Epac1 (Exchange Protein Directly Activated by cAMP 1) Upregulates LOX-1 (Oxidized Low-Density Lipoprotein Receptor 1) to Promote Foam Cell Formation and Atherosclerosis Development.

Objective: The cAMP second messenger system, a major stress-response pathway, plays essential roles in normal cardiovascular functions and in pathogenesis of heart diseases. Here, we test the hypothesis that the Epac1 (exchange protein directly activated by cAMP 1) acts as a major downstream effector of cAMP signaling to promote atherogenesis and represents a novel therapeutic target. Approach and Results: To ascertain Epac1's function in atherosclerosis development, a triple knockout mouse model () was generated by crossing mice with atherosclerosis-prone mice lacking both and .

Phosphatidylinositol-(4,5)-Bisphosphate Regulates Plasma Cholesterol Through LDL (Low-Density Lipoprotein) Receptor Lysosomal Degradation.

TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against or a nontargeting control for 3 to 4 weeks.

A Critical Role of PCSK9 in Mediating IL-17-Producing T Cell Responses in Hyperlipidemia.

We previously demonstrated that atherogenic (LDb) double knockout mice lacking both low-density lipoprotein receptor (LDLR) and apolipoprotein B mRNA-editing catalytic polypeptide-1 (Apobec1) had increased serum IL-17 levels, with T cell programming shifted towards Th17 cells. In this study, we assessed the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in T cell programming and atherogenesis. We deleted the gene from LDb mice to generate (LTp) triple knockout mice.

Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels.

PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases plasma cholesterol levels by promoting LDL receptor degradation. Current antibody inhibitors block the interaction between PCSK9 and LDL receptors, significantly decrease plasma cholesterol levels, and provide beneficial clinical outcomes. To reduce the action of PCSK9 in plasma, a novel strategy that will produce a panel of non-native, conformationally-altered isomers of PCSK9 (X-PCSK9) to develop active immunotherapy targeting of native PCSK9 and inhibiting/blocking the interaction of PCSK9 with LDL receptor, thus decreasing plasma cholesterol levels is proposed.

PCSK9 deficiency reduces atherosclerosis, apolipoprotein B secretion, and endothelial dysfunction.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interacts directly with cytoplasmic apoB and prevents its degradation via the autophagosome/lysosome pathway. This process affects VLDL and LDL production and influences atherogenesis. Here, we investigated the molecular machinery by which PCSK9 modulates autophagy and affects atherogenesis.

Detection and Characterization of the Effect of AB-FUBINACA and Its Metabolites in a Rat Model.

Synthetic cannabinoids were originally developed by academic and pharmaceutical laboratories with the hope of providing therapeutic relief from the pain of inflammatory and degenerative diseases. However, recreational drug enthusiasts have flushed the market with new strains of these potent drugs that evade detection yet endanger public health and safety. Although many of these drug derivatives were published in the medical literature, others were merely patented without further characterization.

Foamy monocytes form early and contribute to nascent atherosclerosis in mice with hypercholesterolemia.

Objective: To examine infiltration of blood foamy monocytes, containing intracellular lipid droplets, into early atherosclerotic lesions and its contribution to development of nascent atherosclerosis.

Approach And Results: In apoE(-/-) mice fed Western high-fat diet (WD), >10% of circulating monocytes became foamy monocytes at 3 days on WD and >20% of monocytes at 1 week. Foamy monocytes also formed early in blood of Ldlr(-/-)Apobec1(-/-) (LDb) mice on WD.

Proatherogenic conditions promote autoimmune T helper 17 cell responses in vivo.

Patients with systemic autoimmune diseases show increased incidence of atherosclerosis. However, the contribution of proatherogenic factors to autoimmunity remains unclear. We found that atherogenic mice (herein referred to as LDb mice) exhibited increased serum interleukin-17, which was associated with increased numbers of T helper 17 (Th17) cells in secondary lymphoid organs.

Long-term Expression of Apolipoprotein B mRNA-specific Hammerhead Ribozyme via scAAV8.2 Vector Inhibits Atherosclerosis in Mice.

Target substrate-specific hammerhead ribozyme cleaves the specific mRNA efficiently and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. The goal of this study is to demonstrate that long-term reduction of apoB gene expression using hammerhead ribozyme would result in inhibition of atherosclerosis development.

Fortilin reduces apoptosis in macrophages and promotes atherosclerosis.

Atherosclerosis, a deadly disease insufficiently addressed by cholesterol-lowering drugs, needs new therapeutic strategies. Fortilin, a 172-amino acid multifunctional polypeptide, binds p53 and blocks its transcriptional activation of Bax, thereby exerting potent antiapoptotic activity. Although fortilin-overexpressing mice reportedly exhibit hypertension and accelerated atherosclerosis, it remains unknown if fortilin, not hypertension, facilitates atherosclerosis.

Proprotein convertase subtilisin/kexin type 9 interacts with apolipoprotein B and prevents its intracellular degradation, irrespective of the low-density lipoprotein receptor.

Objective: proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates the low-density lipoprotein (LDL) receptor (LDLR) in hepatocytes and therefore plays an important role in controlling circulating levels of LDL-cholesterol. To date, the relationship between PCSK9 and metabolism of apolipoprotein B (apoB), the structural protein of LDL, has been controversial and remains to be clarified.

Methods And Results: We assessed the impact of PCSK9 overexpression (≈400-fold above baseline) on apoB synthesis and secretion in 3 mouse models: wild-type C57BL/6 mice and LDLR-null mice (Ldlr(-/-) and Ldlr(-/-)Apobec1(-/-)).

Morelloflavone, a biflavonoid inhibitor of migration-related kinases, ameliorates atherosclerosis in mice.

While macrophages take up modified LDL to form foam cells and multiply to develop fatty streaks, vascular smooth muscle cells (VSMC) migrate from the media to intima, secrete extracellular matrix, and increase the volume of atherosclerotic lesions. A medicinal plant Garcinia dulcis has been used in traditional Thai medicine for centuries to treat various chronic human diseases. Morelloflavone, a biflavonoid and an active ingredient of the plant, has been shown to inhibit VSMC migration through its inhibition of multiple migration-related kinases such as focal adhesion kinase, c-Src, ERK, and RhoA.

Differential expression of genes in the calcium-signaling pathway underlies lesion development in the LDb mouse model of atherosclerosis.

Objective: Atherosclerosis is influenced by the interaction of environmental and genetic susceptibility risk factors. We used global microarray expression profiling to investigate differentially regulated genes in aorta during development of atherosclerosis in a susceptible genetically modified mouse model in response to the interaction between risk factors including hyperlipidemic genotype, shear stress, diet, and age.

Methods And Results: In this study we investigated transcriptional changes in lesion-prone and lesion-resistant regions of aortas in genetically modified mice lacking both genes of the LDL receptor and the apolipoprotein B mRNA editing enzyme (LDb; Ldlr(-/-)Apobec1(-/-)).

Upregulation of ICOS on CD43+ CD4+ murine small intestinal intraepithelial lymphocytes during acute reovirus infection.

Murine intestinal intraepithelial lymphocytes (IELs) can be classified according to expression of a CD43 glycoform recognized by the S7 monoclonal antibody. In this study, we examined the response of S7+ and S7- IELs in mice during acute reovirus serotype 3 (Dearing strain) infection, which was confirmed by virus-specific real-time PCR. In vivo proliferation increased significantly for both S7- and S7+ IELs on day 4 post-infection as determined by BrdU incorporation; however, expression of the inducible costimulatory (ICOS) molecule, which peaked on day 7 post-infection, was upregulated on S7+ CD4+ T cells, most of which were CD4+8- IELs.

Modulation of muscle regeneration, myogenesis, and adipogenesis by the Rho family guanine nucleotide exchange factor GEFT.

Rho family guanine nucleotide exchange factors (GEFs) regulate diverse cellular processes including cytoskeletal reorganization, cell adhesion, and differentiation via activation of the Rho GTPases. However, no studies have yet implicated Rho-GEFs as molecular regulators of the mesenchymal cell fate decisions which occur during development and repair of tissue damage. In this study, we demonstrate that the steady-state protein level of the Rho-specific GEF GEFT is modulated during skeletal muscle regeneration and that gene transfer of GEFT into cardiotoxin-injured mouse tibialis anterior muscle exerts a powerful promotion of skeletal muscle regeneration in vivo.

Simultaneous expression of apolipoprotein B mRNA editing enzyme and scavenger receptor BI mediated by a therapeutic gene expression system.

Cardiovascular diseases are often accompanied by elevated LDL particles and endothelial dysfunction. We have examined the possibility of concurrently reducing LDL levels and modulating endothelial function using a single helper-dependent adenovirus vector system to simultaneously express the apolipoprotein B mRNA editing enzyme (Apobec1) and the scavenger receptor, class B, type I (SR-BI) genes under the control of separate promoters (designated HD-C2). Apobec1 edits apoB mRNA at nucleotide C-6666 to produce truncated apoB48 and is normally expressed in small intestine only.

Maximum immunobioactivity of murine small intestinal intraepithelial lymphocytes resides in a subpopulation of CD43+ T cells.

CD43 has been linked to many function-associated T cell activities. Using mAbs that recognize two different CD43 determinants, we show that, although mouse small intestinal intraepithelial lymphocytes (IELs) expressed the CD43 core molecule reactive with mAb R2/60, only about one-half of the total IELs-including some but not all of the TCRalphabeta and TCRgammadelta cells-expressed the CD43 S7(-) reactive determinant. CD43 S7(+) IELs secreted more IL-2, IL-4, IL-10, IL-17, and IFN-gamma following anti-CD3 stimulation, and were >4-fold more cytotoxic in fresh isolates and >16-fold more cytotoxic after anti-CD3 stimulation, than S7(-) IELs.

Renal expression of the C3a receptor and functional responses of primary human proximal tubular epithelial cells.

Although complement activation and deposition have been associated with a variety of glomerulopathies, the pathogenic mechanisms by which complement directly mediates renal injury remain to be fully elucidated. Renal parenchymal tissues express a limited repertoire of receptors that directly bind activated complement proteins. We report the renal expression of the receptor for the C3 cleavage product C3a, a member of the anaphylatoxin family.

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression.

BACKGROUND: In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo.

Increased plasma non-esterified fatty acids and platelet-activating factor acetylhydrolase are associated with susceptibility to atherosclerosis in mice.

Animal models provide vital tools to explicate the pathogenesis of atherosclerosis. Accordingly, we established two atherosclerosis-prone mice models: (i) mice lacking the LDL (low-density lipoprotein) receptor (LDLR) and the ability to edit apo (apolipoprotein) B mRNA (Apobec1; designated LDb : LDLR-/- Apobec1-/-), and (ii) mice with the LDb background, who also overexpressed human apoB100 (designated LTp : LDLR-/- Apobec1-/- ERhB+/+). Both LDb and LTp mice had markedly elevated levels of LDL and increased levels of NEFAs (non-esterified fatty acids) compared with C57BL/6 wild-type mice.

Paradoxical upregulation of tumor suppressor protein p53 in serum-stimulated vascular smooth muscle cells: a novel negative-feedback regulatory mechanism.

Background: The proliferative response of vascular smooth muscle cells (VSMCs) to various growth stimuli is critical for atherosclerosis and postangioplasty restenosis. Although tumor suppressor protein p53 plays a critical role in the elimination of cancerous cells, recent genetic studies have indicated that it also protects against atherosclerosis and restenosis.

Methods And Results: We examined the levels of p53 protein in normal VSMCs before and after serum stimulation.

Hepatic gene expression profiling reveals perturbed calcium signaling in a mouse model lacking both LDL receptor and Apobec1 genes.

Atherosclerosis is a complex disease that gene and environment interaction influences the progression of atherosclerotic lesion development. Our laboratory used mice lacking both the low density lipoprotein (LDL) receptor and Apobec1 genes (LDLR-/-Apobec1-/-, designated LDb) to investigate gene-gene interaction and the influence of an environmental factor (high-fat diet) on gene networks. LDb mice (males and females) at 5 months of age were fed a chow or high fat diet for 3-month.