SDF-1α inhibits hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells through PI3K/Akt and ERK1/2 signaling pathways

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be a promising cell sources for cardiac regeneration. Poor survival rate of transplanted BMSCs in infarcted myocardium attenuated its clinical application. It’s reported that stromal-derived factor-1 (SDF-1) could protect progenitor cells including endothelial progenitor cells and embryonic stem cells from apoptosis. But little is known whether SDF-1α protein has the same protective effects on BMSCs under conditions of hypoxia and serum deprivation (hypoxia/SD). In present study, we verified that SDF-1α (0.50–2.0 μg/ml) inhibited hypoxia/SD induced apoptosis of BMSCs through mitochondrial pathway. After administration of SDF-1α, the loss of mitochondrial membrane potential and cytochrome c released from mitochondria to cytosol were significantly inhibited, and caspase 3 activity also declined. Furthermore, the effect of SDF-1α on mitochondrial pathway was neutralized by using PI3K inhibitor (Wortmannin) and ERK1/2 inhibitor (U0126). Our observations suggested that SDF-1α inhibits hypoxia/SD induced BMSCs apoptosis through PI3K/Akt and ERK1/2 signaling pathways. These data also imply that the anti-apoptotic effect mediated by SDF-1α may enhance cell survival after cell transplantation.     

Stromal cells cultured from omentum express pluripotent markers,produce high amounts of VEGF,and engraft to injured sites

When rat omentum becomes activated by intraperitoneal injection of inert polydextran particles, these particles are rapidly surrounded by cells that express markers of adult stem cells (SDF–1α, CXCR4, WT–1) and of embryonic pluripotent cells (Oct–4, Nanog, SSEA–1). We have cultured such cells, because they may offer a convenient source of adult stem cells, and have found that they retain stem cell markers and produce high levels of vascular endothelial growth factor for up to ten passages. After systemic or local injection of these cultured cells into rats with acute injury of various organs, the cells specifically engraft at the injured sites. Thus, our experiments show that omental stromal cells can be cultured from activated omentum, and that these cells exhibit stem cell properties enabling them to be used for repair and possibly for the regeneration of damaged tissues.     

CXCR-4 desensitization is associated with tissue localization of hemopoietic progenitor cells

The chemokine stroma-derived factor (SDF)-1, and its receptor, CXCR-4, have been shown to be essential for the translocation of hemopoietic stem cells from the fetal liver to the bone marrow (BM). We hypothesized that if CXCR-4 plays a crucial role in the localization of human hemopoiesis, stem cells from distinct tissue sources should demonstrate distinct CXCR-4 expression or signaling profiles. CD34(+) cells from BM were compared with blood: either mobilized peripheral blood or umbilical cord blood. Unexpectedly, significantly higher levels of CXCR-4 surface expression on CD34(+) cells from blood sources, mobilized peripheral blood, or cord blood were observed compared with BM (p = 0.0005 and p = 0.002, respectively). However, despite lower levels of CXCR-4, responsiveness of the cells to SDF-1 as measured by either calcium flux or transmigration was proportionally greatest in cells derived from BM. Further, internalization of CXCR-4 in response to ligand, associated with receptor desensitization, was significantly lower on BM-derived cells. Therefore, preserved chemokine receptor signaling was highly associated with marrow rather than blood localization. To test the functional effects of perturbing CXCR-4 signaling, adult mice were exposed to the methionine-SDF-1beta analog that induces prolonged down-regulation/desensitization of CXCR-4 and observed mobilization of Lin(-), Sca-1(+), Thy-1(low), and c-kit(+) hemopoietic progenitor cells to the peripheral blood with a >30-fold increase compared with PBS control (p = 0.0007 day 1 and p = 0.004 day 2). These data demonstrate that CXCR-4 expression and function can be dissociated in progenitor cells and that desensitization of CXCR-4 induces stem cell entry into the circulation.     

Expression of tenascin-C and the integrin α9 subunit in regeneration of rat nasal mucosa after chemical injury: involvement in migration and proliferation of epithelial cells

 Nasal mucosa covered by pseudostratified ciliated epithelia can be injured by microbial infection and physical and chemical agents. To elucidate mechanisms of regeneration, erosion of rat nasal mucosa was produced by intranasal instillation of trichloroacetic acid, and tissue specimens were then sequentially obtained after 1–14 days. Since tenascin-C (TN-C) and its receptor, α9β1 integrin, are assumed to play important roles in regeneration of stratified squamous epithelia, their expression was evaluated by immunohistochemistry and in situ hybridization. Three to five days after the injury, TN-C mRNA was found in epithelial cells of migrating fronts and in epithelial sheets recovering ulcerated surfaces between the fronts and normal regions. TN-C deposition was increased under such sheets. Enhanced α9 staining was also evident in the involved epithelium. 5-Bromo-2’-deoxyuridine incorporation assays revealed significant increase in proliferating cells in cell sheets over TN-C deposits at 3–7 days. Therefore, we conclude that regenerating epithelial cells produce and secrete TN-C, associated with an increase in α9 expression, and that interactions between these molecules could regulate migration and proliferation of the epithelial cells in an autocrine manner. Accepted: 18 December 1998     

Stromal cell-derived factor 1alpha (SDF-1alpha) induces gene-expression of early growth response-1 (Egr-1) and VEGF in human arterial endothelial cells and enhances VEGF induced cell proliferation

Abstract. Stromal cell-derived factor-1 (SDF-1), mainly known as a chemotactic factor for haematopoietic progenitor cells, also provides angiogenetic potency. Since the intracellular signalling of SDF-1-induced neovascularization remains unclear, we studied in human umbilical arterial endothelial cells (HUAEC) the influence of SDF-1α on induction of the genes of early growth response-1 (Egr-1) and VEGF, as well as the activation of extracellular regulated kinases (ERK) 1/2, which are all known to be involved in endothelial cell proliferation. We found a time-dependent induction of Egr-1 and VEGF mRNA expression and phosphorylation of ERK1/2 by SDF-1α. Furthermore, we demonstrated that Egr-1 expression is dependent on ERK 1/2 activation. Finally, we tried to confirm the relevance of the induced gene expression by detecting the [3H]thymidine incorporation as a marker for cell proliferation in HUAEC after stimulation with SDF-1α alone or together with VEGF. This particular test showed, that SDF-1α alone has no effect, but is able to significantly enhance VEGF induced DNA synthesis. In summary, SDF-1α is involved in different steps of endothelial cell proliferation, but, since Egr-1 and VEGF offer different functions, it may also play a so far undefined role on other conditions of the endothelium.     

Apelin-13 increases myocardial progenitor cells and improves repair postmyocardial infarction

Apelin is an endogenous ligand for the angiotensin-like 1 receptor (APJ) and has beneficial effects against myocardial ischemia-reperfusion injury. Little is known about the role of apelin in the homing of vascular progenitor cells (PCs) and cardiac functional recovery postmyocardial infarction (post-MI). The present study investigated whether apelin affects PC homing to the infarcted myocardium, thereby mediating repair and functional recovery post-MI. Mice were infarcted by coronary artery ligation, and apelin-13 (1 mg·kg(-1)·day(-1)) was injected for 3 days before MI and for 14 days post-MI. Homing of vascular PCs [CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/stromal cell-derived factor (SDF)-1α(+), and CD133(+)/CXC chemokine receptor (CXCR)-4(+)] into the ischemic area was examined. Myocardial Akt, endothelial nitric oxide synthase (eNOS), VEGF, jagged1, notch3, SDF-1α, and CXCR-4 expression were assessed at 24 h and 14 days post-MI. Functional analyses were performed on day 14 post-MI. Mice that received apelin-13 treatment demonstrated upregulation of SDF-1α/CXCR-4 expression and dramatically increased the number of CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/SDF-1α(+), and c-Kit(+)/CXCR-4(+) cells in infarcted hearts. Apelin-13 also significantly increased Akt and eNOS phosphorylation and upregulated VEGF, jagged1, and notch3 expression in ischemic hearts. This was accompanied by a significant reduction of myocardial apoptosis. Furthermore, treatment with apelin-13 promoted myocardial angiogenesis and attenuated cardiac fibrosis and hypertrophy together with a significant improvement of cardiac function at 14 days post-MI. Apelin-13 increases angiogenesis and improves cardiac repair post-MI by a mechanism involving the upregulation of SDF-1α/CXCR-4 and homing of vascular PCs.     

SDF-1 Enhances Wound Healing of Critical-Sized Calvarial Defects beyond Self-Repair Capacity

Host blood circulating stem cells are an important cell source that participates in the repair of damaged tissues. The clinical challenge is how to improve the recruitment of circulating stem cells into the local wound area and enhance tissue regeneration. Stromal-derived factor-1 (SDF-1) has been shown to be a potent chemoattractant of blood circulating stem cells into the local wound microenvironment. In order to investigate effects of SDF-1 on bone development and the repair of a large bone defect beyond host self-repair capacity, the BMP-induced subcutaneous ectopic bone formation and calvarial critical-sized defect murine models were used in this preclinical study. A dose escalation of SDF-1 were loaded into collagen scaffolds containing BMP, VEGF, or PDGF, and implanted into subcutaneous sites at mouse dorsa or calvarial critical-sized bone defects for 2 and 4 weeks. The harvested biopsies were examined by microCT and histology. The results demonstrated that while SDF-1 had no effect in the ectopic bone model in promoting de novo osteogenesis, however, in the orthotopic bone model of the critical-sized defects, SDF-1 enhanced calvarial critical-sized bone defect healing similar to VEGF, and PDGF. These results suggest that SDF-1 plays a role in the repair of large critical-sized defect where more cells are needed while not impacting de novo bone formation, which may be associated with the functions of SDF-1 on circulating stem cell recruitment and angiogenesis.     

Cellular basis of tissue regeneration by omentum

The omentum is a sheet-like tissue attached to the greater curvature of the stomach and contains secondary lymphoid organs called milky spots. The omentum has been used for its healing potential for over 100 years by transposing the omental pedicle to injured organs (omental transposition), but the mechanism by which omentum helps the healing process of damaged tissues is not well understood. Omental transposition promotes expansion of pancreatic islets, hepatocytes, embryonic kidney, and neurons. Omental cells (OCs) can be activated by foreign bodies in vivo. Once activated, they become a rich source for growth factors and express pluripotent stem cell markers. Moreover, OCs become engrafted in injured tissues suggesting that they might function as stem cells.Omentum consists of a variety of phenotypically and functionally distinctive cells. To understand the mechanism of tissue repair support by the omentum in more detail, we analyzed the cell subsets derived from the omentum on immune and inflammatory responses. Our data demonstrate that the omentum contains at least two groups of cells that support tissue repair, immunomodulatory myeloid derived suppressor cells and omnipotent stem cells that are indistinguishable from mesenchymal stem cells. Based on these data, we propose that the omentum is a designated organ for tissue repair and healing in response to foreign invasion and tissue damage.     

Stromal cell derived factor-1α enhances bone formation based on in situ recruitment: a histologic and histometric study in rabbit calvaria

Histological methods were used to assess whether in situ recruitment using stromal cell derived factor-1α (SDF-1α) enhances bone formation. Four defects were created in the calvarias of 16 rabbits and filled with: (1) a blood clot only (group C); (2) autogenous bone particles (AB, 0.2 ml) (group AB); (3) AB (0.1 ml) + bone marrow derived stromal stem cells (group ABC); or (4) AB (0.1 ml) + SDF-1α (group ABS). Bone formation was significantly greater in groups AB and ABC compared with group ABS after 2 weeks (P < 0.05). Bone formation was similar between groups AB, ABC, and ABS after 4 weeks (P > 0.05). SDF-1α is a promising candidate for in situ recruitment in bone regeneration.     

Human progenitor cells from bone marrow or adipose tissue produce VEGF, HGF, and IGF-I in response to TNF by a p38 MAPK-dependent mechanism

Accumulating evidence suggests that progenitor cells may decrease destructive inflammation and reduce tissue loss by antiapoptotic mechanisms. However, they remain poorly characterized, and many questions remain regarding the mechanisms by which they may positively affect wound healing, tissue remodeling, or tissue regeneration. It has been speculated that various growth factors are responsible, but what components of the wound milieu stimulate progenitor cell production of growth factors and by what mechanisms? We hypothesized that tumor necrosis factor-alpha (TNF-alpha) stimulated progenitor cell secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor I (IGF-I) by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. Human mesenchymal stem cells (hMSCs) and human adipose progenitor cells (hAPCs) were divided into four groups: control, p38 MAPK inhibitor (p38MKI), TNF, and TNF + p38MKI. After 24 h of incubation, supernatants were harvested for ELISA of VEGF, HGF, and IGF-I. Cells were collected for Western blot analysis of p38 MAPK activation. Secretion of VEGF, HGF, and IGF-I in hMSCs and hAPCs was significantly increased by stimulation with TNF and was associated with increased activation of p38 MAPK. The p38 MAPK inhibitor decreased production of TNF-stimulated VEGF, HGF, and IGF-I in hMSCs and hAPCs. However, p38 MAPK inhibitor alone had no effect on production of growth factors. These data demonstrate that progenitor cells are potent sources of VEGF, HGF, and IGF-I. TNF, a prominent tissue cytokine, strongly stimulated production of growth factors by hMSCs and hAPCs via a p38 MAPK-dependent mechanism.     

Overexpression of angiopoietin-1 increases CD133+/c-kit+ cells and reduces myocardial apoptosis in db/db mouse infarcted hearts

Hematopoietic progenitor CD133(+)/c-kit(+) cells have been shown to be involved in myocardial healing following myocardial infarction (MI). Previously we demonstrated that angiopoietin-1(Ang-1) is beneficial in the repair of diabetic infarcted hearts. We now investigate whether Ang-1 affects CD133(+)/c-kit(+) cell recruitment to the infarcted myocardium thereby mediating cardiac repair in type II (db/db) diabetic mice. db/db mice were administered either adenovirus Ang-1 (Ad-Ang-1) or Ad-β-gal systemically immediately after ligation of the left anterior descending coronary artery (LAD). Overexpression of Ang-1 resulted in a significant increase in CXCR-4/SDF-1α expression and promoted CD133(+)/c-kit(+), CD133(+)/CXCR-4(+) and CD133(+)/SDF-1α(+) cell recruitment into ischemic hearts. Overexpression of Ang-1 led to significant increases in number of CD31(+) and smooth muscle-like cells and VEGF expression in bone marrow (BM). This was accompanied by significant decreases in cardiac apoptosis and fibrosis and an increase in myocardial capillary density. Ang-1 also upregulated Jagged-1, Notch3 and apelin expression followed by increases in arteriole formation in the infarcted myocardium. Furthermore, overexpression of Ang-1 resulted in a significant improvement of cardiac functional recovery after 14 days of ischemia. Our data strongly suggest that Ang-1 attenuates cardiac apoptosis and promotes cardiac repair by a mechanism involving in promoting CD133(+)/c-kit(+) cells and angiogenesis in diabetic db/db mouse infarcted hearts.     

VEGF和SDF-1 的协同作用对内皮祖细胞增殖与迁移的影响

目的:研究血管内皮生长因子(VEGF)和基质衍生因子-1(SDF-1)的协同作用对高血压脑出血患者内皮祖细胞(EPCs)增殖迁移能力的影响。方法:采集急性期高血压脑出血患者与健康对照的外周静脉血,分离培养外周血中的EPCs。用分别含有不同VEGF与SDF-1的培养基处理患者外周血分离的EPCs,Western blot检测各组细胞以及患者和对照中VEGF和SDF-1的蛋白表达。MTT法检测细胞增值能力,Transwell法检测细胞迁移能力,分别转染VEGF-si RNA与SDF-1-si RNA至各组细胞观察抑制VEGF和SDF-1对EPCs增殖迁移能力的影响。结果:VEGF和SDF-1在患者中的表达显著高于健康对照组。VEGF和SDF-1对EPCs的增殖和迁移能力有促进作用,且在其协同作用下效果显著。抑制VEGF或SDF-1显著降低VEGF和SDF-1对EPCs增殖迁移能力的促进作用。结论:VEGF和SDF-1的协同作可促进急性期高血压脑出血患者EPCs的细胞增殖能力和迁移能力,对患者血管修复提供新的治疗方向。     

Different physiology of interferon-α/-γ in models of liver regeneration in the rat

Liver regeneration may take place after liver injury through replication of hepatocytes or hepatic progenitor cells called oval cells. Interferons (IFN) are natural cytokines with pleiotrophic effects including antiviral and antiproliferative actions. No data are yet available on the physiology and cellular source of natural IFNs during liver regeneration. To address this issue, we have analyzed the levels and biologic activities of IFN-α/IFN-γ in two models of partial hepatectomy. After 2/3rd partial hepatectomy (PH), hepatic levels of IFN-α and IFN-γ declined transiently in contrast to a transient increase of the IFN-γ serum level. After administration of 2-acetylaminofluorene and partial hepatectomy (AAF/PH model), however, both IFN-α and IFN-γ expression were up-regulated in regenerating livers. Again, the IFN-γ serum level was transiently increased. Whereas hepatic IFN-γ was up-regulated early (day 1–5), but not significantly, in the AAF/PH model, IFN-α was significantly up-regulated at later time points in parallel to the peak of oval cell proliferation (days 7–9). Biological activity of IFN-α was shown by activation of IFN-α-specific signal transduction and induction of IFN-α specific-gene expression. We found a significant infiltration of the liver with inflammatory monocyte-like mononuclear phagocytes (MNP) concomitant to the frequency of oval cells. We localized IFN-α production only in MNPs, but not in oval cells. These events were not observed in normal liver regeneration after standard PH. We conclude that IFN-γ functions as an acute-phase cytokine in both models of liver regeneration and may constitute a systemic component of liver regeneration. IFN-α was increased only in the AAF/PH model, and was associated with proliferation of oval cells. However, oval cells seem not to be the source of IFN-α. Instead, inflammatory MNP infiltrating AAF/PH-treated livers produce IFN-α. These inflammatory MNPs may be involved in the regulation of the oval cell compartment through local expression of cytokines, including IFN-α.     

CD133: Enhancement of Bone Healing by Local Transplantation of Peripheral Blood Cells in a Biologically Delayed Rat Osteotomy Model

Sufficient angiogenesis is crucial during tissue regeneration and therefore also pivotal in bone defect healing. Recently, peripheral blood derived progenitor cells have been identified to have in addition to their angiogenic potential also osteogenic characteristics, leading to the hypothesis that bone regeneration could be stimulated by local administration of these cells. The aim of this study was to evaluate the angiogenic potential of locally administered progenitor cells to improve bone defect healing. Cells were separated from the peripheral blood of donor animals using the markers CD34 and CD133. Results of the in vitro experiments confirmed high angiogenic potential in the CD133(+) cell group. CD34(+) and CD133(+) cells were tested in an in vivo rat femoral defect model of delayed healing for their positive effect on the healing outcome. An increased callus formation and higher bone mineral density of callus tissue was found after the CD133(+) cell treatment compared to the group treated with CD34(+) cells and the control group without cells. Histological findings confirmed an increase in vessel formation and mineralization at day 42 in the osteotomy gap after CD133(+) cell transplantation. The higher angiogenic potential of CD133(+) cells from the in vitro experients therefore correlates with the in vivo data. This study demonstrates the suitability of angiogenic precursors to further bone healing and gives an indication that peripheral blood is a promising source for progenitor cells circumventing the problems associated with bone marrow extraction.     

Informing tendon tissue engineering with embryonic development

Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering.     

17-AAG,a Hsp90 inhibitor,attenuates the hypoxia-induced expression of SDF-1α and ILK in mouse RPE cells

The aim of this study was to investigate the changes of SDF-1α and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1α and ILK. RPE cells were cultured with 200 μmol/L cobalt chloride (CoCl₂) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1α and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1α and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1α and ILK expression in vitro. Our results indicated that SDF-1α and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1α and ILK expression.     

Morphological and cytological diversity of regenerants derived from half-anther cultures of anthurium

Anthurium anther culture was successfully established using half-anthers as explants. Explants were cultured on Winarto–Teixeira basal medium (WT-1) containing 0.01 mg/l α-naphthalene acetic acid (NAA), 0.5 mg/l thidiazuron (TDZ), and 1.0 mg/l 6-benzylaminopurine (BAP), or on New Winarto–Teixeira basal medium (NWT-3) supplemented with 0.02 mg/l NAA, 1.5 mg/l TDZ, and 0.75 mg/l BAP for callus initiation. Regenerated calli produced multiple shoots on WT-1, which were then rooted in NWT-3 supplemented with 1% activated charcoal. Plantlets were acclimatized ex vitro using a mixture of burned rice husk, rice husk, and bamboo peat (1:1:1, v/v/v) as the potting medium. There was considerable morphological and cytological diversity of regenerants derived from anther culture, which are described in detail in this study. The callus cluster color ranged from green to light green and had a high regeneration capacity (7.3 and 4.8 shoots/callus cluster), light reddish-yellow callus showed moderate regeneration (2.6 shoots/callus cluster), while reddish-yellow callus had the lowest regeneration capacity (1.5 shoots/callus cluster). Morphological variations clearly observed in regenerants derived from this technique included alterations in plant size, peduncle length, spathe position compared to leaves, the type and number of buds, spathe and spadix color, and spadix length. There were also cytological variations in both in vitro and ex vitro regenerants of anther culture with 23–29% haploids, 5–10% aneuploids, 56–69% diploids, and 3–4% triploids. The results strengthen other studies in which the development of anther cultures, especially via callus formation, resulted in morphological and cytological alterations. These variations have been discussed to great length in this paper.     

Cell contact interactions in rheumatology, The Kennedy Institute for Rheumatology, London, UK, 1-2 June 2000

The intricate interactions that regulate relationships between endogenous tissue cells and infiltrating immune cells in the rheumatic joint, particularly in rheumatoid arthritis (RA), were the subject of the meeting. A better understanding of these interactions might help to define intervention points that could be used to develop specific therapies. The presentations and discussions highlighted the fact that, once chronic inflammation is established, several pro-inflammatory loops involving tumour necrosis factor (TNF)-α and interleukin (IL)-1β can be defined. Direct cellular contact with stimulated T lymphocytes induces TNF-α and IL-1β in monocytes which in turn induce functions in fibroblast-like synoviocytes. The latter include the production of stromal cell-derived factor-1α (SDF-1α) which enhances the expression of CD40L in T cells, which stimulates SDF-1α production in synoviocytes, which in turn protects T and B cells from apoptosis and enhances cell recruitment thus favoring inflammatory processes. IL-1β and TNF-α also induce IL-15 in fibroblast-like synoviocytes, which induces the production of IL-17 which in turn potentiates IL-1β and TNF-α production in monocyte-macrophages. This underlines the importance of TNF-α and IL-1β in RA pathogenesis, and helps explain the efficiency of agents blocking the activity of these cytokines in RA. Factors able to block the induction of cytokine production (such as apolipoprotein A-I [apo A-I] and interferon [IFN]-β) might interfere more distally in the inflammatory process. Furthermore, stimulated T lymphocytes produce osteoclast differentiation factor (ODF), which triggers erosive functions of osteoclasts. Therefore, factors capable of affecting the level of T lymphocyte activation, such as IFN-β, IL-15 antagonist, or SDF-1α antagonist, might be of interest in RA therapy.     

An<Emphasis Type="Italic">in vitro</Emphasis> method to study the effects of hematopoietic regulators during immune and blood cell development

In adults, hematopoiesis occurs in bone marrow (BM) through a complex process with differentiation of hematopoietic stem cells (HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34. Methods on hematopoietic regulation are presented to show the effects of the chemokine, stromal-derived growth factor (SDF)-1α and the neuropeptide, substance P (SP). SDF-1α production in BM stroma causes interactions with HSCs, thereby retaining the HSCs in regions close to the endosteum, at low oxygen. Small changes in SDF-1α levels stimulate HSC functions through direct and indirect mechanisms. The indirect method occurs by SP production, which stimulates CD34⁺ cells, supported by ligand-binding studies, long-term culture-initiating cell assays for HSC functions, and clonogenic assays for myeloid progenitors. These methods can be applied to study other hematopoietic regulators.     

Inhibition of tumor specific angiogenesis by amentoflavone

The formation of new capillaries from existing blood vessels is critical for tumor growth and metastasis. In this study we report that amentoflavone, a biflavonoid from Biophytum sensitivum, could inhibit the process of angiogenesis. Amentoflavone at nontoxic concentrations (0.05–0.2 μg/ml) showed significant inhibition in the proliferation, migration, and tube formation of endothelial cells, which are key events in the process of angiogenesis. In vivo studies in C57BL/6 mice using amentoflavone showed remarkable inhibition (52.9%) of tumor directed capillary formation. Amentoflavone showed inhibitory effect on the production of various endogenous factors such as IL-1β, IL-6, TNF-α, GM-CSF, and VEGF that control the process of angiogenesis. Amentoflavone treatment could increase the production of IL-2 and TIMP-1, which could successfully shift the equilibrium towards an angiostatic condition. The antiangiogenic activity of amentoflavone was supported by its remarkable suppression in sprouting of microvessels from rat aorta. Our results also show that amentoflavone could inhibit the production of VEGF mRNA in B16–F10 cells. These findings indicate that amentoflavone inhibits angiogenesis by disrupting the integrity of endothelial cells and by altering the endogenous factors that are required for the process of neovascularization. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 2, pp. 258–269.