Different stimuli induce endothelial dysfunction and promote atherosclerosis through the Piezo1/YAP signaling axis.

Vascular endothelial dysfunction is the initial step in atherosclerosis (AS). AS tends to occur at vascular bifurcations and curves, and endothelial cells(ECs) are highly susceptible to injury due to mechanical forces induced by disturbed flow (DF) with inconsistent blood flow directions. However, the pathogenesis of endothelial cell dysfunction in AS remains unclear and needs further study. Here, we found that Piezo1 expression was significantly increased in DF- and oxidized low-density lipoprotein(ox-LDL)-treated HUVECs in vitro and a model of atherosclerotic plaque growth in ApoE-/- mice fed a Western diet. Furthermore, Piezo1 upregulated autophagy levels in the HUVECs model, which was reversed by Piezo1 knockdown with a lentivirus-mediated shRNA system. Mechanistically, the level of Yes-associated protein (YAP), a transcriptional coactivator in the Hippo pathway, was significantly elevated in the DF- and ox-LDL-induced HUVECs model, and this effect was further inhibited by Piezo1 knockdown. Moreover, the Piezo1 agonist Yoda1 inhibited the protein level of microtubule-associated protein 1 light chain 3-II(LC3-II) and increased the protein level of sequestosome1(p62/SQSTM1) in a dose-dependent manner, while significantly promoting the protein expression and nuclear translocation of YAP. The YAP inhibitor CA3 weakened Yoda1-mediated inhibition of autophagy. Our results suggest that Piezo1 may regulate endothelial autophagy by promoting YAP activation and nuclear translocation, thereby contributing to vascular endothelial dysfunction.

Novel interrupted aortic arch: A case report.

BACKGROUND AND AIM OF THE STUDY: Interrupted aortic arch (IAA) is a rare and fatal malformation. Most patients with IAA are diagnosed in early childhood because of the severity of their symptoms. IAA is classified into three morphologic types (A, B, or C), depending on the site of the interruption. In our case, this patient did not have a common brachiocephalic trunk, left carotid artery, or left subclavian artery, IAA classification of this case cannot be judged based on the existing interruption method. METHODS: We present a 6-year-old Chinese boy with a history of neck masses since birth, and an echocardiogram from a local county hospital revealing an IAA without any cardiac anomalies, was referred to our hospital. RESULTS: The patient was feeling good and was nearly asymptomatic. Computed tomography angiography was performed, which indicated an absent aortic arch, likely due to disruption during development, and aortic discontinuity. The ascending aorta gave rise to both carotid arteries, and the descending aorta was supplied by large subclavian arteries. The right vertebral artery was supplied by right large collateral vessels that connected the right carotid artery. The left side was similar in structure to the right side. The descending aorta was supplied by large subclavian arteries. The subclavian arteries and carotid arteries were connected by large collateral vessels. Due to the large collateral vessels, the child's lower body had sufficient blood supplied, so that the typical differential cyanosis did not occur, and the child without symptomatic can survive to now. CONCLUSIONS: This patient did not have a common brachiocephalic trunk, left carotid artery, or left subclavian artery. Maybe, this patient belonged to a new type of IAA.

Small-molecule inhibitor LF3 restrains the development of pulmonary hypertension through the Wnt/ß-catenin pathway.

Pulmonary hypertension (PH) associated with congenital heart disease is a progressive hemodynamic disease that can lead to increased pulmonary vascular resistance, vascular remodeling, and even right heart failure and death. LF3 is a novel inhibitor of the reporter gene activity of ß-catenin/TCF4 interaction in the Wnt/ß-catenin signal pathway. However, whether this action of LF3 can prevent PH development remains unclear. In this study, we investigated the therapeutic effect of LF3 in rat primary pulmonary artery smooth muscle cells (PASMCs) of the PH model. We found that LF3 inhibited the decrease in pulmonary artery acceleration time and ejection time by ultra-high-resolution ultrasound imaging and blocked the increase of pulmonary artery systolic pressure by using the BL420 biological function experimental system and right ventricular hypertrophy index by the electronic scales. Simultaneously, it prevented the increase of α-smooth muscle actin and fibronectin and the decrease of elastin in pulmonary arteries of rats in the PH group, as revealed by an immunohistochemical analysis. Moreover, cell proliferation and migration assays showed that LF3 significantly reduced the proliferation and migration of PASMCs. Western blotting and quantitative real-time polymerase chain reaction analyses revealed that LF3 suppressed the expression of proliferating cell nuclear antigens and Bcl-2 and increased the expression of Bax but did not alter the expressions of ß-catenin and TCF4. Taken together, LF3 can reduce the migration and proliferation of PASMCs and induce their apoptosis to prevent the development of PH. It would be worthwhile to explore the potential use of LF3 in the treatment of PH.

[IL-8 Induces Epithelial-to-mesenchymal Transition of Ovarian Carcinoma Cells: a Preliminary Study].

OBJECTIVE: To explore the effect of IL-8 on the epithelial-to-mesenchymal transition (EMT) in ovarian cancer,which will provide experimental basis for revealing related molecular mechanism in malignant metastasis of ovarian cancer. METHODS: The migration of ovarian cancer cell line SKOV3 cells was explored with Real time label free cell analysis (RTCA) after treatment with recombinant human IL-8.SKOV3 cells were co-cultured with IL-8 for 48 h,proteins involved in EMT were investigated via Western blot to explore the effect of IL-8 on the activation of the EMT. Invasion of SKOV3 cells after treatment with IL-8 were evaluated by transwell assay. RESULTS: According to the results of RTCA,after treatment with IL-8 for 48 h,the migration of SKOV3 cells was in platform phase. The treatment of IL-8 unregulated vimentin and snail and downregulated E-cadherin,which suggested that IL-8 induced EMT in ovarian cancer. The results of transwell test showed that invasive ability of IL-8 pretreated SKOV3 cells was enhanced (P<0.05). CONCLUSION: IL-8 can induce the EMT of ovarian cancer and enhance the invasion and migration of ovarian cancer.

Simultaneous genotyping of HPA-17w to -21w by PCR-SSP in Chinese Cantonese.

Studies have reported the polymorphism of human platelet antigen (HPA)-17w, -18w, -19w, -20w, and -21w. However, the distribution of these five antigens in Chinese Cantonese is still unknown. In this study, we designed new sequence-specific primers for HPA-19w to -21w and used published primers for HPA-17w and -18w to develop a polymerase chain reaction with the sequence-specific primers (PCR-SSP) method for simultaneously genotyping HPA-17w to -21w. A total of 820 unrelated Cantonese apheresis platelet donors in Guangzhou were involved in this study. Among the five HPAs, complete a/a homozygosity was observed for HPA-17w to -20w with an allele frequency of 1.0000. For HPA-21w, nine individuals (9/820, 1.10%) were found to be HPA-21a/bw heterozygous and the allele frequencies of HPA-21a and HPA-21bw were 0.9945 (1631/1640) and 0.0055 (9/1640), respectively. The reliability of the PCR-SSP method was determined by comparing with the genotyping results by DNA sequencing, and no inconsistencies were observed between the two methods. This study provides a reliable PCR-SSP method for simultaneously genotyping HPA-17w to -21w and could improve HPA-matched platelet transfusion in Chinese Cantonese.

[SPIO-labeled rat bone marrow mesenchymal stem cells: alterations of biological activity and labeling efficiency assay in vitro].

This study aimed to characterize and magnetic resonance imaging (MRI) track the mesenchymal stem cells labeled with polylysine-coated superparamagnetic iron oxide (PLL-SPIO). Rat bone marrow derived mesenchymal stem cells (rMSCs) were labeled with 25, 50 and 100 microg/mL PLL-SPIO for 24 hours. The labeling efficiency was assessed by iron content, Prussian blue staining, electron microscopy and in vitro MR imaging. The labeled cells were also analyzed for cytotoxicity and differentiation potential. Electron microscopic observations and Prussian blue staining revealed that 75% -100% of cells were labeled with iron particles. PLL-SPIO did not show any cytotoxicity up to 100 microg/mL concentration. Both 25 microg/mL and 50 microg/mL PLL-SPIO labeled stem cells did not exhibit any significant alterations in the adipo/osteo/chondrogenic differentiation potential compared to unlabeled control cells. The lower concentration of 25 microg/mL iron labeled cells emitted an obvious dark signal in T1W, T2WI and T2 * WI MR image. The novel PLL-SPIO enables to label and track rMSCs for in vitro MRI without cellular alteration. Therefore PLL-SPIO may potentially become a better MR contrast agent especially in tracking the transplanted stem cells and other cells without compromising cell functional quality.

Talin mechanotransduction in disease.

Talin protein (Talin 1/2) is a mechanosensitive cytoskeleton protein. The unique structure of the Talin plays a vital role in transmitting mechanical forces. Talin proteins connect the extracellular matrix to the cytoskeleton by linking to integrins and actin, thereby mediating the conversion of mechanical signals into biochemical signals and influencing disease progression as potential diagnostic indicators, therapeutic targets, and prognostic indicators of various diseases. Most studies in recent years have confirmed that mechanical forces also have a crucial role in the development of disease, and Talin has been found to play a role in several diseases. Still, more studies need to be done on how Talin is involved in mechanical signaling in disease. This review focuses on the mechanical signaling of Talin in disease, aiming to summarize the mechanisms by which Talin plays a role in disease and to provide references for further studies.

Low shear stress induces inflammatory response via CX3CR1/NF-κB signal pathway in human umbilical vein endothelial cells.

Low shear stress (LSS) has been reported to induce atherosclerosis. However, the molecular mechanisms underlying inflammation induced by LSS are still poorly understood. The objective of our study is the comprehensive identification of molecular circuitry involved in low shear stress-induced inflammation in human umbilical vein endothelial cells (HUVECs) through protein profiling and cell function experiment. In this study, Western blotting analyses revealed a significant increase in the expression of CX3CR1, nucleusP65, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and Interleukin-6 (IL-6), while the expression of cytosolic P65 and IκB has significantly decreased in HUVECs treated with low shear stress. CX3CR1 Sh-RNA was use to reveal its effect on LSS-induced inflammation. Further, specific NF-κB P65 inhibitors pyrrolidinedithiocarbamate (PDTC) were used to reveal the downstream NF-κB P65 exclusively involved in LSS-induced inflammation in HUVECs, this effect can be abrogated by CX3CR1 sh-RNA and NF-κB inhibitors. Monocyte adhesion assay and scratch test revealed low shear stress to promotes adhesion of monocytes and migration of cells, this effect can be abrogated by CX3CR1 sh-RNA and NF-κB inhibitors. LSS was involved in the expression of adhesion molecules and chemokines, which are important for the initiation of endothelial inflammation-related atherosclerosis. Therefore, the cell signaling pathways activated by LSS in endothelial cells may represent therapeutic targets of atherosclerosis.

The role of 14-3-3 in the progression of vascular inflammation induced by lipopolysaccharide.

OBJECTIVE: To explore the role of 14-3-3 protein and the Hippo and yes-associated protein 1 (YAP) signaling pathway in lipopolysaccharide (LPS)-induced vascular inflammation. METHODS: Human umbilical vein endothelial cells (HUVECs) and C57B6 mice were treated with LPS to establish cell and animal models of vascular inflammation. Lentiviral transfection, Western blot, qPCR, immunofluorescence, immunohistochemistry, co-immunoprecipitation, and enzyme-linked immunosorbent assays were used to measure inflammatory factors and expression of 14-3-3 protein and phosphorylation of YAP at S127. HUVECs and C57B6 mice were pretreated with a YAP inhibitor, Verteporfin, to observe changes in YAP expression and downstream vascular inflammation. RESULTS: LPS induced acute and chronic inflammatory responses in HUVECs and mice and upregulated the expression of several inflammatory factors. LPS also induced expression of 14-3-3 protein and phosphorylation of YAP at S127 in response to acute vascular inflammation and downregulated these markers in response to chronic vascular inflammation. Verteporfin reduced these LPS-induced effects on vascular inflammation. CONCLUSION: In chronic vascular inflammation, 14-3-3 protein is downregulated, which promotes inflammation by increasing Hippo/YAP nuclear translocation.

Emerging Piezo1 signaling in inflammation and atherosclerosis; a potential therapeutic target.

Purpose of Review: Atherosclerosis is the principal cause of cardiovascular diseases (CVDs) which are the major cause of death worldwide. Mechanical force plays an essential role in cardiovascular health and disease. To bring the awareness of mechanosensitive Piezo1 role in atherosclerosis and its therapeutic potentials we review recent literature to highlight its involvement in various mechanisms of the disease. Recent Findings: Recent studies reported Piezo1 channel as a sensor, and transducer of various mechanical forces into biochemical signals, which affect various cellular activities such as proliferation, migration, apoptosis and vascular remodeling including immune/inflammatory mechanisms fundamental phenomenon in atherogenesis. Summary: Numerous evidences suggest Piezo1 as a player in different mechanisms of cell biology, including immune/inflammatory and other cellular mechanisms correlated with atherosclerosis. This review discusses mechanistic insight about this matter and highlights the drugability and therapeutic potentials consistent with emerging functions Piezo1 in various mechanisms of atherosclerosis. Based on the recent works, we suggest Piezo1 as potential therapeutic target and a valid candidate for future research. Therefore, a deeper exploration of Piezo1 biology and translation towards the clinic will be a novel strategy for treating atherosclerosis and other CVDs.

Mechanosensitive Piezo1 Channel Evoked-Mechanical Signals in Atherosclerosis.

Recently, more and more works have focused and used extensive resources on atherosclerosis research, which is one of the major causes of death globally. Alongside traditional risk factors, such as hyperlipidemia, smoking, hypertension, obesity, and diabetes, mechanical forces, including shear stress, pressure and stretches exerted on endothelial cells by flow, is proved to be crucial in atherosclerosis development. Studies have recognized the mechanosensitive Piezo1 channel as a special sensor and transducer of various mechanical forces into biochemical signals, and recent studies report its role in atherosclerosis through different mechanical forces in pressure, stretching and turbulent shear stress. Based on our expertise in this field and considering the recent advancement of atherosclerosis research, we will be focusing on the function of Piezo1 and its involvement in various cellular mechanisms and consequent involvement in the development of atherosclerosis in this review. Also, we will discuss various functions of Piezo1 involvement in atherosclerosis and come up with new mechanistic insight for future research. Based on the recent findings, we suggest Piezo1 as a valid candidate for novel therapeutic innovations, in which deep exploration and translating its findings into the clinic will be a new therapeutic strategy for cardiovascular diseases, particularly atherosclerosis.

Low Shear Stress Upregulates CX3CR1 Expression by Inducing VCAM-1 via the NF-κB Pathway in Vascular Endothelial Cells.

Atherosclerosis is a significant cause of mortality and morbidity. Studies suggest that the chemokine receptor CX3CR1 plays a critical role in atherogenesis. Shear stress is an important mechanical force that affects blood vessel function. In this study, we investigated the effect of shear stress on CX3CR1 expression in vascular endothelial cells (VECs). First, cells were exposed to different shear stress and then CX3CR1 mRNA and protein were measured by quantitative RT-PCR and western blot analysis, respectively. CX3CR1 gene silencing was used to analyze the molecular mechanisms underlying shear stress-mediated effects on CX3CR1 expression. CX3CR1 mRNA and protein expression were significantly increased with 4.14 dyne/cm2 of shear stress compared with other tested levels of shear stress. We observed a significant increase in CX3CR1 mRNA levels at 2 h and CX3CR1 protein expression at 4 h. CX3CR1-induced VCAM-1 expression in response to low shear stress by activating NF-κB signaling pathway in VECs. Our findings demonstrate that low shear stress increases CX3CR1 expression, which increases VCAM-1 expression due to elevated NF-κB activation. The current study provides evidence of the correlation between shear stress and atherosclerosis mediated by CX3CR1.

IL-8 induces imbalances between nitric oxide and endothelin-1, and also between plasminogen activator inhibitor-1 and tissue-type plasminogen activator in cultured endothelial cells.

Interleukin-8 (IL-8), a member of the CXC chemokine family, plays an important role in the modulation of multiple biological functions in endothelial cells containing the receptors CXCR1 and CXCR2. It has previously been shown that IL-8 directly enhances endothelial cell survival, and stimulates the production of matrix metalloproteinases, which in turn regulates angiogenesis. However, its role in the regulation of the production of vasoactive substances in endothelial cells is less well defined. In this study, we investigate the effects of IL-8 on the proliferation of human umbilical vein endothelial cells (HUVECs). In addition, we also study the effects of IL-8 on the production of vasodilator, vasoconstrictor and fibrinolytic factors in these cells. The results show that recombinant IL-8 (50-200ng/ml) induces neither HUVEC proliferation nor nitric oxide (NO) release. However, it significantly increases the production of endothelin-1 (ET-1) in a concentration-dependent manner. Furthermore, incubation of endothelial cells with IL-8 (200ng/ml) up-regulates the plasminogen activator inhibitor-1 (PAI-1) in HUVECs, while it down-regulates the tissue plasminogen activator (t-PA). These findings suggest that IL-8 offsets the balance between endothelial vasoconstrictors and vasodilators. Furthermore, IL-8 also leads to an imbalance between PAI-1 and t-PA, which causes the ECs to become procoagulative and hypofibrinolytic.

Activation of MAPK participates in low shear stress-induced IL-8 gene expression in endothelial cells.

BACKGROUND: Endothelial cells (ECs) are constantly subjected to blood flow-generated mechanical forces including shear stress and cyclic strain. Shear stress modulates vascular structure and function by regulating the expression of many genes. We have previously demonstrated that low shear stress induces the IL-8 gene expression in ECs. The present study was undertaken to investigate the roles of MAPKs in the regulation of the shear stress-induced IL-8 gene expression in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were exposed to low shear stress (4.2 dyne/cm(2)). The phosphorylation of MAPKs including ERK1/2, JNK and p38, was detected by Western blot. Immunocytochemistry was employed to measure the distribution and intensity of MAPKs. Inhibitors, a dominant negative-p38 and RNAi for JNK, were used to block the MAPK pathways, after which the LightCycler system was employed to assay the IL-8 gene expression. FINDINGS: The activation of ERK1/2, p38 MAPK and JNK1/2 was observed in ECs exposed to low shear stress. Furthermore, phospho-ERK1/2, JNK1/2 and p38 MAPK translocated from the cytoplasm into the nucleus. Inhibition of ERK1/2, JNK1/2 and p38 MAPK with PD98059, SP600125 and SB203580, respectively, led to the suppression of the shear stress-induced IL-8 gene expression (P<0.01), which was also blocked by JNK1/2 siRNA (small interfering RNA) (P<0.01). DN-p38, a dominant negative mutant of p38 MAPK, attenuated the shear stress-induced IL-8 promoter-mediated green fluorescent protein expression (P<0.05). INTERPRETATION: These results suggest that ERK1/2, JNK1/2 and p38 MAPK are all involved in the low shear stress-induced IL-8 gene expression. Understanding the mechanism by which low shear stress regulates IL-8 gene expression may provide insight into the initiation of atherosclerosis.

[The effect of PKC activation and Smac release on inhibition of myocardial cell apoptosis by Sini Decoction].

OBJECTIVE: To investigate the effect of Sini Decoction on myocardial cell apoptosis after MI/R and the possible relation to myocardial cell apoptosis, PKC and Smac. METHODS: Sprage-Dawley rats were divided into four groups: control group, sham group, MI/R group and SND group. The rats in MI/R group, left anterior descending coronary artery (LAD) was occluded for 1 h and reperfused for 1 h. The rats in SND group were pretreated with Sini decoction(5 g/kg x d) for three days, the last treatment was pretreated 24 h before the index occlusion. Cell apoptosis was measured by flow cytometry, the expression of PKC was measured by immunohistochemistry. The expression of Smac was analyzed by Western blotting. The activity of Caspase-3 was also measured. RESULTS: 24 h after Sini Decoction treatment, Myocardial cell apoptosis significantly decreased, PKC was translocated and its expression increased, the release of Smac decreased, the activity of Caspase-3 decreased. CONCLUSION: Sini Decoction treatment can protect myocardial cell from apoptosis caused by MI/R, the mechanism is related to PKC activation, Mitochondria protection and in turn inhibition of Smac release, and inhibition of apoptosis signal transduction.