Assessment of Microvessel Permeability in Murine Atherosclerotic Vein Grafts Using Two-Photon Intravital Microscopy

Plaque angiogenesis and plaque hemorrhage are major players in the destabilization and rupture of atherosclerotic lesions. As these are dynamic processes, imaging of plaque angiogenesis, especially the integrity or leakiness of angiogenic vessels, can be an extremely useful tool in the studies on atherosclerosis pathophysiology. Visualizing plaque microvessels in 3D would enable us to study the architecture and permeability of adventitial and intimal plaque microvessels in advanced atherosclerotic lesions. We hypothesized that a comparison of the vascular permeability between healthy continuous and fenestrated as well as diseased leaky microvessels, would allow us to evaluate plaque microvessel leakiness. We developed and validated a two photon intravital microscopy (2P-IVM) method to assess the leakiness of plaque microvessels in murine atherosclerosis-prone ApoE3*Leiden vein grafts based on the quantification of fluorescent-dextrans extravasation in real-time. We describe a novel 2P-IVM set up to study vessels in the neck region of living mice. We show that microvessels in vein graft lesions are in their pathological state more permeable in comparison with healthy continuous and fenestrated microvessels. This 2P-IVM method is a promising approach to assess plaque angiogenesis and leakiness. Moreover, this method is an important advancement to validate therapeutic angiogenic interventions in preclinical atherosclerosis models.

Changes in endothelial connexin 43 expression inversely correlate with microvessel permeability and VE-cadherin expression in endotoxin-challenged lungs

Endothelial barrier restoration reverses microvessel hyperpermeability and facilitates recovery from lung injury. Because inhibiting connexin 43 (Cx43)-dependent interendothelial communication blunts hyperpermeability in single microvessels, we determined whether endothelial Cx43 levels correlate with changes in microvessel permeability during recovery from lung injury. Toward this, bacterial endotoxin was instilled intratracheally into rat lungs, and at different durations postinstillation the lungs were isolated and blood perfused. Microvessel Cx43 expression was quantified by in situ immunofluorescence and microvessel permeability via a fluorescence method. To supplement the immunofluorescence data, protein levels were determined by immunoblots of lung tissue from endotoxin-instilled rats. Immunofluorescence and immunoblot together revealed that both Cx43 expression and microvessel permeability increased above baseline within a few hours after endotoxin instillation but declined progressively over the next few days. On day 5 postendotoxin, microvessel Cx43 declined to negligible levels, resulting in complete absence of intermicrovessel communication determined by photolytic uncaging of Ca2+. However, by day 14, both Cx43 expression and microvessel permeability returned to baseline levels. In contrast to Cx43, expression of microvessel vascular endothelial (VE)-cadherin, a critical determinant of vascular barrier integrity, exhibited an inverse trend by initially declining below baseline and then returning to baseline at a longer duration. Knockdown of vascular Cx43 by tail vein injection of Cx43 shRNA increased VE-cadherin expression, suggesting that reduction in Cx43 levels may modulate VE-cadherin levels in lung microvessels. Together, the data suggest that endotoxin challenge initiates interrelated changes in microvessel Cx43, VE-cadherin, and microvessel permeability, with changes in Cx43 temporally leading the other responses.

Enhanced permeability responses to inflammation in streptozotocin-induced diabetic rat venules: Rho-mediated alterations of actin cytoskeleton and VE-cadherin

Diabetes is a progressive disease that often leads to microvascular complications. This study investigates the impact of diabetes on microvessel permeability under basal and inflammatory conditions. Streptozotocin-induced diabetic rats were used to mimic type 1 diabetes. Parallel experiments were conducted in intact mesenteric venules in normal rats and diabetic rats experiencing hyperglycemia for 2–3 wk. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). The correlated changes in endothelial intracellular Ca2+ concentration ([Ca2+]i), adherens junctions, and cytoskeleton F-actin were examined with fluorescence imaging. Diabetic vessels showed moderately increased basal Lp, but upon platelet-activating factor (PAF) exposure, these vessels showed an ∼10-fold higher Lp increase than the normal vessels. Concomitantly, we observed higher increases in endothelial [Ca2+]i, enhanced stress fiber formation, vascular endothelial-cadherin separation, and larger gap formation between endothelial cells than those occurring in normal vessels. PAF receptor staining showed no significant difference between normal and diabetic vessels. The application of Rho kinase inhibitor Y27632 did not affect PAF-induced increases in endothelial [Ca2+]i but significantly reduced PAF-induced Lp increases by 90% in diabetic vessels. The application of both Y27632 and nitric oxide (NO) synthase inhibitor attenuated PAF-induced Lp increases more than using one inhibitor alone. Our studies indicate that diabetic conditions prime endothelial cells into a phenotype with increased susceptibility to inflammation without altering receptor expression and that the increased Rho activation and NO production play important roles in exaggerated permeability increases when diabetic vessels were exposed to inflammatory mediators, which may account for the exacerbated vascular dysfunction when diabetic patients are exposed to additional inflammation.

Caveolin-1 scaffolding domain promotes leukocyte adhesion by reduced basal endothelial nitric oxide-mediated ICAM-1 phosphorylation in rat mesenteric venules

Exogenously applied caveolin-1 scaffolding domain (CAV) has been shown to inhibit inflammatory mediator-induced nitric oxide (NO) production and NO-mediated increases in microvessel permeability. However, the effect of CAV on endothelial basal NO that prevents leukocyte adhesion remains unknown. This study aims to investigate the roles of exogenously applied CAV in endothelial basal NO production, leukocyte adhesion, and adhesion-induced changes in microvessel permeability. Experiments were conducted in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). NO was quantified with fluorescence imaging in DAF-2-loaded vessels. Perfusing venules with CAV inhibited basal NO production without affecting basal Lp. Resuming blood flow in CAV-perfused vessels significantly increased leukocyte adhesion. The firmly adherent leukocytes altered neither basal Lp nor adherens junction integrity. Increases in Lp occurred only upon formyl-Met-Leu-Phe application that induces release of reactive oxygen species from the adherent leukocytes. The application of NO synthase inhibitor showed similar results to CAV, and NO donor abolished CAV-mediated leukocyte adhesion. Immunofluorescence staining showed increases in binding of ICAM-1 to an adhesion-blocking antibody concurrent with a Src-dependent ICAM-1 phosphorylation following CAV perfusion. Pre-perfusing vessels with anti-ICAM-1 blocking antibody or a Src kinase inhibitor attenuated CAV-induced leukocyte adhesion. These results indicate that the application of CAV, in addition to preventing excessive NO-mediated permeability increases, also causes reduction of basal NO and promotes ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.