Antibody neutralization of vascular endothelial growth factor inhibits wound granulation tissue formation

OBJECTIVE: The goal of this work was to test the functional role of vascular endothelial growth factor (VEGF) in promoting the vigorous granulation tissue formation, wound fluid accumulation, and angiogenic responses characteristic of this wound model. BACKGROUND: Formation of vessel-rich granulation tissue is central to wound repair and is thought to be regulated by locally liberated angiogenic factors. Despite the clinical importance of granulation tissue formation in the early stage of wound healing, surprisingly little is known about the molecular identity of signals leading to granulation tissue invasion of a wound space. Methods. A ventral hernia, surgically created in the abdominal wall of 15 swine, was repaired using silicone sheeting and skin closure. An osmotic minipump, inserted in a remote subcutaneous pocket, delivered saline (n = 5), an irrelevant control antibody (n = 5), or neutralizing anti-VEGF antibody (n = 5) into the wound environment. Serial ultrasonography on Days 2, 4, 7, 9, 11, and 14 was used to determine the dimensions of the subcutaneous granulation tissue and wound fluid compartment. VEGF and transforming growth factor beta1 (TGF-beta1) levels in serial wound fluid samples were quantitated by ELISA. On Day 14, animals were sacrificed and the abdominal wall was harvested for histologic, biochemical, and molecular analyses. RESULTS: In animals receiving saline or an irrelevant antibody, a nearly linear 4-fold increase in granulation tissue thickness and 7-fold increase in wound fluid volume were measured over the 14-day study interval. In contrast, in animals receiving anti-VEGF neutralizing antibody, Day 14 granulation tissue thickness and wound fluid volume measurements were essentially unchanged from Day 2 values. Moreover, in the anti-VEGF animals, ultrasonography was unable to resolve the "angiogenic zone" typical of both controls, and correspondingly, wound vessel count and vascular surface area estimates derived from image analysis of histological sections were 3-fold lower in the anti-VEGF animals compared with the saline and antibody controls. Finally, VEGF levels in wound fluid detectable by ELISA analysis were strikingly (10-fold) reduced in anti-VEGF animals on Postsurgery Days 7-14. In contrast, TGF-beta1 levels were unaffected by the anti-VEGF treatment. CONCLUSION: Functional VEGF is a key mediator in wound angiogenesis, fluid accumulation, 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

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 N(6) (iminoethyl)-L-lysine (L-NIL; n = 6) into the wound environment. Granulation tissue thickness was determined with ultrasonography, 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-beta(1)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-beta(1)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.     

Serum and drainage fluid vascular endothelial growth factor levels in early surgical wounds

BACKGROUND: Wound healing relies on a coordinated expression and release of growth factors controlling angiogenesis. We measured vascular endothelial growth factor (VEGF) levels in serum and early wound fluid following primary sutured and mesh repair of abdominal hernias. MATERIALS AND METHODS: Thirty-seven patients were studied measuring serum and wound drainage fluid VEGF by enzyme-linked immunosorbent assay preoperatively and on the 1st, 3rd, 5th and 7th postoperative days. RESULTS: Serum and wound fluid VEGF levels increased significantly by the 3rd postoperative day and continued to rise during the conduct of the study with wound fluid concentrations always exceeding serum levels. The kinetics of VEGF increases were similar in both types of hernia repair; however, serum and wound levels rose slightly earlier in the mesh repair group. CONCLUSIONS: Steadily increasing levels of VEGF are detected during the early proliferative phase of wound healing in both serum and wound fluid. It is unknown whether either is predictive for delayed hernia recurrence.     

Local arginine supplementation results in sustained wound nitric oxide production and reductions in vascular endothelial growth factor expression and granulation tissue formation

OBJECTIVE: 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. BACKGROUND: 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. METHODS: 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-beta1 (TGF-beta1), and amino acid concentrations. On day 14, the animals were sacrificed and the abdominal wall was harvested for immunohistochemical and molecular analysis. RESULTS: 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 microM versus 75 microM 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-beta1 levels were unaffected by L-arginine treatment. CONCLUSION: 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.     

Wound repair by bone marrow stromal cells through growth factor production

We have previously shown that treatment with bone marrow stromal cells (BMSCs) augments the healing of fascial wounds in the rat. However, the biochemical mechanism by which BMSCs improve wound healing was not investigated. Growth factors have been shown to play a key role in repairing damaged tissue. In this study, we investigated whether BMSCs are capable of producing growth factors that play a critical role in healing of the damaged tissue. Growth factor expression in BMSCs stimulated with pro-inflammatory cytokines or wound superfusate was measured by RT-PCR and growth factor-specific quantitative sandwich enzyme-linked immunosorbent assay (ELISA). RT-PCR analysis demonstrated that BMSCs are capable of expressing transforming growth factor beta-1 (TGF-beta1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) constitutively or upon stimulation with LPS, IL-1alpha, or TNF-alpha. Quantitative analysis of growth factor production by ELISA showed that BMSCs do not secrete TGF-beta1, EGF or VEGF in response to uninjured fascia tissue superfusate; however, production of these growth factors is significantly increased when cells were stimulated with wound tissue superfusate. The ability of wound to stimulate growth factor production in BMSCs could be detected as early as day 1 and lasted through day 7 after wounding. Thus, growth factor production by BMSCs in response to wound microenvionment suggests that BMSCs might augment wound healing through the responsive secretion of growth factors that enhance angiogenesis and promote wound repair.     

Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats

Bone marrow stem cells participate in tissue repair processes and may have a role in wound healing. Diabetes is characterised by delayed and poor wound healing. We investigated the potential of bone marrow-derived mesenchymal stromal cells (BMSCs) to promote healing of fascial wounds in diabetic rats. After manifestation of streptozotocin (STZ)-induced diabetic state for 5 weeks in male adult Sprague-Dawley rats, healing of fascial wounds was severely compromised. Compromised wound healing in diabetic rats was characterised by excessive polymorphonuclear cell infiltration, lack of granulation tissue formation, deficit of collagen and growth factor [transforming growth factor (TGF-beta), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor PDGF-BB and keratinocyte growth factor (KGF)] expression in the wound tissue and significant decrease in biomechanical strength of wounds. Treatment with BMSC systemically or locally at the wound site improved the wound-breaking strength (WBS) of fascial wounds. The improvement in WBS was associated with an immediate and significant increase in collagen levels (types I-V) in the wound bed. In addition, treatment with BMSCs increased the expression of growth factors critical to proper repair and regeneration of the damaged tissue moderately (TGF-beta, KGF) to markedly (EGF, VEGF, PDGF-BB). These data suggest that cell therapy with BMSCs has the potential to augment healing of the diabetic wounds.     

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

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.     

025Modulation of Wound Repair in the Obese Diabetic Mouse: a Role for VEGF Gene Transfer

Identifying molecular loci of impaired cutaneous healing in diabetes with an eye towards developing targeted therapy to ameliorate dysrepair continues to evolve as a promising area of study. By using an excisional wound model produced on the dorsum of female diabetic C57BL/KsJ db  + / db + mice as well as their normal (WT) & heterozygous (HZ) littermates, we studied the effects of peri‐wound intradermal injection of adeno‐associated viral vector (AAV) expressing the 165‐amino acid isoform of human vascular endothelial growth factor (VEGF) on the following: kinetics of re‐epithelialization, neoangiogenesis and granulation tissue formation, matrix remodelling, collagen deposition, and maturation. One sq. in. full thickness excisional wound was created in the mid‐upper back, rendering half of the wound as either right or left paravertebral. Animals were randomized to receive 1 of 3 treatments via intradermal injection: 1)VEGF (AAV) vector; 2)Adnull vector; 3)PBS. Postoperatively, wounds were examined & photographed on Days 3, 7, 10, 14, 21 & 28. Also, tissue was harvested for histology & immunohistochemistry (PECAM), and snap frozen for protein & RNA analysis. A scoring system was used to grade re‐epithelialization, granulation tissue thickness, matrix density, inflammation, vascular density, epithelial maturity. AAV‐VEGF exerted minimal effect on repair in WT and HZ mice. However, pronounced neovascularization, thickened granulation tissue & increased matrix deposition was noted after VEGF treatment in the db/db mice compared to those that received PBS or adnull vector at all timepoints. While the induction of angiogenesis in VEGF treated db/db mice lagged behind the unimpaired mice by 5–7 days, a global improvement in wound healng was observed.
R Crystal, Dir Inst Genetic Medicine, Weill Med College‐Cornell Univ     

Modulation of α-smooth muscle actin expression in fibroblasts by transforming growth factor-β isoforms: an in vivo and in vitro study

Myofibroblasts are granulation tissue fibroblasts bearing ultrastructural and biochemical features of smooth muscle cells, such as cytoplasmic microfilaments and alpha-smooth muscle actin expression. They appear transiently during wound healing and more permanently during several pathologic situations such as fibrotic diseases. Transforming growth factor-beta1 has been suggested to be an important promoter of the myofibroblastic phenotype. Here we show that (1) transforming growth factor-beta2, like transforming growth factor-beta1, induces myofibroblast formation in vivo and in vitro; (2) transforming growth factor-beta3 acts as a negative regulator of the myofibroblastic phenotype in vivo but not in vitro; and (3) in vitro, the three different transforming growth factor-beta isoforms are equally able to induce alpha-smooth muscle actin messenger RNA and protein expression in growing and quiescent cultured human and rat subcutaneous tissue fibroblasts. These data confirm that in vitro the behavior of the three different transforming growth factor-beta isoforms is similar, whereas in vivo transforming growth factor-beta isoforms possibly play different but complementary roles in myofibroblast modulation during wound repair.     

Retroviral delivery of dominant-negative vascular endothelial growth factor receptor type 2 to murine wounds inhibits wound angiogenesis

Vascular endothelial growth factor (VEGF) is a potent paracrine signal for initiating angiogenesis. Although VEGF can bind to several cell surface receptors, VEGF receptor type 2 (VEGFR2, a.k.a. KDR or flk-1) is the primary receptor responsible for VEGF-induced endothelial cell proliferation. To determine whether the VEGF-VEGFR2 signaling axis has an important role in wound healing angiogenesis, we used a retrovirus to deliver a signaling-defective truncated VEGFR2 (tm VEGFR2) to block VEGF-VEGFR2-induced endothelial cell proliferation in murine wounds. We show that the retroviral construct effectively blocked phosphorylation of VEGFR2 in vitro and we were able to express the truncated receptor in murine wounds. We achieved significant reduction of angiogenesis and granulation tissue formation in murine wounds, but this did not lead to delayed wound closure. In contrast, there was a corresponding increase in wound contraction, showing that functional VEGFR2 intracellular signaling is not critical for normal closure of excisional dermal wounds. Our results show a novel relationship between wound bed vascularity and wound contraction.     

Topical vascular endothelial growth factor reverses delayed wound healing secondary to angiogenesis inhibitor administration

The prevention of new blood vessel growth is an increasingly attractive strategy to limit tumor growth. However, it remains unclear whether anti-angiogenesis approaches will impair wound healing, a process thought to be angiogenesis dependent. Results of previous studies differ as to whether angiogenesis inhibitors delay wound healing. We evaluated whether endostatin at tumor-inhibiting doses delayed excisional wound closure. C57/BL6J mice were treated with endostatin or phosphate-buffered solution 3 days prior to the creation of two full-thickness wounds on the dorsum. Endostatin was administered daily until wound closure was complete. A third group received endostatin, but also had daily topical vascular endothelial growth factor applied locally to the wound. Wound area was measured daily and the wounds were analyzed for granulation tissue formation, epithelial gap, and wound vascularity. Endostatin-treated mice showed a significant delay in wound healing. Granulation tissue formation and wound vascularity were significantly decreased, but reepithelialization was not effected. Topical vascular endothelial growth factor application to wounds in endostatin-treated mice resulted in increased granulation tissue formation, increased wound vascularity, and wound closure approaching that of control mice. This study shows that the angiogenesis inhibitor endostatin delays wound healing and that topical vascular endothelial growth factor is effective in counteracting this effect.     

Mechanisms of TGF-β action in connective tissue repair of rat mesenteric wounds

We have recently reported that transforming growth factor-beta stimulates genuine connective tissue repair in the perforated rat mesentery and that this stimulation is not caused by increased macrophage chemotaxis. To further characterize the effect of transforming growth factor-beta(1) on the enhanced rate of wound closure, we performed a series of morphometric analyses with determination of mitotic index, fibroblast labeling index, cellular density, neovascularization, and scar tissue formation. Actin expression close to the wound margin was also evaluated morphologically. Fibroblast cell proliferation was not stimulated by transforming growth factor-beta(1) in either wounded or unwounded tissue. Transforming growth factor-beta(1) did, however, significantly increase the formation of healing tissue postoperative days 5 to 10 (p < 0.05) and angiogenesis was significantly stimulated by transforming growth factor-beta(1) postoperative days 7 and 10 (p < 0.005). The mean cellular density was significantly increased in unperforated, transforming growth factor-beta(1)-treated membranes from days 3 to 10, and increased expression of actin with time was observed close to the wound margin. Transforming growth factor-beta(1) was thus shown to be a potent stimulator of angiogenesis and healing tissue formation in connective tissue repair, but this stimulation mainly occurred after closure of perforations. The increased cellular density in the absence of stimulated proliferation and increased actin expression in wound cells indicate that contraction may be an important mechanism of connective tissue repair in the perforated rat mesentery.     

Reactive stroma in prostate cancer progression.

PURPOSE: The development of an altered stromal microenvironment in response to carcinoma is a common feature of many tumors. We reviewed the literature describing characteristics of reactive stroma, how reactive stroma affects cancer progression and how carcinoma regulates reactive stroma. Moreover, we present a hypothesis of reactive stroma in prostate cancer and discuss how the biology of reactive stroma may be used in novel diagnostic and therapeutic approaches. MATERIALS AND METHODS: An extensive literature search was performed to review reports of the general features of wound repair stroma, general stromal responses to carcinoma, and stromal biology of normal and prostate cancer tissues. These studies were analyzed and a reactive stroma hypothesis in prostate cancer was developed. RESULTS: Modifications to the stroma of breast, colon and prostate tumors parallel the generation of granulation tissue in wound repair. These changes include stromal cell phenotypic switching, extracellular matrix remodeling and angiogenesis induction. Therefore, it is predicted that a modified wound healing response induces the formation of reactive stroma in cancer to create a tumor promoting environment. Based on its role in wound repair and its over expression in prostate cancer, transforming growth factor-beta stands out as a potential regulator of reactive stroma. CONCLUSIONS: Reactive stroma in prostate cancer and granulation tissue in wound repair show similar biological responses and processes that are predicted to promote cancer progression. Further identification of specific functional and regulatory mechanisms in prostate cancer reactive stroma may aid in the use of reactive stroma for novel diagnostic and therapeutic approaches.     

Wound epithelialization deficits in the transforming growth factor-α knockout mouse

In vitro, transforming growth factor-alpha is an important factor controlling epithelial cell proliferation and migration. However, the transforming growth factor-alpha knockout mouse has shown no wound epithelialization defect in tail amputation and full-thickness back wounds. To resolve this disparity, we combined a full-thickness head wound and a partial-thickness ear wound on the transforming growth factor-alpha knockout mouse for analysis of wound epithelialization with or without granulation tissue formation. Three-millimeter ear wounds were made on the transforming growth factor-alpha knockout and heterozygous control mice. Full-thickness head wounds were made using a 6-mm trephine on the crown of the skull. In the ear model, transforming growth factor-alpha knockout mice had significantly larger epithelial gaps versus control at post-operative day 3 and 5. Epithelial thickness at the wound edge of transforming growth factor-alpha deficient mice was also depressed at post-operative day 3 and post-operative day 5 compared to control mice. On post-operative day 8, most wounds of both groups were epithelialized. In contrast, no difference in epithelial gap or new granulation tissue was found in the head model. The data support the concept that transforming growth factor-alpha plays a significant early role in wound epithelialization in vivo but its deficit is compensated if accompanied by granulation tissue formation. The data further show the importance of appropriate wound models to address the role of vulnerary factors.     

血管内皮生长因子与治疗性血管生成研究进展

血管内皮生长因子(vascular endothelial growtll factor,VEGF)是内皮细胞特异的有丝分裂原,有促进内皮细胞增生、增强血管通透性、加速新血管形成的作用.血管生成是一个具有重要生理、病理意义的过程.在人体的创伤愈合、炎症反应、器官再生过程以及肿瘤生长转移、血管增生性疾病中,血管生成有重要作用.治疗性血管生成是指利用成血管诱导因子或内皮祖细胞,模拟体内血管生成机制,促进新生血管形成,改善侧支循环.本文就VEGF和治疗性血管生成研究进展做一综述.     

Epidermal growth factor increases granulation tissue formation dose dependently

Recent studies have shown that epidermal growth factor (EGF) stimulated the rate of formation of granulation tissue in a model of wound repair (A. Buckley, et al., Proc. Nat. Acad. Sci. USA 82: 7340, 1985). Because pharmacologic doses of EGF were used previously, the relationship of EGF concentration to physiologic effects was determined in this study. Rats were implanted with subcutaneous polyvinyl alcohol sponges containing slow-release pellets formulated to release 0, 0.1, 1.0, or 10 micrograms of EGF/day. Tissue response was judged by the degree of histologic organization and vascularity, as well as several quantitative parameters: wet weight, hydroxyproline content, protein content, and DNA concentration. Each of these parameters showed consistent increases by Day 5 after implantation, when inflammation and edema had subsided. Compared with placebo controls, hydroxyproline (collagen) content was significantly increased by as little as 1 microgram/day of EGF, and DNA content was significantly increased by all dose levels of EGF. Endogenous EGF concentration in experimental granulation tissue was found to be fairly constant (30-40 ng/g wet wt); however, the increasing cellularity of the sponges may have reduced the local concentration of free EGF to low levels. Pellets releasing as little as 4 ng/hr of EGF into the surrounding tissue were able to accelerate wound healing, suggesting that the availability of this growth factor may be a rate-limiting step in wound repair.