当归对高脂血清所致ECV304细胞损伤的保护作用

实验观察了高脂血清对培养的人脐静脉内皮细胞（ECV304）的损伤及传统中药当归的保护作用,以探讨当归的抗动脉粥样硬化作用及其可能机制,培养人脐静脉内皮细胞,以高脂血清作损伤因子,用扫描电镜观察细胞的超微结构,分光光度法检测细胞培养液中一氧化氮（NO）的含量,免疫细胞化学方法检测细胞表面细胞间粘附分子－1（ICAM－1）,碱性成纤维细胞生长因子（bFGF)及转化生长因子β1（TGFβ）的表达,与高脂血清孵育24h后,内皮细胞的超微结构明显收损,且细胞表面ICAM－1,bFGF的表达明显增加,而细胞培养液中NO的量及细胞表达TGFβ1明显减少,加入当归后,高脂血清对内皮细胞的这些作用均可被逆转,当归对内皮细胞中ICAM－1,bFGF,TGFβ及NO表达改变的影响可能与其抗动脉粥样硬化的作用有关。     

Alterations in plasma angiogenic growth factor concentrations after coronary artery bypass graft surgery: relationships with post-operative complications

To determine whether angiogenic growth factor levels are altered during and after cardiac surgery, plasma concentrations of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and transforming growth factor beta1 (TGFbeta1) were measured in 32 patients undergoing coronary artery bypass graft (CABG) surgery with extracorporeal circulation (ECC). EGF levels significantly decreased during ECC and remained low until the 24th post-operative hour with no difference between complicated and uncomplicated patients. TGFbeta1 and bFGF concentrations significantly increased at the end of ECC and after cross-clamp release, and returned to pre-operative values at the 6th post-operative hour suggesting that the source of these elevations are the lungs and heart. After cross-clamp release bFGF levels but not TGFbeta1 ones were higher in patients with respiratory impairments. VEGF values increased significantly at the 6th and 24th post-operative hours. At the 24th post-operative hour plasma VEGF levels were higher in patients with cardiovascular and hematological impairments. In conclusion, these results highlight that the angiogenic network is profoundly altered in patients undergoing cardiopulmonary bypass as previously demonstrated for lipidic, cytokine and haematopoietic growth factor ones and identify an association between specific post-CABG complications and systemic release of bFGF and VEGF.     

Immunoenzymometric analysis for expression and shedding of intercellular adhesion molecule-1 on human endothelial cells stimulated with cytokines or lipopolysaccharide

Unstimulated endothelial cell (EC)cultures express low levels of intercellular adhesion molecule-1 (ICAM-1) and their expression can be enhanced by inflammatory cytokines such as tumor necrosis factor (TNF). Three monoclonal antibodies (MoAbs) highly reactive with TNF-stimulated human ECs were established and defined to recognize a 95 kDa cell surface protein specifically expressed on cytokine-activated ECs, which was immunochemically identified as ICAM-1. The quantitative immunoassay of soluble and insoluble ICAM-1 could be performed with two different MoAbs. Secretion of fibronectin or the von Willebrand factor, was not significantly enhanced with TNF stimulation. Cellular expression of ICAM-1 was drastically induced by TNF or interleukin-1 stimulation, and the moderate expression with delayed-action was observed only by lipopolysaccharide stimulation. A maximal amount of soluble ICAM-1 was released from ECs stimulated only by TNF, apparently in a dose dependent manner, but no significant release of ICAM-1 was induced by thrombin interleukin-2, or lipopolysacchardes. Released levels of soluble ICAM-1 from interleukin-1-stimulated ECs were apparently diminished as compared with those from TNF-stimulated cells. These results suggest that release of soluble ICAM-1 from EC surfaces can be most significantly enhanced by TNF-specific signaling, and prospectively, should be a sensitive indicator of intravascular inflammation in acute endothelium injury.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Dissecting signaling pathways that govern self-renewal of rabbit embryonic stem cells

The pluripotency and self-renewal of embryonic stem cells (ESC) are regulated by a variety of cytokines/growth factors with some species differences. We reported previously that rabbit ESC (rESC) are more similar to primate ESC than to mouse ESC. However, the signaling pathways that regulate rESC self-renewal had not been identified. Here we show that inhibition of the transforming growth factor beta (TGFbeta), fibroblast growth factor (FGF), and canonical Wnt/beta-catenin (Wnt) pathways results in enhanced differentiation of rESC accompanied by down-regulation of Smad2/3 phosphorylation and beta-catenin expression and up-regulation of phosphorylation of Smad1 and beta-catenin. These results imply that the TGFbeta, FGF, and Wnt pathways are required for rESC self-renewal. Inhibition of the MAPK/ERK and PI3K/AKT pathways, which lie downstream of the FGF pathway, led to differentiation of rESC accompanied by down-regulation of phosphorylation of ERK1/2 or AKT, respectively. Long-term self-renewal of rESC could be achieved by adding a mixture of TGFbeta ligands (activin A, Nodal, or TGFbeta1) plus basic FGF (bFGF) and Noggin in the absence of serum and feeder cells. Our findings also suggest that there is a regulatory network consisting of the FGF, Wnt, and TGFbeta pathways that controls rESC pluripotency and self-renewal. We conclude that bFGF controls the stem cell properties of rESC both directly and indirectly through TGFbeta or other pathways, whereas the effect of Wnt on rESC might be mediated by the TGFbeta pathway.     

Attenuation of leukocyte-endothelium interaction by antioxidant enzymes

This report assessed the effect of overexpressing Cu,Zn superoxide dismutase (SOD) and/or catalase on the interaction of mononuclear cells (MNCs) and endothelial cells (ECs). ECs were obtained from the aorta of wild-type mice and transgenic mice overexpressing Cu,ZnSOD and/or catalase. MNCs were obtained from wild-type mice. Treatment of wild-type ECs with CuSO4-oxidized low-density lipoprotein (oxLDL) significantly elevated the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and increased the adherence of MNCs. Overexpression of Cu,ZnSOD and/or catalase in ECs attenuated the adherence of MNCs and the expression of cell adhesion molecules induced by oxLDL. For example, ECs overexpressing Cu,ZnSOD and/or catalase showed significantly less expression of VCAM-1 and ICAM-1 and less number of adherent MNCs than wild-type ECs. Moreover, ECs overexpressing Cu,ZnSOD and catalase in combination showed significantly less expression of VCAM-1 and ICAM-1 and less number of adherent MNCs than those overexpressing either Cu,ZnSOD or catalase alone. These results suggest that combinational overexpression of Cu,ZnSOD and catalase can reduce the expression of cell adhesion molecules and inhibit the adherence of leukocyte to ECs more efficiently than overexpression of Cu,ZnSOD or catalase alone.     

Growth factor-dependent proliferation and invasion of muscle satellite cells require the cell-associated fibrinolytic system

The process of muscle regeneration in normal and dystrophic muscle depends on locally produced cytokines and growth factors and requires the activity of the urokinase plasminogen activator/urokinase plasminogen activator receptor/plasminogen activator inhibitor-1 system. In this study we tested the effect of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and transforming growth factor-beta (TGFbeta) on the fibrinolytic pattern of normal and dystrophic satellite cells, their mitogenic and motogenic activities and the dependence of such activities on the cell-associated fibrinolytic system. We have observed that the urokinase plasminogen activator (u-PA) receptor is weakly upregulated by bFGF in normal satellite cells, while it is strongly up-regulated by TGFbeta, mainly in dystrophic myoblasts. bFGF up-regulated u-PA in both normal and dystrophic myoblasts grown in primary culture, while a striking down-regulation was observed with TGFbeta. TGFbeta was the only growth factor able to exceptionally up-regulate plasminogen activator inhibitor-1 (PAI-1), mainly in dystrophic satellite cells. HGF did not show any activity on the fibrinolytic system. Proliferation and invasion into Matrigel matrices of normal and dystrophic cells occurred regardless of the growth factor-dependent regulation of the fibrinolytic system. Nevertheless, each growth factor required the efficiency of the constitutive cell-associated fibrinolytic system to operate, as shown by impairment of growth factor activity with antagonists of u-PA and of its receptor. Noteworthy, TGFbeta induced a dose-dependent increase of Matrigel invasion only in dystrophic myoblasts. Since TGFbeta-challenged dystrophic myoblasts undergo an exceptional up-regulation of the receptor and of PAI-1, we propose the possibility that the TGFbeta-induced fibrinolytic pattern (low urokinase plasminogen activator, high receptor and high PAI-1) may be exploited to promote survival and spreading of transplanted engineered myoblasts in Duchenne muscular dystrophy.     

Vascular endothelial growth factor up-regulates ICAM-1 expression via the phosphatidylinositol 3 OH-kinase/AKT/Nitric oxide pathway and modulates migration of brain microvascular endothelial cells

Endothelium of the cerebral blood microvessels, which constitutes the major component of the blood-brain barrier, controls leukocyte and metastatic cancer cell adhesion and trafficking into the brain parenchyma. In this study, using rat primary brain microvascular endothelial cells (BMEC), we demonstrate that the vascular endothelial growth factor (VEGF), a potent promoter of angiogenesis, up-regulates the expression of the intracellular adhesion molecule-1 (ICAM-1) through a novel pathway that includes phosphatidylinositol 3 OH-kinase (PI3K), AKT, and nitric oxide (NO), resulting in the migration of BMEC. Upon VEGF treatment, AKT is phosphorylated in a PI3K-dependent manner. AKT activation leads to NO production and release and activation-deficient AKT attenuates NO production stimulated by VEGF. Transfection of the constitutive myr-AKT construct significantly increased basal NO release in BMEC. In these cells, VEGF and the endothelium-derived NO synergistically up-regulated the expression of ICAM-1, which was mediated by the PI3K pathway. This activity was blocked by the PI3K-specific inhibitor, wortmannin. Furthermore, VEGF and NO significantly increased BMEC migration, which was mediated by the up-regulation of ICAM-1 expression and was dependent on the integrity of the PI3K/AKT/NO pathway. This effect was abolished by wortmannin, by the specific ICAM-1 antibody, by the specific inhibitor of NO synthase, N(G)-l-monomethyl-arginine (l-NMMA) or by a combination of wortmannin, ICAM-1 antibody, and l-NMMA. These findings demonstrate that the angiogenic factor VEGF up-regulates ICAM-1 expression and signals to ICAM-1 as an effector molecule through the PI3K/AKT/NO pathway, which leads to brain microvessel endothelial cell migration. These observations may contribute to a better understanding of BMEC angiogenesis and the physiological as well as pathophysiological function of the blood-brain barrier, whose integrity is crucial for normal brain function.     

Bronchial epithelial cell matrix production in response to silica and basic fibroblast growth factor

BACKGROUND: Previous studies show that macrophages, lung fibroblasts, and their soluble mediators are responsible for the onset and development of pulmonary fibrosis. This study was conducted to determine whether airway epithelial cells are also directly involved in response to fibrogenic agents and consequently in the pathogenesis of lung fibrosis. To verify the hypothesis, we determined whether silica acts directly on human bronchial epithelial cells by stimulating cytokine and growth factor release and by modifying matrix production. MATERIALS AND METHODS: An SV40 large T antigen-transformed human airway epithelial cell line, 16HBE14o (16HBE), was used. The expression profile of some proinflammatory interleukins (ILs), such as IL-1alpha, IL-1beta and IL-6 and their modulation by silica, were evaluated by polymerase chain reaction (PCR) analysis. Transforming growth factor beta (TGFbeta) and basic fibroblast growth factor (bFGF) mRNA levels were tested by Northern blotting in the presence and in the absence of silica. The silica- and/or bFGF-induced effects on matrix components (total proteins, collagen, and fibronectin) were also evaluated using radio-labeled precursors. RESULTS: The results demonstrated 16HBE internalized silica particles. Silica induced a little IL-6 secretion, without affecting IL-1 and TGFbeta isoform production and strongly stimulated bFGF mRNA level and bFGF protein secretion. Silica also induced changes in 16HBE production of total proteins, collagen, and fibronectin production. When added in combination with the growth factor, it strengthened bFGF stimulation of matrix component secretion. CONCLUSIONS: These results support the hypothesis that the changes in matrix components are due to a direct effect of silica on bronchial epithelial cells. Silica-induced over-secretion of bFGF suggests that autocrine and paracrine differentiation loops for bFGF may also be operative and that these mechanisms may be involved in the pathogenesis of pulmonary fibrosis. In the future, cytokine-directed therapeutic strategies might find a place in clinical practice.     

LKB1 in endothelial cells is required for angiogenesis and TGFbeta-mediated vascular smooth muscle cell recruitment

Inactivation of the tumor suppressor kinase Lkb1 in mice leads to vascular defects and midgestational lethality at embryonic day 9-11 (E9-E11). Here, we have used conditional targeting to investigate the defects underlying the Lkb1(-/-) phenotype. Endothelium-restricted deletion of Lkb1 led to embryonic death at E12.5 with a loss of vascular smooth muscle cells (vSMCs) and vascular disruption. Transforming growth factor beta (TGFbeta) pathway activity was reduced in Lkb1-deficient endothelial cells (ECs), and TGFbeta signaling from Lkb1(-/-) ECs to adjacent mesenchyme was defective, noted as reduced SMAD2 phosphorylation. The addition of TGFbeta to mutant yolk sac explants rescued the loss of vSMCs, as evidenced by smooth muscle alpha actin (SMA) expression. These results reveal an essential function for endothelial Lkb1 in TGFbeta-mediated vSMC recruitment during angiogenesis.     

Angiogenic interaction between retinal endothelial cells and pericytes from normal and diabetic rabbits, and phenotypic changes of diabetic cells.

Retinal endothelial cells (ECs) and pericytes (PCs) were cloned and cultured from normal and diabetic rabbits to clarify the mechanism of diabetic proliferative retinopathy from the viewpoint of the interaction between ECs and PCs, and phenotypic changes of diabetic cells. PC-conditioned medium (PC-CM) from normal rabbits stimulated in vitro angiogenesis of diabetic ECs more than that of normal ECs. in vitro angiogenesis was also more stimulated in diabetic ECs than in normal ECs by basic fibroblast growth factor (bFGF) or transforming growth factor-beta 1, indicating that diabetic ECs are different from normal ECs in terms of angiogenic potential. One mechanism of this property of diabetic ECs was the acceleration of cell proliferation but not of cell migration, because diabetic ECs grew more rapidly but did not migrate more than normal ECs in response to PC-CM or bFGF. Moreover, PC-CM from diabetic PCs stimulated angiogenesis of normal ECs more than that from normal PCs, indicating that diabetic PCs secreted more angiogenic factor(s) than normal PCs. The angiogenic, mitogenic and migratory activities of PC-CM both from normal and diabetic PCs were similarly inhibited by an anti-bFGF antibody. Western blot analysis revealed this factor to be a bFGF-like molecule. These data indicate that the interaction between ECs and PCs and the phenotypic changes of diabetic ECs and PCs both contribute to the proliferative retinopathy in diabetes.     

串珠素表达下调参与低氧对大鼠心肌微血管内皮细胞增殖的抑制作用

低氧可以抑制内皮细胞增殖,但是其机理目前尚不清楚。串珠素在调节内皮细胞增殖中发挥着重要作用。为了探讨串珠素是否参与低氧对内皮细胞增殖的抑制,将大鼠心肌微血管内皮细胞在低氧或常氧状态下培养12 h后,用实时定量RT-PCR方法检测串珠素mRNA的表达。结果发现：低氧可以明显抑制串珠素mRNA的表达,与常氧状态下串珠素mRNA表达水平比较,差异显著（P〈0．05）。与此同时,低氧状态下或用串珠素抗体中和内源性串珠素,内皮细胞的增殖和对成纤维细胞生长因子的反应明显降低,粘着斑激酶（focal adhesion kinase,FAK）表达和细胞外信号调节激酶1／2（extracellular signal- regulated kinase,ERK1／2）活性明显下降。结果提示,串珠素表达下调可能通过抑制FAK介导的ERK1/2依赖的信号转导途径,参与低氧对大鼠心肌微血管内皮细胞增殖的抑制作用。     

Effects of TGFbeta and bFGF on the differentiation of human bone marrow stromal fibroblasts

Adipocytes and osteoblasts have common origins from fibroblastic stem cells. Consequently, modulation of the processes of adipogenesis and osteogenesis has implications for the possible treatment of metabolic bone diseases, such as osteoporosis, in which medullary fat accumulates and trabecular bone volume decreases. It is likely that the balance between these two systems is affected by particular endogenous growth factors which are known to affect bone metabolism. We have therefore investigated the effects of transforming growth factor beta (TGFbeta), basic fibroblast growth factor (bFGF) and dexamethasone (Dex) on cultured human bone marrow (HBM) fibroblastic cells to observe the effects on adipogenesis and osteogenesis. In the absence of fetal calf serum (FCS), TGFbeta caused a dose-dependent increase in cell growth and alkaline phosphatase activity (AP); however, in the presence of FCS growth was inhibited at high concentrations and AP unaffected. TGFbeta increased matrix proteoglycan and collagen synthesis. bFGF inhibited AP and increased colony number and size, while Dex treatment increased AP activity and colony number, and both factors in combination resulted in an additive increase in growth. Dex-induced adipocyte formation was accelerated but not increased by bFGF. A significant inhibition of adipogenesis by TGFbeta was observed within 7 days. These results demonstrate the importance of biological factors known to be involved in bone remodelling in the regulation of osteogenesis and adipogenesis.     

Microparticle-Induced Activation of the Vascular Endothelium Requires Caveolin-1/Caveolae

Microparticles (MPs) are small membrane fragments shed from normal as well as activated, apoptotic or injured cells. Emerging evidence implicates MPs as a causal and/or contributing factor in altering normal vascular cell phenotype through initiation of proinflammatory signal transduction events and paracrine delivery of proteins, mRNA and miRNA. However, little is known regarding the mechanism by which MPs influence these events. Caveolae are important membrane microdomains that function as centers of signal transduction and endocytosis. Here, we tested the concept that the MP-induced pro-inflammatory phenotype shift in endothelial cells (ECs) depends on caveolae. Consistent with previous reports, MP challenge activated ECs as evidenced by upregulation of intracellular adhesion molecule-1 (ICAM-1) expression. ICAM-1 upregulation was mediated by activation of NF-κB, Poly [ADP-ribose] polymerase 1 (PARP-1) and the epidermal growth factor receptor (EGFR). This response was absent in ECs lacking caveolin-1/caveolae. To test whether caveolae-mediated endocytosis, a dynamin-2 dependent process, is a feature of the proinflammatory response, EC’s were pretreated with the dynamin-2 inhibitor dynasore. Similar to observations in cells lacking caveolin-1, inhibition of endocytosis significantly attenuated MPs effects including, EGFR phosphorylation, activation of NF-κB and upregulation of ICAM-1 expression. Thus, our results indicate that caveolae play a role in mediating the pro-inflammatory signaling pathways which lead to EC activation in response to MPs.     

Expression of cell adhesion molecules and lymphocyte-endothelium interaction under simulated hypogravity in vitro

Using histochemical staining and FACS-analysis we have studied the basal and TNF-alpha induced expression of E-selectin, ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (ECs) exposed to simulated hypogravity. Control ECs did not contain detectable amounts of E-selectin or VCAM-1 but were ICAM-1 positive. As soon as after 6-8 hrs of clinorotation at 5 RPM the cellular content of ICAM- 1 increased. Moreover, hypogravity potentiated the effect of inflammatory cytokines (TNF-alpha and IL-1) on ICAM-1 expression. No increase in E-selectin or VCAM-1 expression was observed in ECs exposed to hypogravity itself. However, hypogravity reduced E-selectin and VCAM-1 expression in cell cultures activated by cytokines, more visible at their low (5-10 U/ml) concentrations. Both, control and clinorotated ECs poorly supported spontaneous lymphocyte adhesion; the adhesion of PMA-activated leukocytes was 15-20-fold higher. The interaction of unstimulated lymphocytes with cytokine-activated endothelium was more noticeable but significantly lower in cultures exposed to hypogravity. Activated blood cells interacted with endothelium more effectively, particularly, under hypogravity. Obtained results suggest that EC adhesion molecule expression and endothelium-lymphocyte interaction are altered under simulated hypogravity conditions in direction of increase of endotlielial adhesiveness for activated blood cells.     

Endothelial microparticles reduce ICAM‐1 expression in a microRNA‐222‐dependent mechanism

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE−/− mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced.     

Culture of endothelial cells by transfection with plasmid harboring vascular endothelial growth factor

Vascular endothelial cells (ECs) are usually difficult to culture in a large scale because of their complicated requirements for cell growth. As the vascular endothelial growth factor (VEGF) is a key growth factor in the EC culture, we transfected human umbilical vein endothelial cells (HUVEC) using a plasmid containing VEGF gene and let them grow in a culture medium eliminated an important supplement, endothelail cell growth supplement (ECGS). The expression of VEGF by HUVEC tansfected with VEGF gene was not enough to stimulate the growth of HUVEC, only 40% of maximum cell density obtainable in the presence of ECGS., However, when the culture medium was supplied with 2.5 ng/mL of basic fibroblast growth factor (bFGF), a synergistic effect of VEGF and bFGF was observed. In this case, the final cell density was recovered up to about 78% of maxium value.