Fibrillar Level Research Articles

Alternatively Activated Macrophages and Collagen Remodeling Characterize the Postpartum Involuting Mammary Gland across Species

Recent pregnancy correlates with decreased survival for breast cancer patients compared with non-pregnancy-associated breast cancer. We hypothesize that postpartum mammary involution induces metastasis through wound-healing programs known to promote cancer. It is unknown whether alternatively activated M2 macrophages, immune cells important in wound-healing and experimental tumorigenesis that also predict poor prognosis for breast cancer patients, are recruited to the normal involuting gland. Macrophage markers CD68, CSF-1R, and F4/80 were examined across the pregnancy and involution cycle in rodent and human mammary tissues. Quantitative immunohistochemistry revealed up to an eightfold increase in macrophage number during involution, which returned to nulliparous levels with full regression. The involution macrophages exhibit an M2 phenotype as determined by high arginase-1 and low inducible nitric oxide synthase staining in rodent tissue, and by mannose receptor expression in human breast tissue. M2 cytokines IL-4 and IL-13 also peaked during involution. Extracellular matrix (ECM) isolated from involuting rat mammary glands was chemotactic for macrophages compared with nulliparous mammary ECM. Fibrillar collagen levels and proteolysis increased dramatically during involution, and denatured collagen I acted as a strong chemoattractant for macrophages in cell culture, suggesting proteolyzed fibrillar collagen as a candidate ECM mediator of macrophage recruitment. M2 macrophages, IL-4, IL-13, fibrillar collagen accumulation, and proteolysis of collagen are all components of tumor promotional microenvironments, and thus may mediate promotion of breast cancers arising in the postpartum setting.

Brain-Specific Deletion of Extracellular Signal-Regulated Kinase 2 Mitogen-Activated Protein Kinase Leads to Aberrant Cortical Collagen Deposition

The mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and 2 are essential intracellular mediators of numerous transmembrane signals. To investigate neural-specific functions of ERK2 in the brain, we used a Cre/lox strategy using Nestin:Cre to drive recombination in neural precursor cells. Nestin:Cre;ERK2(fl/fl) conditional knockout (cKO) mice have architecturally normal brains and no gross behavioral deficits. However, all cKO mice developed early-onset (postnatal day 35 to 40) frontal cortical astrogliosis, without evidence of neuronal degeneration. Frontoparietal cortical gray matter, but not underlying white matter, was found to contain abundant pericapillary and parenchymal reticulin fibrils, which were shown by immunohistochemistry to contain fibrillar collagens, including type I collagen. ERK1 general KO mice showed neither fibrils nor astrogliosis, indicating a specific role for ERK2 in the regulation of brain collagen. Collagen fibrils were also observed to a lesser extent in GFAP:Cre;ERK2(fl/fl) mice but not in CamKII-Cre;ERK2(fl/fl) mice (pyramidal neuron specific), consistent with a possible astroglial origin. Primary astroglial cultures from cKO mice expressed elevated fibrillar collagen levels, providing further evidence that the phenotype may be cell autonomous for astroglia. Unlike most other tissues, brain and spinal cord parenchyma do not normally contain fibrillar collagens, except in disease states. Determining mechanisms of ERK2-mediated collagen regulation may enable targeted suppression of glial scar formation in diverse neurological disorders.

Soluble fibrillar oligomer levels are elevated in Alzheimer's disease brain and correlate with cognitive dysfunction

Recent evidence has suggested a role for soluble oligomeric Aβ species in the pathology of Alzheimer's disease (AD). Fibrillar plaque deposits are present in non-demented individuals and levels of soluble Aβ correlate better with cognitive dysfunction in AD and transgenic mouse models. We have previously reported that there are at least two conformationally distinct types of Aβ oligomers: prefibrillar oligomers that are kinetic intermediates in fibril assembly reactions and are specifically recognized by A11 antibody and fibrillar oligomers that may represent fibril seeds or small pieces of fibrils and are recognized by a fibril specific antibody, OC. We have examined the levels of these two types of oligomers in the PBS soluble fraction of brain tissue from control cases, cases with senile degenerative changes (SDC) and AD patients. We found that the levels of soluble fibrillar oligomers detected by OC antibody are significantly elevated in multiple brain regions of AD patients. The elevated fibrillar oligomer levels were found not to be an artifact of tissue homogenization, nor a result of increased Aβ or APP levels. The concentration of fibrillar oligomers in adjacent brain regions of the same patient can vary widely and were not detected in post-mortem cerebrospinal fluid. In contrast, the level of prefibrillar oligomers are variable in both AD and age matched controls, indicating that they are not correlated with cognitive dysfunction and suggesting that they precede dementia in AD. Significant correlations were found between the levels of fibrillar oligomers and cognitive decline (MMSE scores) as well as the neuropathological hallmarks of AD. These results indicate that fibrillar oligomers may play a key role in the pathology of AD and may be a new target for diagnostic and therapeutic development.

Adenovirus-mediated gene transfer of fibromodulin inhibits neointimal hyperplasia in an organ culture model of human saphenous vein graft disease

Poor long-term graft patency remains a major limitation of coronary artery bypass grafting using saphenous vein aortocoronary grafts. Neointimal hyperplasia (NIH) represents the principal mechanism of graft failure; a substantial body of evidence implicates transforming growth factor-beta1 (TGF-beta1) in the pathogenesis of NIH. The small leucine-rich proteoglycans decorin and fibromodulin possess TGF-beta-antagonist activity to differing extents and with differing avidities for the isoforms of TGF-beta. We compared their ability to inhibit NIH in an ex vivo model of human saphenous vein organ culture following adenovirus-mediated gene transfer. Surgically prepared human saphenous vein segments received adenovirus expressing fibromodulin (Ad5-Fmod), decorin (Ad5-Dcn), beta-galactosidase (Ad5-lacZ) or vehicle-only. Computerized morphometry 14 days after infection revealed significantly reduced neointimal area, neointimal thickness and intima/media ratio in Ad5-Fmod- and Ad5-Dcn-infected veins. Each parameter was significantly smaller in Ad5-Fmod- than in Ad5-Dcn-exposed segments. Fibrillar collagen content and levels of biologically active TGF-beta were lower in vessels receiving Ad5-Fmod or Ad5-Dcn than in those receiving Ad5-lacZ or vehicle-only. Fibromodulin is a more potent inhibitor of NIH in cultured human saphenous vein than decorin and offers potential therapeutic benefits in saphenous vein graft failure (and possibly in other forms of accelerated atherosclerosis) by reduction of associated neointima formation.

Microstructural response and fluid flow mechanisms in cartilage loading: New insights using a novel indentation method

A novel indentation method was used to investigate the response of articular cartilage in the non-directly loaded region. The indenter contained a relief channel that allowed a tissue bulge to develop within it under load. Healthy bovine tissue samples were statically loaded at a nominal compressive stress of 3.6 MPa. The tissue's equilibrium deformed state was chemically fixed. Differential interference contrast microscopy was used to obtain high-resolution images of the deformed microstructure in the region of the tissue bulge. At the submicro-level, the fibrillar resistance to load is highly complex such that regions of relative compression and tension coincide along a single radial direction. This fibrillar level response is manifested as large-scale matrix shear effects within the bulge region. Further, the surface layer, besides being strain-limiting in the tangential direction, has an intrinsic resistance to axial load. Finally, the pattern of load-induced fluid flow is seen to traverse zonal depths and hence suggests an added complexity to the overall permeability in the deformed tissue matrix.

Constitutive modeling of crimped collagen fibrils in soft tissues

A microstructurally oriented constitutive formulation for the hyperelastic response of crimped collagen fibrils existing in soft connective tissues is proposed. The model is based on observations that collagen fibrils embedded in a soft matrix crimp into a smooth three-dimensional pattern when unloaded. Following ideas presented by Beskos and Jenkins [Beskos, D., Jenkins, J., 1975. A mechanical model for mammalian tendon. ASME Journal of Applied Mechanics 42, 755–758] and Freed and Doehring [Freed, A., Doehring, T., 2005. Elastic model for crimped collagen fibrils. Journal of Biomechanical Engineering 127, 587–593] the collagen fibril crimp is approximated by a cylindrical helix to represent the constitutive behavior of the hierarchical organized substructure of biological tissues at the fibrillar level. The model is derived from the nonlinear axial force–stretch relationship of an extensible helical spring, including the full extension of the spring as a limit case. The geometrically nonlinear solution of the extensible helical spring is carried out by an iterative procedure. The model only requires one material parameter and two geometrical parameters to be determined from experiments. The ability of the proposed model to reproduce the biomechanical response of fibrous tissues is demonstrated for fascicles from rat tail tendons, for porcine cornea strips, and for bovine Achilles tendons.

The echinoderm collagen fibril: a hero in the connective tissue research of the 1990s

Collagen fibrils are some of the most-abundant and important extracellular structures in our bodies, yet we are unsure of their shape and size. This is largely due to an inherent difficulty in isolating them from their surrounding tissues. Echinoderms have collagenous tissues that are similar to ours in many ways, yet they can be manipulated to easily relinquish their collagen fibrils, providing an excellent opportunity to study native fibrillar structure. In the early 1990s, they were found to defy the commonly accepted fibrillar model of the time in that they were much shorter, they were shaped like double-ended spindles, and their centers exhibited a reversal in molecular polarity. Realization of these features helped to reform the questions that were being asked about vertebrate fibrils, shifting the focus toward shape and size. Since then, researchers working with both groups (echinoderms and vertebrates) have worked together to find the structure of native fibrils. This information will be fundamental in understanding what holds collagenous tissues together at the fibrillar level, and could have important implications for people with Ehlers-Danlos syndrome.

Real-time and Single Fibril Observation of the Formation of Amyloid β Spherulitic Structures

In Alzheimer disease, amyloid beta, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 microm on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

A novel microstructural approach in tendon viscoelastic modelling at the fibrillar level

Novel applications in rehabilitation, surgery and tissue engineering require the knowledge of the mechanical behaviour of the tissues at microstructural level. The aim of this work is to investigate the viscoelastic properties of the tendon from the interaction of its biological constituents in the fibrillar network. Traction, relaxation and creep in-vitro tests have been performed on porcine flexor digital tendons. A viscoelastic constitutive equation at finite deformation is presented. The fibrillar deformation modes are described through a network of adaptive links between collagen type I and decorin. The theoretical predictions fit accurately the experimental data. The results of the model demonstrate the mechanical importance of glycosaminoglycan chains of decorin for the differential recruitment and the activation of fibrillar collagen.

Effects of Aging on the Toughness of Human Cortical Bone: A Study from Nano to Macro Size-Scales

ABSTRACTAge-related deterioration of both the fracture properties and the architecture of bone, coupled with increased life expectancy, are factors leading to the increasing incidence of bone fracture in the elderly. In order to facilitate the development of treatments which counter this increased fracture risk, a thorough understanding of how fracture properties degrade with age is required. The present study describes ex vivo fracture experiments to quantitatively assess the effects of aging on the fracture toughness of human cortical bone in the longitudinal direction. Because cortical bone exhibits rising crack-growth resistance with crack extension, we depart from most previous studies by evaluating the toughness in terms of resistance-curve (R-curve) behavior, measured for bone taken from donors 34 to 99 years old. Using this approach, both the crack-initiation and crack-growth toughness are determined and are found to deteriorate with age; the initiation toughness decreases ∼40% from 40 to 100 years, while the growth toughness is effectively eliminated over the same age range. Evidence from x-ray synchrotron tomography is provided to support the hypothesis that the reduction in crack-growth toughness is associated primarily with a degradation in the degree of extrinsic toughening, in particular involving crack bridging at the microstructural level in the wake of the crack. Atomic force microscope-based nanoidentation of individual collagen fibers revealed changes at the collagen fibrillar level and deep-ultraviolet Raman spectroscopy showed that the cross-linking at the nanostructural level also changes with age. These results should provide for a better mechanistic understanding of the increased propensity for bone fracture with age.

Altered collagen structure in mouse tail tendon lacking the α2(I) chain

Type I collagen is the most prevalent member of the fibril forming family of collagens in higher vertebrates and its heterotrimeric form is comprised of two α1(I) chains and one α2(I) polypeptide chain. The functional importance of having two distinct chain types in type I collagen is largely undefined. The existence of a mouse model with a Cola-2 gene mutation (termed oim) that results in non-functional proα2(I) chains presents a unique opportunity to explore changes in collagen structure resulting from the complete ( oim/ oim mice) and partial ( oim/+ mice) loss of functional α2(I) chains. Tail tendon is a tissue with a well-defined, hierarchical organization of type I collagen. X-ray diffraction studies on oim/ oim versus control tendons indicate that the total absence of α2(I) chains results in a decrease in the order of axial packing and a loss of crystalline lateral packing. This suggests that the non-equivalence of three chains is an important determinant of lateral interactions between adjacent molecules and may be involved in the long-range axial order in type I collagen-containing tissues. Both homotrimeric and heterotrimeric type I collagen molecules are found in heterozygous oim mice and these appear to be present in the same co-polymeric fibrils, preventing crystalline lateral packing. In addition to these changes at a fibrillar level, the absence of the α2(I) chain results in an increased enzymatic susceptibility at one site. The oim/ oim mice are observed to have reduced body size and smaller tendon bundles, which may be a consequence of these molecular and fibrillar changes in collagen. Furthermore, it is likely that a similar alteration in the molecular packing of collagen in bone fibrils contributes to the osteopenia and decreased bone strength in mice with the oim mutation that are also characteristic of human osteogenesis imperfecta.

Downregulation of sarcoplasmic reticulum Ca(2+)-ATPase during progression of left ventricular hypertrophy.

To determine whether reduced sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) (SERCA2) activity contributes to delayed myocardial relaxation during chronic left ventricular hypertrophy (LVH) progression, LVH was produced in rats by abdominal aortic coarctation. Systolic and diastolic functions were assessed in vivo 8 and 16 wk after surgery, and compositional alterations in LV myocardium [SERCA2 concentration, myosin heavy chain (MHC) isoenzymes, and tissue collagen] were correlated with the development of prolonged isovolumic relaxation and impaired cardiac performance over time. Myocardial relaxation was prolonged in 8-wk banded rats, despite normal isovolumic systolic function and LV end-diastolic pressure (LVEDP). No significant alterations in SERCA2 protein, beta-MHC, or fibrillar collagen levels were observed at this early time point. In contrast, LV SERCA2, beta-MHC, and fibrillar collagen concentrations were all significantly altered in 16-wk banded rats. These late compositional changes were associated with reduced cardiac performance, as manifested by a significant elevation in LVEDP (14 +/- 2 mmHg). The 34% decrease in SERCA2 protein was associated with reduced SR Ca2+ uptake and an even greater reduction (76%) in SERCA2 mRNA. SERCA2 mRNA levels were also significantly reduced to 43 +/- 10% of sham-operated rats 8 wk after banding, despite unchanged SERCA2 protein levels and normal SR Ca2+ uptake. These results argue against a significant contribution of SERCA2 downregulation to the subtle alterations in myocardial relaxation observed in compensated LVH. However, the early reduction in SERCA2 mRNA levels may serve as a molecular marker for impaired cardiac performance during the transition from compensated LVH to heart failure.

Collagen linkage in periodontal connective tissue reattachment. An ultrastructural study in beagle dogs.

Early stages of connective tissue reattachment to surgically denuded cementum and root dentin following citric acid application were studied in fenestration wounds. Block specimens were obtained after seven, 14, and 21 days. Continuity between newly deposited collagen fibrils in the granulation tissue and unmasked dental matrix collagen had been established within seven days by interdigitation of fibrils in an up to 0.5 micron-wide zone at the cementum or dentin surface. Splicing of collagen at the fibrillar level by direct attachment to the severed end of matrix fibrils or by juxtapositioning of new and old fibrils was seen in rare instances only. At 14 and 21 days, distinct bundles of collagen fibrils inserted deeply into the orifice of dentinal tubules. Arrested and reversed surface resorption was encountered at all time points. At these sites as well, new collagen fibrils interdigitated with denuded matrix collagen, while some resorbed surfaces were characterized by absence of collagen continuity. The results lend continued support to the concept of reattachment based on interdigitation of collagen fibrils. However, since attachment is established even in the absence of this feature, a significant role for specific attachment substances not shown can also be postulated.

The biology of wound healing in the corneal stroma. Castroviejo lecture.

The healing and remodeling of a stromal incision in described at the gross, fibrillar, and cellular levels. The possibility that the healing of the wound and the clearing of the scar can be accelerated by biological interventions is discussed. Castroviejo Lecture was given on November 9, 1986.

New aspects of the etiology of tendon rupture. An analysis of time-resolved dynamic-mechanical measurements using synchrotron radiation.

Native collagen fibers were exposed to different dynamic loads to simulate damage to tendons and ligaments relevant clinically and for sports medicine. The results suggest that the rupture of a tendon is caused at the submicroscopic fibrillar level. Not only slow or very fast elongation, but also very fast unloading of stretched fibers seems to be responsible for disseminated damage, which reduces the stability of a fiber. This damage is induced by intrafibrillar sliding processes, which occur only a few seconds before macroscopic slippage takes place. The significance of these events for the beginning and progress of repair in vivo is discussed. The conclusions are supported by simultaneous mechanical and radiological measurements, as well as by light- and electron-microscopic results.

Ultrastructural identification of extension aminopropeptides of type I and III collagens in human skin.

Human skin was labeled with purified antibodies against type II and III collagens and against their extension aminopropeptides by using the ferritin technique. Both aminopropeptides were visualized mainly along thin collagenous fibrils (diameter, 20-40 nm) and rarely in nonfibrillar regions of the skin. The labeling showed a periodicity of 60-65 nm resembling the D (67 nm) stagger of collagen molecules. Blocking of antibodies with aminopropeptides and treatment of tissues with procollagen NH2-terminal protease abolished labeling. Antibodies against type I collagen uniformly labeled approximately equal to 80% of the fibrils (diameter, 20-80 nm), while reaction with antibodies against type III collagen was restricted to thin fibrils. It is currently thought that the aminopropeptides of procollagen molecules are cleaved after they are released from the cell and before fibril formation. Our data indicate that aminopropeptides are removed at the fibrillar level and that fibril growth can be regulated by extracellular procollagen processing.

Hierarchy of fibrillar organization levels in the polytene interphase chromosomes of Chironomus.

ABSTRACT The present study shows a hierarchy of fibrillar organization levels in polytene somatic interphase chromosomes. The chromosomes of several Chironomus species have been examined by electron microscopy in situ, i.e., in the physiological environment of intact salivary glands, and in vitro as spread preparations. In situ, the basic structural element of bands, interbands and constitutive heterochromatin is a DNP fibre (chromatid) which varies in thickness from 11 to 25 nm. The fibre displays a varying degree of coiling in bands and interbands. It runs more or less straight through the interbands but forms aggregates (chromomeres), which have a densely packed fibrillar texture, in the band regions. On the whole there is always an abrupt transition between the fibre and the chromomeres which it connects. Constitutive heterochromatin is also made up of a fibre of the same thickness as in bands and interbands. However, in the heterochromatin the fibre generally measures 20–25 nm. In telomeric heterochromatin the fibre is extremely densely arranged, whereas the centric heterochromatin (chromocentre) of C. melanotus is made up of very loosely bundled fibres. Neither type of heterochromatin exhibits any visible subdivision into chromomeres and interchromomeres. In spread preparations of native isolated polytene chromosomes of different Chironomus tissues seen in vitro the bands and interbands are also composed of a DNP fibre which varies in thickness from 11 to 25 nm. Fibres in spread preparations are at most 20–25 nm thick. The spreading of band regions enables one to recognize more or less tangled, sometimes transcribed 0 · 8–8 · 4 μm loops, which are regarded as unfolded chromomeres. The shape of the 20–25-nm fibre in band (loop) and interband regions is irregular and clearly reflects orderly packing of globular particles (superbeads) embedded in a diffuse matrix. The latter can be removed by spreading the fibres in low salt medium or distilled water, which also makes individual superbeads visible. Arranged in a row, 4 superbeads account for 0·1 μm. Superbeads are structures consisting of subunits which correspond in size (n 0± 10 nm) to nucleosomes. Tentatively, there are 7–12 nucleosomes in one superbead. A row of nucleosomes constitutes the 11–13-nm fibre. This is the basic fibre of polytene chromosomes. The 20–25-nm fibre appears to be a higher order of the basic fibre. Digestion of the 20–25-nm fibre by trypsin and DNase reveals that the fibre is unineme as far as its DNA is concerned. The fact that polytene interphase chromosomes analysed in situ and in vitro both show a 20-25-nm fibre, leads to the conclusion that this fibre represents the highest level of fibrillar organization of the DNP fibre. Presumably the distinct levels of organization revealed by the 20–25-nm fibre in vitro also exist in the chromosomes in situ, but are invisible because the fibres prepared at physiological ionic strength are densely packed.

Thyroidal influence on early muscle growth of chickens

Severe hypothyroidism, produced by the injection of 24 mg of propylthiouracil (PTU) per 100 g body weight per day, significantly reduced body growth during a 4-week period shortly after hatching. Body weight and shank-toe length of PTU-treated cockerels were 16.2% and 12.4% less than controls, and were maintained at normal levels by replacement with as little as 0.25 μg and 0.50 μg of thyroxine (T 4) per 100 g body weight per day, respectively. Muscle growth was also reduced by severe hypothyroidism and returned to normal with low replacement doses of T 4. Gastrocnemius muscle weight and total crude fibrillar protein and DNA levels were reduced 17.9%, 18.0%, and 34.5%, respectively, compared to controls. Administration of 0.50, 1.50, and 3.00 μg of T 4 per 100 g body weight per day to PTU-treated cockerels maintained these muscle parameters at normal levels. Total actin and the concentration of actin, on the basis of fresh weight or crude fibrillar protein content, were unaltered in the gastrocnemius by changes in thyroid status. There was a 29.1% increase in the actin/DNA in the gastrocnemius muscle, however, reflecting the lesser DNA. Sartorius muscle growth was much less affected by changes in thyroid status than the gastrocnemius. Total DNA was reduced 22.1% and actin/DNA increased 26.8% in the PTU-treated cockerels compared to controls. The actin levels in the gastrocnemius, sartorius, and pectoralis muscles were ascertained for the normal growth period from 2 to 63 days of age. Two pertinent observations were noted. First, it was observed that total actin and the concentration of actin increase during the growth of all three muscles; and second, the pattern of accumulation of actin was found to differ in the three muscles.