RAGE antagonist peptide mitigates AGE-mediated endothelial hyperpermeability and accumulation of glycoxidation products in human ascending aortas and in a murine model of aortic aneurysm

Background: Aortic dissection and aneurysm are the result of altered biomechanical forces associated with structural weakening of the aortic wall caused by genetic or acquired factors. Current guidelines recommend replacement of the ascending aorta when the diameter is >5.5 cm in tricuspid aortic valve patients. Aortopathies are associated with altered wall stress and stiffness as well as endothelial cell dysfunction and synthetic vascular smooth muscle cell (VSMC) phenotype. We reported that these mechanisms are mediated by glycoxidation products [Reactive oxygen species (ROS) and Advance Glycation End products (AGE)]. This study addresses the role of glycoxidation on endothelial function and AGE-mediated aortic stiffness. Hypothesis and aims: Here we investigate how circulating glycation products infiltrate the aortic wall via AGE-mediated endothelial hyperpermeability and contribute to both VSMC synthetic phenotype and extracellular matrix (ECM) remodeling in vivo and ex vivo. We also study how RAGE antagonist peptide (RAP) can rescue the effect of AGEs in vitro and in vivo in eNOS-/- vs WT mice. Methods and results: Human ascending aortas (n=30) were analyzed for AGE, ROS, and ECM markers. In vitro glycation was obtained by treating VSMC or human and murine aortas with glyoxal. Endothelial permeability was measured under glycation treatment. Vascular stiffness was measured by a pressure myograph comparing wild-type mice in the absence of presence of glyoxal. eNOS-/- mice, a model of increased endothelial permeability, were treated for 28 days with hyperlipidemic diet and Angiotensin II (1000ng/kg/min) with or without anti-glycation treatment (RAP 20mg/kg). Echo data of aortic diameter were collected. Murine vascular stiffness was measured by a pressure myograph (n=5/group). Glycoxidation products were detected in all human aortas independently of aortic diameter, with stronger accumulation on the lumen and the adventitia layer. AGEs increased endothelial permeability, induce synthetic phenotypic switch in human VSMCs, and inhibit cell migration. RAP pre-treatment rescue the effect of glyoxal on endothelial cells. Ex vivo glycation treatment of murine arteries impacted on ECM and increased stiffness. Aortic stiffness was higher in eNOS-/- vs WT mice. Ang II-mediated aortopathies results in aortic dilation, and AGE/ROS accumulation, which is rescued by RAGE antagonist peptide treatment of eNOS-/- mice. Conclusions: Glycoxidation reaction mediates EC permeability, VSMCs phenotype, and ECM remodeling leading to dysfunctional microstructure of the ascending aorta, altered vascular stiffness and increasing aortic susceptibility to dilation and rupture. Moreover, we show that RAP can mitigate AGE-mediated endothelial hyper-permeability in vitro and impact on ascending aneurysm in vivo

Experimental faecal peritonitis alters response to 5-hydroxytryptamine in mesenteric lymphatic vessels

Aim. To compare the reactions to 5-hydroxytryptamine (5-HT, serotonin) in the mesenteric lymphangions upon endothelial denudation and during experimental faecal peritonitis as compared with the control lymphangions. Materials and Methods. Experiments were performed on the intestinal lymph trunk lymphangions of rats using a pressure myograph system. We examined the changes in contraction frequency and amplitude as well as tonic reactions upon the addition of 5-HT (10-8-10-4 M). Experimental peritonitis was caused by an intraperitoneal injection of feces. Results. Faecal peritonitis reduced contraction frequency and amplitude in mesenteric lymphangions. 5-HT increased contraction amplitude only at low concentration (10-8 M) and did not alter the tonic reactions. Upon endothelial denudation, serotonin inhibited contraction frequency and amplitude in the lymphangions. As 5-HT stimulates motility through 5-НТ2 receptors and α2-adrenoceptors on endothelial cells, faecal peritonitis abates the sensitivity of such receptors to 5-HT. Conclusion. In experimental faecal peritonitis, alterations in lymphatic vessels are reminiscent of those in blood vessels. Endothelial dysfunction disturbs the response of lymphatic vessels to 5-HT.

Superoxide inhibition restores endothelium-dependent dilatation in aging arteries by enhancing impaired adherens junctions

Endothelium-dependent, nitric oxide-mediated dilatation is impaired in aging arteries. The dysfunction reflects increased production of reactive oxygen species (ROS), is reversed by inhibiting superoxide with superoxide dismutase (SOD) mimics, and is assumed to reflect superoxide-mediated inactivation of nitric oxide. However, the dysfunction also reflects Src-dependent degradation and loss of vascular-endothelial (VE)-cadherin from adherens junctions, resulting in a selective impairment in the ability of the junctions to amplify endothelial dilatation. Experiments therefore tested the hypothesis that SOD mimics might restore endothelial dilation in aging arteries by inhibiting Src and protecting endothelial adherens junctions. Tail arteries from young and aging Fisher 344 rats were processed for functional (pressure myograph), biochemical (immunoblot), and morphological (immunofluorescence) analyses. Cell-permeable SOD mimics [manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) or tempol] did not affect acetylcholine-induced dilatation in young arteries but increased responses and restored normal dilator function in aging arteries. In aging arteries, MnTMPyP decreased Src activity (immunoblots of Tyr416 phosphorylated compared with total Src), increased the intensity and width of VE-cadherin staining at endothelial junctions, and increased VE-cadherin levels in Triton X-100-insoluble lysates, which represents the junctional protein. Because of aging-induced junctional disruption, inhibiting VE-cadherin clustering at adherens junctions with a function-blocking antibody does not affect acetylcholine-induced dilatation in aging arteries. However, the antibody prevented SOD mimics from restoring acetylcholine-induced dilatation in aging arteries. Therefore, SOD mimics improve impaired adherens junctions in aging endothelium, which is essential for SOD mimics to restore endothelium-dependent dilatation in aging arteries. The results suggest an important new pathological role for ROS in aging endothelium, namely, disruption of adherens junctions. NEW & NOTEWORTHY Aging-induced endothelial dysfunction is reversed by SOD mimics. This study demonstrates that they improve impaired adherens junctions in aging endothelium and that their restoration of endothelial dilatation is dependent on increased junctional activity. The results suggest a novel role for oxygen radicals in vascular aging, namely, disruption of adherens junctions.

Dynamic micro- and macrovascular remodeling in coronary circulation of obese Ossabaw pigs with metabolic syndrome

Previous studies from our laboratory showed that coronary arterioles from type 2 diabetic mice undergo inward hypertrophic remodeling and reduced stiffness. The aim of the current study was to determine if coronary resistance microvessels (CRMs) in Ossabaw swine with metabolic syndrome (MetS) undergo remodeling distinct from coronary conduit arteries. Male Ossabaw swine were fed normal ( n = 7, Lean) or hypercaloric high-fat ( n = 7, MetS) diets for 6 mo, and then CRMs were isolated and mounted on a pressure myograph. CRMs isolated from MetS swine exhibited decreased luminal diameters (126 ± 5 and 105 ± 9 μm in Lean and MetS, respectively, P < 0.05) with thicker walls (18 ± 3 and 31 ± 3 μm in Lean and MetS, respectively, P < 0.05), which doubled the wall-to-lumen ratio (14 ± 2 and 30 ± 2 in Lean and MetS, respectively, P < 0.01). Incremental modulus of elasticity (IME) and beta stiffness index (BSI) were reduced in CRMs isolated from MetS pigs (IME: 3.6 × 106 ± 0.7 × 106 and 1.1 × 106 ± 0.2 × 106 dyn/cm2 in Lean and MetS, respectively, P < 0.001; BSI: 10.3 ± 0.4 and 7.3 ± 1.8 in Lean and MetS, respectively, P < 0.001). BSI in the left anterior descending coronary artery was augmented in pigs with MetS. Structural changes were associated with capillary rarefaction, decreased hyperemic-to-basal coronary flow velocity ratio, and augmented myogenic tone. MetS CRMs showed a reduced collagen-to-elastin ratio, while immunostaining for the receptor for advanced glycation end products was selectively increased in the left anterior descending coronary artery. These data suggest that MetS causes hypertrophic inward remodeling of CRMs and capillary rarefaction, which contribute to decreased coronary flow and myocardial ischemia. Moreover, our data demonstrate novel differential remodeling between coronary micro- and macrovessels in a clinically relevant model of MetS.

The Effects of Poststroke Captopril and Losartan Treatment on Cerebral Blood Flow Autoregulation in SHRsp with Hemorrhagic Stroke

The ability of captopril and losartan treatment to restore cerebral blood flow (CBF) autoregulation after intracerebral hemorrhagic stroke (HS) was assessed in Kyoto–Wistar stroke-prone hypertensive rats (SHRsp). Laser Doppler techniques assessed CBF autoregulation in the middle cerebral artery (MCA) perfusion domain and a pressure myograph was used to measure pressure-dependent constriction (PDC) in isolated MCAs before and after stroke and after 13, 33, and 63 days of poststroke captopril or losartan treatment. The treatments did not lower blood pressure (BP) and equally suppressed plasma aldosterone after HS. The HS development was associated with the loss of CBF autoregulation, high CBF, increased CBF conductance to elevations in BP, and the loss of PDC in the MCAs. Both treatments restored these functions to prestroke levels within 13 days. The PDC and CBF autoregulation subsequently deteriorated after 63 days of captopril treatment while being maintained at prestroke levels over all durations of losartan treatment. The SHRsp subjected to 35 days of poststroke losartan treatment exhibited less blood–brain barrier (BBB) disruption and brain herniation than captopril-treated SHRsp. The superior ability of losartan to restore CBF autoregulation and myogenic function may have contributed to the more effective attenuation of cerebral damage after HS.

Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220–300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40–90 mg·kg−1·h−1 iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 μm fluorescently labeled microspheres. N/OFQ (0.6–60 nmol/kg iv) caused hypotension (SAP, baseline: 154 ± 11 mmHg, 15 nmol/kg N/OFQ: 112 ± 10 mmHg, P = 0.009), vasodilation (venules: 23.9 ± 1.2 μm, 26.7 ± 1.2 μm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 ± 3.4, 123.5 ± 11.8, P = 0.009), and leukocyte adhesion (2.0 ± 0.9, 15.2 ± 0.9/100 μm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 ± 370 μm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 ± 0.13 vs. 2.06 ± 0.38, P < 0.05). The NOP antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101; 60 and 150 nmol/kg iv), H1 and H2antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg·kg−1·min−1) also abolished vasodilation and macromolecular leak to N/OFQ in vivo ( P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (∼200 μm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10−5 M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: −6.9 ± 3.8%; N/OFQ: 32.6 ± 8.4%; pyrilamine: 31.5 ± 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo.

Extravascular pressure modulates responses of isolated rat coronary arteries to vasodilator, but not vasoconstrictor, stimuli

The aims of the study were to investigate whether elevated extravascular pressure modulates responses of isolated rat coronary arteries to constrictor and dilator stimuli. Isolated segments of rat coronary artery were mounted in a modified pressure myograph system that allowed independent modulation of both intra- and extravascular pressures. The influence of elevated extravascular pressure on stable levels of myogenic tone and on responses to vasoconstrictor and vasodilator stimuli was investigated at constant overall transmural pressures. Stable levels of myogenic tone were independent of the relative levels of intra- and extravascular pressure, as were responses to depolarization and to addition of the thromboxane agonist U-46619. Elevating extravascular pressure, however, significantly reduced dilatory responses to introduction of intraluminal flow and to addition of endothelium-dependent and endothelium-independent vasodilatory agonists. These results support the notion that elevated extravascular pressure may attenuate responses of coronary arteries to a variety of dilatory stimuli. This finding may be of relevance to cardiac disorders associated with elevated ventricular pressures.

Altered circulating hormone levels, endothelial function and vascular reactivity in the testicular feminised mouse

OBJECTIVE: Testicular feminised (Tfm) mice express a non-functional androgen receptor, and also have reduced levels of circulating testosterone. Recent studies support a cardio-protective role for testosterone since it elicits systemic and pulmonary vasodilatation. The aim of the present study was to determine whether androgen insensitivity and hypotestosteronaemia in the Tfm mouse are associated with abnormal vascular reactivity or hormone status. METHODS: Adult male Tfm and littermate control mice were killed and the blood collected. Femoral (diameter range = 183-508 microm) and pulmonary (diameter range = 320-816 microm) arteries were dissected and loaded in either a wire or pressure myograph, at 100 mmHg or 17.5 mmHg respectively. Pharmacological assessment of the vasoreactivity to potassium chloride (KCl, 80 mmol/l) and either noradrenaline (NA, 1 nmol/l-100 micromol/l) and acetylcholine (ACh, 0.1-100 micromol/l) or testosterone (1 nmol/l-100 micromol/l) was then made. RESULTS: Tfm mice had reduced levels of testosterone (1.8+/-0.3 nmol/l) compared with controls (9.3+/-2.0 nmol/l, P<0.001) and elevated levels of cholesterol (3.6+0.1 mmol/l) compared with controls (3.2+0.1 mmol/l, P<0.05). Femoral arteries from Tfm mice exhibited reduced vasoconstriction to 80 mmol/l KCl (3.27+/-0.23 mN/mm) compared with vessels from controls (4.44+/-0.41 mN/mm, P<0.05), and reduced endothelial-dependent vasodilatation to 0.1-100 micromol/l ACh (23.3+/-3.6% relaxation) compared with vessels from controls (41.6+/-5.4% relaxation, P<0.05). Vasoconstriction to NA (1 nmol/l-100 micromol/l) and vasodilatation to testosterone were unaffected. CONCLUSIONS: Androgen receptor deficiency and hypotestosteronaemia in the Tfm mouse reduced endothelial function and impaired voltage-operated calcium channel activity, which may pre-dispose to cardiovascular disease. Testosterone-induced vasodilatation was unaffected, demonstrating no involvement of the androgen receptor in this response.