VEGF-induced Hyperpermeability Research Articles

Abstract 18731: Connexin 43 Hemichannels Contribution to Ischemia-Reperfusion-Induced Hyperpermeability

Introduction: Ischemia-reperfusion (I-R) injury is a significant complication in vascular surgery or organ transplantation. I-R injury involves intracellular Ca2+ overload, nitric oxide (NO), oxidative stress. I-R injury increases endothelial vascular hyperpermeability leading to tissue edema and damage mediated by endothelial Ca2+ increase. The role of Ca2+ permeable channels in I-R is not defined. Connexin (Cx) proteins form hemichannels, allowing the exchange of molecules between intracellular and extracellular spaces. The Cx43 isoform is highly expressed in endothelial cells (EC). Endothelial Ca2+ increase promotes NO-dependent Cx43 S-nitrosylation. However, whether Cx43 hemichannels are involved in I-R-induced hyperpermeability is unknown. Hypothesis: VEGF-induced hyperpermeability causes increased NO-mediated Cx43 S-nitrosylation and opening of endothelial Cx43 hemichannels, leading to Ca2+ entry and exacerbating reperfusion damage. Methods: We used VEGF as I-R related pro-inflammatory agonist. Using primary cultures of mesenteric endothelial cells, we tested Cx43 hemichannel activity by ethidium uptake and Ca2+ influx with Fluo-4 indicator. We evaluated hyperpermeability in vitro and in vivo Results: Results indicate that VEGF stimulation increased hemichannel activity in EC, associated with a rapid rise of Ca2+ influx. Both hemichannel activation and Ca2+ increase were abolished by treatment with specific Cx43 blocker (Gap19) or blocking NO production with NG-monomethyl-L-arginine (LNMMA). Cx43 inhibition blunted VEGF-induced hyperpermeability both in vitro in primary EC culture and in vivo mouse mesenteric vascular bed. To further understand the participation of NO-mediated S-nitrosylation in the VEGF-induced hyperpermeability, we replaced cysteine 271 by serine to create a Cx43C271S mouse that fails to cause S-nitrosylation of Cx43. We demonstrated that this knockin mouse exhibits a decrease in VEGF-stimulated hyperpermeability in vivo. Conclusions: These results indicate that Cx43 hemichannels activation is critical to VEGF (I-R inflammatory environment)-induced hyperpermeability and the S-nitrosylated Cx43 is a key regulator in I-R damage. AHA support: # 932684 and #23DIVSUP1054931

Dissecting VEGF-Induced Acute Versus Chronic Vascular Hyperpermeability: Essential Roles of Dimethylarginine Dimethylaminohydrolase-1

Abnormal vascular leakiness plays crucial roles in several pathogenesis including cancer, macular degeneration, cardiovascular diseases and even in COVID-19-related pulmonary edema. Vascular endothelial cell growth factor (VEGF), also well-known as vascular permeability factor (VPF), activates VEGF receptor 2 (VEGFR2) and its downstream signaling leading to both acute and chronic vascular permeability. While many key downstream components are identified in cultured cells and murine models upon acute VEGF administration, the key molecular mediators of VEGF-induced chronic permeability remain unclear due to lack of appropriate in experimental model. Here we demonstrated that chronic VEGF exposure induces unique pattern of signaling as compared to that of acute exposure, including phosphorylation of VEGFR2, c-Src, and steady increases of nitric oxide (NO). Utilizing heat-inducible VEGF transgenic zebrafish (Danio rerio) and establishing an algorithm incorporating segmentation techniques to precisely quantify the extravasated dye, we monitored acute and chronic VEGF-induced vascular hyperpermeability in real time. Importantly, knockdown of dimethylarginine dimethylaminohydrolase-1 (DDAH1), an enzyme essential for NO generation, significantly altered downstream signaling pathways due to both acute and chronic VEGF exposure in human endothelial cells and the VEGF-inducible zebrafish permeability model. Finally, the effect of DDAH1 on VEGF-induced hyperpermeability was confirmed using DDAH1 transgenic mice as well as DDAH1 knockout mice by Miles assays, which exhibited increased and decreased leakage of Evan’s blue dye, respectively. Taken together, our data reveal novel divergent signaling of VEGF-induced acute versus chronic hyperpermeability and identify DDAH1 as a critical player and potentially a therapeutic target of vascular hyperpermeability-mediated pathogenesis.

Primary tumor-secreted VEGF induces vascular hyperpermeability in premetastatic lung via the occludin phosphorylation/ubiquitination pathway.

Primary tumor can induce the formation of premetastatic niche. The hyperpermeability of the vessels in the premetastatic niche is the first step in the development of metastasis. However, the cellular and molecular mechanisms of vascular hyperpermeability remain to be elucidated. In this study, 4T1 breast cells were injected into the breasts of mice to establish a tumor model. Our results showed that primary tumors induced hyperpermeability of the vessels in the premetastatic lung. Subsequent studies showed that the level of vascular endothelial growth factor (VEGF) was elevated in the tumor-bearing mice serum and the levels of tight junction (TJ) proteins occludin and ZO-1 were decreased in the premetastatic lung. In vitro studies demonstrated that VEGF increased the permeability of dextran and decreased the levels of occludin and ZO-1 in human umbilical vein endothelial cells. Moreover, the hyperpermeability of vessels and the degradation of occludin was blocked by bevacizumab. Overexpression of occludin alleviated the VEGF-induced hyperpermeability. Further investigations revealed that VEGF-induced occludin phosphorylation at Ser-490 and ubiquitination. Finally, we showed that VEGF accelerated the process of occludin degradation through the ubiquitin-proteasome system. In conclusion, primary tumor-secrete VEGF induce the occludin phosphorylation/ubiquitination and downregulation, resulting in the disruption of TJs and hyperpermeability of vessels in premetastatic lung. The occludin phosphorylation/ubiquitination pathway may be the mechanism of VEGF-induced vascular hyperpermeability in the lung premetastatic niche.

Rap1B promotes VEGF-induced endothelial permeability and is required for dynamic regulation of the endothelial barrier.

Vascular endothelial growth factor (VEGF), a key angiogenic and permeability factor, plays an important role in new blood vessel formation. However, abnormal VEGF-induced VEGFR2 signaling leads to hyperpermeability. We have shown previously that Rap1, best known for promoting cell adhesion and vessel stability, is a critical regulator of VEGFR2-mediated angiogenic and shear-stress EC responses. To determine the role of Rap1 role in endothelial barrier dynamics, we examined vascular permeability in EC-specific Rap1A- and Rap1B-knockout mice, cell-cell junction remodeling and EC monolayer resistivity in Rap1-deficient ECs under basal, inflammatory or elevated VEGF conditions. Deletion of either Rap1 isoform impaired de novo adherens junction (AJ) formation and recovery from LPS-induced barrier disruption in vivo However, only Rap1A deficiency increased permeability in ECs and lung vessels. Interestingly, Rap1B deficiency attenuated VEGF-induced permeability in vivo and AJ remodeling in vitro Therefore, only Rap1A is required for the maintenance of normal vascular integrity. Importantly, Rap1B is the primary isoform essential for normal VEGF-induced EC barrier dissolution. Deletion of either Rap1 isoform protected against hyper permeability in the STZ-induced diabetes model, suggesting clinical implications for targeting Rap1 in pathologies with VEGF-induced hyperpermeability.

Angiopoietin-1 Induces Krüppel-like Factor 2 Expression through a Phosphoinositide 3-Kinase/AKT-dependent Activation of Myocyte Enhancer Factor 2

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group have recently shown that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts and further demonstrated that the former selectively induces expression of Krüppel-like factor 2 (KLF2), a transcription factor involved in vascular quiescence. Here, we investigated the mechanism of how Ang1/Tie2 signal induces KLF2 expression to clarify the role of KLF2 in Ang1/Tie2 signal-mediated vascular quiescence. Ang1 stimulated KLF2 promoter-driven reporter gene expression in endothelial cells, whereas it failed when a myocyte enhancer factor 2 (MEF2)-binding site of KLF2 promoter was mutated. Depletion of MEF2 by siRNAs abolished Ang1-induced KLF2 expression, indicating the requirement of MEF2 in KLF2 induction by Ang1. Constitutive active phosphoinositide 3-kinase (PI3K) and AKT increased the MEF2-dependent reporter gene expression by enhancing its transcriptional activity and stimulated the KLF2 promoter activity cooperatively with MEF2. Consistently, inhibition of either PI3K or AKT and depletion of AKT abrogated Ang1-induced KLF2 expression. In addition, we confirmed the dispensability of extracellular signal-regulated kinase 5 (ERK5) for Ang1-induced KLF2 expression. Furthermore, depletion of KLF2 resulted in the loss of the inhibitory effect of Ang1 on vascular endothelial growth factor (VEGF)-mediated expression of vascular cell adhesion molecule-1 in endothelial cells and VEGF-mediated monocyte adhesion to endothelial cells. Collectively, these findings indicate that Ang1/Tie2 signal stimulates transcriptional activity of MEF2 through a PI3K/AKT pathway to induce KLF2 expression, which may counteract VEGF-mediated inflammatory responses.

Distinct signaling pathways confer different vascular responses to VEGF 121 and VEGF 165

Vascular endothelial growth factor (VEGF) is a pleiotropic factor that regulates embryonal vasculogenesis, tumor angiogenesis and vascular permeability. Among eight differential isoforms, VEGF 121 mainly regulates vascular permeability, while VEGF 165 induces angiogenesis. Our previous studies have suggested that VEGF 121 and VEGF 165 are mainly detected in the lesions of psoriasis and atopic dermatitis, especially VEGF 121. VEGF 121 is the most predominant isoform, which plays a major role in the increased vascular permeability in the aforementioned skin lesions. Thus, the differential expression of VEGF isoforms may be critical in determining either an angiogenic or a hyper-permeable state. However, the distinct VEGF signaling pathways that induce angiogenesis and vascular hyper-permeability in endothelial cells have never been demonstrated. To clarify the differential effects elicited by VEGF 121 and VEGF 165, we compared the biological responses and the signaling pathways activated by VEGF 121 and VEGF 165 in human umbilical vein endothelial cells (HUVEC). VEGF 165 significantly increased the level of phosphorylation in the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, whereas VEGF 121 had little to no effect. In contrast, VEGF 121 induced rapid activation of the Src pathway, while VEGF 165 showed a less pronounced and delayed activation of the Src pathway. Furthermore, the VEGF-induced hyper-permeability and cell proliferation of HUVEC were inhibited by a Src inhibitor (PP2) and a MEK inhibitor (PD98059), respectively. These results indicate that distinct signaling pathways confer different vascular responses to VEGF 121 and VEGF 165.

Rho and ROCK Signaling in VEGF‐Induced Microvascular Endothelial Hyperpermeability

Vascular endothelial growth factor (VEGF) plays an important role in the regulation of microvascular permeability under various physiological and pathological conditions. The authors tested the hypothesis that the small GTPase Rho and its downstream effector ROCK (Rho-associated coiled-coil-containing protein kinase) mediate VEGF-induced increases in venular permeability. They also investigated myosin light chain (MLC) phosphorylation and actin polymerization, two well-characterized targets of the Rho-ROCK pathway that are implicated in the regulation of endothelial barrier function. The apparent permeability coefficient of albumin (P(a)) was measured in intact isolated porcine coronary venules and in cultured coronary venular endothelial cell (CVEC) monolayers. RhoA activation was determined using a Rhotekin-agarose pull down assay. MLC phosphorylation was evaluated by immunoblotting with phospho-specific antibodies, and endothelial cellular F-actin was viewed using fluorescence microscopy. VEGF increased P(a) in both isolated coronary venules and CVEC monolayers. The hyperpermeability response occurred in a similar time course to that of Rho activation, MLC phosphorylation, and actin stress fiber formation. Selective blockage of ROCK with Y27632 dose-dependently inhibited VEGF-induced venular hyperpermeability. Moreover, inhibition of either Rho with exoenzyme C3 or ROCK with Y-27632 attenuated VEGF-induced increases in permeability, MLC phosphorylation, and actin-stress fiber formation in CVEC monolayers. Collectively, these findings suggest that the Rho-ROCK signal pathway contributes to VEGF-induced hyperpermeability. Myosin light-chain phosphorylation and actin stress fiber formation occur concomitantly with the increase in permeability upon VEGF stimulation.

Structural Mechanisms in the Abolishment of VEGF-induced Microvascular Hyperpermeability by cAMP

To investigate the structural mechanisms by which elevation of the intraendothelial cAMP levels abolishes or attenuates the transient increase in microvascular permeability by vascular endothelial growth factor (VEGF), we examined cAMP effect on VEGF-induced hyperpermeability to small solute sodium fluorescein (Stokes radius = 0.45 nm) P(sodium fluorescein), intermediate-sized solute alpha-lactalbumin (Stokes radius = 2.01 nm) P(alpha-lactalbumin), and large solute albumin (BSA, Stokes radius = 3.5 nm) P(BSA) on individually perfused microvessels of frog mesenteries. After 20 min pretreatment of 2 mM cAMP analog, 8-bromo-cAMP, the initial increase by 1 nM VEGF was completely abolished in P(sodium fluorescein) (from a peak increase of 2.6+/-0.37 times control with VEGF alone to 0.96+/-0.07 times control with VEGF and cAMP), in P(alpha-lactalbumin) (from a peak increase of 2.7+/-0.33 times control with VEGF alone to 0.76+/-0.07 times control with VEGF and cAMP), and in P(BSA) (from a peak increase of 6.5+/-1.0 times control with VEGF alone to 0.97+/-0.08 times control with VEGF and cAMP). Based on these measured data, the prediction from our mathematical models suggested that the increase in the number of tight junction strands in the cleft between endothelial cells forming the microvessel wall is one of the mechanisms for the abolishment of VEGF-induced hyperpermeability by cAMP.

Vascular endothelial growth factor stimulates differential signaling pathways in in vivo microcirculation.

Vascular endothelial growth factor (VEGF) induces mild vasodilation and strong increases in microvascular permeability. Using intravital microscopy and digital integrated optical intensity image analysis, we tested, in the hamster cheek pouch microcirculation, the hypothesis that differential signaling pathways in arterioles and venules represent an in vivo regulatory mechanism in the control of vascular diameter and permeability. The experimental design involved blocking specific signaling molecules and simultaneously assessing VEGF-induced changes in arteriolar diameter and microvascular transport of FITC-Dextran 150. Inhibition of Akt [indirectly via phosphatidylinositol 3-kinase with LY-294002 or wortmannin] or PKC (with bisindolylmaleimide) reduced VEGF-induced hyperpermeability. However, phosphatidylinositol 3-kinase/Akt inhibition enhanced the early phase and attenuated the late phase of VEGF-induced vasodilation, whereas blocking PKC had no effect. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 (with PD-98059 or AG-126) also reduced VEGF-induced hyperpermeability but did not block VEGF-induced vasodilation. Blockade of endothelial nitric oxide synthase (with N(omega)-monomethyl-l-arginine) inhibited VEGF-induced changes in both permeability and diameter. Furthermore, immunofluorescence studies with human umbilical vein endothelial cells revealed that bisindolylmaleimide, PD-98059, and l-NMMA attenuate VEGF-induced reorganization of vascular endothelial cadherin. Our data demonstrate that 1) endothelial nitric oxide synthase is a common convergence pathway for VEGF-induced changes in arteriolar diameter and microvascular permeability; 2) PKC and ERK-1/2 do not play a major role in VEGF-induced vasodilation in the hamster cheek pouch microcirculation; and 3) Akt, PKC, and ERK-1/2 are elements of the signaling cascade that regulates VEGF-stimulated microvascular hyperpermeability. Our data provide evidence for differential signaling as a regulatory step in VEGF-stimulated microvascular dynamics.

Simultaneous activation of several second messengers in hypoxia‐induced hyperpermeability of brain derived endothelial cells

In vivo, ischemia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Hypoxia-induced vascular endothelial growth factor (VEGF) has been shown to be a key regulator of these permeability changes. However, the signaling pathways that underlie VEGF-induced hyperpermeability are incompletely understood. In this study, we demonstrate that hypoxia- and VEGF-induced permeability changes depend on activation of phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), and protein kinase G (PKG). Inhibition of mitogen-activated protein kinases (MAPK) and of the protein kinase C (PKC) did not affect permeability at all. Paralleling hypoxia- and VEGF-induced permeability changes, localization of the tight junction proteins occludin, zonula occludens-1 (ZO-1), and ZO-2 along the cell membrane changed from a continuous to a more discontinuous expression pattern during hypoxia. In particular, localization of ZO-1 and ZO-2 expression moved from the cell membrane to the cytoplasm and nucleus whereas occludin expression remained at the cell membrane. Inhibition of PLCgamma, PI3-kinase, and PKG abolished these hypoxia-induced changes. These findings demonstrate that hypoxia and VEGF induce permeability through rearrangement of endothelial junctional proteins which involves activation of the PLCgamma and PI3-K/AKT pathway leading to the activation of PKG.

VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide.

We tested the hypothesis that VEGF regulates endothelial hyperpermeability to macromolecules by activating the ERK-1/2 MAPK pathway. We also tested whether PKC and nitric oxide (NO) mediate VEGF-induced increases in permeability via the ERK-1/2 pathway. FITC-Dextran 70 flux across human umbilical vein endothelial cell monolayers served as an index of permeability, whereas Western blots assessed the phosphorylation of ERK-1/2. VEGF-induced hyperpermeability was inhibited by antisense DNA oligonucleotides directed against ERK-1/2 and by blockade of MEK and Raf-1 activities (20 microM PD-98059 and 5 microM GW-5074). These blocking agents also reduced ERK-1/2 phosphorylation. The PKC inhibitor bisindolylmaleimide I (10 microM) blocked both VEGF-induced ERK-1/2 activation and hyperpermeability. The NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (200 microM) and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidiazoline-1-oxyl-3-oxide (100 microM) abolished VEGF-induced hyperpermeability but did not block ERK-1/2 phosphorylation. These observations demonstrate VEGF-induced hyperpermeability involves activation of PKC and NOS as well as Raf-1, MEK, and ERK-1/2. Furthermore, our data suggest that ERK-1/2 and NOS are elements of different signaling pathways in VEGF-induced hyperpermeability.

Significance of Nitric Oxide and Peroxynitrite in Permeability Changes of the Retinal Microvascular Endothelial Cell Monolayer Induced by Vascular Endothelial Growth Factor

Reactive oxygen species (ROS) play an important role in signaling pathways stimulated by growth factors in vascular cells. We investigated whether vascular endothelial growth factor (VEGF), which is upregulated in diabetic retinopathy and atherosclerosis, is able to enhance production of ROS, and if so, whether ROS modulate endothelial permeability. ROS levels in bovine retinal microvascular endothelial cells (BMEC) were measured by the oxidation of 2′,7′-dichlorodihydrofluorescein (DCHF), and permeability was examined by monitoring the passage of albumin through BMEC monolayers. VEGF stimulated oxidation of DCHF in BMEC, an effect which was inhibited by superoxide dismutase (SOD) and the nitric oxide (NO) synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME), but not by D-NAME. Urate, a scavenger of peroxynitrite, attenuated the VEGF-induced oxidation of DCHF. VEGF elicited a significant increase in the macromolecule permeability of BMEC monolayers within 30 min. SOD did not modify the basal or the VEGF-stimulated hyperpermeability, but the combination of SOD and VEGF induced a transient reduction in permeability after 10 min. L-NAME, but not D-NAME, enhanced VEGF-induced hyperpermeability without affecting basal values. Urate did not modify the VEGF-induced changes in permeability. In conclusion, VEGF stimulates oxidation of DCHF, which most likely represents peroxynitrite formation, and induces an increase in permeability of BMEC monolayers. Activation of NO synthase seems to counteract this stimulatory effect of VEGF on endothelial permeability.

VEGF induces hyperpermeability by a direct action on endothelial cells.

Vascular endothelial growth factor (VEGF) is a key regulator of vasculo- and angiogenesis. Earlier studies demonstrated a permeability-increasing effect of VEGF in skin tests, leading to its other name, vascular permeability factor. We wondered whether VEGF-induced hyperpermeability was a direct effect of VEGF on endothelial cells and studied the permeability of human and porcine endothelial cell monolayers in a well-characterized in vitro system. VEGF increased the hydraulic conductivity up to 20-fold and simultaneously decreased the albumin reflection coefficient. This effect occurred after a delay of 150 min, although VEGF-induced early endothelial cell activation was verified by enhanced inositol phosphate accumulation within 5 min and increased P-selectin expression within 15 min. Platelet-derived growth factor and granulocyte-macrophage colony-stimulating factor, two endothelial cell nonspecific mitogens, also stimulated phosphatidylinositol metabolism and P-selectin expression; however, they had no effect on endothelial permeability. The increase in intracellular cyclic nucleotide levels of human endothelial monolayers abolished VEGF-induced endothelial hyperpermeability. In summary, VEGF increased endothelial permeability by a direct action on endothelial cells. Based on the pattern of endothelial cell activation by growth factors, VEGF appears to be a unique stimulus.