Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are critical events in the progression of several vasculopathologies. Adenosine monophosphate-activated protein kinase (AMPK) has been shown to play a pivotal role in cellular proliferation and migration. However, the roles of AMPK in VSMCs migration and its underlying molecular mechanisms remain elusive.
Approach and Results
VSMC migration and the neointima formation were studied in cultured mouse VSMCs or in carotid artery ligation of wild-type (WT) C57BL/6J mice, AMPKα2, AMPKα1 homozygous-deficient (AMPKα2−/−, AMPKα1−/−) mice. Deletion of AMPKα2, but not AMPKα1, led to increased phosphorylation of both IκB kinase α (IκKα) and its downstream target nuclear factor κB2 (NFκB2)/p100 at serine 866/870. Consequently, phosphor-p100 at S866/870 bound with E3 ubiquitin ligase β–transducin repeat-containing protein (β-TrCP) resulting in the proteolytic processing of the p100 precursor and NFκB2/p52 induction. Interestingly, acetylation of histone H3 at lysine 56 (AcH3-K56) mediated by histone deacetylase 3 (HDAC3) reduction was enhanced significantly in AMPKα2−/− VSMCs compared with WT or AMPKα1−/− VSMCs. Moreover, the augmented association of p52/AcH3-K56 with the promoter of ubiquitin E3 ligase, S-phase kinase-associated protein 2 (Skp2), was shown in AMPKα2−/− VSMCs by ChIP assay. Furthermore, AMPKα2 deletion caused Skp2-mediated E-cadherin downregulation. Skp2 siRNA abolished the increased migration of AMPKα2−/− VSMCs via E-cadherin upregulation. Finally, neointima formation after ligation of carotid artery was increased in AMPKα2−/−, but not AMPKα1−/−, mice.
We conclude that deletion of AMPKα2 causes aberrant VSMCs migration with accelerated neointima formation in vivo.
AMPK; VSMCs; migration; Skp2; E-cadherin
Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages.
Highly purified heparanase was added to mouse peritoneal macrophages (MPM) and macrophage-like J774 cells and the levels of TNFα, MMP-9, IL-1, and MCP-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll like receptor (TLR)-2 and -4 knockout mice (KO) were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction (MI), stable angina (SA), and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with SA or acute MI. Addition or over expression of heparanase variants resulted in marked increase in TNFα, MMP-9, IL-1 and MCP-1 levels. MPM harvested from TLR-2 or TLR-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute MI, compared to patients with SA and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared to specimens of stable plaque and controls.
Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression towards vulnerability.
heparanase; macrophages; vulnerable plaque; TLR; TNFα
Protein inhibitor of activated STAT1 (PIAS1) is known to function as SUMO E3 ligase as well as transrepressor. The aim of the study is to elucidate the regulatory mechanisms for these two different functions, especially with respect to endothelial inflammation.
Methods and Results
The MAPK-activated protein kinase-2 (MK2) is pro-inflammatory kinase and phosphorylates PIAS1 at the Ser522 residue. Activation of MK2 enhances p53-SUMOylation, but a PIAS1 phosphorylation mutant, PIAS1-S522A, abolished this p53-SUMOylation, suggesting a critical role for PIAS1 S522 phosphorylation in its SUMO ligase activity. Since nuclear p53 can inhibit KLF2 promoter activity, we investigated the roles for PIAS1 phosphorylation and p53-SUMOylation in the KLF2 and eNOS expression. Both MK2 and PIAS1 overexpression increased KLF2 promoter activity and eNOS expression, which were inhibited by expressing a p53-SUMOylation defective mutant, p53-K386R, and PIAS1-S522A. PIAS1-S522A also abolished the anti-inflammatory effect of wild type (WT) PIAS1 in vitro and also in vivo which was examined by leukocyte rolling in microvessels of skin grafts transduced by adenovirus encoding WT PIAS1 or the S522A mutant.
Our study has identified a novel negative feedback regulatory pathway through which MK2 limits endothelial inflammation via the PIAS1 S522 phosphorylation-mediated increase in PIAS1 transrepression and SUMO ligase activity.
Endothelial inflammation; MK2; NF-κB Transrepression; PIAS1; Vascular biology
Inflammation is essential to combat invading microbial pathogens. In this process, the involvement of multiple immune cell populations is critical in mounting an optimum immune response. In the past decade, a new class of non-coding small RNAs, called miRNAs, has emerged as important regulators in biological processes. The important role of miRNAs in inflammation and immune response is highlighted by studies in which deregulation of miRNAs was demonstrated to accompany diseases associated with excessive or uncontrolled inflammation. In this brief review, we summarize the roles of miRNAs that have been characterized in innate and adaptive immune responses. We discuss the role of miRNAs in macrophage polarization, a molecular event that has clear impact on inflammation.
microRNA; immune response; macrophage polarization
The regulated response of endothelial cells (ECs) to signals in their environment is not only critical for the de novo formation of primordial vascular networks during early development (i.e. vasculogenesis), but is also required for the subsequent growth and remodelling of new blood vessels from pre-existing ones (i.e. angiogenesis). Vascular endothelial growth factors (Vegfs) and their endothelial-cell specific receptors play a crucial role in nearly all aspects of blood vessel growth. How the outputs from these pathways affect and coordinate endothelial behaviour is an area of intense research. Recently, numerous studies have highlighted roles for microRNAs in modulating Vegf signalling output in several different contexts. In this review we will provide an overview of how small RNAs regulate multiple aspects of the Vegf signalling pathway. In particular, we highlight areas where identification of microRNAs and their targets has provided new insight into the role of downstream effectors in modulating Vegf output during development. Since Vegf plays a broad role in multiple aspects of endothelial biology and has become a target for therapeutic manipulation of pathological blood vessel growth, microRNAs that affect Vegf signalling output will undoubtedly be major targets of clinical value.
Saturated fatty acids, such as palmitic and stearic acid, cause detrimental effects in endothelial cells (ECs) and have been suggested to contribute to macrophage accumulation in adipose tissue and the vascular wall in states of obesity and insulin resistance. Long-chain fatty acids are believed to require conversion into acyl-CoA derivatives to exert most of their detrimental effects, a reaction catalyzed by acyl-CoA synthetases (ACSL). The objective of this study was to investigate the role of ACSL1, an ACSL isoform previously shown to mediate inflammatory effects in myeloid cells, in regulating EC responses to a saturated fatty acid-rich environment in vitro and in vivo.
Methods and Results
Saturated fatty acids caused increased inflammatory activation, ER stress, and apoptosis in mouse microvascular ECs. Forced ACSL1 overexpression exacerbated the effects of saturated fatty acids on apoptosis and ER stress. However, endothelial ACSL1-deficiency did not protect against the effects of saturated fatty acids in vitro, nor did it protect insulin resistant mice fed a saturated fatty acid-rich diet from macrophage adipose tissue accumulation or increased aortic adhesion molecule expression.
Endothelial ACSL1 is not required for inflammatory and apoptotic effects of a saturated fatty acid-rich environment.
Acyl-CoA synthetase; Apoptosis; Endothelium; Inflammation
Calcific aortic valve disease (CAVD) is a major public health problem with no effective treatment available other than surgery. We previously showed that mature heart valves calcify in response to retinoic acid (RA) treatment through downregulation of the SRY-transcription factor Sox9. In this study, we investigated the effects of excess vitamin A and its metabolite RA on heart valve structure and function in vivo, and examined the molecular mechanisms of RA signaling during the calcification process in vitro.
Methods and Results
Using a combination of approaches, we defined CAVD pathogenesis in mice fed 200 IU/g and 20 IU/g of retinyl palmitate for 12 months at molecular, cellular and functional levels. We show that mice fed excess vitamin A develop aortic valve stenosis and leaflet calcification associated with increased expression of osteogenic genes and decreased expression of cartilaginous markers. Using a pharmacological approach, we show that RA-mediated Sox9 repression and calcification is regulated by classical RA signaling and requires both RAR and RXR receptors.
Our studies demonstrate that excess vitamin A dietary intake promotes heart valve calcification in vivo. Therefore suggesting that hypervitaminosis A could serve as a new risk factor of CAVD in the human population.
The principle role of the vascular endothelium is to present a semi-impermeable barrier to soluble factors and circulating cells, while still permitting the passage of leukocytes from the bloodstream into the tissue. The process of diapedesis involves the selective disruption of endothelial cell junctions, an event that could in theory compromise vascular integrity. It is therefore somewhat surprising that neutrophil transmigration does not significantly impair endothelial barrier function. We examined whether neutrophils might secrete factors that promote vascular integrity during the latter stages of neutrophil transmigration, and found that neutrophil proteinase 3 (PR3) – a serine protease harbored in azurophilic granules – markedly enhanced barrier function in endothelial cells. PR3 functioned in this capacity both in its soluble form and in a complex with cell-surface NB1. PR3-mediated enhancement of endothelial cell junctional integrity required its proteolytic activity, as well as endothelial cell expression of the protease-activated receptor, PAR-2. Importantly, PR3 suppressed the vascular permeability changes and disruption of junctional proteins induced by the action of PAR-1 agonists. These findings establish the potential for neutrophil-derived PR3 to play a role in reestablishing vascular integrity following leukocyte transmigration, and in protecting endothelial cells from PAR-1-induced permeability changes that occur during thrombotic and inflammatory events.
NB1; PECAM-1; calcium signaling; serine protease
Vessels in brain arteriovenous malformations (bAVM) are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 (HHT2) patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of bAVM than the general population. We tested the hypothesis that vascular endothelial growth factor (VEGF) impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell-coverage.
Methods and Results
Adult Alk11f/2f mice (loxP sites flanking exons 4-6) and wild-type (WT) mice were injected with 2×107 PFU Ad-Cre and 2×109 genome copies of AAV-VEGF to induce focal homozygous Alk1 deletion (in Alk11f/2f mice) and angiogenesis. Brain vessels were analyzed eight weeks later. Compared to WT mice, the Alk1-deficient brain had more fibrin (99±30×103 pixels/mm2 vs. 40±13×103, P=0.001), iron deposition (508±506 pixels/mm2 vs. 6 ±49, P=0.04), and Iba1+ microglia/macrophage infiltration (888±420 Iba1+ cells/mm2 vs. 240±104 Iba1+, P=0.001) after VEGF stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-SMA- vessels (52±9% vs. 12±7%, P<0.001), fewer vascular associated pericytes (503±179/mm2 vs. 931±115, P<0.001), and reduced PDGFR-β expression (26±9%, P<0.001).
Reduction of mural cell coverage in response to VEGF stimulation is a potential mechanism for the impairment of vessel wall integrity in HHT2-associated bAVM.
brain arteriovenous malformation; activin receptor-like kinase 1; pericyte; iron deposition; PDGFR-β
Cell surface localization and intracellular trafficking of ATP-binding cassette transporter A-1 (ABCA1) are essential for its function. However, regulation of these activities is still largely unknown. Brefeldin A (BFA), a uncompetitive inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1 trafficking and cholesterol efflux, and further to explore the potential mechanism.
Methods and Results
By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells and inhibited by 60% cholesterol release. In contrast, BIG1 over-expression increased surface ABCA1 and cholesterol secretion. With partial restoration of BIG1 through over-expression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione cleavage revealed that BIG1 siRNA dramatically decreased the internalization and recycling of ABCA1. This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation of ADP-ribosylation factor 1 (ARF1).
BIG1, through its ability to activate ARF1, regulates cell surface levels and function of ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action.
BIG1; ABCA1; trafficking; cholesterol efflux
Extracellular microRNAs (miRNA) are present in most biological fluids, relatively stable, and hold great potential for disease biomarkers and novel therapeutics. Circulating miRNAs are transported by membrane-derived vesicles (exosomes and microparticles), lipoproteins, and other ribonucleoprotein complexes. Evidence suggests that miRNAs are selectively exported from cells with distinct signatures that have been found to be altered in many pathophysiologies, including cardiovascular disease. Protected from plasma ribonucleases by their carriers, functional miRNAs are delivered to recipient cells by various routes. Transferred miRNAs utilize cellular machinery to reduce target gene expression and alter cellular phenotype. Similar to soluble factors, miRNAs mediate cell-to-cell communication linking disparate cell types, diverse biological mechanisms, and homeostatic pathways. Although, significant advances have been made, miRNA intercellular communication is full of complexities and many questions remain. This review brings into focus what is currently known and outstanding in a novel field of study with applicability to cardiovascular disease.
Abdominal aortic aneurysms (AAAs) are common, but their exact pathogenesis remains unknown and no specific medical therapies are available. We sought to evaluate interleukin-1β (IL-1β) and interleukin-1 receptor (IL-1R) in an experimental AAA model to identify novel therapeutic targets for AAA treatment.
Methods and Results
IL-1β mRNA and protein levels were significantly elevated in abdominal aortas of 8-12 week old male C57Bl/6 mice following elastase aortic perfusion (WT) compared to saline perfusion. Mice with genetic deletion of IL-1β (IL-1β KO) or IL-1R (IL-1R KO) that underwent elastase perfusion demonstrated significant protection against AAA formation, with maximal aortic dilations of 38.0±5.5% for IL-1β KO and 52.5±4.6% for IL-1R KO compared to 89.4±4.0% for WT mice (p<0.005). Correspondingly, IL-1β KO and IL-1R KO aortas had reduced macrophage and neutrophil staining with greater elastin preservation compared to WT. In WT mice pretreated with escalating doses of the IL-1R antagonist anakinra, there was a dose-dependent decrease in maximal aortic dilation (R=−0.676, p <0.0005). Increasing anakinra doses correlated with decreasing macrophage staining and elastin fragmentation. Lastly, WT mice treated with anakinra 3 or 7 days following AAA initiation with elastase demonstrated significant protection against AAA progression and had decreased aortic dilation compared to control mice.
IL-1β is critical for AAA initiation and progression, and IL-1β neutralization through genetic deletion or receptor antagonism attenuates experimental AAA formation. Disrupting IL-1β signaling offers a novel pathway for AAA treatment.
Aortic disease; Aneurysm; Vascular Inflammation; Interleukin-1 Receptor Antagonist; Animal model surgery
Leptin promotes atherosclerosis and vessel wall remodeling. As abdominal aorta aneurysm (AAA) formation involves tissue remodeling, we hypothesized that local leptin synthesis initiates and promotes this process.
Methods and Results
Human surgical AAA walls were analyzed for antigen and mRNA levels of leptin and leptin receptor (ObR), as well as mRNA for matrix metalloproteinases (MMP)-9, and MMP-12. Leptin and ObR antigen were evident in all AAAs, and, leptin, MMP-9, and MMP-12 mRNA was increased relative to age-matched non-dilated controls. To simulate in vivo local leptin synthesis, ApoE-/- mice were subjected to a para-visceral peri-aortic application of low-dose leptin. Leptin-treated aortas exhibited decreased TGFβ and increased MMP-9 mRNA levels 5 days after surgery, and ObR mRNA was up-regulated by day 28. Serial ultrasonography demonstrated accelerated regional aortic diameter growth after 28 days, correlating with local medial degeneration, increased MMP-9, MMP-12 and peri-adventitial macrophage clustering. Furthermore, the combination of local peri-aortic leptin and systemic angiotensin II administration augmented medial MMP-9 synthesis and aortic aneurysm size.
Leptin is locally synthesized in human AAA wall. Para-visceral aortic leptin in ApoE-/- mice induces local medial degeneration, and augments angiotensin II-induced AAA, thus suggesting novel mechanistic links between leptin and AAA formation.
abdominal aortic aneurysm; leptin; vessel wall remodeling; transforming growth factor β; angiotensin II
Apolipoprotein A-II (apo A-II) is the second major apolipoprotein of HDLs, yet its pathophysiological roles in the development of atherosclerosis remain unknown. We aimed to examine whether apo A-II plays any role in atherogenesis and if so, to elucidate the mechanism involved.
Methods and Results
We compared the susceptibility of human apo A-II transgenic (Tg) rabbits to cholesterol diet-induced atherosclerosis with non-Tg littermate rabbits. Tg rabbits developed significantly less aortic and coronary atherosclerosis than their non-Tg littermates while total plasma cholesterol levels were similar. Atherosclerotic lesions of Tg rabbits were characterized by reduced macrophages and smooth muscle cells and apo A-II immunoreactive proteins were frequently detected in the lesions. Tg rabbits exhibited low levels of plasma CRP and blood leukocytes compared to non-Tg rabbits and HDLs of Tg rabbit plasma exerted stronger cholesterol efflux activity and inhibitory effects on the inflammatory cytokine expression by macrophages in vitro than HDLs isolated from non-Tg rabbits. In addition, β-VLDLs of Tg rabbits were less sensitive to copper-induced oxidation than β-VLDLs of non-Tg rabbits.
These results suggest that enrichment of apo A-II in HDL particles has atheroprotective effects and apo A-II may become a target for the treatment of atherosclerosis.
Atherosclerosis; Apolipoproteins; Lipoproteins; Metabolism; Transgenic rabbits
Animals deficient for connexin 45 (Cx45), Cx43, or Cx40 and Cx37 all suffer embryonic or post-natal lethal vascular phenotypes. We developed an in vitro model of blood vessel assembly to dissect the specific roles of these connexins in this process. Previously, we showed that heterocellular gap junction channel formation between endothelial and mesenchymal cells is required for TGF-β activation and endothelial-induced mural cell differentiation, and that Cx43-containing channels support these processes. Developmental studies suggest that Cx45 is required for mural cell development during embryogenesis, although its exact role was not delineated.
The focus of this study was to investigate the role of Cx45 in endothelial-induced mural cell differentiation.
METHODS AND RESULTS
We created mural cell precursors that stably express only Cx45 in Cx43-deficient mesenchymal cells (ReCx45), and used our in vitro model of blood vessel assembly to assess the capacity of this Cx to support endothelial-induced mural cell differentiation. Lucifer Yellow dye injection and dual whole-cell patch clamping revealed that functional gap junctions exhibiting properties of Cx45-containing channels formed amongst ReCx45 transfectants, and between ReCx45 and endothelial cells. Heterocellular Cx45-containing gap junction channels enabled TGF-β activation, and promoted the upregulation of mural cell-specific proteins in the mesenchymal precursors.
These studies reveal a critical role for Cx45 in the regulation of endothelial-induced mural cell differentiation, which is consistent with the phenotype of Cx45-deficient embryos that exhibit dysregulated TGF-β and lack mural cell development.
Gap junction; connexin; mural cell development; TGF-β; endothelial cell
Alterations in the metabolic control of lipid and glucose homeostasis predispose an individual to develop cardiometabolic diseases such as type 2-diabetes and atherosclerosis. Work over the last years has suggested that miRNAs play an important role in regulating these physiological processes. The contribution of miRNAs in regulating metabolism is exemplified by miR-33, an intronic miRNA encoded in the Srebp genes. miR-33 controls cellular cholesterol export and fatty acid degradation while its host genes stimulate cholesterol and fatty acid synthesis. Other miRNAs, such as miR-122, also play a critical role in regulating lipid homeostasis by controlling cholesterol synthesis and lipoprotein secretion in the liver. This review article summarizes the recent findings in the field, highlighting the contribution of miRNAs in regulating lipid and glucose metabolism. We will also discuss how the modulation of specific miRNAs may be a promising strategy to treat metabolic diseases.
On the luminal surface of injured arteries, platelet activation and leukocyte-platelet interactions are critical for the initiation and progression of arterial restenosis. The transcription factor nuclear factor kappa B (NF-κB) is a critical molecule in platelet activation. Here, we investigated the role of the platelet NF-κB pathway in forming arterial neointima after arterial injury.
Methods and Results
We performed carotid artery wire injuries in LDL receptor-deficient (LDLR–/–) mice with a platelet-specific deletion of IκB kinase beta (IKKβ) (IKKβfl/fl/PF4cre/LDLR–/–) and in control mice (IKKβfl/fl/LDLR–/–). The size of the arterial neointima was 61% larger in the IKKβfl/fl/PF4cre/LDLR–/– mice compared to the littermate control IKKβfl/fl/LDLR–/– mice. Compared to the control mice, the IKKβfl/fl/PF4cre/LDLR–/– mice exhibited more leukocyte adhesion at the injured area. The extent of GPIbα shedding after platelet activation was compromised in the IKKβ-deficient platelets. This effect was associated with a low level of the active form of A Disintegrin And Metalloproteinase 17 (ADAM17), the key enzyme involved in mediating GPIbα shedding in activated IKKβ-deficient platelets.
Platelet IKKβ deficiency increases the formation of injury-induced arterial neointima formation. Thus, NF-κB-related inhibitors should be carefully evaluated for use in patients after an arterial intervention.
restenosis; arterial injury; platelets; leukocytes; NF-κB
Estradiol (E2) regulates gene transcription by activating estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Many of the genes regulated by E2 via ERs are repressed, yet the molecular mechanisms that mediate E2-induced gene repression are currently unknown. We hypothesized that E2, acting through ERs, regulates expression of microRNAs (miRs) leading to repression of expression of specific target genes.
Methods and Results
Here, we report that E2 significantly up-regulates the expression of 26 miRs and down-regulates the expression of 6 miRs in mouse aorta. E2 mediated up-regulation of one of these miRs, miR-203, was chosen for further study. In cultured vascular smooth muscle cells (VSMC), E2-mediated up-regulation of miR-203 is mediated by ERα (but not ERβ) via transcriptional up-regulation of the primary miR. We demonstrate that the transcription factors Zeb-1 and AP-1 play critical roles in mediating E2-induced up-regulation of miR-203 transcription. We show further that miR-203 mediates E2-induced repression of Abl1 and p63 protein abundance in VSMC. Finally, knocking-down miR-203 abolishes E2-mediated inhibition of VSMC proliferation, and over-expression of miR-203 inhibits cultured VSMC proliferation, but not vascular endothelial cell proliferation.
Our findings demonstrate that E2 regulates expression of miRs in the vasculature, and support that ER-dependent induction of miRs is a mechanism for E2-mediated gene repression. Furthermore, our findings demonstrate that miR-203 contributes to E2-induced inhibition of VSMC proliferation and highlight the potential of miR-203 as a therapeutic agent in the treatment of proliferative cardiovascular diseases.
estrogen; gene regulation; microRNA; muscle, smooth; proliferation
Atherosclerosis; HDL; apolipoprotein A-II; cholesterol
To determine whether changes in standard and novel risk factors during the ACT NOW trial explained the slower rate of CIMT progression with pioglitazone treatment in persons with prediabetes.
Methods and Results
CIMT was measured in 382 participants at the beginning and up to three additional times during follow-up of the ACT NOW trial. During an average follow-up of 2.3 years, the mean unadjusted annual rate of CIMT progression was significantly (P=0.01) lower with pioglitazone treatment (4.76 × 10−3 mm/year, 95% CI, 2.39 × 10−3 – 7.14 × 10−3 mm/year) compared with placebo (9.69 × 10−3 mm/year, 95% CI, 7.24 × 10−3 – 12.15 × 10−3 mm/year). High-density lipoprotein cholesterol, fasting and 2-hour glucose, HbA1c, fasting insulin, Matsuda insulin sensitivity index, adiponectin and plasminogen activator inhibitor-1 levels improved significantly with pioglitazone treatment compared with placebo (P < 0.001). However, the effect of pioglitazone on CIMT progression was not attenuated by multiple methods of adjustment for traditional, metabolic and inflammatory risk factors and concomitant medications, and was independent of changes in risk factors during pioglitazone treatment.
Pioglitazone slowed progression of CIMT, independent of improvement in hyperglycemia, insulin resistance, dyslipidemia and systemic inflammation in prediabetes. These results suggest a possible direct vascular benefit of pioglitazone.
Carotid atherosclerosis progression; Impaired glucose tolerance; Insulin resistance; Inflammation; Pioglitazone
This review describes the effect of lifestyle change or metformin compared to standard care on incident diabetes and cardiometabolic risk factors in the Diabetes Prevention Program (DPP) and its Outcome Study. The DPP was a randomized, controlled clinical trial of intensive lifestyle and metformin treatments versus standard care in 3234 subjects at high risk for diabetes. At baseline, hypertension was present in 28%, and 53% had the metabolic syndrome, with considerable variation in risk factors by age, gender and race. Over 2.8 years, diabetes incidence fell by 58% and 31% in the lifestyle and metformin groups respectively, and metabolic syndrome prevalence fell by a third with lifestyle change but was not reduced by metformin. In placebo- and metformin-treated subjects the prevalence of hypertension and dyslipidemia increased during the DPP, while lifestyle intervention slowed these increases significantly. During long term follow up using modified interventions, diabetes incidence decreased to about 5% per year in all groups. This was accompanied by significant improvement in CVD risk factors over time in all treatment groups, in part associated with increasing use of lipid lowering and antihypertensive medications. Thus a program of lifestyle change significantly reduced diabetes incidence and metabolic syndrome prevalence in subjects at high risk for diabetes. Metformin had more modest effects.
prediabetes; metabolic syndrome; lifestyle; metformin
Genetic studies might provide new insights into the biological
mechanisms underlying lipid metabolism and risk of CAD. We therefore
conducted a genome-wide association study to identify novel genetic
determinants of LDL-c, HDL-c and triglycerides.
Methods and results
We combined genome-wide association data from eight studies,
comprising up to 17,723 participants with information on circulating lipid
concentrations. We did independent replication studies in up to 37,774
participants from eight populations and also in a population of Indian Asian
descent. We also assessed the association between SNPs at lipid loci and
risk of CAD in up to 9,633 cases and 38,684 controls.
We identified four novel genetic loci that showed reproducible
associations with lipids (P values 1.6 × 10−8 to
3.1 × 10−10). These include a potentially
functional SNP in the SLC39A8 gene for HDL-c, a SNP near
the MYLIP/GMPR and PPP1R3B genes for LDL-c
and at the AFF1 gene for triglycerides. SNPs showing strong
statistical association with one or more lipid traits at the
APOE-C1-C4-C2 cluster, LPL,
ZNF259-APOA5-A4-C3-A1 cluster and
TRIB1 loci were also associated with CAD risk (P values
1.1 × 10−3 to 1.2 ×
We have identified four novel loci associated with circulating
lipids. We also show that in addition to those that are largely associated
with LDL-c, genetic loci mainly associated with circulating triglycerides
and HDL-c are also associated with risk of CAD. These findings potentially
provide new insights into the biological mechanisms underlying lipid
metabolism and CAD risk.
lipids; lipoproteins; genetics; epidemiology
To investigate the role of Pyk2, a proline-rich non-receptor tyrosine kinase, in GPCR agonist, thrombin-induced HASMC growth and migration and injury-induced vascular wall remodeling.
Methods and results
Thrombin, a GPCR agonist, activated Pyk2 in a time-dependent manner and inhibition of its stimulation attenuated thrombin-induced HASMC migration and proliferation. Thrombin also sequentially activated Gab1, p115 RhoGEF, Rac1, RhoA and Pak1 and interference with stimulation of these molecules attenuated thrombin-induced HASMC migration and proliferation. In addition, adenovirus-mediated expression of dominant negative Pyk2 inhibited thrombin-induced Gab1, p115 RhoGEF, Rac1, RhoA and Pak1 stimulation. Balloon injury also caused activation of Pyk2, Gab1, p115 RhoGEF, Rac1, RhoA and Pak1 in the carotid artery of rat and these responses were sensitive to inhibition by dominant negative Pyk2. Furthermore, inhibition of Pyk2 activation resulted in reduced recruitment of SMC onto the luminal surface and their proliferation in the intimal region leading to suppression of neointima formation.
Together, these results demonstrate for the first time that Pyk2 plays a crucial role in GPCR agonist thrombin-induced HASMC growth and migration as well as balloon injury-induced neointima formation.
Syndecan 4 (Sdc4) modulates signal transduction and regulates activity of protein channels. Sdc4 is essential for the regulation of cellular permeability. We hypothesized that Sdc4 may regulate transient receptor potential canonical 6 (TRPC6) channels, a determinant of glomerular permeability, in a RhoA/Rho-associated protein kinase-dependent manner.
Methods and Results
Sdc4 knockout (Sdc4−/−) mice showed increased glomerular filtration rate and ameliorated albuminuria under baseline conditions and after bovine serum albumin overload (each P<0.05). Using reverse transcription–polymerase chain reaction and immunoblotting, Sdc4−/− mice showed reduced TRPC6 mRNA by 79% and TRPC6 protein by 82% (each P<0.05). Sdc4−/− mice showed an increased RhoA activity by 87% and increased phosphorylation of ezrin in glomeruli by 48% (each P<0.05). Sdc4 knockdown in cultured podocytes reduced TRPC6 gene expression and reduced the association of TRPC6 with plasma membrane and TRPC6-mediated calcium influx and currents. Sdc4 knockdown inactivated negative regulatory protein Rho GTPase activating protein by 33%, accompanied by a 41% increase in RhoA activity and increased phosphorylation of ezrin (P<0.05). Conversely, overexpression of Sdc4 reduced RhoA activity and increased TRPC6 protein and TRPC6-mediated calcium influx and currents.
Our results establish a previously unknown function of Sdc4 for regulation of TRPC6 channels and support the role of Sdc4 for the regulation of glomerular permeability.
receptors; signal transduction
mir-33; atherosclerosis; LDLr; HDL-C; antisense