Collagen peptide simulated bending after applied axial deformation.

Structural proteins in the extracellular matrix are subjected to a range of mechanical loading conditions, including varied directions of force application. Molecular modeling suggests that these mechanical forces directly affect collagen's conformation and the subsequent mechanical response at the molecular level is complex. For example, tensile forces in the axial direction result in collagen triple helix elongation and unwinding, while perpendicular forces can cause local triple helix disruption. However, the effects of more complicated mechanical loading, such as the effect of axial pretension on collagen bending and triple helix microunfolding are unknown. In this study we used steered molecular dynamics to first model a collagen peptide under axial tension and then apply a perpendicular bending force. Axial tension causes molecular elongation and increased the subsequent perpendicular bending stiffness, but surprisingly did not increase the predicted collagen triple helix microunfolding threshold. We believe these results elucidate a key potential mechanism by which microscale mechanical loads translate from cellular and micro scales down to the nano and atomistic. Further, these data predict that cryptic force-induced collagen triple helix unwinding is axial-deformation independent, supporting the possibility that cell traction forces could be a key molecular mechanism to alter the cellular matrix microenvironment to facilitate collagen enzymatic degradation and subsequent cellular migration, such as in tumor extravasation.

Glycation cross-linking induced mechanical-enzymatic cleavage of microscale tendon fibers.

Recent molecular modeling data using collagen peptides predicted that mechanical force transmitted through intermolecular cross-links resulted in collagen triple helix unwinding. These simulations further predicted that this unwinding, referred to as triple helical microunfolding, occurred at forces well below canonical collagen damage mechanisms. Based in large part on these data, we hypothesized that mechanical loading of glycation cross-linked tendon microfibers would result in accelerated collagenolytic enzyme damage. This hypothesis is in stark contrast to reports in literature that indicated that individually mechanical loading or cross-linking each retards enzymatic degradation of collagen substrates. Using our Collagen Enzyme Mechano-Kinetic Automated Testing (CEMKAT) System we mechanically loaded collagen-rich tendon microfibers that had been chemically cross-linked with sugar and tested for degrading enzyme susceptibility. Our results indicated that cross-linked fibers were >5 times more resistant to enzymatic degradation while unloaded but became highly susceptible to enzyme cleavage when they were stretched by an applied mechanical deformation.

Deep tissue injury in development of pressure ulcers: a decrease of inflammasome activation and changes in human skin morphology in response to aging and mechanical load.

Molecular mechanisms leading to pressure ulcer development are scarce in spite of high mortality of patients. Development of pressure ulcers that is initially observed as deep tissue injury is multifactorial. We postulate that biomechanical forces and inflammasome activation, together with ischemia and aging, may play a role in pressure ulcer development. To test this we used a newly-developed bio-mechanical model in which ischemic young and aged human skin was subjected to a constant physiological compressive stress (load) of 300 kPa (determined by pressure plate analyses of a person in a reclining position) for 0.5-4 hours. Collagen orientation was assessed using polarized light, whereas inflammasome proteins were quantified by immunoblotting. Loaded skin showed marked changes in morphology and NLRP3 inflammasome protein expression. Sub-epidermal separations and altered orientation of collagen fibers were observed in aged skin at earlier time points. Aged skin showed significant decreases in the levels of NLRP3 inflammasome proteins. Loading did not alter NLRP3 inflammasome proteins expression in aged skin, whereas it significantly increased their levels in young skin. We conclude that aging contributes to rapid morphological changes and decrease in inflammasome proteins in response to tissue damage, suggesting that a decline in the innate inflammatory response in elderly skin could contribute to pressure ulcer pathogenesis. Observed morphological changes suggest that tissue damage upon loading may not be entirely preventable. Furthermore, newly developed model described here may be very useful in understanding the mechanisms of deep tissue injury that may lead towards development of pressure ulcers.

A new paradigm for mechanobiological mechanisms in tumor metastasis.

Tumor metastases and epithelial to mesenchymal transition (EMT) involve tumor cell invasion and migration through the dense collagen-rich extracellular matrix surrounding the tumor. Little is neither known about the mechanobiological mechanisms involved in this process, nor the role of the mechanical forces generated by the cells in their effort to invade and migrate through the stroma. In this paper we propose a new fundamental mechanobiological mechanism involved in cancer growth and metastasis, which can be both protective or destructive depending on the magnitude of the forces generated by the cells. This new mechanobiological mechanism directly challenges current paradigms that are focused mainly on biological and biochemical mechanisms associated with tumor metastasis. Our new mechanobiological mechanism describes how tumor expansion generates mechanical forces within the stroma to not only resist tumor expansion but also inhibit or enhance tumor invasion by, respectively, inhibiting or enhancing matrix metalloproteinase (MMP) degradation of the tensed interstitial collagen. While this mechanobiological mechanism has not been previously applied to the study of tumor metastasis and EMT, it may have the potential to broaden our understanding of the tumor invasive process and assist in developing new strategies for preventing or treating cancer metastasis.

Molecular simulations predict novel collagen conformations during cross-link loading.

Collagen cross-linking mechanically strengthens tissues during development and aging, but there is limited data describing how force transmitted across cross-links affects molecular conformation. We used Steered Molecular Dynamics (SMD) to model perpendicular force through a side chain. Results predicted that collagen peptides have negligible bending resistance and that mechanical force causes helix disruption below covalent bond failure strength, suggesting alternative molecular conformations precede cross-link rupture and macroscopic damage during mechanical loading.

Deformation-dependent enzyme mechanokinetic cleavage of type I collagen.

Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%. However, comparison of the EMK response (T(E) versus epsilon) to collagen's stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsilon<3%), (2) a rapid decrease ( approximately 50%) in the transition of the toe-to-heel region (epsilon congruent with3%) followed by (3) a constant value throughout the heel (epsilon=3-5%) and linear (epsilon=5-10%) regions. This observation suggests that the mechanism for the strain-dependent inhibition of enzyme cleavage of the collagen triple helix may be by a conformational change in the triple helix since the decrease in T(E)(epsilon) appeared concomitant with stretching of the collagen molecule.

Mutational data integration in gene-oriented files of the Hermansky-Pudlak Syndrome database.

Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder characterized by oculocutaneous albinism and prolonged bleeding due to abnormal vesicle trafficking to lysosomes and related organelles such as melanosomes and platelet dense granules. This HPS database (HPSD; http://liweilab.genetics.ac.cn/HPSD/) provides integrated, annotatory, and curative data that is distributed in a variety of public databases or predicted by bioinformatics servers for the recently cloned human and mouse HPS genes, as well as for the genes responsible for HPSrelated syndromes, such as ChediakHigashi Syndrome (CHS), Griscelli syndrome (GS), oculocutaneous albinism (OCA), Usher syndrome type 1B (USH1B), and ocular albinism (OA). The HPSD is designed by using a unique GeneOriented File (GOF) format. Seven blocks (genomic, transcript, protein, function, mutation, phenotype, and reference) are carefully annotated in each userfriendly GOF entry. The HPSD emphasizes paired human and mouse GOF entries. The genes included in this database (currently 58 in total) are arbitrarily divided into four categories: 1) Human and Mouse HPS, 2) Mouse HPS Only, 3) Putative Mouse or Human HPS, and 4) HPS Related Syndromes. All the mutations in these genes are integrated in the GOFs. We expect that these very informative and peerreviewed GOFs will be shortcuts to utilize the webbased information for the emerging interdisciplinary studies of HPS.