Proteins and cytoplasmic organelles undergo degradation and recycling via autophagy; its role in patients with chronic kidney disease (CKD) is still unclear. We hypothesize that impaired kidney function causes autophagy activation failure.
We included 60 patients with stage 5 CKD and 30 age- and sex-matched healthy subjects as controls. Patients with conditions that could affect autophagy were excluded. Leukocytes were isolated and analyzed from peripheral blood samples collected after overnight fasting and 2 h after breakfast.
Overnight fasting induced conversion of microtubule-associated protein-1 light chain 3 I to II (γLC3) and increased mRNA levels of the autophagy-related gene 5 (Atg5) and Beclin-1 in healthy subjects, which were nearly absent in CKD patients (p = 0.0001). Moreover, no significant difference in autophagy activation was observed between CKD patients with or without hemodialysis. Correlation studies showed that γLC3 was negatively associated with the left atrium size. Changes in Atg5 transcript levels were negatively associated with the left ventricular end-diastolic diameter, and changes in Beclin-1 transcript levels were negatively associated with the mitral inflow E- and A-wave sizes.
These data suggest that CKD patients have impaired autophagy activation, which cannot be reversed with hemodialysis and is closely related to their cardiac abnormalities.
Autophagy; Renal failure; Cardiovascular diseases; Light chain 3
Little is known about long-term outcomes among patients who receive percutaneous angioplasty (PTA) for peripheral artery disease (PAD) then undergo below-knee or above-knee amputations. We sought to determine clinical outcomes associated with below-knee or above-knee amputation, along with possible explanatory factors and treatment strategies.
Using data from Taiwan’s National Health Insurance Research Database from 1997 to 2010, 7,568 adult patients were divided into three groups: lower extremity preserved (LE), below-knee amputation (BK) and above-knee amputation (AK). We assessed outcomes including major adverse cardiovascular events (MACE) and associated risk factors.
Overall MACE was significantly higher in the AK group compared to the LE and BK groups, over a mean follow-up of 2.45 years (hazard ratio [HR]: 1.81; 95% confidence interval [CI]: 1.50–2.18 for AK vs. LE; HR: 1.67; 95% CI: 1.36–2.06 for AK vs. BK). However MACE were similar for the BK and LE groups (HR: 1.08; 95% CI: 0.98–1.20). Overall mortality was highest in the AK group (HR: 1.65, 95% CI: 1.34–2.04 for AK vs. BK). As for patient characteristics, atrial fibrillation was more prevalent in the AK group than in the BK group (17% vs. 7%). Independent risk factors associated with death after above- or below-knee amputation included advanced age, heart failure, dialysis, male gender and high patient volume.
The MACE rate was highest in the AK group, whereas the LE and BK groups were similar in this regard. Furthermore, overall mortality increased with larger area of amputation.
Data from a large patient population regarding very long-term outcomes after BMS implantation are inadequate. This study aimed to evaluate the very long-term (8–17 years) clinical and long-term (3–5 years) angiographic outcomes after intracoronary bare-metal stenting (BMS).
Methods and Results
From the Cardiovascular Atherosclerosis and Percutaneous TrAnsluminal INterventions (CAPTAIN) registry, a total of 2391 patients with 2966 lesions treated with 3190 BMSs between November 1995 and May 2004 were evaluated. In total, 1898 patients with 2364 lesions, and 699 patients with 861 lesions underwent 6-month and 3- to 5- year angiographic follow-up, respectively. During a mean follow-up period of 149±51 months, 18.6% of the patients died (including 10.8% due to cardiac death), 6.1% developed reinfarction, 16.2% had target lesion revascularization (including 81% of the patients within the first year), 14.5% underwent new lesion stenting (including 72% of the patients after 3 years), 2.4% underwent coronary bypass surgery, and 1.6% had definite stent thrombosis. The overall cardiovascular event-free survival rate was 58.5%. The 6-month angiographic study indicated a 20% restenosis rate. The minimal luminal diameter increased from 0.65±0.44 mm to 3.02±0.46 mm immediately after stenting, decreased to 2.06±0.77 mm at the 6-month follow-up, and increased to 2.27±0.68 mm at the 3- to 5-year follow-up.
This study provides clinical and angiographic results from a large population of patients who underwent BMS implantations after a long-term follow-up period (149±51 months). The progression of coronary atherosclerosis developed over time, and presented with new lesion required stent implantation. The follow-up angiographic findings reconfirmed the late and sustained improvement in luminal diameter between 6 months and 3–5 years.
Obesity is reaching pandemic proportions in Western society. It has resulted in increasing health care burden and decreasing life expectancy. Obesity is a complex, chronic disease, involving decades of pathophysiological changes and adaptation. Therefore, it is difficult ascertain the exact mechanisms for this long-term process in humans. To circumvent some of these issues, several surrogate models are available, including murine genetic loss-of-function mutations, transgenic gain-of-function mutations, polygenic models, and different environmental exposure models. The mouse model of diet-induced obesity has become one of the most important tools for understanding the interplay of high-fat Western diets and the development of obesity. The diet-induced obesity model closely mimics the increasingly availability of the high-fat/high-density foods in modern society over the past two decades, which are main contributors to the obesity trend in human. This model has lead to many discoveries of the important signalings in obesity, such as Akt and mTOR. The chapter describes protocols for diet induced-obesity model in mice and protocols for measuring insulin resistance and sensitivity.
Obesity; High-fat diet; Insulin resistance; Body weight; Metabolism
Exercise limitation is an important issue in patients with chronic obstructive pulmonary disease (COPD), and it often co-exists with obstructive sleep apnoea (overlap syndrome). This study examined the effects of nocturnal continuous positive airway pressure (CPAP) treatment on walking capacity in COPD patients with or without obstructive sleep apnoea.
Forty-four stable moderate-to-severe COPD patients were recruited and completed this study. They all underwent polysomnography, CPAP titration, accommodation, and treatment with adequate pressure. The incremental shuttle walking test was used to measure walking capacity at baseline and after two nights of CPAP treatment. Urinary catecholamine and heart rate variability were measured before and after CPAP treatment.
After two nights of CPAP treatment, the apnoea-hypopnoea index and oxygen desaturation index significantly improved in both overlap syndrome and COPD patients, however these changes were significantly greater in the overlap syndrome than in the COPD group. Sleep architecture and autonomic dysfunction significantly improved in the overlap syndrome group but not in the COPD group. CPAP treatment was associated with an increased walking capacity from baseline from 226.4 ± 95.3 m to 288.6 ± 94.6 m (P < 0.05), and decreased urinary catecholamine levels, pre-exercise heart rate, oxygenation, and Borg scale in the overlap syndrome group. An improvement in the apnoea-hypopnoea index was an independent factor associated with the increase in walking distance (r = 0.564).
Nocturnal CPAP may improve walking capacity in COPD patients with overlap syndrome.
Chronic obstructive pulmonary disease; Obstructive sleep apnoea; Walking capacity; Autonomic dysfunction; Continuous positive airway pressure
Patients with chronic kidney disease have abnormal energy expenditure and metabolism. The mechanisms underlying altered energy expenditure in uremia are unknown and remain to be elucidated. Irisin is a peroxisome proliferator-activated receptor γ coactivator 1-α–dependent myokine, and it increases energy expenditure in the absence of changes in food intake or activity. We hypothesize that chronic kidney disease patients have altered irisin levels. We measured resting irisin levels in 38 patients with stage 5 chronic kidney disease and in 19 age- and sex-matched normal subjects. Plasma irisin levels were significantly decreased in chronic kidney disease patients (58.59%; 95% CI 47.9%–69.2%, p<0.0001). The decrease in irisin levels was inversely correlated with the levels of blood urea nitrogen and creatinine. Further association analysis revealed that irisin level is independently associated with high-density lipoprotein cholesterol level. Our results suggest that chronic kidney disease patients have lower than normal irisin levels at rest. Furthermore, irisin may play a major role in affecting high-density lipoprotein cholesterol levels and abnormal energy expenditure in chronic kidney disease patients.
Long lesions have been associated with adverse outcomes in percutaneous coronary interventions with bare metal stents (BMS). However, the exact impact of lesion length on the short- and long-term outcomes of drug-eluting stent (DES) implantations is not as clear.
Methods and Results
This study compared the impact of lesion length on angiographic and clinical outcomes of BMS and DES in a single-center prospective registry. Lesion length was divided into tertiles. The primary endpoints were angiographically defined binary in-stent restenosis (ISR) rate and major adverse cardiac event (MACE). Of the 4,312 de novo lesions in 3,447 consecutive patients in the CAPTAIN registry, 2,791 lesions (of 2,246 patients) received BMS, and the remaining 1,521 lesions (of 1,201 patients) received DES. The mean follow-up duration was 4.5 years. The longer the lesion, the higher the ISR rate (14%, 18%, and 29%, p<0.001) and the lower the MACE-free survivals (p = 0.007) in the BMS group. However, lesion length showed no such correlation with ISR rates (4.7%, 3.3%, and 7.8%, p = 0.67) or MACE-free survivals (p = 0.19) in the DES group.
In our single-center prospective registry, lesion length defined in tertiles has no impact on the short-term (ISR) or long-term (MACE) outcomes of patients implanted with DES. In contrast, longer lesion correlates with higher ISR and MACE rates in BMS group.
Aging and age-related diseases can be viewed as the result of the lifelong accumulation of stress insults. The identification of mutant strains and genes which are responsive to stress and can alter longevity profiles provides new therapeutic targets for age-related diseases. Here we reported that a Drosophila strain with reduced expression of ribose-5-phosphate isomerase (rpi), EP2456, exhibits increased resistance to oxidative stress and enhanced lifespan. In addition, the strain also displays higher levels of NADPH. The knockdown of rpi in neurons by double-stranded RNA interference recapitulated the lifespan extension and oxidative stress resistance in Drosophila. This manipulation was also found to ameliorate the effects of genetic manipulations aimed at creating a model for studying Huntington’s disease by overexpression of polyglutamine in the eye, suggesting that modulating rpi levels could serve as a treatment for normal aging as well as for polyglutamine neurotoxicity.
ribose-5-phosphate isomerase; pentose phosphate pathway; neuron; oxidative stress; longevity; polyglutamine toxicity; Drosophila
The signaling mechanisms that regulate the recruitment of bone marrow (BM)-derived cells to the injured heart are not well known. Notch receptors mediate binary cell fate determination and may regulate the function of BM-derived cells. However, it is not known whether Notch1 signaling in BM-derived cells mediates cardiac repair following myocardial injury.
Methods and Results
Mice with postnatal cardiac-specific deletion of Notch1 exhibit similar infarct size and heart function following ischemic injury as control mice. However, mice with global hemizygous deletion of Notch1 (N1+/−) developed larger infarct size and worsening heart function. When the BM of N1+/− mice were transplanted into wild-type (WT) mice, infarct size and heart function were worsened and neovascularization in the infarct border area was reduced compared to WT mice transplanted with WT BM. In contrast, transplantation of WT BM into N1+/− mice lessened the myocardial injury observed in N1+/− mice. Indeed, hemizygous deletion of Notch1 in BM-derived cells leads to decreased recruitment, proliferation, and survival of mesenchymal stem cells (MSC). Compared to WT MSC, injection of N1+/− MSC into the infarcted heart leads to increased myocardial injury, whereas injection of MSC overexpressing Notch intracellular domain leads to decreased infarct size and improved cardiac function.
These findings indicate that Notch1 signaling in BM-derived cells is critical for cardiac repair, and suggest that strategies that increase Notch1 signaling in BM-derived MSC could have therapeutic benefits in patients with ischemic heart disease.
stem cells; gene therapy; myocardial infarction; angiogenesis
Clopidogrel is a prodrug that undergoes in vivo bioactivation to show its antiplatelet effects. Recent studies have shown that cytochrome P450 (CYP), ATP-binding cassette transporters (ABCB1), and paraoxonase-1 (PON1) play crucial roles in clopidogrel bioactivation. Here, we aim to determine the effects of genetic polymorphisms of CYP (CYP 2C19*2, CYP 2C19*3, and CYP 2C19*17), ABCB1 (ABCB1 3435C>T, ABCB1 129T>C, and ABCB1 2677G>T/A), and PON1 (PON1 Q192R, PON1 L55M, and PON1 108C>T) on the development of stent thrombosis (ST) in patients receiving clopidogrel after percutaneous coronary intervention (PCI).
Methods and Results
We evaluated the incidence of ST (0.64%) in 4964 patients who were recruited in the CAPTAIN registry (Cardiovascular Atherosclerosis and Percutaneous TrAnsluminal INterventions). The presence of genetic polymorphisms was assessed in 20 subjects who developed ST after aspirin and clopidogrel therapy and in 40 age- and sex-matched control subjects who did not develop ST, which was documented after 9 months of angiographic follow-up. ST was acute in 5 subjects, subacute in 7, late in 7, and very late in 1. The presence of CYP 2C19*2 allele was significantly associated with ST (adjusted odds ratio [ORadj]: 4.20, 95% confidence interval [CI], 1.263–9.544; P = 0.031). However, genetic variations in PON1 and ABCB1 showed no significant association with ST.
We conclude that in a Taiwanese population, PON1 Q192R genotype is not associated with ST development after PCI. However, the presence of CYP 2C19*2 allele is a risk factor for ST development after PCI.
Autophagy and molecular chaperones both regulate protein homeostasis and maintain important physiological functions. Atg7 (autophagy-related gene 7) and Hsp27 (heat shock protein 27) are involved in the regulation of neurodegeneration and aging. However, the genetic connection between Atg7 and Hsp27 is not known.
The appearances of the fly eyes from the different genetic interactions with or without polyglutamine toxicity were examined by light microscopy and scanning electronic microscopy. Immunofluorescence was used to check the effect of Atg7 and Hsp27 knockdown on the formation of autophagosomes. The lifespan of altered expression of Hsp27 or Atg7 and that of the combination of the two different gene expression were measured.
We used the Drosophila eye as a model system to examine the epistatic relationship between Hsp27 and Atg7. We found that both genes are involved in normal eye development, and that overexpression of Atg7 could eliminate the need for Hsp27 but Hsp27 could not rescue Atg7 deficient phenotypes. Using a polyglutamine toxicity assay (41Q) to model neurodegeneration, we showed that both Atg7 and Hsp27 can suppress weak, toxic effect by 41Q, and that overexpression of Atg7 improves the worsened mosaic eyes by the knockdown of Hsp27 under 41Q. We also showed that overexpression of Atg7 extends lifespan and the knockdown of Atg7 or Hsp27 by RNAi reduces lifespan. RNAi-knockdown of Atg7 expression can block the extended lifespan phenotype by Hsp27 overexpression, and overexpression of Atg7 can extend lifespan even under Hsp27 knockdown by RNAi.
We propose that Atg7 acts downstream of Hsp27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila.
Atg7; Hsp27; Neurodegeneration; Lifespan; Drosophila
Epidemiological studies have shown gender differences in the incidence of congestive heart failure (CHF); however, the role of estrogen in CHF is not known. We hypothesize that estrogen prevents cardiomyocyte apoptosis and the development of CHF.
Methods and Results
17β-Estradiol (E2, 0.5 mg/60-day release) or placebo pellet was implanted subcutaneously into male Gαq transgenic (Gq) mice. After 8 weeks, E2 treatment decreased the extent of cardiac hypertrophy and dilation and improved contractility in Gq mice. E2 treatment also attenuated nicotinamide adenine dinucleotide phosphate oxidase activity and superoxide anion production via downregulation of Rac1. This correlated with reduced apoptosis in cardiomyocytes of Gq mice. The antioxidative properties of E2 were also associated with increased expression of thioredoxin (Trx), Trx reductases, and Trx reductase activity in the hearts of Gq mice. Furthermore, the activation of apoptosis signal-regulating kinase 1 and its downstream effectors, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, in the hearts of Gq mice was reduced by long-term E2 treatment. Indeed, E2 (10 nmol/L)-treated cardiomyocytes were much more resistant to angiotensin II–induced apoptosis. These antiapoptotic and cardioprotective effects of E2 were blocked by an estrogen receptor antagonist (ICI 182,780) and by a Trx reductase inhibitor (azelaic acid).
These findings indicate that long-term E2 treatment improves CHF by antioxidative mechanisms that involve the upregulation of Trx and inhibition of Rac1-mediated attenuated nicotinamide adenine dinucleotide phosphate oxidase activity and apoptosis signal-regulating kinase 1 /c-Jun N-terminal kinase/p38 mitogen-activated protein kinase–mediated apoptosis. These results suggest that estrogen may be a useful adjunctive therapy for patients with CHF.
apoptosis; antioxidants; heart failure; hormones
Obesity and age are important risk factors for cardiovascular disease. However, the signaling mechanism linking obesity with age-related vascular senescence is unknown. Here we show that mice fed a high-fat diet show increased vascular senescence and vascular dysfunction compared to mice fed standard chow and are more prone to peripheral and cerebral ischemia. All of these changes involve long-term activation of the protein kinase Akt. In contrast, mice with diet-induced obesity that lack Akt1 are resistant to vascular senescence. Rapamycin treatment of diet-induced obese mice or of transgenic mice with long-term activation of endothelial Akt inhibits activation of mammalian target of rapamycin (mTOR)–rictor complex 2 and Akt, prevents vascular senescence without altering body weight, and reduces the severity of limb necrosis and ischemic stroke. These findings indicate that long-term activation of Akt-mTOR signaling links diet-induced obesity with vascular senescence and cardiovascular disease.
Alteration of the circadian rhythm and increased vascular senescence are linked to cardiovascular disease. Per2, a circadian gene, is known to regulate endothelium-dependent vasomotion. However, the mechanism by which Per2 affects endothelial function is unknown. We hypothesize that endothelial dysfunction in Per2 mutant (Per2m/m) mice is mediated in part by increased vascular senescence and impaired endothelial progenitor cell (EPC) function.
Methods and Results
Endothelial cells from Per2m/m mice exhibit increased protein kinase Akt signaling, greater senescence, and impaired vascular network formation and proliferation. Indeed, Per2m/m mice have impaired blood flow recovery and developed autoamputation of the distal limb when subjected to hind-limb ischemia. Furthermore, matrigel implantation into Per2m/m mice resulted in less neovascularization. Because EPCs contribute to angiogenesis, we studied the role of Per2 in these cells using bone marrow transplantation. Basal EPC levels were similar between wild-type and Per2m/m mice. However, compared with wild-type bone marrow transplantation mice, EPC mobilization was impaired in Per2m/m bone marrow transplantation mice in response to ischemia or VEGF stimulation. Bone marrow transplantation or infusion of wild-type EPC restored blood flow recovery and prevented autoamputation in Per2m/m mice.
These findings indicate that mutation of Per2 causes Akt-dependent senescence and impairs ischemia-induced revascularization through the alteration of EPC function.
angiogenesis; circadian rhythm; endothelium; ischemia; senescence
Plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolytic activity and mediates vascular atherothrombotic disease. Endothelial cells (ECs) synthesize and secrete PAI-1, but the intracellular signaling pathways that regulate PAI-1 expression are not entirely known. We hypothesize that the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt pathway, which regulates endothelial function, could modulate PAI-1 expression in ECs. Cultured bovine aortic and human saphenous vein ECs were stimulated with TNF-α, ANG II, insulin, or serum, and PAI-1 expression was determined by Northern and Western analyses. Inhibition of PI3K with wortmannin or LY-294002 enhanced PAI-1 expression induced by these extracellular stimuli. Similarly, overexpression of a dominant-negative mutant of PI3K or Akt increased TNF-α- and insulin-induced PAI-1 expression. The increase in PAI-1 was due to transcriptional and posttranscriptional mechanisms as PI3K inhibitors increased PAI-1 promoter activity and mRNA stability. The induction of PAI-1 by TNF-α and insulin is mediated, in part, by ERK and p38 MAPK. PI3K inhibitors augmented TNF-α- and insulin-induced phosphorylation of these MAPKs. Simvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, which is known to activate PI3K/Akt, blocks TNF-α- and insulin-induced PAI-1 expression. Treatment with PI3K inhibitors reversed the inhibitor effects of simvastatin on TNF-α- and insulin-induced PAI-1 expression. These findings indicate that the PI3K/Akt pathway acts as a negative regulator of PAI-1 expression in ECs, in part, through the downregulation of MAPK pathways. These results suggest that factors that activate the PI3K/Akt pathway in ECs may have therapeutic benefits for atherothrombotic vascular disease.
mitogen-activated protein kinase; cardiovascular disease; statins; cholesterol
Rho GTPase and its downstream target, Rho-associated kinase (ROCK), have been implicated in diverse cardiovascular diseases such as cardiac hypertrophy. However, pharmacological inhibitors of ROCK are not entirely specific, nor can they discriminate between the ROCK isoforms ROCK1 and ROCK2. To determine the specific role of ROCK1 in the development of cardiac hypertrophy, we generated ROCK1+/− haploinsufficient mice and determined whether cardiac hypertrophy and remodeling are decreased in these mice.
Methods and Results
Litters of ROCK1−/− mice on C57Bl/6 background were markedly underrepresented, suggesting lethality in utero or postnatally. ROCK1+/− mice, however, are viable and fertile with no obvious phenotypic abnormalities. Basal blood pressure, heart rate, and cardiac dimension and function in ROCK1+/− mice were similar to those in wild-type (WT) littermates. Infusion of angiotensin II (400 ng · kg−1 · min−1 for 28 days) or treatment with NG-nitro-l-arginine methyl ester (1 mg/mL in drinking water for 28 days) caused similar increases in systolic blood pressure, left ventricular wall thickness, left ventricular mass, ratio of heart weight to tibial length, and cardiomyocyte size in ROCK1+/− mice and WT littermates. In contrast, perivascular fibrosis in hearts was increased to a lesser extent in ROCK1+/− mice compared with WT littermates. This was associated with decreased expression of transforming growth factor-β, connective tissue growth factor, and type III collagen. In addition, perivascular fibrosis induced by transaortic constriction or myocardial infarction was decreased in ROCK1+/− mice compared with WT littermates.
These findings indicate ROCK1 is critical for the development of cardiac fibrosis, but not hypertrophy, in response to various pathological conditions and suggest that signaling pathways leading to the hypertrophic and profibrotic response of the heart are distinct.
blood pressure; hypertension; hypertrophy; remodeling; angiotensin
Statins inhibit the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which is required for cholesterol biosynthesis, and are beneficial in the primary and secondary prevention of cardiovascular disease. Most of the benefits of statin therapy are owing to the lowering of serum cholesterol levels. However, by inhibiting HMG-CoA reductase, statins can also inhibit the synthesis of isoprenoids, which are important lipid attachments for intracellular signaling molecules, such as Rho, Rac and Cdc42. Therefore, it is possible that statins might exert cholesterol-independent or ‘pleiotropic’ effects through direct inhibition of these small GTP-binding proteins. Recent studies have shown that statins might have important roles in diseases that are not mediated by cholesterol. Here, we review data from recent clinical trials that support the concept of statin pleiotropy and provide a rationale for their clinical importance.