Inflammatory Lymphangiogenesis Research Articles

Alkali injury-induced pathological lymphangiogenesis in the iris facilitates the infiltration of T cells and ocular inflammation.

Inflammatory lymphangiogenesis is intimately linked to immune regulation and tissue homeostasis. However, current evidence has suggested that classic lymphatic vessels are physiologically absent in intraocular structures. Here, we show that neolymphatic vessels were induced in the iris after corneal alkali injury (CAI) in a VEGFR3-dependent manner. Cre-loxP-based lineage tracing revealed that these lymphatic endothelial cells (LECs) originate from existing Prox1+ lymphatic vessels. Notably, the ablation of iridial lymphangiogenesis via conditional deletion of VEGFR3 alleviated the ocular inflammatory response and pathological T cell infiltration. Our findings demonstrate that iridial neolymphatics actively participate in pathological immune responses following injury and suggest intraocular lymphangiogenesis as a valuable therapeutic target for the treatment of ocular inflammation.

ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation.

The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.

Inflammatory biomarkers, angiogenesis and lymphangiogenesis in epicardial adipose tissue correlate with coronary artery disease

In this study, we explored the relationship between inflammatory adipokine levels and coronary artery disease (CAD). We collected subcutaneous adipose tissues(SAT), pericardial adipose tissues(PAT), and epicardial adipose tissues (EAT) and serum samples from 26 inpatients with CAD undergone coronary artery bypass grafting and 20 control inpatients without CAD. Serum inflammatory adipokines were measured by ELISA. Quantitative real-time PCR and western blot were used to measure gene and protein expression. Adipocyte morphology was assessed by H&E staining. Immunohistochemistry and immunofluorescence were used to measure endothelial and inflammatory markers. Serum pro- and anti-inflammatory adipokine levels were higher and lower, respectively, in the CAD group than those in the control group (P < 0.05). In CAD, the pro-inflammatory adipokine levels via ELISA in EAT and PAT were elevated. Pro-inflammatory adipokine mRNA expression was increased, while anti-inflammatory adipokine mRNA expression decreased, in CAD relative to NCAD in EAT and PAT rather than SAT. In EAT, adipocyte area and macrophage-specific staining were lower, while lymphatic vessel marker expression was higher in CAD. Additionally, the endothelial marker expression in EAT was higher than PAT in CAD. The three tissue types had different blood vessel amounts in CAD. The regulation and imbalance expression of the novel biomarkers, including inflammatory adipokine, macrophage infiltration, angiogenesis, and lymphangiogenesis in EAT and PAT, may be related to the pathogenesis of CAD. The serum levels of inflammatory adipokines may correlate to CAD, which requires large sample size studies to get further validation before clinic practice.

Hydroxysafflor yellow A regulates lymphangiogenesis and inflammation via the inhibition of PI3K on regulating AKT/mTOR and NF-κB pathway in macrophages to reduce atherosclerosis in ApoE-/- mice

BackgroundMacrophage-mediated inflammatory infiltration and pathological lymphangiogenesis around atherosclerotic plaques are newly highlighted treatment targets of atherosclerosis. Although the effect of Hydroxysafflor yellow A(HSYA) on atherosclerosis was clear, few studies focus on the regulation of HSYA on such mechanisms. PurposeThis study aimed to uncover the key site of HSYA on improving atherosclerosis by regulating macrophage-induced inflammation and lymphangiogenesis. Study designThis study was designed to explore the new mechanism of HSYA on alleviating atherosclerosis in vitro and in vivo. MethodsWe determined the expression of vascular endothelial growth factor C(VEGF-C) in Raw264.7 cells and high-fat diet fed ApoE knockout (ApoE−/−) mice. Raw264.7 cells were treated with HSYA under the stimulation of LPS and ox-LDL. HFD induced ApoE−/− mice were given different concentrations of HSYA-saline solution by tail vein injection and ATV-saline suspension by gavage. C57/B6j mice fed with chow diet were used for the control group. H&E, oil red O and immunofluorescence staining analysis were used for visualizing the pathological changes. The biological impact of HSYA was evaluated by body weight, lipid metabolism, inflammation levels, and corresponding function indexes of kidney and liver. RT-qPCR and western blot methods were conducted to determine the expression of the inflammation and lymphangiogenesis factors. Molecular docking and microscale thermophoresis analysis were used to verify the combination of HSYA and PI3K. ResultsIn vivo, HSYA reduced the plaque formation, hepatic steatosis and inflammation-related lymphangiogenesis (IAL). It also changed the serum levels of inflammation (VEGF-C, TNF-α, IL-6, VCAM1, MCP1), lipid indexes (LDL, CHOL, TRIG) and relevant lymphangiogenesis (VEGF-C and LYVE-1) and inflammation (VCAM-1 and IL-6) signals in the aorta. In vitro, HSYA regulated Akt/mTOR and NF-κB activation by the inhibition of PI3K in macrophages. ConclusionHSYA affects inflammation and inflammation-associated lymphangiogenesis via suppressing PI3K to affect AKT/mTOR and NF-B pathway activation in macrophages, showing a comprehensive protective effect on atherosclerosis.

Galectin-8 involves in arthritic condylar bone loss via podoplanin/AKT/ERK axis-mediated inflammatory lymphangiogenesis

The lymphatic system plays a crucial role in the maintenance of tissue fluid homeostasis and the immunological response to inflammation. Galectin-8 (Gal-8) regulates pathological lymphangiogenesis but the effects of which on inflammation-related condylar bone loss in temporomandibular joint (TMJ) have not been well studied. We used TNFα-transgenic (TNFTG) mice and their wildtype (WT) littermates to compare their inflammatory phenotype in TMJs. Next, lymphatic endothelial cells (LECs) were used to examine the effects of which on osteoclast formation, pro-inflammatory factor expression, and inflammatory lymphangiogenesis with or without thiodigalactoside (TDG, a Gal-8 inhibitor) treatment. At last, two murine models (TNFTG arthritic model and forced mouth opening model) were used to explore TDG as a potential drug for the treatment of inflammation-related condylar bone loss. In comparison to WT mice, lymphatic areas of lymphatic vessel endothelial receptor 1 (LYVE1)+/podoplanin (PDPN)+ and Gal-8+/PDPN+, TRAP-positive osteoclast number, and condylar bone loss are increased in TNFTG mice. Inhibition of Gal-8 in LECs by TDG, reduces TNFα-induced osteoclast formation, pro-inflammatory factor expression, and inflammatory lymphangiogenesis. In addition, Gal-8 promotes TNFα-activated AKT/ERK/NF-κB pathways by binding to PDPN. Finally, the administration of TDG attenuates inflammatory lymphangiogenesis, inhibits osteoclast activity, and reduces condylar bone loss in TNFTG arthritic mice and forced mouth opening mice. Our findings reveal the important role of Gal-8-promoted pathological lymphangiogenesis in inflammation-related condylar bone loss.

PS-B12-1: EPLERENONE INHIBITS UUO-INDUCED LYMPHANGIOGENESIS AND CARDIAC FIBROSIS BY ATTENUATING INFLAMMATORY INJURY

Objective: Cardiorenal syndrome (CRS) is the leading cause of death associated with chronic kidney disease (CKD) and end-stage renal disease (ESRD), however, the underlying mechanism of CKD-induced cardiac injury is incompletely understood. We reported that aldosterone is associated with cardiac fibrosis, and that inflammatory injury and lymphangiogenesis are involved. This pathogenic mechanism CKD-induced cardiac fibrosis still needs further investigation. Design and method: The unilateral ureteral obstruction (UUO) model was used to replicate the CRS type 4 model. Following induction of renal injury, the rats received eplerenone for 180-day. For in-vitro studies, we exposed human lymphatic endothelial cells (hLEC) to aldosterone and eplerenone. Changes in cardiac tissue related to lymphangiogenesis and inflammatory were analyzed in vivo. Changes in lymphangiogenesis, LEC phenotypic transformation and activation of mineralocorticoid receptor (MR)/ transforming growth factor (TGF)-beta/ vascular endothelial growth factor C (VEGFC) signaling axis involved in these changes were analyzed in vivo and in vitro. Results: We found that eplerenone ameliorated cardiac inflammation and fibrosis in UUO rats. Moreover, eplerenone inhibited MR activation, attenuated lyphangiogenesis and LEC phenotypic transformation, and regulated TGF-beta/CTGF/VEGFC signaling axis in vivo and in vitro. The levels of podoplanin, VEGFR-3 and VEGFC confirmed the role of lymphangiogenesis and inflammatory injury. Co-expression of LEC marker LYVE-1 and myofibroblast marker alpha-SMA validated EndMT in cardiac fibrosis. Treatment with eplerenone, clarified the role of aldosterone in UUO-induced cardiac fibrosis. Conclusions: We confirmed that eplerenone can alleviate UUO-induced cardiac fibrosis by inhibiting inflammation and lymphangiogenesis via MR/TGF-beta/VEGFC signaling axis. These findings provide an in-depth understanding of pathogenesis of UUO-induced cardiac injury and reveal novel therapeutic targets.

Ocular surface mast cells promote inflammatory lymphangiogenesis.

Mast cells, sentinel immune cells, are most abundantly expressed in vascularized tissues that interface the external environment, such as the skin and ocular surface. Our previous reports have shown mast cells reside closely with vascular endothelial cells and mediate the pathogenic angiogenic response. However, the contribution of mast cells and their underlying mechanisms on lymphangiogenesis have not been fully deciphered. Using a murine model of inflammatory corneal angiogenesis, we observed adjacent migration of activated mast cells with new lymph vessel growth. Our in vitro co-culture assays demonstrate that mast cells express high levels of of VEGF-D and directly promote lymphatic endothelial cell tube formation and proliferation. Moreover, our loss-of-function approaches, using mast cell knockout mice and cromolyn-mediated mast cell inhibition, showed mast cell deficiency suppresses the induction of inflammatory lymphangiogenesis and VEGF-D expression at the ocular surface following corneal tissue insult. Our findings suggest blockade of mast cells as a potential therapeutic strategy to inhibit pathological lymphangiogenesis.

The effect of induced lymphatic circulation on lymphangiogenesis and inflammatory mediators in rats with adjuvant induced arthritis

Abstract Rheumatoid arthritis (RA) is a chronic inflammatory disease involving dysfunctional lymphatic circulation leading to edema. The adjuvant-induced arthritis (AIA) rat model was used to study the effect of lymphatic pump treatment (LPT) on the expression levels of inflammatory cytokines and lymphangiogenesis within the draining popliteal lymph nodes (PLN). PLN from both control and LPT treatment groups were harvested, and their cDNA were analyzed by qPCR to determine the levels of cytokines, VEGF-C, and VEGFR-3. Additionally, immunohistochemistry was used to visualize the expression of VEGF-C and VEGFR-3 and flow cytometry was used to quantify leukocyte cell types in the PLN. The LPT group showed a decrease in ankle circumference and arthritis grade a few days after the initiation of treatment. Flow cytometry of popliteal lymph nodes showed an increase in CD8, CD4, and CD3 cells in arthritic rats, however cell populations were not significantly different between treatment groups. The qPCR data indicated that the LPT group had a slight reduction in levels of IL-17a, IL-4, IL-6, and IL-1β. IHC showed that the PLN from the LPT group had the highest expression area of VEGF-C and the lowest expression area of VEGFR-3 compared to control. In conclusion, The LPT group showed a reduction in ankle circumference and arthritis score, suggesting that LPT helped alleviate edema and inflammation. When compared to control, the LPT group had lower expression of pro-inflammatory cytokines and elevated levels of VEGF-C, an important lymphatic vessel growth factor, in PLN. Thus, the data suggests that LPT may elevate the swelling and inflammation in rat AIA through the reduction of inflammatory cytokines and the elevation of a lymphatic growth factor.

Avian Reticuloendotheliosis Viral Oncogene Related B Regulates Lymphatic Endothelial Cells during Vessel Maturation and Is Required for Lymphatic Vessel Function in Adult Mice

NF-κB signals through canonical transcription factor p65 (RelA)/p50 and noncanonical avian reticuloendotheliosis viral oncogene related B (RelB)/p52 pathways. The RelA/p50 is involved in basal and inflammatory lymphangiogenesis. However, the role of RelB/p52 in lymphatic vessel biology is unknown. Herein, we investigated changes in lymphatic vessels (LVs) in mice deficient in noncanonical NF-κB signaling and the function of RelB in lymphatic endothelial cells (LECs). LVs were examined in Relb-/-, p52-/-, or control mice, and the gene expression profiles in LECs with RelB knockdown. Relb-/-, but not p52-/-, mice exhibited multiple LV abnormalities. They include the following: i) increased capillary vessel diameter, ii) reduced smooth muscle cell (SMC) coverage of mature vessels, iii) leakage, and iv) loss of active and passive lymphatic flow. Relb-/- mature LVs had thinner vessel walls, more apoptotic LECs and SMCs, and fewer LEC junctions. RelB knockdown LECs had decreased growth, survival, and adhesion, and dysregulated signaling pathways involving these cellular events. These results suggest that Relb-/- mice have abnormal LVs, mainly in mature vessels with reduced SMC coverage, leakage, and loss of contractions. RelB knockdown in LECs leads to reduced growth, survival, and adhesion. RelB plays a vital role in LEC-mediated LV maturation and function.

Myeloid-Derived Lymphatic Endothelial Cell Progenitors Significantly Contribute to Lymphatic Metastasis in Clinical Breast Cancer

Lymphatic metastasis is a high-impact prognostic factor for mortality of breast cancer (BC) patients, and it directly depends on tumor-associated lymphatic vessels. We previously reported that lipopolysaccharide-induced inflammatory lymphangiogenesis is strongly promoted by myeloid-derived lymphatic endothelial cell progenitors (M-LECPs) derived from the bone marrow (BM). As BC recruits massive numbers of provascular myeloid cells, we hypothesized that M-LECPs, within this recruited population, are specifically programmed to promote tumor lymphatics that increase lymph node metastasis. In support of this hypothesis, high levels of M-LECPs were found in peripheral blood and tumor tissues of BC patients. Moreover, the density of M-LECPs and lymphatic vessels positive for myeloid marker proteins strongly correlated with patient node status. It was also established that tumor M-LECPs coexpress lymphatic-specific, stem/progenitor and M2-type macrophage markers that indicate their BM hematopoietic-myeloid origin and distinguish them from mature lymphatic endothelial cells, tumor-infiltrating lymphoid cells, and tissue-resident macrophages. Using four orthotopic BC models, we show that mouse M-LECPs are similarly recruited to tumors and integrate into preexisting lymphatics. Finally, we demonstrate that adoptive transfer of invitro differentiated M-LECPs, but not naïve or nondifferentiated BM cells, significantly increased metastatic burden in ipsilateral lymph nodes. These data support a causative role of BC-induced lymphatic progenitors in tumor lymphangiogenesis and suggest molecular targets for their inhibition.

Adenosine A2a receptor promotes lymphangiogenesis and lymph node metastasis

ABSTRACT The formation of new lymphatic vessels, or lymphangiogenesis, is a critical step of the tissue repair program. In pathological conditions involving chronic inflammation or tumorigenesis, this process is often dysregulated and can contribute to disease progression. Yet, lymphangiogenesis is still incompletely understood. In this study, we identified A2a adenosinergic signaling as an important regulator of inflammatory and tumor-associated lymphangiogenesis. Using Adora2a (A2a)-deficient mice, we demonstrated that A2a signaling was involved in the formation of new lymphatic vessels in the context of peritoneal inflammation. We also demonstrated that tumor-associated and sentinel lymph node lymphangiogenesis were impaired in A2a-deficient mice, protecting them from lymph node metastasis. Notably, A2a signaling in both hematopoietic and non-hematopoietic cells contributed to sentinel lymph node metastasis. In A2a-deficient tumor-draining lymph nodes, impaired lymphangiogenesis was associated with a reduced accumulation of B cells and decreased VEGF-C levels. Supporting a role for non-hematopoietic A2a signaling, we observed that primary murine lymphatic endothelial cells (LEC) predominantly expressed A2a receptor and that A2a signaling blockade altered LEC capillary tube formation in vitro. Finally, we observed that Adora2a, Nt5e and Entpd1 gene expression positively correlated with Lyve1, Pdpn and Vegfc in several human cancers, thereby supporting the notion that adenosine production and A2a receptor activation might promote lymphangiogenesis in human tumors. In conclusion, our study highlights a novel pathway regulating lymphangiogenesis and further supports the use of A2a or adenosine blocking agents to inhibit pathological lymphangiogenesis in cancers and block the dissemination of tumor cells through the lymphatic system.

Tyrosinase Is a Novel Endogenous Regulator of Developmental and Inflammatory Lymphangiogenesis

Lymphangiogenesis is critically involved in tissue fluid balance, graft rejection, and tumor metastasis. Endogenous regulation of lymphangiogenesis is poorly understood. Herein, we use the lymphatic vessel architecture at the limbal border of the normally avascular cornea, a quantitative trait under strong genetic influence, as a model system to identify new candidate genes regulating lymphangiogenesis. Comparing low-lymphangiogenic BALB/cN with high-lymphangiogenic C57BL/6N mice, we performed quantitative trait loci analysis of five phenotypes in a large BALB/cN × C57BL/6N intercross (n = 795) and identified three to eight genome-wide significant loci, the strongest on chromosome 7 containing tyrosinase (Tyr). Tyrosinase-negative mice showed significantly increased limbal lymph vascularized areas, a higher number of lymphatic vessel end points, and branching points and increased inflammation-induced lymphangiogenesis. These findings confirm that tyrosinase is a novel lymphangiogenesis regulator in developmental and inflammatory lymphangiogenesis. Our findings link melanin synthesis with lymphangiogenesis and open new treatment options in lymphangiogenesis-related diseases.

Lymphotoxins Promote the Progression of Human Lymphatic Malformation by Enhancing Lymphatic Endothelial Cell Proliferation

Formation of inflammation-related tertiary lymphoid organs promotes human lymphatic malformation (LM) development. However, the role of lymphotoxins (LTs) and LT-related inducible ligand, the crucial mediators for tertiary lymphoid organ formation, is undetermined in LMs. Herein, we show that LTs and LT-related inducible ligand promote LM development by enhancing lymphatic endothelial cell (LEC) proliferation via activating NF-κB pathways. The expression of LTs and their receptors was increased in LMs, especially the infected ones, when compared with normal skins. Nuclear translocation of p65, p52, and RelB in the LECs of LMs indicated the activation of classic and alternative NF-κB pathways. Pearson's correlation and cluster analysis suggested the close relationship between LEC proliferation and NF-κB activation. Moreover, invitro data demonstrated LTs accelerated the proliferation of human dermal LECs (HdLECs) through activation of NF-κB. In addition, lipopolysaccharide (LPS) up-regulated LT receptor expression in HdLECs, leading to increased sensitivity to LTs. Suppression of LT receptors hampered LPS-enhanced HdLEC proliferation, indicating the crucial role of LT pathways in inflammatory lymphangiogenesis. Besides, evidence from the LM rat models demonstrated LTα and LPS enhanced LEC proliferation, therefore promoting LM development. Blocking LT pathways by neutralizing antibodies against LTα and lymphotoxin β receptor may decelerate the growth of the disease. In summary, our present study demonstrated activation of LT signaling pathways in LECs contributed to the progression of LMs.

Blockade of MMP-2 and MMP-9 inhibits corneal lymphangiogenesis.

To investigate the roles of a selective MMP-2 and -9 inhibitor (SB-3CT) in corneal inflammatory lymphangiogenesis. The expression of MMP-2 and -9 in the cornea after suture inplacement, treated with SB-3CT or negative control, was detected by real-time polymerase chain reaction (PCR). Inflammatory corneal neovascularization (NV) was induced by corneal suture placement. Mice were treated with SB-3CT eye drops (twice daily for 1 week, 5 μL per drop; 50, 100, or 200 μM). The outgrowth of blood and lymphatic vessels, and macrophage recruitment were analyzed by immunofluorescence assay. The expressions of vascular endothelial growth factor-C (VEGF-C) and its receptor VEGFR-3 were tested by real-time PCR. MMP-2 and -9 expression were suppressed significantly by treatment with SB-3CT. The data demonstrated, for the first time, that SB-3CT strongly reduced corneal lymphangiogenesis and macrophage infiltration during inflammation. Furthermore, expressions of VEGF-C and its receptor VEGFR-3 were significantly inhibited by SB-3CT during corneal lymphangiogenesis. These novel findings indicated that blockade of MMP-2 and -9 could inhibit lymphangiogenesis. Further investigation of this factor may provide novel therapies for transplant rejection and other lymphatic disorders.

Inhibition of lymphangiogenesis in vitro and in vivo by the multikinase inhibitor nintedanib.

PurposeTo investigate the feasibility of nintedanib, a novel triple angiokinase inhibitor, for inhibiting lymphatic endothelial cell (LEC)-induced lymphangiogenesis in vitro and inflammatory corneal lymphangiogenesis in vivo.Materials and methodsMethylthiazolyldiphenyl-tetrazolium bromide (MTT) test, transwell system, and tube-formation assay were used to evaluate the effects of nintedanib on the proliferation, migration, and tube formation of LECs stimulated by vascular endothelial growth factor-C (VEGF-C), basic fibroblast growth factor (bFGF), or platelet-derived growth factor-BB (PDGF-BB). The murine model of suture-induced corneal neovascularization was used to assess the anti-hemangiogenic and anti-lymphangiogenic effects of nintedanib via systemic and topical applications. Corneal flatmounts were stained with lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and CD31, and the areas of involved blood and lymph vessels were analyzed morphometrically. Corneal cryosections were stained with F4/80 to evaluate inflammatory cell recruitment.ResultsWe observed a significant enhanced effect of LEC proliferation, migration, and tube formation with the administration of VEGF-C, PDGF-BB, and bFGF, respectively, which was diminished by nintedanib. Both topical and systemic applications of nintedanib inhibited suture-induced hemangiogenesis and lymphangiogenesis in the murine cornea. A reduction in F4/80+ cell infiltration was observed at day 14 after corneal suture for both systemic and topical applications of nintedanib. In comparison with controls, 61% of F4/80+ cell recruitment was inhibited via the systemic application of nintedanib, while 49% of F4/80+ cell recruitment was inhibited with the topical application of nintedanib.ConclusionNintedanib was shown to inhibit in vitro lymphangiogenesis stimulated by VEGF-C, bFGF, and PDGF-BB. Applied topically or systemically, it effectively inhibited corneal hemangiogenesis and lymphangiogenesis, accompanied by reduced inflammatory cell recruitment, which represents a new promising treatment for graft rejection after penetrating keratoplasty.

Abstract 1: Topical Tacrolimus for the Treatment of Lymphedema

PURPOSE: Lymphedema is a common, life-long complication of cancer treatment that currently has no cure. Patients with lymphedema have decreased quality of life and suffer recurrent infections, while current treatments are merely palliative and designed to prevent disease progression. Accumulating evidence indicates that T cells play a key role in the pathology of lymphedema by inhibiting lymphangiogenesis and promoting tissue fibrosis. Because the pathophysiology of lymphedema involves primarily the skin and subcutaneous tissues, it may be possible to target T cells locally using topical medications such as tacrolimus without inducing systemic immunosuppression. The purpose of this study was therefore to study the efficacy of topical tacrolimus for prevention and treatment of lymphedema using preclinical mouse models. METHODS: Topical tacrolimus (0.1%) was used to treat mice that underwent tail superficial/deep lymphatic ablation or popliteal lymph node dissection (PLND). To test the hypothesis that this therapy prevents development of lymphedema, mice were treated after a short delay following surgery (2 weeks). Other animals were treated once lymphedema was established (6 weeks postop) to test the hypothesis that topical tacrolimus can be used to treat established lymphedema. Outcomes including fibroadipose tissue deposition, limb volumes, inflammation, lymphangiogenesis, lymphatic transport and pumping capacity were then measured. RESULTS: Treatment with topical tacrolimus did not result in significant systemic absorption or immunosuppression. However, this treatment markedly decreased swelling, soft tissue thickness, and fibrosis in the tail model of lymphedema. In addition, topical tacrolimus treatment significantly decreased T cell infiltration. Preventative treatment eliminated more than 95% of lymphedema-associated swelling while late treatment reduced swelling by more than 70%. Technetium99 lymphoscintigraphy showed that preventative treatment resulted in a 5-fold increase in interstitial fluid transport to regional lymph nodes. This increased transport capacity corresponded to a more than 2 fold increase in collecting lymphatic pumping capacity as measured by near infra-red lymphangiography. Interestingly, tacrolimus application significantly increased formation of collateral superficial lymphatics and increased inflammatory lymphangiogenesis in 2 other in vivo lymphangiogenesis assays. We observed more collateral lymphatic vessels in both the tail and the PLND models. CONCLUSION: Treatment with topical tacrolimus markedly decreased chronic T cell inflammation, significantly improved formation of lymphatic collaterals, and decreased tissue fibrosis. These changes were associated with significant decreases in lymphedema and improved lymphatic function. These results have significant implications for the treatment of lymphedema because there is currently no effective pharmacologic treatment for this chronic disease.

Matrix metalloproteinase 14 participates in corneal lymphangiogenesis through the VEGF-C/VEGFR-3 signaling pathway.

The aim of the present study was to investigate the roles of matrix metalloproteinase 14 (MMP-14) in corneal inflammatory lymphangiogenesis. The expression of MMP-14 in vivo was detected by immunohistochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot assays, under various corneal conditions. pCMV-MMP-14 or empty pCMV vectors were injected into mouse corneal stroma, 3 days after suture placement in a standard suture-induced inflammatory corneal neovascularization assay. The outgrowth of blood and lymphatic vessels and macrophage recruitment were analyzed using immunofluorescence. The expression levels of vascular endothelial growth factor (VEGF) subtypes were tested by RT-qPCR. MMP-14 expression was upregulated significantly following various corneal injuries. The results demonstrated, for the first time, that MMP-14 strongly promotes corneal lymphangiogenesis and macrophage infiltration during inflammation. Furthermore, expression levels of VEGF-C and VEGF receptor-3, but not other VEGF components, were significantly upregulated by the intrastromal delivery of MMP-14 during corneal lymphangiogenesis. In conclusion, this study indicates that MMP-14 is critically involved in the processes of lymphangiogenesis. Inhibition of MMP-14 may provide a viable treatment for transplant rejection and other lymphatic disorders.

CXCL12/CXCR4 axis regulates neovascularization and lymphangiogenesis in sutured corneas in mice.

The present study aimed to determine the plausible functional role of chemokine (C-X-C motif) ligand 12 (CXCL12)/chemokine (C-X-C motif) receptor 4 (CXCR4) in inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo. Corneal hemangiogenesis and lymphangiogenesis were induced by placing an 11-0 nylon suture in an intrastromal position. The expression levels of the vascular endothelial growth factor (VEGF) family, CXCL12 and CXCR4 in the corneas were investigated in the corneas using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Corneal hemangiogenic and lymphangiogenic responses were assessed by immunofluorescence using specific antibodies against cluster of differentiation 31 and lymphatic vessel endothelial hyaluronan receptor-1. Subconjunctival injection of AMD3100 to the sutured corneas was also performed. CXCL12/CXCR4 mRNA and protein expression levels increased markedly in suture-induced corneal neovascularization (CNV) and decreased with AMD3100 treatment. Hemangiogenesis and lymphangiogenesis were captured in images using immunofluorescence and were shown to be markedly increased with suture placement and reduced with AMD3100 treatment. VEGF-A/VEGFR-1 and VEGF-C/VEGFR-3 mRNA expression levels were upregulated in the suture placement and control groups, whereas the expression levels of all the factors were downregulated in the AMD3100 treatment group. The results from the present study demonstrated that CXCL12/CXCR4 interactions regulate hemangiogenesis and lymphangiogenesis in suture-induced CNV. AMD3100 may be a novel therapeutic target for the prevention of blindness.

Pathological lymphangiogenesis is modulated by galectin-8-dependent crosstalk between podoplanin and integrin-associated VEGFR-3.

Lymphangiogenesis plays a pivotal role in diverse pathological conditions. Here, we demonstrate that a carbohydrate-binding protein, galectin-8, promotes pathological lymphangiogenesis. Galectin-8 is markedly upregulated in inflamed human and mouse corneas, and galectin-8 inhibitors reduce inflammatory lymphangiogenesis. In the mouse model of corneal allogeneic transplantation, galectin-8-induced lymphangiogenesis is associated with an increased rate of corneal graft rejection. Further, in the murine model of herpes simplex virus keratitis, corneal pathology and lymphangiogenesis are ameliorated in Lgals8−/− mice. Mechanistically, VEGF-C-induced lymphangiogenesis is significantly reduced in the Lgals8−/− and Pdpn−/− mice; likewise, galectin-8-induced lymphangiogenesis is reduced in Pdpn−/− mice. Interestingly, knockdown of VEGFR-3 does not affect galectin-8-mediated lymphatic endothelial cell (LEC) sprouting. Instead, inhibiting integrins α1β1 and α5β1 curtails both galectin-8- and VEGF-C-mediated LEC sprouting. Together, this study uncovers a unique molecular mechanism of lymphangiogenesis in which galectin-8-dependent crosstalk among VEGF-C, podoplanin and integrin pathways plays a key role.

P-140 Promoting Inflammatory Lymphangiogenesis by VEGF–C Aggravates Intestinal Inflammation in Mice with Experimental Acute Colitis

Angiogenesis and lymphangiogenesis are thought to play a role in the pathogenesis of inflammatory bowel diseases (IBD). However, it is not understood if inflammatory lymphangiogenesis is a pathological consequence or a productive attempt to resolve the inflammation. This study investigated the effect of lymphangiogenesis on intestinal inflammation by overexpressing a lymphangiogenesis factor, vascular endothelial growth factor-C (VEGF-C) in a mouse model of acute colitis. C57BL/6 mice were treated with recombinant adenovirus overexpressing VEGF-C or with recombinant VEGF-C156S protein. Acute colitis was then established by exposing the mice to 5% dextran sodium sulfate (DSS) for 7 days. Mice were evaluated for disease activity index (DAI), colonic inflammatory changes, colon edema, microvessel density, lymphatic vessel density (LVD), and VEGFR-3mRNA expression in colon tissue. When acute colitis was induced in mice overexpressing VEGF-C, there was a significant increase in colonic epithelial damage, inflammatory edema, microvessel density, and neutrophil infiltration compared to control mice. These mice also exhibited increased lymphatic vessel density (73.0 ± 3.9 versus 38.2 ± 1.9, P < 0.001) and lymphatic vessel size (1974.6 ± 104.3 versus 1639.0 ± 91.5, P < 0.001) compared to control mice. Additionally, the expression of VEGFR-3 mRNA was significantly upregulated in VEGF-C156S mice compared to DSS-treated mice after induction of colitis (42.0 ± 1.4 versus 3.5 ± 0.4, P < 0.001). Stimulation of lymphangiogenesis by VEGF-C during acute colitis promoted inflammatory lymphangiogenesis in the colon and aggravated intestinal inflammation. Inflammatory lymphangiogenesis may have pleiotropic effects at different stages of IBD.