PhD theses

Cardiovascular disease remains the leading cause of death worldwide. Recent advances in single-cell transcriptomics (scRNA-seq) have revealed that vascular fibroblasts are far more heterogeneous and dynamic than previously appreciated, and that these cells play key roles in vascular homeostasis and cardiovascular disease. In this dissertation, we combined large-scale scRNA-seq meta-analysis with in vivo lineage tracing and mechanistic perturbation studies to define fibroblast trajectories in atherosclerosis and to uncover their functional roles in vascular aging and hypertension.

The main focus of this thesis was to gain mechanistic insight into how coagulation factors lead to thrombo-inflammatory VSMC changes and arterial remodeling via PAR activation to identify new suitable therapeutic targets to limit/prevent pathological vascular remodeling. We described the PAR-dependent molecular pathways that drive VSMC phenotype switching and their implications in promoting pathological vascular remodeling. The absence of PAR1 on iPSCs-derived VSMCs significantly impairs key processes associated with the transition from a contractile to a synthetic/proinflammatory VSMC phenotype. In here we investigated the role of cleavage-insensitive PAR1 and PAR2 variants in regulating VSMC phenotype switching, focusing on their contributions to pathological vascular remodeling. Finally, we studied the impact of switching from warfarin to rivaroxaban on vascular calcification and plaque progression in atherosclerosis.

This thesis contributes to the knowledge of pharmacological and nutritional interventions at attenuating vascular calcification, with a specific focus on vitamin K2 (menaquinone-7, MK-7) supplementation. A systematic review of the literature revealed that while several drug classes exhibit promising mechanistic or preclinical effects, consistent and clinically relevant reductions in vascular calcification have not yet been demonstrated in humans. These findings underscore the complexity of vascular calcification as a tightly regulated process involving multiple pathways that are likely not sufficiently addressed by single-agent therapy. Our randomized, placebo-controlled VitaK-CAC trial showed the first statistically significant evidence that vitamin K2 supplementation may beneficially modulate calcification processes in early-stage disease, with a more pronounced effect among patients exhibiting a biochemical response (reduction in dp-ucMGP).

The use of betaine as a novel senotherapeutic to combat vascular ageing is quickly emerging. The main pathway for betaine production in human cells is in mitochondria, through the oxidation of choline. Mitochondrial dysregulation occurs with ageing; hence betaine levels can become depleted with a knock-on effect on the maintenance of the epigenetic landscape of ageing and other hallmarks of ageing. We have achieved and exceeded our primary objectives. We have gained a novel mechanistic insight into vascular ageing that is applicable to all non-communicable diseases of ageing. This prototype non-drug approach has been successfully developed to extend lifespan and health span and tested this in two different in vivo models. As betaine is accessible and affordable, further studies should keep on exploring other pathways to achieve better treatment for diseasome of ageing.

This study established a custom, user-friendly computational pipeline for the detection and quantification of intracellular calcium ([Ca²⁺]ᵢ) oscillations, validated in human aldosterone-producing adrenocortical cells and accessible to researchers without prior coding experience. Its application provided the first characterization of [Ca²⁺]ᵢ transients in aldosterone-producing adenoma (APA) and normal adrenocortical (AAC) cells, addressing a critical gap in the field.

Additionally, APA cells showed unexpectedly low mitochondrial reactive oxygen species levels, possibly reflecting a protective adaptation sustaining aldosterone overproduction. Finally, the study introduced a novel in vitro model demonstrating that plasma from APA patients can induce vascular calcification, offering a promising platform for personalized cardiovascular risk assessment and targeted therapeutic development.

Mechanobiology research in (cardio)laminopathy research is crucial for understanding how mechanical forces and cellular structures influence tissue development, disease progression, and overall intra- and inter-cellular function. Mechanotransduction, the process through which cells convert mechanical stimuli into biochemical signals, plays a pivotal role in regulating gene expression and maintaining cellular homeostasis. The use of induced cardiac structures, particularly those derived from patient-specific iPSCs, provides valuable models to study these phenomena in a controlled setting, offering insights into disease mechanisms and potential therapeutic targets. Together, the research described in this dissertation holds immense potential for advancing our understanding of laminopathic behaviour, taking the first steps in in vivo LMNA gene therapy and aid in developing innovative treatments for mechanotransduction-related diseases.