CCM3 Loss-Induced Lymphatic Defect Is Mediated by the Augmented VEGFR3-ERK1/2 Signaling.

OBJECTIVE: Cerebral cavernous malformations (CCMs) can happen anywhere in the body, although they most commonly produce symptoms in the brain. The role of CCM genes in other vascular beds outside the brain and retina is not well-examined, although the 3 CCM-associated genes (CCM1, CCM2, and CCM3) are ubiquitously expressed in all tissues. We aimed to determine the role of CCM gene in lymphatics. Approach and Results: Mice with an inducible pan-endothelial cell (EC) or lymphatic EC deletion of Ccm3 (Pdcd10ECKO or Pdcd10LECKO) exhibit dilated lymphatic capillaries and collecting vessels with abnormal valve structure. Morphological alterations were correlated with lymphatic dysfunction in Pdcd10LECKO mice as determined by Evans blue dye and fluorescein isothiocyanate(FITC)-dextran transport assays. Pdcd10LECKO lymphatics had increased VEGFR3 (vascular endothelial growth factor receptor-3)-ERK1/2 (extracellular signal-regulated kinase 1/2) signaling with lymphatic hyperplasia. Mechanistic studies suggested that VEGFR3 is primarily regulated at a transcriptional level in Ccm3-deficient lymphatic ECs, in an NF-κB (nuclear factor κB)-dependent manner. CCM3 binds to importin alpha 2/KPNA2 (karyopherin subunit alpha 2), and a CCM3 deletion releases KPNA2 to activate NF-κB P65 by facilitating its nuclear translocation and P65-dependent VEGFR3 transcription. Moreover, increased VEGFR3 in lymphatic EC preferentially activates ERK1/2 signaling, which is critical for lymphatic EC proliferation. Importantly, inhibition of VEGFR3 or ERK1/2 rescued the lymphatic defects in structure and function. CONCLUSIONS: Our data demonstrate that CCM3 deletion augments the VEGFR3-ERK1/2 signaling in lymphatic EC that drives lymphatic hyperplasia and malformation and warrant further investigation on the potential clinical relevance of lymphatic dysfunction in patients with CCM.

Microvascular Dysfunction in the Critically Ill.

Microvascular dysfunction is a frequent complication of many chronic and acute conditions, especially in the critically ill. Moreover, the severity of microvascular alterations is associated with development of organ dysfunction and poor outcome. The complexities and heterogeneity of critical illness, especially in the elderly patient, requires more mechanistically oriented clinical trials that monitor the effectiveness of existing therapies and of those to come. Recent advances in the ability to obtain physiologically based assessments of microcirculatory function at the bedside will make microcirculatory-guided resuscitation a point of care reality.

ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4.

RATIONALE: Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown. OBJECTIVE: We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation. METHODS AND RESULTS: Primary endothelial cells from dermal blood and lymphatic vessels (blood vascular endothelial cells and lymphatic endothelial cells [LECs]) were exposed to low-rate steady laminar flow. Shear stress-induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in blood vascular endothelial cells and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the vascular endothelial growth factor (VEGF)-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release-activated calcium channel, was identified to induce the shear stress phenotypes and cell proliferation in LECs responding to the fluid flow. Mechanistically, ORAI1 induced upregulation of Krüppel-like factor (KLF)-2 and KLF4 in the flow-activated LECs, and the 2 KLF proteins cooperate to regulate VEGF-A, VEGF-C, FGFR3, and p57 by binding to the regulatory regions of the genes. Consistently, freshly isolated LECs from Orai1 knockout embryos displayed reduced expression of KLF2, KLF4, VEGF-A, VEGF-C, and FGFR3 and elevated expression of p57. Accordingly, mouse embryos deficient in Orai1, Klf2, or Klf4 showed a significantly reduced lymphatic density and impaired lymphatic development. CONCLUSIONS: Our study identified a molecular mechanism for laminar flow-activated LEC proliferation.

Polydom Is an Extracellular Matrix Protein Involved in Lymphatic Vessel Remodeling.

RATIONALE: Lymphatic vasculature constitutes a second vascular system essential for immune surveillance and tissue fluid homeostasis. Maturation of the hierarchical vascular structure, with a highly branched network of capillaries and ducts, is crucial for its function. Environmental cues mediate the remodeling process, but the mechanism that underlies this process is largely unknown. OBJECTIVE: Polydom (also called Svep1) is an extracellular matrix protein identified as a high-affinity ligand for integrin α9ß1. However, its physiological function is unclear. Here, we investigated the role of Polydom in lymphatic development. METHODS AND RESULTS: We generated Polydom-deficient mice. Polydom-/- mice showed severe edema and died immediately after birth because of respiratory failure. We found that although a primitive lymphatic plexus was formed, it failed to undergo remodeling in Polydom-/- embryos, including sprouting of new capillaries and formation of collecting lymphatic vessels. Impaired lymphatic development was also observed after knockdown/knockout of polydom in zebrafish. Polydom was deposited around lymphatic vessels, but secreted from surrounding mesenchymal cells. Expression of Foxc2 (forkhead box protein c2), a transcription factor involved in lymphatic remodeling, was decreased in Polydom-/- mice. Polydom bound to the lymphangiogenic factor Ang-2 (angiopoietin-2), which was found to upregulate Foxc2 expression in cultured lymphatic endothelial cells. Expressions of Tie1/Tie2 receptors for angiopoietins were also decreased in Polydom-/- mice. CONCLUSIONS: Polydom affects remodeling of lymphatic vessels in both mouse and zebrafish. Polydom deposited around lymphatic vessels seems to ensure Foxc2 upregulation in lymphatic endothelial cells, possibly via the Ang-2 and Tie1/Tie2 receptor system.

An Evolutionarily Conserved Role for Polydom/Svep1 During Lymphatic Vessel Formation.

RATIONALE: Lymphatic vessel formation and function constitutes a physiologically and pathophysiologically important process, but its genetic control is not well understood. OBJECTIVE: Here, we identify the secreted Polydom/Svep1 protein as essential for the formation of the lymphatic vasculature. We analyzed mutants in mice and zebrafish to gain insight into the role of Polydom/Svep1 in the lymphangiogenic process. METHODS AND RESULTS: Phenotypic analysis of zebrafish polydom/svep1 mutants showed a decrease in venous and lymphovenous sprouting, which leads to an increased number of intersegmental arteries. A reduced number of primordial lymphatic cells populated the horizontal myoseptum region but failed to migrate dorsally or ventrally, resulting in severe reduction of the lymphatic trunk vasculature. Corresponding mutants in the mouse Polydom/Svep1 gene showed normal egression of Prox-1+ cells from the cardinal vein at E10.5, but at E12.5, the tight association between the cardinal vein and lymphatic endothelial cells at the first lymphovenous contact site was abnormal. Furthermore, mesenteric lymphatic structures at E18.5 failed to undergo remodeling events in mutants and lacked lymphatic valves. In both fish and mouse embryos, the expression of the gene suggests a nonendothelial and noncell autonomous mechanism. CONCLUSIONS: Our data identify zebrafish and mouse Polydom/Svep1 as essential extracellular factors for lymphangiogenesis. Expression of the respective genes by mesenchymal cells in intimate proximity with venous and lymphatic endothelial cells is required for sprouting and migratory events in zebrafish and for remodeling events of the lymphatic intraluminal valves in mouse embryos.

Efficacy of AdipoDren® in Reducing Interleukin-1-Induced Lymphatic Endothelial Hyperpermeability.

Lymphatic leakage can be seen as a detrimental phenomenon associated with fluid retention and deposition as well as gain of weight. Moreover, lymphatic dysfunction is associated with an inflammatory environment and can be a substrate for other health conditions. A number of treatments can ameliorate lymphatic vasculature: natural substances have been used as treatment options particularly suitable for their consolidated effectiveness and safety profile. Here we report the protective effect of AdipoDren®, an association of a series of plant-derived natural complexes, on lymphatic endothelium permeability promoted by interleukin-1 beta (IL-1ß) and the associated molecular mechanisms. AdipoDren® demonstrated a protective effect on dermal lymphatic endothelial cell permeability increased by IL-1ß. Reduced permeability was due to the maintenance of tight junctions and cell-cell localisation of occludin and zonula occludens-1 (ZO-1). Moreover, AdipoDren® reduced the expression of the inflammatory key element cyclooxygenase-2 (COX-2), while not altering the levels of endothelial and inducible nitric oxide synthases (eNOS and iNOS). The upregulation of antioxidant enzymatic systems (catalase and superoxide dismutase-1, SOD-1) and the downregulation of pro-oxidant markers (p22 phox subunit of NADPH oxidase) were also evident. In conclusion, AdipoDren® would be useful to ameliorate conditions of altered lymphatic vasculature and to support the physiological functionality of the lymphatic endothelium.

Blunted flow-mediated responses and diminished nitric oxide synthase expression in lymphatic thoracic ducts of a rat model of metabolic syndrome.

Shear-dependent inhibition of lymphatic thoracic duct (TD) contractility is principally mediated by nitric oxide (NO). Endothelial dysfunction and poor NO bioavailability are hallmarks of vasculature dysfunction in states of insulin resistance and metabolic syndrome (MetSyn). We tested the hypothesis that flow-dependent regulation of lymphatic contractility is impaired under conditions of MetSyn. We utilized a 7-wk high-fructose-fed male Sprague-Dawley rat model of MetSyn and determined the stretch- and flow-dependent contractile responses in an isobaric ex vivo TD preparation. TD diameters were tracked and contractile parameters were determined in response to different transmural pressures, imposed flow, exogenous NO stimulation by S-nitro-N-acetylpenicillamine (SNAP), and inhibition of NO synthase (NOS) by l-nitro-arginine methyl ester (l-NAME) and the reactive oxygen species (ROS) scavenging molecule 4-hydroxy-tempo (tempol). Expression of endothelial NO synthase (eNOS) in TD was determined using Western blot. Approximately 25% of the normal flow-mediated inhibition of contraction frequency was lost in TDs isolated from MetSyn rats despite a comparable SNAP response. Inhibition of NOS with l-NAME abolished the differences in the shear-dependent contraction frequency regulation between control and MetSyn TDs, whereas tempol did not restore the flow responses in MetSyn TDs. We found a significant reduction in eNOS expression in MetSyn TDs suggesting that diminished NO production is partially responsible for impaired flow response. Thus our data provide the first evidence that MetSyn conditions diminish eNOS expression in TD endothelium, thereby affecting the flow-mediated changes in TD lymphatic function.

Altered reactivity and nitric oxide signaling in the isolated thoracic duct from an ovine model of congenital heart disease with increased pulmonary blood flow.

We have previously shown decreased pulmonary lymph flow in our lamb model of chronically increased pulmonary blood flow, created by the in utero placement of an 8-mm aortopulmonary shunt. The purpose of this study was to test the hypothesis that abnormal lymphatic function in shunt lambs is due to impaired lymphatic endothelial nitric oxide (NO)-cGMP signaling resulting in increased lymphatic vascular constriction and/or impaired relaxation. Thoracic duct rings were isolated from 4-wk-old shunt (n = 7) and normal (n = 7) lambs to determine length-tension properties, vascular reactivity, and endothelial NO synthase protein. At baseline, shunt thoracic duct rings had 2.6-fold higher peak to peak tension and a 2-fold increase in the strength of contractions compared with normal rings (P < 0.05). In response to norepinephrine, shunt thoracic duct rings had a 2.4-fold increase in vascular tone compared with normal rings (P < 0.05) and impaired relaxation in response to the endothelium-dependent dilator acetylcholine (63% vs. 13%, P < 0.05). In vivo, inhaled NO (40 ppm) increased pulmonary lymph flow (normalized for resistance) ∼1.5-fold in both normal and shunt lambs (P < 0.05). Inhaled NO exposure increased bioavailable NO [nitrite/nitrate (NOx); ∼2.5-fold in normal lambs and ∼3.4-fold in shunt lambs] and cGMP (∼2.5-fold in both) in the pulmonary lymph effluent (P < 0.05). Chronic exposure to increased pulmonary blood flow is associated with pulmonary lymphatic endothelial injury that disrupts NO-cGMP signaling, leading to increased resting vasoconstriction, increased maximal strength of contraction, and impaired endothelium-dependent relaxation. Inhaled NO increases pulmonary lymph NOx and cGMP levels and pulmonary lymph flow in normal and shunt lambs. Therapies that augment NO-cGMP signaling within the lymphatic system may provide benefits, warranting further study.

Advances in therapeutic interventions targeting the vascular and lymphatic endothelium in inflammatory bowel disease.

PURPOSE OF REVIEW: The review summarizes the current knowledge of the roles played by the vascular and lymphatic endothelium throughout the gut in the pathogenesis of inflammatory bowel disease (IBD) and gives an update on emerging strategies targeting both vasculatures. RECENT FINDINGS: Enormous efforts have been made to understand the mechanisms underlining the origin, development and maintenance of intestinal chronic inflammation. In particular, new studies focused their attention on the role played by the microvascular and lymphatic endothelium in the pathogenesis of IBD. During inflammation, whereas the microvasculature is responsible for the entry and distribution of immune cells in the mucosa, the lymphatic system controls leukocyte exit, bacterial clearance and edema absorption. The study of these events, which are aberrant during chronic inflammation, has resulted in the identification and validation of several targets for the treatment of experimental colitis, some of which have translated into effective treatments for patients with IBD. SUMMARY: Although much attention has been paid to the microvascular endothelium and to antiangiogenic therapies, specific studies on the lymphatic vasculature and its functions in IBD are still at the initial stage, and other molecular mechanisms, genes, molecules and new pathways must definitely be explored.

Tumor angiogenesis and lymphangiogenesis: tumor/endothelial crosstalk and cellular/microenvironmental signaling mechanisms.

Tumor angiogenesis and lymphangiogenesis are key features of tumor progression and metastasis. The role of tumor cells-derived factors in the promotion of associated angiogenesis and lymphangiogenesis is much studied and, no doubt, very important for the understanding of cancer progression. This review aims to present and discuss the work done on the pro-angiogenic and lymphangiogenic cellular interactions within the tumor microenvironment and the signaling pathways that regulate this crosstalk. Such multifactor studies are critical for the development of future therapeutic approaches for cancer because they take into account the complexities of cellular interactions within the tumor microenvironment.

Increased nitric oxide production in lymphatic endothelial cells causes impairment of lymphatic drainage in cirrhotic rats.

BACKGROUND AND AIM: The lymphatic network plays a major role in maintaining tissue fluid homoeostasis. Therefore several pathological conditions associated with oedema formation result in deficient lymphatic function. However, the role of the lymphatic system in the pathogenesis of ascites and oedema formation in cirrhosis has not been fully clarified. The aim of this study was to investigate whether the inability of the lymphatic system to drain tissue exudate contributes to the oedema observed in cirrhosis. METHODS: Cirrhosis was induced in rats by CCl(4) inhalation. Lymphatic drainage was evaluated using fluorescent lymphangiography. Expression of endothelial nitric oxide synthase (eNOS) was measured in primary lymphatic endothelial cells (LyECs). Inhibition of eNOS activity in cirrhotic rats with ascites (CH) was carried out by L-N(G)-methyl-L-arginine (L-NMMA) treatment (0.5 mg/kg/day). RESULTS: The (CH) rats had impaired lymphatic drainage in the splanchnic and peripheral regions compared with the control (CT) rats. LyECs isolated from the CH rats showed a significant increase in eNOS and nitric oxide (NO) production. In addition, the lymphatic vessels of the CH rats showed a significant reduction in smooth muscle cell (SMC) coverage compared with the CT rats. CH rats treated with L-NMMA for 7 days showed a significant improvement in lymphatic drainage and a significant reduction in ascites volume, which were associated with increased plasma volume. This beneficial effect of L-NMMA inhibition was also associated with a significant increase in lymphatic SMC coverage. CONCLUSIONS: The upregulation of eNOS in the LyECs of CH rats causes long-term lymphatic remodelling, which is characterised by a loss of SMC lymphatic coverage. The amelioration of this lymphatic abnormality by chronic eNOS inhibition results in improved lymphatic drainage and reduced ascites.

Relationships between lymphangiogenesis and angiogenesis during inflammation in rat mesentery microvascular networks.

BACKGROUND: Lymphatic and blood microvascular systems play a coordinated role in the regulation of interstitial fluid balance and immune cell trafficking during inflammation. The objective of this study was to characterize the temporal and spatial relationships between lymphatic and blood vessel growth in the adult rat mesentery following an inflammatory stimulus. METHODS AND RESULTS: Mesenteric tissues were harvested from unstimulated adult male Wistar rats and at 3, 10, and 30 days post compound 48/80 stimulation. Tissues were immunolabeled for PECAM, LYVE-1, Prox1, podoplanin, CD11b, and class III ß-tubulin. Vascular area, capillary blind end density, and vascular length density were quantified for each vessel system per time point. Blood vascular area increased compared to unstimulated tissues by day 10 and remained increased at day 30. Following the peak in blood capillary sprouting at day 3, blood vascular area and density increased at day 10. The number of blind-ended lymphatic vessels and lymphatic density did not significantly increase until day 10, and lymphatic vascular area was not increased compared to the unstimulated level until day 30. Lymphangiogenesis correlated with the upregulation of class III ß-tubulin expression by endothelial cells along lymphatic blind-ended vessels and increased lymphatic/blood endothelial cell connections. In local tissue regions containing both blood and lymphatic vessels, the presence of lymphatics attenuated blood capillary sprouting. CONCLUSIONS: Our work suggests that lymphangiogenesis lags angiogenesis during inflammation and motivates the need for future investigations aimed at understanding lymphatic/blood endothelial cell interactions. The results also indicate that lymphatic endothelial cells undergo phenotypic changes during lymphangiogenesis.

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.

Role of the vascular and lymphatic endothelium in the pathogenesis of inflammatory bowel disease: 'brothers in arms'.

The 'IN' of chronic inflammation-that is, the mechanisms of cell entry into the intestinal mucosa, bacterial and foreign antigen invasion, angiogenesis, and the control of gut inflammation through intestinal microvasculature-has received a great deal of attention in studies of the pathogenesis of inflammatory bowel disease (IBD). This has resulted in the validation of several targets for the treatment of experimental inflammation-both on immune and non-immune cells-some of which have translated into effective treatments for patients with IBD. An important aspect of this has been our growing understanding of the role the intestinal vascular microcirculation plays in the initiation and perpetuation of the inflammatory process, by regulating the migration of leucocytes into the interstitial space. However, it is becoming increasingly clear that it is also important to focus on the 'OUT' of chronic inflammation-that is, the lymphatics and their role in controlling tissue oedema, leucocyte exit, bacterial antigen and inflammatory chemokine clearance. As our understanding of the lymphatics and the role they play grows, another rich source of non-immune cell targets for therapeutic intervention is gradually being revealed. This article describes current knowledge of the roles played by the vascular and lymphatic endothelium throughout the gut in the pathogenesis of IBD, and how this differs from their role under physiological conditions, as well as discussing current and future therapeutic targets that have been identified.

Lymphangiogenesis, lymphatic endothelial cells and lymphatic metastasis in head and neck cancer--a review of mechanisms.

Lymphatic metastasis is a continuous and complicated process. The detailed mechanisms of lymphatic metastasis are still not very clear, despite considerable research efforts in recent years. Previously, it was commonly accepted that there were no lymphatic vessels in the primary tumor. However, recent studies have demonstrated that lymphatic vessels are detectable in certain types of cancer, and more and more evidence has shown that cancer cells invade into local lymph nodes mainly via peritumoral lymphatic vessels. Moreover, activated endothelial cells may also be important, having an influence on lymphatic metastasis of cancer cells. This article, based on recent research findings, provides an in-depth discussion of the relationship between lymphangiogenesis, tumor-derived lymphatic endothelial cells and lymphatic metastasis in head and neck cancer.

Transmural flow modulates cell and fluid transport functions of lymphatic endothelium.

RATIONALE: Lymphatic transport of peripheral interstitial fluid and dendritic cells (DCs) is important for both adaptive immunity and maintenance of tolerance to self-antigens. Lymphatic drainage can change rapidly and dramatically on tissue injury or inflammation, and therefore increased fluid flow may serve as an important early cue for inflammation; however, the effects of transmural flow on lymphatic function are unknown. OBJECTIVE: Here we tested the hypothesis that lymph drainage regulates the fluid and cell transport functions of lymphatic endothelium. METHODS AND RESULTS: Using in vitro and in vivo models, we demonstrated that lymphatic endothelium is sensitive to low levels of transmural flow. Basal-to-luminal flow (0.1 and 1 mum/sec) increased lymphatic permeability, dextran transport, and aquaporin-2 expression, as well as DC transmigration into lymphatics. The latter was associated with increased lymphatic expression of the DC homing chemokine CCL21 and the adhesion molecules intercellular adhesion molecule-1 and E-selectin. In addition, transmural flow induced delocalization and downregulation of vascular endothelial cadherin and PECAM-1 (platelet/endothelial cell adhesion molecule-1). Flow-enhanced DC transmigration could be reversed by blocking CCR7, intercellular adhesion molecule-1, or E-selectin. In an experimental model of lymphedema, where lymphatic drainage is greatly reduced or absent, lymphatic endothelial expression of CCL21 was nearly absent. CONCLUSIONS: These findings introduce transmural flow as an important regulator of lymphatic endothelial function and suggest that flow might serve as an early inflammatory signal for lymphatics, causing them to regulate transport functions to facilitate the delivery of soluble antigens and DCs to lymph nodes.

Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac-1/ICAM-1-dependent mechanism.

The lymphatic system is essential for the generation of immune responses by facilitating immune cell trafficking to lymph nodes. Dendritic cells (DCs), the most potent APCs, exit tissues via lymphatic vessels, but the mechanisms of interaction between DCs and the lymphatic endothelium and the potential implications of these interactions for immune responses are poorly understood. In this study, we demonstrate that lymphatic endothelial cells (LECs) modulate the maturation and function of DCs. Direct contact of human monocyte-derived DCs with an inflamed, TNF-alpha-stimulated lymphatic endothelium reduced expression of the costimulatory molecule CD86 by DCs and suppressed the ability of DCs to induce T cell proliferation. These effects were dependent on adhesive interactions between DCs and LECs that were mediated by the binding of Mac-1 on DCs to ICAM-1 on LECs. Importantly, the suppressive effects of the lymphatic endothelium on DCs were observed only in the absence of pathogen-derived signals. In vivo, DCs that migrated to the draining lymph nodes upon inflammatory stimuli, but in the absence of a pathogen, showed increased levels of CD86 expression in ICAM-1-deficient mice. Together, these data demonstrate a direct role of LECs in the modulation of immune response and suggest a function of the lymphatic endothelium in preventing undesired immune reactions in inflammatory conditions.

TGF-beta1 is a negative regulator of lymphatic regeneration during wound repair.

Although clinical studies have identified scarring/fibrosis as significant risk factors for lymphedema, the mechanisms by which lymphatic repair is impaired remain unknown. Transforming growth factor -beta1 (TGF-beta1) is a critical regulator of tissue fibrosis/scarring and may therefore also play a role in the regulation of lymphatic regeneration. The purpose of this study was therefore to assess the role of TGF-beta1 on scarring/fibrosis and lymphatic regeneration in a mouse tail model. Acute lymphedema was induced in mouse tails by full-thickness skin excision and lymphatic ligation. TGF-beta1 expression and scarring were modulated by repairing the wounds with or without a topical collagen gel. Lymphatic function and histological analyses were performed at various time points. Finally, the effects of TGF-beta1 on lymphatic endothelial cells (LECs) in vitro were evaluated. As a result, the wound repair with collagen gel significantly reduced the expression of TGF-beta1, decreased scarring/fibrosis, and significantly accelerated lymphatic regeneration. The addition of recombinant TGF-beta1 to the collagen gel negated these effects. The improved lymphatic regeneration secondary to TGF-beta1 inhibition was associated with increased infiltration and proliferation of LECs and macrophages. TGF-beta1 caused a dose-dependent significant decrease in cellular proliferation and tubule formation of isolated LECs without changes in the expression of VEGF-C/D. Finally, the increased expression of TGF-beta1 during wound repair resulted in lymphatic fibrosis and the coexpression of alpha-smooth muscle actin and lymphatic vessel endothelial receptor-1 in regenerated lymphatics. In conclusion, the inhibition of TGF-beta1 expression significantly accelerates lymphatic regeneration during wound healing. An increased TGF-beta1 expression inhibits LEC proliferation and function and promotes lymphatic fibrosis. These findings imply that the clinical interventions that diminish TGF-beta1 expression may be useful in promoting more rapid lymphatic regeneration.

Developmental and pathological lymphangiogenesis: from models to human disease.

The lymphatic vascular system, the body's second vascular system present in vertebrates, has emerged in recent years as a crucial player in normal and pathological processes. It participates in the maintenance of normal tissue fluid balance, the immune functions of cellular and antigen trafficking and absorption of fatty acids and lipid-soluble vitamins in the gut. Recent scientific discoveries have highlighted the role of lymphatic system in a number of pathologic conditions, including lymphedema, inflammatory diseases, and tumor metastasis. Development of genetically modified animal models, identification of lymphatic endothelial specific markers and regulators coupled with technological advances such as high-resolution imaging and genome-wide approaches have been instrumental in understanding the major steps controlling growth and remodeling of lymphatic vessels. This review highlights the recent insights and developments in the field of lymphatic vascular biology.

Markers for microscopic imaging of lymphangiogenesis and angiogenesis.

Imaging of lymphangiogenesis and angiogenesis requires robust and unambiguous markers of lymphatic and blood vessels. Although much progress has been made in recent years in identifying molecules specifically expressed on lymphatic and blood vessels, no perfect marker has been found that works reliably in all species, tissues, vascular beds, and in all physiological and pathologic conditions. The heterogeneity of expression of markers in both blood and lymphatic vessels reflects underlying differences in the phenotype of endothelial cells. Use of only one marker can lead to misleading interpretations, but these pitfalls can usually be avoided by use of multiple markers and three-dimensional whole-mount preparations. LYVE-1, VEGFR-3, Prox1, and podoplanin are among the most useful markers for microscopic imaging of lymphatic vessels, but, depending on histologic location, each marker can be expressed by other cell types, including vascular endothelial cells. Other markers, including CD31, junctional proteins, and receptors, such as VEGF-2, are shared by lymphatic and blood vessels.