Cholesteryl ester hydrolase (CEH) is a critical enzyme in cholesterol ester hydrolysis, influencing cholesterol metabolism and efflux. This study demonstrates that CEH overexpression promotes free cholesterol efflux from macrophages, thereby reducing the lipid burden in existing atherosclerotic plaques. To enable targeted delivery, galactose-functionalized polyamidoamine (PAMAM) dendrimeric nanoparticles were utilized as nanocarriers for hepatic delivery of the CEH expression vector. The therapeutic potential of CEH plasmid-loaded dendrimeric nanoparticles was evaluated in Ldlr-/- mice. Results showed a significant reduction in total lesion area (21%) and aortic arch lesion area (23%) compared to baseline. Lesion component analysis revealed marked decreases in total cholesterol (36%), free cholesterol (35%), and cholesterol esters (44%). Collectively, these results support CEH overexpression as an effective strategy to enhance cholesterol efflux and mitigate lipid accumulation in atherosclerotic plaques. Moreover, galactose-functionalized PAMAM dendrimeric nanoparticles demonstrate strong potential as a targeted hepatic gene delivery system for therapeutic intervention in atherosclerosis.

Rationale: Intestinal alkaline phosphatase (IAP) is secreted by enterocytes and is present on the apical surface. It not only detoxifies bacterial endotoxin lipopolysaccharide (LPS) in the gut lumen and limits intestinal inflammation but also restricts translocation of LPS into systemic circulation. Diet-induced intestinal barrier dysfunction and subsequent development of metabolic endotoxemia seen in diabetes and heart disease is associated with reduced IAP levels. To examine the direct effects of increased IAP expression on barrier function and development of metabolic diseases, we developed intestine-specific IAP transgenic mice (IAP Tg ) overexpressing human chimeric IAP. Objective: The aim of this study was to evaluate the effects of intestine-specific IAP overexpression on Western-type diet (WD)–induced atherosclerosis in Ldlr −/ − mice. Methods and Results: IAP Tg mice crossed into Ldlr −/− background (Ldlr −/− IAP Tg ) and Ldlr −/− littermates were fed WD for 16 weeks. Intestinal barrier dysfunction was assessed by monitoring plasma LPS levels and histological examination of colon. Overexpression of IAP attenuated WD-induced disruption of the colonic mucous layer, reducing intestinal barrier dysfunction and plasma LPS levels. Significant reduction in body, liver, and adipose tissue weight was also seen in WD-fed Ldlr −/− IAP Tg mice. Plasma and hepatic lipids were also significantly reduced in WD-fed Ldlr −/− IAP Tg mice. Consistently, intestinal lipid absorption was attenuated in Ldlr −/− IAP Tg mice with reduced expression of apical lipid transporters (CD [cluster of differentiation] 36, FATP4 [fatty acid transport protein 4], and NPC1L1 [Niemann-Pick C-like protein 1]) and intracellular lipid transport proteins (FABP [fatty acid binding protein] 1/2 and SCP2 [sterol carrier protein-2]). Attenuation of WD-induced atherosclerosis in Ldlr −/− IAP Tg mice was demonstrated by significant reduction in arch and total aortic lesions as seen by enface analyses, as well as significantly reduced atherosclerotic lesions in the ascending aorta of these mice. Conclusions: IAP overexpression improves intestinal barrier function by maintaining the integrity of the mucin layer in WD-fed Ldlr −/− IAP Tg mice and attenuates intestinal lipid absorption. Thus, by limiting translocation of gut-derived LPS or reducing plasma lipids, overexpression of IAP attenuates development of WD-induced atherosclerosis.

Reduction of lipoprotein uptake by macrophages and stimulation of cholesterol efflux are two essential steps required for atherosclerotic plaque regression. We used the optimized mannose-functionalized dendrimeric nanoparticle (mDNP)-based platform for macrophage-specific delivery of therapeutics to simultaneously deliver SR-A siRNA (to reduce LDL uptake) and LXR ligand (LXR-L, to stimulate cholesterol efflux) - a novel "Two-pronged" approach to facilitate plaque regression. mDNP-mediated delivery of SR-A siRNA led to a significant reduction in SR-A expression with a corresponding decrease in uptake of oxLDL. Delivery of LXR-L increased expression of ABCA1/G1 and cholesterol efflux. Combined delivery of siRNA and LXR-L led to a significantly greater decrease in macrophage cholesterol content compared to either treatment alone Administration of this in vitro optimized formulation of mDNP complexed with SR-A-siRNA and LXR-L (Two-pronged complex) to atherosclerotic LDLR-/- mice fed western diet (TD88137) led to significant regression of atherosclerotic plaques with a corresponding decrease in aortic cholesterol content.

The intestinal barrier is complex and consists of multiple layers, and it provides a physical and functional barrier to the transport of luminal contents to systemic circulation. While the epithelial cell layer and the outer/inner mucin layer constitute the physical barrier and are often referred to as the intestinal barrier, intestinal alkaline phosphatase (IAP) produced by epithelial cells and antibacterial proteins secreted by Panneth cells represent the functional barrier. While antibacterial proteins play an important role in the host defense against gut microbes, IAP detoxifies bacterial endotoxin lipopolysaccharide (LPS) by catalyzing the dephosphorylation of the active/toxic Lipid A moiety, preventing local inflammation as well as the translocation of active LPS into systemic circulation. The causal relationship between circulating LPS levels and the development of multiple diseases underscores the importance of detailed examination of changes in the "layers" of the intestinal barrier associated with disease development and how this dysfunction can be attenuated by targeted interventions. To develop targeted therapies for improving intestinal barrier function, it is imperative to have a deeper understanding of the intestinal barrier itself, the mechanisms underlying the development of diseases due to barrier dysfunction (eg, high circulating LPS levels), the assessment of intestinal barrier function under diseased conditions, and of how individual layers of the intestinal barrier can be beneficially modulated to potentially attenuate the development of associated diseases. This review summarizes the current knowledge of the composition of the intestinal barrier and its assessment and modulation for the development of potential therapies for barrier dysfunction-associated diseases.

Dietary, environmental or genetic changes contribute to the development of several metabolic and neurological diseases. Extensive research efforts have been directed towards examining how these factors modulate gut bacterial composition. However, the mechanisms by which changes in gut bacterial diversity affect host function are not completely defined. Intestinal barrier dysfunction is being increasingly recognized as an early or initiating step in communicating bacterial diversity-dependent changes to the host. Here, we review the functional composition of the intestinal barrier and describe the consequences of the breach of this barrier. The functional layers of the intestinal barrier provide an initial defense and prevent the infiltration of bacteria or bacterial products from the gut lumen to the underlying lamina propria. Mesenteric lymph nodes subsequently act as the first firewall where dendritic cells from the lamina propria transport the infiltrated bacteria or bacterial products during the early stages of barrier dysfunction. When overwhelmed, the liver functions as the second firewall to detoxify bacterial endotoxins, such as lipopolysaccharide (LPS), and limit systemic involvement. Continuous translocation of LPS from the leaky gut to the liver results in liver damage or dysfunction that leads to the development of type 2 diabetes, atherosclerosis, heart disease, heart failure and also neurological diseases, such as Alzheimer’s and Parkinson’s disease. Thus, targeted restoration of intestinal barrier function represents a novel strategy for the attenuation of multiple diseases.

Background: A Western-type diet (WD), rich in fat and cholesterol but deficient in fiber, induces development of diabetes and atherosclerosis. Colonic bacteria use the gut's mucous lining as an alternate energy source during periods of fiber deficiency, resulting in intestinal barrier erosion. Objective: We hypothesized that supplementing a WD with galactooligosaccharide (GOS) fiber would attenuate WD-induced mucin layer disruption and attenuate development of metabolic diseases. Methods: C57BL/6mice (both sexes, 8-10 wk of age) were fed a standard rodent diet (TD7012, reference) or a high-fat, high-cholesterol-containing WD (TD88137, 21% fat, 0.15% cholesterol, 19.5% caesin) or a WD supplemented with 5% GOS fiber (TD170432, WD + GOS) for 16 wk. WD-fed mice that were gavaged daily with curcumin (100 mg/kg) served as positive controls. Glucose tolerance, colonic mucin layer, gene expression, and circulating macrophage/neutrophil levels were determined. Hyperlipidemic Ldlr(-/-) mice (both sexes, 8-10 wk of age) fed a WD with or without GOS supplementation (for 16 wk) were used to assess plasma LPS and atherosclerosis. Effects of dietary supplementation on different parameters were compared for each genotype. Results: Compared with a WD, glucose tolerance was significantly improved in male C57BL/6 mice fed a WD + GOS (mean +/- SEM: AUC = 53.6 +/- 43.9 compared with 45.4 +/- 33.3 g center dot min/dL; P = 0.015). Continuity of colonic mucin layer (MUC-2 expression) was improved in mice receiving GOS supplementation, indicating improved intestinal barrier. GOS supplementation also reduced circulating macrophages (30% decrease) and neutrophils (60% decrease), suggesting diminished systemic inflammation. In Ldlr(-/-) mice, GOS supplementation significantly reduced plasma LPS concentrations (mean +/- SEM: 0.81 +/- 0.43 EU/mL compared with 0.32 +/- 0.26 EU/mL, P < 0.0001, in females and 0.56 +/- 0.24 EU/mL compared with 0.34 +/- 0.12 EU/mL, P = 0.036, in males), improved glucose tolerance in male mice, and attenuated atherosclerotic lesion area (mean +/- SEM: 54.2% +/- 6.19% compared with 43.0% +/- 35.12%, P = 0.0006, in females and 54.6% +/- 3.99% compared with 43.1% +/- 8.11%, P = 0.003, in males). Conclusions: GOS fiber supplementation improves intestinal barrier in C57BL/6 and Ldlr(-/-) mice and significantly attenuates WD-induced metabolic diseases and, therefore, may represent a novel strategy for management of these diseases.

In an inflammatory setting, macrophages can be polarized to an inflammatory M1 phenotype or to an anti-inflammatory M2 phenotype, as well as existing on a spectrum between these two extremes. Dysfunction of this phenotypic switch can result in a population imbalance that leads to chronic wounds or disease due to unresolved inflammation. Therapeutic interventions that target macrophages have therefore been proposed and implemented in diseases that feature chronic inflammation such as diabetes mellitus and atherosclerosis. We have developed a model for the sequential influx of immune cells in the peritoneal cavity in response to a bacterial stimulus that includes macrophage polarization, with the simplifying assumption that macrophages can be classified as M1 or M2. With this model, we were able to reproduce the expected timing of sequential influx of immune cells and mediators in a general inflammatory setting. We then fit this model to in vivo experimental data obtained from a mouse peritonitis model of inflammation, which is widely used to evaluate endogenous processes in response to an inflammatory stimulus. Model robustness is explored with local structural and practical identifiability of the proposed model a posteriori. Additionally, we perform sensitivity analysis that identifies the population of apoptotic neutrophils as a key driver of the inflammatory process. Finally, we simulate a selection of proposed therapies including points of intervention in the case of delayed neutrophil apoptosis, which our model predicts will result in a sustained inflammatory response. Our model can therefore provide hypothesis testing for therapeutic interventions that target macrophage phenotype and predict outcomes to be validated by subsequent experimentation.

A causal relationship exists between macrophage cholesterol levels and inflammation, for example, Interleukin-1 beta (IL-1 beta) secretion. A decrease in intracellular K+ is essential for inflammasome activation/IL-1 beta secretion and, herein, we examined the hypothesis that cellular cholesterol affects K+-channel activity and K+-efflux using mouse peritoneal macrophages (MPMs) and human/THP1 macrophages. An increase in cellular cholesterol led to a significant increase in K+ currents (> 350% in both MPM and THP1). Enhancing cholesterol efflux returned K+ currents back to basal levels with corresponding increase in intracellular K+ (11.2-14.5%) and reduced IL-1 beta secretion (32-62%). These data demonstrate a novel mechanism by which cellular cholesterol modulates inflammation/inflammasome via regulation of K+-channel activity and intracellular K+ levels. Attenuation of IL-1 beta secretion by Nateglinide/Repaglinide further suggests involvement of Kir6 channels.

IAP represents the first layer of the intestinal barrier and detoxifies bacterial endotoxin LPS in the gut lumen. Reduced levels of IAP are associated with diabetes and heart disease. We developed intestine specific IAP transgenic mice (IAPTg) and demonstrated attenuation of WD-induced intestinal barrier dysfunction and glucose intolerance. In this study, we crossed the IAPTg mice into LDLR-/- background to examine the effects on atherosclerosis. Panel A shows significant reduction in plasma total cholesterol (TC) and triglyceride (TG) in LDLR-/-IAPTg mice. There was a small but statistically non-significant improvement in glucose tolerance (Panel B). Enface analyses (Panel C) showed significant reduction in arch and total aortic lesions (Panel D). Expression of IAP was observed along the entire length of the GI tract including colon (Panel E). Expression of Muc-2, major protein of the mucin or the second layer of the intestinal barrier was enhanced in colon of IAPTg mice. Increased expression of GLP-1 was also noted in the ileum of IAPTg mice. These data suggest that in addition to limiting LPS-mediated intestinal inflammation, IAP also improves intestinal barrier function by increasing Muc-2 expression and IAP-modulated increase in ileal GLP-1 expression may contribute to the improved glucose tolerance. Disclosure S.S. Ghosh: None. J. Wang: None. P.J. Yannie: None. Y.K. Sandhu: None. S. Ghosh: None. Funding American Diabetes Association (1-16-IBS-105 to S.G.)

Intestinal epithelial cell derived alkaline phosphatase (IAP) dephosphorylates/detoxifies bacterial endotoxin lipopolysaccharide (LPS) in the gut lumen. We have earlier demonstrated that consumption of high-fat high-cholesterol containing western type-diet (WD) significantly reduces IAP activity, increases intestinal permeability leading to increased plasma levels of LPS and glucose intolerance. Furthermore, oral supplementation with curcumin that increased IAP activity improved intestinal barrier function as well as glucose tolerance. To directly test the hypothesis that targeted increase in IAP would protect against WD-induced metabolic consequences, we developed intestine-specific IAP transgenic mice where expression of human chimeric IAP is under the control of intestine-specific villin promoter. This chimeric human IAP contains domains from human IAP and human placental alkaline phosphatase, has a higher turnover number, narrower substrate specificity, and selectivity for bacterial LPS. Chimeric IAP was specifically and uniformly overexpressed in these IAP transgenic (IAPTg) mice along the entire length of the intestine. While IAP activity reduced from proximal P1 segment to distal P9 segment in wild-type (WT) mice, this activity was maintained in the IAPTg mice. Dietary challenge with WD impaired glucose tolerance in WT mice and this intolerance was attenuated in IAPTg mice. Significant decrease in fecal zonulin, a marker for intestinal barrier dysfunction, in WD fed IAPTg mice and a corresponding decrease in translocation of orally administered nonabsorbable 4kDa FITC dextran to plasma suggests that IAP overexpression improves intestinal barrier function. Thus, targeted increase in IAP activity represents a novel strategy to improve WD-induced intestinal barrier dysfunction and glucose intolerance.

Imbalance between lipoprotein uptake and cholesterol efflux from macrophages underlies the formation of foam cells and development of fatty streaks, a process that starts early in life. While currently available lipid lowering interventions attenuate the progression of atherosclerotic plaques, no targeted therapy is presently available for reduction of existing plaques, a process dependent not only on limiting further accumulation of cholesterol but also on stimulation of cholesterol efflux. We earlier reported the development of a mannose functionalized dendrimeric nanoparticles (mDNP)-based platform for targeted delivery of therapeutics to arterial plaque associated macrophages. In this study, we used the optimized mDNP platform to simultaneously deliver SR-A siRNA (to knock down SR-A and limit modified LDL uptake) and LXR ligand (LXR-L, to stimulate macrophage cholesterol efflux by inducing expression of ABCA1/G1) - a “ Two Pronged ” approach. Compared to non-specific NS-siRNA, mDNP mediated delivery of SR-A siRNA led to significant reduction in expression of SR-A (A&B) with a corresponding decrease in uptake of DiI-labeled oxLDL (C). Delivery of LXR-L increased expression of ABCA1/G1 (D) as well as cholesterol efflux (E) and simultaneous delivery of SR-A siRNA did not alter these LXR-L effects. However, combined delivery of siRNA and LXR-L led to significantly higher decrease in macrophage cholesterol content compared to either treatment alone (F). Administration of this in vitro optimized formulation of mDNP complexed with SR-A siRNA and LXR-L to atherosclerotic LDLR-/- mice fed western diet (TD88137) led to a dramatic reduction in lesion area compared to single treatment alone (G). In conclusion, targeted delivery of multiple therapeutics using mDNP platform represents a novel strategy to not only reduce lesion size but it can also be used to simultaneously alter many lesion characteristics such as reducing inflammation or enhancing efferocytosis.

Intracellular cholesterol transport proteins move cholesterol to different subcellular compartments and thereby regulate its final metabolic fate. In hepatocytes, for example, delivery of high-density lipoprotein (HDL)-associated cholesterol for bile acid synthesis or secretion into bile facilitates cholesterol elimination from the body (anti-atherogenic effect), whereas delivery for esterification and subsequent incorporation into apolipoprotein B-containing atherogenic lipoproteins (e.g. very-low-density lipoprotein (VLDL)) enhances cholesterol secretion into the systemic circulation (pro-atherogenic effect). Intracellular cholesterol transport proteins such as sterol carrier protein-2 (SCP2) should, therefore, play a role in regulating these pro- or anti-atherosclerotic processes. Here, we sought to evaluate the effects of SCP2 deficiency on the development of diet-induced atherosclerosis. We generated LDLR-/- mice deficient in SCP2/SCPx (LS) and examined the effects of this deficiency on Western diet-induced atherosclerosis. SCP2/SCPx deficiency attenuated atherosclerosis in LS mice by >80% and significantly reduced plasma cholesterol and triglyceride levels. Investigation of the likely underlying mechanisms revealed a significant reduction in intestinal cholesterol absorption (given as an oral gavage) in SCP2/SCPx-deficient mice. Consistently, siRNA-mediated knockdown of SCP2 in intestinal cells significantly reduced cholesterol uptake. Furthermore, hepatic triglyceride/VLDL secretion from the liver or hepatocytes isolated from SCP2/SCPx-deficient mice was significantly reduced. These results indicate an important regulatory role for SCP2 deficiency in attenuating diet-induced atherosclerosis by limiting intestinal cholesterol absorption and decreasing hepatic triglyceride/VLDL secretion. These findings suggest targeted inhibition of SCP2 as a potential therapeutic strategy to reduce Western diet-induced dyslipidemia and atherosclerosis.

Accumulation of cholesterol loaded macrophage foam cells is the hallmark of atherosclerotic lesions where high cellular cholesterol content not only contributes to the plaque volume but is also responsible for the inflammatory milieu. Causal relationship exist between macrophage cholesterol content and inflammatory status involving TLR4, NFκB or cholesterol crystal mediated activation of inflammasome and IL1β secretion. Since K+ efflux is central to inflammasome activation, in this study we examined the hypothesis that cellular cholesterol content directly affects K+ channel activity as well as K+ efflux. K+ currents were monitored in mouse peritoneal macrophages (MPMs) from chow or western diet (WD) fed LDLR-/- or LDLR-/-CEHTg mice. WD feeding led to a significant increase in the K+ currents in MPMs from LDLR-/- mice (A, red). In contrast, WD feeding did not affect K+ currents in MPMs from LDLR-/-CEHTg mice; these MPMs have reduced cellular cholesterol accumulation due to increased efflux. Consistently, cholesterol loading with AcLDL led to an increase in K+ currents in THP1 macrophages (B, red) and this increase was attenuated following cholesterol efflux. The physiological effect of changes in K+ channel activity or K+ efflux on inflammasome activation in MPMs by different activators (K+ ionophore Nigericin, N or ALUM crystals) was examined. Increase in extracellular K+ (+KCl) or inhibition of K+ efflux by glyburide significantly reduced IL1β secretion (C&D). Consistently, cellular K+ levels after LPS+N or LPS+ALUM treatment were significantly reduced (E). While in MPMs only K ATP channel inhibitors reduced IL1β secretion (F), in human THP1 macrophages inhibition of K ATP as well as Na/K pump inhibitors significantly reduced IL1β secretion (G). These data demonstrate a novel mechanism for cholesterol mediated regulation of inflammatory pathways by regulation of K+ channel activity and also illustrate species specific differences in K+ channels involved

WD induces the development of type 2 diabetes (T2DM). In addition to the direct effects on gut dysbiosis, we have shown that WD also affect intestinal barrier function leading to increased release of LPS into circulation resulting in chronic inflammation that underlies the development of T2DM; oral supplementation with curcumin attenuate these WD-induced effects. Herein we examined the effects of GOS supplementation on WD induced changes. C57BL/6 mice were either fed a standard chow or WD or WD+GOS or WD+Curcumin (WD+C) for 16 weeks. Glucose tolerance tests were performed and while no significant differences were seen in female mice (Panels A and C), WD-induced glucose intolerance was significantly attenuated by GOS or curcumin supplementation in male mice (Panels B and D). Although plasma LPS levels were not significantly affected, a significant decrease in circulating macrophages (Panel E) as well as neutrophils (Panel F) was noted with GOS or Curcumin supplementation indicating a reduction in systemic inflammation. Gene expression and histological changes in the ileum and colon indicate GOS or curcumin-mediated increase in mucin production and maintenance of mucous layer integrity. Dietary supplementation with GOS or Curcumin, therefore, represents a simple strategy to ameliorate the metabolic/diabetogenic effects of WD. Disclosure H. He: None. S.S. Ghosh: None. P.J. Yannie: None. J. Wang: None. S. Ghosh: None.

Atherosclerotic cardiovascular diseases remain the number one cause of morbidity and mortality despite significant advances in lipid management. Complex cellular interactions occur within the artery wall requiring timely infiltration/egress of immune cells and lipoproteins within a changing inflammatory milieu that determine the progression of an atherosclerotic plaque. Lack of detailed understanding of the multiple processes involved and their potential interactions has hindered development of targeted therapies. The objective of this study was to develop a computational model of the sequential influx of immune cells in response to a trigger to permit a system-level analyses of the processes involved using ordinary differential equations (ODEs). Thioglycollate induced peritonitis was used as a model to examine the infiltration of immune cells and phenotypic polarization of macrophages in response to a stimulus. Peritoneal exudates obtained at 10 different time-points over 7 days were analyzed by FACS to determine the distribution of neutrophils, macrophages and Ly6C Hi (M1) or Ly6C Lo (M2) polarization. Weighted least squares of the different parameters were used to calibrate the model developed in our laboratory (Panel A) to simulate inflammation/repair during sepsis using the Levenberg-Marquardt algorithm. Panels B-D show the “match” of the experimental data to the calibrated model demonstrating the validity of this model. Since the ODEs are derived from a combination of known and hypothesized kinetics of the biological system, model the changes in physiological variables over time and are based on biological interactions, our calibrated mathematical model will permit the evaluation of changes in one or more targeted parameters in silico on immune cell distribution/phenotype. Efficacy of various macrophage-specific interventions (e.g., reduction in cholesterol content or inflammation) can be predicted prior to preclinical experimentation.