Nitric oxide synthase isoform expression in a porcine model of granulation tissue formation

doi:10.1067/msy.2001.111700

Surgery

Volume 129, Issue 3, March 2001, Pages 341-350

https://doi.org/10.1067/msy.2001.111700 Get rights and content

Abstract

Background. This study was designed to determine whether the nitric oxide (NO) pathway is involved in wound granulation tissue formation. Methods. A section of the pig abdominal wall (excluding the skin) was excised, creating an incisional hernia. The resulting defect was repaired with silicone sheeting in a manner that mimics a temporary abdominal wall closure. During the 14-day experimental period, porcine omentum adhered to the peritoneal edges of the defect and a highly vascularized granulation tissue formed on both sides of the sheeting. Granulation tissue thickness and wound fluid volume were monitored by ultrasonography and epigastric artery flow velocity was monitored by color Doppler flow analysis at days 2, 4, 7, 9, 11, and 14. Fluid was serially harvested from the wound compartment at days 2, 4, 7, 9, 11, and 14 for nitrite/ nitrate (NOx) analysis. Finally, granulation tissue was harvested at day 14 for immunohistochemical and molecular analyses. Results. There was a significant increase in granulation tissue thickness and wound fluid volume during the 14-day study period. Blood flow to the wound increased significantly by day 4 and returned toward baseline by day 14. Wound fluid NOx levels significantly increased from days 7 to 11 and then decreased to near baseline values by day 14. Wound fluid arginine levels significantly decreased when compared with peritoneal fluid and plasma levels at day 14, while wound fluid ornithine levels significantly increased. Immunohistochemical analysis of granulation tissue at day 14 revealed nitric oxide synthase (NOS) 2 was present in the majority of the cells in the granulation tissue. NOS 3 was expressed in endothelial cells only, and NOS 1 expression was not observed in the granulation tissue. Conclusions. This study suggests that NO, NOS 2, and arginine may play critical roles in granulation tissue formation and wound healing. Arginase and NOS 2 may compete for available arginine as a substrate, thereby limiting later NO production in favor of sustained ornithine synthesis. (Surgery 2001;129:341-50.)

Section snippets

Pig model of wound healing

Domestic female Landrace swine weighing 15 to 20 kg were induced with Telazol (2 mg/kg) intramuscularly (IM), xylazine (2 mg/kg) IM, and atropine (0.2 mg) subcutaneously and anesthetized with isoflurane (1.5%-3%) by inhalation through an endotracheal tube. Postoperative analgesia was produced by using buprenorphine hydrochloride (Buprenex, 0.01 mg/kg IM) every 9 to 12 hours for 48 hours. After maintenance of inhalation anesthesia, sterile povidone-iodine (Betadine) preparation, and draping, an

Results

Ultrasonography of the wound site was used to serially measure the increase in external granulation tissue thickness and wound fluid accumulation (Fig 1).

. Ultrasonography of wound site. A, Cross-sectional view of the abdominal wall before surgical intervention. The thickness of the subcutaneous tissue measures 6 mm (cursor). RM, Rectus muscle; PC, peritoneal cavity; SQ, subcutaneous tissue. B, Appearance of the abdominal wall (magnified view) on postoperative day 2 revealing a fluid-filled wound

Discussion

The major findings of this report suggest that NO and specifically NOS 2 may play a critical role in granulation tissue formation in the presence of a foreign body. In addition, it appears that NOS 2 and arginase may compete for the available arginine in the wound environment. Increased NO production resulting from up-regulation of NOS expression could promote granulation tissue formation by several mechanisms. Neovascularization is a key component of normal wound healing, and NO has been shown

Acknowledgements

We thank Dr Jennifer Waller for completing the statistical analyses and Debbie Garner, Jinjdong Xin, Rickey Dent, and Lisa Gordon for their expert technical assistance.

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Because the cell migration and proliferation occur hours to a few days after injuries, a 7-day interval (when the first histologic evaluation has been performed) may be enough to achieve balance in the cell types in all groups, and because of that, no difference in cell types was detected in this study. Pollock et al.23 evaluated the effect of arginine through NO synthesis in granulation tissue formation after experimentally induced abdominal wall herniation and repair with silicone sheeting in a porcine model and showed that arginine may have a role in granulation tissue formation. Akçay et al.24 reported the effect of arginine in granulation tissue formation in experimentally induced burn wounds in a mouse model.
Incomplete repair of the dura mater may result in numerous complications such as cerebrospinal fluid leakage and meningitis. For this reason, accurate repair of the dura mater is essential. In this study, the effect of systemic and local supplementation of l-arginine on dural healing was evaluated.
Thirty male Wistar rats were used and divided into control, local, and systemic l-arginine groups, with 10 rats in each. In each group, a 5-mm experimental incision was made at the lumbar segment of the dura mater and cerebrospinal fluid leakage was induced. Each group was divided into 2 subgroups and at the end of the first and sixth weeks, the rats were killed and the damaged segments of the dura were separated, histologically evaluated and the dural healing indicators including cell types, granulation tissue formation, collagen deposit, and vascularization were compared between groups.
The systematic supplementation of l-arginine showed a significant effect in dural healing compared with the control group. After the first week, granulation formation increased considerably (P < 0.031), and after 6 weeks, collagen deposition and neovascularization were significantly different compared with the control group (P < 0.030; P < 0.009). In comparison between different groups at the end of the first and sixth weeks, maximum changes in healing indicators were observed in the systemic group and the least variations were related to the control group.
The systemic supplementation of l-arginine may accelerate dural healing by increasing the level of granulation tissue formation, collagen deposition, and vascularization.
Role of arginine in superficial wound healing in man
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Arginine supplementation has been identified as advantageous in experimental wound healing. However, the mechanisms underlying this beneficial effect in tissue repair remain unresolved. Animal studies suggest that the beneficial role of arginine supplementation is mediated, at least in part through NO. The latter component mediates processes involved in tissue repair, including angiogenesis, epithelialization and collagen formation. This prospective study is performed to investigate arginine metabolism in acute surgical wounds in man. Expression of enzymes, known to be involved in arginine metabolism, was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation. Plasma and wound fluid levels of arginine metabolites (ornithine, citrulline, nitrate and nitrite = NOx) were measured using High Performance Liquid Chromatography. Expression of iNOS, eNOS, arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue. Arginase-1 concentration was measured in plasma and wound fluid using ELISA. Arginase-2 was determined using Western blot analysis. We observed increased levels of citrulline, ornithine, NOx and arginase-1 in wound fluid when compared with plasma. Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma. iNOS was expressed by neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells upon wounding, whereas eNOS reactivity was observed in endothelial cells and fibroblasts. Arginase-1 was expressed in neutrophils post-wounding, while arginase-2 staining was observed in endothelial cells, keratinocytes, fibroblasts, macrophages and neutrophils. For the first time, human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin.
A biochemical approach to wound healing through the use of modalities
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This use of arginine has been implicated to downregulate NO synthesis via substrate depletion.113 It is necessary to consider that NOS has a Km of 2 to 20 μmol/L, whereas arginase has a Km of 2 to 20 μmol/L, which means that NOS requires a concentration of substrate 1000 times less than arginase.114 Angiogenesis, the creation of new blood vessels, is a multi-step complex component of the wound healing process.115
Wound healing is a complex pathway that is energy dependent. Nonhealing wounds frequently require the use of physical modalities to achieve healing. There is much debate over which treatment modality to use, with varying clinical results in the literature. This review paper describes a common biochemical pathway that helps the clinician understand, at a molecular level, how the transference of energy to a wound can result in positive clinical results. The mechanisms of action for ultraviolet light, electrical stimulation, and ultrasound are reviewed along with a proposed biochemical roadmap. An emphasis on protein biochemistry is supported with an extensive review of the literature.
Infected Chronic Wounds Show Different Local and Systemic Arginine Conversion Compared With Acute Wounds
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Several experimental studies have shown the importance of arginine in wound healing. However, little is known about its role in human wound healing. In this study, we investigated arginine metabolism in impaired wound healing.
Twenty patients with chronic wounds and 10 patients with acute wounds were included in a prospective study. Amino acids, nitrate/nitrite, and arginase concentrations were determined in plasma and wound fluid using high-performance liquid chromatography and enzyme-linked immunosorbent assay. Chronic wounds were divided into two groups: noninfected chronic wounds (n = 11) and infected chronic wounds (n = 9), based on quantitative bacterial analysis of wound fluid samples.
Plasma arginine levels, next to total plasma amino acid levels, were significantly decreased in patients with infected chronic wounds compared with patients having acute or noninfected wounds. Citrulline and ornithine levels were significantly increased in infected chronic wounds and related to decreased nitrate/nitrite levels, whereas wound fluid arginine levels were similar in all groups. In addition, wound fluid arginase levels of infected chronic wounds were significantly enhanced.
This study demonstrates that patients with infected chronic wounds have decreased plasma arginine levels and suggests enhanced arginine conversion in the wound. In contrast to noninfected chronic wounds, arginine seems to be mainly metabolized by arginase in infected chronic wounds. In conclusion, our hypothesis is that impaired wound healing is related to an altered arginine usage.
Local Arginine Supplementation Results in Sustained Wound Nitric Oxide Production and Reductions in Vascular Endothelial Growth Factor Expression and Granulation Tissue Formation
2006, Journal of Surgical Research
The goal of this work was to test the functional role of l-arginine in promotion of nitric oxide (NO) production and the vigorous granulation tissue formation characteristic of this wound model.
Therapeutic use of supplemental arginine has been proposed as a safe and efficacious method to produce NO from nitric oxide synthase (NOS) and to produce proline and polyamines from arginase to improve wound healing. Although NO appears to be necessary to promote wound healing, the preferential metabolism of arginine to NO via NOS 2 may be detrimental if maintained beyond the initial days of healing.
A ventral hernia, surgically created in the abdominal wall of 12 swine, was repaired with silicone sheeting and skin closure. Osmotic infusion pumps, inserted in remote subcutaneous pockets, continuously delivered saline (n = 6) or l-arginine (n = 6) into the wound environment. Granulation tissue thickness was determined by ultrasonography. Fluid was aspirated serially from the wound compartment for measurements of nitrite/nitrate (NOx), vascular endothelial growth factor (VEGF), transforming growth factor-β₁ (TGF-β₁), and amino acid concentrations. On day 14, the animals were sacrificed and the abdominal wall was harvested for immunohistochemical and molecular analysis.
In animals receiving saline, a nearly linear four-fold increase in granulation tissue thickness was measured during the 14-day interval. In contrast, quantitative ultrasound analysis detected significant reductions in l-arginine infused granulation tissue thickness compared with controls between days 4 and 14 (P < 0.05). Wound vessel count and luminal vascular surface area estimates derived from image analysis of histological sections were two- to three-fold lower in the l-arginine animals compared with controls (P < 0.05). Significant and sustained increases in wound fluid NOx levels were noted in l-arginine animals compared to saline controls (230 μm versus 75 μm at day 14, P < 0.05). Conversely, late VEGF levels (days 11 to 14) were reduced in the l-arginine animals compared to controls (7500 pg/ml versus 10,000 pg/ml at day 11, P < 0.05; 7250 pg/ml versus 11,101 pg/ml at day 14, P < 0.05). Arginine concentrations remained two- to four-fold greater in l-arginine treated animals compared with controls over the entire time course (P < 0.05). There were no significant differences in concentrations of ornithine, citrulline, or proline noted between groups over the 14-day period. Finally, TGF-β₁ levels were unaffected by l-arginine treatment.
Although NO appears to be necessary for granulation tissue formation, early supplemental arginine may disturb the reciprocal regulation of NOS 2 and arginase, leading to the preferential metabolism of arginine to excess NO rather than ornithine, with consequent reductions in angiogenesis and granulation tissue formation.
Inhibition of inducible nitric oxide synthase results in reductions in wound vascular endothelial growth factor expression, granulation tissue formation, and local perfusion
2003, Surgery
Citation Excerpt :
Sonography was used to serially image the wound site to measure the thickness of the developing 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).
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-β₁concentrations. 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-β₁levels 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.)

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^☆: Supported by grants from the American Heart Association; (J. S. P., T. R. H.), the McGhan Medical Corporation, Santa Barbara, Calif (T. R. H.); and a Georgia Institute of Technology/Medical College of Georgia Bioengineering Grant (T. R. H.).

^☆☆: Reprint requests: Jennifer S. Pollock, PhD, Vascular Biology Center CB3207, Medical College of Georgia, Augusta, GA 30912-2500.

^★: Surgery 2001;129:341-50.

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Original CommunicationsNitric oxide synthase isoform expression in a porcine model of granulation tissue formation☆,☆☆,★

Abstract

Section snippets

Pig model of wound healing

Results

Discussion

Acknowledgements

J Surg Res

J Surg Res

Nitric Oxide

J Biol Chem

Normoxic wound fluid contains high levels of vascular endothelial growth factor

Ann Surg

Temporary abdominal wall closure in trauma patients: indications, technique, and results

World J Surg

Biosynthesis and homeostatic roles of nitric oxide in the kidney

Am J Physiol

Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer

J Clin Invest

Enhanced collagen accumulation following direct transfection of the nitric oxide synthase gene in cutaneous wounds

Biochem Biophys Res Comm

Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells

Proc Natl Acad Sci U S A

Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase

J Clin Invest

Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells

Cardiovasc Res

Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor-beta 1

Arteriolscler Thromb Vasc Biol

Nitric oxide contributes to the angiogenic properties of vascular endothelial growth factor in human cells

J Clin Invest

Original Communications
Nitric oxide synthase isoform expression in a porcine model of granulation tissue formation☆,☆☆,★