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.

Hepatocyte growth factor prevents pericyte loss in diabetic retinopathy.

Diabetic retinopathy (DR) is a disease that causes blindness due to vascular leakage or abnormal angiogenesis. Hepatocyte growth factor (HGF) is increased in the serum or vitreous fluid in proliferative diabetic retinopathy (PDR) patients, although the effect of HGF on the blood vessels remains unclear. This study focused on the effect of HGF on pericyte (PC) survival and endothelial cell (EC) permeability. It was demonstrated that HGF was increased in the diabetic mouse retina. However, HGF prevented PC apoptosis caused by TNF-α, which increased in the diabetic retinas both in vitro and in vivo. In addition, HGF was involved in PC survival by increasing the Akt signaling pathway. Moreover, HGF strengthened the EC tight junction in co-cultures of PCs and ECs by promoting PC survival, thereby reducing EC permeability. These results suggest that HGF may play a role in the prevention of increased vascular leakage by inhibiting the PC loss that occurs in DR to some extent. However, careful HGF reduction in DR might avoid an increase in PC loss.

Chlamydia pneumoniae infection promotes monocyte transendothelial migration by increasing vascular endothelial cell permeability via the tyrosine phosphorylation of VE-cadherin.

Migration of monocytes into the subendothelial layer of the intima is one of the critical events in early atherosclerosis. Chlamydia pneumoniae (C. pneumoniae) infection has been shown to promote monocyte transendothelial migration (TEM). However, the exact mechanisms have not yet been fully clarified. In this study, we tested the hypothesis that C. pneumoniae infection increases vascular endothelial cell (VEC) permeability and subsequent monocyte TEM through stimulating the tyrosine phosphorylation of vascular endothelial-cadherin (VE-cadherin). Here, we demonstrated that C. pneumoniae infection promoted monocyte TEM in a TEM assay possibly by increasing the permeability of a VEC line EA.hy926 cell as assessed by measuring the passage of FITC-BSA across a VEC monolayer. Subsequently, Western blot analysis showed that C. pneumoniae infection induced VE-cadherin internalization. Our further data revealed that Src-mediated VE-cadherin phosphorylation at Tyr658 was involved in C. pneumoniae infection-induced internalization of VE-cadherin, VEC hyperpermeability and monocyte TEM. Taken together, our data indicate that C. pneumoniae infection promotes monocyte TEM by increasing VEC permeability via the tyrosine phosphorylation and internalization of VE-cadherin in VECs.

Trimethylamine-N-Oxide Instigates NLRP3 Inflammasome Activation and Endothelial Dysfunction.

BACKGROUND/AIM: Plasma trimethylamine-N-oxide (TMAO), a product of intestinal microbial metabolism of dietary phosphatidylcholine has been recently associated with atherosclerosis and increased risk of cardiovascular diseases (CVD) in rodents and humans. However, the molecular mechanisms of how TMAO induces atherosclerosis and CVD progression are still unclear. The present study tested whether TMAO induces NLRP3 inflammasome formation and activation and thereby contributes to endothelial injury initiating atherogenesis. METHODS: Inflammasome formation and activation was determined by confocal microscopy, caspase-1 activity was measured by colorimetric assay, IL-1ß production was measured using ELISA, cell permeability was determined by microplate reader and ZO-1 expression was determined by western blot analysis and confocal microscopy. In in vivo experiments, TMAO was infused by osmotic pump implantation. RESULTS: TMAO treatment significantly increased the colocalization of NLRP3 with Asc or NLRP3 with caspase-1, caspase-1 activity, IL-1ß production, cell permeability in carotid artery endothelial cells (CAECs) compared to control cells. Pretreatment with caspase-1 inhibitor, WEHD or Nlrp3 siRNA abolished the TMAO-induced inflammasome formation, activation and cell permeability in these cells. In addition, we explored the mechanisms by which TMAO activates NLRP3 inflammasomes. TMAO-induced the activation of NLRP3 inflammasomes was associated with both redox regulation and lysosomal dysfunction. In animal experiments, direct infusion of TMAO in mice with partially ligated carotid artery were found to have increased NLRP3 inflammasome formation and IL-1ß production in the intima of wild type mice. CONCLUSION: The formation and activation of NLRP3 inflammasomes by TMAO may be an important initiating mechanism to turn on the endothelial inflammatory response leading to endothelial dysfunction.

Brain-derived neurotrophic factor prevents the endothelial barrier dysfunction induced by interleukin-1ß and tumor necrosis factor-α.

BACKGROUND AND OBJECTIVE: Brain-derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Although a local inflammatory step is required to initiate the subsequent process of tissue regeneration, excessive inflammation may inhibit or delay tissue regeneration. Therefore, the regulation of inflammation is essential for periodontal tissue regeneration. In the present study, we examined the influence of BDNF on the human microvascular endothelial cell (HMVEC) barrier dysfunction induced by interleukin (IL)-1ß or tumor necrosis factor (TNF)-α to determine the effects of BDNF on the regulation of local inflammation in periodontal tissue regeneration. MATERIAL AND METHODS: Endothelial permeability was analyzed using a Dextran transport assay with transwell plates. The expression of vascular endothelial (VE)-cadherin was assessed by immunoblotting and immunofluorescence microscopy. RESULTS: BDNF (25 ng/mL) inhibited increase induced in endothelial permeability by IL-1ß and TNF-α. IL-1ß and TNF-α decreased VE-cadherin protein levels, while BDNF recovered the reduction in HMVECs. BDNF protected the increase induced in endothelial permeability by IL-1ß and TNF-α through TrkB. The single addition of BDNF into the culture increased the expression of VE-cadherin in HMVECs. CONCLUSION: BDNF played an important role in inhibiting endothelial barrier dysfunction, which suggests that it may assist in enhancing periodontal tissue regeneration.

Role of gC1qR as a modulator of endothelial cell permeability and contributor to post-stroke inflammation and edema formation.

Ischemic stroke is a leading cause of death and disability worldwide. A serious risk of acute ischemic stroke (AIS) arises after the stroke event, due to inflammation and edema formation. Inflammation and edema in the brain are mediated by bradykinin, the formation of which is dependent upon a multi-ligand receptor protein called gC1qR. There are currently no preventive treatments for the secondary damage of AIS produced by inflammation and edema. This review aims to summarize recent research regarding the role of gC1qR in bradykinin formation, its role in inflammation and edema following ischemic injury, and potential therapeutic approaches to preventing post-stroke inflammation and edema formation.

miR-152-3p aggravates vascular endothelial cell dysfunction by targeting DEAD-box helicase 6 (DDX6) under hypoxia.

Stroke is a main cause of disability and death worldwide, and ischemic stroke accounts for most stroke cases. Recently, microRNAs (miRNAs) have been verified to play critical roles in the development of stroke. Herein, we explored effects of miR-152-3p on vascular endothelial cell functions under hypoxia. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia to mimic cell injury in vitro. Reverse transcription quantitative polymerase chain reaction revealed that miR-152-3p exhibited high expression in HUVECs treated with hypoxia. The inhibition of miR-152-3p reversed hypoxia-induced decrease in cell viability and the increase in angiogenesis, according to the results of cell counting kit-8 assays and tube formation assays. miR-152-3p inhibition reversed the increase in endothelial cell permeability mediated by hypoxia, as shown by endothelial cell permeability in vitro assays. In addition, the increase in protein levels of angiogenetic markers and the decrease in levels of tight junction proteins induced by hypoxia were reversed by miR-152-3p inhibition. Mechanistically, miR-152-3p directly targets 3'-untranslated region of DEAD-box helicase 6 (DDX6), which was confirmed by luciferase reporter assays. DDX6 is lowly expressed in HUVECs under hypoxic condition, and mRNA expression and protein level of DDX6 were upregulated in HUVECs due to miR-152-3p inhibition. Rescue assays showed that DDX6 knockdown reversed effects of miR-152-3p on cell viability, angiogenesis and endothelial permeability. The results demonstrated that miR-152-3p aggravates vascular endothelial cell dysfunction by targeting DDX6 under hypoxia.

Aminophylline modulates the permeability of endothelial cells via the Slit2-Robo4 pathway in lipopolysaccharide-induced inflammation.

Sepsis and septic shock are the main cause of mortality in intensive care units. The prevention and treatment of sepsis remains a significant challenge worldwide. The endothelial cell barrier plays a critical role in the development of sepsis. Aminophylline, a non-selective phosphodiesterase inhibitor, has been demonstrated to reduce endothelial cell permeability. However, little is known regarding the role of aminophylline in regulating vascular permeability during sepsis, as well as the potential underlying mechanisms. In the present study, the Slit2/Robo4 signaling pathway, the downstream protein, vascular endothelial (VE)-cadherin and endothelial cell permeability were investigated in a lipopolysaccharide (LPS)-induced inflammation model. It was indicated that, in human umbilical vein endothelial cells (HUVECs), LPS downregulated Slit2, Robo4 and VE-cadherin protein expression levels and, as expected, increased endothelial cell permeability in vitro during inflammation. After administration of aminophylline, the protein expression levels of Slit2, Robo4 and VE-cadherin were upregulated and endothelial cell permeability was significantly improved. These results suggested that the permeability of endothelial cells could be mediated by VE-cadherin via the Slit2/Robo4 signaling pathway. Aminophylline reduced endothelial permeability in a LPS-induced inflammation model. Therefore, aminophylline may represent a promising candidate for modulating vascular permeability induced by inflammation or sepsis.

Ophiopogon Saponin C1 Inhibits Lung Tumors by Stabilizing Endothelium Permeability via Inhibition of PKCδ.

As the most frequent cause of cancer-related death worldwide, lung cancer is closely related to inflammation. The interaction between tumor cells and inflammatory cells promotes tumor development and metastasis. During tumor development, vascular endothelial cells form the most important barrier to prevent tumor cell migration to the blood and tissue. Increased vascular permeability provides favorable conditions for the migration of tumor cells, and endothelial tight junctions are an important component of the vascular barrier. Protein kinase C δ is involved in the occurrence of non-small cell lung cancer and regulates vascular permeability and tight junction protein expression. Src kinase was reported to play an important role in TNF-α-induced endothelial inflammation. Ophiopogon Saponin C1 is a new chemical compound isolated from Liriope muscari, but its pharmacological activities have not been fully elucidated. Therefore, we tested the protective effects of C1 on endothelial permeability in a model of TNF-α-induced endothelial inflammation by transendothelial electrical resistance and sodium fluorescein assays and verified these results in a nude mouse model of experimental pulmonary adenocarcinoma metastasis. We further elucidated the mechanism of C1, which was based on the PKCδ and Src proteins, by Western blotting. C1 can inhibit lung cancer in vivo, regulate the level of plasma inflammation in tumor-bearing mice, and protect the pulmonary vascular barrier against injury induced by cancer. It was investigated the expression and distribution of the TJ index protein ZO-1 in mouse vascular endothelium and HUVECs and found that C1 could inhibit the degradation and breakage of the ZO-1 protein. Related signaling experiments confirmed that C1 can inhibit TNF-α and activation of PKCδ and Src kinase. This study laid the foundation for further analysis of new drugs with clear mechanisms and independent intellectual property rights of traditional Chinese medicines.

Neuroprotective mechanism of TMP269, a selective class IIA histone deacetylase inhibitor, after cerebral ischemia/reperfusion injury.

TMP269 is a selective class IIA histone deacetylase inhibitor that has a protective effect on the central nervous system, whose specific mechanism of action is unclear. We aimed to reveal the optimal concentration of TMP269 for protecting against cerebral ischemia/reperfusion injury and its neuroprotective mechanism. Male Sprague-Dawley rats were randomly divided into sham, ischemia/reperfusion, and 1, 4, 10 and 16 mg/kg TMP269 groups. Cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion. TMP269 was intraperitoneally administered at different doses 0.5 hours before ischemia induction. Western blot assay and immunohistochemistry were used to detect effects of TMP269 on histone 2 acetylation. The results showed that the level of histone 2 acetylation was increased 24 hours after TMP269 injection. 2,3,5-Triphenyltetrazolium chloride staining was utilized to examine effect of TMP269 on infarct volume. The results found that different doses of TMP269 could reduce the infarct volume. Western blot assay, immunohistochemistry and Evans blue staining were employed to measure the effect of TMP269 on blood-brain barrier. The results showed that TMP269 counteracted the abnormal endothelial cell permeability changes caused by cerebral ischemia/reperfusion. Western blot assay and immunohistochemistry were used to determine the effect of TMP269 on tissue kallikrein. The results found that TMP269 up-regulated the expression of tissue kallikrein. Western blot assay further determined the optimal concentration to be 4 mg/kg. In conclusion, TMP269 plays a neuroprotective role by up-regulating the level of histone 2 acetylation, alleviating endothelial cell injury after cerebral ischemia/reperfusion, and up-regulating the expression of tissue kallikrein. The experimental protocol was approved in 2014 by the Department of Laboratory Animal Science, Fudan University, China (approval No. 20140143C001).

Extracellular RNA, a Potential Drug Target for Alleviating Atherosclerosis, Ischemia/Reperfusion Injury and Organ Transplantation.

Extracellular RNA (eRNA), composed of mainly rRNA e.g. released upon cell injury, has previously been shown to have three main detrimental functions in the context of cardiovascular disease: (1) to promote tissue edema by activating the VEGF signal transduction cascade, disrupting endothelial tight junctions and increasing intercellular permeability; (2) to induce thrombus formation by activating the contact phase system of intrinsic blood coagulation; and (3) to increase inflammation by stimulating leukocyte adhesion and transmigration and the mobilization of pro-inflammatory cytokines. This review proposes eRNA to be a possible new drug target in cardiovascular disease. The effects of eRNA could potentially be limited by enhancing its degradation through the naturally occurring ribonuclease RNase. In acute settings such as transplantation or ischemia/reperfusion injury after myocardial infarction, this could be achieved by administering RNase intravenously; however, in chronic situations such as atherosclerosis, a new orally administrable chemical compound e.g. blocking the endogenous RNase inhibitor might be developed. In ischemia/reperfusion injury as well as in acute graft rejection, such an intervention would likely reduce edema, thrombosis, inflammation and cellular damage and hence improve survival. In atherosclerosis, antagonizing the RNase inhibitor would presumably reduce inflammation and slow plaque growth. Crucially, toxicological examinations of RNase administration did not find any adverse side effects, denoting it as potentially safe and well-tolerated. Therefore, eRNA appears to be a promising drug target in cardiovascular disease, and further investigations are required for the possible clinical use of an agent limiting its activity.

Gold Nanoparticles Induced Endothelial Leakiness Depends on Particle Size and Endothelial Cell Origin.

The endothelium presents a formidable barrier for cancer nanomedicine, as the intravenously introduced nanomedicine needs to leave the blood vessel at the tumor site. Endothelial permeability and retention effect (EPR) is not dependable since it is derived from tumors. Certain nanoparticles with specific characteristics are able to induce micrometer sized gaps between endothelial cells. This effect is called "nanoparticle induced endothelial leakiness" (NanoEL). NanoEL therefore allows the nanotechnology to control access to the tumor even in the absence of any EPR effect. Morever, NanoEL can be applicable to noncancer issues, thereby expanding its usefulness in other subfields of nanomedicine. In this paper, we have shown that Gold (Au) nanoparticles within the range of 10-30 nm are good NanoEL inducing particles. As not all endothelial cells have the same permeability, we found that human mammary endothelial cells and human skin endothelial cells are sensitive to Au induced NanoEL, while human umbilical vein endothelial cells are insensitive, reflective of their innate nature of endothelial permeability. The size window and endothelial cell type sensitivity then helps the nanotechnologists to design future nanoparticles that either exploit NanoEL as a nanotechnology driven strategy to access immature tumors, which do not induce the EPR effect, or avoid NanoEL as a nanotoxic side effect.

The role of Slit-Robo signaling in the regulation of tissue barriers.

The role of Slit/Robo signaling has extended from initial axon repulsion in the developing nervous system to organ morphogenesis, cancer development and angiogenesis. Slit/Robo signaling regulates similar pathways within these processes. Slit/Robo ensures the homeostasis of the dynamic interaction between cell-cell and cell-matrix interactions. The dysregulation of Slit/Robo signaling damages the tissue barrier, resulting in developmental abnormalities or disease. Here, we summarize how Slit/Robo controls kidney morphogenesis and describe the dual roles of Slit/Robo signaling in the regulation of tumorigenesis and angiogenesis.

Endothelial permeability in vitro and in vivo: protective actions of ANP and omapatrilat in experimental atherosclerosis.

Increased arterial endothelial cell permeability (ECP) is considered an initial step in atherosclerosis. Atrial natriuretic peptide (ANP) which is rapidly degraded by neprilysin (NEP) may reduce injury-induced endothelial cell leakiness. Omapatrilat represents a first in class of pharmacological agents which inhibits both NEP and angiotensin converting enzyme (ACE). We hypothesized that ANP prevents thrombin-induced increases of ECP in human aortic ECs (HAECs) and that omapatrilat would reduce aortic leakiness and atherogenesis and enhance ANP mediated vasorelaxation of isolated aortas. Thrombin induced ECP determined by I(125) albumin flux was assessed in HAECs with and without ANP pretreatment. Next we examined the effects of chronic oral administration of omapatrilat (12 mg/kg/day, n=13) or placebo (n=13) for 8 weeks on aortic leakiness, atherogenesis and ANP-mediated vasorelaxation in isolated aortas in a rabbit model of atherosclerosis produced by high cholesterol diet. In HAECs, thrombin-induced increases in ECP were prevented by ANP. Omapatrilat reduced the area of increased aortic leakiness determined by Evans-blue dye and area of atheroma formation assessed by Oil-Red staining compared to placebo. In isolated arterial rings, omapatrilat enhanced vasorelaxation to ANP compared to placebo with and without the endothelium. ANP prevents thrombin-induced increases in ECP in HAECs. Chronic oral administration of omapatrilat reduces aortic leakiness and atheroma formation with enhanced endothelial independent vasorelaxation to ANP. These studies support the therapeutic potential of dual inhibition of NEP and ACE in the prevention of increased arterial ECP and atherogenesis which may be linked to the ANP/cGMP system.