Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis

Abstract

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF, VEGF-A) is a multifunctional cytokine with important roles in pathological angiogenesis. Using an adenoviral vector engineered to express murine VEGF-A(164), we previously investigated the steps and mechanisms by which this cytokine induced the formation of new blood vessels in adult immunodeficient mice and demonstrated that the newly formed blood vessels closely resembled those found in VEGF-A-expressing tumors. We now report that, in addition to inducing angiogenesis, VEGF-A(164) also induces a strong lymphangiogenic response. This finding was unanticipated because lymphangiogenesis has been thought to be mediated by other members of the VPF/VEGF family, namely, VEGF-C and VEGF-D. The new "giant" lymphatics generated by VEGF-A(164) were structurally and functionally abnormal: greatly enlarged with incompetent valves, sluggish flow, and delayed lymph clearance. They closely resembled the large lymphatics found in lymphangiomas/lymphatic malformations, perhaps implicating VEGF-A in the pathogenesis of these lesions. Whereas the angiogenic response was maintained only as long as VEGF-A was expressed, giant lymphatics, once formed, became VEGF-A independent and persisted indefinitely, long after VEGF-A expression ceased. These findings raise the possibility that similar, abnormal lymphatics develop in other pathologies in which VEGF-A is overexpressed, e.g., malignant tumors and chronic inflammation.

Figure 1.

Lymphangiogenic response induced by Ad-VEGF-A¹⁶⁴ in nude mouse ears. (a) Normal lymphatic in control ear skin (arrow indicates a valve). (b) Lymphatics at 3 d after Ad-VEGF-A¹⁶⁴ are distended from dermal edema but have already enlarged further as the result of endothelial cell division and are transitioning into giant lymphatics. (c–h) Giant lymphatics occupy large portions of the dermis at 7–131 d after Ad-VEGF-A¹⁶⁴. Panel c illustrates giant lymphatics that contain colloidal carbon injected as described in Materials and Methods and illustrated macroscopically in Fig. 5. Lymphatic (L) in g contains fibrin clot whereas three unlabeled lymphatics (top right) are filled with colloidal carbon. (i and j) Giant lymphatics wrap around glomeruloid bodies (GB) 13 d after Ad-VEGF-A¹⁶⁴. (k) Autoradiograph showing [³H]thymidine incorporation by lymphatic endothelial cells (arrows) at 10 d after Ad-VEGF-A¹⁶⁴. L, lymphatics. Giemsa stained 1 μm Epon sections. Bars: a–h, 100 μm; i and j, 50 μm; k, 25 μm.

Figure 2.

Percent of lymphatic endothelial cells labeled by [³H]thymidine (mean ± std error) at intervals after ear injection with Ad-VEGF-A¹⁶⁴ or Ad-PlGF. Values at successive times (based on 3–6 animals per time point) were compared with those at time zero using Dunn's multiple comparisons test. *P < 0.05; **P < 0.01.

Figure 3.

Immunostaining of lymphatics for VEGFR-2 (a–e) and for the hyaluronan receptor LYVE-1 (f and g). Immunoperoxidase staining of control lymphatics (a and b) or giant lymphatics (c–e) at indicated times after Ad-VEGF-A¹⁶⁴. Note bridging of lymphatics by VEGFR-2–positive endothelium (d, arrows). L, lymphatics; v, micro blood vessels whose endothelial cells also stain. (f and g) Immunofluorescence staining of lymphatics with anti-LYVE antibody (green) and micro-blood vessels with CD31 (red), 6 and 14 d after Ad-VEGF-A¹⁶⁴. Bars: a, b, and e, 10 μm; c and d, 20 μm; f and g, 200 μm.

Figure 4.

Confocal microscopy of lymphatic and microvascular plexuses in ears of a control mouse (a–c) and a mouse injected 4 d previously with Ad-VEGF-A¹⁶⁴ (d–g). FITC-dextran (green) was injected intravenously into the tail vein and TMR-dextran (red) was microinjected into the peripheral ear lymphatics. (a) Normal ear lymphatic plexus delineated by TMR-dextran. (b) Normal ear blood microvasculature delineated by FITC-dextran. (c) Composite retains distinct green and red compartments for the most part, indicating little or no lymphatic uptake of FITC-dextran. (d and e) TMR-dextran, reproduced in both black and white and in red, within a giant lymphatic. Centrally the channel is partially obstructed (presumably by fibrin clot, see text and Figs. 6 and 7), such that the upper and lower portions are connected only through narrow channels that allow tracer flow (best illustrated in d). (f) FITC-dextran fills leaky micro-blood vessels and has entered the lymphatic illustrated in d and e. (g) Merged image. Yellow color indicates FITC-dextran extravasated from leaky blood vessels has been taken up by the giant lymphatic infused with TMR-dextran. Bars, 50 μM.

Figure 5.

Ear lymphatics after intravital infusion of colloidal carbon in a control mouse and in mice injected at indicated intervals with Ad-PlGF or Ad-VEGF-A¹⁶⁴. (a) Control ear. Multiple injection sites (black blotches at top) were required to fill the lymphatic network. Note periodic bulbous swellings that identify valves. (b–d) Lymphatic filling in ears of mice injected at indicated times with Ad-PlGF. Injecting micropipette is shown in place in b. Lymphatics retain bulbous valve markings. (e–h) Pattern of lymphatic filling in ears of mice injected previously, as indicated, with Ad-VEGF-A¹⁶⁴. Giant lymphatics are apparent as early as 3 d (e) and persist through day 270. Bulbous valve markings are lost. (i–l) Kinetics of lymphatic filling in ear of a mouse 84 d after injection with Ad-VEGF-A¹⁶⁴. Note widespread filling of lymphatic network by 4 min from a single injection site (that with the micropipette in place). An earlier injection site (to left of pipette) failed to engage the terminal lymphatics. (m–p) Clearance of carbon from control ear lymphatics (m and n) is complete by 20 min but at 35 d after Ad-VEGF-A¹⁶⁴ ear lymphatics still retain abundant tracer after 150 min (o and p).

Figure 6.

Intraluminal fibrin clot formation and transluminal bridging of giant lymphatics at indicated times after Ad-VEGF-A¹⁶⁴ injection. None of the lymphatics illustrated had been injected with carbon or other agents. (a and b) Fibrin clot (*) within giant lymphatics at 10–11 d; note transluminal bridging by lymphatic endothelium. (c–i) Migrating lymphatic endothelial cells formed transluminal bridges across giant lymphatics, dividing their lumens into multiple smaller, endothelium-lined channels. Initially fibrin formed a substrate for endothelial cell migration but was subsequently digested and replaced by collagen. By 22 d (i) bridges had become quite cellular. Note red blood cells within lymphatics (d–f, h). L, giant lymphatics. Giemsa stained 1 μm Epon sections. Bars: a, b, and d–i, 50 μm; c, 25 μm.

Figure 7.

Electron micrographs of giant lymphatics at 21 d (a) and 14 d (b) after Ad-VEGF-A¹⁶⁴ injection, illustrating intraluminal fibrin deposits and transluminal bridging by lymphatic endothelial cells. Black arrows indicate intralymphatic fibrin and open arrows the endothelial cell bridging that followed. (c) Intraluminal fibrin is more clearly demonstrated at higher magnification. L, lymphatic lumens. Bars: a and b, 5 μm; c, 1 μm.