Intraplatelet miRNA-126 regulates thrombosis and its reduction contributes to platelet inhibition.

AIMS: MicroRNA-126 (miR-126), one of the most abundant microRNAs in platelets, is involved in the regulation of platelet activity and the circulating miR-126 is reduced during antiplatelet therapy. However, whether intraplatelet miR-126 plays a role in thrombosis and platelet inhibition remains unclear. METHODS AND RESULTS: Here, using tissue-specific knockout mice, we reported that the deficiency of miR-126 in platelets and vascular endothelial cells significantly prevented thrombosis and prolonged bleeding time. Using chimeric mice, we identified that the lack of intraplatelet miR-126 significantly prevented thrombosis. Ex vivo experiments further demonstrated that miR-126-deficient platelets displayed impaired platelet aggregation, spreading and secretory functions. Next, miR-126 was confirmed to target phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) in platelet, which encodes a negative regulator of the PI3 K/AKT pathway, enhancing platelet activation through activating the integrin αIIbß3-mediated outside-in signaling. After undergoing myocardial infarction (MI), chimeric mice lacking intraplatelet miR-126 displayed reduced microvascular obstruction and prevented MI expansion in vivo. In contrast, overexpression of miR-126 by the administration of miR-126 agonist (agomiR-126) in wild-type mice aggravated microvascular obstruction and promoted MI expansion, which can be almost abolished by aspirin administration. In patients with cardiovascular diseases, antiplatelet therapies, either aspirin alone or combined with clopidogrel, decreased the level of intraplatelet miR-126. The reduction of intraplatelet miR-126 level was associated with the decrease of platelet activity. CONCLUSIONS: Our murine and human data reveal that (i) intraplatelet miR-126 contributes to platelet activity and promotes thrombus formation, and (ii) the reduction of intraplatelet miR-126 contributes to platelet inhibition during antiplatelet therapy.

Single-Cell Transcriptome Analysis Reveals Embryonic Endothelial Heterogeneity at Spatiotemporal Level and Multifunctions of MicroRNA-126 in Mice.

BACKGROUND: Endothelial cells (ECs) play a critical role in angiogenesis and vascular remodeling. The heterogeneity of ECs has been reported at adult stages, yet it has not been fully investigated. This study aims to assess the transcriptional heterogeneity of developmental ECs at spatiotemporal level and to reveal the changes of embryonic ECs clustering when endothelium-enriched microRNA-126 (miR-126) was specifically knocked out. METHODS: C57BL/6J mice embryos at day 14.5 were harvested and digested, followed by fluorescence-activated cell sorting to enrich ECs. Then, single-cell RNA sequencing was applied to enriched embryonic ECs. Tie2 (Tek receptor tyrosine kinase)-cre-mediated ECs-specific miR-126 knockout mice were constructed, and ECs from Tie2-cre-mediated ECs-specific miR-126 knockout embryos were subjected to single-cell RNA sequencing. RESULTS: Embryonic ECs were clustered into 11 groups corresponding to anatomic characteristics. The vascular bed (arteries, capillaries, veins, lymphatics) exhibited transcriptomic similarity across the developmental stage. Embryonic ECs had higher proliferative potential than adult ECs. Integrating analysis showed that 3 ECs populations (hepatic, mesenchymal transition, and pulmonary ECs) were apparently disorganized after miR-126 being knocked out. Gene ontology analysis revealed that disrupted ECs were mainly related to hypoxia, glycometabolism, and vascular calcification. Additionally, in vivo experiment showed that Tie2-cre-mediated ECs-specific miR-126 knockout mice exhibited excessive intussusceptive angiogenesis; reductive glucose and pyruvate tolerance; and excessive accumulation of calcium. Agonist miR-126-3p agomir significantly rescued the phenotype of glucose metabolic dysfunction in Tie2-cre-mediated ECs-specific miR-126 knockout mice. CONCLUSIONS: The heterogeneity of ECs is established as early as the embryonic stage. The deficiency of miR-126 disrupts the differentiation and diversification of embryonic ECs, suggesting that miR-126 plays an essential role in the maintenance of ECs heterogeneity.

The Protective Effect of Cx43 Protein-Mediated Phosphocreatine on Myocardial Ischemia/Reperfusion Injury.

OBJECTIVES: To verify the protective effect of phosphocreatine on myocardium in an ischemic model and the possible mechanism of action. METHODS: The model of myocardial ischemia/reperfusion (I/R) was established by the ligation balloon method. 30 SD rats were randomly divided into three groups, n = 10 in each group. Sham operation group: the coronary artery was not blocked and observed for 120 minutes. The ischemia/reperfusion (I/R) group was given ischemia for 30 minutes and ischemia reperfusion for 90 minutes. Phosphocreatine (PCr) group: after 30 minutes of ischemia, the rats were intraperitoneally injected with PCr (200 mg/kg) for 90 minutes. The animal groups of myocardial ischemia/reperfusion model in vitro were the same as those in vivo. The heart was removed by thoracotomy and washed immediately in H-K buffer solution. Then, the heart was installed on the Langendorff instrument. The concentration of PCr perfusion fluid in the PCr group was 10 mmol/L. The changes in coronary blood flow in isolated myocardium were recorded. The heart rate and electrocardiogram were recorded by RM6240BT. At the end of the experiment, myocardial pathological sections and Cx43 immunofluorescence staining were made, and the contents of malondialdehyde (MDA) in myocardial tissue were detected. RESULTS: Phosphocreatinine treatment improved the myocardial ischemia model, performance in electrocardiogram (ECG) changes (ST segment apparent), and histological changes (decrease in necrotic myocardial cells, inflammatory cell infiltration, and a reduction in myocardial edema). At the same time, MDA decreased, while coronary blood flow and Cx43 expression significantly improved. CONCLUSIONS: Phosphocreatine can improve the electrocardiogram and restore histologic changes in ischemic myocardium and coronary blood flow. The postulated mechanism is by inhibiting the generation of free oxygen radicals and restoring the expression of Cx43 protein.

Vitamin D Enhances Efficacy of Oral Nifedipine in Treating Preeclampsia with Severe Features: A Double Blinded, Placebo-Controlled and Randomized Clinical Trial.

Vitamin D (VD) has exhibited immunomodulatory role in the pathogenesis of preeclampsia. We hypothesize VD potentiate nifedipine treatment for preeclampsia by shortened the time to control blood pressure and prolong time before subsequent hypertensive crisis. We conduct a randomized trial of 683 primigravid women with preeclampsia, who were assigned to different treatment groups, either nifedipine+placebo or nifedipine+VD orally, by random after screening. Primary endpoints include time to control hypertension and time before another hypertensive crisis. Maternal adverse effects including nausea, vomiting, chest pain, mild headache, dizziness, maternal tachycardia, hypotension or shortness of breath, and neonatal parameters including birth weight and Apgar scores, as well as the minimum number of dosages needed to control hypertension were defined as secondary endpoints. Serum levels of cytokines tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were also examined. There was a marked reduction of the time required to control hypertension and a significant lengthening (p = 0.013) of the time before a new hypertensive crisis in participants received nifedipine+VD treatments (41.8 ± 18.3 min), in comparison with the nifedipine+placebo controls (61.1 ± 15.9 min). In women treated with nifedipine+VD, the minimum number of dosages needed to control hypertension was also lower. With regard to adverse effects, no statistical difference was observed between the two treatment groups. Moreover, treatment with VD increased IL-10 and reduced TNF-α serum levels. VD possesses the potential of serving as a safe and effective adjuvant to oral nifedipine in treating women with preeclampsia against hypertension, possibly through the upregulation of IL-10 and the downregulation of TNF-α.

[Attenuated Total Reflection Infrared Spectroscopy for Degradation Profile of High Density Polyethylene after Weathering Aging].

High density polyethylene (HDPE) was widely used as rotational packaging case in the material reserve field. The chemical changes of HDPE, exposed to particular climatic conditions of tropic marine atmosphere for one year-long in Wanning Hainan, were elucidated by the attenuated total reflection infrared spectroscopy (ATR-FTIR). The structural changes were studied qualitatively, mainly from the polymeric chain breaking, branching and oxidation to distinguish the degradation profile. The variations of crystallinity & carbonyl index were also studied quantitatively according to the characteristic peaks intensity & area ratio. Finally, the relationships between structural changes and mechanical properties were investigated. The results showed that the polymeric chain breaking & branching play a leading role before 3 months in the aging progress. Then oxidation phenomena gradually takes place during 3-6 months. The chain branching & oxidation were predominant factors after 6 months. Nine months later, the oxidation was saturated gradually. Furthermore, the aging process is positively correlated to the temperature and irradiation. After 12 months aging, the carbonyl index increased by 112 times and crystallinity was 10% higher than before. The tensile/bending modulus deceased faster than tensile/bending strength of HDPE. The linear degree of tensile modulus and carbonyl index was 0.97. The degree of linearity of tensile strength and crystallinity calculated by feature bands (720-730 cm(-1)) was 0.96. It showed that the mechanical properties of HDPE can be speculated from the structural changes by ATR-FTIR.

Anti-angiogenesis and anti-tumor effects of AdNT4-anginex.

Anginex is a novel artificial peptide that can inhibit angiogenesis. AdNT4-anginex was constructed by inserting the artificial anginex gene into a recombinant adenoviral vector. We demonstrated that AdNT4-anginex inhibited migration of human endothelial cells, angiogenesis and tumor growth in in vitro and in vivo studies. Tumor growth of human H22 hepatoma in mice was inhibited after AdNT4-anginex treatment for 4 weeks, and a significant decrease in tumor size was observed as compared with the control group. Overall, these studies indicate that AdNT4-anginex is an effective anti-tumor agent, and deserves more attention and research.

[The relationship between lymphatic metastasis and serum vascular endothelial growth factor C and cyclooxygenase 2 expression in breast cancer].

OBJECTIVE: To investigate the relationship among the serum vascular endothelial growth factor C (sVEGF-C), expression of cyclooxygenase 2 (COX-2), and lymph vessel density (LMVD), and to discuss their role in tumor progression and lymphatic metastasis. METHODS: sVEGF-C level was detected by ELISA in 68 pre-operation breast cancer patients, 35 breast benign disease patients, and 12 healthy women. Immunohistochemical method was used to detect the expression of COX-2 and lymphatic vessel endothelial hyaluronidase receptor (LYVE)-1 in the breast cancer tissues and benign disease tissues obtained during operation. RESULTS: (1) The sVEGF-C level of the pre-operation breast cancer group was (49 +/- 8) pg/ml, significantly higher than those of the begin tumor group [(8 +/- 4) pg/ml, P = 0.045] and the healthy women [(5 +/- 3) pg/ml, P = 0.003]. (2) The COX-2 overexpression positive rate in the breast cancer tissues was 44.1%, significantly higher than that in the begin disease tissues (11.4%, P = 0.002). Higher than that in the begin disease tissue; the LMVD overexpression in the breast cancer tissues was mainly located in the para-carcinoma tissue (8.7 +/- 4.7), significantly higher than that in the begin disease tissues (1.8 +/- 1.7, P = 0.002). (3) The COX-2 overexpression rate of the patients with a high sVEGF-C level was significantly higher than that of the patients with a low sVEGF-C level (P = 0.024). The LMVD level in the breast cancer of the sVEGF-C high level group was (8.7 +/- 3.9), significantly higher than that of the sVEGF-C normal group (5.6 +/- 3.3, P = 0.039). (4) The LMVD in the COX-2 overexpression breast cancer group was 9.5 +/- 3.7, significantly higher than that in the COX-2 negative group (6.0 +/- 3.8, P = 0.012). (5) The sVEGF-C level, COX-2, and LMVD expression in the breast cancer patients with lymphatic metastasis were significantly higher than those in the patients without lymphatic metastasis (P = 0.025, 0.022, and 0.002 respectively). CONCLUSION: VEGF-C, COX-2, and LMVD play important roles in the lymphatic metastasis of breast cancer. They may be important prognostic factors in evaluating breast cancer lymphatic metastasis.

[Relationship between serum VEGF level and VEGF, COX-2 and MVD expression in breast cancer tissues].

OBJECTIVE: To investigate the relationship between serum VEGF (sVEGF) level and VEGF, COX-2 and MVD expression in breast cancer, and to discuss their role in angiogensis of breast cancer. METHODS: sVEGF level was detected by ELISA in 68 preoperative breast cancer, 35 benign breast disease and 20 healthy women. The expression of VEGF, COX-2 and MVD was detected by immunohistochemical method in tissues of breast cancer and breast benign diseases, and to analyze the relationship of sVEGF, VEGF, COX-2 and MVD. RESULTS: (1) sVEGF level in preoperative breast cancers was 306.51 pg/ml (interquartile range from 190.44 to 442.04 pg/ml), in benign diseases was 150.82 pg/ml (interquartile range from 82.36 to 212.34 pg/ml), and in healthy control was 105.93 pg/ml (interquartile range from 78.54 to 157.77 pg/ml). The sVEGF level of preoperative breast cancer group was significantly higher than that of breast benign disease group and healthy women (P = 0.001). (2) The VEGF expression positive rate in breast cancer (67.65%) was significantly higher than that in breast benign disease (44.12%) (P = 0.015). The COX-2 expression positive rate in breast cancer (42.86%) was significantly higher than that in breast benign disease (11.43%) (P = 0.002). (3) the COX-2 expression positive rate in sVEGF high level patients (56.00%) was significantly higher than that in sVEGF normal level patients (11.11%) (P = 0.024), and MVD in sVEGF high level patients (27.32 +/- 3.40) was also higher than that in sVEGF normal level patients (15.31 +/- 6.16) (P = 0.011). (4) The sVEGF level (322.09 +/- 79.31) of 68 breast cancer patients whose VEGF was positive in breast cancer tissues was significantly higher than that in VEGF negative group (222.47 +/- 73.53) (P = 0.017). (5) The COX-2 expression positive rate in VEGF positive expression group (65.21%) was significantly higher than that in VEGF negative expression group (18.18%) (P = 0.017). The MVD expression in COX-2 positive expression group (22.94 +/- 5.51) was significantly higher than that in COX-2 negative expression group (10.30 +/- 4.42) (P = 0.027). CONCLUSION: sVEGF level in breast cancer is significantly higher than that in breast benign disease and healthy women, and is correlated with the expression of COX-2 and MVD in breast cancer tissues.