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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. [ABSTRACT FROM AUTHOR]

Abstract The development of treatments targeting the vascular endothelial growth factor (VEGF) signaling pathways have traditionally been firstly investigated in oncology and then advanced into retinal disease indications. Members of the VEGF family of endogenous ligands and their respective receptors play a central role in vasculogenesis and angiogenesis during both development and physiological homeostasis. They can also play a pathogenic role in cancer and retinal diseases. Therapeutic approaches have mostly focused on targeting VEGF-A signaling; however, research has shown that VEGF-C and VEGF-D signaling pathways are also important to the disease pathogenesis of tumors and retinal diseases. This review highlights the important therapeutic advances and the remaining unmet need for improved therapies targeting additional mechanisms beyond VEGF-A. Additionally, it provides an overview of alternative VEGF-C and VEGF-D signaling involvement in both health and disease, highlighting their key contributions in the multifactorial pathophysiology of retinal disease including neovascular age-related macular degeneration (nAMD). Strategies for targeting VEGF-C/-D signaling pathways will also be reviewed, with an emphasis on agents currently being developed for the treatment of nAMD.

The development of treatments targeting the vascular endothelial growth factor (VEGF) signaling pathways have traditionally been firstly investigated in oncology and then advanced into retinal disease indications. Members of the VEGF family of endogenous ligands and their respective receptors play a central role in vasculogenesis and angiogenesis during both development and physiological homeostasis. They can also play a pathogenic role in cancer and retinal diseases. Therapeutic approaches have mostly focused on targeting VEGF-A signaling; however, research has shown that VEGF-C and VEGF-D signaling pathways are also important to the disease pathogenesis of tumors and retinal diseases. This review highlights the important therapeutic advances and the remaining unmet need for improved therapies targeting additional mechanisms beyond VEGF-A. Additionally, it provides an overview of alternative VEGF-C and VEGF-D signaling involvement in both health and disease, highlighting their key contributions in the multifactorial pathophysiology of retinal disease including neovascular age-related macular degeneration (nAMD). Strategies for targeting VEGF-C/-D signaling pathways will also be reviewed, with an emphasis on agents currently being developed for the treatment of nAMD. [ABSTRACT FROM AUTHOR]

Current clinical practice primarily relies on surgical intervention to remove hematomas in patients with intracerebral hemorrhage (ICH), given the lack of effective drug therapies. Previous research indicates that simvastatin (SIM) may enhance hematoma absorption and resolution in the acute phase of ICH, though the precise mechanisms remain unclear. Recent findings have highlighted the glymphatic system (GS) as a crucial component in intracranial cerebrospinal fluid circulation, playing a significant role in hematoma clearance post-ICH. This study investigates the link between SIM efficacy in hematoma resolution and the GS. Our experimental results show that SIM alleviates GS damage in ICH-induced rats, resulting in improved outcomes such as reduced brain edema, neuronal apoptosis, and degeneration. Further analysis reveals that SIM's effects are mediated through the VEGF-C/VEGFR3/PI3K-Akt pathway. This study advances our understanding of SIM's mechanism in promoting intracranial hematoma clearance and underscores the potential of targeting the GS for ICH treatment.

Abstract Purpose Our explorative study assessed a panel of molecules for their association with epithelial ovarian carcinomas and their prognostic implications. The panel included tissue expression of VEGF-C, COX-2, Ki-67 and eNOS alongside plasma levels of VEGF-C and nitric oxide. Methods 130 cases were enrolled in the study. Plasma levels were quantified by ELISA and tissue expressions were scored by immunohistochemistry. The Chi square and Fischer’s exact test were applied to examine the impact of markers on clinicopathological factors. Non-parametric Spearman’s rank correlation test was applied to define the association among test factors. Results Plasma VEGF-C levels and COX-2 tissue expression strongly predicted recurrence and poor prognosis (

Purpose: Our explorative study assessed a panel of molecules for their association with epithelial ovarian carcinomas and their prognostic implications. The panel included tissue expression of VEGF-C, COX-2, Ki-67 and eNOS alongside plasma levels of VEGF-C and nitric oxide. Methods: 130 cases were enrolled in the study. Plasma levels were quantified by ELISA and tissue expressions were scored by immunohistochemistry. The Chi square and Fischer's exact test were applied to examine the impact of markers on clinicopathological factors. Non-parametric Spearman's rank correlation test was applied to define the association among test factors. Results: Plasma VEGF-C levels and COX-2 tissue expression strongly predicted recurrence and poor prognosis (< 0.001). Tissue Ki-67 was strongly indicative of late-stage disease (< 0.001). The aforementioned markers significantly associated with clinicopathological factors. Nuclear staining of VEGF-C was intriguing and was observed to correlate with high grade-stage malignancies, highly elevated plasma VEGF-C, and with recurrence. eNOS tissue expression showed no significant impact while nitric oxide associated positively with ascites levels. Tissue expression of VEGF-C did not associate significantly with poor prognosis although the expression was highly upregulated in most of the cases. Conclusion: Plasma VEGF-C holds immense promise as a prognostic marker and the nuclear staining of VEGF-C seems to have some significant implication in molecular carcinogenesis and is a novel finding that commands further robust scrutiny. We present a first such study that assesses a set of biomarkers for prognostic implications in clinical management of epithelial ovarian carcinomas in a pan-Indian (Asian) population. [ABSTRACT FROM AUTHOR]

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. Synopsis: Lymphedema is a disorder of the lymphatic system characterized by massive swelling due to the accumulation of undrained interstitial fluid and fibrotic adipose tissue. Regenerative medicine for lymphedema has to combine molecules to repair both superficial capillaries and deeper collecting vessels. Apelin expression is downregulated in lymphedema tissues from patients. Apelin deficiency increases lymphedema in mice. Apelin restores lymphatic collecting vessel pumping and reduces fibrosis. Transient mRNA delivery of Apelin combined to VEGFC restores function in the entire lymphatic network from capillaries to collecting vessels. Lymphedema is a disorder of the lymphatic system characterized by massive swelling due to the accumulation of undrained interstitial fluid and fibrotic adipose tissue. Regenerative medicine for lymphedema has to combine molecules to repair both superficial capillaries and deeper collecting vessels. [ABSTRACT FROM AUTHOR]

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.

Abstract Background Sepsis-associated encephalopathy (SAE) is an acute cerebral dysfunction caused by sepsis. Neuroinflammation induced by sepsis is considered a potential mechanism of SAE; however, very little is known about the role of the meningeal lymphatic system in SAE. Methods Sepsis was established in male C57BL/6J mice by intraperitoneal injection of 5 mg/kg lipopolysaccharide, and the function of meningeal lymphatic drainage was assessed. Adeno-associated virus 1-vascular endothelial growth factor C (AAV1-VEGF-C) was injected into the cisterna magna to induce meningeal lymphangiogenesis. Ligation of deep cervical lymph nodes (dCLNs) was performed to induce pre-existing meningeal lymphatic dysfunction. Cognitive function was evaluated by a fear conditioning test, and inflammatory factors were detected by enzyme-linked immunosorbent assay. Results The aged mice with SAE showed a significant decrease in the drainage of OVA-647 into the dCLNs and the coverage of the Lyve-1 in the meningeal lymphatic, indicating that sepsis impaired meningeal lymphatic drainage and morphology. The meningeal lymphatic function of aged mice was more vulnerable to sepsis in comparison to young mice. Sepsis also decreased the protein levels of caspase-3 and PSD95, which was accompanied by reductions in the activity of hippocampal neurons. Microglia were significantly activated in the hippocampus of SAE mice, which was accompanied by an increase in neuroinflammation, as indicated by increases in interleukin-1 beta, interleukin-6 and Iba1 expression. Cognitive function was impaired in aged mice with SAE. However, the injection of AAV1-VEGF-C significantly increased coverage in the lymphatic system and tracer dye uptake in dCLNs, suggesting that AAV1-VEGF-C promotes meningeal lymphangiogenesis and drainage. Furthermore, AAV1-VEGF-C reduced microglial activation and neuroinflammation and improved cognitive dysfunction. Improvement of meningeal lymphatics also reduced sepsis-induced expression of disease-associated genes in aged mice. Pre-existing lymphatic dysfunction by ligating bilateral dCLNs aggravated sepsis-induced neuroinflammation and cognitive impairment. Conclusion The meningeal lymphatic drainage is damaged in sepsis, and pre-existing defects in this drainage system exacerbate SAE-induced neuroinflammation and cognitive dysfunction. Promoting meningeal lymphatic drainage improves SAE. Manipulation of meningeal lymphangiogenesis could be a new strategy for the treatment of SAE.

Abstract 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.

Background: Sepsis-associated encephalopathy (SAE) is an acute cerebral dysfunction caused by sepsis. Neuroinflammation induced by sepsis is considered a potential mechanism of SAE; however, very little is known about the role of the meningeal lymphatic system in SAE. Methods: Sepsis was established in male C57BL/6J mice by intraperitoneal injection of 5 mg/kg lipopolysaccharide, and the function of meningeal lymphatic drainage was assessed. Adeno-associated virus 1-vascular endothelial growth factor C (AAV1-VEGF-C) was injected into the cisterna magna to induce meningeal lymphangiogenesis. Ligation of deep cervical lymph nodes (dCLNs) was performed to induce pre-existing meningeal lymphatic dysfunction. Cognitive function was evaluated by a fear conditioning test, and inflammatory factors were detected by enzyme-linked immunosorbent assay. Results: The aged mice with SAE showed a significant decrease in the drainage of OVA-647 into the dCLNs and the coverage of the Lyve-1 in the meningeal lymphatic, indicating that sepsis impaired meningeal lymphatic drainage and morphology. The meningeal lymphatic function of aged mice was more vulnerable to sepsis in comparison to young mice. Sepsis also decreased the protein levels of caspase-3 and PSD95, which was accompanied by reductions in the activity of hippocampal neurons. Microglia were significantly activated in the hippocampus of SAE mice, which was accompanied by an increase in neuroinflammation, as indicated by increases in interleukin-1 beta, interleukin-6 and Iba1 expression. Cognitive function was impaired in aged mice with SAE. However, the injection of AAV1-VEGF-C significantly increased coverage in the lymphatic system and tracer dye uptake in dCLNs, suggesting that AAV1-VEGF-C promotes meningeal lymphangiogenesis and drainage. Furthermore, AAV1-VEGF-C reduced microglial activation and neuroinflammation and improved cognitive dysfunction. Improvement of meningeal lymphatics also reduced sepsis-induced expression of disease-associated genes in aged mice. Pre-existing lymphatic dysfunction by ligating bilateral dCLNs aggravated sepsis-induced neuroinflammation and cognitive impairment. Conclusion: The meningeal lymphatic drainage is damaged in sepsis, and pre-existing defects in this drainage system exacerbate SAE-induced neuroinflammation and cognitive dysfunction. Promoting meningeal lymphatic drainage improves SAE. Manipulation of meningeal lymphangiogenesis could be a new strategy for the treatment of SAE. [ABSTRACT FROM AUTHOR]

[Display omitted] • VEGF-C can be used as a proxy for lymphatic system function in AHF. • Low VEGF-C levels were associated with the clinical picture of congestion (fluid accumulation) in patients hospitalized for AHF. • Low serum VEGF-C levels were independently associated with adverse clinical outcomes: 1-year mortality and composite endpoint (1-year mortality and heart failure rehospitalizations). Although vascular endothelial growth factor C (VEGF-C) is a known lymphangiogenesis modulator, its relationship with congestion formation and outcomes in acute heart failure (AHF) is unknown. Serum VEGF-C levels were measured in 237 patients hospitalized for AHF. The population was stratified by VEGF-C levels and linked with clinical signs of congestion and outcomes. The study's population was divided in VEGF-C tertiles: low (median [Q25–Q75]: 33 [15–175]), medium (606 [468–741]) and high (1141 [968–1442] pg/mL). The group with low VEGF-C on admission presented with the highest prevalence of severe lower-extremity edema (low VEGF-C vs medium VEGF-C vs high VEGF-C): 30% vs 13% vs 20%; P = 0.02); the highest percentage of patients with ascites: 22% vs 9% vs 6%; P = 0.006; and the lowest proportion of patients with pulmonary congestion: 22% vs 30% vs 46%; P = 0.004. The 1-year mortality rate was the highest in the low VEGF-C tertile: 35% vs 28% vs 18%, respectively; P = 0.049. The same pattern was observed for the composite endpoint (death and AHF rehospitalization): 45% vs 43% vs 26%; P = 0.029. The risks of death at 1-year follow-up and composite endpoint were significantly lower in the high VEGF-C group. Low VEGF-C was associated with more severe signs of congestion (signs of fluid accumulation) and adverse clinical outcomes. [ABSTRACT FROM AUTHOR]

Meningeal lymphatic vessels (MLVs) help maintain central nervous system (CNS) homeostasis via their ability to facilitate macromolecule waste clearance and neuroimmune trafficking. Although these vessels were overlooked for centuries, they have now been characterized in humans, non-human primates, and rodents. Recent studies in mice have explored the stereotyped growth and expansion of MLVs in dura mater, the various transcriptional, signaling, and environmental factors regulating their development and long-term maintenance, and the pathological changes these vessels undergo in injury, disease, or with aging. Key insights gained from these studies have also been leveraged to develop therapeutic approaches that help augment or restore MLV functions to improve brain health and cognition. Here, we review fundamental processes that control the development of peripheral lymphatic networks and how these might apply to the growth and expansion of MLVs in their unique meningeal environment. We also emphasize key findings in injury and disease models that may reveal additional insights into the plasticity of these vessels throughout the lifespan. Finally, we highlight unanswered questions and future areas of study that can further reveal the exciting therapeutic potential of meningeal lymphatics. [ABSTRACT FROM AUTHOR]

ITGA5, a fibronectin receptor was highly expressed in laryngeal squamous cell carcinoma (LSCC) samples and was related to poor survival. However, the potential mechanism remains unclear. To elucidate the regulatory role of ITGA5 in LSCC progression, we investigated the effect of ITGA5 expression on lymphangiogenesis, migration, and invasion of LSCC cells in vitro and in vivo using immunohistochemistry, siRNA transfection, qRT-PCR, western blotting, enzyme-linked immunosorbent assay, flow cytometry, transwell co-culture, tube formation, cell migration, and invasion assays, and a subcutaneous graft tumor model. The expression of ITGA5 was higher in the LSCC tissues and linked to lymph node metastasis and T staging. Moreover, ITGA5 expression was significantly positively correlated with VEGF-C expression, and the lymphatic vessel density of patients with high ITGA5 expression was noticeably higher than that of patients with low ITGA5 expression. Additionally, it was found in vitro that downregulation of ITGA5 expression not only inhibited the expression and secretion of VEGF-C, but also suppressed the tube-forming ability of human lymphatic endothelial cells (HLECs) and the migration and invasion ability of LSCC cells, while exogenous VEGF-C supplementation reversed these phenomena. Furthermore, a tumor xenograft assay showed that si-ITGA5 restrained the growth and metastasis of TU212-derived tumors in vivo. Our findings suggested that ITGA5 induces lymphangiogenesis and LSCC cell migration and invasion by enhancing VEGF-C expression and secretion. [ABSTRACT FROM AUTHOR]

Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. There is no cure for the disease. Clinically, a preventive surgical approach called immediate lymphatic reconstruction (ILR) has gained traction. Experimental gene-based therapeutic approaches (e.g., using viral vectors) have had limited translational applicability. Tissue nanotransfection (TNT) technology uses a direct, transcutaneous nonviral vector, gene delivery using a chip with nanochannel poration in response to a rapid (

Background: Secretory factors linked to lymphogenesis, such as vascular endothelial growth factor C (VEGF-C), angiopoietin like protein 4 (ANGPTL4), and activin A (ACV-A), have been recognized as potential markers of chronic inflammatory status and age-related diseases. Furthermore, these factors may also be linked to frailty. The primary objective of this study was to examine the serum VEGF-C, ANGPTL4, and ACV-A levels in young individuals, healthy older individuals, and older individuals with pre-frailty and frailty, and to determine their association with pro-inflammatory factor levels. Methods: We conducted an observational study, enrolling a total of 210 older individuals and 20 young healthy volunteers. Participants were divided into four groups based on the Freid frailty phenotype: healthy young group, older patients without frailty group, pre-frail older group, and frail older group. Plasma and peripheral blood mononuclear cells (PBMCs) were collected from all four groups. ELISA was used to measure the serum levels of VEGF-C, ANGPTL4, ACV-A, and pro-inflammatory cytokines, while RT-qPCR was used to measure the transcription level of VEGF-C, ANGPTL4 and ACV-A in PBMCs. Results: In comparison to healthy young individuals and older participants without frailty, older participants with frailty exhibited lower renal function, higher serum levels and transcription levels of VEGF-C, ANGPTL4, ACV-A, and elevated levels of pro-inflammatory cytokines (CRP, IL-1β, and TNF-α). Multiple linear regression analysis revealed that serum levels of VEGF-C, ANGPTL4, and ACV-A were positively correlated with the frailty index, independent of age, eGFR, and comorbidities. Furthermore, the receiver operating characteristic (ROC) curve analysis demonstrated that serum levels of VEGF-C, ANGPTL4, and ACV-A have great accuracy in predicting frailty. Conclusion: Elevated serum levels of VEGF-C, ANGPTL4, and ACV-A are associated with frailty status.

Recent studies, conducted mainly on the rodent model, have demonstrated that regulatory pathway in the skin provided by glycosaminoglycans, nuclear factor of activated T cells 5 (NFAT5), vascular endothelial growth factor C (VEGF-C) and process of lymphangiogenesis may play an important role in extrarenal regulation of sodium (Na+) balance, body water volume, and blood pressure. We aimed to investigate the concentrations and relations among the main factors of this pathway in human skin to confirm that this regulatory axis also exists in humans. Skin specimens from patients diagnosed with arterial hypertension and from control group were histologically and molecularly examined. The primary hypertensive and control groups did not differ in Na+ ​concentrations in the skin. However, the patients with hypertension and higher skin Na+ concentration had significantly greater density of skin lymphatic vessels. Higher skin Na+concentration was associated with higher skin water content. In turn, skin water content correlated with factors associated with lymphangiogenesis , i.e. NFAT5, VEGF-C, and podoplanin (PDPN) mRNA expression in the skin. The strong mutual pairwise correlations of the expressions of NFAT5, VEGF-C, vascular endothelial growth factor D (VEGF-D) and PDPN mRNA were noted in the skin in all of the studied groups. Our study confirms that skin interstitium and the lymphatic system may be important players in the pathophysiology of arterial hypertension in humans. Based on the results of our study and existing literature in this field, we propose the hypothetical model which might explain the phenomenon of salt-sensitivity. [ABSTRACT FROM AUTHOR]

The cancer-promoting ligand vascular endothelial growth factor-C (VEGF-C) activates VEGF receptor-3 (VEGFR-3). The VEGF-C/VEGFR-3 axis is expressed by a range of human tumor cells in addition to lymphatic endothelial cells. Activating the VEGF-C/VEGFR-3 signaling enhances metastasis by promoting lymphangiogenesis and angiogenesis inside and around tumors. Stimulation of VEGF-C/VEGFR-3 signaling promotes tumor metastasis in tumors, such as ovarian, renal, pancreatic, prostate, lung, skin, gastric, colorectal, cervical, leukemia, mesothelioma, Kaposi sarcoma, and endometrial carcinoma. We discuss and update the role of VEGF-C/VEGFR-3 signaling in tumor development and the research is still needed to completely comprehend this multifunctional receptor. [ABSTRACT FROM AUTHOR]

Purpose of Review: This review aims to summarize the current knowledge regarding the pharmacological interventions studied in both experimental and clinical trials for secondary lymphedema. Recent Findings: Lymphedema is a progressive disease that results in tissue swelling, pain, and functional disability. The most common cause of secondary lymphedema in developed countries is an iatrogenic injury to the lymphatic system during cancer treatment. Despite its high incidence and severe sequelae, lymphedema is usually treated with palliative options such as compression and physical therapy. However, recent studies on the pathophysiology of lymphedema have explored pharmacological treatments in preclinical and early phase clinical trials. Summary: Many potential treatment options for lymphedema have been explored throughout the past two decades including systemic agents and topical approaches to decrease the potential toxicity of systemic treatment. Treatment strategies including lymphangiogenic factors, anti-inflammatory agents, and anti-fibrotic therapies may be used independently or in conjunction with surgical approaches. [ABSTRACT FROM AUTHOR]

Together with the platelet-derived growth factors (PDGFs), the vascular endothelial growth factors (VEGFs) form the PDGF/VEGF subgroup among cystine knot growth factors. The evolutionary relationships within this subgroup have not been examined thoroughly to date. Here, we comprehensively analyze the PDGF/VEGF growth factors throughout all animal phyla and propose a phylogenetic tree. Vertebrate whole-genome duplications play a role in expanding PDGF/VEGF diversity, but several limited duplications are necessary to account for the temporal pattern of emergence. The phylogenetically oldest PDGF/VEGF-like growth factor likely featured a C-terminus with a BR3P signature, a hallmark of the modern-day lymphangiogenic growth factors VEGF-C and VEGF-D. Some younger VEGF genes, such as VEGFB and PGF, appeared completely absent in important vertebrate clades such as birds and amphibia, respectively. In contrast, individual PDGF/VEGF gene duplications frequently occurred in fish on top of the known fish-specific whole-genome duplications. The lack of precise counterparts for human genes poses limitations but also offers opportunities for research using organisms that diverge considerably from humans. Sources for the graphical abstract: 326 MYA and older [1]; 72–240 MYA [2]; 235–65 MYA [3] [ABSTRACT FROM AUTHOR]

Introduction: Surgery and radiotherapy are key cancer treatments and the leading causes of damage to the lymphatics, a vascular network critical to fluid homeostasis and immunity. The clinical manifestation of this damage constitutes a devastating side-effect of cancer treatment, known as lymphoedema. Lymphoedema is a chronic condition evolving from the accumulation of interstitial fluid due to impaired drainage via the lymphatics and is recognised to contribute significant morbidity to patients who survive their cancer. Nevertheless, the molecular mechanisms underlying the damage inflicted on lymphatic vessels, and particularly the lymphatic endothelial cells (LEC) that constitute them, by these treatment modalities, remain poorly understood. Methods: We used a combination of cell based assays, biochemistry and animal models of lymphatic injury to examine the molecular mechanisms behind LEC injury and the subsequent effects on lymphatic vessels, particularly the role of the VEGF-C/VEGF-D/VEGFR-3 lymphangiogenic signalling pathway, in lymphatic injury underpinning the development of lymphoedema. Results: We demonstrate that radiotherapy selectively impairs key LEC functions needed for new lymphatic vessel growth (lymphangiogenesis). This effect is mediated by attenuation of VEGFR-3 signalling and downstream signalling cascades. VEGFR-3 protein levels were downregulated in LEC that were exposed to radiation, and LEC were therefore selectively less responsive to VEGF-C and VEGF-D. These findings were validated in our animal models of radiation and surgical injury. Discussion: Our data provide mechanistic insight into injury sustained by LEC and lymphatics during surgical and radiotherapy cancer treatments and underscore the need for alternative non-VEGF-C/VEGFR-3-based therapies to treat lymphoedema. [ABSTRACT FROM AUTHOR]

Background: Tumor lymphangiogenesis is a critical component in the progression of cancers and specific microRNAs have been reported to be implicated in this process. Recent studies revealed the involvement of miR‐1236 in lymphangiogenic signaling by targeting vascular endothelial growth factor receptor 3 (VEGFR3). However, the prognostic importance of miR‐1236 and its clinical relevance for lymphangiogenesis in ovarian cancer (OC) remains unclear. Methods: The study included 52 ovarian tumors and 28 normal ovarian tissues. Quantitative real‐time PCR was utilized to analyze the VEGFR3, VEGF‐C, LYVE‐1 and PROX1 mRNA expression as well as miR‐1236. VEGFR3 protein expression was measured by immunohistochemistry staining. Immunohistochemistry for the podoplanin marker (D2‐40) was performed to measure lymphatic vessel density (LVD). In addition, diagnostic evaluation based on the receiver‐operating characteristic (ROC) curve was performed. The influence of miR‐1236 on overall survival was evaluated by Kaplan–Meier method. Results: Here, we show that miR‐1236 expression was significantly decreased in ovarian tumors compared with control tissues (p < 0.001) and correlated with advanced clinical stage, lymph node metastasis, distant metastasis and patient survival (All P < 0.05). Moreover, in ovarian tumors, LVD as well as the gene expression of VEGFR3, VEGF‐C and LYVE‐1, but not PROX1, were found to be remarkably higher compared with control tissues. We also detected a more robust positive staining for VEGFR3 in OC tissues than in control tissues. Furthermore, our results demonstrated an inverse association of miR‐1236 expression with LVD, VEGFR3, LYVE‐1 and PROX1 expression in OC tissues. The ROC curve analysis indicated that miR‐1236 expression has the potential to be used as a diagnostic and prognostic biomarker in OC. Survival analysis further verified a lowered overall survival rate in patients with low miR‐1236 expression than in those with high expression. Conclusions: Our results provide evidence for the translational involvement of miR‐1236 in the lymphangiogenesis of OC by regulating lymphangiogenesis‐related factors and support the clinical importance of miR‐1236 as a new diagnostic and prognostic biomarker for OC. [ABSTRACT FROM AUTHOR]

Abstract Background NEAT1 has been shown to play an oncogenic role in many kinds of cancers. However, detailed roles of NEAT1 in bladder cancer are largely unknown. Methods In the present study, the expression of NEAT1, miR-101 and VEGF-C was detected in human bladder cancer samples. The relationship between NEAT1 and the prognosis of patients with bladder cancer was analysed. In vitro experiments explored the effects of NEAT1 on biological behaviours of bladder cancer T24 and 5637 cells. Bioinformatics prediction and luciferase assays were used to assay the regulatory mechanism of action of NEAT1 and miR-101. Loss and gain of the expression of miR-101 and VEGF-C were used to explore the effects of the NEAT1/miR-101/VEGF-C pathway on T24 and 5637 cells. The effect of NEAT1 on the growth of bladder cancer in vivo was explored using an orthotopic tumourigenesis model. Results NEAT1 and VEGF-C were significantly upregulated in bladder cancer samples, and miR-101 was significantly downregulated. NEAT1 upregulation was associated with poorer recurrence-free survival of patients with bladder cancer. Overexpression of NEAT1 promoted the proliferation, migration and invasion of bladder cancer cells. The results of the luciferase assay indicated that miR-101 was a target of NEAT1. The promoting effects of NEAT1 on bladder cancer cells were reversed by miR-101 upregulation, and inhibition of miR-101 enhanced the effects of NEAT1. Overexpression of VEGF-C had a clear synergistic effect with the action of NEAT1. Overexpression of NEAT1 increased tumour growth and induced the development of liver metastasis. Conclusions In conclusion, our data indicated that NEAT1 was expressed at high levels in bladder cancer patients and correlated with unfavourable prognosis. NEAT1 promoted malignant development of bladder cancer in vitro and in vivo by regulating the miR-101/VEGF-C pathway.

Chronic wounds represent a major therapeutic challenge. Lymphatic vessel function is impaired in chronic ulcers but the role of lymphangiogenesis in wound healing has remained unclear. We found that lymphatic vessels are largely absent from chronic human wounds as evaluated in patient biopsies. Excisional wound healing studies were conducted using transgenic mice with or without an increased number of cutaneous lymphatic vessels, as well as antibody-mediated inhibition of lymphangiogenesis. We found that a lack of lymphatic vessels mediated a proinflammatory wound microenvironment and delayed wound closure, and that the VEGF-C/VEGFR3 signaling axis is required for wound lymphangiogenesis. Treatment of diabetic mice (db/db mice) with the F8–VEGF-C fusion protein that targets the alternatively spliced extra domain A (EDA) of fibronectin, expressed in remodeling tissue, promoted wound healing, and potently induced wound lymphangiogenesis. The treatment also reduced tissue inflammation and exerted beneficial effects on the wound microenvironment, including myofibroblast density and collagen deposition. These findings indicate that activating the lymphatic vasculature might represent a new therapeutic strategy for treating chronic non-healing wounds. [ABSTRACT FROM AUTHOR]

Brain edema is the most common and fatal complication after traumatic brain injury (TBI). Meningeal lymphatic vessels (MLVs) are the conduits that transport cerebrospinal fluid (CSF) and macromolecules to deep extracranial cervical lymph nodes (dCLNs). After TBI, the drainage function of MLVs can become impaired. However, the scenario in which the improvement of the function of MLVs can promote brain edema absorption after TBI has not been reported. Therefore, the purpose of this study was to investigate the effects of ketoprofen, 9-cis retinoic acid (RA) and vascular endothelial cell growth factor-C (VEGF-C), which promote the proliferation of peripheral lymphatic vessels, on the cerebellar medullary cistern injection of TBI rats, as well as their mechanism of action on brain edema after TBI. In the experiment, we found that ketoprofen, 9-cisRA, and VEGF-C can improve the function of MLVs, promote the extracranial drainage of CSF and the absorption of brain edema, weaken the neuroinflammatory response, reduce reactive oxygen species (ROS) production, maintain the structural integrity of MLVs, and improve neurological function. In addition, ketoprofen, 9-cisRA, and VEGF-C upregulated the lymphatic-specific proteins VEGF receptor (VEGFR)3, PROX1, forkhead box protein C2 (FOXC2), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1). These results indicate that ketoprofen, 9-cisRA, and VEGF-C may maintain the integrity of the meningeal lymphatic wall and promote lymphatic proliferation by upregulating the expression of lymphatic vessel-specific proteins, improve meningeal lymphatic function after TBI, promote CSF drainage and brain edema absorption, reduce the immune response of the nervous system, and reduce ROS formation, thereby improving prognoses. These findings may provide new ideas for the treatment of brain edema after TBI. [ABSTRACT FROM AUTHOR]

Introduction: Surgery and radiotherapy are key cancer treatments and the leading causes of damage to the lymphatics, a vascular network critical to fluid homeostasis and immunity. The clinical manifestation of this damage constitutes a devastating side-effect of cancer treatment, known as lymphoedema. Lymphoedema is a chronic condition evolving from the accumulation of interstitial fluid due to impaired drainage via the lymphatics and is recognised to contribute significant morbidity to patients who survive their cancer. Nevertheless, the molecular mechanisms underlying the damage inflicted on lymphatic vessels, and particularly the lymphatic endothelial cells (LEC) that constitute them, by these treatment modalities, remain poorly understood.Methods: We used a combination of cell based assays, biochemistry and animal models of lymphatic injury to examine the molecular mechanisms behind LEC injury and the subsequent effects on lymphatic vessels, particularly the role of the VEGF-C/VEGF-D/VEGFR-3 lymphangiogenic signalling pathway, in lymphatic injury underpinning the development of lymphoedema.Results: We demonstrate that radiotherapy selectively impairs key LEC functions needed for new lymphatic vessel growth (lymphangiogenesis). This effect is mediated by attenuation of VEGFR-3 signalling and downstream signalling cascades. VEGFR-3 protein levels were downregulated in LEC that were exposed to radiation, and LEC were therefore selectively less responsive to VEGF-C and VEGF-D. These findings were validated in our animal models of radiation and surgical injury.Discussion: Our data provide mechanistic insight into injury sustained by LEC and lymphatics during surgical and radiotherapy cancer treatments and underscore the need for alternative non-VEGF-C/VEGFR-3-based therapies to treat lymphoedema.

Background: Circulating cell-free DNA (cfDNA) and vascular endothelial growth factor-C (VEGF-C) can be utilized to detect cancer and predict its prognosis. However, their potential application in laryngeal squamous cell carcinoma (LSCC) is unclear. Purpose: This study aimed to identify the diagnostic and prognostic value of cfDNA and VEGF-C in LSCC patients. Methods: The plasma cfDNA of 148 LSCC patients and 43 non-tumor patients were isolated. Quantitative real-time PCR (qRT-PCR) was performed to assess long and short DNA fragments in plasma by amplifying the ALU repeats. ALU-qPCR results (ALU247/ALU115) were used to calculate cfDNA integrity index. Vascular endothelial growth factor-C (VEGF-C) level was detected by ELISA assay. Correlation between cfDNA and clinical features was analyzed. For detecting the sensitivity and specificity of cfDNA and VEGF-C alone or in combination for diagnosing LSCC, receiver operator characteristic (ROC) was established. For evaluating the overall survival (OS) of LSCC, Kaplan-Meier curves were established. Results: LSCC patients had significantly higher levels of plasma cfDNA (ALU115, ALU247, and cfDNA integrity index) and VEGF-C than those without cancer (p

The lymphatic vascular system plays a fundamental role in inflammation by draining interstitial fluid, immune cells, antigens, and inflammatory mediators from peripheral tissues. Site-specific delivery of the lymphangiogenic growth factor VEGF-C alleviates acute inflammation in mouse models of psoriasis and chronic colitis by enhancing local drainage. However, it is unclear whether therapeutically induced lymphangiogenesis is transient or long-lasting and whether it might prevent relapses of inflammation. Here, we investigated the long-term effects of targeted VEGF-C delivery in a chronic dermatitis model in mice. Congruent with our previous results, intravenous injection with a VEGF-C fusion protein targeted to the EDA domain of fibronectin initially resulted in reduced inflammation. Importantly, we found that targeted VEGF-C-mediated expansion of lymphatic vessels in the skin persisted for more than 170 days, long after primary inflammation had resolved. Furthermore, the treatment markedly decreased tissue swelling upon inflammatory re-challenge at the same site. Simultaneously, infiltration of leukocytes, including CD4+ T cells, macrophages, and dendritic cells, was significantly reduced in the previously treated group. In conclusion, our data show that targeted delivery of VEGF-C leads to long-lasting lymphatic expansion and long-term protection against repeated inflammatory challenge, suggesting that it is a promising new approach for the treatment of chronic, recurrent inflammatory diseases. [ABSTRACT FROM AUTHOR]

Plasminogen activating inhibitor‐1 (PAI‐1) is associated with poor clinical outcomes, and elevated levels of PAI‐1 in both tissue and serum are correlated with poor response to therapy in various cancers, including skin cancer. Cutaneous angiosarcoma (CAS) is a vascular tumor histologically characterized by detachment of endothelial cell‐derived tumor cells. Since CAS expresses multiple angiogenic growth factors and has increased expressions of angiogenic receptor tyrosine kinase transcripts including VEGFR1/2/3, angiogenesis‐promoting factors are potential drug targets in CAS. In this study, the expression of PAI‐1 was examined in 31 cases of CAS, and the immunomodulatory effects of PAI‐1 on a human CAS cell line, ISO‐HAS‐B, were evaluated. We found that, of the angiogenesis‐promoting factors, PAI‐1 was expressed in almost all cases of CAS, and PAI‐1 increased the mRNA expressions of IL‐23p19, VEGF‐C, CXCL5 and CCL20 on ISO‐HAS‐B. Moreover, PAI‐1 stimulated ISO‐HAS‐B culture supernatant promoted favourable tube networks, suggesting that these tumor‐derived factors promote the pro‐angiogenic effect on tumor development. In addition, IL‐23p19 was expressed in 61.3% of cases, whereas VEGF‐C was expressed in 41% of cases. The results of the present study suggest that PAI‐1 promotes angiogenesis that results in tumor progression in CAS. [ABSTRACT FROM AUTHOR]

Objective　 To investigate the effect of interfering with the expression of VEGF-C on the radiosensitivity of the nasopharyngeal carcinoma CNE-2 cell transplanted tumor and related signaling pathways. Methods　The mice with transplanted tumors of nasopharyngeal carcinoma CNE-2 were randomly divided into tumor body group，siRNA-NC group， siRNA-VEGF-C group; irradiation group；siRNA-NC+ irradiation group; siRNA-VEGF-C+irradiation group. Tumor growth was observed，and the protein expression of VEGF-C，ATM and P65 were detected by immunohistochemistry. Results　Interference of VEGF-C expression in nasopharyngeal carcinoma CNE-2 cell transplanted tumors showed significantly slower tumor growth after irradiation. Immunohistochemical test results showed that after tumor transplantation of mice interfered with VEGF-C，VEGFC and ATM were significantly decreased （ P < 0.05）， while P65 was increased （ P < 0.05） . After combined irradiation，VEGF-C and ATM were significantly decreased （P < 0.05）. Conclusion　In mouse nasopharyngeal carcinoma CNE-2 cell transplantation tumors，interfering with VEGF-C gene affects downstream ATM by activating NF-kB signaling pathway，thereby increasing the sensitivity of radiotherapy. [ABSTRACT FROM AUTHOR]

Background: NEAT1 has been shown to play an oncogenic role in many kinds of cancers. However, detailed roles of NEAT1 in bladder cancer are largely unknown.Methods: In the present study, the expression of NEAT1, miR-101 and VEGF-C was detected in human bladder cancer samples. The relationship between NEAT1 and the prognosis of patients with bladder cancer was analysed. In vitro experiments explored the effects of NEAT1 on biological behaviours of bladder cancer T24 and 5637 cells. Bioinformatics prediction and luciferase assays were used to assay the regulatory mechanism of action of NEAT1 and miR-101. Loss and gain of the expression of miR-101 and VEGF-C were used to explore the effects of the NEAT1/miR-101/VEGF-C pathway on T24 and 5637 cells. The effect of NEAT1 on the growth of bladder cancer in vivo was explored using an orthotopic tumourigenesis model.Results: NEAT1 and VEGF-C were significantly upregulated in bladder cancer samples, and miR-101 was significantly downregulated. NEAT1 upregulation was associated with poorer recurrence-free survival of patients with bladder cancer. Overexpression of NEAT1 promoted the proliferation, migration and invasion of bladder cancer cells. The results of the luciferase assay indicated that miR-101 was a target of NEAT1. The promoting effects of NEAT1 on bladder cancer cells were reversed by miR-101 upregulation, and inhibition of miR-101 enhanced the effects of NEAT1. Overexpression of VEGF-C had a clear synergistic effect with the action of NEAT1. Overexpression of NEAT1 increased tumour growth and induced the development of liver metastasis.Conclusions: In conclusion, our data indicated that NEAT1 was expressed at high levels in bladder cancer patients and correlated with unfavourable prognosis. NEAT1 promoted malignant development of bladder cancer in vitro and in vivo by regulating the miR-101/VEGF-C pathway. [ABSTRACT FROM AUTHOR]

Lymphedema is a common problem after breast cancer treatment. Lymfactin® is a prolymphangiogenic growth factor vector inducing the expression of human vascular endothelial growth factor C (VEGF-C). It promotes growth and repair of lymphatic vessels. Lymfactin® was combined with microvascular lymph node transfer surgery (VLNT) to study the safety and efficacy of the treatment in breast cancer-related upper limb lymphedema (BCRL) patients. This is a continuation study with a 3 year efficacy and 5 year safety follow-up. Fifteen patients were recruited in the study between June 2016 and February 2018. Three patients received a lower dose (1 × 1010 viral particles (vp)), and 12 patients received a higher dose (1 × 1011 vp) of Lymfactin®, respectively. In the higher dose group, the reduction of excess arm volume was on average 46% after the 12 month follow-up, and the transport index was improved in 7/12 patients. At baseline, removal of the compression garment for 7 days resulted in significant arm swelling (105.7±161.0 ml, p=0.0253). However, at 12 months, there was less and not significant swelling after removal of the garment (84.4±143.0 ml, p=0.0682). Lymphedema Quality of Life Inventory (LQOLI or LyQLI) questionnaire showed significant and sustained improvement of quality of life. During 24 months' of follow-up, the results indicate that Lymfactin® is well tolerated. The most promising findings were a 46% reduction in excess arm volume and a nonsignificant volume increase after garment removal at 12 months, suggesting that there is potential for the reduction of lymphedema. [ABSTRACT FROM AUTHOR]

Current clinical practice primarily relies on surgical intervention to remove hematomas in patients with intracerebral hemorrhage (ICH), given the lack of effective drug therapies. Previous research indicates that simvastatin (SIM) may enhance hematoma absorption and resolution in the acute phase of ICH, though the precise mechanisms remain unclear. Recent findings have highlighted the glymphatic system (GS) as a crucial component in intracranial cerebrospinal fluid circulation, playing a significant role in hematoma clearance post-ICH. This study investigates the link between SIM efficacy in hematoma resolution and the GS. Our experimental results show that SIM alleviates GS damage in ICH-induced rats, resulting in improved outcomes such as reduced brain edema, neuronal apoptosis, and degeneration. Further analysis reveals that SIM's effects are mediated through the VEGF-C/VEGFR3/PI3K-Akt pathway. This study advances our understanding of SIM's mechanism in promoting intracranial hematoma clearance and underscores the potential of targeting the GS for ICH treatment. • Simvastatin intervention accelerates hematoma resolution in intracerebral hemorrhage rats. • Simvastatin repairs the damaged glymphatic system post-intracerebral hemorrhage. • Glymphatic system protection is promising for intracerebral hemorrhage treatment. [ABSTRACT FROM AUTHOR]

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.)

p53 is a tumor suppressor protein that plays an essential role in controlling the cell and vascular endothelial growth factor (VEGF) is one of the most strong and specific angiogenic factors. The main objective of this study was to evaluate the impact of p53 and VEGF-C gene expression in the neoplastic and normal mammary gland of canine as an animal model. Elleven benign and malignant specimens and 5 normal specimens were collected. After RNA extraction and cDNA synthesis, relative quantification of p53 and VEGF-C genes were accomplished by Real-time quantitative PCR (RT-qPCR) based on use of β-actin as a reference gene. The relative mRNA expression of the p53 and VEGF-C genes were analyzed by GLM procedure of SAS software v9.2. The results indicated that the VEGF-C and p53 mRNA expression in neoplastic specimens was over-and down-expressed respectively as compared with normal specimens and p53 mRNA expression was significantly negatively associated with VEGF-C (~4 fold) in neoplastic specimens (P

The liver is a major biosynthetic and detoxifying organ in vertebrates, but also generates 25%–50% of the lymph passing through the thoracic duct and is thereby the organ with the highest contribution to lymph flow. In contrast to its metabolic function, the role of the liver for lymph generation and composition is presently severely understudied. We took a rigorous, volume imaging-based approach to describe the microarchitecture and spatial composition of the hepatic lymphatic vasculature with cellular resolution in whole mount immune stained specimen ranging from thick sections up to entire mouse liver lobes. Here, we describe that in healthy adult livers, lymphatic vessels were exclusively located within the portal tracts, where they formed a unique, highly ramified tree. Ragged, spiky initials enmeshed the portal veins along their entire length and communicated with long lymphatic vessels that followed the path of the portal vein in close association with bile ducts. Together these lymphatic vessels formed a uniquely shaped vascular bed with a delicate architecture highly adapted to the histological structure of the liver. Unexpectedly, with the exception of short collector stretches at the porta hepatis, which we identified as exit point of the liver lymph vessels, the entire hepatic lymph vessel system was comprised of capillary lymphatic endothelial cells only. Functional experiments confirmed the space of Disse as the origin of the hepatic lymph and flow via the space of Mall to the portal lymph capillaries. After entry into the lymphatic initials, the lymph drained retrograde to the portal blood flow towards the exit at the liver hilum. Perinatally, the liver undergoes complex changes transforming from the main hematopoietic to the largest metabolic organ. We investigated the time course of lymphatic vessel development and identified the hepatic lymphatics to emerge postnatally in a process that relies on input from the VEGF-C/VERGFR-3 growth factor—receptor pair for formation of the fully articulate hepatic lymph vessel bed.

The prognosis of cholangiocarcinoma remains poor in spite of the advances in immunotherapy and molecular profiling, which has led to the identification of several targetable genetic alterations. Surgical procedures, including both liver resection and liver transplantation, still represent the treatment with the best curative potential, though the outcomes are significantly compromised by the early development of lymph node metastases. Progression of lymphatic metastasis from the primary tumor to tumor-draining lymph nodes is mediated by tumor-associated lymphangiogenesis, a topic largely overlooked until recently. Recent findings highlight tumor-associated lymphangiogenesis as paradigmatic of the role played by the tumor microenvironment in sustaining cholangiocarcinoma invasiveness and progression. This study reviews the current knowledge about the intercellular signaling and molecular mechanism of tumor-associated lymphangiogenesis in cholangiocarcinoma in the hope of identifying novel therapeutic targets to halt a process that often limits the success of the few available treatments. [ABSTRACT FROM AUTHOR]

The papillary thyroid carcinoma (PTC) metastasizes through lymphatic spread, but the follicular thyroid cancer (FTC) metastasis occurs by following hematogenous spread. To date, the molecular mechanism underlying different metastatic routes between PTC and FTC is still unclear. Here, we showed that specifically androgen-regulated gene (SARG) was significantly up-regulated in PTC, while obviously down-regulated in FTC through analyzing the Gene Expression Omnibus (GEO) database. Immunohistochemistry assay verified that the PTC lymph node metastasis was associated with higher levels of SARG protein in clinical PTC patient samples. SARG-knockdown decreased TPC-1 and CGTH-W3 cells viability and migration significantly. On the contrary, SARG-overexpressed PTC cells possessed more aggressive migratory ability and viability. In vivo , SARG overexpression dramatically promoted popliteal lymph node metastasis of xenografts from TPC-1 cells mouse footpad transplanting. Mechanistically, SARG overexpression and knockdown significantly increased and decreased the expression of vascular endothelial growth factor C (VEGF-C) and VEGF receptor 3 (VEGFR-3), respectively, thereby facilitating or inhibiting the tube formation in HUVECs. The tube formation experiment showed that SARG overexpression and knockdown promoted or inhibited the number of tube formations in HUVEC cells, respectively. Taken together, we showed for the first time the differential expression profile of SARG between PTC and FTC, and SARG promotes PTC lymphatic metastasis via VEGF-C/VEGFR-3 signal. It indicates that SARG may represent a target for clinical intervention in lymphatic metastasis of PTC. [ABSTRACT FROM AUTHOR]

Hepatocyte growth factor (HGF) activator inhibitor type‐1 (HAI‐1), encoded by the SPINT1 gene, is a transmembrane protease inhibitor that regulates membrane‐anchored serine proteases, particularly matriptase. Here, we explored the role of HAI‐1 in tongue squamous cell carcinoma (TSCC) cells. An immunohistochemical study of HAI‐1 in surgically resected TSCC revealed the cell surface immunoreactivity of HAI‐1 in the main portion of the tumor. The immunoreactivity decreased in the infiltrative front, and this decrease correlated with enhanced lymphatic invasion as judged by podoplanin immunostaining. In vitro homozygous deletion of SPINT1 (HAI‐1KO) in TSCC cell lines (HSC3 and SAS) suppressed the cell growth rate but significantly enhanced invasion in vitro. The loss of HAI‐1 resulted in enhanced pericellular activities of proteases, such as matriptase and urokinase‐type plasminogen activator, which induced activation of HGF/MET signaling in the co‐culture with pro‐HGF‐expressing fibroblasts and plasminogen‐dependent plasmin generation, respectively. The enhanced plasminogen‐dependent plasmin generation was abrogated partly by matriptase silencing. Culture supernatants of HAI‐1KO cells had enhanced potency for converting the proform of vascular endothelial growth factor‐C (VEGF‐C), a lymphangiogenesis factor, into the mature form in a plasminogen‐dependent manner. Furthermore, HGF significantly stimulated VEGF‐C expression in TSCC cells. Orthotopic xenotransplantation into nude mouse tongue revealed enhanced lymphatic invasion of HAI‐1KO TSCC cells compared to control cells. Our results suggest that HAI‐1 insufficiency leads to dysregulated pericellular protease activity, which eventually orchestrates robust activation of protease‐dependent growth factors, such as HGF and VEGF‐C, in a tumor microenvironment to contribute to TSCC progression. [ABSTRACT FROM AUTHOR]

In mice, embryonic dermal lymphatic development is well understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4, and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5 and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increase in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis. [ABSTRACT FROM AUTHOR]

• Inflammatory lymphangiogenesis parallels cardiac inflammation in VMC. • VEGF-C/VEGFR3 axis promotes cardiac lymphangiogenesis, improving VMC inflammation and cardiac dysfunction. • Macrophage-derived VEGF-C promoted cardiac lymphangiogenesis in VMC. Cardiac lymphatic vessels are important channels for cardiac fluid circulation and immune regulation. In myocardial infarction and chronic heart failure, promoting cardiac lymphangiogenesis is beneficial in reducing cardiac edema and inflammation. However, the specific involvement of cardiac lymphangiogenesis in viral myocarditis (VMC) has not been studied. Despite the recognized participation of macrophages in lymphangiogenesis, the contribution of macrophages to cardiac lymphangiogenesis in VMC is still unclear. The male Balb/c mice with VMC were grouped according to the time to explore changes in inflammation, cardiac function and lymphangiogenesis. Adeno-associated virus (AAV) was used to determine the effect of cardiac lymphangiogenesis in VMC. Macrophage depletion and VEGF-C C156S treatment were used to investigate the connection between macrophages and cardiac lymphangiogenesis. Cardiac inflammation and lymphatic vessel density were both upregulated, peaking on day 7 following CVB3 infection. After treatment with AAV-sVEGFR3, lymphangiogenesis was inhibited, leading to worsened cardiac dysfunction and aggravated inflammation. However, these effects were reversed by AAV-VEGF-C treatment. Furthermore, macrophages infiltrated the inflamed myocardium and secreted VEGF-C. In vitro, VEGF-C was upregulated when RAW264.7 cells were co-cultured with CVB3. Macrophage depletion in mice with VMC inhibited lymphangiogenesis, while supplementation with VEGF-C C156S depressed it. Collectively, these results indicate that activation of the VEGF-C/VEGFR3 axis exerts a protective effect in CVB3-induced VMC by resolving inflammation and alleviating cardiac dysfunction through increased lymphatic vasculature density, with macrophage-derived VEGF-C partially contributing to this effect. [ABSTRACT FROM AUTHOR]

The papillary thyroid carcinoma (PTC) metastasizes through lymphatic spread, but the follicular thyroid cancer (FTC) metastasis occurs by following hematogenous spread. To date, the molecular mechanism underlying different metastatic routes between PTC and FTC is still unclear. Here, we showed that specifically androgen-regulated gene (SARG) was significantly up-regulated in PTC, while obviously down-regulated in FTC through analyzing the Gene Expression Omnibus (GEO) database. Immunohistochemistry assay verified that the PTC lymph node metastasis was associated with higher levels of SARG protein in clinical PTC patient samples. SARG-knockdown decreased TPC-1 and CGTH-W3 cells viability and migration significantly. On the contrary, SARG-overexpressed PTC cells possessed more aggressive migratory ability and viability. In vivo, SARG overexpression dramatically promoted popliteal lymph node metastasis of xenografts from TPC-1 cells mouse footpad transplanting. Mechanistically, SARG overexpression and knockdown significantly increased and decreased the expression of vascular endothelial growth factor C (VEGF-C) and VEGF receptor 3 (VEGFR-3), respectively, thereby facilitating or inhibiting the tube formation in HUVECs. The tube formation experiment showed that SARG overexpression and knockdown promoted or inhibited the number of tube formations in HUVEC cells, respectively. Taken together, we showed for the first time the differential expression profile of SARG between PTC and FTC, and SARG promotes PTC lymphatic metastasis via VEGF-C/VEGFR-3 signal. It indicates that SARG may represent a target for clinical intervention in lymphatic metastasis of PTC.

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. • The expression of miR-455–3p was decreased in the lung tissue of silicosis rats. • The dual luciferase reporter gene confirmed VEGF-C as the target gene of miR-455–3p. • Lymphangiogenesis plays an important role in the progression of silicosis. • miR-455–3p regulates lymphangiogenesis in silicosis by regulating VEGF-C/VEGFR3. [ABSTRACT FROM AUTHOR]

The aim of this study is to investigate the relationship between age-related macular degeneration (AMD) and lymphangiogenesis biomarkers, namely LYVE-1, Podoplanin, VEGF-C, VEGFR-2 and VEGFR-3. This prospective and interventional study includes 30 patients with AMD which may be dry or wet type and 30 controls for whom vitrectomy and phacoemulsification was indicated due to additional pathologies (epiretinal membrane, macular hole, retinal detachment, and cataract). 0.1–0,2 ml of aqueous humor and 0.5–1 ml of vitreous sample was taken during the operations. Before the operations 1 tube serum was also taken. All the lymphangiogenesis biomarkers in the study are examined by ELISA method. LYVE-1 (p = 0.001) and Podoplanin (p = 0.004) levels in the vitreous for the patient group are found to be significantly lower than the control group. Serum (p = 0.019), vitreous (p = 0.001), aqueous (p < 0.001) levels of VEGF-C for the patient group are significantly higher than the control group. VEGF-C/VEGFR-2 (p < 0.001), VEGF-C/VEGFR-3 (p < 0.001) ratios in the vitreous for the patient group are found to be significantly higher than the control group. Especially in wet AMD patients, LYVE-1 level is significantly lower in the vitreous (p = 0.002) and aqueous (p = 0.002) than the control group. In addition, Podoplanin level is observed as significantly lower in the vitreous (p = 0.014) and serum (p = 0.002) in comparison to control group. In the wet AMD group, VEGF-C level in the vitreous (p < 0.001), aqueous (p < 0.001) and serum (p = 0.001) is higher than the control group. The result of this study indicates a valid relationship between the weakening of lymphangiogenesis and the pathophysiology of AMD, especially for the wet type. It is observed that the levels of receptors that bind VEGF-C (VEGFR-2 and VEGFR-3) do not increase at the same rate as VEGF-C to compensate for the increase in VEGF-C. The absence of an increase in VEGFR-3, which is especially necessary for lymphangiogenesis, also suggests that lymphangiogenesis is weakened or decreased in AMD. In the future interventional studies with larger series, examination of lymphangiogenic biomarkers in inflammatory retinal diseases and glaucoma may reveal unexplored details. • Lymphangiogenesis biomarkers in ocular fluids and serum forAMD with control groups were examined by ELISA method. • It seems that the weakening of lymphangiogenesis may play a role in the pathology of AMD, especially in the wet type. • It is concluded that decreased lymphatic drainage of inflammatory antigens in the eye may cause many eye diseases. • A better understanding of lymphangiogenesis in the eye will open new therapeutic opportunities in ocular diseases. [ABSTRACT FROM AUTHOR]

• This study examines the role of the cerebral lymphatic system in determining the response, both centrally and peripherally, following ischemic stroke. • We used a rat model involving middle cerebral artery occlusion, along with removal of the deep cervical lymph nodes, to obstruct drainage from the meningeal lymphatic system. • We found that blocking drainage from meningeal lymphatic vessels, significantly reduced the infarct area and infiltration by inflammatory cells, and inhibited activation of microglia and astrocytes. • We also observed accompanying changes in the levels of plasma inflammatory factors such as IL-6, IL-10 and TNF-α. • Blockage of cerebral lymphatic drainage was associated with reduced functional deficits. • We believe that our study makes a significant contribution to the literature because our findings suggest that the cerebral lymphatic system may trigger systemic inflammation after ischemic stroke. Reduced blood supply to the brain activates the intracranial inflammatory response, a key contributor to secondary brain damage in ischemic stroke. Post-stroke, activation of peripheral immune cells leads to systemic inflammatory responses. Using in vivo approaches, we investigated meningeal lymphatics' role in central immune cell infiltration and peripheral immune cell activation. The bilateral deep cervical lymph nodes (dCLNs) were removed 7 days before right middle cerebral artery occlusion in Sprague Dawley (SD) rats. At 3, 24, and 72 h post-intervention, brain immune cell infiltration and microglial and astrocyte activation were measured, while immune cells were classified in the spleen and blood. Inflammatory factor levels in peripheral blood were analyzed. Simultaneously, reverse verification was conducted by injecting AAV-vascular endothelial growth factor C (AAV-VEGFC) adenovirus into the lateral ventricle 14 days before middle cerebral artery occlusion (MCAO) induction to enhance meningeal lymph function. Blocking meningeal LVs in MCAO rats significantly reduced infarct area and infiltration, and inhibited microglia and pro-inflammatory astrocytes activation. After removing dCLNs, CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, macrophages, and neutrophils in the spleen and blood of MCAO rats decreased significantly at different time points. The levels of inflammatory factors IL-6, IL-10, IL-1β, and TNF-α in plasma decreased significantly. Tests confirmed the results, and AAV-VEGFC-induced MCAO rats provided reverse validation. [ABSTRACT FROM AUTHOR]

Chronic wounds represent a major therapeutic challenge. Lymphatic vessel function is impaired in chronic ulcers but the role of lymphangiogenesis in wound healing has remained unclear. We found that lymphatic vessels are largely absent from chronic human wounds as evaluated in patient biopsies. Excisional wound healing studies were conducted using transgenic mice with or without an increased number of cutaneous lymphatic vessels, as well as antibody-mediated inhibition of lymphangiogenesis. We found that a lack of lymphatic vessels mediated a proinflammatory wound microenvironment and delayed wound closure, and that the VEGF-C/VEGFR3 signaling axis is required for wound lymphangiogenesis. Treatment of diabetic mice (db/db mice) with the F8–VEGF-C fusion protein that targets the alternatively spliced extra domain A (EDA) of fibronectin, expressed in remodeling tissue, promoted wound healing, and potently induced wound lymphangiogenesis. The treatment also reduced tissue inflammation and exerted beneficial effects on the wound microenvironment, including myofibroblast density and collagen deposition. These findings indicate that activating the lymphatic vasculature might represent a new therapeutic strategy for treating chronic non-healing wounds.