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Am J Physiol Heart Circ Physiol 294: H1326-H1334, 2008. First published January 18, 2008; doi:10.1152/ajpheart.00900.2007
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The resolution of lymphedema by interstitial flow in the mouse tail skin

Joseph Uzarski,1,* Megan B. Drelles,1,* Sara E. Gibbs,1,* Emily L. Ongstad,1 Julie C. Goral,1 Katherine K. McKeown,1 Alisha M. Raehl,1 Melissa A. Roberts,1 Bronislaw Pytowski,2 Martyn R. Smith,1 and Jeremy Goldman1

1Biomedical Engineering Department, Michigan Technological University, Houghton, Michigan; and 2ImClone Systems, New York, New York

Submitted 2 August 2007 ; accepted in final form 14 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lymphangiogenesis is considered a promising approach for increasing fluid drainage during secondary lymphedema. However, organization of lymphatics into functional capillaries may be dependent upon interstitial flow (IF). The present study was undertaken to determine the importance of lymphangiogenesis for lymphedema resolution. We created a lymphatic obstruction that produces lymphedema in mouse tail skin. The relatively scar-free skin regeneration that occurred across the obstruction allowed the progression of lymphangiogenesis to be observed and compared with the evolution of lymphedema. The role of vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3 signaling in lymphedema resolution was investigated by exogenous administration of VEGF-C or neutralizing antibodies against VEGFR-3. VEGF-C protein improved lymphedema at 15 days [reducing dermal thickness from 742 ± 105 to 559 ± 141 µm with 95% confidence intervals (CIs), P < 0.05] without increasing lymphatic capillary coverage (11.6 ± 6.4% following VEGF-C treatment relative to 9.6 ± 6.2% with 95% CIs, P > 0.50). Blocking VEGFR-3 signaling did not inhibit lymphedema resolution at 25 days (dermal thickness of 462 ± 127 µm following VEGFR-3 inhibition relative to 502 ± 87 µm with 95% CIs) or inhibit IF, although VEGFR-3 blocking prevented lymphangiogenesis (reducing lymphatic coverage to 0.2 ± 0.7% relative to 8.7 ± 7.3% with 95% CIs, P < 0.005). A second mouse tail lymphedema model was employed to investigate the ability of VEGF-C to increase fluid drainage across a scar. We found that neither neutralization of VEGFR-3 nor administration of VEGF-C affected the course of skin swelling over 25 days. These findings suggest that resolution of lymphedema in the mouse tail skin may be more dependent upon IF and regeneration of the extracellular matrix across the obstruction than lymphatic capillary regeneration.

lymphatic capillary; lymphangiogenesis; vascular endothelial growth factor-C; vascular endothelial growth factor receptor 3; skin; extracellular matrix

SECONDARY LYMPHEDEMA is a chronic condition of excessive interstitial fluid accumulation that often occurs following surgical disruption of the lymphatic network and radiotherapy during axillary lymph node dissection (13, 16, 20, 30, 34, 41, 42). The compromised lymphatic flow that accompanies lymphatic injury can result in disfiguring thickening of the skin and chronic infection (21, 25, 31, 36, 53). Unfortunately, there is no cure at present for secondary lymphedema. Because compromised lymphatic function leads to secondary lymphedema, it has been widely hypothesized that lymphedema may be resolved by improving lymphatic function (2, 37, 44, 45). Consequently, lymphangiogenesis (the growth of new lymphatic capillaries) has been considered a promising solution for lymphedema, and several studies have investigated the ability of lymphatic growth factors to promote lymphangiogenesis and increase fluid drainage.

One of the most studied of the lymphatic growth factors is vascular endothelial growth factor-C (VEGF-C), a prolymphangiogenic protein that binds to VEGF receptor (VEGFR)-2 and VEGFR-3 (1, 14). Several studies have reported that VEGF-C administration, whether by gene transfer or recombinant protein, dramatically improves edema resolution by augmenting the growth of functional lymphatic capillaries (12, 40, 51, 55). However, it has also been reported that VEGF-C overexpression may merely induce lymphatic hyperplasia [i.e., the proliferation of lymphatic endothelial cells (LECs) within existing lymphatic capillaries] without increasing lymphatic capillary density (i.e., the organization of LECs into new lymphatic capillaries) (17, 23), and that hyperplastic lymphatic capillaries may be less functional than lymphatic capillaries of normal caliber (22, 36). It has recently been shown that LECs can organize into functional lymphatic capillaries in the adult in the absence of signaling through VEGFR-2 or VEGFR-3 (19, 27). It has also been demonstrated in a model of adult lymphangiogenesis in regenerating mouse tail skin that LECs migrate and later organize in the direction of preexisting interstitial flow (IF; see Refs. 6 and 35). When IF was decreased (to simulate the biophysical environment present during lymphedema), the ability of the regenerated LECs to organize into functional capillaries was substantially reduced (18, 35), and these poorly organized LECs failed to increase their organization despite exogenous administration of VEGF-C protein (18).

Because functional lymphatic growth in the adult may largely be dependent upon preexisting IF, the extent to which exogenously induced lymphatic growth can increase IF and improve lymphatic drainage is unclear. Thus it remains uncertain whether increased VEGF-C/VEGFR signaling can promote functional lymphatic growth and ameliorate diseases caused by poor IF. Because we have found that both physiological and therapeutic lymphangiogenesis may be dependent upon preexisting IF, we hypothesized that resolution of lymphedema in an experimental model may be more dependent upon IF than upon lymphangiogenesis across the lymphatic obstruction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
For all studies, 6- to 8-wk-old female BALB/c mice (Harlan) were used. At least four mice were used for each condition at each time point examined. Mice were anesthetized with a subcutaneous injection of ketamine (65 mg/kg), xylazine (13 mg/kg), and morphine (2 mg/kg). All protocols were approved by the Animal Care and Use Committee of Michigan Technological University.

Lymphangiogenesis model 1. To test our hypothesis, we combined a recent model of lymphangiogenesis (6) with an established model of secondary lymphedema (49) to produce a clearly defined region of regenerating mouse tail skin downstream from a region of lymph edematous skin. Because lymphatics are not present in the regenerating region initially, this model allows us to observe the process of lymphatic migration and functional capillary organization, as they occur over time, and for the progression of lymphangiogenesis within the regenerating region to be compared with the evolution of lymphedema in the distal skin. The biomolecular environment can also be altered by systemic administration of neutralizing antibodies for VEGFR-3 or by local administration of recombinant VEGF-C protein. Because lymph in the tail moves in only one direction (i.e., from distal to proximal), by comparing the evolution of lymphangiogenesis in the regenerating region with the degree of swelling in the distal skin, we are uniquely able to determine whether lymphangiogenesis causes lymphedema to resolve or whether resolution precedes the formation of functional lymphatic capillaries and may be independent of lymphatic growth.

The regenerating region of skin was created by excising a 4-mm circumferential band of dermal tissue (which contained the lymphatic capillary network) midway up the tail, leaving the underlying bone, muscle, tendons, and major blood vessels intact. The tail skin began to swell ~1 wk postsurgery. The regenerating region was initially covered with a close-fitting, gas-permeable silicone sleeve and was refitted with a series of larger sleeves as the tail experienced edema and increased in diameter. Because the regenerating region is initially cell free, any LECs or capillaries later observed within the regenerating region are the result of newly initiated cellular migration, proliferation, and organization. Preparation of the 4-mm-long lymphatic defect resulted in lymphedema in the distal tail skin.

Lymphangiogenesis model 2. Because the wound produced in model 1 results in nearly scar-free skin regeneration, a recently characterized model of lymphedema (36) was employed to produce a wound with a more natural healing response. This model was slightly modified to excise a 2-mm-long circumferential band of dermal tissue ~10 mm from the tail base, leaving the underlying bone, muscle, tendons, and major blood vessels intact, and the wound site was left unprotected. The excision was followed by a brief elecrocautery of the exposed dermal wound edges. Preparation of the 2-mm wound resulted in lymphedema of the distal tail skin within 24 h.

VEGF-C therapy. To determine the ability of VEGF-C to augment lymphangiogenesis during lymphedema, mouse tail skin regenerating regions were prepared as described above to induce lymphedema. Mice received injections every 2 days of carrier free recombinant human VEGF-C (R&D Systems) at the site of regenerated skin at 4 µg·dose–1·mouse–1 (12 µl total volume/dose) for a time period that varied for each group. In model 1, one group of mice received injections from day 0 to 10, and another group received injections from day 10 to day 20 postsurgery. In model 2, two groups of mice received injections from day 5 to day 20 postsurgery. Control mice received saline injections (12 µl total volume/dose) over the same period of time.

Neutralizing antibodies. Antagonist antibodies were provided by ImClone Systems (New York, NY). Anti-mouse VEGFR-3 (mF4–31C1) (32) was used to neutralize mouse VEGFR-3 in vivo. In mice receiving the antibodies in model 1, 0.625 mg of the antibody was administered to each mouse intraperitoneally immediately following surgery. In model 2, mice received the antibody immediately following surgery and then two times weekly thereafter.

Detection of functional lymphatics via microlymphangiography. To visualize lymph flow patterns in situ, a 2 mg/ml solution of tetramethylrhodamine-conjugated lysine fixable dextran of 2,000 kDa (Invitrogen, Carlsbad, CA) was injected intradermally in the tip of the tail at a constant pressure where it was taken up and transported through the lymphatics in the proximal direction (47), revealing functional lymphatic vessels and the path of lymph through the regenerating region. The fluid tracer is lysine fixable, allowing the tracer to be immobilized within the extracellular matrix or lymphatic capillaries of the tissue by standard 4% formaldehyde fixation treatment for analysis in tissue sections. Fluorescence images of the microlymphangiographies were captured with a DP71 color camera on a BX51 Olympus fluorescence microscope.

Immunofluorescence and immunohistochemistry. Tail specimens were cut into 10- and 60-µm longitudinal cryosections and immunostained. To detect LECs, a rabbit polyclonal antibody against the lymphatic-specific hyaluronan receptor LYVE-1 (Upstate, Charlottesville, VA) was used along with an Alexa Fluor 488 goat anti-rabbit secondary antibody (Invitrogen). The path taken by lymph through the tail skin during the microlymphangiography was identified by the immobilized tetramethylrhodamine-labeled dextran fluid tracer. Cell nuclei were labeled with 4',6-diamino-2-phenylindole (Vector Laboratories, Burlingame, CA). Fluorescence images in the 60-µm cryosections were captured with a Zeiss MRm camera on a Zeiss Axiovert 200M fluorescence microscope with the Apotome system. This system collects a stack of two- dimensional images that are then compressed into a single image.

Biotinylated antibodies (R&D Systems) were used to detect VEGF-C in 10-µm longitudinal cryosections. Antibody labeling was visualized with an ABC-AP kit and Vector Red substrate (Vector Laboratories). Cell nuclei were counterstained with hematoxylin. Color images were captured with a BX51 Olympus microscope under bright field.

Measurement of lymphatic growth, dermal thickness, and tail diameter. Lymphatic growth was measured with Metamorph software from fluorescence images. The regenerating region was outlined from the upper lymphatic capillaries that were present beneath the epidermis to the lymphatic capillaries present in the lower dermis. A threshold analysis was used on the outlined region to measure the percent coverage of LYVE-1-positive pixels in the regenerating region. Single, unorganized, LYVE-1-positive cells were excluded from the measurement. Because subpopulations of macrophages are known to express LYVE-1 receptor (28, 29) and may contribute to the lymphatic coverage measurements, lymphatic and macrophage double staining (using the F4/80 macrophage marker) was undertaken in regenerating skin to determine the contribution of macrophages to the LYVE-1 signal. The overlap between F4/80 and LYVE-1 signal appeared to be small at the latter stages of wound healing that we evaluated (data not shown), consistent with a recent report that showed the F4/80 signal gradually disappears from the LYVE-1-positive cells over the course of wound healing (28). Dermal thickness of the regenerating region was measured in perpendicular cross sections of skin with Metamorph image analysis software. The regenerating region was outlined from underneath the epidermis to the tendon, and the total surface area of the outlined region was divided by the length of the outlined segment to obtain an average dermal thickness. Tail diameters distal to the wound site were measured in Photoshop from digital images captured with a DP71 color camera mounted to a stereo microscope. Two images were captured from each mouse tail, at a 90 degree rotation from each other, and the two diameters were averaged together.

Statistical methods. At least two sections were measured per specimen. At least four animals were used for each data point. Data are presented as means with 95% confidence intervals (CIs). P values were calculated using a two-tailed or paired Student's t-test or ANOVA, as indicated.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Adult lymphatic regeneration during lymphedema. Previously, we have described a model of adult lymphangiogenesis where a circumferential portion of the mouse tail skin is removed and the wound site is protected with a silicone cuff allowing skin to regenerate in a relatively scar-free manner (6, 17–19, 32, 35, 48). At early times in the regenerating tail skin, before lymphatics have regenerated, interstitial fluid initially collects in the regenerating region from upstream lymphatics and moves through the region interstitially in bulk flow patterns. Over time, upstream lymph that collects in the region moves interstitially through discrete fluid channels that become populated with LECs. Ultimately, lymph moves through the regenerating region almost entirely within regenerated lymphatic capillaries.

To clarify the dependence of lymphedema resolution upon lymphangiogenesis, and VEGF-C/VEGFR signaling, a recent model of adult lymphangiogenesis (6) was modified to monitor lymphatic capillary regeneration across an injury that produced secondary lymphedema. This model was found to reproducibly induce lymphedema of mouse tail skin distal to the region of lymphatic capillary regeneration. Because lymphatic capillaries are not present in the region initially, and IF is always directed from the distal toward proximal regions of the tail (formed from lymph collected by "upstream" lymphatics), this model allows us to compare the spatial distribution of IF through the regenerating region (either within the interstitium or within regenerating lymphatic capillaries) with the evolution of tissue swelling in the distal skin. At 15 days postsurgery, lymphedema (determined by dermal thickness measurements) was evident relative to the normal mouse tail skin dermis (P < 0.05). By 25 days, lymphedema had significantly resolved from day 15 (P < 0.05) (Fig. 1D).


Figure 1
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  		Fig. 1. Untreated lymphatic capillary regeneration during lymphedema. Lymph fluid flows in the proximal direction, right to left. Shown are the regenerating regions of 60-µm mouse tail skin cryosections labeled for LYVE-1 (green), lymph fluid tracer from the microlymphangiography (red), and cell nuclei (blue). In normal skin, lymph fluid tracer is present inside LYVE-1-positive lymphatic vessels (indicated by the yellow arrowheads). There is minimal fluid tracer present in the interstitial space, signifying functional lymphatic capillaries (A). Lymphatic endothelial cells (LECs) are present throughout the regenerating region at 15 days, as shown by LYVE-1 staining. However, fluid tracer is present predominantly in the interstitial space (white arrows) (B). At 25 days, lymph fluid tracer remains present in the interstitium (C). Bar in C = 200 µm. D: dermal thickness was measured from cryosections of mouse tail skin at 15 and 25 days and in normal controls. P < 0.05, significant difference relative to normal skin (*) and significant difference relative to day 15 (#). Graphs depict the mean and 95% confidence intervals from 4 to 5 mice/experiment.

 
Observations of immunostained thick sections of normal skin demonstrated that lymphatic fluid tracer predominantly overlapped with the LEC LYVE-1 receptor marker (Fig. 1A), with low levels of fluid tracer present in the interstitial space, indicative of a functional lymphatic capillary network. In contrast to the lymphatic functional continuity found in the normal skin, most fluid tracer at 15 days in the lymph edematous regenerating region was present in the interstitial space with low levels of fluid tracer located inside the LYVE-1-positive structures. This suggests that the LECs that had migrated into the initially acellular regenerating region had not yet organized into functional lymphatic capillaries. This was the case, even though there was evidence of flow spreading through the interstitium (Fig. 1B). Remarkably, although lymphedema was improved by 25 days postsurgery, and there was some overlap of the LYVE-1 labeling with fluid marker labeling in some areas, most of the lymphatic fluid tracer was still predominantly moving interstitially through the regenerating region, suggesting the continued absence of a functional lymphatic capillary network at 25 days (Fig. 1C).

Endogenous VEGF-C expression in the regenerating region during lymphedema. It has been recently reported that the presence of endogenous VEGF-C in edematous mouse tail skin correlated with tail volume during lymphedema but was preceded by lymphatic hyperplasia (36). To observe endogenous VEGF-C and to correlate endogenous VEGF-C in the regenerating skin with both the progression of lymphangiogenesis and the evolution of lymphedema, we labeled sections of the regenerating region for VEGF-C protein. Although this method is not quantitative, qualitative information can be derived from an inspection of these tissue sections. VEGF-C protein appeared weakly present in sections of normal skin (Fig. 2A). Moderate blushing of VEGF-C was apparent in the regenerating region at 15 days (Fig. 2B), coincident with lymphedema and substantial lymphatic migration and proliferation. VEGF-C appeared more prominently present at day 25 (Fig. 2C), subsequent to the period of lymphatic migration and proliferation. Significant resolution of lymphedema at day 25 correlated temporally with the presence of endogenous VEGF-C protein. Thus these results were similar to previous findings (36).


Figure 2
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  		Fig. 2. Changes in vascular endothelial growth factor-C (VEGF-C) expression over time in untreated regenerating lymphedematous skin. The regenerating regions of 10-µm cryosections were immunostained against VEGF-C (red in each image) in normal tissue and at day 15 and 25 postsurgery. The epidermis is located at the top of each image. VEGF-C is present at very low levels in the control skin (A). VEGF-C is seen at low to moderate levels in the dermis of the regenerating region at 15 days (B) and at high levels at 25 days (C). Bar = 200 µm.

 
Effects of early VEGF-C administration on lymphangiogenesis and lymphedema. It has been recently reported that increased VEGFR-3 signaling with exogenous VEGF-C can induce lymphatic growth and improve lymphedema (15, 23, 37, 40, 51, 55). In contrast, we have found that IF precedes lymphatic regrowth (6), lymphangiogenesis may be dependent upon preexisting IF (18), and continuous expression of excess VEGF-C mainly induces lymphatic hyperplasia of a regenerating lymphatic capillary network (17) (although transient VEGF-C injections cannot sustain lymphatic hyperplasia) without increasing lymphatic organization or function. We have more recently found that LECs can organize in the absence of VEGFR-2 or VEGFR-3 signaling (19). Based on the results from these studies, we hypothesized that increased VEGF-C/VEGFR signaling may not improve lymphedema.

To determine whether VEGF-C protein could improve lymphedema, VEGF-C signaling was increased during experimental lymphedema by exogenous administration of recombinant VEGF-C protein. When assessed at day 15, it was found that 4 µg of VEGF-C injected every other day (12 µl/dose) from day 0 to day 10 postsurgery significantly reduced the tissue swelling relative to saline-treated controls (P < 0.05, 559 with 95% CIs of ±141 µm vs. 742 with 95% CIs of ±105 µm for VEGF-C treated and controls, respectively; Fig. 3E). However, when visualized and measured in thick immunostained sections, it was apparent that VEGF-C had failed to induce lymphatic growth in the regenerating region [P > 0.05, 11.6 with 95% CIs of ±6.4% relative to 9.6 with 95% CIs of ±6.2% for VEGF-C treated and controls, respectively; Fig. 3, A, B, and F; However, continuous VEGF-C expression in the mouse tail has been shown to induce lymphatic hyperplasia without changing lymphatic capillary density (17, 23).]. The inability of excess VEGF-C to increase lymphatic growth relative to the controls may have been due to the abundant physiological lymphatic growth that was occurring in the controls. Fluid tracer was present predominantly in the interstitial space irrespective of saline or VEGF-C treatments (Fig. 3, A and B), although numerous lymphatic capillaries appeared to overlap with fluid tracer, indicative of a lymphatic capillary network that was not yet fully functional. Thus, although VEGF-C injections improved lymphedema, they failed to increase lymphatic growth, suggesting that VEGF-C may improve experimental lymphedema without increasing lymphangiogenesis in the mouse tail skin.


Figure 3
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  		Fig. 3. Lymphatic capillary regeneration and lymphedema following VEGF-C administration. Cryosections (60 µm) were immunostained against the LEC-specific receptor LYVE-1 (green) following microlymphangiography to visualize the spatial distribution of lymph within the skin (red). Cell nuclei were stained with 4',6-diamino-2-phenylindole (DAPI) (blue). Shown are fluorescence images of the regenerating regions, with the proximal direction from right to left. Saline (A) or VEGF-C (B) was administered for the first 10 days postsurgery to tail skin with lymphedema, and tissue was collected at day 15. Several interstitial fluid positive locations are identified with white arrows. Bar in B = 200 µm. C and D represent enlargements from A and B, respectively, to improve visualization of the fluid marker within and near lymphatics. Bar in D = 100 µm. Dermal thickness of the regenerating skin was measured in cryosections of mouse tail skin for each condition (E). Lymphatic growth in the regenerating skin was measured in the cryosections by using a threshold analysis that determined the percentage of LYVE-1-positive pixels in the regenerating region (F). *P < 0.05, significant differences between groups. Graphs depict the mean and 95% confidence intervals from 4 to 5 mice/ experiment.

 
Effects of late VEGF-C administration and VEGFR-3 neutralization on lymphangiogenesis and lymphedema. Because exogenous VEGF-C administration significantly improved lymphedema at day 15 when endogenous VEGF-C appeared to be at moderate levels, we asked whether VEGF-C therapy would continue to be beneficial when administered in the presence of high levels of endogenous VEGF-C protein. Therefore, to determine if VEGF-C protein injections could result in sustained therapeutic benefits, lymphedema was induced in the mouse tail skin and VEGF-C protein was administered from day 10 to day 20. When assessed at day 25, VEGF-C injections did not result in improved lymphatic growth or lymphedema relative to saline-treated controls (Fig. 4, A and B). Although there was some overlap of the LYVE-1 labeling and fluid marker in some areas, most of the fluid marker was seen moving interstitially through the regenerating region, similar to what was found in the normally regenerating controls at this time (Fig. 1C). The thickness of the dermis was similar in both groups (P > 0.50, 473 with 95% CIs of ±66 µm vs. 481 with 95% CIs of ±112 µm for VEGF-C treated and controls, respectively) (Fig. 4G), and the lymphatic coverage was also similar (P > 0.05, 9.0 with 95% CIs of ±8.7% relative to 7.8 with 95% CIs of ±6.5% for VEGF-C treated and controls, respectively), suggesting that exogenous VEGF-C administration may be useful in tissues with low endogenous VEGF-C expression but less effective in tissues with high expression levels of endogenous VEGF-C.


Figure 4
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  		Fig. 4. Lymphedema resolution is not dependent upon lymphangiogenesis of the lymphatic capillaries across the wound. Cryosections (60 µm) were immunostained against the LEC-specific receptor LYVE-1 (green) following microlymphangiography to visualize the spatial distribution of lymph within the skin (red). Cell nuclei were stained with DAPI (blue). Shown are fluorescence images of the regenerating regions, with the proximal direction from right to left. Saline (A) or VEGF-C (B) was administered from day 10 to day 20 postsurgery to tail skin with lymphedema, and tissue was collected at day 25. Several interstitial fluid positive locations are identified with white arrows. Despite the complete lack of any LECs in the regenerating region following VEGF receptor (VEGFR)-3 neutralization, lymph tracer was seen moving interstitially through the region (C). Bar in C = 200 µm. Portions of each image were enlarged for improved visualization of the distribution of fluid marker near or in the lymphatic structures. D, E, and F represent enlargements from A, B, and C, respectively. Bar in F = 50 µm. Dermal thickness of the regenerating skin was measured at day 25 in cryosections of mouse tail skin for each condition (G). Lymphatic growth in the regenerating region was measured in cryosections for each condition by using a threshold analysis that determined the percentage of LYVE-1-positive pixels in the regenerating region (H). *P < 0.05, significant differences between groups. Graphs depict the mean and 95% confidence intervals from 4 to 5 mice/experiment.

 
Although early administration of VEGF-C improved lymphedema, neutralization of VEGFR-3 did not impede lymphedema resolution (P > 0.50, 462 with 95% CIs of ±127 µm vs. 502 with 95% CIs of ±87 µm for VEGFR-3 neutralized and control treated conditions, respectively) (Fig. 4, C and G) or prevent IF (Fig. 4, C and F). In accordance with previous findings, VEGFR-3 neutralization completely prevented lymphangiogenesis initiation (19, 32) as can be seen by the near absence of LECs in the regenerating regions of these mice at 25 days (Fig. 4, C and F) and based upon measurements of the lymphatic growth (P < 0.005, 0.2 with 95% CIs of ±0.7% relative to 8.7 with 95% CIs of ±7.3% for VEGFR-3 neutralized and control treated conditions, respectively). Dermal thickness for all measured groups was not significantly different from each other at day 25 (ANOVA, P > 0.50) (Fig. 4G), suggesting that lymphedema had achieved a similar degree of resolution under all conditions by this time.

Effects of VEGF-C therapy and VEGFR-3 neutralization on lymphedema resolution across a scar. Breast cancer-related lymphedema is a consequence of lymph node dissection in the axilla (34, 50), a surgical process that produces a fibrotic scar across the axilla. Because we found that resolution of lymphedema in the mouse tail skin occurred in the complete absence of lymphangiogenesis as fluid was seen to spread interstitially through the scar-free regenerating skin, we asked whether resolution may be dependent upon the composition of the extracellular matrix that forms across the wound. To this end, we produced a lymphatic obstruction that formed into a scar to determine the ability of lymphedema to resolve across a more physiological obstruction.

Lymphedema was induced in mouse tails as per model 2 (described in MATERIALS AND METHODS) (Fig. 5). Edematous tails that received VEGF-C protein administered locally every other day from day 5 to day 20 were compared with control tails that received saline injections. Because we had found that VEGF-C improved edema without increasing lymphatic growth (Fig. 3) and that VEGFR-3 signaling was not critical for edema resolution (Fig. 4), we created a third group of mice that received both VEGF-C protein and VEGFR-3 blocking antibodies to determine whether the therapeutic effects of increased VEGF-C signaling may occur through VEGFR-2. Measurements of the mouse tail diameter were undertaken to compare mouse tail diameters under the different conditions. An average of 1–2 tails/group (out of the 10 initial subjects/group) was lost between the 2nd and 3rd wk, possibly because of small variations in surgically induced trauma. The loss did not appear to be treatment related and did not affect any of the data analyses. When assessed over the 25-day period, VEGF-C injections did not improve lymphedema relative to controls, and VEGFR-3 blocking did not exacerbate the edematous condition relative to controls. ANOVA conducted on all three groups at each time point showed no significant differences between groups (P > 0.05). Surprisingly, although lymphedema was present in all conditions (P < 0.001, paired t-test, between day 0 and day 5), the lymph edematous tails failed to resolve across the obstructive scar (P > 0.05, t-test, between days 25 and 10). Thus the evolution of the tail diameter was similar in all groups, suggesting that increased VEGF-C/VEGFR signaling may not effectively increase fluid drainage across a scar.


Figure 5
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  		Fig. 5. Resolution of lymphedema and effectiveness of VEGF-C therapy may be dependent upon the composition of the extracellular matrix at the lymphatic obstruction. Lymphedema was produced in mouse tails, as per model 2 in MATERIALS AND METHODS. Edematous mouse tails were subjected to the indicated treatment/blocking conditions with local VEGF-C injections given every other day from day 5 to day 20 and systemic VEGFR-3 blocking injections provided twice weekly to ensure complete neutralization of VEGFR-3 signaling during the observation period (A). Shown are typical images of edematous mouse tails under the three different treatments, with the distal to proximal direction of each tail from top to bottom of the image. Black bar in bottom right panel = 5 mm. The external diameter of each tail was measured distal to the injury from digital images captured every 5 days over a period of 25 days (B). Graphs depict the mean and 95% confidence intervals from 10 mice/condition.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Lymphedema in the human arm is initiated by excision of axillary lymph nodes in women with breast tumors in an effort to prevent metastasis. It has been hypothesized that lymphedema arises due to the absence of adequate lymphatic regrowth at the wound site and that lymphatic growth factors (such as VEGF-C) resolve experimental lymphedema by increasing lymphatic growth at the site of the injury. Using two models of experimental lymphedema in the mouse tail (one with a protected wound site that results in nearly scar-free skin regeneration and the other with an open wound site that produces a more realistic wound healing response), we found that blockage of VEGFR-3 (which completely inhibits lymphatic capillary growth) does not worsen lymphedema over time in either model. Our finding that IF and edema resolution readily occur across the scar-free regenerating skin in the absence of VEGFR-3 signaling and lymphangiogenesis demonstrates that VEGFR-3 signaling and lymphangiogenesis are not required for IF or for the resolution of mouse tail skin lymphedema. This suggests that the resolution may be more dependent upon IF dynamics or fluid filtration dynamics than functional lymphatic growth. Our finding that scarring across the wound, but not neutralization of VEGFR-3, inhibited edema resolution (even in the presence of excess VEGF-C) suggests that IF dynamics across the wound may be dependent upon the composition of the extracellular matrix that reforms at the site of the injury and that IF across the site of injury may be impeded by the formation of scar tissue.

In the present study, VEGF-C protein injections were found to hasten the natural resolution of lymphedema without increasing lymphatic capillary growth in the regenerating region, and VEGFR-3 signaling was not required for resolution of lymphedema. This suggests that the increased resolution produced by excess VEGF-C may be independent of lymphatic capillary growth at the wound site and raises questions about the mechanism of resolution. Because we earlier showed that VEGFR-3 blocking completely prevents the initiation of lymphangiogenesis (32) but that VEGFR-3 was redundant with VEGFR-2 for lymphatic organization (19), it is possible that neutralizing VEGFR-3 may block lymphatic growth but may not prevent lymphatic organization. A reorganization of the existing lymphatics may decrease lymphatic resistance and increase lymph flow rates in the absence of lymphatic capillary growth. VEGF-C has been reported to increase the pumping activity of lymphatic collecting vessels (7). Therefore, excess VEGF-C may increase the flow of lymph through the lymphatic collecting vessels, which may compensate for diminished lymphatic capillary function. Because it is not possible in our experimental model to quantify the relative flow rates through the lymphatics and the interstitium, we were unable to measure the percentages of lymph vs. IFs to determine whether the excess VEGF-C had increased lymph flow rates or IF rates during the resolution. Furthermore, although we found that most of the fluid tracer was present in the interstitium and not inside lymphatic capillaries in the scar-free regenerating region, it is not possible to determine from the tissue sections whether the regenerating lymphatic capillaries had failed to drain the fluid from the interstitium or the lymph had leaked into the interstitium from poorly functioning regenerating lymphatic capillaries. For these reasons, our data do not rule out the possibility that the excess VEGF-C treatments increased the flow rate through the lymphatic capillaries.

However, we found that scar tissue at the wound site strongly inhibited the natural resolution of lymphedema, whereas resolution was able to occur across the scar-free regenerating zone, even in the absence of lymphatic regrowth. This suggests that the resolution we saw may be more dependent upon IF through the extracellular matrix that reforms at the wound site than upon transport through lymphatic capillaries that regenerate across the wound. It is well known that VEGF-C activates LECs by binding to VEGFR-2 and VEGFR-3 (24) and thereby induces lymphangiogenesis. However, VEGF-C also activates macrophages, which can express VEGFR-2 and VEGFR-3 (43, 54), and increased recruitment of macrophages was found in VEGF-C-overexpressing tumors (43). This suggests that macrophages may become activated by exogenous VEGF-C protein and by the high endogenous VEGF-C expression that we found to be present during experimental lymphedema. Although the function of VEGF-C-activated macrophages during lymphedema is unknown, it is possible that these macrophages may act to reduce interstitial resistance to fluid flow by increasing extracellular matrix proteolysis at the wound site. Our finding that scar tissue at the wound site strongly inhibited lymphedema resolution, whereas resolution occurred across the scar-free regenerating zone in the absence of lymphatic capillary regrowth supports the notion that an increased matrix proteolysis/remodeling may reduce interstitial resistance across the obstruction and increase IF. The elevated interstitial fluid pressure that is known to be present in swollen tissues (5, 26) may work in tandem with a reduced interstitial resistance across the wound to increase IF through the reformed extracellular matrix, even in the absence of functional lymphatic capillaries. Although we did not directly demonstrate the role of VEGF-C-activated macrophages in matrix remodeling at the wound site, data from a recent study suggest that downregulation of VEGFR-3 on macrophages may impair wound healing (28).

Because edema depends on a balance of fluid formation and fluid drainage, it is important to consider how VEGF-C may act to reduce fluid formation (i.e., capillary filtration) in addition to considering how VEGF-C may act to increase fluid drainage without increasing lymphatic capillary growth. In addition to decreasing resistance to fluid flow, the increased proteolysis by macrophages may also decrease the interstitial oncotic pressure (9). An increased interstitial fluid pressure and a reduced interstitial oncotic pressure would increase the net pressure opposing capillary blood pressure and may reduce the capillary filtration rate. Supporting this mechanism, a reduced interstitial protein concentration (3, 4) and capillary filtration rate (46) have been found in the edematous human arm, and increased proteolysis of interstitial proteins with Benzopyrones has been shown to slightly improve lymphedema in human clinical trials (8, 10, 11). Thus, in addition to promoting lymphangiogenesis, we speculate that excess VEGF-C may act to reduce interstitial resistance to flow through the regenerating region and/or to oppose capillary filtration by increasing proteolysis of interstitial proteins with macrophages.

Although experimental lymphedema is often used to simulate human secondary lymphedema, there are notable limitations of these experimental models. For example, experimental lymphedema typically resolves within 1 mo after a rapid onset and involves the disruption of dermal lymphatic capillaries, whereas lymphedema in humans worsens over time, has a delayed onset that occurs (or does not occur) on an individual basis, and is caused by lymph node excision and severing of pre- and postnodal collecting lymphatics (34, 50). A critical question is whether experimental models can be used to provide insights into the mechanistic aspects of human lymphedema. Because secondary lymphedema does not resolve in humans, the resolution of edema in the mouse model may represent an opportunity to study the process of endogenous resolution in the mouse for clues to the human disease. It is believed that VEGF-C will be useful as an exogenous agent for lymphangiogenic therapy in diseases resulting from poor IF. Results from several investigations have suggested that, in some tissues, overexpression of one of the VEGFR-3 ligands, VEGF-C or VEGF-D, can generate an increased density of sometimes hyperplastic lymphatic capillaries that improve lymphatic function (12, 15, 33, 38–40, 51, 52, 55). In contrast, other investigations have reported that overexpression of VEGF-C may induce lymphatic hyperplasia without increasing lymphatic density (17, 23, 52) and may induce irregular lymphatic function (22, 36), and that the prolymphangiogenic action of VEGF-C may be dependent upon IF (18). We have recently found that excess VEGF-C can promote lymphatic capillary growth in regions of lymphatic-deficient tissue but cannot sustainably increase lymphatic capillary growth or increase lymphatic function above physiological levels (17, 18).

Our present results confirm the ability of exogenous VEGF-C to hasten the natural resolution of skin swelling induced by disruption of dermal lymphatic capillaries. However, we have found that resolution of lymphedema in the mouse tail skin can occur in the complete absence of lymphatic growth, since fluid was seen spreading interstitially across the obstruction. We have also found that the ability of fluid to flow interstitially across the obstruction may be dependent upon the composition of the regenerated extracellular matrix deposited at the site of injury during wound healing.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
Funding for this work was provided by National Institutes of Health Grants 1R15HL-081102 and 1R21AR-053094.


   ACKNOWLEDGMENTS
 
We thank Dr. Vincent Cimmino, Professor of Surgery at the University of Michigan Health System, for allowing the senior author to observe axillary lymph node and sentinel lymph node dissections.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Goldman, Biomedical Engineering Dept., Michigan Technological Univ., Houghton, MI 49931 (e-mail: jgoldman{at}mtu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* J. Uzarski, M. B. Drelles, and S. E. Gibbs contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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