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Am J Physiol Heart Circ Physiol 295: H2113-H2127, 2008. First published October 10, 2008; doi:10.1152/ajpheart.00879.2008
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TGF-β1 is a negative regulator of lymphatic regeneration during wound repair

Nicholas W. Clavin, Tomer Avraham, John Fernandez, Sanjay V. Daluvoy, Marc A. Soares, Arif Chaudhry, and Babak J. Mehrara

Division of Plastic and Reconstructive Surgery, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York

Submitted 8 August 2008 ; accepted in final form 3 October 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although clinical studies have identified scarring/fibrosis as significant risk factors for lymphedema, the mechanisms by which lymphatic repair is impaired remain unknown. Transforming growth factor -β1 (TGF-β1) is a critical regulator of tissue fibrosis/scarring and may therefore also play a role in the regulation of lymphatic regeneration. The purpose of this study was therefore to assess the role of TGF-β1 on scarring/fibrosis and lymphatic regeneration in a mouse tail model. Acute lymphedema was induced in mouse tails by full-thickness skin excision and lymphatic ligation. TGF-β1 expression and scarring were modulated by repairing the wounds with or without a topical collagen gel. Lymphatic function and histological analyses were performed at various time points. Finally, the effects of TGF-β1 on lymphatic endothelial cells (LECs) in vitro were evaluated. As a result, the wound repair with collagen gel significantly reduced the expression of TGF-β1, decreased scarring/fibrosis, and significantly accelerated lymphatic regeneration. The addition of recombinant TGF-β1 to the collagen gel negated these effects. The improved lymphatic regeneration secondary to TGF-β1 inhibition was associated with increased infiltration and proliferation of LECs and macrophages. TGF-β1 caused a dose-dependent significant decrease in cellular proliferation and tubule formation of isolated LECs without changes in the expression of VEGF-C/D. Finally, the increased expression of TGF-β1 during wound repair resulted in lymphatic fibrosis and the coexpression of {alpha}-smooth muscle actin and lymphatic vessel endothelial receptor-1 in regenerated lymphatics. In conclusion, the inhibition of TGF-β1 expression significantly accelerates lymphatic regeneration during wound healing. An increased TGF-β1 expression inhibits LEC proliferation and function and promotes lymphatic fibrosis. These findings imply that the clinical interventions that diminish TGF-β1 expression may be useful in promoting more rapid lymphatic regeneration.

lymphangiogenesis; scar; lymphedema; vascular endothelial growth factor C; transforming growth factor-β1; excisional wound healing; myofibroblast; fibrosis

LYMPHEDEMA IS A DEBILITATING disorder that occurs commonly after axillary lymph node dissection for breast cancer. Approximately 30% of the two million breast cancer survivors in the United States have some degree of lymphedema (49). Despite the profound impact of lymphedema on the quality of life, very few treatment options exist (52). There is currently no medical or surgical cure. Understanding the molecular mechanisms responsible for abnormal lymphatic regeneration and subsequent lymphedema is an important step in the development of targeted therapeutic and preventative strategies.

Recent studies have elucidated several key molecular mechanisms that regulate lymphangiogenesis in wound healing, inflammation, and in tumors. These studies have demonstrated critical roles for growth factors such as vascular endothelial growth factor C and D (VEGF-C and VEGF-D, respectively) as well as inflammatory cells such as macrophages (38, 41). The coordinated expression of these mediators results in the formation of new lymphatic vessels and lymphatic repair. Although these mechanisms are well described, the defects that prevent a normal lymphatic regeneration and result in a subsequent lymphedema remain largely unknown.

Clinical studies have identified extensive surgery, infections, and radiation therapy as important risk factors for the development of lymphedema (17, 39). The combination of radiation and surgery increases the risk of lymphedema four- to eightfold (1, 53). The common denominator in these risk factors is soft tissue fibrosis and scarring, suggesting that increased scarring is associated with abnormal lymphatic regeneration. This contention is supported by the finding that the induction of lymphedema in animal models requires extensive scarring, often including a full-thickness skin resection in combination with radiation therapy (46).

The molecular mechanisms responsible for soft tissue fibrosis have been the topic of intense investigation. These studies have shown that transforming growth factor -β1 (TGF-β1) is an important regulator of scarring and fibrosis both during wound repair as well as in abnormal organ fibrosis (2, 4, 12, 40, 51). For example, TGF-β1 expression is increased in abnormal wound healing such as keloids or hypertrophic scars as well as in liver, lung, and renal fibrosis (16, 20, 21). Similarly, radiation therapy results in chronically increased TGF-β1 expression that can last several years after injury (23). The inhibition of TGF-β1 signaling decreases scarring during wound repair and reduces organ fibrosis in the lung, liver, and kidney (9, 16, 19). A downregulation of TGF-β1 expression after radiation therapy also decreases soft tissue fibrosis (13, 54). Thus, although it is known that fibrosis is associated with lymphedema and the upregulation of TGF-β1 expression, the effects of this growth factor on lymphatic regeneration during wound repair remain essentially unknown.

A recent study has shown that TGF-β1 has direct inhibitory effects on isolated LECs and inhibits lymphangiogenesis in tumors (30). We therefore hypothesized that the inhibition of scarring would decrease TGF-β1 expression and improve lymphatic regeneration. We demonstrate that decreasing TGF-β1 expression during wound repair using a topical collagen gel treatment accelerates lymphatic regeneration and is associated with an earlier accumulation of LECs, reduced myofibroblast proliferation, and diminished capillary lymphatic fibrosis. In contrast, the increased TGF-β1 expression during abnormal wound repair or with an addition of a supplemental growth factor significantly delayed lymphatic regeneration. Furthermore, we show that TGF-β1 has direct effects on isolated LECs by inhibiting the proliferation and tubule formation without altering the VEGF-C or VEGF-D expression. Collectively, our results suggest that the inhibition of TGF-β1 signaling during wound repair could be a viable means of improving lymphatic regeneration and decrease the incidence of postoperative lymphedema.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse tail excision model and surgical preparation. Female 6–8-wk-old C57B6 mice (Jackson, Bar Harbor, ME) were used for all experiments after approval from the Research Animal Resource Center at Memorial Sloan Kettering Cancer Center. For the creation of acute postsurgical lymphedema, a 2-mm full-thickness skin and subcutaneous excision (thereby removing the superficial lymphatic network) was performed at a distance of 15 mm from the base of the tail as previously described (6). In addition, the collecting lymphatics running adjacent to the lateral tail veins were identified and ligated using a surgical microscope (StereoZoom SZ-4, Leica, Wetzlar, Germany). The mice were divided into two groups. In group 1 (+collagen gel; n = 16), the skin defect was filled with 0.3% type I rat collagen gel (BD Biosciences, Bedford, MA) and the wound was covered with Bacitracin Zinc Ointment USP (Fougera, Melville, NY), Xeroform Petrolatum Dressing (Cardinal Health, Dublin, OH), and Tegaderm (3M, St. Paul, MN) to prevent desiccation. Based on previous experiments, we hypothesized that this treatment would decrease the scar formation and therefore inhibit the TGF-β1 expression (8). Animals in group 2 (–collagen gel; n = 16) were treated surgically and had their wounds covered only with the ointment and dressing to prevent desiccation. To further delineate the role of TGF-β1 during lymphatic repair, additional animals underwent identical procedures with skin and lymphatic ablation and wound coverage with 0.3% type I rat collagen impregnated with a physiological dose of recombinant TGF-β1 (50 ng) (+collagen gel/+TGF-β1; n = 16) (45). The recombinant TGF-β1 used was mature human TGF-β1, which has cross-reactivity with mouse and shares 99% amino acid identity with mouse TGF-β1 (R&D Systems, Minneapolis, MN).

Specimen preparation and histology. Ten-millimeter tail sections centered on the repair site were harvested after 1, 2, 4, and 6 wk, fixed in 4% paraformaldehyde, decalcified, embedded in paraffin, and sectioned at 5 µm. Histochemical staining for scar tissue was performed using picrosirius red (Direct Red 80, Sigma, St. Louis, MO) and hematoxylin and eosin stains as previously described (12). The specimens were examined using polarized light microscopy (Leica TCS AOBSSP2), and the scar index was calculated as previously described (12). The scar index is based on the fact that normal skin is characterized by thin, randomly oriented collagen fibers demonstrating yellow-green birefringence, whereas the scarring is associated with the deposition of thick, parallel collagen bundles with orange-red birefringence (10). To calculate the scar index, representative 70,000-µm2 dermal images within and distal to the wound bed of specimens harvested after 4 or 6 wk were analyzed using Metamorph Offline software (Molecular Devices, Sunnyvale, CA) and the ratio of the number of orange-red pixels to the number of yellow-green pixels was calculated in three high-power fields (12).

Tail volume measurements and lymphoscintigraphy. To calculate the degree of postprocedural acute lymphedema, the tail volumes distal to the site of lymphatic excision were calculated 1, 2, 4, and 6 wk postprocedure (n = 4/group) using the truncated cone formula and compared with baseline (47).

Lymphoscintigraphy was performed using unfiltered [99mTc]- labeled sulfur colloid (100 nm particle size; 400–800 µCi in ~50 µl) injected intradermally at the tip of the tail. Dynamic planar gamma camera images were acquired for 2.5 h postinjection using the X-SPECT (Gamma Medica, Northridge, CA), fitted with low-energy parallel-hole collimators. The resulting images were imported into ASIPro (CTI Molecular Imaging, Knoxville, TN) for analysis. A region of interest analysis was performed to derive the time activity data for the injection site and lymph nodes at the base of the tail. The decay-adjusted isotope uptake in the lymph node basin of the tail was determined.

Immunofluorescence and immunohistochemistry. Immunohistochemical and immunofluorescent staining were performed as previously described (26). Immunofluorescence was used to identify lymphatic vessel endothelial receptor 1 (LYVE-1; Rabbit polyclonal, Abcam, Cambridge, MA), proliferating cell nuclear antigen (PCNA; Rabbit polyclonal, Abcam), and {alpha}-smooth muscle actin ({alpha}-SMA; Rabbit polyclonal, Abcam). Fluorescence detection was performed with fluorescein (R&D Systems) or Cy3 Zymed (Invitrogen Molecular Probes, Carlsbad, CA) and visualized using a fluorescent microscope (Leica TCS).

Immunohistochemistry was used to label TGF-β1 (Rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA) and F4/80 (Acris, Hiddenhausen, Germany). The secondary antibody was from Vectastain ABC Kit (Vector, Burlingame, CA). The images were obtained after 3,3'-diaminobenzidine staining using bright-field microscopy (Leica TCS). For all immunolocalization experiments, the negative controls included tissues treated with no primary antibody or no secondary antibody.

Quantitation of TGF-β1 expression. The relative expression of TGF-β1 was assessed using a visual analog scale after immunostaining (0 = no staining, and 3 = intense staining) by two blinded reviewers. A minimum of three high-powered fields (x100 magnification) from the distal margins of the wound were analyzed for each animal (n = 4) at each time point. The average staining score was calculated.

To confirm the changes in expression of TGF-β1 in group 1 (+collagen) and group 2 (–collagen) animals, tail excision and wound repairs were performed as described in Mouse tail excision model and surgical preparation in three animals per group. The wounds were then harvested 2 wk postoperatively (the time point corresponding to the highest level of TGF-β1 expression by immunohistochemistry), and quantitative (q)PCR was performed using our previously published techniques (43). Briefly, the wounded section and an area spanning ~1 mm proximal and 1 mm distal to the wound were harvested, rapidly frozen in liquid nitrogen, and homogenized in TRIzol Reagent (Invitrogen Molecular Probes), and the total cellular RNA was harvested using the manufacturer's protocol. The RNA quantity and quality of the purified samples were analyzed using a NanoDrop ND 1000 spectrophotometer. Reverse transcription was performed using the TaqMan Reverse Transcription reagents kit (Applied Biosystems) using 1 µg RNA. qRT-PCR was then performed using the LightCycler thermocycler and the TaqMan Universal Mastermix (Roche Diagnostics, Indianapolis, IN), according to manufacturer's directions. A standard curve was prepared using serially diluted control samples and was plotted against the cycle numbers obtained at the log-linear phase of the reaction. The expression of TGF-β1 (Applied Biosystems) was normalized to 18S RNA expression measured concurrently. Experiments were performed in triplicate.

The total cellular protein was isolated from the region surrounding the wounds at 2 wk following surgery. Briefly, the wounded section and an area spanning ~1 mm proximal and 1 mm distal to the wound were harvested, rapidly frozen in liquid nitrogen, and homogenized in TRIzol Reagent, and the total cellular protein was harvested using the manufacturer's protocol. The total protein was quantified using the Bradford method. Western blot analysis for TGF-β1 was performed using a polyclonal antibody mouse TGF-β1 (Santa Cruz Antibodies) and normalized to actin levels. The immunoreactivity was determined using the enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL).

LEC quantification and identification of proliferating cells. LECs in the distal portions of the wound were identified with LYVE-1 antibodies and counted by two blinded reviewers using confocal laser florescence microscopy in three random high-powered fields (x200) for four animals per group at each time point. Individual cells that had both 4,6-diamidino-2-phenylindole-stained nuclei and LYVE-1-stained cell membranes were identified and counted. To identify the proliferating LECs, the sections were double stained with PCNA and LYVE-1. Individual proliferating cells were identified using confocal laser microscopy to identify the nuclear staining of PCNA and the membrane-associated LYVE-1. The cells were quantified in three random high-powered fields (x200) at the distal margin of the wound by two blinded observers for each animal at the 2 wk time point.

Cell culture and reagents. Human LECs were obtained from PromoCell (Heidelberg, Germany) and cultured in endothelial cell growth medium-MV containing 0.4% endothelium cell growth serum-heparin, 5% FCS, 10 ng/ml EGF, 1 µg/ml hydrocortisone, and 50 U/ml penicillin-streptomycin (Invitrogen) and passaged every 48 h. LEC morphology was confirmed with immunofluorescent staining for LYVE-1 and prospero-homebox-1 (not shown). Early passage cells (<10) were used for all experiments.

Proliferation and matrigel tubule formation assay. LECs were plated at a density of 2 x 103 cells in 96-well plates and incubated with 0, 1, or 10 ng/ml of TGF-β1. The relative cell number was measured using the CellTiter96 Aqueous Assay (Promega, Madison, WI) according to the manufacturer's instructions. The mitochondrial conversion of the modified Tyrode solution to formazan (directly proportional to the number of living cells in the culture) was recorded at 490 nm using a plate reader (EL 312e Biokinetics reader, Biotek Instruments, Winooski, VT). For lymphatic tubule formation, the LECs were cultured for 3 days in media supplemented with 0, 1, or 10 ng/ml of recombinant human TGF-β1. The cells were subsequently trypsinized, and tubule formation was observed 24 h after plating 1 x 103 cells/well in 96-well plates coated with growth factor-reduced matrigel matrix (BD Biosciences). The images were acquired using an inverted brightfield microscope (Nikon Eclipse TS-100, Tokyo, Japan) and digitally photographed (Nikon Coolpix p5100). All experiments were performed in quadruplicate.

Human VEGF-C/D ELISA. Sandwich ELISA kits (R&D Systems) were used to determine the levels of expressed VEGF-C and -D in cultured human LECs 24 h after exposure to varying concentrations of TGF-β1 (0, 1, and 10 ng/ml). The optical density was measured at 450 nm and corrected with the optical density measurement at 570 nm. The values obtained were reported (in pg/ml) using a standard curve.

Statistical analysis. Multigroup comparison was performed using one-way ANOVA with the Tukey-Kramer post hoc test. Student's t-test was used for analyzing differences between two groups. The data are presented as means ± SD or SE as noted, with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Treatment of mouse tail skin and lymphatic excision with topical collagen gel inhibits TGF-β1 expression and results in decreased fibrosis. All mouse tails were grossly stable after surgery without any evidence of arterial insufficiency. All mice demonstrated a variable period of acute lymphedema without any wound complications.

The collected tissue specimens were assayed for TGF-β1 expression using immunohistochemistry. The wounds collected from the group 1 animals (+collagen gel) demonstrated little extracellular matrix (ECM) staining for TGF-β1 although the spindle-shaped cells consistent with fibroblasts present deep to and adjacent to the wound stained positively. Furthermore, the low-level TGF-β1 expression noted in the early time points of the group 1 wounds all but disappeared by the later time points. In contrast, wounds from the group 2 animals (–collagen gel) demonstrated a marked increase in TGF-β1 expression in the ECM, in fibroblasts surrounding the excision sites, and in spindle-shaped cells within the granulation tissue. The intensity of TGF-β1 staining was highest in the early time points (1 and 2 wk) in this group and persisted through the later time points (Fig. 1, A and B). The staining for TGF-β1 in group 2 animals (–collagen gel) was demonstrably more intense than in group 1 animals (+collagen) at all time points evaluated. To further demonstrate the decreased TGF-β1 expression in group 1 animals, quantitative PCR and Western blot analysis were performed 2 wk after surgery. These analyses demonstrated a significant decreased expression of TGF-β1 as a result of the collagen gel treatment (Fig. 1, C and D).


Figure 1
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  		Fig. 1. Treatment of mouse tail skin and lymphatic excision with topical collagen gel inhibits transforming growth factor (TGF)-β1 expression. A: representative photomicrographs of TGF-β1 immunostaining in group 1 (+collagen; left, open arrow) and group 2 (–collagen gel; right, closed arrow) wounds at various times after surgery (x100). Note marked increase in TGF-β1 expression both in the ECM and in spindle-shaped cells surrounding the excision sites. Note also that TGF-β1 expression in group 2 persisted in the ECM even at the 6-wk time point. B: relative quantitation of TGF-β1 staining. Note the significant increase in TGF-β1 expression at all time points in the group 2 (–collagen gel) wounds compared with group 1 (+collagen gel) animals (*P < 0.05). C: quantitative PCR for TGF-β1 from total cellular RNA harvested from wounds of mouse in group 1 and group 2 at 2 wk postoperatively (P < 0.002). D: Western blot of TGF-β1 protein expression from wounds of mice in group 1 and group 2 at 2 wk postoperatively. Note significant increase in TGF-β1 expression in group 2 (–collagen) animals.

 
When we evaluated the wound sections histologically 1 wk after wounding, we noted inflammation, dermal hypercellularity, spongiosis, and dilated lymphatic vessels distal to the excision site in all three treatment groups (not shown). By 2 wk, the animals in group 2 (–collagen gel) continued to exhibit hypercellularity and distal lymphatic dilatation, whereas the group 1 animals (+collagen gel) showed decreased cellularity, inflammation, and resolution of distal lymphedema (not shown). The addition of 50 ng TGF-β1 to the collagen gel (+collagen/+TGF-β1) resulted in a histological appearance that was very similar to animals treated without the collagen gel and completely reversed the beneficial effects of the collagen gel dressing on wound repair. At the later time points evaluated, the group 1 (+collagen gel) animals continued to heal rapidly, demonstrating decreased subdermal thickness and collapsed lymphatics, reaching near normal histology by 6 wk (Fig. 2A). In contrast, the animals in group 2 (–collagen gel) and those treated with supplemental TGF-β1 remained hypercellular with a thickened dermal/subdermal tissues and persistent edema distally even at the 6-wk time point. Thus the topical collagen treatment inhibited TGF-β1 expression and was associated with an decreased inflammation and cellularity. In contrast, the exogenous application of TGF-β1 to the collagen gel resulted in an increased inflammation and recapitulated the histological characteristics of the animals treated without the collagen gel.


Figure 2
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  		Fig. 2. Collagen gel treatment and decreased TGF-β1 expression are associated with decreased scarring and fibrosis. A: representative hematoxylin and eosin low-power images (x50) of group 1 (+collagen gel; top, left) and group 2 (–collagen gel; top, middle) and supplemental TGF-β1-treated animals (+collagen gel/+TGF-β1; top, right) 6 wk after surgery. Note increased inflammation, hypercellularity, and tissue edema in group 2 and TGF-β1-treated wounds. B: representative high-power images (x200) of picrosirius red staining in group 1 (bottom, left), group 2 (bottom, middle), and TGF-β1-treated (bottom, right) wounds 6 wk after surgery. Note increased orange/red birefringence (closed arrow) and more parallel deposition of fibers (circle) in group 2 and supplemental TGF-β1-treated wounds compared with those of group 1, indicating more scarring and fibrosis (open arrow). C: scar indexes of wounds in group 1, group 2, and supplemental TGF-β1 (TGF-B1)-treated animals. Note significant decrease in scarring in the group 1 (+collagen) compared with other groups (*P < 0.05). Data are presented as means ± SE of quantification from 3 random high-powered fields per animal (n = 4) at each time point.

 
The decreased inflammation, cellularity, and TGF-β1 expression in group 1 animals (+collagen gel) translated to decreased scarring as demonstrated by picrosirius red staining. The collagen gel-treated animals had a more random (i.e., normal) organization of green-yellow birefringent collagen fibers and nearly a threefold lower scar index than the excision without the collagen gel group and nearly sixfold lower index than the collagen gel supplemented with TGF-β1 group 4 and 6 wk after surgery (P < 0.01; Fig. 2, B and C).

Inhibition of TGF-β1 expression and scarring is associated with increased functional lymphatic transport and decreased acute lymphedema. To evaluate the effect of variable scarring on lymphatic regeneration, we quantified [99mTc]-labeled sulfur colloid lymphatic transport using lymphoscintigraphy by calculating the decay-corrected base of tail lymph node uptake relative to the initial injection at the distal tail tip (Fig. 3, A and B). Although uptake was limited in all groups at 1 wk after surgery, it was significantly higher in the group 1 animals (+collagen gel; nearly sixfold increase; P < 0.05). Similar trends were noted after 2 and 4 wk with the group 1 animals, demonstrating more than a threefold higher technetium uptake compared with the group 2 animals (–collagen gel; P < 0.01 at each time point). Interestingly, at the 6 wk time point, the radionucleotide uptake in the group 1 animals (+collagen gel) returned to baseline levels but remained markedly abnormal in the animals treated without the collagen gel and in those in which the collagen gel was supplemented with TGF-β1. This finding demonstrates that an increased TGF-β1 expression during wound repair inhibits lymphatic regeneration.


Figure 3
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  		Fig. 3. Inhibition of TGF-β1 expression and scarring is associated with increased functional lymphatic transport and decreased acute lymphedema. A: static representative images of nodal uptake of [99mTc]-labeled sulfur colloid in group 1 (+collagen gel; top), group 2 (–collagen gel; middle), and supplemental TGF-β1-treated (+collagen gel/+TGF-β1; bottom) animals. Note radioactive uptake (red hue) in lymph nodes (arrow) at the base of the tail in group 1 (+collagen gel) animals beginning at 2 wk. In contrast, note minimal uptake in group 2 and supplemental TGF-β1 groups even after 6 wk. Arrow marks the tip of the tail. B: quantitation of radionucleotide uptake after lymphoscintigraphy 6 wk after surgery. Note significant increase in both the rate (i.e., slope of the line) and amount of radionucleotide uptake in group 1 animals, demonstrating more rapid lymphatic regeneration (data are presented as means ± SE of uptake calculations from 4 animals per time point; P < 0.05). In contrast, note severe dysfunction in lymphoscintigraphic uptake in the supplemental TGF-β1-treated animals. C: tail volume measurements after surgery. Note significant decrease in tail volume and return to normal by 4 wk in group 1 animals (*P < 0.05 compared with all other groups). In contrast, note increase in tail volume measurements and lymphedema in group 2 and supplemental TGF-β1-treated animals even 6 wk postoperatively.

 
Increased lymphatic transport noted on lymphoscintigraphy was associated with decreased tail volumes and a more rapid resolution of acute lymphedema in the group 1 (+collagen gel) compared with the group 2 (–collagen gel) animals and in those treated with recombinant TGF-β1. Even 2 wk after surgery, the increase in the tail volume of the group 1 animals was nearly fourfold less than that observed in the group 2 animals, as well as animals treated with recombinant TGF-β1 (P < 0.05, Fig. 3C). At 4 wk postoperatively, tail volumes in group 1 returned to baseline, whereas the group 2 animals still had a 40% increase in tail volume. Similarly, the addition of TGF-β1 to the wound reversed the beneficial effects of the collagen gel and resulted in a change in tail volumes almost indistinguishable from the group 2 animals. This trend was maintained at the 6-wk time point with only a minimal improvement in tail volumes in group 2 and the supplemental TGF-β1 group. In addition, the histological sections of wounds and the distal portions of the tails obtained 6 wk after treatment with TGF-β1 demonstrated changes consistent with chronic lymphedema (i.e., fat deposition, thickened dermis/subdermis, and subdermal fibrosis; Fig. 4). Thus the inhibition of TGF-β1 expression during wound repair is associated with improved lymphatic function and decreased acute lymphedema. In contrast, the increased TGF-β1 expression either endogenously (i.e., group 2) or as a result of TGF-β1 supplementation is associated with a delayed lymphatic regeneration and a prolonged acute lymphedema.


Figure 4
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  		Fig. 4. Treatment of wounds with supplemental TGF-β1 results in chronic lymphedema changes distal to the site of surgery. Representative low-power photomicrographs of wounds treated with collagen gel with (left) or collagen gel supplemented with 50 ng of recombinant human TGF-β1 (right). Note subdermal thickening (bottom, braces), dilated lymphatics (open arrow), fibrosis, and hypercellularity in supplemental TGF-β1-treated wounds. Also, note fat deposition (top, right, solid arrow) in the cross sections of the tail harvested distal to the surgical site and treated with TGF-β1.

 
Increased expression of TGF-β1 during wound repair is associated with delayed recruitment and decreased proliferation of LECs. To evaluate the cellular mechanisms by which an increased TGF-β1 expression during wound repair can inhibit lymphatic regeneration, we quantified the number and proliferation of LECs present in the wound. This was based on previous work by Boardman et al. (35) demonstrating that the recruitment of LECs beginning distally and migrating proximally is crucial for lymphatic regeneration in the mouse tail model. Thus the LECs were quantified and microscopic lymphatic regeneration in the distal portions of the wound was analyzed using LYVE-1 immunofluorescence (Fig. 5A). At all time points evaluated, animals in group 1 (+collagen gel) had more LECs present (3–5x increase) in the distal portion of the wound than group 2 (–collagen gel) or animals treated with supplemental TGF-β1 (P < 0.01 at all time points). There was no statistical difference between group 2 and supplemental TGF-β1-treated animals in LEC number.


Figure 5
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  		Fig. 5. TGF-β1 inhibits lymphatic endothelial cell (LEC) recruitment and proliferation. A: number of lymphatic vessel endothelial receptor (LYVE)-1-positive cells (LECs) at the distal margin of the wound at 2, 4, and 6 wk after surgery. Note significant increase in the number of LECs present in the distal portion of group 1 (+collagen gel) compared with group 2 (–collagen gel) and supplemental TGF-β1-treated (+collagen gel/+TGF-β1) animals [means ± SD of 3 high-powered fields/animal (n = 4) per time point]. *P < 0.05. B: identification of proliferating LECs [LYVE-1 and proliferating cell nuclear antigen (PCNA) double-positive stain] in wound sections 2 wk postoperatively (representative high-powered figures shown at x400 magnification). Note red LYVE-1 membrane staining (left), green PCNA nuclear staining (middle), and double-positive cells, indicating proliferating LECs (right, open arrows). C: quantification of proliferating LECs (+PCNA/+LYVE) in wound sections 2 wk postoperatively. Note nearly 6-fold increase in proliferating LECs present in group 1 (+collagen gel) compared with group 2 (–collagen) and supplemental TGF-β1 (+collagen gel/+TGF-β1)-treated animals [data are presented as means ± SD of 3 high-powered fields (HPF)/animal (n = 4) per group]. D: TGF-β1 inhibits LECs proliferation in vitro. Note significant dose-dependent decrease in cellular proliferation with exposure to increasing doses of TGF-β1 (0, 1, 10 ng/ml; means ± SD of triplicate experiments; *P < 0.05). OD, optical density.

 
The reduced number of LECs in group 2 wounds and in animals treated with supplemental TGF-β1 was due in part to the inhibition of LEC proliferation (Fig. 5, B and C). When the number of proliferating LECs was calculated by colocalization of PCNA and LYVE-1, we noted that animals in group 1 (+collagen gel) had nearly six times more proliferating LECs in the distal portion of the wound compared with group 2 (–collagen gel) and animals treated with supplemental TGF-β1 (P < 0.01). There was no statistical difference in the number of proliferating LECs between the group 2 and the supplemental TGF-β1-treated animals.

To evaluate the effects of TGF-β1 on isolated LECs, purified LECs were cultured with varying amounts of TGF-β1 and cellular proliferation was assessed (Fig. 5D). This analysis demonstrated a dose-dependent and statistically significant decrease in LEC proliferation in vitro, thus suggesting that an increased TGF-β1 expression during abnormal wound repair may directly inhibit lymphatic regeneration by inhibiting LEC proliferation.

Increased TGF-β1 during wound repair is associated with abnormal lymphatic architecture and dilated lymphatics. LYVE-1 staining was used to evaluate the architecture of the newly formed lymphatics in each group (Fig. 6A). As expected, in the group 1 animals (+collagen gel), the lymphatic capillary formation was noted at the earliest time points with an infiltration and coalescence of LECs. By 2 wk, the newly formed lymphatics demonstrated a capillary and cylindrical architecture with a mild dilation noted in some vessels. In contrast, the group 2 animals (–collagen gel) and the animals treated with supplemental TGF-β1 demonstrated very little capillary lymphatic regeneration at this time point. The few lymphatics that had formed were markedly abnormal, dilated, and ectatic in appearance. Similar findings were noted in these groups at the 4- and 6-wk time points evaluated with only an occasional normal, collapsed cylindrical capillary lymphatic present. In contrast, the capillary architecture of the group 1 (+collagen gel) animals was similar to normal skin by the 6-wk time point with cylindrical, small collapsed capillary lymphatics.


Figure 6
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  		Fig. 6. Increased TGF-β1 during wound repair is associated with abnormal lymphatic architecture and dilated lymphatics but does not alter VEGF-C or VEGF-D expression. A: representative florescent photomicrographs for LYVE-1 in group 1 (+collagen gel; left), group 2 (–collagen gel; middle), and supplemental TGF-β1 (+collagen/+TGF-β1; right) at various time points after surgery (x50 magnification). Note lymphatic capillary formation in group 1 animals at the earliest time points with cylindrical architecture and mild dilatation progressing to collapse lymphatics by 6 wk (small circle). In contrast, note little capillary lymphatic regeneration in group 2 and supplemental TGF-β1 animals with the presence of markedly dilated, ectatic lymphatics (large circles). B: TGF-β1 inhibits LEC tubule formation in matrigel tubule formation assay in vitro. Representative images (x20) of LECs treated with various doses of TGF-β1 (0, 1, and 10 ng/ml) for 3 days and then plated on Matrigel-coated plates for 24 h. Note dose-dependent inhibition of tubule formation in the TGF-β1-treated LECs. Also, note large ectatic tubule formation in the TGF-β1-treated groups reminiscent of our in vivo findings (bottom: x400 representative images). All experiments were performed in quadruplicate. C and D: expression of VEGF-C and VEGF-D protein by LECs 24 h after treatment with TGF-β1 (0, 1, and 10 ng/ml). Note no significant difference in VEGF-C/D expression by ELISA (means ± SD; P = not significant).

 
To evaluate the effect of TGF-β1 on LEC tubule formation and function, we cultured isolated LECs in varying doses of TGF-β1 and evaluated tubule formation using a matrigel assay (27). LECs cultured in media supplemented with TGF-β1 demonstrated a significant and dose-dependent decrease in the potential for lymphatic tubule formation (Fig. 6B). In the absence of supplemental TGF-β1, the lymphatic tubule formation occurred rapidly and the multiple small tubules were observed after 24 and 48 h. In contrast, large, ectatic and dilated lymphatics similar to those noted in the group 2 (–collagen gel) and supplemental TGF-β1-treated animals were noted in the low-dose TGF-β1 group. The treatment with high-dose TGF-β1 (10 ng/ml) all but abolished the tubule formation in vitro. It should be noted that the high dose of TGF-β1 used in our study is still thought to be within the physiological range of this growth factor in vitro (45). Thus the increased TGF-β1 during abnormal wound repair significantly attenuates the LEC function and tubule formation and may contribute to the delayed lymphatic regeneration and increased acute lymphedema.

To determine whether the increased TGF-β1 causes alterations in LEC proliferation and tubule formation by changing VEGF-C or -D expression, we analyzed the expression of these growth factors in isolated LECs exposed to various doses of TGF-β1 (Fig. 6, C and D). Interestingly, TGF-β1 did not alter the expression of VEGF-C or -D significantly in vitro. This finding is somewhat surprising given that we and others have shown that TGF-β1 is a potent regulator of VEGF-A expression (32, 37). Thus it is likely that the differences we noted in LEC proliferation or tubule formation are not due to changes in VEGF-C or VEGF-D expression by LECs. This conclusion is supported by the finding that little difference in VEGF-C expression was noted between the group 1 (+collagen gel) and group 2 (–collagen gel) animal wounds as assessed by immunohistochemistry in vivo (not shown). It is possible, however, that TGF-β1 can interact indirectly with VEGF-C/D through alterations in the expression of VEGF receptors or other mechanisms.

Increased TGF-β1 expression during wound repair is associated with increased myofibroblastic infiltration and lymphatic fibrosis. TGF-β1 is a known inducer of myofibroblast proliferation and differentiation (28). In addition, recent studies have demonstrated that endothelial-mesenchymal (i.e., fibroblast) differentiation occurs in a number of scenarios (e.g., peritumoral fibrosis and renal fibrosis) and that this effect is mediated by TGF-β1 (29, 31). In these cases, the direct differentiation of microvascular endothelial cells to fibroblastic phenotype (i.e., expression of fibroblast markers such as {alpha}-SMA) leads to tissue fibrosis. Lymphatic fibrosis has been shown in anatomic and histological studies of patients with lymphedema (48). Therefore, we analyzed {alpha}-SMA expression in the wound sections to evaluate the possibility of LEC-fibroblast differentiation as a molecular mechanism of lymphatic fibrosis resulting from the increased TGF-β1 expression during an abnormal wound. This analysis demonstrated that abnormal wound repair as noted in group 2 (–collagen gel) was associated with a marked increase in the number of myofibroblasts in the wound bed (Fig. 7A). In addition, we noted that numerous LECs present in the capillary lymphatics (i.e., LYVE-1 positive) coexpressed {alpha}-SMA at the later time points in group 2. Wounds treated with collagen gel supplemented with TGF-β1 demonstrated an even greater degree of double staining for LYVE-1 and {alpha}-SMA (Fig. 7, B and C). Consistent with previous histological studies of lymphatic fibrosis, the capillary lymphatics that coexpressed the lymphatic and fibroblast markers appeared to be thickened and fibrotic, indicating that an endothelial-fibroblast differentiation may be present. In contrast, the wounds harvested from the group 1 animals (+collagen gel) demonstrated normal lymphatic architecture and no evidence of lymphatic fibrosis or coexpression of LYVE-1 and {alpha}-SMA. It is important to note that capillary lymphatics, unlike collecting lymphatics, are not associated with pericytes, a cell type known to express {alpha}-SMA (50). Therefore, the expression of this fibroblastic marker by the LECs lining the normally thin-walled capillary lymphatics vessels is an abnormal finding. These observations suggest that the increased TGF-β1 expression during the abnormal wound repair may promote lymphatic fibrosis, possibly as a result of direct LEC to fibroblast transdifferentiation. This finding is important and may represent a molecular mechanism for lymphatic fibrosis observed clinically in patients with lymphedema. In addition, fibrosis may be responsible for lymphatic dysfunction by physically preventing the dilation and collapse of lymphatic channels.


Figure 7
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  		Fig. 7. TGF-β1 induces expression of {alpha}-SMA and results in lymphatic fibrosis and coexpression of lymphatic and fibroblast markers by LECs in vivo. A: representative photomicrographs (x200) of {alpha}-SMA staining in group 1 (+collagen gel) and group 2 (–collagen gel) animals 6 wk after surgery. Note significant increase in {alpha}-SMA expression in group 2 animals. Also note few tubular structures in group 2 animals with {alpha}-SMA staining. B: localization LYVE-1 (red; top) and {alpha}-SMA (green; middle) in group 1 (+collagen gel; left), group 2 (–collagen gel; middle), and supplemental TGF-β1 (+collagen gel/+TGF-β1; right) 6 wk postoperatively. Colocalization (bottom) is shown as orange/yellow staining. Note numerous double-stained lymphatic vessels (LYVE-1 and {alpha}-SMA) in group 2 and supplemental TGF-β1-treated animals compared with those in group 1. Also, note that double-stained lymphatics demonstrate enlarged lumens and thickened walls consistent with lymphatic fibrosis (ovals) when compared with single-staining group 1 lymphatics (circle). C: a high-powered image of a lymphatic composed of cells expressing both LYVE-1 and {alpha}-SMA (arrow) as opposed to a lymphatic with no {alpha}-SMA expression (arrowhead).

 
Decreased scarring is associated with increased infiltration of F4/80-positive macrophages. Since macrophages are known to be important regulators of lymphangiogenesis, the differences in macrophage migration were evaluated using F4/80 immunohistochemistry. The wounds in the group 1 animals (+collagen gel) demonstrated an early infiltration of macrophages, with most of the cells localized to the distal margin of the wound in the subdermal tissues. Some of the macrophages could clearly be seen migrating across the margin of the wound into the wound bed itself. At the 2- and 4-wk time points, large numbers of macrophages could be observed in the wound distally infiltrating the distal margin in the deep dermal tissues (Fig. 8). The macrophage number appeared to decrease by 6 wk. In contrast, the group 2 animals (–collagen gel) demonstrated very few macrophages in the distal wound margin in the early time points. The few macrophages present were localized to the deep dermal structures at the junction of the wound, and normal tissues and macrophages invading into the wound were seldom noted (Fig. 8). Thus macrophage migration to the distal wound margin and into the wound bed from this margin appeared to be delayed significantly by increased scarring and fibrosis.


Figure 8
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  		Fig. 8. Decreased scarring is associated with increased infiltration of F4/80-positive macrophages. Representative images the distal portions of group 1 and 2 animals at 4 wk after surgery (distal aspect of the wound is toward right). Low-power (x50; top) and magnified inset (x200; bottom). Note large numbers of F4/80-positive macrophages infiltrating into the distal margin of the group 1 section. In contrast, note paucity of cells in the group 2 section (arrow).

 

   DISCUSSION
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Clinical studies have identified a number of risk factors for lymphedema, including 1) treatment-related factors (e.g., surgery and radiation), 2) disease-related factors (e.g., stage, nodal status, and number of lymph nodes removed), and 3) patient factors (e.g., age, obesity, infection/inflammation, and excessive arm use) (33). It is clear that some of these factors are related and that soft tissue scarring and fibrosis are the common denominator.

Abnormal wound repair and scarring are known to be associated with marked alterations in the expression of profibrotic growth factors such as TGF-β1. Many of the clinical conditions that predispose patients to develop lymphedema, most notably radiation therapy, are known to be associated with increased TGF-β1 expression (5, 23, 42). Furthermore, the inhibition of TGF-β1 or its intermediaries (e.g., SMAD-3) decreases the scarring caused by fibrotic stimuli and radiation and improves wound healing (12–14). Consistent with this, we demonstrated that the treatment of excisional wounds with collagen resulted in significantly decreased scarring and the downregulation of TGF-β1 expression. Furthermore, we show that diminished TGF-β1 expression is correlated both temporally and spatially with decreased scarring and accelerated lymphatic regeneration. The addition of TGF-β1 to the collagen gel recapitulated the fibrosis and deficiencies in the lymphatic regeneration noted in the group treated without the collagen gel. Thus the inhibition of TGF-β1 expression in the collagen gel-treated animals was associated with near normal lymphatic function by 4 wk postoperatively, whereas the lymphatic regeneration in the no collagen group or the group treated with collagen supplemented with a small dose of TGF-β1 remained markedly delayed with continued lymphedema even 6 wk after surgery.

TGF-β1 inhibition of lymphatic regeneration may occur as a consequence of direct inhibitory effects of TGF-β1 on LECs. We found that even low doses of this growth factor significantly attenuate the proliferation of isolated LECs in vitro. This in vitro finding is correlated with significant decreases in the number of LECs and the number of proliferating LECs present in the animals treated without the collagen gel (with a resultant increase in TGF-β1) and those treated with supplemental TGF-β1 compared with animals treated with the collagen gel. In addition, TGF-β1 may have direct negative effects on the potential of LECs to coalesce into lymphatic channels as evidenced by a significant inhibition of tubule formation in vitro and large ectatic vessels in vivo. These findings, together with a recent publication by Oka et al. (30) demonstrating a direct inhibitory role for TGF-β1 during inflammatory and tumor associated lymphangiogenesis, suggest that this profibrotic molecule can directly inhibit lymphatic repair by inhibiting the assembly of lymphatic vessels, reducing LEC proliferation, and inhibiting LEC migration. Our results add to the findings of Oka et al. (30) by demonstrating a dose-dependent inhibition of LEC proliferation and function, as well as demonstrating a negative role for TGF-β1 during wound repair. These findings are important since they suggest that antiscarring strategies in general and the inhibition of TGF-β1 signaling in particular may lead to an improved lymphatic regeneration and possibly represent a potential preventative treatment. Given that no preventative strategies for lymphedema are currently available and that this debilitating condition affects a large number of patients who undergo lymph node dissection for cancer, our study provides an important first step toward a potential treatment to prevent abnormal lymphatic regeneration.

Anatomic studies on cadavers treated with axillary lymph node dissection have recently demonstrated that capillary lymphatics become obliterated due to fibrosis and narrowing (48). Fibrosis contributes to lymphatic dysfunction by preventing physiological dilatation of capillary lymphatic vessels necessary for lymphatic fluid uptake when fluid accumulates within tissues. Consistent with this finding we have demonstrated that the increased scarring is associated with lymphatic fibrosis and an increased number of {alpha}-SMA-positive myofibroblasts. In addition, we found that in contrast to the collagen gel-treated animals, animals treated without the collagen gel or those treated with the collagen gel supplemented with recombinant TGF-β1 demonstrated numerous capillary LECs that coexpressed LYVE-1 and {alpha}-SMA. This finding implies that a direct LEC-mesenchymal cell transdifferentiation may occur in the setting of increased TGF-β1 expression and scarring and may be a putative mechanism for the clinically observed phenomena of lymphatic vessel fibrosis and obliteration in patients with lymphedema or lymphatic dysfunction. This hypothesis is supported by the fact that TGF-β1 causes microvascular endothelial to mesenchymal differentiation both in vivo and in isolated microvascular and macrovascular bovine endothelial cells, resulting in the induction of {alpha}-SMA expression (3, 31, 55). In addition, similar to our study, these in vitro and in vivo studies have demonstrated so-called "transitional" cells coexpressing endothelial and fibroblastic markers ({alpha}-SMA in most cases) (3, 7, 31, 55). Finally, our hypothesis that TGF-β1 causes lymphatic fibrosis is further validated by the findings of a recent study demonstrating that the expression of TGF-β1 by regulatory T-cells in the cortical portions of lymph nodes is responsible for lymph node fibrosis after simian immunodeficiency virus infection (11). Clearly, however, additional work is required to determine conclusively whether abnormal TGF-β1 expression results in LEC transdifferentiation.

Interestingly, we did not find differences in VEGF-C expression as a consequence of the changes in TGF-β1 expression during wound repair. VEGF-C is a critical regulator of lymphangiogenesis by promoting LEC proliferation, migration, and tubule formation (22). Thus it may be that the increased TGF-β1 expression and the resultant soft tissue fibrosis/scarring do not alter VEGF-C expression but instead change the other factors in the wound that promote lymphatic regeneration. This is supported by our finding that TGF-β1 had no effect on VEGF-C or VEGF-D expression in isolated LECs. Similarly, Oka et al. (30) found no differences in the expression of VEGF-C by peritoneal macrophages after the inhibition of TGF-β1 activity in their inflammatory model of lymphangiogenesis. The lack of changes in VEGF-C/D expression further supports a direct inhibitory role for TGF-β1 on lymphatic regeneration. It is possible however, that TGF-β1 reacts indirectly with VEGF-C/D by altering the expression of VEGF receptor 2 or 3, thereby inhibiting lymphatic regeneration during wound repair (15). These interactions require further study.

Soft tissue fibrosis and abnormal scarring may also have indirect effects on lymphatic regeneration. For example, abnormal wound healing may alter the interstitial flow that has been recently identified as an important regulator of tissue morphogenesis and lymphatic regeneration (36). In a series of elegant experiments, Boardman and Swartz (6) demonstrated that interstitial flow is an important mediator of LEC migration leading to a reconnection to proximal lymphatics. Although undoubtedly interstitial flow is important to normal lymphatic regeneration, it is unlikely that changes in interstitial flow alone could explain our findings during abnormal wound healing. This conclusion is supported by our finding that abnormal wound repair did not result in significant changes in VEGF-C expression, whereas Pytowski et al. (34) have shown that interstitial flow-dependent lymphatic regeneration in the tail model is dependent on VEGF-C expression and VEGF receptor 3 signaling. In addition, it is unlikely that there would be significant differences in interstitial flow in animals treated with collagen compared with those treated with collagen supplemented with TGF-β1.

An additional mechanism by which soft tissue fibrosis may delay or inhibit lymphatic regeneration is alterations in the recruitment of macrophages. Numerous studies have implicated macrophages as critical regulators of lymphangiogenesis (18). The macrophage expression of VEGF-C is a major regulator of lymphangiogenesis in some tumors and in inflammation (25, 44). Decreased macrophage number and function are associated with diminished lymphangiogenesis and an impaired wound healing in diabetic mice (24). In addition, macrophages are thought to directly contribute to lymphatic vessel formation by transdifferentiating into LECs. We demonstrated that macrophages were more prominently present in the +collagen gel beginning as early as 1 wk postoperatively. More importantly, we found that more F4/80-stained macrophages infiltrated the distal and proximal portions of the wound in the group 1 animals (+collagen gel) at the early time points compared with the group 2 animals (–collagen gel).

In conclusion, we have shown for the first time that the rate of lymphatic regeneration is inversely proportional to the expression of TGF-β1 and the amount of scarring that is present in a wound. Furthermore, we have shown that this effect appears to be independent of VEGF-C/D. The increased TGF-β1 expression appears to have direct inhibitory effects on lymphatic repair by decreasing LEC recruitment, proliferation, and tubule formation. In addition, TGF-β1 expressed during abnormal wound repair is associated with lymphatic fibrosis and possibly LEC-fibroblast transdifferentiation. These results are critical to the understanding of lymphatic repair after surgical disruption, implying that the methods which decrease scarring/fibrosis or the expression of TGF-β1 may play a role in the prevention of lymphatic dysfunction and subsequent secondary lymphedema.


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This study was funded in part by a grant from the Breast Cancer Alliance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. J. Mehrara, 1275 York Ave., Rm. MRI 1005, New York, NY 10065 (e-mail: mehrarab{at}mskcc.org)

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.


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