Loss of endothelial barrier integrity in mice with conditional ablation of podocalyxin (Podxl) in endothelial cells

Horrillo, Angélica; Porras, Gracia; Ayuso, Matilde Sánchez; González-Manchón, Consuelo

doi:10.1016/j.ejcb.2016.04.006

Cited by 36 publications

(33 citation statements)

References 39 publications

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“…To reduce the magnification and increase the visible area while still viewing individual RNAs, we applied 4-rounds of clampFISH to mouse kidney samples and probed for PODXL mRNA, an gene that is highly expressed in podocytes 18 , and observed specific expression of clampFISH signal in the appropriate regions (Figure 2b, Supplementary Figure 4). Interestingly, clampFISH further revealed that PODXL also expressed in the kidney endothelium, a signal that was only faintly visible by single molecule RNA FISH, but was clearly detected by clampFISH at low magnification ( Figure 2b) consistent with previous findings that PODXL is expressed at low levels in the kidney endothelium 19 .…”

supporting

confidence: 77%

Exponential fluorescent amplification of individual RNAs using clampFISH probes

Rouhanifard

Mellis

Dunagin

et al. 2017

Preprint

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Abstract:Non-enzymatic, high-gain signal amplification methods with single-cell, single-molecule resolution are in great need. We present a new method (click-amplifying FISH; clampFISH) for the fluorescent detection of RNA that combines the specificity of oligonucleotides with bioorthogonal click chemistry in order to achieve high specificity and high-gain (>400x) signal amplification. We show that clampFISH signal enables detection with low magnification microscopy and separation of cells by RNA levels via flow cytometry. Additionally, we show that clampFISH is multiplexable, compatible with expansion microscopy, and works in tissue samples. Main text:Single molecule RNA fluorescence in situ hybridization (RNA FISH), which enables the direct detection of individual RNA molecules 1,2,3 , has emerged as a powerful technique for measuring both RNA abundance and localization in single cells. Yet, while single molecule RNA FISH is simple and robust, the total signal generated by single molecule RNA FISH probes is relatively low, thus requiring high-magnification microscopy for detection. This keeps the assay relatively low throughput and precludes the ability to combine it with flow cytometry. As such, high efficiency, high gain amplification methods for single molecule RNA FISH signal could enable a host of new applications for RNA FISH.A number of different signal amplification techniques are available for RNA FISH, but each suffers from particular limitations. Approaches such as tyramide signal amplification (TSA) 4 , or enzyme ligated fluorescence (ELF) 5 utilize enzymes to catalyze the deposition of fluorescent substrates near the probes. Alternatively, enzymes can catalyze a "rolling circle" nucleic acid amplification to generate a repeating sequence that can subsequently detected using fluorescent probes [6][7][8] . These methods can lead to large gains in fluorescence, but can suffer from poor sensitivity because of the difficulties in getting bulky enzymes through the fixed cellular environment to the target molecule. Meanwhile, there are a number of non-enzymatic amplification methods, most notably the hybridization chain reaction 9-11 and branched DNA methods [12][13][14] . These methods rely only on hybridization to amplify signal by creating larger DNA scaffolds to which fluorescent probes can attach. However, these methods have an inherent tradeoff between stringency of hybridization and wash conditions for specificity and maintaining hybridization to increase signal gain. Thus, our goal was to create a non-enzymatic, exponential . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/222794 doi: bioRxiv preprint first posted online Nov. 21, 2017; amplification scheme with high sensitivity (detection efficiency), gain (signal amplification), and specificity (low background).We first designed probes that would bind with high specificity and sensitivity; specifically, we ...

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supporting

confidence: 77%

Exponential fluorescent amplification of individual RNAs using clampFISH probes

Rouhanifard

Mellis

Dunagin

et al. 2017

Preprint

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“…Intriguingly, although Podxl loss cripples the ability of endothelial cells to form appropriate intact monolayers in vitro, we observed no overt severe vascular defects due to Podxl loss in vivo at steady state. Podxl-deficient mice exhibited only a modest delay in the opening of vascular lumens during embryogenesis and a modest increase in permeability in adulthood (17,44,45). Thus, in vivo there appear to be sufficiently redundant mechanisms to ensure appropriate formation of properly patent vessels to allow survival in the absence of Podxl.…”

Section: Discussionmentioning

confidence: 99%

Podocalyxin is required for maintaining blood–brain barrier function during acute inflammation

Cait

Hughes

Zeglinski

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele ofPodxlin vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood–brain barrier (BBB) inPodxlΔTie2Cremice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primedPodxlΔTie2Cremice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

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“…RhoA via ROCK2b was found to prevent vascular leakage during leucocyte diapedesis and S1P was proposed to enhance barrier function at least in part via RhoA and ROCK . On the other hand, endothelial cells deficient in podocalyxin have delayed barrier recovery and sustained RhoA activation indicating a negative effect of RhoA . Moreover, it was shown by FRET sensors that Rac1 reduces tension applied on AJs, whereas ROCK had the opposite effect indicating that Rac1 but not RhoA stabilizes AJs …”

Section: Maintenance and Stabilization Of The Endothelial Barrier In mentioning

confidence: 99%

“…163,169,170 On the other hand, endothelial cells deficient in podocalyxin have delayed barrier recovery and sustained RhoA activation indicating a negative effect of RhoA. 171 Moreover, it was shown by FRET sensors that Rac1 reduces tension applied on AJs, whereas ROCK had the opposite effect indicating that Rac1 but not RhoA stabilizes AJs. 154 The structural basis for RhoA regulation at cell junctions appears to be sequestration of several GEFs for RhoA by AJ and TJ components.…”

Section: Mechanisms Activating Rac1mentioning

confidence: 99%

Mind the gap: mechanisms regulating the endothelial barrier

2017

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The endothelial barrier consists of intercellular contacts localized in the cleft between endothelial cells, which is covered by the glycocalyx in a sievelike manner. Both types of barrier-forming junctions, i.e. the adherens junction (AJ) serving mechanical anchorage and mechanotransduction and the tight junction (TJ) sealing the intercellular space to limit paracellular permeability, are tethered to the actin cytoskeleton. Under resting conditions, the endothelium thereby builds a selective layer controlling the exchange of fluid and solutes with the surrounding tissue. However, in the situation of an inflammatory response such as in anaphylaxis or sepsis intercellular contacts disintegrate in post-capillary venules leading to intercellular gap formation. The resulting oedema can cause shock and multi-organ failure. Therefore, maintenance as well as coordinated opening and closure of interendothelial junctions is tightly regulated. The two principle underlying mechanisms comprise spatiotemporal activity control of the small GTPases Rac1 and RhoA and the balance of the phosphorylation state of AJ proteins. In the resting state, junctional Rac1 and RhoA activity is enhanced by junctional components, actin-binding proteins, cAMP signalling and extracellular cues such as sphingosine-1-phosphate (S1P) and angiopoietin-1 (Ang-1). In addition, phosphorylation of AJ components is prevented by junction-associated phosphatases including vascular endothelial protein tyrosine phosphatase (VE-PTP). In contrast, inflammatory mediators inhibiting cAMP/Rac1 signalling cause strong activation of RhoA and induce AJ phosphorylation finally leading to endocytosis and cleavage of VE-cadherin. This results in dissolution of TJs the outcome of which is endothelial barrier breakdown.

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Loss of endothelial barrier integrity in mice with conditional ablation of podocalyxin (Podxl) in endothelial cells

Cited by 36 publications

References 39 publications

Exponential fluorescent amplification of individual RNAs using clampFISH probes

Exponential fluorescent amplification of individual RNAs using clampFISH probes

Podocalyxin is required for maintaining blood–brain barrier function during acute inflammation

Mind the gap: mechanisms regulating the endothelial barrier

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