The influence of receptor activity–modifying protein-1 overexpression on angiogenesis in mouse brain capillary endothelial cells

Receptor activity–modifying protein-1 (RAMP1) is highly expressed in the heart and vasculature, indicating that it might be related to the vascular system. However, the effects of RAMP1 on angiogenesis and the intrinsic mechanisms underlying this process remain unclear. Here, we verified that RAMP1 is a critical regulator of angiogenesis in a mouse brain capillary endothelial cell line (bEnd.3). We first constructed a RAMP1 overexpression lentiviral vector system and stably transfected bEnd.3 cells. We further showed that RAMP1 overexpression could lead to bEnd.3 migration and capillary tube formation in Matrigel without exogenous calcitonin gene–related peptide (CGRP) treatment. At the same time, RAMP1 overexpression had little effect on proliferation. More importantly, vascular endothelial growth factor (VEGF) and CGRP expression levels were not significantly higher in RAMP1-overexpressing cells than in control cells (P > 0.05), indicating that RAMP1 did not function through upregulating VEGF or CGRP expression in bEnd.3 cells. Strikingly, RAMP1 transfection increased adrenomedullin 2 (AM2) expression levels ( P < 0.05). Taken together, these data contribute to a better understanding of the molecular mechanisms of RAMP1 in angiogenesis.     

Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes

Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2‐Cre or VE‐cadherin‐Cre constructs to facilitate fate‐mapping of LSECs in liver regeneration. Some YFP‐positive LSECs were observed to convert into hepatocytes following a two‐thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non‐hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte‐like (iHep) cells may provide a new approach to tissue engineering.     

Celecoxib reduces hepatic vascular resistance in portal hypertension by amelioration of endothelial oxidative stress

The balance between endothelial nitric oxide (NO) synthase (eNOS) activation and production of reactive oxygen species (ROS) is very important for NO homeostasis in liver sinusoidal endothelial cells (LSECs). Overexpression of cyclooxygenase-2 (COX-2), a major intravascular source of ROS production, has been observed in LSECs of cirrhotic liver. However, the links between low NO bioavailability and COX-2 overexpression in LSECs are unknown. This study has confirmed the link between low NO bioavailability and COX-2 overexpression by COX-2-dependent PGE2-EP2-ERK1/2-NOX1/NOX4 signalling pathway in LSECs in vivo and in vitro. In addition, the regulation of COX-2-independent LKB1-AMPK-NRF2-HO-1 signalling pathway on NO homeostasis in LSECs was also elucidated. The combinative effects of celecoxib on diminishment of ROS via COX-2-dependent and COX-2-independent signalling pathways greatly decreased NO scavenging. As a result, LSECs capillarisation was reduced, and endothelial dysfunction was corrected. Furthermore, portal hypertension of cirrhotic liver was ameliorated with substantial decreasing hepatic vascular resistance and great increase of portal blood flow. With the advance understanding of the mechanisms of LSECs protection, celecoxib may serve as a potential therapeutic candidate for patients with cirrhotic portal hypertension.     

受体活性修饰蛋白研究进展

受体活性修饰蛋白(receptor activity-modifying proteins,RAMPs)属于单跨膜蛋白家族,分三个结构域,RAMP的N端和跨膜区决定本身的功能和受体表型,胞内C端对于配体的信号传导和受体循环有重要作用。目前发现有三个成员:RAMP1、RAMP2和RAMP3。RAMPs通过改变G蛋白偶联受体的糖基化,作用于配体结合区域来调节受体表型。RAMP1与降钙素受体样受体(calcitonin receptor like receptor,CRLR)结合表现出降钙素基因相关肽(calcitonin gene-related peptide,CGRP)受体表型:RAMP2和RAMP3与CRLR结合则对肾上腺髓质素(adrenomedullin,AM)表现高亲和力,与降钙素受体(calcitonin receptor,CTR)结合则作为胰淀粉样酶(amylin,AMY)受体。由此可见,RAMPs不仅调节受体与配体结合,还影响细胞内的蛋白相互作用调节细胞内信号传导来影响细胞的增殖、迁移、分化等生物学特性。RAMPs还对心血管系统的病理生理有重要调节作用。     

Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.     

Involvement of signaling of VEGF and TGF-β in differentiation of sinusoidal endothelial cells during culture of fetal rat liver cells

Embryonic development of the liver is closely associated with vascular organization. However, little is known about the mechanisms of vascular differentiation during liver development. Our previous study showed that the maturation of sinusoidal endothelial cells (SECs) occurred during embryonic day 13.0 (E13.0) to E15.0. To improve our understanding of SEC differentiation, we examined here the expression of maturation markers, SE-1 and stabilin-2, in fetal livers and also attempted to establish an in vitro SEC differentiation system by culturing E13.5 fetal liver cells. Immunohistochemical examination of SE-1 and stabilin-2 expression during fetal rat liver development revealed that these differentiation markers were co-expressed in SECs in the late stage of liver development, although stabilin-2 was expressed in almost all vascular endothelial cells in the early stage. Liver cells from the E13.5 rat fetus were cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1) and VEGF plus SB-431542 (an inhibitor of the TGF-β1 receptor, activin receptor-like kinase 5 [ALK-5]). In vitro SEC differentiation, as indicated by the appearance of cells co-expressing SE-1 and stabilin-2 and of cells with cytoplasmic fenestrae in endothelial sheets, was induced by the addition of both VEGF and SB-431542, an inhibitor of the phosphorylation of Smad2/3 but not that of Smad1/5/8 in the cultured cells. These results indicate for the first time that both VEGF signaling and the blocking of the ALK-5-Smad2/3 signal pathway are important for the fetal differentiation of SECs.     

Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.     

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.     

Clathrin-coated vesicles form a unique net-like structure in liver sinusoidal endothelial cells by assembling along undisrupted microtubules

Liver sinusoidal endothelial cells (LSECs) are highly active professional scavenger cells using clathrin-mediated endocytosis to clear the blood from macromolecular waste products. Using confocal microscopy, we observed a remarkable net-like distribution of clathrin heavy chain (CHC) in LSECs while all other cell types examined including various primary endothelial cells and cell lines showed the well-known punctuate staining pattern representing clathrin-coated vesicles (CCV). The net-like distribution of CHC in LSECs co-localized fully with microtubules, but not with actin. Upon 3D imaging, the net-like distribution of CHC resolved into numerous CCVs organized along the microtubules. The CCVs only partially co-localized with early endosome antigen 1 (EEA1) and adaptor protein 2 (AP-2). Endocytic vesicles containing ligand destined for degradation (FITC-AHGG) were organized along the clathrin/tubulin net-like structures, whereas transferrin-containing recycling vesicles co-localized to a much lower extent. Disruption of the microtubules by nocodazole treatment caused a collapse of the net-like organization of CCVs as well as a profound redistribution of EEA1, AP-2 and FITC-AHGG-containing vesicles, while transferrin internalization and recycling remained unaffected.     

Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice

Mammalian Genome - Stabilin2 (Stab2) encodes a large transmembrane protein which is predominantly expressed in the liver sinusoidal endothelial cells (LSECs) and functions as a scavenger receptor...     

Lymphatic Endothelial Differentiation in Pulmonary Lymphangioleiomyomatosis Cells

Pulmonary lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm affecting almost exclusively women of childbearing age. LAM belongs to the family of perivascular epithelioid cell tumors, characterized by spindle and epithelioid cells with smooth muscle and melanocytic differentiation. LAM cells infiltrate the lungs, producing multiple, bilateral lesions rich in lymphatic channels and forming cysts, leading to respiratory insufficiency. Here we used antibodies against four lymphatic endothelial markers—podoplanin (detected by D2-40), prospero homeobox 1 (PROX1), vascular endothelial growth factor receptor 3 (VEGFR-3), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)—to determine whether LAM cells show lymphatic differentiation. Twelve of 12 diagnostic biopsy specimens (early-stage LAM) and 19 of 19 explants (late-stage LAM) showed immunopositivity for D2-40 in most neoplastic cells. PROX1, VEGFR-3, and LYVE1 immunoreactivity varied from scarce in the early stage to abundant in the late stage. Lymphatic endothelial, smooth muscle, and melanocytic markers were partially co-localized. These findings indicate that lymphatic endothelial differentiation is a feature of LAM and provide evidence of a previously unidentified third lineage of differentiation in this neoplasm. This study has implications for the histological diagnosis of LAM, the origin of the neoplastic cells, and potential future treatment with drugs targeting lymphangiogenesis.     

Interaction between pericytes and endothelial cells leads to formation of tight junction in hyaloid vessels

The hyaloid vessel is a transient vascular network that nourishes the lens and the primary vitreous in the early developmental periods. In hyaloid vessels devoid of the support of astrocytes, we demonstrate that tight junction proteins, zonula occludens-1 and occludin, are regularly expressed at the junction of endothelial cells. To figure out the factor influencing the formation of tight junctions in hyaloid vessels, we further progress to investigate the interactions between endothelial cells and pericytes, two representative constituent cells in hyaloid vessels. Interestingly, endothelial cells interact with pericytes in the early postnatal periods and the interaction between two cell types provokes the up-regulation of transforming growth factor β1. Further in vitro experiments demonstrate that transforming growth factor β1 induces the activation of Smad2 and Smad3 and the formation of tight junction proteins. Taken together, in hyaloid vessels, pericytes seem to regulate the formation of tight junctions by the interaction with endothelial cells even without the support of astrocytes. Additionally, we suggest that the hyaloid vessel is a valuable system that can be utilized for the investigation of cell-cell interaction in the formation of tight junctions in developing vasculatures.     

小鼠肝血窦内皮细胞分离与鉴定新方法

目的:改进小鼠原代肝血窦内皮细胞的分离方法。方法:经过小鼠肝脏的原位灌洗、消化制备单细胞悬液、差速离心、密度梯度离心以及免疫磁珠分选等步骤,分离获得小鼠原代肝血窦内皮细胞,再通过流式细胞仪鉴定、细胞内吞功能染色以及对细胞超微结构的电子显微镜观察,对分离出的肝血窦内皮细胞进行鉴定。结果:肝血窦内皮细胞的平均得率为5.6×10~6个/只小鼠,细胞活性比率约为96%左右;细胞流式鉴定结果显示新鲜分离出的肝血窦内皮细胞VEGFR3阳性率达到95.8%,VEGFR2+CD31+双阳性细胞阳性率达到93.7%。分选出的LSECs能够有效吞噬FITC-FSA和Dil-Ac-LDL。培养1天后肝血窦内皮细胞的微观结构,可见其特征性的窗孔和筛板。结论:本文总结的分离方法可以稳定、高效地获得小鼠原代肝血窦内皮细胞。     

Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells

Tubulogenic transformation of a nontubulogenic endothelial cell line NP31 by a constitutively activated form of the Flt-1 kinase (NP31/kinase) was accompanied by an increased expression of Nox1 by sixfold over NP31. Overexpression of Nox1 in NP31 cells (NP31/Nox1) stimulated branching morphogenesis in Matrigel but surprisingly cords lacked a lumen. The branching morphogenesis by NP31/kinase and NP31/Nox1 cells was blocked either by N-acetyl-l-cysteine (NAC) or Tiron. Vascular endothelial growth factor (VEGF)-dependent sinusoidal endothelial cells (SEC) in primary culture showed fivefold increase in Nox1 expression 4 days after VEGF stimulation. Interestingly, VEGF-resistant apoptosis in SEC at day 7 was inhibited by NAC or by anti-Nox1 siRNA. These results suggest that Nox1 regulates apoptosis in SEC and can potentially stimulate branching morphogenesis in SEC-derived NP 31 cells.     

Neovessel formation promotes liver fibrosis via providing latent transforming growth factor-β

Aim

Hepatic fibrosis and angiogenesis occur in parallel during the progression of liver disease. Fibrosis promotes angiogenesis via inducing vascular endothelial growth factor (VEGF) from the activated hepatic stellate cells (HSCs). In turn, increased neovessel formation causes fibrosis, although the underlying molecular mechanism remains undetermined. In the current study, we aimed to address a role of endothelial cells (ECs) as a source of latent transforming growth factor (TGF)-β, the precursor of the most fibrogenic cytokine TGF-β.

Methods

After recombinant VEGF was administered to mice via the tail vein, hepatic angiogenesis and fibrogenesis were evaluated using immunohistochemical and biochemical analyses in addition to investigation of TGF-β activation using primary cultured HSCs and liver sinusoidal ECs (LSECs).

Results

In addition to increased hepatic levels of CD31 expression, VEGF-treated mice showed increased α-smooth muscle actin (α-SMA) expression, hepatic contents of hydroxyproline, and latency associated protein degradation products, which reflects cell surface activation of TGF-β via plasma kallikrein (PLK). Liberating the PLK-urokinase plasminogen activator receptor complex from the HSC surface by cleaving a tethering phosphatidylinositol linker with its specific phospholipase C inhibited the activating latent TGF-β present in LSEC conditioned medium and subsequent HSC activation.

Conclusion

Neovessel formation (angiogenesis) accelerates liver fibrosis at least in part via provision of latent TGF-β that activated on the surface of HSCs by PLK, thereby resultant active TGF-β stimulates the activation of HSCs.     

Liver Sinusoidal Endothelial Cells Escape Senescence by Loss of p19ARF

Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestrations under physiological conditions, which are reduced upon chronic liver injury. Cultivation of rodent LSECs associates with a rapid onset of stress-induced senescence a few days post isolation, which limits genetic and biochemical studies ex vivo. Here we show the establishment of LSECs isolated from p19^ARF-/- mice which undergo more than 50 cell doublings in the absence of senescence. Isolated p19^ARF-/- LSECs display a cobblestone-like morphology and show the ability of tube formation. Analysis of DNA content revealed a stable diploid phenotype after long-term passaging without a gain of aneuploidy. Notably, p19^ARF-/- LSECs express the endothelial markers CD31, vascular endothelial growth factor receptor (VEGFR)-2, VE-cadherin, von Willebrand factor, stabilin-2 and CD146 suggesting that these cells harbor and maintain an endothelial phenotype. In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19^ARF-/- LSECs to respond to anti-angiogenic therapeutics. From these data we conclude that loss of p19^ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics. Thus, p19^ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.     

An immortalized human liver endothelial sinusoidal cell line for the study of the pathobiology of the liver endothelium

Background

The endothelium lines blood and lymph vessels and protects underlying tissues against external agents such as viruses, bacteria and parasites. Yet, microbes and particularly viruses have developed sophisticated ways to bypass the endothelium in order to gain access to inner organs. De novo infection of the liver parenchyma by many viruses and notably hepatitis viruses, is thought to occur through recruitment of virions on the sinusoidal endothelial surface and subsequent transfer to the epithelium. Furthermore, the liver endothelium undergoes profound changes with age and in inflammation or infection. However, primary human liver sinusoidal endothelial cells (LSECs) are difficult to obtain due to scarcity of liver resections. Relevant derived cell lines are needed in order to analyze in a standardized fashion the transfer of pathogens across the liver endothelium. By lentiviral transduction with hTERT only, we have immortalized human LSECs isolated from a hereditary hemorrhagic telangiectasia (HHT) patient and established the non-transformed cell line TRP3. TRP3 express mesenchymal, endothelial and liver sinusoidal markers. Functional assessment of TRP3 cells demonstrated a high capacity of endocytosis, tube formation and reactivity to immune stimulation. However, TRP3 displayed few fenestrae and expressed C-type lectins intracellularly. All these findings were confirmed in the original primary LSECs from which TRP3 were derived suggesting that these features were already present in the liver donor. We consider TRP3 as a model to investigate the functionality of the liver endothelium in hepatic inflammation in infection.     

Brivanib,a multitargeted small-molecule tyrosine kinase inhibitor,suppresses laser-induced CNV in a mouse model of neovascular AMD

In age-related macular degeneration (AMD), choroidal neovascularization (CNV), a major pathologic feature of neovascular AMD (nAMD), affects 10% of patients, potentially causing serious complications, including vision loss. Vascular endothelial growth factor receptor 2 (VEGFR2) and fibroblast growth factor receptor 1 (FGFR1) contribute to the pathogenesis of CNV. Brivanib is an oral selective dual receptor tyrosine kinase (RTK) inhibitor of FGFRs and VEGFRs, especially VEGFR2 and FGFR1. In this study, brivanib inhibited zebrafish embryonic angiogenesis without impairing neurodevelopment. In a mouse CNV model, brivanib intravitreal injection blocked phosphorylation of FGFR1 and VEGFR2 and reduced CNV leakage, area, and formation without causing intraocular toxicity. Moreover, brivanib oral gavage reduced CNV leakage and area. Accordingly, brivanib remained at high concentrations (above 14,000 ng/ml) in retinal/choroidal/scleral tissues following intravitreal injection. Similarly, brivanib remained at high concentrations (over 10,000 ng/ml) in retinal/choroidal/scleral tissues following oral gavage. Finally, in vitro cell experiments demonstrated that brivanib inhibited the proliferation, migration and tube formation of microvascular endothelial cells. In conclusion, our study suggested that brivanib treatment could be a novel therapeutic strategy for nAMD.