Feasibility of measuring blood-brain barrier permeability using ultra-short echo time radial magnetic resonance imaging.

BACKGROUND AND PURPOSE: The purpose of this study is to evaluate the feasibility of using 3-dimensional (3D) ultra-short echo time (UTE) radial imaging method for measurement of the permeability of the blood-brain barrier (BBB) to gadolinium-based contrast agent. In this study, we propose to use the golden-angle radial sparse parallel (GRASP) method with 3D center-out trajectories for UTE, hence named as 3D UTE-GRASP. We first examined the feasibility of using 3D UTE-GRASP dynamic contrast-enhanced (DCE)-magnetic resonance imaging (MRI) for differentiating subtle BBB disruptions induced by focused ultrasound (FUS). Then, we examined the BBB permeability changes in Alzheimer's disease (AD) pathology using Alzheimer's disease transgenic mice (5xFAD) at different ages. METHODS: For FUS experiments, we used four Sprague Dawley rats at similar ages where we compared BBB permeability of each rat receiving the FUS sonication with different acoustic power (0.4-1.0 MPa). For AD transgenic mice experiments, we included three 5xFAD mice (6, 12, and 16 months old) and three wild-type mice (4, 8, and 12 months old). RESULTS: The result from FUS experiments showed a progressive increase in BBB permeability with increase of acoustic power (p < .05), demonstrating the sensitivity of DCE-MRI method for detecting subtle changes in BBB disruption. Our AD transgenic mice experiments suggest an early BBB disruption in 5xFAD mice, which is further impaired with aging. CONCLUSION: The results in this study substantiate the feasibility of using the proposed 3D UTE-GRASP method for detecting subtle BBB permeability changes expected in neurodegenerative diseases, such as AD.

Respiratory drive heterogeneity associated with systemic inflammation and vascular permeability in acute respiratory distress syndrome.

BACKGROUND: In acute respiratory distress syndrome (ARDS), respiratory drive often differs among patients with similar clinical characteristics. Readily observable factors like acid-base state, oxygenation, mechanics, and sedation depth do not fully explain drive heterogeneity. This study evaluated the relationship of systemic inflammation and vascular permeability markers with respiratory drive and clinical outcomes in ARDS. METHODS: ARDS patients enrolled in the multicenter EPVent-2 trial with requisite data and plasma biomarkers were included. Neuromuscular blockade recipients were excluded. Respiratory drive was measured as PES0.1, the change in esophageal pressure during the first 0.1 s of inspiratory effort. Plasma angiopoietin-2, interleukin-6, and interleukin-8 were measured concomitantly, and 60-day clinical outcomes evaluated. RESULTS: 54.8% of 124 included patients had detectable respiratory drive (PES0.1 range of 0-5.1 cm H2O). Angiopoietin-2 and interleukin-8, but not interleukin-6, were associated with respiratory drive independently of acid-base, oxygenation, respiratory mechanics, and sedation depth. Sedation depth was not significantly associated with PES0.1 in an unadjusted model, or after adjusting for mechanics and chemoreceptor input. However, upon adding angiopoietin-2, interleukin-6, or interleukin-8 to models, lighter sedation was significantly associated with higher PES0.1. Risk of death was less with moderate drive (PES0.1 of 0.5-2.9 cm H2O) compared to either lower drive (hazard ratio 1.58, 95% CI 0.82-3.05) or higher drive (2.63, 95% CI 1.21-5.70) (p = 0.049). CONCLUSIONS: Among patients with ARDS, systemic inflammatory and vascular permeability markers were independently associated with higher respiratory drive. The heterogeneous response of respiratory drive to varying sedation depth may be explained in part by differences in inflammation and vascular permeability.

Blood-brain barrier permeability for the first 24 hours in hypoxic-ischemic brain injury following cardiac arrest.

BACKGROUND: This study aimed to explore the changes in blood-brain barrier (BBB) permeability and intracranial pressure (ICP) for the first 24 h after the return of spontaneous circulation (ROSC) and their association with injury severity of cardiac arrest. METHODS: This prospective study analysed the BBB permeability assessed using the albumin quotient (Qa) and ICP every 2 h for the first 24 h after ROSC. The injury severity of cardiac arrest was assessed using Pittsburgh Cardiac Arrest Category (PCAC) scores. The primary outcome was the time course of changes in the BBB permeability and ICP for the first 24 h after ROSC and their association with injury severity (PCAC scores of 1-4). RESULTS: Qa and ICP were measured 274 and 197 times, respectively, in 32 enrolled patients. Overall, the BBB permeability increased progressively over time after ROSC, and then it increased significantly at 18 h after ROSC compared with the baseline. In contrast, the ICP revealed non-significant changes for the first 24 h after ROSC. The Qa in the PCAC 2 group was < 0.01, indicating normal or mild BBB disruption at all time points, whereas the PCAC 3 and 4 groups showed a significant increase in BBB permeability at 14 and 22 h, and 12 and 14 h after ROSC, respectively. CONCLUSION: BBB permeability increased progressively over time for the first 24 h after ROSC despite post-resuscitation care, whereas ICP did not change over time. BBB permeability has an individual pattern when stratified by injury severity.

A mechanical modeling framework to study endothelial permeability.

The inner lining of blood vessels, the endothelium, is made up of endothelial cells. Vascular endothelial (VE)-cadherin protein forms a bond with VE-cadherin from neighboring cells to determine the size of gaps between the cells and thereby regulate the size of particles that can cross the endothelium. Chemical cues such as thrombin, along with mechanical properties of the cell and extracellular matrix are known to affect the permeability of endothelial cells. Abnormal permeability is found in patients suffering from diseases including cardiovascular diseases, cancer, and COVID-19. Even though some of the regulatory mechanisms affecting endothelial permeability are well studied, details of how several mechanical and chemical stimuli acting simultaneously affect endothelial permeability are not yet understood. In this article, we present a continuum-level mechanical modeling framework to study the highly dynamic nature of the VE-cadherin bonds. Taking inspiration from the catch-slip behavior that VE-cadherin complexes are known to exhibit, we model the VE-cadherin homophilic bond as cohesive contact with damage following a traction-separation law. We explicitly model the actin cytoskeleton and substrate to study their role in permeability. Our studies show that mechanochemical coupling is necessary to simulate the influence of the mechanical properties of the substrate on permeability. Simulations show that shear between cells is responsible for the variation in permeability between bicellular and tricellular junctions, explaining the phenotypic differences observed in experiments. An increase in the magnitude of traction force due to disturbed flow that endothelial cells experience results in increased permeability, and it is found that the effect is higher on stiffer extracellular matrix. Finally, we show that the cylindrical monolayer exhibits higher permeability than the planar monolayer under unconstrained cases. Thus, we present a contact mechanics-based mechanochemical model to investigate the variation in the permeability of endothelial monolayer due to multiple loads acting simultaneously.

The paracrine isthmin1 transcriptionally regulated by C/EBPß exacerbates pulmonary vascular leakage in murine sepsis.

It is known that pulmonary vascular leakage, a key pathological feature of sepsis-induced lung injury, is largely regulated by perivascular cells. However, the underlying mechanisms have not been fully uncovered. In the present study, we aimed to evaluate the role of isthmin1, a secretory protein originating from alveolar epithelium, in the pulmonary vascular leakage during sepsis and to investigate the regulatory mechanisms of isthmin1 gene transcription. We observed an elevated isthmin1 gene expression in the pulmonary tissue of septic mice induced by cecal ligation and puncture (CLP), as well as in primary murine alveolar type II epithelial cells (ATII) exposed to lipopolysaccharide (LPS). Furthermore, we confirmed that isthmin1 derived from ATII contributes to pulmonary vascular leakage during sepsis. Specifically, adenovirus-mediated isthmin1 disruption in ATII led to a significant attenuation of the increased pulmonary microvascular endothelial cell (PMVEC) hyperpermeability in a PMVEC/ATII coculture system when exposed to LPS. In addition, adeno-associated virus 9 (AAV9)-mediated knockdown of isthmin1 in the alveolar epithelium of septic mice significantly attenuated pulmonary vascular leakage. Finally, mechanistic studies unveiled that nuclear transcription factor CCAAT/enhancer binding protein (C/EBP)ß participates in isthmin1 gene activation by binding directly to the cis-regulatory element of isthmin1 locus and may contribute to isthmin1 upregulation during sepsis. Collectively, the present study highlighted the impact of the paracrine protein isthmin1, derived from ATII, on the exacerbation of pulmonary vascular permeability in sepsis and revealed a new regulatory mechanism for isthmin1 gene transcription.NEW & NOTEWORTHY This article addresses the role of the alveolar epithelial-secreted protein isthmin1 on the exacerbation of pulmonary vascular permeability in sepsis and identified nuclear factor CCAAT/enhancer binding protein (C/EBP)ß as a new regulator of isthmin1 gene transcription. Targeting the C/EBPß-isthmin1 regulatory axis on the alveolar side would be of great value in the treatment of pulmonary vascular leakage and lung injury induced by sepsis.

Determination of Solute Permeability of Microvascular Endothelial Cell Monolayers In Vitro.

The microvascular endothelium has a critical role in regulating the delivery of oxygen, nutrients, and water to the surrounding tissues. Under inflammatory conditions that accompany acute injury or disease, microvascular permeability becomes elevated. When microvascular hyperpermeability becomes uncontrolled or chronic, the excessive escape of plasma proteins into the surrounding tissue disrupts homeostasis and ultimately leads to organ dysfunction. Much remains to be learned about the mechanisms that control microvascular permeability. In addition to in vivo and isolated microvessel methods, the cultured endothelial cell monolayer protocol is an important tool that allows for understanding the specific, endothelial subcellular mechanisms that determine permeability of the endothelium to plasma proteins. In this chapter, two variations of the popular Transwell culture methodology to determine permeability to using fluorescently labeled tracers are presented. The strengths and weaknesses of this approach are also discussed.

Bortezomib Increased Vascular Permeability by Decreasing Cell-Cell Junction Molecules in Human Pulmonary Microvascular Endothelial Cells.

Bortezomib (BTZ), a chemotherapeutic drug used to treat multiple myeloma, induces life-threatening side effects, including severe pulmonary toxicity. However, the mechanisms underlying these effects remain unclear. The objectives of this study were to (1) investigate whether BTZ influences vascular permeability and (2) clarify the effect of BTZ on the expression of molecules associated with cell-cell junctions using human pulmonary microvascular endothelial cells in vitro. Clinically relevant concentrations of BTZ induced limited cytotoxicity and increased the permeability of human pulmonary microvascular endothelial cell monolayers. BTZ decreased the protein expression of claudin-5, occludin, and VE-cadherin but not that of ZO-1 and ß-catenin. Additionally, BTZ decreased the mRNA expression of claudin-5, occludin, ZO-1, VE-cadherin, and ß-catenin. Our results suggest that BTZ increases the vascular permeability of the pulmonary microvascular endothelium by downregulating cell-cell junction molecules, particularly claudin-5, occludin, and VE-cadherin.

Hematoloechus sp. attachment shifts endothelium in vivo from pro- to anti-inflammatory profile in Rana pipiens: evidence from systemic and capillary physiology.

This prospective, descriptive study focused on lung flukes (Hematoloechus sp., H) and their impact on systemic and individual capillary variables measured in pithed Rana pipiens, a long-standing model for studies of capillary physiology. Three groups were identified based on Hematoloechus attachment: no Hematoloechus (No H), Hematoloechus not attached (H Not Att), and Hematoloechus attached (H Att). Among 38 descriptive, cardiovascular, and immunological variables, 18 changed significantly with H. Symptoms of H included weight loss, elevated immune cells, heart rate variability, faster coagulation, lower hematocrit, and fluid accumulation. Important capillary function discoveries included median baselines for hydraulic conductivity (Lp) of 7.0 (No H), 12.4 (H Not Att), and 4.2 (H Att) × 10-7 cm·s-1·cmH2O-1 (P < 0.0001) plus seasonal adaptation of sigma delta pi [σ(πc-πi), P = 0.03]. Pro- and anti-inflammatory phases were revealed for Lp and plasma nitrite/nitrate concentration ([NOx]) in both H Not Att and H Att, whereas capillary wall tensile strength increased in the H Att. H attachment was advantageous for the host due to lower edema and for the parasite via a sustained food source illustrating an excellent example of natural symbiosis. However, H attachment also resulted in host weight loss: in time, a conundrum for the highly dependent parasite. The study increases overall knowledge of Rana pipiens by revealing intriguing effects of H and previously unknown, naturally occurring seasonal changes in many variables. The data improve Rana pipiens as a general scientific and capillary physiology model. Diseases of inflammation and stroke are among the clinical applications.

The impact of pericytes on the stability of microvascular networks in response to nanoparticles.

Recapitulating the normal physiology of the microvasculature is pivotal in the development of more complex in-vitro models and organ-on-chip designs. Pericytes are an important component of the vasculature, promoting vessel stability, inhibiting vascular permeability and maintaining the vascular hierarchical architecture. The use of such co-culture for the testing of therapeutics and nanoparticle safety is increasingly considered for the validation of therapeutic strategies. This report presents the use of a microfluidic model for such applications. Interactions between endothelial cells and pericytes are first explored. We identify basal conditions required to form stable and reproducible endothelial networks. We then investigate interactions between endothelial cells and pericytes via direct co-culture. In our system, pericytes prevented vessel hyperplasia and maintained vessel length in prolonged culture (> 10 days). In addition, these vessels displayed barrier function and expression of junction markers associated with vessel maturation, including VE-cadherin, ß-catenin and ZO-1. Furthermore, pericytes maintained vessel integrity following stress (nutrient starvation) and prevented vessel regression, in contrast to the striking dissociation of networks in endothelial monocultures. This response was also observed when endothelial/pericyte co-cultures were exposed to high concentrations of moderately toxic cationic nanoparticles used for gene delivery. This study highlights the importance of pericytes in protecting vascular networks from stress and external agents and their importance to the design of advanced in-vitro models, including for the testing of nanotoxicity, to better recapitulate physiological response and avoid false positives.

A ketogenic diet improves vascular hyperpermeability in type 2 diabetic mice by downregulating vascular pescadillo1 expression.

The role of pescadillo1 (PES1) in regulating vascular permeability has been unknown. This study probes the role of PES1 and its mediated molecular mechanism in modulating vascular hyperpermeability in diabetic mice. Male C57BL/6J and db/db mice were fed a standard diet and a ketogenic diet (KD). Meanwhile, mouse vascular endothelial cells (MVECs) were treated with ß-hydroxybutyric acid (ß-HB), Pes1 siRNA or a Pes1 overexpression plasmid. Additionally, knockout (KO) of Pes1 in mice was applied. After 12 weeks of feedings, enhanced vascular PES1 expression in diabetic mice was inhibited by the KD. The suppression of PES1 was also observed in ß-HB-treated MVECs. In mice with Pes1 KO, the levels of vascular VEGF and PES1 were attenuated, while the levels of vascular VE-cadherin, Ang-1 and Occludin were upregulated. Similar outcomes also occurred after the knockdown of Pes1 in cultured MVECs, which were opposite to the effects induced by PES1 overexpression in MVECs. In vitro and in vivo experiments showed that high glucose concentration-induced increases in vascular paracellular permeability declined after MVECs were treated by ß-HB or by knockdown of Pes1. In contrast, increases in vascular permeability were induced by overexpression of Pes1, which were suppressed by coadministration of ß-HB in cultured endothelial cells. Similarly declines in vascular permeability were found by Pes1 knockdown in diabetic mice. Mechanistically, ß-HB decreased PES1-facilitated ubiquitination of VE-cadherin. The KD suppressed the diabetes-induced increase in PES1, which may result in vascular hyperpermeability through ubiquitination of VE-cadherin in type 2 diabetic mice.

Edema after CNS Trauma: A Focus on Spinal Cord Injury.

Edema after spinal cord injury (SCI) is one of the first observations after the primary injury and lasts for few days after trauma. It has serious consequences on the affected tissue and can aggravate the initial devastating condition. To date, the mechanisms of the water content increase after SCI are not fully understood. Edema formation results in a combination of interdependent factors related to mechanical damage after the initial trauma progressing, along with the subacute and acute phases of the secondary lesion. These factors include mechanical disruption and subsequent inflammatory permeabilization of the blood spinal cord barrier, increase in the capillary permeability, deregulation in the hydrostatic pressure, electrolyte-imbalanced membranes and water uptake in the cells. Previous research has attempted to characterize edema formation by focusing mainly on brain swelling. The purpose of this review is to summarize the current understanding of the differences in edema formation in the spinal cord and brain, and to highlight the importance of elucidating the specific mechanisms of edema formation after SCI. Additionally, it outlines findings on the spatiotemporal evolution of edema after spinal cord lesion and provides a general overview of prospective treatment strategies by focusing on insights to prevent edema formation after SCI.

KIF13B mediates VEGFR2 recycling to modulate vascular permeability.

Excessive vascular endothelial growth factor-A (VEGF-A) signaling induces vascular leakage and angiogenesis in diseases. VEGFR2 trafficking to the cell surface, mediated by kinesin-3 family protein KIF13B, is essential to respond to VEGF-A when inducing angiogenesis. However, the precise mechanism of how KIF13B regulates VEGF-induced signaling and its effects on endothelial permeability is largely unknown. Here we show that KIF13B-mediated recycling of internalized VEGFR2 through Rab11-positive recycling vesicle regulates endothelial permeability. Phosphorylated VEGFR2 at the cell-cell junction was internalized and associated with KIF13B in Rab5-positive early endosomes. KIF13B mediated VEGFR2 recycling through Rab11-positive recycling vesicle. Inhibition of the function of KIF13B attenuated phosphorylation of VEGFR2 at Y951, SRC at Y416, and VE-cadherin at Y685, which are necessary for endothelial permeability. Failure of VEGFR2 trafficking to the cell surface induced accumulation and degradation of VEGFR2 in lysosomes. Furthermore, in the animal model of the blinding eye disease wet age-related macular degeneration (AMD), inhibition of KIF13B-mediated VEGFR2 trafficking also mitigated vascular leakage. Thus, the present results identify the fundamental role of VEGFR2 recycling to the cell surface in mediating vascular permeability, which suggests a promising strategy for mitigating vascular leakage associated with inflammatory diseases.

Pericytes protect rats and mice from sepsis-induced injuries by maintaining vascular reactivity and barrier function: implication of miRNAs and microvesicles.

BACKGROUND: Vascular hyporeactivity and leakage are key pathophysiologic features that produce multi-organ damage upon sepsis. We hypothesized that pericytes, a group of pluripotent cells that maintain vascular integrity and tension, are protective against sepsis via regulating vascular reactivity and permeability. METHODS: We conducted a series of in vivo experiments using wild-type (WT), platelet-derived growth factor receptor beta (PDGFR-ß)-Cre + mT/mG transgenic mice and Tie2-Cre + Cx43flox/flox mice to examine the relative contribution of pericytes in sepsis, either induced by cecal ligation and puncture (CLP) or lipopolysaccharide (LPS) challenge. In a separate set of experiments with Sprague-Dawley (SD) rats, pericytes were depleted using CP-673451, a selective PDGFR-ß inhibitor, at a dosage of 40 mg/(kg·d) for 7 consecutive days. Cultured pericytes, vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were used for mechanistic investigations. The effects of pericytes and pericyte-derived microvesicles (PCMVs) and candidate miRNAs on vascular reactivity and barrier function were also examined. RESULTS: CLP and LPS induced severe injury/loss of pericytes, vascular hyporeactivity and leakage (P < 0.05). Transplantation with exogenous pericytes protected vascular reactivity and barrier function via microvessel colonization (P < 0.05). Cx43 knockout in either pericytes or VECs reduced pericyte colonization in microvessels (P < 0.05). Additionally, PCMVs transferred miR-145 and miR-132 to VSMCs and VECs, respectively, exerting a protective effect on vascular reactivity and barrier function after sepsis (P < 0.05). miR-145 primarily improved the contractile response of VSMCs by activating the sphingosine kinase 2 (Sphk2)/sphingosine-1-phosphate receptor (S1PR)1/phosphorylation of myosin light chain 20 pathway, whereas miR-132 effectively improved the barrier function of VECs by activating the Sphk2/S1PR2/zonula occludens-1 and vascular endothelial-cadherin pathways. CONCLUSIONS: Pericytes are protective against sepsis through regulating vascular reactivity and barrier function. Possible mechanisms include both direct colonization of microvasculature and secretion of PCMVs.

CD93 maintains endothelial barrier function by limiting the phosphorylation and turnover of VE-cadherin.

Regulation of vascular permeability to plasma is essential for tissue and organ homeostasis and is mediated by endothelial cell-to-cell junctions that tightly regulate the trafficking of molecules between blood and tissue. The single-pass transmembrane glycoprotein CD93 is upregulated in endothelial cells during angiogenesis and controls cytoskeletal dynamics. However, its role in maintaining homeostasis by regulating endothelial barrier function has not been elucidated yet. Here, we demonstrate that CD93 interacts with vascular endothelial (VE)-cadherin and limits its phosphorylation and turnover. CD93 deficiency in vitro and in vivo induces phosphorylation of VE-cadherin under basal conditions, displacing it from endothelial cell-cell contacts. Consistent with this, endothelial junctions are defective in CD93-/- mice, and the blood-brain barrier permeability is enhanced. Mechanistically, CD93 regulates VE-cadherin phosphorylation and turnover at endothelial junctions through the Rho/Rho kinase-dependent pathway. In conclusion, our results identify CD93 as a key regulator of VE-cadherin stability at endothelial junctions, opening up possibilities for therapeutic strategies directed to control vascular permeability.

The role of PLVAP in endothelial cells.

Endothelial cells play a major part in the regulation of vascular permeability and angiogenesis. According to their duty to fit the needs of the underlying tissue, endothelial cells developed different subtypes with specific endothelial microdomains as caveolae, fenestrae and transendothelial channels which regulate nutrient exchange, leukocyte migration, and permeability. These microdomains can exhibit diaphragms that are formed by the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), the only known protein component of these diaphragms. Several studies displayed an involvement of PLVAP in diseases as cancer, traumatic spinal cord injury, acute ischemic brain disease, transplant glomerulopathy, Norrie disease and diabetic retinopathy. Besides an upregulation of PLVAP expression within these diseases, pro-angiogenic or pro-inflammatory responses were observed. On the other hand, loss of PLVAP in knockout mice leads to premature mortality due to disrupted homeostasis. Generally, PLVAP is considered as a major factor influencing the permeability of endothelial cells and, finally, to be involved in the regulation of vascular permeability. Following these observations, PLVAP is debated as a novel therapeutic target with respect to the different vascular beds and tissues. In this review, we highlight the structure and functions of PLVAP in different endothelial types in health and disease.

Dialogue between VE-Cadherin and Sphingosine 1 Phosphate Receptor1 (S1PR1) for Protecting Endothelial Functions.

The endothelial cells (EC) of established blood vessels in adults remain extraordinarily quiescent in the sense that they are not actively proliferating, but they fulfill the necessary role to control the permeability of their monolayer that lines the interior of blood vessels. The cell-cell junctions between ECs in the endothelium comprise tight junctions and adherens homotypic junctions, which are ubiquitous along the vascular tree. Adherens junctions are adhesive intercellular contacts that are crucial for the organization of the EC monolayer and its maintenance and regulation of normal microvascular function. The molecular components and underlying signaling pathways that control the association of adherens junctions have been described in the last few years. In contrast, the role that dysfunction of these adherens junctions has in contributing to human vascular disease remains an important open issue. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator found at high concentrations in blood which has important roles in the control of the vascular permeability, cell recruitment, and clotting that follow inflammatory processes. This role of S1P is achieved through a signaling pathway mediated through a family of G protein-coupled receptors designated as S1PR1. This review highlights novel evidence for a direct linkage between S1PR1 signaling and the mediation of EC cohesive properties that are controlled by VE-cadherin.

Cancer-associated fibroblasts facilitate premetastatic niche formation through lncRNA SNHG5-mediated angiogenesis and vascular permeability in breast cancer.

Background: Metastasis is the leading cause of death in patients with breast cancer (BC). Primary tumors create a premetastatic niche (PMN) in secondary organs for subsequent metastases. Cancer-associated fibroblasts (CAFs) are a predominant stromal component in the tumor microenvironment and serve as a major contributor to tumor metastasis. However, the function and mechanism of primary CAFs in the premetastatic niche of secondary organs remain unclear in BC. Methods: We investigated the expression profiles of lncRNAs in pairs of CAFs and NFs derived from breast tumor tissues using lncRNA microarray. The expression levels of lncSNHG5, ZNF281, IGF2BP2, CCL2 and CCL5 were assessed by qRT-PCR; the protein levels of related genes (e.g., ZNF281, IGF2BP2, CCL2, and CCL5) were analyzed using western blotting and/or ELISA in primary and immortalized CAFs and clinical samples. Tubule formation and three-dimensional sprouting assays and tissue fluorescence staining were conducted to investigate angiogenesis. In vitro permeability assays, trans-endothelial invasion assays, in vivo permeability assays and tissue fluorescence staining were conducted to examine vascular permeability. The regulatory mechanism of lncSNHG5 was investigated by RNA sequencing, fluorescent in situ hybridization, cellular fractionation assay, mass spectrometry, RNA pull-down, RNA immunoprecipitation, gene-specific m6A assay, chromatin immunoprecipitation, dual luciferase reporter assay and actinomycin D treatment in CAFs and NFs. Results: LncSNHG5 was highly expressed in breast CAFs and played an essential role in premetastatic niche formation by promoting angiogenesis and vascular leakiness through regulation of ZNF281 in CAFs. lncSNHG5 enhanced ZNF281 mRNA stability by binding with the m6A reader IGF2BP2. Enhanced ZNF281 transcriptionally regulated CCL2 and CCL5 expression to activate P38 MAPK signaling in endothelial cells. High CCL2 and CCL5 expression was associated with tumor metastasis and poor prognosis in BC patients. The inhibitors RS102895, marasviroc and cenicriviroc inhibited angiogenesis and vascular permeability in the PMN by blocking the binding of CCL2/CCR2 and CCL5/CCR5. The lncSNHG5-ZNF281-CCL2/CCL5 signaling axis plays an essential role in inducing premetastatic niche formation to promote BC metastasis. Conclusions: Our work demonstrates that lncSNHG5 and its downstream signaling ZNF281-CCL2/CCL5 in CAFs play a crucial role in premetastatic niche formation in breast cancer and may serve as potential targets for the diagnosis and treatment of BC metastasis.

Targeting VEGF-A/VEGFR2 Y949 Signaling-Mediated Vascular Permeability Alleviates Hypoxic Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is associated with increased expression of VEGF-A (vascular endothelial growth factor A) and its receptor, VEGFR2 (vascular endothelial growth factor 2), but whether and how activation of VEGF-A signal participates in the pathogenesis of PH is unclear. METHODS: VEGF-A/VEGFR2 signal activation and VEGFR2 Y949-dependent vascular leak were investigated in lung samples from patients with PH and mice exposed to hypoxia. To study their mechanistic roles in hypoxic PH, we examined right ventricle systolic pressure, right ventricular hypertrophy, and pulmonary vasculopathy in mutant mice carrying knock-in of phenylalanine that replaced the tyrosine at residual 949 of VEGFR2 (Vefgr2Y949F) and mice with conditional endothelial deletion of Vegfr2 after chronic hypoxia exposure. RESULTS: We show that PH leads to excessive pulmonary vascular leak in both patients and hypoxic mice, and this is because of an overactivated VEGF-A/VEGFR2 Y949 signaling axis. In the context of hypoxic PH, activation of Yes1 and c-Src and subsequent VE-cadherin phosphorylation in endothelial cells are involved in VEGFR2 Y949-induced vascular permeability. Abolishing VEGFR2 Y949 signaling by Vefgr2Y949F point mutation was sufficient to prevent pulmonary vascular permeability and inhibit macrophage infiltration and Rac1 activation in smooth muscle cells under hypoxia exposure, thereby leading to alleviated PH manifestations, including muscularization of distal pulmonary arterioles, elevated right ventricle systolic pressure, and right ventricular hypertrophy. It is important that we found that VEGFR2 Y949 signaling in myeloid cells including macrophages was trivial and dispensable for hypoxia-induced vascular abnormalities and PH. In contrast with selective blockage of VEGFR2 Y949 signaling, disruption of the entire VEGFR2 signaling by conditional endothelial deletion of Vegfr2 promotes the development of PH. CONCLUSIONS: Our results support the notion that VEGF-A/VEGFR2 Y949-dependent vascular permeability is an important determinant in the pathogenesis of PH and might serve as an attractive therapeutic target pathway for this disease.

The role of protein kinase C in diabetic microvascular complications.

Protein kinase C (PKC) is a family of serine/threonine protein kinases, the activation of which plays an important role in the development of diabetic microvascular complications. The activation of PKC under high-glucose conditions stimulates redox reactions and leads to an accumulation of redox stress. As a result, various types of cells in the microvasculature are influenced, leading to changes in blood flow, microvascular permeability, extracellular matrix accumulation, basement thickening and angiogenesis. Structural and functional disorders further exacerbate diabetic microvascular complications. Here, we review the roles of PKC in the development of diabetic microvascular complications, presenting evidence from experiments and clinical trials.

PLCß2 Promotes VEGF-Induced Vascular Permeability.

BACKGROUND: Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need. METHODS: In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) ß2. RESULTS: Global knock-out of PLCß2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCß2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCß2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCß2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls. CONCLUSIONS: The results implicate PLCß2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCß2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.