Endothelial cell direct reprogramming: Past, present, and future.

Ischemic cardiovascular disease still remains as a leading cause of morbidity and mortality despite various medical, surgical, and interventional therapy. As such, cell therapy has emerged as an attractive option because it tackles underlying problem of the diseases by inducing neovascularization in ischemic tissue. After overall failure of adult stem or progenitor cells, studies attempted to generate endothelial cells (ECs) from pluripotent stem cells (PSCs). While endothelial cells (ECs) differentiated from PSCs successfully induced vascular regeneration, differentiating volatility and tumorigenic potential is a concern for their clinical applications. Alternatively, direct reprogramming strategies employ lineage-specific factors to change cell fate without achieving pluripotency. ECs have been successfully reprogrammed via ectopic expression of transcription factors (TFs) from endothelial lineage. The reprogrammed ECs induced neovascularization in vitro and in vivo and thus demonstrated their therapeutic value in animal models of vascular insufficiency. Methods of delivering reprogramming factors include lentiviral or retroviral vectors and more clinically relevant, non-integrative adenoviral and episomal vectors. Most studies made use of fibroblast as a source cell for reprogramming, but reprogrammability of other clinically relevant source cell types has to be evaluated. Specific mechanisms and small molecules that are involved in the aforementioned processes tackles challenges associated with direct reprogramming efficiency and maintenance of reprogrammed EC characteristics. After all, this review provides summary of past and contemporary methods of direct endothelial reprogramming and discusses the future direction to overcome these challenges to acquire clinically applicable reprogrammed ECs.

A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases.

Research involving α/ß hydrolases, including α-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting α/ß hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a 'hit' or a 'miss,' in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of α/ß hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.

Generation of Directly Reprogrammed Human Endothelial Cells.

Direct reprogramming provides a novel breakthrough for generating functional endothelial cells (ECs) without the need for intermediate stem or progenitor states, offering a promising resource for cardiovascular research and treatment. ETV2 is a key transcription factor that has been identified as a pioneering factor for specifying endothelial lineage. Achieving precise ETV2 induction is essential for effective endothelial reprogramming, and maintaining the reprogrammed cellular phenotype relies on a specific combination of growth factors and small molecules. Thus, we hereby provide a straightforward and comprehensive protocol for generating two distinct types of reprogrammed ECs (rECs) from human dermal fibroblasts (HDFs). Early rECs demonstrate a robust neovascularization property but lack the mature EC phenotype, while late rECs exhibit phenotypical similarity to human postnatal ECs and have a neovascularization capacity similar to early rECs. Both cell types can be derived from human somatic source cells, making them suitable for personalized disease investigations, drug discovery, and disease therapy.

Reduced angiovasculogenic and increased inflammatory profiles of cord blood cells in severe but not mild preeclampsia.

Preeclampsia (PE) is a prevalent pregnancy disorder that leads to high maternal and fetal morbidity and mortality. While defective vascular development and angiogenesis in placenta are known as crucial pathological findings, its pathophysiological mechanism remains elusive. To better understand the effects of PE on angio-vasculogenesis and inflammatory networks in the fetus and to identify their biological signatures, we investigated the quantitative and functional characteristics of cord blood-derived mononuclear cells (CB-MNCs) and CD31-positive MNCs. Flow cytometry analysis demonstrated that the CB-MNCs from the severe PE group had significantly decreased number of cells expressing CD3, CD11b, CD14, CD19, KDR, and CD31 compared with the normal group. Quantitative real time PCR (qRT-PCR) shows down-regulation of the major angiogenic factor VEGFA in MNCs and CD31+ MNCs in severe PE. The major inflammatory cytokines IL1 was highly upregulated in CD31+ CB-MNCs in the severe PE patients. Mild PE patients, however, did not display any significant difference in expression of all measured angiogenic genes and most inflammatory genes. These findings show distinct angiogenic and inflammatory signatures from severe PE, and they may play a significant role in the pathogenesis of vascular defects in placenta of severe PE.