Alveolar macrophage modulation of proteolysis by neutrophil elastase in extracellular matrix

An extracellular connective tissue matrix, made up of components found in the pulmonary alveolar interstitium, was generated in vitro and used as a culture surface and substrate for proteolysis by human alveolar macrophages (AM) and neutrophil elastase (NE). The ability of human AM to modulate NE-mediated degradation of elastin and collagen in the surrounding matrix was studied to gain insights into the inflammatory process that accompanies the pathogenesis of emphysema in humans. Neutrophil elastase that had been internalized by AM showed a diminished but more prolonged time course of matrix proteolysis than did a similar amount of NE added to the matrix in the absence of AM. Collagen and elastin degradation were quantitated by release of hydroxylysine and desmosine, respectively, into the culture medium. Significantly more hydroxylysine and desmosine were released by AM that had internalized NE than by AM or by culture medium alone. When 14 X 10(6) AM were added to the extracellular matrix, followed 2 h later by addition of 2 micrograms of NE, collagen and elastin degradation measured at 24 h were not significantly different from that which occurred when matrix was incubated with NE in the absence of AM. Collagen degradation, determined in the same cultures during the period from 24 to 96 h, was significantly greater when matrix was incubated with both AM and NE. These findings suggest that AM can release previously internalized NE in an enzymatically active form and that AM may enhance collagen degradation in matrix that was also exposed to NE.     

Copper modulation of extracellular matrix synthesis by human articular chondrocytes

OBJECTIVE: The effects of Cu2+ on human articular chondrocytes, arising from both N (normal) and OA (osteoarthritic) cartilage, were investigated "in vitro". METHODS: Chondrocytes, cultured in high density, were incubated with copper chloride (0.01-0.25 microM/mL). Proteoglycan and collagen were assessed by incorporation of [35S]-Sulfate and [3H]-Proline. SDS-PAGE analysis was performed to quantify the ratio of type II to type I collagen. RESULTS: Cu2+ neither increased proteoglycan synthesis by chondrocytes. of origin N or OA, nor influenced their proliferation rate. Collagen synthesis was increased. This effect is time and concentration dependant: in cultures treated for 12 days, collagen synthesis stimulation was +20% and +26% (P < 0.02) in N and OA cultures respectively, the ratio of type II to type I collagen was slightly increased. This effect was more obvious in OA cell lines than in N ones. CONCLUSION: The observations suggest that Cu2+ upregulates collagen anabolism in human articular chondrocytes.     

Bone marrow matrix modulation of HL-60 phenotype

The initiation and maintenance of cellular differentiation for a variety of cell types has been shown to be influenced by the microenvironment. To investigate the influence of bone marrow stroma on leukemic cell differentiation, HL-60 human promyelocytic leukemia cells were grown in the presence of Triton-treated extracellular matrix derived from normal human bone marrow stromal cells. This bone marrow matrix microenvironment had a dramatic impact on the phenotypic expression of this malignant line. HL-60 cellular proliferation, morphology, nonspecific esterase activity, formation of Fc rosettes, and sensitivity to induction by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were all influenced by the presence of matrix molecules. In contrast, stromal cell-conditioned media did not alter HL-60 phenotype. Thus, HL-60 cells appear to retain responsiveness to a human bone marrow stromal cell-derived matrix despite their ability to grow autonomously. Studies of the interaction of leukemic cells and marrow stroma in vitro may provide important information concerning the regulation of leukemic cell behavior.     

Permissive effect of the extracellular matrix on cell proliferation in vitro.

Corneal endothelial cells maintained in tissue culture retain the ability to synthesize and secrete an extracellular matrix (ECM) along their basal cell surface. Treatment of confluent cultures with 0.5% Triton X-100 results in the removal of the cell monolayer, thereby exposing the ECM, which adheres strongly to the tissue culture dish. Dishes coated with ECM were used to study the permissive effect of such a substrate on cell proliferation. The proliferation of bovine granulosa and adrenal cortex cells maintained on plastic tissue culture dishes was compared to that on dishes coated with ECM. Neither cell type, even when exposed to optimal serum concentration, replicated when seeded at low cell density on plastic. In contrast, when seeded on ECM they proliferated actively. None of the cultures maintained on ECM required fibroblast growth factor in order to reach confluence, although when maintained on plastic they were totally dependent on fibroblast growth factor for proliferation. Because cells maintained on plastic do not respond to factors present in serum or plasma, although they do so respond when maintained on ECM, it is likely that the close contact of the cells with the ECM restores their sensitivity to agents present in serum and plasma.     

Human fetal adenohypophysis: morphologic and functional analysis in vitro.

Pituitaries from 24 human fetuses at 7-20 weeks of gestation were studied in culture to assess hormone release, response to adenohypophysiotropic hormones and cytodifferentiation by electron microscopy in cultures lasting 4-8 days and in some cases up to 28 days. At 7 weeks of gestation, ACTH was released by cultured cells which included recognizable corticotrophs. GH was released by cells cultured from 8- to 9-week fetuses and densely granulated somatotrophs were present in the cultures. alpha-Subunit of glycoprotein hormones was present in cultures from 10-week fetuses and TSH and LH were released from 12-week fetuses. FSH was found in cultures of a 13-week female fetus but not before 14 weeks in cultures from males. The levels of FSH and LH were higher in media from cultures of females than from those of males at all ages from detection to 20 weeks, whereas alpha-subunit was slightly higher in media from males. While cells with features of the glycoprotein hormone cell line were found in cultures from 10-week fetuses, no characteristic thyrotrophs or gonadotrophs were recognized. PRL was not measured in basal incubations before 14 weeks. The amounts of all hormones released were proportional to fetal age and decreased with duration of culture. Cortisol suppressed ACTH release in cultures from 7- to 8-week fetuses. Responsiveness of GH release to GRH/SRIH, of ACTH to CRH, and of FSH to GnRH, was found at 12 weeks; LH stimulation by GnRH and TSH response to TRH were documented at 14 weeks. Increments of gonadotropin release during incubation with GnRH were greater in cultures from females than in those from males. PRL release responded to GRH stimulation and to SRIH inhibition in parallel with GH; this behavior is consistent with production of both hormones by mammosomatotrophs. The onset of hormone release by cultured human fetal pituitaries correlates with the detection of hormones biochemically and immunohistochemically. Responsiveness of fetal adenohypophysial cells to hormonal influences indicates functional maturity early in gestation.     

Noncalcium-dependent modulation of in vitro atrial natriuretic factor release by extracellular osmolality

Recent reports have described modulation of atrial natriuretic factor (ANF) secretion by atrial stretch, increased Na+ concentration, and a variety of hormones and neurotransmitters. One problem in the study of ANF secretion has been the isolation of stimulatory effects from interference due to rhythmic myocardial contraction. To avoid this problem, rat atria were dispersed, the myocytes were suspended in a polyacrylamide gel matrix, and the resulting cell column was perifused with a physiological buffer. The secretion of ANF immunoactivity was markedly stimulated by increases in extracellular osmolality, regardless of the solute (NaCl, KCl, or glucose). This effect did not require extracellular Ca2+, nor could it be mimicked by depolarizing concentrations of K+ in an isotonic medium. Functional viability of this model was demonstrated by significant dose-related increases in ANF release in response to as little as 1 nM epinephrine. The dissociation of ANF secretion from depolarization-induced changes in Ca2+ flux is unusual and may represent an adaptation to the dual roles of the atrial myocyte, contraction and secretion.     

Cell survival,activation and apoptosis of hepatic stellate cells: modulation by extracellular matrix proteins

Aim: Cytokines and growth factors released by various hepatic cells exert both paracrine and autocrine effects on hepatic stellate cell (HSC) activation during liver injury. The aim of the present study was to examine whether the surrounding extracellular matrix (ECM) influences the activation, transdifferentiation and survival of HSCs. Methods: An in vitro model system of isolated HSCs maintained in culture on different matrix protein substrata was employed. Results: The rate of loss of HSC‐specific retinol uptake activity and gain of myofibroblast‐like activity such as ³⁵[S] proteoglycan synthesis varied in cells maintained on different matrix proteins and was in the order collagen I > collagen IV ≥ laminin. ³[H]‐thymidine incorporation by HSCs maintained on different matrix proteins varied and was in the order collagen I > collagen IV > laminin. MTT assay revealed that the growth inhibition in response to curcumin was significantly low in cells maintained on collagen I. Apoptotic marker activities such as DNA fragmentation, 4′,6′‐diamidino‐2‐phenylindole dihydrochloride (DAPI) staining, annexin staining and caspase‐3 activities showed that cells maintained on collagen I showed minimal apoptosis than those maintained on collagen IV, laminin and polylysine, showing the influence of ECM on HSC apoptosis. Experiments using blocking antibodies showed that the collagen I effect was mediated through α₂β₁ integrin. Conclusions: These results indicate that ECM influences activation, transdifferentiation and survival of HSCs, and suggest that apart from diffusible factors, the surrounding ECM also influences HSC behavior critical in both the progression of the fibrosis and the restitution of the liver during recovery after hepatic injury.     

生长因子及细胞外基质对肝前体细胞体外增殖及分化的影响

目的 明确多种生长因子对胚胎肝脏前体细胞体外增殖分化的影响。方法 用胶原酶从胎龄14．5d SD大鼠胚胎肝脏分离单个细胞，采用^3H-TdR掺入法检测生长因子对胎肝细胞体外增殖的促进作用，并观察生长因子及细胞外基质成分对胎肝干细胞集落形成的影响。采用免疫细胞双标记及G-6-P酶活性测定以检测肝前体细胞表面标记的表达及向成熟肝细胞的分化能力。结果 肝前体细胞在体外培养时显示克隆样生长的特性。促肝细胞生长因子(HGF)、表皮生长因子(EGF)能促进肝前体细胞的增殖，使DNA合成加速，并促进干细胞集落的形成及角蛋白19、白蛋白、G-6-P的表达。转化生长因子α对肝前体细胞的促增殖作用较弱。转化生长因子β对肝前体细胞的增殖起到抑制作用。细胞基质成分Ⅰ型胶原、Ⅳ型胶原、层黏连蛋白能促进肝干细胞集落的形成，而纤维连接蛋白的作用较弱。肝干细胞单克隆增殖需要生长因子及细胞外基质的共同参与，加入新鲜分离胎肝细胞培养液上清液时单细胞增殖较快，于第5天即形成细胞集落。结论 HGF、EGF对肝前体细胞的增殖分化起重要作用，细胞外基质成分亦参与了其增殖分化过程。肝干细胞单克隆培养除需生长因子和细胞外基质外，可能亦有某些造血细胞、间质细胞分泌的因子参与其增殖分化的调控。     

Maturation of fetal hepatocytes in vitro by extracellular matrices and oncostatin M: induction of tryptophan oxygenase

Previously, we described that embryonic day 14.5 (E14.5) mouse fetal hepatocytes differentiate to express tyrosine amino transferase (TAT) and glucose-6-phosphatase, which are expressed in the perinatal liver, in response to oncostatin M (OSM) or in high-cell-density culture. However, under such conditions, fetal hepatic cells failed to express genes for adult liver-specific enzymes, such as tryptophan oxygenase (TO). Although phenobarbital (PB) and dimethylsulfoxide (DMSO) have been known to maintain the functions of adult hepatocytes in vitro, they failed to induce TO expression in fetal hepatic cells. Thus far, no system has been developed that reproduces terminal differentiation of fetal hepatocytes in vitro. Here, we describe that extracellular matrices derived from Engelbreth-Holm-Swarm sarcoma (EHS) in combination with OSM or high-cell-density culture induced expression of TO as well as cytochrome P450 genes that are involved in detoxification. However, EHS alone was insufficient to induce expression of TO, although it induced TAT expression in fetal hepatocytes. In addition, high-density culture further augmented differentiation. In conclusion, the combination of signals by cytokines, cell-cell contact, and cell-matrix interaction is required for induction of adult liver functions in fetal hepatocytes in vitro. This primary culture system will be useful for studying the mechanism of liver development.     

Human bone marrow angiogenesis: in vitro modulation by substance P and neurokinin A

We have previously described a culture system for human bone marrow endothelial cells that organize into capillary tubes associated to pericytes. In the present work, we used this model to assess the angiogenic properties of tachykinins, which have been demonstrated to be involved in neuro-immuno-haematopoietic interactions. The substance P (SP) and neurokinin A (NKA) were similarly potent at increasing in vitro angiogenesis, via NK1 and NK2 receptors respectively. These mediators were not produced by cells in culture, suggesting that in vivo they may be released by nerve fibres in the bone marrow. Therefore, we looked for in situ innervation of the human bone marrow, unknown to date, using immunohistochemistry techniques. As in rodents, arterioles were largely innervated, associated with between one and 10 nerve fibres. Capillary innervation was more restrictive as a unique thin nerve fibre was found in the vicinity of only 6% of these vessels. Finally, no nerve fibres were observed in the vicinity of sinus walls. In conclusion, both in vitro results and the anatomical display of nerve fibres suggest a role in human bone marrow for the vasoactive neuropeptides SP and NKA, which were secreted into a perivascular location. These neural mediators might modulate blood flow in the bone marrow both in the short term by adjusting vascular tone and in the long term by inducing angiogenesis.     

Monolayer culture of adult rat pancreatic islets on extracellular matrix: modulation of B-cell function by chronic exposure to high glucose.

Studies in vivo indicate that chronic hyperglycemia is deleterious for insulin secretion. We have used an improved islet monolayer culture system to study chronic modulations of B-cell function. Adult rat islets maintained over several weeks on extracellular matrix in the presence of 11.1 mM glucose responded to an acute stimulation with 16.7 mM glucose by a 5- to 8-fold increase in insulin secretion. When cultured in the presence of higher glucose concentrations, the response to an acute glucose stimulus diminished time and dose dependently. In islets desensitized by exposure to 33.3 mM glucose for 1 week, reduction of the glucose level to 11.1 mM reversed the desensitization within 2 weeks. This desensitization was not limited to the glucose stimulus; responses to other nutrient secretagogues, such as glyceraldehyde and alpha-ketoisocaproic acid, were also reduced. In contrast, responses of insulin secretion to nonnutrient stimulators (tolbutamide and quinine) and amplifiers (isobutylmethylxanthine and carbachol) showed no desensitization in islets exposed to 33.3 mM glucose. Desensitization similar to that caused by 33.3 mM glucose could be induced by 11.1 mM glucose together with 0.1 mM isobutylmethylxanthine. High glucose also caused a time-dependent loss in compact monolayer organization with disruption of cell contacts. Our studies suggest that 1) generation of the reduced insulin response may be related to the prolonged high insulin secretion rate; 2) expression of the functional change is specific to the nutrient stimulus-secretion coupling; and 3) modifications in intercellular contacts may be involved in B-cell desensitization.     

Effects of fibroblast extracellular matrix calcification on platelet adhesion in vitro

Calcified atherosclerotic lesions are more prone to rupture during angioplasty than non-calcified lesions and are associated with an increased risk of thrombotic complications following angioplasty. This study investigates the possible role of extracellular matrix (ECM) calcification for platelet adhesion. Human cultured fibroblasts (CRL-1635) were subjected to β-glycerophosphate (10 mM) for 10 to 16 days. Calcification was visualized by von Kossa staining and quantified by the O-cresolphthalein complexone method. Adhesion of calcein-labelled platelets was measured by fluorescence microscopy at static conditions and in a parallel-flow chamber at a shear rate of 1000 s^?1. β-glycerophosphate treatment resulted in a marked calcification of the ECM. In parallel, a small, albeit significant increase in platelet adhesion under static conditions was observed. In contrast, at flow conditions, the area covered by thrombi was significantly lower when calcified ECM was used. The number of thrombi was not significantly different which is compatible with a smaller thrombus size. Taken together, it appears unlikely that calcification of atherosclerotic lesions contributes to thrombotic complications by an increased platelet adhesion.     

Effects of fibroblast extracellular matrix calcification on platelet adhesion in vitro

Calcified atherosclerotic lesions are more prone to rupture during angioplasty than non-calcified lesions and are associated with an increased risk of thrombotic complications following angioplasty. This study investigates the possible role of extracellular matrix (ECM) calcification for platelet adhesion. Human cultured fibroblasts (CRL-1635) were subjected to beta-glycerophosphate (10 mM) for 10 to 16 days. Calcification was visualized by von Kossa staining and quantified by the O-cresolphthalein complexone method. Adhesion of calcein-labelled platelets was measured by fluorescence microscopy at static conditions and in a parallel-flow chamber at a shear rate of 1000 s(-1). beta-glycerophosphate treatment resulted in a marked calcification of the ECM. In parallel, a small, albeit significant increase in platelet adhesion under static conditions was observed. In contrast, at flow conditions, the area covered by thrombi was significantly lower when calcified ECM was used. The number of thrombi was not significantly different which is compatible with a smaller thrombus size. Taken together, it appears unlikely that calcification of atherosclerotic lesions contributes to thrombotic complications by an increased platelet adhesion.     

Identification of extracellular matrix components and their integrin receptors in the human fetal adrenal gland

The development of the human fetal adrenal gland is characterized by a gradient of mitotic activity, cell migration, and cell apoptosis, all of which dictate its particular function. Such plasticity may possibly be under the control of the extracellular environment. The goal of this study was to identify components of the extracellular matrix in second-trimester fetal adrenal glands. Whereas collagen IV was expressed evenly throughout the gland, both fibronectin and laminin demonstrated a mirror-imaged distribution, with higher expression of fibronectin in the central portion and laminin at the periphery of the gland. The integrin subunit alpha1 was found mainly in the definitive zone and the alpha2-subunit mainly in the transitional zone, whereas integrin alpha3 (which binds both fibronectin and laminin) was detected only in the fetal zone. The beta2-subunit was observed solely in chromaffin cells. Such specific gradients of integrin and MEC component expression suggest that the extracellular environment does play a definite role during adrenal gland development. Indeed, compared with that in untreated plastic dishes, ACTH stimulation of dehydroepiandrosterone sulfate and cortisol was enhanced by collagen IV. In addition, fibronectin enhanced dehydroepiandrosterone sulfate but decreased cortisol secretion, compared with collagen IV substrates. These results provide fundamental insight into the contribution of the microenvironment in cellular processes leading to fetal adrenal gland development.     

Control of calcification by extracellular matrix

Physiological calcification begins from crystallization of hydroxyapatite in extracellular matrix of both bones and teeth. Because calcification is exactly the extracellular event, organic components of extracellular matrix play important roles in control of calcification. The authors discuss the molecular regulation of calcification by organic components of extracellular matrix, focusing on the mineral/organic interaction.     

Incorporation of cellular and plasma fibronectins into smooth muscle cell extracellular matrix in vitro.

Fibronectins isolated from the conditioned medium produced by cultures of undifferentiated (monolayer) and differentiated (nodular) swine vascular smooth muscle cells are similar but not identical. In general, the nodular-cell fibronectin has a smaller molecular mass than monolayer-cell fibronectin and appears to lack the COOH-terminal interchain disulfide linkage. We studied the incorporation of cellular and plasma fibronectins into the cell layer. Smooth muscle cells bound 2.5 times more monolayer-cell fibronectin than nodular-cell fibronectin. Polypeptide fragments of human plasma fibronectin were used as a model system to investigate fibronectin incorporation into the cell layer. Only intact molecules were incorporated into the cell layer and subsequently organized into fibers. Polypeptide fragments of molecular mass 205 kDa and 185 kDa were not incorporated even though they retained the collagen-, cell-, and heparin-binding regions. Incorporation appears to require an activity associated with either the NH2-terminal or COOH-terminal domains. We propose that fibronectin activity is lost during differentiation of smooth muscle cells.