The development of cardiac fibrosis in low tissue factor mice is gender-dependent and is associated with differential regulation of urokinase plasminogen activator

Tissue factor (TF) initiates the protease coagulation cascade in response to tissue injury. Homozygous deficiency of murine TF results in embryonic lethality, which is rescued by low-level expression of human TF. These low-TF mice have been shown to develop cardiac fibrosis. We tested the hypothesis that the development of cardiac fibrosis in low-TF mice results from dysregulated protease expression and is affected by gender. Mice were divided into the age groups 2-5, 6-12, 13-18 and 19+ weeks. Fibrosis was assessed by trichrome staining. Protease expression was measured in male and female mice by RT-PCR for mRNA and zymography, ELISA or immunoblot for protein. Urokinase plasminogen activator (uPA) activity was determined by zymography and chromogenic substrate assay. A marked gender effect was noted for the development of fibrosis, with interstitial collagen deposition occurring from 9 weeks in male low-TF mice, but not until 19 weeks in low-TF females. This delayed onset in females was accompanied by delayed up-regulation of molecular markers of injury. Matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 expression were up-regulated in the hearts of male low-TF mice from 6 to 12 weeks and in females from 19 weeks. MMP/TIMP dysregulation was not seen prior to cardiac fibrosis and did not appear to explain the gender differences. However, uPA expression and activity were down-regulated prior to cardiac fibrosis in low-TF females, but were up-regulated in age-matched males. This suggests that the down-regulation of uPA in female low-TF mice protects them from more severe cardiac fibrosis.     

胰腺癌组织中血管内皮生长因子mRNA与尿激酶型纤溶酶原激活物mRNA定量表达的临床意义分析

目的探讨胰腺癌组织中血管内皮生长因子(VEGF)mRNA、尿激酶型纤溶酶原激活物(uPA)mRNA定量表达的临床意义。方法自2008年1月至2011年12月于本科行胰头癌根治术的患者中筛选出经病理证实为导管腺癌的30例患者的完整资料,采用荧光定量PCR(qPCR)检测其胰腺癌组织及6例正常胰腺组织中VEGF mRNA、uPA mRNA定量表达,分析其与临床病理因素之间的关系。结果 VEGF mRNA、uPA mRNA的表达与胰腺癌的组织分化程度、神经侵犯有关。VEGF mRNA在淋巴结转移阳性组中的定量表达高于淋巴结转移阴性组,两组比较差异有统计学意义(t=20.007,P=0.000);uPA mRNA在直径≤2 cm的肿瘤组织中定量水平小于直径2 cm的肿瘤组织,两组比较差异有统计学意义(t=7.539,P=0.000)。uPA mRNA在伴有十二指肠侵犯组中的定量表达高于无十二指肠侵犯组,两组比较差异有统计学意义(t=-2.089,P=0.037)。uPA mRNA在Ⅲ期肿瘤组织中的定量表达高于Ⅰ、Ⅱ期肿瘤组织中的定量表达,两组比较差异有统计学意义(t=-9.450,P=0.000)。VEGF mRNA与uPA mRNA的表达存在正相关(r=0.334,P=0.000)。结论 VEGF mRNA、uPA mRNA在胰腺癌组织中过度表达可为肿瘤细胞的侵润创造条件。     

VEGF、uPA及uPAR与胃癌侵袭和转移的相关性

目的探讨血管内皮生长因子(VEGF)、尿激酶型纤溶酶原激活物(uPA)及其受体(uPAR)与胃癌侵袭、转移的关系及其相关性。方法采用免疫组化SP方法检测198份胃癌组织标本(胃癌组)、60份正常胃黏膜组织标本(对照组)VEGF、uPA、uPAR表达。结果与对照组比较,胃癌组VEGF呈高表达,并与浸润深度、淋巴结转移和临床分期呈正相关,与肿瘤的分化程度呈负相关,P均<0.05;胃癌组uPA和uPAR呈高表达,与病理分级、浸润深度、淋巴转移、临床分期有关,P均<0.05。胃癌组VEGF与uPA表达呈正相关,P<0.01;uPA与uPAR表达呈正相关,P<0.01。结论 VEGF、uPA、uPAR在胃癌发生、发展、侵袭和转移中起促进作用;三者相互促进,相互协调,关系密切。三者均可作为胃癌诊断和预后估计的指标及胃癌治疗的新靶点。     

Stimulation of plasminogen activator and inhibitor in the lymphatic endothelium

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at M(r) of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of approximately 110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph.     

Normal and Perturbed Endothelial Cells from Canine Femoral Arteries and Femoral Veins Exhibit Heterogeneity in Hemostatic Properties and Growth Characteristics

Background: We sought to examine the heterogeneity of endothelial cells from the same anatomic site but different vascular systems and described von Willebrand factor (vWF) release and morphological change in response to injury-associated factor in femoral vessels from canine in vitro. Methods: Levels of hemostatic factors (vWF, plasminogen activator inhibitor type 1(PAI-1), antithrombin III (ATIII), in tissue sections and cultured endothelial cells of canine femoral arteries and canine femoral veins were compared by the immunohistochemistry technique. In addition to comparing cell growth density and cell protein contents, cultured femoral arterial endothelial cells (FAECs) and cultured femoral venous endothelial cells (FVECs) were incubated with a series concentration of basic fibroblast factor (bFGF) (1, 10, 100 ng/ml) for up to 48 hours to test the amount of vWF secretion and morphological change. Results: Both in tissue sections and cultured cells, the levels of vWF are higher in FVECs than in FAECs. We were unable to differentiate the level of PAI-1 and ATIII difference between FAECs and FVECs. bFGF (10 ng/ml) significantly increased vWF secretion from cultured FAECs but not from FVECs. The size of cultured FAECs is smaller than of FVECs; however, FAECs have higher amounts of protein contents than FVECs. Conclusions: These comparative studies provide evidence indicating that the characteristics of FVECs differ from those of FAECs. These differences may be indicated heterogeneity with either inherited or acquired thrombotic disease.     

Neovascularization and angiogenic gene expression following chronic arsenic exposure in mice

Exposure to arsenic in drinking water increases incidence of cardiovascular diseases. However, the basic mechanisms and genetic changes that promote these diseases are unknown This study investigated the effects of chronic arsenic exposure on vessel growth and expression of angiogenic and tissue remodeling genes in cardiac tissues. Male mice were exposed to low to moderately high levels of arsenite (Aslll) for 5, 10, or 20 wk in their drinking water. Vessel growth in Matrigel implants was tested during the last 2 wk of each exposure period. Implant vascularization increased in mice exposed to 5–500 ppb Aslll for 5 wk. Similar increases were seen following exposure to 50–250 ppb of Aslll over 20 wk, but the response to 500 ppb decreased with time. RT-PCR analysis of cardiac mRNA revealed differential expression of angiogenic or tissue remodeling genes, such as vascular endothelial cell growth factor (VEGF), VEGF receptors, plasminogen activator inhibitor-1, endothelin-1, and matrix metalloproteinase-9, which varied with time or amount of exposure. VEGF receptor mRNA and cardiac microvessel density were reduced by exposure to 500 ppb Aslll for 20 wk. These data demonstrate differential concentration and time-dependent effects of chronic arsenic exposure on cardiovascular phenotype and vascular remodeling that may explain the etiology for Aslll-induced disease.     

Pancreas recovery following cerulein-induced pancreatitis is impaired in plasminogen-deficient mice

BACKGROUND & AIMS: The plasminogen (plg) system participates in tissue repair in several organs, but its role in pancreas repair remains poorly characterized. To understand better the role of plg in pancreas recovery following injury, we examined the course of cerulein-induced pancreatitis in plg-deficient and -sufficient mice. METHODS: Pancreatitis was induced by cerulein administration (50 microg/kg, 7 intraperitoneal injections). Mice were killed either at the acute phase (7 hours after the first cerulein injection) or during recovery (at 2, 4, and 7 days). In pancreatic sections, we examined pancreatic morphology, trypsin activation, inflammatory cell infiltration, acinar cell death, cell proliferation, extracellular matrix deposition, activation of stellate cells (PSCs), and components of the plg and metalloproteinase systems. RESULTS: In plg-sufficient mice, pancreatic plg levels and plasmin activity increased during the acute phase and remained elevated during recovery. Pancreatitis resolved in plg-sufficient mice within 7 days. Pancreas recovery involved reorganization of the parenchyma structure, removal of necrotic debris, cell proliferation, transient activation of PSCs, and moderate deposition of extracellular matrix proteins. Acute pancreatitis (7 hours) was indistinguishable between plg-deficient and -sufficient mice. In contrast, pancreas recovery was impaired in plg-deficient mice. Plg deficiency led to disorganized parenchyma, extensive acinar cell loss, poor removal of necrotic debris, reduced cell proliferation, and fibrosis. Fibrosis was characterized by deposition of collagens and fibronectin, persistent activation of PSCs, and up-regulation of pancreatic transforming growth factor beta1. CONCLUSIONS: Plg/plasmin deficiency leads to features similar to those found in chronic pancreatitis such as parenchymal atrophy and fibrosis.     

The endocrine and paracrine control of menstruation

During the reproductive life, the human endometrium undergoes cycles of substantial remodeling including, at menstruation, a massive but delimited tissue breakdown immediately followed by scarless repair. The present review aims at summarizing the current knowledge on the endocrine and paracrine control of menstruation in the light of recent observations that undermine obsolete dogmas. Menstruation can be globally considered as a response to falling progesterone concentration. However, tissue breakdown is heterogeneous and tightly controlled in space and time by a complex network of regulators and effectors, including cytokines, chemokines, proteases and various components of an inflammatory response. Moreover, menstruation must be regarded as part of a complex and integrated mechanism of tissue remodeling including features that precede and follow tissue lysis, i.e. decidualization and immediate post-menstrual regeneration. The understanding of the regulation of menstruation is of major basic and clinical interest. Indeed, these mechanisms largely overlap with those controlling other histopathological occurrences of tissue remodeling, such as development and cancer, and inappropriate control of menstrual features is a major potential cause of two frequent endometrial pathologies (i.e. abnormal uterine bleeding and endometriosis).     

Melatonin promotes angiogenesis during protection and healing of indomethacin‐induced gastric ulcer: role of matrix metaloproteinase‐2

Abstract: Matrix metalloproteinase (MMP)‐2 is considered as a crucial regulator of angiogenesis, a process of new blood vessel formation. We reported previously that melatonin (N‐acetyl‐5‐methoxy tryptamine), an antioxidant and anti‐inflammatory agent, prevents indomethacin‐induced gastric ulcers. Herein, we investigated the effect of melatonin on MMP‐2‐mediated angiogenesis during gastroprotection. Angiogenic properties of melatonin were tested in both rat corneal micropocket assay and in mouse model of indomethacin‐induced gastric lesions. Melatonin augmented angiogenesis that was associated with amelioration of MMP‐2 expression and activity and, upregulation of vascular endothelial growth factor (VEGF) in rat cornea. Melatonin prevented gastric lesions by promoting angiogenesis via upregulation of VEGF followed by over‐expression of MMP‐2. Similarly, healing of gastric lesions was associated with early expression of VEGF followed by MMP‐2. In addition, upregulation of MMP‐2 was parallel to MMP‐14 and inverse to tissue inhibitor of metalloprotease (TIMP)‐2 expression during gastroprotection. Our data demonstrated that melatonin exerts angiogenesis through MMP‐2 and VEGF over‐expression during protection and healing of gastric ulcers. This study highlights for the first time a phase‐associated regulation of MMP‐2 activity in gastric mucosa and an angiogenic action of melatonin to rescue indomethacin‐induced gastropathy.     

Expression of serine protease SNC19/matriptase and its inhibitor hepatocyte growth factor activator inhibitor type 1 in normal and malignant tissues of gastrointestinal tract

AIM: To provide the expression profile of serine protease SNC19/matriptase and its inhibitor hepatocyte growth factor activator inhibitor type 1 (HAI-1) in normal and malignant tissues of gastrointestinal tract at mRNA level for further study on their correlations with tumor progression and metastasis. METHODS: Total RNAs were prepared from 37 samples of colorectal cancer tissues, 40 samples of gastric cancer tissues, and their adjacent normal tissues. The expression of SNC19/matriptase and HAM in these samples was detected by real-time fluorescent quantitative PCR using glyceraldehyde-3-phosphate dehydrogenase as internal standard, and the clinical significance for the correlation with clinicopathological parameters was evaluated. RESULTS: In gastric cancer tissues the expression of HAI-1 and SNC19/matriptase was significantly lower than that in the corresponding adjacent normal tissues (Z=-3.280, P= 0.006; Z=-4.651, P=0.000). HAI-1:SNC19/matriptase ratio showed no difference between normal and malignant tissues (P>0.05). Analysis of clinicopathological parameters showed decreased expression of HAM and HAI-1:SNC19/ matriptase ratio associated with stage Ⅲ/Ⅳ gastric tumors as compared to stage Ⅰ/Ⅱ ones (Z = -2.140, P= 0.031; Z= -2.155, P= 0.031), and with lymph node-positive gastric cancer tissues as compared to lymph node-negative ones (Z= -2.081, P= 0.036; Z= -2.686, P= 0.006). The expression of SNC19/matriptase had no relationship with stages and lymph node metastasis (P>0.05). The expression of HAM and HAM:SNC19/matriptase ratio increased in well-differentiated gastric cancer tissues, but there was no statistical significance (P>0.05). The difference of SNC19/matriptase expression was not significant in gastric cancer tissues of different histological differentiation status (P>0.05). In colorectal cancer tissues, the expression of HAI-1 and SNC19/matriptase was also markedly lower than that in their adjacent normal tissues (Z=-3.100, P=0.002; Z=-2.731, P=0.006), whereas HAI-1:SNC19/matriptase ratio showed no difference. Decreased expression of HAI-1 was associated with increased invasive depth and lymph node metastasis, but there was no statistical significance (P>0.05). The difference of SNC19/matriptase expression and HAI-1: SNC19/matriptase ratio was not significant in different stages and different lymph node metastasis status (P>0.05). The expression of SNC19/matriptase, HAI-1 or HAI-1: SNC19/matriptase ratio showed no difference in colorectal cancer tissues of different histological differentiation status (P>0.05). CONCLUSION: The expressions of SNC19/matriptase and its inhibitor HAI-1 are decreased in gastrointestinal cancer tissues compared to their normal counterparts, and the decreased expression of HAI-1 may correlate with invasion and lymph node metastasis. The possible mechanisms involved need to be further investigated.     

Disruption of tissue-type plasminogen activator gene in mice aggravated liver fibrosis

Background and Aim:  Tissue-type plasminogen activator (tPA) is one of the major components in the matrix proteolytic network whose role in the pathogenesis of liver fibrosis remains unknown. The aim of this study is to investigate the role of tPA in carbon tetrachloride (CCl₄)-induced liver fibrosis.
Methods:  Wild-type and tPA knockout mice (8 mice per group) were injected interperitoneumly with 25% CCl₄ 2 ml/kg twice per week as CCl₄ administration groups and olive oil 2 ml/kg as controls. After 4 weeks, the livers of mice were removed under deep anesthesia and prepared for further studies such as histology, immunostaining, hydroxyproline assay, zymography and western blot analysis.
Results:  Mice lacking tPA developed more severe morphological injury and displayed an increased deposition of collagen in the liver after CCl₄ administration compared with wild-type counterparts. Deficiency of tPA increased α-smooth muscle actin expression in the mice livers. On the other hand, the decrease of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) activities, metalloproteinase-13 (MMP-13) expression and a marked increase of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) expression were found in the liver of CCl₄ administrated tPA^−/− mice compared with wild-type counterparts.
Conclusions:  Deficiency of tPA aggravated liver fibrosis through promoting hepatic stellate cells (HSCs) activation and inhibiting ECM degradation by decreasing MMP-2, MMP-9 activities and disrupting the balance between MMP-13 and TIMP-1.     

缬沙坦对兔自体移植静脉内膜增生及基质金属蛋白酶-2、组织基质金属蛋白酶抑制剂-2表达的影响

目的观察缬沙坦对兔颈部自体移植静脉内膜增生、基质金属蛋白酶-2（MMP-2）、组织基质金属蛋白酶抑制剂-2（TIMP-2）表达的影响，探讨AT1受体拮抗剂干预移植静脉再狭窄的作用和机制。方法雄性新西兰大白兔20只行颈部自体静脉移植手术，对照组予普通饲料饲养，缬沙坦组予普通饲料＋缬沙坦10mg／（kg·d）饲养。4周后取出静脉移植物行血管形态学检查；图像分析系统计算血管腔内膜的厚度、面积及中膜的厚度、面积；免疫组织化学方法检测MMP-2、TIMP-2表达情况。结果对比对照组，缬沙坦组移植静脉新生内膜厚度及面积明显降低，MMP-2表达降低，TIMP-2表达增加。结论缬沙坦可以抑制兔自体移植静脉新生内膜的形成，其机制可能与抑制内膜MMP-2表达、促进TIMP-2表达有关。     

尿激酶对大鼠胸膜成纤维细胞分泌TGF-β1、VEGF、PAI-1的影响

目的探讨尿激酶(urokinase,UK) 对大鼠胸膜的成纤维细胞（fibroblasts，FB）分泌转化生长因子-β₁ （transforming growth factor beta-1，TGF-β₁）、血管内皮生长因子（vascular endothelial growth factor，VEGF）、纤溶酶原激活物抑制剂（plasminogen activator inhibitor-1，PAI-1）的影响。方法取健康雄性SD大鼠胸膜，培养、纯化并鉴定FB。设对照组和实验组。对照组分2组：对照0组由不含胸膜FB的40个样本组成，加入无血清RPMI 1640培养液；对照1组由40个胸膜FB样本组成，加入无血清RPMI 1640培养液，培养24h后，用酶联免疫吸附法测定上清液中TGF-β₁、PAI-1、VEGF的含量；实验组分为4组，共40个样本，分别加入5000、10000、20000和30000IU/ml的UK，培养24h后取上清液，测定其中TGF-β₁、VEGF、PAI-1量。结果对照0组未检测出TGF-β₁、PAI-1、VEGF；对照组1胸膜FB分泌TGF-β₁、VEGF、PAI-1的量为（3135.205±390.975）pg/mL；（22.09±7.48）ng/ml；（1.8775±0.39）ng/ml；加入5000～30000IU/ml尿激酶的试验组， TGF-β1、VEGF、PAI-1的浓度与对照1组相比，有明显下降（P<0.05）。结论胸膜成纤维细胞能分泌TGF-β₁、PAI-1、VEGF；而尿激酶能抑制其分泌。     

Oncogenic K-ras stimulates Wnt signaling in colon cancer through inhibition of GSK-3beta

BACKGROUND & AIMS: Two key genetic events underlying the development of colon cancer are activation of the K-ras and Wnt signaling pathways. We have previously shown that these 2 pathways can cooperate to regulate vascular endothelial growth factor (VEGF) gene expression. The goal of this study was to define the molecular basis for this interaction. METHODS: The effects of K-ras(Val12) on VEGF and T-cell factor 4 (TCF-4) promoter activity, nuclear levels of beta-catenin and beta-catenin/TCF-4 complexes, glycogen synthase kinase 3beta (GSK-3beta) phosphorylation, and GSK-3beta kinase activity were measured. LY294002 and PD98059 were used to define the role of specific ras effector pathways. RESULTS: Oncogenic K-ras up-regulated the activity of the VEGF promoter, and selective mutagenesis of TCF-4 binding sites significantly blocked this induction. K-ras(Val12) also induced the activity of a heterologous TCF-4 reporter construct in Caco-2 and HeLa cells. LY294002 and dominant negative phosphatidylinositol 3-kinase nearly completely blocked this induction. K-ras(Val12) increased the stability of beta-catenin, the levels of nuclear beta-catenin, and the formation of nuclear beta-catenin/TCF-4 complexes, and these effects were also blocked by LY294002. Finally, K-ras(Val12) inhibited the kinase activity of total cellular GSK-3beta and GSK-3beta complexed with Axin. This effect was not mediated through phosphorylation at serine 9 but did depend on phosphatidylinositol 3-kinase. CONCLUSIONS: Our results suggest a unique cooperative interaction between 2 critical oncogenic pathways in colorectal tumorigenesis and highlight the pivotal role of GSK-3beta.     

Expression and function of vascular endothelial growth factor receptors (Flt-1 and Flk-1) in vascular adventitial fibroblasts

Vascular endothelial growth factor receptors (VEGFRs) are previously considered to exist exclusively in endothelial cells. However, little is known if the receptors are expressed in other non-endothelial cells. In this study, we measured activation of two VEGFRs, Flk-1 and Flt-1, and their biological functions in cultured adventitial fibroblasts and injured rat carotid injury arteries induced by balloon angioplasty. Our results indicated that Flt-1, but not Flk-1, existed in adventitial fibroblasts. Angiotensin II increased Flt-1 protein expression in a time- and concentration-dependent manner. Adventitial fibroblast migration stimulated by vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) required Flt-1 expression. The Flt-1-induced adventitial fibroblast migration was blocked by anti-Flt-1 neutralizing antibody and soluble VEGFR1 protein (sFlt-1). However, Flt-1 activation did not enhance cell proliferation. In addition, Flt-1 expression was significantly increased in the neointima and adventitia in injured rat carotid arteries. We concluded that functional expression of Flt-1 in adventitial fibroblasts might be an important mediator in the pathogenesis of vascular remodeling after arterial injury.