The lysosomal trafficking regulator "LYST": an 80-year traffic jam.

Lysosomes and lysosome related organelles (LROs) are dynamic organelles at the intersection of various pathways involved in maintaining cellular hemostasis and regulating cellular functions. Vesicle trafficking of lysosomes and LROs are critical to maintain their functions. The lysosomal trafficking regulator (LYST) is an elusive protein important for the regulation of membrane dynamics and intracellular trafficking of lysosomes and LROs. Mutations to the LYST gene result in Chédiak-Higashi syndrome, an autosomal recessive immunodeficiency characterized by defective granule exocytosis, cytotoxicity, etc. Despite eight decades passing since its initial discovery, a comprehensive understanding of LYST's function in cellular biology remains unresolved. Accumulating evidence suggests that dysregulation of LYST function also manifests in other disease states. Here, we review the available literature to consolidate available scientific endeavors in relation to LYST and discuss its relevance for immunomodulatory therapies, regenerative medicine and cancer applications.

Implanted Tissue-Engineered Vascular Graft Cell Isolation with Single-Cell RNA Sequencing Analysis.

The advent of single-cell RNA sequencing (scRNA-Seq) has brought with it the ability to gain greater insights into the cellular composition of tissues and heterogeneity in gene expression within specific cell types. For tissue-engineered blood vessels, this is particularly impactful to better understand how neotissue forms and remodels into tissue resembling a native vessel. A notable challenge, however, is the ability to separate cells from synthetic biomaterials to generate high-quality single-cell suspensions to interrogate the cellular composition of our tissue-engineered vascular grafts (TEVGs) during active remodeling in situ. We present here a simple, commercially available approach to separate cells within our TEVG from the residual scaffold for downstream use in a scRNA-Seq workflow. Utilizing this method, we identified the cell populations comprising explanted TEVGs and compared these with results from immunohistochemical analysis. The process began with explanted TEVGs undergoing traditional mechanical and enzymatic dissociation to separate cells from scaffold and extracellular matrix proteins. Magnetically labeled antibodies targeting murine origin cells were incubated with enzymatic digests of TEVGs containing cells and scaffold debris in suspension allowing for separation by utilizing a magnetic separator column. Single-cell suspensions were processed through 10 × Genomics and data were analyzed utilizing R to generate cell clusters. Expression data provided new insights into a diverse composition of phenotypically unique subclusters within the fibroblast, macrophage, smooth muscle cell, and endothelial cell populations contributing to the early neotissue remodeling stages of TEVGs. These populations were correlated qualitatively and quantitatively with immunohistochemistry highlighting for the first time the potential of scRNA-Seq to provide exquisite detail into the host cellular response to an implanted TEVG. These results additionally demonstrate magnetic cell isolation is an effective method for generating high-quality cell suspensions for scRNA-Seq. While this method was utilized for our group's TEVGs, it has broader applications to other implantable materials that use biodegradable synthetic materials as part of scaffold composition. Impact statement Single-cell RNA sequencing is an evolving technology with the ability to provide detailed information on the cellular composition of remodeling biomaterials in vivo. This present work details an effective approach for separating nondegraded biomaterials from cells for downstream RNA-sequencing analysis. We applied this method to implanted tissue-engineered vascular grafts and for the first time describe the cellular composition of the remodeling graft at a single-cell gene expression level. While this method was effective in our scaffold, it has broad applicability to other implanted biomaterials that necessitate separation of cell from residual scaffold materials for single-cell RNA sequencing.

Tamoxifen reduces silicone implant capsule formation in a mouse model.

Capsular contracture as a result of the foreign body response (FBR) is a common issue after implant-based breast reconstruction, affecting up to 20% of patients. New evidence suggests that tamoxifen may mitigate the FBR. C57BL/6 female mice were treated with daily tamoxifen or control injections and implanted with bilateral silicone implants in the submammary glandular plane. Implants were removed en bloc after 2 weeks and the implant capsules were evaluated histologically. Tamoxifen treatment decreased capsule thickness, decreased the number of αSMA+ cells (477 ± 156 cells/mm control vs 295 ± 121 cells/mm tamoxifen, p = 0.005 unpaired t test), and decreased CD31+ cells (173.9 ± 96.1 cells/mm2 control vs 106.3 ± 51.8 cells/mm2 tamoxifen, p = 0.043 unpaired t test). There were similar amounts of pro- and anti-inflammatory macrophages (iNOS 336.1 ± 226.3 cells/mm control vs 290.6 ± 104.2 cells/mm tamoxifen, p > 0.999 Mann-Whitney test and CD163 136.6 ± 76.4 cells/mm control vs 94.1 ± 45.9 cells/mm tamoxifen, p = 0.108 unpaired t test). Tamoxifen treatment in the mouse silicone breast implant model decreased capsule formation through modulation of myofibroblasts, neovascularization, and collagen deposition. Tamoxifen may be useful for reducing or preventing capsule formation in clinical breast implantations.

Tissue engineered vascular grafts transform into autologous neovessels capable of native function and growth.

Background: Tissue-engineered vascular grafts (TEVGs) have the potential to advance the surgical management of infants and children requiring congenital heart surgery by creating functional vascular conduits with growth capacity. Methods: Herein, we used an integrative computational-experimental approach to elucidate the natural history of neovessel formation in a large animal preclinical model; combining an in vitro accelerated degradation study with mechanical testing, large animal implantation studies with in vivo imaging and histology, and data-informed computational growth and remodeling models. Results: Our findings demonstrate that the structural integrity of the polymeric scaffold is lost over the first 26 weeks in vivo, while polymeric fragments persist for up to 52 weeks. Our models predict that early neotissue accumulation is driven primarily by inflammatory processes in response to the implanted polymeric scaffold, but that turnover becomes progressively mechano-mediated as the scaffold degrades. Using a lamb model, we confirm that early neotissue formation results primarily from the foreign body reaction induced by the scaffold, resulting in an early period of dynamic remodeling characterized by transient TEVG narrowing. As the scaffold degrades, mechano-mediated neotissue remodeling becomes dominant around 26 weeks. After the scaffold degrades completely, the resulting neovessel undergoes growth and remodeling that mimicks native vessel behavior, including biological growth capacity, further supported by fluid-structure interaction simulations providing detailed hemodynamic and wall stress information. Conclusions: These findings provide insights into TEVG remodeling, and have important implications for clinical use and future development of TEVGs for children with congenital heart disease.

Tissue engineering: Relevance to neonatal congenital heart disease.

Congenital heart disease (CHD) represents a large clinical burden, representing the most common cause of birth defect-related death in the newborn. The mainstay of treatment for CHD remains palliative surgery using prosthetic vascular grafts and valves. These devices have limited effectiveness in pediatric patients due to thrombosis, infection, limited endothelialization, and a lack of growth potential. Tissue engineering has shown promise in providing new solutions for pediatric CHD patients through the development of tissue engineered vascular grafts, heart patches, and heart valves. In this review, we examine the current surgical treatments for congenital heart disease and the research being conducted to create tissue engineered products for these patients. While much research remains to be done before tissue engineering becomes a mainstay of clinical treatment for CHD patients, developments have been progressing rapidly towards translation of tissue engineering devices to the clinic.

The lysosomal trafficking regulator is necessary for normal wound healing.

Non-healing wounds are a major threat to public health throughout the United States. Tissue healing is complex multifactorial process that requires synchronicity of several cell types. Endolysosomal trafficking, which contributes to various cell functions from protein degradation to plasma membrane repair, is an understudied process in the context of wound healing. The lysosomal trafficking regulator protein (LYST) is an essential protein of the endolysosomal system through an indeterminate mechanism. In this study, we examine the impact of impaired LYST function both in vitro with primary LYST mutant fibroblasts as well as in vivo with an excisional wound model. The wound model shows that LYST mutant mice have impaired wound healing in the form of delayed epithelialization and collagen deposition, independent of macrophage infiltration and polarisation. We show that LYST mutation confers a deficit in MCP-1, IGF-1, and IGFBP-2 secretion in beige fibroblasts, which are critical factors in normal wound healing. Identifying the mechanism of LYST function is important for understanding normal wound biology, which may facilitate the development of strategies to address problem wound healing.

Sex and Tamoxifen confound murine experimental studies in cardiovascular tissue engineering.

Tissue engineered vascular grafts hold promise for the creation of functional blood vessels from biodegradable scaffolds. Because the precise mechanisms regulating this process are still under investigation, inducible genetic mouse models are an important and widely used research tool. However, here we describe the importance of challenging the baseline assumption that tamoxifen is inert when used as a small molecule inducer in the context of cardiovascular tissue engineering. Employing a standard inferior vena cava vascular interposition graft model in C57BL/6 mice, we discovered differences in the immunologic response between control and tamoxifen-treated animals, including occlusion rate, macrophage infiltration and phenotype, the extent of foreign body giant cell development, and collagen deposition. Further, differences were noted between untreated males and females. Our findings demonstrate that the host-response to materials commonly used in cardiovascular tissue engineering is sex-specific and critically impacted by exposure to tamoxifen, necessitating careful model selection and interpretation of results.

Evaluating the Longevity of the Fontan Pathway.

Children born with single ventricle physiology who undergo Fontan palliation face a diverse set of long-term complications. However, patient follow-up has in large part been limited to single institutional experiences without uniform application of diagnostic modalities to screen for relevant outcomes. Additionally, the use of different graft materials and variable surgical technique as part of the Fontan procedure has further complicated the evaluation of single ventricle patients. The purpose of this review is to define the changes in the Fontan pathway specific to the graft material used and its relationship to patient outcomes. As a means of introduction, we briefly review the historical evolution of the Fontan procedure with a focus on the intent behind design changes and incorporation of different biomaterials. We further delineate changes to the Fontan pathway which include the development of stenosis, differential growth, thrombosis, and calcification. Ultimately, the recognition of the changes noted within the Fontan pathway need to be assessed relative to their impact on patient hemodynamics, functional capacity, and Fontan-associated comorbidities.

Effects of Braiding Parameters on Tissue Engineered Vascular Graft Development.

Tissue engineered vascular grafts (TEVGs) using scaffolds fabricated from braided poly(glycolic acid) (PGA) fibers coated with poly(glycerol sebacate) (PGS) are developed. The approach relies on in vivo tissue engineering by which neotissue forms solely within the body after a scaffold has been implanted. Herein, the impact of altering scaffold braid design and scaffold coating on neotissue formation is investigated. Several combinations of braiding parameters are manufactured and evaluated in a Beige mouse model in the infrarenal abdominal aorta. Animals are followed with 4D ultrasound analysis, and 12 week explanted vessels are evaluated for biaxial mechanical properties as well as histological composition. Results show that scaffold parameters (i.e., braiding angle, braiding density, and presence of a PGS coating) have interdependent effects on the resulting graft performance, namely, alteration of these parameters influences levels of inflammation, extracellular matrix production, graft dilation, neovessel distensibility, and overall survival. Coupling carefully designed in vivo experimentation with regression analysis, critical relationships between the scaffold design and the resulting neotissue that enable induction of favorable cellular and extracellular composition in a controlled manner are uncovered. Such an approach provides a potential for fabricating scaffolds with a broad range of features and the potential to manufacture optimized TEVGs.

Tissue Engineered Vascular Graft Recipient Interleukin 10 Status Is Critical for Preventing Thrombosis.

Tissue engineered vascular grafts (TEVGs) are a promising technology, but are hindered by occlusion. Seeding with bone-marrow derived mononuclear cells (BM-MNCs) mitigates occlusion, yet the precise mechanism remains unclear. Seeded cells disappear quickly and potentially mediate an anti-inflammatory effect through paracrine signaling. Here, a series of reciprocal genetic TEVG implantations plus recombinant protein treatment is reported to investigate what role interleukin-10, an anti-inflammatory cytokine, plays from both host and seeded cells. TEVGs seeded with BM-MNCs from wild-type and IL-10 KO mice, plus unseeded grafts, are implanted into wild-type and IL-10 KO mice. Wild-type mice with unseeded grafts also receive recombinant IL-10. Serial ultrasound evaluates occlusion and TEVGs are harvested at 14 d for immunohistochemical analysis. TEVGs in IL-10 KO mice have significantly higher occlusion incidence compared to wild-type mice attributed to acute (<3 d) thrombosis. Cell seeding rescues TEVGs in IL-10 KO mice comparable to wild-type patency. IL-10 from the host and seeded cells do not significantly influence graft inflammation and macrophage phenotype, yet IL-10 treatment shows interesting biologic effects including decreasing cell proliferation and increasing M2 macrophage polarization. IL-10 from the host is critical for preventing TEVG thrombosis and seeded BM-MNCs exert a significant anti-thrombotic effect in IL-10 KO mice.

Spontaneous reversal of stenosis in tissue-engineered vascular grafts.

We developed a tissue-engineered vascular graft (TEVG) for use in children and present results of a U.S. Food and Drug Administration (FDA)-approved clinical trial evaluating this graft in patients with single-ventricle cardiac anomalies. The TEVG was used as a Fontan conduit to connect the inferior vena cava and pulmonary artery, but a high incidence of graft narrowing manifested within the first 6 months, which was treated successfully with angioplasty. To elucidate mechanisms underlying this early stenosis, we used a data-informed, computational model to perform in silico parametric studies of TEVG development. The simulations predicted early stenosis as observed in our clinical trial but suggested further that such narrowing could reverse spontaneously through an inflammation-driven, mechano-mediated mechanism. We tested this unexpected, model-generated hypothesis by implanting TEVGs in an ovine inferior vena cava interposition graft model, which confirmed the prediction that TEVG stenosis resolved spontaneously and was typically well tolerated. These findings have important implications for our translational research because they suggest that angioplasty may be safely avoided in patients with asymptomatic early stenosis, although there will remain a need for appropriate medical monitoring. The simulations further predicted that the degree of reversible narrowing can be mitigated by altering the scaffold design to attenuate early inflammation and increase mechano-sensing by the synthetic cells, thus suggesting a new paradigm for optimizing next-generation TEVGs. We submit that there is considerable translational advantage to combined computational-experimental studies when designing cutting-edge technologies and their clinical management.

Results through skeletal maturity of planned fibular nonunion for the treatment of genu varum in achondroplasia: An observational retrospective study.

Current surgical options for treating genu varum in achondroplasia include tibial and fibular osteotomy and growth modulation using plates and screws. However, a single surgeon consistently treated genu varum using a planned fibular nonunion (PFN). The purpose of this study is to describe his surgical technique and report radiographic and clinical outcomes for the cohort studied.This is an observational retrospective review. The cohort studied included patients with achondroplasia who had PFN surgery for the treatment of genu varum at a young age (<13 years) and was followed through to skeletal maturity. The surgery included meticulous closure of the periosteum over the remaining fibula. The surgery was considered a success if the patient did not require subsequent surgery and had acceptable or improved clinical alignment. Radiographic measures used to determine change in genu varum included the anatomic tibio-femoral angle (aTFA), tibia varus, and tibia-fibula ratio. Clinically, changes in lower limb alignment were defined using a plumb line and 6 categories of alignment ranging from extreme varus to valgus. Statistics were used to validate the plumb line categorization to available radiographic measures. Other appropriate statistical methods were used with P < .05 considered significant.Of the 53 PFN cases (27 patients) included in the study, 34 (64%) did not require subsequent surgery and had acceptable or improved alignment. The average age at surgery and follow-up was 6.1 and 17.0 years, respectively. For the 37 limbs (19 patients) with available radiographs, pre- and post-surgery radiographic measures significantly improved including aTFA (3° varus to 2° valgus, P = .003), tibia varus (2° varus to 3° valgus, P = .004), and the tibia-fibula ratio (0.977 to 1.013, P < .001). Clinically, 32 cases (60%) demonstrated significant improvement by translating into an improved alignment and 9 (17%) remained the same (P < .01). Complications were minimal and insignificant. Failures were readily managed by tibia-fibular osteotomies in adolescence and at maturity.PFN for the treatment of genu varum in young achondroplasia patients significantly improved radiographic and clinical measures of lower limb alignment through skeletal maturity with relatively few complications.