Pressure sore, also called decubitus ulcers or bedsores, affect the immobilized (such as patients with paraplegia and quadriplegia), the aged, and the people who are bed-ridden due to illness. These wounds are due to the persisting pressure over bony prominences such as the sacrum and Ischii (Nguyen et al., 2008). This disease has obvious negative effect on quality of life. The cause of this disease is not only the continuing pressure against subcutaneous soft tissue, but also external environmental factors such as temperature, humidity, shear stress, and tissue deformation (Bouten et al., 2003). Pressure ulcers are not the exclusive result of local continuous pressure. Their formation also results from ischemia-reperfusion cycles. Localized external pressure and restriction of blood supply lead to ischemia (Nguyen et al., 2008). This leads to localized tissue ischemia and ultimately results in the ulceration and necrosis of soft tissues (Wang, 2011). The mechanism of ischemia-reperfusion injury is believed to play an important role in the pressure ulcer development.
Angiogenesis, formation of blood vessels from existing blood vessels, is essential for preparing a closed vascular circulatory system in the body, and for providing oxygen and nutrition to tissues (Shibuya, 2008). It has been proven (Stalmans, 2005; Damico, 2007; Mizia-Malarz et al., 2008; Przybylski, 2009; Kaiser et al., 2011) that growth factors play an important role in cell proliferation, differentiation, tissue repair, ulcer formation, and healing. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are recognized as key factors in regulating ischemia and angiogenesis in pressure ulcer.
Evidence accumulating over the last decade has established the fundamental role of VEGF as an important regulator of normal and abnormal angiogenesis (Ciulla and Rosenfeld, 2009). VEGF is a chemotactic agent and a potent endothelial cell mitogen that also influences vascular permeability (Mammoto et al., 2009). VEGF-A is a key player in vasculogenesis, the formation of blood vessels from progenitor cells, as well as angiogenesis (Shibuya, 2008). The biological effects of VEGF are mediated by two high-affinity tyrosine kinase receptors, VEGF-receptor 1 (VEGF-R1) and VEGF-receptor 2 (VEGF-R2) (Larrivee and Karsan, 2000). VEGF is expressed in organs during embryogenesis and to a limited extent in adult organs, such as the circumventricular organs have the expression of VEGF. (Dor et al., 2003). In disease states, VEGF is detected in the synovial pannus of rheumatoid arthritis, various tumor cells, and in keratinocytes in the process of wound healing (Fava et al., 1994; Ryuto et al., 1996).
bFGF was originally isolated and identified from bovine brain and pituitary by its induction of cell proliferation of fibroblasts (Ellman et al., 2008). bFGF is involved in numerous cellular functions in various cell types, including angiogenesis, cell proliferation, migration, differentiation, wound healing, limb formation, tissue remodeling, and tumorigenesis (Bodo et al., 2002; Douwes et al., 2007). bFGF also induces the expression of VEGF (Nugent and Iozzo, 2000). In normal tissue, bFGF is present in the basement membrane and in the extracellular matrix of blood vessels; it has been hypothesized that in wound healing in normal tissues and carcinogenesis, heparan sulfate-degrading enzymes can activate bFGF, which mediates the formation of new blood vessels, a process called angiogenesis (Kuhn et al., 2012).
However, the roles of VEGF and bFGF in the mechanism underlying the development of pressure ulcers have not yet been elucidated. Therefore, in this study, we established an animal model to detect the expression of VEGF and bFGF in different degrees of pressure ulcers. Our results showed that with an increase in the degree of pressure ulcer, the expression of VEGF and bFGF in pressure ulcer tissues are decreased. This leads to a reduction in angiogenesis and may be one of the crucial factors in the pressure ulcers formation.
MATERIALS AND METHODS
Animals and Grouping
This study was approved by the Taizhou University's Institutional Animal Care and Use Committee and was proceeded in compliance with international guidelines of laboratory animal protection for care and use of animal models. Twenty-eight healthy adult male Sprague-Dawley (SD) rats, weighing 230–280 g were used for the study. Animals were caged in a room with temperature controlled at 25 ± 2.0°C and unlimited access to food and water. SD rats were divided randomly into two groups, the untreated group (Group A) and the ischemia-reperfusion group (Group B), with 4 and 24 rats in each group, respectively. The body weight of all rats has no statistical significance.
Instruments and Reagents
The StepOne real-time PCR system was obtained from the Applied Biosystems (ABI). Vertical electrophoresis tank and transfer membranes slot were obtained from Beijing LiuYi Instrument Factory, China. The rat antimouse VEGF monoclonal antibody and the rabbit anti-bFGF polyclonal antibody were purchased from Abcam Company. Two-step immunohistochemistry detection kit and the DAB chromogen kit were obtained from Beijing Golden Bridge Biotechnology Co. The tissue protein extraction reagent (T-PER) was purchased from Thermo Fisher Company. The Trizol RNA extraction kit was obtained from Invitrogen Corporation. The Quanta cDNA synthesis kit for the synthesis of first-strand cDNA and the RealMasterMix (SYBR Green) kit were purchased from Tiangen Biotech Co. (Beijing). Primers were synthesized by Hai Jierui Biological Engineering Co. (China).
Preparation of Animal Model
The rats in Group B were anesthetized intraperitoneally with 10% chloral hydrate (300 mg/kg) and an incision was made in the midline of the back. A blunt dissection was carried out to expose the deep fascia and a magnetic disk (diameter 13 mm, thickness 2 mm, magnetic flux density of 1500 Gauss) was implanted subcutaneously. An external magnet was used to exert intermittent pressure. The external magnet attracted the transplanted disk, thus exerting pressure on the skin and resulting in localized skin ischemia. A cycle of external magnet removal after every 2 hr inducing ischemia, followed by partial restoration of blood flow for 30 min was performed. Each rat underwent three successive cycles per day for four consecutive days.
I-degree: the erythema confounded that cannot be restored to normal, 30 min after removing pressure; II-degree: superficial ulcers with red pink wound; III-degree: darkening and hardening of the skin that did not bleed by needling. For the Group A (control group), the magnetic disk was implanted in the back of rats subcutaneously, and no pressure was exerted from the external magnetic source. For the Group B, after removing the pressure, the rats were killed by cervical dislocation and pressure ulcers tissue were about 10 × 10 mm in size. The distance of pressure ulcers tissue within 3 cm tissue was about 10 × 10 mm. Tissue was obtained from the control group rats directly after they were sacrificed and stored at −80°C.
A total of 28 tissue specimens including the rats with normal skin tissue, different degrees of pressure ulcers tissue, and the tissue surrounding pressure ulcers were stored at −80°C. Each tissue specimen weighed about 50–100 mg. Total RNA was extracted with Trizol reagent, and the RNA concentration and purity were determined by ultraviolet spectroscopy. RNA integrity was evaluated by 1% agarose gel electrophoresis and revealed clear bands of 28S rRNA and 18S rRNA. The value of D 260 /D 280 was 1.8 to 2.0. The cDNA synthesis was performed with reactions (20 μL) containing 2 μg RNA, added 2 μL 10 × RT mix (final concentration of 1 ×), 2 μL dNTP mix (final concentration of 0.25 mmol/L dNTP), 2 μL of the Oligo-dT15 (final concentration of 1 μmol/L), 1 μL of Quant Reverse Transcriptase, then added RNase free ddH2O to a final volume of 20 μL at 37°C for 60 min. Using the fluorescent dye SYBR Green, the real-time PCR was completed according to the manufacturer's instruction. The real-time PCR was carried out separately for different genes, and was repeated three times for each sample.
PCR was performed using the following conditions: initial denaturation at 95°C for 2 min; denaturation at 95°C for 15 sec, annealing at 65°C for 30 sec (annealing temperature of different genes are shown in Table 1), extension at 68°C for 60 sec; final stage of denaturation at 95°C for 15 sec and cooling at 60°C for 1 min, then 0.3°C increase per 15 sec to 95°C, and maintained for 15 sec. The fluorescence signals were collected and the melting curve analysis was performed, respectively. The experiment was independently repeated three times. The relative quantitative results were automatically calculated at the end of the reaction. The primer sequences are shown in Table 1.
Table 1. The primer sequences of VEGF, bFGF, and β-actin
Forward primer sequence
Reverse primer sequence
CAC TGG ACC CTG
CAC TCC AGG GCT
GCT TTA CTG
TCA TCA TTG
CAC ACG TCA AAC
TTC GTT TCA GTG
TAC AGC TCC AAG
CCA CAT ACC A
AGA GGG AAA TCG
AGA GGT CTT TAC
TGC GTG AC
GGA TGT CAA CG
A total of 28 tissue specimens including the rats with normal skin tissue, different degrees of pressure ulcers tissue, and the tissue surrounding pressure ulcers were stored at −80°C. Protein was extracted from 25 mg of each tissue specimen in 500 μL of ice-cold T-PER (containing 1% PMSF), and centrifuged at 10,000g for 5 min. The supernatants were collected. The protein concentration was detected by BCA assay, and 30 μg of total protein was taken for electrophoresis. After separating the protein by SDS-PAGE with 8% separating gel and 5% stacking gel, the protein gel was transferred to a nitrocellulose membrane using a wet transfer method, and incubated with 5% BSA for 1.5 hr. Then, rabbit anti-VEGF (1:1000), bFGF (1:1250), and β-actin was added (1:1000) and incubated overnight. The HRP-conjugated goat antirabbit IgG antibody (1:1000) was used as the secondary antibody and incubated at room temperature for 1.5 hr. The X-ray lithography was performed using the chemiluminescence (ECL) method. The results were analyzed by computer software and data processing systems. The experiment was independently repeated three times.
The data is expressed as mean ± SEM and analyzed by using SPSS 17.0 statistical software. Differences between the three groups were determined by univariate analysis of variance. A correlation between VEGF and bFGF was used by the Pearson's correlation. The value P < 0.05 was considered statistically significant.
Establishment of Rat Pressure Ulcer Models
Rat pressure ulcer models were roughly divided into three degrees. I-degree model with the erythema confound that cannot be restored to normal, 30 min after removing pressure; II-degree model contained superficial ulcers with red pink wound; III-degree model with darkening and hardening of the skin that did not bleed by needling (Fig. 1).
Histopathological Changes of Pressure Ulcers
The skin integrity was normal in the control group rats with the stratified squamous epithelium, clear epithelial structures, and no obvious inflammatory cell infiltration. The skin surface of the rat I-degree pressure ulcer was relatively normal with clear epithelial structures and no obvious inflammatory cell infiltration. The skin surface of the rat II-degree pressure ulcer was defective and there was a small amount of inflammatory cell infiltration in the epithelial tissue. The skin surface of the rat III-degree pressure ulcer was abnormal with thin stratified squamous epithelium, in comparison with normal group and the surrounding tissues. The epithelial structures were not clear with reduced cellular levels and eventual disappearance of the stratified structure. Part of the epithelial structures was defective and more inflammatory cell infiltration was observed in the dermis (Fig. 2).
mRNA Expressions of VEGF and bFGF in Rats with Normal Skin and Pressure Ulcers Tissue
The amplification plots and melting curves of the target genes VEGF, bFGF, and the reference gene β-actin showed that the PCR amplification efficiencies of VEGF, bFGF, and β-actin were constant and have reached the plateau. The corresponding threshold cycle (Ct) values were stable with good reproducibility. The melting curves showed a single peak, demonstrating specific peaks at 84.54°C, 84.50°C, and 87.38°C, respectively. The primers were specific and no nonspecific primers were generated. Therefore, quantitative real-time PCR was performed for target genes VEGF and bFGF.
The mRNA levels of VEGF and bFGF in the tissues of rat I-, II-, and III-degree pressure ulcer, the surrounding tissues, and normal skin tissue of the rats were compared (Fig. 3). The results were calculated as relative quantification with the 2-ΔΔCt method. The mRNA levels of VEGF in the tissues of rat with I- and II-degree pressure ulcer and the surrounding tissues were higher than that in the untreated group (P < 0.05). The mRNA expression of VEGF in the tissues of rat III-degree pressure ulcer was lower than the normal skin tissue and the surrounding tissues (P < 0.05). However, there was no statistical significance between normal skin tissue and surrounding tissues (P > 0.05).
The bFGF mRNA levels in the tissues of rat I-degree pressure ulcer and its surrounding tissues were higher than that in the untreated group(P < 0.05). The expression of bFGF mRNA in the tissues of rat II-degree pressure ulcer was lower than its surrounding tissues and the normal skin tissue (P < 0.05), but the bFGF expression in the surrounding tissues of II-degree pressure ulcer was higher than that of the untreated group (P < 0.05). The bFGF mRNA expression in the tissues of rat III-degree pressure ulcer was lower than its surrounding tissues and the untreated group (P < 0.05). However, there was no statistically significant difference between its surrounding tissues and the normal skin tissue (P > 0.05).
Protein Expressions of VEGF and bFGF in Rats with Normal Skin and Pressure Ulcers Tissue
The expression of VEGF and bFGF results are shown in Table 2. The VEGF expression in I- and II-degree pressure ulcer and its surrounding tissues of rats was higher than the normal skin (P < 0.05) whereas, the VEGF expression in the III-degree pressure ulcer tissues was lower than its surrounding tissues and the normal group (P < 0.05). The expression of bFGF in the tissues of rat I-degree pressure ulcer and the surrounding tissues was higher than the normal skin tissue (P < 0.05) whereas, the expression of bFGF in the II- and III-degree pressure ulcer tissues was lower than the surrounding tissues and the normal group (P < 0.05; Fig. 4).
Table 2. Expressions of VEGF and bFGF determined by Western blot
I degree pressure ulcer tissue
0.8313 ± 0.0263
0.7499 ± 0.0188
Tissue surrounding the I degree pressure ulcer
0.7532 ± 0.0146
0.7246 ± 0.0163
II degree pressure ulcer tissue
0.5237 ± 0.0182
0.3973 ± 0.0076
Tissue surrounding the II degree pressure ulcer
0.5992 ± 0.0125
0.5287 ± 0.0173
III degree pressure ulcer tissue
0.3415 ± 0.0134
0.2947 ± 0.0109
Tissue surrounding the III degree pressure ulcer
0.4515 ± 0.0182
0.4705 ± 0.0307
Correlation between VEGF and bFGF in Rats with Normal Skin and Pressure Ulcers Tissue
The expression of VEGF mRNA and bFGF mRNA was analyzed by bivariate correlation analysis and scatter plots (Fig. 5). A linear correlation was found to exist between the two variables. The Pearson's correlation coefficient analysis (r = 0.959, P < 0.05) showed the presence of a positive correlation between bFGF and VEGF.
Pressure ulcers are chronic skin ulcers commonly encountered in medical practice. Clinically, pressure ulcers can be divided into I-degree (pink red area of the skin phase), II-degree (inflammatory infiltration phase), and III-degree (ulceration phase). Pressure ulcers, once occurred, not only increased the patient's suffering and economic burden, but also affect the disease rehabilitation resulting in prolonged hospitalization (Hoff et al., 2012). In the present study, we utilized magnetic compression to establish a new pressure ulcer model in rat, and to investigate the roles of the angiogenesis factors in formation of skin ulceration. The histological findings of our study showed no changes in the control group, mild changes in the I-degree pressure ulcer tissues (clear epithelial structures and no obvious inflammatory cell infiltration), moderate changes in the II-degree pressure ulcer tissues (small amount of inflammatory cell infiltration in the epithelial tissue), and considerable damage in the III-degree pressure ulcer tissues (thin stratified squamous epithelium, no clear epithelial structures, and disappearance of the stratified structure). It has been proposed that the activation and infiltration of polymorphonuclear cells play an important part in reperfusion injury, which is accompanied by interaction with activated endothelial cells (Gefen, 2008). Our study showed that polymorphonuclear cells were infiltrating in the dermis. Therefore, our study will help to understand the underlying mechanisms involved in the development of pressure ulcers.
We evaluated the angiogenesis factors in the development process of pressure ulcers. Using the qPCR and Western blotting, we detected mRNA and protein expression of VEGF and bFGF in different degrees of pressure ulcers. The expression levels of VEGF and bFGF were shown to be first increase in early stages of pressure ulcer and then decrease in the III-degree pressure ulcer tissues, indicating that VEGF and bFGF may be involved in the early stages of pressure ulcers developing process. One study (Takayama et al., 2010) has shown the concentrations of VEGF165 and FGF-2 were obvious in the exudate and fibrinous sloughs of pressure ulcer patients after polyvinylidene film dressing by ELISA, demonstrating that the VEGF165 would contribute to the spontaneous healing of pressure ulcer patients. Previous study (Pufe et al., 2003) has showed VEGF expression in the granulation tissue of chronic sacral pressure ulcers, suggesting that pressure ulcer itself releases factors to promote wound healing. These studies showed that VEGF and bFGF are expressed in the chronic wound healing, but our study showed that VEGF and bFGF play an important role in the formation of pressure ulcers. It has been reported that the chronic wound is related to the rise of infiltration of inflammatory cells, which are helpful to reduce the production of cytokines, and finally delayed the formation of new skin. However, these cytokines can promote angiogenesis, poor granulation, keloid formation, and ascribe to decreased or increased accumulation of extracellular matrix (Kimura et al., 2011). Our study found that the higher expression levels of VEGF and bFGF in the surrounding tissues, compared to the pressure ulcer tissues, may facilitate the peripheral vascular tissue growth to the center of the pressure ulcer tissues and promote the healing of ulcers. According to our experimental findings, the speed of peripheral angiogenesis slowed down and even stopped with an increase in the degree of pressure ulcers and expansion of the necrotic tissue, leading to decreased expression levels of VEGF and bFGF.
Previous study had found that bFGF can promote angiogenesis in vitro and in vivo. It can increase some important angiogenic growth factors to inhibit endothelial cell apoptosis through the Bcl-2 pathway (Przybylski, 2009). We found that the expression of bFGF in the pressure ulcer tissues were lower than the surrounding tissues and the normal skin, but the expression levels of VEGF in the II-degree pressure ulcer tissues and the surrounding tissues was slightly higher, compared to the normal skin. Thus, the higher expression levels of bFGF in the surrounding tissues may partly contribute to the increased expression of VEGF in the pressure ulcer tissues. Moreover, the correlation analysis showed a positive relationship between VEGF and bFGF, suggesting that VEGF and bFGF may play a synergistic role in the angiogenic process. Some previous studies showed that sequential treatment with granulocyte macrophage colony stimulating factor (GM-CSF) and bFGF can effectively promote the healing of pressure ulcers (Robson et al., 2000). Our previous study (Xue Ling Wang, 2012) found that recombinant human epidermal growth factor (rhEGF) can significantly promote healing in the tissues of rat III-degree pressure ulcer. However, above studies only speculate that VEGF and bFGF can promote the healing of pressure ulcers.
In this work, we established the rat model of pressure ulcer, and detected the expression of VEGF and bFGF in different degrees of pressure ulcers in vivo. Our study found that VEGF and bFGF expressed in the pressure ulcer, the expression levels of VEGF and bFGF were shown to first increase in the early stage of pressure ulcer and then decrease in III-degree pressure ulcers, indicating that VEGF and bFGF may be involved in the early stages of pressure ulcers' developing process. VEGF and bFGF have synergistic effects on pressure ulcer formation. Based on our experimental results, we speculate that in early pressure ulcers (I- and II-degree pressure ulcers), under the stimulation of a variety of factors, there may be a compensatory increase in the secretion of VEGF, bFGF, and many other growth factors from the epithelial cells in order to protect the skin epithelial cells, and prevent further development of pressure ulcers. However, in III-degree pressure ulcers, the tissue ischemia, hypoxia, and other factors exceed the extent of body's compensatory mechanisms, so that the secretion of growth factors cannot be further improved or even decreased, resulting in the continuous development and difficult healing of pressure ulcers. Therefore, we believed that the application of recombinant proteins, genetic modification, and growth factors closely related to the pressure ulcers, such as VEGF and bFGF, could provide new effective treatment options for pressure ulcers.
The authors thank Dr. Liang Yong and Yao Jun for constructive comments on statistical analysis.