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Peyer’s patches (PPs) are secondary lymphoid organs that monitor the intestinal lumen and initiate adaptive immune responses against pathogens. The efferent lymphatic vessels of PPs serve as the primary pathway for lymphocyte egress from the gut, however little is known about their molecular identity and development.

We performed scRNA-seq of mouse intestinal lymphatic endothelial cells (LECs) from gut and PP samples and used a combination of bioinformatic analysis, immunofluorescence and multiparameter flow cytometry to characterize the different gut LEC populations. We also utilized 3D imaging to examine steps of PP lymphatic development and exploited a nanoindentation technique to provide a map of tissue stiffness across the PP.

PPs harbor a molecularly distinct population of LECs with upregulated gene expression programs related to cell adhesion, complement system, lipid metabolism processes and leukocyte migration. We found four subtypes characterized by unique molecular signatures and spatial distributions, which exhibit both similarities to and differences from previously described lymph node LECs.

We provide a three-stage model of PP lymphatic formation during postnatal development, which includes an initial phase of VEGF-C driven lymphatic expansion, followed by VEGF-C independent steps of remodeling and specialization.

Furthermore, PPs feature a spatial gradient in stiffness, attributed to the presence of diverse immune cell types. We also highlight how these differences emerge during development due to the presence of various mechanical cues, which coincide with PP lymphatic remodeling.

Our findings identify distinct subtypes PP-specific LECs with an insight into subset-specific marker genes, location and functions. We propose a role of mechanical forces in PP postnatal lymphatic remodeling and specialization. We aim to provide a better understanding on the roles of PP lymphatics in the promotion of beneficial mucosal and systemic immune surveillance during postnatal development and in pathological conditions.

Europe’s aging population is increasing, with individuals over 65 projected to reach 28.9% of the population by 2050. Aging is associated with a decline in health conditions, an increase in chronic illnesses and growing healthcare expenditures. Among age-related diseases (ARDs), cardiovascular diseases remain the leading cause of mortality and morbidity.
Endothelial dysfunction plays a key role in vascular aging and contributes to multiple ARDs. Heterochronic parabiosis experiments have shown that young blood can modulate endothelial aging hallmarks and improve cardiovascular and cognitive function. However, translating these findings to humans is challenging due to species-specific differences in aging. Developing human-relevant in vitro models that closely mimic endothelial aging could help bridging this gap and advance translational research.

We developed a high-throughput platform (up to 32 samples) to biofabricate 3D human organotypic models using fully characterized dermal microvascular endothelial cells and fibroblasts derived from healthy donor biopsies, embedded in a 3D fibrin hydrogel. The model features mesoscale dimensions (i.e. millimeter-scale) and incorporates organotypic co-culture and unidirectional hemodynamic stimuli, which help to recover the in vivo molecular profile of endothelial cells. The platform enables automated hydrogel seeding, high-content confocal imaging, and perfusability quantification.

Optimized culture conditions supported the formation of self-assembled microvascular networks with physiologically relevant vessel diameters (5-50 micrometer) and lumen formation. Perfusability tests demonstrated flow of 70kDa-dextran through the lumens of the network spanning the entire matrix. Exposure to old sera resulted in a loss of perfusability, suggesting functional microvascular impairment. We are currently validating aging markers (e.g. p16, gamma-h2ax, Sirt1) and microvascular architecture by comparing our model with dermal biopsies from donors of different ages.

Our 3D microvascular model recapitulates key aspects of physiological microvessels and shows a promising differential response to young and old sera. Future work will include identifying how the serum from young and old donors affects the vessels at the molecular and functional level, and developing new organotypic models of human microvasculature (e.g. blood-brain-barrier).

Atherosclerosis and plaque progression involve complex cellular and molecular interactions, with disturbed blood flow critically affecting endothelial cells (ECs). The transcription factor TWIST1 is a key regulator in this process and has been linked to coronary artery disease and stroke. Although endothelial TWIST1 promotes atherogenesis, its role in later stages of the disease remains poorly understood. In this study, we aimed to characterize TWIST1’s involvement in atheroprogression and identify key downstream targets and mechanisms it regulates.

Using ApoE-/- mice fed a Western diet, endothelial-specific Twist1 deletion was induced after plaque formation. Following additional Western diet feeding, ECs were isolated for scRNA-seq, and plaques were histologically analyzed. In parallel, Human aortic ECs exposed to low oscillatory shear stress (LOSS) after TWIST1 silencing underwent RNA-seq to identify downstream targets.

Single-cell RNA-seq analysis revealed that, compared to controls, Twist1 deletion suppressed endothelial cell clusters associated with LOSS markers, endothelial-to-mesenchymal transition (EndMT), proliferation, and extracellular matrix organization. In murine brachiocephalic plaques, endothelial Twist1 expression promoted plaque growth but also collagen deposition, and ACTA2-positive cell accumulation, hallmarks of plaque stability. Conversely, endothelial Twist1 reduced necrosis and macrophage infiltration, features of plaque instability. Cross-species analysis identified conserved TWIST1 targets and supported its regulatory role in EC phenotypic transitions. Mechanistically, TWIST1 promotes EndMT by enhancing endothelial migration and proliferation through the transcriptional coactivator PELP1. It also induces AEBP1 expression, which upregulates COL4A1 to further drive endothelial proliferation. In human carotid plaques, TWIST1 expression correlated with asymptomatic atherosclerosis and was associated with favorable clinical outcomes.

These findings challenge the traditional view of EndMT as a destabilizing process in atherosclerosis, instead suggesting that TWIST1-driven EndMT may contribute to plaque stability and reveal novel avenues for understanding disease progression