccharides (LPS), a key mediator of many pathological processes, thereby reducing the inflammatory response associated with NEC. Furthermore, IAP can help prevent dysbiosis, improve intestinal perfusion, and promote autophagy. In this comprehensive review, we present evidence of the possible connection between IAP and the LPS/Toll-like receptor 4 (TLR4) pathway, impaired gut immunity, and dysbiosis in the preterm gut. Based on these findings, the administration of exogenous IAP might provide promising preventive and therapeutic avenues for the management of NEC., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background Context: Metals from spinal implants are released into surrounding tissues by various mechanisms. Metal ion release has been associated with clinical implant failure, osteolysis, and remote site accumulation with adverse events. Significant corrosion and associated metal ion release has been described with currently used spinal implant alloys. A novel metal alloy, Molybdenum-47.5Rhenium alloy (MoRe®), was approved for use in medical implants in 2019 by the FDA., Purpose: To evaluate the metal ion release profile of MoRe alloy after immersion in both a stable physiologic, as well as in an inflammatory environment., Study Design: In vitro study., Methods: The ion release profile of the MoRe alloy was comprehensively evaluated in-vitro after prolonged immersion in physiologic and inflammatory environments. Ion concentration analyses were then conducted using inductively coupled plasma-mass spectrometry (ICP-MS) methods. Comparative testing of titanium (Ti-6Al-4V) and cobalt chromium (Co-28Cr-6Mo) was also performed., Results: Under baseline physiologic conditions, the MoRe alloy demonstrates very low molybdenum and rhenium ion release rates throughout the 30-day test period. During the first time interval (day 0-1), low levels of molybdenum and rhenium ions are detected (<0.3 μg/cm 2 day) followed by a rapid reduction in the ion release rates to <0.05 μg/cm 2 day during the second time interval (days 1-3) followed by a further reduction to very low steady-state rates <0.01 μg/cm 2 day during the third time interval (days 3-7), which were maintained through 30 days. In the inflammatory condition (H 2 O 2 solution), there was a transient increase in the release of molybdenum and rhenium ions, followed by a return to baseline ion release rates (days 2-4), with a further reduction to low steady-state rates of ∼0.01 μg/cm 2 day (days 4-8). The measured molybdenum and rhenium ion release rates in both steady state (<0.01 μg/cm 2 day), and inflammatory environments (0.01 μg/cm 2 day) were far below the established FDA-permitted daily exposure (PDE) of 1,900 μg/cm 2 day for molybdenum and 4,400 μg/cm 2 day for rhenium. In contrast, titanium and cobalt chromium approached or exceeded their established PDE values in an inflammatory environment., Conclusions: The novel biomaterial MoRe demonstrated a lower metal ion release profile in both a physiologic and inflammatory environment and was well below the established PDE.  Comparative testing of the cobalt-chromium and titanium alloys found higher levels of ion release in the inflammatory environment that exceeded the PDE for cobalt and vanadium., Competing Interests: Declarations of Competing Interests One or more of the authors declare financial or professional relationships on ICMJE-TSJ disclosure forms., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Liver diseases are a leading cause of death worldwide and are rising exponentially due to increasing prevalence of metabolic disorders. Hepatic stellate cells (HSCs) are recognized as a key therapeutic target in liver diseases as these cells, upon activation during liver damage and ongoing liver inflammation, secrete excessive amounts of extracellular matrix that leads to liver tissue scarring (fibrosis) responsible for liver dysfunction (end-stage liver disease) and desmoplasia in hepatocellular carcinoma. Targeting of HSCs to reverse fibrosis progression has been realized by several experts in the field, including us. We have developed strategies to target activated HSCs by utilizing the receptors overexpressed on the surface of activated HSCs. One well-known receptor is platelet derived growth factor receptor-beta (PDGFR-β). Using PDGFR-β recognizing peptides (cyclic PPB or bicyclic PPB), we can deliver biologicals, e.g., interferon gamma (IFNγ) or IFNγ activity domain (mimetic IFNγ), to the activated HSCs that can inhibit their activation and reverse liver fibrosis. In this chapter, we provide the detailed methods and the principles involved in the synthesis of these targeted (mimetic) IFNγ constructs. These methods can be adapted for synthesizing constructs for targeted/cell-specific delivery of peptides/proteins, drugs, and imaging agents useful for various applications including diagnosis and treatment of inflammatory and fibrotic diseases and cancer., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Spinal surgical robots are in the early phases of development and adoption. These systems need to be easier to use, less costly, and more workflow-efficient., Methods: A portable, operating room table-mounted spine robot and camera system are described. Accuracy and workflow efficiency were assessed in comparison to another commonly utilized spinal robotic system., Results: For the surgical task of inserting 4 pedicle screws into 2 adjacent lumbar vertebrae, equivalent accuracy was seen with both systems. The new robotic system was more efficient in terms of total procedure time, system setup time, and screw planning to in-position time (p<0.05)., Conclusions: Spinal robotic systems can be more efficient and less expensive while maintaining accuracy., Clinical Relevance: Spinal robots are being increasingly utilized in clinical practice. Lowering the cost of these systems and increasing their workflow efficiency should help patients and spine surgeons alike., Competing Interests: Declaration of Conflicting Interests: Dr. Pollina has the following disclosures: Alphatec Spine: consultant, advisory board member; Medtronic: consultant, advisory board member. Dr. Poelstra has the following disclosures: Acuity Surgical: consultant; Atlas Spine: consultant; Innovative Surgical Devices: consultant; Flowpharma: distribution group; Kuros: research support; Inion OI: royalties; Stryker: royalties; Camber Spine: scientific advisory board; Society of Minimally Invasive Spine Surgery: scientific advisory board; Medtronic: speaking/teaching arrangements; North American Spine Society: research grant. Dr. Chaudhary has the following disclosures: Omega Fellowship: educational grant; Globus Fellowship: educational grant; Stryker: consultant; Innovative Surgical Design: consultant; Globus: honorarium; Stryker: speaker; North American Spine Society: committee role; American Academy of Orthopedic Surgeons: committee role. Dr. Foley: consultant for Medtronic; direct stock ownership in Accelus, Companion Spine, Discgenics, DuraStat, Medtronic, NuVasive, Practical Navigation, RevBio, Spine Wave, Tissue Differentiation Intelligence, Triad Life Sciences, and True Digital Surgery; patent holder with Medtronic and NuVasive; royalties from Medtronic; and board of directors of Discgenics, DuraStat, RevBio, Tissue Differentiation Intelligence, Triad Life Sciences, and True Digital Surgery. All other authors have no personal, financial, or institutional interest in the materials or devices described in this manuscript., (This manuscript is generously published free of charge by ISASS, the International Society for the Advancement of Spine Surgery. Copyright © 2022 ISASS. To see more or order reprints or permissions, see http://ijssurgery.com.)

Drug targeting and nanomedicine are different strategies for improving the delivery of drugs to their target. Several antibodies, immuno-drug conjugates and nanomedicines are already approved and used in clinics, demonstrating the potential of such approaches, including the recent examples of the DNA- and RNA-based vaccines against COVID-19 infections. Nevertheless, targeting remains a major challenge in drug delivery and different aspects of how these objects are processed at organism and cell level still remain unclear, hampering the further development of efficient targeted drugs. In this review, we compare properties and advantages of smaller targeted drug constructs on the one hand, and larger nanomedicines carrying higher drug payload on the other hand. With examples from ongoing research in our Department and experiences from drug delivery to liver fibrosis, we illustrate opportunities in drug targeting and nanomedicine and current challenges that the field needs to address in order to further improve their success.

Background: Necrotizing enterocolitis (NEC) is a neonatal disease associated with necrosis and perforation of the bowel. We investigated the association between blood group and NEC outcomes and the potential contribution of fetal-maternal blood group incompatibility., Methods: Retrospective study including all preterm-born infants with NEC (≥ Bell's stage IIa) admitted to our NICU between January 2008 and October 2019. We analyzed the association between infants' blood groups and fetal-maternal blood group incompatibility with Bell stage severity, need for surgery, and mortality due to NEC., Results: We included 237 NEC patients. In univariable analyses both AB blood group and fetal-maternal blood group incompatibility increased infants' risk of severe outcomes, with odds ratios (OR) ranging from 6.57 to 12.06 and 1.97 to 2.38, respectively. When adjusted for gestational age only AB blood group remained significant with OR 7.47 (95% confidence interval, 1.95-28.53, P = 0.003), 12.37 (2.63-58.20, P = 0.001), and 8.16 (2.28-29.14, P = 0.001) for NEC Bell's stage III, need for surgery, and NEC related mortality, respectively. Blood group incompatibility adjusted for gestational age was not related to worse outcomes with OR 1.84 (0.87-3.89, P = 0.11, 2.08 (0.98-4.41, P = 0.06) 1.52 (0.68-3.42, P = 0.31), for NEC Bell's stage III, need for surgery, and NEC related mortality, respectively., Conclusion: Our data confirm an association between blood group AB and worse outcomes in NEC infants, but this is not based on fetal-maternal blood group incompatibility., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Robotic-assisted adult deformity surgery has played a rapidly expanding role since its introduction. As robotic spine technologies improve, the potential to limit complications and morbidity is vast. The improvements in instrumentation accuracy combined with the ability to maintain that accuracy in multiple positions allow creative surgical approaches and techniques that can limit operative time, blood loss, and improve outcomes. In the years to come, robotic-assisted spine surgery and navigation will likely play an expanding role that continues to be defined. LEVEL OF EVIDENCE: 5, expert opinion., (This manuscript is generously published free of charge by ISASS, the International Society for the Advancement of Spine Surgery. Copyright © 2021 ISASS.)

Proper regulation of the innate immune response to bone biomaterials after implantation is pivotal for successful bone healing. Pro-inflammatory M1 and anti-inflammatory M2 macrophages are known to have an important role in regulating the healing response to biomaterials. Materials with defined structural and topographical features have recently been found to favourably modulate the innate immune response, leading to improved healing outcomes. Calcium phosphate bone grafts with submicron-sized needle-shaped surface features have been shown to trigger a pro-healing response through upregulation of M2 polarised macrophages, leading to accelerated and enhanced bone regeneration. The present review describes the recent research on these and other materials, all the way from benchtop to the clinic, including in vitro and in vivo fundamental studies, evaluation in clinically relevant spinal fusion models and clinical validation in a case series of 77 patients with posterolateral and/or interbody fusion in the lumbar and cervical spine. This research demonstrates the feasibility of enhancing biomaterial-directed bone formation by modulating the innate immune response through topographic surface features.

Alkaline phosphatase (AP) activity is highly upregulated in plasma during liver diseases. Previously, we demonstrated that AP is able to detoxify lipopolysaccharide (LPS) by dephosphorylating its lipid A moiety. Because a role of gut-derived LPS in liver fibrogenesis has become evident, we now examined the relevance of phosphate groups in the lipid A moiety in this process. The effects of mono-phosphoryl and di-phosphoryl lipid A (MPLA and DPLA, respectively) were studied in vitro and LPS-dephosphorylating activity was studied in normal and fibrotic mouse and human livers. The effects of intestinal AP were studied in mice with CCL4-induced liver fibrosis. DPLA strongly stimulated fibrogenic and inflammatory activities in primary rat hepatic stellate cells (rHSCs) and RAW264.7 macrophages with similar potency as full length LPS. However, MPLA did not affect any of the parameters. LPS-dephosphorylating activity was found in mouse and human livers and was strongly increased during fibrogenesis. Treatment of fibrotic mice with intravenous intestinal-AP significantly attenuated intrahepatic desmin + - and αSMA + -HSC and CD68 + - macrophage accumulation. In conclusion, the lack of biological activity of MPLA, contrasting with the profound activities of DPLA, shows the relevance of LPS-dephosphorylating activity. The upregulation of LPS-dephosphorylating activity in fibrotic livers and the protective effects of exogenous AP during fibrogenesis indicate an important physiological role of intestinal-derived AP during liver fibrosis.

Objective: Augmented reality-mediated spine surgery (ARMSS) is a minimally invasive novel technology that has the potential to increase the efficiency, accuracy, and safety of conventional percutaneous pedicle screw insertion methods. Visual 3D spinal anatomical and 2D navigation images are directly projected onto the operator's retina and superimposed over the surgical field, eliminating field of vision and attention shift to a remote display. The objective of this cadaveric study was to assess the accuracy and precision of percutaneous ARMSS pedicle implant insertion., Methods: Instrumentation was placed in 5 cadaveric torsos via ARMSS with the xvision augmented reality head-mounted display (AR-HMD) platform at levels ranging from T5 to S1 for a total of 113 total implants (93 pedicle screws and 20 Jamshidi needles). Postprocedural CT scans were graded by two independent neuroradiologists using the Gertzbein-Robbins scale (grades A-E) for clinical accuracy. Technical precision was calculated using superimposition analysis employing the Medical Image Interaction Toolkit to yield angular trajectory (°) and linear screw tip (mm) deviation from the virtual pedicle screw position compared with the actual pedicle screw position on postprocedural CT imaging., Results: The overall implant insertion clinical accuracy achieved was 99.1%. Lumbosacral and thoracic clinical accuracies were 100% and 98.2%, respectively. Specifically, among all implants inserted, 112 were noted to be Gertzbein-Robbins grade A or B (99.12%), with only 1 medial Gertzbein-Robbins grade C breach (> 2-mm pedicle breach) in a thoracic pedicle at T9. Precision analysis of the inserted pedicle screws yielded a mean screw tip linear deviation of 1.98 mm (99% CI 1.74-2.22 mm) and a mean angular error of 1.29° (99% CI 1.11°-1.46°) from the projected trajectory. These data compare favorably with data from existing navigation platforms and regulatory precision requirements mandating that linear and angular deviation be less than 3 mm (p < 0.01) and 3° (p < 0.01), respectively., Conclusions: Percutaneous ARMSS pedicle implant insertion is a technically feasible, accurate, and highly precise method.

Osteoprotegerin (OPG) serum levels are associated with liver fibrogenesis and have been proposed as a biomarker for diagnosis. However, the source and role of OPG in liver fibrosis are unknown, as is the question of whether OPG expression responds to treatment. Therefore, we aimed to elucidate the fibrotic regulation of OPG production and its possible function in human and mouse livers. OPG levels were significantly higher in lysates of human and mouse fibrotic livers compared to healthy livers. Hepatic OPG expression localized in cirrhotic collagenous bands in and around myofibroblasts. Single cell sequencing of murine liver cells showed hepatic stellate cells (HSC) to be the main producers of OPG in healthy livers. Using mouse precision-cut liver slices, we found OPG production induced by transforming growth factor β1 (TGFβ1) stimulation. Moreover, OPG itself stimulated expression of genes associated with fibrogenesis in liver slices through TGFβ1, suggesting profibrotic activity of OPG. Resolution of fibrosis in mice was associated with decreased production of OPG compared to ongoing fibrosis. OPG may stimulate fibrogenesis through TGFβ1 and is associated with the degree of fibrogenesis. It should therefore be investigated further as a possible drug target for liver fibrosis or biomarker for treatment success of novel antifibrotics., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The pivotal cell involved in the pathogenesis of liver fibrosis, i.e., the activated hepatic stellate cell (HSC), has a wide range of activities during the initiation, progression and even regression of the disease. These HSC-related activities encompass cellular activation, matrix synthesis and degradation, proliferation, contraction, chemotaxis and inflammatory signaling. When determining the in vitro and in vivo effectivity of novel antifibrotic therapies, the readout is currently mainly based on gene and protein levels of α-smooth muscle actin (α-SMA) and the fibrillar collagens (type I and III). We advocate for a more comprehensive approach in addition to these markers when screening potential antifibrotic drugs that interfere with HSCs. Therefore, we aimed to develop a gene panel for human in vitro and ex vivo drug screening models, addressing each of the HSC-activities with at least one gene, comprising, in total, 16 genes. We determined the gene expression in various human stellate cells, ranging from primary cells to cell lines with an HSC-origin, and human liver slices and stimulated them with two key profibrotic factors, i.e., transforming growth factor β (TGFβ) or platelet-derived growth factor BB (PDGF-BB). We demonstrated that freshly isolated HSCs showed the strongest and highest variety of responses to these profibrotic stimuli, in particular following PDGF-BB stimulation, while cell lines were limited in their responses. Moreover, we verified these gene expression profiles in human precision-cut liver slices and showed similarities with the TGFβ- and PDGF-BB-related fibrotic responses, as observed in the primary HSCs. With this study, we encourage researchers to get off the beaten track when testing antifibrotic compounds by including more HSC-related markers in their future work. This way, potential compounds will be screened more extensively, which might increase the likelihood of developing effective antifibrotic drugs.

Rho kinase activity in hepatic stellate cells (HSCs) is associated with activation, transformation and contraction of these cells, leading to extracellular matrix production and portal hypertension in liver cirrhosis. Inhibition of rho kinase activity can reduce these activities, but may also lead to side effects, for instance systemic hypotension. This can be circumvented by liver-specific delivery of a rho kinase inhibitor to effector cells. Therefore, we targeted the rho kinase inhibitor Y27632 to the key pathogenic cells in liver fibrosis, i.e. myofibroblasts including activated HSCs that highly express the PDGFβ-receptor, using the drug carrier pPB-MSA. This carrier consists of mouse serum albumin (MSA) covalently coupled to several PDGFβR-recognizing moieties (pPB). We aimed to create a prolonged release system of such a targeted construct, by encapsulating pPB-MSA-Y27632 in biodegradable polymeric microspheres, thereby reducing short-lasting peak concentrations and the need for frequent administrations. Firstly, we confirmed the vasodilating potency of PDGFβ-receptor targeted Y27632 in vitro in a contraction assay using HSCs seeded on a collagen gel. We subsequently demonstrated the in vivo antifibrotic efficacy of pPB-MSA-Y27632-loaded microspheres in the Mdr2-/- mouse model of progressive biliary liver fibrosis. A single subcutaneous microsphere administration followed by organ harvest one week later clearly attenuated liver fibrosis progression and significantly suppressed the expression of fibrosis related genes, such as several collagens, profibrotic cytokines and matrix metalloproteinases. In conclusion, we demonstrate that polymeric microspheres are suitable as drug delivery system for the sustained systemic delivery of targeted protein constructs with antifibrotic potential, such as pPB-MSA-Y27632. This formulation appears suitable for the sustained treatment of liver fibrosis and possibly other chronic diseases., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Rho-kinase (ROCK) activation in hepatic stellate cells (HSC) is a key mechanism promoting liver fibrosis and portal hypertension (PTH). Specific delivery of ROCK-inhibitor Y-27632 (Y27) to HSC targeting mannose-6-phosphate-receptors reduces portal pressure and fibrogenesis. In decompensated cirrhosis, presence of ascites is associated with reduced renal perfusion. Since in cirrhosis, platelet-derived growth factor receptor beta (PDGFRβ) is upregulated in the liver as well as the kidney, this study coupled Y27 to human serum albumin (HSA) substituted with PDGFRβ-recognizing peptides (pPB), and investigated its effect on PTH in cirrhotic rats. In vitro collagen contraction assays tested biological activity on LX2 cells. Hemodynamics were analyzed in BDL and CCl 4 cirrhotic rats 3 h, 6 h and 24 h after i.v. administration of Y27pPBHSA (0.5/1 mg/kg b.w). Phosphorylation of moesin and myosin light chain (MLC) assessed ROCK activity in liver, femoral muscle, mesenteric artery, kidney and heart. Three Y27 molecules were coupled to pPBHSA as confirmed by HPLC/MS, which was sufficient to relax LX2 cells. In vivo, Y27pPBHSA-treated rats exhibited lower portal pressure, hepatic vascular resistance without effect on systemic vascular resistance, but a tendency towards lower cardiac output compared to non-treated cirrhotic rats. Y27pPBHSA reduced intrahepatic resistance by reduction of phosphorylation of moesin and MLC in Y27pPBHSA-treated cirrhotic rats. Y27pPBHSA was found in the liver of rats up to 6 hours after its injection, in the HSC demonstrated by double-immunostainings. Interestingly, Y27pPBHSA increased renal arterial flow over time combined with an antifibrotic effect as shown by decreased renal acta2 and col1a1 mRNA expression. Therefore, targeting the ROCK inhibitor Y27 to PDGFRβ decreases portal pressure with potential beneficial effects in the kidney. This unique approach should be tested in human cirrhosis.

Safety is prerequisite for preventive medicine, but non-toxic agents are generally ineffective as clinical chemoprevention. Here we propose a strategy overcoming this challenge by delivering molecular-targeted agent specifically to the effector cell type to achieve sufficient potency, while circumventing toxicity in the context of cancer chemoprevention. Hepatic myofibroblasts drive progressive fibrosis that results in cirrhosis and liver cancer. In a rat model of cirrhosis-driven liver cancer, a small molecule epidermal growth factor receptor inhibitor, erlotinib, was delivered specifically to myofibroblasts by a versatile nanoparticle-based system, targeting platelet-derived growth factor receptor-beta uniquely expressed on their surface in the liver. With systemic administration of erlotinib, tumor burden was reduced to 31%, which was further improved to 21% by myofibroblast-targeted delivery even with reduced erlotinib dose (7.3-fold reduction with equivalent erlotinib dose) and less hepatocyte damage. These findings demonstrate a strategy, cell type-specific kinase inhibition, for more effective and safer precision cancer chemoprevention., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Liver fibrogenesis is associated with excessive production of extracellular matrix by myofibroblasts that often leads to cirrhosis and consequently liver dysfunction and death. Novel protein-based antifibrotic drugs show high specificity and efficacy, but their use in the treatment of fibrosis causes a high burden for patients, since repetitive and long-term parenteral administration is required as most proteins and peptides are rapidly cleared from the circulation. Therefore, we developed biodegradable polymeric microspheres for the sustained release of proteinaceous drugs. We encapsulated the drug carrier pPB-HSA, which specifically binds to the PDGFβR that is highly upregulated on activated myofibroblasts, into microspheres composed of hydrophilic multi-block copolymers composed of poly(l-lactide) and poly ethylene glycol/poly(ϵ-caprolactone), allowing diffusion-controlled release. Firstly, we estimated in mice with acute fibrogenesis induced by a single CCl4 injection the half-life of I125-labeled pPB-HSA at 40 min and confirmed the preferential accumulation in fibrotic tissue. Subsequently, we determined in the Mdr2 −/− mouse model of advanced biliary liver fibrosis how the subcutaneously injected microspheres released pPB-HSA into both plasma and fibrotic liver at 24 h after injection, which was maintained for six days. Although the microspheres still contained protein at day seven, pPB-HSA plasma and liver concentrations were decreased. This reduction was associated with an antibody response against the human albumin-based carrier protein, which was prevented by using a mouse albumin-based equivalent (pPB-MSA). In conclusion, this study shows that our polymeric microspheres are suitable as sustained release formulation for targeted protein constructs such as pPB-HSA. These formulations could be applied for the long-term treatment of chronic diseases such as liver fibrosis.

Injectable sustained release drug delivery systems are an attractive alternative for the intravenous delivery of therapeutic proteins. In particular, for chronic diseases such as fibrosis, this approach could improve therapy by reducing the administration frequency while avoiding large variations in plasma levels. In fibrotic tissues the platelet-derived growth factor receptor beta (PDGFβR) is highly upregulated, which provides a target for site-specific delivery of drugs. Our aim was to develop an injectable sustained release formulation for the subcutaneous delivery of the PDGFβR-targeted drug carrier protein pPB-HSA, which is composed of multiple PDGFβR-recognizing moieties (pPB) attached to human serum albumin (HSA). We used blends of biodegradable multi-block copolymers with different swelling degree to optimize the release rate using the model protein HSA from microspheres produced via a water-in-oil-in-water double emulsion evaporation process. The optimized formulation containing pPB-HSA, showed complete release in vitro within 14days. After subcutaneous administration to mice suffering from renal fibrosis pPB-HSA was released from the microspheres and distributed into plasma for at least 7days after administration. Furthermore, we demonstrated an enhanced accumulation of pPB-HSA in the fibrotic kidney. Altogether, we show that subcutaneously administered polymeric microspheres present a suitable sustained release drug delivery system for the controlled systemic delivery for proteins such as pPB-HSA., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

Exchange protein activated by cAMP (Epac-1) is an important signaling mechanism for cAMP-mediated effects, yet factors that change Epac-1 levels are unknown. Such factors are relevant because it has been postulated that Epac-1 directly affects fibrogenesis. Prostaglandin E 2 (PGE 2 ) is a well-known cAMP activator, and we therefore studied the effects of this cyclo-oxygenase product on Epac-1 expression and on fibrogenesis within the liver. Liver fibrosis was induced by 8 weeks carbon tetrachloride (CCL 4 ) administration to mice. In the last 2 weeks, mice received vehicle, PGE 2 , the cyclo-oxygenase-2 inhibitor niflumic acid (NFA), or PGE 2 coupled to cell-specific carriers to hepatocytes, Kupffer cells, or hepatic stellate cells (HSC). Results showed antifibrotic effects of PGE 2 and profibrotic effects of NFA in CCL 4 mice. Western blot analysis revealed reduced Epac-1 protein expression in fibrotic livers of mice and humans compared with healthy livers. PGE 2 administration to fibrotic mice completely restored intrahepatic Epac-1 levels and also led to reduced Rho kinase activity, a downstream target of Epac-1. Cell-specific delivery of PGE 2 to either hepatocytes, Kupffer cells, or HSC identified the latter cell as the key player in the observed effects on Epac-1 and Rho kinase. No significant alterations in protein kinase A expressions were found. In primary isolated HSC, PGE 2 elicited Rap1 translocation reflecting Epac-1 activation, and Epac-1 agonists attenuated platelet-derived growth factor-induced proliferation and migration of these cells. These studies demonstrate that PGE 2 enhances Epac-1 activity in HSC, which is associated with significant changes in (myo)fibroblast activities in vitro and in vivo. Therefore, Epac-1 is a potential target for antifibrotic drugs., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

WNT-5A is a secreted growth factor that belongs to the noncanonical members of the Wingless-related MMTV-integration family. Previous studies pointed to a connection between WNT-5A and the fibrogenic factor TGF-β warranting further studies into the functional role of WNT-5A in liver fibrosis. Therefore, we studied WNT-5A expressions in mouse and human fibrotic livers and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines, and extracellular matrix proteins. WNT-5A gene and protein expressions were significantly increased in fibrotic mouse and human livers compared with healthy livers. Regression or therapeutic intervention in mice resulted in decreased hepatic WNT-5A levels paralleled by lower collagen levels. Immunohistochemical analysis showed WNT-5A staining in fibrotic septa colocalizing with desmin staining indicating WNT-5A expression in myofibroblasts. In vitro studies confirmed WNT-5A expression in this cell type and showed that TGF-β significantly enhanced WNT-5A expression in contrast to PDGF-BB and proinflammatory cytokines IL-1β and TNF-α. Additionally, TGF-β induces the expression of the WNT receptors FZD2 and FZD8. After silencing of WNT-5A, reduced levels of collagen type I, vimentin, and fibronectin in TGF-β-stimulated myofibroblasts were measured compared with nonsilencing siRNA-treated controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A in vitro and in vivo. IFNγ-treated fibrotic mice showed significantly less WNT-5A expression compared with untreated fibrotic mice. In conclusion, WNT-5A paralleled collagen I levels in fibrotic mouse and human livers. WNT-5A expression in myofibroblasts is induced by the profibrotic factor TGF-β and plays an important role in TGF-β-induced regulation of fibrotic matrix proteins, whereas its expression can be reversed upon treatment, both in vitro and in vivo. NEW & NOTEWORTHY This study describes the localization and functional role of WNT-5A in human and mouse fibrotic livers. Hepatic WNT-5A expression parallels collagen type I expression. In vivo and in vitro, the myofibroblasts were identified as the key hepatic cells producing WNT-5A. WNT-5A is under control of TGF-β and its activities are primarily profibrotic., (Copyright © 2017 the American Physiological Society.)

Renal fibrosis cannot be adequately treated since anti-fibrotic treatment is lacking. Interferon-γ is a pro-inflammatory cytokine with anti-fibrotic properties. Clinical use of interferon-γ is hampered due to inflammation-mediated systemic side effects. We used an interferon-γ peptidomimetic (mimγ) lacking the extracellular IFNγReceptor recognition domain, and coupled it to the PDGFβR-recognizing peptide BiPPB. Here we tested the efficacy of mimγ-BiPPB (referred to as "Fibroferon") targeted to PDGFβR-overexpressing interstitial myofibroblasts to attenuate renal fibrosis without inducing inflammation-mediated side effects in the mouse unilateral ureter obstruction model.Unilateral ureter obstruction induced renal fibrosis characterized by significantly increased α-SMA, TGFβ1, fibronectin, and collagens I and III protein and/or mRNA expression. Fibroferon treatment significantly reduced expression of these fibrotic markers. Compared to full-length IFNγ, anti-fibrotic effects of Fibroferon were more pronounced. Unilateral ureter obstruction-induced lymphangiogenesis was significantly reduced by Fibroferon but not full-length IFNγ. In contrast to full-length IFNγ, Fibroferon did not induce IFNγ-related side-effects as evidenced by preserved low-level brain MHC II expression (similar to vehicle), lowered plasma triglyceride levels, and improved weight gain after unilateral ureter obstruction.In conclusion, compared to full-length IFNγ, the IFNγ-peptidomimetic Fibroferon targeted to PDGFβR-overexpressing myofibroblasts attenuates renal fibrosis in the absence of IFNγ-mediated adverse effects.

In healthy lungs, many macrophages are characterized by IL-10 production, and few are characterized by expression of IFN regulatory factor 5 (formerly M1) or YM1 and/or CD206 (formerly M2), whereas in asthma, this balance shifts toward few producing IL-10 and many expressing IFN regulatory factor 5 or YM1/CD206. In this study, we tested whether redressing the balance by reinstating IL-10 production could prevent house dust mite-induced allergic lung inflammation. PGE2 was found to be the best inducer of IL-10 in macrophages in vitro. Mice were then sensitized and challenged to house dust mites during a 2 wk protocol while treated with PGE2 in different ways. Lung inflammation was assessed 3 d after the last house dust mite challenge. House dust mite-exposed mice treated with free PGE2 had fewer infiltrating eosinophils in lungs and lower YM1 serum levels than vehicle-treated mice. Macrophage-specific delivery of PGE2 did not affect lung inflammation. Adoptive transfer of PGE2-treated macrophages led to fewer infiltrating eosinophils, macrophages, (activated) CD4(+), and regulatory T lymphocytes in lungs. Our study shows that the redirection of macrophage polarization by using PGE2 inhibits development of allergic lung inflammation. This beneficial effect of macrophage repolarization is a novel avenue to explore for therapeutic purposes., (© Society for Leukocyte Biology.)

Cytokines, growth factors, and other locally produced mediators play key roles in the regulation of disease progression. During liver fibrosis, these mediators orchestrate the balance between pro- and antifibrotic activities as exerted by the hepatic cells. Two important players in this respect are the profibrotic mediator platelet-derived growth factor BB (PDGF-BB) and the antifibrotic cytokine interferon gamma (IFNγ). PDGF-BB, produced by many resident and infiltrating cells, causes extensive proliferation, migration, and contraction of hepatic stellate cells (HSCs) and myofibroblasts. These cells are the extracellular matrix-producing hepatic cells and they highly express the PDGFβ receptor. On the other hand, IFNγ is produced by natural killer cells in fibrotic livers and is endowed with proinflammatory, antiviral, and antifibrotic activities. This cytokine attracted much attention as a possible therapeutic compound in fibrosis. However, clinical trials yielded disappointing results because of low efficacy and adverse effects, most likely related to the dual role of IFNγ in fibrosis. In our studies, we targeted the antifibrotic IFNγ to the liver myofibroblasts. For that, we altered the cell binding properties of IFNγ, by delivery of the IFNγ-nuclear localization sequence to the highly expressed PDGFβ receptor using a PDGFβ receptor recognizing peptide, thereby creating a construct referred to as "Fibroferon" (i.e., fibroblast-targeted interferon γ). In recent years, we demonstrated that HSC-specific delivery of IFNγ increased its antifibrotic potency and improved its general safety profile in vivo, making Fibroferon highly suitable for the treatment of (fibrotic) diseases associated with elevated PDGFβ receptor expression. The present review summarizes the knowledge on these two key mediators, PDGF-BB and IFNγ, and outlines how we used this knowledge to create the cell-specific antifibrotic compound Fibroferon containing parts of both of these mediators.

Renal fibrosis is a serious clinical problem resulting in the greatest need for renal replacement therapy. No adequate preventive or curative therapy is available that could be clinically used to target renal fibrosis specifically. The search for new efficacious treatment strategies is therefore warranted. Although in vitro models using homogeneous cell populations have contributed to the understanding of the pathogenetic mechanisms involved in renal fibrosis, these models poorly mimic the complex in vivo milieu. Therefore, we here evaluated a precision-cut kidney slice (PCKS) model as a new, multicellular ex vivo model to study the development of fibrosis and its prevention using anti-fibrotic compounds. Precision-cut slices (200-300 μm thickness) were prepared from healthy C57BL/6 mouse kidneys using a Krumdieck tissue slicer. To induce changes mimicking the fibrotic process, slices were incubated with TGFβ1 (5 ng/ml) for 48 h in the presence or absence of the anti-fibrotic cytokine IFNγ (1 µg/ml) or an IFNγ conjugate targeted to PDGFRβ (PPB-PEG-IFNγ). Following culture, tissue viability (ATP-content) and expression of α-SMA, fibronectin, collagen I and collagen III were determined using real-time PCR and immunohistochemistry. Slices remained viable up to 72 h of incubation, and no significant effects of TGFβ1 and IFNγ on viability were observed. TGFβ1 markedly increased α-SMA, fibronectin and collagen I mRNA and protein expression levels. IFNγ and PPB-PEG-IFNγ significantly reduced TGFβ1-induced fibronectin, collagen I and collagen III mRNA expression, which was confirmed by immunohistochemistry. The PKCS model is a novel tool to test the pathophysiology of fibrosis and to screen the efficacy of anti-fibrotic drugs ex vivo in a multicellular and pro-fibrotic milieu. A major advantage of the slice model is that it can be used not only for animal but also for (fibrotic) human kidney tissue., (© 2015. Published by The Company of Biologists Ltd.)

Renal fibrosis leads to end-stage renal disease demanding renal replacement therapy because no adequate treatment exists. IFN-γ is an antifibrotic cytokine that may attenuate renal fibrosis. Systemically administered IFN-γ causes side effects that may be prevented by specific drug targeting. Interstitial myofibroblasts are the effector cells in renal fibrogenesis. Here, we tested the hypothesis that cell-specific delivery of IFN-γ to platelet-derived growth factor receptor β (PDGFRβ)-expressing myofibroblasts attenuates fibrosis in an obstructive nephropathy [unilateral ureteral obstruction (UUO)] mouse model. PEGylated IFN-γ conjugated to PDGFRβ-recognizing peptide [(PPB)-polyethylene glycol (PEG)-IFN-γ] was tested in vitro and in vivo for antifibrotic properties and compared with free IFN-γ. PDGFRβ expression was >3-fold increased (P < 0.05) in mouse fibrotic UUO kidneys and colocalized with α-smooth muscle actin-positive (SMA(+)) myofibroblasts. In vitro, PPB-PEG-IFN-γ significantly inhibited col1a1, col1a2, and α-SMA mRNA expression in TGF-β-activated NIH3T3 fibroblasts (P < 0.05). In vivo, PPB-PEG-IFN-γ specifically accumulated in PDGFRβ-positive myofibroblasts. PPB-PEG-IFN-γ treatment significantly reduced renal collagen I, fibronectin, and α-SMA mRNA and protein expression. Compared with vehicle treatment, PPB-PEG-IFN-γ preserved tubular morphology, reduced interstitial T-cell infiltration, and attenuated lymphangiogenesis (all P < 0.05) without affecting peritubular capillary density. PPB-PEG-IFN-γ reduced IFN-γ-related side effects as manifested by reduced major histocompatibility complex class II expression in brain tissue (P < 0.05 vs. free IFN-γ). Our findings demonstrate that specific targeting of IFN-γ to PDGFRβ-expressing myofibroblasts attenuates renal fibrosis and reduces systemic adverse effects., (© FASEB.)

Macrophages have been found to both promote liver fibrosis and contribute to its resolution by acquiring different phenotypes based on signals from the micro-environment. The best-characterized phenotypes in the macrophage spectrum are labeled M1 (classically activated) and M2 (alternatively activated). Until now the in situ localization of these phenotypes in diseased livers is poorly described. In this study, we therefore aimed to localize and quantify M1- and M2-dominant macrophages in diseased mouse and human livers. The scarred collagen-rich areas in cirrhotic human livers and in CCl4-damaged mouse livers contained many macrophages. Though total numbers of macrophages were higher in fibrotic livers, the number of parenchymal CD68-positive macrophages was significantly lower as compared to normal. Scar-associated macrophages were further characterized as either M1-dominant (IRF-5 and interleukin-12) or M2-dominant (CD206, transglutaminase-2, and YM-1) and significantly higher numbers of both of these were detected in diseased livers as compared to healthy human and mouse livers. Interestingly, in mouse, livers undergoing resolution of fibrosis, the total number of CD68(+) macrophages was significantly lower compared to their fibrotic counterparts. M2-dominant (YM-1) macrophages were almost completely gone in livers undergoing resolution, while numbers of M1-dominant (IRF-5) macrophages were almost unchanged and the proteolytic activity (MMP9) increased. In conclusion, this study shows the distribution of macrophage subsets in livers of both human and murine origin. The presence of M1- and M2-dominant macrophages side by side in fibrotic lesions suggests that both are involved in fibrotic responses, while the persistence of M1-dominant macrophages during resolution may indicate their importance in regression of fibrosis. This study emphasizes that immunohistochemical detection of M1/M2-dominant macrophages provides valuable information in addition to widely used flow cytometry and gene analysis.

Background: Progressive liver fibrosis is the result of chronic liver injury and is characterized by the excessive accumulation of extracellular matrix that may result in liver failure. Activated hepatic stellate cells are known to play a central role in this process and their elimination is a crucial step towards the resolution and reversion of liver fibrosis. In the present study, we investigated the potential application of an anti-epidermal growth factor receptor single chain fragment variable antibody-tumor necrosis factor-related apoptosis-inducing ligand (scFv425-sTRAIL) fusion protein in the targeted elimination of activated hepatic stellate cells., Methods: Activated hepatic stellate cells (LX2 cells) were treated by adenovirus-derived scFv425-sTRAIL to evaluate its effect on the viability and extracellular matrix production of this type of cells., Results: In vitro treatment of activated hepatic stellate cells with scFv425-sTRAIL induced a significant reduction in viability (up to 100% reduction) and extracellular matrix production (60% reduction), yet no significant effect was observed on hepatic parenchymal cells. Blockage of the epidermal growth factor receptor (EGFR) by a monoclonal antibody significantly reduced the effectiveness of scFv425-sTRAIL in activated hepatic stellate cells, whereas a reduced effectivity was also observed after inhibition of the caspase pathway., Conclusions: Evidence is presented for the successful application of the scFv425-sTRAIL fusion protein in the targeted elimination of activated hepatic stellate cells via EGFR and simultaneous activation of the caspase pathway. scFv425-sTRAIL may thus represent a new therapeutic compound against liver fibrosis., (Copyright © 2014 John Wiley & Sons, Ltd.)

Hepatic stellate cells play a crucial role in the pathogenesis of hepatic fibrosis. Thus, pharmacological inhibition of pro-fibrotic activities of these cells might lead to an effective therapy for this disease. Among the potent anti-fibrotics, interferon gamma (IFNγ), a proinflammatory cytokine, is highly efficacious but it failed in clinical trials due to the poor efficacy and multiple adverse effects attributed to the ubiquitous IFNγ receptor (IFNγR) expression. To resolve these drawbacks, we chemically synthesized a chimeric molecule containing (a) IFNγ signaling peptide (IFNγ peptidomimetic, mimγ) that retains the agonistic activities of IFNγ but lacks an extracellular receptor recognition sequence for IFNγR; coupled via heterobifunctional PEG linker to (b) bicyclic platelet derived growth factor beta receptor (PDGFβR)-binding peptide (BiPPB) to induce internalization into the stellate cells that express PDGFβR. The synthesized targeted IFNγ peptidomimetic (mimγ-BiPPB) was extensively investigated for its anti-fibrotic and adverse effects in acute and chronic CCl4-induced liver fibrosis models in mice. Treatment with mimγ-BiPPB, after the onset of disease, markedly inhibited both early and established hepatic fibrosis as reflected by a reduced intrahepatic α-SMA, desmin and collagen-I mRNA expression and protein levels. While untargeted mimγ and BiPPB had no effect, and native IFNγ only induced a moderate reduction. Additionally, no off-target effects, e.g. systemic inflammation, were found with mimγ-BiPPB, which were substantially observed in mice treated with native IFNγ. The present study highlights the beneficial effects of a novel BiPPB mediated cell-specific targeting of IFNγ peptidomimetic to the disease-inducing cells and therefore represents a highly potential therapeutic approach to treat fibrotic diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hepatic stellate cells (HSCs), following transdifferentiation to myofibroblasts plays a key role in liver fibrosis. Therefore, attempts to attenuate this myofibroblastic phenotype would be a promising therapeutic approach. Interferon gamma (IFNγ) is a potent anti-fibrotic cytokine, but its pleiotropic receptor expression leading to severe adverse effects has limited its clinical application. Since, activated HSC express high-level of platelet derived growth factor beta receptor (PDGFβR), we investigated the potential of PDGFβR-specific targeting of IFNγ and its signaling peptide that lacks IFNγR binding site (mimetic IFNγ or mimIFNγ) in liver fibrosis. We prepared DNA constructs expressing IFNγ, mimIFNγ or BiPPB (PDGFβR-specific bicyclic peptide)-IFNγ, BiPPB-mimIFNγ fusion proteins. Both chimeric proteins alongwith IFNγ and mimIFNγ were produced in E.coli. The expressed proteins were purified and analyzed for PDGFβR-specific binding and in vitro effects. Subsequently, these recombinant proteins were investigated for the liver uptake (pSTAT1α signaling pathway), for anti-fibrotic effects and adverse effects (platelet counts) in CCl4-induced liver fibrogenesis in mice. The purified HSC-targeted IFNγ and mimIFNγ fusion proteins showed PDGFβR-specific binding and significantly reduced TGFβ-induced collagen-I expression in human HSC (LX2 cells), while mouse IFNγ and mimIFNγ did not show any effect. Conversely, mouse IFNγ and BiPPB-IFNγ induced activation and dose-dependent nitric oxide release in mouse macrophages (express IFNγR while lack PDGFβR), which was not observed with mimIFNγ and BiPPB-mimIFNγ, due to the lack of IFNγR binding sites. In vivo, targeted BiPPB-IFNγ and BiPPB-mimIFNγ significantly activated intrahepatic IFNγ-signaling pathway compared to IFNγ and mimIFNγ suggesting increased liver accumulation. Furthermore, the targeted fusion proteins ameliorated liver fibrogenesis in mice by significantly reducing collagen and α-SMA expression and potentiating collagen degradation. IFNγ also induced reduction in fibrogenesis but showed significant decrease in platelet counts, which was restored with targeted proteins. These results suggest that these rationally designed proteins can be further developed as novel anti-fibrotic therapeutics.

Liver fibrosis is a complex disease affecting millions of people world-wide. It involves the activation of several cell types whose activities are tightly controlled by endogenous mediators. No pharmacotherapy is available for this disease, despite the fact that many experimental drugs are very effective in vitro and the liver is easily accessible for most drugs. Our review provides arguments showing that cell-selectivity is essential for most antifibrotics. Several cell-specific drug carriers targeting the key pathogenic liver cells are discussed with special focus on hepatic stellate cells and fibroblast-like cells. Since endogenous mediators represent a powerful set of tools to modify the pathogenic process, this review focuses on these mediators as therapeutics and the problems and pitfalls associated with the use of such biologicals., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Transforming growth factor-β (TGF-β) is a major pro-fibrotic cytokine, causing the overproduction of extracellular matrix molecules in many fibrotic diseases. Inhibition of its type-I receptor (ALK5) has been shown to effectively inhibit fibrosis in animal models. However, apart from its pro-fibrotic effects, TGF-β also has a regulatory role in the immune system and influences tumorigenesis, which limits the use of inhibitors. We therefore explored the cell-specific delivery of an ALK5-inhibitor to hepatic stellate cells, a key cell in the development of liver fibrosis. We synthesized a conjugate of the ALK5-inhibitor LY-364947 coupled to mannose-6-phosphate human serum albumin (M6PHSA), which binds to the insulin-like growth factor II receptor on activated HSC. The effectivity of the conjugate was evaluated in primary HSC and in an acute liver injury model in mice. In vitro, the free drug and the conjugate significantly inhibited fibrotic markers in HSC. In hepatocytes, TGF-β-dependent signaling was inhibited by free drug, but not by the conjugate, thus showing its cell-specificity. In vivo, the conjugate localized in desmin-positive cells in the liver and not in hepatocytes or immune cells. In the acute liver injury model in mice, the conjugate reduced fibrogenic markers and collagen deposition more effectively than free drug. We conclude that we can specifically deliver an ALK5-inhibitor to HSC using the M6PHSA carrier and that this targeted drug reduces fibrogenic parameters in vivo, without affecting other cell-types.

Background & Aims: Rho-kinase activation mediates cell contraction and increases intrahepatic resistance and consequently portal pressure in liver cirrhosis. Systemic Rho-kinase inhibition decreases portal pressure in cirrhosis, but also arterial pressure. Thus, liver-specific Rho-kinase inhibition is needed. The delivery of Rho-kinase inhibitor to activated hepatic stellate cells reduces fibrosis. It might also relax these contractile cells and therewith decrease intrahepatic resistance. We tested this hypothesis by performing acute experiments in cirrhotic rats., Methods: Cirrhosis models were CCl(4)-intoxication and bile duct ligation. Three hours after injection of the Rho-kinase inhibitor (Y26732) coupled with a carrier (mannose-6-phosphate modified human serum albumin), which targets activated hepatic stellate cells, hemodynamics were analyzed by the colored microsphere technique and direct pressure measurements. The delivery site and effect of Rho-kinase inhibitor were investigated by immunohistochemical stainings, as well as Western blot. Experiments with Rho-kinase inhibitor coupled with unmodified human serum albumin served as untargeted control., Results: In both models of cirrhosis, the carrier coupled Rho-kinase inhibitor lowered the portal pressure and decreased the hepatic-portal resistance. Immunohistochemical desmin-staining showed the carrier in hepatic stellate cells. The targeted therapy decreased the expression of the phosphorylated substrate of Rho-kinase (moesin) and abolished myosin light chains phosphorylation in fibrotic septae (collagen-staining). The targeted Rho-kinase inhibitor showed no major extrahepatic effects. By contrast, the untargeted Rho-kinase inhibitor elicited severe systemic hypotension., Conclusions: Activated hepatic stellate cells are crucially involved in portal hypertension in cirrhosis. Targeting of Rho-kinase in hepatic stellate cells not only decreased fibrosis, as previously shown, but also lowers portal pressure acutely without major systemic effects as demonstrated in this study., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

New approaches to block the function of tumor stromal cells such as cancer-associated fibroblasts and pericytes is an emerging field in cancer therapeutics as these cells play a crucial role in promoting angiogenesis and tumor growth via paracrine signals. Because of immunomodulatory and other antitumor activities, IFNγ, a pleiotropic cytokine, has been used as an anticancer agent in clinical trials. Unfortunately only modest beneficial effects, but severe side effects, were seen. In this study, we delivered IFNγ to stromal fibroblasts and pericytes, considering its direct antifibrotic activity, using our platelet-derived growth factor-beta receptor (PDGFβR)-binding carrier (pPB-HSA), as these cells abundantly express PDGFβR. We chemically conjugated IFNγ to pPB-HSA using a heterobifunctional PEG linker. In vitro in NIH3T3 fibroblasts, pPB-HSA-IFNγ conjugate activated IFNγ-signaling (pSTAT1α) and inhibited their activation and migration. Furthermore, pPB-HSA-IFNγ inhibited fibroblasts-induced tube formation of H5V endothelial cells. In vivo in B16 tumor-bearing mice, pPB-HSA-IFNγ rapidly accumulated in tumor stroma and pericytes and significantly inhibited the tumor growth while untargeted IFNγ and pPB-HSA carrier were ineffective. These antitumor effects of pPB-HSA-IFNγ were attributed to the inhibition of tumor vascularization, as shown with α-SMA and CD-31 staining. Moreover, pPB-HSA-IFNγ induced MHC-II expression specifically in tumors compared with untargeted IFNγ, indicating the specificity of this approach. This study thus shows the impact of drug targeting to tumor stromal cells in cancer therapy as well as provides new opportunities to use cytokines for therapeutic application., (©2012 AACR.)

Liver fibrosis represents a scar formation process as a response to chronic injury and a major cause of death worldwide. To date, no drug is available for this condition. Interleukin-10 (IL-10) has potent anti-inflammatory and antifibrotic properties but its short half-life in the circulation hampers its clinical use. Our aim was therefore to modify IL-10 with polyethylene glycol (PEG) to prolong its circulation time and enhance its effectivity. IL-10 was modified with 5 or 20 kDa PEG. The biological activity was preserved after PEGylation as assessed by inhibition of TNF-α production by macrophages. In vivo, during CCl(4)-induced fibrogenesis in mice, both 5PEG-IL-10 and 20PEG-IL-10 showed a longer circulation time compared to IL-10, which was associated with a significant increased liver accumulation. Immunohistochemical analysis of fibrotic livers of mice receiving treatment with IL-10 or its PEGylated forms, revealed a decrease in markers reflecting HSC and KC activation induced by 5PEG-IL10. Transcription levels of IL-6 were decreased upon treatment with IL-10 and both PEGylated forms, whereas IL-1β levels were only down-regulated by 5PEGIL-10 and 20PEGIL-10. We conclude that PEGylation of IL-10 is a good strategy to attenuate liver fibrosis and that 5PEGIL-10 is the most effective conjugate., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Many serious liver diseases affecting millions of people world-wide cannot be treated despite many efforts which warrants a search for new therapeutic strategies. Potent drugs may not be effective enough in vivo or exhibit adverse effects and enhanced delivery into the target cells may improve this significantly. We aim to summarize the available options for drug delivery to the different intrahepatic cell-types. The most relevant target cells are identified for each liver disease and the strategies for drug delivery to these cells are subsequently reviewed. The review describes the use of proteins, viruses, polymers and liposomes for therapeutic purposes in various liver diseases. It is shown that to date, all resident intrahepatic cells can be reached with several different drug carriers. Much progress has been made in recent years to deliver small drug molecules, proteins and nucleic acids specifically to the key pathogenic cells in vivo. The knowledge of drug targeting gained in the past decades, combined with a proper preclinical evaluation, may bring new therapeutics to the clinic in the near future., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Introduction: Kinase inhibitors have been hailed as a breakthrough in the treatment of cancer. Extensive research is now being devoted to the development of kinase inhibitors as a treatment for many nonmalignant diseases. However, the use of kinase inhibitors in both malignant and nonmalignant diseases is also associated with side effects and the development of resistance. It may be worthwhile to explore whether cell-specific delivery of kinase inhibitors improves therapeutic efficacy and reduces side effects., Areas Covered: This review aims to provide an overview of the preclinical studies performed to examine the specific targeting of kinase inhibitors in vitro and in vivo. It gives an introduction to kinase signaling pathways induced during disease, along with the possible problems associated with their inhibition. It also discusses the studies on specific delivery and shows that altering the specificity of kinase inhibitors by targeting methods improves their effectivity and safety., Expert Opinion: Compared with the delivery of cytotoxic compounds, the specific delivery of kinase inhibitors has not yet been studied extensively. The studies discussed in this review provide an insight into methods used to target kinase inhibitors to different organs. The targeting of different kinase inhibitors has improved their therapeutic possibilities, but many questions still remain to be studied.

Excessive accumulation of the extracellular matrix proteins primarily produced by activated hepatic stellate cells (HSC) leads to liver fibrosis. To date, no successful therapeutic intervention is available for the treatment of this disease. Platelet derived growth factor beta receptor (PDGFβR) is highly upregulated on disease-inducing activated HSC and thus can be used for delivery of antifibrotic drugs to increase therapeutic efficacy with reduced adverse effects. Interferon gamma (IFNγ) has been recognized as a potent antifibrotic cytokine; however, poor pharmacokinetics and side effects due to frequent administration have limited its clinical use. For HSC-specific delivery, a PDGFβR-specific drug delivery carrier (PPB-HSA) was developed by modifying albumin with PDGFβR-recognizing cyclic peptides. Subsequently, IFNγ was conjugated to PPB-HSA via bifunctional PEG linkers to synthesize PPB-HSA-PEG-IFNγ. In vitro, PPB-HSA-PEG-IFNγ retained complete biological activity similar to unmodified IFNγ and showed PDGFβR-specific binding to human HSC and primary culture-activated rat HSC. In TGFβ-stimulated mouse fibroblasts and human HSC, PPB-HSA-PEG-IFNγ induced significant reduction in crucial fibrotic parameters. In vivo, the conjugate rapidly accumulated into PDGFβR-expressing HSC in fibrotic livers and activated IFNγ-mediated pstat1α signaling pathway. Furthermore, in a CCl(4)-induced acute liver injury model in mice, treatment with HSC-targeted IFNγ strongly ameliorated hepatic fibrogenesis by inducing significant reduction (about 60%; p < 0.01) in collagen I and α-SMA expression as well as enhanced fibrolysis (increased MMP/TIMP ratio; p < 0.05) while free unmodified IFNγ was ineffective. Furthermore, in contrast to free native IFNγ, the conjugate did not induce macrophage infiltration and IL-1β expression in the liver. In conclusion, these data demonstrate the enhanced antifibrotic efficacy and reduced off-target effects of PPB-HSA-PEG-IFNγ conjugate showing the potential of cell-specific targeting of IFNγ for the treatment of liver fibrosis.

Interferon gamma (IFNγ) is a potent cytokine that displays a variety of anti-viral, anti-proliferative, immunomodulatory, apoptotic and anti-fibrotic functions. However, its clinical use is limited to the treatment of few diseases due to the rapid clearance from the body. PEGylated IFN-alpha formulations are shown to be beneficial in viral hepatitis, but PEGylation of IFNγ to enhance its therapeutic effects in liver fibrosis is not yet explored. Liver fibrosis is characterized by the extensive accumulation of an abnormal extracellular matrix and is the major cause of liver-related morbidity and mortality worldwide. To date, there is no pharmacotherapy available for this disease. We modified IFNγ with different-sized linear PEG molecules (5, 10 and 20kDa) and assessed the biological activity in vitro and in vivo. All PEGylated IFNγ constructs were biologically active and activated IFNγ signaling in vitro as determined with a nitric oxide release assay and a pGAS-Luc reporter plasmid assay, respectively. Similar to IFNγ, all PEGylated IFNγ induced a significant reduction of fibrotic parameters in mouse NIH3T3 fibroblasts as shown with immunohistochemical staining and quantitative PCR analyses. In vivo, the pharmacokinetic profile of radiolabeled (125)I-IFNγ-PEG conjugates revealed a decreased renal clearance and an increased plasma half-life with an increase of PEG size. Moreover, the liver accumulation of PEGylated IFNγ constructs was significantly higher than the unmodified IFNγ, which was also confirmed by increased MHC-II expression in the livers. Furthermore, in a CCl(4)-induced acute liver injury model in mice, PEGylated constructs reduced the early fibrotic parameters more drastically than unmodified IFNγ. Of note, these effects were stronger with higher PEG-sized IFNγ constructs. These data nicely correlated with the pharmacokinetic data. In conclusion, PEGylation significantly improved the pharmacokinetics, liver uptake and anti-fibrotic effects of IFNγ. This study opens new opportunities to exploit the therapeutic applications of PEGylated IFNγ for the treatment of liver fibrosis and other diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Unlabelled: Liver fibrogenesis is a process tightly controlled by endogenous anti- and pro-fibrogenic factors. Interferon gamma (IFNγ) is a potent antifibrogenic cytokine in vitro and might therefore represent a powerful therapeutic entity. However, its poor pharmacokinetics and adverse effects, due to the presence of IFNγ receptors on nearly all cells, prevented its clinical application so far. We hypothesized that delivery of IFNγ specifically to the disease-inducing cells and concurrently avoiding its binding to nontarget cells might increase therapeutic efficacy and avoid side effects. We conjugated IFNγ to a cyclic peptide recognizing the platelet-derived growth factor beta receptor (PDGFβR) which is strongly up-regulated on activated hepatic stellate cells (HSC), the key effector cells responsible for hepatic fibrogenesis. The IFNγ conjugates were analyzed in vitro for PDGFβR-specific binding and biological effects and in vivo in acute (early) and chronic (progressive and established) carbon-tetrachloride-induced liver fibrosis in mice. The targeted-IFNγ construct showed PDGFβR-specific binding to fibroblasts and HSC and inhibited their activation in vitro. In vivo, the targeted-IFNγ construct attenuated local HSC activation in an acute liver injury model. In the established liver fibrosis model, it not only strongly inhibited fibrogenesis but also induced fibrolysis. In contrast, nontargeted IFNγ was ineffective in both models. Moreover, in contrast to unmodified IFNγ, our engineered targeted-IFNγ did not induce IFNγ-related side effects such as systemic inflammation, hyperthermia, elevated plasma triglyceride levels, and neurotropic effects., Conclusion: This study presents a novel HSC-targeted engineered-IFNγ, which in contrast to systemic IFNγ, blocked liver fibrogenesis and is devoid of side effects, by specifically acting on the key pathogenic cells within the liver., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Purpose: Rho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, but the ubiquitous presence of this kinase may hinder examination of its exact role and the therapeutic use of inhibitors. We therefore coupled the Rho-kinase inhibitor Y27632 to a drug carrier that binds the mannose-6-phosphate insulin-like growth factor II (M6P/IGFII)-receptor which is upregulated on activated HSC., Methods: Y27632 was coupled to mannose-6-phosphate human serum albumin (M6PHSA), and in vitro experiments were performed on primary rat HSC. Biodistribution and effect studies were performed in an acute CCl(4) model in mice., Results: Y27-conjugate remained stable in serum, while drug was efficiently released in liver homogenates. Receptor-blocking studies revealed that it was specifically taken up through the M6P/IGFII-receptor on fibroblasts, and it inhibited expression of fibrotic markers in activated HSC. In vivo, liver drug levels were significantly higher after injection of Y27-conjugate as compared to Y27632, and the conjugate accumulated specifically in HSC. After acute CCl(4)-induced liver injury, Y27-conjugate reduced the local activation of HSC, whereas an equimolar dose of free drug did not., Conclusions: We conclude that specific targeting of a Rho-kinase inhibitor to HSC leads to enhanced accumulation of the drug in HSC, reducing early fibrogenesis in the liver.

One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] has been shown to reduce fibrosis in animal models. However, kinase expression is ubiquitous, so any inhibitor may affect many cell types. We hypothesize that cell-specific delivery of a kinase inhibitor will be beneficial. Therefore, we conjugated Y27632 to the carrier mannose-6-phosphate (M6P) human serum albumin (HSA), which is taken up specifically in activated HSC through the M6P/insulin-like growth factor II receptor. This conjugate decreased protein expression of phosphorylated myosin light chain 2 (pMLC2) and vinculin, downstream of Rho kinase, in activated primary HSC and decreased the migration and contraction of HSC. In an ex vivo model, free Y27632 decreased contractility of rat aortas, whereas the Y27-conjugate did not, showing that the Y27-conjugate does not affect nontarget tissue. In chronic CCl(4)-induced liver fibrosis, both free drug and conjugate reduced HSC activation; however, only the Y27-conjugate significantly reduced collagen deposition. Treatment with the Y27-conjugate, but not with free drug, reduced pMLC2 expression in livers 24 h after injection, demonstrating prolonged inhibition of the Rho kinase pathway. The Rho kinase inhibitor Y27632 can be specifically targeted to HSC using M6PHSA, decreasing its effects in nontarget tissues. The targeted drug effectively reduced fibrotic parameters in vivo via the inhibition of the Rho kinase pathway.

Study Design: Cadaveric study., Objective: Identifying anatomic structures at risk for injury during direct lateral transpsoas approach to the spine., Summary of Background Data: Direct lateral transpsoas approach is a novel technique that has been described for anterior lumbar interbody fusion. Potential risks include damage to genitofemoral nerve and lumbar plexus, which are not well visualized during small retroperitoneal exposure. Previous cadaveric studies did not evaluate the direct lateral transpsoas approach, and considering the approach being used in clinical practice, the current study was undertaken in an effort to identify the structures at risk during direct lateral transpsoas approach., Methods: Sixteen dissections were performed on eight fresh-frozen male cadavers. Eight were to localize proximal lumbar nerve roots, ilioinguinal nerves, and genitofemoral nerves and establish their relationship to psoas muscle and disc space. Four simulated direct lateral transpsoas approaches, with K-wire placed into mid-disc space under fluoroscopic guidance, were made. In four dissections, sequential dilators were inserted, disc space was evacuated, and interbody devices were placed. The study was conducted in three phases to identify, in a stepwise progression, which portion of the procedure placed the nerve at greatest risk. RESULTS.: With initial dissections, perforating branches of lumbar nerve roots were identified in anterior, middle, and posterior third of psoas muscle. Sympathetic chain was identified in anterior third of psoas over L1-L4. Distance from the middle of the anterior longitudinal ligament at the level of the disc to the sympathetic chain averaged 9.25 mm. The nerve roots and genitofemoral nerve were placed at risk in all dissections in which the approach was recreated. Damage secondary to K-wire placement occurred in 25% of cases at L3-L4 and L4-L5; in one case, L4 nerve root was pierced, and in another, genitofemoral nerve was pierced. K-wire was posterior to the nerve roots in 25% of cases at L3-L4 and in 50% of cases at L4-L5. The lumbar plexus was placed under tension because of sequential dilator placement., Conclusion: On the basis of our results, there is no zone of absolute safety when using the direct lateral transpsoas approach. The potential for nerve injury exists when using this approach, and consequently, we recommend either direct visualization of the nerve roots intraoperatively and/or the use of neuromonitoring. In our opinion, a higher quality, larger clinical study that examines the outcomes and surgical complications of the direct lateral transpsoas approach is warranted.