With increases in life expectancy, the number of patients requiring joint replacement therapy and experiencing periprosthetic osteolysis, the most common complication leading to implant failure, is growing or underestimated. In this study, we found that osteolysis progression and osteoclast differentiation in the surface of the skull bone of adult mice were accompanied by significant expansion of lymphatic vessels within bones. Using recombinant VEGF-C protein to activate VEGFR3 and promote proliferation of lymphatic vessels in bone, we counteracted excessive differentiation of osteoclasts and osteolysis caused by titanium alloy particles or inflammatory cytokines LPS/TNF-α. However, this effect was not observed in aged mice because adipogenically differentiated mesenchymal stem cells (MSCs) inhibited the response of lymphatic endothelial cells to agonist proteins. The addition of the JAK inhibitor ruxolitinib restored the response of lymphatic vessels to external stimuli in aged mice to protect against osteolysis progression. These findings suggest that inhibiting SASP secretion by adipogenically differentiated MSCs while activating lymphatic vessels in bone offers a new method to prevent periprosthetic osteolysis during joint replacement follow-up.

Lymphatics are involved in the resolution of inflammation and wound healing, but their role in the oral wound healing process after tooth extraction has never been investigated. We therefore sought to evaluate the healing process following the extraction of maxillary molars in two transgenic mouse models: K14-VEGFR3-Ig mice, which lack initial mucosal lymphatic vessels, and K14-VEGFC mice, which have hyperplastic mucosal lymphatics. Maxillary molars were extracted from both transgenic mouse types and their corresponding wild-type (WT) controls. Mucosal and alveolar bone healing were evaluated. A delayed epithelialization and bone regeneration were observed in K14-VEGFR3-Ig mice compared with their WT littermates. The hampered wound closure was accompanied by decreased levels of epidermal growth factor (EGF) and persistent inflammation, characterized by infiltrates of immune cells and elevated levels of pro-inflammatory markers in the wounds. Hyperplastic mucosal lymphatics did not enhance the healing process after tooth extraction in K14-VEGFC mice. The findings indicate that initial mucosal lymphatics play a major role in the initial phase of the oral wound healing process.

Pineal cysts are frequently encountered as incidental findings in magnetic resonance imaging, usually devoid of symptoms, yet some patients exhibit symptomatic manifestations possibly associated with the cyst, even in the absence of hydrocephalus. The etiology of these symptoms remains contentious. This study aims to investigate the presence of lymphatic endothelial cell (LEC) markers and indications of inflammation or immune response within the pineal cysts of patients experiencing symptomatic non-hydrocephalic presentations. Eight patients who underwent surgical excision of their cysts were included in the study. Immunohistochemistry was utilized to assess the expression of LYVE-1, PDPN, and VEGFR3 as LEC markers, alongside IL-6 and CD3 for indications of inflammation or immune activity. Our analysis revealed an absence of inflammatory markers or immune response. However, a distinct expression of VEGFR3 was observed, likely localized to neurons within the pineal cyst tissue. We propose that these VEGFR3+ neurons within the pineal cyst may contribute to the headache symptoms reported by these patients. Further investigations are warranted to substantiate this hypothesis.

Lymphangiogenesis at primary tumor and draining lymph nodes plays a pivotal role in tumor metastasis, which has been demonstrated to be regulated by the vascular endothelial growth factor receptor 3 (VEGFR-3) pathway. However, the effect of molecular imaging peptides, which specifically bind VEGFR-3, in tracing tumors remains unclear. We prepared a novel peptide, TMVP1448, with high-affinity to VEGFR-3. The dissociation constant (KD) of TMVP1448 with VEGFR-3 was 7.07 ×10-7 M. In vitro cellular assay showed that TMVP1448 could bind specifically with VEGFR-3. Near infrared imaging results showed that Cy7-TMVP1448 was able to accurately trace primary and metastatic cancers, and PET/CT results showed that [68Ga]Ga-DOTA-TMVP1448 was superior to commonly used radiotracers 18F-FDG. Additionally, no significant negative effect of TMVP1448 was found in mice. Our results suggested that TMVP1448 had great potential for future clinical applications in fluorescence imaging and nuclear imaging of tumors.

The β-secretase β-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer\'s disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer\'s disease.

Cervical cancer stands as a prevalent gynaecologic malignancy affecting women globally, often linked to persistent human papillomavirus infection. Biomarkers associated with cervical cancer, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E, show upregulation and are linked to angiogenesis and lymphangiogenesis. This research aims to employ in-silico methods to target tyrosine kinase receptor proteins-VEGFR-1, VEGFR-2, and VEGFR-3, and identify novel inhibitors for Vascular Endothelial Growth Factors receptors (VEGFRs). A comprehensive literary study was conducted which identified 26 established inhibitors for VEGFR-1, VEGFR-2, and VEGFR-3 receptor proteins. Compounds with high-affinity scores, including PubChem ID-25102847, 369976, and 208908 were chosen from pre-existing compounds for creating Deep Learning-based models. RD-Kit, a Deep learning algorithm, was used to generate 43 million compounds for VEGFR-1, VEGFR-2, and VEGFR-3 targets. Molecular docking studies were conducted on the top 10 molecules for each target to validate the receptor-ligand binding affinity. The results of Molecular Docking indicated that PubChem IDs-71465,645 and 11152946 exhibited strong affinity, designating them as the most efficient molecules. To further investigate their potential, a Molecular Dynamics Simulation was performed to assess conformational stability, and a pharmacophore analysis was also conducted for indoctrinating interactions.

Cadmium (Cd) is a widespread environmental toxicant that poses significant threat to public health. After intake, Cd is distributed throughout the body via blood and lymphatic circulation. However, the effect of Cd on lymphatic vessels has not been revealed. In this study, mice were exposed to 10 μM cadmium chloride through drinking water immediately after corneal alkali burn. In vivo analyses showed that Cd treatment enhances the alkali burn-induced corneal lymphangiogenesis, which is characterized by increased expression of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), prospero-related homeobox 1 (PROX-1) and vascular endothelial growth factor receptor 3 (VEGFR3). In vitro, the proliferation and migration of human dermal lymphatic endothelial cells (HDLECs) are increased by 1 μM Cd treatment, while inhibited by 10 μM Cd treatment. At a concentration of 1 μM, Cd specifically induces phosphorylation of signal transducer and activator of transcription 3 (STAT3), but has no effect on the MAPK, AKT, or NF-κB signaling pathway. In the presence of the STAT3 inhibitor STATTIC, Cd fails to induce HDLECs proliferation and migration. In addition, Cd upregulates VEGFR3 expression and its gene promoter activity in a STAT3-dependent manner. Our study suggests that low-dose Cd promotes lymphangiogenesis through activation of the STAT3 signaling pathway.

Generalized lymphatic anomaly (GLA) and kaposiform lymphangiomatosis (KLA) are rare congenital disorders that arise through anomalous embryogenesis of the lymphatic system. A somatic activating NRAS p.Q61R variant has been recently detected in GLA and KLA tissues, suggesting that the NRAS p.Q61R variant plays an important role in the development of these diseases. To address this role, we studied the effect of the NRAS p.Q61R variant in lymphatic endothelial cells (LECs) on the structure of the lymphatics during embryonic and postnatal lymphangiogenesis applying inducible, LEC-specific NRAS p.Q61R variant in mice. Lox-stop-Lox NrasQ61R mice were crossed with Prox1-CreERT2 mice expressing tamoxifen-inducible Cre recombinase specifically in LECs. Whole-mount immunostaining of embryonic back skin using an antibody against the LEC surface marker VEGFR3 showed considerably greater lymphatic vessel width in LEC-specific NRAS p.Q61R mutant embryos than in littermate controls. These mutant embryos also showed a significant reduction in the number of lymphatic vessel branches. Furthermore, immunofluorescence staining of whole-mount embryonic back skin using an antibody against the LEC-specific nuclear marker Prox1 showed a large increase in the number of LECs in LEC-specific NRAS p.Q61R mutants. In contrast, postnatal induction of the NRAS p.Q61R variant in LECs did not cause abnormal lymphatic vessel morphogenesis. These results suggest that the NRAS p.Q61R variant in LECs plays a role in development of lymphatic anomalies. While this model does not directly reflect the human pathology of GLA and KLA, there are overlapping features, suggesting that further study of this model may help in studying GLA and KLA mechanisms.

Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases. Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but, unexpectedly, recent studies have revealed that the Tie2 locus has been lost in many vertebrate species, whereas the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells, where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in endothelial cell migration and proliferation, and in lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show that Angpt1/Tie1 signaling constitutes an essential signaling pathway for lymphatic development in zebrafish.

Silicosis is a disease characterized by lung inflammation and fibrosis caused by long-term inhalation of free silicon dioxide (SiO2). Recent studies have found that a large number of lymphatic hyperplasia occurs during the occurrence and development of silicosis. miRNAs play an important role in lymphangiogenesis. However, the regulation and mechanism of miRNAs on lymphangiogenesis in silicosis remain unclear. In this study, lymphangiogenesis was observed in silicosis rats, and VEGF-C-targeted miRNAs were screened, and the effect of miRNAs on the formation of human lymphatic endothelial cells (HLECs) tubular structure was investigated in vitro. The results showed that SiO2 promoted the expressions of Collagen Ι and α-SMA, TNF-α, IL-6 and VEGF-C increased first and then decreased, and promoted the formation of lymphatic vessels. Bioinformatics methods screened miR-455-3p for targeted binding to VEGF-C, and dual luciferase reporter genes confirmed VEGF-C as the target gene of miR-455-3p, and miR-455-3p was down-regulated in the lung tissue of silicosis rats. Transfection of miR-455-3p Inhibitors down-regulated the expression level of miR-455-3p and up-regulated the expression levels of VEGF-C and VEGFR-3 in HLECs, enhanced migration ability and increased tube formation. Transfection of miR-455-3p Mimics showed an opposite trend. These results suggest that miR-455-3p further regulates the tubular structure formation of HLECs by regulating VEGF-C/VEGFR3. Therefore, targeting miR-455-3p may provide a new therapeutic strategy for SiO2-induced silicosis injury.