Urocortin protects coronary endothelial function during ischemia-reperfusion: a brief communication

Urocortin is a vasodilator peptide related to corticotrophin-releasing factor, which may protect myocardium during coronary ischemia-reperfusion. To study whether urocortin also protects coronary endothelial function during ischemia-reperfusion, hearts from Sprague-Dawley rats were perfused at constant flow and then exposed to 15 mins ischemia followed by 15 mins reperfusion. In one series of experiments, we found that the coronary relaxation to urocortin (10(-11) to 10(-8) M) was reduced by ischemia-reperfusion (51 +/- 4% vs. 79 +/- 4% of the active tone, for the 10(-10) Mdose). In other series of experiments, we observed that ischemia-reperfusion reduced the coronary relaxation to a test dose of acetylcholine (10(-6) M) (25 +/- 2% vs. 54 +/- 9% of active tone), without modifying the relaxation to sodium nitroprusside (10(-6) M). Treatment with a low threshold concentration of urocortin (10(-11) M), administered before ischemia and during reperfusion, partly improved the coronary relaxation to acetylcholine (36 +/- 3% of active tone). These results suggest that ischemia-reperfusion impairs the coronary vasodilation to urocortin and produces endothelial dysfunction and that this endothelial dysfunction may be improved by urocortin.     

Role of adhesion and contraction in Rac 1-regulated endothelial barrier function in vivo and in vitro

We demonstrated previously that inhibition of the small GTPase Rac-1 by Clostridium sordellii lethal toxin (LT) increased the hydraulic conductivity (L(p)) of rat venular microvessels and induced gap formation in cultured myocardial endothelial cells (MyEnd). In MyEnd cells, we also demonstrated that both LT and cytochalasin D reduced cellular adhesion of vascular endothelial (VE)-cadherin-coated beads. Here we further evaluate the contribution of actin depolymerization, myosin-based contraction, and VE-cadherin linkage to the actin cytoskeleton to LT-induced permeability. The actin-depolymerizing agent cytochalasin D increased L(p) in single rat mesenteric microvessels to the same extent as LT over 80 min. However, whereas the actin-stabilizing agent jasplakinolide blunted the L(p) increase due to cytochalasin D by 78%, it had no effect on the LT response. This conforms to the hypothesis that the predominant mechanism whereby Rac-1 stabilizes the endothelial barrier in intact microvessels is separate from actin polymerization and likely at the level of the VE-cadherin linkage to the actin cytoskeleton. In intact vessels, neither inhibition of contraction (butanedione monoxime, an inhibitor of myosin ATPase) nor inhibition of Rho kinase (Y-27632) modified the response to LT, even though both inhibitors lowered resting L(p). In contrast butanedione monoxime and inhibition of myosin light chain kinase completely inhibited LT-induced intercellular gap formation and largely reduced the LT-induced permeability increase in MyEnd monolayers. These results support the hypothesis that the contractile mechanisms that contribute to the formation of large gaps between cultured endothelial cells exposed to inflammatory conditions do not significantly contribute to increased permeability in intact microvessels.     

Adrenomedullin stabilizes the lymphatic endothelial barrier in vitro and in vivo

The lymphatic vascular system functions to maintain fluid homeostasis by removing fluid from the interstitial space and returning it to venous circulation. This process is dependent upon the maintenance and modulation of a semi-permeable barrier between lymphatic endothelial cells of the lymphatic capillaries. However, our understanding of the lymphatic endothelial barrier and the molecular mechanisms that govern its function remains limited. Adrenomedullin (AM) is a 52 amino acid secreted peptide which has a wide range of effects on cardiovascular physiology and is required for the normal development of the lymphatic vascular system. Here, we report that AM can also modulate lymphatic permeability in cultured dermal microlymphatic endothelial cells (HMVEC-dLy). AM stimulation caused a reorganization of the tight junction protein ZO-1 and the adherens protein VE-cadherin at the plasma membrane, effectively tightening the endothelial barrier. Stabilization of the lymphatic endothelial barrier by AM occurred independently of changes in junctional protein gene expression and AM^−/− endothelial cells showed no differences in the gene expression of junctional proteins compared to wildtype endothelial cells. Nevertheless, local administration of AM in the mouse tail decreased the rate of lymph uptake from the interstitial space into the lymphatic capillaries. Together, these data reveal a previously unrecognized role for AM in controlling lymphatic endothelial permeability and lymphatic flow through reorganization of junctional proteins.     

The lectin-like domain of complement receptor 3 protects endothelial barrier function from activated neutrophils

The adhesion of neutrophils to endothelial cells is a central event leading to diapedesis and involves the binding of the I-domain of beta(2) integrins (CD11/CD18) to endothelial ICAMs. In addition to the I-domain, the beta(2) integrin complement receptor 3 (CR3) (CD11b/CD18) contains a lectin-like domain (LLD) that can alter leukocyte functions such as chemotaxis and cytotoxicity. The present study demonstrates that, in contrast to the CR3 I-domain, Ab blockade of the CR3 LLD has no role in mediating neutrophil-induced loss of endothelial barrier function. However, activation of CR3 with the LLD agonist beta-glucan protects the barrier function of endothelial cells in the presence of activated neutrophils and reduces transendothelial migration without affecting adhesion of the neutrophils to the endothelium. The LLD site-specific mAb VIM12 obviates beta-glucan protection while activation of the LLD by VIM12 cross-linking mimics the beta-glucan response by both preserving endothelial barrier function and reducing neutrophil transendothelial migration. beta-glucan has no direct effect on endothelial cell function in the absence of activated neutrophils. These findings demonstrate that signaling through the CR3 LLD prevents neutrophil-induced loss of endothelial barrier function and reduces diapedesis. This suggests that the LLD may be a suitable target for oligosaccharide-based anti-inflammatory therapeutics.     

Ena/VASP is required for endothelial barrier function in vivo

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell–cell and cell–matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we establish the importance of Ena/VASP in endothelial junctions in vivo by analysis of Ena/VASP-deficient animals. In the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and late stage embryonic lethality. In endothelial cells, we find that Ena/VASP activity is required for normal F-actin content, actomyosin contractility, and proper response to shear stress. These findings demonstrate that Ena/VASP is critical for actin cytoskeleton remodeling events involved in the maintenance of functional endothelia.     

Spatial variations in endothelial barrier function in disturbed flows in vitro

Hindered barrier function has been implicated in the initiation and progression of atherosclerosis, a disease of focal nature associated with altered hemodynamics. In this study, endothelial permeability to macromolecules and endothelial electrical resistance were investigated in vitro in monolayers exposed to disturbed flow fields that model spatial variations in fluid shear stress found at arterial bifurcations. After 5 h of flow, areas of high shear stress gradients showed a 5.5-fold increase in transendothelial transport of dextran (molecular weight 70,000) compared with no-flow controls. Areas of undisturbed fully developed flow, within the same monolayer, showed a 2.9-fold increase. Monolayer electrical resistance decreased with exposure to flow. The resistance measured during flow and the rate of change in monolayer resistance after removal of flow were lowest in the vicinity of flow reattachment (highest shear stress gradients). These results demonstrate that endothelial barrier function and permeability to macromolecules are regulated by spatial variations in shear stress forces in vitro.     

L-arginine protects from ischemia-reperfusion-induced endothelial dysfunction in humans in vivo.

It has been shown that nitric oxide (NO) protects from myocardial ischemia-reperfusion injury in animal models. The present study investigated whether administration of the NO substrate l-arginine protects against ischemia-reperfusion-induced endothelial dysfunction in humans. Forearm blood flow was measured with venous occlusion plethysmography in 16 healthy male subjects who were investigated on two occasions. Forearm ischemia was induced for 20 min followed by 60-min reperfusion. With the use of a crossover protocol, the subject received a 15-min intrabrachial artery infusion of l-arginine (20 mg/min) and vehicle (saline, n = 12 or d-arginine, n = 4) starting at 15 min of ischemia on two separate occasions. Compared with preischemia, endothelium-dependent increase in forearm blood flow induced by intra-arterial acetylcholine (3-30 microg/min) was significantly impaired at 15 and 30 min of reperfusion when the subjects received saline (P < 0.001). When the subjects received l-arginine, the acetylcholine-induced increase in forearm blood flow was not significantly affected by ischemia-reperfusion. The recovery of endothelium-dependent vasodilatation at 15- and 30-min reperfusion was significantly greater after administration of l-arginine than after saline (P < 0.05). d-Arginine did not affect the response to acetylcholine. Endothelium-independent vasodilatation to nitroprusside was not affected during reperfusion. These results demonstrate that the NO substrate l-arginine significantly attenuates ischemia-reperfusion-induced endothelial dysfunction in humans in vivo. This suggests that l-arginine may be useful as a therapeutic agent in the treatment of ischemia-reperfusion injury in humans.     

Pterostilbene protects vascular endothelial cells against oxidized low-density lipoprotein-induced apoptosis in vitro and in vivo

Vascular endothelial cell (VEC) apoptosis is the main event occurring during the development of atherosclerosis. Pterostilbene (PT), a natural dimethylated analog of resveratrol, has been the subject of intense research in cancer and inflammation. However, the protective effects of PT against oxidized low-density lipoprotein (oxLDL)-induced apoptosis in VECs have not been clarified. We investigated the anti-apoptotic effects of PT in vitro and in vivo in mice. PT at 0.1–5 μM possessed antioxidant properties comparable to that of trolox in a cell-free system. Exposure of human umbilical vein VECs (HUVECs) to oxLDL (200 μg/ml) induced cell shrinkage, chromatin condensation, nuclear fragmentation, and cell apoptosis, but PT protected against such injuries. In addition, PT injection strongly decreased the number of TUNEL-positive cells in the endothelium of atherosclerotic plaque from apoE^−/− mice. OxLDL increased reactive oxygen species (ROS) levels, NF-κB activation, p53 accumulation, apoptotic protein levels and caspases-9 and -3 activities and decreased mitochondrial membrane potential (MMP) and cytochrome c release in HUVECs. These alterations were attenuated by pretreatment with PT. PT inhibited the expression of lectin-like oxLDL receptor-1 (LOX-1) expression in vitro and in vivo. Cotreatment with PT and siRNA of LOX-1 synergistically reduced oxLDL-induced apoptosis in HUVECs. Overexpression of LOX-1 attenuated the protection by PT and suppressed the effects of PT on oxLDL-induced oxidative stress. PT may protect HUVECs against oxLDL-induced apoptosis by downregulating LOX-1-mediated activation through a pathway involving oxidative stress, p53, mitochondria, cytochrome c and caspase protease. PT might be a potential natural anti-apoptotic agent for the treatment of atherosclerosis.     

Activation of estrogen receptor-alpha protects the in vivo rabbit heart from ischemia-reperfusion injury

The estrogen receptor (ER) mediates estrogenic activity in a variety of organs, including those in the reproductive, cardiovascular, immune, and central nervous systems. Experimental studies have demonstrated that 17beta-estradiol (E2) protects the heart from ischemia-reperfusion injury. Two estrogen receptors, ER alpha and ER beta, mediate the actions of estrogen; however, it is not certain which ER mediates the cardioprotective effects of E2. In the present study, the ER-selective agonists 4,4',4'-[4-propyl-(1H)-pyrazole-1,3,5-triyl]tris-phenol (PPT; ER alpha) and 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ER beta) were assessed for their cardioprotective potential in an in vivo rabbit model of ischemia-reperfusion injury. Anesthetized female rabbits were administered PPT (3 mg/kg), DPN (3 mg/kg), E2 (20 microg/rabbit), or vehicle intravenously 30 min before a 30-min occlusion of the left anterior descending coronary artery followed by 4 h of reperfusion. Acute treatment with E2 (17.7 +/- 2.9%; P < 0.001) and PPT (18.1 +/- 2.9%; P < 0.001), but not DPN (45.3 +/- 2.4%) significantly decreased infarct size as a percent of area at risk compared with vehicle (45.3 +/- 2.4%). Coadministration of PPT or E2 with the ER antagonist ICI-182,780 limited the infarct size-sparing effect of the compounds (43.8 +/- 6.6% and 40.6 +/- 5.7% respectively, expressed as a percentage of risk region). PPT reduced the release of cardiac-specific troponin-I and reduced the tissue deposition of the membrane attack complex and C-reactive protein similar to that of E2. The results indicate that activation of ER alpha, but not ER beta, is required for the observed cardioprotective effects of E2.     

Glucocorticoids and endothelial cell barrier function

Glucocorticoids (GCs) are steroid hormones that have inflammatory and immunosuppressive effects on a wide variety of cells. They are used as therapy for inflammatory disease and as a common agent against edema. The blood brain barrier (BBB), comprising microvascular endothelial cells, serves as a permeability screen between the blood and the brain. As such, it maintains homeostasis of the central nervous system (CNS). In many CNS disorders, BBB integrity is compromised. GC treatment has been demonstrated to improve the tightness of the BBB. The responses and effects of GCs are mediated by the ubiquitous GC receptor (GR). Ligand-bound GR recognizes and binds to the GC response element located within the promoter region of target genes. Transactivation of certain target genes leads to improved barrier properties of endothelial cells. In this review, we deal with the role of GCs in endothelial cell barrier function. First, we describe the mechanisms of GC action at the molecular level. Next, we discuss the regulation of the BBB by GCs, with emphasis on genes targeted by GCs such as occludin, claudins and VE-cadherin. Finally, we present currently available GC therapeutic strategies and their limitations.     

Adrenomedullin protects rat cerebral endothelial cells from oxidant damage in vitro

Increased permeability and reduced cerebral endothelial cell (CEC) viability induced by oxidative stress are the hallmarks of the blood-brain barrier disruption. In our experiments hydrogen peroxide (H2O2, 0.5 mM) induced a continuous decrease of the transendothelial electrical resistance (TEER) and resulted in intercellular gap formations in cultured rat CECs. Adrenomedullin (AM) increased TEER, enhanced peripheral localization of F-actin bands and attenuated the increased permeability induced by H2O2. Furthermore, AM treatment preserved mitochondrial membrane potential, attenuated cytochrome c release, and consequently improved CEC viability in H2O2 treated cultures. These results suggest that AM treatment protects CECs against oxidative injury.     

Serotonin,norepinephrine, and histamine mediation of endothelial cell barrier function in vitro

The effects of serotonin (5-hydroxytryptamine, 5-HT), norepinephrine (NE), and histamine on endothelial cell barrier function were examined in vitro. Bovine aortic endothelial (BAE) cells grown to confluence on microcarriers formed a measurable barrier to the passage of a trypan blue dye-bovine serum albumin conjugate (TB-BSA) from the culture medium into the microcarrier matrix. Vascular smooth muscle (VSM) cells or Swiss 3T3 fibroblasts impeded TB-BSA diffusion only 42% and 56%, respectively, relative to BAE cells. These results suggest that barrier formation may be an endothelial cell-specific phenomenon. Treatment of BAE cells with histamine was associated with 2-to 3-fold increases in the rate of TB-BSA diffusion. In contrast, treatment with 5-HT or NE at concentrations ranging from normal to pathophysiological circulating plasma levels significantly impeded TB-BSA diffusion by up to 43% and 33%, respectively, relative to untreated controls. The barrier-modulating effects of the vasoactive amines were dose-dependent, cell-specific, and in some cases appear to be receptor-mediated. These results are consistent with previous reports that histamine increases vascular permeability in part by affecting diffusion between endothelial cells; they support the hypothesis that 5-HT and NE contribute to the maintenance of the endothelial barrier in vivo.     

Cardamonin protects septic mice from acute lung injury by preventing endothelial barrier dysfunction

Cardamonin, a flavone compound isolated from Alpinia katsumadai Heyata seeds, has been reported to possess anti-inflammatory and anticoagulative activities, and it might be beneficial for management of sepsis. This study was conducted to examine the protective effects of cardamonin on experimental sepsis and resultant acute lung injury (ALI). Cardamonin (30 and 100 mg/kg) significantly elevated the survival rate of septic mice, alleviated ALI and lung microvascular leak, and lowered the serum levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6. In vitro, it (25 and 50 μM) concentration dependently inhibited endothelium permeability and downregulated phosphorylation of P38 in rat lung microvascular endothelial cells induced by lipopolysaccharide (LPS). P38 inhibitor inhibited the endothelium permeability. In RAW 264.7 macrophage cells, cardamonin also showed selective inhibition of P38 phosphorylation induced by LPS. These results indicate that cardamonin can protect septic mice from ALI by preventing endothelium barrier dysfunction via selectively inhibiting P38 activation.     

Pyridoxamine protects proteins from damage by hypohalous acids in vitro and in vivo

Diabetes is characterized, in part, by activation of toxic oxidative and glycoxidative pathways that are triggered by persistent hyperglycemia and contribute to diabetic complications. Inhibition of these pathways may benefit diabetic patients by delaying the onset of complications. One such inhibitor, pyridoxamine (PM), had shown promise in clinical trials. However, the mechanism of PM action in vivo is not well understood. We have previously reported that hypohalous acids can cause disruption of the structure and function of renal collagen IV in experimental diabetes (K.L. Brown et al., Diabetes64:2242–2253, 2015). In the present study, we demonstrate that PM can protect protein functionality from hypochlorous and hypobromous acid-derived damage via a rapid direct reaction with and detoxification of these hypohalous acids. We further demonstrate that PM treatment can ameliorate specific hypohalous acid-derived structural and functional damage to the renal collagen IV network in a diabetic animal model. These findings suggest a new mechanism of PM action in diabetes, namely sequestration of hypohalous acids, which may contribute to known therapeutic effects of PM in human diabetic nephropathy.     

Ascorbate protects endothelial barrier function during septic insult: Role of protein phosphatase type 2A

Endothelial barrier dysfunction contributes to morbidity in sepsis. We tested the hypothesis that raising the intracellular ascorbate concentration protects the endothelial barrier from septic insult by inhibiting protein phosphatase type 2A. Monolayer cultures of microvascular endothelial cells were incubated with ascorbate, dehydroascorbic acid (DHAA), the NADPH oxidase inhibitors apocynin and diphenyliodonium, or the PP2A inhibitor okadaic acid and then were exposed to septic insult (lipopolysaccharide and interferon-γ). Under standard culture conditions that depleted intracellular ascorbate, septic insult stimulated oxidant production and PP2A activity, dephosphorylated phosphoserine and phosphothreonine residues in the tight junction-associated protein occludin, decreased the abundance of occludin at cell borders, and increased monolayer permeability to albumin. NADPH oxidase inhibitors prevented PP2A activation and monolayer leak, showing that these changes required reactive oxygen species. Okadaic acid, at a concentration that inhibited PP2A activity and monolayer leak, prevented occludin dephosphorylation and redistribution, implicating PP2A in the response of occludin to septic insult. Incubation with ascorbate or DHAA raised intracellular ascorbate concentrations and mitigated the effects of septic insult. In conclusion, ascorbate acts within microvascular endothelial cells to inhibit septic stimulation of oxidant production by NADPH oxidase and thereby prevents PP2A activation, PP2A-dependent dephosphorylation and redistribution of occludin, and disruption of the endothelial barrier.     

Estrogen receptor-beta signaling protects epidermal cytokine expression and immune function from UVB-induced impairment in mice.

A previous study in the hairless mouse, in which the photoimmune protective properties of a topical phytoestrogen or 17-beta-estradiol were abrogated by the estrogen receptor antagonist ICI 182,780, revealed that estrogen receptor (Er) signaling is involved in the regulation of the suppression of immune function by UVB (290-320 nm) radiation. Here we identify the expression of Er-beta but not Er-alpha mRNA in hairless mouse skin, whereas Er-alpha and Er-beta mRNA were present in normal haired mouse skin. This suggests that the non-classical estrogen target Er-beta is involved in the photoimmune modulation, and is consistent with Er-alpha being more closely associated with hair growth control, as indicated by other studies. In mice with a null mutation for Er-beta, there was a significant exacerbation of the solar simulated UV (290-400 nm)-induced suppression of contact hypersensitivity. Immunohistochemical analysis revealed that the Er-beta deficiency inhibited the normally immunoprotective upregulation by the UVA (320-400 nm) waveband of the epidermal expression of the cytokines IFN-gamma and IL-12. Er-beta deficiency also significantly increased the UVB-induced expression of the immunosuppressive cytokine IL-10. Thus Er signalling via the Er-beta is evidently a major regulator of the UVA and UVB waveband interactions that determine the skin's immune functional status, and achieves this by normalization of the cutaneous cytokine array in the UV-irradiated skin.     

Mechanisms regulating endothelial cell barrier function

Endothelium forms a physical barrier that separates blood from tissue. Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells. Inflammation promotes macromolecular transport by decreasing cell-cell and cell-matrix adhesion and increasing centripetally directed tension, resulting in the formation of intercellular gaps. Inflammation may also increase the selected transport of macromolecules through cells. Significant progress has been made in understanding the molecular and cellular mechanisms that account for constitutive endothelial cell barrier function and also the mechanisms activated during inflammation that reduce barrier function. Current concepts of mechanisms regulating endothelial cell barrier function were presented in a symposium at the 2000 Experimental Biology Conference and are reviewed here.     

RhoA inactivation enhances endothelial barrier function

The modulation of endothelial barrier function is thought to bea function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA). We tested this hypothesis by dissociating SF/FA withClostridium botulinum exoenzyme C3transferase (C3), an inhibitor of the small GTP-binding protein RhoA.Bovine pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombin, thrombin, or no treatment were examined using asize-selective permeability assay and quantitative digital imagingmeasurements of SF/FA. C3 treatment disassembled SF/FA, stimulateddiffuse myosin II immunostaining, and reduced the phosphotyrosine (PY)content of paxillin and 130- to 140-kDa proteins that includedp125^FAK. C3-treated monolayersdisplayed a 60-85% decline in F-actin content and a170-300% increase in EC surface area with enhanced endothelialbarrier function. This activity correlated with reorganization ofF-actin and PY protein(s) to -catenin-containing cell-cell junctions. Because C3 prevented the thrombin-induced formation ofmyosin ribbons, SF/FA, and the increased PY content of proteins, thesecharacteristics were Rho dependent. Our data show that C3 inhibition ofRho proteins leads to cAMP-like characteristics of reduced SF/FA andenhanced endothelial barrier function.

     

cAMP protects endothelial barrier functions by preventing Rac-1 inhibition

cAMP enhances endothelial barrier properties and is protective against various inflammatory mediators both in vivo and in vitro. However, the mechanisms whereby cAMP stabilizes the endothelial barrier are largely unknown. Recently we demonstrated that the Rho family GTPase Rac-1 is required for maintenance of endothelial barrier functions in vivo and in vitro. Therefore, in the present study we investigated the effect of forskolin (5 microM)- and rolipram (10 microM)-induced cAMP increase on reduction of barrier functions in response to Rac-1 inhibition by Clostridium sordellii lethal toxin (LT). Forskolin and rolipram treatment blocked LT (200 ng/ml)-induced hydraulic conductivity (Lp) increase in mesenteric microvessels in vivo. Likewise, LT-induced intercellular gap formation in monolayers of cultured microvascular myocardial endothelial (MyEnd) cells and LT-induced loss of adhesion of vascular endothelial cadherin-coated microbeads were abolished. Inhibition of PKA by myristoylated inhibitor peptide (14-22) of PKA (100 microM) reduced the protective effect of cAMP on LT-induced Lp increase in vivo and gap formation in vitro, indicating that the effect of cAMP on Rac-1 inhibition was PKA dependent. Glucosylation assays demonstrated that cAMP prevents inhibitory Rac-1 glucosylation by LT, indicating that one way that cAMP enhances endothelial barrier functions may be by regulating Rac-1 signaling. Our study suggests that cAMP may provide its well-established protective effects at least in part by regulation of Rho proteins.