Cross­talk between lymphangiogenesis and malignant melanoma cells: New opinions on tumour drainage and immunization (Review).

Malignant melanoma (MM) is a highly aggressive tumour that can easily metastasize through the lymphatic system at the early stages. Lymph node (LN) involvement and lymphatic vessel (LV) density (LVD) represent a harbinger of an adverse prognosis, indicating a strong link between the state of the lymphatic system and the advancement of MM. Permeable capillary lymphatic vessels are the optimal conduits for melanoma cell (MMC) invasion, and lymphatic endothelial cells (LECs) can also release a variety of chemokines that actively attract MMCs expressing chemokine ligands through a gradient orientation. Moreover, due to the lower oxidative stress environment in the lymph compared with the blood circulation, MMCs are more likely to survive and colonize. The number of LVs surrounding MM is associated with tumour-infiltrating lymphocytes (TILs), which is crucial for the effectiveness of immunotherapy. On the other hand, MMCs can release various endothelial growth factors such as VEGF-C/D-VEGFR3 to mediate LN education and promote lymphangiogenesis. Tumour-derived extracellular vesicles are also used to promote lymphangiogenesis and create a microenvironment that is more conducive to tumour progression. MM is surrounded by a large number of lymphocytes. However, both LECs and MMCs are highly plastic, playing multiple roles in evading immune surveillance. They achieve this by expressing inhibitory ligands or reducing antigen recognition. In recent years, tertiary lymphoid structures have been shown to be associated with response to anti-immune checkpoint therapy, which is often a positive prognostic feature in MM. The present review discusses the interaction between lymphangiogenesis and MM metastasis, and it was concluded that the relationship between LVD and TILs and patient prognosis is analogous to a dynamically tilted scale.

Angiopoietin-2 expression and its relationship with lymphangiogenesis and clinicopathological characteristics in cutaneous malignant melanoma.

Objective: The aim of this study was to investigate angiopoietin-2 (Ang-2/ANGPT2) expression and its relationship with lymphangiogenesis and clinicopathological characteristics in cutaneous malignant melanoma (CMM). Methods: Gene expression differences between metastatic melanoma and melanoma in situ in 472 patients from the TCGA database were analyzed. The target gene Ang-2 was screened. A clinical study was conducted to analyze the correlation between Ang-2 expression in CMM and tumor-associated lymphangiogenesis. A total of 42 patients with primary CMM who underwent extended tumor resection procedures at the Affiliated Hospital of Jiangsu University were included in this study. Clinical data (gender, age, lymph node metastasis, Breslow thickness, and clinical stage) were collected. The expression levels of both Ang-2 and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) proteins were detected by immunohistochemistry (IHC). Lymphatic vascular density (LVD) was counted by using LYVE-1 to label lymphatic endothelial cells (LECs) in peritumoral and intratumoral areas per high-magnification field of view. Statistical analysis was performed using the Pearson correlation test and Student's t-test. Results: Using the TCGA database, it was found that the gene expression level of Ang-2 in 368 cases of metastatic melanoma was significantly higher than that in 104 cases of melanoma in situ. Correlation analysis showed a significant relationship between Ang-2 and LYVE-1, and vascular endothelial growth factor receptor 3(VEGFR3) expression, respectively, in CMM. Moreover, the optimal cutoff value of survival analysis showed that high Ang-2 expression in CMM had a worse prognosis, based on data from the TCGA database. Our research showed that Ang-2 was more highly expressed in the group of patients with lymph node metastasis and in the group of stage 3C-4 patients than in the group of patients with no lymph node metastasis and in the group of stage 0-3B patients. Our research also showed that LVD in the group of patients with lymph node metastasis and in the group of stage 3C-4 patients was significantly higher than that in the group of no lymph node metastasis and in the group of stage 0-3B patients, respectively. Breslow thickness also correlated with Ang-2 expression and LVD. Ang-2 expression was not related to sex or age. Ang-2 expression was obviously correlated with LVD. Conclusion: An evaluation of Ang-2 expression and LVD can be used to predict the risk of tumor lymphatic metastasis and determine the prognosis of CMM. These results may also provide a new clinical treatment strategy for CMM.

Sebaceous Carcinoma of the Submandibular Gland a Case Report and Review of the Literature.

Objective: Sebaceous carcinoma (SC) of the submandibular gland is extremely rare. Owing to the low morbidity and nonspecific clinical manifestations, diagnosis is commonly delayed, which increases metastasis and mortality. To date, there have been five reported cases of SC of the submandibular gland. Here, we present a new case and review the relevant literature. Methods and Results: A 36-year-old woman presented with an enlarged left submandibular gland. Clinical features included a non-tender solitary nodular mass with normal overlying skin. There were no special findings on computed tomography or ultrasound examination except for a swollen mass in the left submandibular gland. The patient underwent surgical resection. Pathological examination confirmed the diagnosis of SC with nerve infiltration. Immunohistochemical examination of this case showed positive staining for P63, P40, CK7, CK8/18, MLH1, MSH2, MSH6, and PMS2. The specimen was negative for androgen receptor, CEA, S-100, CK5/6, SOX-10, SOX-11, SMA, and GCDFP-15. The KI-67 labeling index was determined to be 15%. PAS and anti-epithelial membrane antigen were positive in partial area. The patient is still undergoing follow-up, and no metastasis or recurrence has been observed for 2 months. Conclusion: This case highlighted the fact that despite its rarity, SC should be considered as a differential diagnosis for masses located in the head and face. Early and accurate diagnosis, followed by wide surgical excision, has a favorable prognosis. Therefore, clinicians should be familiar with the clinical and pathological features of this disease.

Brain-derived neurotrophic factor preserves intestinal mucosal barrier function and alters gut microbiota in mice.

The intestinal mucosal barrier (IMB) enables the intestine to provide adequate containment of luminal microorganisms and molecules while preserving the ability to absorb nutrients. In this study, we explored the effect of brain-derived neurotrophic factor (BDNF) on IMB function and gut microbiota in mice. BDNF gene knock-out mice (the BDNF+/- group) and wild-type mice (the BDNF+/+ group) were selected. The gut microbiota of these mice was analyzed by denaturing gradient gel electrophoresis (DGGE) assay. The ultrastructure of the ileum and the colonic epithelium obtained from decapitated mice were observed by transmission electron microscopy. The protein expression of epithelial tight junction proteins, zonula occludens-1 (ZO-1) and occludin was detected by immunohistochemistry staining. The protein expression of claudin-1 and claudin-2 was determined by Western blotting. The DGGE band patterns of gut microbiota in the BDNF+/- group were significantly different from that in the BDNF+/+ group, which indicated that the BDNF expression alters the gut microbiota in mice. Compared with the BDNF+/+ group, the BDNF+/- group presented no significant difference in the ultrastructure of ileal epithelium; however, a significant difference was observed in the colonic epithelial barrier, manifested by decreased microvilli, widening intercellular space and bacterial invasion. Compared with the BDNF+/+ group, the expression of ZO-1 and occludin in the BDNF+/- group was significantly decreased. The expression of claudin-1 in the BDNF+/- group was significantly reduced, while the expression of claudin-2 was elevated. These findings indicate that BDNF preserves IMB function and modulates gut microbiota in mice.

Effects of SCN9A gene modification on Na+ channel and the expression of nerve growth factor in a rat model of diarrhea­predominant irritable bowel syndrome.

The aim of the present study was to identify whether the sodium voltage-gated channel alpha subunit 9 (SCN9A) gene modification is a potential treatment for diarrhea­predominant irritable bowel syndrome (D­IBS), via regulating the Na+ channel and the expression of nerve growth factor (NGF). The recombinant adenovirus vector of the SCN9A gene was established, and rat colon cells were isolated for SCN9A gene modification. All subjects were divided into four groups: i) The SCN9A­modified (D­IBS rat model implanted with SCN9A­modified colon cells), ii) negative control (NC; D­IBS rat model implanted with colon cells without SCN9A gene modification), iii) blank (D­IBS rat model without any treatment) and iv) normal (normal rats without any treatment). Western blotting and reverse transcription­quantitative polymerase chain reaction were used to detect the protein and mRNA expression levels of SCN9A, NGF and voltage gated sodium channels (Nav)1.8 and Nav1.9 in rat colon tissues. Compared with the normal group, the rats in the SCN9A, NC and blank groups had significantly elevated mRNA and protein expression levels of NGF, SCN9A, Nav1.8 and Nav1.9. The rats in the SCN9A group demonstrated significantly increased mRNA and protein expression levels of NGF, SCN9A, Nav1.8 and Nav1.9 compared with the NC group and the blank group (all P<0.05). SCN9A gene modification can promote the expression of Nav1.8 and Nav1.9 channels, in addition to NGF which may provide a novel therapeutic basis for treating of D­IBS.

Blockade of angiopoietin-2/Tie2 signaling pathway specifically promotes inflammation-induced angiogenesis in mouse cornea.

AIM: To investigate angiopoietin-2 (Ang-2)/Tie2 signaling pathway involving in inflammatory angiogenesis. METHODS: Three interrupted 11-0 nylon sutures were placed into the corneal stroma of BALB/c mice (6wk old) to induce inflammatory neovascularization. Expression of Ang-2 and Tie2 protein on neovascularization were examined by immunofluorescence. The dynamic expression of Ang-2 mRNA on neovascularization was examined by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, the mouse model of suture-induced corneal neovascularization was used to assess the role of Ang-2/Tie2 signaling pathway in inflammatory angiogenesis by systemic application of L1-10, an Ang-2 specific inhibitor. Mouse corneal hemangiogenesis were evaluated by whole mount immunofluorescence. RESULTS: Both Ang-2 and Tie2 were expressed on newly generated blood vessels in inflammatory cornea. Ang-2 expression was gradually upregulated around 2wk following injury, which was concurrent with an increased number of blood vessels. Blockade of Ang-2/Tie2 signaling pathway obviously promoted angiogenesis in inflammatory cornea. CONCLUSION: Ang-2/Tie2 signaling pathway seems to play an important role during angiogenesis in inflammatory cornea. This may open new therapeutic applications in pathological processes such as corneal graft survival, wound healing and carcinogenesis.

Angiopoietin-2 promotes inflammatory lymphangiogenesis and its effect can be blocked by the specific inhibitor L1-10.

Angiopoietin (Ang)-2, a ligand of the receptor tyrosine kinase Tie2, is known to be involved in the regulation of embryonic lymphangiogenesis. However, the role of Ang-2 in postnatal pathological lymphangiogenesis, such as inflammation, is largely unknown. We used a combination of imaging, molecular, and cellular approaches to investigate whether Ang-2 is involved in inflammatory lymphangiogenesis. We observed strong and continuous expression of Ang-2 on newly generated lymphatic vessels for 2 wk in sutured corneas of BALB/c mice. This expression was concurrent with an increased number of lymphatic vessels. TNF-α expression also increased, with peak TNF-α expression occurring before peak Ang-2 expression was reached. In vitro experiments showed that TNF-α stimulates Ang-2 and Tie2 and ICAM-1 expression on human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs). Ang-2 alone did not affect the biological behavior of LECs, whereas Ang-2 combined with TNF-α significantly promoted the proliferation of LECs but not BECs. In mouse models, blockade of Ang-2 with L1-10, an Ang-2-specific inhibitor, significantly inhibited lymphangiogenesis but promoted angiogenesis. These results clearly indicate that Ang-2 acts as a crucial regulator of inflammatory lymphangiogenesis by sensitizing the lymphatic vasculature to inflammatory stimuli, thereby directly promoting lymphangiogenesis. The involvement of Ang-2 in inflammatory lymphangiogenesis provides a strong rationale for the exploitation of anti-Ang-2 treatment in the prevention and treatment of tumor metastasis and transplant rejection.

[Comparison study of radionuclide lymphoscintigraphy and dynamic magnetic resonance lymphangiography for the diagnosis of extremity lymphedema].

OBJECTIVE: To compare the role of radionuclide lymphoscintigraphy and dynamic magnetic resonance lymphangiography (MRL) for the diagnosis of extremity lymphedema. METHODS: Sixteen patients with primary extremity lymphedema and two with Klippel-Trenaunay syndrome combined with lymphedema were examined by lymphoscintigraphy using the tracer 99Tc-labelled dextran, and also by MRL using gadobenate dimeglumine as contrast agent. The results of morphological abnormalities and functional state of the lymphatic system at affected limbs from the two imaging methods were compared. RESULTS: Lymphatic vessels were imaged in 14 of 18 limbs with lymphedema using MRL, compared with one of 18 using lymphoscintigraphy. MRL detected the inguinal nodes in 16 of 17 patients, whereas lymphoscintigraphy revealed inguinal nodes in only nine cases. MRL revealed more precise information about structural and functional abnormalities of lymph vessels and nodes than lymphoscintigraphy by real-time measurement of lymph flow in vessels and nodes. CONCLUSIONS: Dynamic MRL is more sensitive and accurate than lymphoscintigraphy in the detection of anatomical and functional abnormalities in the lymphatic system in patients with extremity lymphedema.

[Treatment of chronic extremity lymphedema with manual lymph drainage].

OBJECTIVE: To evaluate the effect of manual lymph drainage on chronic extremity lymphedema. METHODS: Fifty patients with chronic lymphedema of extremity were treated with manual lymph drainage (MLD) complex decongestion therapy. Among them, 29 had primary lymphedema, 21 had secondary lymphedema. 42 had lymphedema of lower extremity and 8 had lymphedema of upper limb. The result of treatment was evaluated with measurement of circumference of extremities and edema fluid in tissue with Multiple-frequency bioelectrical impedance analysis. RESULTS: After 1-2 treatment courses, all 50 patients showed significant decrease of circumference of lymphomatous limbs (P < 0.05) and remarkable reduction of accumulated edema fluid in tissue (P < 0. 05). There was highly correlation between the decrease of limb circumference and edema fluid in tissue (r(s) = 0.774, P < 0.01). CONCLUSIONS: MLD complex decongestion therapy is effective for the treatment of chronic lymphedema of extremity.

Lymphatic malformation is a common component of Klippel-Trenaunay syndrome.

OBJECTIVES: Few previous studies have focused on the involvement of the lymphatic system in Klippel-Trenaunay syndrome (KTS), although some evidence suggests that lymphatic abnormalities are associated with the disease. The aim of the present study was to investigate the involvement of the lymphatic system in KTS. METHODS: Magnetic resonance lymphangiography (MRL) with the use of gadobenate dimeglumine as the contrast was performed on 32 patients with KTS involving the extremities to evaluate lymphatic vessels, lymph nodes, and veins. RESULTS: Thirty-one of 32 patients exhibited lymphatic vessel and/or lymph node anomalies, including hyperplasia (11/31), hypoplasia or aplasia (20/31) of lymphatic vessels, and lymphedema (31/31) of the affected limbs. Twenty-two patients showed asymmetry of the inguinal nodes exhibiting either the absence, or an increase or a decrease in number and size of the inguinal nodes. Venous dysplasia was found in 31 patients in superficial and/or deep veins. The results showed a high concomitance of malformations of the lymphatic system and veins in the affected limbs of patients with KTS. CONCLUSIONS: Lymphatic system abnormalities as examined with MRL are commonly associated with KTS and are likely to play a significant role in the disorder.