Loss of vascular endothelial growth factor a activity in murine epidermal keratinocytes delays wound healing and inhibits tumor formation

The angiogenic cytokine vascular endothelial growth factor (VEGF)-A plays a central role in both wound healing and tumor growth. In the skin, epidermal keratinocytes are a major source of this growth factor. To study the contribution of keratinocyte-derived VEGF-A to these angiogenesis-dependent processes, we generated mice in which this cytokine was inactivated specifically in keratin 5-expressing tissues. The mutant mice were macroscopically normal, and the skin capillary system was well established, demonstrating that keratinocyte-derived VEGF-A is not essential for angiogenesis in the skin during embryonic development. However, healing of full-thickness wounds in adult animals was appreciably delayed compared with controls, with retarded crust shedding and the appearance of a blood vessel-free zone underneath the newly formed epidermis. When 9,12-dimethyl 1,2-benzanthracene was applied as both tumor initiator and promoter, a total of 143 papillomas developed in 20 of 23 (87%) of control mice. In contrast, only three papillomas arose in 2 of 17 (12%) of the mutant mice, whereas the rest merely displayed epidermal thickening and parakeratosis. Mutant mice also developed only 2 squamous cell carcinomas, whereas 11 carcinomas were found in seven of the control animals. These data demonstrate that whereas keratinocyte-derived VEGF-A is dispensable for skin vascularization under physiological conditions, it plays an important albeit nonessential role during epidermal wound healing and is crucial for the development of 9,12-dimethyl 1,2-benzanthracene-induced epithelial skin tumors.     

国产虫草素对Hela细胞MMP-9、TIMP-1及TIMP-1 mRNA表达的调节

目的：观察国产虫草素对人宫颈癌Hela细胞生长,MMP-9,TIMP-1及TIMP-1mRNA表达的影响。方法：采用MTr法观察不同浓度的虫草素溶液（0,1,2,4mg／ml）对Hela细胞增殖抑制作用。人MMP-9／TIMP-1ELISA试剂盒及RT—PCR技术检测经不同浓度虫草素溶液（0,1,2,4mg／ml）处理后不同时间点（12,24,48h）Hela细胞MMP-9／TIMP-1及TIMP-1 mRNA表达的影响。结果：国产虫草素以剂量依赖方式抑制Hela细胞生长。不同浓度虫草素处理不同时问的Hela细胞分泌MMP-9呈轻度剂量依赖抑制方式,但未见统计学差异（P〉0．05）。可上调TIMP-1及其mRNA的表达,且呈明显剂量依赖方式,统计学差异显著（P〈0．05）,尤其在24h TIMP-1 mRNA的表达及蛋白分泌达最高峰。结论：国产虫草素可以以剂量依赖方式抑制肿瘤细胞的生长;可以通过上调TIMP—1 mRNA及蛋白的表达发挥潜在的抗肿瘤细胞侵润转移的作用,为国产新型抗肿瘤药物的临床研发及应用提供实验室依据。     

In situ gene expression and localization of metalloproteinases MMP1, MMP2, MMP3, MMP9, and their inhibitors TIMP1 and TIMP2 in human renal cell carcinoma

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) play a major role in the maintenance of extracellular matrix homeostasis. Alterations of MMP and TIMP expressions have been found in several malignant tumour entities. In this study the expression pattern of MMP1, MMP2, MMP3, MMP9, and their inhibitors TIMP1, and TIMP2 were investigated at mRNA and protein levels in human renal cell carcinoma (RCC). Formalin fixed paraffin embedded tumour samples of 10 patients and adjacent non-malignant controls were analysed by radioactive labelled riboprobe in situ hybridisation (isH) and immunohistochemistry. The slides were evaluated semiquantitatively. MMP1-antigen was strongly expressed in tumour epithelium with moderate stroma expression in one case. The gelatinases MMP2 and MMP9 showed moderate to strong signals in tumour epithelial cells at the mRNA and protein level, while the expression in tumour stroma was moderate. MMP3-mRNA and -antigen were expressed moderately to strong in tumour epithelium and focally in stroma cells. mRNA or TIMP1- and TIMP2-mRNA and -antigen were also predominantly expressed in tumour epithelium; only few samples showed positive expression in stroma cells. mRNA expression could be generally correlated to the protein expression in our study group, except for MMP1 (mRNA expression was only expressed in two cases). We found a pronounced expression for the gelatinases MMP2 and MMP9 and for MMP3 in RCC at the mRNA and protein level. The expression of TIMP1 and TIMP2 appears also to be relevant in RCC. Due to the small sample size further investigations need to be done to prove a statistical significant correlation between the MMP/TIMP expression and clinicopathological parameters.     

Effect of radiation and cell implantation on wound healing in a rat model

BACKGROUND AND OBJECTIVES: Having shown that intra-dermal injection of fibroblasts decreases the effect of radiation on healing of superficial wounds, we now test the effect of fibroblasts and syngeneic marrow stromal cells on irradiated deep and superficial wounds. METHODS: Wistar rats received bilateral buttock irradiation followed by partial excision of the gluteus muscle bilaterally. In Protocol 1, one irradiated wound was treated with 1.2 x 10(7) autologous cells injected intra-dermally. In Protocol 2, the experimental side was treated with a fibrin and autologous cell implant (1.2 x 10(7) cells). Twenty-one days later, wound mechanical characteristics were tested. In Protocol 3, the effect of pooled marrow stromal cells on healing of superficial irradiated wounds in Lewis rats was similarly tested. RESULTS: The fibrin-fibroblast implant (Protocol 2) had no effect on wound mechanics. Superficial injection of fibroblasts (Protocol 1) significantly improved wound breaking strength when compared to the control group (mean +/- SEM, breaking strength: treated 504.6 +/- 37.0 g vs. control 353.4 +/- 35.2 g, P = 0.005). The dermal injection of marrow stromal cells also resulted in marked increases in breaking strength (mean +/- SEM, breaking strength: treated 338.5 +/- 39.9 g vs. control 187.1 +/- 12.0 g, P < 0.01). In both Protocols 1 and 3, ultimate tensile strength and toughness were increased in the side receiving cell transplantation. CONCLUSIONS: Cell implantation holds promise for decreasing the effect of radiation on healing of irradiated wounds.     

基质金属蛋白酶及其抑制因子与神经母细胞瘤侵袭转移的关系

目的　探讨基质金属蛋白酶 (MMP) 2、MMP 9及其抑制因子 (TIMP) 2、TIMP 1与神经母细胞瘤侵袭转移的关系。方法　应用免疫组化SABC法检测 35例神经母细胞瘤标本中MMP 2、MMP 9及TIMP 2、TIMP 1的表达情况 ,并比较其在无转移、局部转移及远处转移患者肿瘤组织中的表达程度。结果　在有局部转移的B组和远处转移的C组患儿中 ,MMP 2高表达者分别为 9例和 10例 ,相对于病灶局限的A组明显升高 (P <0 .0 5 )。MMP 9表达仅在A组和C组间有统计学意义 (P <0 .0 5 )。预后良好的A组TIMP 2的阳性表达程度最高 ,随着肿瘤进展表达程度逐渐减弱。各组间TIMP 1表达差异无统计学意义 (P =0 .36 5 2 )。结论　MMP 2、MMP 9的表达与神经母细胞瘤的侵袭转移有关 ,可促进肿瘤进展 ,而TIMP 2则抑制肿瘤细胞的侵袭转移。     

检测基质金属蛋白酶及其抑制物对判断膀胱癌侵袭和转移的意义

目的 :研究基质金属蛋白酶 (MMPs)中MMP 2、MMP 9及MMP 9抑制物TIMP 1在膀胱移行细胞癌中的表达情况及其在判断膀胱癌侵袭转移中的作用。方法 :应用免疫组化LSAB法检测 90例膀胱移行细胞癌 (transitionalcellcarcinomaofbladder,TCCB)组织中MMP 2、MMP 9和TIMP 1蛋白表达水平。结果 :膀胱癌组织中MMP 2、MMP 9和TIMP 1蛋白表达显著高于正常膀胱黏膜。浸润性膀胱癌组织中阳性表达显著高于表浅性膀胱癌组织。阳性表达细胞在肿瘤 -间质界面较多。与病理组织学分级、分期均呈正相关 ;与肿瘤的复发和转移呈正相关。结论 :MMP 2、MMP 9和TIMP 1参与膀胱癌侵袭转移 ,在膀胱移行细胞癌中的表达具有预后价值。     

SU5416 delays wound healing through inhibition of TGF-beta 1 activation

Angiogenesis, development of new blood vessels, is essential for wound healing and tumor growth. A potentially important side effect of anti-angiogenic therapy can be delayed wound healing. In this study we address this issue by using a novel in vivo method utilizing fibrin containing dual porous plexiglass chambers (Fibrin Z-Chambers; F-ZC) to investigate wound healing in rats administered with SU5416 (inhibitor of Flk-1 and Flt-1, at 20 mg/kg i.p.). SU5416 treated F-ZCs developed 45% less granulation tissue (p = 0.0076) and showed a 10% reduction in microvessel density (p = 0.0009) than controls treated with drug carrier alone. The granulation tissue showed distinctly decreased collagen deposition (p = 0.0006) in SU5416 treated animals that was associated with 90% reduction in active TGF-beta 1 level. We found that tissue transglutaminase (TG), a cross-linking enzyme involved in TGF-beta 1 activation and matrix stabilization, was inhibited by SU5416. These results suggest that SU5416 delays wound healing by reducing matrix synthesis and stabilization through inhibition of TGF-beta 1 activation. This study was made feasible via the development of a unique method to study anti-angiogenic compounds that provides highly reproducible and quantitative results. Further studies are needed to evaluate in vivo whether anti-angiogenic agents alter wound healing.     

Increased expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinases 1 and 2 correlate with poor prognostic variables in renal cell carcinoma.

PURPOSE: Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix and are implicated in tissue remodeling and tumor infiltration. Tissue inhibitor of metalloproteinases (TIMPs) inhibit enzymes of the MMP family and preserve stromal integrity, thus inhibiting tumor migration. Although numerous studies on several human carcinomas have demonstrated a role for MMPs in tumor metastasis and patient survival, their prognostic role in patients with renal cell carcinoma (RCC) has not been well defined. More importantly, the recently documented paradoxical functions of TIMPs have not been characterized in these neoplasms. EXPERIMENTAL DESIGN: Five-microm, formalin-fixed, paraffin-embedded tissue sections from 153 RCCs were immunostained using specific antibodies against MMP2, MMP9, (Novocastra, Burlingame, CA) TIMP1, and TIMP2 (NeoMarkers, Fremont, CA) proteins. Immunostaining was semiquantitatively scored based on intensity and distribution, and results were correlated with histological and prognostic variables. RESULTS: The rates of increased expression of MMPs and TIMPs in RCC were as follows: MMP2, 67%; MMP9, 43%; TIMP1, 46%; and TIMP2, 73%. Each of these four markers individually correlated with histological tumor type with a vast majority of papillary and sarcomatoid RCCs expressing these proteins as compared with clear cell tumors (P range, 0.0001-0.003). Significant coexpression of MMPs and TIMPs was observed (P = 0.0001). Increased immunoreactivity for each of these proteins correlated with high tumor grade (P range, 0.0001-0.01). On univariate analysis, expression of each of these markers correlated with shortened survival (P range, 0.004-0.05). On multivariate analysis, including tumor grade, stage, and all four markers, only advanced stage (P = 0.047) and increased TIMP1 expression (P = 0.007) independently predicted shortened survival. CONCLUSION: Increased expression of MMP2, MMP9, TIMP1, and TIMP2 proteins in RCCs correlate with poor prognostic variables including shortened patient survival. The paradoxical poor prognostic implication of TIMP overexpression complements the recently documented dual function of TIMPs and warrants further investigation.     

Activation of MMP‐9 by membrane type‐1 MMP/MMP‐2 axis stimulates tumor metastasis

An artificial receptor for proMMP‐9 was created by fusing tissue inhibitor of MMP‐1 (TIMP‐1) with type II transmembrane mosaic serine protease (MSP‐T1). Expression of MSP‐T1 in 293T cells induced binding of proMMP‐9, which was processed by MMP‐2 activated by membrane type 1 MMP (MT1‐MMP). HT1080 cells transfected with the MSP‐T1 gene produced activated MMP‐9 in collagen gel, and addition of proMMP‐2 to the culture augmented it, which resulted in intensive collagen digestion. These cells metastasized into chick embryonic liver more than control cells. Treatment of HT1080 cells with concanavalin A in the presence of exogenous proMMP‐2 induced activation of not only proMMP‐2 but also proMMP‐9. Knockdown of MT1‐MMP or TIMP‐2 expression with siRNA suppressed activation of both proMMP‐2 and proMMP‐9. Transfection of TIMP‐1 siRNA suppressed cell binding and activation of proMMP‐9, but not proMMP‐2 activation. Knockdown of a disintegrin and metalloproteinase 10 (ADAM10) expression reduced cell binding and processing of proMMP‐9. These results suggest that proMMP‐9, which binds to a receptor complex containing TIMP‐1 and ADAM10, is activated by the MT1‐MMP/MMP‐2 axis, and MMP‐9 thus activated stimulates cellular proteolysis and metastasis.     

胰腺癌和非癌变组织中MMP1和TIMP1表达及其意义

目的研究胰腺癌和非癌病变组织中MMP1和TIMP1表达特征及临床病理意义.方法常规石蜡包埋切片行MMP1和TIMP1多克隆抗体ABC免疫组化法.结果 53例胰腺癌MMP1阳性26例(49%),TIMP1阳性28例(53%);20例慢性胰腺炎导管上皮MMP1阳性1例(5%),TIMP1阳性20例(100%);25例癌旁上皮MMP1阳性3例(12%),TIMP1阳性23例(92%).胰腺癌MMP1阳性率明显高于慢性胰腺炎和癌旁上皮,而TIMP1阳性率明显低于慢性胰腺炎和癌旁上皮,差异均有显著性(P<0.005).高分化腺癌和未转移癌MMP1阳性率明显低于低分化腺癌和转移癌,差异均有显著性(P<0.05),但TIMP1表达则相反.胰腺癌中MMP1与TIMP1表达呈密切相关(P<0.005).结论 MMP1和TIMP1表达与胰腺癌发生发展、生物学行为和转移发生均有密切关系,两者均为重要生物学标记物.     

MiR-886-5p Inhibition Inhibits Growth and Induces Apoptosis of MCF7 Cells

Background and Aims: To explore the molecular mechanisms of miR-886-5p in breast cancer., we examinedroles in inhibiting growth and migration of MCF-7 cells. Methods: MiR-886-5p mimics and inhibitors were usedto express or inhibit MiR-886-5p, respectively, and MTT and clone formation assays were used to determine thesurvival and proliferation. Hoechst 33342/ PI double staining was applied to detect apoptosis. The expressionof caspase-3, caspase-8, caspase-9, MT1-MMP, VEGF-C and VEGF-D was detected by Western blotting, andthe levels of MMP2 and MMP9 secreted from MCF-7 cells were assessed by ELISA. MCF-7 cell migration wasdetermined by wound healing and Transwell assays. Results: We found that the growth of MCF-7 cells wasinhibited upon decreasing miR-886-5p levels. Inhibiting miR-866-5p also significantly induced apoptosis anddecreased the migratory capacity of these cells. The expression of VEGF-C, VEGF-D, MT1-MMP, MMP2, andMMP9 was also found to be decreased as compared to controls. Conclusions: Our data show that downregulationof miR-886-5p expression in MCF-7 cells could significantly inhibit cell growth and migration. This might implythat inhibiting miR-886-5p could be a therapeutic strategy in breast cancer.     

Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues

Extracellular proteases of the matrix metalloproteinase (MMP) and serine protease families participate in many aspects of tumour growth and metastasis. Using quantitative real-time RT-PCR analysis, we have undertaken a comprehensive survey of the expression of these enzymes and of their natural inhibitors in 44 cases of human prostate cancer and 23 benign prostate specimens. We found increased expression of MMP10, 15, 24, 25 and 26, urokinase plasminogen activator-receptor (uPAR) and plasminogen activator inhibitor-1 (PAI1), and the newly characterised serine proteases hepsin and matriptase-1 (MTSP1) in malignant tissue compared to benign prostate tissue. In contrast, there was significantly decreased expression of MMP2 and MMP23, maspin, and the protease inhibitors tissue inhibitor of metalloproteinase 3 (TIMP3), TIMP4 and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) in the cancer specimens. The expression of MMP15 and MMP26 correlated positively with Gleason score, whereas TIMP3, TIMP4 and RECK expression correlated negatively with Gleason score. The cellular localisation of the expression of the deregulated genes was evaluated using primary malignant epithelial and stromal cell cultures derived from radical prostatectomy specimens. MMP10 and 25, hepsin, MTSP1 and maspin showed predominantly epithelial expression, whereas TIMP 3 and 4, RECK, MMP2 and 23, uPAR and PAI1 were produced primarily by stromal cells. These data provide the first comprehensive and quantitative analysis of the expression and localisation of MMPs and their inhibitors in human prostate cancer, leading to the identification of several genes involved in proteolysis as potential prognostic indicators, in particular hepsin, MTSP1, MMP26, PAI1, uPAR, MMP15, TIMP3, TIMP4, maspin and RECK.     

基质金属蛋白酶MMP-9、2及其抑制物TIMP-1、2在侵袭性垂体腺瘤生物学行为中的意义

背景与目的:通常垂体腺瘤在组织学上属良性肿瘤,但仍有部分肿瘤侵犯周围组织,形成侵袭性肿瘤,垂体腺瘤侵袭的生物学机制尚不清楚。垂体腺瘤血管丰富,而血管形成与肿瘤侵袭都需要细胞外基质成份的降解和细胞移动,这一过程中基质金属蛋白酶(matrixmetalloproteinases,MMP)和它们的天然抑制物-组织金属蛋白酶抑制剂(tissueinhibitorsofmetalloproteinases,TIMP)有可能起着关键作用。本研究拟通过检测MMP-9和MMP-2及其抑制因子TIMP-1和TIMP-2在非侵袭性垂体瘤和侵袭性垂体瘤中的表达,探讨垂体腺瘤的侵袭机制。方法:收集垂体瘤手术切除标本61例,分为侵袭组(49例)和非侵袭组(12例)。采用免疫组织化学SP法检测,并根据阳性细胞占肿瘤细胞总数的比率进行半定量非参数检验分析。结果:MMP-9、TIMP-1、MMP-2和TIMP-2在侵袭性垂体瘤中表达的阳性率分别为95.9%(47/49),57.1%(28/49),75.5%(37/49),和89.8%(44/49);在非侵袭性垂体瘤中阳性率分别为100%(12/12),91.7%(11/12),66.7%(8/12)和91.7%(11/12)。TIMP-1和TIMP-2的表达在侵袭性垂体瘤组明显少于非侵袭性垂体瘤组,(P<0.05);MMP-2在侵袭性垂体瘤组有增加的趋势,但无统计学意义,(P>0.05)。结论:在垂体腺瘤的侵袭性生物学机制中组织金属蛋白酶抑制剂TIMP-1和TIMP-2有可     

膀胱癌组织中MMP-2与TIMP-2 mRNA的表达及临床意义

目的 探讨基质金属蛋白酶-2(MMP-2)及其组织抑制因子-2(TIMP-2)mRNA在膀胱癌组织中的表达,以及与膀胱癌侵袭、转移和复发的关系。方法 应用半定量PCR和实时定量PCR(RQ-PCR)方法,检测10例正常膀胱组织和45例膀胱移行细胞癌组织中MMP-2和TIMP-2 mRNA的表达。结果 半定量PCR显示MMP-2、TIMP-2在膀胱正常组织中均呈阴性表达,而在45例膀胱癌组织中,MMP-2阳性率为66.7%(30/45),TIMP-2为57.8%(26/45)。定量PCR检测显示MMP-2表达量随肿瘤病理分级和临床分期的升高而增加,在复发病例中也增加,其组间差异有统计学意义(P＜0.05);TIMP-2的表达量随病理分级和临床分期的升高而降低,而且组间差异有统计学意义(P＜0.05),而在初发复发病例的组间差异无统计学意义(P＞0.05)。结论 MMP-2和TIMP-2对于膀胱癌的侵袭、转移和复发发挥了重要作用;MMP-2可能对判断膀胱癌的预后有一定的指导意义。     

miRNA-218 Inhibits Osteosarcoma Cell Migration and Invasion by Down-regulating of TIAM1, MMP2 and MMP9

Deregulated miRNAs participate in osteosarcoma genesis. In this study, the expression of miRNA-218 inhuman osteosarcomas, adjacent normal tissues and Saos-2 human osteosarcoma cells was first assessed. Then theprecise role of miRNA-218 in osteosarcoma cells was investigated. Upon transfection with a miR-218 expressionvector, the proliferation of Saos-2 human osteosarcoma cells determined using the ATPlite assay was significantlysuppressed, whilw migration of Saos-2 cells detected by wound healing and invasion determined using transwellswere dramatically inhibited. Potential target genes of miR-218 were predicted and T-cell lymphoma invasionand metastasis 1 (TIAM1) and matrix metalloproteinase 2 (MMP2) and 9 (MMP9) were identified. This wasconfirmed by western blotting, which showed that miR-218 expression inhibited TIAM1, MMP2 and MMP9protein expression. Collectively, these data suggest that miR-218 acts as a tumor suppressor in osteosarcomasby down-regulating TIAM1, MMP2 and MMP9 expression.