المؤلفون: Singh, Pragya, Ramanathan, Varshini, Zhang, Yang, Georgakoudi, Irene, Jay, Daniel G.

المصدر: Cancers; Nov2023, Vol. 15 Issue 21, p5237, 27p

مصطلحات موضوعية: PROTEINS, COLLAGEN, IN vitro studies, BIOLOGICAL models, CANCER invasiveness, CONNECTIVE tissues, ANIMAL experimentation, CELL physiology, METASTASIS, CELL survival, DESCRIPTIVE statistics, CELL proliferation, RESEARCH funding, EXTRACELLULAR space, BREAST tumors, MICE

مستخلص: Simple Summary: Breast cancer cells secrete Hsp90, a protein that, inside of cells, regulates the function of hundreds of proteins, but outside of cells, extracellular Hsp90 (eHsp90) can activate a subset of proteins that promote invasion, the first step of metastasis. Blocking eHsp90 in mouse models inhibits metastasis, and we sought to understand how this occurs. Prior studies have predominantly focused on eHsp90 in cancer invasion within the immediate vicinity of the primary tumor, specifically its role in invading outside the epithelial compartment. However, eHsp90's role in cancer invasion across the extended connective tissue after the cells have crossed the boundary of the epithelial compartment remains unknown. We show here that eHsp90 directly binds to and aligns Collagen-1 fibers, a major structural component of connective tissues, which, when aligned, form highways that allow efficient cancer migration. Our study suggests that the Hsp90 dimer, in its open state, binds to Collagen-1 molecules to align the fibers, which results in enhanced breast cancer invasion through the Collagen-1 matrix. Knowing this could help us propose experiments to test eHsp90 inhibitors for therapeutically targeting metastatic breast cancer. Cancer cell-secreted eHsp90 binds and activates proteins in the tumor microenvironment crucial in cancer invasion. Therefore, targeting eHsp90 could inhibit invasion, preventing metastasis—the leading cause of cancer-related mortality. Previous eHsp90 studies have solely focused on its role in cancer invasion through the 2D basement membrane (BM), a form of extracellular matrix (ECM) that lines the epithelial compartment. However, its role in cancer invasion through the 3D Interstitial Matrix (IM), an ECM beyond the BM, remains unexplored. Using a Collagen-1 binding assay and second harmonic generation (SHG) imaging, we demonstrate that eHsp90 directly binds and aligns Collagen-1 fibers, the primary component of IM. Furthermore, we show that eHsp90 enhances Collagen-1 invasion of breast cancer cells in the Transwell assay. Using Hsp90 conformation mutants and inhibitors, we established that the Hsp90 dimer binds to Collagen-1 via its N-domain. We also demonstrated that while Collagen-1 binding and alignment are not influenced by Hsp90's ATPase activity attributed to the N-domain, its open conformation is crucial for increasing Collagen-1 alignment and promoting breast cancer cell invasion. These findings unveil a novel role for eHsp90 in invasion through the IM and offer valuable mechanistic insights into potential therapeutic approaches for inhibiting Hsp90 to suppress invasion and metastasis. [ABSTRACT FROM AUTHOR]

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المؤلفون: Papathanasiou, Evangelos, Scott, Andrew R., Trotman, Carroll Ann, Beale, Corinna, Price, Lori Lyn, Huggins, Gordon S., Zhang, Yang, Georgakoudi, Irene, Van Dyke, Thomas E.

المصدر: Frontiers in Immunology; 4/27/2022, Vol. 13, p1-14, 14p

مصطلحات موضوعية: CLEFT lip, SCARS, TOPICAL drug administration, VELOPHARYNGEAL insufficiency, ANIMAL fibers, HEALING

مصطلحات جغرافية: NEW Zealand

مستخلص: Objective: Residual scarring after cleft lip repair surgery remains a challenge for both surgeons and patients and novel therapeutics are critically needed. The objective of this preclinical experimental study was to evaluate the impact of the methyl-ester of pro-resolving lipid mediator lipoxin A4 (LXA4-ME) on scarring in a novel rabbit model of cleft lip repair. Methods: A defect of the lip was surgically created and repaired in eight six-week old New Zealand white rabbits to simulate human cleft lip scars. Rabbits were randomly assigned to topical application of PBS (control) or 1 ug of LXA4-ME (treatment). 42 days post surgery all animals were euthanized. Photographs of the cleft lip area defect and histologic specimens were evaluated. Multiple scar assessment scales were used to compare scarring. Results: Animals treated with LXA4-ME exhibited lower Visual Scar Assessment scores compared to animals treated with PBS. Treatment with LXA4-ME resulted in a significant reduction of inflammatory cell infiltrate and density of collagen fibers. Control animals showed reduced 2D directional variance (orientation) of collagen fibers compared to animals treated with LXA4-ME demonstrating thicker and more parallel collagen fibers, consistent with scar tissue. Conclusions: These data suggest that LXA4-ME limits scarring after cleft lip repair and improves wound healing outcomes in rabbits favoring the resolution of inflammation. Further studies are needed to explore the mechanisms that underlie the positive therapeutic impact of LXA4-ME on scarring to set the stage for future human clinical trials of LXA4-ME for scar prevention or treatment after cleft lip repair. [ABSTRACT FROM AUTHOR]

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المؤلفون: Huang, Mengqi, Cai, Guoshuai, Baugh, Lauren M., Liu, Zhiyi, Smith, Avi, Watson, Matthew, Popovich, Dillon, Zhang, Tianyue, Stawski, Lukasz S., Trojanowska, Maria, Georgakoudi, Irene, Black, Lauren D., Pioli, Patricia A., Whitfield, Michael L., Garlick, Jonathan

المصدر: Arthritis & Rheumatology; May2020, Vol. 72 Issue 5, p791-801, 11p

مصطلحات موضوعية: COLLAGEN, FIBROBLASTS, GENE expression, GENES, MICROSCOPY, OXIDOREDUCTASES, SKIN, STRUCTURAL models, SYSTEMIC scleroderma, TRANSFORMING growth factors-beta, FIBROSIS, DESCRIPTIVE statistics

مستخلص: Objective: Systemic sclerosis (SSc) is a clinically heterogeneous disease characterized by increased collagen accumulation and skin stiffness. Our previous work has demonstrated that transforming growth factor β (TGFβ) induces extracellular matrix (ECM) modifications through lysyl oxidase–like 4 (LOXL‐4), a collagen crosslinking enzyme, in bioengineered human skin equivalents (HSEs) and self‐assembled stromal tissues (SAS). We undertook this study to investigate cutaneous fibrosis and the role of LOXL‐4 in SSc pathogenesis using HSEs and SAS. Methods: SSc‐derived dermal fibroblasts (SScDFs; n = 8) and normal dermal fibroblasts (NDFs; n = 6) were incorporated into HSEs and SAS. These 3‐dimensional skin‐like microenvironments were used to study the effects of dysregulated LOXL‐4 on ECM remodeling, fibroblast activation, and response to TGFβ stimulation. Results: SScDF‐containing SAS showed increased stromal thickness, collagen deposition, and interleukin‐6 secretion compared to NDF‐containing SAS (P < 0.05). In HSE, SScDFs altered collagen as seen by a more mature and aligned fibrillar structure (P < 0.05). With SScDFs, enhanced stromal rigidity with increased collagen crosslinking (P < 0.05), up‐regulation of LOXL4 expression (P < 0.01), and innate immune signaling genes were observed in both tissue models. Conversely, knockdown of LOXL4 suppressed rigidity, contraction, and α‐smooth muscle actin expression in SScDFs in HSE, and TGFβ‐induced ECM aggregation and collagen crosslinking in SAS. Conclusion: A limitation to the development of effective therapeutics in SSc is the lack of in vitro human model systems that replicate human skin. Our findings demonstrate that SAS and HSE can serve as complementary in vitro skin‐like models for investigation of the mechanisms and mediators that drive fibrosis in SSc and implicate a pivotal role for LOXL‐4 in SSc pathogenesis. [ABSTRACT FROM AUTHOR]

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المؤلفون: Bayan, Christopher, Levitt, Jonathan M., Miller, Eric, Kaplan, David, Georgakoudi, Irene

المصدر: Journal of Applied Physics; May2009, Vol. 105 Issue 10, p102042-102053, 11p, 4 Black and White Photographs, 3 Charts, 7 Graphs

مصطلحات موضوعية: COLLAGEN, SECOND harmonic generation, FIBROBLASTS, IMAGE analysis, ENTROPY, FOURIER transforms

مستخلص: Collagen is the most prominent protein of human tissues. Its content and organization define to a large extent the mechanical properties of tissue as well as its function. Methods that have been used traditionally to visualize and analyze collagen are invasive, provide only qualitative or indirect information, and have limited use in studies that aim to understand the dynamic nature of collagen remodeling and its interactions with the surrounding cells and other matrix components. Second harmonic generation (SHG) imaging emerged as a promising noninvasive modality for providing high-resolution images of collagen fibers within thick specimens, such as tissues. In this article, we present a fully automated procedure to acquire quantitative information on the content, orientation, and organization of collagen fibers. We use this procedure to monitor the dynamic remodeling of collagen gels in the absence or presence of fibroblasts over periods of 12 or 14 days. We find that an adaptive thresholding and stretching approach provides great insight to the content of collagen fibers within SHG images without the need for user input. An additional feature-erosion and feature-dilation step is useful for preserving structure and noise removal in images with low signal. To quantitatively assess the orientation of collagen fibers, we extract the orientation index (OI), a parameter based on the power distribution of the spatial-frequency-averaged, two-dimensional Fourier transform of the SHG images. To measure the local organization of the collagen fibers, we access the Hough transform of small tiles of the image and compute the entropy distribution, which represents the probability of finding the direction of fibers along a dominant direction. Using these methods we observed that the presence and number of fibroblasts within the collagen gel significantly affects the remodeling of the collagen matrix. In the absence of fibroblasts, gels contract, especially during the first few days, in a manner that allows the fibers to remain mostly disoriented, as indicated by small OI values. Subtle changes in the local organization of fibers may be taking place as the corresponding entropy values of these gels show a small decrease. The presence of fibroblasts affects the collagen matrix in a manner that is highly dependent on their number. A low density of fibroblasts enhances the rate of initial gel contraction, but ultimately leads to degradation of collagen fibers, which start to organize in localized clumps. This degradation and reorganization is seen within the first days of incubation with fibroblasts at a high density and is followed by de novo collagen fiber deposition by the fibroblasts. These collagen fibers are more highly oriented and organized than the fibers of the original collagen gel. These initial studies demonstrate that SHG imaging in combination with automated image analysis approaches offer a noninvasive and easily implementable method for characterizing important features of the content and organization of collagen in tissuelike specimens. Therefore, these studies could offer important insights for improving tissue engineering and disease diagnostic efforts. [ABSTRACT FROM AUTHOR]

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المؤلفون: Yildirim, Murat, Quinn, Kyle P., Kobler, James B., Zeitels, Steven M., Georgakoudi, Irene, Ben-Yakar, Adela

المصدر: Scanning; Nov/Dec2016, Vol. 38 Issue 6, p684-693, 10p

مصطلحات موضوعية: IMAGE analysis, COLLAGEN, VOCAL cords, FIBER lasers, HARMONIC generation

مستخلص: The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 ± 0.03) was significantly lower (p < 0.05) than control tissue (0.73 ± 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p < 0.005) fiber density (0.72 ± 0.09) compared to controls (0.18 ± 0.03). In the present study, image analysis of both fiber alignment and density from depth-resolved second-harmonic generation images in epi-detection mode enabled the quantification of the increased collagen fiber deposition and alignment typically observed in fibrosis. The epi-detection geometry is the only viable method for in vivo imaging as well as imaging thick turbid tissues. These quantitative endpoints, clearly differentiating between control and scarred hamster cheek pouches, provide an objective means to characterize the extent of vocal fold scarring in vivo in preclinical and clinical research. In particular, this non-invasive method offers advantages for monitoring scar treatments in live animals and following the effects of scarring-related treatments such as application of steroids or drugs targeting pathways involved in fibrosis. SCANNING 38:684-693, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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المؤلفون: Marturano, Joseph E., Arena, Jeffrey D., Schiller, Zachary A., Georgakoudi, Irene, Kuo, Catherine K.

المصدر: Proceedings of the National Academy of Sciences of the United States of America; 4/16/2013, Vol. 110 Issue 16, p6370-6375, 6p

مصطلحات موضوعية: TENDONS, SECOND harmonic generation, GLYCOSAMINOGLYCANS, PROTEIN crosslinking, COLLAGEN

مستخلص: Tendons have uniquely high tensile strength, critical to their function to transfer force from muscle to bone. When injured, their innate healing response results in aberrant matrix organization and functional properties. Efforts to regenerate tendon are challenged by limited understanding of its normal development. Consequently, there are few known markers to assess tendon formation and parameters to design tissue engineering scaffolds. We profiled mechanical and biological properties of embryonic tendon and demonstrated functional properties of developing tendon are not wholly reflected by protein expression and tissue morphology. Using force volume-atomic force microscopy, we found that nano- and micro-scale tendon elastic moduli increase nonlinearly and become increasingly spatially heterogeneous during embryonic development. When we analyzed potential biochemical contributors to modulus, we found statistically significant but weak correlation between elastic modulus and collagen content, and no correlation with DNA or glycosaminoglycan content, indicating there are additional contributors to mechanical properties. To investigate collagen cross-linking as a potential contributor, we inhibited lysyl oxidasemediated collagen cross-linking, which significantly reduced tendon elastic modulus without affecting collagen morphology or DNA, glycosaminoglycan, and collagen content. This suggests that lysyl oxidase-mediated cross-linking plays a significant role in the development of embryonic-tendon functional properties and demonstrates that changes in cross-links alter mechanical properties without affecting matrix content and organization. Taken together, these data demonstrate the importance of functional markers to assess tendon development and provide a profile of tenogenic mechanical properties that may be implemented in tissue engineering scaffold design to mechanoregulate new tendon regeneration. [ABSTRACT FROM AUTHOR]

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المؤلفون: Quinn, Kyle P., Golberg, Alexander, Broelsch, G. Felix, Khan, Saiqa, Villiger, Martin, Bouma, Brett, Austen, William G., Sheridan, Robert L., Mihm, Martin C., Yarmush, Martin L., Georgakoudi, Irene

المصدر: Experimental Dermatology; Jan2015, Vol. 24 Issue 1, p78-80, 3p

مصطلحات موضوعية: SCARS, BURNS & scalds, COLLAGEN, TISSUE fixation (Histology), DIAGNOSIS, THERAPEUTICS

مستخلص: Standard approaches to evaluate scar formation within histological sections rely on qualitative evaluations and scoring, which limits our understanding of the remodelling process. We have recently developed an image analysis technique for the rapid quantification of fibre alignment at each pixel location. The goal of this study was to evaluate its application for quantitatively mapping scar formation in histological sections of cutaneous burns. To this end, we utilized directional statistics to define maps of fibre density and directional variance from Masson's trichrome-stained sections for quantifying changes in collagen organization during scar remodelling. Significant increases in collagen fibre density are detectable soon after burn injury in a rat model. Decreased fibre directional variance in the scar was also detectable between 3 weeks and 6 months after injury, indicating increasing fibre alignment. This automated analysis of fibre organization can provide objective surrogate endpoints for evaluating cutaneous wound repair and regeneration. [ABSTRACT FROM AUTHOR]

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المؤلفون: Zhang, Yang, Baloglu, Fatma Kucuk, Ziemer, Lauren E. Hillers, Liu, Zhiyi, Lyu, Boyang, Arendt, Lisa M., Georgakoudi, Irene

المصدر: Journal of Biomedical Optics; Jan2020, Vol. 25 Issue 1, p14513-14513, 1p

مصطلحات موضوعية: BREAST cancer, OBESITY, BREAST, TISSUES, COLLAGEN, DISEASE progression

مستخلص: Obesity is associated with a higher risk of developing breast cancer and with worse disease outcomes for women of all ages. The composition, density, and organization of the breast tissue stroma are also known to play an important role in the development and progression of the disease. However, the connections between obesity and stromal remodeling are not well understood. We sought to characterize detailed organization features of the collagen matrix within healthy and cancerous breast tissues acquired from mice exposed to either a normal or high fat (obesity inducing) diet. We performed second-harmonic generation and spectral two-photon excited fluorescence imaging, and we extracted the level of collagen-associated fluorescence (CAF) along with metrics of collagen content, three-dimensional, and two-dimensional organization. There were significant differences in the CAF intensity and overall collagen organization between normal and tumor tissues; however, obesity-enhanced changes in these metrics, especially when three-dimensional organization metrics were considered. Thus, our studies indicate that obesity impacts significantly collagen organization and structure and the related pathways of communication may be important future therapeutic targets. [ABSTRACT FROM AUTHOR]

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المؤلفون: Angheloiu, George O., van de Poll, Sweder W.E., Georgakoudi, Irene, Motz, Jason T., Haka, Abigail S., Podrez, Eugene, Fitzmaurice, Maryann, Dasari, Ramachandra R., Feld, Michael S., Kramer, John R.

المصدر: Applied Spectroscopy. Dec2012, Vol. 66 Issue 12, p1403-1410. 8p.

مصطلحات موضوعية: *FLUORESCENCE spectroscopy, *LASERS, *CORONARY arteries, *ATHEROSCLEROSIS, *ELASTIN, *COLLAGEN

مستخلص: Laser-induced fluorescence (LIF) and intrinsic fluorescence spectroscopy (IFS) have been used experimentally for diagnosing coronary atherosclerosis. In this study, we demonstrated the diagnostic superiority of IFS at 342-nm excitation (IFS342) versus LIF (LIF342) and described a protocol for head-to-head comparison of old (LIF) versus new (IFS) generations of similar diagnostic methods, labeled as “generational comparison model”. IFS342and LIF342were modeled with basis spectra of media, fibrous caps, and superficial foam cells and of their correspondent chemicals (elastin, collagen, and lipoproteins). The average accuracy and receiver operating characteristic area under the curve of IFS342in single-, double-, and triple-parameter diagnostic algorithm iterations, geared toward identifying 84 atherosclerotic specimens from a group of 117 coronary segments, was 90% ± 1% and 0.87 ± 0.025, superior to LIF342(84% ± 3% and 0.84 ± 0.016; P= 0.0002 and 0.02, respectively) in a generational comparison model. [ABSTRACT FROM AUTHOR]