Collagenase‐resistant collagen promotes mouse aging and vascular cell senescence

Collagen fibrils become resistant to cleavage over time. We hypothesized that resistance to type I collagen proteolysis not only marks biological aging but also drives it. To test this, we followed mice with a targeted mutation (Col1a1^r/r) that yields collagenase‐resistant type I collagen. Compared with wild‐type littermates, Col1a1^r/r mice had a shortened lifespan and developed features of premature aging including kyphosis, weight loss, decreased bone mineral density, and hypertension. We also found that vascular smooth muscle cells (SMCs) in the aortic wall of Col1a1^r/r mice were susceptible to stress‐induced senescence, displaying senescence‐associated ß‐galactosidase (SA‐ßGal) activity and upregulated p16^INK4A in response to angiotensin II infusion. To elucidate the basis of this pro‐aging effect, vascular SMCs from twelve patients undergoing coronary artery bypass surgery were cultured on collagen derived from Col1a1^r/r or wild‐type mice. This revealed that mutant collagen directly reduced replicative lifespan and increased stress‐induced SA‐ßGal activity, p16^INK4A expression, and p21^CIP1 expression. The pro‐senescence effect of mutant collagen was blocked by vitronectin, a ligand for αvß3 integrin that is presented by denatured but not native collagen. Moreover, inhibition of αvß3 with echistatin or with αvß3‐blocking antibody increased senescence of SMCs on wild‐type collagen. These findings reveal a novel aging cascade whereby resistance to collagen cleavage accelerates cellular aging. This interplay between extracellular and cellular compartments could hasten mammalian aging and the progression of aging‐related diseases.     

Collagen and the myocardium: fibrillar structure,biosynthesis and degradation in relation to hypertrophy and its regression

The extracellular matrix of the myocardium contains an elaborate structural matrix composed mainly of fibrillar types I and III collagen. This matrix is responsible for the support and alignment of myocytes and capillaries. Because of its alignment, location, configuration and tensile strength, relative to cardiac myocytes, the collagen matrix represents a major determinant of myocardial stiffness. Cardiac fibroblasts, not myocytes, contain the mRNA for these fibrillar collagens. In the hypertrophic remodeling of the myocardium that accompanies arterial hypertension, a progressive structural and biochemical remodeling of the matrix follows enhanced collagen gene expression. The resultant significant accumulation of collagen in the interstitium and around intramyocardial coronary arteries, or interstitial and perivascular fibrosis, represents a pathologic remodeling of the myocardium that compromises this normally efficient pump. This report reviews the structural nature, biosynthesis and degradation of collagen in the normal and hypertrophied myocardium. It suggests that interstitial heart disease, or the disproportionate growth of the extracellular matrix relative to myocyte hypertrophy, is an entity that merits greater understanding, particularly the factors regulating types I and III collagen gene expression and their degradation.     

Immunological identification of types I and III collagen in bovine lens epithelium and its anterior lens capsule

To define the molecular structure of bovine lens epithelium and its anterior lens capsule, we investigated the composition of lens capsule basement membrane proteins. Immunofluorescence and immunogold techniques were used to demonstrate the presence of type I and type III collagen in the lens capsule and in primary explant epithelial cultures grown on protein-binding membranes. Immunofluorescence staining with specific antibodies indicated that type I and type III collagen were constituents of lens basement membrane. We observed that deposition of type III collagen was more than type I collagen. The synthesis of fibrillar collagen by lens epithelium and its deposition in the lens capsule was established by localization of fibrillar collagen by transmission immunoelectron microscopy. These results demonstrate for the first time that normal lens epithelium synthesize fibrillar collagen which is an intrinsic component of the anterior lens capsule basement membrane.     

Corneal and scleral type I collagens: analyses of physical properties and molecular flexibility

The physical properties of type I collagen were studied by electron microscopy of rotary shadowed collagen molecules and laser light scattering techniques. The physical properties, molecular structure and flexibility of type I collagen molecules from two structurally and functionally different connective tissues, cornea and sclera, were similar when measured in HCl, pH 2.0. The molecular weights were 328 and 298 × 10² for corneal and scleral type I collagen, respectively, while the values of T_M were 33.7°C for both preparations. These values were in agreement with those obtained for other type I collagens. The higher level of glycosylation in corneal versus scleral type I collagen did not significantly modify the physical properties of type I collagen in acid solution or the charge distribution along the molecule as determined from the positively stained SLS banding patterns. Our morphological studies indicated that the collagen molecule, although relatively flexible based on electron microscopy, behaved as a long thin rod in solution. The mean end-to-end distances measured from electron micrographs were 253 and 256 nm for corneal and cler type I collagen, respectively, while the molecular contour lengths were 298 and 305 nm. The translational diffusion coefficients (0.849 and 0.857 × 10^?7cm²s^?1) were consistent with the contour lengths while the reported values in the literature for the rotational diffusion coefficient of type I collagen were consistent with the end-to-end distances. The intermediate value for molecular length obtained from the particle scattering factor (277 nm) reflects contributions from all possible molecular configurations.     

Kinetics of hydrolysis of type I, II, and III collagens by the class I and II Clostridium histolyticum collagenases.

The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class IIClostridium histolyticum collagenases (CHC) have been investigated. To facilitate this study, radioassays developed previously for the hydrolysis of these [³H]acetylated collagens by tissue collagenases have been adapted for use with the CHC. While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events. The individual kinetic parametersk _cat andK _M have been determined for the hydrolysis of all three collagens by both class I and class II CHC. The specific activities of these CHC toward fibrillar type I and III collagens have also been measured. In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form. The values of the kinetic parametersk _cat andK _M for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates. Thus, the widely held view that the CHC are more potent collagenases is not strictly correct. As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.     

Interleukin-6: a growth factor stimulating collagen synthesis in human tendon

Human connective tissue, e.g., tendon, responds dynamically to physical activity, with collagen synthesis being increased after both acute and prolonged exercise or training. Markers of collagen synthesis and degradation as well as concentration of several potential growth factors have been shown to increase markedly in the peritendinous tissue around the human Achilles tendon following exercise. Of these potential growth factors interleukin-6 (IL-6) showed the largest fold increase, suggesting that IL-6 may be involved in transforming mechanical loading into collagen synthesis in human tendon tissue. In the present study the tissue levels of type I collagen turnover markers [procollagen type I NH(2)-terminal propeptide (PINP) and C-OOH terminal telopeptide of type I collagen (ICTP)] were measured by the use of microdialysis in peritendinous tissue of the Achilles tendon in 14 male volunteers, who had recombinant human IL-6 (rhIL-6) infused into the peritendinous tissue of the Achilles' tendon in one leg, with the other leg serving as control. Subjects were randomly assigned to either a resting group or an exercise group performing a 1-h treadmill run (12 km/h, 2% uphill) before infusion. In addition to IL-6, serum concentrations of collagen turnover markers PINP, ICTP, and COOH-terminal telopeptide of type I collagen (ICTX) were measured. The peritendinous concentration of PINP rose markedly in response to rhIL-6 infusion in both the exercise and the rest group, demonstrating that infusion of IL-6 significantly stimulates collagen synthesis in the peritendinous tissue in humans. Exercise alone did not result in an increased collagen synthesis. This indicates that IL-6 is involved in the collagen synthesis and supports the hypothesis that IL-6 is an important growth factor of the connective tissue in healthy human tendons.     

Double thermal transitions of type I collagen in acidic solution

Contributed equally to this work. To further understand the origin of the double thermal transitions of collagen in acidic solution induced by heating, the denaturation of acidic soluble collagen was investigated by micro-differential scanning calorimeter (micro-DSC), circular dichroism (CD), dynamic laser light scattering (DLLS), transmission electron microscopy (TEM), and two-dimensional (2D) synchronous fluorescence spectrum. Micro-DSC experiments revealed that the collagen exhibited double thermal transitions, which were located within 31–37?°C (minor thermal transition, T _s?～?33?°C) and 37–55?°C (major thermal transition, T _m?～?40?°C), respectively. The CD spectra suggested that the thermal denaturation of collagen resulted in transition from polyproline II type structure to unordered structure. The DLLS results showed that there were mainly two kinds of collagen fibrillar aggregates with different sizes in acidic solution and the larger fibrillar aggregates (T _p2?=?40?°C) had better heat resistance than the smaller one (T _p1?=?33?°C). TEM revealed that the depolymerization of collagen fibrils occurred and the periodic cross-striations of collagen gradually disappeared with increasing temperature. The 2D fluorescence correlation spectra were also applied to investigate the thermal responses of tyrosine and phenylalanine residues at the molecular level. Finally, we could draw the conclusion that (1) the minor thermal transition was mainly due to the defibrillation of the smaller collagen fibrillar aggregates and the unfolding of a little part of triple helices; (2) the major thermal transition primarily arose from the defibrillation of the larger collagen fibrillar aggregates and the complete denaturation of the majority part of triple helices.     

Shear stress magnitude and duration modulates matrix composition and tensile mechanical properties in engineered cartilaginous tissue

Cartilage tissue‐engineering strategies aim to produce a functional extracellular matrix similar to that of the native tissue. However, none of the myriad approaches taken have successfully generated a construct possessing the structure, composition, and mechanical properties of healthy articular cartilage. One possible approach to modulating the matrix composition and mechanical properties of engineered tissues is through the use of bioreactor‐driven mechanical stimulation. In this study, we hypothesized that exposing scaffold‐free cartilaginous tissue constructs to 7 days of continuous shear stress at 0.001 or 0.1 Pa would increase collagen deposition and tensile mechanical properties compared to that of static controls. Histologically, type II collagen staining was evident in all construct groups, while a surface layer of type I collagen increased in thickness with increasing shear stress magnitude. The areal fraction of type I collagen was higher in the 0.1‐Pa group (25.2 ± 2.2%) than either the 0.001‐Pa (13.6 ± 3.8%) or the static (7.9 ± 1.5%) group. Type II collagen content, as assessed by ELISA, was also higher in the 0.1‐Pa group (7.5 ± 2.1%) compared to the 0.001‐Pa (3.0 ± 2.25%) or static groups (3.7 ± 3.2%). Temporal gene expression analysis showed a flow‐induced increase in type I and type II collagen expression within 24 h of exposure. Interestingly, while the 0.1‐Pa group showed higher collagen content, this group retained less sulfated glycosaminoglycans in the matrix over time in bioreactor culture. Increases in both tensile Young's modulus and ultimate strength were observed with increasing shear stress, yielding constructs possessing a modulus of nearly 5 MPa and strength of 1.3 MPa. This study demonstrates that shear stress is a potent modulator of both the amount and type of synthesized extracellular matrix constituents in engineered cartilaginous tissue with corresponding effects on mechanical function. Biotechnol. Bioeng. 2009; 104: 809–820 © 2009 Wiley Periodicals, Inc.     

Defective calcium pump in the sarcoplasmic reticulum of the hypertrophied rabbit heart.

To evaluate Ca²⁺-uptake in sarcoplasmic reticulum in the hypertrophied rabbit heart, microsomes were prepared from myocardium of rabbits with experimentally induced aortic stenosis. A significant reduction of microsomal Ca²⁺-uptake was observed in hypertrophied left ventricle, 195±10 compared to 280±18 nmol/mg found in control animals. A similar pattern was observed for the Ca²⁺-stimulated ATPase (30±9 and 59±10 nmol/min/mg resp.). A minimal activity difference of the microsomal marker enzyme rotenone-insensitive NADPH cyt. $\text{c}$ reductase was found (7.77±0.05 and 8.17±0.11 nmol/min/mg resp.). The specific activity of the latter enzyme was 5–6 fold increased in microsomes compared to homogenates in both animal groups, which excludes the possibility of increased amounts of contaminant or nonfunctional protein in sarcoplasmic reticulum prepared from hypertrophied myocardium. In addition the yield of microsomal protein did not differ significantly. Maximal phosphorylation by exogenous cyclic AMP and protein kinase increased Ca²⁺-uptake in both microsomal preparations (to 287±27 and 375±26 nmol/mg resp. for hypertrophied and control hearts), but Ca²⁺-transport rate found in pathological hearts remained lower than in controls. These findings indicate that impairment of Ca²⁺-metabolism in the hypertrophied heart is based on a defective Ca²⁺-pump.     

Increased mRNA expression of cardiac renin-angiotensin system and collagen synthesis in spontaneously hypertensive rats

Hypertensive cardiac hypertrophy is associated with the accumulation of collagen in the myocardial interstitium. Previous studies have demonstrated that this myocardial fibrosis accounts for impaired myocardial stiffness and ventricular dysfunction. Although cardiac fibroblasts are responsible for the synthesis of fibrillar collagen, the factors that regulate collagen synthesis in cardiac fibroblasts are not fully understood. We investigated the effects of angiotensin II on cardiac collagen synthesis in cardiac fibroblasts. Cardiac fibroblasts of 10 week old spontaneously hypertensive rats and age-matched Wistar-Kyoto rats were prepared and maintained in culture medium supplemented with 10% fetal calf serum. The expression of mRNA of the renin-angiotensin system (renin, angiotensinogen, angiotensin converting enzyme) was determined by using a ribonuclease protection assay. Basal collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats was 1.6 fold greater than that in the cell of Wistar-Kyoto rats. Angiotensin II stimulated collagen synthesis in cardiac fibroblasts in a dose-dependent manner. The responsiveness of collagen production to angiotensin II was significantly enhanced in cardiac fibroblasts from spontaneously hypertensive rats (100 nM angiotensin II resulted in 185 ± 18% increase above basal levels, 185 ± 18 versus 128 ± 19% in Wistar-Kyoto rats p < 0.01). This effect was receptor-specific, because it was blocked by the competitive inhibitor saralasin and MK 954. These results indicate that collagen production was enhanced in cardiac fibroblasts from spontaneously hypertensive rats, that angiotensin II had a stimulatory effect on collagen synthesis in cardiac fibroblasts, and that cardiac fibroblasts from spontaneously hypertensive rats were hyper-responsive to stimulation by angiotensin II.Level of angiotensin and renin mRNA expressed in ventricles, and angiotensinogen mRNA expressed in fibroblasts from SHR were higher than those from WKY.These findings suggest that the cardiac renin-angiotensin system may play an important role in collagen accumulation in hypertensive cardiac hypertrophy.     

Assay for radiolabeled type IV collagen in the presence of other proteins using a specific collagenase

An enzymatic assay is described which quantitates radiolabeled type IV basement membrane collagen in the presence of large amounts of other proteins. A partially purified neutral protease is used which cleaves type IV collagen into fragments at 37°C which are not precipitated at 1.3% (final concentration) trichloroacetic acid-tannic acid. The kinetics of type IV collagen digestion by this enzyme are not significantly altered by the presence of a 10-fold excess of type III collagen. [¹⁴C]Tryptophan-labeled control proteins prepared from fibroblast cultures are not degraded significantly by this protease in the presence of 2.5 mmN-ethylmaleimide. The proportion of type IV collagen in a mixture of labeled placenta collagenous proteins was calculated after separate digestions with the type IV collagenolytic activity and bacterial collagenase: this value compared favorably with the proportion of type IV collagen estimated by gel electrophoresis.     

Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture

Collagen phenotypes were determined for rabbit articular chondrocytes in cartilage slices and first through fifth monolayer cultures. During the first 24 hr of slice culture, chondrocytes exhibited the following collagen phenotype: 96% type II, 3% X₂Y and 1% type III. In primary monolayer culture, no other types of collagen were added to this differentiated chondrocyte phenotype; however, the synthesis per cell of each of the expressed collagens was stimulated. By the fifth day of primary culture, X₂Y synthesis increased 10 fold, and by the eighth day, a further 4 fold. In contrast, the synthesis of collagen types II and III showed no change by the fifth day, but increased 7 fold by the eighth day. These results suggest independent regulation of X₂Y in this situation. In a separate experiment, first through fifth cultures were studied. The synthesis per cell of type II collagen declined steadily and essentially ceased by the fifth culture, indicating the loss of differentiated function by these chondrocyte progeny. The loss of type II synthesis was not quantitatively replaced by the synthesis of type I trimer and type I collagen which was first detected in the third culture. While these qualitative changes in phenotype occurred, the stimulated rate of type III collagen synthesis did not change and that of X₂Y declined only slightly. Thus the termination of type II synthesis did not significantly alter the synthesis of the other collagens produced by differentiated chondrocytes. The final “de-differentiated” phenotype was 41% type I, 25% X₂Y, 20% type I trimer, 13% type III and 1% type II.     

Reduced type I collagen utilization: a pathogenic mechanism in COL5A1 haplo-insufficient Ehlers-Danlos syndrome

To examine mechanisms by which reduced type V collagen causes weakened connective tissues in the Ehlers-Danlos syndrome (EDS), we examined matrix deposition and collagen fibril morphology in long-term dermal fibroblast cultures. EDS cells with COL5A1 haplo-insufficiency deposited less than one-half of hydroxyproline as collagen compared to control fibroblasts, though total collagen synthesis rates are near-normal because type V collagen represents a small fraction of collagen synthesized. Cells from patients with osteogenesis imperfecta (OI) and haplo-insufficiency for proalpha1(I) chains of type I collagen also incorporated about one-half the collagen as controls, but this amount was proportional to their reduced rates of total collagen synthesis. Collagen fibril diameter was inversely proportional to type V/type I collagen ratios (EDS > control > OI). However, a reduction of type V collagen, in the EDS derived cells, was associated with the assembly of significantly fewer fibrils compared to control and OI cells. These data indicate that in cell culture, the quantity of collagen fibrils deposited in matrix is highly sensitive to reduction in type V collagen, far out of proportion to type V collagen's contribution to collagen mass.     

Collagenase activity in the normal rat myocardium

Summary Fibrillar collagen in the myocardium provides a supportive framework for myocytes and capillaries. Disruption of this organized framework has been observed in certain pathological states. Collagen degradation is primarily mediated by the specific enzyme collagenase, which has been found to exist in various tissues including the myocardium. In this report we describe a method that detects collagenase activity in sections of cardiac tissue. This method is on the basis of degradation of collagen by collagenase on one hand and the visualization of disrupted collagen fibers by immunofluorescence on the other. Frozen rat heart secctions were incubated under optimal conditions for collagenase activity (37°C in the presence of 0.1 M calcium at pH 7.4) for 24 h and 48 h. Subsequently, immunofluorescence staining with antibody to type I collagen was performed and the collagenous structures were visualized by immunofluorescence light microscopy. As control, untreated rat heart sections and sections incubated in the absence of calcium were similarly treated with antibody. After the 24 h of incubation, we found no change in the structural integrity of collagen fibers. Marked disruption of the type I collagen fibers was observed 48 h after incubation. No evidence of collagen fiber disruption was found in control sections. Experiments with exogenous collagenase resulted in similar collagen fiber disruption in the frozen rat heart sections. We conclude that the disruption of collagen type I fibers after 48 h of incubation, under optimal conditions for collagenolytic digestion, is. the result of collagen degradation by intrinsic collagenase of the myocardium.     

New insight into the shortening of the collagen fibril D-period in human cornea

Collagen fibrils type I display a typical banding pattern, so-called D-periodicity, of about 67 nm, when visualized by atomic force or electron microscopy imaging. Herein we report on a significant shortening of the D-period for human corneal collagen fibrils type I (21 ± 4 nm) upon air-drying, whereas no changes in the D-period were observed for human scleral collagen fibrils type I (64 ± 4 nm) measured under the same experimental conditions as the cornea. It was also found that for the corneal stroma fixed with glutaraldehyde and air-dried, the collagen fibrils show the commonly accepted D-period of 61 ± 8 nm. We used the atomic force microscopy method to image collagen fibrils type I present in the middle layers of human cornea and sclera. The water content in the cornea and sclera samples was varying in the range of .066–.085. Calculations of the D-period using the theoretical model of the fibril and the FFT approach allowed to reveal the possible molecular mechanism of the D-period shortening in the corneal collagen fibrils upon drying. It was found that both the decrease in the shift and the simultaneous reduction in the distance between tropocollagen molecules can be responsible for the experimentally observed effect. We also hypothesize that collagen type V, which co-assembles with collagen type I into heterotypic fibrils in cornea, could be involved in the observed shortening of the corneal D-period.