scholarly journals Three-dimensional biomimetic vascular model reveals a RhoA, Rac1, and N-cadherin balance in mural cell–endothelial cell-regulated barrier function

2017 ◽  
Vol 114 (33) ◽  
pp. 8758-8763 ◽  
Author(s):  
Stella Alimperti ◽  
Teodelinda Mirabella ◽  
Varnica Bajaj ◽  
William Polacheck ◽  
Dana M. Pirone ◽  
...  
Keyword(s):  
Barrier Function ◽  
Three Dimensional ◽  
Endothelial Barrier ◽  
Inflammatory Factors ◽  
Mural Cell ◽  
Rhoa Activity ◽  
Mural Cells ◽  
Adhesive Interactions

The integrity of the endothelial barrier between circulating blood and tissue is important for blood vessel function and, ultimately, for organ homeostasis. Here, we developed a vessel-on-a-chip with perfused endothelialized channels lined with human bone marrow stromal cells, which adopt a mural cell-like phenotype that recapitulates barrier function of the vasculature. In this model, barrier function is compromised upon exposure to inflammatory factors such as LPS, thrombin, and TNFα, as has been observed in vivo. Interestingly, we observed a rapid physical withdrawal of mural cells from the endothelium that was accompanied by an inhibition of endogenous Rac1 activity and increase in RhoA activity in the mural cells themselves upon inflammation. Using a system to chemically induce activity in exogenously expressed Rac1 or RhoA within minutes of stimulation, we demonstrated RhoA activation induced loss of mural cell coverage on the endothelium and reduced endothelial barrier function, and this effect was abrogated when Rac1 was simultaneously activated. We further showed that N-cadherin expression in mural cells plays a key role in barrier function, as CRISPR-mediated knockout of N-cadherin in the mural cells led to loss of barrier function, and overexpression of N-cadherin in CHO cells promoted barrier function. In summary, this bicellular model demonstrates the continuous and rapid modulation of adhesive interactions between endothelial and mural cells and its impact on vascular barrier function and highlights an in vitro platform to study the biology of perivascular–endothelial interactions.

Abstract 73: Bone Morphogenetic Protein Activity Modulates Endothelial Barrier Function

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Thomas Helbing ◽  
Elena Ketterer ◽  
Bianca Engert ◽  
Jennifer Heinke ◽  
Sebastian Grundmann ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Barrier Function ◽  
Endothelial Permeability ◽  
Bmp Signaling ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function

Introduction: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are associated with high morbidity and mortality in patients. During the progression of ALI, the endothelial cell barrier of the pulmonary vasculature becomes compromised, leading to pulmonary edema, a characteristic feature of ALI. It is well-established that EC barrier dysfunction is initiated by cytoskeletal remodeling, which leads to disruption of cell-cell contacts and formation of paracellular gaps, allowing penetration of protein-rich fluid and inflammatory cells. Bone morphogenetic proteins (BMPs) are important players in endothelial dysfunction and inflammation but their effects on endothelial permeability in ALI have not been investigated until now. Methods and Results: As a first approach to assess the role of BMPs in acute lung injury we analysed BMP4 and BMPER expression in an infectious (LPS) and a non-infectious (bleomycin) mouse models of acute lung injury. In both models BMP4 and BMPER protein expression levels were reduced demonstrated by western blots, suggesting that BMPs are involved in progression ALI. To assess the role of BMPs on vascular leakage, a key feature of ALI, BMP activity in mice was inhibited by i.p. administration of LDN193189, a small molecule that blocks BMP signalling. After 3 days Evans blue dye (EVB) was administered i.v. and dye extravasation into the lungs was quantified as a marker for vascular leakage. Interestingly, LDN193189 significantly increased endothelial permeability compared to control lungs, indicating that BMP signaling is involved in maintenance of endothelial barrier function. To quantify effects of BMP inhibition on endothelial barrier function in vitro, HUVECs were seeded onto transwell filters and were exposed to LDN193189. After 3 days FITC-dextrane was added and passage into the lower chamber was quantified as a marker for endothelial barrier function. Thrombin served as a positive control. As expected from our in vivo experiments inhibition of BMP signaling by LDN193189 enhanced FITC-dextrane passage. To study specific effects of BMPs on endothelial barrier function, two protagonist of the BMP family, BMP2 and BMP4, or BMP modulator BMPER were tested in the transwell assay in vitro. Interestingly BMP4 and BMPER, but not BMP2, reduced FITC-dextrane passage demonstrating that BMP4 and BMPER improved endothelial barrier function. Vice versa, specific knock down of BMP4 or BMPER increased leakage in transwell assays. Im immuncytochemistry silencing of BMPER or BMP4 induced hyperpermeability as a consequence of a pro-inflammatory endothelial phenotype characterised by reduced cell-cell contacts and increased actin stress fiber formation. Additionally, the pro-inflammatory endothelial phenotype was confirmed by real-time revealing increased expression of adhesion molecules ICAM-1 or proinflammatory cytokines such as IL-6 and IL-8 in endothelial cells after BMPER or BMP4 knock down. Confirming these in vitro results BMPER +/- mice exhibit increased extravasation of EVB into the lungs, indicating that partial loss of BMPER impairs endothelial barrier function in vitro and in vivo. Conclusion: We identify BMPER and BMP4 as local regulators of vascular permeability. Both are protective for endothelial barrier function and may open new therapeutic avenues in the treatment of acute lung injury.

Effect of p18 on Endothelial Barrier Function by Mediating Vascular Endothelial Rab11a-VE-cadherin Recycling

2021 ◽  
Author(s):  
Bo-Wen Xu ◽  
Zhi-Qiang Cheng ◽  
Xu-Ting Zhi ◽  
Xiao-Mei Yang ◽  
Zhi-Bo Yan
Keyword(s):  
Barrier Function ◽  
Adherens Junctions ◽  
Cecal Ligation ◽  
Underlying Mechanism ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction ◽  
Recycling Endosome

Abstract Endothelial barrier integrity requires recycling of VE-cadherin to adherens junctions. Both p18 and Rab11a play significant roles in VE-cadherin recycling. However, the underlying mechanism and the role of p18 in activating Rab11a have yet to be elucidated. Performing in vitro and in vivo experiments, we showed that p18 protein bound to VE-cadherin before Rab11a through its VE-cadherin-binding domain (aa 1–39). Transendothelial resistance showed that overexpression of p18 promoted the circulation of VE-cadherin to adherens junctions and the recovery of the endothelial barrier. Silencing of p18 caused endothelial barrier dysfunction and prevented Rab11a-positive recycling endosome accumulation in the perinuclear recycling compartments. Furthermore, p18 knockdown in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide and cecal ligation puncture. This study showed that p18 regulated the pulmonary endothelial barrier function in vitro and in vivo by regulating the binding of Rab11a to VE-cadherin and the activation of Rab11a.

scholarly journals Modulation of Rac1 Activity by ADMA/DDAH Regulates Pulmonary Endothelial Barrier Function

2009 ◽  
Vol 20 (1) ◽  
pp. 33-42 ◽  
Author(s):  
Beata Wojciak-Stothard ◽  
Belen Torondel ◽  
Lan Zhao ◽  
Thomas Renné ◽  
James M. Leiper
Keyword(s):  
Nitric Oxide ◽  
Actin Cytoskeleton ◽  
Barrier Function ◽  
Endothelial Permeability ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Nitric Oxide Synthase Inhibitor ◽  
Rac1 Activity

Endogenously produced nitric oxide synthase inhibitor, asymmetric methylarginine (ADMA) is associated with vascular dysfunction and endothelial leakage. We studied the role of ADMA, and the enzymes metabolizing it, dimethylarginine dimethylaminohydrolases (DDAH) in the regulation of endothelial barrier function in pulmonary macrovascular and microvascular cells in vitro and in lungs of genetically modified heterozygous DDAHI knockout mice in vivo. We show that ADMA increases pulmonary endothelial permeability in vitro and in in vivo and that this effect is mediated by nitric oxide (NO) acting via protein kinase G (PKG) and independent of reactive oxygen species formation. ADMA-induced remodeling of actin cytoskeleton and intercellular adherens junctions results from a decrease in PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and a subsequent down-regulation of Rac1 activity. The effects of ADMA on endothelial permeability, Rac1 activation and VASP phosphorylation are prevented by overexpression of active DDAHI and DDAHII, whereas inactive DDAH mutants have no effect. These findings demonstrate for the first time that ADMA metabolism critically determines pulmonary endothelial barrier function by modulating Rac1-mediated remodeling of the actin cytoskeleton and intercellular junctions.

Adrenomedullin and endothelial barrier function

2007 ◽  
Vol 98 (11) ◽  
pp. 944-951 ◽  
Author(s):  
Bettina Temmesfeld-Wollbrück ◽  
Andreas Hocke ◽  
Norbert Suttorp ◽  
Stefan Hippenstiel
Keyword(s):  
Septic Shock ◽  
Barrier Function ◽  
Severe Infection ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Oedema Formation ◽  
In Vivo Models ◽  
Sepsis And Septic Shock

SummaryAlthough loss of endothelial barrier function is a hallmark of every acute inflammation and contributes to fatal loss of organ function during severe infections, there is no sufficient therapy for stabilization of endothelial barrier function. Endogenous peptide adrenomedullin (AM) serum levels were shown to be increased during severe infection including sepsis and septic shock. In different in-vitro and in-vivo models AM acted as a potent therapeutic endothelial barrier function-stabilizing agent. Activation of specific receptors by AM results in elevation of second messenger cAMP. AM inhibits actin-myosin based endothelial cell contraction and junctional disassembly, thereby preventing paracellular permeability and oedema formation. The peptide furthermore possesses several protective cardiovascular qualities, including protection of the microcirculation during inflammation, and was proven as an efficient counter-regulatory molecule in various models of sepsis and septic shock. Overall, AM may be an attractive molecule to combat against cardiovascular malfunction during severe infection.

scholarly journals Mural cell SRF controls pericyte migration, vessel patterning and blood flow

2021 ◽  
Author(s):  
Michael Martin Orlich ◽  
Rodrigo Diéguez-Hurtado ◽  
Regine Muehlfriedel ◽  
Vithiyanjali Sothilingam ◽  
Hartwig Wolburg ◽  
...  
Keyword(s):  
Serum Response Factor ◽  
Mural Cell ◽  
Vascular Integrity ◽  
Mural Cells ◽  
Ischemic Retinopathy ◽  
Retinal Angiogenesis ◽  
Serum Response

Rationale: Pericytes (PCs) and vascular smooth muscle cells (vSMCs), collectively known as mural cells(MCs), are recruited through PDGFB-PDGFRB signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions. Objective: Here, we characterize the role of the transcription factor serum response factor (SRF) in MCs and study its function in developmental and pathological contexts. Methods and Results: We generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis. By postnatal day (P)6, PCs lacking SRF were morphologically abnormal and failed to properly co-migrate with angiogenic sprouts. As a consequence, PC-deficient vessels at the retinal sprouting front became dilated and leaky. By P12, also the vSMCs had lost SRF, which coincided with the formation of pathological arteriovenous (AV) shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF co-factors. We further show that MRTF-SRF signaling promotes pathological PC activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone. Conclusions: SRF is crucial for distinct functions in PCs and vSMCs. SRF directs PC migration downstream of PDGFRB signaling and mediates pathological PC activation during ischemic retinopathy. In vSMCs, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of AV shunts. These essential roles in physiological and pathological contexts provide a rational for novel therapeutic approaches through targeting SRF activity in MCs.

scholarly journals Evidence That Cingulin Regulates Endothelial Barrier Function In Vitro and In Vivo

2016 ◽  
Vol 36 (4) ◽  
pp. 647-654 ◽  
Author(s):  
Klaudia Schossleitner ◽  
Sabine Rauscher ◽  
Marion Gröger ◽  
Heinz Peter Friedl ◽  
Richard Finsterwalder ◽  
...  
Keyword(s):  
Barrier Function ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function

scholarly journals Developmental differences in focal adhesion kinase expression modulate pulmonary endothelial barrier function in response to inflammation

2018 ◽  
Vol 315 (1) ◽  
pp. L66-L77 ◽  
Author(s):  
Lihua Ying ◽  
Cristina M. Alvira ◽  
David N. Cornfield
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Barrier Function ◽  
Developmental Differences ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Barrier Disruption ◽  
Adult Mice

Compromised pulmonary endothelial cell (PEC) barrier function characterizes acute respiratory distress syndrome (ARDS), a cause of substantial morbidity and mortality. Survival from ARDS is greater in children compared with adults. Whether developmental differences intrinsic to PEC barrier function contribute to this survival advantage remains unknown. To test the hypothesis that PEC barrier function is more well-preserved in neonatal lungs compared with adult lungs in response to inflammation, we induced lung injury in neonatal and adult mice with systemic lipopolysaccharide (LPS). We assessed PEC barrier function in vivo and in vitro, evaluated changes in the expression of focal adhesion kinase 1 (FAK1) and phosphorylation in response to LPS, and determined the effect of FAK silencing and overexpression on PEC barrier function. We found that LPS induced a greater increase in lung permeability and PEC barrier disruption in the adult mice, despite similar degrees of inflammation and apoptosis. Although baseline expression was similar, LPS increased FAK1 expression in neonatal PEC but increased FAK1 phosphorylation and decreased FAK1 expression in adult PEC. Pharmacologic inhibition of FAK1 accentuated LPS-induced barrier disruption most in adult PEC. Finally, in response to LPS, FAK silencing markedly impaired neonatal PEC barrier function, whereas FAK overexpression preserved adult PEC barrier function. Thus, developmental differences in FAK expression during inflammatory injury serve to preserve neonatal pulmonary endothelial barrier function compared with that of adults and suggest that intrinsic differences in the immature versus pulmonary endothelium, especially relative to FAK1 phosphorylation, may contribute to the improved outcomes of children with ARDS.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Sign in / Sign up

Export Citation Format

Share Document