Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response.

Vasculopathies occur 15 years earlier in individuals with diabetes mellitus (DM) as compared to those without, but the underlying mechanisms driving diabetic vasculopathy remain incompletely understood. Endothelial cells (ECs) and macrophages (MΦ) are critical players in vascular wall and their crosstalk is crucial in diabetic vasculopathy. In diabetes, EC activation enables monocyte recruitment, which transmigrate into the intima and differentiate into macrophages (MΦ). Beyond this established model of diapedesis, EC-MΦ interplay is highly intricate and heterogenous. To capture these highly context dependent EC-MΦ interactions, we leveraged single-cell (sc)RNA-seq in conjunction with spatial transcriptome (ST)-seq profiling to analyze human mesenteric arteries from non-diabetic (ND) and type 2 diabetic (T2D) donors. We provide in this study a transcriptomic map encompassing major arterial vascular cells, e.g., EC, mononuclear phagocyte (MP), and T cells, and their interactions associated with human T2D. Furthermore, we identified Triggering Receptor Expressed on Myeloid Cells 2 ( TREM2) as a top T2D-induced gene in MP, with concomitant increase of TREM2 ligands in ECs. TREM2 induction was confirmed in mouse models of T2D and monocyte/MΦ subjected to DM-mimicking stimuli. Perturbing TREM2 with either an antibody or silencing RNA in MPs led to decreased pro-inflammatory responses in MPs and ECs and increased EC migration in vitro . In a mouse model of diabetes, TREM2 expression and its interaction with ECs are increased in the ischemic, as compared to non-ischemic muscles. Importantly, neutralization of TREM2 using a neutralizing antibody enhanced ischemic recovery and flow reperfusion in the diabetic mice, suggesting a role of TREM2 in promoting diabetic PAD. Finally, we verified that both TREM2 expression and the TREM2-EC-interaction are increased in human patients with DM-PAD. Collectively, our study presents the first atlas of human diabetic vessels with a focus on EC-MP interactions. Exemplified by TREM2, our study provides valuable insights into EC-MΦ interactions, key processes contributing to diabetic vasculopathies and the potential of targeting these interactions for therapeutic development.

Seeing is Believing: Developing Multimodal Metabolic Insights at the Molecular Level.

This outlook explores how two different molecular imaging approaches might be combined to gain insight into dynamic, subcellular metabolic processes. Specifically, we discuss how matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and stimulated Raman scattering (SRS) microscopy, which have significantly pushed the boundaries of imaging metabolic and metabolomic analyses in their own right, could be combined to create comprehensive molecular images. We first briefly summarize the recent advances for each technique. We then explore how one might overcome the inherent limitations of each individual method, by envisioning orthogonal and interchangeable workflows. Additionally, we delve into the potential benefits of adopting a complementary approach that combines both MSI and SRS spectro-microscopy for informing on specific chemical structures through functional-group-specific targets. Ultimately, by integrating the strengths of both imaging modalities, researchers can achieve a more comprehensive understanding of biological and chemical systems, enabling precise metabolic investigations. This synergistic approach holds substantial promise to expand our toolkit for studying metabolites in complex environments.

Quantitative Real-Time Analysis of Living Materials by Stimulated Raman Scattering Microscopy.

Composite materials built in part from living organisms have the potential to exhibit useful autonomous, adaptive, and self-healing behavior. The physicochemical, biological, and mechanical properties of such materials can be engineered through the genetic manipulation of their living components. Successful development of living materials will require not only new methods for design and preparation but also new analytical tools that are capable of real-time noninvasive mapping of chemical compositions. Here, we establish a strategy based on stimulated Raman scattering microscopy to monitor phosphatase-catalyzed mineralization of engineered bacterial films in situ. Real-time label-free imaging elucidates the mineralization process, quantifies both the organic and inorganic components of the material as functions of time, and reveals spatial heterogeneity at multiple scales. In addition, we correlate the mechanical performance of films with the extent of mineralization. This work introduces a promising strategy for quantitatively analyzing living materials, which should contribute to the accelerated development of such materials in the future.

Genetic Deletion of the LINC00520 Homolog in Mouse Aggravates Angiotensin II-Induced Hypertension.

(1) Background: Hypertension is a complex, multifactorial disease that is caused by genetic and environmental factors. Apart from genetic predisposition, the mechanisms involved in this disease have yet to be fully understood. We previously reported that LEENE (lncRNA enhancing endothelial nitric oxide expression, transcribed from LINC00520 in the human genome) regulates endothelial cell (EC) function by promoting the expression of endothelial nitric oxide synthase (eNOS) and vascular growth factor receptor 2 (VEGFR2). Mice with genetic deletion of the LEENE/LINC00520 homologous region exhibited impaired angiogenesis and tissue regeneration in a diabetic hindlimb ischemia model. However, the role of LEENE in blood pressure regulation is unknown. (2) Methods: We subjected mice with genetic ablation of leene and wild-type littermates to Angiotensin II (AngII) and monitored their blood pressure and examined their hearts and kidneys. We used RNA-sequencing to identify potential leene-regulated molecular pathways in ECs that contributed to the observed phenotype. We further performed in vitro experiments with murine and human ECs and ex vivo experiments with murine aortic rings to validate the select mechanism. (3) Results: We identified an exacerbated hypertensive phenotype of leene-KO mice in the AngII model, evidenced by higher systolic and diastolic blood pressure. At the organ level, we observed aggravated hypertrophy and fibrosis in the heart and kidney. Moreover, the overexpression of human LEENE RNA, in part, restored the signaling pathways impaired by leene deletion in murine ECs. Additionally, Axitinib, a tyrosine kinase inhibitor that selectively inhibits VEGFR suppresses LEENE in human ECs. (4) Conclusions: Our study suggests LEENE as a potential regulator in blood pressure control, possibly through its function in ECs.

Methods for Suppressing Hydrogen Sulfide in Biological Systems.

Significance: Hydrogen sulfide (H2S) plays critical roles in redox biology, and its regulatory effects are tightly controlled by its cellular location and concentration. The imbalance of H2S is believed to contribute to some pathological processes. Recent Advances: Downregulation of H2S requires chemical tools such as inhibitors of H2S-producing enzymes and H2S scavengers. Recent efforts have discovered some promising inhibitors and scavengers. These advances pave the road toward better understanding of the functions of H2S. Critical Issues: Precise H2S downregulation is challenging. The potency and specificity of current inhibitors are still far from ideal. H2S-producing enzymes are involved in complex sulfur metabolic pathways and ubiquitously present in biological matrices. The inhibition of these enzymes can cause unwanted side effects. H2S scavengers allow targeted H2S clearance, but their options are still limited. In addition, the scavenging process often results in biologically active by-products. Future Directions: Further development of potent and specific inhibitors for H2S-producing enzymes is needed. Scavengers that can rapidly and selectively remove H2S while generating biocompatible by-products are needed. Potential therapeutic applications of scavengers and inhibitors are worth exploring. Antioxid. Redox Signal. 36, 294-308.

Effect of Loading Method on a Peptide Substrate Reporter in Intact Cells.

Studies of live cells often require loading of exogenous molecules through the cell membrane; however, effects of loading method on experimental results are poorly understood. Therefore, in this work, we compared three methods for loading a fluorescently labeled peptide into cells of the model organism Dictyostelium discoideum. We optimized loading by pinocytosis, electroporation, and myristoylation to maximize cell viability and characterized loading efficiency, localization, and uniformity. We also determined how the loading method affected measurements of enzyme activity on the peptide substrate reporter using capillary electrophoresis. Loading method had a strong effect on the stability and phosphorylation of the peptide. The half-life of the intact peptide in cells was 19 ± 2, 53 ± 15, and 12 ± 1 min, for pinocytosis, electroporation, and myristoylation, respectively. The peptide was phosphorylated only in cells loaded by electroporation. Fluorescence microscopy suggested that the differences between methods were likely due to differences in peptide localization.