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Background/aim: Predicting lymph node metastasis (LNM) in early gastric cancer (EGC) is crucial for making treatment decisions. This study aimed to confirm risk factors for LNM and identify novel auxiliary biomarkers for predicting LNM in EGC., Patients and Methods: We established a training set, comprising 63 patients with LNM-EGC and 274 patients with non-LNM EGC, and a test set, comprising 19 patients with LNM-EGC and 146 non-LNM EGC. Immunohistochemistry for lymphangiogenic and related pathway components (VEGF-C, TGF-β1, SMAD2/3, VEGF-D, pSTAT3, E-cadherin, CD44, c-MET, YAP, and HER2), in situ hybridization for Epstein-Barr virus-encoded small RNAs, and multiplex PCR for microsatellite instability were conducted., Results: In the training set, Lauren's diffuse/mixed classification, stromal desmoplasia, submucosal invasion ≥500 μm, lymphatic invasion, and high VEGF-C and SMAD2/3 expression were independent risk factors for LNM (p<0.05). A large tumor size, mixed histology, submucosal invasion, perineural invasion, and ulceration were determined as risk factors using univariate analysis (p<0.05). The tumor cutoff size for predicting LNM was 2.65 cm, based on a ROC analysis. The test set study verified that stromal desmoplasia, submucosal invasion, and high VEGF-C expression were independent risk factors for LNM (p<0.05). Moreover, mixed histology, lymphatic invasion, ulceration, and high SMAD 2/3 expression were identified as additional risk factors using univariate analysis (p<0.05)., Conclusion: Stromal desmoplasia, submucosal invasion, and high VEGF-C expression are potential biomarkers for LNM in EGC. VEGF-C expression might serve as an adjunct biomarker for predicting LNM on forceps-biopsy tissue at initial diagnosis., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Purpose: Dysfunctional lymphangiogenesis is pivotal for various pathological processes including tumor lymph node metastasis which is a crucial cause of therapeutic failure for ESCC. In this study, we aim to elucidate the molecular mechanisms and clinical relevance of Zinc-finger protein ZNF468 in lymphangiogenesis and lymphatic metastasis in ESCC., Methods: Immunohistochemistry, Western blot, Kaplan-Meier plotter analysis and Gene Set Enrichment Analysis were preformed to detect the association of ZNF468 with lymphangiogenesis and poor prognosis in ESCC patients. Foot-pads lymph node metastasis model, tube formation assay, 3D-culture assay and invasion assay were preformed to verify the effect of ZNF468 on lymphangiogenesis and lymph node metastasis. CUT&Tag analysis, immunoprecipitation and mass spectrometry analysis and ChIP-PCR assay were preformed to study the molecular mechanisms of ZNF468 in lymphangiogenesis., Results: We found that ectopic expression of ZNF468 was correlated with higher microlymphatic vessel density in ESCC tissues, leading to poorer prognosis of ESCC patients. ZNF468 enhanced the capacity of lymphangiogenesis and promoted lymphatic metastasis in ESCC both in vitro and in vivo. However, silencing ZNF468 reversed these phenotypes in ESCC. Mechanically, we demonstrated that ZNF468 recruits the histone modification factors (PRMT1/HAT1) to increase the levels of H4R2me2a and H3K9ac, which then leads to the recruitment of the transcription initiation complex on the VEGF-C promoter, ultimately promoting the upregulation of VEGF-C transcription. Strikingly, the promoting effect of lymphatic metastasis induced by ZNF468 in ESCC was abrogated by targeting PRMT1 using Arginine methyltransferase inhibitor-1 or silencing VEGF-C. Furthermore, we found that the activation of PI3K/AKT and ERK1/2 signaling is required for ZNF468-medicated lymphatic metastasis in ESCC. Importantly, the clinical relevance between ZNF468 and VEGF-C were confirmed not only in ESCC samples and but also in multiple cancer types., Conclusion: Our results identified a precise mechanism underlying ZNF468-induced epigenetic upregulation of VEGF-C in facilitating lymphangiogenesis and lymph node metastasis of ESCC, which might provide a novel prognostic biomarker and potential therapeutic for ESCC patients., (© 2024. Springer Nature Switzerland AG.)

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., (© 2024. The Author(s).)

Ubiquitination plays an essential role in protein stability, subcellular localization, and interactions. Crosstalk between different types of ubiquitination results in distinct biological outcomes for proteins. However, the role of ubiquitination-related crosstalk in lymph node (LN) metastasis and the key regulatory factors controlling this process have not been determined. Using high-throughput sequencing, we found that ubiquitin-conjugating enzyme E2 C (UBE2C) was overexpressed in bladder cancer (BCa) and was strongly associated with an unfavorable prognosis. Overexpression of UBE2C increased BCa lymphangiogenesis and promoted LN metastasis both in vitro and in vivo. Mechanistically, UBE2C mediated sodium-coupled neutral amino acid transporter 2 (SNAT2) monoubiquitination at lysine 59 to inhibit K63-linked polyubiquitination at lysine 33 of SNAT2. Crosstalk between monoubiquitination and K63-linked polyubiquitination increased SNAT2 membrane protein levels by suppressing epsin 1-mediated (EPN1-mediated) endocytosis. SNAT2 facilitated glutamine uptake and metabolism to promote VEGFC secretion, ultimately leading to lymphangiogenesis and LN metastasis in patients with BCa. Importantly, inhibition of UBE2C significantly attenuated BCa lymphangiogenesis in a patient-derived xenograft model. Our results reveal the mechanism by which UBE2C mediates crosstalk between the monoubiquitination and K63-linked polyubiquitination of SNAT2 to promote BCa metastasis and identify UBE2C as a promising target for treating LN-metastatic BCa.

Silicosis is a disease characterized by lung inflammation and fibrosis caused by long-term inhalation of free silicon dioxide (SiO 2 ). 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 SiO 2 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 SiO 2 -induced silicosis injury., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and young adults. Account for 80% of all OS cases, conventional OS are characterized by the presence of osteoblastic, chondroblastic and fibroblastic cell types. Despite this heterogeneity, therapeutic treatment and prognosis of OS are essentially the same for all OS subtypes. Here, we report that DEC2, a transcriptional repressor, is expressed at higher levels in chondroblastic OS compared with osteoblastic OS. This difference suggests that DEC2 is disproportionately involved in the progression of chondroblastic OS, and thus, DEC2 may represent a possible molecular target for treating this type of OS. In the human chondroblastic-like OS cell line MNNG/HOS, we found that overexpression of DEC2 affects the proliferation of the cells by activating the VEGFC/VEGFR2 signalling pathway. Enhanced expression of DEC2 increased VEGFR2 expression, as well as increased the phosphorylation levels at sites Y951 and Y1175 of VEGFR2. On the one hand, activation of VEGFR2 Y1175 enhanced cell proliferation through VEGFR2 Y1175 -PLCγ1-PKC-SPHK-MEK-ERK signalling. On the other hand, activation of VEGFR2 Y951 decreased mitochondria-dependent apoptosis rate through VEGFR2 Y951 -VARP-PI3K-AKT signalling. Activation of these two signalling pathways resulted in enhanced progression of chondroblastic OS. In conclusion, DEC2 plays a pivotal role in cell proliferation and apoptosis-resistance in chondroblastic OS via the VEGFC/VEGFR2 signalling pathway. These findings lay the groundwork for developing focused treatments that target specific types of OS., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cervical cancer (CC) patients with lymph node metastasis (LNM) have a poor prognosis. However, the molecular mechanism of LNM in CC is unclear, and there is no effective clinical treatment. Here, we found that 7-dehydrocholesterol reductase (DHCR7), an enzyme that catalyzes the last step of cholesterol synthesis, was upregulated in CC and closely related to LNM. Gain-of-function and loss-of-function experiments proved that DHCR7 promoted the invasion ability of CC cells and lymphangiogenesis in vitro and induced LNM in vivo. The LNM-promoting effect of DHCR7 was partly mediated by upregulating KN motif and ankyrin repeat domains 4 (KANK4) expression and subsequently activating the PI3K/AKT signaling pathway. Alternatively, DHCR7 promoted the secretion of vascular endothelial growth factor-C (VEGF-C), and thereby lymphangiogenesis. Interestingly, cholesterol reprogramming was needed for the DHCR7-mediated promotion of activation of the KANK4/PI3K/AKT axis, VEGF-C secretion, and subsequent LNM. Importantly, treatment with the DHCR7 inhibitors AY9944 and tamoxifen (TAM) significantly inhibited LNM of CC, suggesting the clinical application potential of DHCR7 inhibitors in CC. Collectively, our results uncover a novel molecular mechanism of LNM in CC and identify DHCR7 as a new potential therapeutic target., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Secondary lymphedema (LD) corresponds to a severe lymphatic dysfunction leading to the accumulation of fluid and fibrotic adipose tissue in a limb. Here, we identified apelin (APLN) as a powerful molecule for regenerating lymphatic function in LD. We identified the loss of APLN expression in the lymphedematous arm compared to the normal arm in patients. The role of APLN in LD was confirmed in APLN knockout mice, in which LD is increased and associated with fibrosis and dermal backflow. This was reversed by intradermal injection of APLN-lentivectors. Mechanistically, APLN stimulates lymphatic endothelial cell gene expression and induces the binding of E2F8 transcription factor to the promoter of CCBE1 that controls VEGF-C processing. In addition, APLN induces Akt and eNOS pathways to stimulate lymphatic collector pumping. Our results show that APLN represents a novel partner for VEGF-C to restore lymphatic function in both initial and collecting vessels. As LD appears after cancer treatment, we validated the APLN-VEGF-C combination using a novel class of nonintegrative RNA delivery LentiFlash® vector that will be evaluated for phase I/IIa clinical trial., (© 2024. The Author(s).)

The purpose of this study was to explore the differential expression of Pax3, Rad51 and VEGF-C in esophageal gastric junction adenocarcinoma and distal gastric adenocarcinoma and their relationship with cancer occurrence and development. 57 patients with gastric cancer were included and divided into esophageal gastric junction adenocarcinoma group (n=28) and distal gastric adenocarcinoma group (n=29). The positive expressions of Pax3, Rad51 and VEGF-C in the control group were lower than those in the esophageal gastric junction adenocarcinoma group and distal gastric adenocarcinoma group respectively (P<0.05). In esophageal gastric junction adenocarcinoma with low differentiation, positive expressions of Pax3, Rad51, and VEGF-C surpassed those in high/medium differentiation (P<0.05). Serosa-infiltrated cases exhibited higher Pax3 and Rad51 expressions compared to non-infiltrated cases (P<0.05). Rad51 and VEGF-C positivity were notably elevated in cases with lymph node metastasis compared to those without (P<0.05). Distal gastric adenocarcinoma displayed higher VEGF expression than middle/low differentiated adenocarcinomas. Rad51 expression was significantly higher in women than in men (P<0.05). The positive rates of Pax3, Rad51, and VEGF-C were markedly increased in esophageal gastric junction adenocarcinoma and distal gastric adenocarcinoma compared to normal gastric tissue, and these were associated with the degree of differentiation, depth of invasion, and lymph node metastasis in patients. Particularly, Rad51 exhibited a positive correlation with cancer cell differentiation, invasion depth, and lymph node metastasis in cancer tissue.

Recent studies have described the importance of lymphatics in numerous organ-specific physiological and pathological processes. The role of meningeal lymphatics in various neurological and cerebrovascular diseases has been suggested. It has also been shown that these structures develop postnatally and are altered by aging and that the vascular endothelial growth factor C (VEGFC)/ vascular endothelial growth factor receptor 3 (VEGFR3) signaling plays an essential role in the development and maintenance of them. However, the molecular mechanisms governing the development and maintenance of meningeal lymphatics are still poorly characterized. Recent in vitro cell culture-based experiments, and in vivo studies in zebrafish and mouse skin suggest that collagen and calcium binding EGF domains 1 (CCBE1) is involved in the processing of VEGFC. However, the organ-specific role of CCBE1 in developmental lymphangiogenesis and maintenance of lymphatics remains unclear. Here, we aimed to investigate the organ-specific functions of CCBE1 in developmental lymphangiogenesis and maintenance of meningeal lymphatics during aging. We demonstrate that inducible deletion of CCBE1 leads to impaired postnatal development of the meningeal lymphatics and decreased macromolecule drainage to deep cervical lymph nodes. The structural integrity and density of meningeal lymphatics are gradually altered during aging. Furthermore, the meningeal lymphatic structures in adults showed regression after inducible CCBE1 deletion. Collectively, our results indicate the importance of CCBE1-dependent mechanisms not only in the development, but also in the prevention of the age-related regression of meningeal lymphatics. Therefore, targeting CCBE1 may be a good therapeutic strategy to prevent age-related degeneration of meningeal lymphatics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)