Abstract
Cigarette smoke has been associated with poor healing in several studies, but the precise mechanisms involving this impairment are still not elucidated. The aim of this work was to investigate cigarette smoke exposure effects on initial phases of cutaneous healing in mice, focusing mainly on gene expression of two molecules involved in wound repair (Ccn2/Ctgf and Tgfb1) and to study if these effects are strain dependent. Mice were exposed to the smoke of nine cigarettes per day, three times per day, for ten days. In the eleventh day an excisional wound was made. The control group was sham-exposed. The cigarette smoke exposure protocol was performed until euthanasia, seven days after wounding. Wound contraction was evaluated. Sections were stained with hematoxylin-eosin, Sirius red, and toluidine blue, and also immunostained for alpha-smooth muscle actin. Gene expression of Ccn2/Ctgf and Tgfb1 was evaluated by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Smoke-exposed animals presented delay in wound contraction; fibroblastic, inflammatory, and mast cell recruitment; re-epithelialization; myofibroblastic differentiation; and Ccn2/Ctgf and Tgfb1 gene expression. Those alterations were strain dependent. This work confirmed the deleterious effects of cigarette smoke exposure on mouse cutaneous healing depending on mouse strain and links these effects to an overexpression of Ccn2/Ctgf.
Introduction
Normal tissue repair follows a sequence of events, namely inflammation, granulation tissue formation, and remodeling, that are coordinated by specialized cell types and by a network of growth factors and cytokines (Singer and Clark 1999).
Transforming growth factor-β (TGF-β) is a multifunctional cytokine, present in high amounts in platelet granules (Assoian et al. 1983), that participates in numerous biological processes, including cell proliferation, differentiation, immune modulation, and extracellular matrix production (Massague 1990; Sporn et al. 1986).
It is known that the growth stimulatory action of TGF-β and the induction of collagen synthesis and deposition occur via a post-TGF-β receptor mechanism mediated by connective tissue growth factor (CCN2/CTGF) (Frazier et al. 1996; Kothapalli et al. 1997).
Ccn2/Ctgf is a member of a gene family known as the CCN family, which includes Ccn2/Ctgf, Cyr61, Nov, Widp1/Elm1, Wisp2/Cop1, and Wisp3 (Brigstock 1999). CCN2/CTGF protein production and secretion is selectively induced by TGF-β, and other growth factors (Leask et al. 2002; Soma and Grotendorst 1989).
CCN2/CTGF exhibits numerous biological properties that are of potential importance in wound healing response, including stimulation of cell proliferation, cell adhesion, chemotaxis, angiogenesis, and production of extracellular matrix components (Wang et al. 2003; Yoshida and Munakata 2006).
The association between poor healing and cigarette smoke is well recognized in clinical practice (Freiman et al. 2005; Frick and Seals 1994; Manassa et al. 2003; Rogliani et al. 2006; Silverstein 1992) and in experimental studies (Cardoso et al. 2007; Poggi et al. 2002; Wong et al. 2004; Wong and Martins-Green 2004).
Cigarette smoke can induce TGF-β release, increase Tgfb1 gene expression, and quickly induce Ccn2/Ctgf gene expression in small airway remodeling (Churg et al. 2006; Wang et al. 2005). However, the effects of cigarette smoke in the expression of Ccn2/Ctgf and Tgfb1 during cutaneous wound healing was not described. Since CCN2/CTGF protein appears to function as an autocrine growth stimulator for connective tissue cells and its production is regulated by TGF-β 1, we examined the expression of both Tgfb1 and Ccn2/Ctgf during wound healing of an excisional cutaneous lesion in C57BL/6 and BALB/c mice strains, exposed or not exposed to cigarette smoke.
Material and Methods
Animals
Adult male C57BL/6 and BALB/c mice (four to six weeks old, body weight 25–30 g) were housed under controlled environmental conditions (light/dark cycle, temperature, and humidity); food and water were provided ad libitum. All animal procedures were approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro.
Cigarette Smoke Exposure and Wounding
In each strain, animals were separated in control (n = 10) and smoke-exposed (n = 14) groups. Smoke-exposed groups had the whole body exposed, in an inhalation chamber, to a smoke–air mixture of commercial filtered Virginia cigarettes, three times per day, seven days per week, during the entire experiment, as previously described (Cardoso et al. 2007; Valenca et al. 2004). The cigarette smoke exposure protocol started ten days before the excisional wound and continued until the end of the experiment. The control group was sham-exposed.
On day 0 (ten days after the beginning of smoke or sham exposure), the animals were anesthetized with ketamine (5 mg/kg, ip) and xylazine (2 mg/kg, ip). The dorsal surface was shaved, and a full-thickness excisional wound (1 cm2) was made. The wound was not sutured or covered and healed by second intention.
Macroscopic Analysis
To evaluate wound contraction, a transparent plastic sheet was placed over the wound, and the wound margins were traced. After digitalization, the wound area was evaluated using Zeiss image-processing system KS400 (Zeiss-Vision, Oberkochen, Germany) (Souza et al. 2005). Wound area was measured soon after wounding and seven days later. Data are expressed as a percentage of the initial wound area.
Tissue Harvesting and Staining
Mice were sacrificed in a CO2 chamber seven days after wounding. Fragments of wound with adjacent skin tissue were formalin-fixed (pH 7.2) and paraffin-embedded. Sections (5 μm) were stained with hematoxylin-eosin, for general evaluation of wound and normal skin and of inflammatory infiltrate, with Sirius red for analysis of collagen fiber organization, and with toluidine blue for analysis and quantification of mast cells. Collagen organization was evaluated in tissue sections stained with Sirius red observed under polarized light where thick collagen fibers are strongly birefringent and yellow to red, whereas thin collagen fibers are weakly birefringent and greenish.
Mast Cell Quantification
The number of mast cells was evaluated in toluidine blue stained sections. Six random fields (0.119 mm2) were analyzed using the x40 objective (Olympus BH-2, Olympus Optical Co. Ltd., Tokyo, Japan), and the average cell count per field was calculated for each animal. The percentage of degranulating mast cells was also evaluated. All analyses were repeated without significant difference among them.
Immunohistochemistry
Myofibroblasts expressing alpha-smooth muscle actin (α-SMA) were localized by immunohistochemistry. To allow the use of a mouse monoclonal antibody in mouse tissue, some modifications in standard technique were performed. Sections (5 μm) were deparaffinized and hydrated, and after washing in phosphate buffered saline (PBS), sections were incubated with the EnVision system (DAKO, Carpinteria, CA, USA) for fifty minutes to allow anti-mouse IgG to bind to tissue, then peroxidase (endogenous and polymer-linked) was inhibited by incubation in 3% H2O2 in methanol for thirty minutes. After washing, sections were incubated with a solution of a monoclonal antibody against α-SMA (DAKO, Carpinteria, CA, USA) and EnVision in phosphate buffered saline/bovine serum albumin (PBS/BSA) 1% overnight. Diaminobenzidine was used as chromogen. Sections were counterstained with Delafield’s hematoxylin. Negative controls were prepared by replacing primary antibody with PBS/BSA, and no labeling was observed.
RNA Isolation and Semiquantitative RT-PCR Procedure
Tissue samples were homogenized in TRIZOL reagent (Invitrogen, Carlsbad, CA, USA), and RNA extraction was performed according to the manufacturer’s directions. Before cDNA synthesis, RNA concentration was measured at 260 nm. For cDNA synthesis, 1 μg of RNA was reverse-transcribed using a SuperScript III kit (Invitrogen) following manufacturer directions for one hour at 42°C. For semiquantitative polymerase chain reaction (PCR), 3 μl of cDNA was mixed with PCR buffer containing 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 μM oligonucleotide sense and anti-sense primers, 0.25 U Taq polymerase (Invitrogen) and nuclease-free water. Oligonucleotide sequences used were Gapdh (571 bp, 25 cycles) forward 5′ ATC ACC ATC TTC CAG GAG CG 3′ and reverse 5′ CCT GCT TCA CCA CCT TCT TG 3,′ Ccn2/Ctgf (236 bp, 30 cycles) forward 5′ GAA GGG CAA AAA GTG CAT CC 3,′ and reverse 5′ GAC AGT TGT AAT GGC AGG CA 3′ (Lin et al. 2002), and Tgfb1 (571 bp, 30 cycles) forward 5′ ATC ATG TTG GAC AAC TGC TC 3′ and reverse 5′ CTG AGT GGC TGT CTT TTG AC 3′ (Hannon et al. 1992).
Statistical Analysis
All data are presented as mean ± standard deviation (SD). Data concerning lesion area, mast cell number, and Tgfb1 and Ccn2/Ctgf mRNA levels were analyzed with a nonparametric Mann-Whitney test. Statistical analysis was done using the software Graph Pad Instat version 3.01 (GraphPad Software Inc., La Jolla, CA, USA).
Results
Macroscopic Analysis
In C57BL/6 mice, seven days after lesion, wound surface was smaller (16.5% ± 4.6%) in control group than in the smoke-exposed group (20.9% ± 7.9%) (p < .05) (Figure 1). In BALB/c mice, seven days after lesion, wound surface was smaller (36.3% ± 12.2%) in the control group than in the smoke-exposed group (60.1% ± 7.4%) (p < .001
General Histology
Among C57BL/6 mice, seven days after wounding, animals from the control group presented fusiform “fibroblast-like” cells arranged mainly parallel to the surface, and a smaller number of inflammatory cells; in the smoke-exposed group, “fibroblast-like” cells were mainly ovoid and located in the deep region, without a particular arrangement (Figures 2a and 2b). In both groups there were no signs of re-epithelialization (keratinocytes from wound borders migrating into the wound bed).
In BALB/c mice, seven days after wounding, animals from the control group presented inflammatory and fibroblastic cells in granulation tissue (Figure 2c). In this group, re-epithelialization was almost complete (Figure 3a). In the smoke-exposed group, the number of inflammatory cells was higher, and re-epithelialization was impaired (Figures 2d and 3b ).
Mast Cells
In all groups, mast cells were sparse and localized in the deep region of the granulation tissue. The majority of mast cells were ovoid, but some tadpole-shaped cells were also observed.
Smoke exposure did not change mast cell distribution in C57BL/6 animals. On the other hand, in BALB/c animals, the smoke-exposed group showed a smaller number of mast cells (−78%) than the control group (p < .05) (Figure 4).
When the number of mast cells was compared among control groups of mouse strains, we observed that C57BL/6 mice presented a higher number of mast cells (+ 159%) than BALB/c mice (p < .05). Comparison between smoke-exposed groups showed a similar response pattern; C57BL/6 mice presented a higher number of mast cells (400% more) than BALB/c mice (p < .05) (Figure 4). Mast cell degranulation was not influenced by cigarette smoke exposure or by strain (Figure 4).
Organization and Distribution of Collagen Fibers
In normal skin, collagen fibers were yellow-red and thick and presented a basketlike pattern in all experimental groups. In the C57BL/6 control group, collagen fibers were localized in the superficial region of the scar and were mainly red fragmented fibers and some greenish, and most of them were parallel to the surface; in the smoke-exposed group, collagen fibers were mainly greenish and very fragmented, and some were yellow-red with a random arrangement (Figure 5).
Seven days after wounding, in the BALB/c control group collagen fibers were mainly yellow-greenish, elongated fibers, and some yellow-red fragmented collagen fibers were also observed in the superficial region of the scar; in the smoke-exposed group, collagen fibers were mainly greenish and fragmented (Figure 5). Thus, collagen fibers were less organized and dense in the smoke-exposed groups than in the control groups (Figure 5).
Analysis of Myofibroblasts
Myofibroblasts were fusiform, parallel to the surface, and homogenously distributed in granulation tissue of both strains and experimental groups. Seven days after wounding, both control groups (C57BL/6 and BALB/c) presented a higher density of myofibroblasts than the smoke-exposed groups (Figure 6).
Ccn2/Ctgf and Tgfb1 Gene Expression
mRNA levels for Ccn2/Ctgf and Tgfb1 genes were evaluated in tissue samples of both strains and groups. In C57BL/6 mice, the Ctgf mRNA levels were lower in the control group than in the smoke-exposed group (p < .05) (Figure 7). In BALB/c mice, Ctgf mRNA levels were lower in the control group than in the smoke-exposed group, but this difference was not statistically significant (Figure 7).
In C57BL/6 mice, the Tgfb1 mRNA levels were lower in the control group than in the smoke-exposed group, but this difference was not statistically significant (Figure 7). Similary, in BALB/c mice, Tgfb1 mRNA levels were lower in the control group than in the smoke-exposed group, but this difference was not statistically significant (Figure 7).
Discussion
This report showed alterations induced by cigarette smoke exposure on Ccn2/Ctgf mRNA expression and other morphological parameters during the proliferative phase of cutaneous wound healing in two mice strains (BALB/c and C57BL/6). Furthermore, these data demonstrate that these alterations are strain dependent.
Deleterious effects of cigarette smoke on cutaneous wound healing have already been shown in clinical and experimental studies (Fang and Svoboda 2005; Manassa et al. 2003; Netscher and Clamon 1994; Wong and Martins-Green 2004). We have previously demonstrated cigarette smoke effects during the remodeling phase of cutaneous wound repair using different mice strains (Cardoso et al. 2007). As the mice have a different wound healing process compared to that of humans, we experienced that this process begins with contraction, followed by epithelialization, and even with administration of any toxic agent, this process will finish in a satisfactory way. We decided to study the effects of cigarette smoke on the proliferative phase of cutaneous wound repair where several growth factors should be acting in a coordinated manner (Colwell et al. 2007; Komi-Kuramochi et al. 2005; Schafer and Werner 2007; Sogabe et al. 2006) to better understand how this process is impaired by cigarette smoke exposure. Within this hypothesis, as in our previous study, C57BL/6 and BALB/c mice presented an evident impairment in the wound healing process seven days after wounding, and we decided to investigate the mechanism of wound healing analyzing Ccn2/Ctgf and Tgfb1 gene expression while studying this time point.
Ccn2/Ctgf is known to be a potent inducer of fibroblast proliferation and deposition of extracellular matrix (Bradham et al. 1991). The most potent Ccn2/Ctgf inducer is Tgfb1, and it has been proposed that Ccn2/Ctgf overproduction plays a major role in pathways that lead to fibrosis (Frazier et al. 1996; Igarashi et al. 1993). In a previous study, Ccn2/Ctgf mRNA levels were analyzed in full-thickness excisional mouse wounds, and Ccn2/Ctgf mRNA was most abundant between twelve hours and one day after wounding and reached basal levels after seven days (Dammeier et al. 1998). Interestingly, another study observed similar kinetics of induction for Tgfb1, a Ccn2/Ctgf inducer (Frank et al. 1996). Recently, studies showed that cigarette smoke caused up-regulation of Tgfb1 and Ccn2/Ctgf gene expression in small airway remodeling (Churg et al. 2006; Wang et al. 2005). Our study showed an overexpression of Ccn2/Ctgf mRNA only in the C57BL/6 mouse strain exposed to cigarette smoke. BALB/c smoke-exposed mice also presented more elevated levels compared to the control group; however, this difference was not statistically significant. The effects on Ccn2/Ctgf gene expression were independent from Tgfb1, since Tgfb1 expression was not altered by cigarette smoke exposure and can be a result of other factors present during the proliferative phase of wound healing.
During wound healing, myofibroblasts are transiently present in the wound bed, where they are believed to play essential roles in wound contraction and granulation tissue formation (Grotendorst et al. 2004). Our results showed a delay in myofibroblastic differentiation since mouse strains exposed to cigarette smoke presented a reduction in the number of myofibroblasts and more immature collagen scaffold seven days after wounding, and an elevated number of myofibroblasts on granulation tissue fourteen days after wounding (Cardoso et al. 2007). The interaction between myofibroblasts and the surrounding extracellular matrix plays an important role in wound contraction, since cigarette smoke may delay myofibroblastic differentiation and reduced collagen deposition, leading to a delay in wound contraction.
Mast cells contribute to cutaneous wound healing with their large repertoire of proinflammatory and growth-promoting mediators such as histamine, leukotrienes, prostaglandins, proteases, and cytokines (Biedermann et al. 2000; Huttunen and Harvima 2005). Mast cells promote the proliferation of fibroblasts, endothelial cells, and keratinocytes during the proliferative phase of wound healing (Katayama et al. 1992) and can promote the conversion of fibroblasts to a myofibroblast phenotype, which facilitates wound closure (Gailit et al. 2001). Our study showed a reduction in mast cell recruitment only in the BALB/c mouse strain. This reduction could be explained by an alteration in the inflammatory phase of the wound healing process, leading to impairment of recruitment of these cells to the granulation tissue. Furthermore, this impairment could justify the alterations in myofibroblastic differentiation observed in cigarette smoke-exposed mice and the presence of fibroblast-like cells with ovoid shape in the granulation tissue of BALB/c mice.
Another study showed that in vitro, cigarette smoke inhibited mast cell degranulation and production of nitric oxide (NO) by mast cells (Wei et al. 2005). The effects of cigarette smoke exposure could not be attributed to the pharmacological activity of nicotine, the main alkaloid from the particulate phase of cigarette smoke, but may be related to oxidative free radicals (Gillissen and Nowak 1998). Our data did not show alterations in mast cell degranulation, but this finding could be owing to different methods employed to study the effects of cigarette smoke. On the other hand, an inhibition of NO and/or histamine synthesis showing an alteration not only in the number but also in the function of these cells could be the reason for the impairment in myofibroblastic differentiation.
Collectively, the data from this study confirm the deleterious effects of cigarette smoke exposure on mouse cutaneous wound healing and link these effects to an overexpression of Ccn2/Ctgf. Our study suggests a potential role of the Ccn2/Ctgf pathway in the wound healing impairment induced by cigarette smoke. Furthermore, we demonstrated that the different patterns of cigarette smoke impairment depend on the mouse strain studied, suggesting the influence of genetic background on the susceptibility to cigarette smoke effects on wound healing.
Footnotes
Figures
Acknowledgments
This study was partially supported by CNPq and FAPERJ. T. P. Amadeu held a postgraduate fellowship from CAPES. F. A. Mendes holds a postgraduate fellowship from the Oncobiology program UFRJ/FUJB, and B. Romana-Souza from UERJ.