CEPT1-Mediated Phospholipogenesis Regulates Endothelial Cell Function and Ischemia-Induced Angiogenesis Through PPARα.

De novo phospholipogenesis, mediated by choline-ethanolamine phosphotransferase 1 (CEPT1), is essential for phospholipid activation of transcription factors such as peroxisome proliferator-activated receptor α (PPARα) in the liver. Fenofibrate, a PPARα agonist and lipid-lowering agent, decreases amputation incidence in patients with diabetes. Because we previously observed that CEPT1 is elevated in carotid plaque of patients with diabetes, we evaluated the role of CEPT1 in peripheral arteries and PPARα phosphorylation (Ser12). CEPT1 was found to be elevated in diseased lower-extremity arterial intima of individuals with peripheral arterial disease and diabetes. To evaluate the role of Cept1 in the endothelium, we engineered a conditional endothelial cell (EC)-specific deletion of Cept1 via induced VE-cadherin-CreERT2-mediated recombination (Cept1Lp/LpCre +). Cept1Lp/LpCre + ECs demonstrated decreased proliferation, migration, and tubule formation, and Cept1Lp/LpCre + mice had reduced perfusion and angiogenesis in ischemic hind limbs. Peripheral ischemic recovery and PPARα signaling were further compromised by streptozotocin-induced diabetes and ameliorated by feeding fenofibrate. Cept1 endoribonuclease-prepared siRNA decreased PPARα phosphorylation in ECs, which was rescued with fenofibrate but not PC16:0/18:1. Unlike Cept1Lp/LpCre + mice, Cept1Lp/LpCre + Ppara -/- mice did not demonstrate hind-paw perfusion recovery after feeding fenofibrate. Therefore, we demonstrate that CEPT1 is essential for EC function and tissue recovery after ischemia and that fenofibrate rescues CEPT1-mediated activation of PPARα.

Macrophage galectin-3 enhances intimal translocation of vascular calcification in diabetes mellitus.

The clinical risks and prognosis of diabetic vascular intimal calcification (VIC) and medial calcification (VMC) are different. This study aims to investigate the mechanism of VIC/VMC translocation. Anterior tibial arteries were collected from patients with diabetic foot amputation. The patients were then divided into VIC and VMC groups. There were plaques in all anterior tibial arteries, while the enrichment of galectin-3 in arterial plaques in the VIC group was significantly higher than that in the VMC group. Furthermore, a macrophage/vascular smooth muscle cell (VSMC) coculture system was constructed. VSMC-derived extracellular vesicles (EVs) was labeled with fluorescent probe. After macrophages were pretreated with recombinant galectin-3 protein, the migration of VSMC-derived EVs and VSMC-derived calcification was more pronounced. And anti-galectin-3 antibody can inhibit this process of EVs and calcification translocation. Then, lentivirus (LV)-treated bone marrow cells (BMCs) were transplanted into apolipoprotein E-deficient (ApoE-/-) mice, and a diabetic atherosclerosis mouse model was constructed. After 15 wk of high-fat diet, ApoE-/- mice transplanted with LV-shgalectin-3 BMCs exhibited medial calcification and a concentrated distribution of EVs in the media. In conclusion, upregulation of galectin-3 in macrophages promotes the migration of VSMC-derived EVs to the intima and induces diabetic vascular intimal calcification.NEW & NOTEWORTHY The clinical risk and prognosis of vascular intimal and medial calcification are different. Macrophage galectin-3 regulates the migration of vascular smooth muscle cell-derived extracellular vesicles and mediates diabetic vascular intimal/medial calcification translocation. This study may provide insights into the early intervention in diabetic vascular calcification.

Trimethylamine-N-Oxide Promotes Vascular Calcification Through Activation of NLRP3 (Nucleotide-Binding Domain, Leucine-Rich-Containing Family, Pyrin Domain-Containing-3) Inflammasome and NF-κB (Nuclear Factor κB) Signals.

OBJECTIVES: Vascular calcification is highly prevalent in patients with chronic kidney disease. Increased plasma trimethylamine N-oxide (TMAO), a gut microbiota-dependent product, concentrations are found in patients undergoing hemodialysis. However, a clear mechanistic link between TMAO and vascular calcification is not yet established. In this study, we investigate whether TMAO participates in the progression of vascular calcification using in vitro, ex vivo, and in vivo models. Approach and Results: Alizarin red staining revealed that TMAO promoted calcium/phosphate-induced calcification of rat and human vascular smooth muscle cells in a dose-dependent manner, and this was confirmed by calcium content assay. Similarly, TMAO upregulated the expression of bone-related molecules including Runx2 (Runt-related transcription factor 2) and BMP2 (bone morphogenetic protein-2), suggesting that TMAO promoted osteogenic differentiation of vascular smooth muscle cells. In addition, ex vivo study also showed the positive regulatory effect of TMAO on vascular calcification. Furthermore, we found that TMAO accelerated vascular calcification in rats with chronic kidney disease, as indicated by Mico-computed tomography analysis, alizarin red staining and calcium content assay. By contrast, reducing TMAO levels by antibiotics attenuated vascular calcification in chronic kidney disease rats. Interestingly, TMAO activated NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome and NF-κB (nuclear factor κB) signals during vascular calcification. Inhibition of NLRP3 inflammasome and NF-κB signals attenuated TMAO-induced vascular smooth muscle cell calcification. CONCLUSIONS: This study for the first time demonstrates that TMAO promotes vascular calcification through activation of NLRP3 inflammasome and NF-κB signals, suggesting the potential link between gut microbial metabolism and vascular calcification. Reducing the levels of TMAO could become a potential treatment strategy for vascular calcification in chronic kidney disease.

Exercise training restores the myogenic response in skeletal muscle resistance arteries and corrects peripheral edema in rats with heart failure.

Impairment of the myogenic response can affect capillary hydrostatic pressure and contribute to peripheral edema and exercise intolerance, which are markers of heart failure (HF). The aim of this study was to assess the effects of exercise training (ET) on myogenic response in skeletal muscle resistance arteries and peripheral edema in HF rats, focusing on the potential signaling pathways involved in these adjustments. Male Wistar rats were submitted to either coronary artery occlusion or a sham-operated surgery. After 4 wk, an exercise test was performed, and the rats were divided into the following groups: untrained normal control (UNC) and untrained HF (UHF) and exercise- trained (on treadmill, 50-60% of maximal capacity) NC (TNC) and exercise-trained HF (THF). Caudal tibial artery (CTA) myogenic response was impaired in UHF compared with UNC, and ET restored this response in THF to NC levels and increased it in TNC. Rho kinase (ROCK) inhibitor abolished CTA myogenic response in the untrained and blunted it in exercise-trained groups. CTA-stored calcium (Ca2+) mobilization was higher in exercise-trained rats compared with untrained rats. The paw volume was higher in UHF rats, and ET decreased this response compared with UNC. Myogenic constriction was positively correlated with maximal running distance and negatively correlated with paw volume. The results demonstrate, for the first time, that HF impairs the myogenic response in skeletal muscle arteries, which contributes to peripheral edema in this syndrome. ET restores the myogenic response in skeletal muscle arteries improving Ca2+ sensitization and handling. Additionally, this paradigm also improves peripheral edema and exercise intolerance. NEW & NOTEWORTHY The novel and main finding of the present study is that moderate intensity exercise training restores the impaired myogenic response of skeletal muscle resistance arteries, exercise intolerance and peripheral edema in rats with heart failure. These results also show for the first time to our knowledge that exercise training improving calcium sensitization through the ROCK pathway and enhancing intracellular calcium handling could contribute to restoration of flow autoregulation to skeletal muscle in heart failure.

RAGE/galectin-3 yields intraplaque calcification transformation via sortilin.

AIMS: Macrocalcification and microcalcification present different clinical risks, but the regulatory of their formation was unclear. Therefore, this study explored the underlying mechanisms of macrocalcification and microcalcification in diabetes mellitus. METHODS: Anterior tibial arteries of amputated diabetic feet were collected. According to the calcium content, patients were divided into less-calcification group and more-calcification group. And calcification morphology in plaques was observed. For further study, an in vivo mouse diabetic atherosclerosis model and an in vitro primary mouse aortic smooth muscle cell model were established. After the receptors for AGEs (RAGE) or galectin-3 were silenced, calcified nodule sizes and sortilin expression were determined. Scanning electron microscopy (SEM) was performed to detect the aggregation of matrix vesicles with the inhibition or promotion of sortilin. RESULTS: Both macro- and microcalcification were found in human anterior tibial artery plaques. Macrocalcification formed after the silencing of RAGE, and microcalcification formed after the silencing of galectin-3. In the process of RAGE- or galcetin-3-induced calcification, sortilin played an important role downstream. SEM showed that sortilin promoted the aggregation of MVs in the early stage of calcification and formed larger calcified nodules. CONCLUSION: RAGE downregulated sortilin and then transmitted microcalcification signals, whereas galectin-3 upregulated sortilin, which accelerated the aggregation of MVs in the early stage of calcification and mediated the formation of macrocalcifications, These data illustrate the progression of two calcification types and suggest sortilin as a potential target for early intervention of calcification and as an effective biomarker for the assessment of long-term clinical risk and prognosis.

[ENDOTHELIAL DYSFUNCTION AND MICROSCIRCULATION FEATURES IN PATIENTS WITH HIGH RISK OF DEVELOPMENT OF REPERFUSION SYNDROME IN CONDITIONS OF RECONSTRUCTION ARTERIAL OPERATIONS].

Aim - to study the state of the microcirculatory bed and the endothelial system in patients at risk of developing reperfusion syndrome and suggest methods for their correction. The work included 29 patients with obliterating diseases of the abdominal aorta and lower limb arteries with a high risk of developing reperfusion complications. Two groups of patients were identified. Group I - 8 patients, preoperative preparation include the generally accepted approaches. Group II - 21 patients whose preoperative preparation included, in addition to preparations for improving rheological blood conditions, prolonged epidural anesthesia, intravenous injection of hydroxyethylstarches, korvetin and alprostadil. In patients of both groups, a study was made of the state of the level of endothelial dysfunction, changes in capillary blood flow and arterio-venular blood shunting. Revascularization of the lower limbs in patients with a high risk of developing reperfusion complications leads to a deepening of endothelial dysfunction. The latter is characterized by a 2.3-fold increase (p<0.001) in the early postoperative period of circulating endothelial cells in the blood, by 2.5 times (p <0.001) endothelin-1, while a 1.3-fold decrease (p<0,05) P-selectin and in 1,7 times (p<0,05) E-selectin. The depth of the lesion of the endothelial system is indicated by a decrease of 29.9 % (p<0.001) in the level of NO. Deepening of endothelial dysfunction after reconstructive-reconstructive surgery is reflected in violations of the function of the microcirculatory bed. It is characterized by a 1.9-fold decrease (p<0.001) of skin perfusion, 2.0 times (p<0.001) in the erythrocyte concentration index and a 14% decrease (p<0.05) in capillary blood flow. This is facilitated by an increase of 14% (p<0.05) in skin perfusion pressure and an increase of 16% (p<0.05) in the resistance index of the microcirculatory bed, which leads to a decrease in tissue oxygenation to a level 3.38±0.14 mm.hg.

Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans.

Understanding the functional consequences of genetic variation, and how it affects complex human disease and quantitative traits, remains a critical challenge for biomedicine. We present an analysis of RNA sequencing data from 1641 samples across 43 tissues from 175 individuals, generated as part of the pilot phase of the Genotype-Tissue Expression (GTEx) project. We describe the landscape of gene expression across tissues, catalog thousands of tissue-specific and shared regulatory expression quantitative trait loci (eQTL) variants, describe complex network relationships, and identify signals from genome-wide association studies explained by eQTLs. These findings provide a systematic understanding of the cellular and biological consequences of human genetic variation and of the heterogeneity of such effects among a diverse set of human tissues.

Association of cardiovascular and biochemical risk factors with tibial artery calcification.

The cardiovascular risk factors that contribute to coronary calcification have been extensively studied while those related to tibial artery calcium are less well defined. We sought to determine the associations between cardiovascular risk factors and tibial artery calcification in a cohort of patients with and without significant peripheral atherosclerosis. A total of 222 patients without end-stage renal disease were identified in a prospectively maintained database containing tibial artery calcification (TAC) scores, and demographic, cardiovascular, and biochemical risk factor information. Patients with prevalent tibial artery calcification were more likely to be older, male, and have a history positive for hypertension, hyperlipidemia, diabetes, and tobacco use. Patients with an abnormal ankle-brachial index (ABI) or symptoms of peripheral artery disease (PAD) were also more likely to have higher calcium values. In analyses using multivariable logistic regression, age, gender, diabetes, and tobacco use maintained their association with prevalent tibial calcification while hypertension, hyperlipidemia and body mass index did not. These associations remained when PAD was added to the model. After adjusting for relevant cardiovascular risk factors, we found that only abnormal ABI, current PAD symptoms, and lower serum calcium values were associated with the presence of tibial artery calcification. In conclusion, in patients without end-stage renal disease, tibial artery calcification has risk factors that are similar but not identical to those for coronary artery calcification and peripheral atherosclerosis.

Differential mechanisms for insulin-induced relaxations in mouse posterior tibial arteries and main mesenteric arteries.

The characteristics of endothelium-dependent relaxations in response to insulin and acetylcholine (ACh) in the mouse posterior tibial artery (PTA) were studied on wire myograph, and compared to those in the mouse main mesenteric artery (MMA). Insulin-induced relaxation in PTA was reversed by PI3K and Akt inhibitors, LY294002 and triciribine, but not by nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) or guanylate cyclase inhibitor, ODQ. The relaxation in PTA was also inhibited by apamin (small-conductance Ca(2+)-activated K(+) channel blocker) plus charybdotoxin (intermediate-conductance Ca(2+)-activated K(+) channel blocker), elevated KCl or ouabain (Na(+)-K(+) ATPase inhibitor) plus BaCl(2) [inwardly rectifying K(+) (K(IR)) channel inhibitor]; whereas L-NAME but not triciribine inhibited ACh-induced relaxation in PTA. On the other hand, nitric oxide and endothelium-derived hyperpolarizing factor albeit to a less extent mediated both insulin- and ACh-induced relaxations in MMA. The present study is for the first time dissecting out the components of endothelium-dependent relaxation in mouse PTA and suggesting differential responses to different agonists in distinctive blood vessels.

BK channels in innate immune functions of neutrophils and macrophages.

Oxygen-dependent antimicrobial activity of human polymorphonuclear leukocytes (PMNs) relies on the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate oxidants. As the oxidase transfers electrons from NADPH the membrane will depolarize and concomitantly terminate oxidase activity, unless there is charge translocation to compensate. Most experimental data implicate proton channels as the effectors of this charge compensation, although large-conductance Ca2+-activated K+ (BK) channels have been suggested to be essential for normal PMN antimicrobial activity. To test this latter notion, we directly assessed the role of BK channels in phagocyte function, including the NADPH oxidase. PMNs genetically lacking BK channels (BK(-/-)) had normal intracellular and extracellular NADPH oxidase activity in response to both receptor-independent and phagocytic challenges. Furthermore, NADPH oxidase activity of human PMNs and macrophages was normal after treatment with BK channel inhibitors. Although BK channel inhibitors suppressed endotoxin-mediated tumor necrosis factor-alpha secretion by bone marrow-derived macrophages (BMDMs), BMDMs of BK(-/-) and wild-type mice responded identically and exhibited the same ERK, PI3K/Akt, and nuclear factor-kappaB activation. Based on these data, we conclude that the BK channel is not required for NADPH oxidase activity in PMNs or macrophages or for endotoxin-triggered tumor necrosis factor-alpha release and signal transduction BMDMs.

Indirect coupling between Cav1.2 channels and ryanodine receptors to generate Ca2+ sparks in murine arterial smooth muscle cells.

In arterial vascular smooth muscle cells (VSMCs), Ca(2+) sparks stimulate nearby Ca(2+)-activated K(+) (BK) channels that hyperpolarize the membrane and close L-type Ca(2+) channels. We tested the contribution of L-type Ca(v)1.2 channels to Ca(2+) spark regulation in tibial and cerebral artery VSMCs using VSMC-specific Ca(v)1.2 channel gene disruption in (SMAKO) mice and an approach based on Poisson statistical analysis of activation frequency and first latency of elementary events. Ca(v)1.2 channel gene inactivation reduced Ca(2+) spark frequency and amplitude by approximately 50% and approximately 80%, respectively. These effects were associated with lower global cytosolic Ca(2+) levels and reduced sarcoplasmic reticulum (SR) Ca(2+) load. Elevating cytosolic Ca(2+) levels reversed the effects completely. The activation frequency and first latency of elementary events in both wild-type and SMAKO VSMCs weakly reflected the voltage dependency of L-type channels. This study provides evidence that local and tight coupling between the Ca(v)1.2 channels and ryanodine receptors (RyRs) is not required to initiate Ca(2+) sparks. Instead, Ca(v)1.2 channels contribute to global cytosolic [Ca(2+)], which in turn influences luminal SR calcium and thus Ca(2+) sparks.

Selective downregulation of endothelin-B receptors in diabetic African-American patients.

The purpose of this study was to investigate whether there are differences in the expression of the endothelin (ET) system in the peripheral vasculature of diabetic African-American (AA) and Caucasian (CA) patients. Tibial artery specimens were obtained from diabetic (MD = 8 and CAD = 5) and non-diabetic (AAND = 6 and CAND = 5) patients undergoing lower limb amputation. The gene expression of ET-1 precursor (PPET-1), ET(A)R and ET(B)R was determined by a reverse transcriptase polymerase chain reaction technique. PPET-1 and ET(A)R expression was up-regulated 4- and 3-fold, respectively, in both AA and CA diabetics (P<.05 vs non-diabetics). ET(B)R mRNA was significantly lower in AA diabetic patients. Function of ET-1 and ET receptors was assessed by vascular contractility assays. Vascular relaxation in response to sodium nitroprusside in arteries precontracted with ET-1 was significantly lower in AA (58% +/- 9) as compared to CA diabetics (74% +/- 5) (P<.05). In conclusion, this study demonstrated that the ET system is altered in favor of the contractile phenotype in AA diabetics and may contribute to the increased incidence of vascular complications in this population.

Increased expression of tumor necrosis factor-alpha in diabetic macrovasculopathy.

Large vessel disease, a common feature of diabetes mellitus, appears to run an aggressive course, but its cellular and molecular aspects remain partially elucidated. Although in common atherosclerosis and especially in other forms of accelerated vasculopathy, immunoinflammatory mechanisms participate in the disease process, it is unclear whether this is present in diabetic vasculopathy. We hypothesized that diabetic macrovasculopathy, compared with classical atherosclerosis, is associated with increased immunoinflammatory features and matrix accumulation. In this study, vessel segments obtained after lower-limb amputation for advanced atherosclerotic disease, from type 2 diabetic patients (n = 20; 68.9+/-10.9 years) and nondiabetic patients (n = 16; 67.1+/-14.6 years) were analyzed. Histological characteristics (extent of intimal proliferation, cellularity, and fibrosis) were semiquantitatively graded in the two lesion types. Using immunohistochemistry, the presence of T cells and macrophages, accumulation of fibronectin, and expression of tumor necrosis factor-alpha was also assessed. Histological features of these advanced atherosclerotic lesions were similar in the two lesions examined. By immunohistochemistry, a similar pattern of T-cell and macrophage infiltration and fibronectin accumulation was observed. Nevertheless, increased expression of tumor necrosis factor-alpha was observed in diabetic lesions (13/19 patients had positive staining), whereas only 2 of 16 lesions from nondiabetic patients had positive staining (p < 0.003), with an odds ratio of 15.17 (confidence interval 2.12-139.5). These data suggest that increased expression of tumor necrosis factor-alpha observed in the diabetic lesions may reflect an enhanced inflammatory activity associated with the development of vascular lesions in type 2 diabetic patients.

Phenotypic characterization of human smooth muscle cells derived from atherosclerotic tibial and peroneal arteries.

PURPOSE: The vascular smooth muscle cell plays a pivotal role in the development of atherosclerosis. The objectives of this study were to characterize smooth muscle cells from the human atherosclerotic tibial artery to determine their phenotypic properties and to examine the contractile reactions of these cells to physiologic and pharmacologic stimuli. METHODS: After below-knee amputations were performed, vascular smooth muscle cells were harvested and cultivated from tibioperoneal source. Characterization was done with transmission electron microscopy and immunocytochemistry. The contractile properties were determined by observing the response to various stimuli. In addition, segments of vessels harvested were submitted to electron microscopy studies for comparison with the cultured cells. RESULTS: Immunofluorescent labeling was positive for alpha-smooth muscle actin. Electron microscopy revealed the presence of a thickened basal laminae and large intracellular lipid vacuoles. The earlier passages revealed cells with a large number of microfilaments characteristic of a contractile cell. As later passages were examined, there was a notable change in character with an increasing amount of rough endoplasmic reticulum and Golgi complexes. The increased thickness of the basal lamina in the cultured cells resembled that found in vessel segments studied by electron microscopy. A rapid contraction response was seen when the cells were incubated with angiotensin II, bradykinin, or endothelin. No response was seen with the addition of isoproterenol, nitroglycerin, or nitroprusside, known smooth-muscle relaxants. CONCLUSION: This model demonstrates the apparent inability of these smooth muscle cells from atherosclerotic tibial arteries to relax to pharmacologic and physiologic stimuli. In addition, as seen by transmission electron microscopy, these cells maintain their atherosclerotic phenotype after multiple passages.

Results of directional peripheral atherectomy with reference to histology, histochemistry, and ultrastructure.

One hundred patients with symptomatic peripheral vascular disease were treated with a directional atherectomy catheter; 153 lesions comprising 98 stenoses and 55 occlusions were located in the iliac (n = 22), superficial femoral (n = 114), popliteal (n = 16), and anterior tibial (n = 1) arteries. The majority of these patients were poor candidates for balloon angioplasty because of the complexity of lesions. There were 70 eccentric and 28 concentric lesions and 55 occlusions (mean length 4.2 +/- 2.9 cm). Acute success rate was 94% for both stenoses and occlusions. Four patients were treated in both lower extremities. The stenoses were reduced from 85 +/- 12% to 12 +/- 10% acutely (occlusions 100% to 9 +/- 9%). Six-month angiographic follow-ups were performed in 81% of treated patients, the others refusing angiography. Mean stenosis after six months was 33 +/- 25% (occlusions 44 +/- 28%). Restenosis (> 50%) was found in 20% of treated lesions: 26% in concentric lesions, 8% in eccentric lesions, and 32% in occluded vessels. Treatment of peripheral vascular disease with the atherectomy device is safe and effective therapy with good long-term results. These results were obtained in complex lesions with 55 occlusions. Atherectomy seems to be particularly beneficial in the treatment of eccentric and complex stenoses and is not limited by occlusion or calcification. Furthermore, insight into the pathogenesis of arteriosclerosis and the development of restenosis is enabled by analysis of removed plaque material.