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Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the US, partly due to the increasing incidence of metabolic syndrome, obesity, and type 2 diabetes. The roles of bile acids and their receptors, such as the nuclear receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, on the development of NASH are not fully clear. C57BL/6J male mice fed a Western diet (WD) develop characteristics of NASH, allowing determination of the effects of FXR and TGR5 agonists on this disease. Here we show that the FXR-TGR5 dual agonist INT-767 prevents progression of WD-induced hepatic steatosis, inflammation, and fibrosis, as determined by histological and biochemical assays and novel label-free microscopy imaging techniques, including third harmonic generation, second harmonic generation, and fluorescence lifetime imaging microscopy. Furthermore, we show INT-767 decreases liver fatty acid synthesis and fatty acid and cholesterol uptake, as well as liver inflammation. INT-767 markedly changed bile acid composition in the liver and intestine, leading to notable decreases in the hydrophobicity index of bile acids, known to limit cholesterol and lipid absorption. In addition, INT-767 upregulated expression of liver p-AMPK, SIRT1, PGC-1α, and SIRT3, which are master regulators of mitochondrial function. Finally, we found INT-767 treatment reduced WD-induced dysbiosis of gut microbiota. Interestingly, the effects of INT-767 in attenuating NASH were absent in FXR-null mice, but still present in TGR5-null mice. Our findings support treatment and prevention protocols with the dual FXR-TGR5 agonist INT-767 arrest progression of WD-induced NASH in mice mediated by FXR-dependent, TGR5-independent mechanisms.

BackgroundImage-based machine learning tools hold great promise for clinical applications in pathology research. However, the ideal end-users of these computational tools (e.g., pathologists and biological scientists) often lack the programming experience required for the setup and use of these tools which often rely on the use of command line interfaces.MethodsWe have developed Histo-Cloud, a tool for segmentation of whole slide images (WSIs) that has an easy-to-use graphical user interface. This tool runs a state-of-the-art convolutional neural network (CNN) for segmentation of WSIs in the cloud and allows the extraction of features from segmented regions for further analysis.ResultsBy segmenting glomeruli, interstitial fibrosis and tubular atrophy, and vascular structures from renal and non-renal WSIs, we demonstrate the scalability, best practices for transfer learning, and effects of dataset variability. Finally, we demonstrate an application for animal model research, analyzing glomerular features in three murine models.ConclusionsHisto-Cloud is open source, accessible over the internet, and adaptable for segmentation of any histological structure regardless of stain.

Diabetic kidney disease continues to be the leading cause of chronic kidney disease, often advancing to end stage kidney disease. In addition to the well characterized glomerular alterations including mesangial expansion, podocyte injury, and glomerulosclerosis, tubulointerstitial fibrosis is also an important component of diabetic kidney injury. Similarly, tubulointerstitial fibrosis is a critical component of any chronic kidney injury. Therefore, sensitive and quantitative identification of tubulointerstitial fibrosis is critical for the assessment of long-term prognosis of kidney disease. Here, we employed phasor approach to fluorescence lifetime imaging, commonly known as FLIM, to understand tissue heterogeneity and calculate changes in the tissue autofluorescence lifetime signatures due to diabetic kidney disease. FLIM imaging was performed on cryostat sections of snap-frozen biopsy material of patients with diabetic nephropathy. There was an overall increase in phase lifetime (τphase) with increased disease severity. Multicomponent phasor analysis shows the distinctive differences between the different disease states. Thus, phasor autofluorescence lifetime imaging, which does not involve any staining, can be used to understand and evaluate the severity of kidney disease.

Nonalcoholic fatty liver disease is a rapidly rising problem in the 21st century and is a leading cause of chronic liver disease that can lead to end-stage liver diseases, including cirrhosis and hepatocellular cancer. Despite this rising epidemic, no pharmacological treatment has yet been established to treat this disease. The rapidly increasing prevalence of nonalcoholic fatty liver disease and its aggressive form, nonalcoholic steatohepatitis (NASH), requires novel therapeutic approaches to prevent disease progression. Alterations in microbiome dynamics and dysbiosis play an important role in liver disease and may represent targetable pathways to treat liver disorders. Improving microbiome properties or restoring normal bile acid metabolism may prevent or slow the progression of liver diseases such as NASH. Importantly, aberrant systemic circulation of bile acids can greatly disrupt metabolic homeostasis. Bile acid sequestrants are orally administered polymers that bind bile acids in the intestine, forming nonabsorbable complexes. Bile acid sequestrants interrupt intestinal reabsorption of bile acids, decreasing their circulating levels. We determined that treatment with the bile acid sequestrant sevelamer reversed the liver injury and prevented the progression of NASH, including steatosis, inflammation, and fibrosis in a Western diet-induced NASH mouse model. Metabolomics and microbiome analysis revealed that this beneficial effect is associated with changes in the microbiota population and bile acid composition, including reversing microbiota complexity in cecum by increasing Lactobacillus and decreased Desulfovibrio The net effect of these changes was improvement in liver function and markers of liver injury and the positive effects of reversal of insulin resistance.

Chronic kidney disease (CKD) is a common disease of aging and increases fracture risk over advanced age alone. Aging and CKD differently impair bone turnover and mineralization. We thus hypothesize that the loss of bone quality would be greatest with the combination of advanced age and CKD. We evaluated bone from young adult (6 mo.), middle-age (18 mo.), and old (24 mo.) male C57Bl/6 mice three months following either 5/6th nephrectomy, to induce CKD, or Sham procedures. CKD exacerbated losses of cortical and trabecular microarchitecture associated with aging. Aging and CKD each resulted in thinner, more porous cortices and fewer and thinner trabeculae. Bone material quality was also reduced with CKD, and these changes to bone material were distinct from those due to age. Aging reduced whole-bone flexural strength and modulus, micrometer-scale nanoindentation modulus, and nanometer-scale tissue and collagen strain (small-angle x-ray scattering [SAXS]. By contrast, CKD reduced work to fracture and variation in bone tissue modulus and composition (Raman spectroscopy), and increased percent collagen strain. The increased collagen strain burden was associated with loss of toughness in CKD. In addition, osteocyte lacunae became smaller, sparser, and more disordered with age for Sham mice, yet these age-related changes were not clearly observed in CKD. However, for CKD, larger lacunae positively correlated with increased serum phosphate levels, suggesting that osteocytes play a role in systemic mineral homeostasis. This work demonstrates that CKD reduces bone quality, including microarchitecture and bone material properties, and that loss of bone quality with age is compounded by CKD. These findings may help reconcile why bone mass does not consistently predict fracture in the CKD population, as well as why older individuals with CKD are at high risk of fragility.

The cloning of the renal NaPi-2a (SLC34A1) and NaPi-2c (SLC34A3) phosphate transporters has made it possible to characterize the molecular and biophysical regulation of renal proximal tubular reabsorption of inorganic phosphate (Pi). Dietary factors, such as Pi and K, and several hormones and phosphatonins, including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and glucocorticoids, regulate the transporters through various transcriptional, translational, and post-translational mechanisms that involve acute trafficking via endocytosis or exocytosis, interactions with PDZ domain proteins, lipid microdomains, and diffusion and clustering in the apical brush border membrane. The visualization of these trafficking events by means of novel microscopy techniques that includes fluorescence lifetime imaging microscopy (FLIM), Förster resonance energy transfer (FRET), fluctuation correlation spectroscopy (FCS), and modulation tracking (MT), is the primary focus of this review.

HIV disease remains prevalent in the United States and is particularly prevalent in sub-Saharan Africa. Recent investigations revealed that mitochondrial dysfunction in kidney contributes to HIV-associated nephropathy (HIVAN) in Tg26 transgenic mice. We hypothesized that nicotinamide adenine dinucleotide (NAD) deficiency contributes to energetic dysfunction and progressive tubular injury. We investigated metabolomic mechanisms of HIVAN tubulopathy. Tg26 and wild-type (WT) mice were treated with the farnesoid X receptor (FXR) agonist INT-747 or nicotinamide riboside (NR) from 6 to 12 wk of age. Multiomic approaches were used to characterize kidney tissue transcriptomes and metabolomes. Treatment with INT-747 or NR ameliorated kidney tubular injury, as shown by serum creatinine, the tubular injury marker urinary neutrophil-associated lipocalin, and tubular morphometry. Integrated analysis of metabolomic and transcriptomic measurements showed that NAD levels and production were globally downregulated in Tg26 mouse kidneys, especially nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway. Furthermore, NAD-dependent deacetylase sirtuin3 activity and mitochondrial oxidative phosphorylation activity were lower in ex vivo proximal tubules from Tg26 mouse kidneys compared with those of WT mice. Restoration of NAD levels in the kidney improved these abnormalities. These data suggest that NAD deficiency might be a treatable target for HIVAN. NEW & NOTEWORTHY: The study describes a novel investigation that identified nicotinamide adenine dinucleotide (NAD) deficiency in a widely used HIV-associated nephropathy (HIVAN) transgenic mouse model. We show that INT-747, a farnesoid X receptor agonist, and nicotinamide riboside (NR), a precursor of nicotinamide, each ameliorated HIVAN tubulopathy. Multiomic analysis of mouse kidneys revealed that NAD deficiency was an upstream metabolomic mechanism contributing to HIVAN tubulopathy. [ABSTRACT FROM AUTHOR]

Increasingly, evidence suggests that exposure to maternal obesity creates an inflammatory environment in utero, exerting long-lasting postnatal signatures on the juvenile innate immune system and microbiome that may predispose offspring to development of fatty liver disease. We found that exposure to a maternal Western-style diet (WD) accelerated fibrogenesis in the liver of offspring and was associated with early recruitment of proinflammatory macrophages at 8-12 weeks and microbial dysbiosis as early as 3 weeks of age. We further demonstrated that bone marrow-derived macrophages (BMDMs) were polarized toward an inflammatory state at 8 weeks of age and that a potent antioxidant, pyrroloquinoline quinone (PQQ), reversed BMDM metabolic reprogramming from glycolytic toward oxidative metabolism by restoring trichloroacetic acid cycle function at isocitrate dehydrogenase. This resulted in reduced inflammation and inhibited collagen fibril formation in the liver at 20 weeks of age, even when PQQ was withdrawn at 3 weeks of age. Beginning at 3 weeks of age, WD-fed mice developed a decreased abundance of Parabacteroides and Lactobacillus, together with increased Ruminococcus and decreased tight junction gene expression by 20 weeks, whereas microbiota of mice exposed to PQQ retained compositional stability with age, which was associated with improved liver health. Conclusion: Exposure to a maternal WD induces early gut dysbiosis and disrupts intestinal tight junctions, resulting in BMDM polarization and induction of proinflammatory and profibrotic programs in the offspring that persist into adulthood. Disrupted macrophage and microbiota function can be attenuated by short-term maternal treatment with PQQ prior to weaning, suggesting that reshaping the early gut microbiota in combination with reprogramming macrophages during early weaning may alleviate the sustained proinflammatory environment, preventing the rapid progression of nonalcoholic fatty liver disease to nonalcoholic steatohepatitis in offspring of obese mothers. (Hepatology Communications 2018;2:313-328).

The phasor approach to auto-fluorescence lifetime imaging was used to identify and characterize a long lifetime species (LLS) (~7.8 ns) in livers of mice fed with a Western diet. The size of the areas containing this LLS species depends on the type of diet and the size distribution shows Western diet has much larger LLS sizes. Combination of third harmonic generation images with FLIM identified the LLS species with fat droplets and the droplet size distribution was estimated. Second harmonic generation microscopy combined with phasor FLIM shows that there is an increase in fibrosis with a Western diet. A new decomposition in three components of the phasor plot shows that a Western diet is correlated with a higher fraction of free NADH, signifying more reducing condition and more glycolytic condition. Multiparametric analysis of phasor distribution shows that from the distribution of phasor points, a Western diet fed versus a low fat diet fed samples of mice livers can be separated. The phasor approach for the analysis of FLIM images of autofluorescence in liver specimens can result in discovery of new fluorescent species and then these new fluorescent species can help assess tissue architecture. Finally integrating FLIM and second and third harmonic analysis provides a measure of the advancement of fibrosis as an effect of diet.

[This corrects the article on p. 3143 in vol. 8.].

All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust assessment procedure independent of observer interpretation and let us create criteria to quantify the extent of fibrosis directly from the image. Thus, the FLIM-SHG technique shows remarkable improvement in quantification of renal fibrosis compared to standard histological techniques.

Phasor approach to fluorescence lifetime microscopy is used to study development of fibrosis in the unilateral ureteral obstruction model (UUO) of kidney in mice. Traditional phasor analysis has been modified to create a multiparametric analysis scheme that splits the phasor points in four equidistance segments based on the height of peak of the phasor distribution and calculates six parameters including average phasor positions, the shape of each segment, the angle of the distribution and the number of points in each segment. These parameters are used to create a spectrum of twenty four points specific to the phasor distribution of each sample. Comparisons of spectra from diseased and healthy tissues result in quantitative separation and calculation of statistical parameters including AUC values, positive prediction values and sensitivity. This is a new method in the evolving field of analyzing phasor distribution of FLIM data and provides further insights. Additionally, the progression of fibrosis with time is detected using this multiparametric approach to phasor analysis.

[This corrects the article DOI: 10.1371/journal.pone.0152877.].

Spaceflight affects numerous organ systems in the body, leading to metabolic dysfunction that may have long-term consequences. Microgravity-induced alterations in liver metabolism, particularly with respect to lipids, remain largely unexplored. Here we utilize a novel systems biology approach, combining metabolomics and transcriptomics with advanced Raman microscopy, to investigate altered hepatic lipid metabolism in mice following short duration spaceflight. Mice flown aboard Space Transportation System -135, the last Shuttle mission, lose weight but redistribute lipids, particularly to the liver. Intriguingly, spaceflight mice lose retinol from lipid droplets. Both mRNA and metabolite changes suggest the retinol loss is linked to activation of PPARα-mediated pathways and potentially to hepatic stellate cell activation, both of which may be coincident with increased bile acids and early signs of liver injury. Although the 13-day flight duration is too short for frank fibrosis to develop, the retinol loss plus changes in markers of extracellular matrix remodeling raise the concern that longer duration exposure to the space environment may result in progressive liver damage, increasing the risk for nonalcoholic fatty liver disease.

In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur. This is a label free approach towards fast automatable detection of fibrosis in tissue samples. FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy. Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated.

P(i) uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary P(i) but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2(BBE) cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1(-/-) mice, but not PDZK1(-/-) mice, had a diminished adaptation of NaPi-2b expression in response to a low P(i) diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.

Cholesterol crystals (ChCs) have been identified as a major factor of plaque vulnerability and as a potential biomarker for atherosclerosis. Yet, due to the technical challenge of selectively detecting cholesterol in its native tissue environment, the physiochemical role of ChCs in atherosclerotic progression remains largely unknown. In this work, we demonstrate the utility of hyperspectral stimulated Raman scattering (SRS) microscopy combined with second-harmonic generation (SHG) microscopy to selectively detect ChC. We show that despite the polarization sensitivity of the ChC Raman spectrum, cholesterol monohydrate crystals can be reliably discriminated from aliphatic lipids, from structural proteins of the tissue matrix and from other condensed structures, including cholesteryl esters. We also show that ChCs exhibit a nonvanishing SHG signal, corroborating the noncentrosymmetry of the crystal lattice composed of chiral cholesterol molecules. However, combined hyperspectral SRS and SHG imaging reveals that not all SHG-active structures with solidlike morphologies can be assigned to ChCs. This study exemplifies the merit of combining SRS and SHG microscopy for an enhanced label-free chemical analysis of crystallized structures in diseased tissue.

The accumulation of lipids, including cholesterol, in the arterial wall plays a key role in the pathogenesis of atherosclerosis. Although several advances have been made in the detection and imaging of these lipid structures in plaque lesions, their morphology and composition have yet to be fully elucidated, particularly in different animal models of disease. To address this issue, we analyzed lipid morphology and composition in the atherosclerotic plaques of two animal models of disease, the low density lipoprotein receptor-deficient (LDLR(-/-)) mouse and the ApoE lipoprotein-deficient (ApoE(-/-)) mouse, utilizing hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy in combination with principal component analysis (PCA). Hyperspectral CARS imaging revealed lipid-rich macrophage cells and condensed needle-shaped and plate-shaped lipid crystal structures in both mice. Spectral analysis with PCA and comparison to spectra of pure cholesterol and cholesteryl ester derivatives further revealed these lipid structures to be pure cholesterol crystals, which were predominantly observed in the ApoE(-/-) mouse model. These results illustrate the ability of hyperspectral CARS imaging in combination with multivariate analysis to characterize atherosclerotic lipid morphology and composition with chemical specificity, and consequently, provide new insight into the formation of cholesterol crystal structures in atherosclerotic plaque lesions.

The kidney is a key regulator of phosphate homeostasis. There are two predominant renal sodium phosphate cotransporters, NaPi2a and NaPi2c. Both are regulated by parathyroid hormone (PTH), which decreases the abundance of the NaPi cotransporters in the apical membrane of renal proximal tubule cells. The time course of PTH-induced removal of the two cotransporters from the apical membrane, however, is markedly different for NaPi2a compared with NaPi2c. In animals and in cell culture, PTH treatment results in almost complete removal of NaPi2a from the brush border (BB) within 1 h whereas for NaPi2c this process in not complete until 4 to 8 h after PTH treatment. The reason for this is poorly understood. We have previously shown that the unconventional myosin motor myosin VI is required for PTH-induced removal of NaPi2a from the proximal tubule BB. Here we demonstrate that myosin VI is also necessary for PTH-induced removal of NaPi2c from the apical membrane. In addition, we show that, while at baseline the two cotransporters have similar diffusion coefficients within the membrane, after PTH addition the diffusion coefficient for NaPi2a initially exceeds that for NaPi2c. Thus NaPi2c appears to remain "tethered" in the apical membrane for longer periods of time after PTH treatment, accounting, at least in part, for the difference in response times to PTH of NaPi2a versus NaPi2c.

We developed an optical imaging method based on a feedback principle in which the specific scan pattern is adapted according to the shape of the sample. The feedback approach produces nanometer-resolved 3D images of very small and moving features in live cells in seconds. We show images of microvilli in live cultured opossum kidney cells expressing NaPi co-transporter proteins with different GFP constructs and images of cell protrusions in a collagen matrix with a resolution of about 20 nm. We found that in the microvilli the NaPi proteins can be found clustered. Along cell protrusions in 3D we identified cellular adhesions to the extracellular matrix. Our approach to super-resolution and to 3D nanoimaging is different than other proposed methods that break the diffraction limit using non-linear effects or are based on single molecule localization.

The sodium-dependent phosphate (Na/P(i)) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of P(i). The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in P(i) reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/P(i) transporters. Pdzk1(-/-) mice adapted to chronic low P(i) diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/P(i) transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/P(i) transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low P(i) concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/P(i) transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c.

Although in vivo and cell culture studies have provided useful information about the regulation of the sodium phosphate (NaPi) cotransporters, such studies are unable to provide information at the molecular level about interactions between proteins. The NaPi proteins are found within both intestinal and renal brush border microvilli, and previous work has shown that these microvilli contain scaffolding proteins (PDZ proteins) and myosin motors. The recent development of several advanced imaging techniques has allowed detailed analysis of how NaPi proteins interact with scaffolding proteins and myosin motors. Using techniques such as apical total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy, raster image correlation spectroscopy, and fluorescence lifetime imaging-Förster resonance energy transfer, we have found that a myosin motor is involved in trafficking of the NaPi cotransporters and also that Npt2a and Npt2c seem to have different affinities for the PDZ protein Na+/H+ exchanger regulatory factor 1. Further application of these techniques will provide additional insights into NaPi trafficking and regulation.

We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis-the ApoE-deficient mouse-by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet-fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.