Imaging Collagen in Scar Tissue: Developments in Second Harmonic Generation Microscopy for Biomedical Applications

The ability to respond to injury with tissue repair is a fundamental property of all multicellular organisms. The extracellular matrix (ECM), composed of fibrillar collagens as well as a number of other components is dis-regulated during repair in many organs. In many tissues, scaring results when the balance is lost between ECM synthesis and degradation. Investigating what disrupts this balance and what effect this can have on tissue function remains an active area of research. Recent advances in the imaging of fibrillar collagen using second harmonic generation (SHG) imaging have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. Here, we review the physical properties of SHG, and the current nonlinear optical microscopy imaging (NLOM) systems that are used for SHG imaging. We provide an extensive review of studies that have used SHG in skin, lung, cardiovascular, tendon and ligaments, and eye tissue to understand alterations in fibrillar collagens in scar tissue. Lastly, we review the current methods of image analysis that are used to extract important information about the role of fibrillar collagens in scar formation.Medicine, Faculty ofOther UBCAnesthesiology, Pharmacology and Therapeutics, Department ofReviewedFacult

Macrophages Regulate Schwann Cell Maturation after Nerve Injury

Summary: Pro-regenerative macrophages are well known for their role in promoting tissue repair; however, their specific roles in promoting regeneration of the injured nerve are not well defined. Specifically, how macrophages interact with Schwann cells following injury during remyelination has been largely unexplored. We demonstrate that after injury, including in humans, macrophages function to clear debris and persist within the nerve microenvironment. Macrophage ablation immediately preceding remyelination results in an increase in immature Schwann cell density, a reduction in remyelination, and long-term deficits in conduction velocity. Targeted RNA-seq of macrophages from injured nerve identified Gas6 as one of several candidate factors involved in regulating Schwann cell dynamics. Functional studies show that the absence of Gas6 within monocyte lineage cells impairs Schwann cell remyelination within the injured nerve. These results demonstrate a role for macrophages in regulating Schwann cell function during nerve regeneration and highlight a molecular mechanism by which this occurs. : Stratton et al. demonstrate that macrophages persist in the injured rodent and human nerve and regulate Schwann cells. Macrophages have a unique transcriptional profile, including the expression of Gas6, that functions to regulate Schwann cell remyelination. Keywords: nerve injury, macrophage, Schwann cell, regeneration, remyelination, population-based RNA-se

Chlamydia muridarum T-Cell Antigens Formulated with the Adjuvant DDA/TDB Induce Immunity against Infection That Correlates with a High Frequency of Gamma Interferon (IFN-γ)/Tumor Necrosis Factor Alpha and IFN-γ/Interleukin-17 Double-Positive CD4+ T Cells▿

Major impediments to developing a Chlamydia vaccine lie in identifying immunologically relevant T-cell antigens and delivery in a manner to stimulate protective immunity. Using an immunoproteomic approach, we previously identified three immunodominant Chlamydia T-cell antigens (PmpG-1, PmpE/F-2, and RplF). Because RplF has high homology to a human ortholog, it may not be suitable for human vaccine development. Therefore, in this study, we evaluated protection against Chlamydia infection in the genital tract in C57BL/6 mice immunized with Chlamydia-specific membrane proteins PmpG-1, PmpE/F-2, and major outer membrane protein (MOMP; as a reference) or a combination of them formulated with one of three adjuvants, CpG oligodeoxynucleotide (CpG-ODN), AbISCO-100 (AbISCO), or DDA/TDB (dimethyldioctadecylammonium bromide/d-(+)-trehalose 6,6′-dibehenate). The results show that immunization with the CpG-ODN formulation failed to provide protection against Chlamydia infection; the AbISCO formulation conferred moderate protection, and the DDA/TDB formulation showed the highest degree of protective efficacy. The combination of PmpG-1, PmpE/F-2, and MOMP proteins formulated with DDA/TDB exhibited the greatest degree of protection among all vaccine groups studied. Moreover, this vaccine combination also engendered significant protection in BALB/c mice, which have a different major histocompatibility complex (MHC) background. We measured cell-mediated immune cytokine responses in mice immunized with PmpG-1 mixed with each of the three adjuvants. The results demonstrate that mice immunized with the DDA/TDB formulation induced the strongest gamma interferon (IFN-γ) and interleukin-17 (IL-17) responses, characterized by the highest frequency of IFN-γ/tumor necrosis factor alpha (TNF-α) and IFN-γ/IL-17 double-positive CD4+ T cells. In conclusion, a Chlamydia vaccine based on the recombinant proteins PmpG-1, PmpE/F-2, and MOMP delivered in a DDA/TDB adjuvant conferred protection against infection that correlated with IFN-γ/TNF-α and IFN-γ/IL-17 double-positive CD4+ T cells

Antibody therapy can enhance AngiotensinII-induced myocardial fibrosis

BACKGROUND: Myocardial fibrosis is a pathological process that is characterized by disrupted regulation of extracellular matrix proteins resulting in permanent scarring of the heart tissue and eventual diastolic heart failure. Pro-fibrotic molecules including transforming growth factor-β and connective tissue growth factor are expressed early in the AngiotensinII (AngII)-induced and other models of myocardial fibrosis. As such, antibody-based therapies against these and other targets are currently under development. RESULTS: In the present study, C57Bl/6 mice were subcutaneously implanted with a mini-osmotic pump containing either AngII (2.0 μg/kg/min) or saline control for 3 days in combination with mIgG (1 mg/kg/d) injected through the tail vein. Fibrosis was assessed after picosirius red staining of myocardial cross-sections and was significantly increased after AngII exposure compared to saline control (11.37 ± 1.41%, 4.94 ± 1.15%; P <0.05). Non-specific mIgG treatment (1 mg/kg/d) significantly increased the amount of fibrosis (26.34 ± 3.03%; P <0.01). However, when AngII exposed animals were treated with a Fab fragment of the mIgG or mIgM, this exacerbation of fibrosis was no longer observed (14.49 ± 2.23%; not significantly different from AngII alone). CONCLUSIONS: These data suggest that myocardial fibrosis was increased by the addition of exogenous non-specific antibodies in an Fc-mediated manner. These findings could have substantial impact on the future experimental design of antibody-based therapeutics

Treatment with activated protein C (aPC) is protective during the development of myocardial fibrosis: an angiotensin II infusion model in mice.

Myocardial fibrosis contributes to the development of heart failure. Activated Protein C (aPC) is a circulating anticoagulant with anti-inflammatory and cytoprotective properties. Using a model of myocardial fibrosis second to Angiotensin II (AngII) infusion, we investigated the novel therapeutic function aPC in the development of fibrosis.C57Bl/6 and Tie2-EPCR mice were infused with AngII (2.0 µg/kg/min), AngII and aPC (0.4 µg/kg/min) or saline for 3d. Hearts were harvested and processed for analysis or used for cellular isolation. Basic histology and collagen deposition were assessed using histologic stains. Transcript levels of molecular mediators were analyzed by quantitative RT-PCR. Mice infused with AngII exhibited multifocal areas of myocardial cellular infiltration associated with significant collagen deposition compared to saline control animals (p<0.01). AngII-aPC infusion inhibited this cellular infiltration and the corresponding collagen deposition. AngII-aPC infusion also inhibited significant expression of the pro-fibrotic cytokines TGF-β1, CTGF and PDGF found in AngII only infused animals (p<0.05). aPC signals through its receptor, EPCR. Using Tie2-EPCR animals, where endothelial cells over-express EPCR and exhibit enhanced aPC-EPCR signaling, no significant reduction in cellular infiltration or fibrosis was evident with AngII infusion suggesting aPC-mediate protection is endothelial cell independent. Isolated infiltrating cells expressed significant EPCR transcripts suggesting a direct effect on infiltrating cells.This data indicates that aPC treatment abrogates the fibrogenic response to AngII. aPC does not appear to confer protection by stimulating the endothelium but by acting directly on the infiltrating cells, potentially inhibiting migration or activation

EPCR Expression on Infiltrating Cells.

<p>Infiltrating cells were isolated from heart exposed to AngII for 3d and placed in culture for 3d (A). The phenotype of cultured cells were determined by staining for CD45 (red), Collagen (green) and nuclei (blue). The overlay appears yellow. Conventional RT-PCR was used on samples isolated from these cultured cells to analyze the expression of EPCR in fibrocytes rich cultures (FC, n = 2 - cultured cells from 2 different animals) isolated from AngII infused hearts (B). Significant EPCR expression was detected in isolated cells as well as faint expression in a sample of whole heart (HRT). 18s was used as a housekeeping gene to ensure equal loading between samples.</p

Putative Mechanism of Action of aPC.

<p>Transcript levels of the pro-survival factor Bcl-2 and the pro-apoptotic factor Bax were measured by qRT-PCR and a ratio of the two genes were generated for each animal assessed from each experimental group (A). No significant difference in the Bcl-2/Bax ratio was measured. To assess the effect of anticoagulation in this model, animals were infused with both AngII and heparin to determine the effects of anticoagulation within our fibrotic model (B). Cellular infiltration and collagen deposition, as measured by H&E (Bi) and Sirius red (Bii) staining respectively, were still evident within the myocardium of animals infused with AngII and heparin. Quantification of both cellular infiltration (Biii) and collagen content (Biv) demonstrated a significant increase beyond the levels of saline controls. Images were taken at 40× magnification. **<i>p</i><0.01.</p

Hemodynamic Measurements.

<p>AngII infusion after 3d was associated with a significant increase in the mean arterial pressure (mmHG) of mice as measured by a non-invasive tail cuff system. Co-infusion with aPC appears to modestly inhibit AngII induced hypertension. *<i>p</i><0.05 **<i>p</i><0.01.</p

Cellular Infiltration.

<p>Representative images of hearts sections stained with H&E from animal infused with saline (A), AngII (B) and AngII and aPC (C). AngII infusion was associated with cellular infiltration within the interstitial and perivascular tissues. Evident cellular infiltration can be observed as non-myocyte cells that are mononuclear with little cytoplasm and are typically found within focal clusters (example in panel B on the left side of the micrograph). Little cellular infiltration was evident when aPC was also infused. Infiltrating cell counts were completed to quantify this observation within an average of 5 field of views/heart (D). Significant infiltration was measured in AngII infused animals compared to saline controls but no significant increase in cell infiltration could be detected between aPC or AngII-aPC and saline infused animals. Images were taken at 40× magnification. *<i>p</i><0.05.</p