Diabetes-impaired healing and reduced wound nitric oxide synthesis: A possible pathophysiologic correlation

Diabetes-impaired healing and reduced wound nitric oxide synthesis: A possible pathophysiologic correlation Michael Gretchen

R. SchHffer, M. Ahrendt,

MD,” MD,

Udaya Tantry, PhD, Francis J. Thornton,

Philip MD,

A. Efron, ~777~1 Adrian

ES, Barbul,

MD,

FACS,

Balfimore, Md.

Background. LITitsic oxide (iV0) is synthesized in wou,nds, bu.t its role in the healing process is not

fulr,l

understood. The inhibition of X0 produ.ction dltm’ng wound healing is acconzpaxied by decreased wound reparative collagen deposition. To @ther define the role of LVO in reparative collagen accu m dation, zue studied its production during diabetes-induced wound hea.ling impairment. Methods. Male Sprague-Dawle) lnts (290 to 310 grit) ulere rendered diabetic 6)) intraperitoneal strepto;otoci,n administrcrtion. Seven days ajter induction of diabetes (blood gtu.cose greater than 300 mg/dl), the rats ~underwent dorsal skin incision, a,nd subcutaneous im$antation of poijvinyl alcohol sponges. Begirrning on the du? of woundin.g, 21 diabetic animals were treated with 3 zc~~its/da~~ insulin via. int rapeliton.ealJy I nzplan ted miniosttzotic pu mps. Ten days after in$.q wound blmki,ng strength was determined, and wound collagen ncacmulution a,nd types I and III collagen gene ex@ssion were measured in su bcu ta neousb inzplan ted polyvinyl alcohol sponges. NO sy thesi.s, as .measared @ nihl’te/rzitsate acczl tnxllation, zuas determined-in wound j&id and in su~ernatan ts oj‘ wound cell cultures. Results.Stn?ptozotocin-induced diabetes markedly irnpaireld wound breaking stren.gth and collagen deposition. A parallA decrease occurred in wound NO synthesis as rejlected 6~ decreased nitrite/nitrate concentration in zoound fluid and in diminished eJx zlivo NO production b? wound cells. Insulin treatnren t partial4 but sign ijicantb i,nzproved wound mechanical stren@h (p c 0.01) and collagen accumu.lation (p c 0.001). Decreased wound NO a.ccumulation and ex viuo NO production lq wound cells were also partinlly restored 0)) insulin treatment. Conclusions.Irnpai,rei dia.betic zuound healing is paralleled b? decreased wound NO synthesi.s, suppol?ing the hypothesis th.at NO plays a signiJjcar~ t role in UIOUnd ,reparative collagen. nccumulation. (Surgeq 1.997;121:.513-9.)

COMPLEX CASCADE of cellular and biochemical events that occurs after injury determines the successful outcome of womld repair. Deposition of collagen lzith subsequent cross-linking provides the principal strength characteristic of most wounds. Although many of the inductive signals for successful wound healing are becoming better defined, our knowledge is still limited in many areas. Nitric oxide (NO) is a short-lived biologic mediator whose role in wound healing is just beginning ‘THE

Supported @j;/1-7 -. Accepted &print Hospital

byagrant for publication

the Deutsche Dec.

Forschungsgemeinschaft

0 1997

0039.6060!97,~~5.00

Dept. of Surgery, Hoppe-Seyler-Str. by Rlosbl+ 0

S&a

10. 1996.

requests: .%drian Barbul, RID, Department of Surgeg of Baltimore, 2435 W. Belvedere Ax-e.. Baltimore. MD

“Present address: Karls-Universitit, Cop!Cght

from

Chirurgische Mink Eberhard3, 72076 Ttibingen, German!.

Year Book, 11/56,‘79963

, Sinai 21215.

[nc.

to be defined. We have previously demonstrated that NO is synthesized in wounds.‘* ‘) The cellular sources of NO during the healing process are multiple but not full! delineated. Inflammatoq cells (i.e., neutrophils and macrophages) and fibroblasts have been shown to express the inducible form of the synthetic enzyme NO svnthase after cytokine or lipopolysaccharide stimulation.3.4 Inhibition of wound NO wnthesis in viva results , in diminished wound reparative collagen accumulation, suggesting that in situ NO production may play a role in normal reparative collagen synthesis and deposition To further define such a regulatory role for wound NO, we chose to study its synthesis and actions in diabetes mellitus, a widely accepted model of impaired healing. Abnormalities in granulation tissue and collagen formation caused by a delayed inflammatory response and direct inhibition of fibrobkast collagen synthesis have been described in this modeLCg Insulin can counSLTRGERY

513

surgery

514 Sch@ii et al.

lmzy 1997

Wounding

I

III1

-6 -4 -2

0

I

I

III

2

4

6

*

Control

-

DIAB

*

INSUL

8 10

Time (days) Fig 1. Serum glucose levels in three experimental

Wounding Control

350 53 2 .g s 0”

INSUL 325 300 -

z

DIAB 215 I -1

I

I

-6 -4 -‘2

b

i

b

fi

i? ;O

Time (days) Fig. 2. Body weights in three experimental

groups before

and after wounding.

teract some of the vulnerary defects that characterize the diabetic state, but it must be given early after injury for maximal efficacy.lO, ” Therefore the purpose of this study was to investigate whether the deleterious effects of diabetes on wound healing are accompanied by altered wound NO synthesis and to determine whether these events are possibly correlated. MATERIAL

AND

METHODS

Animals. Male Sprague-Dawley rats weighing between 290 and 310 gm were individually caged and allowed 1 week to acclimatize to our laboratory conditions. The

groups before and after wounding.

animals were fed a complete pelleted laboratory diet (Teklad LM-485 Diet; Harlan Teklad, Madison, WI) and had access to tap water ad libitum. Seven days before wounding, animals were injected intraperitoneally with either streptozotocin (70 mg/kg in 0.1 mol/L citrate buffer, pH 4) to induce diabetes or with vehicle alone (15 control animals). Glucose levels were determined daily in all rats by means of glucose oxidase strips (Chemstrip bC, Boehringer-Mannheim, Indianapolis, IN) on blood obtained from the tail vein, and diabetic state was defined as levels of blood glucose greater than 300 mg/dl. One week later on the day of wounding, 21 rats were randomly selected to receive treatment with regular human recombinant insulin (Humulin; Eli Lilly and Company, Indianapolis, IN) at a dose of 3 units/day via intraperitoneally implanted miniosmotic pumps (Alza Corp., Palo Alto, CA) (INSUL group). Pilot experiments have shown this insulin treatment to be sufficient to achieve blood glucose levels less than 100 mg/dl. The other 21 nontreated diabetic animals (DIAB group) and 15 control animals underwent sham laparotomy. Wounding. At the time of osmotic pump placement or sham laparotomy all animals underwent a 7 cm midline dorsal skin incision under pentobarbital anesthesia (45 mg/kg body weight intraperitoneally) with clean conditions. Ten preweighed, sterile, saline-moistened polyvinyl alcohol sponges (Unipoint Industries, High Point, NC) were inserted into subcutaneous pockets, and the wounds were then closed with surgical staples (US Surgical Corp., Norwalk, CT). Assessment of wound healing. Ten days after wounding, the animals were killed by using an overdose of pentobarbital and the wound staples were removed.

Schti@-

Fig. 3. Expression of types I and III collagen sponges 10 days after w&ding.

iCOL) mRNLk in subcutaneously

Cardiac blood was obtained by means of open thoracotomy technique for biochemical analyses. The dorsal pelt containing the healing scar was removed and cut into equal strips on a multiblade guillotine. Each strip was centered by a segment of the healing scar. Strips 1, 3, and 5 (cephalad to caudad) were placed in normal saline solution and were used within 30 minutes for assessment of fi-esh breaking strength. Fixed breaking strength of strips 2,4, and 6 was measured after fisati’on in 10% formalin for 4 days, a process that masimall! cross-links the collagen present in the scar. The implanted sponges were retrieved for hydroxyproline determination and for harvesting wound fluid and cells. Hydrox:proline content, an indes of reparative collagen deposition, was measured calorimetrically in the two most cephalad sponges.” The values of the two sponges were averaged for each animal. Wound fluid preparation. Eight sponges from each animal were squeezed with forceps, and the fluid was centrifuged for 10 minutes (MO g) and subsequently for 20 minutes (1600 g) at 4” C for cell and debris separation. After filtering (0.22 mm), the pooled wound fluid from each animal was stored at -70” C. M’ound fluid was tested for sterility in the clinical microbiology laborator! of Sinai Hospital of Baltimore. Wound cell preparation. Squeezed sponges were

implanted

polyinyl

et al.

515

alcohol

I-

Control Fig. 4. Nitrite,‘nitrate concentrations after wounding. *p < 0.001 vs Control. ;\NO\‘A.

DIAB

INSUL

in wound fluid 10 days #p < 0.001 vs INSUL by

minced with iris scissors in hlinimum Essential Medium (hfEhf) (Gibco, Grand Island, NY) containing l%, bovine serum albumin and passed through SO- and 100mesh stainless steel screens. The reco\rered cells were combined with the cell pellet from wound fluid prepa-

516

Schtiffer et al.

60

1 I

Table I. Blood concentrations

and wound fluid (1wj glucose 10 days after wounding

T

BIOOLI (n&W Control DIAB INSUL *,,>< 0.001 ~3 ConW,l

73.1 r 4.3 392.4 +- 15.7* 53.4 2 6.3 and INSUL

WF Ong/dlj 50.8 5 3.5 314.6 I! 9.9”: 41.8 t 3.6

(;\No\:~J.

Table II. Plasma albumin and total protein concentration 10 days after wounding

Control

DIAB

INSUL

Control DIAB INSUL *p < 0.001

60

1*

30 20

10 Control

DIkB

INiUL

Fig. 5. (A) Nitrite/nitrate

and (B) citrulline release into culture supernatants by l&day-old wound cells after 20 hours of incubation in MEM containing 1 mmol/L r-arginine. *p < 0.001 vs Control. #p < 0.01 vs Control by ANOVA.

ration. Red blood cells were lysed with O.S3% ammonium chloride in tris[hydroxymethyl]-aminomethane hydrochloride (Tris) buffer (pH 7.4) (Sigma Chemical Co., St. Louis, MO). Cell incubation. The 1.5 x lo6 viable wound cells (as determined by trypan blue dye exclusion) were cultured in 24-well plates at 37” C for 20 hours in 1 ml MEM containing l.mmol/L L-arginine, 1.0 pCi ~-[2,3-~H] arginine, 1% bovine serum albumin, 100 units/ml penicillin, 100 pg/ml streptomycin, and 0.25 pg/ml amphotericin B. At the end of incubation the plates were centrifuged for 10 minutes (400 @, and the culture

vs Control

2.81 2 0.05 2.51 2 0.03” 2.79 2 0.06 and INSUL

5.03 2 0.07 4.48 2 0.04* 5.04 r+_ 0.10

(.%NO\‘Xj.

supernatants were removed and stored at -70” C for subsequent analysis of nitrite and nitrate accumulation. m7ells containing medium alone were used as controls. Amino acid analysis. Wound fluid, plasma, and cell supernatants were passed through polysulfone filters (Ultrafree-PF, 10 kd; Millipore Corp., Marlborough, MA), mixed with internal standards, and derivatized with polyisothiocyanate (Waters Pica Tag Vacuum Station; Millipore Corp., Milford, Ml). Polyisothiocyanatederivatized amino acids were separated by reverse phase chromatography (HPLC System; Waters Chromatography Div., Millipore Corp.) and quantified by computerized analysis. Separation of radiolabeled argiuiue, citrulliue. The conversion of tritiated arginine to tritiated citrulline was measured as an index of NO synthesis by wound cells in vitro. Radiolabeled amino acids were separated by using cationic exchange chromatography (Dowex AG-50; Bio-Rad Laboratories, Richmond, CA) and quantified in a beta counter (Liquid Scintillation A4nalyzer 1600TR; Packard, Downers Grove, IL).l’ The amount of newly synthesized citrulline was calculated from percentage conversion of tritiated arginine to tritiated citrulline in each well containing 1 mmol/L arginine. Analysis of nitrite and nitrate. Nitrite and nitrate levels, both stable end products of NO biosynthesis, were measured spectrophotometrically in filtered (10 kd Ultrafree-PF filters) serum, wound fluid, and cell supematams. Combined nitrite and nitrate concentrations are reported as an index of NO synthesis. Nitrite concentrations were determined with the Griess reagent.‘” Briefly, 0.1% N-[ 1-Naphthyl] ethylenediamine, 1% sulfanilamide in 5% H:3PO+ and test solutions were mixed

Szcrgeq Volume

121. l\iuml)er

Table

III.

Wound

5

healing

parameters (pg/lOO

Control DLU INSUL

in three experimental

groups

OHP mg sponge,)

FBS

FxBS

(2v)

1409 i 80

3.41 2 0.27

446 c 51* 895 2 679

1.29 20.13" 2.04 i 0.19y

(YJ 13.68 -c 1.29 5.51 2 0.39* 9.86 + 0.65$jll

*p< O.Obl 4 Control.’ -I[> < :p < s/j< lip c

0.05 vs DLkE. 0.001 v.s DLU. 0.01 \ T DLU. 0.05 vs Control.

at a ratio of 1:1:2 (v,i/v), incubated for 10 minutes (25” C) in dimmed light, and measured at 550 nm. Sodium nitrite was used as standard. Nitrate concentrations were quantified with NO3-aspergillus reductase as say.ls Briefly, 0.275 mg, /ml of nicotinamide adenine diuucleotide phosphate (reduced form) in imidazole buffer (pH 6.8)) 0.41 units/ml NOs-aspergillus reductase (Boehringer Mannheim, Indianapolis, IN) in H20, and test solutions were mixed at a ratio of 3:l:l (v/r) and measured immediately and again after 40 minutes at 340 nm. Sodium nitrate was used as standard. DNA concentrations. DNA concentrations in cell cultures were determined after cell lysis with 1 ml of 0.02% sodium dodecyl sulfate (Gibco) in sodium chloride sodium citrate buffer (Gibco) at 37” C for 60 minutes bp Hoechst 33258 dye technique on a TKO 100 minifluorometer (Hoefer, San Francisco, CuL\).I6 Measurement of glucose, albumin, and protein in plasma and wound fluid. Blood glucose levels were measured daily in all rats on blood from the tail vein b) glucose oxidase strips (Chemstrip bG) . At the time of death glucose, albumin, and total protein levels in plasma and wound fluid glucose concentrations were determined by standard techniques in the clinical lab oratory of Sinai Hospital of Baltimore. Total protein levels in wound fluid were measured br; color reaction with Coomassie blue.17 Collagen mRNA expression. The expression of types I and III collagen mRNA was studied in the subcutaneously implanted sponges. Harvested sponges were minced and immediately placed in TRIzol reagent (Life Technologies, Gaithersburg, MD), and total RNA was extracted according to the guanidinium extraction technique. The A260,/A280 ratio was measured to determine purity of samples and the h260 value to calculate the amount of total RNA loaded. Equal amounts of RNA were separated by electrophoresis through a 1% formaldehyde-agarose gel, and equal loading was confirmed by ethidium bromide staining to visualize ribosomal RNA. After transfer onto a nylon membrane, the RNA was cross-linked by incubation at 80” C for 90 minutes. Mouse type I (900 bp) and type III (1800 bp) col-

Table IV. Number of wound cells isolated from subcutaneously implanted sponges and wound cell viabiliq No. of wou nn cells/sponge

Control DL-\B INSUL

3.2 2.5 3.3

t 0.7 x 10” _t 0.6 x 10” t 0.9 x 106

Cell uiability (5%)

2 7 2 4 59 2 8

67 62

lagen cDNA probes (Dr. D. Butler; Celltrix Pharmaceuticals Inc., Santa Clara, CrZ) were radiolabeled with [w3*P] deoxycytidine triphosphate by using an oligolabeling kit (Pharmacia LKB Biotechnology, Piscataway, NJ). Cross-reactivity and specificity of the mouse cDNA probes for rat qpe I and III collagens were demonstrated by running control mouse RNA in parallel during the hybridization process. Membranes were prehybridized overnight at 12” C and later hybridized with denatured [ol-“‘P]-labeled cDNX probe overnight at 42” C. After washings, the membrane was exposed to radiographic film at -70” C. Data analysis. ;Ul data are reported as mean C standard error of the mean (SEM). Statistical analysis was performed by applying ANOVAwith the StatView II statistical package (Abacus Concepts, Berkeley, CA) on a Macintosh (Apple ComputerInc., Cupertino, CX) computer. Statistical significance was achieved at p< 0.05. RESULTS All nontreated diabetic animals (DL%B) were found to have blood glucose levels greater than 300 mg/dl throughout the experiment.. The insulin treatment via intraperitoneally implanted osmotic pumps maintained blood glucose levels at less than 100 mg./dl throughout the postwounding period (Fig. 1). Wound fluid glucose concentrations were significantly increased in the DIAB group and correlated with blood glucose levels at the time of death (Table I). Weight loss was significant in diabetic animals in spite

518

Schiiffer et al.

of marked polyphagia but was reversed by insulin treatment (Fig. 2). In parallel, nutritional indexes such as plasma albumin and total protein levels were decreased in DLm animals (Table II). However, there were no statistically significant differences in individual plasma amino acid concentrations among the three groups (data not shown). There were no wound infections, as assessed visually and by bacteriologic testing of the wound fluid. Wound mechanical strength, as assessed by both fresh and formalin-fixed breaking strengths, and sponge hydroxyproline contents, an index of wound collagen accumulation, were significantly decreased in the DL%B group. Treatment of diabetic animals with insulin (INSUL group) partially restored wound breaking strength and collagen deposition toward normal, although these values were still significantly lower than those observed in the control group (Table III). By contrast, mRNA expression for types I and II collagen was markedly increased in the subcutaneously implanted sponges harvested from DIAB animals; insulin treatment partly reversed the heightened gene expression to levels closer to those observed in control animals (Fig. 3). Arginine levels were undetectable in any wound fluids (less than 1 pmol/L). Levels of the NO end products, nitrite/nitrate, in wound fluid were significantly decreased by diabetes (46.4 + 4.5 pmol/L versus 145.2 t 11.2 pmol,/L in control animals; p< 0.001, ANOVA). The INSUL group had intermediate vahles (86.8 t 8.8 pmol/L) (Fig. 4). The total number of wound cells isolated from the subcutaneously implanted polyvinyl alcohol sponges and their viability, as assessed by trypan blue exclusion, were not significantly different among the three gronps (Table IAT). In vitro nitrite/nitrate and citrulline production by wound cells obtained from DIAB and INSUL animals was significantly lower when compared with synthesis by cells obtained from control animals (Fig. 5). The cells harvested from the INSUL group had synthetic values higher than those observed in the DIAB group.

DISCUSSION Diabetes mellitus is well known to impair wound healing, and this represents a significant clinical problem. The present experiments confirm that streptozotocin-induced diabetes in rats leads to decreased wound breaking strength accompanied by a significantly diminished wound collagen deposition. Somewhat unexpectedly we noted that the decreased wound collagen accumulation in diabetic animals was paralleled by markedly increased types I and III collagen gene expression. Type I collagen constitutes most of the collagen in 10dayold wounds. l8 The lower net wound collagen may be due to a defect in the translational or

posttranslational steps of collagen synthesis, including such essential events as procollagen formation, extrusion, and enzymatic hydroxylation of proline. Conversely, the reduced wound collagen accumulation may reflect increased collagen breakdown. High levels of collagen gene expression in diabetes have been noted in other cell types. Increased types lAT and VI collagen gene expression has been shown in endothelial cells,lg tubular epithelium,” and perineuronal cellszl after in vitro incubation in the presence of high glucose concentrations; the findings for bpe I collagen gene expression were ambiguous.1g, -70 This has been proposed as a mechanism for the vascular basal membrane thickening and accumulation of microfibrils in kidneys and peripheral nerves that are hallmarks of diabetes. Impaired collagen deposition in the wounds of diabetic animals was paralleled by decreased wound NO synthesis. This was documented by the decreased in viva lkrels of nitrite/nitrate in wound fluid, two stable end products of NO synthesis, and by diminished ex vivo synthesis by wound infiltrating cells at 10 days after wounding. Reduced NO synthesis in the wound may be a reflection of either diminished inflammatory response with a reduced number of cells migrating into the wound or a change in wound metabolic activity caused by reduced cellular capacity to produce NO. At death we found similar numbers of infiltrating cells present in each sponge; furthermore, incubation of equal number of cells still demonstrated diminished NO synthesis by diabetic wound cells. This suggests that cells such as macrophages and fibroblasts, which are known to synthesize NO after cytokine stimulation, may be suboptimally stimulated to produce NO or there may be direct inhibition of NO synthesis. Whether the reduced level of NO synthesis reflects less growth factor and cytokine activity in the diabetic wound is not known at present. A very recent study demonstrated that urinary nitrate excretion is elevated after the creation of full-thickness circular wounds in rats and that urinary nitrate excretion is greatly reduced in streptozotocin-induced diabetic animals.” We have previously demonsmated that NO is produced in wounds and that its synthesis appears critical to wound collagen accumulation.‘, a Systemic treatment with competitive inhibitors of the inducible NO synhase, including methylisothiourea and aminoguanidine, decreases collagen deposition and wound breaking strength in murine wound healing.’ More recently we have shown that wound fibroblasts are phenotypically altered to syAesize NO and that inhibition of this activity results in decreased fibroblast collagen production. All these findings strongly indicate that wound NO sj?thesis and collagen deposition are related events. In the diabetes impaired model, as used in the present experiments, as well as in a model of impaired healing by acute protein calorie malnutrition,

Schtifer

reduced wound collagen accumulation correlated directly with reduced wound NO synthesis.‘” What remains to be determined is which steps in collagen synthesis and accumulation are affected by NO. The present experiments strongly suggest that posttranslational events in collagen qnthesis are affected by wound-generated NO. DL%l3 animals lost about 9% of their original body weight throughout the experiment. Plasma nutritional parameters, including albumin and protein concentrations, were also decreased in these animals. Lf\Teight loss associated with poor nutritional intake and lack of individual nutrients, even of short duration, are well known to impair wound healing. ‘, ‘, 25, 26 ,2nimals t.hat received insulin gained weight faster during the postoperative period than the control animals, and plasma albumin and protein concentrations were completely restored to control values. Also, at the time of death no significant differences in body weight were noted between control animals and insulin-treated animals. Preoperative weight loss can impair wound healing, and brief and not necessarily full-target nutritional intervention can reverse or prevent the decreased collagen deposition seen with malnutrition or with postoperative starvation.“, ” =Uthough weight loss may contribute partially to the wound defect observed in the diabetic animals, the finding that insulin treatment only partially reverses the wound healing defect argues against weight loss as the sole mechanism for impaired healing. In summary, our data show that the deleterious effects of diabetes mellitus on wound healing are reflected in decreased wound NO synthesis. Diminished wound collagen deposition and wound mechanical strength in diabetic wounds, as well as wound NO synthesis, are partially restored by insulin treatment. The exact mechanism by which wound NO regulates reparative collagen accumulation remains to be elucidated.

6. Fahey TJ. Sadaty .\. Jones i\%, Barber Diabetes impairs the late inflammatol? ing. J Surg Res 1991;50:308-13.

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