Biology subjects

Franco, C. A.

Publications and source records attributed to Franco, C. A..

10 recordsLinked to original sources

bioRxiv2026

PDGFD-induced pericyte contractility drives pathological neoangiogenesis

Neovascular tufts (NVTs) are a defining feature of proliferative retinopathies such as diabetic retinopathy and retinopathy of prematurity, in which they compromise retinal integrity and vision. Excessive endothelial cell (EC) sprouting underlies their formation, yet the cellular and mechanical mechanisms that shape NVTs, and the contribution of pericytes (PCs) to this process, remain unclear. In this work, we investigated the role of actomyosin contractility in ECs and PCs during pathological angiogenesis and NVT formation. We identified non-muscle myosin IIA (NMIIA) and phosphorylated myosin light chains (MLCs) as a key signature of PCs and ECs in retinopathy and NVTs. Using inducible deletion of Myh9 in the oxygen-induced retinopathy mouse model, we uncover distinct and opposing roles for NMIIA in the two cell types. Endothelial NMIIA promotes neovascularisation and prevents enlargement of NVTs, whilst PC NMIIA drives neovascular tuft formation. Mechanistically, we identify platelet-derived growth factor D (PDGFD), but not PDGFB, as a driver of pathological PC actomyosin contractility through Rho-associated coiled-coil kinase (ROCK). Genetic deletion of Pdgfd partially phenocopies the loss of NMIIA in PCs, reducing NVT formation and lowering NMIIA and phosphorylated MLC levels within NVTs. Our findings establish cell-type-specific actomyosin contractility as a determinant of NVT pathogenesis and identify PDGFD-ROCK-NMIIA signalling in PCs as a therapeutic target in proliferative retinopathies.

cell biology↗

bioRxiv2026

A 3D Tumor-on-a-chip Platform to Identify Drugs that Block Breast Cancer Cell Intravasation

Metastasis is the leading cause of death in breast cancer patients, yet there are no drugs specifically designed to block cancer cell intravasation, an early step of the metastatic cascade that originates circulating tumour cells (CTCs). A major challenge in developing anti-intravasation drugs is the scarcity of relevant in vitro platforms suitable for predictable drug discovery. Intravasation is a fundamental step of metastasis and involves the crossing of cancer cells through an endothelial barrier to enter the blood circulation. Here we developed an intravasation-on-a-chip model with controlled extracellular matrix composition, fluid flow and shear stress, which mimics the dynamic tumour-endothelium interface. The systems allows real-time imaging of intravasation and the isolation and quantification of intravasated cancer cells. As a proof-of-concept for drug testing, we show that perfusion with the PI3K/mTOR inhibitor Dactolisib, significantly reduced intravasation without compromising endothelial cell viability. The system also provides the capability to evaluate inhibitor on-target activity via imaging analysis. This intravasation-on-a-chip model offers a powerful, scalable, and imaging-compatible platform for discovering and evaluating anti-intravasation compounds.

cancer biology↗

bioRxiv2023

3DVascNet: an automated software for segmentation and quantification of vascular networks in 3D

BackgroundAnalysis of vascular networks is an essential step to unravel the mechanisms regulating the physiological and pathological organization of blood vessels. So far, most of the analyses are performed using 2D projections of 3D networks, a strategy that has several obvious shortcomings. For instance, it does not capture the true geometry of the vasculature, and generates artifacts on vessel connectivity. These limitations are accepted in the field because manual analysis of 3D vascular networks is a laborious and complex process that is often prohibitive for large volumes. MethodsTo overcome these issues, we developed 3DVascNet, a deep learning (DL) based software for automated segmentation and quantification of 3D retinal vascular networks. 3DVascNet performs segmentation based on a DL model, and it quantifies vascular morphometric parameters such as the vessel density, branch length, vessel radius, and branching point density. ResultsWe tested 3DVascNets performance using a large dataset of 3D microscopy images of mouse retinal blood vessels. We demonstrated that 3DVascNet efficiently segments vascular networks in 3D, and that vascular morphometric parameters capture phenotypes detected by using manual segmentation and quantification in 2D. In addition, we showed that, despite being trained on retinal images, 3DVascNet has high generalization capability and successfully segments images originating from other datasets and organs. More-over, the source code of 3DVascNet is publicly available, thus it can be easily extended for the analysis of other 3D vascular networks by other users. ConclusionsOverall, we present 3DVascNet, a freely-available software that includes a user-friendly graphical interface for researchers with no program-ming experience, which will greatly facilitate the ability to study vascular networks in 3D in health and disease.

bioinformatics↗

bioRxiv2022

Alternative splicing changes are associated with pre-birth adaptation during lung development

Gas exchanges are ensured by lung alveoli, which are mainly composed by epithelial alveolar type 1 (AT1), alveolar type 2 (AT2) and capillary endothelial cells (ECs). Alveologenesis starts during late embryonic development and continues after birth and relies on extensive biochemical crosstalk between these cell types. How this crosstalk is modulated to anticipate and accommodate the radical changes occurring at birth is still unclear. We investigated the alternative splicing (AS) changes occurring during lung development at the embryonic to postnatal transition by performing RNAseq of mouse lungs at distinct developmental stages. We found that most of the AS changes occur at the embryonic to postnatal transition. In addition, we identified hnRNP A1, Cpeb4 and Elavl2/HuB as putative splicing regulators of this transition. We show that the AS of a major pro- angiogenic chemokine, vascular endothelial growth factor A (VEGFA), is differentially regulated at this transition. Remarkably, we found that there is a switch from the predominance of Vegfa 164 to Vegfa 188 just before birth specifically in AT1 cells, whilst in other cell populations Vegfa does not undergo AS changes. Moreover, we identified a novel Vegfa isoform generated by the retention of intron 5, Vegfa i5. Our results reveal a cell type-specific regulation of Vegfa AS that may constitute a pre- birth adaptation mechanism of the epithelial-endothelial crosstalk, which may be fundamental for the adaptation to breathing and may have implications for pathological conditions.

developmental biology↗

bioRxiv2021

Defective flow-migration coupling causes arteriovenous malformations in hereditary hemorrhagic telangiectasia

BackgroundActivin receptor-like kinase 1 (ACVRL1, hereafter ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia (HHT), a devastating disorder that leads to arteriovenous malformations (AVMs). Here we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. MethodsUsing Cre lines that recombine in different subsets of arterial, capillary-venous or endothelial tip cells, we showed that capillary-venous Alk1 deletion was sufficient to induce AVM formation in the postnatal retina. ResultsALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1 deficient cells exhibited increased integrin signaling interaction with VEGFR2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacological inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1 deficient mice. ConclusionsOur study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of AVM formation in HHT disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in HHT patients.

cell biology↗

bioRxiv2021

Organotypic endothelial adhesion molecules are key for Trypanosoma brucei tropism and virulence

Trypanosoma brucei is responsible for lethal diseases in humans and cattle in Sub-Saharan Africa. These extracellular parasites extravasate from the blood circulation into several tissues. The importance of the vasculature in tissue tropism is poorly understood. Using intravital imaging and bioluminescence, we found that gonadal white adipose tissue and pancreas are the two main parasite reservoirs. We show that reservoir establishment happens before vascular permeability is compromised, suggesting that extravasation is an active mechanism. Blocking endothelial surface adhesion molecules (E-selectin, P-selectins, or ICAM2) significantly reduced extravascular parasite load in all organs and delayed host lethality. Remarkably, blocking CD36 had a specific effect on adipose tissue tropism that was sufficient to delay lethality, suggesting that establishment of the adipose tissue reservoir is necessary for parasite virulence. This works demonstrates the importance of the vasculature in a T. brucei infection and identifies organ-specific adhesion molecules as key players for tissue tropism.

microbiology↗

bioRxiv2020

Endothelial cell invasiveness is controlled by myosin IIA-dependent inhibition of Arp2/3 activity

Sprouting angiogenesis is fundamental for development and contributes to multiple diseases, including cancer, diabetic retinopathy and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on the mechanisms that endothelial tip cells use to invade into tissues. Here, we prove that endothelial tip cells use long lamellipodia projections (LLPs) as the main cellular protrusion for invasion into non-vascular extracellular matrix. We show that LLPs and filopodia protrusions are balanced by myosin-IIA (MIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of MIIA promotes excessive LLPs formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents LLPs development and leads to excessive filopodia formation. We further show that MIIA inhibits Rac1-dependent activation of Arp2/3, by regulating the maturation state of focal adhesions. Our discoveries establish the first comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way towards new strategies to block invasive tip cells during sprouting angiogenesis.

cell biology↗

bioRxiv2020

On the preservation of vessel bifurcations during flow-mediated angiogenic remodelling

During developmental angiogenesis, endothelial cells respond to shear stress by migrating and remodelling the initially hyperbranched plexus, removing certain vessels whilst maintaining others. The key regulator of vessel preservation is cell decision behaviour at bifurcations. At flow-convergent bifurcations where migration paths diverge, cells must finely tune migration along both possible paths if the bifurcation is to persist. Experiments have demonstrated that disrupting the cells ability to sense shear or junction forces transmitted between cells impacts the preservation of bifurcations during the remodelling process. However, how these migratory cues integrate during cell decision making remains poorly understood. Therefore, we present the first agent-based model of endothelial cell flow-mediated migration suitable for interrogating the mechanisms behind bifurcation stability. The model simulates flow in a bifurcated vessel network composed of agents representing endothelial cells arranged into a lumen which migrate against flow. Upon approaching a bifurcation where more than one migration path exists, agents refer to a stochastic bifurcation rule which models the decision cells make as a combination of flow-based and collective-based migratory cues. With this rule, cells favour branches with relatively larger shear stress or cell number. We found that cells must integrate both cues nearly equally to maximise bifurcation stability. In simulations with stable bifurcations, we found competitive oscillations between flow and collective cues, and simulations that lost the bifurcation were unable to maintain these oscillations. The competition between these two cues is haemodynamic in origin, and demonstrates that a natural defence against bifurcation loss during remodelling exists: as vessel lumens narrow due to cell efflux, resistance to flow and shear stress increases, attracting new cells to enter and rescue the vessel from regression. Our work provides theoretical insight into the role of junction force transmission has in stabilising vasculature during remodelling and as an emergent mechanism to avoid functional shunting. Author SummaryWhen new blood vessels are created, the endothelial cells that make up these vessels migrate and rearrange in response to blood flow to remodel and optimise the vessel network. An essential part of this process is maintaining the branched structure of the network; however, it is unclear what cues cells consider at regions were vessels branch (i.e., bifurcations). In this research, we present a computer model of cell migration to interrogate the process of preserving bifurcations during remodelling. In this model, cells at bifurcations are influenced by both flow and force transmitted from neighbouring cells. We found that both cues (flow-based and collective-based) must be considered equally in order to preserve branching in the vessel network. In simulations with stable bifurcations, we demonstrated that these cues oscillate: a strong signal in one was accompanied by a weak signal in the other. Furthermore, we found that these cues naturally compete with each other due to the coupling between blood flow and the size of the blood vessels, i.e. larger vessels with more cells produce less flow signals and vice versa. Our research provides insight into how forces transmitted between neighbouring cells stabilises and preserves branching during remodelling, as well as implicates the disruption of this force transmission as a potential mechanism when remodelling goes wrong as in the case of vascular malformation.

cell biology↗

bioRxiv2017

PolNet Analysis: a software tool for the quantification of network-level endothelial cell polarity and blood flow during vascular remodelling

In this paper, we present PolNet, an open source software tool for the study of blood flow and cell-level biological activity during vessel morphogenesis. We provide an image acquisition, segmentation, and analysis protocol to quantify endothelial cell polarity in entire in vivo vascular networks. In combination, we use computational fluid dynamics to characterise the haemodynamics of the vascular networks under study. The tool enables, for the first time, network-level analysis of polarity and flow for individual endothelial cells. To date, PolNet has proven invaluable for the study of endothelial cell polarisation and migration during vascular patterning, as demonstrated by our recent papers [1, 2]. Additionally, the tool can be easily extended to correlate blood flow with other experimental observations at the cellular/molecular level. We release the source code of our tool under the LGPL licence.

developmental biology↗

bioRxiv2017

YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development

Formation of a hierarchically organized blood vessel network by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How in this process endothelial cells arise in adequate numbers and arrange suitably to shape a functional vascular network is poorly understood. Here we show that YAP and TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP and TAZ increase VE-cadherin turnover at junctions and suppress endothelial Notch and BMP signaling, two key pathways that limit sprouting and endothelial dynamics. Consequently, the loss of YAP and TAZ leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP and TAZ integrate mechanical signals with Notch and BMP signaling to balance endothelial cell distribution in angiogenic vessels.

developmental biology↗