Fibroblasts retain their tissue phenotype when grown in three-dimensional collagen gels

Fibroblasts are responsible for the synthesis, assembly, deposition, and organization of extracellular matrix molecules, and thus determine the morphology of connective tissues. Deposition of matrix molecules occurs in extracellular compartments, where the sequential stages are under cellular control. Cell orientation/polarity is important in determining how the cell orients these extracytoplasmic compartments and therefore how the matrix is assembled and oriented. However, the control of cell orientation is not understood. Fibroblasts from three tissues with different morphologies were studied to determine whether cells maintained their characteristic phenotype. Fibroblasts from cornea, which in vivo are oriented in orthogonal layers along with their matrix; from tendon, a uniaxial connective tissue, where cells orient parallel to each other; and from dermis, a connective tissue with no apparent cellular orientation, were used to study cell morphology and orientation in three-dimensional collagen gels. The different cells were grown for 3 and 7 days in identical three-dimensional collagen gels with a nonoriented matrix. Confocal fluorescence microscopy demonstrated that corneal fibroblasts oriented perpendicular to one another at 3 days, and after 7 days in hydrated gels these cells formed orthogonal sheets. Tendon fibroblasts were shown by the same methods to orient parallel to one another in bundles at both 3 and 7 days, throughout the depth of the gel. Dermal fibroblasts showed no apparent orientation throughout the hydrated gels at either time point examined. The organization of these different cell types was consistent with their tissue of origin as was the cell structure and polarity. These studies imply that cellular and tissue phenotype is innate to differentiated fibroblasts and that these cells will orient in a tissue-specific manner regardless of the extracellular matrix present.     

Effects of pdgf-bb on rat dermal fibroblast behavior in mechanically stressed and unstressed collagen and fibrin gels

The dose-response effects of platelet-derived growth factor BB (PDGF-BB) on rat dermal fibroblast (RDF) behavior in mechanically stressed and unstressed type I collagen and fibrin were investigated using quantitative assays developed in our laboratory. In chemotaxis experiments, RDFs responded optimally (P < 0.05) to a gradient of 10 ng/ml PDGF-BB in both collagen and fibrin. In separate experiments, the migration of RDFs and the traction exerted by RDFs in the presence of PDGF-BB (0, 0.1, 1, 10, or 100 ng/ml) were assessed simultaneously in the presence or absence of stress. RDF migration increased significantly (P < 0.05) at doses of 10 and 100 ng/ml PDGF-BB in collagen and fibrin in the presence and absence of stress. In contrast, the effects of PDGF-BB on RDF traction depended on the gel type and stress state. PDGF-BB decreased fibroblast traction in stressed collagen, but increased traction in unstressed collagen (P < 0.05). No statistical conclusion could be inferred for stressed fibrin, but increasing PDGF-BB decreased traction in unstressed fibrin (P < 0.05). These results demonstrate the complex response of fibroblasts to environmental cues and suggest that mechanical resistance to compaction may be a crucial element in dictating fibroblast behavior.     

Cooperative kinking at distant sites in mechanically stressed DNA

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180° apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.     

Localization of calcium and microfilament changes in mechanically stressed cells

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows:

1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium.
2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips.
3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation.
4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA.

It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.     

Localization of calcium and microfilament changes in mechanically stressed cells.

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows: 1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium. 2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips. 3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation. 4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA. It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.     

Mechanical behavior of fibroblasts included in collagen lattices

Striae distensae are characterized by linear, smooth bands of atrophic-appearing skin. Excessive steroid activity, genetic and mechanical factors and inherited defects of connective tissues are the most frequent causes of this disease. Fibroblasts derived from women presenting striae distensae lesions were included into collagen gels to study their mechanical behavior: capacity to contract free-floating lattices and to produce isometric force in tense lattices. To measure the retracted lattice diameter, the culture dishes were placed on a transparent metric scale. An isometric force system was used to study quantitatively the forces developed during lattice contraction. alpha 2 beta 1 integrins expression (transmembrane receptors) was evaluated by flux cytometry. Striae distensae fibroblasts contract collagen gels slower than normal human fibroblasts but the final contraction is similar. They produce a greater isometric force which is associated with enhanced alpha 2 beta 1 integrins expression. By their mechanical properties, striae distensae fibroblasts appear as a different population from normal fibroblasts.     

Histone deacetylase inhibition alters dendritic cells to assume a tolerogenic phenotype and ameliorates arthritis in SKG mice

Introduction  

The purpose of this study was to elucidate the effects of histone deacetylase inhibition on the phenotype and function of dendritic cells and on arthritis in SKG mice.     

Stable isotope resolved metabolomics of primary human hepatocytes reveals a stressed phenotype

The development of techniques allowing the culturing of primary mammalian hepatocytes has provided great insights into liver physiology. For most applications, it is desirable for hepatocytes in culture to mimic hepatocytes in vivo. We used stable isotope resolved metabolomics (SIRM) to assess glucose and glutamine utilization in primary rat and human hepatocytes maintained in standard culture media. Primary rat hepatocytes readily metabolized ¹³C-glucose and ¹³C-glutamine made evident by ¹³C incorporation into glycogen, glycolytic end products, and Krebs cycle intermediates and responded to insulin and glucagon appropriately. In contrast, no glucose or glutamine consumption was detected in primary human hepatocytes over 4 h of exposure to high media concentrations of ¹³C-glucose or ¹³C-glutamine even in the presence of insulin. Nonetheless, cultured human hepatocytes were metabolically active and viable, as demonstrated by incorporation of media ¹³C-octanoic acid into Krebs cycle intermediates and ketone bodies. The failure to utilize glucose was not due to inhibition of glucokinase (hexokinase IV) since the human hepatocytes could readily incorporate ¹³C-glucose into glucuronic acid, as demonstrated by the production of ¹³C-glucuronide conjugates after addition of acetaminophen to the media. These novel observations support inhibition of phosphofructokinase-1, the other regulatory enzyme in glycolysis. Parts of this phenotype could be reproduced in the rat hepatocytes by replacing insulin with glucagon to the media. We conclude that under standard culture conditions human hepatocytes are in an extreme starved state. We believe this may result from prolonged fasting in the human liver donors combined with exposure to stress hormones such as, epinephrine, glucagon, and cortisol. Efforts should now be exerted to find culture conditions that will reverse this state to achieve more metabolically relevant cultures of human hepatocytes.     

Convergence in a mechanically complex phenotype: detecting structural adaptations for crushing in cichlid fish

Morphological convergence provides strong evidence that evolution is adaptive. However, putatively convergent morphology is often examined in two dimensions with no explicit model of function. In this study, we investigated structural and mechanical similarities of the lower pharyngeal jaw (LPJ) in cichlid fish that have evolved the ability to crush hard-shelled molluscs. Using a novel phylogeny, we demonstrated molluscivory has been gained and/or been lost numerous times in Heroine cichlids. Within this comparative framework, we produced three-dimensional computed tomography (CT) scans for LPJs of both morphotypes in the trophically polymorphic Herichthys minckleyi and six evolutionarily independent pairs of closely related species. Like H. minckleyi , these species exhibit divergence between a molluscivore and a nonmolluscivore. Using the CT scans, we generated finite element models of papilliform H. minckleyi LPJs to determine where stress would concentrate in a jaw not modified to crush molluscs. Then, we examined whether stress in the papilliform LPJ predicted structural modifications in molariform H. minckleyi and other molluscivorous species. Despite potential constraints, stresses imposed during prey processing explain 40% of LPJ variation in mollusc crushing species. The structural and mechanical analyses also suggest divergence found in polymorphic species could provide the substrate for trophic differences found in reproductively isolated cichlids.     

Pre-adsorbed type-I collagen structure-dependent changes in osteoblastic phenotype

Type-I collagen is the most abundant extracellular matrix in bones and modulates various functions of osteoblasts. We prepared two different structures of type-I collagen on tissue culture grade polystylene (TCPS) surfaces, one is feltwork structure of filamentous molecules from acid solutions (ACs) and the other is network structure of fibrils from neutral solutions (NCs), to examine effects of the structures on the maturation process of osteoblast-like cells. No significant differences of cell proliferation were observed between TCPS and ACs, but NCs delayed the proliferation. In initial cell attachment, the cells on ACs had tense lamellipodia with sharp tips, while those on NCs had loose lamellipodia. No detectable differences in levels of expressed integrin alpha2- and alpha5-subunits were observed between the structures. Although the matrix mineralization in NCs was also delayed in comparison with TCPS and ACs, fully mineralized levels in NCs were the same as those of TCPS and ACs. In addition, although we examined the effects of densities of pre-adsorbed collagen molecules on osteoblast maturation, the effects were less serious than those of the structures. This study suggests that the structures of collagen affect proliferation and mineralization of osteoblast-like cells.     

Clacium ion involvement in growth inhibition of mechanically stressed soybean (Glycine max) seedlings

A 40–50% reduction in soybean [ Glycine max (L.) Merr. cv. Century 84] hypocotyl elongation occurred 24 h after application of mechanical stress. Exogenous at 10 m M inhibited growth by 28% if applied with the Ca²⁺ ionophore A23187 to the zone of maximum hypocotyl elongation. La³⁺ was even more inhibitory than Ca²⁺, especially above 5 m M . Treatment with ethyleneglycol-bis-(β3-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) alone had no effect on growth of non-stressed seedlings at the concentrations used but negated stress-induced growth reduction by 36% at 4 m M when compared to non-treated, stressed controls. Treatment with EDTA was ineffective in negating stress-induced growth inhibition. Calmodulin antagonists calmidazolium, chlorpromazine, and 48/80 also negated stress-induced growth reduction by 23, 50, and 35%, respectively.     

Induced monoterpene and lignin production in mechanically stressed and fungal elicited cultured Cupressus lusitanica cells

Cultured Cupressus lusitanica cells induced by various stresses are thought to produce different complexes of defense chemicals to optimize defense. To compare the induced products of two stimulations, we investigated the emission of monoterpenes, biosynthesis of β-thujaplicin, and accumulation of lignin in mechanically stressed and fungal elicited cultured C. lusitanica cells. Both mechanical stress and fungal elicitor caused emission of qualitatively similar monoterpene blends indicating de novo biosynthesis of these compounds after stimulation, while mechanical stress alone is sufficient to induce fungal elicitor-related monoterpene emission. Sabinene and limonene were the dominant compounds over the time course in both volatile blends. Although the emitted volatile blends were qualitatively similar, the time course and the relative ratios of the constituents of the volatile blends differed with the type of stimulation. While fungal elicited cells produced significant amounts of β-thujaplicin over the 5-day time course, no β-thujaplicin was observed in the mechanically stressed cells. The production of β-thujaplicin was the main dissimilarity of the induced products of these two treatments, suggesting that synthesis of β-thujaplicin is not a general response to all types of stresses, but is a specific response and serves as a strong toxic compound against already invaded fungus. Significantly higher amounts of lignin accumulations were observed in the fungal elicited and mechanically stressed cells on the 5th day after induction. Based on these results, we suggest the composition of induced products was dependent on the method of stimulation.     

Design of a synthetic collagen-binding peptidoglycan that modulates collagen fibrillogenesis

The ubiquity of collagen in mammalian tissues, with its host of structural and chemical functions, has motivated its research in many fields, including tissue engineering. The organization of collagen is known to affect cell behavior and the resulting structural integrity of tissues or tissue engineered scaffolds. Of particular interest are proteoglycan (PG) interactions with collagen and their influence on collagen assembly. These natural molecules provide unique chemical and mechanical cues and are known to modulate collagen fibrillogenesis. Research has been limited to PGs extracted and purified from animal sources and has the drawbacks of limited design control and costly purification. Consequently, we have designed a synthetic peptidoglycan based on decorin, a collagen-binding PG. The synthetic peptidoglycan containing a collagen-binding peptide with a single dermatan sulfate side chain specifically binds to collagen, delays fibrillogenesis, and increases collagen gel stiffness as decorin does. This design can be tailored with respect to the peptide sequence and attached glycosaminoglycan chain, offering unique control with relative ease of manufacturing.     

Fibroblasts and monocyte macrophages contract and degrade three-dimensional collagen gels in extended co-culture

Background  

Inflammatory cells are believed to play a prominent role during tissue repair and remodeling. Since repair processes develop and mature over extended time frames, the present study was designed to evaluate the effect of monocytes and fibroblasts in prolonged culture in three-dimensional collagen gels.     

Decreased level of PDGF-stimulated receptor autophosphorylation by fibroblasts in mechanically relaxed collagen matrices

The goal of our studies was to characterize the interrelationship between extracellular matrix organization and fibroblast proliferation in response to growth factors. We compared fibroblasts in monolayer culture with cells in contracted collagen matrices that were mechanically stressed or relaxed. In response to platelet-derived growth factor (PDGF), DNA synthesis by fibroblasts in mechanically relaxed collagen matrices was 80-90% lower than in monolayer culture and 50% lower than in mechanically stressed matrices. Fibroblasts in monolayer and contracted collagen matrix cultures contained similar levels of PDGF receptors, but differed in their autophosphorylation response. Cells in mechanically relaxed matrices showed lowest levels of autophosphorylation, 90% less than cells in monolayer culture. Experiments comparing receptor expression and capacity for PDGF- stimulated autophosphorylation showed that cells in mechanically relaxed collagen matrices never developed normal receptor autophosphorylation. Furthermore, when mechanically stressed collagen matrices were switched to mechanically relaxed conditions, capacity for receptor autophosphorylation decreased within 1-2 h and remained low. Based on immunomicroscopic observations and studies on down-regulation of receptors by PDGF binding, it appeared that most PDGF receptors in monolayer or contracted collagen matrix cultures were localized on the cell surface and accessible to PDGF binding. In related studies, we found that EGF receptors of fibroblasts in mechanically relaxed collagen matrices also showed low levels of autophosphorylation in response to EGF treatment. Based on these results, we suggest that mechanical interactions between cells and their surrounding matrix provide regulatory signals that modulate autophosphorylation of growth factor receptors and cell proliferation.