Expression of Thrombospondin-1 Modulates the Angioinflammatory Phenotype of Choroidal Endothelial Cells

The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age-related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation, has a significant impact on phenotype of ChEC. ChEC from TSP1-deficient (TSP1−/−) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re-expression of TSP1 was sufficient to restore TSP1−/− ChEC migration and capillary morphogenesis. TSP1−/− ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS (iNOS), a marker of inflammation, which was associated with significantly higher level of NO and oxidative stress in these cells. Wild type and TSP1−/− ChEC produced similar levels of VEGF, although TSP1−/− ChEC exhibited increased levels of VEGF-R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.     

Induction of Vascular Endothelial Growth Factor and Matrix Metalloproteinase-9 via CD47 Signaling in Neurovascular Cells

Neurovascular injury comprises a wide spectrum of pathophysiology that underlies the progression of brain injury after cerebral ischemia. Recently, it has been shown that activation of the integrin-associated protein CD47 mediates the development of blood–brain barrier injury and edema after cerebral ischemia. However, the mechanisms that mediate these complex neurovascular effects of CD47 remain to be elucidated. Here, we compare the effects of CD47 signaling in brain endothelial cells, astrocytes, and pericytes. Exposure to 4N1 K, a specific CD47-activating peptide derived from the major CD47 ligand thrombospondin-1, upregulated two major neurovascular mediators, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9), in brain endothelial cells and astrocytes. No changes were detected in pericytes. These findings may provide a potential mechanism for CD47-induced changes in blood–brain barrier homeostasis, and further suggest that CD47 may be a relevant neurovascular target in stroke.     

Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding.     

膜型基质金属蛋白酶-1在不同内皮细胞株中的表达差异

为研究膜型基质金属蛋白酶-1（membrane-type matrix metalloproteinase-1, MT1-MMP）在血管生物学中的作用机制,比较了3株常用的内皮细胞株：人微血管内皮细胞株HMEC-1、人脐静脉内皮细胞株ECV304和EAhy926中MT1-MMP及与其功能相关的MMP-2,TIMP-2的表达差异.实时PCR 和流式细胞术检测HMEC-1、EAhy926和ECV304中MT1-MMP/MMP-2/TIMP-2的表达,明胶酶谱法分析各细胞株上清中MMP-2的酶活.实时PCR结果显示,3株细胞均表达MT1-MMP与TIMP-2,MT1-MMP在EAhy926中表达最高,TIMP-2在ECV304中表达最高,而仅在EAhy926中检测到MMP-2的表达.流式细胞术和酶谱的结果与PCR结果基本一致.MT1-MMP和MMP-2在典型的大血管内皮细胞株EAhy926中高表达可能与该细胞独特的来源、表型特点和功能有关.     

The Effect of Extracellular Matrix Molecules on the in Vitro Behavior of Bovine Endothelial Cells

Extracellular matrix (ECM) is an important mediator of endothelial functions such as adhesion, spreading, migration, proliferation, and maintenance of differentiated functions. Attachment of cultured cells to tissue culture polystyrene (TCPS) is dependent on vitronectin which adsorbs onto the surface from the serum in the culture medium. Vitronectin (VN) will adsorb efficiently to TCPS even if the latter has been coated with another matrix molecule and blocked with albumin. This means that studies of the interactions of cells with individual coated ECM molecules will be confounded by the presence of adsorbed VN if serum is present in the culture medium. In this study, the adhesion, spreading, growth, and output of endogenous matrix molecules by bovine corneal endothelial (BCE) cells were measured on five different matrix substrates using medium which had been depleted of vitronectin to avoid such confounding effects. The same cell adhesion and spreading maxima were achieved on vitronectin, fibronectin (FN), laminin (LM), and types I and IV collagen (col I, col IV). The coating concentrations required to achieve these maxima, however, differed among the substrates, LM needing considerably higher concentrations than the other substrates for both maximal adhesion and spreading and FN needing higher concentrations for cell spreading. When cells were continuously passaged on each of the five substrates coated at concentrations optimal for cell spreading, no differences in cell proliferation rates or cell morphology were observed. Significant differences, however, were observed in the subcellular output of endogenous matrix molecules (FN, LM, col IV, and thrombospondin) between the different substrates. Col I was a poor substrate for the production of all ECM molecules tested over the 10 passages of the experiment, whereas col IV was a consistently good substrate. LM and FN substrates displayed differential effects on the output of different ECM molecules. VN was unique in that BCE cells at early passage on this substrate produced high levels of endogenous matrix molecules, whereas with continued passage on this substrate, a progressive decline in ECM secretion was observed. These results show that incorporation of individual molecules into the ECM by BCE cells in culture is significantly affected by the nature of the substratum. They further suggest that passage of endothelial cells in media containing serum (which results in coating of VN onto the substrate) may result in a progressive reduction of ECM output.     

基质金属蛋白酶- 9, 组织抑制因子- 1 在进展期 胃癌中的表达与意义

目的：研究基质金属蛋白酶-9（matrix metalloproteinase-9,MMP-9）及其组织抑制因子-1（tissue inhibitor of metallopmteinase—1,TMP-1）在进展期胃癌中的表达情况,探讨二者的表达与胃癌侵袭转移闻的关系及二者间的联系。方法：应用免疫组化方法检测70例进展期胃癌标本中MMP-9,TIMP-1的表达,并进行回顾性随访。结果：馒反肌层以上者MMP-9的阳性表达（66．67％）明显高于肿瘤局限于粘膜、粘膜下者（20％P〈0.01）。MMP-9阳性表达与胃癌的淋巴转移与肝转移有相关性（P〈0．01）。TIMP-1的表达随胃癌浸润深度增加而减少,当肿瘤突破浆膜时TIMP-1的表达呈现陡降趋势（P〈0．01）。结论：MMP-9的过阳性表达和TIMP-1的表达失衡可能与胃癌转移行为有关。TIMP-1可能抑制胃癌的浸润转移。     

Expression of Matrix Metalloproteinase-9, Type IV Collagen and Vascular Endothelial Growth Factor in Adamantinous Craniopharyngioma

To explore the expression of matrix metalloproteinase 9 (MMP-9), type IV collagen (Col IV) and vascular endothelial growth factor (VEGF) in adamantinomatous craniopharyngioma (ACP) and analyze the correlation between the level of these markers and adamantimous craniopharyngiomas recurrence. Expressions of MMP-9, Col IV and VEGF were tested by immunohistochemistry (IHC) in 40 cases of ACP, including 24 cases of primary group and 16 cases of recurred group. The expression level of MMP-9 and VEGF in recurred group were significantly higher than primary group (93.7% vs. 41.7%, P < 0.05, 87.5% vs. 45.8%, P < 0.05, respectively). The expression of Col IV in the recurred group was significant different from the primary group (Z = −2.619, P < 0.05). MMP-9, Col IV and VEGF may be the potential specific bio-marker related to the recurrence of ACP.     

基质金属蛋白酶-9及金属蛋白酶组织抑制剂-1在孕鼠胎盘形成中期的表达模式

目的探讨基质金属蛋白酶-9(MMP-9)及金属蛋白酶组织抑制剂-1(TIMP-1)在孕鼠胎盘形成中期的表达模式。方法 HE染色观察孕鼠第9天至第14天(D9~D14)胎盘形态结构的变化,同时免疫组织化学法检测相应天数胎盘中MMP-9和TIMP-1的表达情况。结果 HE染色结果显示孕鼠D11时胎盘的3层结构开始形成,包括蜕膜区(Dec)、海绵滋养细胞层(Sp)和迷路区(Lab);随着妊娠天数的增加,Dec区域逐渐缩小,Sp和Lab区域增大;免疫组化表明,MMP-9和TIMP-1均表达于细胞浆和细胞核中,MMP-9强表达于D9和D10胎盘的外胎盘锥中,D10~D13的Dec、Sp和D11~D13的滋养巨细胞中。MMP-9在D11的Lab,D12的Dec和Lab,D13的Dec、Sp、Lab区域中的阳性细胞的积分光密度值与D14胎盘中对应值比较,差异有统计学意义(P0.05);TIMP-1在D9~D12的Dec区域中的阳性细胞的积分光密度值与D14胎盘中对应值比较,差异有统计学意义(P0.05);MMP-9/TIMP-1在D9、D11的Dec及D12的Lab区域中的比值与D14胎盘中对应值比较,差异有统计学意义(P0.05),在D10、D12的Dec和D13的Lab区域中的比值与D14胎盘中对应值比较,差异有高度统计学意义(P0.01)。结论 MMP-9和TIMP-1在孕鼠胎盘中的协同表达可能参与调节胎盘的形成。     

CD36 Mediates the In Vitro Inhibitory Effects of Thrombospondin-1 on Endothelial Cells

Thrombospondin-1 (TSP-1) is a naturally occurring inhibitor of angiogenesis that is able to make normal endothelial cells unresponsive to a wide variety of inducers. Here we use both native TSP-1 and small antiangiogenic peptides derived from it to show that this inhibition is mediated by CD36, a transmembrane glycoprotein found on microvascular endothelial cells. Both IgG antibodies against CD36 and glutathione-S-transferase–CD36 fusion proteins that contain the TSP-1 binding site blocked the ability of intact TSP-1 and its active peptides to inhibit the migration of cultured microvascular endothelial cells. In addition, antiangiogenic TSP-1 peptides inhibited the binding of native TSP-1 to solid phase CD36 and its fusion proteins, as well as to CD36-expressing cells. Additional molecules known to bind CD36, including the IgM anti-CD36 antibody SM, oxidized (but not unoxidized) low density lipoprotein, and human collagen 1, mimicked TSP-1 by inhibiting the migration of human microvascular endothelial cells. Transfection of CD36-deficient human umbilical vein endothelial cells with a CD36 expression plasmid caused them to become sensitive to TSP-1 inhibition of their migration and tube formation. This work demonstrates that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, may be essential for the inhibition of angiogenesis by thrombospondin-1.     

Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.     

Interferon-Gamma Increases the Ratio of Matrix Metalloproteinase-9/Tissue Inhibitor of Metalloproteinase-1 in Peripheral Monocytes from Patients with Coronary Artery Disease

Acute coronary syndromes (ACS) may be triggered by acute infections. Systemic production of interferon gamma (IFN-γ) is induced during infection and regulates the production of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), both important in plaque stability. This study evaluates the effect of IFN-γ on the MMPs/TIMP-1 ratio in cultured monocytes from 30 patients with stable coronary artery disease (CAD), 30 with unstable angina (UA) or non-ST-segment elevation myocardial infarction (NSTEMI), and 30 healthy blood donors. Supernatant concentrations of MMP-1, -2, -9, and TIMP-1 were measured by enzyme-linked immunoassays. Basal concentration of MMP-1 and TIMP-1 was similar between groups, while MMP-2 was higher in healthy individuals and MMP-9 in patients with UA/NSTEMI. Upon IFN-γ stimulation, MMP-9 secretion increased in all groups, while TIMP-1 decreased only in patients with CAD, which in turn result in a strikingly elevation in their mean MMP-9/TIMP-1 ratio. MMP-1/TIMP-1 and MMP-2/TIMP-1 ratios were <1.0 in basal conditions and after stimulation in all groups. Our results suggest that nonstimulated monocytes from patients with stable CAD show a similar behavior than those from healthy individuals. However, stimulation with IFN-γ induces an increase on the MMP-9/TIMP-1 ratio as high as that found in patients with ACS. Thus, it may bring biological plausibility to the association between acute infections and the development of ACS.     

Decreased Expression of Matrix Metalloproteinase-9 and Increased Expression of Tissue Inhibitors of Matrix Metalloproteinase-1 in Paratuberculosis-Infected Cattle in the ELISA-Negative Subclinical Stage

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24 hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.     

Neutrophil Infiltration and Matrix Metalloproteinase-9 in Lacunar Infarction

We use the modified pial vessel disruption rat model to elucidate the cellular and molecular mechanisms of cavitation as it plays a role in lacunar infarction. Here we discuss the similarities between the genesis of pulmonary cavitation in various animal models and lacunar infarction in the cerebral cortex of rats. Both pathological processes involve the creation of a cavity surrounded by fibroblasts or reactive astrocytes. A crucial step in both, the lung and the cerebral cortex, appears to be the migration of neutrophils across the endothelial barrier into the parenchyma. In the lung and cerebral cortex this involves release of matrix metalloproteinase-9 (MMP-9). Inside the parenchyma neutrophils continue to release MMP-9. In both situations batimastat (BB-94) and minocycline reduce release of MMP-9 and prevent cavitation. In the cerebral cortex MMP-9 release by resident microglia plays an additional role. We therefore advance the hypothesis that cavitation in both tissues is driven by MMP-9 originating from invading neutrophils. Therapeutic intervention has to focus on these blood-borne intruder cells and specific MMP actions. Batimastat and its derivatives (marimastat, BB-1101, mCGS-27023-A, ilomastat, GM6001, CTK8G1150) are already in clinical or experimental use in humans for anti-cancer treatment, and these clinically relevant drugs could be repurposed to act as anti-inflammatory to counter neutrophil contribution to lung or cerebral cortex cavitation.     

In Vitro Angiogenic and Proteolytic Properties of Bovine Lymphatic Endothelial Cells

The aim of the present study was to determine whether angiogenic cytokines, which induce neovascularization in the blood vascular system, might also be operative in the lymphatic system. In an assay of spontaneous in vitro angiogenesis, endothelial cells isolated from bovine lymphatic vessels retained their histotypic morphogenetic properties by forming capillary-like tubes. In a second assay, in which endothelial cells could be induced to invade a three-dimensional collagen gel within which they formed tube-like structures, lymphatic endothelial cells responded to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in a manner similar to what has previously been observed with endothelial cells derived from the blood vascular system. Finally, since angiogenesis is believed to require extracellular proteolytic activity, we investigated the effects of bFGF and VEGF on lymphatic endothelial cell proteolytic properties by focussing on the plasminogen activator (PA) system. bFGF and VEGF increased urokinase, urokinase receptor, and tissue-type PA expression. This was accompanied by an increase in PA inhibitor-l, which is thought to play an important permissive role in angiogenesis by protecting the extracellular matrix against excessive proteolytic degradation. Taken together, these results demonstrate that with respect to in vitro morphogenetic and proteolytic properties, lymphatic endothelial cells respond to the previously described angiogenic factors, bFGF and VEGF, in a manner very similar to what has been described for endothelial cells derived from the blood vascular system.     

Effect of Lumican on the Migration of Human Mesenchymal Stem Cells and Endothelial Progenitor Cells: Involvement of Matrix Metalloproteinase-14

Background

Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful in vivo administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC).

Methodology/Principal Findings

Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression.

Conclusion/Significance

Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration via mechanisms that involve a decrease of MMP-14 expression and activity.     

Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.