Novel extracellular matrix architecture on excitatory neurons revealed by HaloTag-HAPLN1.

The brain's extracellular matrix (ECM) regulates neuronal plasticity and animal behavior. ECM staining shows an aggregated pattern in a net-like structure around a subset of neurons and diffuse staining in the interstitial matrix. However, understanding the structural features of ECM deposition across various neuronal types and subcellular compartments remains limited. To visualize the organization pattern and assembly process of the hyaluronan-scaffolded ECM in the brain, we fused a HaloTag to HAPLN1, which links hyaluronan and proteoglycans. Expression or application of the probe enables us to identify spatial and temporal regulation of ECM deposition and heterogeneity in ECM aggregation among neuronal populations. Dual-color birthdating shows the ECM assembly process in culture and in vivo. Sparse expression in vivo reveals novel forms of ECM architecture around excitatory neurons and developmentally regulated dendritic ECM. Overall, our study uncovers extensive structural features of the brain' ECM, suggesting diverse roles in regulating neuronal plasticity.

Microvilli regulate the release modes of alpha-tectorin to organize the domain-specific matrix architecture of the tectorial membrane.

The tectorial membrane (TM) is an apical extracellular matrix (ECM) in the cochlea essential for auditory transduction. The TM exhibits highly ordered domain-specific architecture. Alpha-tectorin/TECTA is a glycosylphosphatidylinositol (GPI)-anchored ECM protein essential for TM organization. Here, we identified that TECTA is released by distinct modes: proteolytic shedding by TMPRSS2 and GPI-anchor-dependent release from the microvillus tip. In the medial/limbal domain, proteolytically shed TECTA forms dense fibers. In the lateral/body domain produced by the supporting cells displaying dense microvilli, the proteolytic shedding restricts TECTA to the microvillus tip and compartmentalizes the collagen-binding site. The tip-localized TECTA, in turn, is released in a GPI-anchor-dependent manner to form collagen-crosslinking fibers, required for maintaining the spacing and parallel organization of collagen fibrils. Overall, we showed that distinct release modes of TECTA determine the domain-specific organization pattern, and the microvillus coordinates the release modes along its membrane to organize the higher-order ECM architecture.

The release of surface-anchored α-tectorin, an apical extracellular matrix protein, mediates tectorial membrane organization.

The tectorial membrane (TM) is an apical extracellular matrix (ECM) that hovers over the cochlear sensory epithelium and plays an essential role in auditory transduction. The TM forms facing the luminal endolymph-filled space and exhibits complex ultrastructure. Contrary to the current extracellular assembly model, which posits that secreted collagen fibrils and ECM components self-arrange in the extracellular space, we show that surface tethering of α-tectorin (TECTA) via a glycosylphosphatidylinositol anchor is essential to prevent diffusion of secreted TM components. In the absence of surface-tethered TECTA, collagen fibrils aggregate randomly and fail to recruit TM glycoproteins. Conversely, conversion of TECTA into a transmembrane form results in a layer of collagens on the epithelial surface that fails to form a multilayered structure. We propose a three-dimensional printing model for TM morphogenesis: A new layer of ECM is printed on the cell surface concomitant with the release of a preestablished layer to generate the multilayered TM.

Evaluation of the Effects of BioCell Collagen, a Novel Cartilage Extract, on Connective Tissue Support and Functional Recovery From Exercise.

CONTEXT: Little is known about the effect of nutritional supplementation on metabolic optimization for enhancing adaptation and recovery of the connective tissue elements that support musculoskeletal function. OBJECTIVE: The study aimed to determine the potential effect of supplementation with a novel, hydrolyzed chicken sternal cartilage extract-called BioCell Collagen-on biomarkers and functional indices of recovery from intense exercise. DESIGN: The research team designed a randomized, double-blind, placebo-controlled pilot study. SETTING: The study was conducted at the Center for Applied Health Sciences in Stow, OH, USA. PARTICIPANTS: Participants were 8 healthy, recreationally active individuals, with a mean age of 29.3 y. INTERVENTION: Participants ingested either 3 g of a novel, hydrolyzed chicken sternal cartilage extract called BioCell Collagen ("supplement") or 3 g of a placebo daily for 6 wk prior to challenge with an upper-body, muscle-damaging resistance exercise (UBC) on day 43 and a rechallenge on day 46 to assess functional recovery. OUTCOME MEASURES: Primary endpoints were levels of 3 blood biomarkers-creatine kinase (CK), lactate dehydrogenase (LDH), and C-reactive protein (CRP)- and scores on a clinical pain scale and a perceived recovery scale (PRS). RESULTS: The extract attenuated the post-UBC increase in serum markers for muscle tissue damage: CK, LDH, and CRP. For the intervention group vs the placebo group, the mean changes were as follows: (1) an increase in CK of 20 U/L vs 4726 U/L, respectively; (2) a decrease in LDH of 3.5 U/L vs an increase of 82.9 U/L, respectively; and (3) an increase in CRP of 0.07 mg/L vs an increase of 0.7 mg/L, respectively. The performance decrement in bench press repetitions to failure was 57.9% on day 43 and 57.8% on day 46 for the intervention group vs 72.2% on day 43 and 65% on day 46 for the placebo group. The overall trend for the performance decrement, together with the results for the PRS, suggested that a more robust muscular recovery and adaptive response occurred with use of the extract. The supplement was well tolerated. CONCLUSIONS: The study's preliminary data are promising with regard to the beneficial effects of the extract on connective tissue protection and recovery in those engaged in routine resistance training and cardiovascular exercise. A larger study is warranted to confirm and refine these findings.

Ingestion of BioCell Collagen(®), a novel hydrolyzed chicken sternal cartilage extract; enhanced blood microcirculation and reduced facial aging signs.

Skin aging and its clinical manifestation is associated with altered molecular metabolism in the extracellular matrix of the dermis. In a pilot open-label study, we investigated the effect of a dietary supplement, BioCell Collagen(®) (BCC), which contains a naturally occurring matrix of hydrolyzed collagen type II and low-molecular-weight hyaluronic acid and chondroitin sulfate, in 26 healthy females who displayed visible signs of natural and photoaging in the face. Daily supplementation with 1 g of BCC for 12 weeks led to a significant reduction of skin dryness/scaling (76%, P = 0.002) and global lines/wrinkles (13.2%, P = 0.028) as measured by visual/tactile score. Additionally, a significant increase in the content of hemoglobin (17.7%, P = 0.018) and collagen (6.3%, P = 0.002) in the skin dermis was observed after 6 weeks of supplementation. At the end of the study, the increase in hemoglobin remained significant (15%, P = 0.008), while the increase in collagen content was maintained, but the difference from baseline was not significant (3.5%, P = 0.134). This study provides preliminary data suggesting that dietary supplementation with BCC elicits several physiological events which can be harnessed to counteract natural photoaging processes to reduce visible aging signs in the human face. A controlled study is necessary to verify these observations.

Effect of the novel low molecular weight hydrolyzed chicken sternal cartilage extract, BioCell Collagen, on improving osteoarthritis-related symptoms: a randomized, double-blind, placebo-controlled trial.

Osteoarthritis (OA) is a significant source of pain and disability. Current medical and surgical treatments can be costly and have serious side effects. The aim of this randomized, double-blind, placebo-controlled trial was to investigate the tolerability and efficacy of BioCell Collagen (BCC), a low molecular weight dietary supplement consisting of hydrolyzed chicken sternal cartilage extract, in the treatment of OA symptoms. Patients (n = 80) in the study had physician-verified evidence of progressive OA in their hip and/or knee joint. Joint pain had been present for 3 months or longer at enrollment, and pain levels were 4 or higher at baseline as assessed by Physician Global Assessment scores. Subjects were divided into two groups and administered either 2 g of BCC or placebo for 70 days. Other outcome measurements included visual analogue scale (VAS) for pain and Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores taken on days 1, 35, and 70. The tolerability profile of the treatment group was comparable to that of the placebo. Intent-to-treat analysis showed that the treatment group, as compared to placebo, had a significant reduction of VAS pain on day 70 (p < 0.001) and of WOMAC scores on both days 35 (p = 0.017) and 70 (p < 0.001). The BCC group experienced a significant improvement in physical activities compared to the placebo group on days 35 (p = 0.007) and 70 (p < 0.001). BCC was well tolerated and found to be effective in managing OA-associated symptoms over the study period, thereby improving patient's activities of daily living. BCC can be considered a potential complement to current OA therapies.

Active immunotherapy induces antibody responses that target tumor angiogenesis.

The inhibition of VEGF signaling with antibodies or small molecules achieves clinical benefits in diverse solid malignancies. Nonetheless, therapeutic effects are usually not sustained, and most patients eventually succumb to progressive disease, indicating that antiangiogenic strategies require additional optimization. Vaccination with lethally irradiated, autologous tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) and antibody blockade of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) trigger a tumor vasculopathy in some long-term responding subjects. These reactions are characterized by disrupted tumor blood vessels in association with lymphocyte and granulocyte infiltrates and zonal areas of ischemic tumor necrosis. However, the mechanisms underlying this immune-mediated destruction of the tumor vasculature remain to be clarified. Here, we show that GM-CSF-secreting tumor cell vaccines and CTLA-4 blockade elicit a functionally important humoral reaction against multiple angiogenic cytokines. Antibodies to angiopoietin-1 and angiopoietin-2 block Tie-2 binding, downstream signaling, endothelial cell tube formation, and macrophage chemotaxis. Antibodies to macrophage inhibitory factor (MIF) attenuate macrophage Tie-2 expression and matrix metalloproteinase-9 (MMP-9) production. Together, these results delineate an immunotherapy-induced host response that broadly targets the angiogenic network in the tumor microenvironment.

Concerted potent humoral immune responses to autoantigens are associated with tumor destruction and favorable clinical outcomes without autoimmunity.

PURPOSE: The therapeutic importance of immune responses against single versus multiple antigens is poorly understood. There also remains insufficient understanding whether responses to one subset of antigens are more significant than another. Autoantibodies are frequent in cancer patients. They can pose no biological significance or lead to debilitating paraneoplastic syndromes. Autoreactivity has been associated with clinical benefits, but the magnitude necessary for meaningful results is unknown. Autologous tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor generate immune infiltrates in preexisting metastases with associated tumor destruction. We sought to identify targets of responses from this vaccination strategy. EXPERIMENTAL DESIGN: Postvaccination sera used in screening a cDNA expression library prepared from a densely infiltrated metastasis of a long-term surviving melanoma patient identified several autoantigens. Additional autoantigens were identified through similar screenings in non-small cell lung cancer and murine models, and proteins implicated in cancer propagation. ELISAs for several targets were established using recombinant proteins, whereas others were evaluated by petit serologies. RESULTS: Eleven gene products were identified through serologic screening from two patients showing highly favorable clinical outcomes. A subset of antigens revealed significant changes in antibody titers compared with weak responses to other proteins. Time course analyses showed coordinated enhanced titers against several targets as a function of vaccination in responding patients. CONCLUSIONS: This study shows the range of biologically significant antigens resulting from a whole-cell vaccine. Targets include autoantigens that are components of cell cycle regulation. Potent antibody responses against multiple autoantigens are associated with effective tumor destruction without clinical autoimmunity.