Update June 2012

Basic Science

Ando, Y., O. Murai, et al. (2011). “Lymphatic architecture of the human gingival interdental papilla.” Lymphology 44(4): 146–154.

Aschen, S., J. C. Zampell, et al. (2012). “Regulation of adipogenesis by lymphatic fluid stasis: Part II. expression of adipose differentiation genes.” Plast Reconstr Surg 129(4): 838–847.

BACKGROUND: Although fat deposition is a defining clinical characteristic of lymphedema, the cellular mechanisms that regulate this response remain unknown. The goal of this study was to determine how lymphatic fluid stasis regulates adipogenic gene activation and fat deposition. METHODS: Adult female mice underwent tail lymphatic ablation and were euthanized at 1, 3, or 6 weeks postoperatively (n=8 per group). Samples were analyzed by immunohistochemistry and Western blot analysis. An alternative group of mice underwent axillary dissections or sham incisions, and limb tissues were harvested 3 weeks postoperatively (n=8 per group). RESULTS: Lymphatic fluid stasis resulted in significant subcutaneous fat deposition and fibrosis in lymphedematous tail regions (p<0.001). Western blot analysis demonstrated that proteins regulating adipose differentiation including CCAAT/enhancer-binding protein-alpha and adiponectin were markedly up-regulated in response to lymphatic fluid stasis in the tail and axillary models. Expression of these markers increased in edematous tissues according to the gradient of lymphatic stasis distal to the wound. Immunohistochemical analysis further demonstrated that adiponectin and peroxisome proliferator-activated receptor-gamma, another critical adipogenic transcription factor, followed similar expression gradients. Finally, adiponectin and peroxisome proliferator-activated receptor-gamma expression localized to a variety of cell types in newly formed subcutaneous fat. CONCLUSIONS: The mouse-tail model of lymphedema demonstrates pathologic findings similar to clinical lymphedema, including fat deposition and fibrosis. The authors show that lymphatic fluid stasis potently up-regulates the expression of fat differentiation markers both spatially and temporally. These studies elucidate mechanisms regulating abnormal fat deposition in lymphedema pathogenesis and therefore provide a basis for developing targeted treatments.

Babu, S., R. Anuradha, et al. (2012). “TLR- and filarial antigen-mediated, MAPK- and NF-kappaB-dependent regulation of angiogenic growth factors in filarial lymphatic pathology.” Infect Immun. Epub 2012/04/18.

Boyle, M. C., T. A. Crabbs, et al. (2012). “Intestinal lymphangiectasis and lipidosis in rats following subchronic exposure to indole-3-carbinol via oral gavage.” Toxicol Pathol. Epub 2012/02/14.

Bronneke, S., B. Bruckner, et al. (2012). “DNA methylation regulates lineage-specifying genes in primary lymphatic and blood endothelial cells.” Angiogenesis. Epub 2012/03/22.

During embryonic development, the lymphatic system emerges by transdifferentiation from the cardinal vein. Although lymphatic and blood vasculature share a close molecular and developmental relationship, they display distinct features and functions. However, even after terminal differentiation, transitions between blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) have been reported. Since phenotypic plasticity and cellular differentiation processes frequently involve epigenetic mechanisms, we hypothesized that DNA methylation might play a role in regulating cell type-specific expression in endothelial cells. By analyzing global gene expression and methylation patterns of primary human dermal LEC and BEC, we identified a highly significant set of genes, which were differentially methylated and expressed. Pathway analyses of the differentially methylated and upregulated genes in LEC revealed involvement in developmental and transdifferentiation processes. We further identified a set of novel genes, which might be implicated in regulating BEC-LEC plasticity and could serve as therapeutic targets and/or biomarkers in vascular diseases associated with alterations in the endothelial phenotype.

Choi, I., S. Lee, et al. (2012). “The new era of the lymphatic system: no longer secondary to the blood vascular system.” Cold Spring Harb Perspect Med 2(4): a006445.

Datar, S. A., E. G. Johnson, et al. (2012). “Altered lymphatics in an ovine model of congenital heart disease with increased pulmonary blood flow.” Am J Physiol Lung Cell Mol Physiol 302(6): L530–540.

Abnormalities of the lymphatic circulation are well recognized in patients with congenital heart defects. However, it is not known how the associated abnormal blood flow patterns, such as increased pulmonary blood flow (PBF), might affect pulmonary lymphatic function and structure. Using well-established ovine models of acute and chronic increases in PBF, we cannulated the efferent lymphatic duct of the caudal mediastinal node and collected and analyzed lymph effluent from the lungs of lambs with acutely increased PBF (n=6), chronically increased PBF (n=6), and age-matched normal lambs (n=8). When normalized to PBF, we found that lymph flow was unchanged following acute increases in PBF but decreased following chronic increases in PBF. The lymph:plasma protein ratio decreased with both acute and chronic increases in PBF. Lymph bioavailable nitric oxide increased following acute increases in PBF but decreased following chronic increases in PBF. In addition, we found perturbations in the transit kinetics of contrast material through the pleural lymphatics of lambs with chronic increases in PBF. Finally, there were structural changes in the pulmonary lymphatic system in lambs with chronic increases in PBF: lymphatics from these lambs were larger and more dilated, and there were alterations in the expression of vascular endothelial growth factor-C, lymphatic vessel endothelial hyaluronan receptor-1, and Angiopoietin-2, proteins known to be important for lymphatic growth, development, and remodeling. Taken together these data suggest that chronic increases in PBF lead to both functional and structural aberrations of lung lymphatics. These findings have important therapeutic implications that warrant further study.

Hadamitzky, C., H. O. Rennekampff, et al. (2012). “Lymphatic regeneration in meshed skin grafts.” Burns. Epub 2012/02/22.

Hajrasouliha, A. R., Z. Sadrai, et al. (2012). “b-FGF Induces Corneal Blood and Lymphatic Vessel Growth in a Spatially Distinct Pattern.” Cornea. Epub 2012/04/03.

Hall, K. L., L. D. Volk-Draper, et al. (2012). “New model of macrophage acquisition of the lymphatic endothelial phenotype.” PLoS One 7(3): e31794.

Lapinski, P. E., S. Kwon, et al. (2012). “RASA1 maintains the lymphatic vasculature in a quiescent functional state in mice.” J Clin Invest 122(2): 733–747.

RASA1 (also known as p120 RasGAP) is a Ras GTPase-activating protein that functions as a regulator of blood vessel growth in adult mice and humans. In humans, RASA1 mutations cause capillary malformation-arteriovenous malformation (CM-AVM); whether it also functions as a regulator of the lymphatic vasculature is unknown. We investigated this issue using mice in which Rasa1 could be inducibly deleted by administration of tamoxifen. Systemic loss of RASA1 resulted in a lymphatic vessel disorder characterized by extensive lymphatic vessel hyperplasia and leakage and early lethality caused by chylothorax (lymphatic fluid accumulation in the pleural cavity). Lymphatic vessel hyperplasia was a consequence of increased proliferation of lymphatic endothelial cells (LECs) and was also observed in mice in which induced deletion of Rasa1 was restricted to LECs. RASA1-deficient LECs showed evidence of constitutive activation of Ras in situ. Furthermore, in isolated RASA1-deficient LECs, activation of the Ras signaling pathway was prolonged and cellular proliferation was enhanced after ligand binding to different growth factor receptors, including VEGFR-3. Blockade of VEGFR-3 was sufficient to inhibit the development of lymphatic vessel hyperplasia after loss of RASA1 in vivo. These findings reveal a role for RASA1 as a physiological negative regulator of LEC growth that maintains the lymphatic vasculature in a quiescent functional state through its ability to inhibit Ras signal transduction initiated through LEC-expressed growth factor receptors such as VEGFR-3.

Lippi, G., E. J. Favaloro, et al. (2012). “Hemostatic properties of the lymph: relationships with occlusion and thrombosis.” Semin Thromb Hemost 38(2): 213–221.

Lymphatic thrombosis is a rare occurrence, and although its frequency is likely underestimated, its burden remains substantially lower than that of venous or arterial thrombosis. Current evidence suggests that despite measurable levels of fibrinogen, von Willebrand factor and other coagulation factors in the lymph, fibrin generation is substantially inhibited under physiological conditions, essentially making the lymph a hypocoagulable biological fluid. Although factor VIIa-tissue factor-catalyzed activation of factor X is possible in the lymph, fibrin generation is largely counteracted by the unavailability of cell surface anionic phospholipids such as those physiologically present on blood platelets, combined with only low levels of coagulation factors, and the strong inhibitory activity of heparin, antithrombin, and tissue factor pathway inhibitor. Enhanced fibrinolytic activity further contributes to reduce the development and growth of lymph clots. Nevertheless, lymphatic thrombosis is occasionally detected, especially in the thoracic duct, axillary, or inguinal lymphatics. Pathogenetic mechanisms are supported by the release of thromboplastin substances from the injured lymphatic endothelium accompanied by chronic obstruction of lymph flow in the presence of a hypercoagulable milieu, thereby mirroring the Virchow triad that otherwise characterizes venous thrombosis. In theory, any source of lymphatic vessel occlusion, such as internal obliteration, external compression, or increased lymphatic pressure, might predispose to localized lymphatic thrombosis. The leading pathologies that can trigger thrombosis in the lymphatic vessels include cancer (due to external compression, neoplastic obliteration of the lymphatic lumen by metastatic cells, or lymphatic dysfunction after lymph node dissection), infections (especially lymphatic filariasis or sustained by Chlamydia trachomatis, Mycobacterium tuberculosis, Treponema pallidum, or Streptococcus pyogenes), congestive heart failure, chronic edema and inflammation of the distal lower limb, complications of central venous catheterization, coronary artery bypass grafting, thoracic outlet syndrome, and amyloidosis.

Luo, Y., L. Liu, et al. (2012). “Rapamycin inhibits lymphatic endothelial cell tube formation by downregulating vascular endothelial growth factor receptor 3 protein expression.” Neoplasia 14(3): 228–237.

Mammalian target of rapamycin (mTOR) controls lymphangiogenesis. However, the underlying mechanism is not clear. Here we show that rapamycin suppressed insulin-like growth factor 1 (IGF-1)- or fetal bovine serum (FBS)-stimulated lymphatic endothelial cell (LEC) tube formation, an in vitro model of lymphangiogenesis. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE), conferred resistance to rapamycin inhibition of LEC tube formation, suggesting that rapamycin inhibition of LEC tube formation is mTOR kinase activity dependent. Also, rapamycin inhibited proliferation and motility in the LECs. Furthermore, we found that rapamycin inhibited protein expression of VEGF receptor 3 (VEGFR-3) by inhibiting protein synthesis and promoting protein degradation of VEGFR-3 in the cells. Down-regulation of VEGFR-3 mimicked the effect of rapamycin, inhibiting IGF-1- or FBS-stimulated tube formation, whereas over-expression of VEGFR-3 conferred high resistance to rapamycin inhibition of LEC tube formation. The results indicate that rapamycin inhibits LEC tube formation at least in part by downregulating VEGFR-3 protein expression.

Maruyama, K., T. Nakazawa, et al. (2012). “The maintenance of lymphatic vessels in the cornea is dependent on the presence of macrophages.” Invest Ophthalmol Vis Sci. epub 2012/04/19.

Mendez, U., E. M. Brown, et al. (2012). “Functional recovery of fluid drainage precedes lymphangiogenesis in acute murine foreleg lymphedema.” Am J Physiol Heart Circ Physiol. 2012/03/20.

Mu, H., T. L. Calderone, et al. (2012). “Lysophosphatidic acid induces lymphangiogenesis and interleukin-8 production in vitro in human lymphatic endothelial cells.” Am J Pathol. 2012/04/03.

Mundinger, G. S., M. Narushima, et al. (2012). “Infrared fluorescence imaging of lymphatic regeneration in nonhuman primate facial vascularized composite allografts.” Ann Plast Surg 68(3): 314–319.

Prangsaengtong, O., K. Senda, et al. (2012). “Calpain 1 and -2 play opposite roles in cord formation of lymphatic endothelial cells via eNOS regulation.” Hum Cell. Epub 2012/02/09.

Sarkisyan, G., S. Cahalan, et al. (2012). “Real-time differential labeling of blood, interstitium and lymphatic and single-field analysis of vasculature dynamics in vivo.” Am J Physiol Cell Physiol. Epub 2012/02/24.

Schwartz, M. A. and M. Simons (2011). “Lymphatics thrive on stress: mechanical force in lymphatic development.” EMBO J 31(4): 781–782.

Solder, E., B. C. Bockle, et al. (2012). “Isolation and characterization of CD133+CD34+VEGFR-2+CD45- fetal endothelial cells from human term placenta.” Microvasc Res. Epub 2012/04/07.

Song, S. H., K. L. Kim, et al. (2012). “Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells.” Biochem Biophys Res Commun. Epub 2012/02/22.

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.

Takahashi, S., K. Ambe, et al. (2012). “Immunohistochemical investigation of lymphatic vessel formation control in mouse tooth development: Lymphatic vessel-forming factors and receptors in tooth development in mice.” Tissue Cell. Epub 2012/04/03.

Tian, M., L. Yu, et al. (2012). “Correlations between SUVmax and expression of GLUT1 and growth factors inducing lymphangiogenesis.” Acad Radiol 19(4): 420–426.

Truman, L. A., K. L. Bentley, et al. (2012). “ProxTom lymphatic vessel reporter mice reveal prox1 expression in the adrenal medulla, megakaryocytes, and platelets.” Am J Pathol.epub 2012/02/09.

von der Weid, P. Y., S. Rehal, et al. (2012). “Mechanisms of VIP-induced inhibition of the lymphatic vessel pump.” J Physiol. Epub 2012/03/28.

Walsh, D. A., P. Verghese, et al. (2012). “Lymphatic vessels in osteoarthritic human knees.” Osteoarthritis Cartilage. Epub 2012/02/14.

Wang, W., N. Chen, et al. (2012). “Lymphatic transport and catabolism of therapeutic proteins following subcutaneous administration to rats and dogs.” Drug Metab Dispos. Epub 2012/02/14.

Zampell, J. C., S. Aschen, et al. (2012). “Regulation of adipogenesis by lymphatic fluid stasis: part I. Adipogenesis, fibrosis, and inflammation.” Plast Reconstr Surg 129(4): 825–834.

BACKGROUND: Although fat deposition is a defining clinical characteristic of lymphedema, the cellular mechanisms that regulate this response remain unknown. The goals of this two-part study were to determine the effect of lymphatic fluid stasis on adipogenesis and inflammation (part I) and how these changes regulate the temporal and spatial expression of fat differentiation genes (part II). METHODS: Adult female mice underwent tail lymphatic ablation and were euthanized 6 weeks after surgery (n=20). Fat deposition, fibrosis, and inflammation were then analyzed in the regions of the tail exposed to lymphatic fluid stasis as compared with normal lymphatic flow. RESULTS: Lymphatic fluid stasis in the tail resulted in significant subcutaneous fat deposition, with a 2-fold increase in fat thickness (p<0.01). In addition, lymphatic stasis was associated with subcutaneous fat fibrosis and collagen deposition. Adipogenesis in response to lymphatic fluid stasis was associated with a marked mononuclear cell inflammatory response (5-fold increase in CD45 cells; p<0.001). In addition, the authors noted a significant increase in the number of monocytes/macrophages as identified by F4/80 immunohistochemistry (p<0.001). CONCLUSIONS: The mouse-tail model has pathologic findings that are similar to clinical lymphedema, including fat deposition, fibrosis, and inflammation. Adipogenesis in response to lymphatic fluid stasis closely resembles this process in obesity. This model therefore provides an excellent means with which to study the molecular mechanisms that regulate the pathophysiology of lymphedema.

Oncology

Abengozar, M. A., S. de Frutos, et al. (2012). “Blocking ephrin-B2 with highly specific antibodies inhibits angiogenesis, lymphangiogenesis, and tumor growth.” Blood. Epub 2012/03/27.

Membrane-anchored ephrinB2 and its receptor EphB4 are involved in the formation of blood and lymphatic vessels in normal and pathological conditions. Eph/ephrin activation requires cell-cell interactions and leads to bidirectional signaling pathways in both ligand- and receptor-expressing cells. To investigate the functional consequences of blocking ephrinB2 activity, two highly specific human single-chain Fv (scFv) antibody fragments against ephrinB2 were generated and characterized. Both antibody fragments suppressed endothelial cell migration and tube formation in vitro in response to VEGF and provoked abnormal cell motility and actin cytoskeleton alterations in isolated endothelial cells. As only one of them (B11) competed for binding of ephrinB2 to EphB4, these data suggest an EphB-receptor-independent blocking mechanism. Anti-ephrinB2 therapy reduced VEGF-induced neovascularization in a mouse Matrigel plug assay. Moreover, systemic administration of ephrinB2-blocking antibodies caused a drastic reduction in the number of blood and lymphatic vessels in xenografted mice and a concomitant reduction in tumor growth. Our results show for the first time that specific antibody-based ephrinB2 targeting may represent an effective therapeutic strategy to be used as an alternative or in combination with existing antiangiogenic drugs for treating patients with cancer and other angiogenesis-related diseases.

Chen, J., J. S. Alexander, et al. (2012). “Integrins and their extracellular matrix ligands in lymphangiogenesis and lymph node metastasis.” Int J Cell Biol 2012: 853703.

In the 1970s, the late Judah Folkman postulated that tumors grow proportionately to their blood supply and that tumor angiogenesis removed this limitation promoting growth and metastasis. Work over the past 40 years, varying from molecular examination to clinical trials, verified this hypothesis and identified a host of therapeutic targets to limit tumor angiogenesis, including the integrin family of extracellular matrix receptors. However, the propensity for some tumors to spread through lymphatics suggests that lymphangiogenesis plays a similarly important role. Lymphangiogenesis inhibitors reduce lymph node metastasis, the leading indicator of poor prognosis, whereas inducing lymphangiogenesis promotes lymph node metastasis even in cancers not prone to lymphatic dissemination. Recent works highlight a role for integrins in lymphangiogenesis and suggest that integrin inhibitors may serve as therapeutic targets to limit lymphangiogenesis and lymph node metastasis. This review discusses the current literature on integrin-matrix interactions in lymphatic vessel development and lymphangiogenesis and highlights our current knowledge on how specific integrins regulate tumor lymphangiogenesis.

Dimaio, T. A. and M. Lagunoff (2012). “KSHV Induction of Angiogenic and Lymphangiogenic Phenotypes.” Front Microbiol 3: 102.

Kaposi's sarcoma (KS) is a highly vascularized tumor supporting large amounts of neo-angiogenesis. The major cell type in KS tumors is the spindle cell, a cell that expresses markers of lymphatic endothelium. KSHV, the etiologic agent of KS, is found in the spindle cells of all KS tumors. Considering the extreme extent of angiogenesis in KS tumors at all stages it has been proposed that KSHV directly induces angiogenesis in a paracrine fashion. In accordance with this theory, KSHV infection of endothelial cells in culture induces a number of host pathways involved in activation of angiogenesis and a number of KSHV genes themselves can induce pathways involved in angiogenesis. Spindle cells are phenotypically endothelial in nature, and therefore, activation through the induction of angiogenic and/or lymphangiogenic phenotypes by the virus may also be directly involved in spindle cell growth and tumor induction. Accordingly, KSHV infection of endothelial cells induces cell autonomous angiogenic phenotypes to activate host cells. KSHV infection can also reprogram blood endothelial cells to lymphatic endothelium. However, KSHV induces some blood endothelial specific genes upon infection of lymphatic endothelial cells creating a phenotypic intermediate between blood and lymphatic endothelium. Induction of pathways involved in angiogenesis and lymphangiogenesis are likely to be critical for tumor cell growth and spread. Thus, induction of both cell autonomous and non-autonomous changes in angiogenic and lymphangiogenic pathways by KSHV likely plays a key role in the formation of KS tumors.

Hu, Y. Y., M. H. Zheng, et al. (2012). “Notch signaling pathway and cancer metastasis.” Adv Exp Med Biol 727: 186–198.

Cancer metastasis is the leading cause of cancer-related deaths all over the world at present. Accumulated researches have demonstrated that cancer metastasis is composed of a series of successive incidents, mainly including epithelial-mesenchymal transition (EMT), malignant cell migration, resistance to anoikis, and angiogenesis and lymphangiogenesis processes. However, the complicated cellular and molecular mechanisms underlying and modulating these processes have not been well elucidated. Thus, studies on cancer metastasis mechanism may propose possibilities to therapeutically interfere with signaling pathways required for each step of cancer metastasis, therefore inhibiting the outgrowth of distant metastasis of tumors. Recent insights have linked the Notch signaling pathway, a critical pathways governing embryonic development and maintaining tumor stemness, to cancer metastasis. This chapter highlights the current evidence for aberration of the Notch signaling in metastasis of tumors such as osteosarcoma, breast cancer, prostate cancer, and melanoma. In these studies, Notch activity seems to participate in cancer metastasis by modulating the EMT, tumor angiogenesis processes, and the anoikis-resistance of tumor cells. Therefore, manipulating Notch signaling may represent a promising alternative/ complement therapeutic strategy targeting cancer metastasis besides cancer stemness.

Martinez-Corral, I., D. Olmeda, et al. (2012). “In vivo imaging of lymphatic vessels in development, wound healing, inflammation, and tumor metastasis.” Proc Natl Acad Sci U S A 109(16): 6223–6228.

Wang, C. A., P. Jedlicka, et al. (2012). “SIX1 induces lymphangiogenesis and metastasis via upregulation of VEGF-C in mouse models of breast cancer.” J Clin Invest. Epub 2012/04/03.

Yun, S. J., H. Y. Park, et al. (2012). “Clinicopathological correlation of cutaneous metastatic breast carcinoma using lymphatic and vascular markers: lymphatics are mainly involved in cutaneous metastasis.” Clin Exp Dermatol. Epub 2012/02/15.

Clinical

Chang, W. Y., J. L. Cane, et al. (2012). “Clinical utility of diagnostic guidelines and putative biomarkers in lymphangioleiomyomatosis.” Respir Res 13(1): 34.

ABSTRACT: BACKGROUND: Lymphangioleiomyomatosis is a rare disease occurring almost exclusively in women. Diagnosis often requires surgical biopsy and the clinical course varies between patients with no predictors of progression. We evaluated recent diagnostic guidelines, clinical features and serum biomarkers as diagnostic and prognostic tools. METHODS: Serum vascular endothelial growth factor-D (VEGF-D), angiotensin converting enzyme (ACE), matrix metalloproteinases (MMP) −2 and −9, clinical phenotype, thoracic and abdominal computerised tomography, lung function and quality of life were examined in a cohort of 58 patients. 32 healthy female controls had serum biomarkers measured. RESULTS: Serum VEGF-D, ACE and total MMP-2 levels were elevated in patients. VEGF-D was the strongest discriminator between patients and controls (median=1174 vs. 332 pg/ml p<0.0001 with an area under the receiver operating characteristic curve of 0.967, 95% CI 0.93–1.01). Application of European Respiratory Society criteria allowed a definite diagnosis without biopsy in 69%. Adding VEGF-D measurement to ERS criteria further reduced the need for biopsy by 10%. VEGF-D was associated with lymphatic involvement (p=0.017) but not the presence of angiomyolipomas. CONCLUSIONS: Combining ERS criteria and serum VEGF-D reduces the need for lung biopsy in LAM. VEGF-D was associated with lymphatic disease but not lung function.

Maus, E. A., I. C. Tan, et al. (2012). “Near-infrared fluorescence imaging of lymphatics in head and neck lymphedema.” Head Neck 34(3): 448–453.

Mayrovitz, H. N. and S. Davey (2011). “Changes in tissue water and indentation resistance of lymphedematous limbs accompanying low level laser therapy (LLLT) of fibrotic skin.” Lymphology 44(4): 168–177.

Mihara, M., H. Hara, et al. (2011). “Severe lymphedema caused by repeated self-injury.” Lymphology 44(4): 183–186.

Mikami, T., M. Hosono, et al. (2011). “Classification of lymphoscintigraphy and relevance to surgical indication for lymphaticovenous anastomosis in upper limb lymphedema.” Lymphology 44(4): 155–167.

Pittaluga, P. and S. Chastanet (2012). “Lymphatic complications after varicose veins surgery: risk factors and how to avoid them.” Phlebology 27 Suppl 1: 139–142.

Szolnoky, G., E. Varga, et al. (2011). “Lymphedema treatment decreases pain intensity in lipedema.” Lymphology 44(4): 178–182.

Vascular Anomalies

Behen, M. E., C. Juhasz, et al. (2011). “Brain damage and IQ in unilateral Sturge-Weber syndrome: support for a “fresh start” hypothesis.” Epilepsy Behav 22(2): 352–357.

Cahill, A. M., E. Nijs, et al. (2011). “Percutaneous sclerotherapy in neonatal and infant head and neck lymphatic malformations: a single center experience.” J Pediatr Surg 46(11): 2083–2095.

Dai, Y., F. Hou, et al. (2012). “Decreased eNOS protein expression in involuting and propranolol-treated hemangiomas.” Arch Otolaryngol Head Neck Surg 138(2): 177–182.

Hassanein, A. H., J. B. Mulliken, et al. (2012). “Venous malformation: risk of progression during childhood and adolescence.” Ann Plast Surg 68(2): 198–201.

Hoornweg, M. J., M. J. Smeulders, et al. (2012). “The prevalence and risk factors of infantile haemangiomas: a case-control study in the Dutch population.” Paediatr Perinat Epidemiol 26(2): 156–162.

Hou, F., Y. Dai, et al. (2011). “A pilot in vivo model of human microcystic lymphatic malformations.” Arch Otolaryngol Head Neck Surg 137(12): 1280–1285.

OBJECTIVE: To develop an in vivo mouse model of human microcystic lymphatic malformations (LMs) and provide a tool for investigating the biological mechanisms and treatment of microcystic disease. DESIGN: Animal model and histologic analysis. SETTING: Tertiary referral center. SUBJECTS: Fresh microcystic LM from human subjects were harvested and xenografted in the immunologically naive nude mice (Athymic Nude- Foxn1(nu)). MAIN OUTCOME MEASURES: Specimens were divided (5×5×5 mm) and secured in 4 quadrants subcutaneously along the dorsum of 4 nude mice. Weekly observations for volume, color, and texture of the grafts were performed with sequential harvesting from each quadrant at 30-day intervals. All grafts (n=16) were sectioned and stained with hematoxylin-eosin. Comparative pathologic evaluation of the grafts and native LM was performed by 2 blinded pathologists. Immunohistochemical analysis for D2-40 (a known lymphatic endothelial cell marker), Ki-67, and human-specific nuclear antigen was performed. RESULTS: Near complete microcystic LM xenograft survival (n=13 [81%]) was achieved in the mouse irrespective of the period of implantation. Xenografts underwent a brief growth phase to day 20 to 30 and were quiescent until approximately day 65 but ultimately had a gradual loss of volume following transplant. Histologic analysis revealed structural characteristics matching the native LM tissue. Immunohistochemical analysis found that 10 (77%) of the surviving xenografts (77%) were positive for D2-40, 9 (69%) were positive for human-specific nuclear antigen, and 8 (62%) were positive for Ki-67. CONCLUSIONS: This preliminary in vivo model suggests that microcystic LM can survive in the athymic nude mouse. The presence of markers for human antibodies, lymphatic endothelium, and cellular proliferation demonstrates the stability of native tissue qualities within the xenografts.

Itinteang, T., S. T. Tan, et al. (2012). “Infantile haemangioma expresses embryonic stem cell markers.” J Clin Pathol. Epub 2012/03/27.

BACKGROUND: The origin of infantile haemangioma (IH) remains enigmatic. A primitive mesodermal phenotype origin of IH with the ability to differentiate down erythropoietic and terminal mesenchymal lineages has recently been demonstrated. AIMS: To investigate the expression of human embryonic stem cell (hESC) markers in IH and to determine whether IH-derived cells have the functional capacity to form teratoma in vivo. METHODS: Immunohistochemical staining and quantitative reverse transcription PCR were used to investigate the expression of hESC markers in IH biopsies. The ability of cells derived from proliferating IH to form teratomas in a mouse xenograft model was investigated. RESULTS: The hESC markers, Oct-4, STAT-3 and stage-specific embryonic antigen 4 were collectively expressed on the endothelium of proliferating IH lesions, whereas Nanog was not. Nanog was expressed by cells in the interstitium and these cells did not express Oct-4, stage-specific embryonic antigen 4 or STAT-3. Proliferating IH-derived cells were unable to form teratomas in severely compromised immunodeficient/non-obese diabetic mice. CONCLUSION: The novel expression of hESC on two different populations of cells in proliferating IH and their inability to form teratomas in vivo infer the presence of a primitive cellular origin for IH downstream from hESC.

Kadam, S. D., M. Gucek, et al. (2012). “Cell proliferation and oxidative stress pathways are modified in fibroblasts from Sturge-Weber syndrome patients.” Arch Dermatol Res 304(3): 229–235.

Sturge-Weber syndrome (SWS) is defined by vascular malformations of the face, eye and brain and an underlying somatic mutation has been hypothesized. We employed isobaric tags for relative and absolute quantification (iTRAQ-8plex)-based liquid chromatography interfaced with tandem mass spectrometry (LC-MS/MS) approach to identify differentially expressed proteins between port-wine-derived and normal skin-derived fibroblasts of four individuals with SWS. Proteins were identified that were significantly up- or down-regulated (i.e., ratios >1.2 or <0.8) in two or three pairs of samples (n=31/972 quantified proteins) and their associated p values reported. Ingenuity pathway analysis (IPA) tool showed that the up-regulated proteins were associated with pathways that enhance cell proliferation; down-regulated proteins were associated with suppression of cell proliferation. The significant toxicologic list pathway in all four observations was oxidative stress mediated by Nrf2. This proteomics study highlights oxidative stress also consistent with a possible mutation in the RASA1 gene or pathway in SWS.

Katz, M. S., C. M. Finck, et al. (2012). “Vacuum-assisted closure in the treatment of extensive lymphangiomas in children.” J Pediatr Surg 47(2): 367–370.

Kim, S. W., K. Kauvanough, et al. (2011). “Long-term outcome of radiofrequency ablation for intraoral microcystic lymphatic malformation.” Arch Otolaryngol Head Neck Surg 137(12): 1247–1250.