Modifiers of inflammatory angiogenesis in a murine model 3

CHAPTER III RESULTS 3.1 Introduction of corneal and skin injury models 3.1.1 Angiogenesis in the corneal injury model The corneal injury model has been used to investigate angiogenesis (Azar 2006). In this study, we have been able to standardize the extent of injury to allow for a fairly consistent angiogenic response on the cornea. The corneal angiogenesis in BALB/c mice is shown in Figure 3.1. After 24 hours, the pre-existing vessels vasodilated and corneas became cloudy. By 72 hours, the sprouts lengthened and multiplied to produce a rich anastomosing plexus. 72 hours to days vascular sprouts elongated and matured, and the cloudiness of cornea diminished. The angiogenesis reached a peak at day after injury and the vascular sprouts reached the injured sites. From day regression started, and new vessels began to fade. Our results are consistent with those reported by Burger et al (1983) and Sunderkötter et al. (1991). Based on the current data, we have been able to establish that the angiogenesis response is the highest at day after injury. 3.1.2 Sexual dimorphism in the corneal and skin injury model To reduce potential variation in experiments, the differences in angiogenesis and skin wound healing were observed between BALB/c male and female mice. The present study shows that the degree of corneal angiogenesis in female mice was significantly greater than that in 74 male mice (Fig. 3.2). In contrast there was no significant difference in skin wound healing between male and female mice (Fig 3.3). However, skin wound healing in female mice tended to close faster than male mice at days 3, and after injury. As such, only female mice were used in all the subsequent experiments to establish the corneal and skin injury models. 75 Fig. 3.1 Angiogenesis response in the corneal injury model The time course of angiogenesis response in the cornea of BALB/c mouse before injury (a), 0.5 hours (b), day (c), days (d), days (e) and days (f) after injury. 76 A Neovasculized Area (mm ) Female male 2.5 1.5 ** 0.5 B Female Male 0.6 Vessel Length (mm) 0.8 0.4 Fig. 3.2 Comparisons of corneal angiogenesis between BALB/c female and male mice Quantitive analysis of neovascularized area (A) and vessel length (B) at day after corneal injury. The bars show the mean ± SEM of independent experiments (n = 8). ** P < 0.01 compared with female mice. 77 % of original wound area 120 Female Male 100 80 60 40 20 0 11 13 15 Days after wound formation Fig. 3.3 Skin wound healing in BALB/c female and male mice Wound closure was evaluated by morphometrical analysis of the wound areas. Each wound region was digitally photographed at the indicated time points, and the percentage of wound areas to the initial areas was calculated from the photographs. The bars show the mean ± SEM of independent experiments (n = 6). 78 3.2 Key roles of neutrophils in angiogenesis in the corneal injury model 3.2.1 Lymphocytes have little impacts on angiogenesis in the corneal injury model Although lymphocytes has been indicated to produce angiogenic factors (Lingen, 2001), their roles in angiogenesis induced by injury are still unclear. We established the corneal injury model in Rag1KO mice and control mice to study the roles of lymphocytes in inflammatory angiogenesis. As shown in Figure 3.4, there was no difference in corneal angiogenesis between BALB/c and Rag1 KO mice indicating that lymphocytes may not play important roles in natural inflammatory angiogenesis. 79 Fig. 3.4 Comparison of corneal angiogenesis between female BALB/c and Rag1 KO mice A Corneal angiogenesis at day and day after injury in BALB/c and Rag1KO mice. B and C: Quantitative analysis of neovascularization at day after injury. The bars show the mean ± SEM of independent experiments (n = 6). 80 3.2.2 Key roles of neutrophils in angiogenesis in the corneal injury model Neutrophils are the dominant infiltrating leukocyte population in the early stages of inflammation. Neutrophils have been shown to play an important role in the inflammatory response, acting as a first line of self-defense against invading microorganisms (Kasama et al., 2005; Scapini et al., 2000). However, recent studies have indicated that neutrophils may also play an important role in angiogenesis. Isolated human neutrophils are known to release VEGF from preformed stores upon activation (Gaudry et al. 1997). Neutrophils are also associated with angiogenesis in a Matrigel sponge model in vivo induced (Kibbey et al., 1994). The induction of angiogenesis by IL-8/CXCL8 is also neutrophil dependent, the angiogenic response being completely abrogated upon neutrophil depletion (Benelli et al., 2002). However, no direct in vivo evidence relates the neutrophil to natural inflammatory angiogenesis. Thus, we have used a corneal injury model to study the role of neutrophils in the process of natural inflammatory angiogenesis as well as the mechanisms underlying the observed response. I. Efficacy of neutrophil depletion by RB6-8C5 treatment To determine the role of neutrophils in inflammatory angiogenesis, neutrophils were systemically depleted by intraperitoneal administration of RB6-8C5. Neutrophil depletion was confirmed by Giemsa staining of the blood smear. Peripheral blood was collected from mice at days -1, 0, and after injury to determine the extent of neutropenia. Peripheral 81 blood counts are shown in Table 3.1. Generally, very few neutrophils were found in the peripheral blood of RB6-8C5-treated mice. RB6-8C5 treatment resulted in more than 97% reduction in the number of circulating neutrophils during the course of experiment (Table 3.1). This result indicated that RB6-8C5 antibody could efficiently deplete circulating neutrophils, which provides a reproducible systemic neutropenia model for the study of neutrophil function. Neutrophil depletion had minimal effects on the differential count of monocytes. II. Effects of neutrophil depletion on corneal angiogenesis The effect of neutrophil depletion on the corneal angiogenesis was evaluated by the biomicroscopic observations, microvessel counts and measurements in new vessel length and neovascularized area (Fig. 3.5). Figure 3.6 shows corneas at days and after injury in the control and RB6-8C5 treatment groups. In the control group, the limbal vessels began sprouting into the corneas at day after the injury. Subsequently, vascular sprouts matured and elongated to the injury site, reaching it at day 5, with the new vessel length and neovascularized area at 0.68±0.04 mm and 2.08±0.15 mm2 respectively (Fig. 3.5B, 3.5C). Neutrophil depletion severely inhibited corneal angiogenesis: fewer new buds of vessels were present in the corneas of RB6-8C5-treated mice by day 3, and no obvious progression of new vessel growth during the process of angiogenesis was visible (Fig. 3.5A). By quantitative image analysis, neutrophil depletion resulted in more than 90% reduction in new blood vessel length and 82 neovascularized area compared with the control mice at day after injury (Fig. 3.5B, 3.5C). We next assessed the role of neutrophils in angiogenesis by determining the microvessel density. Figure 3.6 demonstrates that neutrophil depletion decreased the microvessel density. In the control group, a 1.5-fold increase occurred in the microvessel density from day to day after injury. Compared with the control group, 75% and 90% reduction in the microvessel density were observed in the RB6-8C5 treatment group at day and day after injury, respectively. Moreover, the microvessel density remained unchanged from day to day in the RB6-8C5 treatment group. III. Effects of neutrophil depletion on corneal inflammation response 24 hours after injury the corneas became cloudy in both control and RB6-8C5-treated groups and the degree of cloudiness was graded. We found that RB6-8C5-treated mice showed significantly less corneal opacity than control mice from day to day (Fig. 3.7). At day after injury the mean scale of control group was 2.2, while it was 1.4 in RB6-8C5 -treated group. At day after injury, the mean scale of control group increased to 2.4; however, in RB6-8C5 treated group, it decreased to 1.1. Current data suggest that neutrophil depletion significantly reduced the opacity in the cornea following injury, which is consistent with the previous work (Oshima et al., 2006). 83 new concepts for the therapeutic intervention of impaired wound healing in human diseases associated with deficient neutrophil function, such as the leukocyte-adhesion deficiency syndrome (Kuijpers et sl., 1997) or disease states of neutrophil actin polymerization (Roos et al., 1993). 4.4 Scar formation and inflammatory cells Skin wound healing occurs via a complex series of events that eventually lead to matrix deposition, fibrosis, and scar formation (Martin et al., 1997; Singer et al., 1999). Clinically, scar formation can cause considerable problems, including restricted joint mobility, impaired growth, and loss of organ function as well as psychological and social detriments. While adult skin always heal with a scar, fetal skin wound heal rapidly with a scarless endpoint throughout the first and second trimesters of development (Tredget et al., 1997; Ross RB, 1987). Although the definitive mechanism of scarless fetal wound healing remains to be elucidated, diminished inflammation in fetal wounds is well recognized. Lower numbers of macrophage recruited into the skin wounds and lack of H-21-A positive B cell recruitment have been implicated as the mechanism underlining the scarless healing in early fetal wound healing (Cowin et al., 1998). In a Gore-tex wound healing model, the paucity of neutrophil recruitment characterized the fetal wounding other than neonatal and adult wound healing (Adzick et al., 1985). It has been shown that fetal scarless wound healing is related to decreased angiogenesis and reduced level of VEGF and TGF-β1 (Wilgus et al., 2008; O’kane and Ferguson, 1997). We examined the role of lymphocytes and neutrophils in scar formation in adult skin injury using Rag1 KO mice 148 and RB6-8C5 treated mice. We found that the scar formation was not affected in Rag1KO mice indicating that lymphocytes are not necessary for scar formation. Although neutrophils were only depleted for days following the injury, it was enough to severely decrease neutrophil recruitment and angiogenesis, and reduce the level of VEGF and TGF-β1 in the skin wounds of RB6-8C5 treated control mice. In the present study, the skin wound in RB6-8C5 treated control mice healed with wider and thinner scar formation compared with control mice. One interpretation of the results is that the monocyte play a key role in influencing the scar formation in the late phase of wound healing. Another interpretation could be that there are yet known important mechanisms mediating the scarless wound healing in the early fetus besides diminished inflammation response. 149 CHAPTER V CONCLUSION The present in vivo corneal and skin injury study suggests that neutrophils play a crucial role in inflammatory angiogenesis, possibly via a mechanism that involves the release of VEGF from preformed stores in the neutrophils. In addition, the secretion of MIP-2 might act in a positive feedback fashion to enhance the process by recruiting neutrophils to the site of injury. Our data also provide the direct in vivo evidence for the first time that neutrophil may play important roles in skin wound healing by promoting angiogenesis, granulation tissue formation and secretion of TGF-β1 from macrophages. In addition, induction of MIP-1α and MIP-2 by neutrophil may enhance the migration of monocytes and neutrophils to skin wounds to enhance the effects of neutrophil in skin wound healing. Lymphocytes not seem playing a significant role in inflammatory angiogenesis. However, they play an important role, other than in adaptive immunity, in skin wound healing. Our findings add support to the emerging notion that attempts to target the contribution of neutrophils therapeutically represent a new aspect in the regulation and control of angiogenesis and may thus have practical applications in the treatment of many chronic inflammatory diseases. Our findings also provide new concepts for the therapeutic intervention of impaired wound healing in human diseases associated with deficient neutrophil function, such as a common disease like diabetes mellitus (Alba-Loureiro et al., 2006) and less common diseases like the leukocyte-adhesion deficiency syndrome 150 (Kuijpers et al., 1997) or disease states of neutrophil actin polymerization (Roos et al., 1993). 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Journal of Leukocyte Biology 65, 671-679. 163 [...]... the cornea at day 1 after injury (original magnification: x 100) b Immunostaining for VEGF in the cornea of the control group at day 1 after injury (original magnification: x 100) c Immunostaining for VEGF in the cornea of the control group at day 1 after injury (original magnification: x 400) d H&E staining in the cornea of the control group (original magnification: x 400, oil) e 93 Immunostaining for... Immunostaining for neutrophils in the cornea of the control group after injury Bar 100 μm (original magnification: ×100) Control 2 H&E staining of neutrophils in the cornea of the control group after injury Bar 30 μm (original magnification: ×400, oil) 90 V Induction and localization of VEGF in cornea VEGF is a potent stimulator of the endothelial cell growth and can stimulate both the physiological and pathological... the pretreatment of CHX The data are representative of 4 separate experiments Each experiment included 3 samples 1 03 3 .3 Important roles of neutrophils and lymphocytes in skin wound healing 3. 3.1 Important roles of lymphocytes in wound healing We found that the wound healing in Rag1KO mice was significantly delayed from day 7 following the injury compared with control mice 9 (Fig .3. 17) In Rag1KO mice... on microvessel density in the cornea A Representative photographs of CD31 immunohistochemistry stain in the corneas of control and RB6-8C5 treatment groups at day 5 after injury (original magnification x 100, bar 100 µm) B Quantitative analysis of CD31 immunohistochemistry stain in the corneas of control and RB6-8C5 treatment groups at days 3 and 5 after injury Bars show the mean ± SEM (n=6) * P < 0.05... protein levels of TNF-α in the corneal injury model Corneas were removed at the indicated time after injury Corneal lysate was prepared and individually assayed by enzyme-linked immunosorbent assay The bars show the mean ± SEM (n=6) 100 Control MCP-1 (pg/cornea) 30 0 RB6-8C5 treatment * 250 200 150 100 50 0 Day 0 Day 1 Day 3 Day 5 Days after injury Fig 3. 13 Time kinetics for protein levels of MCP-1 in. .. was no T cell detected in the normal skin and injured skin across all the time points There was also no T cell infiltration during wound healing in Rag1KO mice Furthermore, there were no significant differences in inflammatory cell infiltration (neutrophil and macrophage) (Fig .3. 18, 3. 19), angiogenesis, and the protein levels of VEGF, MIP- 1a, MCP-1, and TNF-α between control and Rag1KO mice (Fig .3. 18 -3. 23, ... Phorbol-12-myristate 13- acetate (PMA) after preincubation with or without 10 µg/ml of cycloheximide (CHX) for 30 minutes at 37 ºC Culture supernatants were separated from the cells by centrifugation The bars show the mean values ±SEM of the VEGF protein between the cellular and extracellular compartments at unstimulated and stimulated neutrophils, depicted as cell-associated and released The data are representative... al 2001; Fahey et al 1990) MIP-1α is able to promote neutrophil infiltration (Wolpe et al 1988) Therefore, we determined the protein levels of MIP-1α, MIP-2, and TNF-α using ELISA in the in vivo murine corneal injury model (Fig 3. 10 -3. 12) MIP-2 and MIP-1α protein levels were increased significantly at day 1 after injury and decreased markedly by day 5 In the uninjured cornea, the levels of MIP-1α and... for protein levels of MIP-2 in the corneal injury model Corneas were removed at the indicated time after injury Corneal lysate was prepared and individually assayed by enzyme-linked immunosorbent assay The bars show the mean ± SEM (n=6) *P < 0.05 compared with control group 99 Control RB6-8C5 treatment 40 TNF-α (pg/cornea) 30 20 10 0 Day 0 Day 1 Day 3 Day 5 Days after injury Fig .3. 12 Time kinetics for... by 63% and 54% at day 1 and day 3 after injury, respectively (Fig 3. 9A) To determine the cellular source of VEGF in the injured cornea, serial sections of eyeballs taken 1 day after injury were immunostained for VEGF in the control and RB6-8C5 treatment groups Both corneal epithelial cells and the infiltrating neutrophils were positively stained with VEGF in the cornea of the control group (Fig 3. 9B) . important roles in natural inflammatory angiogenesis. 80 Fig. 3. 4 Comparison of corneal angiogenesis between female BALB/c and Rag1 KO mice A Corneal angiogenesis at day 3 and day. Neutrophils have been shown to play an important role in the inflammatory response, acting as a first line of self-defense against invading microorganisms (Kasama et al., 2005; Scapini et al., 2000) neovascularization at day 3 and day 5 after injury in the control and RB6-8C5 treatment groups. B and C Quantitative analysis of neovascularization at day 5 after injury. Bars show the mean ± SEM of