Collagen Cross-link Formation Research Articles

Effects of Raloxifene on Bone Metabolism in Hemodialysis Patients

Effects of Raloxifene on Bone Metabolism in Hemodialysis Patients

A Role for Prolyl 3-Hydroxylase 2 in Post-translational Modification of Fibril-forming Collagens

The fibrillar collagen types I, II, and V/XI have recently been shown to have partially 3-hydroxylated proline (3Hyp) residues at sites other than the established primary Pro-986 site in the collagen triple helical domain. These sites showed tissue specificity in degree of hydroxylation and a pattern of D-periodic spacing. This suggested a contributory role in fibril supramolecular assembly. The sites in clade A fibrillar α1(II), α2(V), and α1(I) collagen chains share common features with known prolyl 3-hydroxylase 2 (P3H2) substrate sites in α1(IV) chains implying a role for this enzyme. We pursued this possibility using the Swarm rat chondrosarcoma cell line (RCS-LTC) found to express high levels of P3H2 mRNA. Mass spectrometry determined that all the additional candidate 3Hyp substrate sites in the pN type II collagen made by these cells were highly hydroxylated. In RNA interference experiments, P3H2 protein synthesis was suppressed coordinately with prolyl 3-hydroxylation at Pro-944, Pro-707, and the C-terminal GPP repeat of the pNα1(II) chain, but Pro-986 remained fully hydroxylated. Furthermore, when P3H2 expression was turned off, as seen naturally in cultured SAOS-2 osteosarcoma cells, full 3Hyp occupancy at Pro-986 in α1(I) chains was unaffected, whereas 3-hydroxylation of residue Pro-944 in the α2(V) chain was largely lost, and 3-hydroxylation of Pro-707 in α2(V) and α2(I) chains were sharply reduced. The results imply that P3H2 has preferred substrate sequences among the classes of 3Hyp sites in clade A collagen chains.

Bone quality in lifestyle-related diseases

Lifestyle-related diseases deteriorate bone quality in terms of material properties. Collagen cross-link formation is thought to be a determinant of material strength. Hyperglycemia, oxidative stress, carbonyl stress, and hyperhomocysteinemia induce the reduction in beneficial enzymatic cross-links and the accumulation of disadvantageous non-enzymatic cross-link, Advanced glycation end products (AGEs, Pentosidine) in bone. In this review, we describe that lifestyle-related diseases are crucial determinants of detrimental crosslinking of bone collagen that have been reported in the literature.

Cervical Softening During Pregnancy: Regulated Changes in Collagen Cross-Linking and Composition of Matricellular Proteins in the Mouse1

A greater understanding of the parturition process is essential in the prevention of preterm birth, which occurs in 12.7% of infants born in the United States annually. Cervical remodeling is a critical component of this process. Beginning early in pregnancy, remodeling requires cumulative, progressive changes in the cervical extracellular matrix (ECM) that result in reorganization of collagen fibril structure with a gradual loss of tensile strength. In the current study, we undertook a detailed biochemical analysis of factors in the cervix that modulate collagen structure during early mouse pregnancy, including expression of proteins involved in processing of procollagen, assembly of collagen fibrils, cross-link formation, and deposition of collagen in the ECM. Changes in these factors correlated with changes in the types of collagen cross-links formed and packing of collagen fibrils as measured by electron microscopy. Early in pregnancy there is a decline in expression of two matricellular proteins, thrombospondin 2 and tenascin C, as well as a decline in expression of lysyl hydroxylase, which is involved in cross-link formation. These changes are accompanied by a decline in both HP and LP cross-links by gestation Days 12 and 14, respectively, as well as a progressive increase in collagen fibril diameter. In contrast, collagen abundance remains constant over the course of pregnancy. We conclude that early changes in tensile strength during cervical softening result in part from changes in the number and type of collagen cross-links and are associated with a decline in expression of two matricellular proteins thrombospondin 2 and tenascin C.

Kinky Hair, Kinky Vessels, and Bladder Diverticula in Menkes Disease

Menkes disease (MD) is an infantile-onset X-linked recessive neurodegenerative disorder caused by deficiency or dysfunction of a copper-transporting ATPase, ATP7A. The effect of altered transportation of copper may affect various enzymatic functions differently. Among all enzymatic functions, lysyl-oxidase enzymatic activity, which is crucial in the formation of the lysine-derived cross-links in collagen and elastin, is the most sensitive to the copper transport alterations. Pili torti, tortuous intracranial vessels and bladder diverticula are clinical aspects strictly related to the connective tissue alterations dependent on the lysyl-oxidase deficiency. Despite a pleiotropic clinical appearance of MD patients, we observed tortuous intracranial vessels and bladder diverticula in 4 consecutive Menkes patients at different stages of the disease. We speculate that these findings are present at early stages and could be considered suggestive findings in MD.

Pyridoxamine prevents age-related aortic stiffening and vascular resistance in association with reduced collagen glycation

An increase in oxidative chemical modifications of tissue proteins by advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) has been implicated in normal aging. Pyridoxamine (PM), one of the three natural forms of vitamin B 6, has been identified as an inhibitor of AGE/ALE products formed during the autoxidation of carbohydrates and peroxidation of lipids. The current study seeks to determine whether PM intervention could prevent the age-related aortic stiffening and vascular resistance through its ability to inhibit the pathogenic cross-linking of glycated collagen. Male Fisher 344 rats at 15 months were treated daily with PM (1 g l − 1 in drinking water) for 5 months and compared with the age-matched, untreated controls at 20 months. Pulsatile aortic pressure and flow signals were measured to perform the vascular impedance analysis. The anti-AGE antibody 6D12 was used to detect glycation-derived modification of aortic collagen, using protein blotting analysis. PM therapy attenuated the age-related increase in total peripheral resistance. An increase in wave transit time and aortic compliance by PM indicated that the drug improved aortic distensibility of the aged vasculature. This paralleled its reduction of AGE-collagen cross-links on aortas. Treatment of the old animals with PM also prevented the age-induced augmentation in vascular load imposed on the heart, as evidenced by an increased wave transit time and a decreased wave reflection factor. These findings suggest a partial role of PM in improving arterial mechanics by targeting the pathogenic formation of AGE-induced aortic collagen cross-links in old rats.

Advanced glycation end-products, a pathophysiological pathway in the cardiorenal syndrome

The prevalence of heart failure (HF) is increasing. A distinction is made between diastolic HF (preserved left ventricular ejection fraction (LVEF)) and systolic HF (reduced LVEF). Advanced glycation end-products (AGEs) are crystallized proteins that accumulate during ageing, but are particularly increased in patients with diabetes mellitus and in patients with renal failure. Through the formation of collagen crosslinks, and by interaction with the AGE-receptor, which impairs calcium handling and increases fibrosis, AGE-accumulation has pathophysiologically been associated with the development of diastolic and renal dysfunction. Interestingly, diastolic dysfunction is a frequent finding in elderly patients, diabetic patients and in patients with renal failure. Taken together, this suggests that AGEs are related to the development and progression of diastolic HF and renal failure. In this review, the role of AGEs as a possible pathophysiological factor that link the development and progression of heart and renal failure, is discussed. Finally, the role of AGE intervention as a possible treatment in HF patients will be discussed.

Tissue advanced glycation end products are associated with diastolic function and aerobic exercise capacity in diabetic heart failure patients

Advanced glycation end products (AGEs) are increased in patients with diabetes and are associated with diastolic dysfunction through the formation of collagen crosslinks in the heart. The association among AGEs, diastolic function, and aerobic capacity in heart failure (HF) patients with and without diabetes is, however, unknown. We therefore studied the association among tissue AGEs, diastolic function, and aerobic capacity in patients with HF with or without diabetes. In chronic HF patients (with and without left ventricular systolic dysfunction), tissue AGEs [skin autofluorescence (AF)], diastolic function (echocardiographic mean E' and E/E'), and aerobic capacity [peak oxygen uptake (VO(2)) on cardiopulmonary exercise testing] were obtained. A total of 49 diabetics and 156 non-diabetics were included. Diabetics were older and had more cardiovascular risk factors, but left ventricular ejection fractions (LVEF) were similar. Tissue AGEs were higher in diabetics compared with non-diabetics (2.8 ± 0.8 vs. 2.3 ± 0.7 a.u.; P < 0.001). Furthermore, there was a correlation between tissue AGEs and mean E' (r = -0.30; P < 0.001, after adjustment for age, r = -0.21; P = 0.004). Aerobic capacity was significantly lower in diabetic patients with HF (peak VO(2): 17.4 ± 5.1 vs. 21.7 ± 6.1 mL/min/kg; P = 0.001), even after adjustment for age and LVEF. Peak VO(2) was related to skin AF (P = 0.03), independent of age, diabetes, LVEF, and New York Heart Association functional class. Patients with diabetes and HF have similar LVEF but poorer exercise capacity compared with non-diabetic HF patients. Our data suggest that these findings might be explained by the observed association among tissue AGE levels, diastolic function, and exercise capacity.

The spectra character of photodegraded the pyridinoline cross-links by Hypocrellin B

Pyridinoline cross-links is one of the cross-link formation in collagen which in cell matrix, many research shown that this cross-link cause the fibrosis. Hypocrellin B (HB) is one of the nature photosensitizers, this work investigated the pyridinoline cross-link in collagen was photodegraded by HB. The result shown HB can degrade the pyridinoline cross-link with photo. This is to say, HB may be use as the photodynamic reagent to study the fibrosis.

Lysyl Oxidase-like-2 (LOXL2) Is a Major Isoform in Chondrocytes and Is Critically Required for Differentiation

The lysyl oxidase family is made up of five members: lysyl oxidase (LOX) and lysyl oxidase-like 1-4 (LOXL1-LOXL4). All members share conserved C-terminal catalytic domains that provide for lysyl oxidase or lysyl oxidase-like enzyme activity; and more divergent propeptide regions. LOX family enzyme activities catalyze the final enzymatic conversion required for the formation of normal biosynthetic collagen and elastin cross-links. The importance of lysyl oxidase enzyme activity to normal bone development has long been appreciated, but regulation and roles for specific LOX isoforms in bone formation in vivo is largely unexplored. Fracture healing recapitulates aspects of endochondral bone development. The present study first investigated the expression of all LOX isoforms in fracture healing. A remarkable coincidence of LOXL2 expression with the chondrogenic phase of fracture healing was found, prompting more detailed analyses of LOXL2 expression in normal growth plates, and LOXL2 expression and function in developing ATDC5 chondrogenic cells. Data show that LOXL2 is expressed by pre-hypertrophic and hypertrophic chondrocytes in vivo, and that LOXL2 expression is regulated in vitro as a function of chondrocyte differentiation. Moreover, LOXL2 knockdown studies in vitro show that LOXL2 expression is required for ATDC5 chondrocyte cell line differentiation through regulation of SNAIL and SOX9, important transcription factors that control chondrocyte differentiation. Taken together, data provide evidence that LOXL2, like LOX, is a multifunctional protein. LOXL2 promotes chondrocyte differentiation by mechanisms that are likely to include roles as both a regulator and an effector of chondrocyte differentiation.

CKD-MBD (Chronic Kidney Disease-Mineral and Bone Disorder). Bone quality in chronic kidney disease : enzymatic and non-enzymatic glycation or oxidation induced cross-links in bone

Collagen cross-link formation in bone plays an important role in bone strength and the proper biological function of bone (Saito M, Osteoporos Int, [REVIEW] 2010). Plasma homocysteine (Hcys) level and oxidative stress are increased in patient with chronic kidney disease (CKD) at stage 3. Such moderately increased plasma content of Hcys and oxidative stress are well known to be an independent fracture risk. In a case-controlled study, a significant reduction in the actual amount of enzymatic cross-links and a marked increased in advanced glycation end products cross-link, pentosidine, in bone from patients with post-menopausal osteoporotic hip fracture showing hyperhomocysteinemia (Saito M, Calcif Tissue Int 2006). Additionally, hyperhomocysteinemia induced animal model also showed the same collagen cross-links abnormalities as patients with hyperhomocysteinemia (Saito M, Osteoporos Int, 2010). In this review, we summarized the recent literature.

Musculoskeletal rehabilitation and bone. Mechanical stress and bone quality : do mechanical stimuli alter collagen cross-link formation in bone? "Yes"

Evidence has accumulated that collagen cross-links also play important roles in bone strength and the proper biological functions of bone. Thus, collagen cross-links may be a factor in determining the material properties of bone. Collagen cross-linking is affected by some growth factors, and tissue age. Collagen cross-links can be roughly divided into two types : lysyl oxidase mediated cross-links (enzymatic immature, divalent and mature, trivalent cross-links) and advanced glycation end-products (AGE ; non-enzymatic, pentosidine cross-links). These two types vary by both the mechanism of formation and by functional differences. Hyper- and micro-gravity, weight-bearing, and low intensity pulsed ultrasound (LIPUS) have distinct biological effects on bone collagen cross-link formation in vitro and in vivo (Saito M, J Bone Miner Res 2003, Bone 2004, Calcif Tissue Int 2004) . We demonstrated in previous studies that physiologic levels of mechanical strain such as hypergravity, weight-bearing, and LIPUS induce the formation of bone-type collagenous matrix into a specific molecular packing arrangement through the formation of characteristic types of cross-links as mineralization begins. Collagen cross-links play important roles in the expression of bone strength and the proper biological function of bone. Thus, collagen cross-links are thought to be a determinant of bone quality. While LIPUS have beneficial effects on collagen enzymatic cross-link formation, mechanical stress may improve bone quality (Saito M, Osteoporos Int, REVIEW, 2010) .

Effect of vitamin K on bone material properties

Collagen cross-links are determinants of bone quality. Because vitamin K is thought to ameliorate bone quality, we summarized the literature regarding the effect of vitamin K such as menatetorenone (MK-4) on bone matrix property in the review. MK-4 seems to stimulate the osteoblastic activity. This results in the increase in collagen accumulation and lysyl oxidase controlled enzymatic cross-links in bone. Furthermore, vitamin K stimulates the secretion of collagen binding protein regulating proper fibrillogenesis such as leucine-rich repeat protein (tsukushi). This kinds of non-collagenous proteins induced by the treatment of vitamin K may also affect proper collagen cross-link formation and show the favorable effect on bone material quality.

Collagen cross-links as a determinant of bone quality: a possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus

Collagen cross-linking, a major post-translational modification of collagen, plays important roles in the biological and biomechanical features of bone. Collagen cross-links can be divided into lysyl hydroxylase and lysyloxidase-mediated enzymatic immature divalent cross-links,mature trivalent pyridinoline and pyrrole cross-links, and glycation- or oxidation-induced non-enzymatic cross-links(advanced glycation end products) such as glucosepane and pentosidine. These types of cross-links differ in the mechanism of formation and in function. Material properties of newly synthesized collagen matrix may differ in tissue maturity and senescence from older matrix in terms of crosslink formation. Additionally, newly synthesized matrix in osteoporotic patients or diabetic patients may not necessarily be as well-made as age-matched healthy subjects. Data have accumulated that collagen cross-link formation affects not only the mineralization process but also microdamage formation. Consequently, collagen cross-linking is thought to affect the mechanical properties of bone. Furthermore,recent basic and clinical investigations of collagen cross-links seem to face a new era. For instance, serum or urine pentosidine levels are now being used to estimate future fracture risk in osteoporosis and diabetes. In this review, we describe age-related changes in collagen cross-links in bone and abnormalities of cross-links in osteoporosis and diabetes that have been reported in the literature.

Nutrition and bone health. Roles of vitamin C and vitamin B as regulators of bone mass and quality

Vitamin C and a vitamin B are essential nutrients to maintain bone density and bone quality. Recent literature clearly shows that vitamin C and B affect bone quality determinant "collagen cross-link formation" . Mildly elevated plasma homocysteine levels induced by vitamin B insufficiency and methylenetetrahydrofolate reductase (MTHFR) deteriorate normal collagen cross-link formation (Saito M, Osteoporos Int 2009 May 30. [Epub ahead of print] , Shiraki M and Saito M, J Bone Miner Metab [6] 2008) . In this review, we describe the effects of vitamin C and vitamin B insufficiency and hyperhomocysteinemia on bone quality in terms of collagen cross-link formation in bone that have been reported in the literature.

The molecular mechanism of photodynamic therapy to fibrosis: Regulation on the pyridinoline cross-link formation in collagen

Fibrotic processes in tissues are characterised by an excessive accumulation of collagen containing increased levels of pyridinoline cross-links, which has been taken as an important criterion to diagnose the fibrosis. Hypocrellin B (HB) is a natural photosensitizer with excellent photodynamic activity but very low dark-toxicity. In the current work, main purpose was to clarify if photodynamic action of HB could be applied to regulating the level of pyridinoline cross-link in collagen. To use gelatin as a model collagen, it was observed that the pyridinoline cross-link formation was effectively degraded by HB photodynamic action in the gelatin, suggesting the level of pyridinoline cross-link formation in collagen may be photodynamically regulated to a normal level. Furthermore, mechanism of the photodynamic action of HB was studied.

The roles of TGF-β1 gene transfer on collagen formation during Achilles tendon healing

Collagen content and cross-linking are believed to be major determinants of tendon structural integrity and function. The current study aimed to investigate the effects of transforming growth factor (TGF)-β1 on the collagen content and cross-linking of Achilles tendons, and on the histological and biomechanical changes occurring during Achilles tendon healing in rabbits. Bone marrow-derived mesenchymal stem cells (BMSCs) transfected with the TGF-β1 gene were surgically implanted into experimentally injured Achilles tendons. Collagen proteins were identified by immunohistochemical staining and fiber bundle accumulation was revealed by Sirius red staining. Achilles tendons treated with TGF-β1-transfected BMSCs showed higher concentrations of collagen I protein, more rapid matrix remodeling, and larger fiber bundles. Thus TGF-β1 can promote mechanical strength in healing Achilles tendons by regulating collagen synthesis, cross-link formation, and matrix remodeling.

A10 TRANSFORMING GROWTH FACTOR-BETA INDUCES PERSISTENT SYNOVIAL FIBROSIS WHILE CONNECTIVE TISSUE GROWTH FACTOR-INDUCED FIBROSIS IS REVERSIBLE. ROLE FOR LYSYS HYDROXYLASE

Purpose: The main characteristics of osteoarthritis are cartilage damage, osteophyte formation and synovial fibrosis. Synovial fibrosis can contribute to the loss of the function of an OA joint. TGF-b is considered one of the main players in fibrotic diseases. However, CTGF, which can be induced by TGF-b, has been reported to be an important player as well, stated to contribute to maintenance of fibrotic tissue. We investigated the underlying mechanism in that leads to the observed difference in fibrosis persistence between TGF-band CTGF. Methods: We injected C57Bl/6 mice intra-articularly with Ad-TGF-b or Ad-CTGF. After 3, 7 and 21 days knee joint synovial tissue was isolated for RNA isolation or whole knee joints for histology. With Q-RT-PCR relative mRNA levels were determined of matrix molecules, proteases, protease inhibitors, and growth factors. To analyze the enzymes involved in collagen cross link formation, mRNA expression of lysyl hydroxylase (LH) was determined. CTGF-levels induced by TGF-b were measured by ELISA in 24 hour patella-synovial wash-outs of C57Bl/6 mice intra-articularly injected with Ad-TGF-b (3, 7 and 14 days). Results: TGF-b and CTGF induced synovial fibrosis in murine knee joints as shown by histology (as observed by an increase in synovial width). However, TGF-b-induced fibrosis was very persistent, whereas CTGFinduced fibrosis resorbed by day 28. TGF-b induced elevated collagen type I and very high levels of aggrecan expression and hardly any changes in collagen type II and III. CTGF induced no clear changes in collagen type I, II and III. CTGF even decreased aggrecan expression (day 21 only). CTGF induced no changes in MMP3, -9, -13, ADAMTS-4 or -5, whereas TGF-b increased MMP-3, -13 and ADAMTS4. Thus elevated levels of matrix degrading enzyme expression cannot explain the less persistent CTGF-induced fibrosis. TIMP1 (associated with matrix accumulation in fibrotic disorders), was highly up regulated by TGF-b (all days) and only slightly up regulated by CTGF (day 7). TGF-b also induced elevated mRNA levels of TGF-b1 and CTGF. Only TGF-b induced high levels of LH expression, especially LH2b, which has been implicated in hard-to-degrade collagen linkage and was found up regulated in fibrotic lesions. As TGF-b can induce CTGF and up regulated CTGF mRNA in our experiments, we measured CTGF levels in 24 hour patella-synovial wash outs and found that TGF-b induced 64 ng CTGF/ml after 3 days and that CTGF levels were still elevated by day 14. Conclusions: TGF-b induced increased collagen type I and aggrecan mRNA levels in synovial tissue, but also high levels of degrading enzymes. The latter does not explain the persistent nature of TGF-b versus CTGFinduced synovial fibrosis. Lack of collagen type I mRNA induced by CTGF corresponds to findings of other groups. Despite this fact, fibrosis is found. Only TGF-b induced high levels of TIMP1 expression, which was previously found associated with strain dependent fibrosis sensitivity in mice and implicated in accumulation of ECM in fibrotic disorders. TGF-b, not CTGF, induced high levels of LH which are crucial for ECM cross linking. Moreover, LH2b was reported by van der Slot et al. to induce harder-to-degrade crosslinks compared to LH2a. The 2b splicing variant was highly expressed upon stimulation with TGF-b. Since TGF-b clearly up regulated MMP expression a dominant role for TIMP appears unlikely. The strong induction of LH2b by TGF-b compared to CTGF, and consequently harder to degrade cross links, appears to be the most likely cause of the induction of irreversible fibrosis by TGF-b. A11 EFFICACY OF A SINGLE ULTRASOUND GUIDED INJECTION IN HIP OSTEOARTHRITIS

Collagen Cross-Linking Influences Osteoblastic Differentiation

Osteoblasts synthesize collagen matrix, which itself regulates the differentiation of precursor cells into mature osteoblasts. They express lysyl oxidase (LOX), which is involved in the collagen cross-linking process. Lathyrogens, like ss-aminopropionitrile (ssAPN), inhibit the formation of a stable matrix. The aim of the present study was to investigate the influence of cross-linking on osteoblastic differentiation. MC3T3-E1 cells were seeded and treated with or without 400 muM ssAPN for 1 week. Thereafter, living cells were removed and, on this extracellular matrix, new MC3T3-E1 cells were seeded and cultured for 1 week without ssAPN. RNA was isolated, and expression of specific marker genes was determined by quantitative reverse transcription-polymerase chain reaction. Changes in specific cross-links after ssAPN treatment were measured with Fourier-transform infrared spectroscopy. The collagen matrix that formed showed a significant reduction of two major cross-links of bone collagen, deH-DHLNL and pyr, compared to control cultures. Gene expression studies showed an increase of collagen alpha1 (I) (COL1A1) to 150%. Expression of LOX and osteocalcin (OCN) mRNA was significantly downregulated to about 75%. When fresh MC3T3-E1 cells were seeded on this altered matrix without ssAPN, COL1A1 mRNA expression was upregulated (140%), OCN was downregulated (60%), and LOX mRNA expression remained unaffected. These results indicate that ssAPN treatment not only disrupts collagen cross-link formation but also affects osteoblastic activity and expression. In conclusion, the disrupted matrix produced in the presence of lathyrogen influences, even in its absence, the expression of osteoblastic genes.

Assessment of bone quality. Effects of bisphosphonates, raloxifene, alfacalcidol, and menatetrenone on bone quality: collagen cross-links, mineralization, and microdamage

Bone loss and impaired bone quality have been proposed as major causes of increased bone fragility in osteoporosis. The proposed determinants of the material properties of bone are the degree of secondary mineralization, microdamage accumulation, and collagen cross-link formation that is affected by bone turnover rate. Recently, we demonstrated that (1) three years bisphosphonate increases the degree of mineralization, collagen maturity, and non-enzymatic cross-link, pentosidine without changing the amount of enzymatic cross-link and (2) the changes in collagen cross-link formation after treatment of bisphosphonate were associated with microdamage accumulation, which were independent of the mineral content (Saito M, Osteoporos Int, in press) and (3) urinary excretion of pentosidine predicts vertebral fracture (Shiraki M, Saito M, et al, J Bone Miner Metab, 2008) . In this review, I described effects of drugs for osteoporosis on bone material properties.