Stimulation of NCAM1-14.3.3.ζδ-derived Peptide Interaction Fuels Angiogenesis and Osteogenesis in Ageing

The skeletal structure and bone marrow endothelium collectively form a critical functional unit essential for bone development, health, and aging. At the core of osteogenesis and bone formation lies the dynamic process of angiogenesis. In this study, we reveal a potent new endogenous anabolic NCAM1-14.3.3.ζδ-derived-Peptide interaction, which stimulates bone angiogenesis and osteogenesis during homeostasis, aging, and age-related bone diseases. Employing high-resolution imaging and inducible cell-specific mouse genetics, our results elucidate the pivotal role of the NCAM1-14.3.3.ζδ-derived-Peptide interaction in driving the expansion of Clec14a+ angiogenic endothelial cells. Notably, Clec14a+ endothelial cells express key osteogenic factors. The NCAM1-14.3.3.ζδ-derived-Peptide interaction in osteoblasts drives osteoblast differentiation, ultimately contributing to the genesis of new bone. Moreover, the NCAM1-14.3.3.ζδ-derived-Peptide interaction leads to a reduction in bone resorption. In age-associated vascular and bone loss diseases, stimulating the NCAM1-14.3.3.ζδ-derived-Peptide interaction not only promotes angiogenesis but also reverses bone loss. Consequently, harnessing the endogenous anabolic potential of the NCAM1-14.3.3.ζδ-derived-Peptide interaction emerges as a promising therapeutic modality for managing age-related bone diseases.


Introduction
The skeleton and the bone marrow endothelium form a functional unit with great relevance in development, health, and ageing (Kusumbe et al., 2014;Salhotra & Shah, 2020).The bone marrow endothelium plays a central role in the maintenance of microenvironments required for regulating osteogenesis and haematopoiesis (Johnson et al., 2020;Owen-Woods & Kusumbe, 2022).Apart from supplying nutrients, the vasculature provides several inductive signals and secretory factors, socalled angiocrine signals/factors to regulate the tissue-specific microenvironments (Singh et al., 2019;Sivan et al., 2019).
Vasculature plays a key role in maintaining skeletal health and a dysregulation of the vasculature is speculated for several bone diseases like osteoporosis and osteoarthritis (Chen et al., 2021;Kumar et al., 2021;Stucker et al., 2020).
Musculoskeletal disorders affect almost one in every two individuals posing a major health, psychological and economic burden on people, health systems and the Government (Lewis et al., 2019).With increasing life expectancy, ageing populations are more severely affected with osteoporosis and fracture (Brown et al., 2021;Karademir et al., 2015;Salari, Darvishi, et al., 2021;Salari, Ghasemi, et al., 2021;Xiao et al., 2022).Globally, osteoporosis is the most prevalent bone disease with a significant impact on health systems (Sözen et al., 2017;Sarafrazi et al., 2021).
Existing therapies have mainly focused on slowing the rate of bone damage, with only a few drugs capable of promoting bone repair approved (Chindamo & Sapino, 2020;Liang & Burley, 2022).Due to issues like poor patient response, drug compliance, and drug-induced microfractures leading to atypical femur fractures (Tile & Cheung, 2020;Xiao et al., 2023), there is a pressing need for new therapies that effectively promote bone repair and regeneration in patients with musculoskeletal diseases, restoring tissue homeostasis and functional integrity.
Clinical management of non-healing fractures, which are characterized by delayed angiogenesis remains challenging (Schlundt et al., 2018;Stewart, 2019).In addition to the bone loss and fracture risk associated with cancer therapy; in bone marrow transplantations, metastatic bone disease, and cancer settings, the re-establishment of bone marrow microenvironments after chemotherapy and radiation is crucial for the effective treatment outcome and clinical management of these conditions (Chen et al., 2012;D'Oronzo et al., 2019;Singh et al., 2019;Tsukamoto et al., 2021;Vi et al., 2018;Wu et al., 2018).The bone vasculature and perivascular mesenchymal stem cells, which can differentiate to generate bone cells, adipocytes and chondrocytes, are essential for bone growth, and hematopoietic stem cell (HSC) maintenance (Hsu et al., 2021;James & Péault, 2019;Kusumbe et al., 2016).The manipulation of the vasculature is sufficient for the improvement of vascular HSC niche function, which suggests the existence of molecular pathways coupling the behaviour of endothelial cells and perivascular mesenchymal stem progenitor cells (MSPCs) in bone (Hanoun et al., 2014;Ramasamy, 2017).Thus, the prospect manipulating vasculature has tremendous potential to advance therapeutic interventions for the management of bone diseases and promote bone formation.
Recognizing that aging is associated with bone loss and bone-related disorders, we extended our investigation to aged mice.Remarkably, a 10-day treatment with 14.3.3.ζδ-derived-Peptideproved sufficient to induce a substantial increase in bone mass, accompanied by elevated expression of Sp7, Ibsp and Bglap genes associated with osteogenesis in the aged mice (Figures 1D-G).
Moreover, administration of 14.3.3.ζδ-derived-Peptide; both in young and aged mice, led to increased significant augmentation of angiogenesis, a process crucial for vascular growth and expansion.Both young and aged mice exhibited increased expression of vascular markers and increased vascular density, validated through imaging thick bone slices and quantitative analysis (Figures 1H-K).This augmented angiogenic response correlated closely with an increased accumulation of Clecl14a + cells near the growth plate (Figure 1H) -a notable marker expressed by highly angiogenic endothelial cells with a pivotal role in embryonic and pathological angiogenesis, as well as adhesion and migration (Khan et al., 2017;Kim et al., 2018;Noy et al., 2015).Recent studies have also emphasized the function of Group 14 Ctype lectins, of which Clec14a is a member (Khan et al., 2019).
The observed structural changes and increased bone mass in 14.3.3.ζδ-derived-Peptide-treatedmice were characterized by an increased abundance of Osterixexpressing osteoblasts and Osteopontin expression (Figures 1L-N).However, no changes were observed in number of adipocytes as analysed by Perilipin immunostaining (Figure 1-figure supplement 1B).Taken together, various vascular and osteoblast cell markers demonstrated a remarkable increase following 14.3.3.ζδderived-Peptidetreatment in both young and aged animals (Figures 1L-N).

14.3.3.ζδ-derived-Peptide Treatment induced Expansion of Clec14a+
Angiogenic Endothelial Cells After 14.3.3.ζδ-derived-Peptidetreatment, our results clearly demonstrated the expansion of Clec14a+ endothelial cells located near the growth plate.Moreover, these Clec14a+ endothelial cells were detected both in mouse and human bone marrow as revealed by the single-cell sequencing analysis (Figures 1O and P).
Clec14a, or C-type lectin domain family 14 member A, is a gene that codes for a protein with a C-type lectin domain.C-type lectins are a family of proteins with carbohydrate-binding domains, and they often play roles in immune response and cell adhesion.As stated above Clec14a expressing cells have been found in tumour endothelium and expression of Clec14a is associated with high angiogenic activity (Mancuso et al., 2014).Therefore, to gain a deeper understanding of the role of these cells in bone development, especially during the 14.3.3.ζδ-derived-Peptide-inducedincrease in bone mass, we conducted genetic lineage tracing of these cells.To achieve this, we generated a tamoxifen-inducible Clec14a Cre ER T2 line in Clec14a PiggyBac-on-BAC Mouse Transgenics (Figure 2-figure supplements 1A-D).The utilization of genetic lineage tracing, a powerful investigative tool, enabled us to explore processes such as cell amplification, differentiation, and migration.Employing Clec14a-CreER T2 x ROSA26-mT/mG double transgenics and administering tamoxifen, we identified Clec14a+ cells strategically positioned near the growth plate, at the angiogenic forefront of the bones in both juvenile and adult mice.In the juvenile mice, these cells were abundant in the metaphysis region of the bones.Notably, these cells exhibited high expression of type H endothelium markers such as Endomucin (Figure 2A).
Further genetic lineage tracing of these cells using Clec14a-CreER T2 x ROSA26-mT/mG double transgenics towards which tamoxifen administration was performed in 3-weeks-old mice, followed by analysis in adult 9-weeks-old mice, revealed that these Clec14a+ cells hierarchically positioned upstream of other endothelial cells (Figure 2A).Clec14a+ cells which expressed the markers for type H endothelium gave rise to and differentiated into various endothelial cell types in adult bones.
To further investigate the impact of Clec14a angiogenic cells on bone formation during 14.3.3.ζδ-derived-Peptidetreatment, we implemented a targeted strategy aimed at directly affecting and depleting these cells.To selectively deplete the Clec14a+ cell population and assess their influence on bone formation during 14.3.3.ζδ-derived-Peptidetreatment, we utilized the Clec14a-Cre ER T2 mouse line crossed with mice expressing a tamoxifen-inducible diphtheria toxin antigen (iDTA) (Figure 2-figure supplement 1F).Tamoxifen injections were administered to both Cre-and Cre+ mice.
As expected, Cre+ iDTA mice exhibited suppressed expansion of Clec14a cells and led to reduced expression of Clec14a expressing cells.Moreover, analysis of the Cre+ iDTA mice demonstrated loss of endothelial cells near the growth plate region (Figure 2B).Moreover, analysis of Clec14a+ endothelial cells had high expression of several osteogenic factors compared to Clec14a-endothelial cell in bones (Figure 2-figure supplement 1E).
Microcomputed tomography (m-CT) analyses of Cre+ iDTA mice, 14.3.3.ζδ-derived-Peptidetreatment demonstrated that the removal of Clec14a+ cells did not lead to an increase in bone mass following 14.3.3.ζδ-derived-Peptidetreatment (Figure 2-figure supplement 1F).These collective findings strongly support the crucial role of Clec14a+ cells in facilitating bone formation during 14.3.3.ζδ-derived-Peptidetreatment.The targeted depletion of these cells during 14.3.3.ζδ-derived-Peptidetreatment not only influenced the expression of key osteogenic markers but also resulted in a noticeable decrease in overall bone density, as confirmed by micro-CT assessments.These results highlight the interplay between Clec14a angiogenic cells and the regulatory mechanisms governing bone homeostasis in the context of 14.3.3.ζδ-derived-Peptidetreatment.
Analysis of endothelial-specific and osteoblast-specific Ncam1-loss-of-function Cre+ mouse bones revealed that this specific loss of Ncam1 in endothelial or osteoblast cells resulted in no increase in bone mass and bone density following 14.3.3.ζδderived-Peptidetreatment (Figures 3B and C).
Tamoxifen was administered to both Cre+ and Cre-mice (Figures 3D and E).Col1a Cre ER T2 X Ncam1 fl/fl mice exhibited a notable reduction in bone mass and bone density (Figure 3E), aligning with prior studies highlighting the regulatory role of NCAM-1 in osteoblast differentiation (Cheng et al., 2021).
Analysis of Clec14a Cre ER T2 X Ncam1 fl/fl mice demonstrated reduced vessel density in Cre+ mouse bones and diminished endothelial cell proliferation (Figure 3D), consistent with earlier studies implicating NCAM-1 in the regulation of endothelial cell proliferation through the PI3K-AKT signalling pathway (Xiang et al., 2021).
Prior research has suggested the involvement of NCAM-1 in the regulation of osteoblast differentiation through the Wnt/β-catenin pathway (Cheng et al., 2021).To explore this further, we assessed beta-catenin expression and localization.

Resorption
Recognizing the intricate regulation of bone homeostasis by osteoblasts and osteoclasts (Kim et al., 2020), our investigation aimed to elucidate whether the observed increase in bone mass was a result of heightened osteoblast activity or diminished osteoclast function.The finely tuned interplay between osteoblasts and osteoclasts involves intricate regulation, with heightened osteoblast activity often matched by increased osteoclastogenesis to maintain balance (Kim et al., 2020;Marahleh et al., 2023).To comprehensively understand NCAM1-14.In light of the substantial pro-angiogenic effects demonstrated by NCAM1-14.3.3.ζδderived-Peptideinteraction, our investigation sought to comprehensively assess its therapeutic efficacy in treating musculoskeletal diseases associated with vascular and bone loss.Osteoporosis, a prevalent musculoskeletal condition linked to cancellous bone loss and an increased risk of fractures (Sözen et al., 2017;Sarafrazi et al., 2021), often lacks interventions targeting vascular and osteoblast-driven repair processes.
NACM1-14.3.3.ζδ-derived-Peptide, with its remarkable pro-angiogenic and osteogenic effects, emerged as a compelling candidate for such intervention to stimulate angiogenesis and osteogenesis.
In a murine model of osteoporosis induced by ovariectomy, 14.3.3.ζδ-derived-Peptidetherapy effectively reduced bone loss compared to the vehicle control (Figure 4A).Further exploration of NACM1-14.3.3.ζδ-derived-Peptideinteraction extended to bone metastasis, where treatment with 14.3.3.ζδ-derived-Peptidereversed bone loss in the bone metastasis mouse model (Figure 4C).Analysis of a rheumatoid arthritis model, characterized by bone loss and decreased vascular density, revealed a significant loss of vessel density in advanced disease stages (Figures 4D and E).This observation contrasted with the reported increase in angiogenesis and endothelial cells during inflammation (Hellenthal & Brabenec, 2022), suggesting a coupled relationship between angiogenesis and osteogenesis even in inflammatory settings.The loss of vasculature in advanced disease as compared to early stage was associated with a subsequent loss of bone mass (Figures 4D and E).Treatment with 14.

Discussion
In this ground-breaking study, employing cell-specific mouse genetics coupled with high-resolution imaging techniques, we not only reveal but also illuminate with unprecedented clarity a hitherto undiscovered and remarkably potent anabolic pathway.The 14.3.3.ζδ-derived-Peptide-NCAM1interaction emerges as the driving force behind the robust expansion of highly angiogenic Clec14a+ endothelial cells, playing a pivotal role in promoting bone osteogenesis and genesis of new bone.
The robustness of our findings is underpinned by the meticulous application of inducible cell-specific mouse genetics to delineate the role of highly angiogenic Clec14a+ endothelial cells and the NCAM1-14.3.3.ζδ-derived-Peptideinteraction in these cells.Furthermore, the use of osteoblast-specific transgenics has allowed us to reveal the role of the NCAM1-14.3.3.ζδ-derived-Peptideinteraction, creating a nexus between angiogenesis and osteogenesis.

Mice
The study employed C57BL/6 mice from Charles River as wild-type subjects for all analyses, unless otherwise specified.The mice were categorized into three age groups: juvenile (1-4 weeks), adult (12-16 weeks), and aged (>65 weeks).Both male and female mice were included in the study, and details regarding transgenic mouse lines can be found in the resources table.In the drug treatment phase, mice were randomly assigned to receive treatment, while littermates were utilized as sham controls.Mice underwent daily intra-peritoneal (IP) injections over a 10-day period.
The injections consisted of either a vehicle control (PBS) or 14.3.

Tamoxifen treatment for inducible gene deletion
Tamoxifen treatment for inducible gene deletion and genetic lineage tracing was conducted following established protocols (Kusumbe et al., 2014).Tamoxifen (Sigma-Aldrich, T5648) was freshly prepared by initially dissolving it in 100% ethanol and subsequently suspending it in corn oil to achieve a final concentration of 5 mg/ml.For tamoxifen-induced genetic lineage tracing, Cre ERT2 mouse lines, as specified in the figure legends, were employed.To activate Cre recombinase, tamoxifen was administered orally at a dose of 50 mg/kg for three consecutive days.Consistent with prior studies (Kusumbe et al., 2014;Kusumbe et al., 2016), tamoxifen injections were performed in both Cre+ and Cre-mice across all experiments in this study.At the designated time points, mice were euthanized via CO2 asphyxiation, and tissues were collected for subsequent analysis.This methodology adhered to the established procedures outlined in previous publications (Biswas et al., 2023).

Generation of Clec14a Cre ERT2 mouse line
The mouse Clec14a gene (NCBI Reference Sequence: NM_025809.5) is located on mouse chromosome 12. 1 exon have been identified, with the ATG start codon in exon 1 and TGA stop codon in exon 1.A BAC containing the upstream regulatory sequence of the mouse Clec14a gene was identified.The "CreERT2-polyA" cassette was placed upstream of the ATG start codon in the BAC.The PiggyBac ITRs were inserted into the BAC backbone flanking the genomic insert to facilitate transposition mediated BAC integration.The modified BAC was co-injected with transposase into single-cell stage fertilized eggs from C57BL/6J mice.The pups were genotyped by PCR for the presence of the modified BAC.The positive founder mice were counter screened for transposition.This Clec14a Cre ERT2 mouse line was generated by using the service from Taconic Biosciences.

Preparation of samples for Immunohistochemistry
To prepare samples for immunohistochemistry, bones were dissected and placed in ice-cold 2% paraformaldehyde (PFA) in PBS, where they were left on ice for 4 hours.
Subsequently, the bones underwent processing following established methods (Kusumbe et al., 2015).After rinsing in PBS, bone samples were immersed in 0.5M EDTA (pH 7.4) for a minimum of 36 hours.Following this, they were dehydrated in a solution containing 20% sucrose (Sigma-Aldrich, S9378) and 2% polyvinylpyrrolidone (PVP, Sigma-Aldrich, PVP360) for 48 hours.The prepared bones were then embedded in a mixture of 20% sucrose, 2% polyvinylpyrrolidone, and 8% gelatin (Sigma-Aldrich, G2625).Sectioning of the samples was accomplished at a thickness of 100 µm using a Leica CM3050 cryostat equipped with low-profile blades (Leica, 14035838382).Subsequently, the sections were air-dried before being placed in the freezer for storage.This sample preparation procedure adhered to the methodology previously described previously (Kusumbe et al., 2015).

Immunostaining of thick bone slices
In the immunostaining process for thick bone slices, meticulous steps were undertaken to ensure precision and reproducibility (Kusumbe et al., 2015).Initially, bone sections underwent a 15-minute air-drying phase followed by a 5-minute rehydration in PBS to optimize the subsequent staining procedure.To enhance permeability, sections were treated with 0.3% Triton X-100 for 10 minutes and subsequently blocked in 5% donkey serum at room temperature (RT).Primary antibodies, critical for target-specific binding, were incubated with the samples after being judiciously diluted in blocking buffer during an overnight incubation at 4°C.The comprehensive list of primary antibodies is thoughtfully documented in the key resources table.To eliminate excess primary antibodies and enhance specificity, samples were subjected to multiple 5-minute washes at RT using PBS.Subsequently, the samples underwent a 2-hour incubation at RT with Alexa fluor-conjugated secondary antibodies, detailed in the key resources table, and the nuclear marker TOPRO-3 or 4',6-Diamidino-2-Phenylindole (DAPI) at a 1:1000 dilution.Following additional PBS washes, the stained samples were carefully mounted with glass coverslips using Fluoromount-G (Invitrogen, 00-4958-02).To validate specificity, negative controls were implemented, involving staining without primary antibodies and exclusively with secondary antibodies.This detailed protocol ensures clarity and methodological transparency in the immunostaining process, allowing for precise analysis of thick bone slices.

Microscope set-up and Imaging acquisition
The confocal imaging setup and image acquisition process were conducted with To capture large regions within the thick sections, the tile scan function was employed, and images with a 10% overlap were stitched together using Zen Black (version 3.1, Zeiss) software.To enhance the visualization of organ boundaries, autofluorescence from the 405 channel was converted into greyscale, and the resultant 30% opaque image was manually overlaid with the corresponding TIFF file generated from Imaris.
Image generation, analysis, and compilation were carried out using Imaris, Adobe Photoshop, and Adobe Illustrator software, ensuring a comprehensive and sophisticated approach to confocal imaging and subsequent data processing.During imaging, bone sections were inspected in Live mode using appropriate channels selected based on the fluorescence spectrum of the respective secondary antibody to identify a region of interest.Exposure time, brightness, and contrast were adjusted for optimal visualization of the brightest sample in the staining panel.In instances of high background signal, exposure was reduced to mitigate unspecific signals from endogenous background structures.Exposure settings were standardized for subsequent samples within an experiment group to ensure comparability of mean fluorescence between samples.Images were captured using the tile scan function, and the resulting tile scans were merged and processed with Thunder Computational Clearing software to enhance image contrast and reduce blurriness.Thunder-processed images were further analysed using Imaris software (version 9.6.0,Bitplane) for subsequent processing and detailed analysis.

RNA isolation and quantitative PCR
For the isolation of total RNA from whole bones, the RNeasy Mini Kit (QIAGEN) manufacturer's protocol was strictly followed.Crushed bones, prepared using a mortar and pestle according to established procedures (Kusumbe et al., 2014), underwent subsequent digestion with a mixture comprising 0.2% collagenase IV, dispase (1.25 U/ml) (Thermo Fisher Scientific, catalog no.171055-041), and DNase I (7.5 mg/ml) (Sigma-Aldrich, catalog no.D4527-10KU) for 45 minutes at 37°C.This digestion step was crucial for effective RNA isolation.
To generate cDNA, 100 ng of isolated RNA per reaction was utilized with the iScript cDNA Synthesis System (Bio-Rad).The resulting cDNA served as the template for quantitative polymerase chain reaction (qPCR) on the ABI PRISM 7900HT Sequence Detection System, using TaqMan gene expression assays.FAM-conjugated TaqMan probes, along with the TaqMan Gene Expression Master Mix (Applied Biosystems, 4369510), were employed for qPCR.The gene expression assays were normalized using endogenous VIC-conjugated Actb probes as standards.RNA samples were promptly processed for complementary DNA preparation using the SuperScript IV First-Strand Synthesis System (Invitrogen, 18091200).

Micro-CT analysis and histomorphometry
Tibiae were collected; the attached soft tissue in the bone was removed thoroughly and fixed in 2% paraformaldehyde.The fixed tibiae were analyzed using micro-CT (µCT 100).Following scan settings were used Voxel size 6.0 μm, FOV 10.236 mm, Image matrix 1706 x 1706 x 550, Slices 550, Scanned region 3.3 mm, X-ray voltage 70 kVp, and Intensity 85 μA, 6 W. Calcein double labeling was performed to calculate Bone Formation Rate (BFR) and mineral apposition rate (MAR).Mice were given intraperitoneal injections of 10 mg/kg calcein (Sigma, C0875) dissolved in 2% sodium bicarbonate solution on the 10 th day and 3 rd day before euthanasia.Bones were fixed in 4% PFA, embedded in 8% gelatin and 2% PVP and cryosectioned.Single plane images were acquired from the sections.Sections were stained with von Kossa method to assess mineralized bone.Representative images show cortical bone (diaphysis about 3 mm proximally from the growth plate).Mineral Apposition Rates were calculated from both cortical and trabecular bones.its (R&D Systems) according to the manual provided by the manufacturer.

Bone Metastasis
For experimental metastasis assays, MDA-MB-231 cells or PC3 (1 x 10 5 cells in 100 µl PBS) were injected into the left cardiac ventricle of NOD-SCID mice with a 26½ gauge needle.MDA-MB-231 cells were always injected into female NOD-SCID mice.
Successful injection was characterized by pumping arterial blood into the syringe.

Osteoporosis, Osteoarthritis and Collagen Induced Arthritis mouse models
Collagen-Induced Arthritis (CIA) was used as the experimental model for rheumatoid arthritis.The mice utilized in the experiments were housed under stringent pathogenfree conditions, ensuring a controlled environment, with ad libitum access to food and water.The induction of arthritis involved the subcutaneous injection of 200 μg of bovine type II collagen in complete Freund's adjuvant (CFA) at the base of the tail in DBA/1 mice, following established protocols (Choudhary et al., 2018).
Destabilization of the medial meniscus (DMM) model of osteoarthritis was used.For the surgical destabilization of the DMM model male mice underwent the procedure as described previously (Glasson et al., 2007).Treatment with 14.3.3.ζδ-derived-Peptide was initiated 12 weeks post-surgery.Ovariectomy was performed induce osteoporosis.16-weeks-old female C57Bl/6J underwent ovariectomy as previously described.2 weeks later, mice were culled to acquire baseline measurements or 14.3.3.ζδ-derived-Peptidetreated was initiated.
Subsequently, the data underwent consolidation and additional processing using the Seurat CRAN package (version 4.0.1)(https://satijalab.org/seurat/)(Satija et al., 2015).Specifically, cells positive for PECAM1 were selectively filtered for subsequent analysis.Following this filtering step, a total of 763 cells remained for the ensuing analysis.The filtered cell data underwent normalization, scaling, and identification of variable genes using SCTransform (Hafemeister & Satija, 2019).Cell clusters were determined using the FindClusters function of the Seurat package with a resolution of 0.4.The resulting cell clustering was visualized using Uniform Manifold Approximation and Projection for Dimension Reduction (umap).Additional visualizations, including umap gene expression overlays, feature plots, and violin plots illustrating cell typespecific marker genes, were generated using Seurat-specific functions.
3.3.ζδ-derived-Peptideinteractionresults in suppression of bone resorption.This nexus between NCAM1-14.3.3.ζδderived-Peptideinteraction in endothelium proves to be instrumental in reversing bone loss in age-related vascular and bone loss diseases.This positions the 14.3.3.ζδderived-Peptide-NCAM1anabolic interaction as a promising and innovative therapeutic target for managing conditions characterized by vascular loss and, consequently, bone loss.Taken together, these findings lay the groundwork for a transformative approach in therapeutic interventions.The NCAM1-14.3.3.ζδ-derived-Peptideanabolic interaction, with its remarkable capacity to drive angiogenesis and bone formation, emerges as a powerful tool in our arsenal to combat conditions marked by vascular and bone insufficiencies, offering hope for a paradigm shift in the management of ageassociated and bone-related disorders.Anabolic endogenous NCAM1-14.3.3.ζδ-derived-Peptideinteraction positions it as a viable candidate for preventing and treating bone loss and fracture.Further studies are urgently required to ascertain and harness the therapeutic potential of NCAM1-14.3.3.ζδ-derived-Peptidepathway in the clinical management of patients with excessive bone loss.
precision and detail using the Zeiss Laser Scanning Confocal Microscope LSM880.Zstacks of immunostained sections were captured utilizing both the 20X Plan Apo/0.8 dry lens and the 10X Plan Apo 0.45 WD=2.0 M27 dry lens.The imaging configuration featured a Zeiss laser scanning microscope 880 equipped with Axio Examiner, incorporating laser lines at 405, 453, 488, 514, 561, 594, and 633 nm.The system included a Colibri 7 epifluorescence light source with LED illumination, four objectives, a fast-scanning stage with PIEZO XY, a 32-channel gallium arsenide phosphide detector (GaAsP) PMT (photomultiplier tube) in addition to a two-channel standard PMT.The acquisition and analysis software encompassed various functionalities, such as measurement, multichannel, panorama, extended manual focus, image analysis, time-lapse, Z Stack, extended focus, autofocus, and incorporated additional modules, including Experiment Designer and Tiles and Position.

For
Imaging with the Leica thunder microscope of immunofluorescent-stained bone sections, the Leica DM6 B Thunder Microscope equipped with LAS X software (Version 3.7.4.23463,Leica) was utilized.The microscope configuration included two HC PL FLUOTAR dry objectives (10X and 20X magnification), one HC PL APO 100X oil objective, a Leica K5 camera for fluorescent imaging, Leica CTR6 LED power supply, Leica EL6000 (120 W metal halide) external light source, and a diode-pumped, solid-state laser (355 nm) with 7 cube filters (LMD-BGR, LMD-GFP band pass, LMD-GFP long pass, LMD-Cy3, LMD-DAPI, LMD-Alexa594, LMD-CFP, LMD-GFP/Cy3, LMD-YFP, LMD-Cy5).Z-stacks were acquired using Leica SmartMove Controls for z (focus) movement and x, y (stage) movement for focusing.The LAS X software facilitated image acquisition, offering tools for adjusting exposure times, contrast, and brightness, Live-imaging-mode, a memory function for two z-positions, tile scan, a subsequent merging function, and Thunder Computational Clearing Software.
typically representing data from multiple independent experiments conducted on different days and involving distinct sets of mice.All the data presented in this study is derived from minimum of three independent experiments.All the sample numbers show biological replicates.It is important to note that sample sizes were not predetermined based on statistical power calculations.Mice were randomly allocated to experiments, and sample processing occurred in an arbitrary order.While blinding techniques were not employed, no animals were excluded from the analyses.Variability within the data was consistently indicated using standard deviation.The derived-Peptide.IG1 domain contain, Fibronectin type -III domain 1(499-598), Fibronectin type -III domain 2(600-695) amino acid.(G) Best predicted model of NCAM 1-14.3.3.ζδ-derived-PeptideInteraction.The colour indicates model confidence.Data Information: Two-tailed unpaired t tests (C).nsp > 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Scale bars: magenda 25 μm; white, 50 μm; yellow, 300 μm.n represents biological replicates.Growth plate: gp; Diaphysis: dp; Metaphysis: mp.