Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells

Human fibroblasts were found to produce a potent mitogen and chemoattractant for fetal bovine aortic endothelial cells. Homogenates from AG1523 and AG1518 foreskin, CCD18Lu lung, and CCD18Co colon fibroblasts produced half-maximal stimulation of endothelial cell growth at concentrations of 1-7 micrograms/ml. The factor was purified from large-scale cultures of the CCD18Co fibroblasts using cation exchange chromatography and heparin-Sepharose chromatography. Such preparations were mitogenic for endothelial cells in vitro at concentrations of about 5-10 ng/ml, and promoted chemotaxis at 0.1-1 ng/ml. Heparinase treatment of the cells prevented the chemotactic response. These properties suggest that the factor may be related to fibroblast growth factor.     

Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein-deficient serum

Recently improved culture conditions for human adult arterial endothelial and smooth muscle cells from a wide variety of donors have been used to study the effects of lipoproteins on proliferation of both cell types in low serum culture medium. Optimal growth of endothelial and smooth muscle cells in an optimal nutrient medium (MCDB 107) containing epidermal growth factor, a partially purified fraction from bovine brain, and 1% (v/v) lipoprotein-deficient serum was dependent on either high- or low-density lipoprotein. High- and low-density lipoprotein stimulated cell growth by three- and five-fold, respectively, over a 6-day period. Optimal stimulation of both endothelial and smooth muscle cell growth occurred between 20 and 60 micrograms/ml of high- and low-density lipoproteins, respectively. No correlation between the activation of 3-hydroxyl-3-methylglutaryl coenzyme. A reductase activity and lipoprotein-stimulated cell proliferation was observed. Lipid-free total apolipoproteins or apolipoprotein C peptides from high-density lipoprotein were partially effective and together with oleic acid effectively replaced native high-density lipoprotein for the support of endothelial cell growth. In contrast, apolipoproteins or apolipoprotein C peptides from high-density lipoprotein alone or with oleic acid had no effect on smooth muscle cell proliferation. The results suggest a functional role of high- and low-density lipoproteins and apolipoproteins in the proliferation of human adult endothelial and smooth muscle cells.     

Acute ethanol exposure disrupts VEGF receptor cell signaling in endothelial cells

Physiological angiogenesis is regulated by various factors, including signaling through vascular endothelial growth factor (VEGF) receptors. We previously reported that a single dose of ethanol (1.4 g/kg), yielding a blood alcohol concentration of 100 mg/dl, significantly impairs angiogenesis in murine wounds, despite adequate levels of VEGF, suggesting direct effects of ethanol on endothelial cell signaling (40). To examine the mechanism by which ethanol influences angiogenesis in wounds, we employed two different in vitro angiogenesis assays to determine whether acute ethanol exposure (100 mg/dl) would have long-lasting effects on VEGF-induced capillary network formation. Ethanol exposure resulted in reduced VEGF-induced cord formation on collagen and reduced capillary network structure on Matrigel in vitro. In addition, ethanol exposure decreased expression of endothelial VEGF receptor-2, as well as VEGF receptor-2 phosphorylation in vitro. Inhibition of ethanol metabolism by 4-methylpyrazole partially abrogated the effect of ethanol on endothelial cell cord formation. However, mice treated with t-butanol, an alcohol not metabolized by alcohol dehydrogenase, exhibited no change in wound vascularity. These results suggest that products of ethanol metabolism are important factors in the development of ethanol-induced changes in endothelial cell responsiveness to VEGF. In vivo, ethanol exposure caused both decreased angiogenesis and increased hypoxia in wounds. Moreover, in vitro experiments demonstrated a direct effect of ethanol on the response to hypoxia in endothelial cells, as ethanol diminished nuclear hypoxia-inducible factor-1alpha protein levels. Together, the data establish that acute ethanol exposure significantly impairs angiogenesis and suggest that this effect is mediated by changes in endothelial cell responsiveness to both VEGF and hypoxia.     

Lipopolysaccharide stimulates monocyte adherence by effects on both the monocyte and the endothelial cell

The effects of the LPS moiety of endotoxin on monocyte adherence to an endothelial cell surface were investigated over times before the development of well described LPS-induced endothelial cell surface adhesive molecules. In an in vitro microtiter adherence assay, LPS in concentrations of 10 ng/ml to 10 micrograms/ml incubated for 20 to 60 min with human monocytes significantly stimulated monocyte adherence to human umbilical vein endothelial cell monolayers (HUVEC) and serum-coated plastic surfaces. The time course and concentration dependence of LPS-stimulated monocyte adherence to glutaraldehyde-fixed HUVEC did not differ significantly from that to unfixed HUVEC or serum-coated plastic surfaces. Pretreatment studies suggested that LPS acted on the monocyte within 25 min to stimulate adherence to untreated endothelial cells but required a minimum of 1.5 to 2 h to render the endothelial cell more adhesive for untreated monocytes. The potential role of TNF-alpha, IL-1 alpha, and IL-1 beta in this system was assessed by determining the ability of these cytokines (+/- cytokine antibodies) to increase monocyte adherence. TNF, but neither IL-1, stimulated early monocyte adherence (1 h). This TNF-stimulated monocyte adherence was abrogated by coincubation with anti-rTNF-alpha polyclonal antibody. However, the anti-rTNF antibody had no effect on LPS-induced monocyte adherence to endothelial cells or serum-coated plastic surfaces. An early action of LPS on the monocyte to induce adherence to endothelial cell surfaces may contribute to the initial localization of peripheral blood monocytes in tissues during endotoxemia. The later effects of LPS on the endothelial cell to stimulate monocyte adherence may then amplify these initial monocyte-endothelial cell interactions to prolong and intensify monocyte adherence prior to migration into tissues.     

Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 microm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-betaII receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.     

The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity

We have previously shown that members of the ELR(+) CXC chemokine family, including IL-8; growth-related oncogenes alpha, beta, and gamma; granulocyte chemotactic protein 2; and epithelial neutrophil-activating protein-78, can mediate angiogenesis in the absence of preceding inflammation. To date, the receptor on endothelial cells responsible for chemotaxis and neovascularization mediated by these ELR(+) CXC chemokines has not been determined. Because all ELR(+) CXC chemokines bind to CXC chemokine receptor 2 (CXCR2), we hypothesized that CXCR2 is the putative receptor for ELR(+) CXC chemokine-mediated angiogenesis. To test this postulate, we first determined whether cultured human microvascular endothelial cells expressed CXCR2. CXCR2 was detected in human microvascular endothelial cells at the protein level by both Western blot analysis and immunohistochemistry using polyclonal Abs specific for human CXCR2. To determine whether CXCR2 played a functional role in angiogenesis, we determined whether this receptor was involved in endothelial cell chemotaxis. We found that microvascular endothelial cell chemotaxis in response to ELR(+) CXC chemokines was inhibited by anti-CXCR2 Abs. In addition, endothelial cell chemotaxis in response to ELR(+) CXC chemokines was sensitive to pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in this biological response. The importance of CXCR2 in mediating ELR(+) CXC chemokine-induced angiogenesis in vivo was also demonstrated by the lack of angiogenic activity induced by ELR(+) CXC chemokines in the presence of neutralizing Abs to CXCR2 in the rat corneal micropocket assay, or in the corneas of CXCR2(-/-) mice. We thus conclude that CXCR2 is the receptor responsible for ELR(+) CXC chemokine-mediated angiogenesis.     

Sphingosine 1-phosphate stimulates cell migration through a G(i)-coupled cell surface receptor. Potential involvement in angiogenesis

Sphingosine 1-phosphate (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (10-100 nM) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-1. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (1999) Exp. Cell Res. 247, 17-28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-1-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-1 and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i)-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-1. Similar to SPP, sphinganine 1-phosphate (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis; whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell G(i)-coupled SPP receptors.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

Rac Regulates Vascular Endothelial Growth Factor Stimulated Motility

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood.

Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF.

These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.     

Endothelial cell migration in stable gradients of vascular endothelial growth factor A and fibroblast growth factor 2: effects on chemotaxis and chemokinesis

Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 microm was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.     

Basic fibroblast growth factor and transforming growth factor beta-1: Synergistic mediators of angiogenesis in vitro

We investigated the relative roles of basic fibroblast growth factor (bFGF) and transforming growth factor beta-1 (TGF-b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two-dimensional Petri dish cultures and a threedimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC₅₀ of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three-dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF, modification of the antiproliferative effect of TGF-b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF-b and 10–20 ng/ml bFGF in two-dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose-dependent manner: invasion was maximal at 0.3–0.7 ng/ml TGF-b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a fibroblast-like appearance with slender cell extensions. We concluded that bFGF and TGF-b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two-dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis. © 1993 Wiley-Liss, Inc.     

New proangiogenic activity on vascular endothelial cells for C-terminal mechano growth factor

Angiogenesis is crucial in wound healing. The administration of the C-terminal 24-a.a. peptide of mechano growth factor (MGF24E) has been previously demonstrated to induce more blood vessels in regenerating bone around defective areas compared with the control. Accordingly, this study aims to determine whether MGF24E promotes bone defect healing through MGF24E-increased angiogenesis and whether MGF24E has positive effects on angiogenesis in vitro. The roles of MGF24E on angiogenesis and the underlying mechanisms were investigated. The cell proliferation, migration, and tubulogenesis of the human vascular endothelial EA.hy926 cells co-treated with 2% serum and MGF24E were determined to assess angiogenesis in comparison with 100 ng/ml of vascular endothelial growth factor 165 (VEGF(165))-positive control or vehicle control (phosphate-buffered saline). MGF24E treatment (10 ng/ml) significantly promoted the biological processes of angiogenesis on EA.hy926 cells compared with the vehicle control. The suppression of vascular endothelial growth factor and angiopoietin-I expressions by 2% serum starvation was reversed by the addition of 10 ng/ml of MGF24E in 2% serum medium. This result suggests that MGF24E has a protective effect on angiogenesis. Moreover, the inhibition of ERK due to PD98050 pretreatment completely abolished and mostly blocked MGF24E-induced proliferation and migration, respectively, whereas the MGF24-induced tubulogenesis and the angiogenic factor expression were only partially inhibited. These new findings suggest that MGF24E promotes angiogenesis by enhancing the expression of angiogenic cytokines which involves the MAPK/ERK-signaling pathway.     

Chemoattractant activity of Entamoeba histolytica for human polymorphonuclear neutrophils

The ability of axenic Entamoeba histolytica trophozoites and amoebic protein preparations to stimulate chemotaxis of human polymorphonuclear neutrophils (PMN) was evaluated. Virulent E. histolytica (strain HM1:IMSS) stimulated chemotaxis (delta distance = 0.55 +/- 0.02 mm, P less than 0.01 vs. control medium). Sonicated (100 micrograms protein/ml) or homogenized (500 micrograms protein/ml) virulent amoebae also significantly stimulated PMN chemotaxis, whereas preparations of the nonpathogenic "Entamoeba-like" Laredo strain did not stimulate chemotaxis. Preparations of subcellular fractions of E. histolytica demonstrated maximal stimulation of PMN chemotaxis existed in nonvesiculated membranes and the supernatant from plasma membranes.     

Monocyte chemotaxis and activating factor production by keratinocytes in response to IFN-gamma

Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.     

Involvement of protein kinase C in the mitogenic and chemotaxis effects of basic fibroblast growth factor on bovine cerebral cortex capillary endothelial cells

Basic fibroblast growth factor is increasingly implicated in cellular growth, differentiation, angiogenesis and oncogenesis. In culture, basic fibroblast growth factor greatly improved the growth rate of bovine brain cortex capillary endothelial cells. Down-regulation of protein kinase C by prolonged treatment with phorbol esters prevented the mitogenic effect of basic fibroblast growth factor on capillary endothelial cells. Furthermore, staurosporine, a potent protein kinase inhibitor, showed strong antiproliferative activity against basic fibroblast growth factor-induced endothelial cell growth. Similarly, the chemotaxis effect of basic fibroblast growth factor on capillary endothelial cells was abolished by down-regulation of protein kinase C or by staurosporine treatment. Therefore, it is suggested that protein kinase C could account for part of the angiogenic effect of basic fibroblast growth factor.     

Isolation and characterization of an inhibitor of neovascularization from scapular chondrocytes

An inhibitor of neovascularization from the conditioned media of scapular chondrocytes established and maintained in serum-free culture has been isolated and characterized. To determine whether this chondrocyte-derived inhibitor (ChDI) was capable of inhibiting neovascularization in vivo, this protein was assayed in the chick chorioallantoic membrane assay. ChDI was a potent inhibitor of angiogenesis in vivo (4 micrograms = 87% avascular zones). This inhibitor is also an inhibitor of fibroblast growth factor-stimulated capillary endothelial cell (EC) proliferation and migration, as well as being an inhibitor of mammalian collagenase. ChDI significantly suppressed capillary EC proliferation in a dose-dependent, reversible manner with an IC50 (the inhibitory concentration at which 50% inhibition is achieved) of 2.025 micrograms/ml. Inhibition by ChDI of growth factor-stimulated capillary EC migration was also observed using a modified Boyden chamber assay (IC50 = 255 ng/ml). SDS-PAGE analysis followed by silver staining of ChDI purified to apparent homogeneity revealed a single band having an M(r) of 35,550. Gel elution experiments demonstrated that only protein eluting at this molecular weight was anti-angiogenic. These studies are the first demonstration that chondrocytes in culture can produce a highly enriched, potent inhibitor of neovascularization which also inhibits collagenase.     

黄芫花提取物对V79细胞和WB肝细胞的生物...：1....

A Chinese herb, wikstroemia Chamaedaphen (WC) extract, recently has been shown to be a potential tumor promoting agent on uterine cervical carcinoma induced by HSV-2 or MCA in mice. To determine whether the tumor promoting effects of WC extract were mediated through inhibition of gap junctional intercellular communication (GJIC) with relation to cellular growth, experiments were conducted on Chinese hamster V79 cells and rat WB liver cells by utilization of SLDT method for GJIC detection and cell growth curve examination, 3H-TdR incorporation, mitotic index (MI) and Flow Cytometry (FCM) methods. TPA was used for comparative purpose. WC extract inhibited GJIC and stimulated cell growth in a dose (2-200 micrograms/ml) and time (0-72 hr)-dependent manner in both cell lines. Both WC extract and TPA treatments increased V79 cell growth rate. The average cell doubling-time was decreased from 36.5 hr in control V79 cells to 28.2 hr in WC extract (10 micrograms/ml) and 20.9 hr in TPA (50 ng/ml) treatment by the 3rd day. Stimulating effect of both drugs on DNA synthesis of V79 cells was demonstrated. The results of FCM and MI indicated that the cell number of M-phase cells was increased after drug treatment. It is suggested that (1) tumor promoting effect of WC extract might be mediated through inhibition of GJIC: (2) inhibition of GJIC is closely correlated with increased cell growth rate and entry of cell division cycle.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

NC1 domain of human type VIII collagen (alpha 1) inhibits bovine aortic endothelial cell proliferation and causes cell apoptosis.

Endostatin, a natural angiogenesis inhibitor, had been identified for years. It opened a new approach for cancer therapy. Sequence analysis revealed that endostatin is the NC1 domain (non-triple-helical domain) of collagen XVIII. In this report, the cDNA of NC1 domain of type VIII collagen (alpha 1) was cloned and expressed as soluble form in Escherichia coli. The recombinant protein was purified with Ni-NTA agarose column and named as vastatin. It inhibited the proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner. The ED(50) of vastatin was 0.6 microg/ml, while the ED(50) of endostatin was 0.5 microg/ml. Treatment of BAE cell with vastatin caused G(0)-G(1) arrest and cell apoptosis. It is interesting that sequence analysis showed that there was only about 12% amino acid sequence homology between vastatin and endostatin. The structure-function relationship of these angiogenesis molecules remains to be elucidated.     

The effect of second-line antirheumatic drugs on interleukin-8 mRNA synthesis and protein secretion in human endothelial cells.

Interactions between interleukin 8 (IL-8) and endothelial cells play an important role in the emigration of mononuclear cells from the blood into areas of inflammation. We examined the ability of specific second-line antirheumatic drugs to regulate (IL-8) gene expression and protein secretion in interleukin 1 (IL-1) stimulated human umbilical vein endothelial cells and peripheral blood mononuclear cells. The drugs sodium aurothiomalate, D-penicillamine and sulphasalazine were all able to modulate IL-8 mRNA synthesis in and protein secretion from endothelial cells. A bimodal effect was observed: at low concentrations IL-8 was suppressed, whereas higher concentrations resulted in an increased IL-8 production. In endothelial cells, treatment with hydrocortisone led to a linear suppression of IL-8 production in concentrations ranging from 0.5 micrograms/ml up to 500 micrograms/ml. Sulphapyridine, auranofin, hydroxychloroquine and methotrexate, had no effect on IL-8 secretion in endothelial cells. By contrast, 5-aminosalicylic acid induced a threefold increase in the IL-8 release. In peripheral blood mononuclear cells it was only possible to suppress the IL-8 production by hydrocortisone treatment. These results indicate that suppression of IL-8 production in endothelial cells could be an important factor in the mode of action for a number of second-line antirheumatic drugs.