ILK-PI3K/AKT pathway participates in cutaneous wound contraction by regulating fibroblast migration and differentiation to myofibroblast.

The interactions between fibroblasts and the extracellular matrix in wound contraction are mainly mediated via integrin signaling. Integrin-linked kinase (ILK) is a key mediator in integrin signal transduction. We investigated the role of ILK in cutaneous wound contraction. We found that ILK was involved in cutaneous wound healing in rats, and ILK and PI3K/AKT inhibitors inhibited wound contraction and re-epithelialization, consequently delaying wound healing in vivo. Further, using in vitro studies, we demonstrated that ILK and PI3K/AKT inhibitors suppressed the contraction of fibroblast-populated collagen lattices, inhibited fibroblast migration, and interrupted the effect of TGF-ß1 on promoting alpha smooth muscle actin (α-SMA) expression in fibroblasts. When ILK expression was directly blocked by ILK small interfering RNA transfection, the migration and α-SMA expression of normal dermal fibroblasts were significantly suppressed as well. The data suggest that the ILK-PI3K/AKT signaling pathway mediates cutaneous wound contraction by regulating fibroblast migration and differentiation to myofibroblasts.

A prospective analysis of the efficacy of phase II autologous skin grafting on deep second­degree burns on the dorsum of the hand.

The aim of the present study was to investigate the possibility of reducing the damage to the donor site while preserving the functional recovery of the dorsum of the hand following burn injury. An attempt was made to analyze the effect of a phased surgery approach on inflammatory indicators. A two-phase treatment was administered on a total of 64 patients with deep second-degree burns on the dorsum of the hand who were admitted to Guangzhou Red Cross Hospital between January 2020 and March 2023. During phase I treatment, the wounds were covered with xenogeneic (porcine) skin, followed by the application of autologous thin intermediate thickness skin grafts for wound repair in phase II treatment 1 week later. The surgical results, complications, patient satisfaction and inflammatory response indicators were then analyzed. The mean wound healing time of these patients was found to be 21.94 days without complications. The mean survival rate was 98.66%, and the overall satisfaction score of the patients was high. Finally, the white blood cell, C-reactive protein and IL-6 levels of these patients were continuously decreased 2 days preoperatively and 2 days postoperatively in phase I, and 2 days preoperatively and 2 days postoperatively in phase II. In combination, the effect of phased autologous skin grafting in patients with severe second-degree burns on the dorsum of the hand was ideal, as it significantly reduced inflammatory response and was beneficial to the functional recovery of the hand. Therefore, phased autologous skin grafting is worthy of wider application.

Reconstructing a full-thickness cheek defect of electrical burn with a folding radial forearm flap.

A 42-year-old male patient sustained severe burns from 10-kV, high-voltage electricity on his right cheek. The wound on the right cheek was 12 × 10 cm and was connected to the oral cavity. The teeth, gums, and mandible were exposed. A left radial forearm free flap was designed according to the size and shape of the cheek wound. The superficial portion of the flap was 13 × 9.5 cm, and the lining portion of the flap, replacing the oral mucosa, was 6 × 5 cm. The end of the radial artery was anastomosed to the facial artery contralaterally through a subcutaneous tunnel under his jaw, because the ipsilateral facial artery had been injured. The end of the radial vein was anastomosed to the external jugular vein ipsilaterally. In conclusion, free folding radial forearm flaps could be a good alternative for reconstruction of full-thickness defects of the cheek.

[Role of integrin-linked kinase signaling pathway in skin lesions and wound healing in diabetic rats].

OBJECTIVE: To investigate the role of integrin-linked kinase (ILK) signaling pathway in the skin lesions and wound healing in diabetic rats. METHODS: Thirty-six SD rats were divided into diabetic wound group (D) and non-diabetic wound group (N) according to the random number table, with 18 rats in each group. 10 g/L streptozocin (60 mg/kg) was intraperitoneally injected in rats in group D, while the rats in group N were given same quantity of sodium citrate buffer. Two weeks after successful reproduction of diabetic model of rats in group D, two full-thickness skin of an area of 2 cm × 2 cm was resected on both sides of back of rats in the two groups. Wounds of three rats of each group were photographed and examined on post injury day (PID) 1, 3, 7, 10, 14, and 21, and the wound healing rates were calculated. The non-injured skin and wound tissue (central part) on back of three rats of the rest 15 rats in the two groups were harvested on PID 3, 7, 10, 14, and 21, respectively. Morphology of the non-injured skin tissue was observed with HE staining, and the thickness of full-thickness skin and epidermis were measured. The mRNA expression levels of ILK, protein kinase B (Akt), and glycogen synthase kinase-3ß (GSK-3ß) in non-injured skin tissue were determined with real-time fluorescent quantitative RT-PCR. The protein expression levels of ILK, Akt, phosphorylated Akt, GSK-3ß, and phosphorylated GSK-3ß in non-injured skin tissue, and ILK, phosphorylated Akt in wound tissue were assessed with Western blotting. Data were processed with two independent-sample t test, one-way analysis of variance, SNK test and analysis of variance of factorial design. RESULTS: (1) After injury, the wound scabs of rats in group N were dry, and red granulation tissue with no excretion were seen when the scabs fell off, and the wound healed fast. After injury, excretion under the wound scabs of rats in group D was seen, and the scabs easily fell off with exposure of pink granulation tissue with much excretion, and the wounds healed slowly. Except for PID 3, the wound healing rate of rats in group D was significantly lower than that in group N on other PIDs (with t values from 3.858 to 13.738, P<0.05 or P<0.01). (2) On PID 3, the hair follicles and blood vessels in the non-injured skin tissue of rats in group N were rich, and the epidermis was composed of stratified cells in form of basal cells and keratinocyte, and the hair follicles and blood vessels in the non-injured skin tissue of rats in group D were scarce, and the epidermis was nearly composed of one-layer of cells. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N was similar from PID 3 to 21, and the thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group D on PID 3 was respectively (1 074 ± 66) and (15.1 ± 3.8) µm, and they gradually thinned out to (785 ± 122) and (9.7 ± 2.1) µm on PID 21, respectively. The thickness of full-thickness skin and epidermis of non-injured skin tissue of rats in group N were significantly thicker than those in group D on each PID (with t values from 4.620 to 23.549, P values below 0.001). (3) From PID 3 to 21, the mRNA expression levels of ILK and Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values respectively 4.779 and 3.440, P values below 0.05), the mRNA expression levels of GSK-3ß in non-injured skin tissue of rats were similar in two groups (t=0.363, P>0.05). (4) From PID 3 to 21, the protein expression levels of ILK, Akt and phosphorylated Akt in non-injured skin tissue of rats in group D were significantly lower than those in group N (with t values from 2.630 to 6.209, P<0.05 or P<0.01); the protein expression levels of GSK-3ß in non-injured skin tissue of rats in two groups were similar (t=0.652, P>0.05); the protein expression level of phosphorylated GSK-3ß in non-injured skin tissue of rats in group D was significantly higher than that in group N (t=4.131, P<0.001). The protein expression levels of ILK in wound tissue of rats in two groups were similar on each PID (with t values from 0.381 to 2.440, P values above 0.05). Except for PID 3, the protein expression levels of phosphorylated Akt in wound tissue of rats in group N were significantly higher than that in group D on other PIDs (with t values from 4.091 to 20.555, P<0.05 or P<0.01). From PID 3 to 21, the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group N were similar (F=2.522, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=117.329, P<0.001); the protein expression levels of ILK in wound tissue and non-injured skin tissue of rats in group D were similar (F=1.337, P>0.05), and the protein expression level of phosphorylated Akt in wound tissue was significantly higher than that in non-injured skin tissue (F=184.120, P<0.001). CONCLUSIONS: The skin lesion of diabetic rats may be related to the declined expression levels of ILK, Akt and phosphorylated Akt in the ILK signaling pathway. The refractory healing of wound in diabetic rats may be related to the declined expression level of phosphorylated Akt.

[Treatment of extensive acute radiation burn and its complications].

This article reports the treatment of a patient suffered from acute radiation burn covering 41% TBSA, with deep partial-thickness and full-thickness injury, produced by exposure to a large-scale industrial electron accelerator. An open wound began to appear and enlarged gradually 10 weeks after the exposure. Serious wound infection with methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, pneumonia, respiratory failure, systemic inflammatory response syndrome, nephropathy and hypoproteinemia developed successively since 3 weeks after the wound formation. Skin grafts failed to survive, resulting in enlargement of the wound. After being treated with proper measures, including parenteral nutrition, respiratory support with a ventilator, appropriate antibiotics, steroid administration for nephropathy, deep debridement for wounds followed by skin grafting, the patient was cured and discharged after undergoing 15 operations in 500 days. The clinical condition of an extensive acute radiation burn is complicated. We should pay close attention to the changes in functions of organs, and strengthen the therapeutic strategies to support the function of organs to reduce the incidence of systemic complications. The control of the infection and the timely and effective repair of the wound are still the key points of the treatment of an extensive local radiation injury.

[Expression of integrin-linked kinase in fibroblasts of scar induced by cobalt chloride and its effect on cell proliferation].

OBJECTIVE: To explore the expression of integrin-linked kinase (ILK) in fibroblasts (Fbs) of scar induced by cobalt chloride (CoCl2) and its effect on cell proliferation. METHODS: The human hypertrophic scar Fbs of seven patients were isolated and cultured in vitro. Cells from the 5th to the 6th passages were used in the experiment. Six bottles of Fbs were obtained from each of the seven patients, and they were respectively cultured with DMEM nutrient solution containing CoCl2 in the concentration of 0, 50, 100, 150, 200, and 250 µmol/L for 24 h. The expression of ILK mRNA was determined with real-time fluorescence quantitative PCR. Fbs were stimulated by CoCl2 in the most suitable concentration (100 µmol/L) and the protein expression of ILK was determined 0, 1, 2, 4, 12, and 24 h after the stimulation. Then the Fbs were divided into control group (cultured with nutrient solution), negative control group (transfected with con-siRNA), and ILK siRNA group (transfected with ILK siRNA). They were cultured with nutrient solution containing CoCl2 in different concentrations 24 h after transfection, with 4 wells for each concentration in each group. The cell proliferation was detected by XTT assay. Data were processed with one-way analysis of variance (ANOVA) and ANOVA for repeated measurement, and LSD method was used in multiple comparisons. RESULTS: The expression level of ILK mRNA was highest in Fbs cultured with 100 µmol/L CoCl2 for 24 h, with significant difference compared with those of Fbs cultured with other concentrations of CoCl2 (F = 50.958, P < 0.001). The expression of ILK protein in Fbs cultured with 100 µmol/L CoCl2 for 1 h (0.243 ± 0.009) was lower than that cultured for 0 h (0.387 ± 0.017), and it started to increase from 2 h (0.361 ± 0.010), and exaggerated at 4 h (0.584 ± 0.028), 12 h (0.730 ± 0.029), and 24 h (0.785 ± 0.031). The expression levels of ILK protein at 1, 4, 12, 24 h were statistically different from that at 0 h (P values all below 0.05). XTT showed that cell proliferation level was highest in control group when cultured with 100 µmol/L CoCl2 (F = 488.026, P < 0.001), which decreased from 150 µmol/L. The cell proliferation level in control group cultured with 250 µmol/L CoCl2 was significantly lower than that with 0 µmol/L (P values all below 0.05). There was no significant change in cell proliferation in ILK siRNA group among different concentrations of CoCl2 (F = 2.542, P = 0.056). The cell proliferation level in ILK siRNA group was significantly lower than that in control group and negative control group (F = 2519.542, P < 0.001). CONCLUSIONS: ILK may be a key protein in response of hypoxia in Fbs. The mild hypoxia can stimulate the expression of ILK and promote the proliferation of Fbs, while severe hypoxia can reduce the expression of ILK and inhibit cell proliferation.

[The effects of intergrin-linked kinase on angiogenesis in hypertrophic scar].

OBJECTIVE: To investigate the effects and regulatory mechanism of ILK on angiogenesis in hypertrophic scar. METHODS: The human scar microvascular endothelial cells (HSMECs) were isolated from 6 patients' hypertrophic scar in vitro. The HSMECs with good condition in 2nd to 4th generation were selected as experimental objectives. (1) HSMECs were divided into the blank control group (treated with routine culture), negative control group (treated with only Lipofectamine 2000), LY294002 group (incubated with 50 nmol/L LY294002), ILK siRNA group (incubated with 20 nmol/L ILK siRNA). RT-PCR and Western Blot were used to detect the expression of ILK mRNA and its protein after transfecion for 48 h. (2) The digested HSMECs of four groups were resuspended with DMEM without serum and then seeded onto the upper compartment of transwell insert which contained complete medium in its lower compartment. The cell migration experiment was stopped in 10 h and then the migrated cells were counted to analyze the effects of different interventions on the migration ability of HSMECs. (3) The thawed ECMatrix was put into each well of pre-colled 48-well tissue culture plate, and then the plate was put into the incubator at 37 degrees C to make it to become gel. The HSMECs of four groups were seeded onto the surface of the ECMatrix gel and were put into incubator. Eight random view-fields per well should be valued by the sheet of pattern recognition about angiogenesis after 8 hours to evaluate the ability of angiogenesis in vitro between four groups. RESULTS: (1) The expression of ILK mRNA (ILK mRNA = 0.829 +/- 0.109, t = 13.151, P = 0.006) and protein (ILK protein = 0.096 +/- 0.049, t = 36.656, P = 0.000) were both inhibited obviously in ILK siRNA group compared with the blank control group (ILK mRNA = 0.829 +/- 0.109, ILK protein = 1). And, the expression of ILK in LY294002 group was slightly lower than that of black control group, but there was no statistical difference. (2) The number of migrated cells in ILK siRNA group (88.111 +/- 3.079) and LY294002 group (138. 667 +/- 2.404) were respectively lower than that in blank control group (322.333 +/- 3.712, P < 0. 05) in 10th hour. (3) Compared to blank control group (4.333 +/- 0.191), the ability of angiogenesis in vitro decreased significantly ILK siRNA group (2.625 +/- 0.125) and LY294002 group (3.125 +/- 0.250), in which, the vascular network structures were not formed perfectly in 8th hour (P < 0.05). CONCLUSIONS: The ability of HSMECs' migration and angiogenesis in vitro are inhibited significantly when the expression of ILK is down-regulated. It reveals that ILK may play an role in the regulation of scar angiogenesis.

[The effect of integrin-linked kinase on VEGF expression in fibroblasts from human hypertrophic scar].

OBJECTIVE: To explore the expression of integrin-linked kinase (ILK) and its effect on VEGF expression in fibroblasts from human hypertrophic scar. METHODS: Fibroblasts were isolated from hypertrophic scar of 8 patients and cultured in vitro. Then the cells were divided into three groups: (1) Cells were cultured only in DMEM containing 10% FCS in the control group; (2) Cells were transfected with empty plasmid in the empty plasmid group; (3) Cells were transfected with plasmid expressing ILKcDNA in the ILK cDNA plasmid transfection group. First, the expression of ILK and VEGF was observed by immunocytochemistry before and after ILK cDNA transfection. Second, ILK and VEGF mRNA expression was investigated by real-time PCR (RT-PCR). Third, the protein expression of ILK and VEGF was detected by Western blot. Finally, the protein level of VEGF in supernatant of fibroblasts was measured by ELISA. RESULTS: Before ILK cDNA transfection, the expression of ILK was positive and the VEGF expression was weak in cytoplasm of fibroblasts . After ILK cDNA transfection, both the expression of ILK and VEGF was enhanced. The level of VEGF mRNA was significantly higher in ILK cDNA transfection group (0.338 +/- 0.060) than that in control group (0.022 +/- 0.001) and empty plasmid group (0.028 +/- 0.005, P < 0.05). The level of VEGF protein was significantly higher in ILK cDNA transfection group (0.819 +/- 0.019) than that in control group (0.607 +/- 0.033) and empty plasmid group (0. 591 +/- 0.024, P<0. 05). Secretion of VEGF increased remarkably in ILK cDNA transfection group comparing with the other two groups (P < 0.05). CONCLUSIONS: ILK could up-regulate the VEGF mRNA and protein level in human scar fibroblasts. It may play an important role in the angiogenesis in hypertrophic scar.

[Expression of integrin-linked kinase in human hypertrophic scar and its relationship with angiogenesis].

OBJECTIVE: To explore the expression of integrin-linked kinase (ILK) in scar in different growth stages, as well as its relationship with angiogenesis. METHODS: (1) Fifteen burn patients with scar formation time shorter than 6 months, ranging from 6 to 12 months, and longer than 12 months were hospitalized from December 2009 to December 2010. They were divided into A, B, and C groups according to the scar formation time, with 5 patients in each group. Scar specimens were harvested for observation of ILK expression with immunohistochemistry method, and ILK mRNA expression with real time fluorescence quantitative RT-PCR. (2) Microvascular endothelial cells (MEC) were isolated from scar tissue in A group and cultured in vitro, and then they were purified by immunomagnetic beads and identified with coagulation factor VIII marked by immunofluorescence (fibroblasts from human normal skin were used as control). The cultured cells in logarithmic growth phase were divided into control group (cultured with M131 medium containing microvascular growth supplement), transfection 1 group (transfected with empty plasmid), and transfection 2 group (transfected with ILK cDNA plasmid) according to the random number table. After 24 hours, the expressions of ILK mRNA, Flt-1 mRNA, and KDR mRNA were determined with real time fluorescence quantitative RT-PCR. Data were processed with one-way analysis of variance. RESULTS: Immunohistochemical observation showed that ILK in A group mainly expressed in the basal layer cells of epidermis, cytoplasm of fibroblasts, and MEC in scar, while ILK in B group only distributed in the basal layer cells of epidermis, but ILK expression in C group was not obvious. The expression of ILK mRNA in A group (0.34 ± 0.16) was significantly higher than those in B and C groups (0.17 ± 0.06, 0.07 ± 0.13, F = 37.007, P = 0.000). MEC grew up showing cobble stone formation after purification. The expression of coagulation factor VIII was positive in cytoplasm of purified MEC, while that was negative in fibroblast of human normal skin. The expressions of ILK mRNA (57.807 ± 5.556), KDR mRNA (0.836 ± 0.014), and Flt-1 mRNA (0.162 ± 0.005) in transfection 2 group were higher than those in control and transfection 1 groups (0.018 ± 0.003, 0.028 ± 0.020, 0.023 ± 0.004 and 0.042 ± 0.005, 0.039 ± 0.007, 0.046 ± 0.003; F(ILK) = 87.110, F(KDR) = 11.241, F(Flt) = 18.199, with P values all below 0.01). CONCLUSIONS: ILK mainly expressed in scar tissue with formation time shorter than 6 months, and it may affect vascularization of scar by regulating gene expressions of KDR and Flt-1 in MEC, which plays an important role in early scar formation.