Lymphatic Vessel Endothelial Hyaluronan Receptor Research Articles

Absence of lymphatic vessels in non-functioning bleb capsules of glaucoma drainage devices.

To evaluate the presence and appearance of blood and lymphatic vessels in non-functioning bleb capsules of glaucoma drainage devices (GDD). Non-functioning (n=14) GDD-bleb capsules of 12 patients were analyzed by immunohistochemistry for blood vessels (CD31, vascular endothelium), lymphatic vessels (lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1] and podoplanin) and macrophages (CD68). CD31+++ blood vessels and CD68+ macrophages were detected in the outer layer of all specimens. LYVE-1 immunoreactivity was registered in single non-endothelial cells in 8 out of 14 (57%) bleb capsule specimens. Podoplanin-immunoreactivity was detected in all cases, located in cells and profiles of the collagen tissue network of the outer and/or the inner capsule layer. However, a colocalization of LYVE-1 and podoplanin as evidence for lymphatic vessels was not detected. We demonstrate the presence of blood-vessels but absence of lymphatic vessels in non-functioning bleb capsules after GDD-implantation. While the absence of lymphatic vessels might indicate a possible reason for drainage device failure, this needs to be confirmed in upcoming studies, including animal experiments.

Reduced Lymphatic Reserve in Heart Failure With Preserved Ejection Fraction

BackgroundMicrovascular dysfunction plays an important role in the pathogenesis of heart failure with preserved ejection fraction (HFpEF). However, no mechanistic link between systemic microvasculature and congestion, a central feature of the syndrome, has yet been investigated. ObjectivesThis study aimed to investigate capillary–interstitium fluid exchange in HFpEF, including lymphatic drainage and the potential osmotic forces exerted by any hypertonic tissue Na+ excess. MethodsPatients with HFpEF and healthy control subjects of similar age and sex distributions (n = 16 per group) underwent: 1) a skin biopsy for vascular immunohistochemistry, gene expression, and chemical (water, Na+, and K+) analyses; and 2) venous occlusion plethysmography to assess peripheral microvascular filtration coefficient (measuring capillary fluid extravasation) and isovolumetric pressure (above which lymphatic drainage cannot compensate for fluid extravasation). ResultsSkin biopsies in patients with HFpEF showed rarefaction of small blood and lymphatic vessels (p = 0.003 and p = 0.012, respectively); residual skin lymphatics showed a larger diameter (p = 0.007) and lower expression of lymphatic differentiation and function markers (LYVE-1 [lymphatic vessel endothelial hyaluronan receptor 1]: p < 0.05; PROX-1 [prospero homeobox protein 1]: p < 0.001) compared with control subjects. In patients with HFpEF, microvascular filtration coefficient was lower (calf: 3.30 [interquartile range (IQR): 2.33 to 3.88] l × 100 ml of tissue–1 × min–1 × mm Hg–1 vs. 4.66 [IQR: 3.70 to 6.15] μl × 100 ml of tissue–1 × min–1 × mm Hg–1; p < 0.01; forearm: 5.16 [IQR: 3.86 to 5.43] l × 100 ml of tissue–1 × min–1 × mm Hg–1 vs. 5.66 [IQR: 4.69 to 8.38] μl × 100 ml of tissue–1 × min–1 × mm Hg–1; p > 0.05), in keeping with blood vascular rarefaction and the lack of any observed hypertonic skin Na+ excess, but the lymphatic drainage was impaired (isovolumetric pressure in patients with HFpEF vs. control subjects: calf 16 ± 4 mm Hg vs. 22 ± 4 mm Hg; p < 0.005; forearm 17 ± 4 mm Hg vs. 25 ± 5 mm Hg; p < 0.001). ConclusionsPeripheral lymphatic vessels in patients with HFpEF exhibit structural and molecular alterations and cannot effectively compensate for fluid extravasation and interstitial accumulation by commensurate drainage. Reduced lymphatic reserve may represent a novel therapeutic target.

Prognostic value of lymphatic vessel density in oral squamous cell carcinoma

AimsLymphatic vessel density (LVD) for the evaluation of tumor metastasis and prognosis remains controversial. The aim of this study was to elucidate the association between tumor cells and lymphatic vessels, and evaluate LVD in oral squamous cell carcinoma (OSCC). Main methods128 OSCC cases were used to determine the expression of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and vascular endothelial growth factor C (VEGF-C). Mann-Whitney or Kruskal-Wallis tests were employed to analyze the association between clinicopathological data and intratumoral LVD (ILVD), peritumoral LVD (PLVD), and VEGF-C; comparisons between ILVD and PLVD were made with t-test. Correlations between LVD and VEGF-C were analyzed by Spearman's correlation coefficient. Disease-specific survival curves were obtained with Kaplan–Meier method and compared using the log-rank test. Cox multiple regression was used to clarify the independent effect of clinicopathological data on clinical outcome. Key findingsTumor tissues were positively stained with LYVE-1 and VEGF-C. Both tumor metastasis and recurrence were associated with ILVD. A significant association between ILVD and VEGF-C expression was observed (P < 0.05). A significant association between high ILVD and poor disease-specific survival was observed (P < 0.05). SignificanceThis study showed that ILVD was significantly associated with increased lymphatic metastasis, tumor recurrence, and reduced disease-specific survival in patients with OSCC. ILVD could be an indicator to predict the prognosis of OSCC.

Microstructural characteristics of lymphatic vessels in skin tissues of acupoints "Taichong" and "Yongquan" in the rat

To provide a new method for investigating the histological characteristics of acupoints by obser-ving the microstructure of the lymphatic vessels in the skin tissue of "Taichong" (LR3) and "Yongquan" (KI1) regions. Six male SD rats were used in the present study. The skin tissue of LR3 and KI1 from the hind foot were taken following transcardial perfusion with 4% paraformaldehyde. The skin tissues were cut into sagittal sections with a freezing microtome and stained by fluorescent immunohistochemistry with lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), calcitonin gene-related peptide (CGRP), and phalloidin for displaying the lymphatic vessels, nerve fibers, and blood vessels, separately. The samples were viewed and recorded using fluorescent microscope and laser scanning confocal microscope. In the skin tissue of LR3 and KI1 regions, the lymphatic vessels, nerve fibers, and blood vessels were labeled with LYVE-1, CGRP and phalloidin, respectively. The lymphatic capillaries were found to start from the enlarged blind end and distribute in the dermis and subcutaneous tissues with various forms, crisscrossing. Abundant blood capillaries at various thickness distributed around the lymphatic capillaries in a parallel or crossed pattern, intermingled with free nerve fibers. The lymphatic capillaries, blood capillaries and nerve fibers extensively distribute in the skin tissues of LR3 and KI1 regions in rats, suggesting an involvement of the immunomodulation in the effects of acupuncture in pathological conditions, despite being not limited to the acupoint regions in the distribution of lymphatic capillaries.

Inhibition of receptor activity-modifying protein 1 suppresses the development of endometriosis and the formation of blood and lymphatic vessels.

Neuroimmune interactions are involved in the development of endometriosis. Here, we examined the role of a neuropeptide, calcitonin gene–related peptide (CGRP), and its receptor, receptor activity–modifying protein (RAMP) 1, in growth of endometrial tissues and the formation of blood and lymphatic vessels in a mouse ectopic endometrial transplantation model. Endometrial fragments from donor wild‐type (WT) mice transplanted into the peritoneal wall of recipient WT mice grew with increased density of blood and lymphatic vessels. When tissues from RAMP1‐deficient (RAMP1−/−) mice were transplanted into RAMP1−/− mice, implant growth and angiogenesis/lymphangiogenesis were decreased. CGRP was up‐regulated in dorsal root ganglia, and CGRP+ nerve fibres were distributed into the implants from the peritoneum. RAMP1 was co‐expressed with CD11b (macrophages) and S100A4 (fibroblasts), but did not co‐localize with blood vessel endothelial cell marker CD31 or lymphatic vessel endothelial hyaluronan receptor (LYVE)‐1. Cultured with CGRP, macrophages up‐regulated vascular endothelial growth factor (VEGF)‐A, VEGF‐C and VEGF‐D, whereas fibroblasts up‐regulated VEGF‐C, but not VEGF‐A or VEGF‐D, in a RAMP1‐dependent manner. CGRP receptor antagonist CGRP8‐37 inhibited growth of and angiogenesis/lymphangiogenesis within endometrial tissue implants. These results suggest that RAMP1 signalling is crucial for growth and angiogenesis/lymphangiogenesis in endometrial tissue. Blockade of RAMP1 is a potential tool for the treatment of endometriosis.

Primary Lymphangiosarcoma of the Urinary Bladder in a Dog

Abdominal ultrasonographical and computed tomography examinations of a 12-year-old neutered female toy poodle revealed a protruding mass, approximately 2cm in diameter, at the apex of the bladder. The mass was firm and haemorrhagic with a homogeneously brownish-yellow cut surface. Microscopically, it was unencapsulated and located in the muscle layer with invasion of the extra-muscular layer. It was composed of spindloid to oval neoplastic cells that formed irregular clefts and diffuse sheets that dissected bundles of collagen. Immunohistochemically, the neoplastic cells were positive for vimentin and lymphatic vessel endothelial hyaluronan receptor 1 antigens, but negative for cytokeratin AE1/AE3, factor VIII-related antigen, CD31, CD34, Prox-1, S100, desmin, α-smooth muscle actin and MyoD1. Negative immunolabelling for laminin antigen supported the absence of evidence of a basal lamina on ultrastructural examination. Based on these findings, this tumour was identified as a lymphangiosarcoma. To the best of our knowledge, this case is the first report of lymphangiosarcoma arising from the bladder in a dog.

FK506 induces lung lymphatic endothelial cell senescence and downregulates LYVE-1 expression, with associated decreased hyaluronan uptake

BackgroundTherapeutic lymphangiogenesis in an orthotopic lung transplant model has been shown to improve acute allograft rejection that is mediated at least in part through hyaluronan drainage. Lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expressed on the surface of lymphatic endothelial cells plays important roles in hyaluronan uptake. The impact of current immunosuppressive therapies on lung lymphatic endothelial cells is largely unknown. We tested the hypothesis that FK506, the most commonly used immunosuppressant after lung transplantation, induces lung lymphatic endothelial cell dysfunction.MethodsLung lymphatic endothelial cells were cultured in vitro and treated with FK506. Telomerase activity was measured using the TRAP assay. Protein expression of LYVE-1 and senescence markers p21 and β-galactosidase was assessed with western blotting. Matrigel tubulation assay were used to investigate the effects of FK506 on TNF-α-induced lymphangiogenesis. Dual luciferase reporter assay was used to confirm NFAT-dependent transcriptional regulation of LYVE-1. Flow cytometry was used to examine the effects of FK506 on LYVE-1 in precision-cut-lung-slices ex vivo and on hyaluronan uptake in vitro.ResultsIn vitro, FK506 downregulated telomerase reverse transcriptase expression, resulting in decreased telomerase activity and subsequent induction of p21 expression and cell senescence. Treatment with FK506 decreased LYVE-1 mRNA and protein levels and resulted in decreased LEC HA uptake. Similar result showing reduction of LYVE-1 expression when treated with FK506 was observed ex vivo. We identified a putative NFAT binding site on the LYVE-1 promoter and cloned this region of the promoter in a luciferase-based reporter construct. We showed that this NFAT binding site regulates LYVE-1 transcription, and mutation of this binding site blunted FK506-dependent downregulation of LYVE-1 promoter-dependent transcription. Finally, FK506-treated lymphatic endothelial cells show a blunted response to TNF-α-mediated lymphangiogenesis.ConclusionFK506 alters lymphatic endothelial cell molecular characteristics and causes lymphatic endothelial cell dysfunction in vitro and ex vivo. These effects of FK506 on lymphatic endothelial cell may impair the ability of the transplanted lung to drain hyaluronan macromolecules in vivo. The implications of our findings on the long-term health of lung allografts merit more investigation.

Primary Cystic Lymphangioma of the Spleen in an Adult Dog

Proliferative disorders of lymphatic origin in animals are mostly congenital or occur within the first few months of life. Involvement of internal organs is extremely infrequent. A seven-year-old entire female mixed-breed dog was presented with apathy and poor appetite. Abdominal ultrasonography revealed a focally enlarged spleen with an anechoic round lesion. Splenectomy was performed and pathological examination demonstrated a sponge-like, compressible tumour composed of endothelium-lined vascular cystic spaces filled with eosinophilic proteinaceous material lacking erythrocytes. Immunohistochemical stains showed that cyst-lining cells were strongly positive for CD31 and factor VIII and focally positive for lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Based on these findings, the lesion was identified as splenic cystic lymphangioma. To the best of our knowledge, this is the first description of solitary lymphangioma of the spleen in animals.

Prox1 induces new lymphatic vessel formation and promotes nerve reconstruction in a mouse model of sciatic nerve crush injury.

The peripheral nervous system lacks lymphatic vessels and is protected by the blood–nerve barrier, which prevents lymphocytes and antibodies from entering the neural parenchyma. Peripheral nerve injury results in degeneration of the distal nerve and myelin degeneration causes macrophage aggregation, T lymphocyte infiltration, major histocompatibility complex class II antigen expression, and immunoglobulin G deposition in the nerve membrane, which together result in nerve edema and therefore affect nerve regeneration. In the present paper, we show myelin expression was absent from the sciatic nerve at 7 days after injury, and the expression levels of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1) and Prospero Homeobox 1 (Prox1) were significantly increased in the sciatic nerve at 7 days after injury. The lymphatic vessels were distributed around the myelin sheath and co‐localized with lymphatic endothelial cells. Prox1 induces the formation of new lymphatic vessels, which play important roles in the elimination of tissue edema as well as in morphological and functional restoration of the damaged nerve. This study provides evidence of the involvement of new lymphatic vessels in nerve repair after sciatic nerve injury.

The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

Temporal and dynamic changes in gingival blood flow during progression of ligature-induced periodontitis.

To evaluate temporal changes in gingival blood flow (GBF) during progression of periodontitis in rats using alaser Doppler flowmeter (LDF) approach and to characterize morphological and biochemical features inthe periodontium associated with GBF. Forty-two Wistar rats were divided into aligature-induced periodontitis group and acontrol group. To induce periodontitis, ligatures were tied around maxillary first molars bilaterally. GBF was measured in palatal gingiva at pretreatment and following ligature placement after 30min, 1, 3, 7, 14, 21, and 28days using LDFwith a non-contact probe. Bone loss and gene expression in gingival tissues were assessed using micro-computed tomography (μCT) and quantitative polymerase chain reaction (PCR), respectively. Immunostaining for vascular endothelial growth factor (VEGF) in themaxilla was also histologically evaluated. GBF in the ligature group increased significantly compared with the control group 30min after ligation. However, on days 3 and 7, GBF decreased in the ligature group. Also, after day 10, there was no difference in GBF between groups. The levels of alveolar bone loss, gene expression (interleukin-6, cluster of differentiation-31, VEGF-A, and lymphatic vessel endothelial hyaluronan receptor-1), and immunostained VEGF-positive vessels correlated well with changes in GBF. CONCLUSIONPROGRESSION OF PERIODONTITIS: In rats was associated with atriphasic pattern of GBF, consisting of ashort initial increase, followed by arapid decrease, and then agradual plateau phase.

Telocytes and lymphatic endothelial cells: Two immunophenotypically distinct and spatially close cell entities

Telocytes (TCs) have recently emerged as a peculiar type of stromal cells located in both perivascular and interstitial compartments of multiple anatomical sites in humans, other mammals and vertebrates. Pioneer electron microscopy studies have ultrastructurally defined TCs as “stromal cells with telopodes” (i.e. very long and thin cell processes with a moniliform morphology conferred by the irregular alternation of slender segments and small, bead-like, dilated portions), whereupon it has become apparent that TCs largely correspond to the CD34+ stromal/interstitial cells detectable by immunohistochemical assays. Besides CD34, TCs are also characterized by the expression of platelet-derived growth factor receptor (PDGFR)α. Interestingly, recent works recommended that lymphatic endothelial cell (LEC) markers should be routinely assessed to discriminate with certainty TCs from LECs, because these two cell types may exhibit similar morphological traits, especially when initial lymphatics are sectioned longitudinally and appear as vascular profiles with no obvious lumen. Furthermore, it has been argued that lymphatic microvessels immunostained for the small mucin-type transmembrane glycoprotein podoplanin (PDPN), which is widely used as lymphatic endothelial marker, can be easily misidentified as TCs. Nevertheless, surprisingly these assumptions were not based on double tissue immunostaining for TC and LEC markers. Therefore, the present morphological study was undertaken to precisely investigate the mutual spatial organization and putative relationships of TCs and lymphatic vessels in tissues from different human organs. For this purpose, we carried out a series of double immunofluorescence analyses simultaneously detecting the CD34 or PDGFRα antigen and a marker of LECs, either PDPN or lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). In the connective tissue compartment of different organs, TCs were CD34+/PDGFRα+/PDPN−/LYVE-1− while LECs were CD34−/PDGFRα−/PDPN+/LYVE-1+, thus representing two definitely distinct, though spatially close, cell entities. The arrangement of telopodes to intimately surround the abluminal side of LECs suggests a possible role of TCs in the regulation of lymphatic capillary functionality, which is worth investigating further.

LCZ696, an Angiotensin Receptor-Neprilysin Inhibitor, Improves Cardiac Hypertrophy and Fibrosis and Cardiac Lymphatic Remodeling in Transverse Aortic Constriction Model Mice.

Cardiac hypertrophy and ventricular remodeling following heart failure are important causes of high mortality in heart disease patients. The cardiac lymphatic system has been associated with limited research, but it plays an important role in the improvement of myocardial edema and the promotion of fluid balance. LCZ696 is a novel combination of angiotensin and neprilysin inhibitors. Here, we studied the role played by LCZ696 during transverse aortic constriction (TAC) induced cardiac hypertrophy and changes in the lymphatic system. Mice undergoing aortic coarctation were constructed to represent a cardiac hypertrophy model and then divided into random groups that either received treatment with LCZ696 (60 mg/kg/d) or no treatment. Cardiac ultrasonography was used to detect cardiac function, and hematoxylin and eosin (H&E) and Masson staining were used to detect myocardial hypertrophy and fibrosis. The proinflammatory factors interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) were detected in the blood and heart tissues of mice. The protein expression levels of lymphatic-specific markers, such as vascular endothelial growth factor C (VEGF-C), VEGF receptor 3 (VEGFR3), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) were detected in mouse heart tissues. We also examined the colocalization of lymphatic vessels and macrophages by immunofluorescence. The results showed that LCZ696 significantly improved heart dysfunction, cardiac hypertrophy, and fibrosis and inhibited the expression of proinflammatory factors IL-6, IL-1β, and TNF-α in the circulating blood and heart tissues of mice. LCZ696 also decreased the protein expression levels of VEGF-C, VEGFR3, and LYVE-1 in mouse heart tissues, ameliorated the transport load of lymphatic vessels to macrophages, and improved the remodeling of the lymphatic system in the hypertrophic cardiomyopathy model induced by TAC.

Inhibition of Tumor Lymphangiogenesis is an Important Part that EGFR-TKIs Play in the Treatment of NSCLC.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used to treat non-small cell lung cancer (NSCLC) because they inhibit tumour growth and metastasis. However, the underlying mechanisms are not fully understood. Here, we investigate whether anti-lymphangiogenesis mechanisms contribute to the anti-tumour effects of EGFR-TKIs. Three different EGFR-TKIs (Gefitinib, Afatinib, and AZD9291) were used to determine the possible biological effects of EGFR-TKIs on lymphangiogenesis in vitro and in vivo. EGFR-TKIs inhibited human lymphatic endothelial cells (HLEC) proliferation, migration and tube formation at the indicated concentrations. Conditioned medium from human lung adenocarcinoma HCC827 cells treated with EGFR-TKIs also inhibited HLEC migration and tube formation. EGFR-TKIs inhibited VEGFC secretion, which further influenced HLEC behaviour in vitro. Afatinib inhibited tumour growth and lymphangiogenesis in the HCC827 xenograft mouse model. The densities and tube diameters of the lymphatic vessels were decreased in a dose-dependent manner, as shown by lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) staining. EGFR-TKIs also inhibited the expression of important lymphangiogenesis regulatory factors vascular endothelial growth factor 2/3 (VEGF2/3), VEGFC, and chemokine receptor 7 (CCR7) as shown by immunocytochemistry (IHC) staining. Additional assays confirmed that the JAK/STAT3 signalling pathways play important roles in the anti-lymphangiogenesis process induced by EGFR-TKIs. Inhibition of lymphangiogenesis is another important role that the three EGFR-TKIs play in the treatment of lung cancer and the Janus kinase/signal transducers and activators of transcription 3 (JAK/STAT3) maybe an important signalling pathway regulating lymphangiogenesis, which provides a new idea for clinical therapy of lung cancer.

Expression of factors related with lymph‐/hem‐angiogenesis in the cornea and conjunctiva during dry eye induction

AbstractPurposeCorneal lymphangiogenesis is known to play an important role in the pathogenesis of dry eye disease (DED). And the low‐grade inflammation associated with DED is known as an inducer of lymphangiogenesis without accompanying hemangiogenesis. This study is to evaluate the expression of known factors related with lymph‐/hem‐angiogenesis in the cornea and conjunctiva in the murine DED model.MethodsDED was induced by housing 8‐week‐old female C57BL/6 mice in a controlled environmental chamber that allowed for the relative low humidity (&lt; 25%), continuous regulation of airflow, and temperature (21 to 23ºC) for 3 weeks. Corneal fluorescein score was evaluated to confirm the efficacy of DED induction. Fluorescence images from the corneal whole mounts after immunohistochemical stain for lymphatic vessel endothelial hyaluronan receptor (lyve)‐1 were used for analysis. The expression of m‐RNA of the VEGF‐A, VEGF‐C, VEGF‐D (Figf), Angiopoietin‐2 (Angpt2), and VEGFR3 (flt4) were evaluated by real‐time PCR in the cornea and conjunctiva.ResultsPercentage of the area of lyve‐1+ area of the whole corneal area increased significantly in DED group (5.55 ± 1.45% in DED group versus 3.88 ± 0.74% in control, P=0.015, Mann‐Whitney U test). The expression of Angiopoietin‐2 increased significantly in the cornea (1.38 folds, 95% confidence interval [CI]: 1.03~1.84) and in the conjunctiva (1.36 folds, 95% CI: 1.04~1.77). And the expression of VEGF‐C increased significantly only in the conjunctiva (1.64 folds, 95% CI: 1.21~2.21). However, the expression of VEGF‐D significantly decreased both in the cornea (0.40 folds, 95% CI: 0.32~0.49) and in the conjunctiva (0.44 folds, 95% CI: 0.37~0.52).ConclusionsUnique lymphangiogenesis without hemangiogenesis in DED may be associated with increased expression of Angiopoietin‐2 and VEGF‐C and decreased expression of VEGF‐D. However, the exact interaction of these key factors should be studied further.

Comparison of the Therapeutic Efficacies of Topical Rivoceranib and Topical Bevacizumab in a Murine Model of Corneal Neovascularization

Background and Objectives: Corneal neovasculariziation (CNV) is a serious vision-threatening complication; however, all therapeutics have their clinical limitations. The aim of this study is to investigate the efficacy of topical rivoceranib compared with topical bevacizumab in a murine model of corneal neovascularization (CNV). Materials and Methods: Murine CNV was induced by means of total de-epithelization and alkali burn. Mice were divided into five groups according to topical treatment: untreated control, phosphate-buffered saline (PBS), 0.1% and 0.5% rivoceranib, and 0.5% bevacizumab. CNV area and index were measured 7 and 14 days after treatment. After corneal tissues were excised at day 14, the blood and lymphatic vessels were quantified by cluster of differentiation 31 (CD31) and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) immunofluorescence, respectively. Results: After 14 days, treatment groups with 0.1% and 0.5% rivoceranib and 0.5% bevacizumab showed a decrease in CNV area and index compared with the untreated and PBS groups (all p < 0.01). Blood and lymphatic vascularization significantly decreased in the 0.5% rivoceranib and 0.5% bevacizumab groups, as measured by CD31 and LYVE1 immunofluorescence. There was no significant difference of vascularization between the 0.5% rivoceranib and bevacizumab groups. Conclusions: Topical application of rivoceranib could effectively decrease CNV equivalent to topical bevacizumab in a murine model.

Fenestral diaphragms and PLVAP associations in liver sinusoidal endothelial cells are developmentally regulated

Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.

Endogenous Calcitonin Gene–Related Peptide Deficiency Exacerbates Postoperative Lymphedema by Suppressing Lymphatic Capillary Formation and M2 Macrophage Accumulation

Lymphedema is a chronic condition caused by disruption of lymphatic vessels, which often occurs after invasive surgery. Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene (Calca). CGRP was initially identified as a neuropeptide released primarily from sensory nerves and involved in regulating pathophysiological nociceptive pain. However, recent studies have shown CGRP is also released from a variety of other cells and possesses multiple functions. In this study, CGRP knockout (-/-) mice were used to show the actions of endogenous CGRP in postoperative lymphedema. After generating a mouse postoperative tail lymphedema model, the edema was observed to be more severe in CGRP-/- mice than in wild-type mice. Numbers of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)-positive lymphatic capillaries were decreased and lymphatic capillary formation-related factors were down-regulated in CGRP-/- mice. In addition, accumulation of M2 but not M1 macrophages was selectively reduced in the edematous tissue of CGRP-/- mice. Selective depletion of M2 macrophages decreased lymphatic capillary formation and worsened lymphedema in wild-type mice but not CGRP-/- mice, where numbers of M2 macrophages were already diminished. These findings suggest that endogenous CGRP acts to ameliorate postoperative lymphedema by enhancing lymphatic capillary formation and that M2 macrophages play critical roles. CGRP may be a useful therapeutic target for the treatment of postoperative lymphedema.

Blockade of Lymphangiogenesis Shapes Tumor-Promoting Adipose Tissue Inflammation

Tumor-associated lymphangiogenesis correlates with lymph node metastasis and poor outcome in several human malignancies. In addition, the presence of functional lymphatic vessels regulates the formation of tumor inflammatory and immune microenvironments. Although lymphatic structures are often found deeply integrated into the fabric of adipose tissue, the impact of lymphangiogenesis on tumor-associated adipose tissue (AT) has not yet been investigated. Using K14-VEGFR3-Ig mice that constitutively express soluble vascular endothelial growth factor receptor (VEGFR) 3-Ig in the skin, scavenging VEGF-C and VEGF-D, the role of lymphangiogenesis in the generation of an inflammatory response within tumor-associated AT was studied. Macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 were found within peritumoral adipose tissue from melanoma-bearing K14-VEGFR3-Ig mice, which were further enriched with alternatively activated macrophages based on surface marker CD301/C-type lectin domain family 10 member A expression. The blockade of lymphangiogenesis also resulted in accumulation of the cytokine IL-6, which correlated with enhanced macrophage proliferation of the alternatively activated phenotype. Furthermore, melanomas co-implanted with freshly isolated adipose tissue macrophages grew more robustly than melanomas growing alone. In human cutaneous melanomas, adipocyte-selective FABP4 transcripts closely correlated with gene signatures of CLEC10A and were associated with poor overall survival. These data suggest that the blockade of pathways regulating lymphatic vessel formation shapes an inflammatory response within tumor-associated AT by facilitating accumulation of tumor-promoting alternatively activated macrophages.

Serum sLYVE-1 is not associated with coronary disease but with renal dysfunction: a retrospective study

Recent evidence has indicated that the lymphatic vessel endothelial hyaluronan receptor (LYVE-1) is implicated in chronic inflammation and the lymphatic immune response. The soluble form of LYVE-1 (sLYVE-1) is produced by ectodomain shedding of LYVE-1 under pathological conditions including cancer and chronic inflammation. In this study, 1014 consecutive patients who underwent coronary angiography from May 2015 to September 2015 were included to investigate whether serum sLYVE-1 is associated with coronary artery disease (CAD) and its concomitant diseases includes chronic kidney disease (CKD). Results showed that there was no significant difference in sLYVE-1 levels between patients with CAD and without. However, a significantly higher level of sLYVE-1 was seen in patients with renal dysfunction compared to those with a normal eGFR. Results were validated in a separate cohort of 259 patients who were divided into four groups based on their kidney function assessed by estimated glomerular filtration rate (eGFR). Simple bivariate correlation analysis revealed that Lg[sLYVE-1] was negatively correlated with eGFR (r = −0.358, p < 0.001) and cystatin C (r = 0.303, p < 0.001). Multivariable logistic regression analysis revealed that the increase in Lg[sLYVE-1] was an independent determinant of renal dysfunction (odds ratio = 1.633, p = 0.007). Therefore, renal function should be considered when serum sLYVE-1 is used as a biomarker for the detection of pathological conditions such as chronic inflammation and cancer. Further study is required to elucidate the exact role of sLYVE-1 in renal function.