Expression of matrix metalloproteinase and its tissue inhibitor in haemangioma.

The action mechanism of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in the genesis, development and degeneration of haemangioma was investigated by detecting their expression in the tissue of haemangioma in different phases by using the immunohistochemistry. Fifty paraffin-embedded specimens of skin capillary haemangioma were collected, which were documented in the Department of Pathology, Renmin Hospital of Wuhan University from 2000 to 2006. All samples were stained by regular HE method, and proliferative cell nuclear antigen (PCNA) was tested by immunohistochemical S-P method. The samples were classified according to the Mulliken criteria and the expression pattern of PCNA. Immunohistochemical S-P method was applied to detect the expression of MMP-2 and TIMP-2 in proliferative and degenerative phases of cutaneous capillary haemangioma, and in normal skin tissues. In combination with the detection of the expression of factor VIII-related antigen, it was verified that in haemangioma tissues, the cells expressing MMP-2 and TIMP-2 were vascular endothelial cells. The MMP-2 and TIMP-2 expression was quantitatively analyzed by image analysis system (HPIAS-1000), and one-way ANOVA(107) and SNK(q) test were done to analyze average absorbance (A) and positive area rate of immunohistochemically positive particles by using SPSS11.5. The results showed: (1) Among 50 samples of haemangioma, there were 26 proliferative haemangiomas, and 24 degenerative haemangiomas, respectively; (2) The expression of MMP-2 was weak in normal vascular endothelial cells, cytoplasm of connective tissues and extracellular matrix around blood vessels. The expression of MMP-2 in proliferative group was significantly higher than in degenerative group and control group (normal skin) (P<0.05), but there was no statistically significant difference between the latter two groups; (3) TIMP-2 was highly expressed in normal tissues, degenerative vascular endothelial cells, cytoplasm of connective tissues and extracellular matrix around blood vessels. The expression level of TIMP-2 in proliferative phase was significantly lower than in degenerative phase (P<0.05), and the expression of TIMP-2 in proliferative phase was significantly different from that in degenerative phase and normal tissues (P<0.05). It was concluded that in proliferative phase of haemangioma, MMP-2 may promote over-proliferation of endothelial cells of haemangioma, and in degenerative phase, TIMP-2 can inhibit the proliferation of endothelial cells of haemangioma. The two substances play important roles in the genesis, development and degeneration of haemangiomas.

Treatment of hemangioma by transfection of antisense VEGF gene.

The effect of transfection of antisense vascular endothelial growth factor (VEGF) gene on the growth of hemangioma was studied. A total of 49 cases of capillary hemangiomas of the skin were collected. Immunohistochemical method was used to detect the expression of PCNA in hemangioma tissues. According to the finding, 49 cases of hemangiomas fell into proliferating phase (27 cases) and involuting phase (22 cases) respectively. Another 5 cases of normal skin tissues adjacent to the tumor tissues served as control. Immunohistochemical staining was performed to detect the expression of VEGF in the tumor tissues and the normal tissues. The average absorbance (A) values and the average positive area rate of VEGF were measured by image analysis system (HPIAS-2000). Endothelial cells from the tumor tissues in proliferating phase were cultured. Eukaryotic expression vector was constructed by sub-cloning, and transfected into human hemangioma endothelial cells by using cation liposome as vector. The expression of VEGF mRNA and protein was detected by RT-PCR and indirect immunofluorescence assay (IFA), respectively, and the biological characteristics of the transfected endothelial cells were examined by MTT assay and flow cytometry (FCM) after transfection. Immunohistochemical results showed that the expression of VEGF in proliferating endothelial cells was remarkably higher than those in involuting endothelial cells and normal endothelial cells (P<0.01), but there was no significant difference in the expression of VEGF between involuting endothelial cells and normal ones (P>0.01). Electrophoresis and sequencing indicated that the eukaryotic expression vector containing antisense VEGF gene, i.e. pcDNA3.1-VEGF, was successfully constructed. After VEGF antisense RNA recombinant was transfected into hemangioma endothelial cells, RT-PCR revealed that the expression of VEGF mRNA in pcDNA-VEGF (V) group and blank group was obviously higher than that in pcDNA-VEGF (A) group, and that the expression of endogenous VEGF mRNA in pcDNA-VEGF (A) group was significantly inhibited. Immunohistochemical result demonstrated that, compared with blank group, there was statistically significant difference between pcDNA-VEGF (A) and pcDNA-VEGF (V) groups (P<0.01), but there was no significant difference between pcDNA-VEGF (V) group and blank group (P>0.05). The activity of endothelial cell proliferation was reduced significantly after transfection, and obvious apoptosis occurred in hemangioma endothelial cells after transfection of antisense VEGF. It was suggested that VEGF plays an important role in the pathological change of hemangiomas by promoting endothelial cell proliferation and angiogenesis. Antisense VEGF gene transfection could effectively inhibit the growth of hemanioma endothelial cells.

Treatment of Hemangioma by Transfection of Antisense VEGF Gene / 华中科技大学学报（医学）（英德文版）

nioma endothelial cells.

Expression of Matrix Metalloproteinase and Its Tissue Inhibitor in Haemangioma / 华中科技大学学报（医学）（英德文版）

The action mechanism of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in the genesis, development and degeneration of haemangioma was in-vestigated by detecting their expression in the tissue of haemangioma in different phases by using the immunohistochemistry. Fifty paraffin-embedded specimens of skin capillary haemangioma were col-lected, which were documented in the Department of Pathology, Renmin Hospital of Wuhan University from 2000 to 2006. All samples were stained by regular HE method, and proliferative cell nuclear anti-gen (PCNA) was tested by immunohistochemical S-P method, The samples were classified according to the Mulliken criteria and the expression pattern of PCNA. Immunohistochemical S-P method was ap-plied to detect the expression of MMP-2 and TIMP-2 in proliferative and degenerative phases of cuta-neous capillary haemangioma, and in normal skin tissues. In combination with the detection of the ex-pression of factor Ⅷ-related antigen, it was verified that in haemangioma tissues, the cells expressing MMP-2 and TIMP-2 were vascular endothelial cells. The MMP-2 and TIMP-2 expression was quantita-tively analyzed by image analysis system (HPIAS-1000), and one-way ANOVA(107) and SNK(q) test were done to analyze average absorbance (A) and positive area rate of immunohistochemically positive particles by using SPSS11.5. The results showed: (1) Among 50 samples of haemangioma, there were 26 proliferative haemangiomas, and 24 degenerative haemangiomas, respectively; (2) The expression of MMP-2 was weak in normal vascular endothelial cells, cytoplasm of connective tissues and extracellu-lar matrix around blood vessels. The expression of MMP-2 in proliferative group was significantly higher than in degenerative group and control group (normal skin) (P<0.05), but there was no statisti-cally significant difference between the latter two groups; (3) TIMP-2 was highly expressed in normal tissues, degenerative vascular endothelial cells, cytoplasm of connective tissues and extracellular matrix around blood vessels. The expression level of TIMP-2 in proliferative phase was significantly lower than in degenerative phase (P<0.05), and the expression of TIMP-2 in proliferative phase was signifi-cantly different from that in degenerative phase and normal tissues (P<0.05). It was concluded that in proliferative phase of haemangioma, MMP-2 may promote over-proliferation of endothelial cells of haemangioma, and in degenerative phase, TIMP-2 can inhibit the proliferation of endothelial cells of haemangioma. The two substances play important roles in the genesis, development and degeneration of haemangiomas.

[Expression and clinical significance of cyclin H and CDK7 in human hemangiomas].

OBJECTIVE: To investigate the expression and clinical significance of cyclin H and cyclin-dependent kinase 7 (CDK7) in human hemangiomas. METHODS: Immunohistochemistry technique was used to measure the expression of cyclin H and CDK7 proteins in proliferative, involuting hemangiomas and normal skin tissues. Immunohistochemical technique for factor VIII-related antigen was used to prove that the cells which expressed cyclin H and CDK7 were endothelial cells. Average optical density and positive area of the expression of cyclin H and CDK7 proteins in proliferative, involuting hemangiomas and normal skin tissues were measured by image analysis (HPIAS-1000). RESULTS: The expression of cyclin H and CDK7 protein in proliferating hemangiomas was significantly higher than that in involuting hemangiomas and normal skin tissues (P < 0.01). But no significant difference was found in the expression of cyclin H and CDK7 protein between involuting hemangiomas and normal skin tissues (P > 0.05). CONCLUSIONS: cyclin H and CDK7 may play an important role in the generation and development of human hemangiomas.

Angiogenic effect of intercellular adhesion molecule-1.

UNLABELLED: In order to investigate the angiogenic effect of intercellular adhesion molecule-1 (ICAM-1), two parts of experiment were performed. Chick embryo chorioallantoic membrane (CAM) assay was used for in vivo angiogenic research. The chick embryos were divided into 4 groups: ICAM-1 group (divided into 3 subgroups, I, II and III) for screening the angiogenic effect of ICAM-1 by adding different concentrations of ICAM-1 (0.1, 0.2 and 0.3 microg/microL) 5 microL into the chick embryo CAMs on the day 10 after incubation for every subgroup; Anti-ICAM-1 group A (divided into 2 subgroups, I and II) by adding different concentrations of Anti-ICAM-1 (1:100, 1:50) 5 microL into the chick embryo CAMs on the day 10 after incubation for every subgroup to evaluate the effect of ICAM-1 on the survival of microvessels through observing whether Anti-ICAM-1 could induce involution of the microvessels on CAMs; Anti-ICAM-1 group B (divided into 2 subgroups, I and II) by adding different concentrations of Anti-ICAM-1 (1:100, 1:50) 5 microL into the chick embryo CAMs on the day 6 after incubation for every subgroup to evaluate whether ICAM-1 involved in embryonic angiogenesis through observing the growth of microvessels on CAMs; CONTROL GROUP: ICAM-1 or Anti-ICAM-1 was substituted by PBS 5 muL on the day 10 or day 6 after incubation. Three days later, the CAMs were photographed in vivo, excised, sectioned and the number of microvessels was counted. In ICAM-1 group, there was increased number of microvessels arranged radially with "spoked-wheel" pattern around the gelatin sponges. The new microvessels growing perpendicularly to gelatin sponges were observed. The number of the microvessels growing in the CAM mesenchymes around the sponges in 3 subgroups was higher than that in control group (P<0.01), however, there was no significant difference among the 3 subgroups (P>0.05). In anti-ICAM-1 group A, the radially arranged microvessels were very unclear around the sponges contrast to that of ICAM-1 group. Few new microvessels were detected in the center of the sponges. The number of the microvessels growing in the CAM mesenchymes around the sponges in subgroup II was lower than that in control group (P<0.01). There was no significant difference in the number of the microvessels around the sponges between subgroup I and control group (P>0.05). In anti-ICAM-1 group B, the radially arranged microvessels were very unclear around the sponges contrast to that of control group. New microvessels were very scarce in the center of the sponges. The number of the microvessels growing in the CAM mesenchymes around the sponges in the 2 subgroups were less than that in control group (P<0.01), and there was significant difference between the 2 subgroups (P<0.05). It was suggested that ICAM-1 could induce angiogenesis and support the survival of microvessels, and ICAM-1 was involved in embryonic angiogenesis.

[Reconstruction of the remnant penis: a 52-case report].

OBJECTIVE: To explore the effects of the combined method of abdominal axial flap transposition and penile elongation for the treatment of the remnant penis. METHODS: Fifty-two cases of the remnant penis treated with the combined method from 1984 April to February 2004 were analyzed retrospectively. Follow-up ranged from 0.5 to 20 years postoperatively. RESULTS: The lengths (both in normal and erectile conditions) and the circumferences of the penis gained after operation were (5.6 +/- 1.4) cm, (6.8 +/- 2.5 cm and (6.9 +/- 2.3) cm respectively. The recovery rates of the sensory function were 94.2% and 100% in the glans (immediately and 3 months after operation) and 32.7%, 51.9% and 75% in the flap area (3, 6 and 12 months postoperatively). The two-point distinguishing sense in the glans and the flap area was (5.1 +/- 0.9) mm and(7.9 +/- 1.3) mm 5 years after operation. Early complications included distant flap necrosis (3 cases), disruption of the wound (2 cases), part necrosis of the skin graft in the abdominal wall (2 cases) and poor contours occurred in 4 cases in the later period because of the thickness of the flaps. All of them were corrected with satisfactory results. CONCLUSION: The combined method of abdominal axial flap transposition and penile elongation was recommendable for the treatment of the remnant penis because of its positive effects and less complications.

[The expression of p73 and c-fos protein in hemangioma and its significance].

OBJECTIVE: To investigate the expression of p73 and c-fos protein and its significance in the development of children hemangioma. METHODS: The quantitative expressions of p73 and c-fos protein in hemangioma and normal skin were detected by immunohistochemistry. RESULTS: The expressions of p73 and c-fos protein were strong in proliferative hemangioma while they were very weak in involutional hemangioma and normal skin. There were significant differences between the proliferative and involutional hemangioma or the normal skin in the expressions of p73 and c-fos (P < 0.01). No statistical significances of p73 or c-fos P73 expressions were observed between involutional hemangioma and normal skin (P > 0.05). CONCLUSIONS: P73 and c-fos may play an important role in the development and involution of skin hemangioma.

[Investigation of the influence of nitric oxide on the survival of a random pattern skin flap].

OBJECTIVE: To investigate the changes of nitric oxide concentration in the distal portion of a random pattern skin flap and the influence of the exogenous L-arginine on the survival of the random pattern skin flap. METHODS: A random pattern skin flap (7 cm x 2 cm) was cranially designed and elevated on the back of a Wistar rat. An image analysis technology was used to evaluate the survival rate of the skin flap, while a biochemistry method was used to test the concentrations of the NO in the tissue. RESULTS: The survival area of the flap in the L-arginine-treated group was significantly enlarged (63.83 +/- 5.13)% (P < 0.01) in seven days postoperatively, compared with the control group (43.26 +/- 2.86)%. The NO concentration in the tissue was no statistic difference between all of the groups immediately after the operation (P > 0.05). But, the NO concentration in the control was decreasing at the beginning and then increasing slightly to reach the high level in 12 hours after the operation. It was thereafter slumped down to the baseline in 72 hours after the surgery. Although the changes in the L-arginine-treated group were quite similar to the control excepting of the extent, the NO concentration was kept in a higher level in the sequential time after the operation (P < 0.01, P < 0.01, P < 0.05 and P < 0.01). CONCLUSIONS: The NO concentration in skin flap tissue after the elevation was going up slightly for a short time. The exogenous L-arginine could promote the NO concentration in the random pattern skin flap to protect it from ischemic injury.

Prevention and treatment of postoperative complications of the penile elongation.

To explore the causes of the postoperative complications of the penile elongation and the measures to prevent them in order to raise the success rate of the penile elongation. 1,000 patients who had received the penile elongation were reviewed and analyzed for the causes of postoperative complications, and the measures of prevention and treatment were discussed. Our results showed that, of the 1,000 cases, 64 had the postoperative complications, including 20 cases of edema of prepuce, 15 cases of flap necrosis, 12 hematoma, 9 infections, and 8 cases of fat and clumsy penis. It is concluded that correct operative manipulation, strict aseptic measures and necessary postoperative care and management could avoid or reduce the postoperative complications. When complications happened, a satisfactory result can be achieved with timely and correct treatment in the majority of the patients.

[Correlation between tissue ultrastructure changes of small testis and sex hormone].

OBJECTIVES: To investigate the tissue ultrastructure changes of small testis and sex hormone and their correlation. METHODS: The patients were divided into small tests (n = 8) and control group(n = 12). FSH, LH, T were determined by radioimmunassay. Diameter and wall thickness of convoluted seminiferous tubule were investigated with light microscope and electro microscopy on small testis tissue morphology and ultrastructure. RESULTS: FSH, LH, T of small testis and control group were (21.05 +/- 9.15) IU/L vs (6.74 +/- 3.52) IU/L, (22.88 +/- 6.25) IU/L vs (6.60 +/- 1.48) IU/L and (0.30 +/- 0.04) nmol/L vs (17.55 +/- 9.25) nmol/L, respectively. Seminiferous tubule diameter and wall thickness were(37.33 +/- 6.80) microns vs (198.46 +/- 29.84) microns and (10.30 +/- 1.82) microns vs (2.95 +/- 0.20) microns. Small testis tissue ultrastructure changed significantly. CONCLUSIONS: Pathologic changes of small testis tissue in many parts such as seminiferous tubule, germinal epithelium, Sertoli cell, Leydig cell, limiting membrance and blood vessel may relate with genetics and immunoreaction.