Endothelial progenitor cell transplantation improves the survival following liver injury in mice

BACKGROUND & AIMS: Neovascularization, which is vital to the healing of injured tissues, recently has been found to include both angiogenesis, which involves in mature endothelial cells, and vasculogenesis, involving endothelial progenitor cells. The aim of this study was to clarify the possible roles of endothelial progenitor cells during postnatal liver regeneration. METHODS: To determine how endothelial progenitor cells participate in liver regeneration, human or mouse endothelial progenitor cells were transplanted into the mice with carbon tetrachloride-induced acute liver injury. Survival rate of the mice in endothelial progenitor cell-transplanted and control groups was calculated. Separately, livers removed temporally from both groups were examined. RESULTS: At an early stage, transplanted human endothelial progenitor cells were seen mainly surrounding hepatic central veins where hepatocytes showed extensive necrosis; later, the transplanted cells formed tubular structures. More of these cells were observed along hepatic sinusoids. Transplantation of human or mouse endothelial progenitor cells improved survival of the mice following liver injury (from 28.6% to 85.7%, P < .0005 and from 33.3% to 80.0%, P < .001, respectively), accompanied by greater proliferation of hepatocytes. Human endothelial progenitor cells produced several growth factors, such as hepatocyte growth factor, transforming growth factor-alpha, heparin-binding epidermal growth factor-like growth factor, and vascular endothelial growth factor, and also elicited endogenous growth factors. CONCLUSIONS: Endogenous and exogenous growth factors and direct neovascularization after endothelial progenitor cell transplantation promoted liver regeneration, thus improving survival after liver injury. Transplantation of endothelial progenitor cells could represent a new therapeutic strategy for promoting liver regeneration.     

Angiogenesis in liver regeneration and fibrosis: “a double-edged sword”

Angiogenesis, defined as the formation of new microvasculature from preexisting blood vessels and mature endothelial cells, plays a major role in wound healing and scar formation, and it is associated with inflammatory responses. Angiogenesis can occur in physiological conditions, such as during liver regeneration, and in pathological situations, such as during the progression of fibrosis to cirrhosis and also during tumor angiogenesis. Cellular cross-talk among liver sinusoidal endothelial cells (LSECs), hepatic stellate cells and hepatocytes is believed to play an important role in the angiogenesis process during both liver regeneration and development of cirrhosis. In addition to mature endothelial cells, bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs) have been recently identified for their contribution to post-natal vasculogenesis/angiogenesis. In vivo, EPCs are mobilized into the peripheral blood in response to tissue ischemia or traumatic injury, migrate to the sites of injured endothelium and differentiate into mature endothelial cells. In our recent studies, we have explored the role of EPC-mediated angiogenesis in liver regeneration and/or cirrhosis. Results have demonstrated significantly increased endogenous levels of circulating EPCs in cirrhotic patients in comparison to the controls. Also, EPCs from cirrhotic patients have been observed to stimulate substantial angiogenesis by resident LSECs in vitro via paracrine factors such as vascular endothelial growth factor and platelet-derived growth factor. This review gives an overview of the angiogenesis process in liver regeneration and disease and discusses a new mechanism for intrahepatic angiogenesis mediated by BM-derived EPCs.     

The mechanisms of hepatic sinusoidal endothelial cell regeneration: A possible communication system associated with vascular endothelial growth factor in liver cells

Vascular endothelial growth factor (VEGF) has been shown to induce proliferation of sinusoidal endothelial cells in primary culture. To elucidate the mechanisms of sinusoidal endothelial cell regeneration in vivo, mRNA expression of VEGF and its receptors, flt-1 and KDR/flk-1, were studied in rat livers. Northern blot analysis revealed that VEGF-mRNA was expressed in hepatocytes immediately after isolation from normal rats. In contrast, non-parenchymal cells, including sinusoidal endothelial cells, expressed VEGF receptor-mRNA. Vascular endothelial growth factor-mRNA expression in hepatocytes was decreased during primary culture, but increased following a peak of DNA synthesis, induced by addition of epidermal growth factor or hepatocyte growth factor to the culture medium at 24 h of plating. In a 70% resected rat liver, VEGF-mRNA expression increased with a peak at 72 h after the operation, and mRNA expression of VEGF receptors between 72 and 168 h. In such a liver, mitosis was maximal in hepatocytes at 36 h and in sinusoidal endothelial cells at 96 h. Also, mRNA expression of both VEGF and its receptors was significantly increased in carbon tetrachloride-intoxicated rat liver compared with normal rat liver. Vascular endothelial growth factor expression was minimal in Kupffer cells isolated from normal rats, but marked in activated Kupffer cells and hepatic macrophages from the intoxicated rats. Vascular endothelial growth factor-mRNA expression was also increased in activated stellate cells from these rats and in the cells activated during primary culture compared with quiescent cells. We conclude that increased levels of VEGF expression in regenerating hepatocytes may contribute to the proliferation of sinusoidal endothelial cells in partially resected rat liver, probably through VEGF receptors up-regulated on the cells. Also, VEGF derived from activated Kupffer cells, hepatic macrophages and stellate cells may be involved in this proliferation in injured rat liver.     

Sulfated polysaccharides identified as inducers of neuropilin-1 internalization and functional inhibition of VEGF165 and semaphorin3A

Neuropilin-1 (NRP1) and NRP2 are cell surface receptors shared by class 3 semaphorins and vascular endothelial growth factor (VEGF). Ligand interaction with NRPs selects the specific signal transducer, plexins for semaphorins or VEGF receptors for VEGF, and promotes NRP internalization, which effectively shuts down receptor-mediated signaling by a second ligand. Here, we show that the sulfated polysaccharides dextran sulfate and fucoidan, but not others, reduce endothelial cell-surface levels of NRP1, NRP2, and to a lesser extent VEGFR-1 and VEGFR-2, and block the binding and in vitro function of semaphorin3A and VEGF(165). Administration of fucoidan to mice reduces VEGF(165)-induced angiogenesis and tumor neovascularization in vivo. We find that dextran sulfate and fucoidan can bridge the extracellular domain of NRP1 to that of the scavenger receptor expressed by endothelial cells I (SREC-I), and induce NRP1 and SREC-I coordinate internalization and trafficking to the lysosomes. Overexpression of SREC-I in SREC-I-negative cells specifically reduces cell-surface levels of NRP1, indicating that SREC-I mediates NRP1 internalization. These results demonstrate that engineered receptor internalization is an effective strategy for reducing levels and function of cell-surface receptors, and identify certain sulfated polysaccharides as "internalization inducers."     

血管内皮生长因子促进脑缺血后血管和神经发生

血管内皮生长因子是一种强有力的血管生成因子.近年来还发现其具有强大的促进神经再生能力,在治疗缺血性脑血管病中具有潜在价值.新生的血管内皮细胞可形成"血管龛",通过释放某些神经营养因子促进神经发生.同样,新生的神经细胞也可促进血管发生,二者之间存在"交叉对话",血管内皮生长因子在其中发挥着非常重要的媒介作用.文章对血管内皮生长因子促进脑缺血后血管和神经发生方面的研究做了综述.     

Selective induction of neuropilin-1 by vascular endothelial growth factor (VEGF): A mechanism contributing to VEGF-induced angiogenesis

Neuropilin (NRP) 1, previously identified as a neuronal receptor that mediates repulsive growth cone guidance, has been shown recently to function also in endothelial cells as an isoform-specific receptor for vascular endothelial growth factor (VEGF)(165) and as a coreceptor in vitro of VEGF receptor 2. However, its potential role in pathologic angiogenesis remains unknown. In the present study, we first show that VEGF selectively up-regulates NRP1 but not NRP2 via the VEGF receptor 2-dependent pathway. By NRP1 binding analysis, we showed that its induction by VEGF accompanies functional receptor expression. Endothelial proliferation stimulated by VEGF(165) was inhibited significantly by antibody perturbation of NRP1. In a murine model of VEGF-dependent angioproliferative retinopathy, intense NRP1 mRNA expression was observed in the newly formed vessels. Furthermore, selective NRP1 inhibition in this model suppressed neovascular formation substantially. These results suggest that VEGF cannot only activate endothelial cells directly but also can contribute to robust angiogenesis in vivo by a mechanism that involves up-regulation of its cognate receptor expression.     

Protein-protein interaction map is a key gateway into liver regeneration

Recent studies indicate that the process of liver regeneration involves multiple signaling pathways and a variety of genes,cytokines and growth factors. Protein-protein interactions(PPIs)play a role in nearly all events that take place within the cell and PPI maps should be helpful in further understanding the process of liver regeneration.In this review,we discuss recent progress in understanding the PPIs that occur during liver regeneration especially those in the transforming growth factorβsignaling path...     

Neuropilin structure governs VEGF and semaphorin binding and regulates angiogenesis

Neuropilins (NRP) play a central role in neuronal and blood vessel development as receptors for two ligand types, the semaphorin (SEMA) family of axon guidance modulators and the VEGF family of angiogenesis stimulators. The role of NRPs in axon guidance is well documented but a role in blood vessel development is less so. NRPs mediate normal developmental angiogenesis as shown in mouse and zebrafish models, and pathological angiogenesis in tumors and retinal disease. The ability of two disparate ligand families to bind to the same receptor is unusual but may be explainable by analysis of neuropilin structure. There are two NRP genes, nrp1 and nrp2. The NRPs have a relatively large extracellular domain consisting of sub domains, which are ligand binding sites. VEGF₁₆₅ binds to the b1b2 subdomain, SEMA3A and SEMA3F also bind to b1b2 but to a1a2 as well. Mutagenesis studies have identified NRP amino acids that bind VEGF₁₆₅ but not SEMA3F. These NRP structural elements might dictate differential SEMA and VEGF₁₆₅ binding properties, which in turn regulate angiogenesis. This article reviews the latest information of NRP structure and how structure influences angiogenesis. In addition, the role of NRPs in human cancer is addressed. We dedicate this article to the memory of Dr. Judah Folkman, the leading pioneer of angiogenesis research.     

血管内皮生长因子受体与肝细胞癌血管生成关系的研究进展

新生血管是肿瘤生长和转移的基础.肝细胞肝癌是典型的多血管肿瘤,其发生、发展、转移、侵袭都和血管生成密切相关.血管的生成主要依靠血管生长因子和血管生长抑制因子的调控,其中研究最多也是最重要的是血管内皮生长因子(VEGF)及其受体(VEGFR).VEGFR在机体内作用不同,参与肝癌血管生长的主要是VEGFR-1(flt-1)和VEGFR-2(flk-1).针对VEGFR的抗肿瘤血管治疗在实验室取得了不错的疗效,部分已经进入了临床试验.     

Liver cancer: Targeted future options

Hepatocellular carcinoma(HCC)has a poor prognosis an d systemic chemotherapies have disappointing results.The increasing knowledge of the molecular biolog y of HCC has resulted in novel targets,with the vascular endothelial growth factor and epidermal growth factor receptor(EGFR)-related pathways being of special interest.New blood vessel formation(angiogenesis)is essential for the growth of solid tumors.Anti-angiogenic strategies have become an important therapeutic modality for solid tumors.Several agents targeting angiogenesis-related pathways have entered clinical trials or have been already approved for the treatment of solid tumors.These include monoclonal antibodies,receptor tyrosine kinase inhibitors and immunomodulatory drugs.HCC is a highly vascular tumor,and angiogenesis is be-lieved to play an important role in its development and progression.This review summarizes recent advances in the basic understanding of the role of angiogenesis in HCC as well as clinical trials with novel therapeutic approaches targeting angiogenesis and EGFR-related pathways.     

Growth factor- and cytokine-driven pathways governing liver stemness and differentiation

Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/pro-genitor cell expansion and differentiation, and the rel-evance of these signals in liver development, regen...     

Targeting the vascular endothelial growth factor in hematologic malignancies

There exists increasing evidence that apart from solid tumors, angiogenic growth factors also play important roles in the development and/or maintenance of hematolymphoid malignancies. Thus, in these cancers, angiogenesis and bone marrow microvessel density often correlate with prognosis and disease burden. Several reports speculated on the role of angiogenesis and the resulting possible therapeutic options in hematologic malignancies. The most prominent angiogenic factor, vascular endothelial growth factor (VEGF), is expressed in a number of established leukemic cell lines as well as in freshly isolated human leukemias and lymphomas, and several human leukemias express VEGF receptor 1 and/or VEGF receptor 2. VEGF/VEGF‐receptor interactions are also involved in proliferation, migration, and survival of leukemic cells by autocrine and paracrine mechanisms. As a consequence, a possible drugable effect by inhibiting VEGF signaling in different hematologic malignancies has been discussed. This review focuses on angiogenesis‐independent effects of VEGF on survival and proliferation of leukemic or lymphoma cells and on possible therapeutic approaches using anti‐VEGF/VEGF‐receptor therapies to inhibit proliferation or induce apoptosis of malignant cells in hematologic diseases.     

血管内皮生长因子及其受体的生物学特点

<正>血管内皮生长因子(vascular endothelial growth factor,VEGF)是胚胎形成、骨骼生长和生殖功能过程中血管生成的重要调节因子,与肿瘤、眼内新生血管性等疾病有关[1]。VEGF的生物学活性受2种酪氨酸激酶受体(tyrosine kinases)的调节:VEGFR-1和VEGF-2,这2种受体的信号学活性差异非常大。目前,多种VEGF抑制因子在进行恶性肿瘤临床实验,人们试图通过抑制VEGF降低血管生成、血管渗漏等。VEGF和VEGFR     

Translation of PDGF cardioprotective pathways

Vascular function in the aging heart is impaired and may underlie the increased morbidity and mortality associated with ischemic heart disease in older individuals. This vascular dysfunction is due, in part, to impairment of plateletderived growth factor (PDGF)-mediated pathways in senescent cardiac microvascular endothelial cells. Restoration of these pathways by intramyocardial injection of growth factor transiently rescues senescent cardiac angiogenesis. Longer-term reconstitution can be achieved experimentally by transplantation of young bone marrow-derived stem cells to promote senescent cardiac angiogenic function in the murine host. Moreover, enhancement of PDGF pathways is cardioprotective, markedly reducing the extent of myocardial injury following coronary occlusion. The clinical translation of these findings for treatment of ischemic heart diseases must overcome the limitation of the proatherosclerotic actions of PDGF, as well as the generation of autologous stem/precursor cell approaches, for the aging cardiovascular system. Strategies targeting growth factor and/or stem-cell homing to gene products downstream of PDGF in the cardiac microvasculature may provide positive feedback loops to enhance cardiac angiogenesis and protection from myocardial infarction and may offer a foundation for developing novel therapies for the prevention and treatment of cardiovascular disease associated with aging.     

Hepatocyte growth factor: Molecular structure and implications for a central role in liver regeneration

Hepatocyte growth factor (HGF) is a most potent factor for mature parenchymal hepatocytes in primary culture and may act as a trigger for liver regeneration. We purified HGF from rat platelets to homogeneity and cloned both human and rat HGF cDNA. HGF is a heterodimer molecule composed of the 69 kDa alpha-subunit and the 34 kDa beta-subunit. HGF has no amino acid sequence homology with other known peptide growth factors and possesses the highest potential among known growth factors to stimulate proliferation of hepatocytes in primary culture. HGF is derived from a single chain precursor of 728 amino acid residues and the precursor is proteolytically processed to form a two-chain mature HGF. The alpha-subunit of HGF contains 4 kringle structures and HGF has a homology (38%) with plasmin. Biologically active recombinant human HGF could be expressed from COS-1 cells and CHO cells transfected with cloned cDNA. HGF activity and the HGF mRNA level are markedly increased in the liver following insult such as hepatitis, by the administration of hepatotoxins, ischaemia, physical damage and partial hepatectomy. Moreover, HGF mRNA is induced in the lung and kidney, in the presence of liver injury. In situ hybridization revealed that HGF-producing cells in liver are non-parenchymal liver cells, presumably Kupffer and sinusoidal endothelial cells. Therefore, HGF from neighbouring cells (Kupffer and sinsuoidal endothelial cells) and distal organs (lung and kidney) may function as a trigger for liver regeneration by both a paracrine mechanism and an endocrine mechanism. HGF has mitogenic activity for renal tubular epithelial cells, epidermal melanocytes and keratinocytes as well as mature hepatocytes, and has the potential to promote cell migration for some epithelial cells, including normal human keratinocytes. Since cell growth and cell motility are relevant to tissue repair and embryogenesis, HGF may well have important roles in tissue repair and embryogenesis as well as in liver regeneration.     

Biologic therapy of inflammatory bowel disease

Advancing knowledge regarding the biology of chronic inflammation has led to the development of specific biologic therapies that mechanistically target individual inflammatory pathways. Many biologic therapies are being evaluated for the treatment of the chronic inflammatory bowel diseases, Crohn's disease and ulcerative colitis. Biologic compounds proven to be effective for Crohn's disease include monoclonal antibodies to tumor necrosis factor (infliximab and CDP571) and to the leukocyte adhesion molecule alpha4 integrin (natalizumab). Other biologic compounds for which there is insufficient evidence to judge efficacy for inflammatory bowel disease include: p55 tumor necrosis factor binding protein (onercept); interferon alpha; interferon beta-1a; anti-interferon gamma antibody; anti-interleukin 12 antibody; p65 anti-sense oligonucleotide (blocks NF-kappaB); granulocyte colony stimulating factor, and granulocyte macrophage colony stimulating factor; anti-interleukin 2 receptor antibody; epidermal growth factor; keratinocyte growth factor 2 (repifermin); human growth hormone; anti-CD4 antibody; and anti-alpha4beta7 antibody. Biologic therapies that have been proven ineffective for inflammatory bowel disease include: interleukin 10; interleukin 11; anti-sense intercellular adhesion molecule-1; and the tumor necrosis factor receptor fusion protein etanercept. Based on the early successes of infliximab, CDP571 and natalizumab, it seems certain that biologic therapy will play an important role in the future treatment of inflammatory bowel disease.     

神经纤毛蛋白-1调控血管内皮细胞增殖迁移的功能作用和潜在机制研究

目的 研究神经纤毛蛋白-1（Neuropilin-1,NRP1）在血管内皮细胞（Vascular Endothelial Cells, VECs）增殖迁移中的功能作用及潜在机制。 方法 通过基因干扰腺病毒靶向抑制NRP1的表达,行CCK-8实验研究NRP1下调后对VECs增殖活力的影响,采用流式细胞术研究NRP1下调后对VECs凋亡的影响,行Transwell实验研究NRP1下调后对VECs迁移能力的影响;此外,通过Western Blot检测NRP1下调后对其下游信号通路蛋白表达的影响。 结果 CCK-8实验结果显示下调NRP1表达可抑制大鼠VECs的增殖活力,流式细胞凋亡实验结果显示下调NRP1表达可促进大鼠VECs的凋亡,Transwell实验结果显示下调NRP1表达可抑制大鼠VECs的迁移能力。通过Western Blot检测发现NRP1下调后,下游信号通路蛋白Akt、ERK1/2及NF-kB的磷酸化水平均显著降低。 结论 NRP1可能通过激活PI3K/Akt、MAPK/ERK及NF-kB信号通路促进VECs的增殖与迁移,在静脉桥再内皮化进程中发挥潜在的促进作用。     

Angiogenesis: multiple masks in hepatocellular carcinoma and liver regeneration

Hepatocellular carcinoma (HCC) is naturally resistant to radiotherapy and cytotoxic chemotherapy, leaving surgery as the mainstream therapeutic approach. However, the 5-year recurrence rate after curative resection is as high as 61.5%. The background hepatitis B- or C-induced cirrhosis and the presence of micrometastases at the time of surgery have been regarded as two main causes of recurrence. Recently, accumulating evidence suggests that growth factors and cytokines released during the physiological process of post-surgical liver regeneration could induce the activation of dormant micrometastatic lesions. The establishment of neovasculature to support either liver regeneration or HCC growth involves multiple cell types including liver sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and circulating endothelial progenitors. The crosstalks among these cells are driven by multiple molecules and signaling pathways, including vascular endothelial growth factors and their receptors, platelet-derived growth factor, the angiopoietin/Tie family, hepatocyte growth factor/c-Met signaling, and others. Anti-angiogenic agent targeting liver cancer vasculature has been reported to be able to generate limited survival benefit of the patients. In this review, discussions are focused on various angiogenic mechanisms of HCC and liver regeneration, as well as the prevailing anti-angiogenic strategies.