Interactions of heat shock protein 47 with collagen and the stress response: an unconventional chaperone model?

Heat shock proteins (HSPs) are upregulated and manifested upon cellular stress and possess chaperoning functions. HSP47 is an endoplasmic reticulum (ER)-resident, collagen-specific chaperone and plays a key role in collagen biosynthesis and its structural assembly. The collagen scaffold is a primary structural target of recent interest due to its applications in tissue engineering and drug delivery and in treatment of clinical disorders. This review highlights the fundamental aspects of HSPs in protein folding and quality control, in the elicitation of a stress response in connective tissue and in the characterization of HSP47 in collagen folding and assembly. The significant features of HSP47 which are distinct in its cellular capabilities are discussed. We propose that targeting the stress response is a key factor in identifying connective tissue biomarkers. We also address the issues and strategies involved in the stress response of connective tissue diseases. In conclusion, we describe the prospects of collagen biochemistry in correlation to the science of HSPs.     

Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.     

IGF-II and IGFBP-6 regulate cellular contractility and proliferation in Dupuytren's disease

Dupuytren's disease (DD) is a common and heritable fibrosis of the palmar fascia that typically manifests as permanent finger contractures. The molecular interactions that induce the development of hyper-contractile fibroblasts, or myofibroblasts, in DD are poorly understood. We have identified IGF2 and IGFBP6, encoding insulin-like growth factor (IGF)-II and IGF binding protein (IGFBP)-6 respectively, as reciprocally dysregulated genes and proteins in primary cells derived from contracture tissues (DD cells). Recombinant IGFBP-6 inhibited the proliferation of DD cells, patient-matched control (PF) cells and normal palmar fascia (CT) cells. Co-treatments with IGF-II, a high affinity IGFBP-6 ligand, were unable to rescue these effects. A non-IGF-II binding analog of IGFBP-6 also inhibited cellular proliferation, implicating IGF-II-independent roles for IGFBP-6 in this process. IGF-II enhanced the proliferation of CT cells, but not DD or PF cells, and significantly enhanced DD and PF cell contractility in stressed collagen lattices. While IGFBP-6 treatment did not affect cellular contractility, it abrogated the IGF-II-induced contractility of DD and PF cells in stressed collagen lattices. IGF-II also significantly increased the contraction of DD cells in relaxed lattices, however this effect was not evident in relaxed collagen lattices containing PF cells. The disparate effects of IGF-II on DD and PF cells in relaxed and stressed contraction models suggest that IGF-II can enhance lattice contractility through more than one mechanism. This is the first report to implicate IGFBP-6 as a suppressor of cellular proliferation and IGF-II as an inducer of cellular contractility in this connective tissue disease.     

Effect of some organosilicon compounds (silatranes) on the biosynthesis of collagen in cartilagenous tissue of chick embryo]

Effect of some organosilicon compounds from the class of silatranes on the biosynthesis of collagen in cartilagenous tissue of chick embryos in vitro was studied. The criteria for assessing the intensity of collagen biosynthesis was the formation of peptide bond 14C-hydroxyproline from 14C-proline of incubation medium. All the compounds studied (methylsilatrane, ethoxysilatrane and chloromethylsilatrane) stimulated total protein biosynthesis and collagen biosynthesis in cartilagenous tissue of chick embryos. The most pronounced stimulation of the biosynthesis of collagen was observed at 3-10(-3) M concentration of silatranes within 180 min. of incubation (14C-proline was added to the incubation medium 60 min after the beginning of the incubation). The activity of partly purified collagen prolyl-hydroxylase in the presence of silatranes was also studied.     

Biomechanics of the lung parenchyma: critical roles of collagen and mechanical forces.

The biomechanical properties of connective tissues play fundamental roles in how mechanical interactions of the body with its environment produce physical forces at the cellular level. It is now recognized that mechanical interactions between cells and the extracellular matrix (ECM) have major regulatory effects on cellular physiology and cell-cycle kinetics that can lead to the reorganization and remodeling of the ECM. The connective tissues are composed of cells and the ECM, which includes water and a variety of biological macromolecules. The macromolecules that are most important in determining the mechanical properties of these tissues are collagen, elastin, and proteoglycans. Among these macromolecules, the most abundant and perhaps most critical for structural integrity is collagen. In this review, we examine how mechanical forces affect the physiological functioning of the lung parenchyma, with special emphasis on the role of collagen. First, we overview the composition of the connective tissue of the lung and their complex structural organization. We then describe how mechanical properties of the parenchyma arise from its composition as well as from the architectural organization of the connective tissue. We argue that, because collagen is the most important load-bearing component of the parenchymal connective tissue, it is also critical in determining the homeostasis and cellular responses to injury. Finally, we overview the interactions between the parenchymal collagen network and cellular remodeling and speculate how mechanotransduction might contribute to disease propagation and the development of small- and large-scale heterogeneities with implications to impaired lung function in emphysema.     

Regulation of integrin-type cell adhesion receptors by cytokines.

Integrin heterodimers which share a common beta 1 subunit are the major cellular receptors for many extracellular matrix proteins. Here, we show that two inflammatory mediators, interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), can regulate the expression of the alpha 1 beta 1 integrin heterodimer, known to be a laminin and collagen receptor. In human skin fibroblasts 10 units/ml IL-1 beta increase the biosynthesis of the alpha 1 integrin subunit an average of 4.5-fold. Furthermore, IL-1 beta can turn on alpha 1 subunit expression in MG-63 human osteosarcoma cells even in conditions where the untreated MG-63 cells do not express it in detectable amounts. The effect of TNF-alpha on alpha 1 subunit expression is similar. Both IL-1 beta and TNF-alpha increased MG-63 cell adhesion on laminin. The effect of transforming growth factor-beta 1 (TGF-beta 1) on integrin expression in MG-63 cells has been previously described (Heino, J., and Massagué, J. (1989) J. Biol. Chem. 264, 21806-21811). TGF-beta 1 decreases the biosynthesis of alpha 3 subunit but increases the production of alpha 2 subunit. IL-1 beta potentiates the effects of TGF-beta 1. Furthermore, in the presence of TGF-beta 1 the increase in the expression of alpha 1 subunit by IL-1 beta is even larger. Thus, IL-1 beta and TGF-beta 1, which usually have antagonistic functions in connective tissue, can regulate integrin expression in a synergistic way.     

Effect of cyclic adenosine-3,5-monophosphate and its dibutyryl analog on macromolecule biosynthesis in actively proliferating cells

The effect of cAMP and its dibutyryl analogue on the biosynthesis of nucleic acids and protein in active proliferating cells was studied. It was shown that cAMP (10(-3)--10(-4)M) caused stimulation of the biosynthesis of DNA and RNA in Ehrlich's ascites carcinoma (EAC) cells and intensification of collagen biosynthesis in the chick embryo cartilage tissue in vitro. Dibutyryl -- cAMP (10(-3)--10(-4)M) has an inhibitory action on the biosynthesis of macromolecules both in EAC cells and embryonic cartilage tissue. Addition of cAMP phosphodiesterase inhibitors together with cAMP to the incubation media prevents the stimulation of macromolecular biosynthesis observed under the influence of cAMP. Studies on cAMP metabolism revealed that this compound is rapidly catabolized to AMP and adenosine. The latter enters the cells and incorporates into the adenyl nucleotide intracellular pool. The stimulant action of exogenous cAMP is related to its extracellular metabolism rather than to the intracellular effects of the nucleotide.     

Inositol phospho-oligosaccharide stimulates cell proliferation in the early developing inner ear

The ability of an inositol phospho-oligosaccharide (POS) to mimic the mitogenic effects of nerve growth factor (NGF) and insulin on the early development of the inner ear was investigated. POS (10 microM) stimulated the incorporation of [3H]thymidine into the cochleovestibular ganglion by 3.9-fold. NGF (50 ng/ml) stimulation was 4.7-fold. POS and NGF showed no additivity. Cells induced to proliferate by POS overlapped with those expressing NGF receptors. POS, like insulin, potentiated the mitogenic effect of bombesin on the otic vesicle epithelium. DNA synthesis in the presence of bombesin (100 nM) plus POS (10 microM) was increased by 6.4-fold. POS stimulation was not additive with insulin. The results suggest that POS may play a role in growth factor regulation of cell proliferation during embryonic development.     

Effect of ionizing radiation on the structure of the collagen fibers of human tendons

In studying the effect of ionizing radiation on the properties of human Achilles tendon collagen fibres, the following parameters were analyzed: hydrothermal contraction temperature, module of elasticity, the number of cross-links, free and bound water levels, acids-soluble fraction content, and ultrastructure. With radiation doses of 2-10 Gy no changes in the collagen status were noted. An increased (from 5 to 25 Gy) radiation dose caused changes in physicochemical properties which was indicative of the formation, in the connective tissue collagen, of radiation-induced intermolecular cross-links stabilizing the biopolymer structure.     

Inhibition of collagen biosynthesis and increases in low molecular weight IGF-I binding proteins in the skin of fasted rats

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen biosynthesis and prolidase activity in connective tissue cells. The disturbances in skin collagen metabolism (reflected by significant decrease in skin collagen content, collagen biosynthesis and prolidase activity) in fasted rats were accompanied by decrease in serum IGF-I level. Fasted rat serum was found to contain about 58% of IGF-I (101.6 +/- 15.4 ng/ml) as compared to control rat serum (175.7 +/- 19.8 ng/ml), while the skin of control and fasted rats contained similar concentrations of IGF-I (about 77 ng/g tissue). The insulin-like growth factor binding proteins (IGFBPs) of sera and tissue extracts (known to regulate IGF-I activity) were analysed by ligand blotting. In the serum of control rats one IGFBP band of about 46 kDa (corresponding to the acid-dissociated IGFBP-3) was detected. In the serum of fasted rats the 46 kDa IGFBP was not observed, however, an other IGFBP of about 30 kDa (corresponding to low molecular weight IGFBPs, e.g. IGFBP-1 or IGFBP-2) was found. The intensity of IGF-I binding to the 30 kDa IGFBP was much higher than that of IGFBP-3, found in control rat serum. Control and fasted rat skin contained similar IGFBPs, however their IGF-I binding abilities were much lower, compared to their serum counterparts. It was found that 46 kDa and 30 kDa proteins, observed in ligand blotting represent IGFBP-3 and IGFBP-1 or IGFBP-2. respectively as demonstrated by western immunoblot analysis. An increase in IGF-binding to 30 kDa IGFBP-1 and/or IGFBP-2 (known as an inhibitors of IGF-dependent functions) in the skin of fasted rats may explain the mechanism of reduced collagen biosynthesis and deposition in tissues during fasting.     

Variations in the metabolism and maturation of collagen after fluorine ingestion

This study described the effect of fluoride ingestion (10 mg NaF/kg body weight per day) for up to 180 days, on the biosynthesis, maturation and degradation of rabbit skin collagen. Higher intake of fluoride interferes with the collagen biosynthesis resulting in a reduction in the collagen content (in terms of hydroxyproline). Fluoride administration increases the solubility of collagen by reducing the amount of cross-link precursors, thus impairing the cross-linking and maturation of tissue collagen fibers. Collagen degradation by the collagen-bound collagenase is increased due to the accumulation of higher pools of soluble collagen.     

Connective tissue cells, cell proliferation and synthesis of extracellular matrix-a review.

The ubiquitous connective tissues contain a wide range of cells including fibroblasts, osteoblasts and chondroblasts. Recently it has been demonstrated that another principal cell of the connective tissue is the smooth muscle cell in several organ systems. These have been shown to be responsible for the synthesis of the connective tissue matrix components of the uterine myometrium and of the arterial system, including collagen, both elastic fibre proteins and glycosaminoglycan. Microtubule inhibitors such as colchicine and vinblastine, and iron chelators such as alpha,alpha -dipyridyl have been used to study their morphologic and chemical effects on collagen synthesis and secretion. Colchicine produces an increase in large Golgi-associated vacuoles, which sometimes contain material reminiscent of aggregates of collagen macromolecules. Vinblastine produces alterations in the endoplasmic reticulum cisternae similar to alterations seen in ascorbic acid deficiency, and alpha,alpha-dipyridyl increases the frequency of regions in cells, interpretable as potential sites of communication of rough endoplasmic reticulum cisternae with the cell surface. Ferritin conjugated anti-procallagen sera were used to localize procollagen in cells and demonstrated procollagen not only in the cisternae of rough endoplasmic reticulum but in all of the elements of the Golgi complex as well. The studies reported in this review have shown that in cell culture arterial smooth muscle will produce not only the microfibrillar protein of the elastic fibre but soluble and/or insoluble elastin as well. Recent studies on serum factors responsible for the proliferation of connective tissue cells have demonstrated that at least one of the principal factors responsible for fibroblast and/or smooth muscle cell proliferation in culture is derived from thrombocytes. Medium containing serum derived from cell-free plasma lacks most of this proliferative effect which can be reinstated when platelets are present during recalcification to form serum. This effect is due to the platelet release reaction as shown by combining supernatant factors derived from platelets exposed to purified thrombin to cell-free, plasma derived serum. Studies with macrophages have also suggested that phagocytic macrophages release factor(s) into a cell culture medium that may also participate in stimulating fibroblasts to proliferate in vitro. The means by which these factors stimulate fibroblast proliferation and connective tissue synthesis remains to be elucidated.     

Radiation-induced changes in collagen isotypes I, III, and IV in the lung of LAF1 mouse: effects of time, dose, and WR-2721

Alterations in the amount and distribution of pulmonary connective tissue are commonly observed subsequent to thoracic radiotherapy. The extent to which these changes are important in the expression of radiation damage and its repair remains unclear. We have quantitated changes in the parenchymal levels of collagen types I, III, and IV in the lungs of LAF1 mice at intervals to 1 year, following doses of 0-14 Gy, 300 kV X rays, or 0-18 Gy in the presence of the radioprotective compound, WR-2721. The method of quantitation, which involves video image analysis of fluorescent antibody stained, cryostat tissue sections, provides both quantitative and morphological information for the three collagen isotypes. Type I collagen peaked in tissue content at 15 and 30 weeks postirradiation (p.i.), with transient return to control values 20-25 weeks p.i. Type III collagen peaked at 15 and 25 weeks p.i. and declined in tissue content at 20 and 30 weeks. Type IV peaked 15-20 weeks following irradiation, returned to control levels at 25 weeks, and reached a plateau above control values after 30 weeks. Fluctuations in collagen levels in the parenchyma were dose dependent but were not simultaneous, indicating a radiation response characterized by alpha-chain-specific regulation of collagen biosynthesis and breakdown. In general, WR-2721, which enhanced postirradiation survival (DMF, 1.3), reduced the magnitude and altered the timing of collagen fluctuations; again, the effects were type specific. The results clearly demonstrate that the postirradiation response of the connective tissue is dose dependent, is specific to each macromolecule, and involves both deposition and removal of extracellular matrix. These processes are independently influenced by the presence during irradiation of WR-2721.     

Biopolymer gel matrix as acellular scaffold for enhanced dermal tissue regeneration

Biological grafts have drawbacks such as donor scarcity, disease transmission, tissue infection, while the scaffolds of either collagen or chitosan fabrics fail to become part of the tissue at the wound site, though they favor the formation of connective tissue matrix. This study developed a novel composite consisting of the combination of atelocollagen and chitosan in order to provide a biodegradable molecular matrix in gel form as a biomimetic surface for cell attachment, to promote the wound healing in excision wounds. We found that the topical application of biopolymer composite on the wound promoted cell proliferation, migration and collagen deposition overtime. The enhanced cellular activity in the collagen-chitosan treated wound tissue was also assed by increased levels of Platelet derived growth factor (PDGF) and Nerve growth factor (NGF) associated with elevated levels of antioxidants and decreased level of lipid peroxidation. The acellular matrix-like topical application material is designed to guide the eventual re-establishment of an anatomically normal skin. The results of this study demonstrate the feasibility of multi-cell regeneration on a molecular system that mimics tissue engineering in vivo.     

Combined use of bFGF and GDF-5 enhances the healing of medial collateral ligament injury

Basic fibroblast growth factor (bFGF) and growth and differentiation factor (GDF)-5 stimulate the healing of medial collateral ligament (MCL) injury. However, the effect of isolated and combined use of bFGF/GDF-5 remains still unclear. We investigated cellular proliferation and migration responding to bFGF/GDF-5 using rabbit MCL fibroblasts. Rabbit MCL injury was treated by bFGF and/or GDF-5 with peptide hydrogels. Gene expression and deposition of collagens in healing tissues were evaluated. bFGF/GDF-5 treatment additively enhanced cell proliferation and migration. bFGF/GDF-5 hydrogels stimulated Col1a1 expression without increasing Col3a1 expression. Combined use of bFGF/GDF-5 stimulated type I collagen deposition and the reorganization of fiber alignment, and induced better morphology of fibroblasts in healing MCLs. Our study indicates that combined use of bFGF/GDF-5 might enhance MCL healing by increasing proliferation and migration of MCL fibroblasts, and by regulating collagen synthesis and connective fiber alignment.     

Role of the arterial smooth muscle cell in the pathogenesis of atherosclerosis

The development of an atherosclerotic lesion is characterised by a proliferation of arterial smooth muscle cells and an accumulation of cholesterol, cholesteryl esters and connective tissue. The main connective tissue components of an atherosclerotic lesion, i.e. acidic glycosaminoglycans and collagen, are synthesized by the smooth muscle cells. Cholesterol is chiefly derived from plasma lipoproteins, but there is an enhanced intracellular esterification of cholesterol in the cells of the lesions. The important role of the arterial smooth muscle cell in the development of atherosclerotic lesions has resulted in cultures of these cells being used as experimental models to study the pathogenesis of atherosclerosis. Such studies have revealed many blood-derived and other substances affecting proliferation, as well as lipid and connective tissue metabolism of arterial smooth muscle cells. In this way certain risk factors for cardiovascular disease have turned out to be associated with the metabolic disturbances of atherogenesis at the cellular level. Studies with cultured arterial smooth muscle cells have also demonstrated other factors for example one derived from aggregating platelets that may significantly contribute to the development of atherosclerotic lesions. On the other hand, certain inherent features of the smooth muscle cells of the lesions, such as enhanced proliferation and synthesis of glycosaminoglycans, may also contribute to the pathological changes.