enzyme activities in the repairing epithelium during wound healing

7
ENZYME ACTIVITIES IN THE REPAIRING EPITHELIUM DURING WOUND HEALING MICHAEL J. C. IM, PH.D., AND JOHN E. HOOPES, M.D. EPITHELIAL WOUND REPAIR is com- posed of three phases: cell multiplication, cell migration, and cell maturation. It is known that cell multiplication takes place at the margin of the wound and that the migratory cells do not participate in this activity [ 61. Little is known about the biochemical charac- ter of each of the particular phases of epithelialization because of difficulties of sampling. Microchemical techniques have partially overcome this difllculty and can provide accurate biochemical analyses from limited areas, such as proliferating or migrat- ing wound epithelium. Quantitative enzyme analyses can serve as a baseline for further studies regarding metabolism during wound healing. In the presnt study, several enzymes of carbohydrate metabolism have been analyzed quantitatively in an attempt to characterize proliferating, migrating, and mature epithe- lium. MATERIALS AND METHODS Unilateral, longitudinal, linear incisions 1 cm in length were made with a scalpel on the backs of three guinea pigs, and the wounds were left open. Skin biopsies were Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, St. Louis, Missouri 63110. Submitted for publication Sept. 2, 1969. obtained under anesthesia from the wound sites and from symmetrical control sites at in- tervals of 1, 2, 3, 6, and 10 days following in- jury. The tissues were quick frozen in liquid nitrogen, sectioned 18 p in thickness at -20°C in a cryostat, and dried in vacua in the frozen state overnight [17]. This procedure for tissue preparation preserved the enzymes and pre- vented their diffusion or loss. Lyophilized samples were stored in vacuum tubes at -20°C until the enzyme analyses were made. Unstained, unfixed sections demonstrated structural integrity of the skin. The epithelium at the wound margin and the migrating or repaired epithelium in the central portion of the wound were isolated by dissection under the microscope. The epithelium of the wound margin was obtained within 0.5 mm of the incision. The keratin layer, which has prac- tically no enzyme activity, was excluded from the dissected specimens. The techniques of enzyme assays were essentially those of Lowry et al. [18] with some modifications. The fol- lowing enzymes were assayed on OS-pg. samples: hexokinase [ 11, phosphofructokinase [2], and lactate dehydrogenase [ 151 for gly- colysis; phosphorylase (microadaptation of [3] ) for glycogen degradation; glucose 8 phosphate dehydrogenase [15] for the pen- tose phosphate shunt; and isocitrate dehy- drogenase [ 91 and malate dehydrogenase [ 151 for tricarboxylic acid cycle. The microdis- sected epithelium was incubated in a total volume of 5-15 uliters of an appropriate sub- 173

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ENZYME ACTIVITIES IN THE REPAIRING EPITHELIUM DURING WOUND HEALING

MICHAEL J. C. IM, PH.D., AND JOHN E. HOOPES, M.D.

EPITHELIAL WOUND REPAIR is com- posed of three phases: cell multiplication, cell migration, and cell maturation. It is known that cell multiplication takes place at the margin of the wound and that the migratory cells do not participate in this activity [ 61. Little is known about the biochemical charac- ter of each of the particular phases of epithelialization because of difficulties of sampling. Microchemical techniques have partially overcome this difllculty and can provide accurate biochemical analyses from limited areas, such as proliferating or migrat- ing wound epithelium. Quantitative enzyme analyses can serve as a baseline for further studies regarding metabolism during wound healing.

In the presnt study, several enzymes of carbohydrate metabolism have been analyzed quantitatively in an attempt to characterize proliferating, migrating, and mature epithe- lium.

MATERIALS AND METHODS

Unilateral, longitudinal, linear incisions 1 cm in length were made with a scalpel on the backs of three guinea pigs, and the wounds were left open. Skin biopsies were

Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, St. Louis, Missouri 63110.

Submitted for publication Sept. 2, 1969.

obtained under anesthesia from the wound sites and from symmetrical control sites at in- tervals of 1, 2, 3, 6, and 10 days following in- jury. The tissues were quick frozen in liquid nitrogen, sectioned 18 p in thickness at -20°C in a cryostat, and dried in vacua in the frozen state overnight [17]. This procedure for tissue preparation preserved the enzymes and pre- vented their diffusion or loss. Lyophilized samples were stored in vacuum tubes at -20°C until the enzyme analyses were made. Unstained, unfixed sections demonstrated structural integrity of the skin. The epithelium at the wound margin and the migrating or repaired epithelium in the central portion of the wound were isolated by dissection under the microscope. The epithelium of the wound margin was obtained within 0.5 mm of the incision. The keratin layer, which has prac- tically no enzyme activity, was excluded from the dissected specimens. The techniques of enzyme assays were essentially those of Lowry et al. [18] with some modifications. The fol- lowing enzymes were assayed on OS-pg. samples: hexokinase [ 11, phosphofructokinase [2], and lactate dehydrogenase [ 151 for gly- colysis; phosphorylase (microadaptation of [3] ) for glycogen degradation; glucose 8 phosphate dehydrogenase [15] for the pen- tose phosphate shunt; and isocitrate dehy- drogenase [ 91 and malate dehydrogenase [ 151 for tricarboxylic acid cycle. The microdis- sected epithelium was incubated in a total volume of 5-15 uliters of an appropriate sub-

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JOURNAL OF SURGICAL RESEARCH VOL. 10 NO. 4, APRIL 1970

Table 1. Constituents of Reaction Mixture@ and Assay Conditions

Enzyme

Substrate and Auxilliary Enzymes

Cofactors Buffer (mM) ( W

Incubation Final Time

Volume (min) ( pliters) at 37°C

HK Glucose, 4 mM GBPDH, pgm./ml.

NADP. 0.3 ATP, 3 M&L 5 NADP, 0.3 MgC12, 2.5 EDTA, 0.5 NADH, 1 MgC12, 2.5 ATP, 2.5

Tris. 0.1. nH 8.0 , ‘I

9.1 60

AMP,, 0.1, pH 8.8 9.1 60

Imidazole, 0.05, pH 7.5

15.0 30

GGPDH Glucose 6-P, 2 m M

PFK

LDH ICDH

Fructose 6-P, 10 mM Aldolase, 15 lrgn-~./

ml. Triose isomerase,

3 ugm./ml. a GOPDH, 7.5

pgm./ml. Pyruvate, 1 mA4 Isocitrate, 5 mM

MDH

PHLase

Oxaloacetic acid, 1mM

Glycogen, 15 m M PGM, 3 pgm./ml. GBPDH, ugm./ml.

NADH, 2 NADP, 2 MnCl,, 0.25 NADH, 2

AMP, 1 MgCI,, 5 NADP, 0.2 EDTA, 0.5 KSHPOI, 5

Tris, 0.1, pH 7.9 Tris, 0.1, pH 7.9

Tris, 0.1, pH 8.6

Imidazole, 0.1, pH 7.0

14.4 30 9.6 60

14.4 30

7.3 120

aEach reaction mixture contains bovine serum albumin (0.02% ) as enzyme stabilizer Abbreviations: HK = hexokinase; GGPDH = glucose 6-phosphate dehydrogenase; PFK = phosphofructokinase; LDH = lactate dehy- drogenase; ICDH = isocitrate dehydrogenase; MDH= malate dehydrogenase; PHLase = phosphorylase; o GOP- DH = a glycerophosphate dehydrogenase; PGM = phosphoglucomutase; NADP = nicotinamide adenine dinu- cleotide phosphate; NADH = reduced nicotinamide adenine dinucleotide; ATP = adenosine Striphosphate; AMP- adenosine 5-monophosphate; AMP, = 2-amino-e-methyl-l, 3-propandiol buffer; EDTA = ethylenediamine tetra- acetate.

strate reagent containing an excess of sub- strate and cofactors to provide optimal assay conditions for each enzyme. The constituents of the reaction mixture and the assay condi- tions are summarized in Table 1.

The reduced nicotinamide adenine dinu- cleotide or reduced nicotinamide adenine dinucleotide phosphate produced by enzyme reaction is measured in a fluorometer (Far- rand ratio fluorometer) utilizing Coming glass No. 5860 (360 rnp) as the primary filter and Nos. 4308, 5562, and 3387 (460 mu) as the secondary filters. Reduced nucleotide is diluted and stabilized in 1 ml. of 0.1 M carbonate buffer, pH 10. Nicotinamide ade- nine dinucleotide formed by lactate dehy- drogenase and malate dehydrogenase is de-

174

veloped to its fluorescence product in 100 pliters of 6.6 N NaOH for 30 minutes at 37°C after the excess reduced nicotinamide adenine dinucleotide in the reaction mixture has been destroyed by treatment with HCl. The sample is diluted with 1 ml. of redistilled water, and the fluorescence is measured in a fluorometer with the same filter system. Control tubes were reagent blank without tissue. The tissue blank was very low and negligible with this microtechnique. Appropriate internal stan- dards (0.150 mpmoles per tube) were run simultaneously.

The content of deoxyribonucleic acid (DNA) was measured fluorometrically on l-2 pg. frozen-dried samples by the method of Kissane and Robins [16] modified for cutane-

IM AND HOOPES: EPITHELIUM WOUND REPAIR

ous epithelium [ 231. The optical filter system used was Coming filters 4308, 3060, and 5970 (405 mu) as the primary filter and Coming filters 3384 and 5031 (520 rnp) as the secon- dary.

RESULTS

The epithelium at the margin of the wound is thicker than normal epithelium. In un- stained frozen-dried preparations, the migra- tion of epithelium into the wound was ob- served on the samples obtained 1, 2, and 3 days following injury. The repaired epithelium of day 6 and 10 samples is thicker than that of the wound margin and exhibits a thin keratin layer. The enzyme activities expressed as moles of substrate converted per hour per kilogram dry weight in normal epithelium are as follows: hexokinase 0.28, phosphofructo- kinase 0.64, lactate dehydrogenase 11.4, phosphorylase 0.07, glucose 6-phosphate de- hydrogenase 0.23, isocitrate dehydrogenase 1.2, and malate dehydrogenase 22.7. The various enzyme activities observed during wound healing are summarized in Figs. 1, 2, 3, and 4. Enzyme activities are higher than normal in both proliferating and migrating epithelium during the period of 10 days fol- lowing injury. The degree of increase in activity is higher in the migrating than in the proliferating epithelium.

Enzyme Activity in the Proliferating Epithelium

The epithelium at the margin of a wound is known to be actively multiplying and synthesizing DNA [5]. The changes of glyco- lytic activity in proliferating epithelium is denoted by the dotted lines in Fig. 1 and 2.

In general, glycolytic enzyme activity in- creased 2-fold and maintained the increased level during the 10 day period of observation, Phosphorylase exhibited a delayed increase in activity, and maximum increase in this enzyme activity was found day 6 following injury. Phosphorylase activity in the epithe- lium at the wound margin is higher than in the migrating epithelium. Among the enzymes

Epithelium:

o------o Prolhrotmy

- Migrating A. :w.....z Norma/

I I I I I t

0 2 4 6 8 10 DAYS FOLLOWING INJURY

Fig. 1. Changes of hexokinase (HK) and phos- phorylase activity during wound healing. Each point is an average of 6-10 determinations I+ standard error.

LDH

Eplthelium: Eplthelium: 0. - - 0 P,olrfereting 0. - - 0 P,olrfereting

1 I I I I

0 2 4 6 a IO DAYS FOLLOWING INJURY

Fig. 2. Changes of lactate dehydrogenase (LDH) and phosphofructokinase (PFK) activity during wound healing. Each point is an average of 6-10 determinations + standard error.

175

JOURNAL OF SURGICAL RESEARCH VOL. 10 NO. 4, APRIL 1970

I I I I I I

0 2 4 6 8 IO DAYS FOLLOWING INJURY

Fig. 3. Change of glucose 6-phosphate dehy- drogenase activity during wound healing. Each point is an average of 6-10 determinations + standard error.

analyzed, glucose 6-phosphate dehydrogenase shows the greatest change (Fig. 3). Glucose 6-phosphate dehydrogenase activity increased to 0.34 moles/hr./kg. dry weight (3.5 times normal) on day 3 following injury and main- tained a 3-fold increase at days 6 and 10. Isocitrate dehydrogenase and malate dehy- drogenase of the tricarboxylic acid cycle re- acted differently from the glycolytic enzymes (Fig. 4). Isocitrate dehydrogenase activity decreased by 2030% (P 0.5-0.01) of the normal, whereas malate dehydrogenase showed 40% increase (P 0.01) on day 3 and 6.

DNA content of normal epithelium is 10.9 gm./kg. dry weight. The content of DNA in tbe wound margin decreased markedly to 9.1 and 6.9 gm./kg. dry weight on days 2 and 3, respectively, following injury (dotted line in Fig. 5). This may indicate cell enlargement.

Enzyme Activity in the Migrating Epithelium

Enzyme activities in the migrating, and subsequently repaired, epithelium are de- noted by the solid lines in Figs. 1, 2, 3, and 4. All enzymes except for phosphorylase exhibit higher activities in the epitbelium covering the wound site than in the epithelium at the wound margin.

Hekokinase and phosphofructokinase ex-

176

Eprthslwm:

I

I I * I , I , I

0 2 4 6 a 10

DAYS FOLLOWING INJURY

Fig. 4. Changes of malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) activity during wound healing. Each point is an average of 6-10 determinations + standard error.

hibit their maximum increase of 4- and 3-fold normal, respectively, on day 3 following in- jury (Figs. 1 and 2). Phosphorylase activity in migrating epithelium is the same as in normal epithelium during the first 3 days fol- lowing injury. Phosphorylase activity is in- creased 2-fold on day 6 and remains 40% (P 0.01) above normal on day 10 (Fig. 1). Glucose 6-phosphate dehydrogenase also ex-

Epithelium:

o-----a Prolrferofmg - Migro/ing --...,:.-.-f /tJormo/

I , I I I

0 2 4 6 8 10 DAYS FOLLOWING tNJURY

Fig. 5. Change of DNA content of epidermis during wound healing. Each Point is an average of six determinations +- standard error.

hibited the greatest increase in activity (6.5 times normal) on day 2 (Fig. 3). Isocitrate dehydrogenase remained unchanged during healing except a 20% increase (I’ 0.05) on day 6 and malate dehydrogenase activity in- creased 20-30% (I’ 0.01) of normal (Fig. 4).

DNA content in the migrating or repaired epithelium remained unchanged (ranging from 10.1 to 12.8 gm./kg. dry weight during healing) (solid line in Fig. 5).

DISCUSSION

The increased enzyme activities found in this study reflect the increased metabolic function in vitro essential to maintaining the homeostatic dynamic equilibrium for the changed environmental conditions. Express- ing enzyme activity in terms of epithelial DNA, rather than on a tissue dry weight basis, magnifies the increase in activity. Dramatic change is observed in glucose 6-phosphate dehydrogenase and in the gly- colytic enzymes (hexokinase, phosphofruc- tokinase, and lactate dehydrogenase) in both proliferating (marginal) and migrating epithelium. The migrating epithelium exhibits a greater mcrease in enzyme activity than either the proliferating or the repaired epithe- hum. It appears that migrating epithelium is considerably more active metabolically than proliferating or maturing epithelium. The mitotic response to injury of the epithelial cells is greatest at the wound margin and does not extend more than 2 mm into normal tis- sue [6]. A slight increase above normal in enzyme activity in the epithelium 2 mm from

IM AND HOOPES: EPITHELIUM WOUND REPAIR

the wound margin (Table 2) may indicate a falling gradient of metabolic activity from wound to normal tissue.

Histochemically, the activity of some of the hydrolytic enzymes, alkaline and acid phos- phatase, has been shown to increase during wound healing [22]. Enzyme induction oc- curs by traumatic stimulation during liver regeneration [4]. The increased enzyme ac- tivities found in this study may represent an example of enzyme adaptation induced by tissue damage. Recently, the contribution of the epithelium to the synthesis of collagenase during wound healing has been demonstrated in both guinea pig [12] and human skin [lo, 131. It is believed that less differentiated, probably migrating, epithelium may be re- sponsible for the synthesis of collagenolytic enzymes [lo]. The increase in glucose 6-phos- phate dehydrogenase and glycolytic enzymes may indicate that glucose is utilized by the repairing tissue to provide both energy adenosine triphosphate and certain basic sub- stances for the increased cellular activities of biosynthesis, mitosis, and locomotion.

No dramatic change in the enzyme activi- ties of the tricarboxylic acid cycle is found both in proliferating and migrating cells. Isocitrate dehydrogenase activity even de- creases in the proliferating epithelium. The finding of the lack of increased activities in the enzymes of the tricarboxylic acid cycle is puzzling, since oxygen uptake has been re- ported to increase to five times normal in repairing tissue during wound healing [ZO] and the measurement of oxygen taken up by a tissue can indicate the mitochondrial oxida-

Table 2. Topographic Distribution of Enzyme Activities in 2-Day-Old Wound Skina

Epithelium Marginal 2 mm from Epithelium

Normal Wound (within Epithelium

Migratory Margin 0.5 mm) Epithelium

HK 0.28 t 0.03 0.31 -c 0.02 0.53 t 0.03 0.96 + 0.05 GBPDH 0.22 * 0.01 0.35 &I 0.03 0.78 t- 0.02 1.52 +- 0.07 LDH 11.4 A 0.7 19.4 + 1.0 26.8 f 1.8 39.4 +- 1.3

aThe results are expressed as moles per hour per kilogram dry weight. Each entry represents the mean of 6 to 10 samples + the standard error.

177

JOURNAL OF SURGICAL RESEARCH VOL. 10 NO. 4, APRIL 1970

tive phosphorylation to provide adenosine triphosphate for the cells.

The results presented here clearly indicate that the contribution of glycolysis is greater than that of the tricarboxylic acid cycle to the energy production for the repairing epithe- hum. Intense glycolysis in the presence of oxygen is a specific feature of cutaneous epithelial structure [S, 11, 211. This is in con- trast to most tissues where lactic acid is produced for energy only when oxygen is not available. Further studies on the overall metabolic pathway and the steady-state mea- surement of several metabolic intermediates are underway by means of microrespirometer [7], microradioisotope [3], and enzyme cycl- ing techniques [19] and should provide more meaningful interpretation of the data obtained in this study.

SUMMARY

Fluorometric microchemical methods pro- vided a quantitative assay of enzyme activity in OS-pg. samples obtained from the epithelial cells participating in the different processes of epithelialization during skin wound heal- ing.

In the proliferating epithelium at the wound margin, hexokinase, phosphofructo- kinase, lactate dehydrogenase, and glucose 6-phosphate dehydrogenase increased e-fold on day 1 after wounding and maintained an increased level of activity during the lo-day period of observation. Phosphorylase activity increased by 30-120% of normal, with the maximum increase occurring on day 6. There was no increase in isocitrate dehydrogenase activity, whereas malate dehydrogenase ac- tivity increased by 40% after 3 days healing.

The migrating epithelium, during the first 3 days of healing, exhibited an even more dramatic enzymatic response: 6.5fold in- crease in glucose 6-phosphate dehydrogenase, and 3-4-fold increase in glycolytic enzymes. No significant changes were observed in the enzymes of the tricarboxylic acid cycle or phosphorylase.

In the repaired epithelium covering the

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wound site at days 6 and 10, the enzyme ac- tivities of glycolysis and the pentose shunt were reduced from the increased level of activities during migration but still higher than in normal or the epithelium at the wound margin. The activities of the enzymes of the tricarboxylic acid cycle increased by 2030% and the activity of phosphorylase increased by 2-fold during the late period of healing. DNA content of the proliferating epithelium at the wound margin decreased to 9.1 and 6.9 gm./kg. dry weight on days 2 and 3, respec- tively, after wounding. No significant change of DNA content was observed in the migrat- ing or repaired epithelium.

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