Overweight patients with cardiovascular disease (CVD) tend to survive longer than normal-weight patients, a phenomenon known as the "obesity paradox". The phenotypic characteristics of adipose distribution in these patients (who survive longer) often reveal a larger proportion of subcutaneous white adipose tissue (scWAT), suggesting that the presence of scWAT is negatively associated with all-cause mortality and that scWAT appears to provide protective benefits in patients facing unhealthy states. Exercise-mediated browning is a crucial aspect of the benign remodeling process of adipose tissue (AT). Reduced accumulation, reduced inflammation, and associated adipokine secretion are directly related to the reduction in CVD mortality. This paper summarized the pathogenetic factors associated with AT accumulation in patients with CVD and analyzed the possible role and pathway of exercise-mediated adipose browning in reducing the risk of CVD and CVD-related mortality. It is suggested that exercise-mediated browning may provide a new perspective on the "obesity paradox"; that is, overweight CVD patients who have more scWAT may gain greater cardiovascular health benefits through exercise.

Previous reports have described a statistical association between high-density lipoproteins (HDL) subclasses and the expression of genes coding for pro-calcifying proteins in the epicardial adipose tissue of patients with coronary artery disease (CAD) and aortic valvular stenosis (AVS). These results suggest a causal relationship between HDL and the regulation of gene expression in epicardial adipose tissue. However, there is no experimental evidence that supports this causal relationship. Therefore, we explored the effect of HDL isolated from CAD or AVS patients on the expression of OPN, BMP2, and BMP4, genes coding for proteins related to calcification, osteopontin, and bone morphogenetic proteins -2 and -4, respectively, and LEP, UCP, and PER, coding for leptin, uncoupling protein-1, and perilipin-2, respectively, proteins that confer phenotypic characteristics to adipocytes. The experiments were performed using a novel model of cardiac adipocytes differentiated in vitro from stromal cells of rabbit cardiac adipose tissue. AVS or CAD patients' HDL differentially modulated the expression of BMP4 and LEP, whereas HDL from both kinds of patients upregulated the OPN gene expression. A high concentration of triglycerides associated to small HDL and a higher concentration of phospholipids of large HDL from CAD patients than those from AVS individuals were the most remarkable structural differences. Finally, we demonstrated that cholesterol from reconstituted HDL was internalized to the adipocytes. The regulation of genes related to the secretory activity of cardiac adipocytes mediated by HDL has clinical implications as a potential therapeutic target for the prevention and treatment of CAD and AVS. In summary, the HDL isolated from the CAD and AVS patients differentially regulated gene expression in adipocytes by a mechanism that seems to be dependent on HDL lipid internalization to the cells and structural characteristics of the lipoproteins.

Current research has found that adipose tissue is not only involved in energy metabolism, but also a highly active endocrine organ that secretes various adipokines, including adiponectin, leptin, resistin and apelin, which are involved in the regulation of physiology and pathology of tissues and organs throughout the body. With the yearly increasing incidence, obesity has become a risk factor for a variety of pathological changes, including inflammation and metabolic syndrome in various system (endocrine, circulatory, locomotor and central nervous system). Thus these symptoms lead to multi-organ dysfunctions, including the heart, liver, kidneys, brain and joints. An in-depth summary of the roles of adipokines in the regulation of other tissues and organs can help to provide more effective therapeutic strategies for obesity-related diseases and explore potential therapeutic targets. Therefore, this review has retrospected the endocrine function of adipose tissue under obesity and the role of dysregulated adipokine secretion in related diseases and the underlying mechanisms, in order to provide a theoretical basis for targeting adipokine-mediated systemic dysregulation.

This research aimed to assess the correlation between the Adjusted Body Shape Index (ABSI) and the presence of abdominal aortic calcification (AAC) among middle-aged and older American adults.

Employing a cross-sectional design, this study analyzed data from the 2013-2014 National Health and Nutrition Examination Survey (NHANES), focusing on 3077 participants aged 40 and above. AAC detection was conducted using dual-energy X-ray absorptiometry (DXA). ABSI was determined based on waist circumference (WC), weight, and height data. The association between ABSI and AAC was examined through multiple linear regression, smoothed curve analysis, threshold effect evaluation, subgroup analysis, and interaction testing.

The study encompassed 3077 individuals aged 40 and above. Findings indicated a noteworthy positive relationship between ABSI and AAC when adjusting various covariates. Analysis of threshold effects identified a K-point at 0.0908, showing no significant effect to its left but a significant effect to its right. Further, subgroup and interaction analyses highlighted the ABSI-AAC connection specifically within different age groups and among individuals with diabetes.

Higher ABSI was correlated with higher AAC score.

Fibro-calcific aortic valve disease (FCAVD) is a progressive disorder characterized by the thickening and calcification of the aortic valve, eventually leading to aortic stenosis. Adiponectin and leptin, known for their anti-inflammatory and proinflammatory properties, respectively, have been implicated in cardiovascular diseases, but their associations with FCAVD are controversial. This meta-analysis aims to evaluate the relationships between adiponectin and leptin levels and FCAVD, particularly in patients with severe aortic stenosis (AS).

A systematic search was conducted across the PubMed, Scopus, and Web of Science databases to identify studies on adiponectin and leptin levels in FCAVD. The methodological quality of each study was assessed using the Newcastle-Ottawa Scale. Standardized mean differences (SMDs) and 95% confidence intervals (CIs) were calculated, and publication bias was evaluated using Egger's test and funnel plots.

Out of 191 articles identified, 10 studies involving 2360 patients (989 with FCAVD and 1371 controls) were included. The analysis suggested trends in the associations of lower adiponectin levels (SMD = -0.143, 95% CI: -0.344, 0.057, p = 0.161) and higher leptin levels (SMD = 0.175, 95% CI: -0.045, 0.395, p = 0.119) with FCAVD. The association remained a trend for low adiponectin but showed a significant correlation with high leptin in severe AS patients (SMD = 0.29, 95% CI: 0.036, 0.543, p = 0.025).

This meta-analysis indicates a potential association between elevated leptin levels and severe aortic stenosis, while the relationship with adiponectin levels remains inconclusive. These findings highlight the need for further and dedicated research to clarify the roles of these adipokines in the pathogenesis of FCAVD and their potential roles as biomarkers for disease progression.

Calcific Aortic Valve Disease (CAVD) is prevalent among the elderly as the most common valvular heart disease. Currently, no pharmaceutical interventions can effectively reverse or prevent CAVD, making valve replacement the primary therapeutic recourse. Extensive research spanning decades has contributed to the establishment of animal and in vitro cell models, which facilitates a deeper understanding of the pathophysiological progression and underlying mechanisms of CAVD. In this review, we provide a comprehensive summary and analysis of the strengths and limitations associated with commonly employed models for the study of valve calcification. We specifically emphasize the advancements in three-dimensional culture technologies, which replicate the structural complexity of the valve. Furthermore, we delve into prospective recommendations for advancing in vivo and in vitro model studies of CAVD.

To observe fat tissue and the expression of adipokines in rheumatic heart valves and explore the possible role of fat tissue and adipokines in the pathology of rheumatic heart disease (RHD).

In this retrospective study, a total of 29 patients who received mitral valve replacement surgery were included. The study group consisted of 25 patients with RHD while the control group consisted of 4 patients with secondary mitral insufficiency caused by coronary heart disease (CAD). The clinical data of the patients including medical history, age, body mass index (BMI), fasting blood glucose (FBG), total triglycerides (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), apolipoprotein(a) [apo(a)], apolipoprotein(b) [apo(b)] were collected and compared. Cardiac ultrasonography was used to assess valve conditions before surgery. The removed valves were collected. The hematoxylin-eosin (HE) staining, oil-red O staining, and Masson's trichrome staining were adopted to evaluate the histological changes in the mitral valve. Immunohistochemical (IMC) staining was performed to evaluate the expression of adiponectin, leptin, and chemerin.

There was no significant difference in general information and blood lipid levels between the two groups (all p > .05). Preoperative ultrasonography showed adipose tissue in the mitral valve of RHD patients. In the study group, rheumatic mitral valve samples showed thickening, adherence at the junction of the leaflets, calcification, and yellowish or fat mass by naked observation. The HE staining showed that there was calcification, inflammatory cell infiltration, fibrous tissue arranged disorder, and neovascularization. The oil-red O staining suggested fatty infiltration. Masson's trichrome staining suggested disorderly arrangement of collagen fiber and elastic fiber in rheumatic lesions, and the lesions were dominated by collagen fiber hyperplasia and less elastic fiber hyperplasia. The results of IMC indicated that chemerin was not expressed in valves of the control group. Most of the valve samples from the study group also did not show leptin and the leptin was seen in only a few rheumatic mitral valves with vascular hyperplasia. Adiponectin was not found in the valves of the study group and the control group.

Adipose tissue in the rheumatic mitral valve could be observed by ultrasound. The fat mass and adipokines existed in rheumatic mitral valves, the adipocytokine chemerin is involved in the progression of the pathology in RHD.

Adipokines have been associated with increased risk of cardiovascular disease. Our aim was to determine if adipokine levels are associated with coronary artery calcification (CAC) as well as all-cause mortality in incident dialysis patients.

In patients new to dialysis, we prospectively investigated the association of adiponectin, leptin and resistin with coronary artery calcification measured by ECG-gated computer tomography. Participants were recruited a median of two months after starting dialysis.

The mean age was 50.0 (12.6) years and 31.1% were women. About 42% percent had BMI > 30. Higher adiponectin levels were inversely associated with CAC progression as change in Agatston score [-155.1 (-267.9, -42.2), p = 0.008] or change in CAC volumes between scans [-2.8 (-4.9, -0.6), p = 0.01]. Higher leptin levels were associated with CAC progression [110.4 (34.3-186.6), p = 0.005]. Decreased leptin [HR 0.5 (0.3-0.9), p = 0.05] was associated with all-cause mortality in adjusted models. There was no significant association between all-cause mortality and adiponectin [1.4 (0.6-3.4), p = 0.4] or resistin [HR 1.7 (0.5-5.0), p = 0.4].

High adiponectin protects against CAC progression, but is not associated with increased all-cause mortality. Higher leptin, as well as higher leptin to adiponectin ratio, is associated with CAC progression. Lower leptin levels were associated with all-cause mortality. The association of adipokines and cardiovascular disease in individuals on dialysis is complex and requires further study.

The obesity epidemic is on the rise, and while it is well known that obesity is associated with an increase in cardiovascular risk factors such as type 2 diabetes mellitus, hypertension, and obstructive sleep apnea, recent data has highlighted that the degree and type of fat distribution may play a bigger role in the pathogenesis of cardiovascular disease (CVD) than body mass index (BMI) alone. We aim to review updated data on adipose tissue inflammation and distribution and CVD.

We review the pathophysiology of inflammation secondary to adipose tissue, the association of obesity-related adipokines and CVD, and the differences and significance of brown versus white adipose tissue. We delve into the clinical manifestations of obesity-related inflammation in CVD. We discuss the available data on heterogeneity of adipose tissue-related inflammation with a focus on subcutaneous versus visceral adipose tissue, the differential pathophysiology, and clinical CVD manifestations of adipose tissue across sex, race, and ethnicity. Finally, we present the available data on lifestyle modification, medical, and surgical therapeutics on reduction of obesity-related inflammation. Obesity leads to a state of chronic inflammation which significantly increases the risk for CVD. More research is needed to develop non-invasive VAT quantification indices such as risk calculators which include variables such as sex, age, race, ethnicity, and VAT concentration, along with other well-known CVD risk factors in order to comprehensively determine risk of CVD in obese patients. Finally, pre-clinical biomarkers such as pro-inflammatory adipokines should be validated to estimate risk of CVD in obese patients.

Evidence links osteoporosis and cardiovascular disease but the cellular and molecular mechanisms are unclear. Here we identify skeleton-secreted platelet-derived growth factor-BB (PDGF-BB) as a key mediator of arterial stiffening in response to aging and metabolic stress. Aged mice and those fed high-fat diet (HFD), relative to young mice and those fed normal chow food diet, respectively, had higher serum PDGF-BB and developed bone loss and arterial stiffening. Bone/bone marrow preosteoclasts in aged mice and HFD mice secrete an excessive amount of PDGF-BB, contributing to the elevated PDGF-BB in blood circulation. Conditioned medium prepared from preosteoclasts stimulated proliferation and migration of the vascular smooth muscle cells. Conditional transgenic mice, in which PDGF-BB is overexpressed in preosteoclasts, had 3-fold higher serum PDGF-BB concentration and developed simultaneous bone loss and arterial stiffening spontaneously at a young age. Conversely, in conditional knockout mice, in which PDGF-BB is deleted selectively in preosteoclasts, HFD did not affect serum PDGF-BB concentration; as a result, HFD-induced bone loss and arterial stiffening were attenuated. These studies confirm that preosteoclasts are a main source of excessive PDGF-BB in blood circulation during aging and metabolic stress and establish the role of skeleton-derived PDGF-BB as an important mediator of vascular stiffening.

Increasing adipose tissue mass in obesity directly correlates with elevated circulating leptin levels. Leptin is an adipokine known to play a role in numerous biological processes including regulation of energy homeostasis, inflammation, vascular function and angiogenesis. While physiological concentrations of leptin may exhibit multiple beneficial effects, chronically elevated pathophysiological levels or hyperleptinemia, characteristic of obesity and diabetes, is a major risk factor for development of atherosclerosis. Hyperleptinemia results in a state of selective leptin resistance such that while beneficial metabolic effects of leptin are dampened, deleterious vascular effects of leptin are conserved attributing to vascular dysfunction. Leptin exerts potent proatherogenic effects on multiple vascular cell types including macrophages, endothelial cells and smooth muscle cells; these effects are mediated via an interaction of leptin with the long form of leptin receptor, abundantly expressed in atherosclerotic plaques. This review provides a summary of recent in vivo and in vitro studies that highlight a role of leptin in the pathogenesis of atherosclerotic complications associated with obesity and diabetes.

Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on TSP-1 (thrombospondin-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. Approach and Results: Seventeen-weeks-old ApoE-/- and TSP-1-/-/ApoE-/- double knockout mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5 µg/g bwt, IP) once daily for 3 weeks. Using aortic root morphometry and en face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area, and collagen accumulation in aortic roots of ApoE-/- mice, shown via Oil red O, hematoxylin and eosin, and Masson trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE-/- mice. Moreover, these effects were concomitant to changes in VLDL (very low-density lipoprotein)-triglyceride and HDL (high-density lipoprotein)-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB (phospho-cyclic AMP response element-binding protein) accompanied with augmented smooth muscle-myosin heavy chain expression in aortic vessels of leptin-treated double knockout versus leptin-treated ApoE-/-; also confirmed in aortic smooth muscle cells from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE-/- on western diet, independent of plasma lipid alterations.

The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced vascular smooth muscle cell phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.

Adipose tissue (AT), a critical endocrine gland, is capable of producing and secreting abundant adipokines. Adipokines act on distant or adjacent organ tissues via paracrine, autocrine, and endocrine mechanism, which play attractive roles in the regulation of glycolipid metabolism and inflammatory response. Increasing evidence shows that adipokines can connect obesity with cardiovascular diseases by serving as promoters or inhibitors in vascular calcification. The chronic hypoxia in AT, caused by the adipocyte hypertrophy, is able to trigger imbalanced adipokine generation, which leads to apoptosis, osteogenic differentiation of vascular smooth muscle cells (VSMCs), vascular inflammation, and abnormal deposition of calcium and phosphorus in the vessel wall. The objectives of this review aim at providing a brief summary of the crucial influence of major adipokines on the formation and development of vascular calcification, which may contribute to better understanding these adipokines for establishing the appropriate therapeutic strategies to counteract obesity-associated vascular calcification.

In patients with advanced chronic kidney disease (CKD), the accumulation of uremic toxins, caused by a combination of decreased excretion secondary to reduced kidney function and increased generation secondary to aberrant expression of metabolite genes, interferes with different biological functions of cells and organs, contributing to a state of chronic inflammation and other adverse biologic effects that may cause tissue damage. Several uremic toxins have been implicated in severe vascular smooth muscle cells (VSMCs) changes and other alterations leading to vascular calcification (VC) and early vascular ageing (EVA). The above mentioned are predominant clinical features of patients with CKD, contributing to their exceptionally high cardiovascular mortality. Herein, we present an update on pathophysiological processes and mediators underlying VC and EVA induced by uremic toxins. Moreover, we discuss their clinical impact, and possible therapeutic targets aiming at preventing or ameliorating the harmful effects of uremic toxins on the vasculature.

The cardiorenal syndrome relates to the detrimental interplay between the vascular system and the kidney. The uremic milieu induced by reduced kidney function alters the phenotype of vascular smooth muscle cells (VSMC) and promotes vascular calcification, a condition which is strongly linked to cardiovascular morbidity and mortality. Biological mechanisms involved include generation of reactive oxygen species, inflammation and accelerated senescence. A better understanding of the vasotoxic effects of uremic retention molecules may reveal novel avenues to reduce vascular calcification in CKD. The present review aims to present a state of the art on the role of uremic toxins in pathogenesis of vascular calcification. Evidence, so far, is fragmentary and limited with only a few uremic toxins being investigated, often by a single group of investigators. Experimental heterogeneity furthermore hampers comparison. There is a clear need for a concerted action harmonizing and standardizing experimental protocols and combining efforts of basic and clinical researchers to solve the complex puzzle of uremic vascular calcification.

Background The pathophysiological process of calcific aortic valve disease (CAVD) is similar to that of atherosclerosis. Leptin accelerates the process of atherosclerosis. We sought to examine the relationship between leptin and CAVD. Methods and Results Serum leptin was measured in 397 consecutive patients undergoing standard transthoracic echocardiography and Doppler flow imaging. Multiple logistic regression analyses were used to assess the association between leptin and CAVD. Western blotting was performed to detect the expression of phosphorylated and total extracellular signal-regulated kinase. Serum leptin (median) was higher in 200 patients with CAVD than that in 197 non-CAVD controls (20.07 versus 9.03 ng/mL; P<0.01). Leptin correlated positively with age (r=0.37, P<0.01) and negatively with estimated glomerular filtration rate (r=-0.37, P<0.01). Multivariate analysis indicated that elevated leptin was an independent determinant for the presence of CAVD (P<0.01). Receiver-operating characteristic curve analysis of leptin to detect the presence of CAVD showed that the area under the curve was 0.74 (95% CI, 0.69-0.79; P<0.01). The diagnostic value of leptin for the detection of CAVD was higher among younger patients (aged ≤65 years) or those with at least mildly reduced renal function (estimated glomerular filtration rate ≤82.06 mL/min per 1.73 m²). The activation of extracellular signal-regulated kinase 1/2 was stronger in calcific aortic valves than in normal aortic valves. Conclusions Elevated leptin is associated with the presence of CAVD, especially among younger patients or those with renal dysfunction.

Vascular calcification is a complication of diseases and conditions such as chronic kidney disease, diabetes, and aging. Previous studies have demonstrated that high concentrations of inorganic phosphate (Pi) can induce oxidative stress and vascular smooth muscle cell calcification. KEAP1 (Kelch-like ECH-associated protein 1)/NF-E2-related factor 2 (NRF2) signaling has been shown to play important roles in protecting cells from oxidative stress. The current study aims to investigate the possible involvement of the KEAP1/NRF2/P62 -mediated antioxidant pathway in vascular calcification induced by high Pi levels. Exposure of vascular smooth muscle cells (VSMCs) to high Pi concentrations promoted the accumulation of reactive oxygen species (ROS) and the nuclear translocation of NRF2, along with an increase in P62 levels and a decrease in KEAP1 levels. A classic NRF2 activator, tert-butylhydroquinone (tBHQ), significantly decreased ROS levels and calcium deposition in VSMCs by promoting the nuclear translocation of NRF2 and upregulating P62 and KEAP1 expression. In contrast, silencing NRF2 and P62 with siRNAs increased the levels of ROS and calcium deposition in VSMCs. In conclusion, VSMC calcification can be alleviated by the activation of the KEAP1/NRF2/P62 antioxidative pathway, which could have a protective role when it is exogenously activated by tBHQ.

Vascular calcification is strongly associated with increased cardiovascular mortality and morbidity. C1q/TNF-related protein-13 (CTRP13) is a secreted adipokine that plays important roles in the cardiovascular system. However, the functional role of CTRP13 in the development of vascular calcification has yet to be explored. In this study, we collected blood samples from patients with chronic renal failure (CRF) and from rats with adenine-induced CRF. We found that the serum CTRP13 levels were decreased in patients and rats with CRF and were negatively associated with calcium deposition in the abdominal aorta. Compared to those of the controls, ectopic CTRP13 treatment significantly attenuated the calcium accumulation and alkaline phosphatase activity in the abdominal aorta of CRF rats, and β-glycerophosphate induced the formation of arterial rings and of vascular smooth muscle cells (VSMCs) and decreased the number of VSMCs that transitioned from a contractile to an osteogenic phenotype. The overexpression of Runx2 blocked CTRP13-reduced VSMC calcification. Mechanistically, CTRP13 repressed the phosphorylation of tristetraprolin (TTP), thereby activating TTP and increasing the TTP binding to the 3'untranslated region of the Runx2 mRNA, accelerating the Runx2 mRNA destabilization and degradation. In summary, these findings reveal that CTRP13 regulation is a novel method for the prevention of vascular calcification, representing a novel mechanism of the regulation of Runx2 expression in VSMCs.-Li, Y., Wang, W., Chao, Y., Zhang, F., Wang, C. CTRP13 attenuates vascular calcification by regulating Runx2.

Severe abdominal aortic calcification (AAC) points to high cardiovascular risk and leptin stimulates arterial calcification; however, clinical data on their association are scarce. We studied the link between serum leptin and AAC severity and progression, and the effect of smoking and lipid levels, on this association in men.

At baseline, 548 community-dwelling men aged 50-85 years underwent blood collection and lateral lumbar spine radiography. In 448 men, X-ray was repeated after 3 and 7.5 years. AAC was assessed using Kauppila's semiquantitative score. In multivariable models, high leptin was associated with higher odds of severe AAC (odds ratio [OR]=1.71 per SD, 95% confidence interval [CI]: 1.22-2.40). The odds of severe AAC were the highest in men who had elevated leptin levels and either were ever-smokers (OR=9.22, 95% CI: 3.43-24.78) or had hypertriglyceridemia (vs. men without these characteristics). Higher leptin was associated with greater AAC progression (OR=1.34 per SD, 95% CI: 1.04-1.74). The risk of AAC progression was the highest in men who had elevated leptin levels and either were current smokers or had high low-density lipoprotein-cholesterol levels (OR=5.91, 95% CI: 2.46-14.16 vs. men without these characteristics). These links remained significant after adjustment for baseline AAC and in subgroups defined according to smoking and low-density lipoprotein-cholesterol levels.

In older men, high leptin levels are associated with greater severity and rapid progression of AAC independent of smoking, low-density lipoprotein-cholesterol or triglycerides.

Calcifications occur during the development of healthy bone, and at the onset of calcific aortic-valve disease (CAVD) and many other pathologies. Although the mechanisms regulating early calcium deposition are not fully understood, they may provide targets for new treatments and for early interventions. Here, we show that two-photon excited fluorescence (TPEF) can provide quantitative and sensitive readouts of calcific nodule formation, in particular in the context of CAVD. Specifically, by means of the decomposition of TPEF spectral images from excised human CAVD valves and from rat bone prior to and following demineralization, as well as from calcific nodules formed within engineered gels, we identified an endogenous fluorophore that correlates with the level of mineralization in the samples. We then developed a ratiometric imaging approach that provides a quantitative readout of the presence of mineral deposits in early calcifications. TPEF should enable non-destructive, high-resolution imaging of three-dimensional tissue specimens for the assessment of the presence of calcification.

Atherosclerosis is a chronic inflammatory disease that is promoted, among others, by pro-inflammatory cytokines such as IL-1β and IL-18 produced by NLRP 3 inflammasome. Development of atherosclerotic lesions is also affected by leptin. Furthermore, inflammasome's action is interfered with other inflammatory diseases, like diabetes. On the other hand, colchicine is reported to act as anti-inflammatory agent inhibiting inflammasome's action and stabilizing atherosclerotic lesions. The purpose of this study is to investigate the effect of per os colchicine on the de novo formation of atherosclerotic lesions and on the levels of IL-18, leptin and insulin in cholesterol-fed rabbits.

Twenty-three male, 2 months old New Zealand White rabbits, were seperated in 3 groups and were fed with different types of diet for 7 weeks: standard, cholesterol 1% w/w and cholesterol 1% w/w plus colchicine 2 mg/kg body weight. Blood was collected for biochemical measurements and conduction of ELISA for leptin, IL-18 and insulin. Histologic examination of stained with eosin and hematoxylin aorta specimens was performed. Aortic intimal thickness was evaluated using image analysis. The statistical analysis included non-parametric tests: a) paired-sample Wilcoxon test, b) Spearman correlation coefficient and c) Kruscal-Wallis test.

Triglerycide levels were decreased in cholesterol plus colchicine group in the end of the experiment (p < 0.05), whereas the cholesterol group had increased levels. No statistical differences were observed in the levels of IL-18, leptin and insulin between groups. Likewise, there was neither any correlation between IL-18, leptin and intima thickness nor between IL-18 and glucose and between leptin and weight. In cholesterol and colchicine group there was a strong positive correlation between IL-18 and insulin levels in the 4th week (r s = .66, n = 10, p < 0.05), whereas in the 7th week this correlation became strong negative (r s = -.86, n = 10, p < 0.05). Finally, intima thickness in the ascending and thoracic aorta of the cholesterol and colchicine group was significantly greater than that of the other groups (p < 0.05).

Per os administration of colchicine did not influence atherosclerosis progression in cholesterol-fed rabbits, levels of IL-18, insulin and leptin. We encountered the attenuating role of colchicine on TG levels.

Calcific aortic valve disease (CAVD) affects 2-6% of the population over 65 years, and age, gender, smoking, overweight, dyslipidemia, diabetes contribute to the development of this disease. CAVD results, in part, from the osteoblast differentiation of human valvular interstitial cells (VICs). This study aims to elucidate the effects of leptin on osteoblast phenotype of VICs and the signalling pathways involved.

Patients who underwent aortic valve replacement for CAVD (n = 43) were included in this study. Patients with coronary artery disease (CAD) without CAVD (n = 129) were used as controls.

Patients with CAVD had higher serum leptin concentrations than CAD patients (p = 0.002). Leptin was found in calcific aortic valves, with higher concentrations in calcified versus non-calcified zones (p = 0.01). Chronic leptin stimulation of human VICs enhanced alkaline phosphatase (ALP) activity and ALP, BMP-2 and RUNX2 expression and decreased osteopontin expression. Moreover, inhibiting Akt or ERK during leptin stimulation lowered the expression of osteoblast markers in VIC.

Taken together, these findings indicate that leptin plays a critical role in CAVD development by promoting osteoblast differentiation of human aortic VICs in an Akt- and ERK-dependent manner. This study highlights the role of leptin in CAVD development, and further studies are needed to determine whether reducing circulating leptin levels or blocking leptin actions on VICs is efficient to slow CAVD progression.

Obesity is causally linked with the development of cardiovascular disorders. Accumulating evidence indicates that cardiovascular disease is the collateral damage of obesity-driven adipose tissue dysfunction that promotes a chronic inflammatory state within the organism. Adipose tissues secrete bioactive substances, referred to as adipokines, which largely function as modulators of inflammation. The microenvironment of adipose tissue will affect the adipokine secretome, having actions on remote tissues. Obesity typically leads to the upregulation of proinflammatory adipokines and the downregulation of anti-inflammatory adipokines, thereby contributing to the pathogenesis of cardiovascular diseases. In this review, we focus on the microenvironment of adipose tissue and how it influences cardiovascular disorders, including atherosclerosis and ischemic heart diseases, through the systemic actions of adipokines.

We previously reported that high pathophysiological concentrations of leptin, the adipocyte-secreted peptide, upregulate the expression of a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in vascular smooth muscle cells. Moreover, this regulation was found to occur at the level of transcription; however, the underlying molecular mechanisms remain unknown. The goal of the present study was to investigate the specific transcriptional mechanisms that mediate upregulation of TSP-1 expression by leptin. Primary human aortic smooth muscle cell cultures were transiently transfected with different TSP-1 gene (THBS1) promoter-linked luciferase reporter constructs, and luciferase activity in response to leptin (100 ng/ml) was assessed. We identified a long THBS1 promoter (-1270/+750) fragment with specific leptin response elements that are required for increased TSP-1 transcription by leptin. Promoter analyses, protein/DNA array and gel shift assays demonstrated activation and association of transcription factors, interferon regulatory factor-1 (IRF-1) and cAMP response element-binding protein (CREB), to the distal fragment of the THBS1 promoter in response to leptin. Supershift, chromatin immunoprecipitation, and coimmunoprecipitation assays revealed formation of a single complex between IRF-1 and CREB in response to leptin; importantly, recruitment of this complex to the THBS1 promoter mediated leptin-induced TSP-1 transcription. Finally, binding sequence decoy oligomer and site-directed mutagenesis revealed that regulatory elements for both IRF-1 (-1019 to -1016) and CREB (-1198 to -1195), specific to the distal THBS1 promoter, were required for leptin-induced TSP-1 transcription. Taken together, these findings demonstrate that leptin promotes a cooperative association between IRF-1 and CREB on the THBS1 promoter driving TSP-1 transcription in vascular smooth muscle cells.

Obesity has recently been linked with reduced fertility, and the mechanisms underpinning this effect are currently unknown. The adipokine leptin is dysregulated in obesity and affects reproductive tracts; therefore, we investigated the dose-dependent effects of leptin on Leydig cell function and spermatogenesis. Eight-week-old leptin-deficient obese (ob/ob) male mice were treated with subphysiological (0.1- or 0.5-mg/kg body weight [BW]/d) or physiological (3.0-mg/kg BW/d) doses of leptin or saline for 12 weeks (chronic treatment) or 72 hours (acute treatment). We then evaluated male reproductive function markers. Mean testis weight increased significantly in the 0.1- and 3.0-mg/kg BW/d groups compared with saline controls (both P < .05). Intratesticular testosterone levels relative to testis weight significantly increased in the 0.5-mg/kg BW/d group compared with saline controls (P < .05). FSH levels increased in a dose-dependent manner with leptin treatment, whereas LH levels did not change. Leptin treatment significantly up-regulated both mRNA and protein expression of the steroidogenic enzyme cytochrome P450 17A1. Spermatogenesis improved in leptin-treated animals. Significantly more seminiferous tubules were observed in stages I-VIII (P < .01), and there were fewer abnormal seminiferous tubule structures (P < .01). Acute treatment with physiological leptin doses partially improved male reproductive markers without changing BW. Administration of subphysiological to physiological doses of leptin improves Leydig cell function and spermatogenesis.

Sulforaphane (SFN), a dietary phase-2 enzyme inducer that mitigates cellular oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2) activation, is known to exhibit beneficial effects in the vessel wall. For instance, it inhibits vascular smooth muscle cell (VSMC) proliferation, a major event in atherosclerosis and restenosis after angioplasty. In particular, SFN attenuates the mitogenic and pro-inflammatory actions of platelet-derived growth factor (PDGF) and tumor necrosis factor-α (TNFα), respectively, in VSMCs. Nevertheless, the vasoprotective role of SFN has not been examined in the setting of obesity characterized by hyperleptinemia and insulin resistance. Using the mouse model of western diet-induced obesity, the present study demonstrates for the first time that subcutaneous delivery of SFN (0.5mg/Kg/day) for~3weeks significantly attenuates neointima formation in the injured femoral artery [↓ (decrease) neointima/media ratio by~60%; n=5-8]. This was associated with significant improvements in metabolic parameters, including ↓ weight gain by~52%, ↓ plasma leptin by~42%, ↓ plasma insulin by~63%, insulin resistance [↓ homeostasis model assessment of insulin resistance (HOMA-IR) index by~73%], glucose tolerance (↓ AUCGTT by~24%), and plasma lipid profile (e.g., ↓ triglycerides). Under in vitro conditions, SFN significantly decreased leptin-induced VSMC proliferation by~23% (n=5) with associated diminutions in leptin-induced cyclin D1 expression and the phosphorylation of p70S6kinase and ribosomal S6 protein (n=3-4). The present findings reveal that, in addition to improving systemic metabolic parameters, SFN inhibits leptin-induced VSMC proliferative signaling that may contribute in part to the suppression of injury-induced neointima formation in diet-induced obesity.

Conflicting evidence concerning leptin in atherosclerosis has been published. Furthermore, dose-dependent effects of leptin on atherogenesis have not been studied.

Leptin-deficient low-density lipoprotein receptor (LDLR) knockout (LDLR(-/-);ob/ob) mice were treated with saline, 0.1, 0.5, or 3.0mg/kg body weight (BW)/d recombinant leptin over 12weeks starting at 8weeks of age. Aortic root and brachiocephalic artery (BCA) atherosclerotic lesions were analyzed by oil red O staining. Furthermore, glucose homeostasis, lipid metabolism, and liver function including tissue studies were assessed in all animals.

Leptin treatment dose-dependently decreased BW in LDLR(-/-);ob/ob mice as compared to saline. Mice in the 0.1 and 0.5mg/kgBW/d groups remained heavier (i.e. subphysiological leptin dose) and in the 3.0mg/kgBW/d group had similar weight (i.e. physiological leptin dose) as compared to non-leptin-deficient LDLR(-/-) animals. Recombinant leptin dose-dependently reduced plaque area in the aortic root and the BCA by 36% and 58%, respectively. Leptin-mediated reductions of plasma total and LDL-cholesterol (Chol) remained independent predictors for aortic root plaque area. Chol content in liver, as well as hepatic expression of key lipid and proinflammatory genes, were dose-dependently regulated by leptin. Furthermore, leptin treatment increased circulating levels and adipose tissue mRNA expression of the adipokine adiponectin.

Leptin administration within the subphysiological to physiological range diminishes atherosclerotic lesions. Leptin appears to mediate its antiatherogenic effects indirectly through reduction of hypercholesterolemia and liver steatosis, as well as upregulation of insulin-sensitizing and atheroprotective adiponectin.

Epidemiological evidence has shown that abdominal obesity is closely associated with the development of cardiovascular (CV) disease, suggesting that it might be considered as an independent CV risk factor. However, the pathophysiological mechanisms responsible for the association between these 2 clinical entities remain largely unknown. Adipocytes are considered able to produce and secrete chemical mediators known as "adipokines" that may exert several biological actions, including those on heart and vessels. Of interest, a different adipokine profile can be observed in the plasma of patients with obesity or metabolic syndrome compared with healthy controls. We consider the main adipokines, focusing on their effects on the vascular wall and analyzing their role in CV pathophysiology.

The link between vascular calcification (VC) and increased mortality is now well established. Over time, as clinical importance of this phenomenon has begun to be fully considered, scientists have highlighted more and more physiopathological mechanisms and signaling pathways that underlie VC. Several conditions such as diabetes, dyslipidemia and renal diseases are undoubtedly identified as predisposing factors. But even if the process is better understood, many questions still remain unanswered. This review briefly develops the various theories that attempt to explain mineralization genesis. Nonetheless, the main purpose of the article is to provide a profile of the various existing biomarkers of VC. Indeed, in the past years, a lot of inhibitors and promoters, which form a dense and interconnected network, were identified. Given importance to assess and control mineralization process, a focusing on accumulated knowledge of each marker seemed to be necessary. Therefore, we tried to define their respective role in the physiopathology and how they can contribute to calcification risk assessment. Among these, Klotho/fibroblast growth factor-23, fetuin-A, Matrix Gla protein, Bone morphogenetic protein-2, osteoprotegerin, osteopontin, osteonectin, osteocalcin, pyrophosphate and sclerostin are specifically discussed.

Worldwide, cardiovascular diseases (CVDs) are a leading cause of mortality. While in many westernized societies there has been a decrease prevalence of smoking and that a special emphasis has been put on the urgency to control the, so called, classical risk factors, it is more and more recognized that there remains a residual risk, which contributes to the development of CVDs. Imaging studies conducted over two decades have highlighted that the accumulation of ectopic visceral fat is associated with a plethora of metabolic dysfunctions, which have complex and intertwined interactions and participate to the development/progression/events of many cardiovascular disorders. The contribution of visceral ectopic fat to the development of coronary artery disease (CAD) is now well established, while in the last several years emerging evidence has pointed out that accumulation of harmful ectopic fat is associated with other cardiovascular disorders such as calcific aortic valve disease (CAVD), atrial fibrillation and left ventricular dysfunction. We review herein the key molecular processes linking the accumulation of ectopic fat to the development of CVDs. We have attempted, whenever possible, to use a translational approach whereby the pathobiology processes are linked to clinical observations.

Calcification can occur in fat in multiple clinical conditions including in the dermis, breasts and in the abdomen in calciphylaxis. All of these are more common in patients with advanced kidney disease. Clinically, hyperphosphatemia and obesity are risk factors. Thus we tested the hypothesis that adipocytes can calcify in the presence of elevated phosphorus and/or that adipocytes exposed to phosphorus can induce vascular smooth muscle cell (VSMC) calcification.

3T3-L1 preadipocytes were induced into mature adipocytes and then treated with media containing high phosphorus. Calcification was assessed biochemically and PCR performed to determine the expression of genes for osteoblast and adipocyte differentiation. Adipocytes were also co-cultured with bovine VSMC to determine paracrine effects, and the efficacy of sodium thiosulfate was determined.

The results demonstrated that high phosphorus induced the calcification of differentiated adipocytes with increased expression of osteopontin, the osteoblast transcription factor Runx2 and decreased expression of adipocyte transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (CEBPα), indicating that high phosphorus led to a phenotypic switch of adipocytes to an osteoblast like phenotype. Sodium thiosulfate, dose dependently decreased adipocyte calcification and inhibited adipocyte induced increase of VSMC calcification. Co-culture studies demonstrated that adipocytes facilitated VSMC calcification partially mediated by changes of secretion of leptin and vascular endothelial growth factor (VEGF) from adipocytes.

High phosphorus induced calcification of mature adipocytes, and adipocytes exposed to elevated phosphorus can induce calcification of VSMC in a paracrine manner. Sodium thiosulfate inhibited this calcification and decreased the secretin of leptin and VEGF from adipocytes. These results suggest that adipocyte exposure to elevated phosphorus may be a pathogenic factor in calcification observed in the skin in calciphylaxis and other diseases.

Arterial calcification is a complex and active regulated process, which results from a process of osteoblastic differentiation of vascular smooth muscle cells (VSMCs). Leptin, the product of the ob gene, mainly regulates food intake and energy expenditure and recently has been considered to be correlated with the arterial calcification. However, the mechanisms of the effects of leptin on osteoblastic differentiation of VSMCs are unknown. We used calcifying vascular smooth muscle cells (CVSMCs) as a model to investigate the relationship between leptin and the osteoblastic differentiation of CVSMCs and the signaling pathways involved. Our experiments demonstrated that leptin could increase expression of receptor activator of nuclear factor-κB ligand (RANKL) and bone morphogenetic protein 4 (BMP4), as well as alkaline phosphatase (ALP) activity, runt-related transcription factor 2 expression, calcium deposition, and the formation of mineralized nodules in CVSMCs. Suppression of RANKL with small interfering RNA abolished the leptin-induced ALP activity and BMP4 expression in CVSMCs. Leptin could activate the ERK1/2 and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Furthermore, pretreatment with the ERK inhibitor PD98059 and the PI3K inhibitor LY294002 abolished leptin-induced RANKL expression and blocked the promotion of ALP activity of CVSMCs. Silencing of the leptin receptor OB-Rb with small interfering RNA abolished leptin-induced activation of ERK and Akt and the expression of RANKL and reversed the effects of leptin on ALP activity. Meanwhile, addition of Noggin (the BMP4 inhibitor) blunted the effect of leptin on ALP activity. These results show that leptin can promote osteoblastic differentiation of CVSMCs by the OB-Rb/ERK1/2/RANKL-BMP4 and OB-Rb/PI3K/Akt/RANKL-BMP4 pathways.

Apolipoprotein E (apoE) may act as a vasculoprotective factor by promoting plasma lipid clearance and cholesterol efflux. Moreover, apoE accumulates at sites of vascular injury and modulates the effect of growth factors on smooth muscle cells (SMCs). Experimental data suggested that hypothalamic apoE expression is reduced in obesity and associated with leptin resistance. In this study, we examined the role of apoE in mediating the effects of leptin on vascular lesion formation.

Leptin was administered to apoE knockout (apoE-/-) mice via osmotic pumps to increase its circulating levels. Morphometric analysis revealed that leptin did not alter neointima formation and failed to increase α-actin- or PCNA-immunopositive SMCs after vascular injury. Similar findings were obtained after analysis of atherosclerotic lesions. Comparison of apoE-/-, wild-type, or LDL receptor-/- mice and functional analyses in aortic SMCs from WT or apoE-/- mice or human arterial SMCs after treatment with small interfering (si)RNA or heparinase revealed that leptin requires the presence of apoE, expressed, secreted and bound to the cell surface, to fully activate leptin receptor signalling and to promote SMC proliferation and neointima formation. Mechanistically, leptin induced the phosphorylation and membrane translocation of caveolin (cav)-1, and apoE down-regulation or caveolae disruption inhibited the leptin-induced p47phox activation, ROS formation and SMC proliferation. Finally, leptin failed to increase neointima formation in mice lacking cav-1.

Our findings suggest that apoE mediates the effects of leptin on vascular lesion formation by stabilizing cav-1-enriched cell membrane microdomains in SMCs, thus allowing NADPH oxidase assembly and ROS-mediated mitogenic signalling.

Leptin promotes atherosclerosis and vessel wall remodeling. As abdominal aortic aneurysm (AAA) formation involves tissue remodeling, we hypothesized that local leptin synthesis initiates and promotes this process.

Human surgical AAA walls were analyzed for antigen and mRNA levels of leptin and leptin receptor, as well as mRNA for matrix metalloproteinases (MMP)-9 and MMP-12. Leptin and leptin receptor antigen were evident in all AAAs, and leptin, MMP-9, and MMP-12 mRNA was increased relative to age-matched nondilated controls. To simulate in vivo local leptin synthesis, ApoE(-/-) mice were subjected to a paravisceral periaortic application of low-dose leptin. Leptin-treated aortas exhibited decreased transforming growth factor-β and increased MMP-9 mRNA levels 5 days after surgery, and leptin receptor mRNA was upregulated 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 periadventitial macrophage clustering. Furthermore, the combination of local periaortic leptin and systemic angiotensin II administration augmented medial MMP-9 synthesis and aortic aneurysm size.

Leptin is locally synthesized in human AAA wall. Paravisceral 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.

In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 μg/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3β on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of β-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3β (Ad-GSK-3β S9A) resulted in a >2-fold increase in GSK-3β activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3β activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

The role of microRNAs (miRNAs) in vascular calcification is currently unclear. To examine how miRNAs are involved in vascular smooth muscle cell (VSMC) calcification, we explored the alteration of miRNAs in VSMC calcification in vitro and in vivo. Klotho homozygous mutant mice (kl/kl) display vascular calcification and have perturbations of calcium handling. We therefore hypothesized that the calcium perturbations in VSMCs could be mediated by miRNAs. Using an miRNA array analysis, we demonstrated that miRNAs are aberrantly expressed in the aortic media of 3-week-old kl/kl mice compared with wild-type (WT) mice. The expression levels of miR-135a(*), miR-762, miR-714, and miR-712(*) in the aortic media of kl/kl mice were significantly higher than in WT mice. We used quantitative real-time reverse transcriptase polymerase chain reaction to further confirm that these miRNAs were increased in the aortic media of kl/kl mice and in cultured VSMCs treated with high phosphate and calcium. A search of the miRNA database indicated that the Ca(2+) efflux proteins NCX1, PMCA1, and NCKX4 frequently appeared as potential targets of these miRNAs. The transfection of miRNA mimics into cultured VSMCs reduced the protein levels of each potential target. Conversely, miRNA inhibitors reduced phosphate and calcium-induced VSMC calcification. Furthermore, these inhibitors decreased the intracellular Ca(2+) concentration in cultured VSMCs after treatment with phosphate and calcium. Our results suggest that increased expression of miR-135a(*), miR-762, miR-714, and miR-712(*) in VSMCs may be involved in VSMC calcification by disrupting Ca(2+) efflux proteins.

Vascular calcification, bone loss and increased fracture risk are age-associated disorders. Several epidemiological studies have suggested a relationship between vascular calcification, impaired bone metabolism and increased mortality. So far, this relationship had been under-estimated as osteoporosis and vascular calcification have been considered non-modifiable disorders of aging. Recent data suggest that this association is not simply an artefact of age, stressing that the co-incidence of vascular calcification with low bone activity and osteoporosis could be biologically linked. During the development of vascular calcification, the transition of vascular smooth muscle cells towards an osteoblast-like phenotype promotes the release of the vesicular structures and mineralization within these structures is promoted by several players, including those related to mineral metabolism, like phosphorus, calcium or parathyroid hormone, which influence either the supersaturation within the structure or the expression of osteogenic factors. However, an intriguing question is whether the presence of vascular calcification impacts bone metabolism, thus demonstrating true crosstalk between these tissues. Evidence is now emerging, suggesting that some inhibitors of the Wnt pathway, such as secreted frizzled Proteins 2 and 4 and Dickkopf related protein-1 (DKK-1), may play a role linking vascular calcification and bone loss. An additional important question to answer, from the patient's perspective, is whether or not progression of vascular calcification can be prevented or restricted and whether altering this progression we can efficiently impact patients' outcomes. Much evidence suggests that the control of the chronic kidney disease-mineral and bone disorder components, particularly serum phosphorus, are the main targets to maintain normal bone turnover and protect against vascular calcification.