Genetic analysis of blood vessel formation role of endothelial versus smooth muscle cells

Formation of new blood vessels is vital during embryogenesis, essential for reproduction and wound healing during adulthood, and required to rescue tissue during ischemia. Neovascularization may, however, also contribute to the pathogenesis of several disorders, including tumorigenesis, diabetic vasculopathy, and chronic inflammation. Initially, blood vessels form as endothelium-lined channels by in situ differentiation of endothelial cells. Subsequently, they sprout and remodel into a highly organized and interconnected vascular network. During further maturation of the blood vessels, a sheet of primitive vascular smooth muscle cells surrounds the endothelium-lined channels, which controls endothelial cell function and provides structural support. Recent molecular analyses have identified candidate molecules that affect these processes. Their in vivo role has been further established by targeted gene manipulation in transgenic mice. This review highlights recent developments in the genetic analysis of blood vessel formation, as deduced from analysis of gene-inactivated mice. (Trends Cardiovasc Med 1997;7:271–281). © 1997, Elsevier Science Inc.     

Scatter factor induces blood vessel formation in vivo.

Scatter factor (also known as hepatocyte growth factor) is a glycoprotein secreted by stromal cells that stimulates cell motility and proliferation. In vitro, scatter factor stimulates vascular endothelial cell migration, proliferation, and organization into capillary-like tubes. Using two different in vivo assays, we showed that physiologic quantities of purified native mouse scatter factor and recombinant human hepatocyte growth factor induce angiogenesis (the formation of new blood vessels). The angiogenic activity was blocked by specific anti-scatter factor antibodies. Scatter factor induced cultured microvascular endothelial cells to accumulate and secrete significantly increased quantities of urokinase, an enzyme associated with development of an invasive endothelial phenotype during angiogenesis. We further showed that immunoreactive scatter factor is present surrounding sites of blood vessel formation in psoriatic skin. These findings suggest that scatter factor may act as a paracrine mediator in pathologic angiogenesis associated with human inflammatory disease.     

Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation

Granulation tissue formation is an example of new tissue development in an adult. Its rich vascular network has been thought to derive via angiogenic sprouting and extension of preexisting vessels from the surrounding tissue. The possibility that circulating cells of hematopoietic origin can differentiate into vascular endothelial cells (ECs) in areas of vascular remodeling has recently gained credibility. However, no quantitative data have placed the magnitude of this contribution into a physiological perspective. We have used hematopoietic chimeras to determine that 0.2% to 1.4% of ECs in vessels in control tissues derived from hematopoietic progenitors during the 4 months after irradiation and hematopoietic recovery. By contrast, 8.3% to 11.2% of ECs in vessels that developed in sponge-induced granulation tissue during 1 month derived from circulating hematopoietic progenitors. This recruitment of circulating progenitors to newly forming vessels would be difficult to observe in standard histological studies, but it is large enough to be encouraging for attempts to manipulate this contribution for therapeutic gain.     

Fibroblasts potentiate blood vessel formation partially through secreted factor TIMP-1

During wound repair, new blood vessels form in response to angiogenic signals emanating from injured tissues. Dermal fibroblasts are known to play an important role in wound healing, and have been linked to angiogenesis; therefore, we sought to understand the mechanisms through which these cells control blood vessel formation. Using a three-dimensional angiogenesis assay we demonstrate that dermal fibroblasts enhance the tube-forming potential of endothelial cells, and this augmentation is partially due to secreted factors present in conditioned media. Interestingly, we identified tissue inhibitor of metalloproteinase-1 (TIMP-1) as a factor uniquely secreted by fibroblasts, and addition of exogenous TIMP-1 increased vessel assembly. The enhancing activity of TIMP-1 was matrix metalloproteinase (MMP)-dependent, since a mutant version of TIMP-1 was unable to promote angiogenesis. Consistent with this, chemical inhibition of MMP-2/9 showed a similar increase in angiogenesis, and addition of exogenous MMP-9 blocked the enhancing effect of TIMP-1. We further demonstrated that TIMP-1 inhibits the production of tumstatin, an anti-angiogenic fragment of collagen IV that is produced by MMP-9 cleavage. Our results support the notion that dermal fibroblasts regulate blood vessel formation through multiple mediators, and provide novel evidence that fibroblast-derived TIMP-1 acts on endothelial cells in a pro-angiogenic capacity.     

Characterization of peripheral blood stem cells in mice

Peripheral blood stem cells (PBSCs) were mobilized in mice by treatment with cytosine-arabinoside on day 0, followed by the administration by injection of granulocyte colony-stimulating factor for 4 days. There were remarkable increases in the numbers of cells with lineage-negative (Lin-) c-kit+ markers, cells with colony-forming unit-cell (CFU-C) and colony-forming unit-spleen (CFU-S) activities, and cells with marrow- repopulating ability (MRA) in the extramedullary sites (the spleen, peripheral blood, and liver) on day 5, whereas the number of these immature hematopoietic cells decreased in the bone marrow (BM) on day 5. This finding suggests the mobilization of immature hematopoietic cells from the BM to the extramedullary sites. Three-color flow cytometric analyses showed that CD4 antigen was not expressed on the Lin-Sca-1+ cells in the mobilized PB cells (PBCs), although CD4lo cells were found in those of normal BM cells. Lin-c-kit+ cells in the mobilized PBCs contained more cells with immature phenotypes (Sca-1+, Thy1.2lo, CD71-, and Rh123dull) than in normal BMCs, indicating an alteration of the hierarchical composition of the Lin-c-kit+ cells. The Lin-c-kit+Sca-1+ cells in the mobilized PBCs had similar CFU-C and CFU- S activities to those in normal BMCs. Electron microscopic studies of these cells in the mobilized PBCs showed that only 10% to 20% of these cells had a thin rim of cytoplasm with poorly developed organelles. Allogeneic transplantation [B6 --> C3H] of PBSCs showed long-term reconstituting activity across the major histocompatibility complex barrier 24 weeks after transplantation, although longer observation is necessary.     

Autografts with peripheral blood stem cells

This paper reports a clinical and laboratory experience of peripheral blood stem cell (PBSC) autografts at a single institute. Twenty-eight children with various types of cancer underwent a total of 90 leukaphereses to collect PBSC and 17 of them subsequently received marrow-ablative therapy and PBSC autografts. We found that frozen-thawed progenitor dose is important in determining the rate of hematopoietic recovery after transplantation; in 11 patients who received more than 1 x 10(5) CFU-GM/kg, the granulocyte count reached to 0.5 x 10(9)/L in two weeks. With conditioning chemotherapy without total body irradiation, 6 of 14 patients with high-risk acute leukemia or non-Hodgkin's lymphoma have survived disease-free 3 to 25 months posttransplant. This approach may have a potential to induce prolonged remission-interval and ultimate cure.     

Non-haematological uses of cord blood stem cells

Embryonic stem (ES) cell therapies are often promoted as the optimal stem cell source for regenerative medicine applications because of their ability to develop into any tissue in the body. Unfortunately, ES cell applications are currently limited by ethical, political, biological and regulatory hurdles. However, multipotent non-ES cells are available in large numbers in umbilical cord blood (CB). CB stem cells are capable of giving rise to hematopoietic, epithelial, endothelial and neural tissues both in vitro and in vivo . Thus, CB stem cells are amenable to treat a wide variety of diseases including cardiovascular, ophthalmic, orthopaedic, neurological and endocrine diseases. In addition, the recent use of CB in several regenerative medicine clinical studies has demonstrated its pluripotent nature. Here we review the latest developments in the use of CB in regenerative medicine. Examples of these usages include cerebral palsy and type I diabetes. The numbers of individuals affected with each of these diseases are estimated at 10 000 infants diagnosed with cerebral palsy annually and 15 000 youths diagnosed with type 1 diabetes annually. A summary of the initial results from such clinical studies using autologous cord blood stem cells will be presented.     

Microbial contamination of cord blood stem cells

BACKGROUND AND OBJECTIVES: After storage, low levels of contaminating bacteria in standard blood components can reach bacteraemic levels, causing severe transfusion-associated sepsis. For cord blood (CB), the significance of low levels of contaminating bacteria and the optimal detection method is unknown and not supported by available guidelines. MATERIALS AND METHODS: Spiking experiments and testing of various subfractions of CB units were used to determine the behaviour of bacteria during centrifugation, freezing and thawing. For routine testing of CB, different volumes were compared for the detection of potential pathogens and micro-organisms of low pathogenicity. RESULTS: Centrifugation, as applied to CB fractionation, does not show concentration of bacteria in any particular fraction and supports the possibility of culture of waste fractions. Dimethylsulphoxide (DMSO) and freezing does not affect the viability of bacteria under the conditions used in this study. Owing to the low contamination level, a large sample volume of 20 ml was more sensitive than a 10-ml sample volume. Eighty five per cent of the isolated strains can be considered to be of low pathogenicity. CONCLUSION: When an optimal waste fraction sample volume of 20 ml was cultured, the contamination rate of CB was found to be approximately 13%, with low levels of < 1 colony-forming unit (CFU)/ml. Such levels of bacteria of low pathogenicity are expected to be of clinical importance only when CB is expanded in vitro for a prolonged period of time.     

Properties of peripheral blood and cord blood stem cells.

Mobilized peripheral blood and cord blood are used for transplantation in adults and children. Currently methods which assess the engraftment potential of these cells rely on nucleated cell count, clonogenic colony assays (GM-CFC) and CD34+ cell enumeration. However, data have accumulated which indicate that the cells responsible for short-term and long-term engraftment are different and may be identified by a variety of techniques, including immunophenotyping, in vitro and in vivo assays. There is also evidence that primitive cells in peripheral blood progenitor cell grafts and cord blood are heterogeneous, as cells with similar functional behaviour express different phenotypes. Despite intensive research, the isolation and identification of a homogeneous population of human stem cells is still elusive. Nevertheless, it is possible to obtain CD34+ subpopulations enriched in primitive cells with many of the properties expected of stem cells. Using these cell fractions, the cytokines that induce proliferation, amplification, differentiation and self-renewal are being defined in order to develop improved protocols for expansion of specific populations. From these studies a number of interesting facts have emerged. Certain growth factors frequently used for progenitor cell expansion and gene transduction studies also induce differentiation and impair long-term engraftment. Further, the cytokines required for progenitor cell expansion are probably different to those which favour expansion of the primitive cells, with both the cell cycle status of CD34+ cells as well as the implication of telomere shortening probably needing to be considered where ex vivo manipulation is contemplated.     

外周造血干细胞转化为肝样干细胞的研究

目的探讨外周血造血干细胞在体外转化为肝样干细胞的诱导条件。方法选取自愿行造血干细胞移植符合入选标准患者,用血细胞分离机采集经粒细胞集落刺激因子(recombinant human granulocytecolony stimulating factor,G-CSF)+化疗药物动员后患者的外周血单个核细胞,通过流式细胞仪及免疫磁珠法筛选出CD34+的单个核细胞。将CD34+的单个核细胞分为对照组、肝细胞生长因子(hepatic growth factor,HGF)组、成纤维细胞生长因子4(fibroblast growth factor,FGF4)组、FGF4+HGF组、HGF+FGF4+巨噬细胞集落刺激因子(macrophage colony stimulating factor,MCSF)组。动态观察细胞形态学变化,15 d后免疫细胞化学检测Oct4、ALB、AFP、c-Kit、CK19及CD34在各组细胞中的表达。结果 HGF+FGF4+MCSF组:见大量类圆形细胞,贴壁生长,核仁明显,核质比例大,部分细胞串珠样生长; FGF4+HGF组:见少量类圆形细胞,余细胞形变明显;对照组、HGF组、FGF4组:仅个别细胞呈类圆形,大部分死亡。HGF+FGF4+MCSF组:Oct4、c-Kit、CK19、CD34阳性表达,AFP弱阳性,ALB阴性; FGF4+HGF组:细胞AFP、ALB阳性表达,CK19、CD34弱阳性表达,Oct4、c-Kit阴性;对照组、HGF组、FGF4组:检测指标均无表达。结论经HGF、FGF4及MCSF联合诱导后的外周血CD34+单个核细胞更易转化为肝样干细胞。