PerspectiveDevelopment

Lymphatics Make the Break

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Science  10 Jan 2003:
Vol. 299, Issue 5604, pp. 209-210
DOI: 10.1126/science.1081345

Our body has two distinct vascular networks: one composed of blood vessels and the other of lymphatic vessels. Blood vessels deliver nutrients and immune cells to all tissues in the body. In contrast, lymphatic vessels collect extravasated immune cells (which have migrated across blood vessel walls) as well as fluid leaked from blood vessels, and, after traversing a series of lymph nodes, return them to the blood circulation. These two networks separate from a common origin during embryonic development, thus providing complementary yet distinct pathways for maintaining the integrity of tissues (see the figure).

Imagine the consequences if these two systems did not separate during development. Blood flow to tissues would be compromised because blood would bypass the small capillaries and flow directly into the lymphatic vessels, a hallmark of human congenital diseases involving arteriovenous malformations (AVMs). Furthermore, drainage of excess fluid from tissues would be impaired, leading to edema and accumulation of fluid in the peritoneal cavity (ascites). On page 247 of this issue, Abtahian et al. (1) reveal that mice with mutations in the protein tyrosine kinase Syk or its substrate, the adaptor protein SLP-76, exhibit edema and ascites as well as AVMs. Building on the work of Turner et al. (2), who first observed the formation of lipid-rich ascites in Syk-deficient mice, the authors elucidate a hematopoietic signaling pathway that is involved in the separation of the blood and lymphatic networks. But how does the Syk/SLP-76 signaling pathway fit into our molecular view of the separation of these two networks? What are the other molecular and cellular players involved in lymphatic development, and what are the medical implications of the new work?

Embryonic lymphatic vessels originate primarily from blood vessels (3, 4). In the early embryo, endothelial cells of the cardinal vein express the receptors LYVE-1 and VEGFR-3, molecules found primarily (but not exclusively) on normal adult lymphatic vessels (see the figure, step 1). A signal, as yet unidentified, triggers the polarized expression of the homeobox gene Prox-1 by the cardinal vein endothelial cells, such that different regions of the cardinal vein express different amounts of Prox-1. This marks the first stage of commitment to the lymphatic lineage (step 2). Cells expressing LYVE-1, VEGFR-3, and Prox-1 then start to form buds, again in a polarized fashion (step 3). At this stage, these early lymphatic endothelial cells start to produce the secondary lymphoid chemokine (SLC) and increase their expression of VEGFR-3 (step 4), both markers of mature lymphatic endothelial cells. In this way, the lymphatic system starts to form (step 5). Members of the angiopoietin family (Ang-2) and its receptor (Tie-2) are presumably involved in the maturation and patterning of these newly formed lymphatic vessels (5).

Breaking up is hard to do.

The molecules that orchestrate lymphatic vessel development include members of the VEGF family and their receptors (VEGF-A, -C, and -D, VEGFR-2, and VEGFR-3), members of the angiopoietin family and their receptors (Ang-1, Ang-2, and Tie-2), neuropilin-2 (Nrp-2, a co-receptor for VEGFR-3), Prox-1 (a homeobox protein), LYVE-1, SLC, FOXC2, and α9 integrin (3-5). New additions to this list include Syk (spleen tyrosine kinase) and its substrate the adaptor protein SLP-76 (SH2 domain-containing leukocyte protein, molecular mass 76 kD) (1, 10). Mice lacking SLP-76 fail to show separation of the lymphatic and blood systems during development (1); they do not produce mature T lymphocytes but do produce mature B cells (2). Syk-deficient animals also have an aberrant lymphatic phenotype, and produce mature B (but not T) cells. Although SLP-76 and Syk do not collaborate to control lymphocyte maturation, they do work together to regulate platelet degranulation and the separation of the embryonic lymphatic and blood systems.

CREDIT: KATHARINE SUTLIFF/SCIENCE

In the SLP-76- or Syk-deficient mice studied by Abtahian et al., development of the lymphatic vascular system appears to progress normally through at least step 2, after which it becomes aberrant. The authors were able to recapitulate the aberrant lymphatic phenotypes of the SLP-76- and Syk-deficient mice by rescuing lethally irradiated wild-type mice with bone marrow cells derived from the knockout animals. This suggested that cells arising from bone marrow rather than lymphatic endothelial cells per se may have been responsible for the aberrant lymphatic phenotype of the wild-type mice and their inability to repair radiation-induced vascular damage. But what is the signal from normal bone marrow-derived hematopoietic cells that triggers separation of the lymphatic and blood systems during development? SLP-76 and Syk are important modulators of platelet degranulation and the production of the cytokine interleukin-2. Could molecules released in response to activation of the Syk/SLP-76 pathway signal endothelial cells of a developing lymphatic vessel to stop growing toward blood vessels and remain blind-ended (see the figure, step 5)? Alternatively, could extravasated immune cells in which the Syk/SLP-76 signaling pathway is activated be responsible for the separation of the blood and lymphatic networks? If so, then the absence of these signaling molecules in SLP-76- or Syk-deficient animals might result in fusion of the peripheral lymphatic vessels with blood vessels resulting in the vascular malformations observed in the knockout mice.

Although Abtahian et al. argue to the contrary, another possibility is that the bone marrow-derived cells they used to rescue the lethally irradiated wild-type mice included lymphatic endothelial progenitor cells (6). These circulating cells differentiate into endothelial cells expressing lymphatic markers in vitro (see the figure, step 5). They may be important for lymphatic development as well as for postnatal lymphangiogenesis under physiological or pathological conditions. Could signaling through the SLP-76/Syk pathway trigger the early differentiation of a multipotent bone marrow stem cell into a more differentiated lymphatic endothelial progenitor cell? Because SLP-76 or Syk could not be detected in the endothelial cells of the lethally irradiated wild-type mice, the expression of these molecules may be down-regulated before lymphatic endothelial progenitor cells become incorporated into the developing lymphatic vessels.

The careful functional studies of Abtahian et al. have broad implications. First, the identification of SLP-76 and Syk during the separation of the blood and lymphatic systems implicates these molecules in AVMs. The new findings may help to elucidate other proteins involved in various congenital AVM syndromes, thus speeding development of improved treatment strategies. Second, the Abtahian et al. work focuses attention on early events in the development of lymphatic vessels and the part played by cells derived from bone marrow. It is becoming clear that multiple molecules are necessary to ensure proper differentiation and patterning of lymphatic endothelial cells into a functional lymphatic system. By expanding the roster of molecules, many more targets for therapeutic intervention will emerge beyond VEGFR-3 and its ligands, yielding more possibilities for new treatments for lymphedema. Third, the new work may offer fresh insights into the dysregulation of both the vascular and lymphatic systems during tumor formation, specifically through the SLP-76/Syk pathway. Syk expression is lower in breast tumors than in normal mammary tissue and correlates with an increase in metastases and a poor prognosis (7, 8). Could reduced Syk expression lead to abnormalities in the developing tumor vasculature, hence boosting metastasis? Could mutations in SLP-76, Syk, or other pathway components also explain the finding that blood-filled vessels associated with some tumors bear lymphatic markers (9)? Blood vessels and lymphatic vessels present metastatic tumor cells with two major routes for dispersal. Thus, strategies to selectively eradicate these vessels may provide a potent weapon against cancer. Increased understanding of the signaling pathways driving lymphatic vessel development should boost the impact of therapeutics on diseases associated with the lymphatic system.

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