Omega-3 fatty acid diglyceride emulsions as a novel injectable acute therapeutic in neonatal hypoxic-ischemic brain injury.

Hypoxic-ischemic encephalopathy (HIE), resulting from a lack of blood flow and oxygen before or during newborn delivery, is a leading cause of cerebral palsy and neurological disability in children. Therapeutic hypothermia (TH), the current standard of care in HIE, is only beneficial in 1 of 7-8 cases. Therefore, there is a critical need for more efficient treatments. We have previously reported that omega-3 (n-3) fatty acids (FA) carried by triglyceride (TG) lipid emulsions provide neuroprotection after experimental hypoxic-ischemic (HI) injury in neonatal mice. Herein, we propose a novel acute therapeutic approach using an n-3 diglyceride (DG) lipid emulsions. Importantly, n-3 DG preparations had much smaller particle size compared to commercially available or lab-made n-3 TG emulsions. We showed that n-3 DG molecules have the advantage of incorporating at substantially higher levels than n-3 TG into an in vitro model of phospholipid membranes. We also observed that n-3 DG after parenteral administration in neonatal mice reaches the bloodstream more rapidly than n-3 TG. Using neonatal HI brain injury models in mice and rats, we found that n-3 DG emulsions provide superior neuroprotection than n-3 TG emulsions or TH in decreasing brain infarct size. Additionally, we found that n-3 DGs attenuate microgliosis and astrogliosis. Thus, n-3 DG emulsions are a superior, promising, and novel therapy for treating HIE.

Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.

Lipid and smooth muscle architectural pathology in the rabbit atherosclerotic vessel wall using Q-space cardiovascular magnetic resonance.

BACKGROUND: Atherosclerosis is an arterial vessel wall disease characterized by slow, progressive lipid accumulation, smooth muscle disorganization, and inflammatory infiltration. Atherosclerosis often remains subclinical until extensive inflammatory injury promotes vulnerability of the atherosclerotic plaque to rupture with luminal thrombosis, which can cause the acute event of myocardial infarction or stroke. Current bioimaging techniques are unable to capture the pathognomonic distribution of cellular elements of the plaque and thus cannot accurately define its structural disorganization. METHODS: We applied cardiovascular magnetic resonance spectroscopy (CMRS) and diffusion weighted CMR (DWI) with generalized Q-space imaging (GQI) analysis to architecturally define features of atheroma and correlated these to the microscopic distribution of vascular smooth muscle cells (SMC), immune cells, extracellular matrix (ECM) fibers, thrombus, and cholesteryl esters (CE). We compared rabbits with normal chow diet and cholesterol-fed rabbits with endothelial balloon injury, which accelerates atherosclerosis and produces advanced rupture-prone plaques, in a well-validated rabbit model of human atherosclerosis. RESULTS: Our methods revealed new structural properties of advanced atherosclerosis incorporating SMC and lipid distributions. GQI with tractography portrayed the locations of these components across the atherosclerotic vessel wall and differentiated multi-level organization of normal, pro-inflammatory cellular phenotypes, or thrombus. Moreover, the locations of CE were differentiated from cellular constituents by their higher restrictive diffusion properties, which permitted chemical confirmation of CE by high field voxel-guided CMRS. CONCLUSIONS: GQI with tractography is a new method for atherosclerosis imaging that defines a pathological architectural signature for the atheromatous plaque composed of distributed SMC, ECM, inflammatory cells, and thrombus and lipid. This provides a detailed transmural map of normal and inflamed vessel walls in the setting of atherosclerosis that has not been previously achieved using traditional CMR techniques. Although this is an ex-vivo study, detection of micro and mesoscale level vascular destabilization as enabled by GQI with tractography could increase the accuracy of diagnosis and assessment of treatment outcomes in individuals with atherosclerosis.

Survival intravascular photoacoustic imaging of lipid-rich plaque in cholesterol fed rabbits.

Intravascular photoacoustic (IVPA) imaging is a promising modality for quantitative assessment of lipid-laden atherosclerotic plaques. Yet, survival IVPA imaging of the same plaque in the same animal is not demonstrated. Here, using a sheathed IVUS/PA catheter of 0.9 mm in diameter, we demonstrate MRI-guided survival IVPA imaging of same plaque in an aorta of a well-established rabbit model mimicking atherosclerosis in human patients. The IVUS/PA results were confirmed by histology. These advances open the opportunity to evaluate the effectiveness of a therapy that aims to reduce the size of atherosclerotic plaques and demonstrates the potential of translating the IVPA catheter into clinic for detection of lipid-rich plaques that are at high risk for thrombosis.

Aging-induced microbleeds of the mouse thalamus compared to sensorimotor and memory defects.

The aim of this study is to investigate the relationship between aging and brain vasculature health. Three groups of mice, 3, 17-18, and 24 months, comparable to young adult, middle age, and old human were studied. Prussian blue histology and fast imaging with steady precession T2∗-weighted magnetic resonance imaging were used to quantify structural changes in the brain across age groups. The novel object recognition test was used to assess behavioral changes associated with anatomical changes. This study is the first to show that the thalamus is the most vulnerable brain region in the mouse model for aging-induced vascular damage. Magnetic resonance imaging data document the timeline of accumulation of thalamic damage. Histological data reveal that the majority of vascular damage accumulates in the ventroposterior nucleus and mediodorsal thalamic nucleus. Functional studies indicate that aging-induced vascular damage in the thalamus is associated with memory and sensorimotor deficits. This study points to the possibility that aging-associated vascular disease is a factor in irreversible brain damage as early as middle age.

The brains of aged mice are characterized by altered tissue diffusion properties and cerebral microbleeds.

BACKGROUND: Brain aging is a major risk factor in the progression of cognitive diseases including Alzheimer's disease (AD) and vascular dementia. We investigated a mouse model of brain aging up to 24 months old (mo). METHODS: A high field (11.7T) MRI protocol was developed to characterize specific features of brain aging including the presence of cerebral microbleeds (CMBs), morphology of grey and white matter, and tissue diffusion properties. Mice were selected from age categories of either young (3 mo), middle-aged (18 mo), or old (24 mo) and fed normal chow over the duration of the study. Mice were imaged in vivo with multimodal MRI, including conventional T2-weighted (T2W) and T2*-weighted (T2*W) imaging, followed by ex vivo diffusion-weighted imaging (DWI) and T2*W MR-microscopy to enhance the detection of microstructural features. RESULTS: Structural changes observed in the mouse brain with aging included reduced cortical grey matter volume and enlargement of the brain ventricles. A remarkable age-related change in the brains was the development of CMBs found starting at 18 mo and increasing in total volume at 24 mo, primarily in the thalamus. CMBs presence was confirmed with high resolution ex vivo MRI and histology. DWI detected further brain tissue changes in the aged mice including reduced fractional anisotropy, increased radial diffusion, increased mean diffusion, and changes in the white matter fibers visualized by color-coded tractography, including around a large cortical CMB. CONCLUSIONS: The mouse is a valuable model of age-related vascular contributions to cognitive impairment and dementia (VCID). In composite, these methods and results reveal brain aging in older mice as a multifactorial process including CMBs and tissue diffusion alterations that can be well characterized by high field MRI.

SSO and other putative inhibitors of FA transport across membranes by CD36 disrupt intracellular metabolism, but do not affect FA translocation.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Novel ANO5 mutation c.1067G>T (p.C356F) identified by whole genome sequencing in a big family with atypical gnathodiaphyseal dysplasia.

BACKGROUND: Gnathodiaphyseal dysplasia (GDD) is a rare skeletal disorder that has not been well studied. METHODS: Sanger sequencing, whole-genome sequencing (WGS), and bioinformatics and structural modeling analyses were performed. RESULTS: A family with patients with fibro-osseous lesions of the jawbones were initially diagnosed with cherubism. Sequencing of SH3BP2, which is the causal gene of cherubism, revealed no pathogenic mutation. Through WGS, we identified a novel mutation c.1067G>T (p.C356F) in ANO5, and bioinformatics analyses and structural modeling showed that the mutation was deleterious. Because ANO5 is the gene responsible for GDD, we reappraised the clinical data of the patients, and the diagnosis was corrected to atypical GDD. A review of the literature showed that 67% of GDD cases confirmed by molecular testing were initially misdiagnosed. CONCLUSIONS: The novel mutation c.1067G>T (p.C356F) in ANO5 is responsible for the atypical GDD observed in our patients. GDD should be included in the differential diagnosis for patients with fibro-osseous lesions.

Immunization with cell-free-generated vaccine protects from Porphyromonas gingivalis-induced alveolar bone loss.

INTRODUCTION: Periodontal diseases (PD) are complex oral inflammatory diseases initiated by keystone bacteria such as Porphyromonas gingivalis. A vaccine for PD is desirable as clinical treatment involves protracted maintenance strategies aimed to retain dentition. Although prior immunization approaches targeting P. gingivalis have reported variable success in limiting facets of disease such as oral bone loss, it remains that a vaccine for this disease may be attainable. AIM: To investigate cell-free protein synthesis (CFPS) as a platform to produce vaccinable targets suitable for efficacy testing in a P. gingivalis-induced murine oral bone loss model. MATERIALS AND METHODS: Recombinantly generated P. gingivalis minor fimbriae protein (Mfa1), RgpA gingipain hemagglutinin domain 1 (HA1), and RgpA gingipain hemagglutinin domain 2 (HA2) were combined in equivalent doses in adjuvants and injected intramuscularly to immunize mice. Serum levels of protein-specific antibody were measured by ELISA, and oral bone levels were defined by morphometrics. RESULTS: Recombinantly generated P. gingivalis proteins possessed high fidelity to predicted size and elicited protein-specific IgG following immunization. Importantly, immunization with the vaccine cocktail protected from P. gingivalis elicited oral bone loss. CONCLUSION: These data verify the utility of the CFPS technology to synthesize proteins that have the capacity to serve as novel vaccines.

MRI of atherosclerosis and fatty liver disease in cholesterol fed rabbits.

BACKGROUND: The globally rising obesity epidemic is associated with a broad spectrum of diseases including atherosclerosis and non-alcoholic fatty liver (NAFL) disease. In the past, research focused on the vasculature or liver, but chronic systemic effects and inter-organ communication may promote the development of NAFL. Here, we investigated the impact of confined vascular endothelial injury, which produces highly inflamed aortic plaques that are susceptible to rupture, on the progression of NAFL in cholesterol fed rabbits. METHODS: Aortic atherosclerotic inflammation (plaque Gd-enhancement), plaque size (vessel wall area), and composition, were measured with in vivo magnetic resonance imaging (MRI) in rabbits fed normal chow or a 1% cholesterol-enriched atherogenic diet. Liver fat was quantified with magnetic resonance spectroscopy (MRS) over 3 months. Blood biomarkers were monitored in the animals, with follow-up by histology. RESULTS: Cholesterol-fed rabbits with and without injury developed hypercholesterolemia, NAFL, and atherosclerotic plaques in the aorta. Compared with rabbits fed cholesterol diet alone, rabbits with injury and cholesterol diets exhibited larger, and more highly inflamed plaques by MRI (P < 0.05) and aggravated liver steatosis by MRS (P < 0.05). Moreover, after sacrifice, damaged (ballooning) hepatocytes and extensive liver fibrosis were observed by histology. Elevated plasma gamma-glutamyl transferase (GGT; P = 0.014) and the ratio of liver enzymes aspartate and alanine aminotransferases (AST/ALT; P = 0.033) indicated the progression of steatosis to non-alcoholic steatohepatitis (NASH). CONCLUSIONS: Localized regions of highly inflamed aortic atherosclerotic plaques in cholesterol-fed rabbits may contribute to progression of fatty liver disease to NASH with fibrosis.

Immuno-pathogenesis of Periodontal Disease: Current and Emerging Paradigms.

Periodontal disease (PD) is a highly complex disease involving many factors; however, two principal facets central to initiation and progression of the majority of PD are the composition of the microbes in the sub-gingival plaque, and the host immune response to these organisms. Numerous studies point to the complexity of PD, and to the fact that despite innate and adaptive immune activation, and resultant inflammation, our immune response fails to cure disease. Stunning new findings have begun to clarify several complexities of the host-pathogen interaction of PD pointing to key roles for microbial dysboisis and immune imbalance in the pathogenesis of disease. Furthermore, these investigations have identified novel translational opportunities to intercede in PD treatment. In this review we will highlight a select few recent findings in innate and adaptive immunity, and host pathogen interactions of PD at a micro-environmental level that may have profound impact on PD progression.

Inflammatory response to Porphyromonas gingivalis partially requires interferon regulatory factor (IRF) 3.

Innate immune activation with expression of pro-inflammatory molecules such as TNF-α is a hallmark of the chronic inflammation associated with periodontal disease (PD). Porphyromonas gingivalis, a bacterium associated with PD, engages TLRs and activates MyD88-dependent and TIR-domain-containing adapter-inducing IFN-ß (TRIF)-dependent signaling pathways. IFN regulatory factor (IRF) 3 is activated in a TRIF-dependent manner and participates in production of cytokines such as TNF-α; however, little is known regarding IRF3 and the host response to PD pathogens. We speculated that IRF3 participates in the host inflammatory response to P. gingivalis. Our results show that bone marrow macrophages (MØ) from WT mice respond to P. gingivalis with activation and nuclear translocation of IRF3. Compared with WT, MØ from IRF3(-/-), TRIF(-/-), and TLR4(-/-) mice responded with reduced levels of TNF-α on P. gingivalis challenge. In addition, full expression of IL-6 and RANTES by MØ to P. gingivalis was dependent on IRF3. Lastly, employing MØ from IRF3(-/-) and IRF7(-/-) mice we observed a significant role for IRF3 and a modest role for IRF7 in the P. gingivalis-elicited TNF-α response. These studies identify a role for IRF3 in the inflammatory response by MØ to the periodontal pathogen P. gingivalis.

CD36 enhances fatty acid uptake by increasing the rate of intracellular esterification but not transport across the plasma membrane.

CD36 is a multifunctional protein that enhances cellular fatty acid (FA) uptake, a key step in energy metabolism, and its dysregulation in multiple tissue sites is central to obesity-linked diabetes, a risk factor for atherosclerosis. Although CD36 has been implicated in FA uptake in a correlative way, the molecular mechanisms are not known. Their elucidation in cells is confounded by receptor-mediated uptake of low-density lipoprotein by CD36 and the competitive and/or contributive effects of other proteins involved in FA transport and metabolism, which include caveolin(s), fatty acid transport protein (FATP), intracellular fatty acid binding protein, and enzymes involved in the conversion of FAs to esters. Here we utilized a simpler cellular system (HEK cells), which lack caveolin-1, CD36, and FATP and metabolize FAs slowly compared to the time frame of transmembrane FA movement. Our previous studies of HEK cells showed that caveolin-1 affects FA binding and translocation across the plasma membrane and but not FA esterification [Simard, J. R., et al. (2010) J. Lipid Res. 51 (5), 914-922]. Our key new finding is that CD36 accelerates FA uptake and extensive incorporation into triglycerides, a process that is slower (minutes) than transmembrane movement (seconds). Real-time fluorescence measurements showed that the rates of binding and transport of oleic acid into cells with and without CD36 were not different. Thus, CD36 enhances intracellular metabolism, i.e., esterification, and thereby increases the rate of FA uptake without catalyzing the translocation of FA across the plasma membrane, suggesting that CD36 is central to FA uptake via its effects on intracellular metabolism.

Fatty acids are rapidly delivered to and extracted from membranes by methyl-beta-cyclodextrin.

We performed detailed biophysical studies of transfer of long-chain fatty acids (FAs) from methyl-beta-CD (MBCD) to model membranes (egg-PC vesicles) and cells and the extraction of FA from membranes by MBCD. We used i) fluorescein phosphatidylethanolamine to detect transfer of FA anions arriving in the outer membrane leaflet; ii) entrapped pH dyes to measure pH changes after FA diffusion (flip-flop) across the lipid bilayer; and iii) soluble fluorescent-labeled FA binding protein to measure the concentration of unbound FA in water. FA dissociated from MBCD, bound to the membrane, and underwent flip-flop within milliseconds. In the presence of vesicles, MBCD maintained the aqueous concentration of unbound FA at low levels comparable to those measured with albumin. In studies with cells, addition of oleic acid (OA) complexed with MBCD yielded rapid (seconds) dose-dependent OA transport into 3T3-L1 preadipocytes and HepG2 cells. MBCD extracted OA from cells and model membranes rapidly at concentrations exceeding those required for OA delivery but much lower than concentrations commonly used for extracting cholesterol. Compared with albumin, MBCD can transfer its entire FA load and is less likely to extract cell nutrients and to introduce impurities.

Fatty acid transport and metabolism in HepG2 cells.

The mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that the addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (seconds) and dose-dependent decrease in intracellular pH (pH(in)), indicating diffusion of fatty acids across the plasma membrane. pH(in) returned to its initial value with a time course (in min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with the inhibitors phloretin or triacsin C had no effect on the rapid pH(in) drop after the addition of oleate but greatly suppressed pH(in) recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pH(in) recovery and metabolism. We then used a dual-fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pH(in) decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pH(in) drop or its slow recovery but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.