doi: 10.1161/01.RES.0000069361.79586.12
© 2003 American Heart Association, Inc.
Editorials |
Lessons From Lymph
Flow-Guided Vessel Formation
From the Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Richard T. Lee, MD, Partners Research Facility, 65 Landsdowne St, Room 279, Cambridge, MA 02139. E-mail rlee{at}rics.bwh.harvard.edu
Key Words: lymph • angiogenesis • mechanotransduction • endothelium
Lymphangiogenesis doesn’t get much attention compared with its better-known cousin, angiogenesis. Perhaps that’s because lymphatic vessels don’t carry blood or platelets, and next to arteries or even veins, lymphatics are flimsy weaklings that are barely able to withstand 20 mm Hg before bursting.1 Yet clinical disorders of the lymphatic system occur commonly. Lymphedema, including the common postsurgical and postradiation forms and the rare inherited forms, has no successful therapy. Cancer metastasis occurs frequently by lymphatics, and lymphatic destruction by the parasitic filariasis diseases is among the leading cause of worldwide chronic disability.
The lymphatic system starts as an open-ended transport system in interstitial spaces, in contrast with the closed-loop circulation for blood.R2-128041 2,3 Protein-rich fluid is gathered by open lymphatic capillaries that drain into progressively larger lymphatic vessels. Collecting lymphatics converge into two major ducts, the thoracic duct, which drains most of the body’s lymph into the left subclavian vein, or the smaller right lymphatic duct, which drains into the right subclavian vein. Along the way back to the blood circulation, lymph passes through at least one lymph node, where immunological presentation of antigens and filtering can occur.
Despite the obvious macroscopic differences between the lymphatic and blood circulations, there are close parallels as well (Table). Larger lymphatics have valves like veins and also have smooth muscle–regulated tone that is nitric oxide–responsive.4 In fact, Sabin proposed in 1902 that the lymphatic system arises by endothelial budding from embryonic veins.5 Although others have proposed that the lymphatic system develops independently from mesenchymal tissue, recent studies of the Prox1 homeobox gene demonstrate that murine lymphatics arise from budding vascular endothelium.6
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Molecular studies of lymphatics have been hampered by several factors. The lymphatics are fragile, collapse easily, and may be difficult to visualize in tissues. Animal models have been challenging. Halsted, who pioneered radical mastectomy for breast cancer, was concerned about postoperative lymphedema but struggled to produce an animal model for this complication almost a century ago.7 In the past few decades, lymphedema models have been developed in species like dogs and rats, but models in mice have only recently emerged. In 1999, Slavin and colleagues developed the mouse tail model of lymphedema, potentially allowing the study of this process in genetically engineered mice.8 In 2002, the Folkman laboratory reported that FGF-2 implantation into the mouse cornea below the concentration threshold to induce angiogenesis leads to lymphangiogenesis.9 These mouse models will help enable specific molecular studies of lymphangiogenesis and lymphedema. For example, Yoon and colleagues recently used these animal models to show that gene therapy with VEGF-C, a VEGF family member associated with lymphangiogenesis,10 improved lymphedema in both a rabbit ear model and the mouse tail model.11 These experiments are consistent with the effects observed by Szuba and colleagues.12
Perhaps the greatest hurdle to progress in this field has been a paucity of specific molecular markers for the lymphatic endothelium. However, two important markers, VEGFR-3 and Prox1, have been identified as substantially specific for lymphatic cells.R6-128041 6,13 VEGFR-3, also known as Flt4, is a tyrosine kinase receptor for VEGF-C and VEGF-D, which are both lymphangiogenic factors. Prox1 is a homeobox gene that appears to be required for budding and sprouting of venous endothelial cells to form the lymphatic vasculature; mice with targeted deletion of Prox1 are devoid of lymphatic vessels.14 Furthermore, overexpression of Prox1 in lymphatic endothelial cells can induce genes that are commonly seen in lymphatic endothelium and suppress blood vascular endothelial genes.R14-128041 14,15 Transcriptional profiling experiments indicate that blood and lymphatic endothelial cells have many other distinct differences, and these experiments and others will undoubtedly yield more molecular markers for distinguishing these two different types of endothelium.15
In this issue of Circulation Research, Boardman and Swartz extend their previous work on lymphatic flow in the mouse tail16 by developing a new model that provides novel insights in lymphangiogenesis.17 They removed a small band of dermis from the mouse tail, leaving subcutaneous blood vessels intact. Normally, this loss of dermal lymphatics would lead to accumulation of interstitial fluid and subsequent edema in the distal tail. By applying a type I collagen gel on this site, the interstitial fluid in the distal tail can flow through the collagen, allowing visualization of flow and identification of newly formed lymphatic vessels in the same mouse.
Initially, a pool of lymph flowed through the collagen gel, but over 60 days a complete network of lymphatic channels reformed. These new lymphatic vessels stained positively for Flt4 and LYVE-1 (the lymphatic endothelial hyaluronan receptor, a homologue of CD44) indicating that these channels were indeed lymphatics. Furthermore, the new lymphatic channels formed with the same repetitive hexagonal pattern as native lymphatics. Blood vessels that stained positively for CD31 developed more rapidly in the collagen gel, with complete vascularization by 10 days.
A surprising finding in this mouse model is that fluid channels formed before the new lymphatic endothelial cells formed continuous vessels. Furthermore, VEGF-C expression was seen at the distal end of the collagen gel, where flow initiated, but not the proximal end. These data suggest that fluid flow drives not only channel formation but also expression of a primary lymphangiogenic endothelial mitogenic stimulus and vessel formation by lymphatic endothelium. This contrasts with blood vessel angiogenesis, where endothelial cells can form substantial capillary-like structures before blood begins to flow.
Mechanical signals undoubtedly play key roles in both blood vessel angiogenesis and lymphangiogenesis.18 Endothelial cells can form capillary-like channels in the absence of flow, and cells can align without touching one another, suggesting that direct intercellular communication is not necessary to guide the alignment. These findings suggest that mechanical forces in the extracellular matrix are sensed by the endothelial cells as they form channels.19 In the case of lymphatic endothelium, do the precursor cells require flow, and is there some difference in mechanotransduction between lymphatic and blood endothelium? The directionality of VEGF-C expression in the study of Boardman and Swartz suggests that the answer is no, and that flow-guided lymphangiogenesis is driven by flow-dependent expression of growth factors.
The juxtaposition of blood vessel angiogenesis preceding flow and flow preceding lymphangiogenesis reminds us that biomechanical forces and cellular biology are inseparable. Indeed, a new study of the developing zebrafish heart has demonstrated that proper cardiogenesis is dependent on intracardiac fluid forces.20 At least in some cases, like formation of the heart or repair of damaged lymphatics, the interplay of biomechanical forces and cellular responses may be essential for optimizing tissue function. Circulation Research has wisely recognized the importance of mechanotransduction and signaling in myocardium by commissioning a thematic series on this topic edited by Peter F. Davies, in which three relevant papers have appeared.R21-128041 R22-128041 21–23 Ultimately, we will probably learn that these same mechanotransduction mechanisms determine bone strength, integrity of skin, muscle strength, and many other fundamental tissue properties.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
1. Ohhashi T. Comparison of viscoelastic properties of walls and functional characteristics of valves in lymphatic and venous vessels. Lymphology. 1987; 20: 219–223.[Medline] [Order article via Infotrieve]
2. Baldwin ME, Stacker SA, Achen MG. Molecular control of lymphangiogenesis. Bioessays. 2002; 24: 1030–1040.[CrossRef][Medline] [Order article via Infotrieve]
3. Jussila L, Alitalo K. Vascular growth factors and lymphangiogenesis. Physiol Rev. 2002; 82: 673–700.
4. Shirasawa Y, Ikomi F, Ohhashi T. Physiological roles of endogenous nitric oxide in lymphatic pump activity of rat mesentery in vivo. Am J Physiol Gastrointest Liver Physiol. 2000; 278: G551–G556.
5. Sabin FR. On the origin of the lymphatic system from the veins and the development of the lymph hearts and thoracic duct in the pig. Am J Anat. 1902; 1: 367–391.[CrossRef]
6. Wigle JT, Oliver G. Prox1 function is required for the development of the murine lymphatic system. Cell. 1999; 98: 769–778.[CrossRef][Medline] [Order article via Infotrieve]
7. Halsted WS. The swelling of the arm after operation for cancer of the breast, elephantiasis chirurgica, its causes and prevention. Bull J Hopk Hosp. 1921; 32: 309–313.
8. Slavin SA, Van den Abbeele AD, Losken A, Swartz MA, Jain RK. Return of lymphatic function after flap transfer for acute lymphedema. Ann Surg. 1999; 229: 421–427.[CrossRef][Medline] [Order article via Infotrieve]
9. Chang L, Kaipainen A, Folkman J. Lymphangiogenesis new mechanisms. Ann N Y Acad Sci. 2002; 979: 111–119.[Medline] [Order article via Infotrieve]
10. Jeltsch M, Kaipainen A, Joukov V, Meng X, Lakso M, Rauvala H, Swartz M, Fukumura D, Jain RK, Alitalo K. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science. 1997; 276: 1423–1425.
11. Yoon YS, Murayama T, Gravereaux E, Tkebuchava T, Silver M, Curry C, Wecker A, Kirchmair R, Hu CS, Kearney M, Ashare A, Jackson DG, Kubo H, Isner JM, Losordo DW. VEGF-C gene therapy augments postnatal lymphangiogenesis and ameliorates secondary lymphedema. J Clin Invest. 2003; 111: 717–725.[CrossRef][Medline] [Order article via Infotrieve]
12. Szuba A, Skobe M, Karkkainen MJ, Shin WS, Beynet DP, Rockson NB, Dakhil N, Spilman S, Goris ML, Strauss HW, Quertermous T, Alitalo K, Rockson SG. Therapeutic lymphangiogenesis with human recombinant VEGF-C. FASEB J. 2002; 16: 1985–1987.
13. Kaipainen A, Korhonen J, Mustonen T, van Hinsbergh VW, Fang GH, Dumont D, Breitman M, Alitalo K. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci U S A. 1995; 92: 3566–3570.
14. Hong YK, Harvey N, Noh YH, Schacht V, Hirakawa S, Detmar M, Oliver G. Prox1 is a master control gene in the program specifying lymphatic endothelial cell fate. Dev Dyn. 2002; 225: 351–357.[CrossRef][Medline] [Order article via Infotrieve]
15. Petrova TV, Makinen T, Makela TP, Saarela J, Virtanen I, Ferrell RE, Finegold DN, Kerjaschki D, Yla-Herttuala S, Alitalo K. Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J. 2002; 21: 4593–4599.[CrossRef][Medline] [Order article via Infotrieve]
16. Swartz MA, Kaipainen A, Netti PA, Brekken C, Boucher Y, Grodzinsky AJ, Jain RK. Mechanics of interstitial-lymphatic fluid transport: theoretical foundation and experimental validation. J Biomech. 1999; 32: 1297–1307.[CrossRef][Medline] [Order article via Infotrieve]
17. Boardman KC, Swartz MA. Interstitial flow as a guide for lymphangiogenesis. Circ Res. 2003; 92: 801–808.
18. Ingber DE. Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. Circ Res. 2002; 91: 877–887.
19. Vernon RB, Angello JC, Iruela-Arispe ML, Lane TF, Sage EH. Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro. Lab Invest. 1992; 66: 536–547.[Medline] [Order article via Infotrieve]
20. Hove JR, Koster RW, Forouhar AS, Acevedo-Bolton G, Fraser SE, Gharib M. Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis. Nature. 2003; 421: 172–177.[CrossRef][Medline] [Order article via Infotrieve]
21. Shyy J, Chien S. Role of integrins in endothelial mechanosensing of shear stress. Circ Res. 2002; 91: 769–775.
22. Sussman M, McCulloch A, Borg TK. Dance band on the Titanic: biomechanical signaling in cardiac hypertrophy. Circ Res. 2002; 91: 888–898.
23. Davies PF, Zilberberg J, Helmke BP. Spatial microstimuli in endothelial mechanosignaling. Circ Res. 2003; 92: 359–370.
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