Karen M. Reiser

Enzymatic and nonenzymatic cross-linking of collagen and elastin.

Knowledge regarding the steps and mechanisms related to the intra‐ and interchain cross‐linking of collagen and elastin has evolved steadily during the past 30 years. Recently, effort has been directed at identifying the location and types of cross‐links that are found in collagen and elastin. There are two major groups of cross‐links: those initiated by the enzyme lysyl oxidase and those derived from nonenzymatically glycated lysine and hydroxylysine residues. The formation of enzymatic cross‐links depends on specific enzymes, amino acid sequences, and quaternary structural arrangements. The cross‐links that are derived nonenzymatically occur more adventitiously and are important to pathobiological processes. Considerable progress has been made in elucidating the pathways of synthesis for several of the enzymatically mediated cross‐links, as well as possible mechanisms regulating the specificity of cross‐linking. Although less is known about the chemistry of cross‐links arising from nonenzymatically glycated residues, recent progress has also been made in understanding possible biosynthetic pathways and control mechanisms. This review focuses on such progress and hopes to underscore the biological importance of collagen and elastin cross‐linking.—Reiser, K.; McCormick, R. J.; Rucker, R. B. Enzymatic and nonenzymatic cross‐linking of collagen and elastin. FASEB J. 6: 2439‐2449; 1992.

Polarization-Modulated Second Harmonic Generation in Collagen

Collagen possesses a strong second-order nonlinear susceptibility, a nonlinear optical property characterized by second harmonic generation in the presence of intense laser beams. We present a new technique involving polarization modulation of an ultra-short pulse laser beam that can simultaneously determine collagen fiber orientation and a parameter related to the second-order nonlinear susceptibility. We demonstrate the ability to discriminate among different patterns of fibrillar orientation, as exemplified by tendon, fascia, cornea, and successive lamellar rings in an intervertebral disc. Fiber orientation can be measured as a function of depth with an axial resolution of approximately 10 microm. The parameter related to the second-order nonlinear susceptibility is sensitive to fiber disorganization, oblique incidence of the beam on the sample, and birefringence of the tissue. This parameter represents an aggregate measure of tissue optical properties that could potentially be used for optical imaging in vivo.

Polarization-dependent optical second-harmonic imaging of a rat-tail tendon.

Using scanning confocal microscopy, we measure the backscattered second harmonic signal generated by a 100 fs laser in rat-tail tendon collagen. Damage to the sample is avoided by using a continuous scanning technique, rather than measuring the signal at discrete points. The second harmonic signal varies by about a factor of 2 across a single cross section of the rat-tail tendon fascicle. The signal intensity depends both on the collagen organization and the backscattering efficiency. This implies that we cannot use intensity measurements alone to characterize collagen structure. However, we can infer structural information from the polarization dependence of the second harmonic signal. Axial and transverse scans for different linear polarization angles of the input beam show that second harmonic generation (SHG) in the rat-tail tendon depends strongly on the polarization of the input laser beam. We develop an analytical model for the SHG as a function of the polarization angle in the rat-tail tendon. We apply this model in determining the orientation of collagen fibrils in the fascicle and the ratio gamma between the two independent elements of the second-order nonlinear susceptibility tensor. There is a good fit between our model and the measured data.

Nonenzymatic Glycation of Collagen in Aging and Diabetes

Summary and Conclusions How can we summarize the progress that has been made in the field since this topic was previously reviewed? Should we feel optimistic that the pieces of the puzzle are falling into place, as suggested recently (118), or do we have a disquieting sense that as we work out the details in one area—all the reaction pathways, products, side reactions—the perspective shifts and the puzzle of glycation, like a fractal, regenerates complexity? Perhaps both are appropriate. Glyco-oxidative damage represents the interface between the orderly workings of genomic intelligence and the disorganizing force of stochastic events. This interface, the cusp of our mortality, is the Faustian bargain of evolution. The passage from the unconscious immortality of unicellular existence to the complexities of sentient life required the development of an oxidative metabolism, the slow fire that consumes us. Until now we have only been able to watch it and study it; whether we can change it remains to be seen.

Quantitative second-harmonic generation microscopy in collagen

The second-harmonic signal in collagen, even in highly organized samples such as rat tail tendon fascicles, varies significantly with position. Previous studies suggest that this variability may be due to the parallel and antiparallel orientation of neighboring collagen fibrils. We applied high-resolution second-harmonic generation microscopy to confirm this hypothesis. Studies in which the focal spot diameter was varied from approximately 1 to approximately 6 microm strongly suggest that regions in which collagen fibrils have the same orientation in rat tail tendon are likely to be less than approximately 1 microm in diameter. These measurements required accurate determination of the focal spot size achieved by use of different microscope objectives; we developed a technique that uses second-harmonic generation in a quartz reference to measure the focal spot diameter directly. We also used the quartz reference to determine a lower limit (dXXX > 0.4 pm/V) for the magnitude of the second-order nonlinear susceptibility in collagen.

Silicosis and fibrogenesis: Fact and artifact

Although the pulmonary and extrapulmonary manifestations of silicosis in humans have been extensively documented, the mechanisms by which the fibrogenic effects of silica are manifested remain obscure. In this review, both in vitro and in vivo models of silicosis are discussed, with emphasis on the potential methodological pitfalls of each. In animal models, for example, species variability, silica type and route of administration all effect the results obtained. Tissue culture work has provided evidence that the fibroblast-macrophage interaction is a key event in fibrogenesis. However, critical variables in experimental design make it difficult to compare the often conflicting results of different workers. Experimental conditions that directly affect collagen chain biosynthesis and subsequent hydroxylation of proline appear to be of particular importance. It is concluded that, in part because of methodological difficulties, there are insufficient data to draw firm conclusions regarding the effect of silica-exposed macrophages on collagen biosynthesis by fibroblasts in vitro; there are few, if any, data concerning the role of the macrophage that has ingested silica in human or animal models of silicosis.

Collagen structure and nonlinear susceptibility: Effects of heat, glycation, and enzymatic cleavage on second harmonic signal intensity†

Helical macromolecules such as collagen and DNA are characterized by nonlinear optical properties, including nonlinear susceptibility. Because collagen is the predominant component of most biological tissues, as well as the major source of second harmonic generation (SHG), it is reasonable to assume that changes in harmonic signal can be attributed to structural changes in collagen. The purpose of this study is to determine whether various modifications of collagen structure affect second harmonic intensity.

Bleomycin-induced pulmonary fibrosis: Correlation of biochemical, physiological, and histological changes

Abstract Rats (275–300 g) were intratracheally instilled with 0.38, 0.75, or 1.5 units of bleomycin sulfate. One, two, and three weeks later lung damage was evaluated using a variety of indices, including body weight, right apical lung lobe wet weight, protein- and collagen-synthesis rates by lung minces, lung hydroxyproline content, lung compliance, and lung histology. Several parameters in the high-dose bleomycin group also were evaluated 5 and 8 weeks after instillation. Most of the assays showed a dose-response effect. However, biochemical assays did not correlate with physiological changes 5 and 8 weeks after instillation.

Effect of Aminoguanidine on the Frequency of Neuroaxonal Dystrophy in the Superior Mesenteric Sympathetic Autonomic Ganglia of Rats With Streptozocin-Induced Diabetes

Aminoguanidine, which prevents formation of advanced glycation end products and is a relatively selective potent inhibitor of the inducible (versus constitutive) isoform(s) of nitric oxide synthase, has been reported to ameliorate structural and functional abnormalities in peripheral somatic nerves in rats with streptozocin (STZ)-induced diabetes. In the present studies, the effects of aminoguanidine treatment on ultrastructural changes in the autonomic nervous system of rats with STZ-induced diabetes were examined. The frequency of neuroaxonal dystrophy, the neuropathological hallmark of sympathetic autonomic neuropathy in diabetic rats, increased 9- to 11-fold in the superior mesenteric ganglia of 7- and 10-month STZ-diabetic rats compared with that in age-matched controls. Administration of aminoguanidine continuously from the time of induction of diabetes at a dose equal to or in excess of that providing a salutary effect in the diabetic somatic peripheral nervous system did not alter the severity of diabetes as assessed by plasma glucose level, 24-h urine volume, and levels of glycated hemoglobin. Chronic aminoguanidine therapy did not diminish the frequency or affect the ultrastructural appearance of neuroaxonal dystrophy in diabetic or age-matched control rat sympathetic ganglia after 7 or 10 months of continuous administration. Our findings (under these experimental conditions) do not support a role for aminoguanidine-sensitive processes in the development of sympathetic neuroaxonal dystrophy in diabetic rats. Glycation-linked aminoguanidine-insensitive processes, however, such as the formation of early glucose adducts (Schiff bases and Amadori products) with intracellular and/or extracellular proteins and amine-containing lipids, superoxide anion generation during subsequent autoxidation of these glucose adducts, and non-glycative processes, remain potential pathogenetic mechanisms for diabetic autonomic neuropathy.

Biochemical assays in lung homogenates: artifacts caused by trapped blood after perfusion.

The amounts of trapped whole blood in well-perfused homogenates of rat lung (from rat lungs weighing approximately 1–1.5 g wet weight) were quantitatively determined by a method which utilizes the difference in hemoglobin (Hb) absorbance at 415 nm after reduction of HbO 2 to deoxyHb by dithionite. By adding known amounts of whole blood to unfiltered crude lung homogenates, we constructed standard curves which allowed us to determine residual blood contents of perfused homogenates. Homogenates contained a mean of 20 ± 2.4 (SE) μ l of blood/lung. These data were used to calculate the effects that contaminating whole blood might make upon measured values of various biochemical parameters in lung cytosols and homogenates. Depending on the specific parameter evaluated, blood contamination can contribute a major (e.g., catalase, β - N -acetylglucosaminidase and protein), a trivial (e.g., lysozyme, DNA) or an intermediate (e.g., superoxide dismutase, glutathione peroxidase) fraction of the total amount of a given component present in lung homogenate or cytosol. In studies in which the overall magnitudes of measured lung biochemical changes have been ascribed mechanistic significance (such as in lung damage, repair, protective, or adaptative processes) or in which lung enzyme specific activities are being expressed, the effects of blood contamination must be taken into account.