Fluorescence,aggregation properties and FT-IR microspectroscopy of elastin and collagen fibers

Histological and histochemical observations support the hypothesis that collagen fibers can link to elastic fibers. However, the resulting organization of elastin and collagen type complexes and differences between these materials in terms of macromolecular orientation and frequencies of their chemical vibrational groups have not yet been solved. This study aimed to investigate the macromolecular organization of pure elastin, collagen type I and elastin–collagen complexes using polarized light DIC-microscopy. Additionally, differences and similarities between pure elastin and collagen bundles (CB) were investigated by Fourier transform-infrared (FT-IR) microspectroscopy. Although elastin exhibited a faint birefringence, the elastin–collagen complex aggregates formed in solution exhibited a deep birefringence and formation of an ordered-supramolecular complex typical of collagen chiral structure. The FT-IR study revealed elastin and CB peptide NH groups involved in different types of H-bonding. More energy is absorbed in the vibrational transitions corresponding to CH, CH₂ and CH₃ groups (probably associated with the hydrophobicity demonstrated by 8-anilino-1-naphtalene sulfonic acid sodium salt [ANS] fluorescence), and to νCN, δNH and ωCH₂ groups of elastin compared to CB. It is assumed that the α-helix contribution to the pure elastin amide I profile is 46.8%, whereas that of the B-sheet is 20% and that unordered structures contribute to the remaining percentage. An FT-IR profile library reveals that the elastin signature within the 1360–1189 cm⁻¹ spectral range resembles that of Conex–Toray aramid fibers.     

Interaction Between Proteoglycans and α-Elastin in Construction of Extracellular Matrix of Human Yellow Ligament

One type of large proteoglycan and three types of small proteoglycans (decorin, decorin-subtype, and biglycan) were purified by chromatography, and α-elastin was isolated by alkali treatment from human yellow ligaments taken at the time of operation. The interaction of the proteoglycans with immobilized α-elastin on a sensor was analyzed by surface plasmon resonance, and we confirmed that each of the small proteoglycans exhibited a specific binding to α-elastin. The binding sites of small proteoglycans were contained in the protein cores. In addition, the differences in the interaction of the small proteoglycans with α-elastin of normal and ossified ligaments were compared. The interactions of the small proteoglycans with α-elastin of the ossified ligaments were lower than those with α-elastin of the normal ligaments. In the ossified ligaments, neodesmosine, one of the cross-linking amino acids, was significantly less than in the normal ligaments (p < .05).     

Comparison of the effects of semicarbazide and β-aminopropionitrile on the arterial extracellular matrix in the Brown Norway rat

To investigate a putative role for semicarbazide-sensitive amine oxidase (SSAO) in arterial extracellular matrix (ECM) organization, we compared arteries of growing Brown Norway (BN) rats after chronic administration of semicarbazide (SCZ) and β-aminopropionitrile (BAPN), two inhibitors with different properties and relative specificities for SSAO and lysyl oxidase (LOX). The BN model is particularly well adapted to evaluating effects of toxic compounds on the arterial elastic network. We measured aortic LOX and SSAO activities and quantified several ECM parameters. After a pilot study comparing doses previously studied and testing for additivity, we studied low and high equimolar doses of SCZ and BAPN. Both compounds similarly inhibited LOX, whereas SCZ inhibited SSAO far more effectively than BAPN. Both decreased carotid wall rupture pressure, increased tail tendon collagen solubility, decreased aortic insoluble elastin (% dry weight) and dose-dependently increased defects in the internal elastic lamina of abdominal aorta, iliac and renal arteries. Our results suggest that either these effects are mediated by LOX inhibition, SCZ being slightly more effective than BAPN in our conditions, or SSAO acts similarly to and in synergy with LOX on ECM, the greater SCZ effect reflecting the simultaneous inhibition of both enzymes. However, the high SCZ dose increased aortic collagen and ECM proteins other than insoluble elastin markedly more than did equimolar BAPN, possibly revealing a specific effect of SSAO inhibition. To discriminate between the two above possibilities, and to demonstrate unequivocally a specific effect of SSAO inhibition on ECM formation or organization, we must await availability of more specific inhibitors.     

ARFI Imaging for Noninvasive Material Characterization of Atherosclerosis Part II: Toward In Vivo Characterization

Seventy percent of cardiovascular disease (CVD) deaths are attributed to atherosclerosis. Despite their clinical significance, nonstenotic atherosclerotic plaques are not effectively detected by conventional atherosclerosis imaging methods. Moreover, conventional imaging methods are insufficient for describing plaque composition, which is relevant to cardiovascular risk assessment. Atherosclerosis imaging technologies capable of improving plaque detection and stratifying cardiovascular risk are needed. Acoustic radiation force impulse (ARFI) ultrasound, a novel imaging method for noninvasively differentiating the mechanical properties of tissue, is demonstrated for in vivo detection of nonstenotic plaques and plaque material assessment in this pilot investigation. In vivo ARFI imaging was performed on four iliac arteries: (1) of a normocholesterolemic pig with no atherosclerosis as a control, (2) of a familial hypercholesterolemic pig with diffuse atherosclerosis, (3) of a normocholesterolemic pig fed a high-fat diet with early atherosclerotic plaques and (4) of a familial hypercholesterolemic pig with diffuse atherosclerosis and a small, minimally occlusive plaque. ARFI results were compared with spatially matched immunohistochemistry, showing correlations between elastin and collagen content and ARFI-derived peak displacement and recovery time parameters. Faster recoveries from ARFI-induced peak displacements and smaller peak displacements were observed in areas of higher elastin and collagen content. Importantly, spatial correlations between tissue content and ARFI results were consistent and observable in large and highly evolved as well as small plaques. ARFI imaging successfully distinguished nonstenotic plaques, while conventional B-mode ultrasound did not. This work validates the potential relevance of ARFI imaging as a noninvasive imaging technology for in vivo detection and material assessment of atherosclerotic plaques. (E-mail: russbehler@unc.edu)     

Ontogenic Development of the Elastic Component of the Aortic wall in Spontaneously Hypertensive Rats

In the neonatal stage of development in spontaneously hypertensive rats (SHR), previous studies have shown that arterial pressure is already significantly increased over that of normotensive WKY controls and that other hypertensive characteristics of the cardiovascular system are also in evidence. The present study describes early development of the elastic component of the aortic wall in fetal (days 17,19,21–22 of gestation) and neonatal (days 1,7,14,21 of age) SHR and WKY, to determine whether the early pattern of elastin accumulation differs significantly in hypertensive and normotensive animals. The data indicate that in SHR there is a greater concentration of elastin in the aortic wall, a larger cross-sectional area and an increase in the number of lamellar units, both pre- and postnatally. We conclude that the differences in arterial wall structure which are associated with genetic hypertension are established early in development.     

弹性纤维与皮肤衰老

成熟的弹性纤维主要由弹性蛋白、原纤维蛋白微纤维及与弹性纤维有关的蛋白组成。内源性老化的皮肤弹性和柔韧度降低,弹性纤维网断裂和衰退。光老化皮肤不仅是富含原纤维蛋白的微纤维在表皮真皮交界处丢失,弹性蛋白变性,更重要的是在真皮深层混乱的弹性纤维蛋白物质的沉积,弹性蛋白的功能也受到影响。弹性纤维的修复可归纳为促进组成蛋白表达、改善组装条件、减少破坏因素3方面。     

Photobleaching as a tool to measure the local strain field in fibrous membranes of connective tissues

Connective tissues are complex structures which contain collagen and elastin fibers. These fiber-based structures have a great influence on material mechanical properties and need to be studied at the microscopic scale. Several microscopy techniques have been developed in order to image such microstructures; among them are two-photon excited fluorescence microscopy and second harmonic generation. These observations have been coupled with mechanical characterization to link microstructural kinematics to macroscopic material parameter evolution. In this study, we present a new approach to measure local strain in soft biological tissues using a side-effect of fluorescence microscopy: photobleaching. Controlling the loss of fluorescence induced by photobleaching, we create a pattern on our sample that we can monitor during mechanical loading. The image analysis allows three-dimensional displacements of the patterns at various loading levels to be computed. Then, local strain distribution is derived using the finite element discretization on a four-node element mesh created from our photobleached pattern. Photobleaching tests on a human liver capsule have revealed that this technique is non-destructive and does not have any impact on mechanical properties. This method is likely to have other applications in biological material studies, considering that all collagen–elastin fiber-based biological tissues possess autofluorescence properties and thus can be photobleached.     

Stress urinary incontinence following vaginal trauma involves remodeling of urethral connective tissue in female mice

Objective

The molecular mechanisms underlying stress urinary incontinence (SUI) are not clear. This study was conducted to evaluate molecular alterations in the urethras of mice with experimentally induced SUI.

Study design

Eighteen virgin female mice were equally distributed into three groups as follows: two groups undergoing vaginal distension (VD) for 1 h with 3 mm and 8 mm dilators each, and a non-instrumented control group. Changes in leak point pressure (LPP), morphology, lysyl oxidase (LOX) expression and the metabolism of urethral connective tissue were assessed.

Results

The LPP was significantly decreased in the 3 mm and 8 mm VD groups compared with that in the control group. Collagen and elastin expression in the urethra was significantly decreased in the 8 mm VD group compared with that in the control group, while LOX expression was significantly enhanced.

Conclusions

SUI following vaginal trauma involves over-expression of LOX and decreased synthesis of extracellular matrix components or increased proteolysis in the urethra.     

Elastin peptide receptor-directed monocyte chemotactic polysaccharides derived from seaweed sporophyll and from infectious fungus

We discovered that a seaweed sporophyll-derived polysaccharide of brown alga, Wakame (Undaria pinnatifida) bound to monocytes and attracted them in vitro and in vivo. Physicochemical properties, affinity to a lectin-bead column and sugar composition of the chemotactic polysaccharide indicated this molecule to be a highly sulfated fucogalactan. We then identified the monocyte receptor of the sulfated fucogalactan as the elastin peptide receptor by prophylactic inhibition of the binding and the chemoattraction with lactose and the synthetic elastin peptide, Val-Gly-Val-Ala-Pro-Gly. We assume that the galactose-binding lectin, which is a component of the elastin peptide receptor complex, would recognize a Gal residue of the sulfated fucogalactan. We also observed a similar chemoattracting polysaccharide in a pathogenic fungus, Candida albicans, although the content of it was much lower than in the case of seaweed sporophyll. We speculate that the chemotactic response of monocytes to the sulfated fucogalactan is part of the innate immune system to fungal infection.     

腰椎间盘弹性蛋白五肽的测定及临床意义

为探讨腰椎间盘退变突出的原因，用高效液相色谱法（ＨＰＬＣ）分别对１０个成人正常腰椎间盘和５个病理腰椎间盘的弹性蛋白水解产生的具有特征性的五肽ＶＧＶＰＧ的量进行了测定，从而确定腰椎间盘内弹性蛋白的含量。结果显示：弹性蛋白仅占正常椎间盘干重的１．７４１％±０．２４７％，且其在椎间盘内分布极不均匀。病理性腰椎间盘弹性蛋白的含量仅占其干重的０．９５６％±０．４９２％，含量较正常人明显减少（Ｐ＜０．０１），且弹性蛋白在椎间盘内各部分的分布也发生了明显变化，这可能是腰椎间盘退变突出的重要原因之一。