Quantitative Evaluation Of Myocardial Fibrosis Using Polarization-video Microscopy

Fibrosis is an essential feature of tissue repair and is characterized by the deposition of collagen. Fibrosis in the myocardium is prevalent among patients with heart disease and can significantly impair cardiac function. The evaluation and treatment of myocardial scarring is however limited by a lack of quantitative and practical methods for studying fibrosis. My thesis was that both the amount and the relative maturity of fibrotic collagen could be quantified directly from histologic sections by taking advantage of the birefringent properties of collagen and assessing these with computer-based image analysis. A system, using polarized light microscopy and video microscopy, was therefore developed.;To quantify fibrosis, the high brightness intensity of collagen stained with picrosirius red was exploited. Using color filtering and image processing, a digital video image of myocardium was generated in which only collagen was visible. The collagen was quantified as the area-fraction of visible pixels. This video approach was compared to that of hydroxyproline analysis using samples from 14 autopsy hearts. A strong correlation (r = 0.98) between the two techniques was demonstrated. Also, in 22 endomyocardial biopsy specimens, the collagen content determined by the video technique was shown to correlate well with the collagen content determined by stereology (r = 0.95).;To quantify the relative maturity of fibrosis, the increasing birefringence of maturing fibrotic collagen was utilized. The median grey-level of all pixels depicting collagen served as the index of collagen brightness and reflected fiber birefringence. I measured the brightness of collagen in the scar that formed after superficial injury to the rat gracilis muscle. In this model, the diffuse arrangement of the collagen fibers was similar to that in interstitial myocardial fibrosis. Collagen brightness increased progressively over a 63-day period. I also measured brightness in the scar that formed after myocardial infarction in dogs and found that collagen brightness in 6-week infarcts was greater than that in 3-week infarcts (p {dollar}\u3c{dollar} 0.01).;The clinical utility of the system was confirmed by two studies in which fibrosis in the human transplanted heart was studied. In the first study, I examined the relationship between the length of time the heart was not perfused during transport from donor to recipient (graft ischemic time), and the amount of fibrosis present one week after transplantation. Collagen content was measured in endomyocardial biopsy samples from 36 transplant recipients and found to be linearly related to graft ischemic time (r = 0.60). In the second study, I examined the relationship between allograft rejection and the amount and activity of fibrosis in five transplant patients. Serial biopsy samples, taken over a period of up to 1.5 years after transplantation, were evaluated. The collagen content of samples taken one year after surgery did not correlate with the frequency or severity of rejection. However, the activity of fibrosis was greatest in the early weeks ({dollar}\u3c{dollar}12) after transplantation which was time when most of the rejection episodes had occurred.;In conclusion, both the content and the activity of myocardial fibrosis can be quantified using the polarization-video microscopy technique

Dual Oxidase Maturation factor 1 (DUOXA1) overexpression increases reactive oxygen species production and inhibits murine muscle satellite cell differentiation

Background: Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate. Results: To determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H2O2 production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype. Conclusions: This study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1. © 2014 Sandiford et al.; licensee BioMed Central Ltd

Dual Oxidase Maturation factor 1 (DUOXA1) overexpression increases reactive oxygen species production and inhibits murine muscle satellite cell differentiation

Background: Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate. Results: To determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H2O2 production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype. Conclusions: This study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1. © 2014 Sandiford et al.; licensee BioMed Central Ltd

Collectivization of Vascular Smooth Muscle Cells via TGF-β-Cadherin-11-Dependent Adhesive Switching.

OBJECTIVE: Smooth muscle cells (SMCs) in healthy arteries are arranged as a collective. However, in diseased arteries, SMCs commonly exist as individual cells, unconnected to each other. The purpose of this study was to elucidate the events that enable individualized SMCs to enter into a stable and interacting cell collective. APPROACH AND RESULTS: Human SMCs stimulated to undergo programmed collectivization were tracked by time-lapse microscopy. We uncovered a switch in the behavior of contacting SMCs from semiautonomous motility to cell-cell adherence. Central to the cell-adherent phenotype was the formation of uniquely elongated adherens junctions, ≤60 μm in length, which appeared to strap adjacent SMCs to each other. Remarkably, these junctions contained both N-cadherin and cadherin-11. Ground-state depletion super-resolution microscopy revealed that these hybrid assemblies were comprised of 2 parallel nanotracks of each cadherin, separated by 50 nm. Blocking either N-cadherin or cadherin-11 inhibited collectivization. Cell-cell adhesion and adherens junction elongation were associated with reduced transforming growth factor-β signaling, and exogenous transforming growth factor-β1 suppressed junction elongation via the noncanonical p38 pathway. Imaging of fura-2-loaded SMCs revealed that SMC assemblies displayed coordinated calcium oscillations and cell-cell transmission of calcium waves which, together with increased connexin 43-containing junctions, depended on cadherin-11 and N-cadherin function. CONCLUSIONS: SMCs can self-organize, structurally and functionally, via transforming growth factor-β-p38-dependent adhesive switching and a novel adherens junction architecture comprised of hybrid nanotracks of cadherin-11 and N-cadherin. The findings define a mechanism for the assembly of SMCs into networks, a process that may be relevant to the stability and function of blood vessels

3D vessel-wall virtual histology of whole-body perfused mice using a novel heavy element stain

© 2019, The Author(s). Virtual histology – utilizing high-resolution three-dimensional imaging – is becoming readily available. Micro-computed tomography (micro-CT) is widely available and is often coupled with x-ray attenuating histological stains that mark specific tissue components for 3D virtual histology. In this study we describe a new tri-element x-ray attenuating stain and perfusion protocol that provides micro-CT contrast of the entire vasculature of an intact mouse. The stain – derived from an established histology stain (Verhoeff’s) – is modified to enable perfusion through the vasculature; the attenuating elements of the stain are iodine, aluminum, and iron. After a 30-minute perfusion through the vasculature (10-minute flushing with detergent-containing saline followed by 15-minute perfusion with the stain and a final 5-minute saline flush), animals are scanned using micro-CT. We demonstrate that the new staining protocol enables sharp delineation of the vessel walls in three dimensions over the whole body; corresponding histological analysis verified that the CT stain is localized primarily in the endothelial cells and media of large arteries and the endothelium of smaller vessels, such as the coronaries. The rapid perfusion and scanning protocol ensured that all tissues are available for further analysis via higher resolution CT of smaller sections or traditional histological sectioning

A Method for 3D Histopathology Reconstruction Supporting Mouse Microvasculature Analysis.

Structural abnormalities of the microvasculature can impair perfusion and function. Conventional histology provides good spatial resolution with which to evaluate the microvascular structure but affords no 3-dimensional information; this limitation could lead to misinterpretations of the complex microvessel network in health and disease. The objective of this study was to develop and evaluate an accurate, fully automated 3D histology reconstruction method to visualize the arterioles and venules within the mouse hind-limb. Sections of the tibialis anterior muscle from C57BL/J6 mice (both normal and subjected to femoral artery excision) were reconstructed using pairwise rigid and affine registrations of 5 µm-thick, paraffin-embedded serial sections digitized at 0.25 µm/pixel. Low-resolution intensity-based rigid registration was used to initialize the nucleus landmark-based registration, and conventional high-resolution intensity-based registration method. The affine nucleus landmark-based registration was developed in this work and was compared to the conventional affine high-resolution intensity-based registration method. Target registration errors were measured between adjacent tissue sections (pairwise error), as well as with respect to a 3D reference reconstruction (accumulated error, to capture propagation of error through the stack of sections). Accumulated error measures were lower (

Type i collagen cleavage is essential for effective fibrotic repair after myocardial infarction

Efficient deposition of type I collagen is fundamental to healing after myocardial infarction. Whether there is also a role for cleavage of type I collagen in infarct healing is unknown. To test this, we undertook coronary artery occlusion in mice with a targeted mutation (Col1a1 r/r) that yields collagenase-resistant type I collagen. Eleven days after infarction, Col1a1 r/r mice had a lower mean arterial pressure and peak left ventricular systolic pressure, reduced ventricular systolic function, and worse diastolic function, compared with wild-type littermates. Infarcted Col1a1 r/r mice also had greater 30-day mortality, larger left ventricular lumens, and thinner infarct walls. Interestingly, the collagen fibril content within infarcts of mutant mice was not increased. However, circular polarization microscopy revealed impaired collagen fibril organization and mechanical testing indicated a predisposition to scar microdisruption. Three-dimensional lattices of collagenase-resistant fibrils underwent cell-mediated contraction, but the fibrils did not organize into birefringent collagen bundles. In addition, time-lapse microscopy revealed that, although cells migrated smoothly on wild-type collagen fibrils, crawling and repositioning on collagenase-resistant collagen was impaired. We conclude that type I collagen cleavage is required for efficient healing of myocardial infarcts and is critical for both dynamic positioning of collagen-producing cells and hierarchical assembly of collagen fibrils. This seemingly paradoxical requirement for collagen cleavage in fibrotic repair should be considered when designing potential strategies to inhibit matrix degradation in cardiac disease. © 2011 American Society for Investigative Pathology

Peroxisome proliferator-activated receptor δ agonist GW1516 attenuates diet-induced aortic inflammation, insulin resistance, and atherosclerosis in low-density lipoprotein receptor knockout mice

OBJECTIVE - The peroxisome proliferator-activated receptor (PPAR) δ regulates systemic lipid homeostasis and inflammation. However, the ability of PPARδ agonists to improve the pathology of pre-established lesions and whether PPARδ activation is atheroprotective in the setting of insulin resistance have not been reported. Here, we examine whether intervention with a selective PPARδ agonist corrects metabolic dysregulation and attenuates aortic inflammation and atherosclerosis. APPROACH AND RESULTS - Low-density lipoprotein receptor knockout mice were fed a chow or a high-fat, high-cholesterol (HFHC) diet (42% fat, 0.2% cholesterol) for 4 weeks. For a further 8 weeks, the HFHC group was fed either HFHC or HFHC plus GW1516 (3 mg/kg per day). GW1516 significantly attenuated pre-established fasting hyperlipidemia, hyperglycemia, and hyperinsulinemia, as well as glucose and insulin intolerance. GW1516 intervention markedly reduced aortic sinus lesions and lesion macrophages, whereas smooth muscle α-actin was unchanged and collagen deposition enhanced. In aortae, GW1516 increased the expression of the PPARδ-specific gene Adfp but not PPARα- or γ-specific genes. GW1516 intervention decreased the expression of aortic proinflammatory M1 cytokines, increased the expression of the anti-inflammatory M2 cytokine Arg1, and attenuated the iNos/Arg1 ratio. Enhanced mitogen-activated protein kinase signaling, known to induce inflammatory cytokine expression in vitro, was enhanced in aortae of HFHC-fed mice. Furthermore, the HFHC diet impaired aortic insulin signaling through Akt and forkhead box O1, which was associated with elevated endoplasmic reticulum stress markers CCAAT-enhancer-binding protein homologous protein and 78kDa glucose regulated protein. GW1516 intervention normalized mitogen-activated protein kinase activation, insulin signaling, and endoplasmic reticulum stress. CONCLUSIONS - Intervention with a PPARδ agonist inhibits aortic inflammation and attenuates the progression of pre-established atherosclerosis. © 2013 American Heart Association, Inc