Vascularity during wound maturation correlates with fragmentation of serum albumin but not ceruloplasmin, transferrin, or haptoglobin

Vascularity during wound maturation correlates withfragmentation of serum albumin but not ceruloplasmin,transferrin, or haptoglobin

Christine Bolitho, PhD; Munira Xaymardan, PhD; Garry W. Lynch, PhD; Hans Zoellner, PhD

Cellular and Molecular Pathology Research Unit, Department of Oral Pathology and Oral Medicine, University of Sydney, Westmead Centre for Oral

Health, Westmead Hospital, Westmead, Australia

Reprint requests:Hans Zoellner, Cellular and Molecular

Pathology Research Unit, Department of

Oral Pathology and Oral Medicine, The

University of Sydney, Westmead Centre for

Oral Health, Westmead Hospital, NSW,

2145, Australia.

Tel: 161 2 9845 7892;

Fax: 161 2 9893 8671;

Email: [email protected]

Manuscript received: March 28, 2009

Accepted in final form: December 22, 2009

DOI:10.1111/j.1524-475X.2010.00572.x

ABSTRACT

Reduced vascularity during wound maturation is mediated by endothelial apo-ptosis. Albumin has an anti-apoptotic activity for endothelium, which increasesup to 100-fold on albumin fragmentation (AF). We now report that levels of AFcorrelate with changing vascularity during wound maturation. Both scarring andadipogenic wound-healing models were established in mice. Western blots ofgranulation tissue revealed AF concurrent with periods of high vascularity as de-termined by thin-section microscopy, with reduced AF on wound maturation(p < 0.02). In profiling AF, the levels of 27.5 and 39 kDa fragments were reducedon maturation of both scarring and adipogenic wounds (p < 0.005), as were thelevels of an additional 17.5 kDa fragment prominent only in adipogenic wounds(p < 0.001). A 49 kDa albumin fragment was found to be reduced during matu-ration of scarring (p < 0.001) but not adipogenic wounds. For comparison, weprobed for transferrin, ceruloplasmin, and haptoglobin fragmentation on the ba-sis that like albumin, these are considered acute-phase transport proteins. Min-imal fragmentation of transferrin and ceruloplasmin was seen, along with partialdissociation of haptoglobin subunits, but these did not correlate with AF or vas-cularity. Our findings are consistent with a role for AF in regulating granulationtissue vascularity during healing.

Endothelial cells form the inner lining of the microcircula-tion and endothelial apoptosis is central to both physio-logical and pathological microvascular remodelling.1–4

Conventional soft tissue wound healing involves the for-mation of highly vascular reparative granulation tissue,followed by maturation of this to scar tissue containingvery few blood vessels. Of particular importance to thecurrent work is that endothelial apoptosis is responsiblefor reducing the vascularity of reparative granulation tis-sue during wound maturation5,6 A number of cytokine,chemical, and physical factors induce endothelial apopto-sis,7–9 while the endothelium becomes apoptotic if de-prived of adhesion.10,11 However, the relevance of thesefactors to microvascular remodeling in vivo remains un-clear, particularly during wound maturation, where the re-ducing vascularity of reparative granulation tissue ismarked.

In vivo observations indicate that microvascular seg-ments in granulation tissue and elsewhere undergo ap-optotic degeneration when there is little or no bloodflow.1,3 This coupling between blood flow and endothelialapoptosis appears to optimize microvascular shape, al-though the precise mechanism is unknown. Serum depri-vation causes rapid endothelial apoptosis in both isolatedcell culture and tissue explants.10–14 This has led to thesuggestion that chemical plasma factors contribute to mi-crovascular remodelling by inhibiting endothelial apopto-sis, and serum albumin is one plasma protein known tostrongly inhibit endothelial apoptosis under serum-free

conditions.11–14 Laminar shear stress also inhibits endo-thelial cell apoptosis, providing a further mechanismthrough which blood flow may regulate microvascularform.4,15,16

The anti-apoptotic activity of albumin for the endothe-lium is identical for both recombinant and native protein,while maximal activity is seen at physiological concentra-tions.11–14 This activity is not due to serum contaminants,nonspecific protein effects, bound lipids, or radical scav-enging,11,12 and is confirmed for endothelium in both ratand human tissue explants.13 While the precise endothelialreceptor responsible for mediating the anti-apoptotic ef-fect of albumin is not known, it does appear to work via aG-protein-coupled receptor and a PI-3 kinase-dependentmechanism.12 Also, the receptor-binding domain in albu-min is only exposed by unfolding of the native protein in atransient conformational subpopulation of molecules(Figure 1A).12,14 Related to this and important for the cur-rent work is our recent report that fragmentation of hu-man serum albumin (HSA) by CNBr at Met residues

AF Albumin fragmentation

BSA Bovine serum albumin

ECL Enhanced chemiluminescence

HRP Horseradish peroxidase

HSA Human serum albumin

MSA Mouse serum albumin

Wound Rep Reg (2010) 18 211–222 c� 2010 by the Wound Healing Society 211

Wound Repair and Regeneration

mailto:[email protected]

increases the anti-apoptotic activity for endothelium up to100-fold (Figure 1B).12 This contrasts with the oppositeeffect of CNBr fragmentation in bovine serum albumin(BSA).11 The presence of a single CNBr cleavage site atMet 208 in BSA, as opposed to Leu 209 in the homologous

position in HSA, strongly implicates this protein domainas important in the antiapoptotic activity for endotheli-um.11 Separately, the N terminal 14 kDa CNBr fragmentof HSA has been experimentally excluded from mediatinganti-apoptotic activity for endothelium.12 Also, inhibition

Figure 1. Diagram illustrating the

role of transient conformational

change and protein fragmentation in

mediating the anti-apoptotic activity

of serum albumin for endothelium

(A–D), as well as the hypothesis ad-

dressed in this paper that changes in

reparative granulation tissue vascular-

ity during wound maturation reflect

changes in AF, coupled with the pres-

ence (black arrows) or absence of

blood flow in individual vessel seg-

ments (I, II, III). (A) Most molecules of

native HSA are folded to obscure the

protein domain responsible for acti-

vating the anti-apoptotic receptor.

However, intramolecular movement

generates a small population of tran-

siently unfolded HSA molecules with

the receptor-binding domain exposed

in such a way that the endothelial

anti-apoptotic receptor can be bound

and activated. Once activated, a

G-protein-coupled receptor inhibits

endothelial apoptosis via a PI3 Kin-

ase-dependent mechanism.12 Be-

cause only a few HSA molecules are

in the active transient conformational

form at any one time, high but phys-

iologically relevant concentrations of

HSA are required to achieve sufficient

levels of the active conformational

form to protect endothelium, so that

native HSA has low activity on a Mo-

lar basis.11–14 (B) Fragmentation of

HSA by CNBr exposes the antiap-

optotic receptor-binding domain,

greatly increasing availability to the

receptor so that the anti-apoptotic ac-

tivity of HSA is increased as ex-

pressed on a Molar basis.12,14 (C)

AGE formation in HSA with internal

cross-linkage reduces intramolecular

movement,40 and this reduces ac-

cess of the anti-apoptotic receptor-binding domain to its receptor, with consequent loss of anti-apoptotic activity.14 (D) Anti-apopto-

tic activity is restored to otherwise inactive HSA-AGE by CNBr fragmentation to expose the relevant receptor-binding domain.14 (I, II,

III) Vessels in newly formed reparative granulation tissue are highly permeable to albumin and formed in apparent excess, while

vessels with little or no flow are preferentially lost by endothelial apoptosis during wound maturation,1,3 due to loss of function-

associated intravascular survival signals including albumin and laminar shear stress.11–16 (I) It is hypothesized that albumin escaping

from permeable newly formed vessels is fragmented by extravascular proteinases, to expose the protein domain responsible for

binding the anti-apoptotic receptor. Activation of the endothelial anti-apoptotic receptor by albumin fragments containing the exposed

binding site would thus maintain endothelium in vessels with no flow, while intravascular albumin and laminar shear stress would

signal survival for endothelium in vessels where there is flow (black arrows).4,11,13,15,16 (II) Maturation of vessels in granulation tissue

reduces vascular permeability, so that albumin can no longer be fragmented in the extravascular compartment, depriving endothe-

lium in vessels lacking blood flow from an albumin-associated anti-apoptotic signal. (III) Apoptosis of endothelium in vessels lacking

blood flow would then optimize the structure of the microcirculation.1,3

Wound Rep Reg (2010) 18 211–222 c� 2010 by the Wound Healing Society212

Albumin fragmentation in wounds Bolitho et al.

of intramolecular movement by nonenzymatic glycosyla-tion inactivates the anti-apoptotic activity of albumin, whilefragmentation of albumin inactivated in this way restoresactivity, apparently by reexposing the anti-apoptotic recep-tor-binding domain (Figure 1C, D).14

The dependence of endothelium upon blood flow toavoid apoptosis poses a problem for the vasculature inearly wound healing, because developing microvascularsegments have little, if any, regular flow.1,3 It seems likelythat strong and as yet unidentified anti-apoptotic agentsprotect endothelium in granulation tissue early duringwound healing, before sufficient blood flow is establishedfor vascular flow to maintain vessels. Such anti-apoptoticfactors would then need to reduce in amount, allowing vas-cular form to be optimized by flow-associated factors.1,3

In light of the greatly increased anti-apoptotic activity ofalbumin upon albumin fragmentation (AF),12 here, we in-vestigate the possibility that AF occurs in wound healingand that this correlates with tissue vascularity. Our modelfor the possible role of AF in the regulation of reparativegranulation tissue vascularity is illustrated in Figure 1. Inbrief, we consider the possibility that albumin escapinghighly permeable and newly formed blood vessels in earlyreparative granulation tissue may be fragmented by extra-vascular proteinases, thus exposing the anti-apoptotic albu-min protein domain and protecting the endothelium inpoorly perfused vessels from apoptosis (Figure 1,I). We fur-ther suggest that reduced vascular permeability accompa-nying maturation of vessels in granulation tissue wouldreduce the exit of albumin from vessels into the extravascu-lar compartment (Figure 1,II), with accompanying reduc-tion in AF and induction of apoptosis of vessels having alow flow insufficient to maintain endothelium (Figure 1,III).The current study investigates this possibility by comparingAF at different stages of wound healing and maturation intwo separate soft tissue wound-healing models.

The acute-phase response is a systemic component ofotherwise localized inflammation, usually lasting approxi-mately three weeks after injury, and involving substantialchanges in the circulating levels of proteins important forthe host response.17,18 Most acute-phase proteins are ‘‘pos-itive’’ in that they increase in concentration during inflam-mation, while ‘‘negative’’ acute-phase proteins decrease inconcentration. Some acute-phase proteins are recognizedas primarily binding and transporting metals or other com-pounds.17,18 Serum albumin is accepted as a negative acutephase transport protein. In this study, we contrast AF inwounds with the fate of transferrin, which is another neg-ative acute-phase transport protein, as well as with the pos-itive acute-phase transport proteins ceruloplasmin andhaptoglobin.17,19 Most serum albumin occurs as a 66kDamonomer and a single free cysteine accounts for the for-mation of some circulating albumin dimmer (130 kDa).19

Transferrin (75 kDa) and ceruloplasmin (120 kDa) bindand transport iron and copper, respectively.17,20 Hapto-globin (73 kDa in the mouse) binds hemoglobin and occursas a tetramer of two a (27 kDa) and two b-chains (10 kDa),while free thiol groups allow polymerization and the for-mation of complexes with other proteins.21

Earlier work in this laboratory demonstrated an unex-pected pattern of wound maturation in which adiposerather than scar tissue develops from reparative granula-tion tissue.22 This unusual response is accompanied by the

differentiation of lipoblasts to adipocytes and is seen onlywhen space-occupying implants are placed into the muscletissue of mice. In recognition of the appearance of maturefat tissue, this pattern of wound maturation has beentermed ‘‘adipogenic healing.’’ With regard to the currentwork, the vascularity of granulation tissue in adipogenichealing is initially high and then reduces to low levels,comparable to vascular events in conventional healing byscarring.22 AF during adipogenic healing is compared inthe current study with events in an additional conventionalmouse wound-healing model, in which wound maturationprogresses to scar tissue in subcutaneous sponge implantssimilar to model systems described by others.5,23 For thepurpose of this paper, tissues from this sponge implantmodel system are termed ‘‘scarring wounds’’ to differenti-ate them from the adipogenic wound-healing model.22

MATERIALS AND METHODS

Establishment of wounds by polyvinyl sponge and

nylon mesh implantation

Animal experiments were approved by theWestmeadHos-pital Animal Ethics Committee. Female Balb/C mice, agedbetween 6 and 10 weeks, were used for all experiments.Mice were separated into two groups and anesthetized byintraperitoneal injection of Ketamine (8.5mg/kg) (ParnellLaboratories, Alexandria, NSW, Australia) and Rompom(2.0mg/kg) (Troy Laboratories Pty Ltd, Smithfield, Aus-tralia). Vertical incisions, approximately 15mm in length,were made over the shaved base of the tail area to exposethe underlying muscle. Adipogenic wounds were estab-lished by intramuscular implantation of nylon mesh tubes(Corning, Costar, NY), as described previously.22 Scarringwounds were established in separate mice through similarincisions by placing single fragments of polyvinyl spongematerial (Becton Dickinson Acute Care, NJ) measuringapproximately 3�3�2mm into the potential space be-tween the skin and the underlying muscle.23 Skin woundswere closed with continuous silk sutures and Temgesic(0.05mg/kg) (Reckitt & Colman, Hull, UK) was adminis-tered subcutaneously in the neck region to alleviate post-operative pain. Mice were fed ad libitum with standardlaboratory chow (Glenn Forest, Australia).

Harvesting reparative granulation tissue

Mice were sacrificed by CO2 asphyxiation from 1 to 12weeks after surgery. Implants from both scarring and ad-ipogenic lesions for paraffin histology were excised en blocand fixed with 10% neutral-buffered formalin. Granula-tion tissues for protein analysis were lysed in 250mL vol-umes of lysis buffer (Tris 50mM pH 7.4, sodium dodecylsulfate [SDS] (0.5%) with the proteinase inhibitors: apro-tinin (10mg/mL), leupeptin (10 mg/mL), pepstatin A(10 mg/mL), phenylmethylsulfonyl fluoride (1mM), soy-bean trypsin inhibitor (100 mg/mL), and N-a-p-tosyl-L-ar-ginine methyl ester (10mM) (Sigma, St Louis, MO). Solidmaterial was then separated from tissue extracts by cen-trifugation. In some cases, blood was collected from theinferior vena cava for confirmation of circulating acute-phase proteins by Western blot.


Albumin fragmentation in woundsBolitho et al.

Quantitation of vascularity in granulation tissue

Identification of vessels in 4mm paraffin sections of ad-ipogenic tissues was facilitated by Bandeira simplicofolialectin histochemistry.13 In the case of scarring tissues, un-acceptable levels of background staining, seemingly re-lated to the sponge material used, precluded the use ofspecific endothelial markers to facilitate vessel counts.However, vessels in scarring tissues were readily identifiedin 4mm paraffin sections stained using the Gomori Tri-chrome technique. Four specimens from each time pointwere examined to quantify changes in vascularity overtime. Histometric counts were performed on eight ran-domly selected fields of view in each section studied, usinga 100 square photographic graticule and a�40 objective tostandardize counts, similar to the approach reported inearlier studies.22

Western blotting for plasma proteins in granulation

tissue

The total protein concentrations of tissue extracts were de-termined using the Bradford Assay with Protein AssayReagent (BioRad, Hercules, CA). Tissue extracts wereequilibrated with regard to the total protein concentrationand run on 8–16% SDS-polyacrylamide gel electrophore-sis (PAGE) gels (BioRad). Equivalent protein loading wasconfirmed by Silver Staining (Novex Experimental Tech-nology, San Diego, CA). Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes (Per-kin-Elmer, Boston, MA) and membranes were blocked ineither 5% nonfat milk in Tris-buffered saline (Tris, 20mMand NaCl, 15mM, pH 7.5) (TBS) with Tween 20 (0.5%) orwith casein (5% in PBS pH 7.5) for blots probing for al-bumin. Polyclonal sera were used to allow detection ofprotein fragments from across differing protein domains.Primary polyclonal antibodies for mouse albumin raised inrabbit (ICN Biomedical, Aurora, OH), haptoglobin raisedin goat, transferrin raised in goat (ICN Cappel, OH), andceruloplasmin raised in rabbit (DAKO Corporation, Car-pinteria, CA) were diluted 1 : 1000 in blocking buffer andincubated with membranes for 1 hour at room tempera-ture. Secondary antibodies, consisting of HRP conjugateddonkey anti-rabbit or HRP-conjugated rabbit anti-goat(Biosource International, Camarillo, CA), were diluted(1 : 10,000) in blocking buffer and bound secondary anti-bodies were detected by ECL as per the manufacturer’s in-structions (Perkin-Elmer). Positive controls consisted ofWestern blots of mouse serum probing for all of the pro-teins under study, while negative controls consisted of par-allel Western blots probed without application of specificprimary antibody. Western blots were digitally recordedusing the GeneGenius gel documentation system (Syn-Gene, Cambridge, UK). Molecular mass determination ofprotein bands was performed using GeneTools analysissoftware (Syngene) with reference to standard curves de-termined from molecular mass markers.

Densitometry of western blots

Quantitation of specific protein bands by densitometry wasperformed using GeneTools analysis software. Densitome-try was performed on Western blots of tissues from four

groups of eight animals each, having either scarring or ad-ipogenic wounds harvested at 1 and 4 weeks, respectively,as well as at the 12-week time point. To ensure equivalencyof staining conditions, all 16 scarring wounds were exam-ined on a single Western blot, while all 16 adipogenicwounds analyzed were on a separate single Western blot.All samples were equilibrated for protein content beforeSDS-PAGE. To enable comparison of densitometric ana-lyses across separate blots and experiments, readings wereexpressed as relative percentages with reference to themeandensitometric reading of the native protein at the 1-weektime point in scarring wounds and the 4-week time point inadipogenic wounds, found to be times of maximum vascu-larity in these respective wound-healing models.

Molecular modeling of albumin structures

Molecular modeling was performed to evaluate the effectof AF in mouse tissues with reference to the knowneffect of CNBr fragmentation of HSA and BSA uponanti-apoptotic activity for endothelium.11,12 In brief, theExPASy Proteomics Server made available by the SwissInstitute of Bioinformatics at http://au.expasy.org/tools/was used to obtain: Pdb Viewer X ray crystalographic co-ordinates for HSA; BLAST sequence alignment for HSA,BSA and mouse albumin; predicted CNBr cleavage sitesfor HSA and BSA; HSA, BSA and mouse albumin-predicted albumin fragment molecular masses; as well asDeep View/Swiss-Pdb Viewer protein modeling software.In addition, Persistence of Vision Raytracer softwareavailable from http://www.povray.org/ was used for thepreparation of images. Based on the high structuralhomology of BSA and mouse albumin with HSA includ-ing the location and topology of disulfide bonds, thestructural effects of protein fragmentation at discretehomologous amino acid residues in BSA and mousealbumin were modeled using HSA in Swiss-Pdb Viewer.The topological arrangement of disulfide bonds wasassumed to be unaffected by mouse AF in vivo, and wastaken into consideration when predicting the possiblemouse albumin cleavage domains responsible for thefragments observed.

Statistical analysis

Data were expressed as means� standard errors. A two-tailed Student’s T-test was used to assess the statistical sig-nificance of changes in vascularity, while the statisticalsignificance of densitometric data was determined usingthe two-tailed Mann–Whitney U-test. p values of < 0.05were considered significant.

RESULTS

Scarring and adipogenic patterns of healing were

reproducibly induced in subcutaneous and

intramuscular implants, respectively

In subcutaneous polyvinyl sponge implants, a fibrinousmatrix was seen impregnating sponges within the firstweek of implantation, with reparative granulation tissuegrowing into sponge spaces from the periphery. A foreign



http://au.expasy.org/tools/

http://www.povray.org/

body giant cell response was seen with giant cells sur-rounding sponge material, while maturation of granula-tion tissue was characterized by reducing cellularity andincreased collagen deposition typical of scar tissue forma-tion (Figure 2A and B). In the case of intramuscular nylonmesh implants, highly vascular reparative granulation tis-sue also replaced fibrinous matrix, while maturation of thisgranulation tissue involved the formation of adipose tissue(Figure 2C). Consistent with adipogenic healing in intra-muscular implants, lipoblasts were prevalent in granula-tion tissue by week 2, and were often closely associatedwith blood vessels (Figure 2D).

Fragmentation of albumin occurred during healing and

reduced over time in both scarring and adipogenic

wounds

When granulation tissue extracts from scarring and ad-ipogenic wounds were subjected to Western blotting andprobed for albumin, bands were seen indicative of exten-sive AF in both patterns of healing. Native (66 kDa) anddimmer (130 kDa) forms of albumin were readily distin-guished in brief ECL exposures, as were a number ofsmaller bands that became more clear upon longer ECLexposure. Bands indicative of AF with molecular massesof 27.5, 39, and 49 kDa were observed in both scarring andadipogenic wounds, while an additional fragment of17.5 kDa was also seen in adipogenic lesions but onlyclearly in prolonged ECL exposures. AF was more prom-inent early during healing, and reduced more slowly in ad-

ipogenic as compared with scarring tissues. Minor bandsof 90 and 250 kDa were also observed, seemingly repre-senting high-molecular-mass complexes (Figure 3).

Both AF and high granulation tissue vascularity

persisted longer in adipogenic healing compared with

healing by scarring

In scarring subcutaneous wounds, maximal vascularitywas achieved by the conclusion of the first week of heal-ing, and then reduced from week 2 to the lowest level byweek 4 (p < 0.05). However, in the case of adipogenic in-tramuscular wounds, maximal vascularity of granulationtissue was only achieved by week 4 of healing, and vascu-larity reduced more slowly to a minimum by week 12(p < 0.05) (Figure 4A). The prolonged high vascularity ofadipogenic compared with scarring wounds was accompa-nied by similarly persistent high levels of AF (Figure 4B).

Western blot observations similar to those shown inFigures 3 and 4 were made with tissues from a total of 45animals with scarring wounds at 1 week (10 mice), 2 weeks(10 mice), 3 weeks (3 mice), 4 weeks (3 mice), 6 weeks (3mice), 8 weeks (3 mice), 10 weeks (3 mice), and 12 weeks(10 mice). Also, observations similar to those shown inFigures 3 and 4 were made in adipogenic wounds from 38animals at 1 week (2 mice), 2 weeks (3 mice), 3 weeks (4mice), 4 weeks (9 mice), 5 weeks (1 mouse), 6 weeks (3mice), 8 weeks (3 mice), 9 weeks (2 mice), 10 weeks (1mouse), 11 weeks (1 mouse), and 12 weeks (9 mice).

Figure 2. Photomicrographs of par-

affin sections showing wound heal-

ing in mouse subcutaneous sponge

implants at weeks 8 (A) and 3 (B), and

mouse intramuscular adipogenic im-

plants at weeks 6 (C) and 2 (D). (A)

The epithelium (Ep) and underlying

muscle (M) were separated by a

mass of maturing granulation tissue

(GT) penetrating into subcutaneously

implanted sponge material (arrow

heads), while collagen fibers stained

blue using the Gomori trichrome

method. (B) Blood vessels (black ar-

rows) were readily identified in Go-

mori trichome-stained sections of

subcutaneous implants. Clefts in tis-

sues occupied by sponge material

(arrow heads) were surrounded by

multinucleated foreign body giant

cells (blue arrows). (C) Granulation

tissue matured to adipose tissue

(Ad) in intramuscular implants, while

the implant mesh material (arrows)

was associated with fibrosis (F) and

separated the newly formed adipose tissue (Ad) from surrounding muscle (M). (D) Multivacuolated lipoblasts (arrows) were prom-

inent in week 2 granulation tissues of adipogenic lesions, and preceded the appearance of mature adipose tissue. These adipocyte

precursor cells were often closely associated with vessels, which stained brown in Bandeira simplicifolia lectin histochemistry. (A

and B, Gomori trichrome stain; C, H&E; D, B. simplicifolia lectin histochemistry with hematoxylin counterstain; Bars5300 mm for A,

100mm for B, 500mm for C, 50mm for D).



Native albumin levels did not change during wound

maturation but dimer albumin levels were reduced in

amount in mature scarring wounds

Some variation between animals was apparent in Westernblots, and for this reason, it was important to establish thestatistical significance of changes noted in an experimentusing samples randomly selected for probing in singleWestern blots comparing the times of interest in both scar-ring and adipogenic wounds. Figure 5 shows the results ofWestern blots and densitometric analysis for albumin in 32separate animals comparing times of high tissue vascularityin scarring (1 week) and adipogenic (4 weeks) wounds, withthe 12-week time point that had low vascularity in both an-imal model systems. Native albumin levels were compara-ble in granulation tissues at times of high and lowvascularity in both scarring and adipogenic wounds. How-ever, the amount of the dimer form of albumin was reducedat the 12-week time point in scarring (p < 0.002) but notadipogenic wounds. There was proportionately less of thedimer form of albumin in adipogenic wounds at week 4,compared with scarring wounds in week 1 (p < 0.005).

Only 27.5 and 39 kDa albumin fragments reduced in

amount with wound maturation in both scarring and

adipogenic healing

The 27.5 kDa albumin fragment reduced in amount withwound maturation in both scarring (p < 0.005) and ad-ipogenic healing (p < 0.001), as did the 39 kDa fragment

in both wound-healing models studied (p < 0.001) (Figure5). The 27.5 kDa albumin fragment was proportionatelymore prominent in adipogenic compared with scarringwounds early during wound healing (p < 0.03) as well asat the latest time point studied (p < 0.02). Also, the39 kDa fragment persisted to the 12-week time point inadipogenic wounds, but was not seen in scarring woundsat this time point (p < 0.001) (Figure 5).

The 49 and 17.5 kDa albumin fragments reduced in

amount with wound maturation in only the scarring

and adipogenic wounds, respectively

Although wound maturation was accompanied by a sub-stantial reduction in the intensity of the 49 kDa band inscarring wounds (p < 0.001), this was not seen in ad-ipogenic healing. There was proportionately more of the49 kDa albumin fragment at week 1 in scarring lesions,compared with week 4 in adipogenic wounds (p < 0.001).With regard to the 17.5 kDa fragment, adipogenic woundsdisplayed a marked reduction in the intensity of this AFband during wound maturation (p < 0.001), and this con-trasted with the absence of the 17.5 kDa fragment in all buta single week 1 scarring specimen (Figure 5).

Densitometry confirmed reduced AF with wound

maturation in both scarring and adipogenic healing

The data shown in Figure 5 are consistent with reduced AFduring wound healing, although this is complicated by the

Figure 3. Western blots using an

antibody preparation specific for

mouse albumin in extracts of granu-

lation tissue from scarring and ad-

ipogenic wounds at increasing times

during healing as seen in brief (i) and

prolonged (ii) ECL film exposures.

Bands representing native protein

(66 kDa) and dimer (130 kDa) were

present, as were larger bands of 90

and 250 kDa interpreted as high-

molecular-mass complexes. Bands

consistent with fragmentation of al-

bumin were seen in both wound-

healing models, with fragments hav-

ing molecular masses of 27.5, 39,

and 49 kDa in tissues from both mod-

els, while an additional fragment of

17.5 kDa was observed in adipogenic

wounds. AF appeared more promi-

nent at early times during wound

healing, and seemed to decrease

with wound maturation. Similar re-

sults were obtained with tissues col-

lected at times ranging from 1 to 12

weeks, from 45 animals with scar-

ring wounds as well as from 38 mice

with adipogenic wounds.



absence of the 17.5 kDa fragment at week 1 in scarringwounds, as well as the prolonged high levels of the 49kDafragment during adipogenic healing. Differing levels of thedimer form of albumin also complicate interpretation ofoverall AF. To control for these potentially confoundingfactors, we performed further analysis of the densitometricdata in which the total densitometric readings for 49, 39,27.5, and 17.5 kDa fragments in each sample were calculatedas percentage values relative to the total albumin bands de-tected, including dimer, native, and fragmented forms.

Figure 6 shows a summary of this analysis, which dem-onstrates significantly reduced AFwith wound maturationin both scarring (p < 0.001) and adipogenic (p < 0.02)healing. There was no difference in the extent of AF atweek 1 of scarring healing compared with week 4 of ad-

ipogenic healing. However, when the two wound-healingmodels were compared at the 12-week time point, ad-ipogenic healing had more AF relative to mature scarringwounds (p < 0.001).

In addition, data shown in Figure 6 reveal that a verylarge proportion of total albumin detected byWestern blotwas fragmented early during wound healing, so that at the4-week time point in adipogenic wounds, between 22%and 50% of total albumin had been subjected to AF. Sim-ilarly, between 19% and 51% of total albumin detected in1 week scarring wounds was subject to AF. Data confirmreduced AF with wound maturation and also indicate adegree of specificity with regard to the pattern of AF, de-pendent on the specific wound setting involved.

Minimal fragmentation and dissociation of the acute-

phase transport proteins transferrin, ceruloplasmin,

and haptoglobin did not compare with that seen for

albumin in either extent or time course

All proteins studied were detected in samples of serum(data not shown), supporting the specificity of the anti-body preparations used. Figure 7 shows Western blots fortransferrin, ceruloplasmin, and haptoglobin of extractsfrom scarring and adipogenic wounds over time. Nativetransferrin (75 kDa) was present at all time points studied,and although there was no evidence for fragmentation ofthe protein in adipogenic wounds, a single band labelingwith the specific antibody was found at 65 kDa in scarringtissues from weeks 1 to 8, and this was most prominent atweek 3. Native ceruloplasmin (120 kDa) was seen in all thespecimens studied, and while no bands consistent withfragmentation were seen in adipogenic wounds, single veryfaint 75 kDa bands were seen at weeks 3 and 4 in scarringwounds only. It is of note that no second lower molecularmass bands were seen in the 10 kDa range for transferrinor the 50 kDa range in the case of ceruloplasmin, as wouldbe expected with fragmentation of these two proteins intothe 65 and 75 kDa bands seen. Native haptoglobin wasseen at 73 kDa in all tissues and time points studied, while

Figure 4. Tissue vascularity in scarring (-& -) and adipogenic (-

&-) wounds over time expressed as vessel profile number per

graticule field (A) as well as a Western blot for albumin in gran-

ulation tissue extracts at 1, 4, and 12 weeks from both scarring

and adipogenic wounds (B). (A) Tissue vascularity was maximal

at 1–2 weeks in scarring wounds (p < 0.05) and 3–4 weeks in

adipogenic wounds (p < 0.05), and this was followed by a sig-

nificant reduction in vascularity accompanying wound matura-

tion (p < 0.05). (B) Bands consistent with native protein

(66 kDa) and dimer (130 kDa) were present. Bands consistent

with albumin fragments of 27.5, 39, and 49 kDa were observed

in both scarring and adipogenic wounds, while an additional

fragment of 17.5 kDa was seen in adipogenic tissue only. In

scarring wounds, AF was most prominent at week 1, being

considerably reduced at later times. In adipogenic wounds, AF

was prominent at both weeks 1 and 4, before reducing to lower

levels by week 12 of healing. The prolonged high vascularity of

adipogenic as compared with scarring wounds was accompa-

nied by prolonged AF.



the presence of very faint 27 and 10 kDa bands in ad-ipogenic, as well as 10 kDa bands in scarring wounds, wasconsistent with minimal dissociation of the a and b sub-units. None of the minor fragmentation or dissociationnoted appeared comparable in extent or temporal patternto that seen for albumin. High-molecular-mass bands withmasses exceeding those of native proteins were seen, andinterpreted as either protein complexes or dimer in the caseof haptoglobin (Figure 7).

The predicted structures of 27.5 and 39 kDa mouse

albumin fragments are consistent with exposure of a

protein domain important for mediating the

anti-apoptotic effect for endothelium

Because the 27.5 and 39 kDa fragments correlated withvascularity, it was interesting to evaluate the effect onmouse albumin structure of fragmentation yielding pep-tides of these approximate molecular masses. Figure 8

shows the known effect of CNBr fragmentation on HSAand BSA, and in particular, the location of the Met 209CNBr cleavage site in BSA that is homologous to Leu 209in HSA, recalling that CNBr fragmentation partially inac-tivates BSA but greatly increases the anti-apoptotic activ-ity for endothelium of HSA.11,12 The alpha helical proteindomain containing these important amino acid residues isseen to be deeply buried within the protein, although tran-sient exposure by opening at a hinge region indicated inFigure 8 would make this domain available to its receptor.Also shown in Figure 8 are the effects of mouse AF as pre-dicted at residues 277 and 393, to yield peptides with mo-lecular masses of 28.7 and 37.0, or 41.7 and 24.0 kDa,respectively. Recognizing the variability inherent in mo-lecular mass determination by Western Blot, fragmenta-tion of mouse albumin close to either of these two siteswithout interruption of disulfide bonds would account forthe 27.5 and 39 kDa fragments observed. Importantly, ex-amination of both cases outlined above demonstrates thatany division of mouse albumin into two fragments in the

Figure 5. Western blots for albumin

comparing scarring wounds after 1

and 12 weeks of healing, as well as

adipogenic wounds after 4 and 12

weeks of healing, together with scat-

tergrams showing the results of a

densitometric analysis. Data are from

32 separate animals in total, tissue

extracts from eight separate animals

at each time point being studied in

both adipogenic and scarring pat-

terns of healing, while molecular

mass markers are indicated on the

right of each blot and the molecular

mass of protein bands binding the al-

bumin antibody are indicated on the

left. The intensity of individual bands

is expressed as a percentage relative

to the mean intensity of native albu-

min bands (66 kDa) at the 1-week

time point in the case of scarring

wounds, and the 4-week time point

in adipogenic wounds. Native albu-

min (66 kDa) levels did not change

appreciably over time in either pat-

tern of healing, but there was a mod-

est reduction in the dimer form of

albumin (130 kDa) in scarring wounds

only (p < 0.002). The 27.5 kDa band

reduced in intensity with healing by

scarring (p < 0.005) as well as by

adipogenesis (p < 0.001). Similarly,

there was a reduction in the 39 kDa

band in both scarring and adipogenic

(p < 0.001) healing. Although there

was no change in the levels of the

49 kDa fragment in adipogenic lesions, there was a significant reduction in the levels of this albumin species over time in scarring

wounds (p < 0.001). Separately, while the 17.5 kDa fragment was only present in a single week 1 scarring lesion, this albumin

fragment was present during adipogenic healing and reduced in intensity over time (p < 0.001). Data support reduced AF during

wound maturation, and also reveal differences in the pattern of AF over time between the two wound-healing models studied.



size range observed in the current study would expose theprotein domain thought to be important for anti-apoptoticactivity.

DISCUSSION

Wound maturation was essentially identical to that ex-pected from the literature in both the scarring and the ad-ipogenic models.5,6,22,23 Adipogenic healing was confirmed

as identical to that reported earlier for intramuscular im-plants in mice,22 and lipoblasts associated with vessels inearly reparative granulation tissue were noted. The use ofpolyclonal antibodies as against monoclonal preparationsallowed detection of protein fragments from across multi-ple protein domains. Extensive AF was observed in gran-ulation tissue extracts from both wound-healing models,with increased AF during times of high vascularity, andreduced AF associated with decreased vascularity. Thedifference in the time course of vascularity between scar-ring and adipogenic wounds provided an additional op-portunity to correlate changes in tissue vascularity withAF. Although the active anti-apoptotic site in albumin re-sponsible for inhibiting endothelial apoptosis has not yetbeen identified, the current study does support a role forAF in maintaining high levels of tissue vascularity earlyduring wound healing, and is consistent with the greatlyincreased anti-apoptotic activity of albumin upon CNBrfragmentation.12,14 It is important to note that unlike an-giogenic factors, which result in both endothelial prolifer-ation and inhibition of apoptosis,24 albumin and itsfragments inhibit apoptosis alone without any prolifera-tive effect.11,12 Vascularity was maximal at a later time inadipogenic compared with scarring wounds, and althoughthe basis for this is unknown, this observation is consistentwith our model of AF maintaining poorly perfused vesselsat early time points during healing, with vessel numberonly reducing when AF is also reduced.

Four major albumin fragments were identified inwounds with approximate molecular masses of 49, 39,27.5, and 17.5 kDa. It is tempting to speculate that thefragments identified represent the outcome of cleavage attwo separate sites in the native 66 kDa albumin protein,such that one cleavage site would yield fragments of 49 and17.5 kDa, and the other would produce fragments of 39and 27.5 kDa. Although overall AF reduced over time inboth wound-healing models, production of the 49 kDa

Figure 6. Scattergrams of a densitometric analysis showing

the percentage of albumin appearing as fragments relative to

total albumin detected in Western blots of individual tissue ex-

tracts from scarring and adipogenic wounds at 1 and 4 weeks,

respectively, as well as at the 12-week time point. Although the

pattern of change for individual albumin bands differed be-

tween adipogenic and scarring wounds (Figure 5), there was a

consistent reduction in the total AF over time in both scarring

(p < 0.001) and adipogenic (p < 0.02) healing. While the levels

of total AF were similar for both adipogenic and scarring

wounds at times of high vascularity, at the 12-week time point,

there were nonetheless higher levels of AF in adipogenic com-

pared with scarring wounds (p < 0.001).

Figure 7. Western blots of tissue ex-

tracts from adipogenic and scarring

wounds at increasing time points

during healing, probed for the binding

of antibody preparations specific for

transferrin, cereloplasmin, and hap-

toglobin. Bands consistent with na-

tive transferrin (75 kDa),

ceruloplasmin (120 kDa), and hap-

toglobin (73 kDa) were present at all

time points studied in both ad-

ipogenic and scarring wounds. There

was little clear change in the levels of

these proteins over time, although

ceruloplasmin bands did appear to

be more intense during the first 4

weeks of both scarring and ad-

ipogenic healing. High-molecular-

mass bands greater in size than native protein were interpreted as protein complexes (C) or in the case of haptoglobin the dimer

form of protein (Dim). A 65 kDa band suggestive of transferrin fragmentation was seen in scarring but not adipogenic wounds from

weeks 1 to 8, and this was most prominent at week 3. Similarly, a faint 75 kDa band suggestive of ceruloplasmin fragmentation was

seen during weeks 3 and 4 during scarring but not adipogenic healing. Faint 27 and 10 kDa bands in blots probed for haptoglobin

were consistent with minor dissociation of the native protein into a and b subunits. The minor changes in these acute-phase trans-

port proteins during wound healing did not parallel those seen in albumin.



fragment appeared to be independent of wound matura-tion and vascularity in adipogenic healing, so that this al-bumin fragment would seem unlikely to contribute to theproposed anti-apoptotic role of albumin fragments inwound healing. The absence of the 17.5 kDa fragmentfrom all but one single week 1 scarring specimen alsoseems to exclude this fragment from an important anti-apoptotic role. Expression of the 39 and 27.5 kDa albuminfragments did, however, correlate well with changes in tis-sue vascularity, and this is consistent with the possibilitythat these two fragments represent the products of cleav-age at a single protein site during wound healing. Unfor-tunately, it was not possible to determine the presence orabsence of internal cleavage sites within the albumin frag-ments identified, because reduction with mercaptoethanolto dissociate putative fragments held together by disulfidebonds destroyed the ability of albumin-specific antibodyto bind native and fragmented albumin (data not shown).

With regard to the other acute-phase proteins studied,the lack of significant fragmentation or correlation of thiswith tissue vascularity indicates specificity in AF and isfurther consistent with a specific biological role of suchfragmentation in maintaining the high vascularity of earlyreparative granulation tissue (Figure 1).

Injury increases the catabolism of proteins,25 while frag-mentation of vascular endothelial growth factor and insu-

lin has been detected in wounds.26,27 AF has been detectedin other tissues and circumstances, such as in brain edemafollowing trauma,28,29 supporting a role for AF duringwound healing. Interestingly, albumin fragments have alsobeen detected in serum and urine with molecular massessimilar to the albumin fragments detected in thisstudy.30,31 It seems possible that albumin fragments re-ported as present in urine represent a mechanism for re-moval of fragments leaking into the circulation from sitesof wound healing or elsewhere.

With regard to the greatly increased anti-apoptotic ac-tivity of HSA upon CNBr fragmentation, it is interestingto note that CNBr produces three large human albuminproducts of molecular mass comparable to those seen dur-ing healing in the current study.12 While the precise loca-tion of the active site is unknown, partial separation ofCNBr digests of HSA has excluded activity from thesmaller 14 kDa CNBr fragment that has the single freecysteine residue of the protein. Also, there is a single CNBrcleavage site at Met 208 of BSA that is not represented in

Figure 8. Molecular models of HSA (A–C), BSA (D), and mouse

serum albumin (MSA) (E, F) indicating known CNBr cleavage

products for HSA and BSA, as well as two possible MSA frag-

mentation products (MSA-(A), MSA-(B)) consistent with 27.5

and 39 kDa fragments observed in the current study. N and C

terminal regions are indicated, as is the location of the single

Met residue susceptible to CNBr fragmentation in BSA but not

present in HSA, and known to partially inactivate BSA upon

cleavage (orange ribbon and arrows). (A) CNBr fragments of

HSA are indicated with color such that the 14 kDa fragment is

purple, the 20 kDa fragment is red, and the 32.5 kDa fragment is

colored green, while CNBr fragmentation sites internal to these

major fragments held together by disulfide bonds are indicated

with blue. An apparent hinge region (Hinge) is noted, which, if

opened, would expose the domain known to be important in

mediating anti-apoptotic activity.11,12 (B) The deeply buried lo-

cation of the protein domain important for anti-apoptotic activity

is most readily appreciated when the molecule is viewed from

above. (C) Separation of CNBr fragments in HSA demonstrates

the way in which the putative active site is exposed by CNBr

fragmentation, accounting for the greatly increased anti-apopto-

tic activity of HSA following CNBr fragmentation.12 (D) This

contrasts significantly with the effect of CNBr fragmentation on

BSA, which produces two main fragments each containing in-

ternal cleavage sites (blue) but held together by disulfide bonds.

Notably, the two main fragments are interrupted (orange n and

arrow) in a protein domain not disrupted by CNBr in HSA, and

this reduces anti-apoptotic activity so that this alpha helical do-

main seems to be important in mediating the protection of en-

dothelium.11 (E) The structure of MSA fragments with

predicted molecular masses of 28.7 and 37 kDa reveals expo-

sure of the protein domain thought to be important for activity

(orange arrow). (F) Similarly, theoretical fragmentation of MSA

into 41.7 and 24 kDa fragments also demonstrates exposure of

the active protein domain (orange arrow). This analysis indi-

cates that any MSA fragmentation into two fragments consis-

tent with the size observed by Western blot in the current study

would expose an albumin protein domain important in mediat-

ing the anti-apoptotic activity for endothelium.



human albumin, so that the partial inactivation of bovinealbumin by CNBr fragmentation suggests a role for thisdomain in mediating the anti-apoptotic activity of albu-min for endothelium.11 While experimental confirmationof increased anti-apoptotic activity for 27.5 or 35 kDaalbumin fragments awaits purification of these albuminspecies from wounds together with bioassays in vitro, it isinteresting to note that theoretical cleavage of albumin toyield fragments of comparable size exposes the candidateactive site. The structural analysis described in this paperassumes AF in an extracellular oxidizing environment, sothat retention of disulfide bonds was considered importantin evaluating the structure of potential mouse albuminfragments by molecular modeling. However, it remainspossible that AF occurs in the reducing intracellular envi-ronment or with the assistance of an extracellular disulfideisomerase, in which case theoretical peptide fragmentscould be predicted with molecular masses even moreclosely approximating those measured by SDS-PAGE inthe current study.

There are several mechanisms through which AF mayoccur in wounds. Albumin degradation may involve up-take of the protein into lysosomes after initial binding toendothelial scavenger receptors such as gp18 and gp31.32

Also, albumin catabolism in rat kidney cortical lysosomeshas been described and is attributed to the actions ofaspartic and cysteine proteinases,33 while chymase, se-creted by mast cells, is also able to degrade albumin.34 Fi-broblasts also catabolize albumin in skin and muscle,35 sothat it is possible that wound fibroblasts may play a role inAF. In addition, numerous proteinases are present duringwound healing that may contribute to AF.36

Consistent with the anti-apoptotic role of albumin forendothelium11–14 is that serum albumin levels correlatewith good clinical outcomes, especially for cardiovasculardisease.37,38 Of relevance to the current study is that albu-min concentration has been used to predict clinical out-come for healing and nonhealing wounds, with pooroutcomes correlating with low serum albumin concentra-tions.39

We suggest that low intravascular flow in microvascularsegments early during wound healing precludes exploita-tion of the usual flow-associated anti-apoptotic signalsused by endothelium to circumvent apoptosis,1,3,11–13,15,16

so that newly formed vessels with low blood flow arevulnerable to loss by apoptosis. We also suggest that AFwith associated increased anti-apoptotic activity12,14 mayprotect vulnerable endothelium early during wound heal-ing from apoptosis, till such time that adequate blood flowis established for intravascular flow-associated anti-apoptotic factors to take effect (Figure 1). Current datademonstrating AF at times of high vascularity early duringwound healing support these suggestions.

ACKNOWLEDGMENTS

We thank the National Health and Medical ResearchCouncil of Australia, the NSW Dental Board, and theAustralian Dental Research Foundation for their supportof this work. We also thank the Australian Post-graduateAward Scheme for their support of the first author.

REFERENCES

1. Sandison JC. Observations on the growth of blood vessels as

seen in the transparent chamber introduced into the rabbit’s

ear. Am J Anat 1928; 41: 475–96.2. Walker NI, Bennett RE, Kerr JF. Cell death by apoptosis

during involution of the lactating breast in mice and rats. Am

J Anat 1989; 185: 19–32.3. Meeson A, Palmer M, Calfon M, Lang R. A relationship be-

tween apoptosis and flow during programmed capillary re-

gression is revealed by vital analysis. Development 1996; 122:

3939–48.4. Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angio-

genesis and vessel regression. Circ Res 2000; 87: 434–9.5. Desmouliere A, Redard M, Darby I, Gabbiani G. Apoptosis

mediates the decrease in cellularity during the transition be-

tween granulation tissue and scar.Am J Pathol 1995; 146: 56–

66.6. Darby IA, Bisucci T, Hewitson TD, MacLellan DG. Ap-

optosis is increased in a model of diabetes-impaired wound

healing in genetically diabetic mice. Int J Biochem Cell Biol

1997; 29: 191–200.7. Robaye B, Mosselmans R, Fiers W, Dumont JE, Galand P.

Tumor necrosis factor induces apoptosis (programmed cell

death) in normal endothelial cells in vitro. Am J Pathol 1991;

138: 447–53.8. Bannerman DD, Goldblum SE. Mechanisms of bacterial lip-

opolysaccharide-induced endothelial apoptosis.Am J Physiol

Lung Cell Mol Physiol 2003; 284: L899–914.9. Hermann C, Zeiher AM, Dimmeler S. Shear stress inhibits

H2O2-induced apoptosis of human endothelial cells by mod-

ulation of the glutathione redox cycle and nitric oxide syn-

thase. Arterioscler Thromb Vasc Biol 1997; 17: 3588–92.10. Zoellner H, Bielek E, Vanyek E, Fabry A,Wojta J, HoflerM,

Binder BR. Canalicular fragmentation of apoptotic human

endothelial cells. Endothelium 1996; 4: 177–88.11. Zoellner H, Hofler M, Beckmann R, Hufnagl P, Vanyek E,

Bielek E, Wojta J, Fabry A, Lockie S, Binder BR. Serum al-

bumin is a specific inhibitor of apoptosis in human endothe-

lial cells. J Cell Sci 1996; 109: 2571–80.12. Bolitho C, Bayl P, Hou JY, Lynch G, Hassel AJ, Wall AJ,

Zoellner H. The anti-apoptotic activity of albumin for endo-

thelium is mediated by a partially cryptic protein domain and

reduced by inhibitors of G-coupled protein and PI-3 kinase,

but is independent of radical scavenging or bound lipid.

J Vasc Res 2007; 44: 313–24.13. Zoellner H, Hou JY, Lovery M, Kingham J, Srivastava M,

Bielek E, Vanyek E, Binder BR. Inhibition of microvascular

endothelial apoptosis in tissue explants by serum albumin.

Microvasc Res 1999; 57: 162–73.14. Zoellner H, Siddiqui S, Kelly E, Medbury H. The anti-ap-

optotic activity of albumin for endothelium is inhibited by

advanced glycation end products restricting intramolecular

movement. Cell Mol Biol Lett 2009; 14: 575–86.15. Kaiser D, Freyberg M, Friedl P. Lack of hemodynamic

forces triggers apoptosis in vascular endothelial cells. Bio-

chem Biophys Res Commun 1997; 231: 586–90.16. Bartling B, Tostlebe H, Darmer D, Holtz J, Silber R, Mora-

wietz H. Shear stress-dependant expression of apoptosis-reg-

ulating genes in endothelial cells. Biochem Biophys Res

Commun 2000; 278: 740–6.



17. Ceciliani F, Giordano A, Spagnolo V. The systemic reaction

during inflammation: the acute-phase proteins. Protein Pep

Lett 2002; 9: 211–33.18. Gabay C, Kushner I. Acute-phase proteins and other sys-

temic responses to inflammation. N Engl J Med 1999; 340:

448–54.19. Peters T. All about albumin – biochemistry, genetics and med-

ical applications. San Diego: Academic Press, 1996.20. Hellman NE, Gitlin JD. Ceruloplasmin metabolism and

function. Annu Rev Nutr 2002; 22: 439–58.21. Van Vlierberghe H, Langlois M, Delanghe J. Haptoglobin

polymorphisms and iron homeostasis in health and in dis-

ease. Clin Chim Acta 2004; 345: 35–42.22. Xaymardan M, Gibbins JR, Zoellner H. Adipogenic healing

in adult mice by implantation of hollow devices in muscle.

Anat Rec 2002; 267: 28–36.23. Bailey PJ. Sponge implants as models. Methods Enzymol

1988; 162: 327–34.24. Spyridopoulos I, Brogi E, Kearney M, Sullivan AB, Cetrulo

C, Isner JM, Losordo DW. Vascular endothelial growth fac-

tor inhibits endothelial cell apoptosis induced by tumor ne-

crosis factor-alpha: balance between growth and death

signals. J Mol Cell Cardiol 1997; 29: 1321–30.25. Biolo G, Toigo G, Ciocchi B, Situlin R, Iscra F, Gullo A,

Guarnieri G. Metabolic response to injury and sepsis:

changes in protein metabolism. Nutrition 1997; 13: 52S–7S.26. Lauer G, Sollberg S, Cole M, Flamme I, Sturzebecher J,

Mann K, Krieg T, Eming SA. Expression and proteolysis of

vascular endothelial growth factor is increased in chronic

wounds. J Invest Dermatol 2000; 115: 12–8.27. Shearer JD, Coulter CF, Engeland WC, Roth RA, Caldwell

MD. Insulin is degraded extracellularly in wounds by insulin-

degrading enzyme (EC 3.4.24.56). Am J Physiol 1997; 273:

E657–64.28. Bodsch W, Hossmann K. 125I-Antibody autoradiography

and peptide fragments of albumin in cerebral edema. J Neu-

rochem 1983; 41: 239–43.29. Liu HM, Sturner WQ. Extravasation of plasma proteins in

brain trauma. Forensic Sci Int 1988; 38: 285–95.

30. Kshirsagar B, Wilson B, Wiggins RC. Polymeric complexesand fragments of albumin in normal human plasma. ClinChim Acta 1984; 143: 265–73.

31. Wiggins RC, Kshrisagar B, Kelsch RC, Wilson BS. Frag-mentation and polymeric complexes of albumin in humanurine. Clin Chim Acta 1985; 149: 155–63.

32. Schnitzer JE, Oh P. Albondin-mediated capillary permeabil-ity to albumin. Differential role of receptors in endothelialtranscytosis and endocytosis of native and modified albu-mins. J Biol Chem 1994; 269: 6072–82.

33. Baricos WH, Zhou Y, Fuerst RS, Barrett AJ, Shah SV. Therole of aspartic and cysteine proteinases in albumin degrada-tion by rat kidney cortical lysosomes. Arch Biochem Biophys1987; 256: 687–91.

34. Raymond WW, Ruggles SW, Craik CS, Caughey GH. Albu-min is a substrate of human chymase. Prediction by combi-natorial peptide screening and development of a selectiveinhibitor based on the albumin cleavage site. J Biol Chem2003; 278: 34517–24.

35. Strobel JL, Cady SG, Borg TK, Terracio L, Baynes JW,Thorpe SR. Identification of fibroblasts as a major site of al-bumin catabolism in peripheral tissues. J Biol Chem 1986;261: 7989–94.

36. Barrick B, Campbell EJ, Owen CA. Leukocyte proteinases inwound healing: roles in physiological and pathological pro-cesses. Wound Repair Regen 1999; 7: 410–22.

37. Don BR, Kaysen G. Serum albumin: relationship to inflam-mation and nutrition. Semin Dial 2004; 17: 432–7.

38. Schillinger M, Exner M, Mlekusch W, Amighi J, Sabeti S,Schlager O, Wagner O, Minar E. Serum albumin predictscardiac adverse events in patients with advanced atheroscle-rosis – interrelation with traditional cardiovascular risk fac-tors. Thromb Haemost 2004; 91: 610–8.

39. James TJ, Hughes MA, Cherry GW, Taylor RP. Simple bio-chemical markers to assess chronic wounds. Wound RepairRegen 2000; 8: 264–9.

40. Zoellner H, Hou JY, Hochgrebe T, Poljak A, Duncan MW,Golding J, Henderson T, Lynch G. Fluorometric and massspectrometric analysis of nonenzymatic glycosylated albu-min. Biochem Biophys Res Commun 2001; 284: 83–9.



Vascularity during wound maturation correlates with fragmentation of serum albumin but not ceruloplasmin, transferrin, or haptoglobin

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