Biology subjects

Furtado, J.

Publications and source records attributed to Furtado, J..

7 recordsLinked to original sources

bioRxiv2025

Restoring compromised Blood-Retina-Barrier integrity with Netrin-1 overexpression

The blood-retina barrier (BRB) protects retinal neuronal function and enables vision. A compromised, leaky BRB is a hallmark of vision-threatening retinal diseases such as diabetic retinopathy (DR) and wet age-related macular degeneration (AMD) that affect millions of persons worldwide. Strategies to enhance BRB integrity hold promise as therapeutic interventions to prevent vision loss. Previous studies identified Netrin-1 (NTN1) as a key regulator of BRB stability and revealed reduced Netrin-1 signaling in DR patients, suggesting that Netrin-1 supplementation could help preserve BRB function and prevent disease progression. Herein, we used genetic NTN1 overexpression to investigate effects on BRB development and maintenance. We show that global NTN1 overexpression converted leaky vessels at the P5 angiogenic front into a non-leaky state. In pathological settings, NTN1 overexpression reinforced BRB integrity in oxygen- induced retinopathy (OIR), improving electroretinogram (ERG) amplitudes and rescued vascular leak in laser-induced choroidal neovascularization (CNV). Mechanistically, we determined that NTN1 overexpression minimally and transiently affected retinal angiogenesis in an Unc5b independent manner, while Netrins barrier stabilizing effects were Unc5b dependent. These findings establish Netrin-1 as a promising therapeutic target for preventing BRB breakdown in retinal vascular diseases and suggest that reinforcing the Netrin-1/Unc5b signaling pathway may provide a strategy to selectively stabilize the BRB.

molecular biology↗

bioRxiv2024

Early exposure to Western diet exacerbates visual outcomes in female mice.

Obesity, a growing pandemic in Western societies, significantly impacts metabolic health and contributes to visual disorders. While the systemic consequences of obesity, such as chronic inflammation and insulin resistance, are well-studied in adults, its early-life effects on retinal health remain underexplored. Using a maternal Western Diet (WD) exposure model, we investigated the developmental impact of early-life metabolic disturbances on retinal and cognitive function. Our findings reveal that WD exposure from gestation to early adulthood accelerates the onset of features resembling diabetic retinopathy, including increased retinal vascularization, inflammation, and compromised blood-retina barrier integrity, observed within just four months. Females exhibited heightened vulnerability, showing pronounced ocular defects such as anophthalmia, microphthalmia, and congenital cataracts. These results underscore a critical developmental window during which metabolic disruptions predispose to sex-specific retinal and neurovascular pathologies. This work bridges the link between pediatric and adult obesity, highlighting the urgent need for early interventions to mitigate long-term visual impairments that could further impair recognition memory.

physiology↗

bioRxiv2024

Polycomb Repressive Complex 2 promotes atherosclerotic plaque vulnerability

Key findings1. PRC2 regulates EC shear stress responses. 2. PRC2 governs Klf2/4 suppression downstream of Pcdhg. 3. High PRC2 in ASCVD-prone arterial regions suppresses Klf2/4 to promote ASCVD. 4. Athero-protective Klf2/4 induction upon PRC2 inhibition requires Notch signaling. 5. Tazemetostat, an FDA approved PRC2 inhibitor, slows ASCVD progression and improves markers of plaque stability. Atherosclerotic cardiovascular disease (ASCVD), the leading cause of mortality worldwide, is driven by endothelial cell inflammatory activation and counter-balanced by anti-inflammatory transcription factors Klf2 and Klf4 (Klf2/4). Understanding vascular endothelial inflammation to develop effective treatments is thus essential. Here, we identify, Polycomb Repressive Complex (PRC) 2, which blocks gene transcription by trimethylating histone3 Lysine27 in gene promoter/enhancers, as a potent, therapeutically targetable determinant of vascular inflammation and ASCVD progression. Bioinformatics identified PRC2 as a direct suppressor of Klf2/4 transcription. Klf2/4 transcription requires Notch signaling, which reverses PRC2 modification of Klf2/4 promoter/enhancers. PRC2 activity is elevated in human ASCVD endothelium. Treating mice with established ASCVD with tazemetostat, an FDA approved pharmacological inhibitor of PRC2, slowed plaque progression by 50% and drastically improved markers of plaque stability. This study elucidates a fundamental mechanism of vascular inflammation, thus identifying a potential method for treating ASCVD and possibly other vascular inflammatory diseases.

cell biology↗

bioRxiv2024

PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations

BackgroundHereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder characterized by arteriovenous malformations (AVMs). Loss-of-function mutations in Activin receptor-like kinase 1 (ALK1) cause type 2 HHT and Alk1 knockout (KO) mice develop AVMs due to overactivation of VEGFR2/PI3K/AKT signaling pathways. However, the full spectrum of signaling alterations in Alk1 mutants remains unknown and means to combat AVM formation in patients are yet to be developed. MethodsSingle-cell RNA sequencing of endothelial-specific Alk1 KO mouse retinas and controls identified a cluster of endothelial cells (ECs) that was unique to Alk1 mutants and that overexpressed fluid shear stress (FSS) signaling signatures including upregulation of the mechanosensitive ion channel PIEZO1. PIEZO1 overexpression was confirmed in human HHT lesions, and genetic and pharmacological PIEZO1 inhibition was tested in Alk1 KO mice, as well as downstream PIEZO1 signaling. ResultsPharmacological PIEZO1 inhibition, and genetic Piezo1 deletion in Alk1-deficient mice effectively mitigated AVM formation. Furthermore, we identified that elevated VEGFR2/AKT, ERK5-p62-KLF4, hypoxia and proliferation signaling were significantly reduced in Alk1-Piezo1 double ECKO mice. ConclusionsPIEZO1 overexpression and signaling is integral to HHT2, and PIEZO1 blockade reduces AVM formation and alleviates cellular and molecular hallmarks of ALK1-deficient cells. This finding provides new insights into the mechanistic underpinnings of ALK1-related vascular diseases and identifies potential therapeutic targets to prevent AVMs.

molecular biology↗

bioRxiv2024

Endothelial Rho kinase controls blood vessel integrity and angiogenesis

BackgroundThe Rho kinases 1 and 2 (ROCK1/2) are serine-threonine specific protein kinases that control actin cytoskeleton dynamics. They are expressed in all cells throughout the body, including cardiomyocytes, smooth muscle cells and endothelial cells, and intimately involved in cardiovascular health and disease. Pharmacological ROCK inhibition is beneficial in mouse models of hypertension, atherosclerosis, and neointimal thickening that display overactivated ROCK. However, the consequences of endothelial ROCK signaling deficiency in vivo remain unknown. To address this issue, we analyzed endothelial cell (EC) specific ROCK1 and 2 deletions. MethodsWe generated Cdh5-CreERT2 driven, tamoxifen inducible loss of function alleles of ROCK1 and ROCK2 and analyzed mouse survival and vascular defects through cellular, biochemical, and molecular biology approaches. ResultsWe observed that postnatal or adult loss of endothelial ROCK1 and 2 was lethal within a week. Mice succumbed to multi-organ hemorrhage that occurred because of loss of vascular integrity. ECs displayed deficient cytoskeletal actin polymerization that prevented focal adhesion formation and disrupted junctional integrity. Retinal sprouting angiogenesis was also perturbed, as sprouting vessels exhibited lack of polymerized actin and defective lumen formation. In a three-dimensional endothelial sprouting assay, combined knockdown of ROCK1/2 or knockdown or ROCK2 but not ROCK1 led to reduced sprouting, lumenization and cell polarization defects caused by defective actin and altered VE-cadherin dynamics. The isoform specific role of endothelial ROCK2 correlated with ROCK2 substrate specificity for FAK and LIMK. By analyzing single and three allele mutants we show that one intact allele of ROCK2 is sufficient to maintain vascular integrity in vivo. ConclusionEndothelial ROCK1 and 2 maintain junctional integrity and ensure proper angiogenesis and lumen formation. The presence of one allele of ROCK2 is sufficient to maintain vascular growth and integrity. These data indicate the need of careful consideration for the use of ROCK inhibitors in disease settings.

developmental biology↗

bioRxiv2023

Netrin-1 binding to Unc5B regulates Blood-Retina Barrier integrity

BackgroundThe blood brain barrier (BBB) preserves neuronal function in the central nervous system (CNS) by tightly controlling metabolite exchanges with the blood. In the eye, the retina is likewise protected by the blood-retina barrier (BRB) to maintain phototransduction. We showed that the secreted guidance cue Netrin-1 regulated BBB integrity, by binding to endothelial Unc5B and regulating canonical {beta}-catenin dependent expression of BBB gene expression ObjectiveHere, we investigated if Netrin-1-binding to endothelial Unc5B also controlled BRB integrity, and if this process involved Norrin/{beta}-catenin signaling, which is the major known driver of BRB development and maintenance. MethodsWe analyzed Tamoxifen-inducible loss- and gain-of-function alleles of Unc5B, Ntn1 and Ctnnb1 in conjunction with tracer injections and biochemical signaling studies. ResultsInducible endothelial Unc5B deletion, and inducible global Ntn1 deletion in postnatal mice reduced phosphorylation of the Norrin receptor LRP5, leading to reduced {beta}-catenin and LEF1 expression, conversion of retina endothelial cells from a barrier-competent Claudin-5+/PLVAP-state to a Claudin-5-/PLVAP+ leaky phenotype, and extravasation of injected low molecular weight tracers. Inducible Ctnnb1 gain of function rescued vascular leak in Unc5B mutants, and Ntn1 overexpression induced BRB tightening. Unc5B expression in pericytes contributed to BRB permeability, via regulation of endothelial Unc5B. Mechanistically, Netrin-1-Unc5B signaling promoted {beta}-catenin dependent BRB signaling by enhancing phosphorylation of the Norrin receptor LRP5 via the Discs large homologue 1 (Dlg1) intracellular scaffolding protein. ConclusionsThe data identify Netrin1-Unc5B as novel regulators of BRB integrity, with implications for diseases associated with BRB disruption.

cell 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↗