hyaluronidase treatment of acute lymphedema in a …

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Lymphology 46 (2013) 160-172

HYALURONIDASE TREATMENT OF ACUTE LYMPHEDEMA IN A MOUSE TAIL MODEL

H.J. Jeong, K.H. Roh, G.C. Kim, Y.O. Kim, J.H. Lee, M.J. Lee, Y.-J. Sim

Department of Physical Medicine and Rehabilitation (HJJ,KHR,GCK,JHL,MJL,YJS), Departmentof Pathology(YOK), Kosin University Gospel Hospital, Kosin University College of Medicine, Busan,Korea

ABSTRACT

The purpose of this study was to investi-gate the impact of hyaluronidase (HAase) onlymphedema using an acute mouse taillymphedema model. Six-week old mice servedto produce acute lymphedema and were theneither treated with HAase injection or used as operative controls. An additional group ofunmanipulated normal mice was used forcomparison. Tail volumes were measured for23 days and histological changes examined.Western blot analysis was conducted to quan-tify lymphatic vessel endothelial hyaluronanreceptor (LYVE)-1, tumor necrosis factor(TNF)-α, transforming growth factor (TGF)-ß1, podoplanin, CD 44, and vascularendothelial growth factor receptor3 (VEGFR3)expression levels. The operative control groupshowed an increase in thickness of the dermisand subdermis, microlymphatic dilatation, and an increase in neutrophils. In contrast,the HAase treated group exhibited alleviationof inflammation evidenced by a decline inmicrolymphatic dilatation and neutrophils andan overall increase in microlymphatic vessels.Western blot analysis demonstrated that TNF-α and TGF-ß1 expression declined butCD44 expression increased in the HAasetreated group. Levels of LYVE1, podoplanin,and VEGFR3 also increased significantly inthe HAase group. Our results indicate thatHAase treatment in the acute mouse tail

model reduced lymphedema volume possiblythrough degradation of HA trafficking, whichreduced inflammation and fibrosis in tissuesand stimulated lymphangiogenesis.

Keywords: lymphedema, inflammation,lymphangiogenesis, fibrosis, hyaluronidase,mouse tail

The lymphatic system consists of a keynetwork of vessels which maintains tissuefluid homeostasis and prompt regionalinflammatory and immune responses (1).When the anatomical and functional integrityof lymphatic vessels is impaired, disruption in fluid transport causes abnormal fluidaccumulation in skin and subcutaneoustissues termed lymphedema (2). Lymphedemacan be classified into primary and secondarylymphedema forms with secondary lymphe-dema attributable to various factors such asinfection, malignant tumor, surgery, radiationtherapy, chronic venous insufficiency, andtrauma (1).

Despite continuing developments inconservative and surgical treatmentapproaches, secondary lymphedematreatment options have been limited (2).Some investigations have found that growthfactors can induce lymphangiogenesis to treat secondary lymphedema, but clinicalapplication remains limited (1). Althoughsecondary lymphedema has been defined

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as a high protein edema, Liu and colleagues(3) found in some lymphedema patients thatprotein concentration levels were lower in thelymphedema tissues than normal tissues andthat the level of hyaluronic acid (HA) wasclearly higher in the lymphedema tissuescompared to normal. Moreover, in lymphe-dema, macromolecules such as HA build upin the interstitium hampering cell traffickingand triggering cytokine release. These eventsresult in the trophic changes associated withchronic lymphedema. Therefore, lymphedema,which is viewed as a high protein edema,actually shows stronger correlation to HAthan to protein level. In addition, HA ispartly metabolized in lymph nodes beforeentering the general circulation and rapidlydegrading in the liver. Therefore, the lymphcirculation is the main transport pathway for HA from tissue, and an impaired lymphcirculation promotes interstitial fluidstagnation and changes the metabolism ofHA (4). In the tissues, HA acceleratesextravasation of lymphocytes and macro-phages from the blood circulation andpromotes their migration to sites ofinflammation. Impaired trafficking of HAcan initiate inflammation in edematoustissues and affect collagen metabolism therecausing fibrosis, which is a key pathologicfeature of chronic lymphedema (5).

Recently, studies in secondary lymphe-dema examining structural and functionaltissue changes found higher gene expressionof proinflammatory cytokines in untreatedpatients compared to those treated withcomplex decongestive physiotherapy (6).Tabibiazar et al (7) observed in an experi-mental acute lymphedema model that thedermis and subdermis exhibited intense acuteinflammatory changes and used transcrip-tional profiling to demonstrate upregulationof genes related to acute inflammation,immune response, complement activation,wound healing, fibrosis, and oxidative stressresponse. Accordingly, inflammation waspostulated to be central to the pathogenesis of lymphedema, creating fibrosis in the

transition phase from acute to chroniclymphedema or triggering trophic changes intissues, and further, that inflammation shouldbe a very important factor evaluated in futurestudies of various lymphedema treatments.

Hyaluronidase (HAase) is one of fourglycosaminoglycans that build the dermalextracellular matrix and a class of enzymeswhich degrade HA. Luagier et al (8) found inboth in vivo and in vitro studies that topicalHAase significantly reduces HA in theepidermis and subdermis layers. Theirfindings suggest that HAase might be helpfulin alleviating HA stagnation in the dermisand subdermis of secondary lymphedema.Therefore, the use of HAase in lymphedemapatients would be expected to promote celltrafficking and a reduction in lymphedemavolume. However, few studies have beenconducted on the effect of HAase on secon-dary lymphedema and there have been nostudies that we are aware of focusing on tissuechanges resulting from HAase treatment.

Therefore, in a mouse tail model of acute secondary lymphedema, we investi-gated whether HAase injection can: 1) reduce lymphedema volume; 2) alleviateinflammation and fibrosis in the dermis andsubdermis; and further 3) serve as alymphangiogenesis factor.

METHODS

Materials and Study Plan

Six-week-old ICR female mice weighing25-30 grams were housed and maintained ona 12-hour day-night cycle at 23ºC ambienttemperature and supplied with water andfeed ad libitum. Mice were placed into one ofthe three groups: HAase treatment group,untreated operative control group, and anormal unmanipulated group. Tail volumeswere obtained for all three groups and tissueswere examined at multiple time points toexamine histological changes. Animal studiesconformed to the Guide for the Care and Useof Laboratory Animals published by the US


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National Institutes of Health (NIH PublicationNo. 85-23, revised 1996), and the study wasapproved by the Animal ExperimentationEthics Committee of Kosin University.

The unmanipulated normal mice wereobserved for 23 days without any clinicalinterventions. To create the mouse model of acute lymphedema, operative procedures(see below) were performed on mice in bothHAase and untreated control groups, andlymphedema was allowed to develop for ninedays. On postoperative day 9, the HAasegroup was given HAase injections in the tail.Histological changes for 14 days (to 23 dayspost-op) were observed in both HAase andcontrol groups (Fig. 1).

METHODS

Acute Lymphedema Model

To create a mouse tail model of acutesecondary lymphedema, ICR female mice(25-30 grams) were anesthetized by intra-peritoneal injection of tiletamine/zolazepam(Zoletil® 50; Virbac, France, 30mg/kg) andxylazine hydrochloride (Rompun®; Bayer,Germany, 10mg/kg). Dermis and subcuta-neous tissues (5-10 mm) near the base of thetail were removed without injuring the bloodvessels on both side of the tail, and antibioticcream was applied to prevent infection.Lymphedema volume in the tails was

Fig. 1. Operative procedures were performed on tails of mice to produce acute lymphedema. On postoperative day9, mice were divided into either a HAase treatment group with two injections of HAase into the tail distal to theoperative site or to the control untreated group. Mice were then followed for 14 days until postoperative day 23.


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previously reported to be most pronounced onpostoperative day 9 and persisted for 23 daysafter operation (9).

Hyaluronidse administration

150 IU/0.1ml of HAase(BMHYLUNIDASE INJ®; BMI KOREA,Korea) was injected with a 26 gauge needleon both dorsal and ventral sides approxi-mately 15mm distal from tail base (distal tothe operative area) and observations weremade for 14 days thereafter.

Measurements of Volume in Mouse TailLymphedema

For each group, the diameter of mousetails was measured with an electronic caliperfor 23 days after the surgery at intervals of10mm up to 70mm distal from tail base.Diameters were converted to circumferencesand a standard truncated cone formula wasused to determine volume (10).

Lymphoscintigraphy

To examine function of lymphaticvessels, two mice were randomly selectedfrom both HAase and operative controlgroups on postoperative days 0, 9, and 23 andimaged with lymphoscintigraphy. Underanesthesia, 99mTc-phytate (100 uCi/0.02 ml)was intradermally injected 70mm distal fromthe base of the tail. Images were obtained at10, 30 and 60 minutes after injection using amicroSPECT INFINIA gamma camera (GEmedical system, USA) equipped with a LEGP(low energy general purpose) collimator.

Histology

To examine histological changes, threemice from the HAase and control groupswere euthanized by carbon dioxide asphyxi-ation on postoperative day 9, 16 or 23, andtails were removed for immediate fixation in formalin. In addition, 3 mice from the

unmanipulated normal group were euthan-ized and their tails collected as above. Onemm-thick cross-sectional pieces of tissue wereobtained from the area located 15mm fromthe base of the tail for tissue examination. Alltissues were embedded in paraffin, sectioned,and stained with hematoxylin and eosin forevaluation of histological changes.

Immunohistochemistry

To identify lymphatic vessels, 5µmparaffin sections were cut and mounted onorganosilane-coated glass slides for immuno-histochemical (IHC) staining. After deparaf-finizing and rehydrating, antigen retrievalwas performed in citrate buffer for 10minutes at 121ºC. Sections were blocked andincubated overnight at 4ºC with LYVE-1(Abcam, Cambridge, UK) followed bysecondary horseradish peroxidase-conjugatedantibody (DAKO, Glostrup, Denmark) forone hour at RT. Slides were then developedwith DAB (DAKO) and counter-stained with Mayer’s hematoxylin (DAKO) andimages captured with a Nikon ECLIPSE 50imicroscope (Nikon, Tokyo, Japan).

Immunofluorescence

To confirm the presence of lymphaticvessels through immunofluorescence analysis,LYVE-1 (Abcam), Podoplanin (Santa Cruz,CA, USA), and PROX-1 (Millipore, CA,USA) were used as primary antibodies withAlexa Fluor 488 goat anti-rabbit or AlexaFluor 594 donkey anti-mouse (Invitrogen,CA, USA) secondary antibodies. Images werecaptured with a laser scanning microscope(LSM510 META, Carl Zeiss, Jena, Germany).

Western Blot analysis

To measure changes in levels of CD44,tumor necrosis factor-α (TNF-α), trans-forming growth factor-β1 (TGF β1), LYVE-1,podoplanin, and vascular endothelial growthfactor receptor 3 (VEGFR3) expression, at


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least 10 mice from each group wereeuthanatized with carbon monoxide onpostoperative day 0, 9, 16, or 23 and tailswere removed for immediate formalinfixation. RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodiumdeoxycholate, 2.5 mM sodium pyrophosphate,1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM PMSF) was used toseparate proteins in tail tissues. BCA proteinassay kit (Pierce, Rockford, IL, USA) wasused to perform protein quantification, andsodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) was employedto separate proteins by electrophoresis. Afterthe protein transfer from SDS-PAGE gels toPVDF membrane (Roche Diagnostics GmbH,Mannheim, Germany), the membrane wasblocked (1X TBS, 0.1% Tween-20 with 5%w/v nonfat dry milk) for one hour at roomtemperature to prevent non-specific antibodyinteractions. Primary antibody was incubatedovernight at 4ºC and then rinsed three timeswith TBS-Tween-20 solution. The blots wereincubated in a peroxidase-conjugated secon-dary antibody for 1 hour at room temperatureand then rinsed three times with TBS-Tween-20 before their exposure to X-ray film(FUJI FILM Co. Tokyo, Japan) equippedwith ECL kit (Amersham Pharmacia Biotech,Piscataway, NJ, USA) to confirm the proteinexpression. Primary antibodies LYVE1 andTNF-α were purchased from Abcam (Abcam,Cambridge, UK) and TGF-β1, podoplanin, CD 44, VRGFR3, actin and secondaryantibodies were obtained from Santa CruzBiotechnology (Santa Cruz, CA, USA).

Statistical Analysis

SPSS for windows (version 14.0) wasused to conduct statistical analysis forchanges in diameter and volume of mousetails and to calculate average and standarddeviation of the three groups. One-wayANOVA was used to compare volumechanges between the three groups. To

measure fibrosis, inflammation, andlymphatic vessels, the levels of TGF-β1, TNF-α, CD44, LYVE-1, podoplanin,VEGFR3 in the HAase group were monitoredover time with one-way ANOVA. Student’s t-test was used to compare the HAase andcontrol groups. A p-value of 0.05 was used as a cutoff for all statistical significance.

RESULTS

Lymphedema Is Reduced by Hyaluronidase

Tail diameter and volumes weremeasured to 70mm distal from tail base in the HAase (N=8), control (N=8), and normalmice groups (N=8). The HAase and controlgroups exhibited significant increase involume starting from postoperative day 5compared to the normal group (p<0.05). In particular, HAase and control groupsdisplayed nearly twice the volume comparedwith the normal group at postoperative day 9.Thereafter, tail volumes declined in theHAase treated group (a 47% decrease fromthe maximum volume of 1.115±0.089 cm3 to0.702±0.080 cm3; p<0.05) but remainedunchanged in the control group (Fig. 2A).Comparison between the HAase and controlgroups on post-operation days 16 and 23demonstrated that tail volumes in the HAasegroup significantly declined compared to the control group. At day 23, tail volume in the control group was unchanged while tailvolume in the HAase treated group hadreturned to near normal levels (Fig. 2). In addition, the thickness of dermis andsubdermis clearly reduced in the HAasegroup on postoperative days 16 and 23 com-pared to the control group and returned tonear normal on postoperative day 23 (Fig. 2).

Hyaluronidase Therapy Can ImproveHistological Changes in SecondaryLymphedema

Qualitative and histomorphometricanalysis of paraffin-embedded, hematoxylin/


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Fig. 2. Tail volume changes following acutelymphedema operation. HAase and control groupsdisplayed nearly twice the volume increase comparedwith unmanipulated normal group at postoperationday 9. Tail volume of the HAase treated groupgradually decreased to near the normal group, butvolume of the control group was maintained (A).The HAase treated group displayed a decrease ininterstitial fluid volume compared with controlgroup at days 16 and 23 (H & E stain) (scale bar =1mm) (B) and a decrease in the number ofinflammatory cells at day 23 (H & E stain) (C).

Fig. 3. Immunofluorescence images using LYVE-1,Podoplanin, and PROX-1 demonstrate cross-sectional morphology of lymphatic vessels (scale bar= 20um) (A). An increase in size and dilation of thelymphatic vessels was seen by LYVE-1 immuno-histochemistry in the control (arrows), and thedilation was reduced in the HAase treated group atday 23 (B).

eosin-stained dermis and subdermis sectionsobtained on postoperative day 9, 16 or 23revealed acute inflammatory changes. Theoperative control group exhibited a 2- foldexpansion of tissue between the bone and theepidermis, and dermis and subdermis showeda high degree of neutrophil infiltration (Fig. 2). These changes remained until day23. Fibroblasts, a possible cause of fibrosis,

were observed in the subdermis. HAaseinjection treatment led to a decrease ininflammation (reduction in neutrophils) onpostoperative day 16 and near normalhistological appearance (thickness of dermisand subdermis) at postoperative day 23 (Fig. 2C).

The histological similarity between bloodvessels and lymphatic vessels made it difficultto differentiate them. To clearly identifylymphatic vessels, double staining wasconducted with combinations of either anti-LYVE-1 and anti-prox1 antibodies or anti-LYVE-1 and anti-podoplanin antibodies.Immunofluorescence images showed lym-phatic endothelial vessels were double-stainedwith anti-LYVE-1 and anti-podoplanin


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antibodies and anti-LYVE-1 and anti-prox1antibodies at postoperative day 23.Lymphatic vessels were marked by irregularround shapes (Fig. 3A).

Immuohistochemistry was performed toidentify lymphatic vessel changes resultingfrom HAase injection. At day 9, lymphaticvessels exhibited dilatation and more irregu-lar shapes. HAase injections clearly increasedthe number of lymphatic vessels and reducedthe dilatation in lymphatic vessels of theHAase group at 23 days (Fig. 3B).

Hyaluronidase Therapy ReducesInflammation and Fibrosis

Western blot analysis confirmed expression of tumor necrosis factor-α (TNF-α), aprominent inflammatory mediator inlymphedema. TNF-α expression increased on postoperative day 9 compared to day 0(p<0.05). Thereafter, the operative controlgroup did not exhibit significant changes inTNF-α expression while the HAase groupshowed a significant reduction (p<0.05) in

Fig. 4. The HAase treated group showed a significant reduction in TNF-α (A) and TGF-ß1 (C) and a significantincrease in CD44(B) expression over time while the control group remained stable. The HAase treated group alsodemonstrated a significant decline in TNF-α (A) and TGF-ß1 (C) and a significant increase in CD44(B) expressionon postoperative days 16 and 23 compared to the untreated control group. (*p<0.05 compared with control, †p<0.05change over time).


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expression on postoperative days 16 and 23from both day 9 and compared to the controlsat each day (p<0.05) (Fig. 4A). In addition,expression of CD44 (a HA receptor) wasincreased after day 9 and continued to riseafter HAase injection at days 16 and 23(p<0.05). This increase was also significantlydifferent from the untreated controls at days16 and 23 (p<0.05) (Fig. 4B).

Expression of TGF-β1, a negativeregulator of lymphatic regeneration, wasquantified to examine the level of fibrosis.

TGF-β1 expression was significantlyincreased (p<0.05) on postoperative day 9compared to day 0. Thereafter, both controland HAase groups declined in TGF-β1expression over time with statisticallysignificant declines in the HAase group aswell as a significant difference between thegroups at days 16 and 23 (p<0.05) (Fig. 4C).

Hyaluronidase Therapy PromotedLymphangiogenesis

Fig. 5. The HAase treated group shows a significant increase in LYVE-1 (A), podoplanin (B), VEGFR3 (C)expression over time while the untreated control group remained stable. The HAase treated group also experienceda significant increase in LYVE-1(A), podoplanin (B), VEGFR3 (C) expression on postoperative day 23 compared tothe untreated control group. (*p<0.05 compared with control, †p<0.05 change over time).


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Western blot analysis to quantifylymphatic regeneration found that comparedto day 0: LYVE-1 expression increased onpostoperative days 16 and 23 after HAaseinjection (significantly at day 23); podoplaninexpression reached a peak on postoperativeday 16 (also significant at day 23, p<0.05);and levels of VEGFR3 expression signifi-cantly increased on postoperative day 16 and23 (p<0.05). In comparison to the untreatedcontrols, LYVE-1 and podoplanin expressionswere higher in the HAase group at days 16and 23 (significant at day 23), and VEGFR3expression was significantly higher at bothdays 16 and 23 (p<0.05) (Fig. 5).

Hyaluronidase Therapy Improved LymphaticFunction

Lymphoscintigraphy performed onpostoperative day 9 showed that the controlgroup had a slower flow of lymph fluid/tracerand a lower level of lymphatic drainage thanthe normal unmanipulated group with renalnodes starting to appear at 30 minutes afterradionuclide injection. The presence of dermalbackflow was observed in areas where dermaland sub-dermal tissues were removed andwhere lymphedema was found in the mousetails (Fig. 6A). Lymphoscintigraphy tests ofthe untreated control group conducted onpostoperative day 23 showed that radionuclideuptake did not increase at lymph nodes 1hour after radionuclide injection, indicatingthat the lymph flow rate of the control grouphad deteriorated further. In contrast, thelymph flow of the HAase group had improvedand was restored close to the normalunmanipulated group on postoperative 23day (Fig. 6B).

DISCUSSION

The acute mouse tail model of lymphaticcirculatory failure causes lymph stasis in the interstitium. It is thought that blood flowmay increase as a compensatory responsethat reduces fluid overload in the tissues,

but this would not significantly improve thetransport of excess water from the interstitium.As lymphedema continues, the exact causes

Fig. 6. Lymphoscintigraphy was performed using anintradermal injection of 100 uCi/0.02 ml of filtered99mTc phytate. Dynamic and static images wereacquired using a low energy general purposecollimator in a microSPECT INFINIA gammacamera. At day 9, renal nodes (arrow head) werevisualized early in the unmanipulated normal groupwhile in the untreated control group renal nodeswere visualized 30 minutes after injection anddermal back flow (arrow) was observed (A).Lymphatic flow in HAase treated group at day 23demonstrated improved flow and nodal imagingcompared to the untreated control (B).


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of water retention remain uncertain (11).Possible factors include protein accumulationwithin tissues or impaired lymph flow. Inother words, lymphedema is known to be ahigh protein edema in which protein accumu-lation in the interstitium and increasedoncotic pressure stimulates more edema.However, recent studies found that in theabsence of inflammatory conditions the levelof protein in lymphedema-affected areas didnot increase compared to that of healthycounterparts but instead showed a higher HAconcentration demonstrating the importanceof HA in the development and pathogenesisof lymphedema (11). As a large polymer ofN-acetyl-D-glucosamine and D-glucuronicacid, HA maintains tissue integrity andfacilitates cell migration during inflammation,wound repair, and embryonic developmentperiods (11).

The development of lymphedemainvolves a more complicated pathogenesisthan simple defects in lymph vessel function.Among the most widely discussed pathologicevents include inflammatory changes andfibrosis-caused chronic inflammation asoligosaccharide breakdown products of HAhave inflammatory properties (macrophagesrelease chemokines and cytokines) (12,13).Lymphatic vessels also serve as channels forinflammatory cell migration as well as HA,and this process may be facilitated by adhe-sion between CD44 receptor of inflammatorycells and the LYVE-1 receptor on lymphaticendothelial cells mediated by HA (5). Assuch, HA trafficking caused by defectivelymphatic vessels has little to do with bloodcirculation but its accumulation is solelydriven by lymphedema fluid, which may leadto prolonged inflammation and increasedtissue fibrosis and cause trophic changes inthe tissues (3).

HAase is an enzyme that causes naturaldegradation of HA (14). Clinically, HAase isknown to facilitate the absorption and disper-sion of drugs, and the FDA has approved its use for 1) retrobulbar anesthetic block ineye surgery and 2) hypodermoclysis (15).

We aimed to test whether HAase can reduceinflammation, one of the primary pathogenicfeatures of lymphedema, and also examinetherapeutic potential of HAase treatment tolymphedema tissues. If HAase causes HAdegradation and thus HA trafficking decreasesin tissues, interactions between HA-mediatedreceptor CD44 and LYVE-1 should change,reducing inflammation and promotinglymphangiogenesis.

This study found that HAase treatmentin the mouse tail model led to a visible declinein the swelling of lymphedema and a returnto near-normal conditions two weeks after thetreatment. In addition, lymphoscintigraphyshowed that lymphatic function improvedafter the HAse injection. We believe theexplanation for the reduction of lymphedemaand improvement of lymphatic functionfollowing HAase treatment is: (a) a decline inHA trafficking and suppressed interactionwith CD44 and reduced acute inflammation,(b) a reduction of TGF-β1, a key inhibitor oflymphatic regeneration leading to a decline in fibrosis; and (c) an increase in lymphangio-genesis through VEGFR3. This hypothesis is supported by H&E staining demonstratinga reduction in the thickness of dermis andsubdermis and a clear reduction in inflamma-tory cells. Further, immunohistochemistryfindings show an increase in lymphaticvessels and a reduction in lymphaticdilatation compared to the peak onset oflymphedema.

TNF-α expression is significant in thisstudy. Tabibiazar et al (7) found an increasein inflammatory mediators such as TNF-αin whole tissue homogenates from mice withlymphedema, and Nakamura et al found thatanti-inflammatory Ketoprofen is an effectivetreatment to reduce lymphedema (1). ButNakamura et al argued that direct TNF-αinhibition did not lead to lymphedemareduction, and inflammation reductioncombined with TNF-α activity preservationreduced lymphedema through VEGF-Csignaling (1). This study found that TNF-αexpression fell in the HAase treated group


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compared to the control on postoperativedays 16 and 23 indicating that HAase injectionalleviated inflammation and contradictingexisting data that TNF-α preservation playeda key role in reducing lymphedema. Furtherstudies are therefore needed to determine therole of TNF-α in reducing inflammation andlymphangiogenesis.

The HAase treated group exhibited avisible decline in TGF-β1 expressioncompared to the control group, and thisfinding is supported by prior researchdemonstrating that inhibition of TGF-β1expression stimulated lymphatic regenera-tion, and increased TGF-β1 expressionreduced LEC proliferation and function andstimulated fibrosis (16). This finding indicatesthat HAase may reduce fibrosis in lymphe-dema and improve LEC proliferation andlymphatic function.

Studies on the molecular treatment oflymphedema have concentrated on thepromotion of lymphangiogenesis. In partic-ular, many investigators believe in the use ofrecombinant growth factors in redevelopinglymphatic vessels as a new approach tolymphedema treatment. Vascular endothelialgrowth factor C (VEGFC) and vascularendothelial growth factor receptor 3(VEGFR3) are key factors in lymphangio-genesis. VEGF gene therapy and exogenousVEGFC administration have been reported to resolve experimental lymphedema bystimulating lymphangiogenesis (17-19). Ourstudy found that VEGFR3 increased in theHAase treated group, suggesting a correlationbetween HAase treatment and VEGFR3 inlymphedema. After HAase injection,lymphatic specific markers such as VEGFR3,LYVE-1, and podoplanin also increased inthe HAase group supporting that HAasetreatment caused lymphangiogenesis andthus improved lymphedema.

This study supported the role of HAasetreatment in creating lymphangiogenesis andreducing inflammation, and there are threeplausible mechanisms: (a) HAase reducedinflammation and stimulated lymphangio-

genesis; (b) the reduction of inflammationstimulated the activity of VEGF-C/VEGFR3and thus caused lymphangiogenesis; and (c) HAase increased VEGFR3 directly andVEGFR3 reduced inflammation. Althoughprior studies found that VEGF-C has an anti-inflammatory effect through VEGFR3,and the activation of VEGFR3 has a potentanti-inflammatory effect in a psoriasis murinemodel (20,21), CD44 also increased in theHAase group. This effect is possibly attrib-utable to a negative feedback loop due to HA reduction. Given other prior researchfindings that an increase in CD44 plays acritical role in reducing lung inflammation(22), we cannot rule out the possibility thatan increase in CD44 directly reduced inflam-mation in our model.

Recent investigations of lymphedematreatments have also focused on therapeuticlymphangiogenesis with various growthfactors, and stem cells are used as treatmentmethods (23, 24). However, considering thatthe efficacy of stem cells is maximized whencombined with growth factors (23,24) and the use of growth factors is subject to clinicalconsiderations concerning promotion oftumor metastasis (25), clinically applicabletreatments are not in place. So far, only a fewstudies have been conducted on modulationof drug-induced lymphangiogenesis, but mosthave shown inhibition of lymphangiogenesis(26). Therefore, HAase treatment could playan important role in increasing lymphangio-genesis and reducing inflammation andfibrosis. In addition, future studies are alsoneeded to clarify the relationship(s) betweeninflammation and lymphangiogenesis.

In mammals, HA exists in high-,intermediate-, and low-molecular weightforms. HA can be depolymerized into smalloligosaccharides via oxygen radicals andenzymatic degradation by hyaluronidases,beta-glucuronidases, chondroitinases, andhexosaminidases (27). The various molecularweight forms of HA have vastly differenteffects on inflammation, with high molecularweight HA appearing to attenuate the


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inflammatory response (28). In this study, we investigated the impact of HAase onlymphedema using an acute mouse taillymphedema model. However, we did notexamine whether HAase itself has a directeffect or indirectly through HA fragmentsproduced by degradation. This issue shouldbe clarified in future studies.

In summary, these study results in theacute mouse tail model indicate that HAasetreatment reduces lymphedema volume, andthe underlying mechanism may be a reduc-tion in HA trafficking in lymphedema tissues,which in turn reduces inflammation andfibrosis and stimulates lymphangiogenesisthrough increased expression of VEGFR3.These promising results regarding HAasetreatment should be further explored as apossible treatment strategy for patients withlymphedema.

ACKNOWLEDGMENT

This research was supported by BasicScience Research Program through theNational Research Foundation of Korea(NRF) funded by the Ministry of Education,Science and Technology (2011-0008852).

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Young Joo Sim, M.D.Department of Physical Medicine

and Rehabilitation Kosin University College of Medicine 34, Amnam-Dong, Seo-Gu,Busan, 602-702, Korea Phone: 82-51-990-6156 E-mail:[email protected]


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