Signaling via fibroblast growth factor receptor-1 is dependent on extracellular matrix in capillary endothelial cell differentiation

Differentiation of endothelial cells, i.e., formation of a vessel lumen, is a prerequisite for angiogenesis. The underlying molecular mechanisms are ill defined. We have studied a brain capillary endothelial cell line (IBEC) established from H-2Kb-tsA58 transgenic mice. These cells form hollow tubes in three-dimensional type I collagen gels in response to fibroblast growth factor-2 (FGF-2). Culture of IBEC on collagen gels in the presence of FGF-2 protected cells from apoptosis and allowed tube formation (i.e., differentiation) but not growth of the cells. FGF-induced differentiation, but not cell survival, was inhibited by treatment of the cells with an anti-beta1-integrin IgG. Changes in integrin expression in the collagen-gel cultures could not be detected. Rather, cell-matrix interactions critical for endothelial cell differentiation were created during the culture, as indicated by the gradual increase in tyrosine phosphorylation of focal adhesion kinase in the collagen-gel cultures. Inclusion of laminin in the collagen gels led to FGF-2-independent formation of tube structures, but cells were not protected from apoptosis. These data indicate that FGF receptor-1 signal transduction in this cell model results in cell survival. Through mechanisms dependent on cell-matrix interactions, possibly involving the alpha3beta1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells.     

Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells

Vascular endothelial cells (ECs) seeded sparsely on extracellular matrix (ECM) will proliferate in the absence of exogenous basic fibroblast growth factor (bFGF). This ECM will also stimulate neurite outgrowth in PC12 cells in the absence of exogenous growth factors. We have previously shown that bFGF is found in subendothelial ECM (Vlodavsky, I., J. Folkman, R. Sullivan, R. Fridman, R. Ishai-Michaeli, J. Sasse, and M. Klagsburn. 1987. Proc. Natl. Acad. Sci. USA. 84:2292-2296) and in basement membranes (Folkman, J., M. Klagsburn, J. Sasse, M. Wadzinski, D. Ingber, and I. Vlodavsky. 1988. Am. J. Pathol. 130:393-400). The actual requirement of ECM-associated bFGF for the growth of ECs and differentiation of PC12 cells was shown in two ways. First, polyclonal anti-bFGF antibodies added to subendothelial ECM inhibited both EC proliferation and PC12 neurite outgrowth. Secondly, PF-HR-9 cells, which do not synthesize bFGF and which produce an ECM not permissive for EC proliferation and PC12 neurite outgrowth, were transfected with bFGF cDNA. PF-HR-9 cells transfected with bFGF, but not with the dominant selectable marker SV2-neomycin, were found to express bFGF and to produce an ECM which did support both EC proliferation and PC12 differentiation. The ECM-mediated stimulatory effects were inhibited by anti-bFGF antibodies but not by anti-nerve growth factor antibodies or nonimmune rabbit IgG. These results indicate that bFGF associated with ECM is a required ECM component for ECM-mediated cell proliferation and differentiation.     

Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix

The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern-regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical loads. Thus, we propose that FGF-stimulated endothelial cells may be "switched" between growth, differentiation, and involution modes during angiogenesis by altering the adhesivity or mechanical integrity of their ECM.     

Ataxia-telangiectasia: a variant with altered in vitro phenotype of fibroblast cells

The clinical and cellular phenotype of ataxia telangiectasia (AT) has been extensively documented in numerous patients of different ethnic groups and is characterized by several specific laboratory hallmarks, such as chromosomal instability, profound radiosensitivity and radioresistant DNA synthesis. Several recent reports have, however, shown variations on this theme. This article describes 2 Turkish siblings with AT, who showed a typical but somewhat more prolonged clinical course of the disease and altered characteristics of fibroblasts cells, compared to the ‘classical’ AT cellular phenotype. Fibroblasts strains derived from these patients showed a normal cellular life span, moderate degrees of chromosomal instability and sensitivity to the lethal effects of X-rays and neocarzinostatin, and lack of radioresistant DNA synthesis.

A compilation of the literature on ‘AT variants’ and ‘AT-like’ syndromes shows that in addition to the internal variability of AT, this disease occupies a limited segment within a large spectrum of clinical and cellular features, which are common to a variety of syndromes. Each of these syndromes covers a different segment in this spectrum. The genetic basis of this family of disorders might be complex.     

eNOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1

Reports on the role of AMP-activated protein kinase (AMPK) in thrombin-mediated activation of endothelial nitric-oxide synthase (eNOS) in endothelial cells have been conflicting. Previously, we have shown that under culture conditions that allow reduction of ATP-levels after stimulation, activation of AMPK contributes to eNOS phosphorylation and activation in endothelial cells after treatment with thrombin. In this paper we examined the signaling pathways mediating phosphorylation and activation of eNOS after stimulation of cultured human umbilical vein endothelial cells (HUVEC) with histamine and the role of LKB1-AMPK in the signaling. In Morgan's medium 199 intracellular ATP was lowered by treatment with histamine or the ionophore A23187 while in medium RMPI 1640 ATP was unchanged after identical treatment. In medium 199 inhibition of Ca^+ 2/CaM kinase kinase (CaMKK) by STO-609 only partially inhibited AMPK phosphorylation but after gene silencing of LKB1 with siRNA there was a total inhibition of AMPK phosphorylation by STO-609 after treatment with either histamine or thrombin, demonstrating phosphorylation of AMPK by both upstream kinases, LKB1 and CaMKK. Downregulation of AMPK with siRNA partially inhibited eNOS phosphorylation caused by histamine in cells maintained in medium 199. Downregulation of LKB1 by siRNA inhibited both phosphorylation and activity of eNOS and addition of the AMPK inhibitor Compound C had no further effect on eNOS phosphorylation. When experiments were carried out in medium 1640, STO-609 totally prevented the phosphorylation of AMPK without affecting eNOS phosphorylation. AMPKα2 downregulation resulted in a loss of the integrity of the endothelial monolayer and increased expression of GRP78, indicative of endoplasmic reticular (ER) stress. Downregulation of AMPKα1 had no such effect. The results show that culture conditions affect endothelial signal transduction pathways after histamine stimulation. Under conditions where intracellular ATP is lowered by histamine, AMPK is activated by both LKB1 and CaMKK and, in turn, mediates eNOS phosphorylation in an LKB1 dependent manner. Both AMPKα1 and − α2 are involved in the signaling. Under conditions where intracellular ATP is unchanged after histamine treatment, CaMKK alone activates AMPK and eNOS is phosphorylated and activated independent of AMPK.     

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca²⁺ on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca²⁺ on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca²⁺ (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca²⁺ stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca²⁺ significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.     

Molybdenum trioxide enhances viability,osteogenic differentiation and extracellular matrix formation of human bone marrow-derived mesenchymal stromal cells

BackgroundMetals and their ions allow specific modifications of the biological properties of bioactive materials that are intended for application in bone tissue engineering. While there is some evidence about the impact of particles derived from orthopedic Cobalt-Chromium-Molybdenum (Co-Cr-Mo) alloys on cells, there is only limited data regarding the influence of the essential trace element Mo and its ions on the viability, osteogenic differentiation as well as on the formation and maturation of the primitive extracellular matrix (ECM) of primary human bone marrow-derived stromal cells (BMSCs) available so far.MethodsIn this study, the influence of a wide range of molybdenum (VI) trioxide (MoO₃), concentrations on BMSC viability was evaluated via measurement of fluorescein diacetate metabolization. Thereafter, the impact of three non-cytotoxic concentrations of MoO₃ on the cellular osteogenic differentiation as well as on ECM formation and maturation of BMSCs was assessed.ResultsMoO₃ had no negative influence on BMSC viability in most tested concentrations, as viability was in fact even enhanced. Only the highest concentration (10 mM) of MoO₃ showed cytotoxic effects. Cellular osteogenic differentiation, measured via the marker enzyme alkaline phosphatase was enhanced by the presence of MoO₃ in a concentration-dependent manner. Furthermore, MoO₃ showed a positive influence on the expression of relevant marker genes for osteogenic differentiation (osteopontin, osteocalcin and type I collagen alpha 1) and on the formation and maturation of the primitive ECM, as measured by collagen deposition and ECM calcification.ConclusionMoO₃ is considered as an attractive candidate for supplementation in biomaterials and qualifies for further research.     

Effect of extracellular matrix proteins on in vitro testosterone production by rat Leydig cells

The aim of this study was to detect the effect of extracellular matrix (ECM) proteins on rat Leydig cell shape, adhesion, expression of integrin subunits and testosterone production, in vitro. Leydig cells isolated from adult rats were cultured on plates uncoated or coated with different concentrations of laminin-1, fibronectin, or type IV collagen in the presence or absence of hCG for 3 or 24 hr. A significant increase of cell adhesion and of alpha3, alpha5, and beta1 integrin subunit expression was observed when cells were cultured on ECM proteins, compared to those grown on uncoated plates. Leydig cells cultured on glass coverslips coated with ECM proteins for 24 hr exhibited elongated shapes with long cell processes (spreading), while cells cultured on uncoated plates showed few cell processes. A significant decrease in testosterone production was observed when basal and hCG-stimulated Leydig cells were cultured for 3 or 24 hr on plates coated with type IV collagen (12 and 24 microg/cm(2)) compared to uncoated plates. A significant though a slighter decrease in testosterone production was also observed in cells cultured on plates coated with fibronectin (12 and 24 microg/cm(2)), compared to uncoated plates. Laminin-1 did not modify testosterone production under basal or hCG stimulated conditions. These results suggest that ECM proteins are able to modulate Leydig cell steroidogenesis, in vitro.     

Mechanism of thrombin mediated eNOS phosphorylation in endothelial cells is dependent on ATP levels after stimulation

Conflicting results have been reported concerning the role of AMP-activated protein kinase (AMPK) in mediating thrombin stimulation of endothelial NO-synthase (eNOS). We examined the involvement of two upstream kinases in AMPK activation in cultured human umbilical endothelial cells, LKB1 stimulated by a rise in intracellular AMP/ATP ratio, and Ca(+2)/CaM kinase kinase (CaMKK) responding to elevation of intracellular Ca(+2). We also studied the effects of AMPK activation on the downstream target eNOS. In culture medium 1640 the level of intracellular ATP was unchanged after thrombin stimulation and the CaMKK inhibitor STO-609 totally inhibited phosphorylation of AMPK and acetyl coenzyme A carboxylase (ACC) but not eNOS. In Morgan's medium 199 thrombin caused a significant lowering of intracellular ATP and STO-609 only partially inhibited the phosphorylation of AMPK, ACC and eNOS. Inhibition of AMPK by Compound C or AMPK downregulation using siRNA partially inhibited the phosphorylation of eNOS in medium 199 but not in 1640, underscoring a clear difference in the pathways mediating thrombin-stimulated eNOS phosphorylation in different culture media. Thus, conditions subjecting endothelial cells to a fall in ATP after thrombin stimulation facilitate activation of pathways partly dependent on AMPK causing downstream phosphorylation of eNOS. In contrast, under culture conditions that do not facilitate a fall in ATP after stimulation, AMPK activation is exclusively mediated by CaMKK and does not contribute to the phosphorylation of eNOS.     

Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences

Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium. When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of beta-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of beta-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the beta-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, beta-casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (Millipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions alpha 1-, alpha 2-, and beta-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of differentiation markers in human choriocarcinoma cells by extracellular matrix: on the role of a three-dimensional matrix structure

Abstract. During spontaneous or chemically induced differentiation human choriocarcinoma cells express typical characteristics of the normal differentiating trophoblast: 1) increased production of peptide and steroid hormones (chorionic gonadotropin, placental lactogen, estrogens, progesterone); 2) increased activity of cellular alkaline phosphatase; 3) morphological transition from cytotrophoblast to syncytiotrophoblast-like cells; and 4) arrested cell proliferation. Since the extracellular matrix is known to control gene expression we have examined the effects of different substrates composed of matrix macromolecules on the differentiation of BeWo choriocarcinoma cells. Matrices tested were; fibronectin, laminin, collagens type I and type IV, the basement membrane-like complex matrix Matrigel, and a complex matrix extracted from human term placenta. Irrespective of the type of molecule(s), it was consistently found that, whenever the matrix molecules were presented as threedimensional structures (as opposed to protein coatings on tissue culture plastic) the response of affected differentiation markers monitored was highly pronounced. Morphology was changed from monolayers to rounded colonies, cell proliferation was reduced, and the secretion of chorionic gonadotropin was increased up to tenfold. Heterogeneous effects were observed on progesterone secretion and on the activity of cellular alkaline phosphatase. Cell adhesion to matrix molecules, however, did not depend on the structure of the matrix. This study demonstrates that gene expression in these tumor cells can be modified by extracellular matrix and highlights that not only the presence of effector molecules in the matrix but also the three-dimensional structure of the matrix is important for the induction of differentiation.     

Immunologic responses to a murine mammary adenocarcinoma: in vitro production of specific killer cells is dependent on active T lymphocytes.

The mode of production of specifically armed monocytic killer cells was investigated with the T1699 mammary adenocarcinoma in syngeneic DBA/2 mice. After overnight in vitro incubation of cells from the spleen but not from the lymph nodes, blood, or from the peritoneal cavity produced specific killer cells. The activation of spleen cells was inhibited by pretreatment with anti-theta serum and C; however, already activated specific killer cells were not sensitive to the same treatment. Removal of phagocytic cells did not significantly affect the cytotoxicity of the splenic killer cells whereas removal of rayon-wool adherent cells greatly reduced both the total cytotoxicity, and to a lesser extent, the cytotoxicity indices. Overnight co-cultivation of normal peritoneal-exudate cells with the lymph node cells from tumor-bearers, although neither class of cells alone was cytotoxic to T1699 cells in vitro, produced specific monocytic killer cells, through steps dependent on active T lymphocyte function. Culture spupernatants of tumor-bearer's spleen cells also contained factor(s) which induced cytotoxicity mediated by normal peritoneal-exudate cells against T1699 cells in vitro; and the production of the factor(s) was also inhibited by pretreatment of the spleen cells with anti-theta serum but not by anti-mouse IgG or anti-mouse whole gamma-globulins serum and C.     

Lipids promote survival, proliferation, and maintenance of differentiation of rat liver sinusoidal endothelial cells in vitro

Primary rat liver sinusoidal endothelial cells (LSEC) are difficult to maintain in a differentiated state in culture for scientific studies or technological applications. Relatively little is known about molecular regulatory processes that affect LSEC differentiation because of this inability to maintain cellular viability and proper phenotypic characteristics for extended times in vitro, given that LSEC typically undergo death and detachment around 48-72 h even when treated with VEGF. We demonstrate that particular lipid supplements added to serum-free, VEGF-containing medium increase primary rat liver LSEC viability and maintain differentiation. Addition of a defined lipid combination, or even oleic acid (OA) alone, promotes LSEC survival beyond 72 h and proliferation to confluency. Moreover, assessment of LSEC cultures for endocytic function, CD32b surface expression, and exhibition of fenestrae showed that these differentiation characteristics were maintained when lipids were included in the medium. With respect to the underlying regulatory pathways, we found lipid supplement-enhanced phosphatidylinositol 3-kinase and MAPK signaling to be critical for ensuring LSEC function in a temporally dependent manner. Inhibition of Akt activity before 72 h prevents growth of SEC, whereas MEK inhibition past 72 h prevents survival and proliferation. Our findings indicate that OA and lipids modulate Akt/PKB signaling early in culture to mediate survival, followed by a switch to a dependence on ERK signaling pathways to maintain viability and induce proliferation after 72 h. We conclude that free fatty acids can support maintenance of liver LSEC cultures in vitro; key regulatory pathways involved include early Akt signaling followed by ERK signaling.     

Effects of interleukin-1 on fibroblast extracellular matrix, using a 3-dimensional culture system

This study describes the alterations induced by Interleukin-1 alpha and -beta (IL-1 alpha and IL-1 beta) on fibroblast-synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen-coated Terasaki wells for 3 or 6-9 weeks to create 3-dimensional cell-containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL-1 alpha or IL-1 beta (10(-9) M) at 37 degrees C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL-1 alpha and IL-1 beta induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross-sectional area and an increased density of the matrices. Three-week matrices were reduced 26% and 45% by using IL-1 alpha and IL-1 beta, respectively. Comparable values obtained by using 6-week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL-beta than with IL-1 alpha. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3-week matrix following IL-1 beta incubation. There was also a decrease in collagen type 1 staining of 9-week matrices treated with IL-1 alpha or IL-1 beta. These studies show that IL-1 has an effect on fibroblast-synthesized extracellular matrix and indicate that the effects of IL-1 alpha and IL-1 beta may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.     

The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitro

An in vitro bovine mammosphere model was characterized for use in lactational biology studies using a functional genomics approach. Primary bovine mammary epithelial cells cultured on a basement membrane, Matrigel, formed three-dimensional alveoli-like structures or mammospheres. Gene expression profiling during mammosphere formation by high-density microarray analysis indicated that mammospheres underwent similar molecular and cellular processes to developing alveoli in the mammary gland. Gene expression profiles indicated that genes involved in milk protein and fat biosynthesis were expressed, however, lactose biosynthesis may have been compromised. Investigation of factors influencing mammosphere formation revealed that extracellular matrix (ECM) was responsible for the initiation of this process and that prolactin (Prl) was necessary for high levels of milk protein expression. CSN3 (encoding κ-casein) was the most highly expressed casein gene, followed by CSN1S1 (encoding αS1-casein) and CSN2 (encoding β-casein). Eighteen Prl-responsive genes were identified, including CSN1S1 , SOCS2 and CSN2, however, expression of CSN3 was not significantly increased by Prl and CSN1S2 was not expressed at detectable levels in mammospheres. A number of novel Prl responsive genes were identified, including ECM components and genes involved in differentiation and apoptosis. This mammosphere model is a useful model system for functional genomics studies of certain aspects of dairy cattle lactation.     

体外培养所形成的细胞外基质对MC3T3-E1细胞生长和分化的影响

细胞外基对组织细胞起支持、保护、营养作用,对细胞的增殖、分化有重要影响,在细胞和组织工程中,应该充分考虑细胞外基质的作用。本研究首先脱去培养板中融合培养的原代小鼠心肌成纤维细胞和成骨细胞,获得两种体外形成的细胞外基质包被的培养板,其中成骨细胞细胞外基质中含有骨形成蛋白2。然后将MC3T3-E1成骨前体细胞接种在这种培养板中,发现成纤维细胞胞外基质包被的培养板中的细胞增殖活性最高,而成骨细胞胞外基质包被的培养板中细胞的碱性磷酸酶活性、骨形成蛋白2和骨桥蛋白的相对蛋白表达量最高,细胞外钙沉积量比其他组高1倍左右。结果表明：包被在培养板上的这两种细胞外基质有不同的生物活性,成纤维细胞胞外基质可促进成骨前体细胞增殖,成骨细胞胞外基质可促进成骨前体细胞骨向分化。     

Luteolytic effect of prolactin is dependent on the degree of differentiation of luteal cells in the rat

We studied the morphological and quantitative changes in cyclic corpora lutea (CCL) and in CL of pregnancy (CLP) during structural luteolysis. Elimination of CCL takes several cycles, and cell death occurs as successive apoptotic bursts, from 2100 h in proestrus to 1300 h in estrus. Each apoptotic burst determined a 60% decrease in the CL volume and an 80% decrease in the number of steroidogenic cells (SC). All these changes were inhibited by blocking the preovulatory prolactin (PRL) surge with bromocryptine (CB154). Neither apoptotic cells nor changes in the number of SC were found in regressing CLP from Day 21 of pregnancy to Day 2 postpartum, although there was a 50% decrease in the CLP volume and a 30% decrease in the mean cross-sectional area of SC. Treatment with CB154 on the day of parturition did not modify these regressive changes. On Day 5 postpartum, the volume of the CLP and the number of SC were equivalent in lactating rats (showing high PRL concentrations induced by pup suckling) and nonlactating noncycling rats (in which cyclicity and, therefore PRL surges, were blocked by treatment with LHRH antagonist). However, on Day 10 postpartum, the CLP volume and the number of SC were significantly decreased in lactating rats, and apoptotic cells were frequent. In postpartum cycling rats, the CLP did not show apoptotic cells on the day of the second postpartum estrus (on Day 5 postpartum), whereas on the day of the third postpartum estrus (on Day 9 postpartum), apoptotic cells were abundant. These results indicate that PRL does not induce apoptosis in the CLP before Day 5 postpartum and strongly suggest that the proapoptotic effect of PRL is dependent on the degree of differentiation of luteal cells.     

Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.