Agonist-induced Piezo1 activation promote mitochondrial-dependent apoptosis in vascular smooth muscle cells.

OBJECTIVE: Mechanical damage plays an essential role in the progression of atherosclerosis. Piezo1 is a new mechanically sensitive ion channel. The present study investigated the vascular smooth muscle cells (VSMCs) apoptosis induced by Piezo1 activation and explored its underlying mechanism. METHODS: We evaluated cell viability and apoptosis rate with cell counting kit-8 (CCK-8) and Annexin V-FITC/PI flow cytometry assay, respectively. And then Western blot was performed to measure the relative protein. Reactive oxygen species (ROS) and intracellular Ca2+ were assessed via fluorescence microscope, and the mitochondrial transmembrane potential was monitored by JC-10 staining. RESULTS: Our in vitro study revealed that mice in the ApoE-/- group compared with control mice showed higher Piezo1 expression(P < 0.05). Besides, Yoda1, a Piezo1 agonist, triggered Ca2+ overload, mitochondrial damage, accumulation of ROS, and VSMCs apoptosis in a dose-depend manner. Furthermore, BAPT-AM (an intracellular Ca2+ chelator) and NAC (an antioxidant) suppressed the mitochondrial damage and attenuated the VSMCs apoptosis. CONCLUSION: Our study suggested that Piezo1 induced VSMCs apoptosis because of Ca2+ overload, excessive ROS generation, and mitochondrial dysfunction, which indicated that Piezo1 has potential value in treating vascular diseases.

Nε-carboxymethyl-lysine-induced PI3K/Akt signaling inhibition promotes foam cell apoptosis and atherosclerosis progression.

Advanced glycation end products (AGEs) are closely associated with diabetic macrovascular complications. The present study aimed to investigate the effects of Nε-Carboxymethyl-Lysine (the key active component of AGEs) in diabetic atherosclerosis on foam cell apoptosis and to explore the underlying mechanisms. Tissue sections were collected from 12 Type 2 diabetic patients and 4 control patients who underwent amputation surgery following a car accident. Peritoneal injection of streptozotocin in ApoE-/- mice was used to generate a diabetic model in vivo, and Raw 264.7 cells treated with CML and 740Y-P (a PI3K/AKT signaling agonist) were used to explore the effect of PI3K/AKT signaling in CML-induced foam cell apoptosis in vitro. The anterior tibial section of diabetic amputees contained a thinner fiber cap, higher lipid content, and more apoptotic cells than were found in control patients. in vitro studies using Raw 264.7 cell-derived foam cells and in vivo studies using diabetic ApoE-/- mice showed that CML levels dose-dependently reduced cell vitality, induced foam cell apoptosis and regulated apoptosis related protein. Furthermore, CML significantly decreased the phosphorylation of PI3K/AKT signaling, and restoration of PI3K/AKT signaling by 740Y-P decreased the CML-induced foam cell apoptosis. In conclusion, our results showed CML induced foam cell apoptosis in diabetic atherosclerosis through inhibiting the PI3K/AKT pathway.

Dynamic Ghrelin and GH serum levels during combined simultaneous arginine clonidine stimulation test in children with dwarfism.

BACKGROUND: Combined simultaneous arginine clonidine stimulation (CSACS) test represents a more appropriate stimulus to detect Ghrelin, for it does not affect glucose metabolism. METHODS: Fifty prepubertal children with dwarfism were recruited and further classified into normal growth hormone (NGH) and growth hormone deficiency (GHD) group with growth hormone (GH) peak cut-off value of 10 µg/l. In both groups, GH and Ghrelin serum levels were determined after the GH provocation test at 30, 60, and 120 min and the height standard deviation score (SDS) for bone age was measured six months later. RESULTS: The participants were classified into NGH (n = 24) and GHD group (n = 26). A decrease in the circulating Ghrelin levels prior to the GH peak was observed in the NGH children, whereas both GH and Ghrelin levels demonstrated a rise in the GHD children. Ghrelin level in GHD group was higher compared with NGH group and the GH peak in GHD group is lower than NGH group. The 6 months CSACS treatment could increase the height SDS in both groups. CONCLUSION: Although analogous changes were not detected in GHD group, the inverse correlation between GH and Ghrelin in NGH indicates a negative feedback lying between GH and Ghrelin.

Cardiac resynchronization therapy for heart failure induced by left bundle branch block after transcatheter closure of ventricular septal defect.

A 54-year-old female patient with congenital heart disease had a persistent complete left bundle branch block three months after closure by an Amplatzer ventricular septal defect occluder. Nine months later, the patient suffered from chest distress, palpitation, and sweating at daily activities, and her 6-min walk distance decreased significantly (155 m). Her echocardiography showed increased left ventricular end-diastolic diameter with left ventricular ejection fraction of 37%. Her symptoms reduced significantly one week after received cardiac resynchronization therapy. She had no symptoms at daily activities, and her echo showed left ventricular ejection fraction of 46% and 53%. Moreover, left ventricular end-diastolic diameter decreased 6 and 10 months after cardiac resynchronization therapy, and 6-min walk distance remarkably increased. This case demonstrated that persistent complete left bundle branch block for nine months after transcatheter closure with ventricular septal defect Amplatzer occluder could lead to left ventricular enlargement and a significant decrease in left ventricular systolic function. Cardiac resynchronization therapy decreased left ventricular end-diastolic diameter and increased left ventricular ejection fraction, thereby improving the patient's heart functions.

Bisoprolol reverses epinephrine-mediated inhibition of cell emigration through increases in the expression of ß-arrestin 2 and CCR7 and PI3K phosphorylation, in dendritic cells loaded with cholesterol.

The effect of bisoprolol on dendritic cell (DC) migration was investigated, including the analysis of protein expression, cytokine secretion and activation of the PI3K-pathway. The chemotactic cell numbers in cholesterol-loaded DCs treated with epinephrine were significantly decreased by 26.66±6.29% (6h), 35.67±2.91% (12h) and 29.33±1.09% (24h). This effect was significantly reversed by 46.00±10.65% (6h), 64.25±6.77% (12h) and 55.74±5.51% (24h) when bisoprolol and epinephrine were both present. In cholesterol-loaded DCs, treatment with epinephrine significantly increased AR-ß1 protein expression by 56.99±4.87%, but inhibited ß-arrestin 2 and CCR7 protein expression by 30.51±4.22% and 25.31±0.04%, respectively. These effects were reversed by bisoprolol by 36.87±4.40%, 41.47±3.95% and 30.14±0.54%, respectively. TNF-α and MMP9 levels were decreased by 68.33±4.00% and 39.57±9.21% in cholesterol-loaded DCs treated with epinephrine. In contrast, when bisoprolol and epinephrine were administered together, the secretion of these proteins was significantly increased by 233.81±37.06 % and 76.66±14.21%, respectively. Treatment with epinephrine decreased PI3K-phosphorylation by 31.88±2.79%, 40.24±5.69% and 30.93±4.66% at 15, 30 and 60min, respectively, whereas the effect of epinephrine on the expression of phosphorylated PI3K was reversed by 49.49±12.12%, 70.93±16.14% and 47.62±6.00%, respectively, when cells were treated with both bisoprolol and epinephrine. Wortmannin inhibited the effects of bisoprolol on PI3K-phosphorylation (38.63±6.12%), the expression of CCR7 (23.4±2.72%), the secretion of TNF-α (69.46±4.48%) and MMP9 (43.15±4.63%), and the number of chemotactic cells (36.84±5.22%). This is the first study to establish a signaling pathway, epinephrine-AR-ß1-ß-arrestin2-PI3K-MMP9/CCR7, which plays a critical role in the migration of DCs.

The relationship between CD40 gene polymorphism and unstable coronary atherosclerotic plaques.

BACKGROUND: Recently, CD40 polymorphism was found to be associated with acute coronary syndromes. However, few study was involved in the relationship between CD40 polymorphism and the risk of the vulnerable plaque to rupture so far. MATERIALS AND METHODS: A total of 699 patients who have received coronary angiography were divided into 3 groups according to the morphological division of the plaques: complex lesions (343 cases), smooth lesions (131 cases), and control group (225 cases).The gene polymorphism was measured by the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and identified by sequencing. RESULTS: The CC genotype and C allele frequency of the CD40 gene in the complex lesions group is significantly higher than in the smooth lesions group and the control group (P < .001), while the TT genotype frequency in the complex lesions group is significantly lower than that of the smooth lesions group and the control group (P < .001). The C allele increased disruption risk of the plaques in the complex lesions group as compared with the smooth lesions group (odds ratio [OR]: 1.697, 95% confidence interval [CI]: 1.273-2.261). No significant differences in genotypes or allele frequencies were found between the smooth lesions group and the control group. CONCLUSION: Our results suggested that CD40 (-1C/T) polymorphism was associated with unstable coronary atherosclerotic plaques. The C allele frequency increased the risk of disruption of the coronary atherosclerotic plaques.

Association analysis of CD40 gene polymorphism with acute coronary syndrome.

Current evidence shows that the CD40-CD40 ligand (CD40-CD40L) system plays a crucial role in the development, progression and outcome of acute coronary syndrome (ACS). The aim of this study is to investigate whether a CD40 gene (-1C/T) single nucleotide polymorphism (SNP) is associated with ACS and CD40 expression. We included controls (n = 163) and patients with either ACS (n = 210) or stable angina (SA, n = 189) in the study. The gene polymorphism was measured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and identified by sequencing. CD40 expression on platelets was detected by flow cytometry. The results showed that the frequency of the CC genotype of CD40 gene in the ACS group was significantly higher than in the SA and control groups (P < 0.001), while the TT genotype frequency in the ACS group was significantly lower than that of the SA and control groups (P < 0.001). The C-allele frequency of the ACS group was higher than that of the SA and control groups (P < 0.001). The C allele increased the risk of ACS when compared with the SA group (OR = 1.841, 95% CI: 1.390-2.437) and the control groups (OR = 1.877, 95% CI: 1.402-2.515). No significant differences in genotypes or allele frequencies were found between the SA and control groups. CD40 expression on platelets in patients with C-allele carriers was significant higher than in the T-allele carriers in each group. In conclusion, our results suggested that the CD40 gene (-1C/T) polymorphism was associated with ACS in Chinese people. The -1C-allele gene carriers had a higher expression of CD40 and an increased risk of suffering from ACS.

Upregulation of OX40-OX40 ligand system on T lymphocytes in patients with acute coronary syndromes.

OBJECTIVE: To investigate whether upregulation of OX40-OX40 ligand (OX40L) system is related to stability of coronary atherosclerotic plaque in patients with coronary heart diseases. METHODS: Thirty normal controls and 250 patients, including 80 with stable angina (SA), 110 with unstable angina (UA), and 60 with acute myocardial infarction (AMI), were enrolled in our study. The expression of OX40 and OX40L in peripheral CD4 T lymphocytes were analyzed by flow cytometry. Serum soluble OX40L (sOX40L) and C-reactive protein levels were measured by commercially available enzyme-linked immunosorbent assay kit. RESULTS: The expression of OX40 and OX40L in peripheral CD4 T lymphocytes in patients with UA [26.7 +/- 3.4 and 45.5 +/- 8.1 mean fluorescence intensity (MFI)] and AMI (27.4 +/- 4.6 and 55.7 +/- 9.4 MFI) were significantly higher than those in patients with SA (6.5 +/- 1.4 and 12.4 +/- 3.2 MFI) and controls (7.3 +/- 1.5 and 11.9 +/- 6.1 MFI). sOX40L in patients with UA (38.7 +/- 6.9 ng/mL) and AMI (39.5 +/- 7.3 ng/mL) were significantly higher than those in patients with SA (8.4 +/- 1.4 ng/mL) (P < 0.01) and controls (8.9 +/- 2.3 ng/mL) (P < 0.01). C-reactive protein level in serum in patients with UA (14.6 +/- 3.3 ng/mL) and AMI (15.0 +/- 4.3 ng/mL) were also higher than those in patients with SA (1.4 +/- 0.4 ng/mL) and controls (1.3 +/- 0.3 ng/mL). It was interesting that there was a peak level of sOX40L at 12 hours after AMI in patients with AMI. A positive correlation was found between sOX40L and serum C-reactive protein levels (r = 0.71; P < 0.0001). CONCLUSIONS: Patients with acute coronary syndromes showed increased coexpression of OX40 system, which may create a proinflammatory and prothrombotic milieu for aggravating the development of atherosclerosis and instability of atherosclerotic plaques, and sOX40L is a potential marker for predicting the severity of coronary heart diseases.

OX40-OX40 ligand interaction may activate phospholipase C signal transduction pathway in human umbilical vein endothelial cells.

Studies have recently supported the emerging role of OX40/OX40L interaction in atherosclerosis. The mechanism of OX40/OX40L interaction may be related to a variety of signal pathways. The most important signal pathway involves the activation of phospholipase C (PLC) which induces diacylglycerol-protein kinase C (DAG-PKC) and the inositol trisphosphate (IP(3))-intracellular free calcium ([Ca(2+)](i)) pathway. The aim of this work was to investigate whether OX40-OX40L interaction can stimulate the PLC signal pathway in human umbilical vein endothelial cells (HUVEC). The DAG and IP(3) level in HUVEC were measured by radio-enzymatic assay. The activity of PKC was detected by its ability to transfer phosphate from [gamma-(32)P]ATP to lysine-rich histone. [Ca(2+)](i) concentrations were measured by flow cytometric analysis. Results showed that the DAG level was markedly increased in a concentration-dependent, biphasic manner in HUVEC induced by OX40. The early phase was rapid, peaking at 30 s. The late phase reached the maximum level at 15 min and decayed slowly. OX40 increased PKC activity in a dose-dependent manner with two peaks at 40-50 s and 12-16 min, then decreased slowly, yet maintained a high level for at least 30 min. PKC activity was mainly in cytosol at rest and translocated from cytosol to membrane when stimulated by OX40. Similarly, OX40-induced rapid IP(3) formation coincided with the peak of DAG level. Moreover, OX40 also induced peak [Ca(2+)](i) responses including the rapid transient phase and the sustained phase. Anti-OX40L antibody significantly suppressed OX40-induced DAG-PKC and IP(3)-[Ca(2+)](i) signal pathway activation in HUVEC. In conclusion, the data suggested that OX40-OX40L interaction induced a robust stimulation of phospholipase C signal transduction pathway in HUVEC.

The clinical implications of increased OX40 ligand expression in patients with acute coronary syndrome.

BACKGROUND: Increasing evidence show that OX40 ligand (OX40L), also known as tumor necrosis factor superfamily member 4 (TNFSF4), plays an important role in the pathogenesis of atherosclerosis. We investigated whether expression levels of soluble OX40L in serum and of membrane OX40L on platelets were related to serum concentrations of matrix metalloproteinases (MMPs) and stability of coronary atherosclerotic plaque in patients with acute coronary syndrome (ACS). METHODS: We included healthy controls (n=30), patients with stable angina (SA) (n=40) and patients with ACS, including unstable angina (UA) (n=70) and acute myocardial infarction (AMI) (n=40). The expression of OX40L on platelets (pOX40L) was analyzed with flow cytometry whereas serum concentrations of soluble OX40L (sOX40L), MMP-9 and MMP-3 were determined with ELISA. All coronary stenoses with >or=30% diameter reduction were assessed by angiographic coronary stenosis morphology. RESULTS: The expression of OX40L on platelets were significantly higher in patients with ACS (61.5+/-11.5) compared with healthy controls (28.9+/-7.4) or with the group of patients with SA (31.2+/-8.1) (mean fluorescence intensity+/-SD) (p<0.001). Similarly, we observed higher sOX40L concentrations in patients with ACS (34.6+/-9.3) compared with controls (10.2+/-4.7) or patients with SA (11.4+/-5.8) (ng/ml+/-SD) (p<0.001). Serum MMP-3 and MMP-9 levels in patients were two times greater than those in the control group. A positive correlation was observed between OX40L expression on platelets and MMP-9 and MMP-3 serum concentrations. OX40L expression on platelets were furthermore correlated with soluble OX40L in serum and with complex coronary stenoses (r1=0.61, r2=0.57, p<0.001). CONCLUSION: Patients with ACS show increased OX40L system (pOX40L and sOX40L) expression which may create a proinflammatory milieu for aggravating the development of atherosclerosis, and may be a valuable marker for predicting the severity of ACS.

Relationship between CD40 ligand expression and B type natriuretic peptide levels in patients with chronic heart failure.

BACKGROUND: Inflammation plays a pathogenic role in the development of chronic heart failure (CHF). Increasing evidence shows that CD40-CD40 ligand (CD40L) interaction plays a pathogenic role in inflammatory disorders. We assessed whether CD40 ligand expression was abnormal in patients with CHF. METHODS: Twenty normal controls and 86 patients with CHF were investigated. The expression of CD40L on platelets was analyzed by indirect-immunofluorescence flow cytometry, and the soluble CD40L (sCD40L) level was determined by a commercially available enzyme-linked immunosorbent assay (ELISA). B type natriuretic peptide (BNP) was measured by radioimmunoassay. RESULTS: All patients with CHF showed a significant increased expression of CD40L (32.3+/-13.9 MFI) on platelets and sCD40L levels (20.5+/-8.6 microg/l) compared with controls(p<0.0001). CD40L expression on platelets and sCD40L levels positively correlated with New York Heart Association (NYHA) functional class, left ventricular ejection fraction and BNP levels in CHF. CONCLUSIONS: Patients with CHF showed increased expression of CD40L system, which may create a pathogenic role in the development and progression of CHF.

Losartan inhibited expression of matrix metalloproteinases in rat atherosclerotic lesions and angiotensin II-stimulated macrophages.

AIM: To explore whether the angiotensin II (Ang II) receptor 1 (AT1) antagonist, losartan could reduce activity and expression of matrix metalloproteinases (MMPs) in rat atherosclerotic plaques. METHODS: Male Wistar-Kyoto rats were ip injected a single dose of vitamin D3 600 kU x kg(-1) x month(-1) and fed an atherogenic diet for 4 months to induce experimental atheroma. Then either placebo or losartan 50 mg x kg(-1) x d(-1) was administered in rats for another 2 months. In vitro, the effect of losartan 0.1-10 micromol/L on the expression of MMP-2 and MMP-9 was investigated in Ang II-stimulated rat peritoneal macrophages. The expression and activity of MMP-2 and MMP-9 were monitored by Western blot, RT-PCR, and SDS-PAGE zymography analysis. RESULTS: High levels of MMP-2 and MMP-9 were expressed in rat atherosclerotic lesions. Losartan significantly reduced the activity and expression of MMP-2 and MMP-9 compared with the placebo group (MMP-2, 5861+/-539 vs 8991+/-965, P<0.05; MMP-9,10527+/-1002 vs 14623+/-2462, P<0.01). In cultured rat peritoneal macrophages, Ang II 0.1 micromol/L elicited an increase in MMP-2 and MMP-9 activity and expression that were prevented by losartan in a dose-dependent manner (P<0.01). But the AT2 receptor antagonist PD123319 had no effect. CONCLUSION: Losartan reduced the expression and activity of MMP-2 and MMP-9 in rat atherosclerotic lesions. The anti-atherogenic effects of losartan were due to the direct inhibition of Ang II bioactivity.