THE COLLAGEN DISEASES

The collagen diseases have in common certain relatively specific alterations in the tissues that are derived from the mesenchyme.In reviewing the development of this concept, the nature and the pathogenesis of these changes and the blocking effect of cortisone and ACTH are discussed.The term “diseases of the collagen system” includes a number of clinical states within a broad spectrum of variation in the basic alterations of collagenous and vascular tissues. In rheumatoid arthritis the modifications are chiefly in the somatic connective tissue; in periarteritis nodosa the blood vessel involvement is preeminent; dermatomyositis and disseminated lupus erythematosus present more intermediate mixtures of these changes.     

Effect of estriol on the structure and organization of collagen in the lamina propria of the immature rat uterus.

Estradiol produces both hypertrophic and hyperplastic changes in the uterus, and these changes are associated with alterations in the structure of collagen in the lamina propria. Estriol induces only hypertrophic responses in the immature rat uterus; its effects on collagen structure were characterized in this study. Light micrographs of Masson's trichrome-stained sections revealed that the intensity of the collagen stain in the lamina propria of the rat uterus was profoundly reduced, relative to that in controls, 4 h after estriol (40 micrograms/kg) administration. These changes were not evident 24 h after estriol administration. In control uteri, transmission electron micrographs revealed that the collagen fibers surrounding stromal cells formed dense collections of bundles that were seen throughout the extracellular matrix, whereas in tissues exposed to estriol 4 h earlier, large regions of the extracellular spaces were devoid of collagen bundles. The 4-h changes in collagen were eliminated when animals were pretreated with actinomycin D (8 mg/kg) or cycloheximide (4 mg/kg). Dense collections of collagen bundles were present in tissues 24 h after estriol treatment, and their appearance was not altered by actinomycin D or cycloheximide treatment. Alterations in collagen 4 h after hormone administration appeared to be estrogen-specific since dexamethasone (600 micrograms/kg) and dihydrotestosterone (400 micrograms/kg) had no effect. These data provide evidence that the changes in collagen structure in the uterus are associated with events that function during the hypertrophic growth responses induced by estrogens.     

Extracellular matrix and vascular ageing

The extracellular matrix provides a structural framework essential for the functional properties of tissues. In each tissue, the three-dimensional organisation of the extracellular matrix molecules--elastin, collagens, proteoglycans and structural glycoproteins--synthesized during development and growth is optimal for these functions. In adult tissues, proteases are constitutively expressed but have a very low activity and the turn-over of elastic and collagen fibers is very low. During ageing, the interaction of environmental factors (glucose, lipids, calcium...) and modifications of the biosynthesis and degradation processes lead to modifications of extracellular matrix homeostasis and consequently to alterations of tissue functionality. These alterations are increased during pathological processes such as cardiovascular diseases.     

Radiological aspects of collagen diseases

The collagen diseases, an ill-defined group of clinical entities, have as their basis a generalized alteration of the connective tissue, especially of its extracellular components. They include periarteritis nodosa, disseminated lupus erythematosus, dermatomyositis, scleroderma, rheumatic fever and rheumatoid arthritis. The radiological findings in a series of cases of these diseases were reviewed. In 28 cases of periarteritis, 20 cases showed some abnormal findings in the thorax. These included pleural effusions, pulmonary changes, pericardial effusions and cardiac enlargement. In 32 cases of disseminated lupus erythematosus, thoracic findings were noted in 21. They resembled the changes found in periarteritis. In some 25 cases of scleroderma, diverse radiological findings were noted. These included "cystic" changes in the lungs (one case) and pulmonary "hives." In the intestinal tract esophageal and small bowel alterations were found, both ectatic and stenotic. In the soft tissues of the "pressure areas" variable degrees of calcification were observed. Dermatomyositis is the rarest of the collagen disease group; only one autopsy-proven case is available for study. Chest x-rays taken a year before death showed slight cardiac enlargement. The lungs were clear. In acute rheumatic fever, x-ray examination may disclose pericardial or pleural effusion, and so-called rheumatic pneumonitis; the latter has no specific diagnostic features. Soft tissue swellings may develop around some of the joints. In rheumatoid arthritis, joint changes are numerous and fairly characteristic, and are followed in many cases by fibrous or bony ankylosis and deformities of considerable degree. Awareness of the commoner radiological changes in this entire group of diseases should result in earlier establishment of diagnosis, especially in the more obscure examples.     

Mathematical modeling of collagen turnover in biological tissue

We present a theoretical and computational model for collagen turnover in soft biological tissues. Driven by alterations in the mechanical environment, collagen fiber bundles may undergo important chronic changes, characterized primarily by alterations in collagen synthesis and degradation rates. In particular, hypertension triggers an increase in tropocollagen synthesis and a decrease in collagen degradation, which lead to the well-documented overall increase in collagen content. These changes are the result of a cascade of events, initiated mainly by the endothelial and smooth muscle cells. Here, we represent these events collectively in terms of two internal variables, the concentration of growth factor TGF- $\beta $ and tissue inhibitors of metalloproteinases TIMP. The upregulation of TGF- $\beta $ increases the collagen density. The upregulation of TIMP also increases the collagen density through decreasing matrix metalloproteinase MMP. We establish a mathematical theory for mechanically-induced collagen turnover and introduce a computational algorithm for its robust and efficient solution. We demonstrate that our model can accurately predict the experimentally observed collagen increase in response to hypertension reported in literature. Ultimately, the model can serve as a valuable tool to predict the chronic adaptation of collagen content to restore the homeostatic equilibrium state in vessels with arbitrary micro-structure and geometry.     

Synthesis and functional characterization of a fluorescent peptide probe for non invasive imaging of collagen in live tissues

Targeted molecular imaging to detect changes in the structural and functional organization of tissues, at the molecular level, is a promising approach for effective and early diagnosis of diseases. Quantitative and qualitative changes in type I collagen, which is a major component in the extra cellular matrix (ECM) of skin and other vital organs like lung, liver, heart and kidneys, are often associated with the pathophysiology of these organs. We have synthesized a fluorescent probe that comprises collagelin, a specific collagen binding peptide, coupled to fluorescent porphyrin that can effectively detect abnormal deposition of collagen in live tissues by emitting fluorescence in the near infra red (NIR) region. In this report we have presented the methodology for coupling of 5-(4-carboxy phenyl)-10, 15, 20-triphenyl porphyrin (C-TPP) to the N-terminal of collagelin or to another mutant peptide (used as a control). We have evaluated the efficacy of these fluorescent peptides to detect collagen deposition in live normal and abnormal tissues. Our results strongly suggest that porphyrin-tagged collagelin can be used as an effective probe for the non invasive in vivo detection of tissue fibrosis, especially in the liver.     

Characterization of cell-free synthesis of collagen by lung polysomes in a heterologous system.

In normal lung growth, post-pneumonectomy lung growth, and in possibly several lung disorders, there are marked alterations in the density of collagen and changes in the rate of synthesis of collagen relative to the synthesis of other lung proteins. To provide a technology to begin to understand these changes at the molecular level, polysomes were prepared from rabbit lung and translated in a heterologous cell-free system including rabbit reticulocyte 0.5 M KCl ribosomal wash fraction and liver tRNA. Collagen was shown in the cell-free product by collagenase sensitivity, hydroxylation of incorporated proline by peptidyl prolyl hydroxylase, agarose gel chromatography, and sodium dodecyl sulfate acrylamide gel electrophoresis. The cell-free system was optimized with respect to K+, Mg2+, amino acids, and ribosomal wash fraction and used under conditions where total protein synthesis and collagen synthesis are linear with respect to time and amount of polysomes. Under these conditions, collagen synthesis was directed almost entirely by polysomes derived from the endoplasmic reticulum. Polysomes isolated from late fetal lung directed collagen synthesis at twice the rate (per polysome) as those polysomes isolated from adult lung. Similar changes were seen if lung tRNA replaced liver tRNA and if lung ribosomal wash fraction replaced reticulocyte wash fraction. Although these changes in cell-free lung collagen synthesis with tissue explants, further studies will have to be carried out to determine whether, in fact, age-related alterations in control of lung collagen synthesis are truly explained by these findings.     

The Effect of Diabetes On Lysyl Oxidase Activity and Extractability of Newly Synthesized Collagen in Rat Gingiva and Skin1

Lysyl oxidase activity and the extractability of recently synthesized collagen were measured in gingiva and skin, two tissues which exhibit altered pathophysiologic responses during diabetes. Both tissues showed a reduced content and extractability of newly synthesized, ¹⁴C-Hydroxyproline labeled, collagen after induction of streptozotocin-induced diabetes. Diabetes increased lysyl oxidase activity in skin and gingiva but the effect on gingiva was much less dramatic. The altered characteristics of interstitial collagen in the diabetic rat, including aging-like changes, appear to reflect multiple alterations in collagen metabolism.     

Rheology of heterotypic collagen networks

Collagen fibrils are the main structural element of connective tissues. In many tissues, these fibrils contain two fibrillar collagens (types I and V) in a ratio that changes during tissue development, regeneration, and various diseases. Here we investigate the influence of collagen composition on the structure and rheology of networks of purified collagen I and V, combining fluorescence and atomic force microscopy, turbidimetry, and rheometry. We demonstrate that the network stiffness strongly decreases with increasing collagen V content, even though the network structure does not substantially change. We compare the rheological data with theoretical models for rigid polymers and find that the elasticity is dominated by nonaffine deformations. There is no analytical theory describing this regime, hampering a quantitative interpretation of the influence of collagen V. Our findings are relevant for understanding molecular origins of tissue biomechanics and for guiding rational design of collagenous biomaterials for biomedical applications.     

Effect of drinking green tea on age-associated accumulation of Maillard-type fluorescence and carbonyl groups in rat aortic and skin collagen.

Tea catechins and other flavonoids have been shown to potentially protect against chronic cardiovascular diseases such as coronary heart disease and atherosclerosis. In this study, 6-month-old female Sprague-Dawley rats were fed green tea extract (50 mg/100 ml in drinking water) up to the age of 22 months, and the age-associated changes in Maillard-type fluorescence and carbonyl groups in the aortic and skin collagen were compared with those occurring in the water-fed control animals. Collagen-linked Maillard-type fluorescence was found to increase in both the aortic and skin tissues as animals aged. The age-associated increase in the fluorescence in the aortic collagen was remarkably inhibited by the green tea extract treatment, while that occurring in the skin collagen was not significantly inhibited by the treatment. The collagen carbonyl content also increased in both the aortic and skin tissues as animals aged. In contrast with the case of Maillard-type fluorescence, however, the age-associated increase in the carbonyl content was not inhibited by the green tea extract treatment either in the aortic or skin collagen. These results suggest that the inhibition of AGE formation in collagen is an important mechanism for the protective effects of tea catechins against cardiovascular diseases.     

Age-dependent changes of airway and lung parenchyma in C57BL/6J mice.

In the current study, we hypothesize that senescent-dependent changes between airway and lung parenchymal tissues of C57BL/6J (B6) mice are not synchronized with respect to altered lung mechanics. Furthermore, aging modifications in elastin fiber and collagen content of the airways and lung parenchyma are remodeling events that differ with time. To test these hypotheses, we performed quasi-static pressure-volume (PV) curves and impedance measurements of the respiratory system in 2-, 20-, and 26-mo-old B6 mice. From the PV curves, the lung volume at 30 cmH(2)O pressure (V(30)) and respiratory system compliance (Crs) were significantly (P < 0.01) increased between 2 and 20 mo of age, representing about 80-84% of the total increase that occurred between 2 and 26 mo of age. Senescent-dependent changes in tissue damping and tissue elastance were analogous to changes in V(30) and Crs; that is, a majority of the parenchymal alterations in the lung mechanics occurred between 2 and 20 mo of age. In contrast, significant decreases in airway resistance (R) occurred between 20 and 26 mo of age; that is, the decrease in R between 2 and 20 mo of age represented only 29% (P > 0.05) of total decrease occurring through 26 mo. Morphometric analysis of the elastic fiber content in lung parenchyma was significantly (P < 0.01) decreased between 2 and 20 mo of age. To the contrary, increased collagen content was significantly delayed until 26 mo of age (P < 0.01, 2 vs. 26 mo). In conclusion, our data demonstrate that senescent-dependent changes in airway and lung tissue mechanics are not synchronized in B6 mice. Moreover, the reduction in elastic fiber content with age is an early lung remodeling event, and the increased collagen content in the lung parenchyma occurs later in senescence.     

Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia.

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the non-dystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of proteins in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Structural integrity of collagen and elastin in SynerGraft® decellularized-cryopreserved human heart valves

SynerGraft® (SG) decellularized–cryopreserved cardiac valve allografts have been developed to provide a valve replacement option that has reduced antigenicity, retained structural integrity, and the ability to be stored long-term until needed for implantation. However, it is critical to ensure that both the SG processing and cryopreservation of these allografts do not detrimentally affect the extracellular matrix architecture within the tissue. This study evaluates the effects of SG decellularization and subsequent cryopreservation on the extracellular matrix integrity of allograft heart valves. Human aortic and pulmonary valves were trisected, with one-third of each either left fresh (no further processing after dissection), decellularized, or decellularized and cryopreserved. Two-photon laser scanning confocal microscopy was used to visualize collagen and elastin in leaflets and conduits. The optimized percent laser transmission (OPLT) required for full dynamic range imaging of each site was determined, and changes in OPLT were used to infer changes in collagen and elastin signal intensity. Collagen fiber crimp period and collagen and elastin fiber diameter were measured in leaflet tissue. Statistically significant differences in OPLT and the dimensional characteristics of collagen and elastin in study groups were determined through single factor ANOVA. The majority of donor-aggregated average OPLT observations showed no statistically significant differences among all groups, indicating no difference in collagen or elastin signal strength. Morphometric analysis of collagen and elastin fibers revealed no significant alterations in treated leaflet tissues relative to fresh tissues. Collagen and elastin structural integrity within allograft heart valves are maintained through SynerGraft® decellularization and subsequent cryopreservation.     

Response of rat connective tissues to streptozotocin-diabetes. Tissue-specific effects on collagen metabolism

We assessed the effect of streptozotocin-diabetes on in vivo collagen metabolism in skin, aorta and intestine by injecting [³H]proline into rats, 20 days after administering the diabetogen, streptozotocin. One day after [³H]proline injection, diabetic and control animals were killed, their tissues analyzed for both ³H-labeled and unlabeled hydroxyproline and results expressed per entire tissue. Thereby, the effect of diabetes on net collagen synthesis and tissue collagen mass, respectively, was evaluated.Diabetes resulted in a lower content of [³H]collagen in skin and aorta, suggesting decreased net collagen synthesis. This decrease in net synthesis was accompanied by a decrease of collagen mass in skin, whereas aortic collagen mass was unaffected. Consequently, an acceleration of collagen degradation in skin is postulated to have accompanied the expected depression of collagen synthesis; alterations of the physiochemical properties of skin from diabetic rats support this interpretation. For intestine, both net collagen synthesis and mass increased in diabetic rats, reflecting increased collagen synthesis—possibly associated with polyphagy.In conclusion, with regard to collagen metabolism, representative connective tissues respond differently to experimental diabetes, and we suggest that this insight will be useful in future studies aimed at understanding the pathophysiology of connective tissues affected by diabetes.     

Effect of targeted mutation in collagen V alpha 2 gene on development of cutaneous hyperplasia in tight skin mice.

Collagen V plays a major regulatory role in the formation of heterotypic fibers of the dermis and cartilaginous tissues as well as in the assembly of extracellular matrix. The pN/pN mouse, which is defective in collagen V alpha 2 gene, exhibits skeletal abnormalities, skin fragility, and alterations in the collagen fiber organization, whereas the TSK/+ mouse, which is defective in fibrillin-1, the major component of microfibrils present in the extracellular matrix, develops cutaneous hyperplasia and autoimmunity. We have studied the role of collagen V in the formation of heterotypic collagen fibers in F1 mice, which are obtained by breeding pN/pN with TSK/+ mice. Our results show that F1 progeny neither develop cutaneous hyperplasia nor produce anti-topoisomerase I autoantibodies, unlike TSK/+ mice. The diameter of the collagen fibrils in the skin is also comparable to that found in control mice. Thus, the phenotypic changes observed in the TSK mouse could be reversed by genetic complementation with a collagen V-defective mouse.     

Altered cellular interactions between endothelial cells and nonenzymatically glucosylated laminin/type IV collagen.

Laminin and type IV collagen are two major basement membrane glycoproteins. In previous studies it has been shown that nonenzymatic glucosylation induces structural alterations of these macromolecules and also reduces their ability to self-associate. In the present study, endothelial cells were tested for their ability to adhere and spread on nonenzymatically glucosylated laminin and type IV collagen. Adhesion and spreading were reduced when glucosylated macromolecules were used as substrates. Glucosylation-induced changes in adhesion and spreading may be an important initial event signaling other phenotypic modifications of cells in the microvasculature and may be a crucial factor in order to understand the pathogenesis of diabetic microangiopathy at the molecular level.     

Phase transformations in a model mesenchymal tissue

Connective tissues, the most abundant tissue type of the mature mammalian body, consist of cells suspended in complex microenvironments known as extracellular matrices (ECMs). In the immature connective tissues (mesenchymes) encountered in developmental biology and tissue engineering applications, the ECMs contain varying amounts of randomly arranged fibers, and the physical state of the ECM changes as the fibers secreted by the cells undergo fibril and fiber assembly and organize into networks. In vitro composites consisting of assembling solutions of type I collagen, containing suspended polystyrene latex beads ( approximately 6 microm in diameter) with collagen-binding surface properties, provide a simplified model for certain physical aspects of developing mesenchymes. In particular, assembly-dependent topological (i.e., connectivity) transitions within the ECM could change a tissue from one in which cell-sized particles (e.g., latex beads or cells) are mechanically unlinked to one in which the particles are part of a mechanical continuum. Any particle-induced alterations in fiber organization would imply that cells could similarly establish physically distinct microdomains within tissues. Here we show that the presence of beads above a critical number density accelerates the sol-gel transition that takes place during the assembly of collagen into a globally interconnected network of fibers. The presence of this suprathreshold number of beads also dramatically changes the viscoelastic properties of the collagen matrix, but only when the initial concentration of soluble collagen is itself above a critical value. Our studies provide a starting point for the analysis of phase transformations of more complex biomaterials including developing and healing tissues as well as tissue substitutes containing living cells.     

Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the nondystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of protein in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Molecular and functional defects in kidneys of mice lacking collagen alpha 3(IV): implications for Alport syndrome

Collagen IV is a major structural component of all basal laminae (BLs). Six collagen IV alpha chains are present in mammals; alpha 1 and alpha 2(IV) are broadly expressed in embryos and adults, whereas alpha 3- 6(IV) are restricted to a defined subset of BLs. In the glomerular BL of the kidney, the alpha 1 and alpha 2(IV) chains are replaced by the alpha 3-5(IV) chains as development proceeds. In humans, mutation of the collagen alpha 3, alpha 4, or alpha 5(IV) chain genes results in a delayed onset renal disease called Alport syndrome. We show here that mice lacking collagen alpha 3(IV) display a renal phenotype strikingly similar to Alport syndrome: decreased glomerular filtration (leading to uremia), compromised glomerular integrity (leading to proteinuria), structural changes in glomerular BL, and glomerulonephritis. Interestingly, numerous changes in the molecular composition of glomerular BL precede the onset of renal dysfunction; these include loss of collagens alpha 4 and alpha 5(IV), retention of collagen alpha 1/2(IV), appearance of fibronectin and collagen VI, and increased levels of perlecan. We suggest that these alterations contribute, along with loss of collagen IV isoforms per se, to renal pathology.     

Chaperoning osteogenesis: new protein-folding disease paradigms

Recent discoveries of severe bone disorders in patients with deficiencies in several endoplasmic reticulum chaperones are reshaping the discussion of type I collagen folding and related diseases. Type I collagen is the most abundant protein in all vertebrates and a crucial structural molecule for bone and other connective tissues. Its misfolding causes bone fragility, skeletal deformity and other tissue failures. Studies of newly discovered bone disorders indicate that collagen folding, chaperones involved in the folding process, cellular responses to misfolding and related bone pathologies might not follow conventional protein folding paradigms. In this review, we examine the features that distinguish collagen folding from that of other proteins and describe the findings that are beginning to reveal how cells manage collagen folding and misfolding. We discuss implications of these studies for general protein folding paradigms, unfolded protein response in cells and protein folding diseases.