Biology subjects

Hordijk, P. L.

Publications and source records attributed to Hordijk, P. L..

6 recordsLinked to original sources

bioRxiv2026

Marfan Patient iPSC-Derived Endothelial Cells Carrying FBN1 Variants Reveal Endothelial Dysfunction

Marfan syndrome (MFS) is an inherited connective tissue disorder caused by pathogenic variants in FBN1, encoding fibrillin-1, with life-threatening aortic complications arising in part from endothelial cell (EC) dysfunction. To study this in a human model, we generated hiPSC-derived ECs from three MFS patients (iMFS-ECs). We show that iMFS-ECs recapitulate known disease phenotypes, including impaired alignment in the direction of flow. Moreover, we found that iMFS-ECs do not recover from TNF--induced loss of barrier integrity, due to sustained EC contractility. iMFS-ECs exhibited TNF--induced ICAM1 upregulation and NF-{kappa}B activation comparable to healthy donor-derived hiPSC-ECs by bulk RNA-seq, while expression of genes linked to cytoskeletal arrangements, cell signaling and ECM remodeling were dysregulated. In conclusion, we show that hiPSC derived ECs can serve as a model to investigate MFS pathology. These findings establish a human iPSC platform for MFS endothelial research and suggest impaired inflammatory resolution as a novel therapeutic target.

cell biology↗

bioRxiv2025

Pericytes and Wnt signaling induce functional blood-brain barrier phenotype in human iPSC-based model

The blood-brain barrier (BBB), formed by brain microvascular endothelial cells (BMECs), restricts vascular permeability through tight junctions, selective transporters, and low transcytosis. BBB dysfunction contributes to cerebrovascular and neurodegenerative disease, yet current human in vitro models recapitulate only a subset of BMEC features. Here, we describe a strategy generate BMECs (hiBMECs) from human induced pluripotent stem cell-derived endothelial cells by co-culture with isogenic brain pericytes and activation of Wnt/{beta}-catenin signaling. The resulting hiBMECs display barrier properties, active efflux transporters, and appropriate inflammatory responses. Transcriptomic profiling revealed convergence of pericyte-derived cues and Wnt/{beta}-catenin activation on ETS1, SMAD3/4, and PPAR{gamma} transcriptional networks, establishing a gene signature closely matching the adult human BBB. Downstream analysis revealed that hiBPC cues engaged sphingosine-1-phosphate, TGF-{beta}, and angiopoietin/Tie2 pathways, which were further regulated by canonical Wnt activation. These findings uncover a synergistic mechanism by which brain pericytes and Wnt/{beta}-catenin signaling orchestrate BMEC differentiation and function, providing mechanistic insight into human BBB development and an improved hiPSC-derived BBB model for future drug screening and disease modeling. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=36 SRC="FIGDIR/small/678752v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@186ba4corg.highwire.dtl.DTLVardef@310cc7org.highwire.dtl.DTLVardef@3e1970org.highwire.dtl.DTLVardef@f80f5e_HPS_FORMAT_FIGEXP M_FIG C_FIG The paper explainedO_ST_ABSPROBLEMC_ST_ABSThe blood-brain barrier (BBB) protects the brain by tightly regulating the passage of molecules and cells. Its dysfunction contributes to disorders such as stroke, dementia, and multiple sclerosis. Yet, existing human in vitro models fail to capture the full complexity of BBB biology, limiting our ability to study disease mechanisms or test brain-targeted drugs. RESULTSWe discovered that two signals are essential for generating functional human BBB endothelial cells from stem cells: cues from brain pericytes and activation of the Wnt/{beta}-catenin pathway. Together, these signals enabled endothelial cells to form tight barriers, operate transporters, and mount appropriate responses to inflammation. Transcriptomic analyses of the resulting cells revealed a gene signature closely matching the adult human BBB and identified how pericyte- and Wnt-activated pathways converge on specific transcriptional programs driving BBB identity. IMPACTThis study provides both a molecular framework for in vitro BBB development and a reliable and reproducible human BBB model. This platform can be applied to explore BBB dysfunction in neurological disease and to accelerate the development of drugs that need to reach the brain or target the brain vasculature.

bioengineering↗

bioRxiv2025

Vessel-on-Chip Model Of The Microcirculation In Abdominal Aortic Aneurysms

Abdominal aortic aneurysms (AAA) are pathological dilations of the abdominal aorta. To date, surgical intervention is the only option for managing large AAAs, with no pharmacological therapies to prevent growth of small aneurysms. A current limitation in investigating further pharmacological avenues is the translatability of results from animal models, or from patient trials that are limited by co-morbidities and disease severity. To bridge this knowledge gap, we created a novel, patient-specific vessel-on-chip (VoC) model of the microcirculation in AAA (AAA-VoC). We found that co-culture of both C (control)-VSMCs and AAA-patient derived VSMCs with healthy, hiPSC-derived ECs generate lumenized and perfusable microvascular networks. We show that AAA-VoCs are characterized by an enlarged average vascular diameter. We furthermore found that AAA-VSMCs show phenotypical deviations from C- VSMCs after 7 days in co-culture such as increased number and surface area, indicative of a preserved pathological phenotype in our in vitro model. Lastly, we demonstrate that AAA-VoCs showed an increased level of pro-inflammatory cytokine expression over C-VoCs and displayed an impaired endothelial barrier function, resulting in vascular leakage. With this study, we show that AAA-VSMCs affect microvascular networks formed by healthy hiPSC-ECs and that a AAA phenotype is preserved in 3D co-culture, making this model valuable for future studies investigating treatments for AAA.

cell biology↗

bioRxiv2024

NUAK1 regulates contraction in vascular smooth muscle cells derived from abdominal aortic aneurysm patients

AimAbdominal aortic aneurysms (AAA) are defined as a dilatation of the aortic wall. Impaired contractile ability of vascular smooth muscle cells (vSMC) is a hallmark of AAA. This study investigates the underlying mechanism of altered in vitro contractility of vSMC derived from AAA patients (AAA-SMC) compared to control vSMC (C-SMC). MethodsContractility of AAA-SMC (n=24) and C-SMC (n=8) was measured using Electric Cell- substrate Impedance Sensing. Large variability in AAA-SMC contraction was observed compared to C-SMC contraction, and AAA-SMC were therefore subdivided into low, intermediate (i.e. comparable to C-SMC contraction) and high contracting. To identify novel proteins involved in altered AAA-SMC contraction, a phosphoproteomic analysis was performed. ResultsThe proteomics data showed that Thrombospondin-1, PDZ and LIM domain protein 4 and ATPase plasma membrane Ca2+ transporting 1 expression correlated with vSMC contraction, but knockdown (KD) of these targets did not affect contraction. Next, the phosphoproteomics data identified NUAK family kinase 1 (NUAK1) as a potential regulator of contraction, since its kinase activity correlated with AAA-SMC contraction. NUAK1 regulates Myosin phosphatase targeting subunit 1 (MYPT1) activity by phosphorylation, as confirmed by the correlation between NUAK1 activity and phosphorylation levels of Ser445 and Ser910 on MYPT1. NUAK1 protein and RNA expression correlated with AAA-SMC contraction. Moreover, NUAK1 KD decreased contraction in AAA-SMC, combined with reduced phosphorylation levels of Myosin light chain (pMLC) and Vinculin gene expression, and increased F-actin cytoskeletal filament levels. ConclusionsNUAK1 regulates AAA-SMC contraction. Low NUAK1 expression decreased pMLC, potentially by higher MYPT1 activity, and subsequently reduced contraction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/629332v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@41c0f0org.highwire.dtl.DTLVardef@e1dd1borg.highwire.dtl.DTLVardef@7b36d7org.highwire.dtl.DTLVardef@14d65b5_HPS_FORMAT_FIGEXP M_FIG C_FIG Elements were modified from Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic License. https://smart.servier.com/; https://creativecommons.org/licenses/by/3.0/ HighlightsO_LIQuantification of in vitro vascular smooth muscle cells (vSMC) contractile capacity revealed large variation in contraction of vSMC derived from human AAA tissue (AAA- SMC) compared to vSMC derived from healthy aortic biopsies (C-SMC). C_LIO_LIPhosphoproteomic analysis identified NUAK family kinase 1 (NUAK1) as a novel regulator of contraction in AAA-SMC, since its kinase activity and expression levels correlated with AAA-SMC contraction. C_LIO_LINUAK1 regulates contraction in AAA-SMC by regulating Myosin phosphatase targeting subunit 1 (MYPT1) activity through phosphorylation, which subsequently affects phosphorylation levels of Myosin light chain (pMLC), and by affecting Vinculin expression and F-actin cytoskeletal filament levels. C_LIO_LIGaining more insight into NUAK1 function and mechanisms leading to aortic wall weakening can ultimately contribute to better AAA disease prediction and treatment. C_LI

cell biology↗

bioRxiv2022

Rapid proteostasis controls monolayer integrity of quiescent endothelium

Endothelial monolayer permeability is regulated by actin dynamics and vesicular traffic. Recently, ubiquitination was also implicated in the integrity of quiescent endothelium, as it differentially controls the localization and stability of adhesion- and signaling proteins. We found that inhibition of E1 ubiquitin ligases induces a rapid, reversible loss of integrity in quiescent, primary human endothelial monolayers, accompanied by increased F-actin stress fibers and the formation of intercellular gaps. Concomitantly, total protein and activity of the actin-regulating GTPase RhoB, but not its close homologue RhoA, increase [~]10-fold in 5-8 h. The depletion of RhoB, but not of RhoA, the inhibition of actin contractility and the inhibition of protein synthesis all significantly rescue the loss of cell-cell contact induced by E1 ligase inhibition. Our data suggest that in quiescent human endothelial cells, the continuous and fast turnover of short-lived proteins that negatively regulate cell-cell contact, is essential to preserve monolayer integrity.

molecular biology↗

bioRxiv2019

Selectivity of Guanine Nucleotide Exchange Factor-mediated Cdc42 activation in primary human endothelial cells

The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signaling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the full-length GEFs. Our data reveal a specific GEF dependent activation profile, with most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and pRex1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may activate Cdc42, which will be of great value for the field of vascular biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/541219v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@96ad9forg.highwire.dtl.DTLVardef@60aa3borg.highwire.dtl.DTLVardef@4811f0org.highwire.dtl.DTLVardef@191cfd1_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗