Downregulation of stromal interaction molecule-1 is implicated in the age-associated vasoconstriction dysfunction of aorta, intrarenal, and coronary arteries.

The contractile function of vascular smooth muscle cells (VSMCs) typically undergoes significant changes with advancing age, leading to severe vascular aging-related diseases. The precise role and mechanism of stromal interaction molecule-1 (STIM1) in age-mediated Ca2+ signaling and vasocontraction remain unclear. The connection between STIM1 and age-related vascular dysfunction was investigated using a multi-myograph system, immunohistochemical analysis, protein blotting, and SA-ß-gal staining. Results showed that vasoconstrictor responses in the thoracic aorta, intrarenal artery, and coronary artery decreased with age. STIM1 knockdown in the intrarenal and coronary arteries reduced vascular tone in young mice, while no change was observed in the thoracic aorta. A significant reduction in vascular tone occurred in the STIM1 knockout group with nifedipine. In the thoracic aorta, vasoconstriction significantly decreased with age following the use of nifedipine and thapsigargin and almost disappeared after STIM1 knockdown. The proportion of senescent VSMCs increased significantly in aged mice and further increased in sm-STIM1 KO aged mice. Moreover, the expression of senescence markers p21, p16, and IL-6 significantly increased with age, with p21 expression further increased in the STIM1 knockdown aged group, but not p16 or IL-6. These findings indicate that different arteries exhibit distinct organ-specific features and that STIM1 downregulation may contribute to age-related vasoconstrictive dysfunction through activation of the p21 pathway.

Population pharmacokinetics and safety of eptifibatide in healthy Chinese volunteers and simulations on the dose regimens approved for a Western population.

OBJECTIVE: This study was designed to evaluate the pharmacokinetics (PK) and safety of eptifibatide in healthy Chinese volunteers and provide information for the further study in the Chinese population. METHODS: 30 healthy volunteers (15 male) were enrolled in the study and divided into three dose groups (45 µg x kg⁻¹, 90 µg x kg⁻¹, and 180 µg x kg⁻¹). Plasma and urine samples were drawn after one single-bolus administration and measured by LC-MS/MS. The plasma and urine data were analyzed simultaneously by the population approach using the NONMEM software and evaluated by the visual predicted check (VPC) and bootstraping. The PK profiles of dose regimens approved for a Western population in the Chinese population were simulated. RESULTS: A two-compartment model adequately described the PK profiles of eptifibatide. The clearance (CL) and the distribution volume (V1) of the central compartment were 0.128 L x h⁻¹ x kg⁻¹ and 0.175 L x kg⁻¹, respectively. The clearance (Q) and V2of the peripheral compartment were 0.0988 L x h⁻¹ x kg⁻¹ and 0.147 L x kg⁻¹, respectively. The elimination fraction from plasma to urine (F0) was 17.2%. No covariates were found to have a significant effect. Inter-individual variabilites were all within 33.9%. The VPC plots and bootstrap results indicated good precision and prediction of the model. The simulations of the approved regimens in the Chinese population showed much lower steady-state concentrations than the target concentration obtained from the Western clinical trials. No severe safety events were found in this study. CONCLUSIONS: The PK model of eptifibatide was established and could provide PK information for further studies in the Chinese population.

MicroRNA 16 enhances differentiation of human bone marrow mesenchymal stem cells in a cardiac niche toward myogenic phenotypes in vitro.

AIM: Upregulation of microRNA 16 (miR-16) contributed to the differentiation of human bone marrow mesenchymal stem cells (hMSCs) toward myogenic phenotypes in a cardiac niche, the present study aimed to determine the role of miR-16 in this process. MAIN METHODS: hMSCs and neonatal rat ventricular myocytes were co-cultured indirectly in two chambers to set up a cardiac microenvironment (niche). miRNA expression profile in cardiac-niche-induced hMSCs was detected by miRNA microarray. Cardiac marker expression and cell cycle analysis were determined in different treatment hMSCs. Quantitative real-time PCR and Western blot were used to identify the expression of mRNA, mature miRNA and protein of interest. KEY FINDINGS: miRNA dysregulation was shown in hMSCs after cardiac niche induction. miR-16 was upregulated in cardiac-niche-induced hMSCs. Overexpression of miR-16 significantly increased G1-phase arrest of the cell cycle in hMSCs and enhanced the expression of cardiac marker genes, including GATA4, NK2-5, MEF2C and TNNI3. Differentiation-inducing factor 3 (DIF-3), a G0/G1 cell cycle arrest compound, was used to induce G1 phase arrest in cardiac-niche-induced hMSCs, and the expression of cardiac marker genes was up-regulated in DIF-3-treated hMSCs. The expression of CCND1, CCND2 and CDK6 was suppressed by miR-16 in hMSCs. CDK6, CCND1 or CCND2 knockdown resulted in G1 phase arrest in hMSCs and upregulation of cardiac marker gene expression in hMSCs in a cardiac niche. SIGNIFICANCE: miR-16 enhances G1 phase arrest in hMSCs, contributing to the differentiation of hMSCs toward myogenic phenotypes when in a cardiac niche. This mechanism provides a novel strategy for pre-modification of hMSCs before hMSC-based transplantation therapy for severe heart diseases.

Transcription factor Ap-1 mediates proangiogenic MIF expression in human endothelial cells exposed to Angiotensin II.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with the atherosclerotic process and atherosclerotic plaque stability. MIF was shown to be highly expressed in advanced atherosclerotic lesions. Neutralizing MIF with a blocking antibody induced a regression of established atherosclerotic lesions. In this study, we investigated the mechanism underlying the proangiogenic effect of MIF in human umbilical vein endothelial cells (HUVECs). We showed that MIF induced the expression of angiogenesis-related genes in HUVECs. We also showed that MIF induced tube formation of HUVECs in vitro and in vivo. Angiotensin II (Ang II) could specifically up-regulate MIF expression in HUVECs. Using a luciferase reporter assay, we demonstrated that the AP-1 response element in the 5'-UTR of the MIF gene played a role in Ang II-induced MIF expression. Small hairpin RNA (shRNA) targeting c-Jun, a component of AP-1, and the AP-1 inhibitor CHX both efficiently inhibited MIF expression. The consistent result of electrophoretic mobility shift assay (EMSA) showed that Ang II specifically increased AP-1 activation in HUVECs. Our results suggest that AP-1 mediates Ang II-induced MIF expression which contributes to atherosclerotic plaque destabilization in human endothelial cells.

Improved liquid chromatography-tandem mass spectrometry method for the analysis of eptifibatide in human plasma.

A rapid, selective and highly sensitive high performance liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C(18) column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6-->m/z 646.4 and m/z 931.6-->m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1-1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within +/-7.6% of nominal values. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 microg/kg bolus of eptifibatide to 8 healthy volunteers.