Novel insights into the multifaceted and tissue-specific roles of the endocytic receptor LRP1.

Receptor-mediated endocytosis provides a mechanism for the selective uptake of specific molecules thereby controlling the composition of the extracellular environment and biological processes. The low-density lipoprotein receptor-related protein 1 (LRP1) is a widely expressed endocytic receptor that regulates cellular events by modulating the levels of numerous extracellular molecules via rapid endocytic removal. LRP1 also participates in signalling pathways through this modulation as well as in the interaction with membrane receptors and cytoplasmic adaptor proteins. LRP1 SNPs are associated with several diseases and conditions such as migraines, aortic aneurysms, cardiopulmonary dysfunction, corneal clouding, and bone dysmorphology and mineral density. Studies using Lrp1 KO mice revealed a critical, nonredundant and tissue-specific role of LRP1 in regulating various physiological events. However, exactly how LRP1 functions to regulate so many distinct and specific processes is still not fully clear. Our recent proteomics studies have identified more than 300 secreted proteins that either directly interact with LRP1 or are modulated by LRP1 in various tissues. This review will highlight the remarkable ability of this receptor to regulate secreted molecules in a tissue-specific manner and discuss potential mechanisms underpinning such specificity. Uncovering the depth of these "hidden" specific interactions modulated by LRP1 will provide novel insights into a dynamic and complex extracellular environment that is involved in diverse biological and pathological processes.

iRhom2 regulates ectodomain shedding and surface expression of the major histocompatibility complex (MHC) class I.

Proteolytic release of transmembrane proteins from the cell surface, the so called ectodomain shedding, is a key process in inflammation. Inactive rhomboid 2 (iRhom2) plays a crucial role in this context, in that it guides maturation and function of the sheddase ADAM17 (a disintegrin and metalloproteinase 17) in immune cells, and, ultimately, its ability to release inflammatory mediators such as tumor necrosis factor α (TNFα). Yet, the macrophage sheddome of iRhom2/ADAM17, which is the collection of substrates that are released by the proteolytic complex, is only partly known. In this study, we applied high-resolution proteomics to murine and human iRhom2-deficient macrophages for a systematic identification of substrates, and therefore functions, of the iRhom2/ADAM17 proteolytic complex. We found that iRhom2 loss suppressed the release of a group of transmembrane proteins, including known (e.g. CSF1R) and putative novel ADAM17 substrates. In the latter group, shedding of major histocompatibility complex class I molecules (MHC-I) was consistently reduced in both murine and human macrophages when iRhom2 was ablated. Intriguingly, it emerged that in addition to its shedding, iRhom2 could also control surface expression of MHC-I by an undefined mechanism. We have demonstrated the biological significance of this process by using an in vitro model of CD8+ T-cell (CTL) activation. In this model, iRhom2 loss and consequent reduction of MHC-I expression on the cell surface of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line dampened activation of autologous CTLs and their cell-mediated cytotoxicity. Taken together, this study uncovers a new role for iRhom2 in controlling cell surface levels of MHC-I by a dual mechanism that involves regulation of their surface expression and ectodomain shedding.

Development of a Proteomic Workflow for the Identification of Heparan Sulphate Proteoglycan-Binding Substrates of ADAM17.

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g., HB-EGF) are known to bind to heparan sulphate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-treated secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e., HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting.

Staged vs concurrent hardware removal in total ankle arthroplasty.

INTRODUCTION: Ankle osteoarthritis is more commonly posttraumatic. Consequently, dealing with hardware removal is quite frequent when performing a total ankle arthroplasty (TAA). The purpose of this study is to compare outcomes regarding either a staged or concurrent hardware removal when performing TAA. MATERIALS AND METHODS: 275 consecutive patients with TAA previously treated with internal fixation were retrospectively reviewed. Finally, 57 patients were enrolled based on exclusion criteria, and were differentiated into two groups considering the timing of hardware removal (staged-group A vs concurrent-group B) to compare: neurovascular and wound complications, time to recover full weight bearing, scar-tissue esthetic, and surgical time. Moreover, a subgroup comparison considering the surgical approach (single approach, minor additional approach, major additional approach) was performed between the group A and group B. RESULTS: No statistically significant difference other that longer surgical time (p < 0.05) was observed between group A and group B. When considering surgical approach subgroups, statistically significant higher surgical wound complications and revision rate were reported in group B (concurrent) major additional approach subgroup, and a statistically significant shorter time to full weight bearing was reported in group A (staged) major additional approach subgroup. CONCLUSIONS: When performing TAA requiring hardware removal, no clear superiority of staged over concurrent hardware removal was observed. However, when considering a subgroup of patients requiring a separate major incision, a staged approach has shown reduced surgical time, less risk of wound complications, and shorter recovery to full weight bearing. LEVEL OF EVIDENCE: III.

Custom-made total ankle arthroplasty with patient-specific instrumentation for severe bone loss conditions: a case series.

PURPOSE: Management of bone loss around the ankle is a challenging condition. This retrospective study describes the design process, the surgical technique, and the preliminary results of custom-made total ankle arthroplasties (TAA) with patient-specific instrumentation (PSI) for different severe bone loss conditions. METHODS: Consecutive patients that underwent custom-made TAA for severe bone loss conditions were included. The primary outcome was to describe the implant design in relation to the bone defect. Moreover, pre-operative and final follow-up clinical scores were compared. RESULTS: Seven patients were included. Post-operative radiographs showed good correspondence between the pre-operative planning and the prosthesis alignment in all patients. Improvement in clinical scores was observed in all patients at the final follow-up. One patient developed a deep infection. CONCLUSION: Short-term results reported herein are encouraging suggesting that custom-made TAA implants with PSI may represent an effective solution for ankle bone loss conditions.

First Metatarsal Hemiepiphysiodesis for the Treatment of Juvenile Hallux Valgus: A Systematic Review.

INTRODUCTION: Juvenile hallux valgus (JHV) is a pediatric deformity characterized by the varus deviation of the first metatarsal and valgus deviation of the proximal phalanx. Among the several surgical techniques available, hemiepiphysiodesis consists of the unilateral growth arrest of the first metatarsal physis. Despite this technique has been proposed over 70 years ago, only a few studies including clinical and radiological outcomes have been published, making the procedure unclear in terms of results. This systematic review aimed to evaluate the outcomes of hemiepiphysiodesis of the first metatarsal in the treatment of JHV. METHODS: Google Scholar, Embase, PubMed, and Cochrane databases were searched for all the articles reporting on clinical and radiological results of hemiepiphysiodesis of the first metatarsal in the treatment of JHV. The selected articles were reviewed to extract demographic data, surgical techniques, complications, clinical outcomes, and radiological parameters. RESULTS: Six articles were included in the qualitative analysis. A total of 85 patients with 144 halluces valgus were treated through hemiepiphysiodesis of the first metatarsal. The mean age at surgery was 10.7 years (range 5 to 15). The mean follow-up was 2.7 years (range 1 to 7.5). Hemiepiphysiodesis was performed through 2 different techniques. Eighteen (12.5%) complications occurred. The mean American Orthopaedic Foot and Ankle Society (AOFAS) score increased from 70.6 (range 49 to 93) preoperatively to 89.4 (range 72 to 100) postoperatively. The mean HVA improved from 28.3 (range 14 to 46) to 24.03 degrees (range 0 to 54), and the mean IMA improved from 13 (range 8 to 33) to 10.9 degrees (range 8 to 33). CONCLUSIONS: This review showed that hemiepiphysiodesis of the first metatarsal is a safe treatment for JHV. Improvement in both clinical and radiological results has been observed in all the studies, sometimes being statistically significant. Despite the improvement, mean postoperative radiological angles remained altered and consistent with mild-moderate hallux valgus. This suggests that hemiepiphysiodesis plays a bigger role in preventing the worsening of the deformity rather than correcting it. Randomized controlled trials with longer follow-up and a larger number of patients are needed to further investigate the efficacy and safety of this treatment.

Surgical management of clubfoot overcorrection: a case series.

INTRODUCTION: Overcorrection is a possible complication of clubfoot treatment, whose prevalence varies from 5 to 67%. Overcorrected clubfoot usually presented as a complex flatfoot with different degrees of hindfoot valgus, flat top talus, dorsal bunion, and dorsal navicular subluxation. The management of clubfoot overcorrection is challenging, and both conservative and surgical treatments are available. This study aims to present our experience in the surgical management of overcorrected clubfoot and to provide an overview of actual treatment options for each specific sub-deformity. MATERIALS AND METHODS: A retrospective cohort study of patients surgically treated for an overcorrected clubfoot from 2000 to 2015 at our Institution was conducted. Surgical procedures were tailored to the type and symptomatology of the deformity. A medializing calcaneal osteotomy or subtalar arthrodesis was performed for hindfoot valgus. Subtalar and/or midtarsal arthrodesis were considered in cases of dorsal navicular subluxation. The first metatarsus elevatus was addressed through a proximal plantarflexing osteotomy, sometimes associated with a tibialis anterior tendon transfer. Clinical scores and radiographic parameters were obtained pre-operatively and at the last follow-up. RESULTS: Fifteen consecutive patients were enrolled. The series included 4 females and 11 males, with a mean age at surgery of 33,1 (18-56) years, and a mean follow-up of 4,46 (2-10) years. Seven medializing calcaneal osteotomies, 5 subtalar arthrodesis, 11 first metatarsal plantarflexing osteotomies, and 7 anterior tibialis tendon transfers were performed. A statistically significant improvement in both clinical and radiographic scores was observed. CONCLUSIONS: Management of overcorrected clubfoot involves many surgical techniques because of the high interpersonal variability of the deformities. The surgical approach showed positive results, as long as the indication is based on clinical symptoms and functional impairment rather than morphological alterations and radiographic findings.

Retrograde intramedullary nail entry point for tibio-talo-calcaneal arthrodesis: a review of anatomical studies.

PURPOSE: Tibio-talo-calcaneal arthrodesis (TTCA) is considered a safe and valuable option for end-stage tibiotalar and subtalar arthritis, and usually is performed with a retrograde intramedullary nail. Although the good results reported, potential complications may be related to retrograde nail entry point. Aim of this systematic review is to analyze in cadaveric studies the risk of iatrogenic injuries related to different entry points and different retrograde intramedullary nail design when performing TTCA. METHODS: According to PRISMA, a systematic review of the literature was performed on PubMed, EMBASE and SCOPUS databases. A subgroup analysis comparing different entry point location (anatomical or fluoroscopic guided) and different nail design (straight vs. valgus curved nails) was performed. RESULTS: Five studies were included, for a total of 40 specimens. Superiority of anatomical landmark-guided entry points was observed. Different nail designs did not seem to influence nor iatrogenic injuries neither hindfoot alignment. CONCLUSION: Retrograde intramedullary nail entry point should be placed in the lateral half of the hindfoot in order to minimize the risk of iatrogenic injuries.

Quantitative Proteomics Reveals Changes Induced by TIMP-3 on Cell Membrane Composition and Novel Metalloprotease Substrates.

Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies.

Strategies to Target ADAM17 in Disease: From its Discovery to the iRhom Revolution.

For decades, disintegrin and metalloproteinase 17 (ADAM17) has been the object of deep investigation. Since its discovery as the tumor necrosis factor convertase, it has been considered a major drug target, especially in the context of inflammatory diseases and cancer. Nevertheless, the development of drugs targeting ADAM17 has been harder than expected. This has generally been due to its multifunctionality, with over 80 different transmembrane proteins other than tumor necrosis factor α (TNF) being released by ADAM17, and its structural similarity to other metalloproteinases. This review provides an overview of the different roles of ADAM17 in disease and the effects of its ablation in a number of in vivo models of pathological conditions. Furthermore, here, we comprehensively encompass the approaches that have been developed to accomplish ADAM17 selective inhibition, from the newest non-zinc-binding ADAM17 synthetic inhibitors to the exploitation of iRhom2 to specifically target ADAM17 in immune cells.