Modifiers of inflammatory angiogenesis in a murine model 2

CHAPTER I INTRODUCTION 1.1 Introduction Wound healing is the body’s response to injury, in which angiogenesis is one of the key elements. Immune cells have been shown to play roles in both angiogenesis and wound healing. Angiogenesis is important in a number of normal physiological processes, including embryogenesis, reproduction and wound healing. Uncontrolled angiogenesis contributes to a variety of pathologies. Wound healing is a complex chain of cellular and biochemical events designed to restore tissue integrity and function under tight regulation. However, there are instances whereby the process is less well regulated and injury can lead to a chronic wound (non-healing) or fibrosis (excessive scar formation), which remain common medical problems. Thus, understanding the mechanisms of tissue repair, new vessel formation in the context of the immune response will help lay the foundation for better treatment of pathologies related to aberrant angiogenesis and wound healing. Neutrophils are the main components in innate immunity. Traditionally, neutrophils are regarded mainly as phagocytic cells, providing the first line of defense against invading pathogens. However, there is increasing evidence indicating that neutrophils may have more functions beyond that. A previous study showed that neutrophils could release VEGF, one of the most potent angiogenic factors, and MIP-1α and MIP-2, the potent chemokines for neutrophil, macrophage and lymphocytes. Furthermore, it has been shown that angiogenesis is dependent on the neutrophil in an in vitro model and matrigel model. However, there is no direct in vivo evidence to relate the neutrophil to the natural inflammatory angiogenesis. Similarly, there is evidence showing that lymphocytes may contribute to angiogenesis in a number of pathologic settings; their effects on the inflammation-induced angiogenesis are still unclear. The role of the neutrophil in wound healing was first addressed in 1972. The exact mechanism was uncertain at that time. It is well accepted that lymphocytes also play roles in skin wound healing, but the mechanisms also need to be further elucidated. Although it is well known that the foetal scarless healing follows a minimized inflammatory response, the effects of immune cells (neutrophils and lymphocytes) in adult scar formation are unknown. In this section, the basic knowledge of angiogenesis, wound healing and scar formation will be introduced. Some important cytokines will be also reviewed, including MIP-1α, MIP-2, MCP-1, TNF-α, VEGF, and TGF-β1. Subsequently, the role of neutrophil in angiogenesis and wound healing is reviewed after a brief introduction of neutrophil biology. Finally, the roles of lymphocytes and monocyte/macrophages in angiogenesis and wound healing are also reviewed. 1.2 General introduction of angiogenesis and wound healing 1.2.1 Angiogenesis Angiogenesis is defined as the formation of new vessels from the existing vessels. This process is characterized by a combination of sprouting of new vessels from the sides and ends of pre-existing ones, or by longitudinal division of existing vessels with periendothelial cells, either of which may then split and branch into precapillary arterioles and capillaries (Conaway et al., 2001). The classical angiogenesis process consists of the following three overlapping phases: the initiation of the angiogenic response, endothelial cells migration and proliferation, and maturation of neovasculature (Griffioen et al., 2000). Angiogenesis is a complicated and highly regulated process. In adult, angiogenesis under tight control are found in the female reproductive system and during wound healing (Liekens et al., 2001). Unregulated angiogenesis can be divided into insufficient angiogenesis and excessive angiogenesis. Insufficient angiogenesis may result in tissue ischemia and delayed wound healing; excessive angiogenesis may result in pathologies that include cancer (both solid and hematologic tumors), chronic inflammation (rheumatoid arthritis, Crohn's disease), diabetes (diabetic retinopathy) and psoriasis. With increasing insight of the role of angiogenesis in these pathologies, modulation of angiogenesis is now regarded as a therapeutic target in these diseases (Griffioen et al., 2000). Further delineating the regulation of angiogenesis will lead hopefully to more effective angiogenic and anti-angiogenic treatment approaches to some diseases. I. Angiogenesis process i) Initiation of angiogenesis To initiate the formation of new capillaries, endothelial cells of existing blood vessels must degrade the underlying basement membrane and invade into the stroma of the neighbouring tissue. These processes of endothelial cell invasion and migration require the cooperative activity of the plasminogen activator (PA) system and the matrix metalloproteinases (MMPs) (Conaway et al., 2001; Liekens et al., 2001). Angiogenesis is rapidly initiated by hypoxic or ischemic conditions. vascular endothelial growth factor (VEGF), transcriptionally upregulated in part by hypoxia, mediates an increase in vascular permeability and extravasation of plasma proteins including plasminogen, which can be converted to plasmin by plaminogen activators- urokinase plasminogen activators (uPAs) and tissue plasminogen activators (tPAs) (Conaway et al., 2001). Plasmin has a broad trypsin-like specificity and degrades fibronectin, laminin, and the protein core of proteoglycans. In addition, plasmin activates certain metalloproteinases. Plasmin is believed to be the most important protease for the mobilization of fibroblast growth factor-2 (FGF-2 or basic FGF) from the extracellular matrix (ECM) pool. MMPs play a central role in degrading extracellular membranes and basement membrane structures, allowing endothelial cells to migrate (Conaway et al., 2001; Liekens et al., 2001). ii) Endothelial cell migration and proliferation Following proteolytic degradation of the ECM, “leader” endothelial cells start to migrate through the degraded matrix. They are followed by proliferating endothelial cells, which are stimulated by a variety of growth factors, some of which are released from the degraded ECM. At this step, interplay between VEGF, angiopoeitin, FGFs and their receptors are responsible for mediating the process of angiogenesis. Additionally, there are other factors that have also been implicated in the process, such as tumor necrosis factor alpha (TNF-α) and some chemokines (Conaway et al., 2001; Liekens et al., 2001). iii) Maturation of neovasculature As endothelial cell migrate into the extracellular matrix, they assemble into solid cord, and subsequently acquire a lumen. To form a stable vasculature, the interaction between endothelial cell with ECM and mesenchymal cells occurs, regulated by platelet derived growth factor (PDGF), Transforming growth factor-beta ( TGF-β), and the interaction of angiopoeitin with its receptors, tyrosine kinase Tie1 and Tie2 (Conaway et al., 2001; Griffioen and Molema, 2000). II. Angiogenesis and inflammatory diseases Rheumatoid arthritis (RA) is a chronic systemic disease characterised by an inflammatory erosive synovitis. Early changes in the synovium are marked by revascularization, inflammatory cell infiltration, and associated synoviocyte hyperplasia, which produce a pannus of inflammatory vascular tissue. This pannus covers and erodes articular cartilage, eventually leading to joint destruction. Angiogenesis is now recognized as a key event in the formation and maintenance of the pannus in RA. This suggests that targeting blood vessels in RA may be an effective future therapeutic strategy. Disruption of the formation of new blood vessels would not only prevent delivery of nutrients to the inflammatory site, but could also lead to vessel regression and possibly reversal of disease (Pandya et al., 2006). Psoriasis is a common chronic dermatosis occurring in 2% of the population and associated with an inflammatory arthritis–psoriatic arthritis (PsA)–in up to 40% of cases. PsA accounts for approximately 15% of patients attending early synovitis clinics, therefore it represents the second most common diagnostic category after rheumatoid arthritis. Significant abnormalities of vascular morphology and angiogenic growth factors have been described in psoriasis and PsA. Angiogenesis appears to be a fundamental inflammatory response early in the pathogenesis (Pandya et al., 2006). 1.2.2 Wound healing Wound healing is a highly dynamic process and involves complex interactions of ECM molecules, soluble mediators, various resident cells, and infiltrating leukocyte subtypes. The ultimate goal in repair is to restore tissue integrity and function. The healing process consists of three phases that overlap in time and space: inflammation, proliferation, and tissue remodeling (Diegelmann et al., 2004; Harding et al., 2002). Unregulated wound healing results in chronic wound (non-healing wound) or fibrosis (excessive scar formation). Chronic wound and fibrosis represent the major health burden and threat (Bayat et al., 2003; Harding et al., 2002). To fully understand the mechanisms underlying the wound healing and scar formation will shed light on the treatments of chronic wound and fibrosis. I. Wound healing process Wound healing generally involves the initiation and integration of a biological response, coordinating the migration, proliferation, and differentiation of a heterogeneous group of cells to achieve restoration of tissue integrity and function. In skin full length injury, progress toward healing follows a complex series of events, characterized by: inflammation, proliferation and tissue remodeling (Williams et al., 2003). Although inflammation, proliferation and maturation have been described as separate processes, in reality the phases of repair can overlap so that all three can be observed in different regions of a large dead space wound (Arnold and West, 1991). i) Inflammation phase Inflammation phase occurs immediately after injury. In the first 24 hours, this commences as the formation of the hemostatic primary platelet plug stimulated by thrombin and exposed fibrilla collagen and the influx of neutrophils from the blood (Williams et al., 2003). Neutrophils are attracted to wound sites within a few hours after injury by chemotactic mediators generated at the site. Within a day or two of injury, tissue monocytes enter the wound and differentiate into mature tissue macrophages. Macrophages are thought to play an integral role in a successful outcome of wound healing through the generation of growth factors that promote not only cell proliferation and protein synthesis but also components of the extracellular matrix. Macrophages also stimulate lymphocyte proliferation and cytokine release in response to specific antigens. In the late inflammatory phase of wound repair, T lymphocytes (T cells) appear in the wound bed. (Szpaderska and DiPietro, 2005). ii) Proliferation The proliferative phase is characterized by re-epithelialization, angiogenesis, fibroplasia, and wound contraction (Kirsner et al., 1993). Re-epithelialization One of the major goal of the body during cutaneous wound repair is the restoration of the skin as a functional barrier. The epidermis reacts to the injury within 24 hours. The keratinocytes initially migrate from the free edge of the wound. Approximately 12 hours after wound formation, epidermal cells become flattened and developed pseudopod-like projections (Kirsner et al., 1993). The migrating epidermal part is characterized by the loss of tight binding between these epidermal cells and the basement membrane and underlying dermis. Fibronectin appears to be instrumental in allowing continual migration of these cells. Fibronectin produced from plasma initially, and from plasma and fibroblast later, may also be derived from migrating keratinocytes. This suggests that the migrating epithelial cells may provide its own lattice for continual migration (Williams et al., 2003; Kirsner et al., 1993) The basement membrane zone changes in other ways after wound formation. Two important basement membrane proteins, laminin and type IV collagen, which normally mediate epidermal-dermal adhesion, disappear. Within 7-9 days after the reformation of the functional barrier of the skin, the basement membrane zone returns to normal. It has been shown that growth factors also play a role in signaling cell migration, such as TGF-β (Williams et al., 2003; Kirsner et al., 1993). While the migrating epithelial cells continue their journey across the wound to reestablish the functional barrier of the skin, cells just proximal to these are actively proliferating. Until the wound is closed, a zone of proliferating cells remains between the migrating epithelium and the normal cells at the wound edge. Although the stimulus for cells undergoing rapid proliferation is not known, several growth factors may be involved, including EGF (Williams et al., 2003; Kirsner et al., 1993). Angiogenesis Wound healing cannot occur without angiogenesis. The vasculature comprises up to 60% of repair tissue (Dyson et al., 1991), and the original name of granulation tissue is derived from the prominence of its vessels (Arnold and West, 1991). An abundant blood supply is necessary to meet the enormous local metabolic demands of debridement and fibroplasia (Arnold and West, 1991). Fibroplasia The increase in fibroblast numbers and the matrix they produce coincide with the formation of granulation tissue. They arise from both in situ cell proliferation and migration from adjacent areas (Williams et al., 2003). Fibroblasts migrate into the wound between 48 and 72 hours (Kirsner et al., 1993). Their function is to synthesize structural proteins, reorganize the wound matrix and promote wound contraction. In addition to collagen, they produce tenascin, fibronectin and glycosaminoglycans. Fibroblasts migrate to wound by pulling themselves along a fibronectin matrix. This migration occurs by contraction of intracellualar microfilaments. The loose extracellular matrix made of fibronectin is laid down by the migrating fibroblasts themselves (Kirsner et al., 1993). Fibroblasts differentiate into myofibroblasts by development of cytoskeletal protein, including actin, myosin heavy chain and MyoD (Williams et al., 2003). This affords these cells contractile properties that enable the cells to promote wound edge contraction (Williams et al., 2003). Wound contraction In full-thickness wounds, the wound contraction is an important part of wound healing, accounting for up to a 40% decrease in the size of wound (Kirsner et al., 1993). The contractile forces produced by granulation tissue in the wound are derived from myofibroblasts that contain contractile proteins. Myofibroblasts within the wound align along the lines of contaction and differ from the other cellular components, including 10 At days 1, 5, 9, 13 after injury, 12 mice were euthanized by CO2 at each time point for each group, among which, mice were used for histological studies and mice for ELISA studies. For histological analysis, wound specimens were collected and cut from the middle; half of which were embedded in tissue freezing medium (Leica Microsystems GmbH, Wetzlar, Germany), frozen in liquid nitrogen and cryosectioned to a thickness of µm for immunohistochemistry staining. The other half of wound specimens were fixed in 10% formalin, embedded in paraffin and sectioned to a thickness of µm for H&E and trichrome staining. For ELISA study, wound specimens were snap frozen in liquid nitrogen and kept in a -80ºC freezer. At day 20 after skin injury, wound specimens of mice from each group were collected after the mice were sacrificed. Subsequently, the specimens were fixed in 10% formalin, embedded in paraffin and sectioned to a thickness of µm for H&E and trichrome stain. 58 BALB/c male mice BALB/c female mice Skin wound formation Macroscopic observation Skin wound healing Fig. 2.4 Skin injury model in BALB/c male and female mice 59 -8C RB6-control group (Rag1+/-) 2b IgG IgG 2b Rag1KO group RB Control group (Rag1+/-) RB6-Rag1KO group C5 RB Skin wound formation Macroscopic observation Wound healing Inflammatory cell infiltration Neutrophil: IHC H&E Macrophage: IHC Lymphocytes: IHC Microvessel density IHC of CD31 VEGF Expression of cytokines Expression: ELISA ELISA Fig. 2.5 Skin wound healing model in control, Rag1KO, RB6-control and RB6-Rag1KO mice 60 2.5.3 Neutrophil depletion in skin injury model Control and Rag1KO mice were injected intraperitoneally with 0.1 mg rat anti-mouse RB6-8C5 monoclonal antibody (SouthernBiotech, Alabama, USA) two times. The first dose of RB6-8C5 was injected day before injury; the second dose was injected days after injury. Control mice received an equal quantity of rat IgG2b (isotype control antibody, SouthernBiotech, Alabama, USA). Each experiment was repeated three times to ensure the reproducibility of results. Neutrophil depletion was confirmed by Giemsa staining of the blood smear. 2.6 Immuno-histochemcial techniques 2.6.1 Immunohistochemistry staining of neutrophil, F4/80, CD3 and CD31 I. Procedures Rat anti mouse neutrophil, F4/80, CD3ε and CD31 monoclonal antibodies (Caltag, Burlingame, CA) were used to detect neutrophils, macrophages, T cells and vascularization. F4/80 is a transmembrane protein present on the cell-surface of mouse macrophages, which are derived from the myeloid lineage (Leenen et al., 1994). CD3ε polypeptide, which together with CD3-gamma, -delta and -zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T cell receptor-CD3 complex (Gold et al., 1986). CD31, an adhesion molecule expressed by endothelial cells, leukocytes, and 61 platelets, is used as a marker of normal and neoplastic vascularization (Sapino et al., 2001). For immunohistochemistry, frozen sections were fixed in -20°C acetone for minutes and immersed in 0.3% H2O2 followed by incubation with the selected primary antibody for one hour at room temperature. The sections were then incubated with HRP-conjugated rabbit anti-rat IgG F(ab’)2 . Finally, the slides were incubated with a freshly prepared 3, 3′-Diaminobenzidine (DAB) solution and counterstained with Mayer’s hematoxylin (Sigma-Aldrich, Missouri, USA). Negative controls were prepared by substituting primary antibodies with rat IgG. II. Evaluation of stain results in corneas Neutrophils, macrophages and T cells were counted by microscopic evaluation of the cornea in of the most intensively stained fields of one section under x400 magnification. Neutrophil, macrophage and T cell infiltration were determined as an average count of three sections of one sample and expressed as counts per mm2. CD31 antibody was used for detection of microvessel density. All positively stained discrete cells or cell clusters with or without visible lumina were counted as one microvessel. The microvessel was assessed by enumeration of the total amount of blood vessels in the whole stroma area of one section. The microvessel density was determined as an average count of three sections of one sample and expressed as counts per mm2. III. Evaluation of stain results in skin wounds 62 The wound bed was defined as the area surrounded by unwounded skin and fascia, regenerated epidermis and eschar. Two visual fields(x 400) were chosen from each edge of the wound bed; while three others were chosen from the middle of the wound bed. The numbers of neutrophils, F4/80-positive macrophages, CD3ε-positive T cells and CD31-positive vessels were enumerated and averaged on visual fields (x 400) in the wounded bed at the indicated intervals. All measurements were performed blinded form. The infiltration of neutrophils, macrophages and T cells infiltration were determined as an average count of sections from one sample and expressed as counts per high power field. CD31 immunoactivity was used for detection of microvessel density. All positively stained discrete cells or cell clusters with or without visible lumina were counted as one microvessel. The microvessel density was determined as an average count of sections from one sample and expressed as counts per high power field (HPF). 2.6.2 Immunohistochemical staining of VEGF in paraffin embedded slides Eyeballs were fixed in 10% neutral buffered formalin, paraffin embedded, and cut into μm sections. The paraffin-embedded tissue sections were deparaffined by changes of xylene for minutes each. The slides were then rinsed in double distilled water (ddH2O). The antigen retrieval solution of citric acid was heated to 96ºC for 10 minutes in a coplin jar. The slides were maintained at 96ºC for 10 minutes. Next, the slides were allowed to cool for another 20 minutes at room temperature. Subsequently, the slides were stained with rabbit anti-mouse VEGF polyclonal antibody (Acris, Hiddenhausen, Denmark) and the EnVision+ System-HRP (Dako Cytomation, California, USA) according to the 63 manufacturers’ recommendations. Negative controls were prepared by substituting primary antibodies with rat or rabbit IgG. After counter staining with Mayer’s hematoxylin (Sigma-Aldrich, Missouri, USA), the slides were rinsed in ddH2O and dehydrated in changes of alcohol (75%, 95%, and 100%) for 5minutes each. The slides were then cleared in changes of xylene and cover applied. 2.7 Staining techniques 2.7.1 Blood smear preparation and evaluation A 2mm3 drop of blood, collected from a mouse tail, was placed 1cm from one end of the slide. A second slide (spreader slide) was held and the edge of the spreader slide was placed in front of the drop of blood. The spreader slide was pulled backward to touch the drop of blood. After the blood had spread along the edge of the spreader slide, the spreader slide was pushed forward at a 30º angle with a rapid, even motion. The slide was then air dried and subjected to Giemsa stain. All slides were evaluated in the same manner to assure that consistent information is obtained. The following approaches were performed: an examination at lower power was performed to evaluate the quality of the smear and an optimal area for evaluation at higher magnification (X400) was chosen. A differential count of at least 150 white blood cells was performed. 64 2.7.2 Hematoxylin and Eosin (H&E) staining Sections were stained with hematoxylin and eosin for morphology analysis. The sections were deparaffinized with changes of xylene for minutes each and rehydrated in changes of absolute alcohol, 95% alcohol and 70% alcohol. After rehydration, the slides were washed briefly in distilled water and stained in Harris hematoxylin solution for minutes. The sections were then differentiated in 1% acid alcohol following brief washing with running tap water, developed in 0.2% ammonia water and washed again. The sections were counterstained in eosin Y solution for 30 seconds followed by dehydration through graded alcohol. After clearing in changes of xylene, the sections were mounted and cover applied. 2.7.3 Giemsa Staining Stock Giemsa stain was prepared as follows: 30 ml glass beads, absolute methanol (acetone free 270 ml), 3.0 g Giemsa stain powder and 140 ml glycerol were put into a 500 ml bottle according to the order listed. The bottle was then subjected to moderate shaking for 30 to 60 minutes daily for at least 14 days. Shaking was also done prior to the use of Giemsa stain. 10% working Giemsa stain was prepared in Giemsa stain buffer (4.1 mM Na2HPO4, 26 mM NaH2PO4, 0.05% Triton-X). Blood smear slides were fixed in methanol for minutes. After drying, the slides were incubated in working Giemsa stain for 10 minutes. The smear slides were removed from the working Giemsa stain and rinsed by dipping 3-4 times in the Giemsa stain buffer, and placed upright in a rack until dry. 65 2.7.4 Trichrome Staining Following deparaffinization and rehydration, the sections were re-fixed in Bouin’s solution. After re-fixation, the sections were stained with Weigert’s iron hematoxylin working solution for 10 minutes, washed with distilled water and stained in Biebrich scarlet-acid funsin solution for 10-15 minutes. After staining, the sections were differentiated in phosphomolybdic-phosphotungstic acid solution for 10-15 minutes or until collagen staining is not red after brief washing. The sections were next transferred directly to a light green solution to stain for 5-10 minutes, rinsed briefly, then differentiated in 1% acetic acid solution for 2-5 minutes and subjected to brief washing. Lastly, the sections were dehydrated through 95% ethyl alcohol and absolute ethyl alcohol, cleared in xylene, and mounted with mounting medium. Collagen will be stained green, and nuclei stained black. However, cytoplasma, keratin, and muscle fibers will be stained red. 2.8 Enzyme-linked immunosorbent assay (ELISA) 2.8.1 Measurement of MCP-1 level The protein levels of MCP-1 were measured using a commercial ELISA kit (Pierce Endogen, IL, USA) according to the manufacturer’s instruction. Briefly, standards or tissue samples were pipetted into the precoated 96-well plates containing assay diluents and incubated for hour at room temperature. The wells were then washed times with wash buffer, and they were incubated for hour at room temperature with enzyme 66 conjugated antibody reagent. Samples were then washed again, substrate buffer was added, and the samples were incubated for 20 minutes at room temperature. The reaction was then stopped, and the absorption measured in an ELISA reader (MWG, Ebersberg, Germany) at 450 nm. All measurements were performed in duplicate. The tissue sample concentration was calculated from the standard curve and corrected for protein concentration. The lower detection limit of the ELISA was pg/ml. In the corneal injury model, data was expressed as cytokine or growth factor in picograms per cornea. In the skin injury model, data was expressed as cytokine or growth factor in picograms per milligram protein. 2.8.2 Measurement of MIP-1, MIP-2, VEGF and TNF-α level The protein levels of MIP-1, MIP-2，VEGF, and TNF-α were measured using commercial ELISA kits (Quantikine MIP-1, MIP-2, VEGF, and TNF-α, R&D Systems, MN, and USA) according to the manufacturer’s instruction. Briefly, 100 µl of diluted capture antibody was pipetted into the 96 well plate and incubated overnight. After incubation, the plate was washed times with washing buffer and bocking reagent (1%BSA in PBS) was added into the plates and incubated for hours and washed. Then, standards and tissue samples (100µl) were added into the plate and incubated for hours at room temperature. After washing, diluted detection antibody was pipetted into the plate and incubated for hours followed by washing and incubation with substrate for 20 minutes. Then the reaction was stopped, and the absorption was measured in an ELISA reader (MWG, Ebersberg, Germany) at 450 nm rectified at 540 nm. All measurements were performed in duplicate. The detection limits for each cytokine were as follows: 67 MIP-1, 1.5pg/mL; VEGF, 3.0 pg/mL; MCP-1, pg/mL, MIP-2, 1.5 pg/mL and TNF-α, 5.1pg/ml. In the corneal injury model, data was expressed as cytokine or growth factor in picograms per cornea. In skin injury model, data was expressed as cytokine or growth factor in picograms per milligram protein. 2.8.3 Measurement of TGF-β1 level To detect the active protein level of TGF-β1, the samples were activated according to the manufacturer’s instruction. The skin lysis buffer was mixed with 2.5N Acetic Acid/ 10M Urea and incubated for 10minutes. After incubation, 2.7N NaOH/1 M HEPES was added to the mixture for neutralization. Prior to the assay, the sample was diluted 4-fold with reagent diluents, and the procedure to measure the active TGF-β1 protein level was performed using the same procedure to measure MIP-1α, MIP-2, VEGF and TNF-α levels. The detection limit of TGF-β1 was 4.61 pg/ml. In the skin injury model, data was expressed as cytokine or growth factor in picograms per protein in milligrams. 2.9 Protein assay Bio-Rad protein assay was carried out according to the manufacturer’s instructions. Briefly, dye reagent was prepared by diluting part Dye Reagent Concentrate (Bio-Rad laboratory, Singapore) with parts distilled water and filtered through a Whatman #1 filter (or equivalent) to remove particulates. Three to five dilutions of a known concentration of protein standard were then prepared. The linear range of this microtiter plate assay was 68 0.05 mg/ml to approximately 0.5 mg/ml. Protein solutions were assayed in duplicate or triplicate. Ten μl of each standard or sample solution was pipetted into separate microtiter plate wells followed by 200μl of diluted dye reagent added to each well. After the sample and reagent were mixed thoroughly and incubated at room temperature for at least minutes, the absorbance was measured at 595 nm using an ELISA reader (MWG, Ebersberg, Germany) 2.10 Isolation of murine neutrophils and neutrophil activation study 2.10.1 Preparation of murine neutrophils Murine neutrophils were isolated from peripheral blood. Blood was collected from anesthetized mice by cardiac puncture with a heparinized syringe. Erythrocytes were then removed by two rounds of hypotonic lysis. Neutrophils were isolated from the resulting cell suspension using Histopaque 1077 centrifugation. The entire isolation was performed at 4°C. Purified neutrophils were suspended in IMDM at a concentration of 4.0 x 106 cells/ml and were kept on ice till needed. Neutrophils were >95% viable and >90% pure, as determined by cytospin centrifugation followed by Giemsa Stain. 2.10.2 Neutrophil activation study The neutrophils were cultured in 96-well plates (Costar, Cambridge, America) at a density of 1X106 cells/well. Neutrophils were preincubated with or without 10 µg/ml of cycloheximide (CHX) for 30 minutes at 37ºC and then further stimulated with or without 69 Phorbol-12-myristate 13-acetate (PMA) (100 ng/ml) for hours. After stimulation, cell-free supernatant was separated from the cell pellets and then stored at –80C. Cell pellets were lysed with PBS containing 0.5% NP40, mM EDTA and Complete Protease Inhibitor mixture (Roche Diagnostics, Basel, Switzerland), followed by a quick spin to remove cell debris and stored at -80C. Both the cell-free supernatant and cell pellet lysate were used to determine the protein levels of VEGF, MIP-1α and MIP-2 using commercial ELISA kits (Quantikine MIP-1, MIP-2 and VEGF, R&D Systems, MN, USA) according to the manufacturer’s instructions. Data was expressed as cytokine or growth factor in picogams per 106 cells. 2.11 Statistics For the corneal injury model, the data is presented as means  SEM (the standard error of the mean). Statistical analysis was performed using the unpaired student’s t-test with P < 0.05 accepted as statistically significant. For the skin injury model, the results are presented as mean ± SEM (the standard error of the mean) for the total number of observations. Statistical comparison between groups was performed using one-way ANOVA and unpaired student’s t-test. The results are considered statistically significant when P < 0.05. 2.12 Buffers Proteinase K buffer 50mM Tris PH 8.0, 100mM NaCl, 1% SDS 70 10XPCR buffer 100mM Tris-HCl pH 8.6, 500mM KCl, 15mM MgCl2 Tissue lysis buffer 10mM PBS, 0.1% SDS, 1% IGEPAL, 5mM EDTA 10mM PBS 1.9mM NaH2PO4, 8.1mM Na2HPO4, 0.9% NaCl 10% neutral Buffered Formalin 10% Formalin, 4g/L NaH2PO4.H2O, 6.5g/L Na2HPO4 Giemsa stain buffer 4.1 mM Na2HPO4, 26mM NaH2PO4, 0.05% (v/v) Triton Buffers for Masson’s Trichrome stain Bouin’s solution 72.5% (v/v) saturated picric acid, 22.5% (v/v) formaldehyde, 5% (v/v) glacial acetic acid Weighter’s iron hematoxylin solution 50% (v/v) stock solution A, 50% (v/v) stock solution B Stock solution A 71 1% (w/v) hematoxylin in 95% Alcohol Stock solution B ml 29% Ferric chloride, 1ml concentrated hydrochloric acid, add dH2O to 100ml Biebrich Scarlet-Acid Fuchsin Solution 90ml 1% aqueous biebrich scarlet, 10ml 1% aqueous acid fuchsin, 1ml glacial acetic acid Phosphomolybdic-phosphotungstic Acid Solution (50ml) 25ml 5% phosphomolybdic acid, 25ml 5% phosphotungstic acid 1% Acetic acid solution Light green solution 2% (w/v) light green, 2% (v/v) glacial acetic acid Buffers for ELISA 10X PBS buffer 1.37M NaCl, 27mM KCl, 81mM Na2HPO4, 15mM KH2PO4 Washing Buffer for ELISA 0.05% Tween-20 in PBS Regent Diluents: 1% BSA in PBS 72 Activation Buffer for TGF-β1 2.5N Acetic acid, 10M Urea 2.7N NaOH, 1M HEPES Buffers for Immunohistochemistry 1X Tris Buffered Saline (TBS) 20mM Tris base, 137mM NaCl, PH 7.6 Antibody Dilution Buffer 1%BSA in TBS with 1% Rabbit serum or mouse serum Washing Buffer 0.05% Tween-20 in 1XTBS Buffers for Nuetrophil isolation Hanks balanced salt solution (HBSS) 137mM NaCl, 5.4mM KCl, 0.25mM Na2HPO4, 0.44mM KH2PO4, 4.2mM NaHCO3 Washing Buffer 1%BSA in HBSS Red cell lysis buffer 8.3g/L NH4Cl, 0.85g/L NaHCO3, 0.1mM EDTA 73 [...]... presence of the myofibroblast population in the scar and therefore the increase in contraction of the wound and synthesis of the matrix Many cytokines are implicated in these processes but certain molecules, notably the TGF-β family are particularly important (Muir, 1990) 1.3 Cytokines in angiogenesis and wound healing 1.3.1 Chemokines in angiogenesis and wound healing Chemokines are a family of small secreted... granules are part of the family of peroxidase negative granules Specific granules and gelatinase granules differ significantly from each other with respect to protein content and secretory properties Specific granules are larger and rich in antibiotic substances, participating mainly in the antimicrobial activities of neutrophils Gelatinase granules are smaller and easily exocytosed, acting primarily... repair and serve as a scaffold for cellular migration and tissue support (Kirsner et al., 1993) The total amount of collagen increases early in the repair process, reaching a maximum between 2 and 3 weeks after injury (Kirsner et al., 1993) Tensile strength increase to 40% 1 month after injury and may continue to increase for as long as a year after injury (Kirsner et al., 1993) Changes in the types of. .. an important role in the inflammatory response, and has been implicated as an important factor in mediating monocytic infiltration of tissues in wounds and inflammatory diseases (Charo et al., 1994; DiPietro et al., 20 01; Gibran et al., 1997) MCP-1 is produced by macrophages, fibroblasts, endothelial cells, keratinocytes and smooth muscle cells in response to inflammatory stimuli (Gibran et al., 1997)... angiogenesis in the adult VEGF was detected in the ovary during corpus luteum formation and in the uterus during growth of endometrial vessels and at the site of embryo implantation VEGF therefore plays a pivotal role in angiogenesis by contributing the multiple steps of angiogenesis (Bao et al., 20 08) Vasodilation VEGF has the ability to increase vascular permeability and induce vascular leakage It binds... peroxidase-positive granules, which is defined by the high content of myeoloperoxidase (MPO) Azurophilic granules are formed in early promylocytes The matrix of azurophil granules contains microbicidal proteins and acid hydrolase involved in oxidative and nonoxidative killing of bacteria and fungi (Borregaard and Coeland, 1997; Faurschou and Borregaard, 20 03) Specific (secondary) and gelatinase (tertiary)... normal surrounding tissue (Beanes et al., 20 03; Harty et al., 20 02) In the past 20 years, tremendous progress has been achieved in understanding the cellular and molecular events of wound repair, but the tendency among vertebrates for scarring rather than regeneration remains unexplained (Harty et al., 20 02) In contrast to the adult repair process, skin wounds in first- and second-trimester fetuses heal... primarily as a reservoir of matrix degrading enzymes and membrane receptors needed during neutrophil extravasation and diapedesis.Peroxidase-negative granules also contain three 27 metalloproteases with great physiological and pathophysiological significance, namely collagenase (matrix metalloproteinase-8 (MMP-8)), gelatinase (MMP-9), and the recently discovered leukolysin (MMP -25 ) (Borregaard and Coeland,... MMPs are capable of degrading major structural components of the extracellular matrix including collagens, fibronectin, proteoglycans, laminin and gelatin, which is required for the initiation of angiogenesis (Liekens et al., 20 01) III Roles of neutrophils in angiogenesis Neutrophils are traditionally regarded as rapidly recruited sacrificial cells that, upon stimulation by generic pathogen pattern... degranulate to release proteases, activate phagocytosis and oxidative burst generation before dying However, the concept that alternative activation and plasticity of neutrophils can be a significant source of cytokines and chemokines to orchestrate physiological and pathological processes is now becoming more appreciated (Kasama et al., 20 05) Recent studies have indicated that neutrophils may play an . in psoriasis and PsA. Angiogenesis appears to be a fundamental inflammatory response early in the pathogenesis (Pandya et al., 20 06). 1 .2. 2 Wound healing Wound healing is a highly dynamic. plasminogen, which can be converted to plasmin by plaminogen activators- urokinase plasminogen activators (uPAs) and tissue plasminogen activators (tPAs) (Conaway et al., 20 01). Plasmin has a. vascular endothelial growth factor (VEGF), transcriptionally upregulated in part by hypoxia, mediates an increase in vascular permeability and extravasation of plasma proteins including plasminogen,