Update September 2017

Lymphatic endothelium serves as a barrier to control fluid balance and immune cell trafficking to maintain tissue homeostasis. Long-term alteration of lymphatic vasculature promotes edema and fibrosis, which is an aggravating factor in the onset of cardiovascular diseases such as myocardial infarction. Apelin is a bioactive peptide that plays a central role in angiogenesis and cardiac contractility. Despite an established role of apelin in lymphangiogenesis, little is known about its function in the cardiac lymphatic endothelium. Here, we show that apelin and its receptor APJ were exclusively expressed on newly formed lymphatic vasculature in a pathological model of myocardial infarction. Using an apelin-knockout mouse model, we identified morphological and functional defects in lymphatic vasculature associated with a proinflammatory status. Surprisingly, apelin deficiency increased the expression of lymphangiogenic growth factors VEGF-C and VEGF-D and exacerbated lymphangiogenesis after myocardial infarction. Conversely, the overexpression of apelin in ischemic heart was sufficient to restore a functional lymphatic vasculature and to reduce matrix remodeling and inflammation. In vitro, the expression of apelin prevented the alteration of cellular junctions in lymphatic endothelial cells induced by hypoxia. In addition, we demonstrated that apelin controls the secretion of the lipid mediator sphingosine-1-phosphate in lymphatic endothelial cells by regulating the level of expression of sphingosine kinase 2 and the transporter SPNS2. Taken together, our results show that apelin plays a key role in lymphatic vessel maturation and stability in pathological settings. Thus, apelin may represent a novel candidate to prevent pathological lymphatic remodeling in diseases.

The mammalian intestine is richly supplied with lymphatic vasculature, which has functions ranging from maintenance of interstitial fluid balance to transport of antigens, antigen‐presenting cells, dietary lipids and fat‐soluble vitamins. In this Review, we provide in‐depth information concerning the organization and structure of intestinal lymphatics, the current view of their developmental origins, as well as molecular mechanisms of intestinal lymphatic patterning and maintenance. We will also discuss physiological aspects of intestinal lymph flow regulation and the known and emerging roles of intestinal lymphatic vessels in human diseases, such as IBD, infection and cancer.

Lymph is transported along collecting lymphatic vessels by intrinsic and extrinsic pumping. The walls have muscle of a type intermediate between blood‐vascular smooth muscle and myocardium; a contracting segment between two valves (a lymphangion) constitutes a pump. This intrinsic mechanism is investigated ex vivo in isolated, spontaneously contracting, perfused segments subjected to controlled external pressures. The reaction to varying afterload is probed by slowly ramping up the outlet pressure until pumping fails. Often the failure occurs when the contraction raises intra‐lymphangion pressure insufficiently to overcome the outlet pressure, open the outlet valve and cause ejection, but many segments fail by other means, the mechanisms of which are not clear. We here elucidate those mechanisms by resort to a numerical model. Experimental observations are paired with comparable findings from computer simulations, using a lumped‐parameter model that incorporates previously measured valve properties, plus new measurements of active contractile and passive elastic properties, and the dependence of contraction frequency on transmural pressure, all taken from isobaric twitch contraction experiments in the same vessel. Surprisingly, the model predicts seven different possible modes of pump failure, each defined by a different sequence of valve events, with their occurrence depending on the parameter values and boundary conditions. Some, but not all, modes were found experimentally. Further model investigation reveals routes by which a vessel exhibiting one mode of failure might under altered circumstances exhibit another.

OBJECTIVE: The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood‐filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. APPROACH AND RESULTS: We generated an inducible, lymphatic‐specific podoplanin knockout mouse model (PdpnDeltaLEC) and induced gene deletion postnatally. PdpnDeltaLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, PdpnDeltaLEC mice had blood‐filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood‐filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence‐activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in PdpnDeltaLEC mice. CONCLUSIONS: These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes.

Lymphatic dysfunction is involved in many diseases including lymphedema, hypertension, autoimmune responses, graft rejection, atherosclerosis, microbial infections, cancer and cancer metastasis. Expanding our knowledge of lymphatic system function can lead to a better understanding of these disease processes and improve treatment options. Here, optical coherence tomography (OCT) methods were used to reveal intraluminal valve dynamics in 3 dimensions, and measure lymph flow and vessel contraction simultaneously in 3 neighboring lymphangions of the afferent collecting lymphatic vessels to the popliteal lymph node in mice. Flow measurements were based on Doppler OCT techniques in combination with exogenous lymph labeling by Intralipid. Through these imaging methods, it is possible to study lymphatic function and pumping more comprehensively. These capabilities can lead to a better understanding of the regulation and dysregulation of lymphatic vessels in health and disease. The image depicts the dynamic measurements of lymphatic valves, lymphatic vessels cross‐sectional area and lymph velocity simultaneously measured in vivo with optical coherence tomography.

Lymphatic dysfunction is associated with the progression of many cardiovascular disorders due to their role in maintaining tissue fluid homeostasis. Promoting new lymphatic vessels (lymphangiogenesis) is a promising strategy to reverse these cardiovascular disorders via restoring lymphatic function. Vascular endothelial growth factor (VEGF) members VEGF‐C and VEGF‐D are both potent candidates for stimulating lymphangiogenesis, though maintaining spatial and temporal control of these factors represents a challenge to developing efficient therapeutic lymphangiogenic applications. Injectable alginate hydrogels have been useful for the controlled delivery of many angiogenic factors, including VEGF‐A, to stimulate new blood vasculature. However, the utility of these tunable hydrogels for delivering lymphangiogenic factors has never been closely examined. Thus, the objective of this study was to utilize ionically cross‐linked alginate hydrogels to deliver VEGF‐C and VEGF‐D for potential lymphangiogenic applications. We demonstrated that lymphatic endothelial cells (LECs) are sensitive to temporal presentation of VEGF‐C and VEGF‐D but with different responses between the factors. The greatest LEC mitogenic and sprouting response was observed for constant concentrations of VEGF‐C and a high initial concentration that gradually decreased over time for VEGF‐D. Additionally, alginate hydrogels provided sustained release of radiolabeled VEGF‐C and VEGF‐D. Finally, VEGF‐C and VEGF‐D released from these hydrogels promoted a similar number of LEC sprouts as exogenously added growth factors and new vasculature in vivo via a chick chorioallantoic membrane (CAM) assay. Overall, these findings demonstrate that alginate hydrogels can provide sustained and bioactive release of VEGF‐C and VEGF‐D which could have applications for therapeutic lymphangiogenesis.

PURPOSE: To investigate the roles of a selective MMP‐2 and ‐9 inhibitor (SB‐3CT) in corneal inflammatory lymphangiogenesis. METHODS: 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 muL per drop; 50, 100, or 200 muM). 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. RESULTS: 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. CONCLUSIONS: 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.

The collagen‐ and calcium‐binding EGF domains 1 (CCBE1) protein is necessary for lymphangiogenesis. Its C‐terminal collagen‐like domain was shown to be required for the activation of the major lymphangiogenic growth factor VEGF‐C (Vascular Endothelial Growth Factor‐C) along with the ADAMTS3 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs‐3) protease. However, it remained unclear how the N‐terminal domain of CCBE1 contributed to lymphangiogenic signaling. Here, we show that efficient activation of VEGF‐C requires its C‐terminal domain both in vitro and in a transgenic mouse model. The N‐terminal EGF‐like domain of CCBE1 increased VEGFR‐3 signaling by colocalizing pro‐VEGF‐C with its activating protease to the lymphatic endothelial cell surface. When the ADAMTS3 amounts were limited, proteolytic activation of pro‐VEGF‐C was supported by the N‐terminal domain of CCBE1, but not by its C‐terminal domain. A single amino acid substitution in ADAMTS3, identified from a lymphedema patient, was associated with abnormal CCBE1 localization. These results show that CCBE1 promotes VEGFR‐3 signaling and lymphangiogenesis by different mechanisms, which are mediated independently by the two domains of CCBE1: by enhancing the cleavage activity of ADAMTS3 and by facilitating the colocalization of VEGF‐C and ADAMTS3. These new insights should be valuable in developing new strategies to therapeutically target VEGF‐C/VEGFR‐3‐induced lymphangiogenesis.

Cerebrospinal fluid (CSF) is known to be reabsorbed by the lymphatic vessels and drain into the lymph nodes (LNs) through peripheral lymphatic vessels. In the peripheral lymphatics, the contractile pumping action of lymphangions mediates lymph drainage; yet it is unknown whether lymphatic vessels draining cranial and spinal CSF show similar function. Herein, we used non‐invasive near‐infrared fluorescence imaging (NIRFI) to image (i) indocyanine green (ICG) distribution along the neuraxis and (ii) routes of ICG‐laden CSF outflow into the lymphatics following intrathecal lumbar administration. We demonstrate lymphatic contractile function in peripheral lymphatics draining from the nasal lymphatics to the mandibular LNs. In addition, we observed afferent sciatic lymphatic vessels, which also show contractile activity and transport spinal CSF into the sciatic LNs. This drainage pattern was also visualized by NIRFI following intrathecal thoracic injection. In situ intravital imaging following intrathecal lumbar injection of blue dye shows similar distributions to that seen in vivo with ICG. NIRFI could be used as a tool to probe CSF pathology including neurological disorders by imaging CSF outflow dynamics to lymphatics.

Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC‐2 is found on platelets. Both podoplanin and CLEC‐2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood‐lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC. Addition of platelets or antibody‐mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF‐A or VEGF‐C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co‐culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro‐migratory effect of VEGF‐A and VEGF‐C. Inhibition of RhoA or Rho‐kinase reduced LEC migration induced by VEGF‐C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF‐receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF‐mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.

Lymphatic vessels are the primary route of communication from peripheral tissues to the immune system; as such, they represent an important component of local immunity. In addition to their transport functions, new immunomodulatory roles for lymphatic vessels and lymphatic endothelial cells have come to light in recent years, demonstrating that lymphatic vessels help shape immune responses in a variety of ways: promoting tolerance to self‐antigens, archiving antigen for later presentation, dampening effector immune responses, and resolving inflammation, among others. In addition to these new biological insights, the growing field of immunoengineering has begun to explore therapeutic approaches to utilize or exploit the lymphatic system for immunotherapy.

AIDS‐related lymphomas (ARLs) are expected to increase in the future since combined antiretroviral therapy (cART) enhances the life expectancy of HIV‐1‐infected (HIV+) patients but does not affect the occurrence of ARLs to the same extent as that of other tumors. Lymphangiogenesis is essential in supporting growth and metastatic spreading of ARLs. HIV‐1 does not infect the neoplastic B cells, but HIV‐1 proteins have been hypothesized to play a key role in sustaining a prolymphangiogenic microenvironment in lymphoid organs. The HIV‐1 matrix protein p17 is detected in blood and accumulates in the germinal centers of lymph nodes of HIV+ patients under successful cART. The viral protein displays potent lymphangiogenic activity in vitro and in vivo This is, at least in part, mediated by the secretion of the lymphangiogenic factor endothelin‐1, suggesting that activation of a secretory pathway sustains the lymphangiogenic activity of p17. Here, we show that the p17 lymphangiogenic activity occurs on human lymph node‐derived lymphatic endothelial cells (LN‐LECs) under stress conditions only and relies entirely on activation of an autophagy‐based pathway. In fact, induction of autophagy by p17 promotes lymphangiogenesis, whereas pharmacological and genetic inhibition of autophagy inhibits p17‐triggered lymphangiogenesis. Similarly, the vasculogenic activity of p17 was totally inhibited in autophagy‐incompetent mice. Our findings reveal a previously unrecognized role of autophagy in lymphangiogenesis and open the way to identify novel treatment strategies aimed at inhibiting aberrant tumor‐driven lymphangiogenesis in HIV+ patients.IMPORTANCE AIDS‐related lymphomas (ARLs) are the most common malignancies in HIV‐1‐infected (HIV+) patients after the introduction of combined antiretroviral therapy (cART). Lymphangiogenesis is of critical importance in sustaining growth and metastasis of ARLs. Indeed, enhanced lymphangiogenesis occurs in the lymph nodes of HIV+ patients under successful cART. The HIV‐1 matrix protein p17 is detected in blood and accumulates in the lymph node germinal centers even in the absence of virus replication. Several findings suggest a key role for p17 as a microenvironmental factor capable of promoting lymphangiogenesis. Here, we show that p17 promotes lymphangiogenesis of human lymph node‐derived lymphatic endothelial cells (LN‐LECs). The lymphangiogenic activity of p17 is sustained by an autophagy‐based pathway that enables LN‐LECs to release prolymphangiogenic factors into the extracellular microenvironment. Our findings indicate that specific targeting of autophagy may provide an important new tool for treating ARLs.

OBJECTIVES: Magnetic resonance lymphography (MRL) in small animals is a promising but challenging tool in preclinical lymphatic research. In this study, we compared the gadolinium (Gd)‐based nanoparticle AGuIX with Gd‐DOTA for interstitial MRL in healthy rats and in a chronic rat hindlimb lymphedema model. MATERIALS AND METHODS: A comparative study with AGuIX and Gd‐DOTA for interstitial MRL was performed in healthy Lewis rats (n = 6). For this purpose, 75 muL of 3 mM AGuIX (containing 30 mM Gd‐DOTA side residues) and 75 muL 30 mM Gd‐DOTA were injected simultaneously in the right and left hindlimbs. Repetitive high‐resolution, 3‐dimensional time‐of‐flight gradient recalled echo MRL sequences were acquired over a period of 90 minutes using a 9.4‐T animal scanner. Gadofosveset‐enhanced MR angiography and surgical dissection after methylene blue injection served as supportive imaging techniques. In a subsequent proof‐of‐principle study, AGuIX‐based MRL was investigated in a hindlimb model of chronic lymphedema (n = 4). Lymphedema of the right hindlimbs was induced by means of popliteal and inguinal lymphadenectomy and irradiation with 20 Gy. The nonoperated left hindlimbs served as intraindividual controls. Six, 10, and 14 weeks after lymphadenectomy, MRL investigations were performed to objectify lymphatic reorganization. Finally, skin samples of the lymphedematous and the contralateral control hindlimbs were analyzed by means of histology and immunohistochemistry. RESULTS: AGuIX‐based MRL resulted in high‐resolution anatomical depiction of the rodent hindlimb lymphatic system. Signal‐to‐noise ratio and contrast‐to‐noise ratio of the popliteal lymph node were increased directly after injection and remained significantly elevated for up to 90 minutes after application. AGuIX provided significantly higher and prolonged signal intensity enhancement as compared with Gd‐DOTA. Furthermore, AGuIX‐based MRL demonstrated lymphatic regeneration in the histopathologically verified chronic lymphedema model. Collateral lymphatic vessels were detectable 6 weeks after lymphadenectomy. CONCLUSIONS: This study demonstrates that AGuIX is a suitable contrast agent for preclinical interstitial MRL in rodents. AGuIX yields anatomical imaging of lymphatic vessels with diameters greater than 200 mum. Moreover, it resides in the lymphatic system for a prolonged time. AGuIX may therefore facilitate high‐resolution MRL‐based analyses of the lymphatic system in rodents.

Chylous pleural effusion (chylothorax) frequently accompanies lymphatic vessel malformations and other conditions with lymphatic defects. Although retrograde flow of chyle from the thoracic duct is considered a potential mechanism underlying chylothorax in patients and mouse models, the path chyle takes to reach the thoracic cavity is unclear. Herein, we use a novel transgenic mouse model, where doxycycline‐induced overexpression of vascular endothelial growth factor (VEGF)‐C was driven by the adipocyte‐specific promoter adiponectin (ADN), to determine how chylothorax forms. Surprisingly, 100% of adult ADN‐VEGF‐C mice developed chylothorax within 7 days. Rapid, consistent appearance of chylothorax enabled us to examine the step‐by‐step development in otherwise normal adult mice. Dynamic imaging with a fluorescent tracer revealed that lymph in the thoracic duct of these mice could enter the thoracic cavity by retrograde flow into enlarged paravertebral lymphatics and subpleural lymphatic plexuses that had incompetent lymphatic valves. Pleural mesothelium overlying the lymphatic plexuses underwent exfoliation that increased during doxycycline exposure. Together, the findings indicate that chylothorax in ADN‐VEGF‐C mice results from retrograde flow of chyle from the thoracic duct into lymphatic tributaries with defective valves. Chyle extravasates from these plexuses and enters the thoracic cavity through exfoliated regions of the pleural mesothelium.

Immunoglobulin Y (IgY) is the predominant antibody found in hen's (Gallus domesticus) egg yolk. This antibody, developed against several microorganisms in hen egg yolk, has been successfully used as an alternative to immunoglobulins from mammals for use in immunodiagnostics and immunotherapy. Enteropathogenic Escherichia coli (E.coli) F4 is the main etiological agent associated with swine neonatal diarrhea, and it causes notable economic losses in swine production. The aim of the present study was to evaluate the relationship between humoral immune response and the activation of gut‐associated lymphoid tissue (GALT) in laying hens intramuscularly immunized with E. coli F4. Adult laying Shaver hens were immunized with a bacterin based on an inactivated lysate E. coli F4 strain that was originally isolated from neonatal piglet diarrhea, following a recommended schedule. The percentage of B lymphocytes in blood and spleen homogenates was determined by flow cytometry. Villi histomorphometry and the size of germinal centers (GC) activated in GALT and the spleen were measured in histological samples either stained with hematoxylin/eosin or through immunofluorescence. Antibody and isotype‐specific antibodies in serum and egg yolk were measured using indirect enzyme‐linked immunosorbent assay (ELISA). Secretory and serum immunoglobulin A (IgA) were measured by ELISA tests. Laying hen with intramuscular immunization with E. coli F4 lysate, activated both mucosal and systemic protection. Mucosal protection was provided through B lymphocytes, and most of them were activated on Peyer's patches and esophageal tonsils, in GALT. Furthermore, increased B lymphocyte number in the lamina propria of the gut, and increased intraepithelial plasmatic cell number, produced high levels of mucosal IgA. Activated B lymphocytes interacted with absorptive cells, immune cells, and microbiota in the gut, producing signals that were translated into a powerful physical defense by producing a greater volume of mucin from an increased number of goblet cells. Systemic protection was provided through B lymphocyte activation of spleen GC, which produced hugely specific IgY serum levels. One week later, this specific IgY was deposited in the yolk. This suggests that GALT is a key immunologic tissue inside the mucosal immune system, acting as the “command center” for humoral reaction.

OBJECTIVE: The purpose of this study was to investigate the role of Fat4 and Dachsous1 signaling in the lymphatic vasculature. APPROACH AND RESULTS: Phenotypic analysis of the lymphatic vasculature was performed in mice lacking functional Fat4 or Dachsous1. The overall architecture of lymphatic vasculature is unaltered, yet both genes are specifically required for lymphatic valve morphogenesis. Valve endothelial cells (Prox1high cells) are disoriented and failed to form proper valve leaflets. Using Lifeact‐GFP mice, we revealed that valve endothelial cells display prominent actin polymerization. Finally, we showed the polarized recruitment of Dachsous1 to membrane protrusions and cellular junctions of valve endothelial cells in vivo and in vitro. CONCLUSIONS: Our data demonstrate that Fat4 and Dachsous1 are critical regulators of valve morphogenesis. This study highlights that valve defects may contribute to lymphedema in Hennekam syndrome caused by Fat4 mutations.

Lymphatic vasculature is an important part of the cardiovascular system with multiple functions, including regulation of the return of interstitial fluid (lymph) to the bloodstream, immune responses, and fat absorption. Consequently, lymphatic vasculature defects are involved in many pathological processes, including tumor metastasis and lymphedema. BRG1 is an important player in the developmental window when the lymphatic system is initiated. In the current study, we used tamoxifen inducible Rosa26CreERT2‐BRG1floxed/floxed mice that allowed temporal analysis of the impact of BRG1 inactivation in the embryo. The BRG1floxed/floxed/Cre‐TM embryos exhibited edema and hemorrhage at embryonic day‐13 and began to die. BRG1 deficient embryos had abnormal lymphatic sac linings with fewer LYVE1 positive lymphatic endothelial cells. Indeed, loss of BRG1 attenuated expression of a subset of lymphatic genes in‐vivo. Furthermore, BRG1 binds at the promoters of COUP‐TFII and LYVE1, suggesting that BRG1 modulates expression of these genes in the developing embryos. Conversely, re‐expression of BRG1 in cells lacking endogenous BRG1 resulted in induction of lymphatic gene expression in‐vitro, suggesting that BRG1 was both required and sufficient for lymphatic gene expression. These studies provide important insights into intrinsic regulation of BRG1‐mediated lymphatic‐gene expression, and further an understanding of lymphatic gene dysregulation in lymphedema and other disease conditions.

BACKGROUND: Myeloid‐derived lymphatic endothelial cells (M‐LECP) are induced by inflammation and play an important role in adult lymphangiogenesis. However, the mechanisms driving M‐LECP differentiation are currently unclear. We previously showed that activation of Toll‐like receptor‐4 (TLR4) induces myeloid‐lymphatic transition (MLT) of immortalized mouse myeloid cells. Here the goals were to assess the potential of different TLR4 ligands to induce pro‐lymphatic reprogramming in human and mouse primary myeloid cells and to identify transcriptional changes regulating this process. METHODOLOGY/PRINCIPAL FINDINGS: Human and mouse myeloid cells were reprogrammed to the lymphatic phenotype by TLR4 ligands including lipopolysaccharide (LPS), recombinant high mobility group box 1 protein (HMGB1), and paclitaxel. TLR4 induced similar MLT in cells from mice of different strains and immune status. Commonly induced genes were detected by transcriptional profiling in human and mouse myeloid cells from either immunocompetent or immunodeficient mice. Shared trends included: (1) novel expression of lymphatic‐specific markers vascular endothelial growth factor receptor‐3 (VEGFR‐3), lymphatic vessel endothelial hyaluronan receptor‐1 (LYVE‐1) and podoplanin (PDPN) largely absent prior to induction; (2) lack of notable changes in blood vessel‐specific markers; (3) transient expression of VEGFR‐3, but sustained increase of vascular endothelial growth factor‐C (VEGF‐C) and a variety of inflammatory cytokines; (4) dependency of VEGFR‐3 upregulation and other LEC genes on NF‐kappaB; and (5) novel expression of lymphatic‐specific (e.g., PROX1) and stem/progenitor (e.g., E2F1) transcription factors known for their roles in adult and embryonic vascular formation. M‐LECP generated by TLR4 ligands in vitro were functional in vivo as demonstrated by significantly increased lymphatic vessel density and lymphatic metastasis detected in orthotopic breast cancer models. CONCLUSIONS/SIGNIFICANCE: We established a novel TLR4‐dependent protocol for in vitro production of functionally competent M‐LECP from primary human or mouse myeloid cells and identified many potential regulators of this process. This information can be further exploited for research and therapeutic purposes.

BACKGROUND: Lymphaticovenular anastomosis (LVA) is a useful treatment option for compression‐refractory lower extremity lymphedema (LEL), but its efficacy largely depends on severity of lymphosclerosis. To maximize LVA efficacy, it is important to elucidate factors associated with severe lymphosclerosis (SLS). METHODS: Medical charts of 134 LEL patients who underwent preoperative indocyanine green (ICG) lymphography and LVA were reviewed to obtain data of clinical demographics, ICG lymphography findings, and intraoperative findings. Based on intraoperative findings of lymphatic vessels, severity of lymphosclerosis were classified into s0, s1, s2, and s3. Lymphatic vessels with s3 sclerosis was defined as SLS. Logistic regression analysis was used to identify independent factors associated with SLS. RESULTS: In total, 962 lymphatic vessels were analyzed, among which SLS was observed in 97 (10.1%). Multivariate analysis revealed that independent factors associated with SLS were higher body mass index (odds ration [OR], 1.803; 95% confidence interval [CI], 1.041–3.123; P = 0.035), incision site in the thigh/foot compared with in the groin (OR, 2.355/4.471; 95% CI 1.201–4.617/2.135–9.362; P = 0.013/< 0.001), and S‐region/D‐region on ICG lymphography compared with L‐region (OR, 83.134/1441.126; 95% CI, 11.296–611.843/146.782–14149.195; P < 0.001/< 0.001). Inverse associations were observed in positive history of radiation (OR 0.461; 95% CI, 0.269–0.788; P = 0.005). CONCLUSIONS: Independent factors associated with SLS were clarified. ICG lymphography pattern had the strongest association with SLS. D‐region on ICG lymphography should be avoided for LVA.

High salt (HS) diet can accelerate the progress of hypertensive left ventricular (LV) remodeling. But the detailed mechanism remains poorly understood. We hypothesized HS intake could impact cardiac lymphangiogenesis through tonicity‐responsive enhancer binding protein (TonEBP)/vascular endothelial growth factor‐C (VEGF‐C) signaling pathway which might play an important role in HS intake accelerated LV remodeling. Eight‐week‐old male spontaneously hypertensive rats (SHR) and Wistar‐Kyoto rats (WKY) were randomized to 0.5% NaCl (Low salt, LS) and 8% NaCl (high salt, HS) diets for 12 weeks. LV remodeling was determined by echocardiography. LV invasive hemodynamic analysis and morphologic staining (cardiomyocyte hypertrophy, collagen deposition, TonEBP expression, macrophage infiltration and lymphatic density) were performed at the time of sacrifice. The blood pressure of SHR‐HS group was significantly increased compared to SHR‐LS and WKY groups. Meanwhile, The LV chamber size was markedly enlargement, LV function apparently compromised accompanied with a severe macrophage infiltration, and fibrosis in the perivascular and interstitium of LV compared with SHR‐LS group. Furthermore, the expression levels of VEGF‐C, TonEBP, and lymphatic markers in SHR‐HS group were significantly increased parallel with apparent lymphangiogenesis compared with SHR‐LS group. Our work indicates that TonEBP/VEGF‐C signaling pathway was up‐regulated in HS intake accelerated hypertensive LV remodeling process that may be valuable for further investigation.

BACKGROUND: Patients with secondary lower extremity lymphedema (LEL) often develop suprapubic lymphedema. We developed a novel surgical method of shaping the lower abdomen and debulking suprapubic lymphedema with simultaneous reconstruction of lymphatic flow in case of LEL. METHODS: A skin incision in a fleur‐de‐lis pattern was performed for horizontal and vertical abdominal skin and fat resection. A caudal‐based isosceles triangular flap was created on the central suprapubic region to reduce wound tension at the intersection of the horizontal and vertical incisions. After resection and debulking of the suprapubic region, a lymphaticovenular anastomosis (LVA) between the efferent lymphatic vessel of the groin node and the superficial inferior epigastric vein was created or vascularized lymph node transfer (LNT) to the groin region was performed to restore lymphatic flow. LVA and LNT were also performed at the lower extremities to improve LEL. Perioperative change in limb volume was evaluated using the LEL index, and lymphatic function was evaluated by lymphoscintigraphy. RESULTS: Simultaneous abdominoplasty and reconstructive lymphatic microsurgery were performed in 11 patients. The LEL index improved perioperatively, with a significant difference (p < 0.01). In eight patients who underwent lymphoscintigraphy before and after surgery, the lymphatic function was found to have not deteriorated in any limb. CONCLUSION: When simultaneous lymphatic microsurgeries and careful observation for complications were performed, abdominoplasty resulted in good outcomes in patients with LEL and suprapubic lymphedema without worsening of lymphedema.

PURPOSE: The purpose of the present study was to analyse the performance of non‐contrast MR lymphography for the classification of primary lower limb lymphoedema in 121 consecutive patients with 187 primary lower limb lymphoedemas. MATERIALS AND METHODS: 121 consecutive patients with clinically diagnosed primary lower limb lymphoedema underwent non‐contrast MR lymphography with a free‐breathing 3D fast spin‐echo sequence with a very long TR/TE (4000/884 ms). MR examinations were retrospectively reviewed for severity of lymphoedema (absent, mild, moderate, severe) and characteristics of inguinal lymph nodes and iliac and inguinal lymphatic trunks graded as aplasic (no lymph nodes or lymphatic trunks), hypoplasic (less lymph nodes or lymphatic trunks), normal and hyperplasic (more lymph nodes or more and/or dilated trunks). RESULTS: There was an excellent correlation between clinical stage and severity of lymphoedema (Cramer's V of 0,73 (p < 0.001)). Differentiation was feasible between inguinal lymphatic vessel aplasia (21%), hypoplasia (15%), normal pattern (53%) and hyperplasia (11%). Severe lymphoedema was observed in 46% of aplasic patterns and in 37% of hyperplasic patterns, but in only 15% of hypoplasic patterns and never observed in normal patterns (p < 0.001). CONCLUSION: Non‐contrast MR lymphography is able to classify primary lower limb lymphoedemas into hyperplasic, aplasic, hypoplasic and normal patterns. KEY POINTS: * Non‐contrast MR lymphography is able to classify primary lower limb lymphoedemas. * Lymphoedema can be classified in hyperplasic, aplasic, hypoplasic and normal patterns. * Non‐contrast MR lymphography can optimize clinical management of primary lower limb lymphoedemas.

Lymphatic leak is a well‐documented complication following neck dissection surgeries. When conservative methods fail to control the leak, thoracic duct embolization becomes an option. Transabdominal access is the standard for this procedure; however, it is not always feasible. We discuss a technique of selective lymphatic vessel embolization utilizing retrograde transvenous access.

Renal lymphangiectasia is a rare benign condition of the lymphatic system. Primarily, the diagnosis is based upon characteristic imaging findings along with biochemical analysis of aspirated fluid. No definitive algorithm exists for treating this condition owing to its rarity. The literature describes various modes of management ranging from conservative management to nephrectomies. We present a case of bilateral renal lymphangiectasia managed with percutaneous drainage along with sclerotherapy. Our initial findings show a favorable outcome with this approach.

BACKGROUND: This study evaluated the long‐term clinical outcomes among different vascularized lymph node transfers (VLNT) used at our institution. METHODS: Between July 2010 and July 2016, all patients with International Society of Lymphology (ISL) stages II‐III who underwent VLNT were evaluated. Demographic and clinical data (limb circumference, infectious episodes, lymphoscintigraphic studies) were recorded pre‐operatively. Clinical outcomes, complications, and additional excisional procedures were analyzed post‐operatively. At least 2‐year follow‐up was required for inclusion. RESULTS: Overall, 83 patients (Stage II:47, Stage III:36) met the inclusion criterion. Mean follow‐up was 32.8 months (range, 24–49). Lymph node flaps used were groin (n = 13), supraclavicular (n = 25), gastroepiploic (n = 42), ileocecal (n = 2), and appendicular (n = 1). Total mean circumference reduction rate was 29.1% (Stage II) and 17.9% (Stage III) (P < 0.05). A paired t‐test showed that VLNT significantly decreased the number of infections (P < 0.05). Three patients reported no improvement of the symptoms. Major complications included one flap loss and one donor site hematoma. After the period of follow‐up, 18 patients (21.7%) underwent additional excisional procedures. CONCLUSION: VLNT is a promising technique used for the treatment of lymphedema and appears to be more effective in moderate stages (Stage II). Patients with advanced stage lymphedema (Stage III) may benefit from additional excisional procedures.

Chronic lymphoedema is a disease caused by a congenital or acquired damage to the lymphatic system and characterized by complex chains of pathophysiologic events such as lymphatic fluid stasis, chronic inflammation, lymphatic vessels impairment, adipose tissue deposition and fibrosis. These events seem to maintain and reinforce themselves through a positive feedback loop: regardless of the initial cause of lymphatic stasis, the dysfunctional adipose tissue and its secretion products can worsen lymphatic vessels' function, aggravating lymph leakage and stagnation, which can promote further adipose tissue deposition and fibrosis, similar to what may happen in obesity. In addition to the current knowledge about the tight and ancestral interrelation between immunity system and metabolism, there is evidence for similarities between obesity‐related and lymphatic damage‐induced lymphoedema. Together, these observations indicate strong reciprocal relationship between lymphatics and adipose tissue and suggest a possible key role of the adipocyte in the pathophysiology of chronic lymphoedema's vicious circle.

Secondary lymphedema of the upper and lower extremities related to prior oncologic therapies, including cancer surgeries, radiation therapy, and chemotherapy, is a major cause of long‐term morbidity in cancer patients. For the upper extremities, it is most commonly associated with prior oncologic therapies for breast cancer, while for the lower extremities, it is most commonly associated with oncologic therapies for gynecologic cancers, urologic cancers, melanoma, and lymphoma. Both non‐surgical and surgical management strategies have been developed and utilized, with the primary goal of all management strategies being volume reduction of the affected extremity, improvement in patient symptomology, and the reduction/elimination of resultant extremity‐related morbidities, including recurrent infections. Surgical management strategies include: (i) ablative surgical methods (i.e., Charles procedure, suction‐assisted lipectomy/liposuction) and (ii) physiologic surgical methods (i.e., lymphaticolymphatic bypass, lymphaticovenular anastomosis, vascularized lymph node transfer, vascularized omental flap transfer). While these surgical management strategies can result in dramatic improvement in extremity‐related symptomology and improve quality of life for these cancer patients, many formidable challenges remain for successful management of secondary lymphedema. It is hopeful that ongoing clinical research efforts will ultimately lead to more complete and sustainable treatment strategies and perhaps a cure for secondary lymphedema and its devastating resultant morbidities.

PURPOSE: Despite extensive previous investigations regarding the efficacy of lymphaticovenular anastomosis (LVA) for the treatment of lymphedema, few studies have reported the long‐term clinical course of individual patients undergoing LVA. The present study presented our experience of LVA and to report serial postoperative courses. METHODS: A retrospective chart review was performed for 17 patients (2 male and 15 female) with lymphedema refractory to conservative treatment who underwent LVA and were followed‐up for at least postoperative 24 months. Mean age was 39 years. There were 7 cases with primary lymphedema and 10 with secondary lymphedema. Three patients had lymphedema in upper extremity and the other 14 had lesions in lower extremity. Outcomes were evaluated via qualitative assessment and quantitative volume measurement conducted preoperatively and 6, 12, and 24 months postoperatively. RESULTS: LVA was performed in the distal extremities with a mean number of 2.5 (1–3). No postoperative complications developed. Average follow‐up period was 41.8 months. Of 17 patients, 10 showed improvement in excess volume consistently during the follow‐up period, whereas 3 patients exhibited temporary improvement at postoperative 6 months and subsequent aggravation at the latter follow‐ups. No improvement was observed throughout the follow‐up period in 4 patients. The requirements of complex decongestive physical therapy significantly decreased from 1.7 to 0.77 per patient per year. The frequency of cellulitis also decreased from 0.31 to 0.16 per patient per year. CONCLUSION: Our results suggest that LVA may have long‐term effectiveness and provide a favorable clinical course in the long term.

BACKGROUND: Lymphoscintigraphy is used to confirm the diagnosis of lymphedema; pathologic findings are abnormal transit time to regional nodes and dermal backflow. A universal protocol for the test does not exist. The purpose of this study was to determine whether the clinical severity of lymphedema correlates with lymphoscintigraphy findings. STUDY DESIGN: Patients treated in our Lymphedema Program between 2009 and 2017 were reviewed. Diagnosis of lymphedema was determined by history, physical examination, and lymphoscintigraphy. Severity was defined by increased volume of the limb as follows: mild (<20%), moderate (20% to 40%), and severe (>40%). Candidate variables included location (arm, leg), age, duration of symptoms, infection history, and lymphedema type (primary, secondary). An association between lymphedema severity and lymphoscintigraphy findings was determined using the Pearson chi‐square test and multivariate logistic regression. RESULTS: One hundred and thirty‐four patients with 181 affected extremities (24 upper, 157 lower) were included. Clinical severity was as follows: 54% mild, 30% moderate, and 16% severe. Delayed tracer transit to the regional nodes was as follows: 45 minutes (34%), 2 hours (18%), and 4 hours or longer (48%). Thirty‐six percent of extremities demonstrated dermal backflow. Abnormal transit time or dermal backflow was identified in 97% of extremities by 45 minutes and in 3% of limbs by 2 hours. Transit time and dermal backflow were not predictive of clinical severity when adjusting for candidate variables (p > 0.1). CONCLUSIONS: Clinical severity of lymphedema is not associated with lymphoscintigraphy findings. A lymphoscintigram should be interpreted as normal or abnormal, and does not need to exceed 2 hours.

The discovery of the lymphatic system has a long and fascinating history. The interest in anatomy and physiology of this system paralleled that of the blood cardiocirculatory system and has been maybe obscured by the latter. Paradoxically, if the closed blood system appeared open in Galen's anatomy and physiology, and took a very long time to be correctly described in terms of pulmonary and general circulation by ibn Al‐Nafis/Michael Servetus/Realdo Colombo and William Harvey, respectively, the open lymphatic system was incorrectly described as a closed circuit connected with arteries and veins. In ancient times only macroscopic components of the lymphatic system have been described, although misinterpreted, including lymph nodes and lacteals, the latter being easily identified because of their milk‐like content. For about 15 centuries the dogmatic acceptance of Galen's notions did not allow a significant progress in medicine. After Vesalius' revolution in anatomical studies, new knowledge was accumulated, and the 17th century was the golden age for the investigation of the lymphatic system with several discoveries: gut lacteals (Gaspare Aselli), cloacal bursa (Hieronimus Fabricius of Acquapendente), reservoir of the chyle (Jean Pecquet), extra‐intestinal lymphatic vessels (Thomas Bartholin and Olaus Rudbeck dispute), hepatic lymph circulation (Francis Glisson). In the Enlightenment century Frederik Ruysch described the function of lymphatic valves, and Paolo Mascagni provided a magnificent iconography of the lymphatic network in humans. In recent times, Leonetto Comparini realized three‐dimensional reconstructions of the liver lymphatic vessels, and Kari Alitalo discovered the lymphatic growth factor/receptor system. Far from a complete understanding of its anatomy and function, the lymphatic system still needs to be profoundly examined.

BACKGROUND: Lymphatic filariasis (LF) is a neglected tropical disease transmitted by mosquitoes. Nepal has implemented a national effort to eliminate LF by 2020 through mass drug administration (MDA) using diethylcarbamazine (DEC) and albendazole (ALB). We assessed the impact of MDAs on LF in selected districts of Nepal after the recommended six MDA rounds had been completed. METHODOLOGY AND PRINCIPAL FINDINGS: Baseline surveys were conducted in seven districts and mapping data were used as baseline in the other three districts before starting MDA in 2009. LF antigen (Ag) prevalence ranged from 1.06% to 20% among districts included in the baseline and mapping study. The number of people who received DEC and ALB were recorded during each MDA round and population‐based cluster surveys were conducted at least once in each district during the life of the program. The reported MDA coverage in five districts was consistently at least 65%. Two districts achieved the targeted coverage in four out of five rounds and the rest three districts achieved the target only in the first round. A pre‐transmission assessment survey (pre‐TAS) was conducted in one sentinel site and at least one spot check site in each of the districts after five MDA rounds. In pre‐TAS, all the sites of five districts (Pyuthan, Arghakhanchi, Kaski, Bhaktapur, and Kathmandu) and all but one spot check site of Lalitpur district had LF Ag <2% (ranging from 0.0% to 1.99%). Transmission assessment survey (TAS) was conducted in six evaluation units (EUs) consisting of six districts qualified on pre‐TAS. Though MDA coverage of 65% was not achieved in three districts (Kathmandu, Lalitpur and Bhaktapur), Nepal government in consultation with World Health Organization (WHO) decided to conduct TAS. All six EUs achieved the LF Ag threshold required to stop MDA in TAS, despite the low reported MDA coverage in those three districts. CONCLUSIONS: Although Nepal has achieved significant progress towards LF elimination, five rounds of MDA were not sufficient to disrupt the transmission cycle in all districts, probably because of high baseline prevalence.

BACKGROUND: Studies of monogenic gastrointestinal diseases have revealed molecular pathways critical to gut homeostasis and enabled the development of targeted therapies. METHODS: We studied 11 patients with abdominal pain and diarrhea caused by early‐onset protein‐losing enteropathy with primary intestinal lymphangiectasia, edema due to hypoproteinemia, malabsorption, and less frequently, bowel inflammation, recurrent infections, and angiopathic thromboembolic disease; the disorder followed an autosomal recessive pattern of inheritance. Whole‐exome sequencing was performed to identify gene variants. We evaluated the function of CD55 in patients' cells, which we confirmed by means of exogenous induction of expression of CD55. RESULTS: We identified homozygous loss‐of‐function mutations in the gene encoding CD55 (decay‐accelerating factor), which lead to loss of protein expression. Patients' T lymphocytes showed increased complement activation causing surface deposition of complement and the generation of soluble C5a. Costimulatory function and cytokine modulation by CD55 were defective. Genetic reconstitution of CD55 or treatment with a complement‐inhibitory therapeutic antibody reversed abnormal complement activation. CONCLUSIONS: CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein‐losing enteropathy (the CHAPLE syndrome) is caused by abnormal complement activation due to biallelic loss‐of‐function mutations in CD55. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Congenital pulmonary lymphangiectasia (CPL) is a rare developmental disorder of the lung, characterized by dilation of pulmonary subpleural, interlobar, perivascular and peribronchial lymphatics. The incidence of CPL among stillborn and neonates was estimated to be <1%. The etiology of CPL is unknown. However, it has been suspected to be of a genetic background. Recent basic studies revealed that it might be caused by the FOXC2, Vegfr‐3 and integrin alpha9beta1gene mutations. A clinical diagnosis of CPL can be made much easier in full‐term neonates who present with respiratory distress, pleural (especially chylous) effusions with or without generalized edema. In infancy, the diagnosis seems to be more difficult due to the nonspecific respiratory symptoms like persistent tachypnea, cough and wheeze. Lung biopsy with subsequent histological and immunohistochemical studies is the golden diagnostic method of CPL. Immunohistochemical staining for endothelial cell markers CD31, CD34 and D2‐40 confirms lymphatic origin. Therapeutic strategies include supportive, nutritional, investigational, aggressively interventional and surgical regimens, most of which have shown promising outcomes. Although CPL was once regarded as a disorder of very poor prognosis in neonatal onset cases, teenager and adult patients have shown good outcomes upon long‐term follow‐up.Die angeborene pulmonale Lymphangiektasie (CPL) ist eine seltene Entwicklungsstorung der Lunge, die durch eine Dilatation der pulmonalen subpleuralen, interlobaren, perivaskularen und peribronchialen Lymphgefasse charakterisiert ist. Die Inzidenz der CPL bei Totgeburten und Neugeborenen wird <1% geschatzt. Die Atiologie der CPL ist unbekannt. Allerdings wird ein genetischer Hintergrund vermutet. Neuere Grundlagenstudien zeigten, dass die CPL durch FOXC2, Vegfr‐3 und Integrin alpha9beta1‐Genmutationen verursacht sein konnte. Die klinische Diagnose der CPL ist sehr viel einfacher in Reifgeborenen zu stellen, die Atemnot, Pleuraergusse (vor allem chylose) mit und ohne generalisiertem Odem aufweisen. In der fruhen Kindheit ist die Diagnose aufgrund der unspezifischen respiratorischen Symptomatik wie persistierende Tachypnoen, Husten oder Rocheln schwerer zu stellen. Die Lungenbiopsie mit anschliessenden histologischen und immunhistochemischen Untersuchungen ist der Goldstandard fur die Diagnose der CPL. Die immunhistochemische Farbung der Endothelzellmarker CD31, CD34 und D2‐40 bestatigt den lymphatischen Ursprung. Die Behandlungsstrategien umfassen unterstutzende, alimentare, in Erprobung befindliche, aggressiv‐interventionelle und chirurgische Behandlungsplane, von denen die meisten ermutigende Ergebnisse zeigten. Obwohl die CPL einst bei Fallen mit Ausbruch im Neugeborenenalter als Erkrankung mit sehr schlechter Prognose galt, zeigen Teenager und erwachsene Patienten in der Langzeit‐Nachbeobachtung gute Verlaufe.

The commonly used modalities for therapy of limb lymphedema are manual lymphatic drainage, manual devices moving edema fluid and intermittent pneumatic compression (IPC). What seems to be necessary for validation of the effect of the compression procedure is imaging of the mobilized moving edema fluid. Picture of edema fluid flow would allow the therapist to use force adjusted to the tissue volume and stiffness differing in various limb regions as well as identify sites of abundant accumulation of fluid requiring more compression. The purpose of the present study was visualise tissue edema fluid flow during manual drainage, Linforoll massage, intermittent pneumatic compression and bandaging. To obtain data how high compression pressures should be used to mobilize ICG stained fluid, concomitantly tissue fluid pressure measurements were done. The following observations were obtained: a) the possibility of real time observation of edema fluid movement using various compression modalities, b) the threshold pressures necessary to move edema fluid to be over 80 mmHg in the compression device and over 40 mmHg in the tissue fluid, c) inefficacy of compression in some cases despite of applying high compression force. These observations point to the need of ICG lymphangiography before compression therapy in each patient. The images observed during the compression procedure give an insight into the distribution of edema fluid, sites of its accumulation and efficacy of applied external force on fluid mobilization.

Clinical studies have found that the incidence of cancer metastasis through the lymphatic vessels are 3–5 times higher than that through the blood vessels. These findings suggest the potency of anti‐lymphangiogenic therapy in reducing the incidence of cancer metastasis. Previously, we reported LHbisD4, which is the conjugate of low molecular weight heparin (LMWH) and four bis‐deoxycholates as a potent anti‐angiogenic drug with less toxicity and orally active property. Here, we show that LHbisD4 could also suppress the formation of new lymphatic vessels and attenuate the incidence of metastasis by blocking VEGF‐C signaling pathway. LHbisD4 significantly enhanced binding affinity with VEGF‐C when compared with LMWH, which enables LHbisD4 to suppress the proliferation, migration and formation of tubular structures of human dermal lymphatic endothelial cells(HDLECs) in in vitro condition even in the presence of excessive amounts of VEGF‐C. Similarly, we found that the density of lymphatic vessels in the primary tumor tissue in breast cancer bearing mice was significantly diminished when LHbisD4 was administered compared with the control group. Also, the incidence of axillary lymph nodes and distant organ metastasis was significantly reduced in the LHbisD4 administered group, which demonstrates that LHbisD4 could successfully lower the incidence of metastasis through blocking VEGF‐C induced lymphangiogenesis. Based on these results, we propose LHbisD4 as a potent anti‐cancer drug that can reduce the incidence of metastasis by suppressing lymphangiogenesis through blocking VEGF‐C signaling pathway.

BACKGROUND: Lymphatic metastasis is one of the leading causes of death in patients with different types of cancer and is the main prognostic factor for the disease survival. The formation of new lymphatic vessels (lymphangiogenesis) in primary tumors facilitates tumor cell dissemination to regional lymph nodes and correlates with distant metastases. Lymphangiogenesis has thus emerged as a suitable therapeutic target to block metastases, but no anti‐lymphangiogenic compounds have been approved for clinical use to date. Therefore, new or improved therapies blocking lymphatic metastases are urgently required. METHODS: We established murine breast tumors to assess the effect of AD0157 on tumor growth, lymphangiogenesis, and lymphatic dissemination. Then, a battery of in vivo (mouse corneal neovascularization and ear sponges), ex vivo (mouse lymphatic rings and rat mesentery explants), and in vitro (proliferation, tubulogenesis, wound‐healing, Boyden chambers, and spheroids) assays was used to give insight into the lymphangiogenic steps affected by AD0157. Finally, we investigated the molecular pathways controlled by this drug. RESULTS: AD0157 was found to inhibit the growth of human breast cancer xenografts in mice, to strongly reduce tumor‐associated lymphangiogenesis and to block metastatic dissemination to both lymph nodes and distant organs. The high anti‐lymphangiogenic potency of AD0157 was further supported by its inhibitory activity at low micromolar range in two in vivo pathological models and in two ex vivo assays. In addition, AD0157 inhibited lymphatic endothelial cell proliferation, migration and invasion, cellular sprouting, and tube formation. Mechanistically, this compound induced apoptosis in lymphatic endothelial cells and decreased VEGFR‐3/‐2, ERK1/2, and Akt phosphorylations. CONCLUSIONS: These findings demonstrate the suitability of AD0157 to suppress tumor‐associated lymphangiogenesis. Beyond discovering a new potent anti‐lymphangiogenic drug that is worth considering in future clinical settings, our study supports the interest of designing anti‐lymphangiogenic therapies to avoid distant metastatic processes.

It was already in the 18th century when the French surgeon LeDran first noted that breast cancer patients with spread of tumor cells to their axillary lymph nodes had a drastically worse prognosis than patients without spread (LeDran et al.,). Since then, metastatic spread of cancer cells to regional lymph nodes has been established as the most important prognostic factor in many types of cancer (Carter et al., ; Elston and Ellis,). However, despite its clinical importance, lymph metastasis remains an underexplored area of tumor biology. Fundamental questions, such as when, how, and perhaps most importantly, why tumor cells disseminate through the lymphatic system, remain largely unanswered. Accordingly, no treatment strategies exist that specifically target lymph metastasis. The identification of epithelial‐mesenchymal transition (EMT) as a mechanism, which allows cancer cells to dedifferentiate and acquire enhanced migratory and invasive properties, has been a game changer in cancer research. Conceptually, EMT provides an explanation for why epithelial cancers with poor differentiation status are generally more aggressive and prone to metastasize than more differentiated cancers. Inflammatory cytokines, such as TGF‐beta, which are produced and secreted by tumor‐infiltrating immune cells, are potent inducers of EMT. Thus, reactivation of EMT also links cancer‐related inflammation to invasive and metastatic disease. Recently, we found that breast cancer cells undergoing TGF‐beta‐induced EMT acquire properties of immune cells allowing them to disseminate in a targeted fashion through the lymphatic system similar to activated dendritic cells during inflammation. Here, we review our current understanding of the mechanisms by which cancer cells spread through the lymphatic system and the links to inflammation and the immune system. We also emphasize how imaging techniques have the potential to further expand our knowledge of the mechanisms of lymph metastasis, and how lymph nodes serve as an interface between cancer and the immune system.

BACKGROUND: Peritoneal dissemination and retroperitoneal lymph node involvement are main routes for progression of epithelial ovarian cancer (EOC). Vascular endothelial growth factor (VEGF) mediated angiogenesis has been identified as an important mechanism promoting tumour progression. METHODS: Tumour tissue of 100 patients with EOC was analysed for protein expression of vascular endothelial growth factor (VEGF)‐A, ‐C, ‐D by Western Blot analysis. Expression patterns in patients with ‘extensive intraperitoneal' metastases (pT3c pN1 and pT3b‐pT3c pN0, n = 80) were compared to patients with ‘predominantly retroperitoneal' metastases (pT1a‐pT3b, pN1, n = 20). Overall and progression‐free survival was analysed by Kaplan‐Meier method. RESULTS: While no significant differences in expression levels among the different modes of metastases were noted for VEGF‐A and ‐D, VEGF‐C expression was significantly higher in the group of predominantly retroperitoneal metastases compared to the group with extensive intraperitoneal metastases. Patients with high VEGF‐C expression had a significantly worse overall survival compared to patients with low expression levels. CONCLUSIONS: Retroperitoneal tumour progression in EOC patients is associated with high VEGF‐C expression. VEGF‐C may serve as a molecular marker to identify patients with assumed high risk for lymphatic metastases, who might benefit from specific treatment strategies.

Photodynamic therapy (PDT) has been shown to destroy tumour‐associated lymphatic vessels. Therefore, we sought to investigate the functional outcomes of PDT‐mediated damage to the lymphatic vessels. We observed that PDT with verteporfin, completely but transiently, blocks the functional lymphatic drainage in the orthotopic mammary tumour models. Sustained inhibition of lymphatic vessels regeneration induced by lenalidomide or the soluble form of vascular endothelial growth factor receptor 3 (sVEGFR3) that neutralises lymphangiogenic vascular endothelial growth factor C (VEGF‐C), significantly impaired antitumour efficacy of PDT. Antilymphangiogenic compounds also significantly inhibited the ability of intratumourally inoculated dendritic cells (DCs) to translocate to local lymph nodes and diminished the number of tumour‐infiltrating interferon‐gamma‐secreting or tumour antigen‐specific CD8+ T cells. Lenalidomide also abrogated antitumour effects of the combination immunotherapy with PDT and anti‐programmed death‐ligand 1 (PD‐L1) antibodies. Altogether, these findings indicate that PDT‐mediated damage to the lymphatic vessels negatively affects development of antitumour immunity, and that drugs that impair lymphatic vessel regeneration might not be suitable for the use in combination with PDT.

BACKGROUND: Breast cancer‐related lymphedema (BCRL) is a significant complication for women undergoing treatment. We assessed BCRL incidence and risk factors in a large population‐based cohort. METHODS: We utilized the Olmsted County Rochester Epidemiology Project Breast Cancer Cohort from 1990–2010 and ascertained BCRL and risk factors. The cumulative incidence estimator was used to estimate the rate of BCRL; competing risks regression was used for multivariable analysis. RESULTS: A total of 1794 patients with stage 0–3 breast cancer with a median of 10 years follow‐up were included. The cumulative incidence of BCRL diagnosis within 5 years was 9.1% [95% confidence interval (CI) 7.8–10.5%]. No BCRL events occurred among patients without axillary surgery. In the axillary surgery subset (n = 1512), the 5‐year incidence of BCRL was 5.3% in sentinel lymph node (SLN) surgery and 15.9% in axillary dissection (ALND) patients (p < 0.001). In patients treated with surgery only, BCRL rates were not different between ALND versus SLN (3.5 and 4.1% at 5 years, p = 0.36). Addition of breast or chest wall radiation more than doubled the BCRL rate in ALND patients (3.5 vs. 9.5% at 5 years, p = 0.01). The groups with highest risk (>25% at 5 years) all involved ALND with nodal RT and/or anthracycline/cytoxan + taxane chemotherapy. In multivariable analysis of patients with any axillary surgery factors significantly associated with BCRL were ALND, chemotherapy, radiation, and obesity. CONCLUSIONS: BCRL is a sequelae of multimodal breast cancer treatment and risk is multifactorial. BCRL rates are higher in patients receiving chemotherapy, radiation, ALND, more advanced disease stage, and higher body mass index.

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle‐associated proteins secreted by tumours and/or their stroma have been proposed to condition pre‐metastatic sites in patients with melanoma. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre‐metastatic niches. Injectable lymphatic tracers have been developed, but their limited diffusion precludes whole‐body imaging at visceral sites. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive 'lymphoreporter' because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole‐body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre‐metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin‐binding factor midkine is a systemic inducer of neo‐lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a ‘MetAlert' discovery platform for drivers and inhibitors of metastasis.

Remodelling of lymphatic vessels in tumours facilitates metastasis to lymph nodes. The growth factors VEGF‐C and VEGF‐D are well known inducers of lymphatic remodelling and metastasis in cancer. They are initially produced as full‐length proteins requiring proteolytic processing in order to bind VEGF receptors with high affinity and thereby promote lymphatic remodelling. The fibrinolytic protease plasmin promotes processing of VEGF‐C and VEGF‐D in vitro, but its role in processing them in cancer was unknown. Here we explore plasmin's role in proteolytically activating VEGF‐D in vivo, and promoting lymphatic remodelling and metastasis in cancer, by co‐expressing the plasmin inhibitor alpha2‐antiplasmin with VEGF‐D in a mouse tumour model. We show that alpha2‐antiplasmin restricts activation of VEGF‐D, enlargement of intra‐tumoural lymphatics and occurrence of lymph node metastasis. Our findings indicate that the fibrinolytic system influences lymphatic remodelling in tumours which is consistent with previous clinicopathological observations correlating fibrinolytic components with cancer metastasis.

OBJECTIVES: To investigate the relevance of lymphangiogenic gene expression in primary and liver metastasis of colorectal cancer (CRC) and identify determinants of lymphatic invasion. BACKGROUND: Lymphatic development promoting vascular endothelial growth factor C (VEGFC) is associated with poor outcome in primary CRC. For colorectal liver metastasis (CRLM), intrahepatic lymph invasion and lymph node metastasis are poor prognostic factors. Exact biological factors promoting lymphatic involvement remain elusive, just as the association with molecular subtypes of CRC. METHODS: We designed a lymphangiogenic gene set (VEGFC, Nrp‐2, PDPN, LYVE‐1, MRC1, CCL‐21) and applied it to large datasets of CRC. Gene expression of the lymphangiogenic signature was assessed in resected CRLM specimens by Rt‐QPCR. In vitro experiments were performed with colon cancer cell line Colo320 (high Nrp‐2 expression) and human dermal microvascular lymphatic endothelial cells (LECs). RESULTS: Lymphangiogenic gene expression was associated with poor prognosis in both primary and liver metastasis of CRC. CRLM with high expression of consensus molecular subtype‐4 identifier genes also exhibited high lymphangiogenic gene expression. Lymph node recurrence following CRLM resection was associated with high expression of VEGFC and Nrp‐2. Blocking Nrp‐2 significantly reduced invasion of Colo320 cells through an LEC monolayer. CONCLUSIONS: Lymphangiogenic gene expression is correlated with worse prognosis and consensus molecular subtype‐4 in both primary and liver metastatic CRC. VEGFC and Nrp‐2 expression may be predictive of lymph node involvement in recurrence after resection of CRLM. Nrp‐2, expressed on both tumor and LECs, may have a mechanistic role in lymphatic invasion and is a potential novel target in CRC.

Background ‐Most AVMs are localized and occur sporadically; however they also can be multifocal in autosomal dominant disorders, such as Hereditary Hemorrhagic Telangiectasia (HHT) and Capillary Malformation‐Arteriovenous Malformation (CM‐AVM). Previously, we identified RASA1 mutations in 50% of patients with CM‐AVM. Herein we studied non‐RASA1 patients to further elucidate the pathogenicity of CMs and AVMs. Methods ‐We conducted a genome‐wide linkage study on a CM‐AVM family. Whole exome sequencing was also performed on 9 unrelated CM‐AVM families. We identified a candidate‐gene and screened it in a large series of patients. The influence of several missense variants on protein function was also studied in vitro. Results ‐We found evidence for linkage in two loci. Whole‐exome sequencing data unraveled four distinct damaging variants in EPHB4 in five families that co‐segregated with CM‐AVM. Overall, screening of EPHB4 detected 47 distinct mutations in 54 index patients: 27 lead to a premature stop codon or splice‐site alteration, suggesting loss of function. The other 20 are non‐synonymous variants that result in amino‐acid substitutions. In vitro expression of several mutations confirmed loss of function of EPHB4. The clinical features included multifocal CMs, telangiectasias, and AVMs. Conclusions ‐We found EPHB4 mutations in patients with multifocal CMs associated with AVMs. The phenotype, CM‐AVM2, mimics RASA1‐related CM‐AVM1 and also HHT. RASA1 encoded p120RASGAP is a direct effector of EPHB4. Our data highlights the pathogenetic importance of this interaction and indicts EPHB4‐RAS‐ERK signaling pathway as a major cause for arterio‐venous malformations.

Soblet et al. describe cis mutations in TEK/Tie‐2 in blue rubber bleb nevus and sporadic vascular malformations. This suggests that the remaining normal allele is required for the phenotype. Second, it suggests therapeutic approaches to treatment signal transduction inhibition.

BACKGROUND AND PURPOSE: There is a high prevalence of right‐to‐left shunting pulmonary arteriovenous malformations (PAVMs), which are stroke risk factors, in hereditary hemorrhagic telangiectasia (HHT) patients. While the prevalence of ischemic complications in HHT patients is known, the prevalence of silent brain infarcts (SBI) remains unknown. The purpose of this study was to determine the prevalence and risk factors for SBI in HHT patients. MATERIALS AND METHODS: Our institutional HHT database was queried to identify HHT patients who received a baseline screening brain MRI from January 2000 to February 2017. This study group was further refined by excluding patients who had a history of clinical ischemic disease as defined by having a stroke or transient ischemic attack (TIA). Brain MRIs were reviewed for SBI. Baseline data on demographics, Curacao criteria, presence of PAVMs, and cardiovascular risk factors were collected. The primary outcome was SBI prevalence. We also examined which baseline patient characteristics were associated with SBI through univariate chi‐square and Student t tests and multivariate logistic regression analyses. RESULTS: Three hundred fifty three consecutive HHT patients from January 2000 to February 2017 with a screening brain MRI and no prior history of stroke/TIA were included. SBI prevalence was 9.9% (35/353). SBI patients were more likely to have PAVMs than non‐SBI patients (80.6 vs. 53.1%, p = 0.005). The median age was 66 in the SBI group and 52 in the non‐SBI group (p = 0.006). SBI patients had higher prevalence of hyperlipidemia (34.3 vs. 9.8%, p < 0.0001), hypertension (48.6 vs. 22.0%, p = 0.005), and tobacco use (25.7 vs. 9.8%, p = 0.005). No patients under 30 had SBI. In the 60–69 age group, the prevalence of SBI was 18.8% with rates of 28.6% in the PAVM group and 10.5% in the non‐PAVM group. For patients >/ = 70 years old, the prevalence of SBI was 21.4% overall and 27.6% in the PAVM group and 10.5% in the non‐PAVM group. On multivariate analysis, PAVMs (OR 3.62, 95% CI 1.46–10.40) and increasing age (OR 1.04, 95% CI 1.01–1.07) were independently associated with SBI. CONCLUSIONS: Overall, a similar 10% SBI prevalence in the HHT cohort was noted as compared to the general population. However, the prevalence of SBI was higher in HHT patients with PAVMs when compared to that of the general population, particularly among patients than 60 years old. These findings highlight the need to accurately identify, and when appropriate, treat PAVMs in the HHT population especially given the multiple significant, clinical consequences of SBI.

Treatment of infantile hemangiomas (IH) with propranolol was first reported in 2008. Regressions of lymphatic malformations combined with pulmonary hypertension was first reported in 2012 after three children received treatment with oral sildenafil, which serves as an antagonist of phosphodiesterase isoform‐5 (PDE‐5). A marked expression of endothelial cells in the cytoplasm of IH tissues was obtained in our previous study. Therefore, the present study hypothesized that the antagonist of PDE‐5, sildenafil, may lead to the regression of hemangiomas. To assess this hypothesis, the proliferation and apoptosis of specimen‐derived hemangioma endothelial cells (HemECs) was determined in vitro by an MTT assay and flow cytometry, respectively, following treatment with sildenafil. The potential mechanisms underlying the mRNA and protein expression levels of inhibitor of differentiation 1 (Id‐1) were determined by reverse transcription‐quantitative polymerase chain reaction and western blotting. The results demonstrated that 5 microM sildenafil suppressed the proliferation of HemECs and significantly enhanced the rate of apoptosis after 24 h. Additionally, the mRNA and protein expression levels of Id‐1 were downregulated following treatment with sildenafil. Therefore, the present study concluded that PDE‐5 may be a potential therapeutic target for hemangiomas and Id‐1 may serve a vital role in the associated signaling transduction pathways.

Patients with overgrowth and complex vascular malformation syndromes, including Proteus syndrome have an increased risk of thromboembolism. Proteus syndrome is a mosaic, progressive overgrowth disorder involving vasculature, skin, and skeleton, and caused by a somatic activating mutation in AKT1. We conducted a comprehensive review of the medical histories and hematologic evaluations of 57 patients with Proteus syndrome to identify potential risk factors for thrombosis. We found that six of ten patients, who were deceased, died secondary to deep venous thrombosis and/or pulmonary embolism. Of the remaining 47 living patients, six had thromboembolic events that all occurred postoperatively and in an affected limb. Eleven of 21 patients had an abnormal hypercoagulable panel including Factor V Leiden heterozygotes, antithrombin III deficiency, positive lupus anticoagulant, or Protein C or S deficiencies. We observed that eight of 17 patients had an abnormal D‐dimer level >0.5 mcg/dl, but deep venous thromboses occurred in only four of those with D‐dimer >1.0 mcg/dl. We conclude that the predisposition to thrombosis is likely to be multifaceted with risk factors including vascular malformations, immobility, surgery, additional prothrombotic factors, and possible pathophysiologic effects of the somatic AKT1 mutation on platelet function or the vascular endothelium. The D‐dimer test is useful as a screen for thromboembolism, although the screening threshold may need to be adjusted for patients with this disorder. We propose developing a registry to collect D‐dimer and outcome data to facilitate adjustment of the D‐dimer threshold for Proteus syndrome and related disorders, including PIK3CA‐Related Overgrowth Spectrum.

BACKGROUND: Congenital cystic lymphangiomas are benign malformations due to a developmental disorder of lymphatic vessels. Besides surgical excision, sclerosant therapy of these lesions by intracavitary injection of OK‐432 (Picibanil(R)), a lyophilized mixture of group A Streptococcus pyogenes, is a common therapeutical option. For an appropriate application of OK‐432, a detailed knowledge about the structure and composition of the congenital cystic lymphangioma is essential. SonoVue(R) is a commercially available contrast agent commonly used in sonography by intravenous and intracavitary application. CASE PRESENTATION: Here we report the case of 2 month old male patient with a large thoracic congenital cystic lymphangioma. Preinterventional imaging of the malformation was performed by contrast‐enhanced ultrasound after intracavitary application of SonoVue(R) immediately followed by a successful sclerotherapy with OK‐432. CONCLUSIONS: Contrast agent‐enhanced ultrasound imaging offers a valuable option to preinterventionally clarify the anatomic specifications of a congenital cystic lymphangioma in more detail than by single conventional sonography. By the exact knowledge about the composition and especially about the intercystic communications of the lymphangioma sclerosant therapy becomes safer and more efficient.

The relationship between lymphatic and venous malformations in Klippel‐Trenaunay syndrome is difficult to assess. Herein the authors describe near‐infrared fluorescence lymphatic imaging to assess the lymphatics of a subject with a large port‐wine stain and right leg edema. Although lymphatic vessels in the medial, affected knee appeared dilated and perhaps tortuous, no definitive abnormal lymphatic pooling or propulsion was observed. The lymphatics in the affected limb were well defined but less numerous than in the contralateral limb, and active, contractile function was observed in all vessels. As demonstrated, near‐infrared fluorescence lymphatic imaging enables the clinical assessment of lymphatics in lymphovenous malformations.

Purpose: Proteus syndrome is a rare mosaic overgrowth disorder that is associated with severe complications. While anecdotal data have suggested that the life span of affected patients is reduced, this has not been measured. Mortality data on rare diseases is critical for assessing treatments and other interventions. Methods: To address this we used the clinical research records of 64 patients in a longitudinal natural history cohort at the National Institutes of Health to ascertain the data in an organized manner and estimate survival using a Kaplan‐Meier approach. Results: The median age of diagnosis was 19 months. Based on this analysis, there was 25% probability of death by 22 years of age. Ten of the 11 patients who died were younger than 22 years of age, and there was only a single death after this age. Conclusion: These data quantify the risk of premature death in Proteus syndrome, which can be used to support interventions and trials. Although the risk of death is substantial, the fact that only one patient died after 22 years of age supports anecdotal evidence that the disease process moderates after the end of adolescence. Interventions to reduce mortality should be targeted to the pediatric age range. GENETICS in MEDICINE advance online publication, 29 June 2017; doi:10.1038/gim.2017.65.

BACKGROUND: Current treatment options for lymphatic malformations (LMs) are multimodal. Recently, the effectiveness of treating LMs with Eppikajyutsuto (TJ‐28) has been reported. TJ‐28 is a kind of oral herbal medicine classified as the traditional Japanese Kampo medicine. CASE PRESENTATION: A 12‐year‐old girl was admitted to our hospital for intermittent upper abdominal pain. Radiological examinations revealed a large (9.5 × 5.8 × 10.0 cm) retroperitoneal LM, which was suspected to adhering and stretching both pancreas head and duodenum. The large retroperitoneal tumor resection might induce involving complications because of the size and the location. Therefore, we used TJ‐28 in order to diminish the tumor size before surgery. The patient received oral doses of 7.5 g/day (2.5 g × 3 times/day) of TJ‐28. Six months after the medication, the tumor decreased markedly to 3.5 × 1.5 × 1.2 cm in size. Thereafter, the mass was sub‐totally resected (95%) via a 3 cm trans‐umbilical incision without any surgical complications. CONCLUSIONS: We reported a case of successfully treated retroperitoneal LM with the combination treatment of TJ‐28 and surgery. Based on our experience, this TJ‐28 treatment option may be very useful in treating cases of LMs having surgical difficulties because of size and/or location.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by arteriovenous malformations in the brain, liver, and lungs. Pulmonary hypertension (PH) is increasingly recognized as a severe complication of HHT. However, there are no studies describing the prevalence of PH in HHT compared to HHT‐negative controls. OBJECTIVE: To assess the estimated prevalence of PH in patients with HHT compared to HHT‐negative controls. METHODS: All consecutive subjects screened for HHT with available genetic testing and echocardiography‐based peak tricuspid regurgitation velocity (TRV) measurement were included. Increased‐probability PH was defined as a TRV >2.8 m/s. RESULTS: In 578 subjects, both echocardiography and genetic testing were available. A reliable TRV was measured in 383 (66.3%), of whom 127 had HHT type 1 (HHT1), 150 had HHT type 2 (HHT2), and 106 were HHT‐negative controls, with a mean TRV of 2.3 +/‐ 0.4, 2.4 +/‐ 0.5, and 2.2 +/‐ 0.3 m/s, respectively (p = 0.008 and p < 0.001 vs. controls). Increased‐probability PH was found in 42 subjects (8.7% in HHT1, 18.0% in HHT2, and 3.8% in HHT‐negative controls). HHT2 and hepatic arteriovenous malformations (HAVMs) were the most important predictors for increased‐probability PH (odds ratio 5.6, p = 0.002, and odds ratio 11.3, p < 0.001, respectively). Heritable pulmonary arterial hypertension (HPAH) was diagnosed in 2 patients (0.7%) and only found in HHT2 (1.3%). CONCLUSION: The estimated prevalence of PH is higher in HHT patients compared to HHT‐negative controls. This increase is especially present in HHT2 and mainly associated with the presence of HAVMs. HPAH appears to be rare in HHT patients and was only diagnosed in HHT2.

Capillary malformation‐arteriovenous malformation (CM‐AVM) syndrome, due to inactivating mutations in RASA1 in 68% of cases, is characterized by the development of cutaneous capillary malformations and arteriovenous malformations or fistulas; no known genetic etiology has been identified in patients with CM‐AVM syndrome without RASA1 mutations. We present the case of a child with RASA1‐negative CM‐AVM syndrome with a de novo missense mutation in EPHB4, a transmembrane tyrosine kinase receptor essential for vasculogenesis. Inactivating the mutation in EPHB4 has been shown to upregulate the mitogen‐activated protein kinase pathway and the mammalian target of rapamycin complex 1, possibly contributing to the development of vascular malformations.