Chenghong Li

AMP-Activated Protein Kinase Promotes the Differentiation of Endothelial Progenitor Cells

Objective—Endothelial progenitor cells (EPCs) can differentiate into endothelial cells (ECs) and participate in postnatal vasculogenesis, but the mechanism of EPC differentiation remains largely unknown. We investigated the role of AMP-activated protein kinase (AMPK) in EPC differentiation and functions. Methods and Results—Vascular endothelial growth factor caused the phosphorylation of AMPK, acetyl-coenzymeA (CoA) carboxylase (ACC), and eNOS in human cord blood-derived EPCs. The expression of EC markers, including VE-cadherin and intercellular adhesion molecule1 (ICAM-1), was also increased but blocked by Compound C, an AMPK inhibitor. AICAR, an AMPK agonist, increased the phosphorylation of ACC and eNOS and the expression of EC markers in a time- and dose-dependent manner, which reinforces the positive effect of AMPK on EPC differentiation. The effects of AICAR could be blocked by treatment with L-NAME, an eNOS inhibitor. Functionally, AICAR increased but Compound C decreased the angiogenesis of EPCs in vitro and in vivo. Furthermore, lovastatin promoted the activation of AMPK and eNOS, the expression of EC markers, tube formation, adhesion, and in vivo vasculogenesis of EPCs, which could be blocked by treatment with Compound C. Conclusion—The activation of eNOS by AMPK during EPC differentiation provides a novel mechanism for the pleiotropic effects of statins in benefiting the cardiovascular system.

Mouse patatin-like phospholipase domain-containing 3 influences systemic lipid and glucose homeostasis†‡

Human patatin‐like phospholipase domain‐containing 3 (PNPLA3) is associated with increased liver fat content and liver injury. Here, we show that nutritional status regulates PNPLA3 gene expression in the mouse liver. Sterol response element binding protein‐1 (SREBP‐1) activated PNPLA3 gene transcription via sterol regulatory elements (SREs) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that SREBP‐1 proteins bound to the identified SREs. Furthermore, SREBP‐1c mediated the insulin and liver X receptor agonist TO901317‐dependent induction of PNPLA3 gene expression in hepatocytes. Adenovirus‐mediated overexpression of mouse PNPLA3 increased intracellular triglyceride content in primary hepatocytes, and knockdown of PNPLA3 suppressed the ability of SREBP‐1c to stimulate lipid accumulation in hepatocytes. Finally, the overexpression of PNPLA3 in mouse liver increased the serum triglyceride level and impaired glucose tolerance; in contrast, the knockdown of PNPLA3 in db/db mouse liver improved glucose tolerance. Conclusion: Our data suggest that mouse PNPLA3, which is a lipogenic gene directly targeted by SREBP‐1, promotes lipogenesis in primary hepatocytes and influences systemic lipid and glucose metabolism. (HEPATOLOGY 2011;)

Cholesterol increases adhesion of monocytes to endothelium by moving adhesion molecules out of caveolae.

Caveolae and its structural protein caveolin-1 (Cav-1) are abundant in vascular endothelial cells (ECs). We examined whether caveolae are involved in monocyte adhesion to ECs responding to a synergy of hypercholesterolemia and inflammation. Treating human umbilical vein ECs with cholesterol enhanced endotoxin lipopolysaccharide (LPS)-induced monocyte adhesion. Use of isolated caveolae-enriched membranes revealed that cell adhesion molecules (CAMs), including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), co-localized with Cav-1 in caveolae. LPS upregulated CAMs expression and increased the co-localization. Cholesterol exposure decreased the level of CAMs in the caveolae. Co-immunoprecipitation and confocal microscopy revealed that ICAM-1 interacted with Cav-1. Electron microscopy showed that ICAM-1 was mainly located in caveolae. Cholesterol exposure decreased this interaction and drove ICAM-1 out of caveolae. Knockdown of Cav-1 reduced the synergistic effects of cholesterol and inflammation. In vivo, ICAM-1 and Cav-1 co-localization was lower in the aortic endothelium of ApoE(-)(/)(-) mice than in that of wild-type controls. Cav-1 negatively regulates monocyte adhesion by the co-localization of CAMs in caveolae, which is disturbed by cholesterol. Thus, our study suggests a molecular basis underlying the synergistic effects of hypercholesterolemia and inflammation in atherogenesis.

Prostaglandin E2 promotes endothelial differentiation from bone marrow-derived cells through AMPK activation.

Prostaglandin E2 (PGE2) has been reported to modulate angiogenesis, the process of new blood vessel formation, by promoting proliferation, migration and tube formation of endothelial cells. Endothelial progenitor cells are known as a subset of circulating bone marrow mononuclear cells that have the capacity to differentiate into endothelial cells. However, the mechanism underlying the stimulatory effects of PGE2 and its specific receptors on bone marrow-derived cells (BMCs) in angiogenesis has not been fully characterized. Treatment with PGE2 significantly increased the differentiation and migration of BMCs. Also, the markers of differentiation to endothelial cells, CD31 and von Willebrand factor, and the genes associated with migration, matrix metalloproteinases 2 and 9, were significantly upregulated. This upregulation was abolished by dominant-negative AMP-activated protein kinase (AMPK) and AMPK inhibitor but not protein kinase, a inhibitor. As a functional consequence of differentiation and migration, the tube formation of BMCs was reinforced. Along with altered BMCs functions, phosphorylation and activation of AMPK and endothelial nitric oxide synthase, the target of activated AMPK, were both increased which could be blocked by EP4 blocking peptide and simulated by the agonist of EP4 but not EP1, EP2 or EP3. The pro-angiogenic role of PGE2 could be repressed by EP4 blocking peptide and retarded in EP4+/− mice. Therefore, by promoting the differentiation and migration of BMCs, PGE2 reinforced their neovascularization by binding to the receptor of EP4 in an AMPK-dependent manner. PGE2 may have clinical value in ischemic heart disease.

A Novel Mechanism of γ/δ T-Lymphocyte and Endothelial Activation by Shear Stress The Role of Ecto-ATP Synthase β Chain

Rationale: Endothelial cells (ECs) have distinct mechanotransduction mechanisms responding to laminar versus disturbed flow patterns. Endothelial dysfunction, affected by imposed flow, is one of the earliest events leading to atherogenesis. The involvement of γ/δ T lymphocytes in endothelial dysfunction under flow is largely unknown. Objective: To investigate whether shear stress regulates membrane translocation of ATP synthase β chain (ATPSβ) in ECs, leading to the increased γ/δ T-lymphocyte adhesion and the related functions. Method and Results: We applied different flow patterns to cultured ECs. Laminar flow decreased the level of membrane-bound ATPSβ (ecto-ATPSβ) and depleted membrane cholesterol, whereas oscillatory flow increased the level of ecto-ATPSβ and membrane cholesterol. Incubating ECs with cholesterol or depleting cellular cholesterol with β-cyclodextrin mimicked the effect of oscillatory or laminar flow, respectively. Knockdown caveolin-1 by small interfering RNA prevented ATPSβ translocation in response to laminar flow. Importantly, oscillatory flow or cholesterol treatment elevated the number of γ/δ T cells binding to ECs, which was blocked by anti-ATPSβ antibody. Furthermore, the incubation of γ/δ T cells with ECs increased tumor necrosis fact α and interferon-γ secretion from T cells and vascular cell adhesion molecule-1 expression in ECs. In vivo, γ/δ T-cell adhesion and ATPSβ membrane translocation was elevated in the aortic inner curvature and disturbed flow areas in partially ligated carotid arteries of ApoE−/− mice fed a high-fat diet. Conclusions: This study provides evidence that disturbed flow and hypercholesterolemia synergistically promote γ/δ T-lymphocyte activation by the membrane translocation of ATPSβ in ECs and in vivo in mice, which is a novel mechanism of endothelial activation. # Novelty and Significance {#article-title-34}Rationale: Endothelial cells (ECs) have distinct mechanotransduction mechanisms responding to laminar versus disturbed flow patterns. Endothelial dysfunction, affected by imposed flow, is one of the earliest events leading to atherogenesis. The involvement of &ggr;/&dgr; T lymphocytes in endothelial dysfunction under flow is largely unknown. Objective: To investigate whether shear stress regulates membrane translocation of ATP synthase &bgr; chain (ATPS&bgr;) in ECs, leading to the increased &ggr;/&dgr; T-lymphocyte adhesion and the related functions. Method and Results: We applied different flow patterns to cultured ECs. Laminar flow decreased the level of membrane-bound ATPS&bgr; (ecto-ATPS&bgr;) and depleted membrane cholesterol, whereas oscillatory flow increased the level of ecto-ATPS&bgr; and membrane cholesterol. Incubating ECs with cholesterol or depleting cellular cholesterol with &bgr;-cyclodextrin mimicked the effect of oscillatory or laminar flow, respectively. Knockdown caveolin-1 by small interfering RNA prevented ATPS&bgr; translocation in response to laminar flow. Importantly, oscillatory flow or cholesterol treatment elevated the number of &ggr;/&dgr; T cells binding to ECs, which was blocked by anti-ATPS&bgr; antibody. Furthermore, the incubation of &ggr;/&dgr; T cells with ECs increased tumor necrosis fact &agr; and interferon-&ggr; secretion from T cells and vascular cell adhesion molecule-1 expression in ECs. In vivo, &ggr;/&dgr; T-cell adhesion and ATPS&bgr; membrane translocation was elevated in the aortic inner curvature and disturbed flow areas in partially ligated carotid arteries of ApoE−/− mice fed a high-fat diet. Conclusions: This study provides evidence that disturbed flow and hypercholesterolemia synergistically promote &ggr;/&dgr; T-lymphocyte activation by the membrane translocation of ATPS&bgr; in ECs and in vivo in mice, which is a novel mechanism of endothelial activation.

Suppression of 2,3-Oxidosqualene Cyclase by High Fat Diet Contributes to Liver X Receptor-α-mediated Improvement of Hepatic Lipid Profile

The liver X receptors (LXRs) sense oxysterols and regulate genes involved in cholesterol metabolism. Synthetic agonists of LXRs are potent stimulators of fatty acid synthesis, which is mediated largely by sterol regulatory element-binding protein-1c (SREBP-1c). Paradoxically, an improved hepatic lipid profile by LXR was observed in mice fed a Western high fat (HF) diet. To explore the underlying mechanism, we administered mice normal chow or an HF diet and overexpressed LXRα in the liver. The HF diet with tail-vein injection of adenovirus of LXRα increased the expression of LXR-targeted genes involved in cholesterol reverse transport but not those involved in fatty acid synthesis. A similar effect was also observed with the use of 22R-hydroxycholesterol, an LXR ligand, in cultured hepatocytes. Consequently, SREBP-1c maturation was inhibited by the HF diet, which resulted from the induction of Insig-2a. Importantly, increased cholesterol level suppressed the expression of 2,3-oxidosqualene cyclase (OSC), which led to an increase in endogenous LXR ligand(s). Furthermore, siRNA-mediated knockdown of OSC expression enhanced LXR activity and selectively up-regulated LXR-targeted genes involved in cholesterol reverse transport. Thus, down-regulation of OSC may account for a novel mechanism underlying the LXR-mediated lipid metabolism in the liver of mice fed an HF diet.

Proteomic analysis of endothelial lipid rafts reveals a novel role of statins in antioxidation.

As inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, statins have pleiotropic vascular-protective effects, such as anti-inflammatory and antioxidative effects. We investigated the short-term beneficial effects of statins on modulating the translocation of lipid-raft-related proteins in endothelial cells (ECs). Human umbilical vein ECs were treated with atorvastatin for 30 min or 2 h; lipid-raft proteins were isolated and examined by quantitative proteome assay. Functional classification of identified proteins in lipid rafts revealed upregulated antioxidative proteins; downregulated proteins were associated with inflammation and cell adhesion. Among proteins verified by Western blot analysis, endoplasmic reticulum protein 46 (ERp46) showed increased level in lipid rafts with atorvastatin. Further, atorvastatin inhibited the activation of membrane-bound NADPH oxidase in both untreated and angiotensin II-treated ECs, as shown by reduced reactive oxygen species production. Co-immunoprecipitation and immunofluorescence experiments revealed that atorvastatin increased the association of ERp46 and Nox2, an NADPH oxidase isoform, in lipid rafts, thereby inhibiting Nox2 assembly with its regulatory subunits, such as p47phox and p67phox. Our results reveal a novel antioxidative role of atorvastatin by promoting the membrane translocation of ERp46 and its binding with Nox2 to inhibit Nox2 activity in ECs, which may offer another insight into the pleiotropic functions of statins.

LIF maintains progenitor phenotype of endothelial progenitor cells via Krüppel-like factor 4

BACKGROUND Endothelial progenitor cells (EPCs) participate in post-natal vasculogenesis. Maintaining the preliminary progenitor phenotype and good proliferation capacity of EPCs is key to their use in treating cardiovascular ischemic diseases. However, transcriptional regulation in EPCs remains largely unknown. We investigated the effect of leukemia inhibitory factor (LIF) combined with vascular endothelial growth factor (VEGF) on EPCs and the potential roles of Krüppel-like transcription factors (KLFs). METHODS AND RESULTS Co-treatment with LIF and VEGF (100 ng/ml each) (V+L) could increase EPC colony-forming units and CD34 expression, which reflects the EPC progenitor phenotype and alleviated differentiation of EPCs. The effect was associated with Akt activation and increased expression of KLF4. Upregulation of KLF4 induced by V+L could be inhibited by transfection with dominant-negative Akt adenovirus. Furthermore, overexpression of KLF4 in EPCs enhanced the expression of CD34 and alleviated cell differentiation but did not increase the phosphorylation of Akt. CONCLUSIONS LIF combined with VEGF can maintain the preliminary, progenitor phenotype of EPCs and alleviate cell differentiation by upregulating KLF4, which may provide new insights into transcriptional regulation in EPCs.

A Novel Mechanism of γ/δ T-Lymphocyte and Endothelial Activation by Shear Stress

Rationale: Endothelial cells (ECs) have distinct mechanotransduction mechanisms responding to laminar versus disturbed flow patterns. Endothelial dysfunction, affected by imposed flow, is one of the earliest events leading to atherogenesis. The involvement of γ/δ T lymphocytes in endothelial dysfunction under flow is largely unknown. Objective: To investigate whether shear stress regulates membrane translocation of ATP synthase β chain (ATPSβ) in ECs, leading to the increased γ/δ T-lymphocyte adhesion and the related functions. Method and Results: We applied different flow patterns to cultured ECs. Laminar flow decreased the level of membrane-bound ATPSβ (ecto-ATPSβ) and depleted membrane cholesterol, whereas oscillatory flow increased the level of ecto-ATPSβ and membrane cholesterol. Incubating ECs with cholesterol or depleting cellular cholesterol with β-cyclodextrin mimicked the effect of oscillatory or laminar flow, respectively. Knockdown caveolin-1 by small interfering RNA prevented ATPSβ translocation in response to laminar flow. Importantly, oscillatory flow or cholesterol treatment elevated the number of γ/δ T cells binding to ECs, which was blocked by anti-ATPSβ antibody. Furthermore, the incubation of γ/δ T cells with ECs increased tumor necrosis fact α and interferon-γ secretion from T cells and vascular cell adhesion molecule-1 expression in ECs. In vivo, γ/δ T-cell adhesion and ATPSβ membrane translocation was elevated in the aortic inner curvature and disturbed flow areas in partially ligated carotid arteries of ApoE−/− mice fed a high-fat diet. Conclusions: This study provides evidence that disturbed flow and hypercholesterolemia synergistically promote γ/δ T-lymphocyte activation by the membrane translocation of ATPSβ in ECs and in vivo in mice, which is a novel mechanism of endothelial activation. # Novelty and Significance {#article-title-34}Rationale: Endothelial cells (ECs) have distinct mechanotransduction mechanisms responding to laminar versus disturbed flow patterns. Endothelial dysfunction, affected by imposed flow, is one of the earliest events leading to atherogenesis. The involvement of &ggr;/&dgr; T lymphocytes in endothelial dysfunction under flow is largely unknown. Objective: To investigate whether shear stress regulates membrane translocation of ATP synthase &bgr; chain (ATPS&bgr;) in ECs, leading to the increased &ggr;/&dgr; T-lymphocyte adhesion and the related functions. Method and Results: We applied different flow patterns to cultured ECs. Laminar flow decreased the level of membrane-bound ATPS&bgr; (ecto-ATPS&bgr;) and depleted membrane cholesterol, whereas oscillatory flow increased the level of ecto-ATPS&bgr; and membrane cholesterol. Incubating ECs with cholesterol or depleting cellular cholesterol with &bgr;-cyclodextrin mimicked the effect of oscillatory or laminar flow, respectively. Knockdown caveolin-1 by small interfering RNA prevented ATPS&bgr; translocation in response to laminar flow. Importantly, oscillatory flow or cholesterol treatment elevated the number of &ggr;/&dgr; T cells binding to ECs, which was blocked by anti-ATPS&bgr; antibody. Furthermore, the incubation of &ggr;/&dgr; T cells with ECs increased tumor necrosis fact &agr; and interferon-&ggr; secretion from T cells and vascular cell adhesion molecule-1 expression in ECs. In vivo, &ggr;/&dgr; T-cell adhesion and ATPS&bgr; membrane translocation was elevated in the aortic inner curvature and disturbed flow areas in partially ligated carotid arteries of ApoE−/− mice fed a high-fat diet. Conclusions: This study provides evidence that disturbed flow and hypercholesterolemia synergistically promote &ggr;/&dgr; T-lymphocyte activation by the membrane translocation of ATPS&bgr; in ECs and in vivo in mice, which is a novel mechanism of endothelial activation.

A Novel Mechanism of γ/δ T-Lymphocyte and Endothelial Activation by Shear StressNovelty and Significance: The Role of Ecto-ATP Synthase β Chain

Rationale: Endothelial cells (ECs) have distinct mechanotransduction mechanisms responding to laminar versus disturbed flow patterns. Endothelial dysfunction, affected by imposed flow, is one of the earliest events leading to atherogenesis. The involvement of γ/δ T lymphocytes in endothelial dysfunction under flow is largely unknown. Objective: To investigate whether shear stress regulates membrane translocation of ATP synthase β chain (ATPSβ) in ECs, leading to the increased γ/δ T-lymphocyte adhesion and the related functions. Method and Results: We applied different flow patterns to cultured ECs. Laminar flow decreased the level of membrane-bound ATPSβ (ecto-ATPSβ) and depleted membrane cholesterol, whereas oscillatory flow increased the level of ecto-ATPSβ and membrane cholesterol. Incubating ECs with cholesterol or depleting cellular cholesterol with β-cyclodextrin mimicked the effect of oscillatory or laminar flow, respectively. Knockdown caveolin-1 by small interfering RNA prevented ATPSβ translocation in response to laminar flow. Importantly, oscillatory flow or cholesterol treatment elevated the number of γ/δ T cells binding to ECs, which was blocked by anti-ATPSβ antibody. Furthermore, the incubation of γ/δ T cells with ECs increased tumor necrosis fact α and interferon-γ secretion from T cells and vascular cell adhesion molecule-1 expression in ECs. In vivo, γ/δ T-cell adhesion and ATPSβ membrane translocation was elevated in the aortic inner curvature and disturbed flow areas in partially ligated carotid arteries of ApoE−/− mice fed a high-fat diet. Conclusions: This study provides evidence that disturbed flow and hypercholesterolemia synergistically promote γ/δ T-lymphocyte activation by the membrane translocation of ATPSβ in ECs and in vivo in mice, which is a novel mechanism of endothelial activation. # Novelty and Significance {#article-title-34}Rationale: Endothelial cells (ECs) have distinct mechanotransduction mechanisms responding to laminar versus disturbed flow patterns. Endothelial dysfunction, affected by imposed flow, is one of the earliest events leading to atherogenesis. The involvement of &ggr;/&dgr; T lymphocytes in endothelial dysfunction under flow is largely unknown. Objective: To investigate whether shear stress regulates membrane translocation of ATP synthase &bgr; chain (ATPS&bgr;) in ECs, leading to the increased &ggr;/&dgr; T-lymphocyte adhesion and the related functions. Method and Results: We applied different flow patterns to cultured ECs. Laminar flow decreased the level of membrane-bound ATPS&bgr; (ecto-ATPS&bgr;) and depleted membrane cholesterol, whereas oscillatory flow increased the level of ecto-ATPS&bgr; and membrane cholesterol. Incubating ECs with cholesterol or depleting cellular cholesterol with &bgr;-cyclodextrin mimicked the effect of oscillatory or laminar flow, respectively. Knockdown caveolin-1 by small interfering RNA prevented ATPS&bgr; translocation in response to laminar flow. Importantly, oscillatory flow or cholesterol treatment elevated the number of &ggr;/&dgr; T cells binding to ECs, which was blocked by anti-ATPS&bgr; antibody. Furthermore, the incubation of &ggr;/&dgr; T cells with ECs increased tumor necrosis fact &agr; and interferon-&ggr; secretion from T cells and vascular cell adhesion molecule-1 expression in ECs. In vivo, &ggr;/&dgr; T-cell adhesion and ATPS&bgr; membrane translocation was elevated in the aortic inner curvature and disturbed flow areas in partially ligated carotid arteries of ApoE−/− mice fed a high-fat diet. Conclusions: This study provides evidence that disturbed flow and hypercholesterolemia synergistically promote &ggr;/&dgr; T-lymphocyte activation by the membrane translocation of ATPS&bgr; in ECs and in vivo in mice, which is a novel mechanism of endothelial activation.