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The lymphatic system is implicated in interstitial fluid balance regulation, immune cell trafficking, oedema and cancer metastasis. However, the sequence of events that initiate and coordinate lymphatic vessel development (lymphangiogenesis) remains obscure. In effect, the understanding of physiological regulation of lymphatic vasculature has been overshadowed by the greater emphasis focused on angiogenesis, and delayed by a lack of specific markers, thereby limiting this field to no more than a descriptive characterization. Recently, new insights into lymphangiogenesis research have been due to the discovery of lymphatic-specific markers and growth factors of vascular endothelial growth factor (VEGF) family, such as VEGF-C and VEGF-D. Studies using transgenic mice overexpressing VEGF-C and VEGF-D have demonstrated a crucial role for these factors in tumour lymphangiogenesis.Knowledge of lymphatic development has now been redefined at the molecular level, providing an interesting target for innovative therapies. This review highlights the recent insights and advances into the field of lymphatic vascular research, outlining the most important aspects of the embryo development, structure, specific markers and methods applied for studying lymphangiogenesis. Finally, molecular mechanisms involved in the regulation of lymphangiogenesis are described.

IntroductionMultiple myeloma (MM) remains an incurable disease, despite conventional and high-dose chemotherapies. 1 Molecules targeting not only plasma cells, but also the bone marrow (BM) microenvironment are needed to overcome drug resistance.Pathologic angiogenesis is a constant component of the MM microenvironment. 2 The vascular endothelial growth factor (VEGF)/ VEGF receptor-2 (VEGFR-2) pathway greatly contributes to MM angiogenesis and growth, 3 and mediates proliferation and capillarogenesis in MM endothelial cells (MMECs) through an autocrine loop. 4 VEGF 165 is the most abundant and effective isoform. 5 It binds simultaneously to its cognate receptors VEGFR-1 and VEGFR-2 and to the coreceptor neuropilin-1 (NRP1), a cellsurface glycoprotein expressed on axons in the developing nervous system as well. 6 NRP1 is the ligand-binding subunit of the receptor complex for class 3 semaphorins, a family of secreted proteins that mediate neuronal guidance. 7 Binding of secreted semaphorin 3A (SEMA3A) to NRP1 induces the collapse of neuronal growth cones 8 by activating the signal-transducing subunit(s) of the receptor complex, that is, class A plexins, a family of transmembrane proteins whose cytoplasmic domain is endowed with an R-Ras GAP activity that inhibits integrin function. 9 NRP1 is also expressed on ECs, 10 where it acts as an isoform-specific receptor for VEGF 165 , and enhances by 4-to 6-fold its affinity for VEGFR-2, as well as VEGF 165 -induced cell chemotaxis and proliferation. 11 By competing with VEGF 165 for binding to NRP1 12 and by activating class A plexins, 13 SEMA3A inhibits integrinbased EC adhesion and migration and capillary sprouting. Autocrine loops of endothelial SEMA3A play a self-limiting role in angiogenesis and regulate EC behavior during its physiologic development. 13 Here we analyze the expression levels of VEGF 165 , SEMA3A, and their receptors NRP1 and plexin-A1 in BM ECs isolated from patients with MM and MGUS (MMECs and MGECs), and from the human umbilical vein (HUVECs). We show that overangiogenic MMECs display a high VEGF 165 /SEMA3A ratio and behave like MGECs and HUVECs upon exposure to exogenous SEMA3A, which seems as effective as an anti-VEGFR-2 antibody. Our observations point to SEMA3A as a potential antiangiogenic Patients, materials, and methods PatientsThirty-two patients fulfilling the International Myeloma Working Group diagnostic criteria 14 for MM (n ϭ 18) and MGUS (n ϭ 14) were studied at diagnosis. The MM patients (12 male, 6 female) were aged 44 to 81 years (median 68.5 years) and staged 15 as IIA (n ϭ 4), IIB (n ϭ 2), IIIA (n ϭ 10), and IIIB (n ϭ 2); the M-component was IgG (n ϭ 12), IgA (n ϭ 4), and or (n ϭ 2). The MGUS patients (8 male, 6 female) were aged 42 to 79 years (median 70.6 years) and were IgG (n ϭ 10) or IgA (n ϭ 4). The study was approved by the local ethics committee of the University of Bari Medical School, Italy, and all patients gave their informed consent in accordance with the Declaration of Helsinki. Separation and culture of ECs and...

SummaryBone marrow endothelial cells (EC) from patients with multiple myeloma (MM) were found to express and secrete higher amounts of the CXCchemokines CXCL8/interleukin (IL)-8, CXCL11/interferon-inducible T-cell alpha chemoattractant (I-TAC), CXCL12/stromal cell-derived factor (SDF)-1a, and CCL2/monocyte chemotactic protein(MCP)-1 than EC from human umbilical vein (HUVEC), considered as a healthy counterpart. Paired plasma cells and several MM cell lines expressed cognate receptors of each chemokine to a variable extent. When cells were exposed to chemokines, CXCL8/IL-8 and CXCL12/SDF-1a stimulated their proliferation and all chemokines stimulated cell chemotaxis. It is suggested that angiogenesis also favours MM progression through the release of CXC-chemokines.

Inhibition of multiple myeloma (MM) plasma cells in their permissive bone marrow microenvironment represents an attractive strategy for blocking the tumor/ vessel growth associated with the disease progression. However, target speci-ficity is an essential aim of this approach. Here, we identified platelet-derived growth factor (PDGF)-receptor beta (PDGFR) and pp60c-Src as shared constitutively activated tyrosine-kinases (TKs) in plasma cells and endothelial cells (ECs) isolated from MM patients (MMECs). Our cellular and molecular dissection showed that the PDGF-BB/PDGFR kinase axis promoted MM tumor growth and vessel sprouting by activating ERK1/2, AKT, and the transcription of MMEC-released proangio-genic factors, such as vascular endothe-lial growth factor (VEGF) and interleukin-8 (IL-8). Interestingly, pp60c-Src TK-activity was selectively induced by VEGF in MM tumor and ECs, and the use of small-interfering (si)RNAs validated pp60c-Src as a key signaling effector of VEGF loop required for MMEC survival, migration, and angiogenesis. We also assessed the antitumor/vessel activity of dasatinib, a novel orally bioactive PDGFR/Src TK-inhibitor that significantly delayed MM tumor growth and angiogenesis in vivo, showing a synergistic cytotoxicity with conventional and novel antimyeloma drugs (ie, melphalan, prednisone, bor-tezomib, and thalidomide). Overall data highlight the biologic and therapeutic relevance of the combined targeting of PDGFR/c-Src TKs in MM, providing a framework for future clinical trials. (Blood. 2008;112:1346-1356) Introduction Multiple myeloma (MM) is characterized by a clonal proliferation of immunoglobulin-secreting plasma cells in the bone marrow (BM), with clinical manifestations including lytic bone lesions, anemia, renal failure, immunodeficiency, and hypercalcemia. 1 For patients treated with conventional and high-dose chemotherapies, the median survival time from diagnosis is 3 to 4 years. 2 Approximately one-half of patients with newly diagnosed MM achieve remission from these therapies, but options are more limited for primary resistant or relapsing disease. Partial remission occurs in only 20% of resistant patients and in 45% of relapsing patients. Survival time improves in approximately 50% of those younger than 60 who are able to undergo high-dose chemotherapy followed by autologous stem cell transplantation, but approximately 30% of them are late in the graft intake or even develop myelodysplasia that prevents additional chemotherapy while relapse or resistant disease occurs. Induction of MM plasma cells is thought to involve genetic lesions (ie, translocations between immunoglobulin enhancers and oncogenes) and secondary events underlying the activation of bidirectional MM tumor-microenvironment interactions. 3-5 Indeed, homing and survival of MM plasma cells are sustained by overangiogenic sprouting of microvascular endothelial cells (ECs), 6,7 as well as by osteoclasts, fibroblasts, monocytes, macrophages, and mast cells, 4,8,9 thus resulting in a multiplicity of autocrine/...