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PROJECT TITLE: Impact of high glucose in valvular endothelial cells-monocyte crosstalk: molecular signatures and role in early valvular dysfunction

PROJECT CODE: PN-III-P1-1.1-PD-2021-0498

CONTRACT NO.: PD 35/2022

PROJECT LEADER: PhD. Monica Ţucureanu

PROJECT MENTOR: PhD. Ileana Mânduțeanu

PROJECT ACRONYM: VALDYSIGN

IMPLEMENTATION PERIOD: 01.04.2022 – 31.03.2024

BUDGET: 250.000 lei

IMPLEMENTATION TEAM: Monica Ţucureanu, Ileana Mânduțeanu

ABSTRACT. Aortic valve disease (AVD) and diabetes are progressive diseases that represent world-wide health problems. Diabetes is a risk factor for AVD, predicts faster disease progression and accelerates AVD. The mechanisms of early aortic valve dysfunction are still unclear, but evidence suggest that valvular endothelial cells (VEC) and monocytes (Mo) play key roles in the process. Our working hypothesis is that diabetic conditions (high glucose-HG) alter normal VEC-Mo interaction by progressively inducing molecular changes and function alteration in both cell types. The main goal of this project is to reveal the molecular signatures of VEC and Mo upon their interaction in HG conditions, to evaluate the role of key molecules in cell dysfunction and to propose novel mechanisms of early valvular dysfunction in diabetes. Our hypothesis will be tested by characterizing the molecular profile induced by HG in interacted VEC-Mo and by evaluating the function of relevant HG-induced molecules in endothelial permeability and monocyte adhesion/transmigration. HG-induced VEC and monocyte transcriptome will be analyzed, and key molecules will be validated at gene and protein level. The role of relevant molecules will be investigated in endothelial permeability and monocyte adhesion/transmigration, early processes of valvular dysfunction in diabetes. Our project would provide important knowledge of early AVD in diabetes and might indicate new possible therapeutic targets for AVD in diabetes.

Working hypothesis. In normal glucose conditions (A), monocytes (Mo) and valvular endothelial cells (VEC) crosstalk contribute to valvular homeostasis, while in high glucose (HG) conditions (B), cells crosstalk is altered and HG progressively induces molecular changes in VEC and Mo, their interaction leading to changes in cytoskeletal and junctional proteins, thus influencing endothelial permeability, the expression of inflammatory mediators and cellular adhesion molecules (CAM), determining adhesion and transmigration of monocytes, key events in aortic valve pathology. The goal of the proposed project is to uncover molecules and mechanisms involved in early changes that lead to aortic valve dysfunction in diabetic conditions.

OBJECTIVES:

Objective 1. To characterize the molecular profile induced by short high glucose exposure in VEC-Mo co-culture.

Activity 1.1. Whole-transcriptome sequencing to identify molecules involved in early modifications induced in VEC and Mo after interaction in HG conditions;

Activity 1.2. Validation of key molecules by RealTime PCR;

Activity 1.3. Investigation of protein expression of relevant molecules induced by HG conditions in VEC-Mo co-culture.

Objective 2. To evaluate the function of relevant molecules found modified in O1 in endothelial permeability and monocyte adhesion and transmigration.

Activity 2.1. Investigation of endothelial permeability in HG conditions: role of changes in junctional and cytoskeletal proteins;

Activity 2.2. Establishing the role of diabetes on monocyte adhesion and transmigration in dynamic conditions;

Activity 2.3. Analysis of the role of identified molecules in endothelial permeability and monocyte adhesion and transmigration in dynamic conditions.

SCIENTIFIC REPORT on the implementation of the project in the period April 2022 – December 2022 (Stage 1)

DISSEMINATION: Participation in the International Conference and XXXIX Annual Scientific Session of the Romanian Society of Cell Biology, 21-23 October 2022, Cluj-Napoca, Romania - MOLECULAR SIGNATURES OF VALVULAR ENDOTHELIAL CELLS AND MONOCYTES CROSS-TALK IN EARLY DIABETIC CONDITIONS. Monica Tucureanu, Letitia Ciortan, Ileana Manduteanu– poster presentation.

RESULTS. Aortic valve disease (BVA) and diabetes are progressive diseases that represent health problems worldwide. Diabetes is a risk factor for BVA, predicts a faster progression of the disease and accelerates BVA. The early mechanisms of aortic valve dysfunction are still unclear, but valve endothelial cells (VEC) and monocytes (Mo) play key roles in this process. The mechanisms by which diabetes contributes to early dysfunction of the aortic valve are not yet well known, so our hypothesis is that elevated glucose alters the normal interaction of valvular endothelial cells with monocytes, progressively inducing molecular changes, phenotypic changes and functional alterations of both cell types. The objective of the VALDYSIGN project is to highlight the molecules modified in VEC and Mo in their communication under high glucose (HG) conditions, to assess the role of key molecule in cell dysfunction and to propose new mechanisms of early valve dysfunction in diabetes. In the first stage of the VALDYSIGN project, the molecular profile induced by short exposure to HG in VEC-Mo interaction was characterized by sequencing the transcriptome of both cell types. Differentially expressed genes (DEG) in different experimental conditions were analyzed functionally by associating them with the available databases (KEGG maps). Molecules involved in: focal adhesions, tight junctions, adherens junctions, cell adhesion molecules, matrix-receptor interactions, cytoskeletal regulation and endothelial transmigration have been identified in VECs interacting with Mo in HG conditions, suggesting changes in endothelial permeability and changes in the cell cytoskeleton. Moreover, in Mo interacting with VECs in HG conditions were identified molecules involved in: matrix-receptor interactions, transendothelial migration, cell adhesion molecules and signaling pathways TLR, NF-kB, JAK-STAT, TNFα and chemokines, suggesting increased adherence and transmigration of monocytes to the endothelium and an inflammatory phenotype. These data suggest that early exposure to high glucose conditions progressively induces molecular and phenotypic changes in both cell types, thus contributing to valve dysfunction in diabetes.

SCIENTIFIC REPORT on the implementation of the project in the period January 2023 – December 2023 (Stage 2)

DISSEMINATION:

- Bioinformatic analysis of molecular mechanisms underlying the aortic valvular dysfunction in diabetes. Monica Tucureanu. Participation in the conference “Crossing bridges between bioinformatics and clinical research – Genetoberfest GO2023”, from 16-19 October 2023, Munich, Germany – oral presentation;

- Molecular mechanisms underlying the aortic valvular dysfunction in diabetes. Monica Tucureanu, Letitia Ciortan, Razvan Macarie, Andreea Mihaila, Elena Butoi, Ileana Manduteanu. Participation in the 44th ANNUAL SCIENTIFIC SYMPOSIUM OF THE INSTITUTE OF CELLULAR BIOLOGY AND PATHOLOGY “NICOLAE SIMIONESCU” held jointly with the 40th Annual Scientific SESSION OF THE ROMANIAN SOCIETY FOR CELL BiOLOGY, in the period 16-17 November 2023, Bucharest, Romania – poster presentation.

RESULTS. The valve endothelium is an important protective barrier against metabolic, mechanical and inflammatory aggression and is plays important roles in valve homeostasis and in interactions with cells and molecules in circulating blood. The regulation of the endothelial barrier function is orchestrated by the quantity and arrangement of the intercellular junctions, in particular adherens and tight, the organization of the actin cytoskeleton and the focal adhesions. Together, these elements govern the permeability of valve tissue. There is evidence that the phenotype of valve endothelial cells (VEC) is progressively modified in diabetes, but the specific mechanisms by which diabetes contributes to early endothelial dysfunction remain unelucidated. However, it is doubtful that both VECs and monocytes (Mo) play an important role, and the diabetic environment disturbs cellular homeostasis. Therefore, the hypothesis of our study is that the interaction between monocytes and VECs in diabetic conditions triggers molecular changes that lead to altered endothelial integrity and monocyte adhesion and transmigration. The aim of this study is to establish the molecular signatures of VECs after their short interaction with monocytes in high glucose (HG) conditions and to propose new mechanisms that underlie early valve dysfunction in diabetes. In the second stage of the VALDYSIGN project, the gene and protein expression of molecules modified in VECs by their interaction with monocytes in HG conditions was investigated, focusing on the molecules involved in cell-extracellular matrix and cell-cell interactions, cell adhesion and cytoskeleton regulation (identified by mRNA sequencing analysis). The results showed that the interaction between VECs and monocytes in HG conditions modifies focal adhesions by decreasing the phosphorylation of focal adhesion kinase, decreased paxillin expression, internalization of vinculin and decreased expression of integrins functioning as receptors for the extracellular matrix. Moreover, VEC-monocyte interaction in HG conditions activates PI3K and RhoA-ROCK1 signaling pathways, reduces intercellular junctions by reducing cadherin-2, increases the expression of E-selectin and α4 integrin on the cell surface. Because our study identified changes in cell-cell and cell-matrix interactions, we investigated the functional role of these changes in cell permeability. Our data show that the permeability of the valve endothelium increases in interaction with monocytes in HG, compared to control VEC. Furthermore, VECs interacting with monocytes in HG are less adhesive to collagen I, fibronectin and vitronectin, which corresponds to observations on integrin expression. Our data also suggest that HG conditions lead to increased monocyte adhesion and transmigration, which may contribute to the progression of aortic valve lesions.