- Email: [email protected]
Inhibition of inducible nitric oxide synthase results in reductions in wound vascular endothelial growth factor expression, granulation tissue formation, and local perfusion
Inhibition of inducible nitric oxide synthase results in reductions in wound vascular endothelial growth factor expression, granulation tissue formation, and local perfusion Thomas R. Howdieshell, MD, Whitney L. Webb, MD, Sathyanarayana, MD, and Paul L. McNeil, PhD, Albuquerque, NM; and Augusta, Ga
Background. Wound repair results from a series of highly orchestrated cellular and biochemical events, including increased synthesis of the bioregulatory molecule nitric oxide (NO). The goal of this work was to test the functional role of NO in promotion of vascular endothelial growth factor (VEGF) production and the vigorous granulation tissue formation characteristic of this wound model. Methods. A ventral hernia, surgically created in the abdominal walls of 12 swine, was repaired with silicone sheeting and skin closure. An osmotic infusion pump, inserted in a remote subcutaneous pocket, delivered saline solution (n = 6) or the selective inducible NO synthase inhibitor N6 (iminoethyl)-L-lysine (L-NIL; n = 6) into the wound environment. Granulation tissue thickness was determined with ultrasongraphy, and local wound perfusion was measured with laser Doppler analysis for 2 weeks. Fluid was aspirated serially from the wound compartment for measurement of nitrite/nitrate, VEGF, and transforming growth factor–β1 concentrations. On day 14, the animals were killed and the abdominal wall was harvested for immunohistochemical and molecular analysis. Results. In animals that received saline solution, a nearly linear 4-fold increase in granulation tissue thickness was measured during the 14-day interval. In contrast, in animals that received L-NIL, day 14 granulation tissue thickness was essentially unchanged from the day 2 values of saline solution–treated animals. Moreover, in the L-NIL animals, ultrasonography was unable to resolve the angiogenic zone typical of controls, and correspondingly, wound vessel count and vascular surface area estimates derived from image analysis of histologic sections were 2-fold to 3-fold lower in the L-NIL animals compared with controls. Reductions in basal (2-fold) and heat-provoked (2.5-fold) wound perfusion were noted in L-NIL animals. Wound fluid nitrite/nitrate and VEGF levels were strikingly (4-fold and 5-fold, respectively) reduced in L-NIL animals on days 9 to 14. Immunochemistry results showed reduced VEGF protein content in granulation tissue and keratinocytes within the hyperproliferative epithelium at wound edge. Finally, transforming growth factor–β1 levels were unaffected by L-NIL treatment. Conclusion. VEGF production in granulation tissue is dependent on the presence of functionally active inducible NO synthase and hence, the production of NO. NO and VEGF are therefore defined as key regulators of granulation tissue formation. (Surgery 2003;133:528-37.) From the Department of Surgery, University of New Mexico Health Sciences Center, Albuquerque, NM; and the Departments of Surgery, Radiology, and Anatomy and Cellular Biology, Medical College of Georgia, Augusta, Ga
Accepted for publication January 18, 2003. Supported by American Heart Association Southeast Affiliate Grant (Dr Howdieshell). Reprint requests: Thomas R. Howdieshell, MD, FACS, Trauma/Surgical Critical Care, Department of Surgery ACC-2, University of New Mexico Health Sciences Center, 915 Camino de Salud, Albuquerque, NM 87131. © 2003 Mosby, Inc. All rights reserved. 0039-6060/2003/$30.00 + 0 doi:10.1067/msy.2003.128
528 SURGERY
WOUND HEALING IS a highly ordered and coordinated process that involves inflammation, proliferation, matrix deposition, and tissue remodeling. After injury, new tissue formation begins with reepithelialization and is followed by granulation tissue formation. The latter process encompasses macrophage accumulation, fibroblast ingrowth, matrix formation, and angiogenesis.1 Inflammation, reepithelialization, and granulation tissue formation are driven
Surgery Volume 133, Number 5
in part by a complex interaction of growth factors and cytokines, coordinately released into the area of injury.2,3 In a previous study, we showed that vascular endothelial growth factor (VEGF) is an essential regulator of granulation tissue formation in a porcine abdominal wound model.2 In addition to these proteins, evidence exists for an important role of small diffusible molecules such as nitric oxide (NO) in wound repair.4,5 NO can be produced by 1 of 3 known NO synthase (NOS) isoforms. The cytokine-inducible or inflammatory NOS (NOS2) produces NO in a sustained manner independent of intracellular calcium concentration and is expressed in many inflammatory conditions, including wound healing.4,6 The biologic activities of NO in human skin include regulation of epithelialization, vasodilatation and melanogenesis, and protection against invading pathogens.7,8 Little is known about the functional role of NO during the healing process. However, in vitro study results reveal that NO affects expressional regulation of many genes, including the gene encoding for VEGF.9,10 These results suggest that a key role of NO in wound healing is to upregulate VEGF production in granulation tissue. A central prediction of this hypothesis is that inhibition of NO production with N6 (iminoethyl)-L-lysine (L-NIL) will inhibit VEGF production and, consequently, granulation tissue formation. METHODS Pig model of wound healing. All animals were treated humanely in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals as part of a protocol approved by the Medical College of Georgia’s animal review committee. Domestic female Landrace swine (weight, 15 to 20 kg) underwent induction with Telazol, xylazine hydrochloride, and atropine and anesthesia with isoflurane (1.5% to 3%) via inhalation with an endotracheal tube. Postsurgical analgesia was provided with buprenorphine hydrochloride for 48 hours. After maintenance of inhalation anesthesia and sterile preparation (providine-iodine) and draping, an infraumbilical diagnostic peritoneal lavage was performed (Arrow DPL Kit, Arrow International, Reading, Pa) with the percutaneous Seldinger technique to obtain preincision peritoneal fluid for biochemical analysis. A sample of plasma for enzyme-linked immunosorbent assay was obtained from an ear vein. Next, a supraumbilical midline laparotomy was performed, and biopsy samples of omentum, peritoneum, and portions of abdominal wall were
Howdieshell et al 529
taken to be used as controls. A full-thickness section of the abdominal wall (8 12 cm), excluding the skin, was surgically excised to create a hernia defect. A similar-sized piece of silicone sheeting (Bioplexus Corp, Saticoy, Calif) was sutured to the fascial edges with monofilament suture to effect abdominal wall closure. In previous work, we observed vigorous growth of granulation tissue on both sides of the silicone implant removed from animals on day 14. The sheeting does not incorporate into adjacent tissues but rather becomes encapsulated, creating an excellent scaffold for granulation tissue formation.2,4 Two osmotic minipumps with attached polyethylene tubing were surgically implanted into remote abdominal wall subcutaneous pockets (model ML2; Alzet Corp, Palo Alto, Calif). The attached tubing, used to deliver saline solution or L-NIL into the wound environment, entered the wound compartment via a subcutaneous tunnel. To ensure uniform delivery of L-NIL or saline solution within the aqueous wound compartment, the tubing contained multiple side holes and was looped to increase surface area. The pump was manufactured to deliver 5 mL/h for 14 days. Before implantation, the pump was loaded with saline solution or L-NIL with sterile conditions and maintained at 37°C in an incubator to ensure optimal delivery. After pump insertion, the midline and pocket skin incisions were closed in 2 layers to cover the biomaterial and pumps and thus complete wound closure. Each animal was housed for 14 days after surgery and received water and food ad libitum. On days 2, 4, 7, 9, 11, and 14 after surgery, each animal underwent sedation and was placed supine, and abdominal ultrasonography was performed with an Acusom Ultrasound Imager with 7.5-mHz linear array and 3.5-mHz sector probe (Acusom Corp, Mountain View, Calif). At each time point, images recorded electronically for later analysis included granulation tissue thickness measured in millimeters with a cursor at multiple consistent sites. Each day, a sample of wound fluid located in the compartment between the skin and silicone sheeting was percutaneously aspirated with a 23gauge needle with sterile conditions and ultrasound scan guidance. On day 14 after surgery, the animals underwent sedation, and an infraumbilical diagnostic peritoneal lavage was performed to obtain presacrifice peritoneal fluid for assay. A blood sample was obtained from an ear vein. Each animal then was killed with pentobarbital, and the abdominal wall was harvested en bloc. The osmotic minipumps were removed from the subcutaneous pockets, and their
530 Howdieshell et al
residual volume was measured to confirm complete delivery. Histologic and biochemical analyses were performed on blood, peritoneal fluid, local wound fluid, and subcutaneous granulation tissue. Laser Doppler measurement. Local wound perfusion was determined with the Periflux 5010/5020 laser Doppler device (Perimed, Inc, North Royalton, Ohio) equipped with standard (408) and heater (452) probes.11,12 Perfusion measurements were recorded at 6 consistently spaced and reproducible reference sites located from cephalad to caudad, 3 on each side of the abdominal incision, all overlying the wound compartment. A temperature-control module in the Periflux System measured and enhanced local skin temperature. Perfusion was initially determined at basal temperature with the standard probe. Then, the heater probe was heated to 44°C, a temperature high enough to elicit maximal blood flow without burning the skin, and the measurements were repeated.13 Perfusion was measured for 10 to 15 minutes to ensure equilibration. Testing took place in a room controlled at an ambient temperature of 24°C. Values are reported as means of the 6 reference sites on days 0, 2, 4, 7, 9, 11, and 14. The summated data were stored and analyzed with the Perisoft software package, version V5.10C2 (Perimed, Inc). Enzyme-linked immunosorbent assay measurement. VEGF and transforming growth factor–β1 (TGF-β1) were assayed in plasma and wound and peritoneal fluids with quantitative sandwich immunoassay kits (R&D Systems, Minneapolis, Minn) according to the manufacturer’s instructions. All fluids were centrifuged immediately after harvest and then frozen at –70°C until assayed. Samples for TGF-β1 analysis were acid-treated to activate latent TGF-β1 to the immunoreactive form (according to kit instructions) before application to microtiter plate wells. Each sample was run in duplicate and at pertinent dilutions, with mean values reported. Nitrite/nitrate measurement. Nitrite and nitrate (NOx) are the oxidation products of NO in biologic fluids and are measured as an index of NOS activity. NOx was assayed in wound and peritoneal fluids with the Bioxytech NO nonenzymatic assay (Oxis International, Inc, Portland, Ore) according to manufacturer’s instructions. All fluids were centrifuged immediately after harvest and frozen at –70°C until assayed. Each sample was run in duplicate and at pertinent dilutions, with mean values reported. Vascular density and morphometric determinations. Granulation tissue vessel count and vascular cross-sectional surface area were determined with
Surgery May 2003
image analysis of von Willebrand’s factor– immunostained sections. Multiple consistent wound biopsies were analyzed. The boundaries of the granulation tissue region to be analyzed were the wound fluid compartment and a distinct subcutaneous tissue interface. Immunohistochemical analysis was performed with the avidin-biotin method as previously reported.14 A Zeiss microscope with attached digital camera was used for image analysis. The magnified image (200) of the slide section was acquired with Adobe Photoshop and analyzed with IPLab Spectrum software on a MacIntosh computer. Vessels in each section were defined by the circular or ovoid image of the brown endothelial walls. Capillaries, arterioles, and venules were counted. Vessels were identified and marked by an observer with no knowledge of treatment group. The software program was used to count the number of vessels marked in each section. For confirmation of these estimates of vascular density, the cross-sectional luminal area of vessels was computed. Closed vessels would alter the numeric count but only minimally affect the estimate of vascular area. Thus, the determination of luminal area constituted a separate estimate of vascular density. For determination of area, the minimum and maximum major axis of each vessel was measured. The calibration scale was set with a stage micrometer to match computer pixels to a micrometer scale. The cross-sectional area of each measurement field was computed with careful outlining of the previously reported boundaries of granulation tissue.14 For confirmation of ultrasonographic measurements, granulation tissue thickness was also determined with morphometric analysis of histologic specimens. The wound biopsies used for vascular density determination were scanned with the previously described image analysis techniques. Granulation tissue thickness was measured in millimeters by cursor-defined boundaries with established calibration scale and software computations. Immunohistochemical staining of tissues. Serial sections (4 to 5 µm) of formalin-fixed, paraffinembedded tissue were dewaxed in xylene, taken through graded ethanol, and then hydrated in phosphane-buffered saline solution. Sections were treated with DAKO Target Retrieval Solution (S1700; DAKO Corp, Carpinteria, Calif) and steam heated for 30 minutes to improve antigen retrieval. The sections were incubated for 20 minutes with 0.75% hydrogen peroxide in methanol to block endogenous peroxidase activity, washed, incubated with DAKO Protein Block serum-free for 30 min-
Surgery Volume 133, Number 5
utes to reduce nonspecific binding, and incubated with primary antibody, polyclonal rabbit anti-VEGF (A-20, 1:500; Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) for 30 minutes at room temperature in a humidified chamber. Next, the sections were incubated with a secondary antibody with the DAKO Envision Plus System and DAB kit, according to the manufacturers instructions. The sections were counterstained with Vector Hematoxylin QS (Vector Laboratories, Burlingame, Calif) with quick immersion. The 3, 3'-diaminobenzidine substrate-chromogen resulted in a brown-colored precipitate at the antigen site. For confirmation of the specificity of the immunochemical staining, secondary antibody only, normal rabbit immunoglobulin G, and antigen excess stainings with blocking peptides were performed. L-NIL inhibition of NOS2. N6 (iminoethyl)-Llysine, purchased from Biomol Research Laboratories (Plymouth Meeting, Pa) has been shown to be 33 to 50 times more selective for inhibition of NOS2 than constitutive endothelial NOS.15,16 Each osmotic infusion pump (2 per pig) was loaded with either 2 mL of saline solution (n = 6) or a 2-mL solution containing 25 mg/mL of L-NIL in saline solution as a carrier (n = 6). Each pump was manufactured to deliver 5 µL/h for 14 days. The L-NIL dose was based on the previously stated L-NIL characteristics and the predicted serial NOx levels and day 14 wound fluid volume.4 Statistical analysis. All data are expressed as the mean ± the standard error of the mean. Differences among groups and between baseline and subsequent time points were determined with repeated-measures analysis of variance with Tukey significant difference test used for post hoc analysis. A P value of .05 or less was considered statistically significant. RESULTS Granulation tissue growth, angiogenesis, and wound perfusion in a large-scale abdominal wall wound model. Delivery of saline solution into the wound compartment did not alter the predicted wound responses.2,4 The skin incision was completely epithelialized by day 14, without evidence of induration, erythema, or early dermal hypertrophy. The subcutaneous granulation tissue harvested at day 14 from animals that received saline solution was thick, tough, red, and stratified into zones (Fig 1, A), appearing to correlate with the predicted tripartite histologic profile of granulation tissue, zones of inflammation, angiogenesis, and fibroplasia.1,2 Sonography was used to serially image the wound site to measure the thickness of the devel-
Howdieshell et al 531
oping granulation tissue. In ultrasonographic images, the developing granulation tissue of saline solution–treated animals appeared to be stratified into 3 layers.4 A nearly linear increase in granulation tissue thickness was measured from serially acquired images, and by day 14, the granulation tissue had reached a thickness of 19.2 ± 1.2 mm in saline solution–treated animals (Fig 2). Histologically, control group granulation tissue (day 14) was observed to consist of a presumptive neutrophil/macrophage-rich zone that bordered on the wound fluid compartment, a microvasculature-rich zone whose endothelial cell components could be stained with an antibody against von Willebrand’s factor, and a presumptive fibroblastrich, most mature zone. In saline solution–treated pigs, basal wound perfusion increased in nearly linear fashion to reach a maximal, 5-fold (30 ± 10 PU to 150 ± 22 PU) increase from baseline by day 7 and then decreased to near baseline values by day 14 (Fig 3, A). Before surgical incision, heat provocation to 44°C produced a 2.7-fold (30 ± 7 PU to 80 ± 15 PU) increase in wound perfusion from basal values. Heat-provoked wound perfusion increased to reach a maximal value on day 7, a 33% increase from day 7 basal values (150 ± 20 PU to 200 ± 20 PU), and returned to baseline by day 14 (Fig 3, B). Effects of L-NIL on wound NOS2 activity. As shown in Fig 4, wound NOx levels were reduced on days 9 to 14 (P = .042, P = .008, and P = .05, respectively), clearly indicating an inhibitory effect of LNIL on NOS2 enzyme activity at time points when NOS2 activity was highest during wound repair. Reductions in granulation tissue growth, vascularity, VEGF content, and local wound perfusion with NOS2 inhibition. The day 14 granulation tissue harvested from L-NIL–infused wounds was, compared with saline solution–infused wounds, remarkably thin and friable, uniformly white in color, and without visible stratification when viewed in cross section (Fig 1, B). All skin incisions healed without evidence of wound infection. Moreover, with an ultrasonographic view, the L-NIL wound granulation tissue lacked the zonation characteristic of the control group—that is, a dark band, presumed to be a highly vascularized zone, sandwiched between 2 lighter bands, presumed to represent the inflammatory and fibroplasia zones. Quantitative ultrasound scan analysis detected significant reductions in L-NIL granulation tissue thickness compared with controls between days 4 and 14 (Fig 2). Morphometric measurement of day 0 soft tissue and day 14 granulation tissue in L-NIL and control animals confirmed the accuracy of
532 Howdieshell et al
Surgery May 2003
Fig 2. Ultrasonographic measurements of granulation tissue thickness. Day 0 values are preincision skin and subcutaneous tissue thickness. Bars are standard error of mean. Saline solution–treated animals are represented with solid circles, and L-NIL–treated animals with open circles. (*P < .05 versus controls.)
Fig 1. (A) Harvested silicone implantation site from saline solution–treated animal viewed from peritoneal surface with skin margin down, revealing wound fluid compartment (WFC) and stratification of granulation tissue (GT) into zones. (B) Harvested L-NIL silicone implantation site. Note marked reduction in granulation tissue thickness (arrows) and lack of rich, red appearance. MZ, Macrophage-neutrophil rich-zone; AZ, angiogenic zone; FZ, fibroblast-rich zone; SQ, skin and subcutaneous tissue; S, silicone sheeting.
ultrasonography in determination of granulation tissue thickness. Consistent with the ultrasound scan images, histologic analysis revealed an absence of stratification
within the L-NIL granulation tissue. The tissue was composed primarily of inflammatory cells and was strikingly devoid of vasculature (compare Figs 5, A and B). Counts from sections immunostained for von Willebrand’s factor, in which endothelial cells could be unambiguously identified, confirmed that L-NIL treatment resulted in 2-fold to 3-fold reductions in granulation tissue vessel count (control, 2850 ± 310; L-NIL, 1010 ± 290; P < .05) and crosssectional surface area (control, 1.28 ± 0.22 mm2; LNIL, 0.04 ± 0.15 mm2; P < .05) compared with control wounds. In saline solution–infused wounds, VEGF levels progressively increased over the 14-day interval, reaching a maximal value of 11,101 ± 1910 pg/mL. In the L-NIL wounds, VEGF levels peaked on day 7 at a much lower level (3310 ± 820 pg/mL) and remained at significantly reduced concentrations over days 9 to 14 (Fig 6, A). In contrast, progressive increases in levels of TGF-β1 were noted in control and L-NIL wound fluids, with no difference shown between groups (Fig 6, B).
Surgery Volume 133, Number 5
Howdieshell et al 533
Fig 4. NOx concentrations in serial wound fluid. Bars are standard error of mean. Saline solution–treated animals are represented with solid circles, and L-NIL–treated animals with open circles. (*P < .05 vs controls.)
Fig 3. Laser Doppler flowmetry of basal (A) and heat-provoked (B) wound perfusion. Bars are standard error of mean. Saline solution–treated animals are represented with solid circles, and L-NIL–treated animals with open circles. (*P < .05 vs controls.)
Immunohistochemical analysis of day 14 wound tissue showed differences in VEGF protein content between control and L-NIL animals (Figs 7, A and B). In saline solution–treated animals, large numbers of keratinocytes within the hyperproliferative epithelium were characterized by an intense staining, indicating a large content of VEGF protein. In addition, intense staining was noted in granulation tissue fibroblasts, macrophages, neutrophils, and endothelium. In contrast, in L-NIL–treated animals, the hyperproliferative epithelium and granulation tissue were characterized by a dramatic reduction in VEGF protein content. Finally, basal and heat-provoked wound perfusions were reduced in L-NIL–treated animals (Figs 3, A and B). Reductions in basal perfusion (1.5-fold to 2.5-fold) were recorded in L-NIL animals between days 2 and 11, with a return to baseline by day 14. Heat provocation produced 1.7-fold to 2.5fold reductions in wound perfusion in L-NIL animals compared with saline solution controls throughout the 14-day study period.
534 Howdieshell et al
Fig 5. Histologic comparison of day 14 saline solution (A) and L-NIL (B) granulation tissue shows marked reduction in vascularity in L-NIL specimen. Arrows denote blood vessels. Hematoxylin and eosin, 400.
DISCUSSION The biologic activities of NO are numerous and complex. For example, in human skin, NO regulates epithelial migration, vasodilatation, and melanogenesis and protects against invading pathogens.7,8 Recent studies have implicated NO in the pathogenesis of several skin diseases. NOS2 has been shown to be overexpressed in psoriasis, a
Surgery May 2003
Fig 6. Serial measurement of growth factor concentrations in wound fluid after abdominal wall injury. (A) VEGF. (B) TGF-β1. Saline solution–treated animals are represented with solid circles, and L-NIL–treated animals with open circles. Bars are standard error of mean. Day 0 values are from peritoneal fluid. (*P < .05 vs controls at day 14 (maximal) value.)
chronic inflammatory skin disease characterized by hyperproliferation and incomplete differentiation of epidermal keratinocytes in the involved lesions.17,18 The evidence for its role in skin wound healing has been mentioned previously.
Surgery Volume 133, Number 5
Vascular endothelial growth factor exerts a pivotal role in normal and pathologic angiogenesis. Its production by stromal and epithelial cells is sufficient to trigger angiogenesis, and inactivation of the corresponding gene results in abnormal blood vessel development and embryonic lethality in mice.19 VEGF expression has been shown in granulation tissue macrophages and fibroblasts and proliferating keratinocytes at the wound edge during cutaneous repair.2,20,21 We have reported previously that antibody neutralization of VEGF results in significant reductions in granulation tissue formation, verifying the functional role of VEGF in wound healing.14 Recent evidence indicates that NO regulates the expression of VEGF. Production of angiogenic activity by human monocytes has been found to depend on NO, and NO-generating compounds have been shown to stimulate VEGF gene transcription in human glioblastoma and hepatoma cells in culture.22,23 Furthermore, a strong positive correlation between NOS activity, cyclic guanosine monophosphate levels, and tumor angiogenesis has been described in head and neck and gynecologic cancers.24,25 We reported previously that NOS2 plays a critical role in wound granulation tissue formation.4 Although systemic administration of L-NIL has been well documented to almost completely block the in vivo activity of NOS2 in lymph nodes and skin in mice,15,16 we show the local inhibitory activity of the selective NOS2 inhibitor in our porcine wound healing model. In this study, we provide in vivo evidence for dramatic reductions in wound fluid and tissue VEGF protein content during healing in L-NIL–treated pigs. The lack of effect of LNIL on TGF-β1 levels, a mediator important in wound fibrogenesis, indicates that nonspecific inhibition of a wide range of wound-related processes did not occur as a result of L-NIL infusion. Ultrasonography and morphometric analysis of histology depicted significant reductions in granulation tissue growth in L-NIL animals. Furthermore, immunohistochemistry revealed several-fold reductions in wound vessel count and vascular surface area estimates. Histology does not permit an evaluation of dynamic changes in microvasculature. Therefore, laser Doppler flowmetry was used to measure serially basal and heat-provoked wound perfusion. The microvasculature of human and animal skin is not uniform. Most skin locations have primarily nutritive perfusion by small capillaries with low flow and high resistance. However, areas such as the pulp of the fingertip or toe have large contributions of
Howdieshell et al 535
Fig 7. VEGF immunoreactivity (brown) in saline solution (A) and L-NIL (B) granulation tissue.
arterioles and venules, which are large diameter microvessels with low resistance and high flow.26,27 Previous work has shown that nutritively perfused skin sites such as the abdominal wall have flow properties markedly different from those with substantial arteriolar and venular perfusion.28 The differences are accentuated by local skin heating to 44°C, which causes maximal vasodilation. With local heating, perfusion may increase 50% to 100% at nutritive sites and by as much as 300% to 500% at arteriolar sites.29 In L-NIL–treated animals, laser Doppler analysis showed striking decreases in basal and temperature-provoked wound perfusion resulting from reductions in VEGF-dependent vascular density and NO-dependent vasomotor tone.30 Peak production of wound fluid NOx occurred later in time than peak increases in basal and heatprovoked wound perfusion in control animals, suggesting complex regulation of microvascular blood flow in wound healing. Endothelin and one of its receptors, endothelin receptor B, are expressed in the skin and produce vasoconstriction in cutaneous vasculature.31,32 Increased production of endothelin and endothelin receptor B might account for the decline in week 2 wound perfusion despite sustained NO production at days 9 through 11.33 In L-NIL–treated animals, early reductions in granulation tissue thickness and wound perfusion occurred before reductions in wound fluid NOx production, possibly resulting from an effect of LNIL other than inhibition of NOS2 or the inadequacy of wound fluid NOx levels to predict tissue NO concentration. Recent evidence suggests that VEGF gene expression in hypoxic cells is characterized by its transcriptional activation, primarily through the hypoxia-response element that includes cis-acting DNA elements recognized by multiple transactiva-
536 Howdieshell et al
tors.34 Hypoxia-inducible factor–1 (HIF-1) is the best characterized regulator of VEGF transcription. In its active form, it is a dimer composed of 2 distinct subunits, both of which belong to the basic helix-loop-helix protein family, HIF-1α and HIF-1β. With hypoxic conditions or in the presence of NO, HIF-1α protein levels, HIF-1 DNA binding, and VEGF promoter activity and messenger RNA expression are upregulated in various human cell lines.35 Therefore, NO and hypoxia may share common features in the pathways of VEGF induction. Our data implicate an important role for NO in regulating growth factor–mediated processes during wound repair and therefore identify NO as a potential therapeutic target molecule to improve disorders of wound healing. Repair of excisional wounds is impaired in circumstances associated with reduced NOS2 expression and NO availability, such as steroid administration and diabetes.36,37 Dietary supplementation of arginine or topical application of NO donors might increase wound levels of NO with resultant increases in VEGF production. Gene transfer with NOS2 offers potential advantages over other therapeutic approaches in NO-deficient states. Topical application of targeted delivery of the NOS2 gene in an efficient vector should increase local NO production without systemic effects.38 Unlike the constitutive NOS isoforms, NOS2 shows activity independent of intracellular calcium elevations and produces high-level sustained NO synthesis. Even low-efficiency transfer could provide a constant source of adequate local NO to influence healing rates. Finally, recent evidence suggests that statins, in addition to their ability to improve serum lipid profile, exert beneficial effects on endothelial function through an increase in NO production or bioavailability, underscoring the potential of these drugs to promote neovascularization in impaired healing states.39
REFERENCES 1. Clark RAF. Wound repair: overview and general considerations. In: Clark RAF, editor. The molecular and cellular biology of wound repair. New York: Plenum Press; 1996. p. 3-50. 2. Howdieshell TR, Rieger C, Gupta V, Callaway D, McNeil P. Normoxic wound fluid contains high levels of vascular endothelial growth factor. Ann Surg 1998;228:707-15. 3. Martin P. Wound healing: aiming for the perfect skin regeneration. Science 1997;276:75-81. 4. Pollock JS, Webb W, Callaway D, Sathyanarayana, O’Brien W, Howdieshell TR. Nitric oxide synthase isoform expression in a porcine model of granulation tissue formation. Surgery 2001;129:341-50. 5. Albina JE, Mills CD, Henry JL Jr, Caldwell MD. Temporal expression of different pathways of L-arginine metabolism in healing wounds. J Immunol 1990;144:3877-80.
Surgery May 2003
6. Morris SM Jr, Billiar TR. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol 1994;266: E829-39. 7. Goldsmith PC, Leslie TA, Hayes NA, Levell NJ, Foreman JC. Inhibitors of nitric oxide synthase in human skin. J Invest Dermatol 1996;106:113-8. 8. Romero-Graillet C, Aberdam E, Clement M, Ortonne JP, Balloti R. Nitric oxide produced by ultraviolet-irradiated keratinocytes stimulates melanogenesis. J Clin Invest 1997;99:635-42. 9. Bogdan C. Nitric oxide and the regulation of gene expression. Trends Cell Biol 2001;11:66-75. 10. Tsurumi Y, Murohara T, Krasinski K, Chen D, Witzenbichler B, et al. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nature Med 1997;3:879-86. 11. Nilsson GE, Tenland T, Oberg PA. A new instrument for continuous measurement of tissue blood flow by light beating spectroscopy. IEEE Trans Biomed Eng 1980;27:12-9. 12. Vongsavan N, Matthews B. Some aspects of the use of laser Doppler flow meters for recording tissue blood flow. Exp Physiol 1993;78:1-14. 13. Rendell MS, Milliken BK, Finney DE, Healy JC, Bonner RF. The microvascular composition of the healing wound compared at skin sites with nuritive versus arteriovenous perfusion. J Surg Res 1998;80:373-80. 14. Howdieshell TR, Callaway D, Webb WL, Gaines MD, Proctor CD Jr, Sathyanarayana, et al. Antibody neutralization of vascular endothelial growth factor inhibits wound granulation tissue formation. J Surg Res 2001;96:173-82. 15. Connor JR, Manning PT, Settle SL, Moore WM, Webber RK, Currie MG. Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. Eur J Pharmacol 1995;273:15-24. 16. Stenger S, Thuring H, Rollinghoff M, Manning P, Bogdan C. L-N6-(1-Iminoethyl)-lysine potently inhibits inducible nitric oxide synthase and is superior to NG-monomethyl-arginine in vitro and in vivo. Eur J Pharmacol 1995;294:703-12. 17. Omerud AD, Weller R, Copeland P, Benjamin N, Ralston SH, Herriot R. Detection of nitric oxide and nitric oxide synthase in psoriasis. Arch Dermatol Res 1998;290:3-8. 18. Wang R, Ghahary A, Shen YJ, Scott PG, Tredget EE. Human dermal fibroblasts produce nitric oxide and express both constitutive and inducible nitric oxide synthase isoforms. J Invest Dermatol 1996;106:419-27. 19. Carmeliet P, Ferreira V, Breier G, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435-9. 20. Nissen NN, Polverini PJ, Koch AE, Volin MV, Gamelli RL, DiPietro LA. Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing. Am J Pathol 1998;152:1445-52. 21. Klagsbrun M, D’Amore PA. Vascular endothelial growth factor and its receptors. Cytokine Growth Factor Rev 1996;7:249-57. 22. Leibovich SJ, Polverini PJ, Fong TW, Koch AE. Production of angiogenic activity by human monocytes requires an Larginine/nitric oxide synthase-dependent effector mechanism. Proc Natl Acad Sci U S A 1994;91:4190-4. 23. Chin K, Kurashima Y, Ogura T, Yoshida S, Esumi H. Induction of vascular endothelial growth factor by nitric oxide in human glioblastoma and hepatocellular carcinoma cells. Oncogene 1997;15:437-42. 24. Gallo O, Masini E, Morbidelli L, Franchi A, Vergari WA, Ziche M. Role of nitric oxide in angiogenesis and tumor progression in head and neck cancer. J Natl Cancer Inst 1998;90:587-96.
Surgery Volume 133, Number 5 25. Thomson LL, Lawton FG, Beesley JE, Moncada S. Nitric oxide synthase activity in human gynecological cancer. Cancer Res 1994;54:1352-4. 26. Popoff N. The digital vascular system. Arch Path 1934;18: 295-305. 27. Rendell MS, Milliken BK, Finnegan MF, Finney DA, Healy JC. The skin blood flow response in wound healing. Microvasc Res 1997;53:222-9. 28. Rendell MS, Giiter M, Bamisedun O, Davenport K, Schultz R. The laser Doppler analysis of posturally induced changes in skin blood flow at elevated temperature. Clin Physiol 1992;12:241-53. 29. Rendell MS, Kelly ST, Bamisedun O, Luu T, Finney DA, Knox S. The effect of increasing temperature on skin blood flow and red cell deformability. Clin Physiol 1993;13:235-45. 30. Rendell MS, Johnson ML, Smith D, Finney D, Lancaster S. Skin blood flow response in the rat model of wound healing: expression of vasoactive factors. J Surg Res 2002;107:18-26. 31. Bull HA, Dowd PM. Endothelin-1 in human skin. Dermatology 1993;187:1-15. 32. Tao W, Liou GI, Wu X, Abney TO, Reinach PS. ETB and epidermal growth factor receptor stimulation of wound closure in bovine corneal epithelial cells. Invest Ophthalmol Vis Sci 1995;36:2614-25.
Howdieshell et al 537
33. Lipa JE, Neligan PC, Perreault TM, Baribeau J, Knowlton RJ, Pang CY. Vasoconstrictor effect of endothelin-1 in human skin: role of ETA and ETB receptors. Am J Physiol 1999;276:H359-70. 34. Kimura H, Weisz A, Kurashima Y, Ogura T, Esumi H. Hypoxia response element of human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood 2000;95:189-97. 35. Semenza GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 1999; 15:551-78. 36. Bulgrin JP, Shabani M, Chakravarthy D, Smith DJ. Nitric oxide synthesis is suppressed in steroid-impaired and diabetic wounds. Wounds 1995;7:48-57. 37. Schaffer MR, Tantry U, Enron PA, Thornton FJ, Barbul A. diabetes-impaired healing and reduced wound nitric oxide synthesis: a possible pathophysiologic correlation. Surgery 1997;121:513-9. 38. Yamasaki K, Edington HDJ, McClosky C, Tzeng E, Steed DL, Billiar TR. Reversal of impaired wound repair in iNOSdeficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest 1998;101:967-71. 39. Brovet A, Sonveaux P, Dessy C, Balligand JL, Feron O. Hsp 90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins. Circ Res 2001;89:866-73.
Background. Wound repair results from a series of highly orchestrated cellular and biochemical events, including increased synthesis of the bioregulatory molecule nitric oxide (NO). The goal of this work was to test the functional role of NO in promotion of vascular endothelial growth factor (VEGF) production and the vigorous granulation tissue formation characteristic of this wound model. Methods. A ventral hernia, surgically created in the abdominal walls of 12 swine, was repaired with silicone sheeting and skin closure. An osmotic infusion pump, inserted in a remote subcutaneous pocket, delivered saline solution (n = 6) or the selective inducible NO synthase inhibitor N6 (iminoethyl)-L-lysine (L-NIL; n = 6) into the wound environment. Granulation tissue thickness was determined with ultrasongraphy, and local wound perfusion was measured with laser Doppler analysis for 2 weeks. Fluid was aspirated serially from the wound compartment for measurement of nitrite/nitrate, VEGF, and transforming growth factor–β1 concentrations. On day 14, the animals were killed and the abdominal wall was harvested for immunohistochemical and molecular analysis. Results. In animals that received saline solution, a nearly linear 4-fold increase in granulation tissue thickness was measured during the 14-day interval. In contrast, in animals that received L-NIL, day 14 granulation tissue thickness was essentially unchanged from the day 2 values of saline solution–treated animals. Moreover, in the L-NIL animals, ultrasonography was unable to resolve the angiogenic zone typical of controls, and correspondingly, wound vessel count and vascular surface area estimates derived from image analysis of histologic sections were 2-fold to 3-fold lower in the L-NIL animals compared with controls. Reductions in basal (2-fold) and heat-provoked (2.5-fold) wound perfusion were noted in L-NIL animals. Wound fluid nitrite/nitrate and VEGF levels were strikingly (4-fold and 5-fold, respectively) reduced in L-NIL animals on days 9 to 14. Immunochemistry results showed reduced VEGF protein content in granulation tissue and keratinocytes within the hyperproliferative epithelium at wound edge. Finally, transforming growth factor–β1 levels were unaffected by L-NIL treatment. Conclusion. VEGF production in granulation tissue is dependent on the presence of functionally active inducible NO synthase and hence, the production of NO. NO and VEGF are therefore defined as key regulators of granulation tissue formation. (Surgery 2003;133:528-37.) From the Department of Surgery, University of New Mexico Health Sciences Center, Albuquerque, NM; and the Departments of Surgery, Radiology, and Anatomy and Cellular Biology, Medical College of Georgia, Augusta, Ga
Accepted for publication January 18, 2003. Supported by American Heart Association Southeast Affiliate Grant (Dr Howdieshell). Reprint requests: Thomas R. Howdieshell, MD, FACS, Trauma/Surgical Critical Care, Department of Surgery ACC-2, University of New Mexico Health Sciences Center, 915 Camino de Salud, Albuquerque, NM 87131. © 2003 Mosby, Inc. All rights reserved. 0039-6060/2003/$30.00 + 0 doi:10.1067/msy.2003.128
528 SURGERY
WOUND HEALING IS a highly ordered and coordinated process that involves inflammation, proliferation, matrix deposition, and tissue remodeling. After injury, new tissue formation begins with reepithelialization and is followed by granulation tissue formation. The latter process encompasses macrophage accumulation, fibroblast ingrowth, matrix formation, and angiogenesis.1 Inflammation, reepithelialization, and granulation tissue formation are driven
Surgery Volume 133, Number 5
in part by a complex interaction of growth factors and cytokines, coordinately released into the area of injury.2,3 In a previous study, we showed that vascular endothelial growth factor (VEGF) is an essential regulator of granulation tissue formation in a porcine abdominal wound model.2 In addition to these proteins, evidence exists for an important role of small diffusible molecules such as nitric oxide (NO) in wound repair.4,5 NO can be produced by 1 of 3 known NO synthase (NOS) isoforms. The cytokine-inducible or inflammatory NOS (NOS2) produces NO in a sustained manner independent of intracellular calcium concentration and is expressed in many inflammatory conditions, including wound healing.4,6 The biologic activities of NO in human skin include regulation of epithelialization, vasodilatation and melanogenesis, and protection against invading pathogens.7,8 Little is known about the functional role of NO during the healing process. However, in vitro study results reveal that NO affects expressional regulation of many genes, including the gene encoding for VEGF.9,10 These results suggest that a key role of NO in wound healing is to upregulate VEGF production in granulation tissue. A central prediction of this hypothesis is that inhibition of NO production with N6 (iminoethyl)-L-lysine (L-NIL) will inhibit VEGF production and, consequently, granulation tissue formation. METHODS Pig model of wound healing. All animals were treated humanely in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals as part of a protocol approved by the Medical College of Georgia’s animal review committee. Domestic female Landrace swine (weight, 15 to 20 kg) underwent induction with Telazol, xylazine hydrochloride, and atropine and anesthesia with isoflurane (1.5% to 3%) via inhalation with an endotracheal tube. Postsurgical analgesia was provided with buprenorphine hydrochloride for 48 hours. After maintenance of inhalation anesthesia and sterile preparation (providine-iodine) and draping, an infraumbilical diagnostic peritoneal lavage was performed (Arrow DPL Kit, Arrow International, Reading, Pa) with the percutaneous Seldinger technique to obtain preincision peritoneal fluid for biochemical analysis. A sample of plasma for enzyme-linked immunosorbent assay was obtained from an ear vein. Next, a supraumbilical midline laparotomy was performed, and biopsy samples of omentum, peritoneum, and portions of abdominal wall were
Howdieshell et al 529
taken to be used as controls. A full-thickness section of the abdominal wall (8 12 cm), excluding the skin, was surgically excised to create a hernia defect. A similar-sized piece of silicone sheeting (Bioplexus Corp, Saticoy, Calif) was sutured to the fascial edges with monofilament suture to effect abdominal wall closure. In previous work, we observed vigorous growth of granulation tissue on both sides of the silicone implant removed from animals on day 14. The sheeting does not incorporate into adjacent tissues but rather becomes encapsulated, creating an excellent scaffold for granulation tissue formation.2,4 Two osmotic minipumps with attached polyethylene tubing were surgically implanted into remote abdominal wall subcutaneous pockets (model ML2; Alzet Corp, Palo Alto, Calif). The attached tubing, used to deliver saline solution or L-NIL into the wound environment, entered the wound compartment via a subcutaneous tunnel. To ensure uniform delivery of L-NIL or saline solution within the aqueous wound compartment, the tubing contained multiple side holes and was looped to increase surface area. The pump was manufactured to deliver 5 mL/h for 14 days. Before implantation, the pump was loaded with saline solution or L-NIL with sterile conditions and maintained at 37°C in an incubator to ensure optimal delivery. After pump insertion, the midline and pocket skin incisions were closed in 2 layers to cover the biomaterial and pumps and thus complete wound closure. Each animal was housed for 14 days after surgery and received water and food ad libitum. On days 2, 4, 7, 9, 11, and 14 after surgery, each animal underwent sedation and was placed supine, and abdominal ultrasonography was performed with an Acusom Ultrasound Imager with 7.5-mHz linear array and 3.5-mHz sector probe (Acusom Corp, Mountain View, Calif). At each time point, images recorded electronically for later analysis included granulation tissue thickness measured in millimeters with a cursor at multiple consistent sites. Each day, a sample of wound fluid located in the compartment between the skin and silicone sheeting was percutaneously aspirated with a 23gauge needle with sterile conditions and ultrasound scan guidance. On day 14 after surgery, the animals underwent sedation, and an infraumbilical diagnostic peritoneal lavage was performed to obtain presacrifice peritoneal fluid for assay. A blood sample was obtained from an ear vein. Each animal then was killed with pentobarbital, and the abdominal wall was harvested en bloc. The osmotic minipumps were removed from the subcutaneous pockets, and their
530 Howdieshell et al
residual volume was measured to confirm complete delivery. Histologic and biochemical analyses were performed on blood, peritoneal fluid, local wound fluid, and subcutaneous granulation tissue. Laser Doppler measurement. Local wound perfusion was determined with the Periflux 5010/5020 laser Doppler device (Perimed, Inc, North Royalton, Ohio) equipped with standard (408) and heater (452) probes.11,12 Perfusion measurements were recorded at 6 consistently spaced and reproducible reference sites located from cephalad to caudad, 3 on each side of the abdominal incision, all overlying the wound compartment. A temperature-control module in the Periflux System measured and enhanced local skin temperature. Perfusion was initially determined at basal temperature with the standard probe. Then, the heater probe was heated to 44°C, a temperature high enough to elicit maximal blood flow without burning the skin, and the measurements were repeated.13 Perfusion was measured for 10 to 15 minutes to ensure equilibration. Testing took place in a room controlled at an ambient temperature of 24°C. Values are reported as means of the 6 reference sites on days 0, 2, 4, 7, 9, 11, and 14. The summated data were stored and analyzed with the Perisoft software package, version V5.10C2 (Perimed, Inc). Enzyme-linked immunosorbent assay measurement. VEGF and transforming growth factor–β1 (TGF-β1) were assayed in plasma and wound and peritoneal fluids with quantitative sandwich immunoassay kits (R&D Systems, Minneapolis, Minn) according to the manufacturer’s instructions. All fluids were centrifuged immediately after harvest and then frozen at –70°C until assayed. Samples for TGF-β1 analysis were acid-treated to activate latent TGF-β1 to the immunoreactive form (according to kit instructions) before application to microtiter plate wells. Each sample was run in duplicate and at pertinent dilutions, with mean values reported. Nitrite/nitrate measurement. Nitrite and nitrate (NOx) are the oxidation products of NO in biologic fluids and are measured as an index of NOS activity. NOx was assayed in wound and peritoneal fluids with the Bioxytech NO nonenzymatic assay (Oxis International, Inc, Portland, Ore) according to manufacturer’s instructions. All fluids were centrifuged immediately after harvest and frozen at –70°C until assayed. Each sample was run in duplicate and at pertinent dilutions, with mean values reported. Vascular density and morphometric determinations. Granulation tissue vessel count and vascular cross-sectional surface area were determined with
Surgery May 2003
image analysis of von Willebrand’s factor– immunostained sections. Multiple consistent wound biopsies were analyzed. The boundaries of the granulation tissue region to be analyzed were the wound fluid compartment and a distinct subcutaneous tissue interface. Immunohistochemical analysis was performed with the avidin-biotin method as previously reported.14 A Zeiss microscope with attached digital camera was used for image analysis. The magnified image (200) of the slide section was acquired with Adobe Photoshop and analyzed with IPLab Spectrum software on a MacIntosh computer. Vessels in each section were defined by the circular or ovoid image of the brown endothelial walls. Capillaries, arterioles, and venules were counted. Vessels were identified and marked by an observer with no knowledge of treatment group. The software program was used to count the number of vessels marked in each section. For confirmation of these estimates of vascular density, the cross-sectional luminal area of vessels was computed. Closed vessels would alter the numeric count but only minimally affect the estimate of vascular area. Thus, the determination of luminal area constituted a separate estimate of vascular density. For determination of area, the minimum and maximum major axis of each vessel was measured. The calibration scale was set with a stage micrometer to match computer pixels to a micrometer scale. The cross-sectional area of each measurement field was computed with careful outlining of the previously reported boundaries of granulation tissue.14 For confirmation of ultrasonographic measurements, granulation tissue thickness was also determined with morphometric analysis of histologic specimens. The wound biopsies used for vascular density determination were scanned with the previously described image analysis techniques. Granulation tissue thickness was measured in millimeters by cursor-defined boundaries with established calibration scale and software computations. Immunohistochemical staining of tissues. Serial sections (4 to 5 µm) of formalin-fixed, paraffinembedded tissue were dewaxed in xylene, taken through graded ethanol, and then hydrated in phosphane-buffered saline solution. Sections were treated with DAKO Target Retrieval Solution (S1700; DAKO Corp, Carpinteria, Calif) and steam heated for 30 minutes to improve antigen retrieval. The sections were incubated for 20 minutes with 0.75% hydrogen peroxide in methanol to block endogenous peroxidase activity, washed, incubated with DAKO Protein Block serum-free for 30 min-
Surgery Volume 133, Number 5
utes to reduce nonspecific binding, and incubated with primary antibody, polyclonal rabbit anti-VEGF (A-20, 1:500; Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) for 30 minutes at room temperature in a humidified chamber. Next, the sections were incubated with a secondary antibody with the DAKO Envision Plus System and DAB kit, according to the manufacturers instructions. The sections were counterstained with Vector Hematoxylin QS (Vector Laboratories, Burlingame, Calif) with quick immersion. The 3, 3'-diaminobenzidine substrate-chromogen resulted in a brown-colored precipitate at the antigen site. For confirmation of the specificity of the immunochemical staining, secondary antibody only, normal rabbit immunoglobulin G, and antigen excess stainings with blocking peptides were performed. L-NIL inhibition of NOS2. N6 (iminoethyl)-Llysine, purchased from Biomol Research Laboratories (Plymouth Meeting, Pa) has been shown to be 33 to 50 times more selective for inhibition of NOS2 than constitutive endothelial NOS.15,16 Each osmotic infusion pump (2 per pig) was loaded with either 2 mL of saline solution (n = 6) or a 2-mL solution containing 25 mg/mL of L-NIL in saline solution as a carrier (n = 6). Each pump was manufactured to deliver 5 µL/h for 14 days. The L-NIL dose was based on the previously stated L-NIL characteristics and the predicted serial NOx levels and day 14 wound fluid volume.4 Statistical analysis. All data are expressed as the mean ± the standard error of the mean. Differences among groups and between baseline and subsequent time points were determined with repeated-measures analysis of variance with Tukey significant difference test used for post hoc analysis. A P value of .05 or less was considered statistically significant. RESULTS Granulation tissue growth, angiogenesis, and wound perfusion in a large-scale abdominal wall wound model. Delivery of saline solution into the wound compartment did not alter the predicted wound responses.2,4 The skin incision was completely epithelialized by day 14, without evidence of induration, erythema, or early dermal hypertrophy. The subcutaneous granulation tissue harvested at day 14 from animals that received saline solution was thick, tough, red, and stratified into zones (Fig 1, A), appearing to correlate with the predicted tripartite histologic profile of granulation tissue, zones of inflammation, angiogenesis, and fibroplasia.1,2 Sonography was used to serially image the wound site to measure the thickness of the devel-
Howdieshell et al 531
oping granulation tissue. In ultrasonographic images, the developing granulation tissue of saline solution–treated animals appeared to be stratified into 3 layers.4 A nearly linear increase in granulation tissue thickness was measured from serially acquired images, and by day 14, the granulation tissue had reached a thickness of 19.2 ± 1.2 mm in saline solution–treated animals (Fig 2). Histologically, control group granulation tissue (day 14) was observed to consist of a presumptive neutrophil/macrophage-rich zone that bordered on the wound fluid compartment, a microvasculature-rich zone whose endothelial cell components could be stained with an antibody against von Willebrand’s factor, and a presumptive fibroblastrich, most mature zone. In saline solution–treated pigs, basal wound perfusion increased in nearly linear fashion to reach a maximal, 5-fold (30 ± 10 PU to 150 ± 22 PU) increase from baseline by day 7 and then decreased to near baseline values by day 14 (Fig 3, A). Before surgical incision, heat provocation to 44°C produced a 2.7-fold (30 ± 7 PU to 80 ± 15 PU) increase in wound perfusion from basal values. Heat-provoked wound perfusion increased to reach a maximal value on day 7, a 33% increase from day 7 basal values (150 ± 20 PU to 200 ± 20 PU), and returned to baseline by day 14 (Fig 3, B). Effects of L-NIL on wound NOS2 activity. As shown in Fig 4, wound NOx levels were reduced on days 9 to 14 (P = .042, P = .008, and P = .05, respectively), clearly indicating an inhibitory effect of LNIL on NOS2 enzyme activity at time points when NOS2 activity was highest during wound repair. Reductions in granulation tissue growth, vascularity, VEGF content, and local wound perfusion with NOS2 inhibition. The day 14 granulation tissue harvested from L-NIL–infused wounds was, compared with saline solution–infused wounds, remarkably thin and friable, uniformly white in color, and without visible stratification when viewed in cross section (Fig 1, B). All skin incisions healed without evidence of wound infection. Moreover, with an ultrasonographic view, the L-NIL wound granulation tissue lacked the zonation characteristic of the control group—that is, a dark band, presumed to be a highly vascularized zone, sandwiched between 2 lighter bands, presumed to represent the inflammatory and fibroplasia zones. Quantitative ultrasound scan analysis detected significant reductions in L-NIL granulation tissue thickness compared with controls between days 4 and 14 (Fig 2). Morphometric measurement of day 0 soft tissue and day 14 granulation tissue in L-NIL and control animals confirmed the accuracy of
532 Howdieshell et al
Surgery May 2003
Fig 2. Ultrasonographic measurements of granulation tissue thickness. Day 0 values are preincision skin and subcutaneous tissue thickness. Bars are standard error of mean. Saline solution–treated animals are represented with solid circles, and L-NIL–treated animals with open circles. (*P < .05 versus controls.)
Fig 1. (A) Harvested silicone implantation site from saline solution–treated animal viewed from peritoneal surface with skin margin down, revealing wound fluid compartment (WFC) and stratification of granulation tissue (GT) into zones. (B) Harvested L-NIL silicone implantation site. Note marked reduction in granulation tissue thickness (arrows) and lack of rich, red appearance. MZ, Macrophage-neutrophil rich-zone; AZ, angiogenic zone; FZ, fibroblast-rich zone; SQ, skin and subcutaneous tissue; S, silicone sheeting.
ultrasonography in determination of granulation tissue thickness. Consistent with the ultrasound scan images, histologic analysis revealed an absence of stratification
within the L-NIL granulation tissue. The tissue was composed primarily of inflammatory cells and was strikingly devoid of vasculature (compare Figs 5, A and B). Counts from sections immunostained for von Willebrand’s factor, in which endothelial cells could be unambiguously identified, confirmed that L-NIL treatment resulted in 2-fold to 3-fold reductions in granulation tissue vessel count (control, 2850 ± 310; L-NIL, 1010 ± 290; P < .05) and crosssectional surface area (control, 1.28 ± 0.22 mm2; LNIL, 0.04 ± 0.15 mm2; P < .05) compared with control wounds. In saline solution–infused wounds, VEGF levels progressively increased over the 14-day interval, reaching a maximal value of 11,101 ± 1910 pg/mL. In the L-NIL wounds, VEGF levels peaked on day 7 at a much lower level (3310 ± 820 pg/mL) and remained at significantly reduced concentrations over days 9 to 14 (Fig 6, A). In contrast, progressive increases in levels of TGF-β1 were noted in control and L-NIL wound fluids, with no difference shown between groups (Fig 6, B).
Surgery Volume 133, Number 5
Howdieshell et al 533
Fig 4. NOx concentrations in serial wound fluid. Bars are standard error of mean. Saline solution–treated animals are represented with solid circles, and L-NIL–treated animals with open circles. (*P < .05 vs controls.)
Fig 3. Laser Doppler flowmetry of basal (A) and heat-provoked (B) wound perfusion. Bars are standard error of mean. Saline solution–treated animals are represented with solid circles, and L-NIL–treated animals with open circles. (*P < .05 vs controls.)
Immunohistochemical analysis of day 14 wound tissue showed differences in VEGF protein content between control and L-NIL animals (Figs 7, A and B). In saline solution–treated animals, large numbers of keratinocytes within the hyperproliferative epithelium were characterized by an intense staining, indicating a large content of VEGF protein. In addition, intense staining was noted in granulation tissue fibroblasts, macrophages, neutrophils, and endothelium. In contrast, in L-NIL–treated animals, the hyperproliferative epithelium and granulation tissue were characterized by a dramatic reduction in VEGF protein content. Finally, basal and heat-provoked wound perfusions were reduced in L-NIL–treated animals (Figs 3, A and B). Reductions in basal perfusion (1.5-fold to 2.5-fold) were recorded in L-NIL animals between days 2 and 11, with a return to baseline by day 14. Heat provocation produced 1.7-fold to 2.5fold reductions in wound perfusion in L-NIL animals compared with saline solution controls throughout the 14-day study period.
534 Howdieshell et al
Fig 5. Histologic comparison of day 14 saline solution (A) and L-NIL (B) granulation tissue shows marked reduction in vascularity in L-NIL specimen. Arrows denote blood vessels. Hematoxylin and eosin, 400.
DISCUSSION The biologic activities of NO are numerous and complex. For example, in human skin, NO regulates epithelial migration, vasodilatation, and melanogenesis and protects against invading pathogens.7,8 Recent studies have implicated NO in the pathogenesis of several skin diseases. NOS2 has been shown to be overexpressed in psoriasis, a
Surgery May 2003
Fig 6. Serial measurement of growth factor concentrations in wound fluid after abdominal wall injury. (A) VEGF. (B) TGF-β1. Saline solution–treated animals are represented with solid circles, and L-NIL–treated animals with open circles. Bars are standard error of mean. Day 0 values are from peritoneal fluid. (*P < .05 vs controls at day 14 (maximal) value.)
chronic inflammatory skin disease characterized by hyperproliferation and incomplete differentiation of epidermal keratinocytes in the involved lesions.17,18 The evidence for its role in skin wound healing has been mentioned previously.
Surgery Volume 133, Number 5
Vascular endothelial growth factor exerts a pivotal role in normal and pathologic angiogenesis. Its production by stromal and epithelial cells is sufficient to trigger angiogenesis, and inactivation of the corresponding gene results in abnormal blood vessel development and embryonic lethality in mice.19 VEGF expression has been shown in granulation tissue macrophages and fibroblasts and proliferating keratinocytes at the wound edge during cutaneous repair.2,20,21 We have reported previously that antibody neutralization of VEGF results in significant reductions in granulation tissue formation, verifying the functional role of VEGF in wound healing.14 Recent evidence indicates that NO regulates the expression of VEGF. Production of angiogenic activity by human monocytes has been found to depend on NO, and NO-generating compounds have been shown to stimulate VEGF gene transcription in human glioblastoma and hepatoma cells in culture.22,23 Furthermore, a strong positive correlation between NOS activity, cyclic guanosine monophosphate levels, and tumor angiogenesis has been described in head and neck and gynecologic cancers.24,25 We reported previously that NOS2 plays a critical role in wound granulation tissue formation.4 Although systemic administration of L-NIL has been well documented to almost completely block the in vivo activity of NOS2 in lymph nodes and skin in mice,15,16 we show the local inhibitory activity of the selective NOS2 inhibitor in our porcine wound healing model. In this study, we provide in vivo evidence for dramatic reductions in wound fluid and tissue VEGF protein content during healing in L-NIL–treated pigs. The lack of effect of LNIL on TGF-β1 levels, a mediator important in wound fibrogenesis, indicates that nonspecific inhibition of a wide range of wound-related processes did not occur as a result of L-NIL infusion. Ultrasonography and morphometric analysis of histology depicted significant reductions in granulation tissue growth in L-NIL animals. Furthermore, immunohistochemistry revealed several-fold reductions in wound vessel count and vascular surface area estimates. Histology does not permit an evaluation of dynamic changes in microvasculature. Therefore, laser Doppler flowmetry was used to measure serially basal and heat-provoked wound perfusion. The microvasculature of human and animal skin is not uniform. Most skin locations have primarily nutritive perfusion by small capillaries with low flow and high resistance. However, areas such as the pulp of the fingertip or toe have large contributions of
Howdieshell et al 535
Fig 7. VEGF immunoreactivity (brown) in saline solution (A) and L-NIL (B) granulation tissue.
arterioles and venules, which are large diameter microvessels with low resistance and high flow.26,27 Previous work has shown that nutritively perfused skin sites such as the abdominal wall have flow properties markedly different from those with substantial arteriolar and venular perfusion.28 The differences are accentuated by local skin heating to 44°C, which causes maximal vasodilation. With local heating, perfusion may increase 50% to 100% at nutritive sites and by as much as 300% to 500% at arteriolar sites.29 In L-NIL–treated animals, laser Doppler analysis showed striking decreases in basal and temperature-provoked wound perfusion resulting from reductions in VEGF-dependent vascular density and NO-dependent vasomotor tone.30 Peak production of wound fluid NOx occurred later in time than peak increases in basal and heatprovoked wound perfusion in control animals, suggesting complex regulation of microvascular blood flow in wound healing. Endothelin and one of its receptors, endothelin receptor B, are expressed in the skin and produce vasoconstriction in cutaneous vasculature.31,32 Increased production of endothelin and endothelin receptor B might account for the decline in week 2 wound perfusion despite sustained NO production at days 9 through 11.33 In L-NIL–treated animals, early reductions in granulation tissue thickness and wound perfusion occurred before reductions in wound fluid NOx production, possibly resulting from an effect of LNIL other than inhibition of NOS2 or the inadequacy of wound fluid NOx levels to predict tissue NO concentration. Recent evidence suggests that VEGF gene expression in hypoxic cells is characterized by its transcriptional activation, primarily through the hypoxia-response element that includes cis-acting DNA elements recognized by multiple transactiva-
536 Howdieshell et al
tors.34 Hypoxia-inducible factor–1 (HIF-1) is the best characterized regulator of VEGF transcription. In its active form, it is a dimer composed of 2 distinct subunits, both of which belong to the basic helix-loop-helix protein family, HIF-1α and HIF-1β. With hypoxic conditions or in the presence of NO, HIF-1α protein levels, HIF-1 DNA binding, and VEGF promoter activity and messenger RNA expression are upregulated in various human cell lines.35 Therefore, NO and hypoxia may share common features in the pathways of VEGF induction. Our data implicate an important role for NO in regulating growth factor–mediated processes during wound repair and therefore identify NO as a potential therapeutic target molecule to improve disorders of wound healing. Repair of excisional wounds is impaired in circumstances associated with reduced NOS2 expression and NO availability, such as steroid administration and diabetes.36,37 Dietary supplementation of arginine or topical application of NO donors might increase wound levels of NO with resultant increases in VEGF production. Gene transfer with NOS2 offers potential advantages over other therapeutic approaches in NO-deficient states. Topical application of targeted delivery of the NOS2 gene in an efficient vector should increase local NO production without systemic effects.38 Unlike the constitutive NOS isoforms, NOS2 shows activity independent of intracellular calcium elevations and produces high-level sustained NO synthesis. Even low-efficiency transfer could provide a constant source of adequate local NO to influence healing rates. Finally, recent evidence suggests that statins, in addition to their ability to improve serum lipid profile, exert beneficial effects on endothelial function through an increase in NO production or bioavailability, underscoring the potential of these drugs to promote neovascularization in impaired healing states.39
REFERENCES 1. Clark RAF. Wound repair: overview and general considerations. In: Clark RAF, editor. The molecular and cellular biology of wound repair. New York: Plenum Press; 1996. p. 3-50. 2. Howdieshell TR, Rieger C, Gupta V, Callaway D, McNeil P. Normoxic wound fluid contains high levels of vascular endothelial growth factor. Ann Surg 1998;228:707-15. 3. Martin P. Wound healing: aiming for the perfect skin regeneration. Science 1997;276:75-81. 4. Pollock JS, Webb W, Callaway D, Sathyanarayana, O’Brien W, Howdieshell TR. Nitric oxide synthase isoform expression in a porcine model of granulation tissue formation. Surgery 2001;129:341-50. 5. Albina JE, Mills CD, Henry JL Jr, Caldwell MD. Temporal expression of different pathways of L-arginine metabolism in healing wounds. J Immunol 1990;144:3877-80.
Surgery May 2003
6. Morris SM Jr, Billiar TR. New insights into the regulation of inducible nitric oxide synthesis. Am J Physiol 1994;266: E829-39. 7. Goldsmith PC, Leslie TA, Hayes NA, Levell NJ, Foreman JC. Inhibitors of nitric oxide synthase in human skin. J Invest Dermatol 1996;106:113-8. 8. Romero-Graillet C, Aberdam E, Clement M, Ortonne JP, Balloti R. Nitric oxide produced by ultraviolet-irradiated keratinocytes stimulates melanogenesis. J Clin Invest 1997;99:635-42. 9. Bogdan C. Nitric oxide and the regulation of gene expression. Trends Cell Biol 2001;11:66-75. 10. Tsurumi Y, Murohara T, Krasinski K, Chen D, Witzenbichler B, et al. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nature Med 1997;3:879-86. 11. Nilsson GE, Tenland T, Oberg PA. A new instrument for continuous measurement of tissue blood flow by light beating spectroscopy. IEEE Trans Biomed Eng 1980;27:12-9. 12. Vongsavan N, Matthews B. Some aspects of the use of laser Doppler flow meters for recording tissue blood flow. Exp Physiol 1993;78:1-14. 13. Rendell MS, Milliken BK, Finney DE, Healy JC, Bonner RF. The microvascular composition of the healing wound compared at skin sites with nuritive versus arteriovenous perfusion. J Surg Res 1998;80:373-80. 14. Howdieshell TR, Callaway D, Webb WL, Gaines MD, Proctor CD Jr, Sathyanarayana, et al. Antibody neutralization of vascular endothelial growth factor inhibits wound granulation tissue formation. J Surg Res 2001;96:173-82. 15. Connor JR, Manning PT, Settle SL, Moore WM, Webber RK, Currie MG. Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. Eur J Pharmacol 1995;273:15-24. 16. Stenger S, Thuring H, Rollinghoff M, Manning P, Bogdan C. L-N6-(1-Iminoethyl)-lysine potently inhibits inducible nitric oxide synthase and is superior to NG-monomethyl-arginine in vitro and in vivo. Eur J Pharmacol 1995;294:703-12. 17. Omerud AD, Weller R, Copeland P, Benjamin N, Ralston SH, Herriot R. Detection of nitric oxide and nitric oxide synthase in psoriasis. Arch Dermatol Res 1998;290:3-8. 18. Wang R, Ghahary A, Shen YJ, Scott PG, Tredget EE. Human dermal fibroblasts produce nitric oxide and express both constitutive and inducible nitric oxide synthase isoforms. J Invest Dermatol 1996;106:419-27. 19. Carmeliet P, Ferreira V, Breier G, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435-9. 20. Nissen NN, Polverini PJ, Koch AE, Volin MV, Gamelli RL, DiPietro LA. Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing. Am J Pathol 1998;152:1445-52. 21. Klagsbrun M, D’Amore PA. Vascular endothelial growth factor and its receptors. Cytokine Growth Factor Rev 1996;7:249-57. 22. Leibovich SJ, Polverini PJ, Fong TW, Koch AE. Production of angiogenic activity by human monocytes requires an Larginine/nitric oxide synthase-dependent effector mechanism. Proc Natl Acad Sci U S A 1994;91:4190-4. 23. Chin K, Kurashima Y, Ogura T, Yoshida S, Esumi H. Induction of vascular endothelial growth factor by nitric oxide in human glioblastoma and hepatocellular carcinoma cells. Oncogene 1997;15:437-42. 24. Gallo O, Masini E, Morbidelli L, Franchi A, Vergari WA, Ziche M. Role of nitric oxide in angiogenesis and tumor progression in head and neck cancer. J Natl Cancer Inst 1998;90:587-96.
Surgery Volume 133, Number 5 25. Thomson LL, Lawton FG, Beesley JE, Moncada S. Nitric oxide synthase activity in human gynecological cancer. Cancer Res 1994;54:1352-4. 26. Popoff N. The digital vascular system. Arch Path 1934;18: 295-305. 27. Rendell MS, Milliken BK, Finnegan MF, Finney DA, Healy JC. The skin blood flow response in wound healing. Microvasc Res 1997;53:222-9. 28. Rendell MS, Giiter M, Bamisedun O, Davenport K, Schultz R. The laser Doppler analysis of posturally induced changes in skin blood flow at elevated temperature. Clin Physiol 1992;12:241-53. 29. Rendell MS, Kelly ST, Bamisedun O, Luu T, Finney DA, Knox S. The effect of increasing temperature on skin blood flow and red cell deformability. Clin Physiol 1993;13:235-45. 30. Rendell MS, Johnson ML, Smith D, Finney D, Lancaster S. Skin blood flow response in the rat model of wound healing: expression of vasoactive factors. J Surg Res 2002;107:18-26. 31. Bull HA, Dowd PM. Endothelin-1 in human skin. Dermatology 1993;187:1-15. 32. Tao W, Liou GI, Wu X, Abney TO, Reinach PS. ETB and epidermal growth factor receptor stimulation of wound closure in bovine corneal epithelial cells. Invest Ophthalmol Vis Sci 1995;36:2614-25.
Howdieshell et al 537
33. Lipa JE, Neligan PC, Perreault TM, Baribeau J, Knowlton RJ, Pang CY. Vasoconstrictor effect of endothelin-1 in human skin: role of ETA and ETB receptors. Am J Physiol 1999;276:H359-70. 34. Kimura H, Weisz A, Kurashima Y, Ogura T, Esumi H. Hypoxia response element of human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood 2000;95:189-97. 35. Semenza GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 1999; 15:551-78. 36. Bulgrin JP, Shabani M, Chakravarthy D, Smith DJ. Nitric oxide synthesis is suppressed in steroid-impaired and diabetic wounds. Wounds 1995;7:48-57. 37. Schaffer MR, Tantry U, Enron PA, Thornton FJ, Barbul A. diabetes-impaired healing and reduced wound nitric oxide synthesis: a possible pathophysiologic correlation. Surgery 1997;121:513-9. 38. Yamasaki K, Edington HDJ, McClosky C, Tzeng E, Steed DL, Billiar TR. Reversal of impaired wound repair in iNOSdeficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest 1998;101:967-71. 39. Brovet A, Sonveaux P, Dessy C, Balligand JL, Feron O. Hsp 90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins. Circ Res 2001;89:866-73.