Objective

To determine the effect of the apolipoprotein A-I (ApoA-I) mimetic peptide, D-4F, on atherosclerosis development in a pre-existing diabetic condition.

Research design and methods

We induced hyperglycemia in 6-week-old apoE(-/-) female mice using streptozotocin. Half of the diabetic apoE(-/-) mice received D-4F in drinking water. Ten weeks later, plasma lipids, glucose, insulin levels, atherosclerotic lesions, and lesion macrophage content were measured.

Results

Diabetic apoE(-/-) mice developed ∼300% more lesion area, marked dyslipidemia, increased glucose levels, and reduced plasma insulin levels when compared with nondiabetic apoE(-/-) mice. Atherosclerotic lesions were significantly reduced in the D-4F-treated diabetic apoE(-/-) mice in whole aorta (1.11 ± 0.73 vs. 0.58 ± 0.44, percentage of whole aorta, P < 0.01) and in aortic roots (36,038 ± 18,467 μm²/section vs. 17,998 ± 12,491 μm²/section, P < 0.01) when compared with diabetic apoE(-/-) mice that did not receive D-4F. Macrophage content in atherosclerotic lesions from D-4F-treated diabetic apoE(-/-) mice was significantly reduced when compared with nontreated animals (78.03 ± 26.1 vs. 29.6 ± 15.2 P < 0.001, percentage of whole plaque). There were no differences in glucose, insulin, total cholesterol, HDL cholesterol, and triglyceride levels between the two groups. Arachidonic acid, PGE₂, PGD₂, 15-HETE, 12-HETE, and 13-HODE concentrations were significantly increased in the liver tissue of diabetic apoE(-/-) mice compared with nondiabetic apoE(-/-) mice and significantly reduced by D-4F treatment.

Conclusions

Our results suggest that oral D-4F can prevent atherosclerosis development in pre-existing diabetic mice and this is associated with a reduction in hepatic arachidonic acid and oxidized fatty acid levels.

Background

Follistatin (Fst) promotes brown adipocyte characteristics in adipose tissues.

Methods

Abdominal fat volume (CT scan), glucose clearance (GTT test), and metabolomics analysis (mass spectrometry) of adipose tissues from Fst transgenic (Fst-Tg) and wild type (WT) control mice were analyzed. Oxygen consumption (Seahorse Analyzer) and lipidomics (gas chromatography) was analyzed in 3T3-L1 cells.

Results

Fst-Tg mice show significant decrease in abdominal fat content, increased glucose clearance, improved plasma lipid profiles and significant changes in several conventional metabolites compared to the WT mice. Furthermore, overexpression of Fst in 3T3-L1 cells resulted in up regulation of key brown/beige markers and changes in lipidomics profiles.

Conclusion

Fst modulates key factors involved in promoting metabolic syndrome and could be used for therapeutic intervention.

Objective

We previously reported that hemopexin (Hx), a heme scavenger, is significantly increased and associated with proinflammatory high-density lipoprotein under atherogenic conditions. Although it is established that Hx together with macrophages plays a role in mitigating oxidative damage, the role of Hx in the development of atherosclerosis is unknown.

Approach and results

We used Hx and apoE double-knockout mice (HxE(-/-)) to determine the role of Hx in the development of atherosclerosis. HxE(-/-) mice had significantly more free heme, reactive oxygen species, and proinflammatory high-density lipoprotein in their circulation, when compared with control apoE(-/-) mice. Atherosclerotic plaque area (apoE(-/-)=9.72±2.5×10(4) μm(2) and HxE(-/-)=27.23±3.6×10(4) μm(2)) and macrophage infiltration (apoE(-/-)=38.8±5.8×10(3) μm(2) and HxE(-/-)=103.4±17.8×10(3) μm(2)) in the aortic sinus were significantly higher in the HxE(-/-) mice. Atherosclerotic lesions in the aortas were significantly higher in the HxE(-/-) mice compared with apoE(-/-) mice. Analysis of polarization revealed that macrophages from HxE(-/-) mice were more M1-like. Ex vivo studies demonstrated that HxE(-/-) macrophage cholesterol efflux capacity was significantly reduced when compared with apoE(-/-) mice. Injection of human Hx into HxE(-/-) mice reduced circulating heme levels and human Hx pretreatment of naive bone marrow cells ex vivo resulted in a shift from M1- to M2-like macrophages.

Conclusions

We conclude that Hx plays a novel protective role in alleviating heme-induced oxidative stress, improving inflammatory properties of high-density lipoprotein, macrophage phenotype and function, and inhibiting the development of atherosclerosis in apoE(-/-) mice.

Background

Proper functioning of high-density lipoprotein (HDL) is necessary for protection against sepsis. However, previous work has demonstrated that HDL becomes oxidized and dysfunctional (Dys-HDL) during sepsis. Older (aged >65 years) patients are at particularly high risk of sepsis and poor outcomes from sepsis.

Study objective

The aim of the study was to compare functional properties of HDL (cholesterol efflux capacity and paraoxonase enzyme 1 [PON-1] activity) and Dys-HDL between older (aged >65 years) sepsis patients and older healthy volunteers.

Methods

This was a subanalysis of a prospective study in which patients with sepsis were prospectively enrolled from the emergency department within the first 24 h. Serum and plasma samples were drawn from septic patients and age- and sex-matched control subjects. Percent cholesterol efflux, HDL inflammatory index, and PON1 activity were measured. Data were analyzed using Student t test or Wilcoxon rank-sum test.

Results

Ten sepsis and 10 healthy controls were analyzed. Mean age of sepsis patients (80 ± 2 years [SD]) and control subjects (77 ± 2 years) was similar (P = 0.31). Mean systolic blood pressures were significantly different in sepsis patients (113 ± 8 mmHg) compared with controls (133 ± 6 mmHg) (P = 0.049). Median SOFA scores for sepsis patients were 5.5 (interquartile range [IQR] 4-9). Mean percent cholesterol efflux was significantly reduced in sepsis (24.1 ± 1.2%) compared with controls (31.5 ± 1.0%) (P < 0.001). HDL inflammatory index was also significantly elevated in septic patients (1.63, IQR 1.3-2.34) compared with controls (0.62, IQR 0.56-0.67) (P < 0.001). However, PON1 activity was not significantly different between septic patients (70.3 ± 16.3 nmol/min/mL) and control subjects (88.8 ± 18.3 nmol/min/mL).

Conclusions

Cholesterol efflux capacity seems to be significantly impaired in sepsis patients who also exhibited a higher index of Dys-HDL. The findings suggest that HDL function may be impaired in older individuals with sepsis.

Mouse chow supplemented with lysophosphatidylcholine with oleic acid at sn-1 and a hydroxyl group at sn-2 (LysoPC 18:1) increased LysoPC 18:1 in tissue of the jejunum of LDL receptor (LDLR)-null mice by 8.9 ± 1.7-fold compared with chow alone. Western diet (WD) contained dramatically less phosphatidylcholine 18:1 or LysoPC 18:1 compared with chow, but feeding WD increased LysoPC 18:1 in the jejunum by 7.5 ± 1.4-fold compared with chow. Feeding LysoPC 18:1 or feeding WD increased oxidized phospholipids in the jejunum by 5.2 ± 3.0-fold or 8.6 ± 2.2-fold, respectively, in LDLR-null mice (P < 0.0004), and 2.6 ± 1.5-fold or 2.4 ± 0.92-fold, respectively, in WT C57BL/6J mice (P < 0.0001). Adding 0.06% by weight of a concentrate of transgenic tomatoes expressing the 6F peptide (Tg6F) decreased LysoPC 18:1 in the jejunum of LDLR-null mice on both diets (P < 0.0001), and prevented the increase in oxidized phospholipids in the jejunum in LDLR-null and WT mice on both diets (P < 0.008). Tg6F decreased inflammatory cells in the villi of the jejunum, decreased dyslipidemia, and decreased systemic inflammation in LDLR-null and WT mice on both diets. We conclude that Tg6F reduces diet-induced inflammation by reducing the content of unsaturated LysoPC and oxidized phospholipids in the jejunum of mice.

Ovarian cancer (OC) is most lethal malignancy among all gynecological cancer. Large bodies of evidences suggest that mitochondrial-derived ROS play a critical role in the development and progression of OC. Paraoxonase 2 (PON2) is a membrane-associated lactonase with anti-oxidant properties. PON2 deficiency aggravates mitochondrial ROS formation, systemic inflammation, and atherosclerosis. The role of PON2 in cancer development remains unknown. In this report, in human, we identified that PON2 expression is higher in early stages (but not in late stages) of OC when compared to normal tissue. Using a mouse xenograft model of OC, we demonstrate that overexpression of PON2 prevents tumor formation. Mechanistically, PON2 decreases OC cell proliferation by inhibiting insulin like growth factor-1 (IGF-1) expression and signaling. Intriguingly, PON2 reduces c-Jun-mediated transcriptional activation of IGF-1 gene by decreasing mitochondrial superoxide generation. In addition, PON2 impairs insulin like growth factor-1 receptor (IGF-1R) signaling in OC cells by altering cholesterol homeostasis, which resulted in reduced caveolin-1/IGF-1R interaction and IGF-1R phosphorylation. Taken together, we report for the first time that PON2 acts as a tumor suppressor in the early stage of OC by reducing IGF-1 production and its signaling, indicating PON2 activation might be a fruitful strategy to inhibit early stage ovarian tumor.

Objective

High density lipoprotein (HDL) is important for defense against sepsis but becomes dysfunctional (Dys-HDL) during inflammation. We hypothesize that Dys-HDL correlates with organ dysfunction (sequential organ failure assessment (SOFA) score) early sepsis.

Methods

A prospective cohort study of adult ED sepsis patients enrolled within 24 hours.

Results

Eighty eight patients were analyzed. Dys-HDL (expressed as HDL inflammatory index (HII)) correlated with SOFA at enrollment (r = 0.23, p = 0.024) and at 48 hours (r = 0.24, p = 0.026) but HII change over the first 48 hours did not correlate with change in SOFA (r = 0.06, p = 0.56). Enrollment HII was significantly different in patients with most severe organ failure (2.31, IQR 1.33-5.2) compared to less severe organ failure (1.81, IQR 1.23-2.64, p = 0.043). Change in HII over 48 hours was significantly different for in-hospital non-survivors (-0.45, IQR-2.6, -0.14 p = 0.015) and for 28-day non-survivors (-1.12, IQR -1.52, 0.12, p = 0.044). In a multivariable linear regression equation (R2 = 0.13), for each unit HII increase, 48-hour SOFA increased by 0.72 (p = 0.009).

Conclusion

HII correlated with SOFA and predicted 48-hour SOFA score in early sepsis. Future studies are needed to delineate potential mechanisms.

Trial registration

NCT02370186. Registered February 24, 2015.

After crossing floxed stearoyl-CoA desaturase-1 (Scd1 ^fl/fl) mice with LDL receptor-null (ldlr ^-/-) mice, and then Villin Cre (VilCre) mice, enterocyte Scd1 expression in Scd1 ^fl/fl/ldlr ^-/-/VilCre mice was reduced 70%. On Western diet (WD), Scd1 ^fl/fl/ldlr ^-/- mice gained more weight than Scd1 ^fl/fl/ldlr ^-/-/VilCre mice (P < 0.0023). On WD, jejunum levels of lysophosphatidylcholine (LysoPC) 18:1 and lysophosphatidic acid (LPA) 18:1 were significantly less in Scd1 ^fl/fl/ldlr ^-/-/VilCre compared with Scd1 ^fl/fl/ldlr ^-/- mice (P < 0.0004 and P < 0.026, respectively). On WD, Scd1 ^fl/fl/ldlr ^-/-/VilCre mice compared with Scd1 ^fl/fl/ldlr ^-/- mice had lower protein levels of lipopolysaccharide-binding protein (LBP), cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR4), and myeloid differentiation factor-88 (MyD88) in enterocytes and plasma, and less dyslipidemia and systemic inflammation. Adding a concentrate of tomatoes transgenic for the apoA-I mimetic peptide 6F (Tg6F) to WD resulted in reduced enterocyte protein levels of LBP, CD14, TLR4, and MyD88 in Scd1 ^fl/fl/ldlr ^-/- mice similar to that seen in Scd1 ^fl/fl/ldlr ^-/-/VilCre mice. Adding LysoPC 18:1 to WD did not reverse the effects of enterocyte Scd1 knockdown. Adding LysoPC 18:1 (but not LysoPC 18:0) to chow induced jejunum Scd1 expression and increased dyslipidemia and plasma serum amyloid A and interleukin 6 levels in Scd1 ^fl/fl/ldlr ^-/- mice, but not in Scd1 ^fl/fl/ldlr ^-/-/VilCre mice. We conclude that enterocyte Scd1 is partially responsible for LysoPC 18:1- and WD-induced dyslipidemia and inflammation in ldlr ^-/- mice.

We previously reported that i) a Western diet increased levels of unsaturated lysophosphatidic acid (LPA) in small intestine and plasma of LDL receptor null (LDLR(-/-)) mice, and ii) supplementing standard mouse chow with unsaturated (but not saturated) LPA produced dyslipidemia and inflammation. Here we report that supplementing chow with unsaturated (but not saturated) LPA resulted in aortic atherosclerosis, which was ameliorated by adding transgenic 6F tomatoes. Supplementing chow with lysophosphatidylcholine (LysoPC) 18:1 (but not LysoPC 18:0) resulted in dyslipidemia similar to that seen on adding LPA 18:1 to chow. PF8380 (a specific inhibitor of autotaxin) significantly ameliorated the LysoPC 18:1-induced dyslipidemia. Supplementing chow with LysoPC 18:1 dramatically increased the levels of unsaturated LPA species in small intestine, liver, and plasma, and the increase was significantly ameliorated by PF8380 indicating that the conversion of LysoPC 18:1 to LPA 18:1 was autotaxin dependent. Adding LysoPC 18:0 to chow increased levels of LPA 18:0 in small intestine, liver, and plasma but was not altered by PF8380 indicating that conversion of LysoPC 18:0 to LPA 18:0 was autotaxin independent. We conclude that i) intestinally derived unsaturated (but not saturated) LPA can cause atherosclerosis in LDLR(-/-) mice, and ii) autotaxin mediates the conversion of unsaturated (but not saturated) LysoPC to LPA.

The site and mechanism of action of the apoA-I mimetic peptide 4F are incompletely understood. Transintestinal cholesterol efflux (TICE) is a process involved in the clearance of excess cholesterol from the body. While TICE is responsible for at least 30% of the clearance of neutral sterols from the circulation into the intestinal lumen, few pharmacological agents have been identified that modulate this pathway. We show first that circulating 4F selectively targets the small intestine (SI) and that it is predominantly transported into the intestinal lumen. This transport of 4F into the SI lumen is transintestinal in nature, and it is modulated by TICE. We also show that circulating 4F increases reverse cholesterol transport from macrophages and cholesterol efflux from lipoproteins via the TICE pathway. We identify the cause of this modulation of TICE either as 4F being a cholesterol acceptor with respect to enterocytes, from which 4F enhances cholesterol efflux, or as 4F being an intestinal chaperone with respect to TICE. Our results assign a novel role for 4F as a modulator of the TICE pathway and suggest that the anti-inflammatory functions of 4F may be a partial consequence of the codependent intestinal transport of both 4F and cholesterol.