Recurrent spontaneous abortion, a common condition in reproductive medicine, often arises from complex factors and lacks specific treatments. While studies have associated folate metabolism abnormalities with poor embryonic development, research on methionine metabolism, particularly homocysteine levels, has been limited.
In this study, we analyzed blood samples from women with RSA and those with normal pregnancies.
We observed elevated homocysteine levels in women with RSA, which were correlated with increased total plasma microparticles, endothelial microparticles (EMPs), and free plasma DNA. Furthermore, we exposed human umbilical vein endothelial cells (HUVECs) to varying concentrations of homocysteine (0, 0.5, 1, 2, 4, 8 mmol/L). We found that higher homocysteine levels exacerbated its cytotoxic effects on HUVECs. Flow cytometry revealed that homocysteine compromised cell membrane integrity, enhanced membrane permeability, and promoted EMPs release. Our findings suggest that elevated homocysteine levels in women with RSA may induce significant endothelial cell apoptosis, leading to endothelial dysfunction and an increase in released microparticles and free DNA, potentially contributing to miscarriages.
This study may contribute to understanding and exploring the underlying mechanisms of unexplained recurrent spontaneous abortion (URSA).
© 2025 Qi et al.

This study focuses on dose-response investigation using a codon-optimized and de novo-synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug enabling of subsequent clinical studies.
Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia. Understanding the dose for effective gene delivery is crucial for future Investigational New Drug-enabling studies.
Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved 3 cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly in divided aliquots, ranging from 2 × 10 9 VG to 2 × 10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery, and therapeutic angiogenesis were assessed.
Codon-optimized E-Sel/AAV2 gene therapy exhibits a superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2 × 10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber scores, increased running stamina, and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied.
E-Sel/AAV2 Vascular Regeneration Gene Therapy holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2 × 10 11 VG aliquots injected intramuscularly.
Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.

The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the anti-inflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomain-containing protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent anti-inflammatory effects of depolymerizing microtubule-targeting agents.

Genome-wide association studies have identified >250 genetic variants associated with coronary artery disease (CAD), but the causal variants, genes and molecular mechanisms remain unknown at most loci. We performed pooled CRISPR screens to test the impact of sequences at or near CAD-associated genetic variants on vascular endothelial cell functions. Using CRISPR knockout, inhibition and activation, we targeted 1998 variants at 83 CAD loci to assess their effect on three adhesion proteins (E-selectin, ICAM1, VCAM1) and three key endothelial functions (nitric oxide and reactive oxygen species production, calcium signalling). At a false discovery rate ≤10%, we identified significant CRISPR perturbations near 42 variants located within 26 CAD loci. We used base editing to validate a putative causal variant in the promoter of the FES gene. Although a few of the loci include genes previously characterized in endothelial cells (e.g. AIDA, ARHGEF26, ADAMTS7), most are implicated in endothelial dysfunction for the first time. Detailed characterization of one of these new loci implicated the RNA helicase DHX38 in vascular endothelial cell senescence. While promising, our results also highlighted several limitations in using CRISPR perturbations to functionally dissect GWAS loci, including an unknown false negative rate and potential off-target effects.
Copyright: © 2023 Wünnemann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Endothelial microparticles (EMPs) are partly associated with the progress of chronic obstructive pulmonary disease (COPD). We sought to measure the levels of EMPs in COPD patients and in human pulmonary microvascular endothelial cells (HPMECs) exposed to cigarette smoking extract (CSE) to elucidate the potential mechanisms of their action.
We obtained prospectively blood EMPs from 30 stable COPD patients and 20 non-COPD volunteers. EMP subpopulations were determined by flow cytometry in platelet-free plasma according to the expression of membrane specific antigens. Cell growth, proliferation, apoptosis and the expression of protein kinase B (Akt) in HPMECs after exposure to PECAM EMPs were assessed. After intervention with an antioxidant (Eukarion-134, EUK-134), apoptosis and the expression of Akt in HPMECs were also measured.
Unlike those of MCAM EMPs, VE-cadherin, PECAM and E-selectin EMP values were significantly higher in the stable COPD patients than in the non-COPD volunteers (p<0.05). Only PECAM EMPs were higher in HPMECs exposed to CSE (p<0.05). Further, in vitro studies showed that the apoptosis rate and expression of cleaved caspase 3/9 in HPMECs increased in a dose- and time-independent manner with PECAM EMPs. The expression of phospho-Akt (p-Akt) decreased in a time-independent manner with PECAM EMPs (p<0.05). Compared with the control group, the early apoptosis rate of HPMECs was higher, and the expression of p-Akt was lower in both the PECAM EMP group and EUK-134 + PECAM EMP group (p<0.05). The apoptosis rate declined markedly, and the expression of p-Akt was higher in the EUK-134 + PECAM EMP group, compared with the PECAM EMPs group (p<0.05).
The present results suggest that PECAM EMPs positively regulate apoptosis in HPMECs in COPD, likely by decreasing Akt phosphorylation and can be protected by antioxidants.
© 2022 Zeng Y. et al.