Project 1

Figure legend:  Characterization of human and mouse adipose tissue MVECs. (A) Experimental design showing the workflow for the isolation of ECs utilizing an inducible YFP reporter mouse under the control of the Cdh5-Cre promoter (B-D) HFD reduces tissue specific EC heterogeneity in arteries and capillaries. Data represented are from both mesenteric (Mes) and epididymal fat (Epi) from mice fed either a normal chow (NC) and high fat diet (HFD). Cells were subsetted and re-clustered into UMAPS based on their location in the vascular tree. B Distributions of these cells for each vascular subtype was quantified and graphed (C). Differentially expressed genes between NC and HFD in each vascular subset (Mes and Epi ECs combined) is shown as a percentage of total genes expressed). P-values were calculated with Chi-squared tests. (D) Volcano plot showing differential gene expression in human adipose ECs from obese individuals relative to lean individuals; genes are displayed as up- (red) or down-regulated (blue). (E) UMAP of human EC CAV1 expression across  vascular clusters. (F) Dot plot showing human CAV1 expression among ECs of different vascular origins from both lean and obese humans

Background and preliminary work: The endothelium constitutes the interface between circulating blood and organ parenchyma. As such, ECs control the exchange of nutrients, metabolites, hormones, and other molecules in an organ-specific manner, thereby controlling organ functions. ECs regulate the intake of circulating fat in adipose tissue. Accumulating evidence indicates that excessive fatty acid cargo transported into the endothelium can disrupt the delicate equilibrium of fatty acid storage and handling, thereby promoting endothelial dysfunction ^{1, 2}. Consequently, lipids begin to accumulate within the endothelium, inducing a state of lipotoxicity that further contributes to changes in cellular morphology and function and exacerbates endothelial dysfunction ^3-5. This dysregulation not only compromises the physiological functions of ECs but also sets the stage for the development of various cardiovascular diseases associated with obesity ^{6, 7}.The mechanisms that inhibit the onset and progression of endothelial dysfunction are still unknown and require further investigation ⁸. 

In a project-specific preliminary study ⁹ (Fig.), we performed single-cell mRNAseq analysis of EC subpopulations of adipose tissues and found that microvascular endothelial cells from visceral adipose tissues present very specific molecular characteristics as compared to other endothelial cell types and may participate in the regulation of fat trafficking and accumulation. Moreover, we observed that microvascular EC from mice fed a high-fat diet showed a reduction in Cav1 ¹⁰. In a published scRNA-seq dataset of human adipose tissue from both healthy individuals and patients with metabolic syndrome, we found that Cav1 expression was decreased in adipose ECs from obese patients ¹⁰. These data suggest that Cav1 may be an important component of lipid handling in endothelial cells during obesity. By using EC-specific caveolin-1 KO mice fed with a high-fat diet, we found that the lack of Cav-1 inhibited visceral fat accumulation and was linked to a disturbed subcellular localization of eNOS, uncontrolled increase in NO synthesis, fat accumulation, and nitrosation of the fatty acid transporter CD36 ¹⁰. Surprisingly, these mice showed low circulating lipid levels. Thus, lipid accumulation and plasma lipid levels may be controlled by eNOS/caveolin trafficking, specifically in adipose tissue microvascular endothelial cells. 

Hypothesis: We hypothesize that EC heterogeneity and plasticity play a fundamental role in adipose tissue metabolic activity and fat accumulation, both under homeostatic conditions and in cardiometabolic disease. We poise that a continued excess of fat and caloric intake induce maladaptation of fat handling in microvascular endothelial cells (MVECs) and according to our preliminary work is exacerbated by lack of Cav1/defective eNOS trafficking via NO-mediated regulation of CD36 activity, making Cav1-mediated eNOS trafficking as one possible pathway regulating fat handling of MVECs. 

Work program: The major aim of this study is to identify and characterize the cell-specific role of MVECs in adipose tissue for metabolic regulation and fat homeostasis by applying both non targeted and targeted approaches. Here, we will combine single-cell omics untargeted approaches in human and mouse fat tissue MVECs with functional targeted analysis of EC-specific eNOS KO and knock-in (KI) ¹¹ mice and EC-specific Cav-1 KO mice fed with a high-fat diet and normal chow diet ^{9, 10}. The working program is organized in 3 working packages.

WP1: Molecular and metabolic characteristics of adipose tissue MVECs in homeostatic conditions and HFD (non-targeted approach). The aim of WP1 is to analyze the acute and chronic effects of HFD in molecular and metabolic characteristics of MVECs from human and mice white adipose tissue. WT will undergo treatment with HFD for 1 day, 4 weeks, and 3 months according to previously published protocols ^9 10. MVECs will be isolated from adipose tissue by using magnetic assisted cell sorting (MACS) and will undergo single cell mRNA seq. Changes in molecular characteristics and cell phenotype of MVECs will be compared among the different time points. In addition, changes in glucose tolerance, systemic and adipose tissue insulin resistance, in vivo lipid trafficking, and CV hemodynamics will be analyzed for each time point and compared to chow-treated mice. Human MVECs from people with obesity or controls will be isolated using magnetic assisted cell sorting (MACS) from white adipose tissues obtained by liposuction or surgery from three individuals each, pooled, and subjected to bulk RNA sequencing. The molecular and cellular characteristics of healthy human MVECs will be compared to obese MVECs. To map specific adipose tissue MVECs characteristics, we will also compare healthy human and mouse MVECs, as well as MVECs from obese individuals and mice receiving HFD for three months. Common features will be further tested using a single-cell mRNA seq approach. The results of this WP will provide a deeper understanding of the nature, function, and dysfunction of adipose tissue MVECs.

WP2. Role of NO signaling and metabolism in the function and dysfunction of adipose tissue MVECs (targeted and non-targeted approaches). Global eNOS KO are more prone to fat accumulation in adipose tissue and liver. In preliminary work we found that Cav1-mediated eNOS trafficking as one possible pathway regulating fat handling in adipose tissue MVECs ¹⁰. Therefore we aim to analyze whether endothelial dysfunction induced by lack of eNOS or eNOS trafficking dysregulation may affect molecular characteristics, phenotype and metabolic profile in MVECs, in EC-specific Cav1 KO-mice and EC-eNOS KO/KI mice treated with HFD or chow for 3 months. To analyze acute changes a further time point (1 day or 4 weeks) will be chosen according to the analysis of transcripts of genes belonging to the NO signaling pathways obtained in WP1. In these mouse cohorts, we will perform spatial transcriptomics of adipose tissue slices, focusing on MVECs and surrounding stromal and inflammatory cells, as well as analysis of EC lipotoxicity, systemic and adipose tissue insulin resistance, in vivo lipid trafficking, and CV hemodynamics. The results of these studies will reveal how lipotoxicity and eNOS signaling/trafficking affect MVEC function and phenotype.

WP3: Role of Cav1 and eNOS localization in CD36 nitrosation, fat uptake, and homeostasis in cultured MVECs and organoids (targeted approach). In preliminary work we identified CD36 nitrosation as one possible mechanistic link between Cav1-mediated regulation of eNOS trafficking and fat handling in adipose tissue MVECs ¹⁰. Therefore in this WP we aim to analyze the specific role of Cav1-mediated control of eNOS localization and activity and CD36 nitrosation in human microvascular endothelial cells in the regulation of fat homeostasis in human MVECs co-cultured with adipocytes in a Transwell (similar to previous co-culture models of the Isakson lab ¹² as well as in adipose tissue organoids. In this experiments we will use CRISPR/CAS to modify specific cysteine of CD36, which we found to be targeted by NO¹⁰ to produce a redox death mutant by substituting the targeted cysteine with an alanine. In this context, we will analyze lipid accumulation and trafficking in MVECs into adipocytes, lipotoxicity, eNOS activity/expression/localization, insulin signaling, mitochondrial activity, and redox status. Further experiments will focus on other targets identified by analyzing the results of WP-1 by using the same approach as described for Cav1/eNOS/CD36 pathway. The results of this WP will allow us to understand how lipid accumulation and EC eNOS/Cav- dependent trafficking are controlled and regulated and how excessive lipids may affect the regulatory mechanisms. Taken together, this study will reveal the role of microvascular endothelial cells in adipose tissue homeostasis at the single-cell level, and will potentially provide new molecular targets for the regulation of adipose tissue function in health and disease.

Added value of the collaboration: The collaboration between the Cortese-Krott and Isakson labs represents a synergistic scientific partnership, based on complementary expertise in eNOS/NO biology and endothelial cell function ^{11, 12}. The Cortese-Krott lab specializes in the biological chemistry of NO, including the formation of NO metabolites, their interaction with cysteine and sulfur species, and on NO species analytics ¹³. Additionally, they have developed cell-specific eNOS loss-of-function and gain-of-function models ¹¹. The Isakson lab focuses on the function of resistive vessels¹², endothelial hemoglobin and microvascular endothelial cells ^{14, 15}, with extensive expertise in mRNA sequencing, bioinformatics, and the study of metabolic derangements due to high-fat diet, utilizing EC Cav-1 KO mice and human samples ^{9, 10}, which now should be extended in the EC eNOS KO/KI mice. Over ten years, this collaboration yielded over 12 joint manuscripts on different aspects of NO signaling and function, demonstrating its productivity, impact and added value. The PhD student from the German lab will perform experiments with human samples in the Isakson lab, and with EC eNOS KO/KI in the Cortese-Krott lab. The PhD student in the Isaskon lab will perform experiments with EC Cav-1 KO in his lab, and will establish and perform experiments with co-cultures and organoids during the exchange in the Cortese-Krott lab. Data will be fully shared between both laboratories.