Endothelial Cell-cell Junctions Research Articles

Site-specific m6A-miR-494-3p, not unmethylated miR-494-3p, compromises blood brain barrier by targeting tight junction protein 1 in intracranial atherosclerosis.

Intracranial atherosclerosis is one of the most common causes of ischaemic stroke. However, there is a substantial knowledge gap on the development of intracranial atherosclerosis. Intracranial arteries are characterized by an upregulation of tight junctions between endothelial cells, which control endothelial permeability. We investigated the role of N6-methyladenosine (m6A), a common RNA modification, on endothelial integrity, focusing on the pro-atherogenic microRNA miR-494-3p and tight junction proteins TJP1 and PECAM1. We assessed the m6A landscape, along with the expression of miR-494-3p, TJP1 and PECAM1 in postmortem human vertebral arteries (VA), internal carotid arteries (ICA), and middle cerebral arteries (MCA) with various stages of intimal thickening and plaque formation. The interactions between m6A-modified miR-494-3p mimics, TJP1 and PECAM1, were investigated in vitro using primary human (brain) endothelial cells. Increased m6A expression was observed in the luminal lining of atherosclerosis-affected VAs, accompanied by reduced TJP1 and PECAM1, but not VE-cadherin, expression. Colocalization of m6A and miR-494-3p in the luminal lining of VA plaques was confirmed, indicating m6A methylation of miR-494-3p in intracranial atherosclerosis. Moreover, site-specific m6A-modification of miR-494-3p led to repression specifically of TJP1 protein expression at cell-cell junctions of brain microvascular endothelial cells, while unmodified miR-494-3p showed no effect. This study highlights increasing m6A levels during intracranial atherogenesis. Increases in m6A-miR-494-3p contribute to the observed decreased TJP1 expression in endothelial cell-cell junctions. This is likely to have a negative effect on endothelial integrity and may thus accelerate intracranial atherosclerosis progression.

Controversy in mechanotransduction - the role of endothelial cell-cell junctions in fluid shear stress sensing.

Fluid shear stress (FSS) from blood flow, sensed by the vascular endothelial cells (ECs) that line all blood vessels, regulates vascular development during embryogenesis, controls adult vascular physiology and determines the location of atherosclerotic plaque formation. Although a number of papers have reported a crucial role for cell-cell adhesions or adhesion receptors in these processes, a recent publication has challenged this paradigm, presenting evidence that ECs can very rapidly align in fluid flow as single cells without cell-cell contacts. To address this controversy, four independent laboratories assessed EC alignment in fluid flow across a range of EC cell types. These studies demonstrate a strict requirement for cell-cell contact in shear stress sensing over timescales consistent with previous literature and inconsistent with the newly published data.

Junctions at the crossroads: the impact of mechanical cues on endothelial cell-cell junction conformations and vascular permeability.

Cells depend on precisely regulating barrier function within the vasculature to maintain physiological stability and facilitate essential substance transport. Endothelial cells achieve this through specialized adherens and tight junction protein complexes, which govern paracellular permeability across vascular beds. Adherens junctions, anchored by vascular endothelial (VE)-cadherin and associated catenins to the actin cytoskeleton, mediate homophilic adhesion crucial for barrier integrity. In contrast, tight junctions composed of occludin, claudin, and junctional adhesion molecule A interact with Zonula Occludens proteins, reinforcing intercellular connections essential for barrier selectivity. Endothelial cell-cell junctions exhibit dynamic conformations during development, maturation, and remodeling, regulated by local biochemical and mechanical cues. These structural adaptations play pivotal roles in disease contexts such as chronic inflammation, where junctional remodeling contributes to increased vascular permeability observed in conditions from cancer to cardiovascular diseases. Conversely, the brain microvasculature's specialized junctional arrangements pose challenges for therapeutic drug delivery due to their unique molecular compositions and tight organization. This commentary explores the molecular mechanisms underlying endothelial cell-cell junction conformations and their implications for vascular permeability. By highlighting recent advances in quantifying junctional changes and understanding mechanotransduction pathways, we elucidate how physical forces from cellular contacts and hemodynamic flow influence junctional dynamics.

Lenti-viral overexpression of RhoGEF Trio reduces CD8 T cell extravasation into atherosclerotic vein graft lesions in mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Rembrandt Institute for Cardiovascular Sciences Background In advanced atherosclerotic plaques, such as vein graft lesions, neovessels grow into the hypoxic plaque. This intraplaque angiogenic neovessels lack proper endothelial cell-cell junctions and are therefore susceptible to extravasation of leukocytes, which results in inflammation and atherosclerosis. T cells are the largest leukocyte subset found in atherosclerotic plaques and are key drivers of inflammation and plaque progression. RhoGEF Trio improves endothelial barrier function by strengthening endothelial cell-cell junctions in vitro, but its effect on T cell transmigration and its therapeutic potential to impede atherogenesis remains unknown. Methods ApoE3*Leiden mice (n=10/group) underwent bypass surgery in which a donor caval vein was interpositioned into the arterial circulation at the site of the right common carotid artery. Immediately post-operatively, a pluronic gel containing either GFP-Lentivirus (LV) (control) or Trio-GFP-LV (Trio) was locally applied around the vein graft to induce local overexpression of Trio. At post-op day 28, vein grafts were harvested and processed for morphometric and compositional analysis of the atherosclerotic lesions. Additionally, CD4 and CD8 T cells were isolated from healthy human donors and used in vitro to assess the role of Trio on trans-endothelial migration over in vitro-cultured HUVECs. Results At post-op day 28, both luminal and neovessel endothelial cells were GFP positive indicating successful transfection. CD8 T cell infiltration into the atherosclerotic plaque was reduced in the Trio group (45%, p=0.0426), whilst plaque size and the number of neovessels were unaffected. Mechanistically, we observed that adherence of CD8, but not CD4 T cells to the endothelial monolayer was reduced in vitro upon transfection with Trio-LV. This resulted in a decrease in the absolute number of transmigrated CD8 T cells (56%, p=0.0197), whilst the number of transmigrated CD4 T cells was unaffected. Interestingly, CD8 T cells exhibited no difference trans-/paracellular diapedesis, whilst for CD4 T cells Trio overexpression induced transcellular, rather than paracellular, transmigration. Conclusions LV transfection of vein grafts by local application of a pluronic gel holds promise a therapeutic strategy to improve bypass surgery outcomes. LV transfection with RhoGEF Trio did reduce CD8 T cell transmigration (in vivo and in vitro), but not CD4 T cell transmigration (in vitro) into the atherosclerotic plaque, whilst plaque size was not affected. Ongoing investigations focus on the effect of Trio on micro-vascular leakage as well as infiltration of other immune cells into the plaque.

The role of endothelial junctions in the regulation of the extravasation of tumor cells. A historical reappraisal.

Endothelial cells lining the vessel wall are connected by adherent, tight and gap junctions. Adherent junctions are common to all endothelial cells, whereas tight and gap junctions graduate within different vascular segments. Endothelial cell-cell junctions sustain vascular homeostasis and to control the transendothelial migration of inflammatory cells. Tumor cells need to weaken endothelial cell-cell junctions to penetrate the endothelial barrier and transendothelial migration and metastasis of tumor cells are tightly controlled by endothelial cell-cell junctions.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

3D spheroid-microvasculature-on-a-chip for tumor-endothelium mechanobiology interplay

During the final stage of cancer metastasis, tumor cells embed themselves in distant capillary beds, from where they extravasate and establish secondary tumors. Recent findings underscore the pivotal roles of blood/lymphatic flow and shear stress in this intricate tumor extravasation process. Despite the increasing evidence, there is a dearth of systematic and biomechanical methodologies that accurately mimic intricate 3D microtissue interactions within a controlled hydrodynamic microenvironment. Addressing this gap, we introduce an easy-to-operate 3D spheroid-microvasculature-on-a-chip (SMAC) model. Operating under both static and regulated flow conditions, the SMAC model facilitates the replication of the biomechanical interplay between heterogeneous tumor spheroids and endothelium in a quantitative manner. Serving as an in vitro model for metastasis mechanobiology, our model unveils the phenomena of 3D spheroid-induced endothelial compression and cell-cell junction degradation during tumor migration and expansion. Furthermore, we investigated the influence of shear stress on endothelial orientation, polarization, and tumor spheroid expansion. Collectively, our SMAC model provides a compact, cost-efficient, and adaptable platform for probing the mechanobiology of metastasis.

The therapeutic value of adipose-derived pericyte transplantation after intracerebral hemorrhage in rats.

Intracerebral hemorrhage (ICH) is a devastating cerebrovascular associated with inflammation and BBB disruption. Pericytes plays a critical role in neurological diseases, while whether pericytes could be utilized to treat ICH remains to be elucidated. Here, we isolated CD146+CD34- pericytes from rat adipose tissues (ADPs). Fluorescence-activated cells maintained their cell morphology and differentiation potential and expressed pericytes markers (CD146, NG2, and PDGFRβ) but not endothelial markers (CD31, CD34, and CD45). ADPs transplantation improved the neuro-behavioral functions in ICH rats and resulted in decreased hematoma volume and neuron loss after ICH. Besides, ADPs graft restrained the infiltration of neutrophils and reactive microgliosis after ICH injury around the peri-hematoma area of rats, as evidenced by increased Iba1- and MPO immunoreactivity. The transplanted pericytes were covered on endothelial cells, and promoted angiogenesis and vascular basement membrane formation in the peri-hematoma area of ICH rats, as shown by double staining of PDGFRβ and CD31/CollagenIV. The decreased brain water content and Evans Blue leakage proved the protective role of ADPs graft on BBB permeability. Finally, transplanted ADPs increased the expression of VE-cadherin, ZO-1, and claudin-5, leading to stable endothelial cell-cell adhesion and tight junction. In conclusion, the transplantation of APDs improved neuronal after ICH, which involved different mechanisms including neuroinflammation regulation and BBB dysfunction recovery. Our results supported that ADPs might be the ideal cell type for ICH therapy and provided insights into the potential cell therapy for further ICH treatment.

Donepezil is emerging as a potential therapeutic treatment in improving endothelial barrier function to prevent and treat coronary artery disease in Diabetes Mellitus.

Abstract Current treatments have shown limited success in reversing DM induced vascular endothelial barrier dysfunction. Vascular endothelium forms the inner most lining of the blood vessels, regulates variety of biological processes such as angiogenesis, wound healing and host defense mechanisms. During inflammatory conditions, such as diabetes mellitus and myocardial infarction endothelial cell-cell junctions start to disrupt because of the internalization of the junctional proteins, such as vascular endothelial (VE) cadherin. This leads to the formation of minute inter-endothelial gaps, and the infiltration of protein-rich fluid and immune cells in the interstitial space. If remains unchecked, the persistent buildup of edema underlying the endothelial lining sets the stage for the serious life-threatening complications. Therefore, we hypothesized that the donepezil prevents high glucose-induced endothelial barrier dysfunction by preventing the destabilization of junctional proteins. We investigated the potential protective role of Donepezil in high glucose induced increase in human coronary artery endothelial (HCAE) permeability and wound healing process. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. To investigate the signaling mechanism involved in the protection offered by Donepezil, we found that Donepezil prevented loss of VE-cadherin in endothelial junctions, and abrogated stress fiber formation in endothelial cell exposed to high-glucose solution. In conclusion, our study shows that Donepezil maintained endothelial barrier function and improved wound healing process in endothelial cells exposed to high glucose. Chicago State University CTRE grant to Professor Nadeem Fazal, MD, PhD

Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.

Paradigms of endothelial stiffening in cardiovascular disease and vascular aging.

Endothelial cells, the inner lining of the blood vessels, are well-known to play a critical role in vascular function, while endothelial dysfunction due to different cardiovascular risk factors or accumulation of disruptive mechanisms that arise with aging lead to cardiovascular disease. In this review, we focus on endothelial stiffness, a fundamental biomechanical property that reflects cell resistance to deformation. In the first part of the review, we describe the mechanisms that determine endothelial stiffness, including RhoA-dependent contractile response, actin architecture and crosslinking, as well as the contributions of the intermediate filaments, vimentin and lamin. Then, we review the factors that induce endothelial stiffening, with the emphasis on mechanical signals, such as fluid shear stress, stretch and stiffness of the extracellular matrix, which are well-known to control endothelial biomechanics. We also describe in detail the contribution of lipid factors, particularly oxidized lipids, that were also shown to be crucial in regulation of endothelial stiffness. Furthermore, we discuss the relative contributions of these two mechanisms of endothelial stiffening in vasculature in cardiovascular disease and aging. Finally, we present the current state of knowledge about the role of endothelial stiffening in the disruption of endothelial cell-cell junctions that are responsible for the maintenance of the endothelial barrier.

Septin and actin contributions to endothelial cell-cell junctions and monolayer integrity.

Septins in endothelial cells (ECs) have important roles supporting the integrity of the endothelial monolayer. Cell-cell junctions in EC monolayers are highly dynamic, with continuous retractions and protrusions. Depletion of septins in ECs leads to disruption of cell-cell junctions, which are composed of VE-cadherin and other junctional proteins. In EC monolayers, septins are concentrated at the plasma membrane at sites of cell-cell contact, in curved- and scallop-shaped patterns. These membrane-associated septin accumulations are located in regions of positive membrane curvature, and those regions are often associated with and immediately adjacent to actin-rich protrusions with negative membrane curvature. EC septins associate directly with plasma membrane lipids, based on findings with site-specific mutations of septins in ECs, which is consistent with biochemical and cell biological studies in other systems. Loss of septins leads to disruption of the EC monolayer, and gaps form between cells. The number and breadth of cell-cell contacts and junctions decreases, and the number and frequency of retractions, ruffles, and protrusions at cell edges also decreases. In addition, loss of septins leads to decreased amounts of F-actin at the cortical membrane, along with increased amounts of F-actin in stress fibers of the cytoplasm. Endothelial monolayer disruption from loss of septins is also associated with decreased transendothelial electric resistance (TEER) and increased levels of transendothelial migration (TEM) by immune and cancer cells, owing to the gaps in the monolayer. A current working model is that assembly of septin filaments at regions of positive membrane curvature contributes to a mechanical footing or base for actin-based protrusive forces generated at adjoining regions of the membrane. Specific molecular interactions between the septin and actin components of the cytoskeleton may also be important contributors. Regulators of actin assembly may promote and support the assembly of septin filaments at the membrane, as part of a molecular feedback loop between the assembly of septin and actin filaments.

BRAF increases endothelial cell stiffness through reorganization of the actin cytoskeleton.

The dynamics of the actin cytoskeleton and its connection to endothelial cell-cell junctions determine the barrier function of endothelial cells. The proper regulation of barrier opening/closing is necessary for the normal function of vessels, and its dysregulation can result in chronic and acute inflammation leading to edema formation. By using atomic force microscopy, we show here that thrombin-induced permeability of human umbilical vein endothelial cells, associated with actin stress fiber formation, stiffens the cell center. The depletion of the MEK/ERK kinase BRAF reduces thrombin-induced permeability prevents stress fiber formation and cell stiffening. The peripheral actin ring becomes stabilized by phosphorylated myosin light chain, while cofilin is excluded from the cell periphery. All these changes can be reverted by the inhibition of ROCK, but not of the MEK/ERK module. We propose that the balance between the binding of cofilin and myosin to F-actin in the cell periphery, which is regulated by the activity of ROCK, determines the local dynamics of actin reorganization, ultimately driving or preventing stress fiber formation.

WNK1 collaborates with TGF-β in endothelial cell junction turnover and angiogenesis

Angiogenesis is essential for growth of new blood vessels, remodeling existing vessels, and repair of damaged vessels, and these require reorganization of endothelial cell-cell junctions through a partial endothelial-mesenchymal transition. Homozygous disruption of the gene encoding the protein kinase WNK1 results in lethality in mice near embryonic day (E) 12 due to impaired angiogenesis. This angiogenesis defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase OSR1. We show that inhibition of WNK1 kinase activity not only prevents sprouting of endothelial cells from aortic slices but also vessel extension in inhibitor-treated embryos ex vivo. Mutations affecting TGF-β signaling also result in abnormal vascular development beginning by E10 and, ultimately, embryonic lethality. Previously, we demonstrated cross-talk of WNK1 with TGF-β-regulated SMAD signaling, and OSR1 was identified as a component of the TGF-β interactome. However, molecular events jointly regulated by TGF-β and WNK1/OSR1 have not been delineated. Here, we show that inhibition of WNK1 promotes TGF-β-dependent degradation of the tyrosine kinase receptor AXL, which is involved in TGF-β-mediated cell migration and angiogenesis. We also show that interaction between OSR1 and occludin, a protein associated with endothelial tight junctions, is an essential step to enable tight junction turnover. Furthermore, we show that these phenomena are WNK1 dependent, and sensitive to TGF-β. These findings demonstrate intimate connections between WNK1/OSR1 and multiple TGF-β-sensitive molecules controlling angiogenesis and suggest that WNK1 may modulate many TGF-β-regulated functions.

Abstract 6002: Invasiveness-dependent mechanical plasticity of cancer cells

Abstract Introduction: The capability of living cells to undergo deformations is critical for them to perform different biological functions, such as migration, spreading and endocytosis. Interestingly, recent evidence has also suggested that the mechanical response of cancer cells is intimately related to their pathological state. Physically, a reduced modulus will make it easier for cancer cells to change their morphology when squeezing through tight endothelial cell-cell junctions or pores in the extracellular matrix. However, the deformed cell will fully restore to its original shape if the response is purely elastic or viscoelastic. In this regard, the capability of cells to retain irreversible deformations, and hence effectively “memorize” the characteristics of the physical confinement that they have encountered, could greatly facilitate their subsequent passage through similar barriers, which is a common scenario in tumor cell invasion and metastasis. Here, we developed a method, for the first time to the best of our knowledge, to quantitatively measure the apparent plastic response of tumor cells and examine its correlation with their invasiveness. Materials and Methods: A microfluidic device was designed to deform the cell and then extract its viscoelastic and plastic characteristics from its shape recovery. The chip was casted by molding of silicon wafer which was fabricated using photo etching method. During the test, cells were pushed to squeeze through the deformation channel of the chip. Deformations of the cell and its nucleus were monitored under a fluorescent microscope. Two breast cancer cell lines (MDA-MB-231 and MCF-7), one nasopharyngeal cell line (NP69), and one carcinoma counterpart (HONE-1) were used. To verify the correlation of plastic response with cytoskeleton damage, Latrunculin A was added to block F-actin formation. SYTO fluorescent dye was used for live imaging of the cell nucleus. Results: After being deformed in the narrow channel for a few seconds, cells were released free and recovered the deformation. Interestingly, the highly invasive MDA-MB-231 and HONE-1 cells underwent irreversible deformation even after 10 minutes. However, NP69 and the poorly invasive MCF-7 cells fully recovered to the original round shape in a few minutes. But after blocking the F-actin formation with Latrunculin A, MCF-7 cells retained partial plastic strain. The nucleus elongated when the cell deformed, but fully recovered in 5 minutes. The results indicated the irreversible deformation of cells originated from cytoskeleton damage, but not the nucleus deformation. Conclusions: No apparent irreversible deformation was observed in the less invasive cell lines, suggesting that the plastic response of tumor cells might be correlated with their invasiveness. In addition, it was found that the plastic deformation of highly invasive cancer cells is caused by their cytoskeleton damage rather than plasticity of the nucleus. Citation Format: Xingyu Xia, Zishen Yan, William C. Cho, Yuan Lin. Invasiveness-dependent mechanical plasticity of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6002.

Therapeutic Validation of GEF-H1 Using a De Novo Designed Inhibitor in Models of Retinal Disease.

Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFβ-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach.

Transport of Maternally Administered Pharmaceutical Agents Across the Placental Barrier In Vitro.

To understand the transport of pharmaceutical agents and their effects on developing fetus, we have created a placental microsystem that mimics structural phenotypes and physiological characteristic of a placental barrier. We have shown the formation of a continuous network of epithelial adherens junctions and endothelial cell-cell junctions confirming the integrity of the placental barrier. More importantly, the formation of elongated microvilli under dynamic flow condition is demonstrated. Fluid shear stress acts as a mechanical cue triggering the microvilli formation. Pharmaceutical agents were administered to the maternal channel, and the concentration of pharmaceutical agents in fetal channel for coculture and control models were evaluated. In fetal channel, the coculture model exhibited about 2.5 and 2.2% of the maternal initial concentration for naltrexone and 6β-naltrexol, respectively. In acellular model, fetal channel showed about 10.5 and 10.3% of the maternal initial concentration for naltrexone and 6β-naltrexol, respectively. Gene expressions of epithelial cells after direct administration of naltrexone and 6β-naltrexol to the maternal channel and endothelial cells after exposure due to transport through placental barrier are also reported.

Role of endothelial cell junctions in transendothelial cancer cell migration. A review

During metastasis, tumour cells must become migratory and travel towards a capillary within the tumour. They then degrade the matrix surrounding the pericytes and endothelial cells, insert themselves between endothelial cells, transverse the capillary wall, to then enter the blood stream. This process depends on the motile behaviour of the tumour cells as well as the role of endothelial cell-cell junctions, both including adherens junctions and tight junctions. Circulating tumour cells must next, adhere to the walls of the capillary at the site of secondary tumour formation. Here, they again traverse the capillary wall to enter tissues distant from the primary tumour. This review aim to discuss the basic architecture of the endothelial junctional complex as well as the role played by these components towards the transendothelial migration of cancer cells from the primary site to the secondary site. Proper understanding of the role played by each of these components could invariably lead to the development of novel adjuvant cancer chemotherapy.

Inhibition of MicroRNA-92a Improved Erectile Dysfunction in Streptozotocin-Induced Diabetic Rats via Suppressing Oxidative Stress and Endothelial Dysfunction.

To determine whether microRNA could be a therapy target of erectile dysfunction (ED) and the underlying mechanisms. Eight-week-old fasting male SD rats were intraperitoneally injected with streptozotocin to construct diabetic rat models. Diabetic ED rats were treated with miRNA-92a inhibitor. The cavernous nerves were electrically stimulated to measure the intracavernous pressure and mean arterial pressure of rats in each group. After the detection, the penile cavernous tissues are properly stored for subsequent experiments. Rat aortic endothelial cells were used in in vitro studies. The expression of miR-92a was significantly increased in the corpus cavernosum of Streptozocin (STZ)-induced diabetic rats and injection of miR-92a antagomir into the corpus cavernosum of diabetic rats significantly increased eNOS/NO/cGMP signaling pathway activities, cavernous endothelial cell proliferation, endothelial cell-cell junction protein expression and decreased the levels of oxidative stress. These changes restored erectile function in STZ-induced diabetic rats. Moreover, in vitro study demonstrated that the miR-92a expression increased significantly in endothelial cells treated with high glucose, inhibiting AMPK/eNOS and AMPK/Nrf2/HO-1 signaling pathways in rat aortic endothelial cells via targeting Prkaa2, causing endothelial dysfunction and overactive oxidative stress, miR-92a inhibitor can improve the above parameters. miRNA-92a inhibitor could exert an inhibition role on oxidative stress and endothelial dysfunction to improve diabetic ED effectively.

A 3D printable perfused hydrogel vascular model to assay ultrasound-induced permeability.

The development of an in vitro model to study vascular permeability is vital for clinical applications such as the targeted delivery of therapeutics. This work demonstrates the use of a perfusion-based 3D printable hydrogel vascular model as an assessment for endothelial permeability and its barrier function. Aside from providing a platform that more closely mimics the dynamic vascular conditions in vivo, this model enables the real-time observation of changes in the endothelial monolayer during the application of ultrasound to investigate the downstream effect of ultrasound-induced permeability. We show an increase in the apparent permeability coefficient of a fluorescently labeled tracer molecule after ultrasound treatment via a custom MATLAB algorithm, which implemented advanced features such as edge detection and a dynamic region of interest, thus supporting the use of ultrasound as a non-invasive method to enhance vascular permeability for targeted drug therapies. Notably, live-cell imaging with VE-cadherin-GFP HUVECs provides some of the first real-time acquisitions of the dynamics of endothelial cell-cell junctions under the application of ultrasound in a 3D perfusable model. This model demonstrates potential as a new scalable platform to investigate ultrasound-assisted delivery of therapeutics across a cellular barrier that more accurately mimics the physiologic matrix and fluid dynamics.