TGF-�1 is a negative regulator of lymphatic regeneration during wound repair

TGF-β1 is a negative regulator of lymphatic regeneration

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TGF-β1 is a negative regulator of lymphatic regeneration during wound

repair

Running head: TGF-B is a negative regulator of lymphatic regeneration

Key words: Lymphangiogenesis, scar, lymphedema, VEGF-C, TGF-β1, excisional wound healing, myofibroblast, fibrosis

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

From: The Division of Plastic and Reconstructive Surgery and The Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10021

Correspondence: Babak J. Mehrara, MD FACS

1275 York Avenue Room MRI 1005 New York, NY 10065 212-639-8639 212-717-3677 (Fax) [email protected]

Articles in PresS. Am J Physiol Heart Circ Physiol (October 10, 2008). doi:10.1152/ajpheart.00879.2008

Copyright © 2008 by the American Physiological Society.


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Disclosure None of the authors have any commercial associations or financial relationships that

would create a conflict of interest with the work presented in this article.


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Abstract

Introduction: Although clinical studies have identified scarring/fibrosis as significant risk

factors for lymphedema, the mechanisms by which lymphatic repair is impaired remain

unknown. 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.

Methods: Acute lymphedema was induced in mouse tails by full thickness skin excision and

lymphatic ligation. TGF-β1 expression and scarring were modulated by repairing wounds with

or without topical collagen gel. Lymphatic function and histologic analyses were performed at

various time points. Finally, the effects of TGF-β1 on lymphatic endothelial cell (LEC) in vitro

were evaluated. Results: 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. 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, increased expression of TGF-β1 during wound repair resulted in

lymphatic fibrosis and co-expression of α-SMA and LYVE-1 in regenerated lymphatics.

Conclusions: Inhibition of TGF-β1 expression significantly accelerates lymphatic regeneration

during wound healing. Increased TGF-β1 expression inhibits LEC proliferation and function,

and promotes lymphatic fibrosis. These findings imply that clinical interventions that diminish

TGF-β1 expression may be useful in promoting more rapid lymphatic regeneration.

Key words: Lymphangiogenesis, scar, lymphedema, VEGF-C, excisional wound healing, fibrosis


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Introduction

Lymphedema is a debilitating disorder that occurs commonly after axillary lymph node

dissection (ALND) for breast cancer. Approximately 30% of the two million breast cancer

survivors in the US have some degree of lymphedema.(49) Despite the profound impact of

lymphedema on 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, VEGF-D) as well as inflammatory cells such as macrophages.(38, 41) 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 normal

lymphatic regeneration and result in 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 4-8 fold.(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 full thickness skin resection in combination with radiation therapy.(46)


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The molecular mechanisms responsible for soft tissue fibrosis have been the topic of

intense investigation. These studies have shown that 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) Inhibition of TGF-β1 signaling decreases scarring during wound repair and reduces

organ fibrosis in the lung, liver, and kidney.(9, 16, 19) Down-regulation 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 up-regulation 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 lymphatic

endothelial cells and inhibits lymphangiogenesis in tumors.(30) We therefore hypothesized that

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 lymphatic endothelial cells (LECs), reduced myofibroblast proliferation, and diminished

capillary lymphatic fibrosis. In contrast, increased TGF-β1 expression during abnormal wound

repair or with addition of supplemental growth factor significantly delayed lymphatic

regeneration. Furthermore, we show that TGF-β1 has direct effects on isolated LECs by

inhibiting proliferation and tubule formation without altering VEGF-C or VEGF-D expression.

Collectively, our results suggest that inhibition of TGF-β1 signaling during wound repair could


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be a viable means of improving lymphatic regeneration and decrease the incidence of

postoperative lymphedema.

Materials and Methods

Mouse Tail Excision Model and Surgical Preparation

Female 6-8 week old C57B6 mice (The Jackson Laboratory, Bar Harbor, ME) were used

for all experiments after approval by Resource Animal Research Center at Memorial Sloan

Kettering Cancer Center. For creation of acute post-surgical lymphedema, a 2mm full thickness

skin and subcutaneous excision (thereby removing the superficial lymphatic network) was

performed at a distance of 15mm from the base of the tail as previously described.(6) In

addition, collecting lymphatics running adjacent to the lateral tail veins were identified and

ligated using a surgical microscope (Leica, StereoZoom SZ-4, 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 scar

formation and therefore inhibit TGF-β1 expression.(8) Animals in group 2 (- collagen gel; n=16)

were treated surgically, and had their wounds covered only with ointment and dressing to

prevent desiccation. In order 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 physiologic dose of recombinant

TGF-β1 (50ng), a physiologic dose (+collagen gel/+TGF-β1; n=16). (45) The recombinant


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TGF-β1 used was mature human TGF-β1which 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

weeks, fixed in 4% paraformaldehyde, decalcified, embedded in paraffin and sectioned at 5

microns. Histochemical staining for scar tissue was performed using picro-sirius red (Direct Red

80, Sigma St. Louis, MO) and hematoxylin and eosin stains as previously described.(12)

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 while scarring is associated with 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 weeks

were analyzed using Metamorph™ Offline software (Molecular Devices Corporation,

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 post procedural acute lymphedema, tail volumes distal to the

site of lymphatic excision were calculated 1, 2, 4, and 6 weeks post procedure (n=4/group) using

the truncated cone formula and compared to baseline.(47)

Lymphoscintigraphy was performed using unfiltered 99mTc-sulfur colloid (100nm particle

size; 400-800µCi in ~50 µl) injected intradermally at the tip of the tail. Dynamic planar gamma

camera images were acquired for 2.5 hours post injection using the X-SPECT™ (Gamma


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Medica, Northridge, CA) fitted with low-energy parallel-hole collimators. The resulting images

were imported into ASIPro™ (CTI Molecular Imaging, Knoxville, Tennessee) for analysis.

Region-of-interest (ROI) analysis was performed to derive the time-activity data for the injection

site and lymph nodes at the base of the tail. 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 (SMA; Rabbit Polyclonal,

Abcam). Fluorescence detection was performed with fluorescein (R&D Systems, Minneapolis,

MN) 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). Secondary antibody

was from VECTASTAIN® ABC Kit (Vector, Burlingame, CA). Images were obtained after

DAB staining using bright-field microscopy (Leica TCS). For all immunolocalization

experiments, negative controls included tissues treated with no primary antibody or no secondary

antibody.

Quantitation of TGF-β1 Expression

Relative expression of TGF-β1 was assessed using a visual analog scale after

immunostatining (0=no staining, 3=intense staining) by two blinded reviewers. A minimum of


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three (3) high powered fields (100X magnification) from the distal margins of the wound were

analyzed for each animal (n=4) at each time point. Average staining score was calculated.

In order 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 above

in 3 animals per group. The wounds were then harvested 2 weeks postop (the time point

corresponding to the highest level of TGF-β1 expression by immunohistochemistry) and

quantitative PCR was performed using our previously published techniques.(43). Briefly, the

wounded section and an area spanning approximately 1mm proximal and 1mm distal to the

wound were harvested, rapidly frozen in liquid nitrogen, homogenized in Trizol Reagent

(Invitrogen Molecular Probes, Carlsbad, CA), and total cellular RNA was harvested using the

manufacturer’s protocol. RNA quantity and quality of the purified samples was analyzed using a

NanoDrop ND 1000 Spectrophotometer. Reverse transcription was performed using the

TaqMan Reverese Trasncription reagents Kit (Applied Biosystems, Foster City, CA) using 1 μg

RNA. qRT-PCR was then performed using the LightCycler thermocycler and the TaqMan

Universal Mastermix (Roche Diagnostics Corp., Indianapolis, IN), according to manufacturer’s

directions. A standard curve was prepared using serially diluted control samples and was plotted

against cycle numbers obtained at log-linear phase of the reaction. Expression of TGF-β1

(Applied Biosystems) was normalized to 18S RNA expression measured concurrently.

Experiments were performed in triplicate.

Total cellular protein was isolated from the region surrounding the wounds at 2 weeks

following surgery. Briefly, the wounded section and an area spanning approximately 1mm

proximal and 1mm distal to the wound were harvested, rapidly frozen in liquid nitrogen,

homogenized in Trizol Reagent, and total cellular protein was harvested using the


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manufacturer’s protocol. 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. Immunoreactivity was determined using the ECL

chemiluminescence detection system (Amersham, Arlington Heights, Il).

Lymphatic endothelial cell (LEC) quantification and identification of proliferating cells

LECs in the distal portions of the wound were identified with LYVE-1 antibodies and

counted by 2 blinded reviewers using confocal laser florescence microscopy in 3 random high-

powered fields (200x) for 4 animals per group at each time point. Individual cells which had

both DAPI stained nuclei and LYVE-1 stained cell membranes were identified and counted. To

identify proliferating LECs, 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 membrane associated LYVE-1. Cells were quantified in 3 random high powered

fields (200x) at the distal margin of the wound by 2 blinded observers for each animal at the 2

week time point.

Cell Culture and Reagents

Human Lymphatic Endothelial Cells (HLEC) were obtained from PromoCell

(Heidelberg, Germany) and cultured in ECGM-MV containing 0.4% ECGS/H, 5% FCS, EGF

(10ng/ml), hydrocortisone (1μg/ml), and Penicillin-Streptomycin (50U/ml) (Invitrogen) and

passaged every 48 hours. LEC morphology was confirmed with immunofluorescent staining for

LYVE-1 and prox-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 10ng/ml of TGF-β1. Relative cell number was measured using the CellTiter96 Aqueous


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Assay (Promega, Madison, WI) according to manufacturer’s instructions. The mitochondrial

conversion of MTS to formazan (directly proportional to the number of living cells in the

culture) was recorded at 490nm using a plate reader (EL 312e Biokinetics reader, Biotek

Instruments, Winooski, VT). For lymphatic tubule formation, LECs were cultured for 3 days in

media supplemented with 0, 1, or 10ng/ml of recombinant human TGF-β1. Cells were

subsequently trypsinized and tubule formation was observed 24 hours after plating 1x103

cells/well in 96-well plates coated with growth factor reduced matrigel matrix (BD Biosciences).

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 VEGFC/D ELISA

Sandwich ELISA kits from R&D systems were used to determine the levels of expressed

VEGFC and D in cultured human LECs 24 hours after exposure to varying concentrations of

TGF-β1 (0, 1, 10ng/ml). Optical density was measured at 450nm and corrected with OD

measurement at 570nm. The values obtained were reported in picograms/mL using a standard

curve.

Statistical analysis

Multi-group comparison was performed using one-way ANOVA with the Tukey-Kramer

post-hoc test. Students T-test was used for analyzing differences between 2 groups. Data are

presented as mean ± standard deviation or standard error as noted with p<0.05 considered

significant.

Results


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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 evidence of arterial

insufficiency. All mice demonstrated a variable period of acute lymphedema without any wound

complications.

Collected tissue specimens were assayed for TGF- β1 expression using

immunohistochemistry. Wounds collected from group 1 animals (+collagen gel) demonstrated

little extracellular matrix (ECM) staining for TGF-β1 although 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 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 weeks) in this group and persisted

through the later time points (Figure 1A,B). Staining for TGF-β1 in group 2 animals (-collagen

gel) was demonstrably more intense than group 1 animals (+collagen) at all time points

evaluated. In order to further demonstrate decreased TGF-β1 expression in group 1 animals

quantitative PC and western blot analysis were performed 2 weeks after surgery. These analyses

demonstrated significant decreased expression of TGF-β1 as a result of collagen gel treatment

(Figure 1C,D).

When we evaluated the wound sections histologically 1 week after wounding, we noted

inflammation, dermal hypercellularity, spongiosis, and dilated lymphatic vessels distal to the

excision site in all 3 treatment groups (not shown). By 2 weeks, animals in group 2 (-collagen


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gel) continued to exhibit hypercellularity and distal lymphatic dilatation, while group 1 animals

(+collagen gel) showed decreased cellularity, inflammation, and resolution of distal lymphedema

(not shown). The addition of 50ng TGF-β1 to the collagen gel (+collagen/+TGF-β1) resulted in

a histological appearance that was very similar to animals treated without collagen gel and

completely reversed the beneficial effects of collagen gel dressing on wound repair. At later

time points evaluated, group 1 (+collagen gel) animals continued to heal rapidly demonstrating

decreased subdermal thickness and collapsed lymphatics, reaching near normal histology by six

weeks (Figure 2A.). In contrast, 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 week time point. Thus, topical collagen treatment

inhibited TGF-β1 expression and was associated with decreased inflammation and cellularity. In

contrast, exogenous application of TGF-β1 to the collagen gel resulted in increased inflammation

and recapitulated the histological characteristics of the animals treated without collagen gel.

Decreased inflammation, cellularity, and TGF-β1 expression in group 1 animals

(+collagen gel) translated to decreased scarring as demonstrated by picro-sirius red staining. The

collagen gel treated animals had a more random (i.e. normal) organization of green-yellow

birefringent collagen fibers and nearly 3-fold lower scar index than the excision without collagen

gel group and nearly 6-fold lower index than the collagen gel supplemented with TGF-β1 group

4 and 6 weeks after surgery (p<0.01; Figure 2B,C).

Inhibition of TGF-β1 expression and scarring is associated with increased functional

lymphatic transport and decreased acute lymphedema

In order to evaluate the effect of variable scarring on lymphatic regeneration, we

quantified 99mTc-sulfur colloid lymphatic transport using lymphoscintigraphy by calculating


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decay corrected base of tail lymph node uptake relative to initial injection at the distal tail tip

(Figure 3 A,B.). Although uptake was limited in all groups 1 week after surgery, it was

significantly higher in group 1 animals (+collagen gel; nearly 6 fold increase; p<0.05). Similar

trends were noted after 2 and 4 weeks with group 1 animals demonstrating more than threefold

higher technetium uptake as compared with group 2 animals (-collagen gel; p< 0.01 at each time

point). Interestingly, at the 6 week time point the radio-nucleotide uptake in group 1 animals

(+collagen gel) returned to baseline levels but remained markedly abnormal in the animals

treated without collagen gel and in those in which collagen gel was supplemented with TGF-β1.

This finding demonstrates that increased TGF-β1 expression during wound repair inhibits

lymphatic regeneration.

Increased lymphatic transport noted on lymphoscintigraphy was associated with

decreased tail volumes and more rapid resolution of acute lymphedema in group 1 (+collagen

gel) animals as compared with group 2 (-collagen gel) and in animals treated with recombinant

TGF-β1. Even 2 weeks after surgery, the increase in tail volume of the group 1 animals was

nearly 4 fold less than that observed in group 2 animals as well as animals treated with

recombinant TGF-β1 (p<0.05, Figure 3C). By 4 weeks postoperatively, tail volumes in group 1

returned to baseline where as 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 collagen gel, and resulted

in change in tail volumes almost indistinguishable from group 2 animals. This trend was

maintained at the 6 week time point with only minimal improvement in tail volumes in group 2

and the supplemental TGF-β1 group. In addition, histologic sections of wounds and the distal

portions of the tails obtained 6 weeks after treatment with TGF-β1 demonstrated changes

consistent with chronic lymphedema (i.e. fat deposition, thickened dermis/subdermis, and


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subdermal fibrosis; Figure 4). Thus, inhibition of TGF-β1 expression during wound repair is

associated with improved lymphatic function and decreased acute lymphedema. In contrast,

increased TGF-β1 expression either endogenously (i.e. group 2) or as a result of TGF-β1

supplementation is associated with delayed lymphatic regeneration and prolonged acute

lymphedema.

Increased expression of TGF- β1 during wound repair is associated with delayed recruitment

and decreased proliferation of lymphatic endothelial cells

In order to evaluate the cellular mechanisms by which increased TGF-β1 expression

during wound repair can inhibit lymphatic regeneration, we quantified the number and

proliferation of lymphatic endothelial cells (LEC) present in the wound. This was based on

previous work by Boardman et al demonstrating that the recruitment of LECs beginning distally

and migrating proximally is crucial for lymphatic regeneration in the mouse tail model. (35)

Thus, LECs were quantified and microscopic lymphatic regeneration in the distal portions of the

wound was analyzed using LYVE-1 immunofluorescence (Figure 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.

The reduced number of LECs in group 2 wounds and in animals treated with

supplemental TGF-β1 was due in part to inhibition of LEC proliferation (Figure 5B,C). When

the number of proliferating LECs was calculated by co-localization of PCNA and LYVE-1, we

noted that animals in group 1 (+collagen gel) had nearly 6 times more proliferating LECs in the

distal portion of the wound as compared to group 2 (-collagen gel) and animals treated with


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supplemental TGF- β1 (p<0.01). There was no statistical difference in the number of

proliferating LECs between group 2 and 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 (Figure 5D). This analysis

demonstrated a dose dependent and statistically significant decrease in LEC proliferation in vitro

thus suggesting that 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 (Figure 6 A). As expected, in group 1 animals (+ collagen gel), lymphatic capillary

formation was noted at the earliest time points with infiltration and coalescence of LECs. By 2

weeks, the newly formed lymphatics demonstrated a capillary and cylindrical architecture with

mild dilation noted in some vessels. In contrast, group 2 animals (- collagen gel) and 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 week time

points evaluated with only an occasional normal, collapsed cylindrical capillary lymphatic

present. In contrast, the capillary architecture of group 1 (+collagen gel) animals was similar to

normal skin by the 6 week time point with cylindrical, small collapsed capillary lymphatics.

In order 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


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significant and dose-dependent decrease in the potential for lymphatic tubule formation (Figure

6B). In the absence of supplemental TGF-β1, lymphatic tubule formation occurred rapidly and

multiple small tubules were observed after 24 and 48 hours. In contrast, large, ectatic and dilated

lymphatics similar to those noted in group 2 (-collagen gel) and supplemental TGF-β1 treated

animals were noted in the low dose TGF-β1 group. Treatment with high dose TGF-β1,

(10ng/ml) all but abolished 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 physiologic range of this growth factor

in vitro.(45) Thus, increased TGF- β1 during abnormal wound repair significantly attenuates

LEC function and tubule formation and may contribute to delayed lymphatic regeneration and

increased acute lymphedema.

In order to determine whether 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 (Figure 6 C,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 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.


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Increased TGFβ1 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 (ie. fibroblast)

differentiation occurs in a number of scenarios (e.g. peri-tumoral fibrosis, renal fibrosis) and that

this effect is mediated by TGF-β1 (29, 31). In these cases, direct differentiation of microvascular

endothelial cells to fibroblastic phenotype (i.e. expression of fibroblast markers such as alpha-

smooth muscle actin (SMA) leads to tissue fibrosis. Lymphatic fibrosis has been shown in

anatomic and histologic studies of patients with lymphedema.(48) Therefore, we analyzed alpha

SMA expression in wound sections in order to evaluate the possibility of LEC-fibroblast

differentiation as a molecular mechanism of lymphatic fibrosis resulting from increased TGF-β1

expression during 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 (Figure 7A). In addition, we noted that numerous LECs

present in the capillary lymphatics (ie LYVE-1 positive) co-expressed 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 (Figure

7B,C). Consistent with previous histologic studies of lymphatic fibrosis, the capillary lymphatics

that co-expressed lymphatic and fibroblast markers appeared to be thickened and fibrotic

indicating that endothelial-fibroblast differentiation may be present. In contrast, wounds

harvested from group 1 animals (+collagen gel) demonstrated normal lymphatic architecture and

no evidence of lymphatic fibrosis or co-expression of LYVE-1 and α-SMA. It is important to

note that capillary lymphatics, unlike collecting lymphatics, are not associated with pericytes, a


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cell type known to express alpha-SMA. (50) Therefore, expression of this fibroblastic marker by

the LECs lining the normally thin walled capillary lymphatics vessels is an abnormal finding.

These observations suggest that increased TGF-β1 expression during 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 dilation and collapse of

lymphatic channels.

Decreased scarring is associated with increased infiltration of F4/80 positive macrophages

Since macrophages are known to be important regulators of lymphangiogenesis,

differences in macrophage migration were evaluated using F4/80 immunohistochemistry.

Wounds in group 1 animals (+collagen gel) demonstrated 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 week time points, large numbers of macrophages could be observed in

the wound distally infiltrating the distal margin in the deep dermal tissues (Figure 8).

Macrophage number appeared to decrease by 6 weeks. In contrast, 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 (Figure

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.

Discussion


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Clinical studies have identified a number of risk factors for lymphedema including: 1.

Treatment related factors (e.g. surgery, radiation); 2. Disease related factors (eg. stage, nodal

status, number of lymph nodes removed); and 3. Patient factors (eg. 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 is 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, inhibition of TGF-β1 or

its intermediaries (e.g. SMAD-3), decreases scarring caused by fibrotic stimuli and radiation, and

improves wound healing. (12-14) Consistent with this, we demonstrated that treatment of

excisional wounds with collagen resulted in significantly decreased scarring and down-regulation

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

lymphatic regeneration noted in the group treated without collagen gel. Thus, inhibition of TGF-

β1 expression in the collagen gel treated animals was associated with near normal lymphatic

function by 4 weeks postoperatively, whereas 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 weeks after surgery.

TGF-β1 inhibition of lymphatic regeneration may occur as a consequence of direct

inhibitory effects of TGF-β1 on lymphatic endothelial cells. We found that even low doses of

this growth factor significantly attenuate the proliferation of isolated LECs in vitro. This in vitro


21

finding is correlated with significant decreases in the number of LECs and the number of

proliferating LECs present in the animals treated without collagen gel (with resultant increase in

TGF-β1) and those treated with supplemental TGF-β1 as compared with animals treated with

collagen gel. In addition, TGF-β1 may have direct negative effects on the potential of LECs to

coalesce into lymphatic channels as evidenced by significant inhibition of tubule formation in

vitro, and large ectatic vessels in vivo. These findings, together with a recent publication by Oka

et al demonstrating a direct inhibitory role for TGF-β1 during inflammatory and tumor

associated lymphangiogenesis (30) suggest that this pro-fibrotic molecule can directly inhibit

lymphatic repair by inhibiting assembly of lymphatic vessels, reducing LEC proliferation, and

inhibiting LEC migration. Our results add to the findings of Oka et al 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 anti-

scarring strategies in general and inhibition of TGF-β1 signaling in particular, may lead to

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 towards 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 physiologic dilatation of capillary

lymphatic vessels necessary for lymphatic fluid uptake when fluid accumulates within tissues.

Consistent with this finding we have demonstrated that increased scarring is associated with


22

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 collagen gel

or those treated with collagen gel supplemented with recombinant TGF-β1 demonstrated

numerous capillary lymphatic endothelial cells that co-expressed LYVE-1 and alpha-SMA. This

finding implies that 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 co-expressing 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 if abnormal TGF-β1 expression results in

LEC transdifferentiation. These studies are currently underway in our laboratory.

Interestingly, we did not find differences in VEGF-C expression as a consequence of

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 increased TGF-β1 expression and resultant soft tissue fibrosis/scarring do not alter

VEGF-C expression but instead change other factors in the wound that promote lymphatic


23

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 found no differences in the expression of

VEGF-C by peritoneal macrophages after inhibition of TGF-β1 activity in their inflammatory

model of lymphangiogenesis.(30) 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 interstitial flow which has been

recently identified as an important regulator of tissue morphogenesis and lymphatic

regeneration.(36) In a series of elegant experiments, Boardman et al demonstrated that interstitial

flow is an important mediator of LEC migration leading to re-connection to proximal

lymphatics.(6) 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 where as Swartz et al

have shown that interstitial flow dependent lymphatic regeneration in the tail model is dependent

on VEGF-C expression and VEGF-R3 signaling.(34) In addition, it is unlikely that there would

be significant differences in interstitial flow in animals treated with collagen as 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


24

macrophages as critical regulators of lymphangiogenesis.(18) Macrophage expression of VEGF-

C is a major regulator of lymphangiogenesis in some tumors and in inflammation.(25, 44)

Decreased macrophage number and function is associated with diminished lymphangiogenesis

and impaired wound healing in diabetic mice.(24) In addition, macrophages are thought to

directly contribute to lymphatic vessel formation by trans-differentiating into LECs. We

demonstrated that macrophages were more prominently present in the +collagen gel beginning as

early as 1 week 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 as compared with 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 amount of scarring that is present in a

wound. Further, we have shown that this effect appears to be independent of VEGF-C/D.

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 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.


25

Figure Legends

Figure 1. Treatment of mouse tail skin and lymphatic excision with topical collagen gel

inhibits 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 (100X). Note marked increase in TGF-β1expression 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 week 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 as compared with group 1 (+ collagen gel) animals (*p<0.05). C.

Quantitative PCR for TGF-B1 from total cellular RNA harvested from wounds of mouse in

group 1 and group 2 two weeks postoperatively (*p<0.002). D. Western blot of TGF-B1

protein expression from wounds of mice in group 1 and group 2 two weeks postoperatively.

Note significant increase in TGF-B1 expression in group 2 (-collagen) animals.

Figure 2. Collagen gel treatment and decreased TGF-β1 expression are associated with

decreased scarring and fibrosis.

A. Representative H&E low-power images (50X) of group 1 (+collagen gel; upper left) and

group 2 (-collagen gel; upper middle), and supplemental TGF-β1 treated animals (+collagen

gel/+TGF-β1; upper right) 6 weeks after surgery. Note increased inflammation, hypercellularity,

and tissue edema in group 2 and TGF-β1 treated wounds. B. Representative high-power images

(200x) of picro-sirius red staining in group 1 (lower left), group 2 (lower middle) and TGFβ1-

treated (lower right) wounds 6 weeks after surgery. Note increased orange/red birefringence


26

(closed arrow) and more parallel deposition of fibers (circle) in group 2 and supplemental TGF-

β1 treated wounds when compared to group 1 indicating more scarring and fibrosis. C. Scar

indices of wounds in group 1, group 2, and supplemental TGF-β1 treated animals. Note

significant decrease in scarring in the group 1 (+collagen) as compared with other groups

(*p<0.05). Data are presented as mean + SEM of quantification from 3 random high powered

fields per animal (n=4) at each time point.

Figure 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-sulfur colloid in group 1 (+collagen

gel; upper panels), group 2 (-collagen gel; middle panel), and supplemental TGFβ1 treated

(+collagen gel/+TGF-β1; lower panel) 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 weeks. In

contrast, note minimal uptake in group 2 and supplemental TGF-β1 groups even after 6 weeks.

Arrow marks the tip of the tail. B. Quantitation of radio-nucleotide uptake after

lymphoscintigraphy 6 weeks after surgery. Note significant increase in both the rate (i.e. slope

of the line) and amount of radio-nucleotide uptake in group 1 animals demonstrating more rapid

lymphatic regeneration (data are presented as mean + SEM 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 weeks in group 1 animals (*p <0.05

as compared to all other groups). In contrast, note increase in tail volume measurements and

lymphedema in group 2 and supplemental TGF-β1 treated animals even 6 weeks postoperatively.


27

Figure 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

panels) or collagen gel supplemented with 50ng of recombinant human TGF-β1 (right panels).

Note subdermal thickening (brackets in lower figures), dilated lymphatics (open arrow), fibrosis,

and hypercellularity in supplemental TGF-β1 treated wounds. Also, note fat deposition (filled

black arrow upper right panel) in the cross sections of the tail harvested distal to the surgical site

and treated with TGF-β1.

Figure 5. TGF-β1 inhibits LEC recruitment and proliferation.

A. Number of LYVE-1 positive cells (LECs) at the distal margin of the wound 2, 4, and 6 weeks

after surgery. Note significant increase in the number of LECs present in the distal portion of

group 1 (+collagen gel) as compared with group 2 (-collagen gel) and supplemental TGF-β1

treated (+collagen gel/+TGF-β1) animals (mean + standard deviation of 3 high powered

fields/animal (n=4) per time point). B. Identification of proliferating LECs (LYVE-1 and PCNA

double positive stain) in wound sections 2 weeks postoperatively (representative high powered

figures shown at 400x 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 weeks

postop. Note nearly 6 fold increase in proliferating LECs present in group 1 (+collagen gel) as

compared to group 2 (-collagen) and supplemental TGF-β1 (+collagen gel/+TGF-β1) treated

animals (*p<0.01; data are presented as mean + standard deviation of 3 high powered

fields/animal (n=4 per group). D. TGF-β1 inhibits LECs proliferation in vitro. Note


28

significant, dose dependent decrease in cellular proliferation with exposure to increasing doses of

TGF-β1 (0, 1, 10ng/ml; Mean + SD of triplicate experiments; *p<0.05).

Figure 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 (50X 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 weeks (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 (20x) of LECs treated with

various doses of TGF-β1 (0, 1, 10ng/ml) for 3 days then plated on Matrigel coated plates for 24

hours. Note dose dependent inhibition of tubule formation in the TGF-β1 treated LECs. Also,

not large ectatic tubule formation in the TGF-β1 treated groups reminiscent of our in vivo

findings (below panels: 400X representative images). All experiments were performed in

quadruplicate. C, D. Expression of VEGF-C and VEGF-D protein by LECs 24 hours after

treatment with TGF-β1 (0, 1, 10ng/ml). Note no significant difference in VEGF-C/D expression

by ELISA (mean + SD; p=NS).

Figure 7. TGF-β1 induces expression of alpha-SMA and results in lymphatic fibrosis and

co-expression of lymphatic and fibroblast markers by LECs in-vivo.


29

A. Representative photomicrographs (200x) of alpha-SMA staining in group 1 (+ collagen gel)

and group 2 (- Collagen gel) animals 6 weeks 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; upper panels) and alpha-SMA (green;

middle panel) in group 1 (+ ollagen; left), group 2 (-collagen gel; middle), and supplemental

TGF-β1 (+collagen gel/+TGF-β1; right) 6 weeks postoperatively. Co-localization (lower panels)

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 as compared to group 1.

Also, note that double stained lymphatics demonstrate enlarged lumens and thickened walls

consistent with lymphatic fibrosis (large ovals). C. A high powered image of a lymphatic

composed of cells expressing both LYVE and alpha-SMA (open arrow) as opposed to a

lymphatic with no alpha-SMA expression (open arrowhead).

Figure 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 weeks after surgery

(distal aspect of the wound is towards the right of the figures). Upper row demonstrates low-

power (50X) and lower row demonstrates magnified insert (200X). 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.


30

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TGF-�1 is a negative regulator of lymphatic regeneration during wound repair