UBE2C-induced crosstalk between mono- and polyubiquitination of SNAT2 promotes lymphatic metastasis in bladder cancer.

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.

High expression of DEC2 distinguishes chondroblastic osteosarcoma and promotes tumour growth by activating the VEGFC/VEGFR2 signalling pathway.

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 VEGFR2Y1175 enhanced cell proliferation through VEGFR2Y1175-PLCγ1-PKC-SPHK-MEK-ERK signalling. On the other hand, activation of VEGFR2Y951 decreased mitochondria-dependent apoptosis rate through VEGFR2Y951-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.

Eplerenone reduces lymphangiogenesis in the contralateral kidneys of UUO rats.

Inflammation and fibrosis often occur in the kidney after acute injury, resulting in chronic kidney disease and consequent renal failure. Recent studies have indicated that lymphangiogenesis can drive renal inflammation and fibrosis in injured kidneys. However, whether and how this pathogenesis affects the contralateral kidney remain largely unknown. In our study, we uncovered a mechanism by which the contralateral kidney responded to injury. We found that the activation of mineralocorticoid receptors and the increase in vascular endothelial growth factor C in the contralateral kidney after unilateral ureteral obstruction could promote lymphangiogenesis. Furthermore, mineralocorticoid receptor activation in lymphatic endothelial cells resulted in the secretion of myofibroblast markers, thereby contributing to renal fibrosis. We observed that this process could be attenuated by administering the mineralocorticoid receptor blocker eplerenone, which, prevented the development of fibrotic injury in the contralateral kidneys of rats with unilateral ureteral obstruction. These findings offer valuable insights into the intricate mechanisms underlying kidney injury and may have implications for the development of therapeutic strategies to mitigate renal fibrosis in the context of kidney disease.

miR-455-3p regulates lymphangiogenesis in silicosis by regulating VEGF-C/VEGFR3.

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.

VEGF-C and 5-Fluorouracil Improve Bleb Survival in a Rabbit Glaucoma Surgery Trabeculectomy Model.

Purpose: To evaluate VEGF-C-induced lymphoproliferation in conjunction with 5-fluorouracil (5-FU) antimetabolite treatment in a rabbit glaucoma filtration surgery (GFS) model. Methods: Thirty-two rabbits underwent GFS and were assigned to four groups (n = 8 each) defined by subconjunctival drug treatment: (a) VEGF-C combined with 5-FU, (b) 5-FU, (c) VEGF-C, (d) and control. Bleb survival, bleb measurements, and IOP were evaluated over 30 days. At the end, histology and anterior segment OCT were performed on some eyes. mRNA was isolated from the remaining eyes for RT-PCR evaluation of vessel-specific markers (lymphatics, podoplanin and LYVE-1; and blood vessels, CD31). Results: Qualitatively and quantitatively, VEGF-C combined with 5-FU resulted in blebs which were posteriorly longer and wider than the other conditions: vs. 5-FU (P = 0.043 for longer, P = 0.046 for wider), vs. VEGF-C (P < 0.001, P < 0.001) and vs. control (P < 0.001, P < 0.001). After 30 days, the VEGF-C combined with 5-FU condition resulted in longer bleb survival compared with 5-FU (P = 0.025), VEGF-C (P < 0.001), and control (P < 0.001). Only the VEGF-C combined with 5-FU condition showed a negative correlation between IOP and time that was statistically significant (r = -0.533; P = 0.034). Anterior segment OCT and histology demonstrated larger blebs for the VEGF-C combined with 5-FU condition. Only conditions including VEGF-C led to increased expression of lymphatic markers (LYVE-1, P < 0.001-0.008 and podoplanin, P = 0.002-0.011). Expression of CD31 was not different between the groups (P = 0.978). Conclusions: Adding VEGF-C lymphoproliferation to standard antimetabolite treatment improved rabbit GFS success and may suggest a future strategy to improve human GFSs.

Angpt1 binding to Tie1 regulates the signaling required for lymphatic vessel development in zebrafish.

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.

Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

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.

Examining the correlation of lymphangiogenesis biomarkers with clinical condition in Age-Related Macular Degeneration (AMD).

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.

Ligation of cervical lymphatic vessels decelerates blood clearance and worsens outcomes after experimental subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is characterized by the extravasation of blood into the subarachnoid space, in which erythrocyte lysis is the primary contributor to cell death and brain injuries. New evidence has indicated that meningeal lymphatic vessels (mLVs) are essential in guiding fluid and macromolecular waste from cerebrospinal fluid (CSF) into deep cervical lymph nodes (dCLNs). However, the role of mLVs in clearing erythrocytes after SAH has not been completely elucidated. Hence, we conducted a cross-species study. Autologous blood was injected into the subarachnoid space of rabbits and rats to induce SAH. Erythrocytes in the CSF were measured with/without deep cervical lymph vessels (dCLVs) ligation. Additionally, prior to inducing SAH, we administered rats with vascular endothelial growth factor C (VEGF-C), which is essential for meningeal lymphangiogenesis and maintaining integrity and survival of lymphatic vessels. The results showed that the blood clearance rate was significantly lower after dCLVs ligation in both the rat and rabbit models. DCLVs ligation aggravated neuroinflammation, neuronal damage, brain edema, and behavioral impairment after SAH. Conversely, the treatment of VEGF-C enhanced meningeal lymphatic drainage of erythrocytes and improved outcomes in SAH. In summary, our research highlights the indispensable role of the meningeal lymphatic pathway in the clearance of blood and mediating consequences after SAH.

Age-related changes in meningeal lymphatic function are closely associated with vascular endothelial growth factor-C expression.

Meningeal lymphatic vessels (MLVs) have crucial roles in removing metabolic waste and toxic proteins from the brain and transporting them to the periphery. Aged mice show impaired meningeal lymphatic function. Nevertheless, as the disease progresses, and significant pathological changes manifest in the brain, treating the condition becomes increasingly challenging. Therefore, investigating the alterations in the structure and function of MLVs in the early stages of aging is critical for preventing age-related central nervous system degenerative diseases. We detected the structure and function of MLVs in young, middle-aged, and aged mice. Middle-aged mice, compared with young and aged mice, showed enhanced meningeal lymphatic function along with MLV expansion and performed better in the Y maze test. Moreover, age-related changes in meningeal lymphatic function were closely associated with vascular endothelial growth factor-C (VEGF-C) expression in the brain cortex. Our data suggested that the cerebral cortex may serve as a target for VEGF-C supplementation to ameliorate meningeal lymphatic dysfunction, thus providing a new strategy for preventing age-related central nervous system diseases.

Compartmentalized ocular lymphatic system mediates eye-brain immunity.

The eye, an anatomical extension of the central nervous system (CNS), exhibits many molecular and cellular parallels to the brain. Emerging research demonstrates that changes in the brain are often reflected in the eye, particularly in the retina1. Still, the possibility of an immunological nexus between the posterior eye and the rest of the CNS tissues remains unexplored. Here, studying immune responses to herpes simplex virus in the brain, we observed that intravitreal immunization protects mice against intracranial viral challenge. This protection extended to bacteria and even tumours, allowing therapeutic immune responses against glioblastoma through intravitreal immunization. We further show that the anterior and posterior compartments of the eye have distinct lymphatic drainage systems, with the latter draining to the deep cervical lymph nodes through lymphatic vasculature in the optic nerve sheath. This posterior lymphatic drainage, like that of meningeal lymphatics, could be modulated by the lymphatic stimulator VEGFC. Conversely, we show that inhibition of lymphatic signalling on the optic nerve could overcome a major limitation in gene therapy by diminishing the immune response to adeno-associated virus and ensuring continued efficacy after multiple doses. These results reveal a shared lymphatic circuit able to mount a unified immune response between the posterior eye and the brain, highlighting an understudied immunological feature of the eye and opening up the potential for new therapeutic strategies in ocular and CNS diseases.

Decoding the synergistic potential of MAZ-51 and zingerone as therapy for melanoma treatment in alignment with sustainable development goals.

Melanoma, an invasive class of skin cancer, originates from mutations in melanocytes, the pigment-producing cells. Globally, approximately 132,000 new cases are reported each year, and in South Africa, the incidence stands at 2.7 per 100,000 people, signifying a worrisome surge in melanoma rates. Therefore, there is a need to explore treatment modalities that will target melanoma's signalling pathways. Melanoma metastasis is aided by ligand activity of transforming growth factor-beta 1 (TGF-ß1), vascular endothelial growth factor-C (VEGF-C) and C-X-C chemokine ligand 12 (CXCL12) which bind to their receptors and promote tumour cell survival, lymphangiogenesis and chemotaxis. (3-(4-dimethylaminonaphthelen-1-ylmethylene)-1,3-dihydroindol-2-one) MAZ-51 is an indolinone-based molecule that inhibits VEGF-C induced phosphorylation of vascular endothelial growth factor receptor 3 (VEGFR-3). Despite the successful use of conventional cancer therapies, patients endure adverse side effects and cancer drug resistance. Moreover, conventional therapies are toxic to the environment and caregivers. The use of medicinal plants and their phytochemical constituents in cancer treatment strategies has become more widespread because of the rise in drug resistance and the development of unfavourable side effects. Zingerone, a phytochemical derived from ginger exhibits various pharmacological properties positioning it as a promising candidate for cancer treatment. This review provides an overview of melanoma biology and the intracellular signalling pathways promoting cell survival, proliferation and adhesion. There is a need to align health and environmental objectives within sustainable development goals 3 (good health and well-being), 13 (climate action) and 15 (life on land) to promote early detection of skin cancer, enhance sun-safe practices, mitigation of environmental factors and advancing the preservation of biodiversity, including medicinal plants. Thus, this review discusses the impact of cytostatic cancer drugs on patients and the environment and examines the potential use of phytochemicals as adjuvant therapy.

DHCR7 promotes lymph node metastasis in cervical cancer through cholesterol reprogramming-mediated activation of the KANK4/PI3K/AKT axis and VEGF-C secretion.

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.

Profibrotic VEGFR3-Dependent Lymphatic Vessel Growth in Autoimmune Valvular Carditis.

BACKGROUND: Rheumatic heart disease is the major cause of valvular heart disease in developing nations. Endothelial cells (ECs) are considered crucial contributors to rheumatic heart disease, but greater insight into their roles in disease progression is needed. METHODS: We used a Cdh5-driven EC lineage-tracing approach to identify and track ECs in the K/B.g7 model of autoimmune valvular carditis. Single-cell RNA sequencing was used to characterize the EC populations in control and inflamed mitral valves. Immunostaining and conventional histology were used to evaluate lineage tracing and validate single-cell RNA-sequencing findings. The effects of VEGFR3 (vascular endothelial growth factor receptor 3) and VEGF-C (vascular endothelial growth factor C) inhibitors were tested in vivo. The functional impact of mitral valve disease in the K/B.g7 mouse was evaluated using echocardiography. Finally, to translate our findings, we analyzed valves from human patients with rheumatic heart disease undergoing mitral valve replacements. RESULTS: Lineage tracing in K/B.g7 mice revealed new capillary lymphatic vessels arising from valve surface ECs during the progression of disease in K/B.g7 mice. Unsupervised clustering of mitral valve single-cell RNA-sequencing data revealed novel lymphatic valve ECs that express a transcriptional profile distinct from other valve EC populations including the recently identified PROX1 (Prospero homeobox protein 1)+ lymphatic valve ECs. During disease progression, these newly identified lymphatic valve ECs expand and upregulate a profibrotic transcriptional profile. Inhibiting VEGFR3 through multiple approaches prevented expansion of this mitral valve lymphatic network. Echocardiography demonstrated that K/B.g7 mice have left ventricular dysfunction and mitral valve stenosis. Valve lymphatic density increased with age in K/B.g7 mice and correlated with worsened ventricular dysfunction. Importantly, human rheumatic valves contained similar lymphatics in greater numbers than nonrheumatic controls. CONCLUSIONS: These studies reveal a novel mode of inflammation-associated, VEGFR3-dependent postnatal lymphangiogenesis in murine autoimmune valvular carditis, with similarities to human rheumatic heart disease.

Efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS.

The lymphatic vasculature is the natural pathway for the resolution of inflammation, yet the role of pulmonary lymphatic drainage function in sepsis-induced acute respiratory distress syndrome (ARDS) remains poorly characterized. In this study, indocyanine green-near infrared lymphatic living imaging was performed to examine pulmonary lymphatic drainage function in septic mouse models. We found that the pulmonary lymphatic drainage was impaired owing to the damaged lymphatic structure in sepsis-induced ARDS. Moreover, prior lymphatic defects by blocking vascular endothelial growth factor receptor-3 (VEGFR-3) worsened sepsis-induced lymphatic dysfunction and inflammation. Posttreatment with vascular endothelial growth factor-C (Cys156Ser) (VEGF-C156S), a ligand of VEGFR-3, ameliorated lymphatic drainage by rejuvenating lymphatics to reduce the pulmonary edema and promote draining of pulmonary macrophages and neutrophils to pretracheal lymph nodes. Meanwhile, VEGF-C156S posttreatment reversed sepsis-inhibited CC chemokine ligand 21 (CCL21), which colocalizes with pulmonary lymphatic vessels. Furthermore, the advantages of VEGF-C156S on the drainage of inflammatory cells and edema fluid were abolished by blocking VEGFR-3 or CCL21. These results suggest that efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS. Our findings offer a therapeutic approach to sepsis-induced ARDS by promoting lymphatic drainage function.

CCBE1 regulates the development and prevents the age-dependent regression of meningeal lymphatics.

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.

Inhibition of PFKFB3 Expression Stimulates Macrophage-Mediated Lymphangiogenesis Post-Acute Myocardial Infarction.

BACKGROUND: The dilation of lymphatic vessels plays a critical role in maintaining heart function, while a lack thereof could contribute to heart failure (HF), and subsequently to an acute myocardial infarction (AMI). Macrophages participate in the induction of lymphangiogenesis by secreting vascular endothelial cell growth factor C (VEGF-C), although the precise mechanism remains unclear. METHODS: Intramyocardial injections of adeno-associated viruses (AAV9) to inhibit the expression of VEGFR3 (VEGFR3 shRNA) or promote the expression of VEGFR3 (VEGFR3 ORF) in the heart; Myh6-mCherry B6 D2-tg mice and flow cytometry were used to evaluate the number of myocellular debris in the mediastinal lymph nodes; fluorescence staining and qPCR were used to evaluate fluorescence analysis; seahorse experiment was used to evaluate the level of glycolysis of macrophages; Lyz2𝐶𝑟𝑒, VEGFCfl/fl, and PFKFB3fl/fl mice were used as a model to knock out the expression of VEGF-C and PFKFB3 in macrophages. RESULTS: The escalation of VEGFR3 in cardiac tissue can facilitate the drainage of myocardial debris to the mediastinal lymph nodes, thereby improving cardiac function and reducing fibrosis after reperfusion injury. Conversely, myeloid VEGF-C deficiency displayed an increase in macrophage counts and inflammation levels following reperfusion injury. The inhibition of the critical enzyme PFKFB3 in macrophage glycolysis can stimulate the manifestation of VEGF-C in macrophages. A deficiency in myeloid PFKFB3 is associated with induced lymphangiogenesis following reperfusion injury. CONCLUSIONS: Our initial investigations suggest that the suppression of PFKFB3 expression in macrophages could potentially stimulate the production of VEGF-C in these immune cells, which in turn may facilitate lymphangiogenesis and mitigate the inflammatory effects of I/R injury.

VEGF-C-expressing TAMs rewire the metastatic fate of breast cancer cells.

The expression of pro-lymphangiogenic VEGF-C in primary tumors is associated with sentinel lymph node metastasis in most solid cancer types. However, the impact of VEGF-C on distant organ metastasis remains unclear. Perivascular tumor-associated macrophages (TAMs) play a crucial role in guiding hematogenous spread of cancer cells by establishing metastatic pathways within the tumor microenvironment. This process supports breast cancer cell intravasation and metastatic dissemination. We show here that VEGF-C-expressing TAMs reduce the dissemination of mammary cancer cells to the lungs while concurrently increasing lymph node metastasis. These TAMs express podoplanin and interact with normalized tumor blood vessels expressing VEGFR3. Moreover, clinical data suggest inverse association between VEGF-C-expressing TAMs and breast cancer malignancy. Thus, our study elucidates the paradoxical role of VEGF-C-expressing TAMs in redirecting cancer cells to preferentially disseminate to lymph nodes rather than to lungs, partially achieved by normalizing tumor blood vessels and promoting lymphangiogenesis.

Vascular endothelial growth factor (VEGF) -A, -C and VE-cadherin as potential biomarkers in early breast cancer patients.

BACKGROUND: Vascular endothelial growth factor (VEGF) -A and -C act as multifunctional molecules and growth factors, while VE-cadherin (cadherin 5, CDH5) is the endothelial junction protein. AIM: To assess the relationship between intratumoral VEGF -A, -C and CDH5 levels and clinical outcome, in primary, early-stage, breast cancer patients. PATIENTS AND METHODS: The study included 69 node-negative (N0) breast cancer patients, all of whom had not received any prior hormonal or chemotherapeutic systemic therapy that would affect the course of disease. The median follow-up period was 144 months. Intratumoral mRNA levels of VEGF -A, -C and CDH5 were determined by RT-qPCR. Prognostic performance was evaluated by Cox proportional hazards regression, Kaplan-Meier analysis, as well as by the multivariable approach based on the least absolute shrinkage and selection operator (LASSO) logit regression. Classification of patients into the low and high subgroups was performed using the outcome-oriented cut-off point categorization approach. RESULTS: Of the measured mRNAs, only CDH5 mRNA (t = -2.17; p = 0.04) and VEGF-C mRNA (t = -2.41; p = 0.03) showed significant differences between values in patient subgroups with distant metastasis and those without recurrences, respectively. These t-test results were in agreement with the Cox regression by which CDH5 mRNA reached the most pronounced hazard ratio (HR=2.07; p = 0.05), followed by VEGF-C mRNA (HR=1.59; p = 0.005). HR values above 1.0 indicate that high levels of either CDH5 or VEGF-C mRNAs associated with a higher risk of poor clinical outcome. Distant recurrence incidence was 26% for the CDH5high and 3% for the CDH5low subgroup (Kaplan-Meier analysis). Distant recurrence incidence was 23% for the VEGF-Chigh and 0% for VEGF-Clow subgroup. The independent prognostic value of VEGF-C mRNA was confirmed by LASSO regression. CONCLUSION: Intratumoral VEGF-A levels did not associate with disease outcome in primary, early-stage, breast cancer patients, whilst raised levels of either CDH5 or VEGF-C prognosticated a high risk of distant metastasis.

Immunoregulation mechanism of VEGF signaling pathway inhibitors and its efficacy on the kidney.

Angiogenesis and immunosuppression are closely related pathophysiologic processes. Widely prescribed in malignant tumor and proliferative retinal lesions, VEGF signaling pathway inhibitors may cause hypertension and renal injury in some patients, presenting with proteinuria, nephrotic syndrome, renal failure and thrombotic microangiopathy. VEGF signaling pathway inhibitors block the action of both VEGF-A and VEGF-C. However, VEGF-A and VEGF-C produced by podocytes are vital to maintain the physiological function of glomerular endothelial cells and podocytes. There is still no effective treatment for kidney disease associated with VEGF signaling pathway inhibitors and some patients have progressive renal failure even after withdrawal of the drug. Recent studies reveal that blocking of VEGF-A and VEGF-C can activate CD4 +and CD8+ T cells, augment antigen-presenting function of dendritic cells, enhance cytotoxicity of macrophages and initiate complement cascade activation. VEGF and VEGFR are expressed in immune cells, which are involved in the immunosuppression and cross-talk among immune cells. This review summarizes the expression and function of VEGF-A and VEGF-C in the kidney. The current immunoregulation mechanisms of VEGF signaling pathway inhibitors are reviewed. Finally, combinate strategies are summarized to highlight the proposal for VEGF signaling pathway inhibitors.